^lc.^.^^q^^^^ - tdl

CALCIUM TRANSPORT IN THE PHOTOSYNTHETIC PURPLE

BACTERIUM CHROMATIUM VINOSUM

by

CINDY RENE' PEACH, B.S.

A THESIS

IN

CHEMISTRY

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for

the Degree of

MASTER OF SCIENCE

Approved

• C - ' ^ ^ ^ - j ^ ^ U«t-^

'^Lc.^.^^Q^^^^ Accepted

Dean o ^ t h e Graduate School

May, 1986

FOREWORD

Kalzium macht alles.

L.V. Heilbrunn (1883)

We dance round in a ring and suppose

But the Secret sits in the middle and knows

Robert Frost

11

ACKNOWLEDGMENTS

My deepest gratitude and love to my parents, brothers,

and grandparents, with whose love and constant encouragement

made this accomplisment possible.

I wish to thank the members of my committee. Dr. John

A. Anderson and Dr. Richard A. Bartsch, for their assistance

in the preparation of this thesis. I would like to thank

Dr. Charlotte Kampf for her assistance in growing the

Chromatium vinosum,, advice, and many helpful discussions.

I offer my admiration and heart felt gratitude to Dr.

David B. Knaff for his guidence and assistance throughout my

research and graduate studies.

Ill

CONTENTS

FOREWORD ii

ACKNOWLEDGMENTS iii

TABLES V

FIGURES vi

NOMENCLATURE vii

CHAPTER

I. INTRODUCTION 1

II. MATERIALS AND METHODS 5

Materials 5

Growth of Chromatium vinosum 5

Calcium Transport Experiments 5

Methods 6

Reagent and Solution Preparation 6

Growth and Preparation of Q.. vinosum Whole Cells 7

Preparation of Q.. vinosum Chromatophores . . 9

Filter Assay Technique 9

Flow Dialysis Technique 11

III . RESULTS AND DISCUSSION 15

Ca "*" Uptake Experiments with Whole Cells.... 15

Ca "*" Efflux Experiments with Illuminated Whole Cells 34

Ca Uptake Experiments with Illuminated Chromatophores 42

IV. SUMMARY, CONCLUSION AND DISCUSSION 60

Summary 60

Conclusion and Discussion 62

REFERENCES 64

IV

TABLES

Defined Synthetic Culture Medium for the Growth of the Photosynthetic Bacterium Chromatium vinosum 8

The Effect of Ruthenium Red on 204r[.3 + ^^^ 22 ^ + Uptake by Q.. vinosum Cells in the Dark 27

The Effect of La " on 204rp-L+ ^^^ ^^ua+ Uptake by £. vinosum Cells in the Dark 31

The Effect of Inhibitors and lonophores on ^ Ca2''" Uptake by £. vinosum Cells in the Dark 32

The Effect of Phenothiazine Drugs on ^ Ca2''" Uptake by C. vinosum Cells in in the Dark 35

The Effect of Ruthenium Red and La-"*" on ^^Ca2"^ Efflux from Illuminated Q.. vinosum Cells 37

The Effect of Phenothiazine Drugs on 204.p-j + ^ ^ ^^Na"^ Efflux by Illuminated Q.. vinosum Cells 40

V

FIGURES

1. Schematic representation of the flow dialysis apparatus 12

2. Time course for ^^Ca2'^ uptake by Q.. vinosum cells in the dark 16

3. Lineweaver-Burk Plot for ^ Ca2''" uptake by £. vinosum cells in the dark 19

4. The structures of four calcium antagonists: A. Ruthenium Red, B. Phenothiazine, C. Chlorpromazine, and D. Trifluoperazine 21

5. The effect of ruthenium red on ^ Ca "*" uptake by Q.. vinosum cells in the dark 24

6. The effect of La""*" on ^ Ca "*" uptake by £. vinosum cells in the dark 28

7. The effect of phenothiazine drugs on ^ Ca2''' efflux from illuminated Q. vinosum cells 38

8. The effect of Ca' on proton efflux from £. vinosum chromatophores 43

9. Light-dependent ^ Ca2''" uptake by Q.. vinosum chromatophores 4 6

10. The effect of chlorpromazine on ^ Ca2''' uptake by illuminated Q.. vinosum chromatophores 48

11. The effect of phenothiazine on ^ Ca2''" uptake by illuminated £.. vinosum chromatophores 50

12. The effect of trifluoperazine on ' Ca2''" uptake by illuminated £. vinosum chromatophores 52

13. The effect of ruthenium red on ^^Ca^'^ uptake by illuminated C vinosum chromatophores 55

14. The effect of La3+ on ^ Ca "*" uptake by illuminated Q.. vinosum chromatophores 57

VI

NOMENCLATURE

Bacteriochlorophyll BChl

Calcium Ion Ca "*"

Carbonylcyanide-m-

chlorophenylhydrazine CCCP

Chlorpromazine CPZ

Counts Per Minute cpm

Dimethylsulf oxide DMSO

Disintegrations Per Minute dpm

Electrochemical Proton Gradient M-H"*"

Electrical Membrane Potential A ^

50% Inhibition I^Q

Initial Velocity v^

Inorganic Arsenate Asj^

Lanthanum Ion La^"^

Maximum Velocity V max

Michaelis Constant Kj^

2[N-Morpholino]ethane Sulfonic Acid MES

N,N'-Dicyclohexylcarbo-

diimide DCCD

Phenazine Methosulfate PMS

Phenothiazine PTZ

Potassium Phosphate Buffer KPj_

Potassium Valinomycin K"*" val

Vll

Ruthenium Red RR

Transmembrane Proton Gradient ApH

Trifluoperazine TFP

Vlll

CHAPTER I

INTRODUCTION

It is well established that in eukaryotic systems the

intracellular calcium ion concentration is tightly

controlled and that Ca2"*' plays an important regulatory role

in many cellular functions. However, in prokaryotic

systems, the role of Ca "*" is less well understood and little

firm evidence was available for Ca "*" having any regulatory

functions in these organisms. More recently, evidence has

been obtained that in some microorganisms Ca2"'" fluxes are

involved in the maintenance of appropriate levels of O I

internal Ca^ , chemotactic responses, sporulation,

activation of extracellular enzymes and the assembly of the

cell wall (1-3). As many cellular activities depend on

changes in the free cytosolic Ca "*" ion concentration, it

would be to the organism's advantage to have the means O 1

available to control its internal Ca^ ion concentration.

When needed by an organism, there are two basic mechanisms

by which the concentration of cytosolic Ca2''"can be

increased:

(1) by releasing Ca2"'' ions from intracellular storage

sites,and

(2) by moving Ca "*" ions from the extracellular space,

throught the surface membrane and into the cell.

In general, when transporting any substrate across the cell

membrane, as in the latter case, an organism may use one or

more of the following four mechanisms. The first three

involve what is known as active transport, that is, a

substrate molecule is transported across the cell membrane

against a concentration gradient. (1) Group tr n.qi oration is a process which couples the

2

transport of molecules across the cell membrane to a

chemical modification.

(2) Membrane-bound transport systems involve proteins

or permeases firmly bound to the cell membrane. Substrate

molecules are transported with no modification against a

concentration gradient.

(3) Binding protein transport systems also transport

substrate molecules against a concentration gradient with no

change; however, these transport systems require a soluble

protein component or components which specifically bind the

substrate to be transported. Processes (2) and (3) are

called carrier-mediated transport processes because the

molecular transport is facilitated by a protein carrier.

(4) Simple diffusion involves moving a molecule from

higher concentration to an area of lower concentration

through the cellular membrane. This requires that the

molecule transported be somewhat hydrophobic to enable it to

be soluble in the lipid bilayer. The first three can be

differentiated from the fourth by transport kinetics

utilizing double-reciprocal (Lineweaver-Burk) plots.

The most important structures controlling transport

systems of the second type are the proteins embedded in the

lipid bilayer of the membrane, which function as channels or

pores through which substrate may move. These pores or

channels which transport substrate may also co-transport

another substrate in a variety of manners. If the substrate

of interest is transported into the organism while another

is simultaneously exported, then the channel is considered

to be an antiport (anti=opposite, port=to carry). However,

if the substrate is obligatorily co-transported into the

cell along with another substrate or ion, then the channel

is called a symport (sym=same, port=to carry). In some

cases, the substrate may be moved into the organism without

an additonal substrate or ion involved, in this situation

the channel is named an uniport (uni=one, port=to carry).

3

It has been known for some time that there exists a whole

variety of substrate-specific transport systems for a number

of amino acids, dicarboxylic acids, sugars, cations, and

protons in several different types of bacteria (4).

Recently, many substrate-specific transport systems have

been well characterized in the photosynthetic purple sulfur

bacterium Chromatium vinosum (5-7).

The chemiosmotic hypothesis suggests that bacteria use

an electrochemical proton gradient (A|j.jj+) to drive active

transport. In phototrophic bacteria, A\i^+ is created by

electron flow through the photosynthetic apparatus which is

obligatorily coupled to proton extrusion. The

electrochemical proton gradient consists of two components:

(1) the transmembrane proton gradient (ApH), and

(2) the electrical membrane potential (A^) .

There is strong evidence that most of the membrane-bound

transport systems in bacteria utilize one or both of the two

components of A}ijj+ to supply energy for active transport

(8-14). Antiports, symports and uniports were proposed by

Mitchell (15) as a means of coupling transport to Aiijj+.

Relatively little information is available concerning

the mechanism or mechanisms of cation transport in

photosynthetic bacteria (16,17). There is substantial

evidence demonstrationg the existence of sodium dependent

transport systems in several bacteria (for a detailed review

see reference 4) . Sodium-proton antiports, driven by A\i^+,

have been discovered in the non-photosynthetic bacterium

Barillus alrolophilus (18-20) and the photosynthetic

bacterium Alteromonas baloplanktis (21,22). The presence of

a potassium proton antiport has also been demonstrated in E.

alcolophilus (23).

Recently it has become clear that there exists a number

of potential-dependent Ca "*" channels, each with distict

properties (24). Evidence has been obtained for the

presence of Na' '/H'*' and Ca ' '/H' antiports in the

4

photosynthetic purple non-sulfur bacterium Rhodospirillium

rub rum (25,26). A Ca2"'"/H"'' antiport, as well as Na''"/H' and

K /H"'" antiports, have been shown to be present in Q.. vinosum

(25-27) . In previous experiments performed in this

laboratory, the £. vinosum Ca ' /H''' antiport was shown to be

driven by the electrochemical proton gradient (A|XH+) (2 6) .

Q.. vinosum^ a facultative anaerobe and autotroph (28) ,

was the organism utilized in these studies. The purpose of

the work was further characterization of the Ca2''"/H'*"

antiporter, in particular its inhibitor sensitivity.

Evidence was also obtained to suggest that Q.. vinosum has a

second Ca2'^ transport system, distinct from the Ca2''"/H''"

antiporter. This second tranport system provides a

mechanism for Ca2" entry into the cells. The work presented

in this thesis will address these two Ca "*" transport

systems.

CHAPTER II

MATERIALS AND METHODS

Materials

Growth of Chromatium vinosnm

Potassium phosphate (K2HPO4 and KH2PO4), potassium

chloride (KCl), ammonium chloride (NH4CI), magnesium sulfate

(MgS04•7H2O), disodium ethylenediaminetetraacetic acid

(Na2-EDTA), ferrous chloride (FeCl2*4H2O), zinc chloride

(ZnCl2) f zinc sulfate (ZnS04 •7H2O) , "manganese chloride

(MnCl2•6H2O), boric acid (BH3O3), nickel chloride

(NiCl2*6H2O), sulfuric acid (H2SO4), sodium bicarbonate

(NaHC03), and sodium thiosulfate (Na2S203*5H2O) were

obtained from Fisher Scientific. Calcium chloride

(CaCl2*7H20), sodium molybdate (Na2Mo04•2H2O) and sodium

sulfide (Na2S•9H2O) were purchased from MCB Reagents.

Cobalt chloride (CoCl2*6H20) was purchased from American

Drug and Chemical Company. Cupric chloride (CuCl2'2H20) was

obtained from Merck and Company. DL-Malic acid was obtained

from Sigma Chemical Company.

Membrane filters (0.2 |Xm) used for membrane filtration

sterilization were purchased from Millipore Corporation.

Nitrogen gas (N2) was purchased from Liquid Air Corporation.

Calcium Transport Experiments

[" Ca] Calcium chloride (CaCl2) specific activity, 36.2

mCi/mg Ca, was obtained from ICN Radioisotope Division.

[^^Na]Sodium chloride (NaCl), specific activity, 200 mCi/mg

Na, and [^^^11] thallium sulfate (TI2SO4), specific

activity, 5.3 mCi/mg Tl, were obtained from Amersham

Corporation. [^^C]Methylamine hydrochloride, specific

activitry, 40.0 mCi/mmol, was obtained from New England

6

Nuclear. Ruthenium red (RR) and lanthanum chloride (LaCl3)

were generous gifts from Prof. Pill-Soon Song.

Chlorpromazine (CPZ), trifluoperazine(TFP), phenothiazine

(PTZ), carbonylcyanide-m-chlorophenylhydrazine (CCCP),

N,N'-dicyclohexylcarbodiimide (DCCD), phenazine methosulfate

(PMS) , potassium valinomycin (K"*" valinomycin) , sodium

arsenate (As^), dimethylsulfoxide (DMSO), potassium

phosphate (K2HPO4 and KH2PO4), calcium chloride

(CaCl2*7H20), lithium chloride (LiCl), lithium nitrate

(LiN03), 2[N-morpholino]ethane sulfonic acid (MES), and

choline were purchased from Sigma Chemical Company and used

without futher purification.

Membrane filters (0.45 |im) were purchased from

Millipore Corporation or Amicon. Packard "Filter Count" or

National Diagnostics "Filtron-X" was used as the

scintillation cocktail for filter assays. Spectropore No. 2

dialysis tubing was purchased from Sigma Chemical Company.

Packard "Picofluor" or INC Radiochemicals "Universol" was

used as the scintillation cocktail for aqueous samples.

Methods

Reagent and Solution Preparation

All solutions were made using doubly-distilled water.

All chemicals were reagent grade and obtained from

commercial sources. In transport assays, inhibitors were

added as small aliquots of concentrated stock solutions

using either water or dimethylsulfoxide (DMSO) as the

solvent. The final volume of DMSO was always < 2% (v/v), at

which concentrations DMSO itself was shown to have no effect

on cation transport.

7 Growth and Preparation of C. vinosum Whole Cells

Chromatium vinosum was grown on a malate-containing

medium described by Pfennig at. ai- (2 9) as modified by Kampf

(30) . This method utilizes a medium with increased sulfide

concentration compared to that (31) previously utilized in

our laboratory. With increased sulfide, contaminants such

as Rhodospri11ium rubrum find the environment too harsh for

growth. Table 1 shows the defined synthetic culture medium

used to grow £. vinosum cells. New cultures were inoculated

with 6% of a log phase culture. These cultures were then

allowed to equilibrate for 6 hours in the dark after which

they were placed in the light. All experiments were carried

out on cultures near the end of log phase, that is, cells

that were harvested after illumination at a light intensity

of 7.5 X 10"^ erg'cm~^*s~^ for 2.5 days after inoculation.

Cells were harvested using a Beckman J-21B preparative

centrifuge with a type JA-20 rotor at 4,000 x g for 5

minutes at 5*C. Cells were washed twice and resuspended in

a minimum volume of the buffer to be used in the subsequent

experiment. The resuspended cells were then stored on ice

under a N2 atmosphere prior to experimental use.

The bacteriochlorophyll (BChl) concentration was

determined by the method of Clayton (32), using a 7:2 (v/v)

acetone:methanol solution for extraction. A 0.1 mL volume

of cells was extracted with 4.9 mL of solvent and filtered

through a Whatman No. 1 analytical grade filter paper. The

absorbance of the filtrate was measured at 772 nm using a

Bausch and Lomb Model 710 UV-Visable 710 Spectrophotometer.

BChl concentrations were calculated from the Beer-Lambert

Law, using an extinction coefficient for BChl of 75

mM"^-cm"^ (33).

8

Table 1. Defined Synthetic Culture Medium for the of the Photosynthetic vinosum

Component

1. Macroelements

2. Trace Elements

3. Buffer

4. Reducing

•

Ion

(P04)3" K+ (NH.) + Mg2^ (SQ4)2-Ca2^

Fe2 + Zn2 + Mn2 + (BQ^)^-

Ni2 + Cu2 + (Mo04)2-

(HCO3)"

S2-

Growth : Bacterium Chromatium

Chemical Source Concentration

KH2PO4 KCl NH4CI MgS04-7H20 MgS04*7H20 CaCl2*2H20

FeCl2*4H20 ZnCl2 MnCl2 H3BO3 CoCl2'6H20 NiCl2*6H20 CuCl2*2H20 Na2Mo04•2H2O

NaHC03

Na2S'9H20

2 mM 4 mM 6 mM 2 mM 2 mM 1 mM

7.5 |iM 0.5 \iM 0.5 |XM 1.0 \m 0.8 p.M 0.1 }iM 0.1 |XM 0.15 IM

60 mM

1-2 mM Agent

5. Vitamin

6. Substrates

Cyanocobalamine 15 nM

(S2O3) Malate

2- Na-Na'

1^203' • -Mali

•5H2O c Acid

10 mM 10 mM

All solutions were made from reagent grade chemicals and doubly-distilled water under sterile conditions. Reducing agent (S ) stock solution was replinished every 2 months.

Preparation of C. vinosum rhromatophores

Chromatophores (inside-out vesicles of photosynthetic

bacterial whole cells) were prepared essentially as

described by Knaff and Buchanan (33). £. vinosum cells,

washed twice with potassium phosphate buffer and resuspended

to a concentration of approximately 5 mg cell weight/mL

buffer, were sonicated using a Branson Model 200 Sonifier at

50% duty cycle at a power setting of 7 for a total of 6

minutes, alternating 30 seconds with power on and 30 seconds

with power off. To prevent overheating during sonication,

the cells were placed in an ice bath. Sonicated cells were

centrifuged at 16,000 x g for 10 minutes. The supernatant

was then centrifuged at 300,000 x g for 35 minutes using a

Beckman L5-65 Ultracentrifuge and type 65 rotor. The

resulting chromatophore pellet was resuspended in a minimum

volume of buffer to be used in the flow dialysis experiment.

The resuspended chromatophores maintained activity for up to

2 hours when kept at ice temperature.

Filter Assay Technique

A filter assay technique (85) was used, where

indicated, as a means of monitoring calcium, sodium or

thallium transport in cells. Transport studies were

performed under anaerobic conditions in stoppered test tubes

flushed with water-saturated N2. In dark uptake

experiments, £. vinosum cells (ecjuivalent to [BChl] = 150

|XM) were placed in a reaction mixture (final volume = 100

|XL) with the appropriate assay buffer, inhibitors,

uncouplers or ionophores, and allowed to incubate for 5

minutes (except for samples containing arsenate which were

allowed to incubate for 30 minutes) in the dark under

water-saturated N2 prior to the onset of the experiment.

The experiment was initiated by the addition of 10 mM cation

( Ca"*", 22jjQ+ 204rj,2+j Reaction samples were allowed a 75

minute dark-loading period, after which the internal content

10

of a particular cation was determined by trapping cells on

membrane filters. A 1.0 mL aliquot of 0.1 M LiCl in the

cases of Ca "*" and Na"*", or 0.1 M LiN03 in the case of Tl" ,

was added to each tube to quench the reaction. The reaction

mixture was then filtered using a Millipore filter assay

apparatus fitted with either a Millipore or Amicon 0.45 |im

pore size filter. An additional 1.0 mL alicjuot of LiCl or

LiN03 solution was then used to rinse the test tube and

filter. The filter was then dissolved in 4.5 mL of either

Packard "Filter Count" or National Diagnostics "Filtron-X"

scintillation cocktail. The samples were counted using a

Beckman LS-7000 Liquid Scintillation Counter. To determine

nonspecific binding of radioactive cations to the filter,

samples were prepared as described previously, except the

first 1.0 mL of lithium salt solution was added prior to the

addition of the radionuclide, followed by immediate

filtration of the reaction mixture.

The percent counting efficiency was calculated from the

equation:

(measured cpm/dpm) x 100 = % efficiency.

This correction factor was then used to convert counts to

moles of cation transported. In samples containing

[ Ca "*"], [ Na" ] , or [204^1+], counting efficiency was

determined in this fashion: Samples were prepared in an

identical manner to the actual reaction mixtures, except no

radionuclide was added prior to filtration. The filter was

treated as before and a known amount of isotope, with known

dpm (disintegrations per minute) was added to the

scintillation vial.

Kj and V^^^ values for dark uptake experiments were

determined using Lineweaver-Burk, double-reciprocal plots.

Initial velocity was measured as a function of substrate

concentration. Values were determined by averaging the

11

results of repeated experiments, using uptake during the

first 40 minutes to estimate VQ, and calculated using a

linear least-squares graphics program.

Prior to the calcium efflux experiments, cells were

loaded with ^ Ca "*" by incubation with 10 mM [' Ca]CaCl2

(specific activity of 1 x 10^ dpm/nmol Ca) for 75 minutes in

the dark. Prior to the sodium efflux measurements, cells

were loaded with ^^Ua"^ by incubation with 10 mM [22Na]NaCl

(specific activity of 4.4 x 10" dpm/nmol Na) for 75 minutes

in the dark. Prior to the thallium efflux measurements,

cells were loaded with 204rj,2_+ j-y incubation with 10 mM

[204^1]TISO4 (specific activity of 3.7 x 10^ dpm/nmol Tl)

for 75 minutes in the dark. All reaction mixtures were kept

on ice under a water-saturated N2 atmosphere. After the 75

minute dark loading period, samples were illuminated by a 75

watt bulb to provide a light intensity of 7.5 x 10"^

ergs'cm'^'s" , for 20 minutes. Light intensities were

measured using a Yellow Springs Instruments Model 65A

Radiometer. All inhibitors, uncouplers, or ionophores were

added directly after the 75 minute pre-incubation, prior to

the 20 minute illumination period. Samples were quenched

and counted as described for dark uptake experiments.

Flow Dialysis Technique

Flow dialysis was performed as described by Ramos ot.

al. (9), except that the sample chamber was continuously

flushed with water-saturated N2 gas. Figure 1 shows a

schematic representation of the flow dialysis apparatus.

This technique was used to monitor the light-dependent

uptake of calcium by £. vinosum chromatophores. The upper

sample chamber of the flow dialysis cell contained

chromatophores (equivalent to 150 IM BChl) along with the

appropriate inhibitor, cation and buffer. The total

reaction volume was 500 L. Because £. vinosum

chromatophores lose the soluble electron carrier, cytochrome

12

rD„:

Upper Chamber: Chromatophores, Ra(Jionucli(de and Inhibitor

13

Buffer bubbled with Water-saturated Nitrogen

Dialysis Membrane

Fraction Collector

1 cm

14

^551' during preparation (34,35), it was necessary to add

the non-physiological electron carrier phenazine

methosulfate (PMS) in order to restore cyclic electron flow.

The lower chamber contained the flow-through buffer, which

was continually flushed with N2 gas, as was the upper

chamber which contained the reaction mixture. The

flow-through buffer also contained PMS (5 \iM) and the

inhibitor in a concentration equivalent to that in the

reaction mixture. Approximately, 30 fractions were

collected before turning on the light to start uptake.

These fractions were collected to establish an adequate

baseline prior to the initiation of uptake. Illumination

was provided by a Unitron Microscope lamp at an intensity of

7.5 X 10"^ erg'cm"- • s"-'-. Fractions of 1.5 mL were taken at

a flow rate of 6.0 mL/min. From the fractions collected,

1.0 mL aliquots were taken and combined with 3.0 mL of

"Universol" or "Picofluor" scintillation cocktail for

aqueous samples and counted as described. Uptake in

chromatophores would be represented as a deviation from the

established baseline.

CHAPTER III

RESULTS AND DISCUSSION

^a^"^ Uptake Experiments with Wholf C^lls

Whole cells of purple photosynthetic bacteria show

light-dependent proton ejection (34,36,37), leading to the

formation of a ApH (outside acidic) and a membrane potential

A^ (outside positive). It has been shown previously in this

laboratory, that Chromatium vinosum possesses a

calcium-proton antiport (2 6). It was also found that the

operation of this antiport is electrogenic, with H"'":Ca2'*" >

2. Electrogenic transport processes are those in which net

electrical charge is transported across the membrane (4,38).

In these prior experiments, Q.. vinosum cells were loaded

with ^ Ca2''" using [^^Ca]CaCl2f during a 75 minute

pre-incubation in the dark under a N2 atmosphere. It was

not known whether this loading occurred through a O I

non-specific, limited Ca permeability across the membrane O 1

or was catalyzed by a specific Ca transport system. In

order to investigate this question, one of the methods used

involved transport kinetics. Transport proteins, like

enzymes, take part in substrate binding and release and are

therefore saturable [the rate of substrate transport

increases with increasing substrate concentration from an

initial velocity (VQ) to a maximum velocity (V ^ ^ ) ] , and

substrate-specific (39,40). If a particular substrate is

transported by a number of transport systems, as appears to

be the case with Ca "*", then multiple Kj values may be

obtained.

To examine the Ca2" loading mechanism, the effect of

varying the Ca " concentration on the initial rate of Ca

uptake in the dark was studied. The data presented in

Figure 2, represent the results of a time course study of

15

17

180

160

140

120

100

o 00

o CO

o

o CM

(min)

Time

Dark

(gOl X Ludo)

(l/NjLUOl) ep!JO|M0 ujnioiBO (BQ^^J

18

the uptake of ^^Ca2+ by Q. vinosum cells in the dark. As

can be seen, the uptake is appreciable and becomes saturated

with increased time in the dark. The uptake was observed to

be linear with time for approximately 60 minutes. The

initial velocity (v^) was estimated from the uptake at 40

minutes. In a subsequent experiment, the initial velocity

was measured as a function of ^^Ca2''" concentration. It was

found that the rate became saturated with increasing Ca2"^

concentration. The property of saturation is indicative of

a carrier-mediated process. Figure 3 shows the data in the

form of a Lineweaver-Burk, double-reciprocal plot. As can

be seen, the uptake of ^^Ca2'*" in the dark obeyed

Michaelis-Menten kinetics . Kj^ and Vj^^^^ values were

determined by averaging the results of repeated experiments

and using a linear least-squares graphics program. The

values of Kj^ and Vj^^^^ were calculated to be 7.0 ± 0.7 mM and

150 ± 25 nmol Ca2'^-min"-^ "ixmol BChl"-'-, respectively.

Sensitivity to inhibitors of a transport system is also

a property of a carrier-mediated system. Utilized in this

study were a class of inhibitors often referred to as

calcium antagonists (41), but also called calcium entry

blockers, calcium influx blockers, slow channel blockers,

and calcium blockers. Figure 4 shows the structures of the

four calcium antagonists employed in this work. They

include ruthenium red (RR)(42) and three antipyschotic

drugs, chlorpromazine (CPZ) (43), phenothiazine (PTZ) (43),

and trifluoperazine (TFP) (43) . Each of these reagents

provides a powerful tool in the study of the translocation

of Ca2''" across biological membranes.

Ruthenium red has become a fundamental tool for 2 +

functional separation of the pathway for Ca"" uptake from

that for Ca "*" release (44) . The dye is a hexavalent cation 2 +

which interferes specifically with components of the Ca

uptake system (45). It has been shown to be an inhibitor of

Ca "*" fluxes through Ca^'^'-specific channels in eukaryotic

19

!7> *i'

it KJ

20

. 0 1 . J i ^

22

A. Ruthen- jnm PP^H

[(NH3)5 Ru - O • Ru(NH3)4 • O - Ru(NH3)5]6+

B. Phenothiazinp

Q^ Chlorpromazine

D_. Trifluoperazine

(GH2)3N(CH3).

(CH2)3N NCH

I

23

membranes (46-48). Ruthenium red is known to be a powerful

inhibitor of Ca2+ transport in mitochondria (45-51). It is

also a specific inhibitor of (Ca2++ Mg2+)-ATPase of red

blood cells (52,53) and of Ca2+ binding of the (Ca2++ 2 +

Mg^ )-ATPase of sarcoplasmic reticulum (54-56). In

mitochondria, the uptake of Ca2"'" is mediated by a specific

ruthenium red-sensitive uniporter (52,57-59); however the

nature of the inhibition of the Ca2+ transport mechanism is

uncertain.

Figure 5 shows the effect of ruthenium red on ' Ca2 +

uptake by Q. vinosum cells in the dark. Uptake of ^^Ca2+ in

dark loaded jQ. vinosiim cells is markedly inhibited by

ruthenium red, with an I50 (50% inhibition) for uptake

observed in the range between 60-65 p,M ruthenium red

concentration. Although ruthenium red concentrations up to

2 mM were tested, maximal inhibition of uptake was typically

in the 65-70% range. This result suggests that while 65-70%

of the observed ^ Ca2'*' uptake by Q.. vinosum whole cells in

the dark occurres via a specific, ruthenium red-sensitive

channel, a portion of the uptake (-30-35%) may occur via

non-specific leakage across the cell membrane. If this

conclusion is correct, 50% inhibition of ^ Ca2"'" uptake in

the dark by the ruthenium red-sensitive channel occurs at a

ruthenium red concentration of 20 |XM. However, in ruthenium

red-sensitive eukaryotic systems, the concentration of

ruthenium red required to give 50% inhibiton of Ca

transport is much lower, in the range of 5 |IM or less (4 6) .

It is possible that the outer cell wall of £. vinosum whole

cells may impose some permeability barrier against this

inhibitor and that the ruthenium red-sensitive £. vinosum

channel is in fact of comparable sensitivity to that in

eukaryotic systems. This possibility can be supported by

the observation that several ionophores (e.g., valinomycin,

nigericin, monensin and CCCP) require 10-fold higher

concentrations for effectiveness in whole cells of

24

u.

25

loaiNoo %

26

£. vinosum compared to that in cell wall-free chromatophores

(26,27,33) .

Although, ruthenium red had been shown to be specific 2 + for Ca'' transport in eukaryotic systems, it seemed

important to test its specificity in a prokaryotic system.

Table 2 presents the effect of ruthenium red on 204rp-|_+ ^^^

^ Na"*" uptake by £. vinosum cells in the dark. C. vinosum

has been previously shown to possess both a Na"*'/H''" antiport

and a K'*"/H''" antiport (25-27) . Tl"*" serves as a K"*" analog in

the Q.. vinosum K''"/H''' antiport and in an ATP-energized K"*"

uptake system (27,60). As can be seen, ruthenium red had no

effect (< 10% inhibition), even at a concentration of 1 mM,

on the uptake of either 22 ^ + ^^ 204rp-j_+ ^^ £^ vinosum cells

in the dark. These results suggest that ruthenium red is

inhibiting a Ca2''"-specific transport system in Q.. vinosum

cells.

La" " is another well-investigated inhibitor of

carrier-mediated Ca2''" fluxes (46,61,62) and has been used to

probe biological systems that normally bind Ca^ ions.

Lanthanides, like ruthenium red, are inhibitors of Ca'

transport because they are structural Ca'' analogs which can

mimic the substrate and compete for binding (63). In most

instances, the lanthanide ions displace membrane-bound Ca^

and inhibit Ca2''"-mediated membrane fuction, but unlike Ca2"'",

these ions do not appear to be transported across cellular

membranes (64). Lanthanum ion has been shown to

specifically destroy the permeselectivity for calcium ion 2 +

(65) and it appears to fuction in the same manner as Ca 2 +

because it has an ionic radius very near that of Ca (63).

Figure 6 shows the effect of La " on ' Ca "'" uptake by (I.

vinosum cells in the dark. It appears that La "*" effects

" Ca " transport by £. vi nosum cells in the dark in a manner

similar to that observed for ruthenium red. LaCl3 is a

potent inhibitor of Ca " uptake in the dark, but as was the

case with ruthenium red, 100% inhibition of Ca uptake in

27

Table 2. 204,p2_+ The Effect of Ruthenium Red on Uptake by £. vinosum Cells in the Dark.

and 22Na''"

Sample Percent of Control for Uptake

A.

B.

204

1 . 2 . 3 . 4 . 5 .

22

1 . 2 . 3 . 4 . 5 .

Tl"^

C o n t r o l II

II

II

II

Na"*"

C o n t r o l II

II

II

II

+ + + +

+ + + +

100|XM RR 150|IM RR 200^M RR

ImM RR

lOOjXM RR 1 5 0 p ^ RR 200^M RR

ImM RR

100 94

105 96 97

(±4) (±2) (±2) (±2)

100 90 94

101 98

(±3) (±4) (±2) (±0)

Transport was assayed using the filter assay technique described in Methods. Cells were harvested and washed twice with 50 mM potassium phosphate (pH 6.5) for ^ a ^ transport experiments or 50 mM MES-choline (pH 6.5) for 204 .j.3_+ transport experiments. The reaction mixtures (100 |IL)

[22NalNaCl (specific acivity = 4.4 x mM [^^^T1]T1S04 (specific activity = , 50 mM buffer and cells ecjuivalent

contained either 10 mM 10* dpm/nmol Na) or 10 3.7 X 10- dpm/nmol Tl) to 150 \iM. BChl. Cells atmosphere for 75 minnutes. The results represent the average of three experiments with the standard diviations given in parenthesis.

were pre-incubated at O'C under a N2

?SWf;

28

29

o o o o o o o o o o O 0 ) 0 0 r * - < 0 U > ^ C 0 C M r-

10H1N00 %

30

the dark could not be obtained, even at La "*" concentrations

as high as 500 iM. However, in typical experiments, 80 to

85% inhibition was observed with half-maximal inhibition

observed at 35 |IM La- "'"concent rat ion. These results are

consistent with the earlier hypothesis that the majority of

the observed uptake (-80%) occurs via a Ca2''"-specific

carrier and the remainder (-20%) occurs through a

non-specific leak across the membrane.

The data in table 3 examine the effect of La-"*" on

204.j.j_+ ^j^^ 22j + uptake by C- vinosum cells in the dark. It

was necessary to ascertain the effect of La "*" on the Na' '/H'

and K"**/H"'" antiports known to exist in £. vinosum (25-27) .

As can be seen, La""*" had no effect (<10% inhibition) , even

at a concentration far above that which is needed to produce

50% Ca2''" inhibition on the uptake of either 22 "*" or 204rp-L+

by Q.. vinosum cells in the dark. These results imply that

La "*" inhibits a Ca -specific transport system in H. vinosum

cells in the dark.

Ca2'^ uptake via ruthenium red-sensitive channels in

eukaryotes is electrogenic (46) and thus can be driven by a

membrane potential (A^) , outside positive. Since Q.. vinosum

cells can utilize ATP hydrolysis via a DCCD-sensitive

F^F^^-ATPase (66) to maintain a A^ of +80 mV (outside

positive) in the dark (34,66), it was of interest to

investigate the effect of reagents known to eliminate A^ in

£. vinosum cells on " Ca " uptake in the dark. It has been

previously shown in work from this laboratory that CCCP,

valinomycin (in the presence of K"*") , as well an DCCD will

collapse A^ in £. vinosum (16) . Table 4 shows the effect of

inhibitors and ionophores known to collapse A^ in £. vinosum

on Ca "*" uptake by £. vinsoum cells in the dark. Valinomycin

is an ionophore which catalyzes K"*" equilibrium across the

plasma membrane (67,68). The equilibration of positively

charged potassium ions collapses both the potassium ion

gradient ([K" ]in > [K lout ^^^ ^® membrane potential.

31

Table 3. The Effect of La "*" on 204. 3 + ^nd 22^3+ Uptake by C vinosum Cells in the Dark.


A.

B.

204.j,3_+

1. Control 2. " + 3. " + 4. " +

22Na" 1. Control 2. " + 3. " + 4. " +

100 150 200

100 150 200

IM ^M IM

^M ^M llM

La La La

3+ 3+ 3+

La La La

3-f 3+ 3+

100 92 94 91

100 98 95

102

(±2) (±1) (±3)

(±1) (±1) (±3)

Transport was assayed using the filter assay technique described in Methods. Reaction conditions were the same as those given in Table 2, except that various concentrations of LaCl3 were added, as indicated.

32

Table 4. The Sffect of Inhibitors and lonophores on ^Ca-' Uptake by £. vinosum Cells in the Dark.


1. Control 100

2. " + 20 LM valinomycin 75 (±6)

3. " + 30 |IM CCCP 7 9 (±8)

4. " + 300 ^M DCCD 84 (±5)

5. " + 5 mM arsenate 73 (±2)

Transport was assayed using the filter assay technique described in Methods. Reaction conditions were the same as those given in Figure 2, except that the inhibitors in the indicated concentrations were added and the specific activity of[^^Ca]CaCl2 was 2.97 x lo" dpm/nmol Ca. Uptake was measured after 75 minutes. The results represent the average of five experiments with the standard deviations given in paratheses.

33

Carbonylcyanide-m-chlorophenylhydrazine (CCCP) is an

uncoupler. CCCP is a lipophilic weak acid which catalyzes

proton equilibration across the bacterial plasma membrane

(69). Proton equilibration abolishes both components of the

electrochemical proton gradient (A^IH+) , the proton gradient

(ApH, outside acidic) and the membrane potential (A^,

outside positive) (27) . In the dark, A jj+ is generated by

ATP hydrolysis coupled to proton efflux by the

membrane-bound ATPase (27,34). Arsenate depletes the ATP

pool in £. vinosum (26), thereby depleting the cell of its

energy source for proton efflux.

N,N'-Dicyclohexylcarbodiimide (DCCD) has been shown to

form adducts with proton-translocating glutamate residues in

the membrane-bound ATPase (16,27), thereby blocking

ATP-driven proton efflux. Table 4 shows that elimination of

A^ by inhibition of the ATPase with DCCD, by depleting the

internal pool of ATP with arsenate or by using ionophores

that collapse AT in £. vinosum (16,34) by rendering the cell

membranes permeable to either protons (CCCP) or K"*"

(valinomycin)(68,69), had only a small inhibitory effect on

Ca2"'' uptake in Q.. vinosum cells in the dark. These

observations suggest little if any involvement of the

membrane potential (AT) in providing the energy for Ca2"*"

uptake in the dark. The other component of the

electrochemical proton gradient (A}Xjj+) is the transmembrane

proton gradient (ApH). It has been shown previously that Q.

vinosum cells do not maintain a ApH in the dark (16).

Therefore ApH cannot serve as a possible energy source for

Ca "*" uptake in £. vinosum whole cells in the dark. It thus 2 +

appears the A jj+ plays little part in dark uptake of Ca by

Q.. vinosum cells. 2 +

In an attempt to provide further evidence for a Ca

transport system in £. vi nosum distinct from the Ca /H 2 +

antiport, the effect of other inhibitors on Ca uptake in

the dark was investigated. Three phenothiazine drugs were

34

tested: Chlorpromazine (CPZ), phenothiazine (PTZ) and

trifluoperazine (TFP). These antipsychotic drugs are

well-known for blocking the calcium-binding protein

calmodulin (70-74). Table 5 presents the effect of

phenothiazine drugs on ^ Ca2''' uptake by Q.. vinosum cells in

the dark. These drugs have been shown to influence Ca2"'"

movements in other organisms, but their mechanisms are still

unknown (64,7 6). It has been suggested by Fleischer ot. 2±-

that these drugs inhibit Ca2''" loading and (Ca2'''+ 2 + Mg )-ATPase activity in sarcoplasmic reticulum, yet have no

effect on unidirectional Ca2"'" efflux from vesicles loaded

either activtely or passively with Ca2''' (64,76). The data

presented in Table 5 shows that even at very high

concentrations [100 }XM], these drugs did not inhibit (< 5%

inhibition) Ca'' uptake by £. vinosum cells in the dark. In

fact, one of the phenothiazines, PTZ actually produced an

approximate 3-fold stimulation in the observed extent of

Ca2"'' uptake. This is in contrast to their effect on Ca2"'"

efflux in the light (vide infra).

Ca "*" Efflux Experiments with niuminated Whole Cells

Whole cells of Q.. vinosum have been shown to exhibit

energy-dependent Ca2''" efflux via the Ca2' /H''" antiport (2 6)

and the properties of the Ca "'"/H"'" antiport have been well

documented in earlier studies from this laboratory. It has

been observed that the driving force for Ca " efflux arises

from the A|ijj+ which, in turn, results form the proton efflux

coupled to light-dependent cyclic electron flow, [see

Davidson and Knaff (26)]. It would be, therefore,

interesting to ascertain the effect of the inhibitors (e.g.,

ruthenium red, La " , phenothiazines) on light-dependent Ca

efflux from £. vinosum whole cells.

£. vinosum cells were allowed to pre-incubate for 75

minutes in the dark at O'C under a N2 atmosphere with 10 mM

35

Table 5. The Effect of by £. vinosnm

Phenothiazine Drugs Cells in the Dark.

on 45ca2+ Uptake


A. Chlorpromazine (CPZ)

Control 1, 2 3 4 5 6 7 8

B. Phenothiazine (PTZ)

+ " + " + " + " + " + " +

5 ^M CPZ 10 \m CPZ 15 IXM CPZ 20 lM CPZ 25 |iM CPZ 50 |1M CPZ 100 ^M CPZ

1. 2. 3. 4. 5. 6. 7.

Control II

II

II

II

II

II

+ + + + + +

5 \1JA PTZ 10 |iM PTZ 15 JIM PTZ 20 ^M PTZ 25 ^M PTZ 100 ^M PTZ

C. Trifluoperazine (TFP)

1. 2. 3. 4. 5. 6. 7.

Control II

It

•1

tl

It

It

+ + + + + +

5 lM TFP 10 |IM TFP 15 IM TFP 20 M.M TFP 25 lM TFP 100 |XM TFP

100 99 102 100 102 100 101 98

100 177 297 273 269 305 287

100 96 95 98 94 96 104

(±2) (±1) (±0) (±1) (±2) (±3) (±2)

(±13) (±17) (±24) (±15) (±31) (±29)

(±4) (±2) (±2) (±3) (±2) (±4)

Transport was assayed using the filter assay technique described in Methods. Reaction conditions were as in Figure 2, except that various concentrations of chlorpromazine (CPZ), phenothiazine (PTZ) and trifluoperazine (TFP) were added as indicated. Uptake was measured after 75 minutes. The results represent the average of four experiments with the standard deviations given in parenthesis.

36

[ Ca]CaCl2 in the absence of inhibitors. Inhibitors, at

the specified concentrations, were then introduced into the

rection mixtures and allowed to incubate for 5 minutes

before the light was turned on. The reaction mixtures,

still under a N2 atmosphere, were illuminated for 20 minutes

in the light. Table 6 shows the effect of ruthenium red and

La " on ' 5ca2+ efflux from Q.. vinosum cells in the light.

As can be seen, neither ruthenium red nor La-"*" had any

inhibitory effect on ^^Ca^'^ efflux from pre-loaded £.

vinosum cells in the light. These inhibitors were tested at

concentrations where they had shown potent inhibition of

Ca "*" uptake in the dark. The observation that La""*" and

ruthenium red inhibit Ca2"*" uptake without affecting Ca2'''

efflux strongly suggests that two separate Ca2''' systems

exist in C vinosum: One system sensitive to La "*" and

ruthenium red that results in Ca2''" uptake in the dark and a

second system, unaffected by ruthenium red or La-"*", that

catalyzes Ca^ efflux during illumination. As light-driven

Ca2" efflux from Q. vinosum cells had previously been shown

to occur via an electrogenic Ca'' /H antiport (26) , it can pi 1

be concluded that the Q.. vinosum Ca^ /H antiport is -3 J.

inhibited neither by ruthenium red nor La~' .

The effect of several phenothiazine drugs on the £..

vinosum Ca2"'"/H" antiport was examined next. Figure 7

illustrates the effect of these various phenothiazine drugs

on ^^Ca^"^ efflux from illuminated £. vinosum whole cells.

The three phenothiazines (PTZ, TFP, CPZ) were potent

inhibitors of Ca "*" efflux from pre-loaded £. vinosum cells

in the light, with 50% inhibition of efflux being observed

at 12 |J,M, 14|XM, and 25 |i.M concentrations of phenothiazine,

trifluoperazine, and chlorpromazine, respectively. Table 7

summarizes the effect of the phenothiazine drugs on ° Tl"*"

and 22^3+ efflux from illuminated Q.. vinosum cells. Even at

concentrations as high as 100 |IM, the three phenothiazine

drugs, had no inhibitory effect (< 5% inhibition) on either

37

Table 6. ,3+ The Effect of Ruthenium Red and La-'' on Efflux from Illuminated Q. vinosum Cells.

45ca2+

Sample Percent of Control for Efflux

Ruthenium Red (RR) 1 2 3 4 5 6 7

Control

B. Lanthanum Ion (La* )

+ 100 nM RR + 500 nM RR + 50 ^M RR + 100 ^M RR + 500 lM RR + 1 mM RR

3 + . 1 2 3 4 5

C o n t r o l

I t

I t

+ 25 ^M La + 50 lM La + 100 jiM La + 200 \IM La

3 + 3 + 3 + 3 +

100 100 95

104 103 102 105

100 82 78 90 92

(±1) (±3) (±2) (±1) (±0) (±2)

(±4) (±6) (±3) (±4)

Transport was assayed using the filter assay technique described in Methods. Reaction conditions were as Figure 7, except ruthenium red (RR) and LaCl3 (La^"^) m various concentrations replaced the phenothiazine drugs. The results represent the average of three experiments with the standard deviations given in parenthesis.

38

It'

. 1 •' •

39

lU

z ^^ N < s o GC Q. flC o z o

UJ z N < cc Ul CL O 3

IFL

oc 1-

UJ

z N < Z H O z UJ

z Q. O D <

h* (O LO ^

noaiNOO %

40

Table 7 . The Effect of Phenothiazine Drugs on 204,j.i+ nd 22Na''" Efflux by Illuminated £. vinosum Cells.


A. 204^1+

1. Chlorpromazine (CPZ) i) Control ii) " + 25 ^M CPZ iii) " + 50 IM CPZ iv) " + 100 \iM CPZ

2. Phenothiazine (PTZ) i) Control 11) " + 25 |XM PTZ iii) " + 50 |XM PTZ iv) " + 100 ^M PTZ

3. Trifluoperazine (TFP) i) Control ii) " + 10 IM TFP iii) " + 25 H^ TFP iv) " + 100 |1M TFP

100 99 100 98

100 100 97 100

100 100 99 100

(±1) (±1) (±2)

(±2) (±0) (±3)

(±2) (±1) (±2)

B. 22jj^+

1. Chlorpromazine (CPZ) i) Control ii) " + 25 IM CPZ iii) " + 50 |XM CPZ iv) " + 100 IM CPZ

2. Phenothiazine (PTZ) i) Control ii) " + 25 lM PTZ iii) " + 50 lM PTZ iv) " + 100 ^M PTZ

100 99 101 100

100 98 98 99

(±3) (±0) (±1)

(±0) (±1) (±1)

41

Table 7. Continued.


3. Trifluoperazine (TFP) i) Control 100 ii) " + 10 M TFP 99 (±2) iii) " + 25 IM TFP 99 (±1) iv) " + 100 M.M TFP 102 (±2)

Transport was assayed using the filter assay technique described in Methods. Reaction conditions were the same as those given in Figure 7, except either 10 mM [22Na]NaCl (specific activity = 4.4 x 10^ dpm/nmol Nal or 10 mM [2'J4TI]T1S04 (specific activity = 3.7 x 10- dpm/nmol Tl) replaced the 10 mM [ Ca]CaCl2- The results represent the average of three experiments with the standard deviations given in parenthesis.

42

light-driven Na" efflux via the £. vinosum Na' /H" antiport

(25) or Tl" efflux via the £. vinosum K" /H" antiport (27) .

These results are expected if a Ca2+-specific system was

being inhibited by the phenothiazines.

The observation that light-driven Ca2'*" efflux via the

£.. vinosum Ca2"'"/H"'" antiport is affected by the phenothiazine

drugs, while Ca2+ uptake in the dark shows the opposite

response to the two classes of inhibitors, further supports

the presence of two separate Ca2"'' transport systems in £.

vinosum.

ila"*" Uptake Experiments with Illuminat-f ri Chromatophores

The demonstration that whole cells of £. vinosum can

exchange internal Ca2''" for external protons though the

Ca ' /H" antiport (26), leads to the prediction that

chromatophores prepared from these bacteria, because of

their opposite membrane sidedness compared to intact cells

(36,37), should be able to exchange external Ca2'*' for

internal protons. This prediction was investigated by pi

following the effect of Ca" on transmembrane proton

movements in £. vinosum chromatophores. Flow dialysis was

performed with Q.. vinosum chromatophores using the membrane

permeable weak base [^^C]methylamine to monitor the

transmembrane proton gradient (ApH)(75). In order to

discern the effect of ApH from A^, 2 fiM K"*" valinomycin was

also added to the reaction chamber, in order to completely

collapse A^ in the chromatophores (81,82). The results of

one such experiment are shown in Figure 8. Upon

illumination, the chromatophores generate ApH (inside

acidic), as demonstrated by the uptake of methylamine. In

flow dialysis experiments, a decrease in the amount of

[ " C]-label in the dialysate, which indicates uptake of the

labeled material by the chromatophores, is reflected in a

deviation below the baseline. With the addition of CaCl2

44

O

o

o CO

E

C

o

o cc

o CM

_ O

Q T CO

( luclo 6o|)

9;BsA|B!a Uj 9U!LUB|Aq;9|/\j | Q ^ J

45

(10 mM), where indicated, there results an efflux of

protons, as indicated by an efflux of methylamine. This is

as expected, for if a Ca2+/H"'" antiport existed in £.

vinosum, the addition of Ca2+ to the external medium would

create [Ca2+]^^^^^^^^ > [Ca2+] internal' thereby Ca2+ would

enter the chromatophores down the electrochemical gradient. 2 +

As Ca enters the chromatophores there would be

simultaneous proton efflux. This provides further evidence p i I

for the Ca'' /H antiport operating in Q.. vinosum cells.

With the demonstration of Ca2"'"-dependent H"*" efflux in

£. vinosum chromatophores, Ca2''" uptake was next

re-investigated (27) . Figure 9 shows light-dependent ^ Ca2"'"

uptake by £.. vinosum chromatophores. As can be seen,

energization of the chromatophores by illumination, results pi

in Ca uptake by the chromatophores. Again, in flow

dialysis experiments, a decrease in the amount of

['* Ca2" ]-label in the dialysate is reflected in a deviation

below the baseline, which indicates uptake by the

chromatophores. This again demonstrates the operation of

the Ca2'*'/H"'" antiport in Q.. vinosum chromatophores.

With direct evidence of the light-dependent Ca2"'"/H"'"

antiport operating in £.. vinosum chromatophores, it seemed

of interest to examine the effects of the phenothiazine

drugs which had been found to be potent inhibitors of that

antiport in whole cells, in £. vinosum chromatophores. The

results of such experiments are shown in Figures 10, 11 and

12, for CPZ, PTZ and TFP, respectively. As can be seen, it

appears that the three phenothiazines (CPZ, PTZ, TFP)

indeed, have an inhibitory effect on light-driven Ca2+ ion

uptake in £. vinosum chromatophores. Figure 10 shows the

effect of chlorpromazine on ^^Ca^"^ uptake by illuminated £. 2 +

vinosum chromatophores. While complete inhibition of Ca

uptake in the light is not seen, the amount of uptake is

substantially less than in the control experiment without

chlorpromazine (see Figure 9). The effect of phenothiazine

47

O

o

o CO

0)

n E

C O

o CO k .

LL O CM

«o

1 ^

CO

i

o CO

1 ^

CM

T 00 CM

(uudo Bo|)

9;BsA|B!a U! 9PUOIM0 LunioiBQ (^Og^)

49

O

o

o CO

0

n E

C O

"o CO

o CM

CO o CO

1 ^

CM

r CM

(uudo Bo | )

51

O

o

o CO

o CM

0)

E

C O

o CO

I CM

CO

T—

CO o CO

(oido 6o| )

eiBsAiBiQ u! 9P!J0|L|0 uunioiBQ pOg^j

53

. O

o

o CO

CD

n E

C O

o CO

o CM

. O

1

CO

1

o CO

1 G) CM

1 00 CM CM

CD CM

(Oido B o | )

9 ; B s A | B ! a u! 9p!JO|L|o lunjOiBQ (^Og^j

54

on Ca uptake by illuminated £. vinosum chromatophores is

seen in Figure 11. Again, there is much less uptake of Ca2"*'

than in controls. Figure 12 examines the effect of

trifluoperazine on " ^ 2+ uptake by illuminated C- vinosum

chromatophores. As can be seen, there is nearly complete

inhibition of Ca2''" uptake by the chromatophores. All of the

results obtained from flow dialysis experiments with the

phenothiazine drugs are the results expected.

Chlorpromazine, trifluoperazine and phenothiazine were

observed to be potent inhibitors of the Ca2"'"/H"'" antiport in

£. vinosum whole cells as well as inhibitors of the p 1

light-driven Ca^ uptake in inside-out vesicles. These

results lend further evidence to the hypothesis that the

phenothiazine drugs which are known to influence

light-driven Ca2''" fluxes in other organisms (64,70-74,76),

also affect light-driven Ca2'*" movements via the Ca2'''/H"

antiport in Q.. vinosum.

As a last control, the effect of ruthenium red and La""*" p 1

on light-driven Ca'' uptake by Q.. vinosum chromatophores was

examined. Figure 13 shows the effect of ruthenium red on

^ Ca "'" uptake in illuminated Q.. vinosum chromatophores.

While ruthenium red has been shown to be a potent inhibitor

of Ca "*" uptake in Q.. vinosum whole cells in the dark, it had

no effect on light-dependent Ca2' efflux via the Ca2''"/H'

antiport in illuminated £. vinosum whole cells (77) . As is

evident, ruthenium red had no effect on light-driven Ca2'

uptake in Q.. vinosum chromatophores. This is further P I 1

evidence that ruthenium red does not inhibit the Ca^ /H

antiport previously described (26). Figure 14 establishes

the effect of La""*" on ^^Ca^"^ uptake by illuminated £.

vinosum chromatophores. Again, La " had been shown to be an

effective inhibitor of Ca "*" uptake in whole cells, but had

no effect on light-dependent Ca "*" efflux from whole cells.

La " had no inhibitory effect on the light-driven Ca "*" 2 + uptake in £.. vinosum chromatophores. The amount of Ca

56

O

o

o CO

o CM

_o

0

n E

C O

o CO

1 T

(Oido Bo|)

9;BSA|B!a U! 9PU01LJ0 LUnplBQ [BQg^)

58

O LO

o

o CO

0)

n E

C

q o CO

CM rl= ' ^

1 O Tt

1 o>. CO

£• CO

o u-CM

_o

^

I

CO

1 ^

CO

9JBSA|B!a Uj

(ludo 6o|)

9PU0IM0 uunioiBQ (BOgt.1

59

uptake seen in Figures 13 and 14 is comparable to the

control experiment where no ruthenium red or La-"*" was added

(Figure 9). All of the above evidence suggests that there

are two separate calcium transport systems in the purple

phototrophic bacterium Chromatium vinosum. One of the

systems is the previously described Ca^'^/R'^ antiport which

is sensitive to phenothiazine drugs, but insensitve to

ruthenium red and La "*". The second system appears to be a pi

Ca^ uptake system in the dark, which is insensitive to the o J.

phenothiazines, but sensitive to ruthenium red and La" .

CHAPTER IV

SUMMARY, CONCLUSIONS AND DISCUSSION

Summary

1. Substantial evidence has been presented (see Figure

2) to document the fact that Chromatium vinosum possesses a

Ca uptake system unique to the Ca2"'"/H"'" antiport previously

described (2 6). The uptake system obeyed Michaelis-Menten

kinetics with Kj^=7.0±0.7mM and Vj^^^ = ^^O ± 25 nmol

Ca2'^-m"^-^mol BChl"^ (see Figure 3) and the uptake could be

inhibited by calcium antagonists. All of the above

observations are indicative of a carrier-mediated transport

system.

2. Ruthenium red and LaCl3, both of which are known to p 1

influence Ca^ fluxes in eukaryotic membranes (44-48,51,

57-59,61-63), were shown to markedly inhibit Ca2"'" uptake in

the dark by £. vinosum cells (see Figures 5 and 6).

However, neither ruthenium red nor La* had any inhibitory

effect on the uptake of either Na"*" or the K"*" analog Tl"*" by

Q.. vinosum cells in the dark (see Tables 2 and 3) . This

suggests that both ruthenium red as well as La-'

specifically inhibit a calcium transport system.

3. It has been demonstrated that A^, the membrane

potential, plays little or no part in driving the Ca2"'"

uptake by Q., vinosum cells in the dark. It had been shown

that elimination of A^ by depleting the internal pool of ATP

with arsenate (16,34), inhibiting the ATPase with DCCD

(16,27), or by using ionophohores that collapes A^ in Q.

vinosum cells (16,34) by rendering the membrane permeable to

either H"*" ion (CCCP) (69) or K"*" (K" valinomycin) (68), had

only a small inhibitory effect on Ca " uptake by Q.. vinosum

cells in the dark (see Table 4). These results suggest

little if any involvement of A^ in providing the energy for

60

61 2 +

Ca uptake in the dark. Previosly, the other component of

AHjj+, ApH has been shown to be zero in £. vinosum cells in

the dark (27), which eliminates ApH as a possible Ca2"*"

uptake energy source.

4. The phenothiazine drugs (chlorpromazine,

phenothiazine, trifluoperazine) were shown not to inhibit p I

the Ca uptake system in the dark in £. vinosum cells (see Table 5). These results suggest that the antipsychotic

phenothiazines have little effect on the Ca2+ uptake in £.

vinosum cells.

5. The £. vinosum Ca2" /H"*" antiport previously

identified in this laboratory (26), was further

characterized. Light-dependent Ca2''' efflux via the Ca2''"/H'

antiport in £. vinosum whole cells was shown to be inhibited

by three phenothiazines: Chlorpromazine, trifuoperazine and

phenothiazine (see Figure 7). Also examined was the effect

of the phenothiazine drugs on the previously described

Na"'"/H"'" and K"*"/H"*' antiports which are also found in Q..

vinosum (25-27), (see Table 7). Evidence presented in this

thesis suggests there is no effect of phenothiazines on the

light-dependent efflux of either Na"*" or Tl"*" catalyzed by the

£. vinosum Na"'"/H"'" or K"'"/H''" antiports, respectively. This

suggests that the phenothiazine drugs studied work

specifically on the Ca "'"/H"'" antiport and not on other

cation/H"*" antiports found in Q.. vinosum-

6. Neither ruthenium red nor La "*" inhibit Ca2''" efflux

via the Ca2"'"/H' antiport in £. vinosum cells (see Table 6) .

This evidence lends further support to the hypothesis that

separate systems catalyze Ca "*" uptake and efflux.

7. The inside-out vesicles (chromatophores) of C-

vinosum were observed to pump protons out of the membrane as

Ca "*" was added to the external medium (see Figure 8) . Also P 1

it was shown that the chromatophores will take up Ca in an energy-dependent fashion. This was further evidence that

the Ca2"'"/H"*" antiport was operative in £. vinosum

62 chromatophores (see Figure 9) .

8. The effects of the three phenothiazine drugs on the p i 1

Ca /H antiport in £. vinosnm chromatophores was examined.

Chlorpromazine, phenothiazine, and trifluoperazine were

shown to inhibit the light-driven Ca2''" efflux from Q..

vinosum whole cells as well as light-driven Ca2"'" uptake in

£. vinosum chromatophores (see Figures 10, 11 and 12). The

data provide additional evidence for the inhibition of the p i 1

Ca*' /H" antiport by these drugs.

9. As a last control, the effect of ruthenium red and

La" " on the Q.. vinosum Ca2'*"/H"'" antiport in chromatophores

was examined. It was shown that while these compounds

produced potent inhibition of Ca2"*" uptake in the dark, they

had no effect on Ca2"'" efflux by the Ca2'*"/H"'" antiport in

whole cells. The same situation was observed for pi

light-dependent Ca^ uptake in chromatophores (see Figures

13 and 14) . Ruthenium red and lanthanum ion had no effect

on Ca uptake via the Ca2" /H"*" antiport shown to be

operative in chromatophores.

Conolusion and Discussion

The results presented above provide strong evidence

that the photosynthetic purple bacterium Chromatium vinosum

has two calcium transporting systems available to transport

Ca2"*" ions across the lipid bilayer. One is a phenothiazine-

sensitive, but ruthenium red and La- "*'-insensitive Ca2''"/H"'"

antiport for Ca "*" efflux in the light. The second is a

ruthenium red and La^"^-sensitive, but phenothiazine-

insensitive, Ca "*" uptake system.

The data presented provide the first evidence for

multiple Ca2" transport systems in a photosynthetic

bacterium. However, there is precedent for a similar

situation in a non-photosynthetic bacterium as well as

mitochrondria. For example, fi7,n-t-nhar.ter vlnel^andii contains

a ruthenium red-insensitive Ca^'^/n'^ antiport that catalyzes

63

energy-dependent Ca2+ efflux and a ruthenium red and La "

sensitive transport system that catalyzes Ca2- uptake (83) .

Unlike the case with £. vinosum, the ruthenium

red-sensitive, A. vinelandii Ca2- uptake system appeared to

be driven by A^ (outside positive) . The data in Table 4,

suggest that while A^ may make a small contribution to

driving Ca uptake by £. vinosum in the dark, the ruthenium •3 I

red. La -sensitive system in Q. vinosum may catalyze Ca2"

equilibration across the membrane without coupling A^ to

Ca2''" accumulation.

The sensitivity of the £. vinosum Ca2" /H" antiport to

phenothiazine drugs suggests that the antiport may have some

structural similarities to other proteins involved in Ca2"*"

transport or binding such as the Ca2''" ATPase of the

sarcoplasmic reticulum and calmodulin, since these proteins

are also affected by phenothiazines (78-80). Calmodulin is

a heat stable, acidic polypeptide that contains four 2 + Ca'' -binding sites. The protein appears ubiquitous in

eukaryotes and mediates many of the Ca2''" effects (84) . The

binding of Ca2"*" alters the conformation of calmodulin,

increasing its helical content and exposing hydrophic

regions. It appears that the hydrophobic phenothiazines act

by competing with calcodulin's normal target enzymes for

hydrophobic portions of calmodulin (79,80). It may be

possible that these inhibitors act in a similar fashion in

so far as the £. vinosum Ca2"'"/H"*" antiport in concerned.

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