Partial purification and characterization of a binding factor specific for initiator tRNA and ribosomal 40S subunits and of an aminoacyl-tRNA hydrolase specific for 40S-bound Met-tRNAf from rat liver

McCuiston, James; Parker, Riley David; Moldave, Kivie

doi:10.1016/0003-9861(76)90090-4

Cited by 20 publications

(17 citation statements)

References 30 publications

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“…This finding supports previous reports that Met-tRNAy"' is bound to ribosomes [1, 3,7]. The activity in the crude initiation factors varied with different preparations, as much as threefold.…”

Section: Purification Of Met-trnafmet Deucylusesupporting

confidence: 91%

“…The ionic requirements for the enzyme show that it is active at physiological salt and pH conditions. The enzyme is inhibited at high MgC12 concentrations, under which condition it is reported to deacylate Met-tRNAF' bound to 40-S ribosomal subunits [I, 3,7].…”

Section: Discussionmentioning

confidence: 99%

“…EjJei.C.1 o/ rihosomal subunits and crude initiation fuctor preparation on the M e t -t R N A y ' deacylase 10-pl pre-assay mixtures contained 5 p1 Met-tRNAy" deacylase (stage F), 1 p140-S subunits ( 3 cm-' at 260 nm), 1 pI 60-S subunits (6 cm-' at 260 nm) and 0.1 p1 crude initiation factor (IF) preparation where indicated. The activity of the pre-assay mixtures were measured by the Met-tRNAfl" dedcylase assay deacylase acting on Met-tRNA:" bound to 40-S subunits in the presence of 60-S subunits in high magnesium concentrations [1,7]. As seen in Table 3, no significant effect on the Met-tRNA:" deacylase was found after remixing it with crude initiation factor preparation, from which the Met-tRNA:" deacylase was purified.…”

Section: Efpct Of' M E T -T R N a R E T Deacylase On Translational Inmentioning

confidence: 99%

“…The Met-tRNAy" deacylase activity has been reported to be specific for Met-t RNAy" [2,3,7,8] although some investigations found specificity for both the Met-tRNAy" and Met-tRNAp' species [ 1,5].…”

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confidence: 99%

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Methionyl‐tRNA_f^Met Deacylase

Andersen¹

1979

European Journal of Biochemistry

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A methionyl‐tRNAfMet deacylase was found in ribosomal salt wash from cultured human cells of the HeLa line. This enzyme was purified by the use of DEAE‐cellulose, ammonium sulfate precipitation, gel filtration and isoelectric focusing, and appears to be a protein with a native molecular weight of 80000, which consists of two 40000‐Mr subunits. The mechanism of the Met‐tRNAfMet deacylase is shown to involve end‐product inhibition by the deacylated form of MettRNAfMet. The methionyl‐tRNAfMet deacylase is rather specific for Met‐tRNAfMet as opposed to Met‐tRNAmMet, has a KCl optimum of 85 mM, is inhibited by MgCl2 and is inhibited by GTP and NAD+ at physiological concentration. 40‐S and 60‐S subunits inhibit the enzyme, possibly by binding to it. The stability of translational initiation complexes, containing methionyl‐tRNAfMet, was investigated in the presence of the enzyme. Purified ternary complex was slowly broken down by the enzyme, while the 40‐S‐subunit · Met‐tRNAfMet complex was stable in the presence of enzyme. The 80‐S complex formed with A‐U‐G trinucleotide as the message molecule was broken down, whereas the 80‐S complex formed with globin mRNA was stable in the presence of the enzyme. The physiological role of this enzyme is unclear, but it might act to regulate initiation by deacylating Met‐tRNAfMet.

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Section: Purification Of Met-trnafmet Deucylusesupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Efpct Of' M E T -T R N a R E T Deacylase On Translational Inmentioning

confidence: 99%

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confidence: 99%

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Methionyl‐tRNA_f^Met Deacylase

Andersen¹

1979

European Journal of Biochemistry

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“…Several papers have dealt with the purification of eukaryotic initiation factors from different sources, i.e., rabbit reticulocytes [l -71, Krebs I1 ascites tumor cells [8,9], Arfemiu sulinu [10,11] and rat liver [12]. All procedures have in common that they start with a 0.5-M-KCI wash of the crude ribosomal preparation, which removes, besides the initiation factors, a large number of other proteins from the ribosomes, including several inhibitors of initiation [13,14] and ribosomal proteins (cf.…”

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confidence: 99%

Initiation of Protein Synthesis in Eukaryotes. Binding to Sepharose-heparin and Partial Purification of Initiation Factors from Krebs II Ascites Cells

et al. 1977

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By means of affinity chromatography of lysates from Krebs I1 ascites cells and rabbit reticulocytes on Sepharose-heparin an active fraction of initiation factors has been obtained.The fraction is eluted from the column at 350 mM KC1 using a linear gradient and displays a number of activities, i. e. binding of Met-tRNAy" to form a ternary complex with GTP ; transferring this complex to 40-S subunits in an A-U-G-independent step and finally coupling of the 40-S initiation complex to the 60-S subunit, a reaction which is completely A-U-G-dependent. Moreover, MettRNA is bound into the P-site as is indicated by its puromycin sensitivity. The method is very suitable for large-scale preparation. Further purification and characterization of the factors have been carried out on DEAE-cellulose and phosphocellulose columns.Evidence is presented that the polysomes present in a lysate that has been passed through the Sepharose-heparin column can only complete their nascent chains, initiation of new polypeptides is completely dependent on addition of initiation factors.

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