The primary structure of aspartate aminotransferase from pig heart muscle. Partial sequences determined by digestion with thermolysin and elastase

Abstract

Peptides produced by thermolytic digestion of aminoethylated aspartate aminotransferase and of the oxidized enzyme were isolated and their amino acid sequences determined. Digestion by elastase of the carboxymethylated enzyme gave peptides representing approximately 40% of the primary structure. Fragments from these digests overlapped with previously reported sequences of peptides obtained by peptic and tryptic digestion (Doonan et al., 1972), giving ten composite peptides containing 395 amino acid residues. The amino acid composition of these composite peptides agrees well with that of the intact enzyme. Confirmatory results for some of the present data have been deposited as Supplementary Publication 50018 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.

The primary structure of aspartate aminotransferase from pig heart muscle determined in part using a protease with specificity for lysine

“…1; some acid and amide side cha/ns remain to be assigned. The letters A to J refer to the ten major fragments previously described [ 1 ]. The symbols P8-16 and TL-33 refer to a peptic peptide [2] and a thermolytic peptide [ I] respectively which were not previously incorporated into the partial sequences.…”

“…We have recently reported [1] partial amino acid sequences of the cytoplasmic aspartate aminotransferase from pig heart muscle, based on studies of peptides produced by digestion of the protein with pepsin and trypsin [2] and with thermolysin and elastase [ 1]. This work gave ten major fragments containing 395 amino acid residues.…”

“…Leu-Glu-Ala-Leu-Lys -Thr-Pro -Gly-Thr-Trp-Asx-Hi s -Ile-Thr-Asp-Gl~ -Ile-Gly-Met-Phe- Where an arrow under a peptide is partially dotted, the residues above the solid part were determined by the dansyl-Edman method and those above the dotted part from the amino acid composition by comparison with known sequences [ 1 ]. librated with acetic acid (30% v/v).…”

“…librated with acetic acid (30% v/v). Final purification of peptides was by paper chromatography and high voltage paper electrophoresis [ 1,2] except in the case of peptide L-4b which was purified by ion exchange chromatography on SP-Sephadex C-25 at pH 2.8. Sampies of purified peptides were hydrolysed by the procedure of Sanger and Thompson [6] and amino acid compositions were determined using either a Technicon Auto Analyzer or a Biocal BC200 automatic analyzer.…”

Homology of the primary structures of cytoplasmic and mitochondrial aspartate aminotransferases from pig heart

“…A sample of the protein was amino-ethylated and digested with trypsin and a second sample was reduced with sodium borohydride, carboxymethylated and digested with thermolysin; the methods have been described previously [6,7]. Peptides were mainly isolated by gel filtration through Sephadex followed by chromatography and electrophoresis on paper [6,7] ; some of the thermolytic peptides were purified by ion exchange chromatography using SP-Sephadex C-25. Sequences of peptides were determined using the dansyl-Edman method as described by Hartley [8].…”