Hydrodynamic studies on the self association of vertebrate skeletal muscle myosin.

Sedimentation velocity studies on myosin A solutions at high ionic strength combined with computer-simulation of the concentration dependence of the sedimentation coefficient for a rapidly reversible monomer-dimer equilibrium have confirmed that if such an equilibrium does exist it has an equilibrium constant of less than 10 ml/g. A new hydrodynamic treatment has been used to calculate the molecular weight of myosin from s0 and ks alone and has yielded a value of 470 000. Combination of viscosity and sedimentation velocity results has shown that the myosin molecule displays little swelling (Vs/v = 1.1 +/- 0.1). A new picture of the myosin molecule is presented in which a conformational change in the head region is suggested to account for the variation in published s 0 values.

Frictional properties and molecular weight of native and synthetic myosin filaments from vertebrate skeletal muscle.

1. The molecular weights of a series of synthetic myosin filaments have been measured, using the transport-concentration dependence theory of Rowe, A.J. [Biopolymers, 1977, 16, 2595--2611]. It is shown that for preparations of narrow length distribution (0.60--0.77 micrometer), N, the number of myosin molecules/14.3 nm varies between 3 and 6. 2. The reduced specific viscosity of synthetic myosin filaments has been measured as a function of both concentration and shear rate. From the concentration dependence at zero rate of shear, a value for the "swelling" of the filaments Vs/-v = 2.3 has been calculated. 3. The frictional coefficient of synthetic myosin filaments has been shown to be anomalously but reproducibly high, as compared to that of prolate ellipsoids of the same length and mass. This additional frictional drag has been numerically characterised by a "frictional increment", fi = 1.76 +/- 0.11. 4. A procedure has been devised whereby for any elongated structure which can be assumed to show the same (or other known) fi value, the molecular weight can be estimated from s0 (extrapolated sedimentation coefficient) and 2b (length) alone. 5. An s0 value for natural A-filaments, isolated from rabbit psoas muscle, has been determined by the active enzyme centrifugation technique. From this value, s0 = 132 +/- 3 S, a molecular weight of 1.20 . 10(8) has been computed by the new procedure, for preparations of average length 1.27 micrometer. 6. Contingent upon the validity of the assumptions used (see 4 above) the N value is computed as 3.1 +/- 0.2, consistent with the native, fully intact A-filament having three-fold symmetry, containing 294 myosin molecules, and having a molecular weight based upon myosin and C-protein of 1.31 . 10(8).

Ecto-protein kinase activity in rabbit peritoneal polymorphonuclear leucocytes.

An ectoprotein kinase activity has been identified on intact rabbit peritoneal polymorphonuclear leucocytes and the time course of phosphate incorporation into proteins has been followed at different ATP levels. Saturation is reached at around 3 mM ATP and the activity is inhibited by p-chloromercuribenzoate. The possibility that the observed protein phosphorylation arises through the action of a membrane ATPase liberating phosphate for transfer into the cell, incorporation into ATP and its utilisation by endogenous kinases, has been excluded by studying both enzymes concomitantly and measuring the rate of [32P]orthophosphate uptake. Lactate dehydrogenase measurements in the extracellular media also exclude the possibility of kinase liberation from lysed cells. Moreover, the pattern of 32P-labelling of polypeptides when intact cells are exposed to [32P]ATP is quite different from that when homogenates are incubated with [32P]ATP or intact cells with [32P]-orthophosphate. We have been unable to demonstrate any cAMP dependency for this ectokinase activity.

Preparation of a highly purified surface membrane fraction from rabbit polymorphonuclear leucocytes by high-voltage free-flow electrophoresis.

A surface membrane fraction of high purity and good yield has been prepared from homogenates of rabbit peritoneal polymorphonuclear leucocytes, using a preliminary sorbitol density gradient sedimentation followed by preparative high voltage electrophoresis in a thin flowing buffer film. Enrichment values for the plasma membrane marker enzyme 5'-nucleotidase and 125I-labelled Lens culinaris lectin, after the latter had been applied at the whole cell level, were 18-fold and 6-fold, respectively. Contamination of the surface membrane fraction by other organelles was negligible and approximately 1 mg of surface membrane protein can be obtained from 2 . 10(9) leucocytes. A triacylglycerol-rich, protein-poor fraction that lacks any definable structure in electron microscopy separates discretely from the surface membrane vesicles during electrophoresis. It is considered that this may be a contaminant not previously recognized as present in membrane fractions prepared by more conventional procedures.