Des progres majeurs ont permis le renouvellement des connaissances dans le domaine des « moteurs biologiques » : les deux premieres structures cristallines de la tete de la myosine viennent d'etre publiees ; de nouveaux tests de mobilite permettent de determiner le mouvement et la force developpee par une molecule de myosine unique le long du filament d'actine ; les progres de l'ingenierie des proteines et des echanges entre sous-unites de myosine mettant a disposition des molecules chimeres de myosine permettent l'etude de la fonction des chaines lourdes et legeres de la myosine et du role de certains domaines. La decouverte de mutations ponctuelles dans la chaine lourde de myosine ventriculaire de patients atteints de cardiomyopathie hypertrophique familiale donne une nouvelle dimension a ce domaine accessible maintenant a de nouvelles methodes d'exploration.

The first two crystalline structures of the myosin head were recently published. The myosin head is made of two main domains. The motor domain comprises the first 600 N-terminal residues of the myosin head heavy chain; it contains the ATP-binding site and the main interface with actin. The transmission domain is composed of the long helicoidal C-terminal part of the myosin head heavy chain, along with essential and regulatory light chains. New motility assays demonstrated the movement and force produced by a single myosin molecule along the actin filament. Genetics and protein engineering led to the production of chimeric myosin molecules. These combined techniques enabled to demonstrate that the motor domain of the myosin head in skeletal muscles is self-sufficient for hydrolyzing ATP and inducing movement, and that the presence of light chains is crucial for motility efficiency by stabilizing the transmission domain. In contrast, light chains in non-striated muscle or non-muscle myosins determine motor activity by regulating ATP hydrolysis but do not markedly influence motile performances of the corresponding muscles. Such studies on the myosin structure and structure-function relationships between heavy and light myosin subunits helped to redefine the specific role of certain myosin domains. All these advances have generated renewed interest in the field of biological motor molecules and myosin isoforms. The discovery of point mutations in the ventricular myosin heavy chains from patients with familial hypertrophic cardiomyopathy has accelerated new research projects in this promising field!

Nine monoclonal antibodies were used to test calcium and EDTA effects on the molecular conformation of ventricular VLC2 within myosin. Antibody epitopes were located in six domains of VLC2 using recombinant proteins. The apparent association constants of these antibodies were measured in solution in the presence of calcium or EDTA. An immunofluorescence study was performed to establish whether the observed effects would occur in more integrated systems, as compared to isolated proteins in solution. Our results showed (1) a slight effect of calcium on isolated VLC2, located in the aa 29-45 domain, (2) a clear-cut effect of calcium on VLC2 within myosin, only in the aa 45-59 domain, and (3) in the presence of EDTA, antibody affinities for VLC2 within myosin similar to the affinities for isolated VLC2. These results are discussed in terms of spatial arrangements and binding mechanisms between HC and VLC2. They suggest that there are two processes for stabilizing HC/VLC2 complex formation: one binding via calcium chelation and another involving hydrophobic interactions.

A cDNA clone coding for an internal fragment of slow-cardiacβ-myosin heavy chain was isolated from aλgt10 human skeletal muscle library. Six overlapping cDNA subclones, which span myosin heavy chain subregions and presumably interact with actin, were derived from this clone, fused to aβ-galactosidase vector and expressed inEscherichia coli. Three of the subclones were obtained by PCR (polymerase chain reaction) which enables gene or cDNA fragments to be amplified independently of preexisting restriction sites. Initially, various experiments were carried out using a long MHC (myosin heavy chain) fusion protein containing the 50 kDa-20 kDa connecting region, the whole 20 kDa region and the short subfragment 2 region. This MHC fusion protein was chemically or proteolytically cleaved in the same conditions as the native myosin molecule. Whole and truncated forms of the MHC fusion protein were separated on polyacrylamide gels, electroblotted on nitrocellulose sheets and renatured. They were then assayed in overlay experiments with F-actin and/or myosin light chains in solution. Specific antibodies were used to detect interactions between heavy chain fragments and F-actin or light chains. We thus observed that one long heavy chain fragment synthesized byE. coli behaved like proteolytic or chemical MHC preparations made from native myosin molecules. Two chymotryptic fragments of the MHC fusion protein, which are soluble at low ionic strength, cosedimented with F-actin in solution. Our results demonstrate that, in actin overlay experiments with whole fusion proteins, interactions seem to be due to the heavy chain fragment, not to the bacterial component. All interactions were non ATP-sensitive. We further investigated the possible participation of the six recombinant MHC fragments in contributing to the actomyosin interfaces on the 50 kDa-20 kDa regions of the human cardiacβ-MHC. The present procedure, which enables the synthesis of any MHC fragment independent of any protease site, is a powerful new tool for studying structure-function relationships within the myosin molecule family.

The synthesis of coagulation factor V was investigated in isolated rat hepatocytes maintained in long-term primary culture. Two culture conditions were compared. A clotting assay and an immunoprecipitation experiment with rabbit anti rat factor V IgG were used to demonstrate not only the presence of factor V in the cells but also active secretion into the culture medium. Both the inhibition of the clotting reaction in presence of the antibody and absence of thrombin in culture media confirmed the specificity of the clotting assays. Electron microscopic examination located factor V in the endoplasmic reticulum and Golgi apparatus of hepatocytes in common with other liver specific plasma proteins. Examination of liver tissue sections confirmed the production of factor V in hepatocytes but not in hepatic endothelial cells although it did not exclude a transit pathway of factor V through these cells. Addition of Russell viper venom factor V activating enzyme to the culture medium had no effect on the factor V activity. In contrast, treatment of cell extracts did increase the coagulant activity. This suggests that hepatocytes contained principally an unactivated form or procofactor, whereas factor V present into the culture medium was mainly in an activated form. These data provide evidence for synthesis and secretion of an hepatocytic factor V.

Abstract In a previous paper, we demonstrated that deep hypothermia in dogs provokes a release of a heparin‐like factor. In the present study, we investigated some properties of this anticoagulant activity and compared it with exogenous heparin activity. The endogenous anticoagulant inhibited factors IIa and Xa; it was hydrolysed by heparinase and was AT III dependent. However, it differed from heparin in so far as it was adsorbed on cation exchange gel at neutral pH, its inhibition was decreased in the presence of neuraminidase, and it could not be neutralized with Polybrene or protamine. A release of heparan sulphate is suggested but remains to be demonstrated.

1.1. An original method was applied to purify rat factor V. The final preparation had a sp. act. of 45 U/ml for a 2500-fold purification with a yield of 43%.2.2. The final product is partially activated since it is 4.7-fold activable by RVV-VAE vs 6.3 in plasma. It can explain the presence of some of the four slightly stained additional bands found in SDS-electrophoresis.3.3. Finally, results of the purification suggest that rat factor V is a 338,000 single chain glycoprotein with a strong molecular asymmetry.4.4. A factor V deficient fraction was produced and used to adsorb an anti-factor V antiserum. This adsorbed antiserum was found monospecific against purified rat factor V.

Immunolocalization of the factor V was performed in adult and fetal rat liver by means of an indirect peroxidase labelling. This could be done owing to the production in our laboratory of a mono-specific antiserum anti rat factor V. In all the cases (perfused and non-perfused adult liver and fetal one) the observation of the sections has revealed an intense circular or granular labelling into all the hepatocytes whatever was their localization in the hepatic lobule. Hepatic endothelial cells seemed to be negative for factor V and this aspect of our results was discussed.

1. The course of haemostasis defects was investigated in dimethylnitrosamine (DMNA) acute liver necrosis.