Purification and characterization of a multicatalytic proteinase from crustacean muscle: comparison of latent and heat-activated forms.
A high-molecular-weight (Mr 740,000) multicatalytic proteinase (MCP) was purified over 3100-fold from soluble extracts of lobster claw and abdominal muscles. The enzyme was extracted from muscle in a latent state; brief (3 min) heating of an ammonium sulfate fraction (45-65% saturation) at 60 degrees C irreversibly activated the proteinase while denaturing about 55% of the protein. MCP was further purified by chromatography on two sequential arginine-Sepharose columns and a Mono Q column with a yield of 60%. About 1.12 mg MCP was obtained for every 100 g tissue. In addition to [14C]methylcasein, the MCP hydrolyzed synthetic peptide substrates of trypsin and chymotrypsin at pH 7.75. Serine protease inhibitors (diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, aprotinin, benzamidine, soybean trypsin inhibitor, chloromethyl ketones), leupeptin, antipain, hemin, sulfhydryl-blocking reagents (N-ethylmaleimide, mersalyl acid, p-chloromercurisulfonic acid, iodoacetamide) suppressed activity while Ep-475, a specific inhibitor of cysteine proteinases, had no effect, suggesting the MCP is a serine proteinase with one or more cysteine residues indirectly involved in catalysis. The latent MCP was purified using the same procedure as that for the active form, except that thermal activation was omitted. The elution characteristics of latent MCP from the arginine-Sepharose and Mono Q columns were identical to those of active MCP. Since the purified latent form could still be activated by heating, activation did not involve denaturation of an endogenous inhibitor or substrate. Subunit compositions of both forms were identical in two-dimensional polyacrylamide gels; each was composed of eight polypeptides with molecular weights between 25,000 and 32,500 and a ninth polypeptide with a molecular weight of 41,000. Electron microscopy of negatively stained material showed that each form was a cylinder-shaped particle (approximately 10 x 15 nm) consisting of a stack of four rings with a hollow center; no differences in shape, dimensions, or submolecular structure were observed. These results suggest that activation probably involved small conformational changes rather than covalent modifications or rearrangement of subunits within the complex.
Mykles
D L
DL
Department of Biology, Colorado State University, Fort Collins 80523.
D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D006358:Hot Temperature; D007700:Kinetics; D008854:Microscopy, Electron; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009132:Muscles / Q000201:enzymology*; D008121:Nephropidae; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex