The rapid removal of Ca2+ ions from the cytosol, necessary for the efficient relaxation of cardiac muscle cells, is performed by the Ca2+-pumping ATPase of the sarcoplasmic reticulum. The calcium pump is activated by cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban, an integral membrane protein of the sarcoplasmic reticulum. Using a heterobifunctional crosslinking agent which can be cleaved and photoactivated, we provide evidence for a direct interaction between the two proteins. Only the non-phosphorylated form of phospholamban interacts with the ATPase, demonstrating that phospholamban is an endogenous inhibitor that is removed from the ATPase by phosphorylation. Non-phosphorylated phospholamban interacts only with the calcium-free conformation of the ATPase and is released when it is converted to the calcium-bound state. We localized the site of interaction to a single peptide isolated after cyanogen bromide cleavage of the ATPase. The peptide derives from a domain just C-terminal to the aspartyl phosphate of the active site. This domain is unique to ATPases of the sarcoplasmic reticulum in that it has no homology with any other phosphorylation-type ion pump. The domain occurs in both slow- and fast-twitch isoforms of the ATPase, even though phospholamban is not expressed in fast-twitch muscles.

alpha B-Crystallin is a 20-kd peptide highly homologous to the small heat-shock proteins. This protein forms soluble homomultimeric complexes (M(r), 300-700 kd) and is very abundant in cardiac muscle cells. In vitro experiments (affinity column chromatography and binding studies with isolated proteins) have shown that alpha B-crystallin interacts directly with actin and, in particular, with desmin filaments. The immunocytochemical localization of alpha B-crystallin within the cardiomyocytes showed that the protein is distributed exclusively in the central region of the I bands (Z lines), where desmin is localized. In vitro studies have further shown that the binding affinity of alpha B-crystallin to actin and desmin filaments increases considerably at slightly acidic pH (6.5) or after a heat treatment (45 degrees C). Moreover, alpha B-crystallin was found to prevent effectively the tendency of actin filaments to form aggregates (i.e., paracrystals) at acidic pH. These in vitro data suggest a protective role of alpha B-crystallin during stress conditions such as ischemia of the heart. Crystallin could prevent the aggregation of filaments, which might occur during the acidification of the cytosol and lead eventually to irreversible structural damage.

mRNA levels of the ob gene product, leptin, were investigated by quantitative competitive RT-PCR in a mouse cell line (3T3-L1) which can be induced to differentiate into adipocytes. During conversion to fat cells, the level of leptin mRNA increased several-fold and in parallel to that for typical adipocyte markers like lipoprotein lipase, adipsin and glycerophosphate dehydrogenase. Leptin transcription, however, did not correlate with the size of the adipocytes measured as total triglycerides. On the other hand, mRNA levels for leptin in fully differentiated adipocytes were increased 2-3 fold by insulin. In contrast, free fatty acids exerted a concentration-dependent inhibition of leptin transcription while the corticosteroid dexamethasone and an elevation of intracellular cAMP displayed only marginal inhibitory effects on leptin mRNA levels.

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