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Biomedical subjects

R Roskoski

Publications and source records attributed to R Roskoski.

At least 91 records · Page 5Linked to original sources
PubMed1975

Bovine brain adenosine 3',5'-monophosphate dependent protein kinase. Mechanism of regulatory subunit inhibition of the catalytic subunit.

Adenosine 3',5'-monophosphate (cAMP) dependent protein kinase (EC 2.7.1.37) catalyzes the phosphorylation of serine and threonine residues of a number of proteins according to the following chemical equation: ATP + protein leads to phosphoprotein + ADP. The DEAE-cellulose peak II holoenzyme from bovine brain, which is composed of regulatory and catalytic subunits, is resistant to ethoxyformic anhydride inactivation. After adding cAMP, the protein kinase becomes susceptible to ethoxyformic anhydride inhibition. Ethoxyformic anhydride (2mM) inhibits the enzyme 50% (5 min, pH 6.5, 30 degrees) in the presence of 10 muM cAMP, but less than 5% in its absence. The substrate, Mg2+-ATP, protects against inactivation suggesting that inhibition is associated with modification of the active site. Addition of regulatory subunit or Mg2+-ATP to the isolated catalytic subunit also prevents ethoxyformic anhydride inactivation. These results suggest that the regulatory subunit shields the active site of the catalytic subunit thereby inhibiting it. In contrast to the bovine brain or muscle DEAE-cellulose peak II holoenzyme, the bovine muscle peak I holoenzyme is susceptible to ethoxyformic anhydride inactivation in the absence of cAMP.

Animals↗
PubMed1975

In vitro acetylcholine biosynthesis in normal and failing guinea pig hearts.

Choline acetyltransferase activity, which is rate limiting in acetylcholine biosynthesis, was measured in the four heart chambers of guinea pigs subjected to (1) sham surgery, (2) constriction of the ascending aorta, (3) constriction of the descending thoracic aorta, and (4) constriction of the pulmonary artery. After 30 days when hypertrophy and heart failure were fully established, choline acetyltransferase was quantified in vitro by a radiochemical assay. In the sham-operated group, enzyme activity expressed in terms of unit weight of cardiac tissue was greatest in the right atrium and the right ventricle and lower in th left atrium and the left ventricle (3.62 plus or minus 0.30, 2.96 plus or minus 0.52, 1.64 plus or minus 0.15, and 1.67 plus or minus 0.22 nmoles/min g-1, respectively). Enzyme activity was reduced (P less than 0.05) in the right atria and the right ventricles of guinea pigs with constriction of the pulmonary artery (1.68 plus or minus 0.37 and 1.31 plus or minus 0.29 nmoles/min g-1, respectively). Enzyme activity also tended to be reduced in the left atria and the left ventricles of guinea pigs with constriction of the aorta. These changes represented a relative dilution of enzyme activity per unit weight but not an absolute depletion, since choline acetyltransferase activity per ventricle was not reduced. The absence of significant changes in the total amount of the neuronal enzyme, choline acetyltransferase, per ventricle contrasted with the observed increases in the myocardial enzyme, carnitine acetyltransferase. These results confirm the presence of significant parasympathetic innervation of the ventricles as well as the atria but do not demonstrate alterations in parasympathetic neurotransmitter biosynthesis in hypertrphied and failing myocardium. The absence of absolute reductions in choline acetyltransferase activity in hypertrophied and failing ventricle contrasts strikingly with the previously reported reductions in tyrosine hydroxylase, which is rate limiting in sympathetic neurotransmitter biosynthesis.

Acetyl Coenzyme A↗
PubMed1974

Protein kinase induction in Escherichia coli by bacteriophage T7.

After bacteriophage T7 infection, a protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) activity can be demonstrated in E. coli in vivo by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cell-free extracts catalyzed the transfer of the terminal phosphoryl group of [(gamma)-(32)P]ATP to endogenous protein acceptor or to added histone. The bond between phosphate and protein shows the characteristics of serine phosphate: it is stable in 1 N HCl (100 degrees ) and cleaved by 1 N KOH (37 degrees ) and by alkaline phosphatase treatment. Moreover, after partial acid hydrolysis, radiophosphate migrates with marker O-phosphoserine on polyethyleneimine-cellulose thin-layer chromatograms. Enzyme activity in uninfected cells is negligible. Ultraviolet irradiation of the phage genome prevents the appearance of the protein kinase; irradiation of the host genome does not. The enzyme activity occurs 4 min after infection and its gene maps in the early region (promoter proximal to gene 1). Ribosomal proteins are phosphorylated in vivo and are substrates in vitro. Enzyme activity in vitro is not changed by addition of cyclic AMP or cyclic GMP.

Adenosine Triphosphate↗
PubMed1971

Pantetheine-linked peptide intermediates in gramicidin S and tyrocidine biosynthesis.

To study the function of pantetheine in gramicidin S and tyrocidine biosynthesis, pepsin digests of the polymerizing enzymes, of which only the heavy ones contain pantetheine, were analyzed. The digests of gramicidin S enzymes charged with either [(14)C]proline or with D-phenylalanyl-[(14)C]proline, were analyzed by thin-layer chromatography; only the dipeptide showed a derivative associated with pantetheine. Similar results were obtained from the heavy tyrocidine enzyme charged with either [(14)C]asparagine alone or with the pentapeptide D-Phe-Pro-Phe-D-Phe-[(14)C]Asn. Several radioactive products appeared on the thin-layer chromatograms of both these digests; association with pantetheine was found only in the case of the pentapeptide. Exposure of the chromatogram from the pentapeptide-labeled digest to performic acid and development in a second direction separated the peptide from pantetheine, indicating that a nascent peptide was originally linked to the cofactor by a thioester bond. The connection of pantetheine only with peptide residues appears to confirm its role in transpeptidation during peptide chain growth.

Asparagine↗