The goal of this communication is to show that

(i) actomyosins with different calcium sensitivity interact with each other; (ii) the desensitized actomyosin interacts with the natural actomyosin and modifies its calcium sensitive system. This modification consists both in a decrease of the natural actomyosin calcium sensitivity and in its complete disappearance. However, this disappearance is reversible; (iii) the character of interaction of the desensitized actomyosin with natural actomyosin is unusual and is worth studying in detail.

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7

Buffer system:KCl, 90 mMKH

2PO

4, 0.008 mM

Na2HPO

4, 0.006 mM

pH 6.7DissociationAggregation

Dependence of actomyosin particle size from ionic strenght (scheme)

Size

of

parti

cles

Ionic strenght

Removing of Ca2+-sensitive complex by washing

Preparation of desensitized actomyosin (dAM) was done by washing nAM at 4-6°C in pure water containing a minimal amount of Tris-buffer, pH 8.5-9.0

(Schaub, M.C. and Perry, S.V. 1969, Biochem. J. 115: 993)

Preparation of two-component suspensions of actomyosin

Two-component suspension

Desensitized AMNatural AM

Superprecipitation of nAM, dAM, and their mixtures was carried out at 20°C in phosphate buffer containing 0.5 mM Ca2+ or 0.5 mM EGTA and 0.25 mM MgATP at a protein concentration of 0.1 mg/ml.

The reaction of superprecipitation was recorded by the method of 90°-angle light scattering at 450 nm.

Sensitivity to Ca2+ (Sca) was expressed quantitatively as a light scattering decrease (LSD): LSD = I0-I, in the presence both of 0.5 mM Ca2+ (LSDca) and of 0.5 mM EGTA (LSDegta), where I0 is the initial and I, the final level of the light scattering (in arbitrary units).

SScaca = LSD = LSDcaca/LSD/LSDegtaegta. .

As a rule, three measurements of Sca were done for each actomyosin sample.

0 20 40 60 80 1000,5

1,0

1,5

2,0

2,5

3,0

Interacting gels

dAM only

nAMonly

Without nAM-dAM interaction

Ca2+-sensitvity of two-componentsuspensions

Ca2+

-sen

sitiv

ity, a

rbitr

ary

units

Desensitized actomyosin content, w/w %

Mechanical contact is needed to modify Ca2+-sensitivity of nAM

Test tubeTwo-component suspention

Two-layer system modeling the interaction of the particles

dAM, 40%

nAM, 60%

Particles of n- and dAM came into collision

To form an actomyosin gel with non-uniform distribution of the TT-complex, a two-layer system was prepared in 10-ml centrifuge tubes, using the following steps. First, 30 mg of the dAM in suspension were placed in each tube and precipitated by centrifugation for 20 min at 10,000 g (with the final protein concentration of 30-40 mg/ml in the precipitate). Then the supernatant was removed; different amounts of the nAM suspension were added very carefully, and the centrifugation was repeated. The second centrifugation produced a two-layer system with the overlying supernatant that remained in the tubes during the entire experiment.

Formation of two-layer gels

0 20 40 60 80 100

1,0

1,5

2,0

2,5

3,0

3,5

dAM

nAM

Ca2+-sensitivity of the natural actomyosin,

arranged as the upper part of the two-layer system

Ca2+

-sen

sitiv

ity, a

rbitr

ary

units

Desensitized actomyosin content, w/w %0 20 40 60 80 100

1,0

1,5

2,0

2,5

3,0

3,5

Threshold = 18±5 % of dAM Asymptote

The hyperbolic relation with X-axis asymptote as the threshold for thenAM-dAM interaction

dAM

nAM

Threshold manner of nAM-dAM interaction

Ca2+

-sen

sitiv

ity, a

rbitr

ary

units

Desensitized actomyosin content, w/w %

Threshold manner of nAM-dAM interaction in the two-layer system

Formation of two-tube systems for electrophoretical analysis of tropomyosin and troponin T distribution.

The apparent diffusion coefficient for the TT-complex in dAM gel is about (1-4)·10-4 cm2/sec,

i.e. three orders higher than the same values for protein diffusion in water.

========================

In our conditions, diffusion of tropomyosin-troponin complex began in 6 hour’s time after creation of the two-tube systems and came to an end after 12 hours.So, as a whole the diffusion takes up about 6 hours.

Diffusion direction

Diffusion direction

Diffusion direction

dAM

dAM

dAM

nAM

nAM

nAM

0 20 40 60 80 100

0

5

10

15

20

25

30

35

Trop

onin

T

Tropomyosin

The tropomyosin and troponin T contents in the natural actomyosin

Rel

ativ

e co

nten

t, %

to

actin

Desensibilized actomyosin content, w/w %

Linear

range

Range of insensibility

Saturation range

Opt

ical

den

sity

of

band

Protein content in band

Keeping of actin band density in the linear part of the dependency is the main problem

of the electrophoretical study

0 20 40 60 80 100

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

5,0

Desensitized actomyosin content, w/w %

Lim

it of

dye

ads

orpt

ion,

mm

ole/

kg

0,8

1,0

1,2

1,4

1,6

1,8

2,0

2,2

2,4

Limit of dye adsorption

2+

Ca

2+-sensitivity, a.u.

0 20 40 60 80 100

0,6

0,8

1,0

1,2

1,4

1,6

1,8

2,0

Limit of dye adsorption

Desensitized actomyosin content, w/w %

Lim

it of

dye

ads

orpt

ion,

mm

ole/

kg

0,0

0,5

1,0

1,5

2,0

2,5

3,0

Ca2+-sensitivity

Ca

2+-sensitivity, a.u.

Structural changes in actomyosin gel as a result of interaction of nAM with dAM. Structural changes were tested by the dye,

neutral red.

The Ca2+-sensitivity of the suspension mixture containing 60% of nAM and 40% of dAM plotted as a function

of n-propanol or ethanol concentration after 15-20 h incubation of the protein mixture with the alcohols.

0 1 2 3 4 50

1

2

3

4

5

6 nAM (60%) + dAM (40%)

Ethanol

n-Propanol

Ca2+

-sen

sitiv

ity, a

rbitr

ary

units

Alcohol concentration, mol/l

CONCLUSIONSCONCLUSIONS The studied protein complexes interact in the gel The studied protein complexes interact in the gel

statestate as colloidal particlesas colloidal particles. . In this reason the interaction begins In this reason the interaction begins at a surfaceat a surface

of interacting gel particles and extends over the of interacting gel particles and extends over the entire gel volume. entire gel volume.

the interaction of actomyosin gels the interaction of actomyosin gels reversiblyreversibly disturbs normal operation of the Cadisturbs normal operation of the Ca2+2+-sensitivity -sensitivity system and can change the troponin-tropomyosinsystem and can change the troponin-tropomyosin complex complex distributiondistribution in the whole gel volume. in the whole gel volume.

The revealed peculiarities of the interaction allow The revealed peculiarities of the interaction allow claiming a new type of protein-protein interaction. claiming a new type of protein-protein interaction.

AcknowledgementsAcknowledgements The author wishes to thank sincerely The author wishes to thank sincerely

Dr. A. A. Vereninov for a decisive Dr. A. A. Vereninov for a decisive support of this study.support of this study.

Dr. L. Z. Pevzner for useful criticismDr. L. Z. Pevzner for useful criticism

The project home page:http://actomyosin.narod.ru