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

Abstract

Peptides obtained by tryptic digestion of carboxymethylated and maleylated aspartate aminotransferase and of the aminoethylated enzyme were isolated and the complete amino acid sequences of most of them were determined. Digestion of the carboxymethylated protein with pepsin produced a complex mixture of peptides that allowed some overlapping of the tryptic peptides (Fig. 4); in addition, peptides were obtained that had not been found in either of the tryptic digests. From these studies about 400 amino acid residues were identified. Experimental details and confirmatory data for the results presented here are given in a supplementary paper that has been deposited as Supplementary Publication 50011 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. (1972) 126, 5.

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

“…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. Overlap between fragments A and B is provided by a peptide (RP, residues 148-157) produced by restricted digestion with pepsin.…”

“…Carboxymethylated aspartate aminotransferase [2] was used for all enzymic digests. For digestion with A. mellea protease, 2 g of the modified protein was suspended in N-ethylmorpholine-HC1 buffer (0.2 M, pH 7.5,200 ml); 5 ml of a solution of the protease (approx.…”

“…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.…”

“…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].…”