Comparative study of kallikrein-like serine proteinases from rat submandibular glands.
The present investigation describes the comparative properties, particularly the substrate specificity of three kallikrein-like serine proteinases (I, II and III) purified from rat submandibular gland extract (Bedi, G.S., Prep. Biochem. 22,67-81, 1992). The physico-chemical and immunological properties of three proteinases were compared by Western blot analysis, immunodiffusion, immuno-electrophoresis, amino terminal sequence analysis, molecular weight determination and isoelectric focusing. Detailed substrate specificity of these proteinases was determined using chromogenic substrates, synthetic peptides and native proteins. The chromogenic substrate tosyl-gly-pro-arg-pNA was hydrolyzed preferentially by Proteinase I. The replacement of pro at the P2 position with bulky hydrophobic residues phe and leu completely abolished the hydrolysis by Proteinase I. The hydrolysis of the chromogenic substrates by Proteinase II was also affected by the amino acid residue present at the P2 position in the order of pro > gly > val > leu > phe. Neither Proteinase I nor Proteinase II hydrolyzed substrates in which arg was replaced with lys at the P1 position. Proteinase III was reactive against all the chromogenic substrates with arg or lys at the P1 position. Synthetic polypeptides T-kinin-leu and insulin B chain were resistant to cleavage by both Proteinase I and II and were cleaved specifically at arg-X peptide bond by Proteinase III. Tonin-like activity of Proteinase II was confirmed by cleavage of the angiotensin 1-14 at phe-his linkage to generate two fragments DRVYIHPF and HLLVYS respectively. All three proteinases cleaved human high molecular weight kininogen but only Proteinase III could cleave T-kininogen. Proteinase III was also reactive towards human and bovine fibronectin, fibrinogen and gelatin. Several other salivary and serum proteins were resistant to cleavage by these proteinases. Although the three enzymes are immunologically related, they differ with respect to size, isoelectric point, amino terminal sequence and inhibition profile.
Bedi
G S
GS
Department of Microbiology and Immunology, Medical College of Pennsylvania, Philadelphia 19129, USA.
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism; D007610:Kallikreins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D007704:Kininogens / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D012471:Salivary Proteins and Peptides / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D013363:Submandibular Gland / Q000201:enzymology*; D013379:Substrate Specificity
D007328:Insulin; D007704:Kininogens; D012471:Salivary Proteins and Peptides; D000804:Angiotensin II; D007610:Kallikreins; D012697:Serine Endopeptidases