Molecular and Biochemical Parasitology, 15 (1985) 189-201 189

Elsevier

MBP 00540

A HIGH AFFINI'I~Y Ca2+-DEPENDENT ATPase IN THE SURFACE MEMBRANE OF THE BLOODSTREAM STAGE OF TRYPANOSOMA RHODESIENSE

JOHN M c L A U G H L I N

Department of Microbiology and Immunology, School of Medicine, University of MiamL P.O. Box 016960, MiamL FL 33101, U.S.A.

(Received 29 October 1984; accepted 2 January 1985)

Addition of Ca 2÷ (0.01-1 mM) to a s tandard Trypanosoma rhodesiense Mg2+-ATPase assay failed to elicit

any increase in activity. However, in the absence of externally added Mg 2÷ and using calcium-EGTA or

calc ium-CDTA to precisely maintain free metal ion concentration, it was possible to measure a specific

Ca2+-ATPase. Cell fractionation studies revealed this ATPase to be predominantly associated with subcel-

lular particles having an equilibrium density of 1.22 g c m -3 and identified as surface membrane. Using a

discontinuous sucrose gradient, a surface membrane enriched (SME)fraction, only slightly contaminated

with mitochondria as judged by dichlorophenolindophenol-l inked a-glycerophosphate dehydrogenase

activity, was prepared. The SME fraction exhibited Ca2+-ATPase activity, using 200 nM free Ca 2+, of 90 and

21 mU mg -t protein, respectively, using C DT A and EGTA as buffering ligands. This latter result was most

unexpected and indicated that the Ca2÷-ATPase, in addition to having no Mg 2÷ requirement, was inhibited

by submicromolar levels of Mg 2+.

The Ca2+-ATPase was found to have a K0. 5 = 128 + 22 nM free Ca 2÷, the response to increasing Ca 2÷

concentration displaying an extremely high degree of co-operativity (Hill number (nil) = 4.9). The enzyme

was found to be highly substrate-specific for ATP with K0. 5 = 6.2 4- 0.61 pM ATP. A Hill plot of the reaction

velocity as a function of ATP concentration indicated two substrate binding sites (n H = 1.55). A range of

potential modulators of ATPase activity were investigated, with only vanadate (V20~) having any effect:

47% inhibition at 5.0 pM. The Ca2+-ATPase was unaffected by the calmodulin antagonists chlorpromazine

(50 I.tM) and trifluoperazine (50 laM), whilst addition of calmodulin failed to produce any stimulation of

activity. It is concluded that the kinetic properties of this ATPase are compatible with a potential role in the

regulation of intracellular Ca 2÷ in bloodstream T. rhodesiense.

Key words: Trypanosoma rhodesiense; Surface membrane; High affinity Ca2+-ATPase

INTRODUCTION

There is now an extensive literature describing various regulatory functions of the

Abbreviations: DCPIP, dichlorophenolindophenol; EGTA, ethylene glycol bis ([~-aminoethyl)-N,N,N',N',- tetraacetic acid; CDTA, trans-cyclohexane-l,2-diamine-N,N,N'N'-tetraacetic acid; K0. 5 = apparent half

saturation constant; n H = Hill number.

0166-6851/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

190

cell that are wholly or in part responsive to submicromolar changes in the concentra- tion of free cytosolic Ca ~÷ [1]. For this reason resting levels of cytosolic Ca 2÷ are maintained within strict limits, a role attributable in most higher eukaryotic cells to

the operation of mitochondrial [2] plasma membrane and to a.lesser extent endo- plasmic reticulum control mechanisms [3]. Since there is a continuous passive entry of Ca 2÷ into the cell, ultimate control is a surface membrane function: either by means of a Ca2+/Na 2÷ exchanger [4] or actively through the operation of a Ca2+-ATPase pump [5].

For protozoan parasites there is an extremely limited amount of information regarding either the nature or subcellular sites of any of these control mechanisms. It might be expected that for certain of these organisms where mitochondria are either

totally absent (i.e. Entamoeba, Giardia, Trichomonads) or greatly repressed (LS blood stages of African trypanosomes) surface membrane control of cytosolic Ca 2÷ levels would be especially important. Indeed a high affinity Ca2+-ATPase, that ap- pears to be an authentic surface membrane component [6], has been described for Entamoeba histolytica trophozoites [7].

In the bloodstream stage of Trypanosoma brucei there is some indirect evidence [8] which suggests a limited mitochondrial role in regulating cytosolic Ca 2÷ levels. More recently a brief report appeared [9] describing a Ca2+-Mg2÷-ATPase in T. brucei, however, little detail was provided. Most importantly, no K0. 5 value for Ca 2÷ is given to permit evaluation of the measured activity as an authentic high affinity Ca2+-ATPase. One particularly important aspect of the control of intracellular Ca 2÷ levels in African trypanosomes could relate to the release of surface coat (variant specific antigen), there being some indirect evidence that this is a Ca 2÷ mediated process [10].

In the present report an ATPase activity responsive to submicromolar changes in free Ca 2÷ has been detected in the surface membrane ofTrypanosoma rhodesiense. The activity is unusual, both for the high degree of co-operativity indicated and lack of a Mg 2÷ requirement, as well as the absence of any demonstrable dependence on calmo- dulin.

METHODS

The Wellcome CT strain ofT. rhodesiense was isolated from infected rat blood and a whole cell homogenate prepared by grinding with glass beads as previously described [111.

Cellfractionation. A whole cell homogenate, prepared in buffered sucrose (250 mM sucrose, 1.5 mM EDTA, 1.0 mM KCI, 5.0 mM HEPES, pH 7.5) was subjected to differential centrifugation, as previously described [12], yielding particle fractions Pa (7 720 × g for 10 min) and Pb (48 200 × g for 1.0 h). Since differential centrifugation indicated most of the Ca2+-ATPase activity to be in fraction Pa (see Results) this material was further examined by isopycnic sucrose gradient centrifugation using a

191

Sorvall SV288 vertical rotor following a previously established protocol [ 11]. The data obtained were plotted as a series of frequency distribution histograms [13].

In certain instances the washed trypanosomes were surface labelled with the fluoro- genic compound fluorescamine (Fluram, Roche Diagnostics), either free [ 12] or as the 13-cyclodextrin complex [14,15]. The distribution of label after isopycnic gradient centrifugation was expressed as relative fluorescence and measured as previously described [12].

A surface membrane enriched fraction was prepared by layering 3-4 ml of fraction Pa in buffered sucrose over a discontinuous sucrose gradient: 8 m135% (1.112); 12 ml 42% (1.187) and 7.5 ml 54% (1.252). The figures in parentheses denote the specific gravity (g cm -3) of the sucrose solutions and were chosen based on previously established values for the equilibrium densities of flagellar pocket membrane mito- chondria and surface membrane [12]. After centrifugation for 2.0 h at 18 000 r.p.m. (33000 X g) in a SV 288 vertical rotor, the material sedimenting to the 42/54% interface was removed, resuspended in buffered sucrose and centrifuged for 30 min at 18 000 r.p.m. (42 500 X g) in a SS-34 rotor. This surface membrane enriched (SME) fraction was resuspended in 2-3 ml buffered sucrose using 3-4 light strokes of an all glass dounce homogenizer.

Enzyme assays. For the assay of the low affinity mitochondrial and surface mem- brane Mg2+-ATPases (see ref. 12) present in the fractions recovered after gradient centrifugation the point assay [16] as previously described [12] was employed. In an attempt to measure Ca 2+ dependent stimulation of the basal Mg2*-ATPase in whole cell homogenates, released orthophosphate was measured [6]. The assay of other enzyme activities followed methods used in previous investigations: NAD*-linked ct-glycerophosphate dehydrogenase and dichlorophenolindophenol (DCPIP)-linked ct-glycerophosphate dehydrogenase [11] and carboxypeptidase and acid phosphatase [ 15], the latter using p-nitrophenylphosphate as substrate.

The standard assay for measuring high affinity Ca2+-ATPase activity in the SME fraction contained 250 la1100 mM glycyl-glycine-NaOH, pH 8.5,250 ~tl 4 mM ethylene glycol bis (~-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) or trans-cyclohexane- 1,2-diamine-N,N,N',N'-tetraacetic acid (CDTA) (both pH 8.5) and 25 Ixl SME fraction (4.5 mg ml -~ protein). Sufficient 1 mM calcium acetate and /o r MgCI2, calculated as described below, was added to produce the required concentration of free Ca 2÷ and /or Mg 2÷. The reaction was initiated by adding 100 lai 10 mM ATP (Na salt, vanadate free, Sigma Chemical Co.), the final assay volume being made up to 1.0 ml with distilled water. After 20 min at 30°C, 250 lal ice-cold 6% sulfosalicylic acid was added to stop the reaction and released Pi determined [6].

Enzyme activities are all expressed as mU mg -~ protein where 1 U ---- 1.0 lamol product formed per minute at 30°C. Protein determinations were performed using the Folin-Lowry procedure as previously modified [11].

192

Preparation of Ca2+/Mg 2+ buffers. In order to precisely maintain micromolar and sub-micromolar concentrations of free Ca 2÷ and Mg 2÷, EGTA and CDTA were used as buffering ligands [ 17,18]. From established values of K (association constant) for each ligand [19], the apparent association constant (K-) was calculated for the prevailing assay conditions (pH 8.5) using the relationship:

K / K - = 1 + [H÷]k4 + [Hq 2.k4.k3 + ( . . . . . subsequent terms insignificant)

where k3 and k4 are the third and fourth dissociation constants, respectively, for the ligand used.

From the value of K- calculated above the total amount of calcium (or magnesium), Met, required to produce a given concentration of free metal ion [Me] could then be determined, since, if Me t > > [Me],

K- [Me] Me t = L T + [Me]

1 + K- [Me]

Values for both [Me] and L T (total ligand) can be assigned. Since all assays are performed above pH 8.0, only values of K for the fully dissociated forms of the above ligands need be considered. Furthermore, since K for Ca2+-ATP is several orders of magnitude less than either calcium-EGTA or calcium-CDTA, the role of ATP in complexing Ca 2÷ was found to be insignificant. Where both Mg 2÷ and Ca ~÷ levels were to be set, free Ca 2+ was established using a calcium selective electrode (Orion, Cam- bridge, MA) after calculating the total amount of Mg required.

RESULTS

Mg:+-ATPase activity. In a previous investigation [12] a low affinity Mg2+-ATPase was described having a whole cell homogenate specific activity of 60 mU mg -~ protein. Addition of Ca 2÷ (0.01-1 mM) directly to the basic Mg2+-ATPase assay failed to elicit any additional activity. As confirmed in this study (see below) isopycnic gradient centrifugation revealed this basic Mg2*-ATPase activity to be primarily of surface membrane and mitochondrial origin.

A Ca2+-ATPase was demonstrable but only in the absence of externally added Mg 2÷ and through the use of calcium-EGTA buffers to control the concentration of free Ca 2+. At 200 nM free Ca 2+, activity in a whole cell homogenate was 5.75 + 0.68 mU mg -t protein. Subsequent investigation revealed, as described below, that the use of CDTA as buffering ligand caused an unexpected 3-4 fold increase in activity. It was apparent that a Ca2+-ATPase was present which required sub-micromolar amounts of Ca 2÷ and was normally masked in whole cell homogenates by the overwhelming presence of surface membrane and mitochondrial Mg2÷-ATPases.

5

1(

10

.I ~ . C ~ ° ~ l ~ i ~ m '

S

1

Relative Fluorescence

Arid Phosphatase

l

f Mg ATPase

30

I0

40

20

1 0 145 1-20 1-z5 D E N S I T Y - - g/cm a

193

Fig. 1. Distribution profile of the high affinity Ca2*-ATPase and other known marker enzymes in T. rhodesiense. A large particle fraction (Pa) was subjected to isopycnic centrifugation using a 23-56% linear sucrose gradient prepared in 1 mM EDTA, 5 mM HEPES, pH 7.2. Recoveries ranged from 88% (carboxy- peptidase) to 100% (Ca2+-ATPase). Relative fluorescence of fluorescamine-labelled cell fractions was measured as previously described [ 12]. Plots represent the average of three separate experiments.

194

Subcellular distribution of Ca~*-ATPase. Differential centrifugation of a T. rhode- siense whole cell homogenate [ 12] revealed 32% of the recovered Ca2÷-ATPase activ- ity, assayed using a calcium-CDTA buffer at 200 nM free Ca 2÷ to be in fraction Pa" This represents a 2.2-fold enrichment in specific activity compared to 1.12 for fraction Pb and 0.53 for the final soluble fraction.

To further characterize the subcellular distribution of the Ca2÷-ATPase, fraction Pa was subjected to isopycnic sucrose gradient centrifugation (Fig. 1). It is evident that CaZ÷-ATPase is associated predominantly with particles that equilibrated in the densest part of the gradient (P = 1.22 g cm-3). This coincides with a portion of the Mg2*-ATPase and most of the relative fluorescence. The use of fluorescamine to surface label T. rhodesiense has been reported previously [12,16]. Most of the label (relative fluorescence) is found associated with both low density particles (9 = 1.120 g cm-3), identified as flagellar pocket membrane, and the much higher density surface membrane (P = 1.220 g cm-3). This unusually high density is a feature of trypanosoma- tid surface membranes, including T. brucei [20], and is attributable to the presence of an attached subpellicular microtubular network. The established glycosomal marker NAD*-linked ct-glycerophosphate dehydrogenase [11,21] was found to equilibrate at a density of 1.2025 g cm -3, somewhat less dense than surface membrane.

A substantially less pronounced peak of Ca/+-ATPase activity is also present having a distribution co-incidental with that of DCPIP-linked ct-glycerophosphate dehydro- genase, a mitochondrial enzyme [I 1]. By contrast more than 50% of the Mg2*-ATPase exhibits a similar distribution to DCPIP-linked ct-glycerophosphate dehydrogenase. Although this Mg2*-ATPase is presumably mitochondrial it exhibits no inhibition in the presence of oligomycin, in contrast to the bloodstream T. brucei isolate used by Opperdoes et al. [22]. Interestingly, it has been found (McLaughlin, J., unpublished observations) that this mitochondrial MgZ+-ATPase is fully active toward GTP. A loss of nucleotide specificity has been observed for the F1 portion of mitochondrial Mg2+-ATPase, as compared to sub-mitochondrial particles and correlated with the loss of the oligomycin sensitivity-conferring protein (OSCP) [23] from the enzyme complex.

In view of the above results, further characterization of the CaZ÷-ATPase used a SME fraction prepared from fraction Pa by discontinuous sucrose gradient centrifuga- tion (see Methods). The SME fraction was 4.75-5.2-fold enriched in Ca2+-ATPase (24% of original homogenate) and exhibited no detectable carboxypeptidase (lyso- some) or acid phosphatase (flagellar pocket) activity. Most importantly, there was only slight mitochondrial contamination, 6.95% of the total DCPIP-linked ct-glycero- phosphate dehydrogenase being present in the SME fraction. The principal contam- ination was with glycosomes (32% of the total NAD÷-linked a-glycerophosphate dehydrogenase activity). There is, however, no previous evidence for any ATPase activity being associated with glycosomes, and the present investigation (Fig. 1) has revealed neither any peak or shoulder of Ca2÷-ATPase activity that could be attributed to glycosomes.

195

For all of the Ca2÷-ATPase assays, no detergent (0.075% Triton A-100) was present since activity measured in SME fractions exhibited no increase in the presence of

detergent. With whole cells, however, 91% of the activity was latent, indicating as would be expected that the enzyme is probably localized on the cytoplasmic aspect of the surface membrane. The SME fraction Ca2+-ATPase, assayed as described in the 'Methods ' with 200 nM free Ca 2÷ (calcium-CDTA), was linear with respect to time for at least 30 min.

Because of the pronounced influence [H ÷] has on the value for K-, and therefore on the concentration of free Ca 2+, it was not possible to accurately determine the pH opt imum for the Ca2+-ATPase (see ref. 17). A comparison of activities at pH 7.0 and pH 8.5 revealed the latter to give 1.4-fold greater activity.

Establishing the Ca2÷/Mg 2÷ requirements of the A TPase. As was found for the whole homogenate, Ca2÷-ATPase activity in the SME fraction was manifest without any

external source of Mg ~÷. From the results plotted in Fig. 2a, showing surface mem- brane ATPase activity as a function of the Ca 2÷ concentration, it is evident that the use of CDTA as a buffering iigand results in a far more pronounced response. Since CDTA has a much higher affinity constant than E G T A for Mg z÷ and both have similar affinity constants for Ca ~÷ [ 19], previous investigators [ 17,24-29] have compar- ed ATPase progress curves using each ligand to establish the existence of a submicro- molar requirement for Mg 2÷. If sufficient endogenous Mg 2÷ is available in the presence of E G T A to satisfy any Mg 2÷ requirement, then the use of C D T A should

I 00

2 C

A

- 4 - 3 2

B

o

Ioo 20o I I I

- I 300

Fig. 2. Effect of increasing concentrations of free divalent metal ion (Ca 2÷ or Mg2+), established through the

use of high affinity buffering ligands, on the surface membrane ATPase activity. (a) A comparison of calc ium-CDTA (* e) and calc ium-EGTA (,~ *) as buffering system. In both instances free Ca 2÷ levels

were set to increase from 7.5 X 10 -5 to 3.2 × 10 -2 mM. (b) A comparison ofsub-micromolar levels of free

Ca 2÷ (D o) and Mg 2÷ (o o), prepared using C DT A as the buffering ligand, in st imulating ATPase

activity.

196

complex most or all of this Mg 2÷ with a resulting decrease in ATPase activity. The results obtained (Fig. 2a) are the opposite of what would be expected i fsub-micromo- lar Mg 2÷ was indeed essential for the t rypanosome Ca2÷-ATPase. In addition to being unnecessary for Ca2÷-ATPase activity, Mg 2÷ appears to be inhibitory.

Above 20 ~tM free Ca 2÷, there was evidence of a low affinity Ca2÷-ATPase (Fig. 2a). However, it was the activity assayed between 100 and 250 nM free Ca 2+ (CDTA as

ligand) that was potentially of more physiological significance. From Fig. 2b it can be seen that very little ATPase activity was apparent using the same range of Mg 2÷ concentrations. The plot reached an asymptote at 230 nM Ca 2+ and gave a value for

K0. 5 = 128 4- 23 nM Ca 2÷. Of particular note was the 8-fold increase in activity that occurred for less than a 3-fold increase in effector (Ca 2÷) concentration. In view of this rapid increase in activity it was not surprising to find that the data failed to conform to the Michaelis-Menten equation, a double reciprocal plot yielding a curve with an

upward concavity, suggestive of positive co-operativity. Use of the Hill equation enabled a straight line fit of the data to be obtained ( r 2 ~- 0.96), the slope (nil) of 4.9, again circumstantial evidence for a high degree of co-operativity.

Fig. 4 shows the effect of assaying SME fraction ATPase using the same range of free Ca 2÷ concentrations as for Fig. 2b but in the presence of a fixed level (1.0 ~M) of free Mg 2+. It is evident that the response to increasing Ca 2+ is much less pronounced than in the absence of added Mg 2÷. On considering these results, together with previous data which demonstrated both the absence of a specific high affinity Mg 2+- ATPase (Fig. 2b) and the lack of any Mg 2÷ requirement for the high affinity Ca2+-ATP -

i

4 2.6

~ 80

~40 .~.

0 4~ ob 1~o 1~o

Fig. 3. Hill plot of surface membrane ATPase activity as a function of increasing free Ca 2÷ concentration

using l mM CDTA as buffering ligand. Data fitted using the T.I. STI-08 linear regression analysis program.

Fig. 4. Effect of a constant fixed level of free Mg 2÷ (1.0 m) on surface membrane ATPase activity assayed in

the range 75-200 nM free Ca 2÷. The amount 1.0 M MgCI2, using a fixed volume of 1 mM CDTA, was

calculated as described, after determining K- for magnesium-CDTA. The free Ca 2+ concentration was then

set by titrating with I mM calcium acetate using an Orion calcium-selective electrode.

197

ase, it was concluded that a separate CaZ÷-Mg2÷-ATPase must be present. Further characterization of this activity has not been attempted during the present investiga- tion.

Nucleotide specificity. Using a concentration of 200 nM free Ca 2÷, prepared using calcium-CDTA, activity toward ADP, GTP and UTP (all lmM) was less than 5% of that obtained using 1 mM ATP.

A double reciprocal plot of velocity versus substrate concentration (1-25 laM ATP) was non-linear and above 30 laM ATP no further increase in activity was evident. A Hill plot of the data is shown in Fig. 5, having a slope (nil) of 1.55 (r 2 = 0.93). The curve for reaction velocity as a function of substrate concentration gave K0. 5 = 6.2 + 0.61 laM ATP. The Hill number indicates two binding sites for ATP and is similar to other high affinity Ca2*-ATPases [25,27], though in these instances, contrary to evidence above for the trypanosome enzyme, Mg2+-ATP 2- is probably the true substrate.

Effect of selected agents on Ca2+-ATPase activity. The effect of various agents known to influence the activity of other ATPase activities was assessed and revealed the following to be without effect: oubain (1-5 mM); carbonyl cyanide m-chlorophenyl- hydrazone (10 laM); NaN3 (2 mM); oligomycin ( 10 p.g ml -~) and diethylstibesterol (0.05 mM). Orthovanadate (V20~-) was the only effective inhibitor of the SME fraction Ca2÷-ATPase, 5 laM producing a 47% inhibition of activity. This is comparable to inhibition observed for a variety of ion-motive ATPases, including the erythrocyte Ca2÷-Mg2+-ATPase [30]. The two phenothiazine drugs chlorpromazine and trifluoper- azine are known to inhibit a range of calmodulin dependent enzymes including certain high affinity Ca~÷-ATPases [27,30,31]. However, even at levels well above

,~-Oga

nH.l .f~

l.n ATP

Fig. 5. Substrate dependent increase in Ca2÷-ATPase activity is a cooperative process. Hill plot for data

obtained between 1.0 and 30 M ATP in the presence of 200 nM Ca z÷ using I mM C D T A as buffering ligand.

198

those known to be inhibitory (50 laM), both compounds were without effect. In agreement with this finding, addition of calmodulin (Sigma, from bovine brain) at 1-5.0 g ml -t failed to stimulate activity. This was also found after washing the SME fraction with 2 mM E G T A [29] in an attempt to remove attached calmodulin.

As a preliminary aspect of the future purification of the Ca2*-ATPase, attempts were made to dissociate the enzyme from the SME fraction. The use of 0.2% Zwitter- gent 3-12 (Calbiochem) was found most successful, solubilizing 65% of the total activity. This same detergent has been found most effective in releasing adenylate kinase f rom T. rhodesiense glycosomal membranes (McLaughlin, J., in press [32]). By contrast, 0.25% Triton X-100 released only 39% of the total Ca2÷-ATPase.

DISCUSSION

The results of this study firmly establish the presence of a high affinity Ca2+-ATPase associated with a high density subcellular particle fraction, identified on the basis of fluorescamine labelling as surface membrane (see ref. 12). Noticeable was the lack of CaE+-ATPase in the flageilar pocket membrane, which is thought to be an important site for uptake/ t ranspor t processes [33]. The comparatively low specific activity

agrees with findings for other Ca2+-ATPases, and is in accord with the fact that t ransport of only small amounts of Ca 2÷ are required to produce profound changes in intracellular Ca 2÷ levels.

The K0. 5 (128 nM free C a 2÷) falls within the range recorded for other high affinity CaE+-ATPases [17,24-27,29] and would be compatible with involvement in some physiological role such as Ca 2÷ extrusion. The localization of the enzyme on the inner surface of the membrane, as indicated by latency studies with whole cells, also suggests a role in Ca 2÷ extrusion.

From a consideration of the properties of both erythrocyte and surface membrane high affinity Ca2+-ATPases, Penniston [5] established certain features common to most of these enzyme activities. However, it is becoming more apparent that certain high affinity Ca2+-ATPases diverge from this erythrocyte-like enzyme type. This is most evident with respect to the absence of any requirement for externally supplied Mg 2÷ [17,24-29] for activity. In the majority of these instances it has been found that if E G T A is replaced by CDTA (which has a 10-fold greater association constant for Mg 2+) to control free Ca 2÷, very little ATPase activity is now detectable. This loss of activity is due to" the removal by C D T A of endogenous Mg 2÷ essential for enzyme activity. The T. rhodesiense Ca2+-ATPase together with an enzyme activity described in rat liver plasma membrane [26] appear to be the only two authenticated exceptions to this requirement for Mg 2÷.

In other instances where Mg 2÷ appears not to be required [6,34,35] rigorous precau- tions to exclude its presence (i.e. the use of CDTA as a buffering iigand) were not in evidence. In one of these latter instances, which concerned a surface membrane Ca2+-ATPase in E. histolytica trophozoites [6], there was, as found in the present

199

study, evidence for Mg 2÷ being inhibitory. The effect, however, was observed at much higher levels than the micromolar concentration of Mg 2÷ that appears capable of

inhibiting the T. rhodesiense enzyme. The effect of elevated Mg 2÷ concentrations on the erythrocyte Ca2*-MgE÷-ATPase results in a complex form of inhibition [36].

Even in the above instances where Mg 2÷ was found essential, a role for the Ca 2÷ binding protein calmodulin remains unproven for most of these high affinity C a 2÷-

ATPases. Thus, as in the present investigation, the two phenothiazine compounds, chlorpromazine and trifluoperazine, which are known to antagonize the effect of calmodulin [37] were found to have no effect on these ATPases [24,25,29]. Addition of calmodulin, even to EGTA-washed surface membrane failed to cause any stimulation of activity. Calmodulin has been found to occur ubiquitously in the animal kingdom and is present in T. brucei [38] and at least two other protozoan parasites (E. histolytica and Giardia lamblia; Weinbach, E.C., personal communication). It is possible that calmodulin is very tightly bound to the t rypanosome surface membrane, though this could not readily explain the lack of inhibition of CaE÷-ATPase in the presence of the

phenothiazine drugs. The ostensibly high degree of co-operativity of the T. rhodesiense enzyme, a value of

n H = 4.9 as compared to 1.5-2.0 for other CaE+-ATPases [17,25] is suggestive of some auxiliary Ca 2÷ binding protein. In at least one instance, involving liver cell plasma membrane, a calcium binding protein quite distinct from calmodulin has been detect-

ed [23] and found essential for the full expression of CaE÷-ATPase activity. Compared to the reports for metazoan cells there is a dearth of information o n C a 2÷

regulation in the protozoa. Since there is no reliable evidence of a Na*-K÷-Mg 2÷- ATPase in any of the parasitic protozoa, including African t rypanosomes [12,39],

there would appear to be no means by which a Na ÷ gradient could be established to en- able a Na÷/Ca 2÷ antiporter to function. In most bacteria a CaE÷/H ÷ antiporter func- tions by relying on an electrogenic surface membrane proton pump [40]. So far the only evidence of a surface membrane H*-ATPase in any protozoan parasite comes from a study of intra-erythrocytic Plasmodium chabaudi. However, rather than being involved in Ca 2÷ extrusion, this enzyme has been implicated in the marked accumula- tion of Ca 2÷ that occurs in the intra-erythrocytic stages of the malaria parasite [41].

The properties of the Ca2*-ATPase described in this report make it an excellent candidate for a principal role in regulating intracellular Ca 2÷ levels in T. rhodesiense. In

only a comparatively few instances have attempts been made to correlate the proper- ties of a given high affinity CaE*-ATPase with Ca 2÷ transport. Such studies have used either surface membrane vesicles [27,29] or in the case of erythrocyte Ca2÷-Mg 2÷- ATPase, liposome reconstituted enzyme [30]. Future work will explore the feasibility of these approaches as a means of establishing the role of the T. rhodesiense Ca 2÷-

ATPase in intracellular Ca 2÷ regulation.

200

ACKNOWLEDGEMENTS

T h e a u t h o r is m o s t i n d e b t e d to D r . A r b a A g e r , J r . a n d t he s t a f f o f t h e R a n e

L a b o r a t o r y ( U . M . ) f o r p r o v i d i n g T. rhodesiense i n f e c t e d r a t b l o o d . T h e t e c h n i c a l

a s s i s t a n c e o f M r s . G l a d y s G u e r r a is g r a t e f u l l y a c k n o w l e d g e d . T h i s i n v e s t i g a t i o n w a s

s u p p o r t e d b y U.S . A r m y c o n t r a c t D A M D 17-79C-9038 .

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