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.
Three pepsinogens, namely, pepsinogens A, C-1, and C-2, were purified from gastric mucosa of adult house musk shrew (Suncus murinus) by conventional chromatographic and gel filtration procedures. The molecular masses were 40, 39, and 41 kDa for pepsinogens A, C-1, and C-2, respectively. Pepsinogen C-2 contains an Asn-linked carbohydrate chain(s) of about 2 kDa. Each pepsinogen was converted to pepsin through an intermediate form under acidic conditions. By NH2-terminal sequence analysis of these protein species, the amino acid sequences of activation segments (proparts) of pepsinogens A and C-1 were determined to be LYKVPLVKKKSLRQNLIENGLLKDFLAKHNVNPASKYFPTE and KVTKVTLKKFKSIRENLREQGLLEDFLKTNHYDPAQKYHFGDF, respectively. The similarity of these two sequences is nearly 50%. Each pepsin cleaved preferentially peptide bonds between hydrophobic and aromatic amino acids, or bonds on either side of these amino acids. Although each activation segment had several sites susceptible to pepsin action, activation proceeded by limited cleavages of the segment, presumably due to the steric inflexibility of the segment in native pepsinogen. The activity of pepsin A was inhibited completely in the presence of a more than equimolar amount of pepstatin, while a hundred-molar excess amount of pepstatin was needed for the complete inhibition of the activity of pepsins C-1 and C-2.
A latent protease has been identified in column fractions obtained during the purification of the porcine ovarian serine protease follipsin. The latent enzyme was readily activated by trypsin treatment. The trypsin-activated enzyme was purified using a benzamidine-Sepharose 6B column and was shown to be composed of two distinct, covalently associated polypeptides with M , of 45000 and 32000. This polypeptide chain composition, together with its substrate specificity, inhibition profile using various protease inhibitors, cross-reactivity with anti-follipsin antibody, and ability to activate single-chain precursor tissue plasminogen activator, indicated its identity as porcine follipsin. The activation of the enzyme with trypsin was found to occur by the hydrolysis of an internal peptide bond resulting in a two-chain structure. Thus, we conclude that the latent enzyme is the inactive precursor form (profollipsin) of follipsin. The present study also shows that the follicular fluid of porcine ovary contains a profollipsin-activating enzyme activity. We have previously identified an additional enzyme activity in the follicular fluid of porcine ovaries that is capable of hydrolyzing synthetic, arginine-containing peptide 4-methylcoumaryl-7-amide (-NH-Mec) substrates. Purification and characterization studies revealed that the enzyme follipsin is a serine protease structurally similar to human plasma kallikrein [ 14, 151. Subsequently, the enzyme has been shown to efficiently convert human single-chain precursor tissue-type plasininogen activator (sctPA) to its mature form (tPA) in vitro [16].Follipsin consists of two distinct polypeptide chains with M , of 45 000 and 32 000, associated covalently, and is thought to be synthesized as a single polypeptide chain precursor protein [IS]. The question is thus raised as to where the precursor enzyme undergoes conversion to active two-chain follipsin. In an attempt to approach this problem, we noted that trypsin treatment of certain fractions, which were obtained in the column chromatoCorve,~poi?tl~,rzae fo T. Takahashi,
A cDNA clone of a new mouse tissue kallikrein, designated mKlk27, was isolated from an adult mouse testis cDNA library. mKlk27 was expressed in the submaxillary glands and testis of the mouse. In testis, mKlk27 gene was expressed exclusively in the Leydig cells of the adult mouse. Active recombinant mKlk27 exhibited chymotrypsin-like cleavage specificity. A single amino-acid substitution of Gly for Asp at position 209 in mKlk27 resulted in complete loss of its chymotryptic activity but acquisition of tryptic activity. mKlk27 effectively hydrolyzed casein, gelatin and fibronectin. Insulin-like growth factor binding protein-3 was also hydrolyzed by recombinant mKlk27. These results suggest that mKlk27 plays an important role in association with the function of the adult mouse testis.
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