Michel Savart

Association of calpains 1 and 2 with protein kinase C activities.

Calpains 1 and 2 co‐eluted with protein kinase C activities after hydrophobic (phenyl‐Sepharose) and anion‐exchange (Mono Q) chromatographies of a 100 000 × g supernatant which was defined as cytosol. After centrifugation of the cytosol at 200 000 × g for 16 h, the major part of calpain 1 and of its associated protein kinase C activity was recovered in the pellet, when the major part of calpain 2, also associated to a protein kinase C activity, was present in the resulting supernatant. Polyacrylamide gel electrophoresis of the fractions eluted from the Mono Q column, which contained calpains 1 or 2 and their associated protein kinase C activities, revealed two main bands with a molecular mass of 80 and 28 kDa.

A shear horizontal acoustic plate mode (SH-APM) sensor for biological media

Abstract Acoustic wave devices are often used as chemical sensors. Those described in the literature are merely based on bulk waves or on surface waves (SAW), also called Rayleigh waves. In this paper we describe another type of device, based on shear-horizontal acoustic plate mode (SH-APM) waves. Experimental results with various modes concerning the influence of the temperature, the viscosity and the concentration of NaCl and tris(hydroxymethyl)aminomethane (Tris) in aqueous solution are presented: they show the better sensitivity of higher-order modes. These results show that this device can be used as a sensor in a biological medium.

Isolation and identification of a μ-calpain-protein kinase Cα complex in skeletal muscle

A μ‐calpain‐PKC complex was isolated from rabbit skeletal muscle by ultracentrifugation and by anion‐exchange chromatography. The PKC associated to μ‐calpain was stimulated by calcium, phosphatidylserine and diacylglycerol, and corresponds to a conventional PKC (cPKC). This complex presents an apparent molecular mass close to 190 kDa and is composed of one μ‐calpain molecule and of one cPKC molecule. Using monoclonal antibodies specific for the different cPKC isoforms, the isoenzyme associated to μ‐calpain was identified as cPKCα. Immunofluorescence staining reveals a co‐localization of μ‐calpain and cPKCα on the muscle fibre plasma membranes.

Specific binding of dexamethasone to plasma membranes from skeletal muscle

A procedure was developed to isolate plasma membranes from rabbit skeletal muscle. K+-dependent phosphatase activity was used as marker enzyme for plasma membranes and was determined in the presence of CHAPS (3-( (3-cholamidopropyl)dimethylammonio)-1-propanesulfonate), a zwitterionic detergent. Ca2+-ATPase and succinate dehydrogenase activities were used as marker enzymes for sarcoplasmic reticulum and mitochondria, respectively. Electron-microscopy revealed that plasma membranes were in the form of vesicles. Significant proteolysis of membrane proteins was observed during extraction, which was inhibited by EGTA and 20 mM molybdate. SDS-polyacrylamide gel electrophoresis revealed the disappearance of an intense 96 kDa protein band when membranes were purified in the absence of EGTA and molybdate. Specific binding sites for [3H]dexamethasone were identified in plasma membranes after freezing and incubation with CHAPS. Dithiothreitol was essential for steroid binding and ATP increased it. Under standardized assay conditions, binding was complete with 50 min a 37 degrees C. No binding occurred at 0 degrees C, nor if EGTA and molybdate were absent from the extraction medium.

Degradation of protein kinase Mα by μ-calpain in a μ-calpain-protein kinase Cα complex

Abstract In previous studies, we isolated and identified a μ-calpain-PKCα complex from rabbit skeletal muscle. At the same time we pointed out that an association between μ-calpain and PKCα could occur at the level of the plasma membrane of muscle cells, and that PKCα could thus be considered as a potential μ-calpain substrate. In the present study, using the μ-calpain-PKCα complex as a model, we report that μ-calpain is activated in the combined presence of physiological calcium concentrations (less than 1 μM) and phosphatidylserine. Furthermore our data also show that: (1) there exists a correlation between the appearance of autolyzed μ-calpain forms and PKCα hydrolysis which leads to the formation of PKMα; (2) in certain experimental conditions, autolyzed μ-calpain forms are able to hydrolyze PKMα independently of the presence of diacylglycerol.

Calpain 1-protein kinase C complex: effect of calpain inhibitors after dissociation.

A calpain 1-protein kinase C (PKC) complex was isolated from rabbit skeletal muscle by hydrophobic interaction chromatography on phenyl-sepharose and by strong anion exchange chromatography on Q-Sepharose. Calpain 1 and kinase activities were then dissociated on a phenyl-Sepharose matrix using gradients of decreasing ionic strength. The purified PKC obtained corresponded to conventional PKC and was recognized by a monoclonal antibody specific for alpha and beta isotypes. Leupeptin, calpain inhibitor II, and the more selective calpain inhibitors calpeptin and MDL 28170 did not block the activation of the purified PKC by Ca2+ and phosphatidylserine.

Inhibition of endogenous RNA polymerase activity from thymus chromatin by transcortin-hydrocortisone complex

A transortin-hydrocortisone complex has been isolated from human serum by affinity chromatography on oxidized corticosterone coupled to AH-Sepharose 4B. The influence of this complex and of hydrocortisone alone on endogenous RNA polymerase activity from thymus chromatin have been tested. Results show that hydrocortisone alone has no effect on RNA polymerase activity from thymus chromatin. Under the same experimental conditions, The transcortin-hydrocortisone complex induces an important decrease in the incorporation of UMP into RNA. The dose response of thymic RNA polymerase to transcortin-hydrocortisone complex and the effects of alpha-amanitin on this response are also reported.