Effects of 20,25-diazacholesterol on cholesterol synthesis in cultured chick muscle cells: a radiogas chromatographic and mass spectrometric study of the post-squalene sector.

Radiogas chromatography, used in conjunction with mass spectrometry, has been used to analyze the sterol content of cultured chick muscle cells. Seven sterols, plus lanosterol, were detected. These sterols conformed to a linear biosynthetic pathway linking lanosterol and cholesterol. The reaction sequence is: C-14 demethylation, C-4 demethylation, delta 8 leads to delta 5 double bond rearrangement, delta 24 double bond reduction. When chick cells were treated with increasing concentrations of 20,25-diazacholesterol, components of this pathway and aberrant products accumulated. These accumulations suggest that diazacholesterol affects reductases, double bond isomerases and the C-14 demethylation enzymes of sterol biosynthesis.

Radiogas chromatography mass spectrometry in the selected ion monitoring mode.

The value of selected ion monitoring in analyzing biological radio isotope incorporation experiments by radiogas chromatography mass spectrometry is illustrated with reference to the biosynthesis of the mycotoxin mycophenolic acid and the mode of action of the anticholesterolemic drug 20,25-diazacholesterol. It is shown that the increased sensitivity and specificity of the selected ion monitoring mode detector permits straightforward detection and identification of the relatively small cellular pools associated with metabolic intermediates. The computer program RADSIM is described. Problems that still exist in using radiogas gas chromatography mass spectrometry technology to analyse isotope incorporation experiments are discussed.

Partial purification and characterization of a protein lysine methyltransferase from plasmodia of Physarum polycephalum.

Plasmodia of Physarum polycephalum have an active protein lysine methyltransferase (S-adenosylmethionine:protein-lysine methyltransferase, EC 2.1.1.43). This enzyme has been purified 40-fold with a 13% yield, and it catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to the epsilon-amino group of lysine residues with formation of N epsilon-mono-, N epsilon-di-, and N epsilon-trimethyllysines in a molar ratio of 4:1:1 based on [14C]methyl incorporation into the methylated lysines. The ratio remains unchanged at all stages of the partial purification, as well as after fractionation by sucrose density gradient centrifugation and gel electrophoresis. The rate of protein methylation is time dependent, enzyme concentration dependent, and requires the presence of a sulfhydryl reducing agent for optimal activity. The enzyme has optimal activity at pH 8 and is inhibited by S-adenosyl-L-homocysteine and EDTA. Lysine-rich and arginine-rich histones serve as the most effective exogenous protein acceptors; P. polycephalum actomyosin is inactive, and chick skeletal myofibrillar proteins are 25% as effective as exogenous mixed histones as substrates. Lysine, polylysine, ribonuclease A, cytochrome c, and bovine serum albumin are not methylated.

Sites of biological methylation of proteins in cultured chick muscle cells.

The methylation of myosin and other proteins has been studied using primary cultures of 12-day-old embryonic chick leg muscle. The methyl group of [Me-3H] methionine is incorporated into basic amino acid residues with the formation of Nepsilon-monomethyllysine, Nepsilon-dimethyllysine, Nepsilon-trimethyllysine, 3-methylhistidine, NG-monomethylarginine, and NG-dimethylarginine which are isolated from acid hydrolysates of purified myosin, and of proteins from polysomes and from the cytosol of the cultured muscle cells. In the presence of 0.1 mM cycloheximide, incorporation of [Me-3H] methionine into the polysome-bound proteins was decreased to 16.3% of control levels with no change in the pattern of incorporation into the basic amino acid residues, although protein synthesis was inhibited 97.5%. When protein synthesis was allowed to resume in such cultures by the removal of cycloheximide, polypeptides containing labeled N-methylated residues were released from polysomes into the soluble fraction. Polypeptides containing N-methylated amino acids were also released from polysomes following treatment with 2 mM puromycin. Peptidyl-tRNA, isolated from ribosomes after exposure of cultures to [Me-3H] methionine, contained labeled N-methylated amino acids. When [Me-3H] methionine was incorporated in the presence of cycloheximide, the isolated peptidyl-tRNA still contained N-methylated amino acids although the amount of methylation was 22.4% of control levels. These experiments demonstrate that N-methylation of basic amino acid residues in proteins may occur while the polypeptide is still being synthesized on the ribosome. In addition, N-methylation can occur on the nascent polypeptides in the absence of protein synthesis.