Porcine muscle prolyl endopeptidase and its endogenous substrates.
Prolyl endopeptidase [EC 3.4.21.26] was purified 4,675-fold with a yield of 26.3% from porcine muscle. The purified enzyme was shown to be very similar to the liver enzyme with respect to its molecular weight (72,000-74,000), antigenicity, substrate specificity, and susceptibility to protease inhibitors. Among several bioactive peptides, angiotensins I, II, and III had the lowest Km of 0.6 to 3 microM with the lowest kcat of 0.19 to 0.85 s-1, while thyrotropin-releasing hormone had the highest Km of 98 microM with the highest kcat of 14.4 s-1. Interestingly, mastoparan was hydrolyzed at alanyl bonds, but insulin was only slightly hydrolyzed and glucagon was not hydrolyzed although the latter two peptides contain prolyl and/or alanyl bonds. Muscle prolyl endopeptidase failed to hydrolyze proteins with high molecular weight such as albumin, immunoglobulin G, elastin, collagen, and muscle soluble and insoluble proteins. However, 8 of 14 peptides with molecular weights lower than 3,000, which were isolated from muscle extract, were digested by this enzyme, and they were proved to contain prolyl and/or alanyl residues in their molecules. The data suggest that they are probable endogenous substrates for prolyl endopeptidase.
Moriyama
A
A
Department of Biochemistry, Nagoya City University Medical School, Aichi.
Nakanishi
M
M
Sasaki
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D006882:Hydroxyapatites; D009132:Muscles / Q000201:enzymology*; D000085822:Prolyl Oligopeptidases; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D013552:Swine
D000596:Amino Acids; D006882:Hydroxyapatites; D011480:Protease Inhibitors; D017886:Durapatite; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases