Specific proteolytic modification of creatine kinase isoenzymes. Implication of C-terminal involvement in enzymic activity but not in subunit-subunit recognition.
We are using the isoenzymes of creatine kinase (CK) to investigate the effect of specific proteolytic modification on the abilities of enzyme subunits to establish precise subunit-subunit recognition in vitro. Previous work by others has shown that treatment of the MM isoenzyme of rabbit CK with Proteinase K results in a specific proteolytic modification and inactivation of the enzyme. In the present work, we show that both the MM and BB isoenzymes of chicken CK are also specifically modified by Proteinase K, resulting in over 98% loss of catalytic activity and approx. 10% decreases in subunit molecular masses of the enzymes. Similar reactions appear to occur when the isoenzymes are treated with Pronase E. Limited amino acid sequence analysis of intact and Proteinase K-modified MM-CK suggests that the proteolytic modification results from a single peptide-bond cleavage occurring between alanine residues 328 and 329, about 50 amino acid residues from the C-terminal end; the active-site cysteine residue was recovered in the large protein fragment of modified M-CK subunits. Proteolytically modified M-CK and B-CK subunits were able to refold and reassociate into dimeric structures after treatment with high concentrations of LiCl and at low pH. Thus the proteolytically modified CK subunits retain their ability to refold and to establish precise subunit-subunit recognition in vitro.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002645:Chickens; D002712:Chlorides / Q000494:pharmacology; D003402:Creatine Kinase; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D010450:Endopeptidases / Q000494:pharmacology*; D007527:Isoenzymes; D008094:Lithium / Q000494:pharmacology; D018021:Lithium Chloride; D009132:Muscles / Q000201:enzymology; D011402:Pronase / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects
D002712:Chlorides; D007527:Isoenzymes; D008094:Lithium; D003402:Creatine Kinase; D010450:Endopeptidases; D019286:Endopeptidase K; D011402:Pronase; D018021:Lithium Chloride