Tyrosyl-tRNA Synthetase from Baker's Yeast. Rapid Isolation by Affinity Elution, Molecular Weight of the Enzyme, and Determination of Essential Sulfhydryl Groups

Abstract

Tyrosyl‐tRNA synthetase (EC 6.1.1.1) has been isolated from baker's yeast with an overall purification factor of more than 5000. After opening the cells, pH 4.8 precipitation, ammonium sulfate fractionation, removal of the nucleic acids with DEAE‐cellulose and chromatography on CM‐Sephadex, the critical purification step is the elution of the cation‐exchanger‐bound tyrosyl‐tRNA synthetase with tRNATyr. The homogeneous enzyme exhibits a molecular weight of 40000 as estimated by sedimentation equilibrium centrifugation and dodecylsulfate‐gel electrophoresis under reducing and non‐reducing conditions. Gel filtration experiments show a molecular weight of about 100000 indicating the existence of an active dimeric form. The possibility of proteolytic cleavage of the enzyme is excluded. The reaction of tyrosyl‐tRNA synthetase with p‐chloromercuribenzoate and N‐ethylmaleimide reveals two rapidly reacting sulfhydryl groups per subunit of molecular weight 40000, as demonstrated by the inhibition of aminoacylation and the isolation of enzyme‐inhibitor complexes. In addition an efficient purification method is described for isolating tRNATyr from soluble ribo‐nucleic acid from baker's yeast in three chromatographic steps in a yield of 28%.

Isoleucyl-tRNA Synthetase from Baker’s Yeast. Influence of Substrate Concentrations on Aminoacylation Pathways, Discrimination between tRNAIIeand tRNAVal, and between Isoleucine and Valine

“…The concentration of [ 14 C]ATP was 0.2mM; that of [ 14 C]isoleucine 0.023 or 0.00076mM, and that of tRNA Ite 0.0045mM or 0.0007 ImM according to the concentration range applied. For measuring the aminoacylation rates samples of 0.05 ml in the case of isoleucine and 0.15m/ in experiments with valine were taken in intervals of l min and 3 min and treated as in refJ 15 !. For determination of AMP formation rates samples of 0.005 ml from the same reaction mixture were spotted onto PEI-cellulose F sheets (Merck, D-6100 Darmstadt) at intervals of 1 or 3 min.…”

“…F r die richtigen Substrate Isoleucin und tRNA Ile wurden jeweils zwei K m -Werte bestimmt, die sich etwa um den Faktor f nf unterscheiden; die hohen K m -Werte wurden bei Konzentrationen h her als ΙμΜ, die niedrigeren unterhalb von ΙμΜ Isoleucin oder tRNA Ile gefunden. Bei Substrat-Konzentrationen unterhalb von ΙμΜ Substrate specificities of enzymes involved in protein biosynthesis have been regarded as crucial in error theories of aging 1 1-41 ; in this connection especially the selection of cognate substrates by aminoacyl-tRNA synthetases was investigated extensively 15 " 71 . In our recent works^8' 9i we studied the variations of accuracy by changing pH values, addition of pyrophosphatase, elongation factor Tu-GTP complex, and spermine in the two following mischarging reactions:…”

Isoleucyl‐tRNA Synthetase from Baker's Yeast

“…The velocity of the esterification reaction was measured by the amount of [14C]isoleucine incorporated into tRNA as described in [25]. The standard reaction mixture (0.1 ml) contained 0.15 M Tris/HCl buffer pH 7.6, 0.15 M KC1, 0.05 M MgS04, and the appropriate concentrations of ATP, [14C]isoleucine and tRNA which are given in the legends to the figures.…”

N-Acetyl-tyrosine at the 3' End of Brome Mosaic Virus RNA has Little Effect on Infectivity

“…BMV RNA was tyrosylated in vitro (Kohl & Hall, 1977), using a wheat germ synthetase preparation enriched in tyrosyl-tRNA synthetase (Faulhammer & Cramer, 1977) that routinely yielded viral RNA which was 70 to 100% charged. The conditions for synthesis of the acetylating agent (the N-hydroxysuccinimide ester of acetic acid) and for acetylation of BMV tyr RNA were as described previously (Kohl & Hall, 1977).…”

“…The messenger function of the viral RNA appeared to be unaffected by the modification, but a significant loss of infectivity was observed. We now describe a set of experiments that involve the removal in vitro of N-acetyl-tyr from the 3' terminus of the viral genome, and the subsequent assay of the biological activity of the repaired BMV RNA.BMV RNA was tyrosylated in vitro (Kohl & Hall, 1977), using a wheat germ synthetase preparation enriched in tyrosyl-tRNA synthetase (Faulhammer & Cramer, 1977) that routinely yielded viral RNA which was 70 to 100% charged. The conditions for synthesis of the acetylating agent (the N-hydroxysuccinimide ester of acetic acid) and for acetylation of BMV tyr RNA were as described previously (Kohl & Hall, 1977).…”