Complete purification and studies on the structural and kinetic properties of two forms of yeast valyl-tRNA synthetase.
Two forms of baker's yease valyl-tRNA synthetase have been purified to apparent homogeneity by classical methods. It was demonstrated that one of the two forms of the enzyme originates from the other by proteolysis, the respective amounts of each form depending on the physiological state of the yeast. The species mainly isolated from exponential growing yeast cells is a monomer of 130,000 daltons molecular weight. In stationary phase cells or in commercial yeast the major species is a degraded monomer of 120,000 daltons molecular weight ; however when the purification is carried out in the presence of phenylmethyl-sulphonyl fluoride, or diisopropylfluorophosphate large amounts of the not - degreded monomer can be obtained. Of great practical usefulness is the fact that large amounts of the native enzyme can be obtained pure after only two chromatographic steps on DEAE-cellulose and hydroxylapatite. The kinetic constants for valine, ATP and tRNAVal were determined, as well as the optimum aminoacylation conditions. It was found that the specific activity of the nondegraded valyl-tRNA synthetase is higher than that of the proteolysed enzyme for the aminoacylation reaction. On the contrary, both forms have the same ATP-pyroposphate exchange activity. The amino acids composition of the native enzyme was established. The tryptic fingerprints of the two valyl-tRNA synthetases were studied. Essentially similar maps were obtained. The number of the spots in the fingerprints indicates that the enzymes contain a high proportion of repeated sequences.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D002455:Cell Division; D011756:Diphosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride; D012343:RNA, Transfer / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014357:Trypsin; D014633:Valine / Q000378:metabolism; D014637:Valine-tRNA Ligase / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D011756:Diphosphates; D007531:Isoflurophate; D010664:Phenylmethylsulfonyl Fluoride; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D014357:Trypsin; D000604:Amino Acyl-tRNA Synthetases; D014637:Valine-tRNA Ligase; D014633:Valine; D008274:Magnesium