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.

Amidohydrolases↗

PubMedDate not supplied

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.

1-Deoxynojirimycin↗

PubMedDate not supplied

The role of the mature part of secretory proteins in translocation across the plasma membrane and in regulation of their synthesis in Escherichia coli.

Presently available data are reviewed which concern the role of the mature parts of secretory precursor proteins in translocation across the plasma membrane of Escherichia coli. The following conclusions can be drawn; i) signals, acting in a positive fashion and required for translocation do not appear to exist in the mature polypeptides; ii) a number of features have been identified which either affect the efficiency of translocation or cause export incompatibility. These are: alpha) protein folding prior to translocation; beta) restrictions regarding the structure of N-terminus; gamma) presence of lipophilic anchors; delta) too low a size of the precursor. Efficiency of translocation is also enhanced by binding of chaperonins (SecB, trigger factor, GroEL) to precursors. Binding sites for chaperonins appear to exist within the mature parts of the precursors but the nature of these sites has remained rather mysterious. Mutant periplasmic proteins with a block in release from the plasma membrane have been described, the mechanism of this block is not known. The mature parts of secretory proteins can also be involved in the regulation of their synthesis. It appears that exported proteins are already recognized as such before they are channelled into the export pathway and that their synthesis can be feed-back inhibited at the translational level.

Bacterial Proteins↗

PubMedDate not supplied

Expression in Escherichia coli K12 of the cloned genes for a major outer membrane protein (OmpA protein) from Shigella dysenteriae, Enterobacter aerogenes and Serratia marcescens.

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Bacterial Outer Membrane Proteins↗