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Peptide thioesters and 4-nitroanilides as substrates for porcine pancreatic kallikrein.

ABSTRACT: A series of 14 4-nitroanilide substrates and 17 thioester substrates have been used to measure kinetic constants with porcine pancreatic kallikrein. All of the substrates have a P1 arginine residue. The 4-nitroanilide substrates consist of seven P2-glycine and seven P2-phenylalanine tripeptides. As expected from previous results, the phenylalanine series substrates were generally 100-fold 'better' than those in the glycine series. The S3 subsite was found to 'prefer' lysine or phenylalanine, whereas glutamic acid in this position was distinctly unfavourable. The thioester substrates consisted of various thioester derivatives of arginine as well as 12 dipeptides. These substrates exhibited kcat./Km values generally 1000 times higher than the P2-phenylalanine 4-nitroanilides. With the thioesters, a P2 phenylalanine or tryptophan residue yielded the best substrates, but some of the simple derivatives of arginine were nearly as good. A comparison of the kinetic constants of the thioester substrates between the porcine enzyme and human plasma kallikrein provides further evidence that these enzymes have a similar preference for bulky P2 residues, but otherwise are quite different enzymes. The thioester substrates are nearly as reactive as oxygen ester substrates such as acetylphenylalanylarginine methyl ester for the porcine enzyme [Levison & Tomalin (1982) Biochem. J. 203, 299-302; Fiedler (1983) Adv. Exp. Med. Biol. 156A, 263-274], and owing to the greater ease in assaying with the thioesters, they should find use in routine assays for the glandular kallikreins.

SUBMITTER: Powers JC

PROVIDER: S-EPMC1153661 | biostudies-other | 1984 Jun

REPOSITORIES: biostudies-other

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Project description:BackgroundIn a previous report we have demonstrated that the chymotryptic-like serine protease kallikrein 7 (KLK7/hK7) is overexpressed in pancreatic cancer. In normal skin, hK7 is thought to participate in skin desquamation by contributing in the degradation of desmosomal components, such as desmogleins. Thus, the ability of hK7 to degrade desmogleins was assessed and the effect of hK7 expression on desmoglein 2 was examined in cultured pancreatic cancer cells.MethodsThe expression of Dsg1, Dsg2, and Dsg3 in pancreatic tissues was examined by immunohistochemistry and their expression in two pancreatic cancer cell lines, BxPC-3 and Panc-1, was determined by western blot analysis. The ability of hK7 to degrade Dsg1 and Dsg2 was investigated using in vitro degradation assays. BxPC-3 cells stably transfected to overexpress hK7 were used to examine the effect of hK7 on cell-surface resident Dsg2.ResultsThe levels of immunoreactive Dsg1 and Dsg2 were reduced in pancreatic adenocarcinomas compared with both normal pancreatic and chronic pancreatitis tissues. Among the desmosomal proteins examined, Dsg2 exhibited robust expression on the surface of BxPC-3 cells. When hK7 was overexpressed in this cell line, there was a significant increase in the amount of soluble Dsg2 released into the culture medium compared with vector-transfected control cells.ConclusionA reduction in the amount of the cell adhesion components Dsg1 and Dsg2 in pancreatic tumors suggests that loss of these desmosomal proteins may play a role in pancreatic cancer invasion. Using in vitro degradation assays, both Dsg1 and Dsg2 could be readily proteolyzed by hK7, which is overexpressed in pancreatic adenocarcinomas. The enforced expression of hK7 in BxPC-3 cells that express significant amounts of Dsg2 resulted in a marked increase in the shedding of soluble Dsg2, which is consistent with the notion that aberrant expression of hK7 in pancreatic tumors may result in diminished cell-cell adhesion and facilitate tumor cell invasion.

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Peptide thioesters and 4-nitroanilides as substrates for porcine pancreatic kallikrein.

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