Primary cultures of cardiac muscle cells as models for investigation of protein glycosylation.

Primary cardiac cell cultures of newborn rats containing approximately 50% (by cell number) spontaneously contracting cardiomyocytes were used to study the role of protein N-glycosylation for the binding of dihydropyridine (DHP) to the voltage-dependent L-type calcium channel. This binding is not influenced by the accompanying non-muscle cells. Exposure of the cells up to 6 micrograms/ml of the N-glycosylation inhibitor tunicamycin for a 44 h period resulted in a decrease of the specific DHP binding sites (Bmax) to 46.0 +/- 17.2% of the untreated control. Similar effects were observed after enzymatic deglycosylation using N-glycosidase F (PNGase F). The results suggest that a posttranslational modification of parts of the cardiac L-type Ca+2 channel by N-glycosylation is an important determinant for the binding of Ca+2 antagonists of the DHP-type to the alpha 1 subunit which itself is not glycosylated. The results suggest a participation of N glycosylation in the assembling of the subunits to the functional channel and/or its turnover. However, a possible effect of tunicamycin on the expression of the Ca channel as an alternative mechanism cannot be excluded.

Influence of glycosylation inhibitors on dihydropyridine binding to cardiac cells.

In primary cultures of neonatal rat heart cells we found a linear correlation between the number of L-type calcium channel-specific dihydropyridine (DHP) binding sites and spontaneous beating frequency (v). Formation of glycoproteins in tissue culture was suppressed by different inhibitors of N-glycosylation. This inhibition alters to a different extent the binding of the DHP ligand (+)-[methyl-3H]PN 200-110 and v. The most severe but reversible effect occurs at 6 micrograms/ml tunicamycin (Bmax approximately 45% and v approximately 6%, resp., of control), a slight increase in Bmax at 0.1-0.5 mM castanospermine and 0.05-2.5 mM deoxymannojirimycin. The other inhibitors gave no significant alteration of Bmax.

Bead cellulose derivatives as supports for immobilization and chromatographic purification of proteins.

Characteristic data are presented for Divicell, a macroporous bead cellulose with excellent flow parameters. The preparation of Divicell derivatives and their properties are described with respect to their application as chromatographic supports. The ion exchangers Divicell DEAE and Divicell CM were manufactured in two types with different exclusion limits and an available capacity for proteins of up to 100 mg/ml gel. Divicell Blue is a bead cellulose with covalently bound Cibacron Blue F3G-A and was found to be a very suitable adsorbent for the selective separation and purification of human serum albumin. Activation of Divicell with sodium periodate, epichlorohydrin and 5-norbornene-2,3-dicarboximido carbonochloridate provided activated supports used for immobilization of ligands in organic solvents and in aqueous solutions. Coupling of amines, diamines, amino acids, carbohydrates and proteins is described. The immobilized ligands retained their biological activity as determined by their specific adsorption of proteins. Divicell alkyl derivatives were tested in hydrophobic interaction chromatography with bovine serum albumin as a model. Examples are presented of the application of Divicell derivatives to the purification of biomacromolecules such as immunoglobulins and lectins by affinity chromatography. The results were comparable to those obtained using the corresponding Sepharose-derived absorbents.

A rapid procedure for the purification of cardiac 1,4-dihydropyridine receptors from porcine heart.

Highly purified porcine cardiac sarcolemma was used as a source for purification of mammalian cardiac 1,4-dihydropyridine receptors associated with the voltage-dependent Ca2+ channel. The cardiac digitonin-solubilized receptor prelabeled with (+)-[3H]PN 200-110 was enriched at least 236-fold using an improved, rapid three-step purification protocol which could be completed within 12 h. The purity of the preparation was at least 22%, the yield of the receptors 24%. Photoaffinity labeling experiments with (-)-[3H]azidopine allowed the identification of the cardiac alpha 1 subunit. In contrast to the purified rabbit or guinea-pig skeletal muscle Ca2+ channel complex, none of the purified polypeptides underwent rapid and substantial phosphorylation by the catalytic subunit of the cyclic AMP-dependent protein kinase in vitro.

An enzyme immunoassay for phospholamban.

Phospholamban, a phosphorylatable protein of the cardiac sarcoplasmic reticulum, has been estimated by a semi-quantitative immunoassay. It can be determined in purified membrane preparations as well as in crude fractions of cardiac muscle membranes, regardless of the phosphorylation state of the phosphoprotein. The content of phospholamban in mammalian heart muscle membrane vesicles correlates with the activity of the calcium/magnesium-dependent ATPase with the exception of the oxalate-loaded membrane preparations. This observation indicates that phospholamban and the calcium transporting enzyme are localized at different sites in cardiac sarcoplasmic reticulum.

Reconstitution of phospholamban--an approach to its function.

Isolated cardiac Ca,Mg-dependent ATPase of the sarcoplasmic reticulum and purified phospholamban, a proteolipid involved in the regulation of the calcium transport systems of the heart muscle, were reconstituted into soybean lecithin liposomes. Whereas the enzymatic activity of the CaATPase in the obtained liposomes was unaffected as well as by unphosphorylated and cAMP-dependent phosphorylated phospholamban, the capacity of oxalate-supported calcium uptake was lowered in the presence of phospholamban. The proteolipid is discussed not as a regulatory protein of the enzyme but as a mediator of the calcium storage in the lumen of the liposomes or sarcoplasmic reticulum network.

Differences in phosphorylation of isolated phospholamban in dependence on the preparation procedure.

Phospholamban was purified by two different preparation protocols. The products of both preparations are immunochemical similar. They differ, however, with regard to their phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. It is suggested that the lipid environment plays a crucial role in the exposition of the phosphorylation sites of phospholamban.

The putative role of phospholamban in the regulation of the heart muscle.

We describe the 50fold purification of phospholamban, the production of antibodies against it and preliminary experiments of reconstitution of purified phospholamban into vesicles of skeletal sarcoplasmic reticulum (SR). Purified phospholamban migrates in a modified Laemmli SDS polyacrylamide gel electrophoresis (PAGE) system with a relative molecular mass (Mr) of 22 kD and 6 kD. The higher Mr form is detectable by immunoreaction on Western blots only in heart SR preparations, whereas the low Mr form is also present in sarcolemmal preparations. No immunoreactivity was found in SR of skeletal muscle. Reconstituted skeletal SR vesicles were not influenced by phospholamban in respect to their Ca++-ATPase activity and calcium accumulation rate, but the obtained data suggest that phospholamban alters the calcium storage capacity of SR.

Indirect technique for the estimation of cAMP-dependent and Ca2+/calmodulin-dependent phospholamban phosphorylation state in canine heart in vivo.

An indirect technique was employed to estimate the in vivo phosphorylation state of phospholamban in preparations from dog hearts depleted from catecholamines and from dog hearts treated with isoproterenol. This method allows the separate detection of cAMP-dependent and Ca2+/calmodulin-dependent phospholamban phosphorylation. The data obtained demonstrate the phosphorylation of both the cAMP-dependent and the Ca2+/calmodulin-dependent phosphorylatable site of phospholamban in response to the beta-adrenergic agonist isoproterenol in canine heart in vivo.

Purification of phospholamban and characterization of its antibodies.

Phospholamban was extracted from pig heart microsomal membranes with a methanol-chloroform (2:1) mixture. The proteolipid was further purified to electrophoretic homogeneity by chromatography in organic solvents and in SDS-containing medium. Purification was about 50-fold with respect to specific 32P-phospholamban radioactivity. Antisera were produced in rabbits against phospholamban. Electroblot analysis demonstrates specific binding of the antisera to purified phospholamban and to phospholamban in preparations of cardiac sarcoplasmic reticulum and sarcolemma.

Characterization of endogenous phosphorylation in isolated cardiac sarcolemma.

The cardiac sarcolemma contains kinases which catalyze the incorporation of 32P-phosphate into acid stable and acid precipitable membrane components of low molecular weight. The phosphorylation is not influenced by cyclic AMP or calmodulin. Analysis of phosphorylation products using proteolytic digestion, organic solvent extraction, thin layer chromatography and gel filtration reveals both polypeptides and lipids as kinase substrates. Polypeptides are phosphorylated at their serine and threonine residues, while lipid phosphorylation gives rise to 32P-labelled phosphatidylinositol phosphates and some nonidentified compounds. Phosphorylated polypeptides and phosphorylated lipids do not separate in SDS polyacrylamide gel electrophoresis. On the basis of the fast time course of 32P-phosphate incorporation, it may be supposed that endogenous phosphorylation may play a role in the short term regulation of the cardiac sarcolemmal function.