Probing conformational changes within LC2 domains of cardiac myosin.

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.

Probing myosin light chain 1 structure with monoclonal antibodies.

Five monoclonal antibodies that react with different regions of myosin light chain 1 from human ventricular myocardial muscle were used to obtain information on interactions between the light chain 1 and heavy chains and generally on the tertiary structure of the light chain 1 within the myosin head. We performed Western blot assays of the five antibodies with myosins from different cardiac and skeletal muscles, with different proteolytic fragments of bovine ventricular myosin light chain 1 (LC1) and to different recombinant fragments of human ventricular LC1 and rat fast skeletal light chain LC1/LC3. The five antibodies were mapped in three different regions of the light chain 1: two antibodies mapped within the first eight amino-terminal residues, two between residues 71 and 74, and one between residues 129 and 134.

Expression of human beta-myosin heavy chain fragments in Escherichia coli; localization of actin interfaces on cardiac myosin.

A cDNA clone coding for an internal fragment of slow-cardiac beta-myosin heavy chain was isolated from a lambda gt10 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 a beta-galactosidase vector and expressed in Escherichia 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.

Biosynthesis of factor V by normal adult rat hepatocytes.

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.

Characterization of a heparin-like activity released in dogs during deep hypothermia.

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.