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The nucleotide sequence of cysteine transfer ribonucleic acid from baker's yeast. Identification of the products from partial degradation of the molecule and derivation of the complete sequence.

ABSTRACT: 1. A series of large oligonucleotide fragments derived from tRNA Cys, were separated chromatographically and the sequence of each was deduced by examination of the products of digestion with pancreatic and T1 ribonucleases. 2. The location of the specific cleavage points in the nucleotide chain was similar to that produced by brief treatment with pancreatic ribonuclease. 3. The fragments could be arranged into two alternative sequences. The correct sequence was deduced by the sequential removal and identification of the first nine nucleotides from the 3'-end of the terminal half of the molecules.

SUBMITTER: Holness NJ

PROVIDER: S-EPMC1172592 | biostudies-other | 1976 Feb

REPOSITORIES: biostudies-other

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Project description:Endosomes have recently been identified as one major site of glucagon degradation in intact rat liver. In this study, a cell-free system has been used to assess the role of ATP-dependent acidification in endosomal glucagon degradation and identify the glucagon products generated. Percoll gradient fractionation of Golgi-endosomal fractions prepared 10-30 min after injection of [125I]iodoglucagon showed a time-dependent shift of the radioactivity towards high densities. Regardless of time, the radioactivity was less precipitable by trichloroacetic acid (Cl3Ac) at high densities than at low densities. Chloroquine treatment slightly increased the density shift of the radioactivity and decreased its Cl3Ac-precipitability throughout the gradient. Incubation of endosomal fractions containing [125I]iodoglucagon in 0.15 M-KCl at 30 degrees C resulted in a time- and pH-dependent generation of Cl3Ac-soluble radioactivity, with a maximum at pH 4 (t1/2, 7 min). At pH 5, 1,10-phenanthroline, bacitracin and p-chloromercuribenzoic acid partially inhibited [125I]iodoglucagon degradation. At pH 6-7, ATP stimulated [125I]iodoglucagon degradation by 5-10-fold and caused endosomal acidification as judged from Acridine Orange uptake. The effects of ATP were inhibited by chloroquine, monensin, N-ethylmaleimide and dansylcadaverine. Poly(ethylene glycol) (PEG) precipitation of the radioactivity associated with endosomes showed that lowering the pH below 5.5 caused dissociation of the glucagon-receptor complex, and that, regardless of incubation conditions, all degraded [125I]iodoglucagon diffused extraluminally. On h.p.l.c., at least three products less hydrophobic than [125I]iodoglucagon were identified in incubation mixtures along with monoiodotyrosine. Radiosequence analysis of the products revealed one major cleavage located C-terminally to Tyr-13 and two minor cleavages affecting Thr-5-Phe-6 and Phe-6-Thr-7 bonds. It is concluded that glucagon degradation in liver endosomes is functionally linked to ATP-dependent endosomal acidification and involves several cleavages in the glucagon sequence.

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The nucleotide sequence of cysteine transfer ribonucleic acid from baker's yeast. Identification of the products from partial degradation of the molecule and derivation of the complete sequence.

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