Purification and characterization of Pz-peptidase from rabbit muscle.
Pz-peptidase was purified from rabbit muscle by acid precipitation of tissue homogenate followed by cation- and anion-exchange chromatography, gel chromatography, and immunoadsorption. In analytical gel chromatography, one single peak of protein with corresponding Pz-peptidase activity was obtained. The enzyme had an apparent Mr of 74,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was eluted at pH 4.8 in chromatofocusing. No metals were detectable in the protein by neutron activation analysis. Purified Pz-peptidase hydrolyzed Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys (Km 7.2 microM) most effectively in the presence of 5 mM 2-mercaptoethanol and 10 mM CaCl2. No inhibition was observed with inhibitors of serine proteinases, aspartic proteinases, or metalloproteinases, apart from some nonspecific reversible inhibition by 1,10-phenanthroline. The activation by Ca2+ was reversed by EDTA. The enzyme was not inhibited by E-64, cystatin, or leupeptin, but was irreversibly inactivated by iodoacetate, iodoacetamide, and N-ethylmaleimide. It was therefore concluded that rabbit muscle Pz-peptidase is not a typical member of any of the four recognized catalytic classes of proteinases, but may be an atypical cysteine endopeptidase. The peptidase was not bound by alpha 2-macroglobulin. No hydrolysis of gelatin or fibronectin by the enzyme was detected, nor was there any activation of latent collagenase.
Tisljar
U
U
Biochemistry Department, Strangeways Research Laboratory, Cambridge, United Kingdom.
Barrett
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008666:Metalloendopeptidases; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D009842:Oligopeptides; D011480:Protease Inhibitors / Q000494:pharmacology; D011817:Rabbits; D013379:Substrate Specificity
D009842:Oligopeptides; D011480:Protease Inhibitors; D010450:Endopeptidases; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase