Limited proteolysis of pig heart citrate synthase by subtilisin, chymotrypsin, and trypsin.

TitleLimited proteolysis of pig heart citrate synthase by subtilisin, chymotrypsin, and trypsin.
Publication TypeJournal Article
Year of Publication1980
AuthorsBloxham, DP, Ericsson, LH, Titani, K, Walsh, KA, Neurath, H
JournalBiochemistry
Volume19
Issue17
Pagination3979-85
Date Published1980 Aug 19
ISSN0006-2960
KeywordsAmino Acid Sequence, Animals, Chymotrypsin, Citrate (si)-Synthase, Immunodiffusion, Kinetics, Macromolecular Substances, Molecular Weight, Myocardium, Oxo-Acid-Lyases, Peptide Fragments, Substrate Specificity, Subtilisins, Swine, Trypsin
Abstract

Pig heart citrate synthase was subjected to limited proteolytic attack by subtilisin, chymotrypsin, and trypsin in the presence of palmitoyl-CoA. Initial proteolysis by all three proteolytic enzymes resulted in cleavage of the monomeric subunit (Mr 45 000 +/- 3000) into a large (Mr 35 000-38 500) and a small (Mr 9000 +/- 3000) into a large (Mr 35 000-38 500) and a small (Mr 9000-12 000) fragment. Further proteolysis of the large subunit produced a secondary fragment (Mr 31 000-36 000). The small (Mr 9000-12 000) fragment was stable in the presence of subtilisin but was substantially degraded by both chymotrypsin and trypsin. The actual molecular weight of fragments varied with the choice of the proteolytic enzyme. Limited proteolysis was absolutely dependent on the presence of palmitoyl-CoA and resulted in complete inhibition of the catalytic activity of the enzyme. Citrate, ammonium sulfate, and especially oxaloacetate provided complete protection against proteolysis whereas acetyl-CoA, CoASH, NADH, and ATP were ineffective. Reaction of rabbit anti-citrate synthase with citrate synthase and its proteolytic fragments indicated that the main antigenic region lay primarily in the small fragment. The products of subtilisin cleavage were isolated by gel filtration under denaturing conditions. The large (Mr 35 000-38 500) fragment contained the amino-terminal (approximately)336 amino acids and the small fragment contained the remaining carboxyl-terminal amino acids. The results are discussed in relation to the structure of citrate synthase.

Alternate JournalBiochemistry
Citation KeyPMID6773558
PubMed ID6773558
Grant ListGM-15731 / GM / NIGMS NIH HHS / United States