Purification of rat urinary kallikrein: comparative studies with rat submandibular gland kallikrein-like serine protease.
A 427-fold purification of rat urinary kallikrein (RUK) was achieved in three steps involving chromatography on columns of DEAE-Sepharose CL-6B, gel filtration on Sephadex G-100 and affinity chromatography on a column of benzamidine-Sepharose. Purified enzyme showed a single band on SDS-PAGE with an estimated molecular weight of 43,000. The amino-terminal sequences of the first 25 residues of RUK resemble the reported sequence for true kallikrein and share 80% identity with rat submandibular gland (RSMG) kallikrein-like serine protease. The RUK is highly reactive towards kallikrein substrates Bz-pro-phe-arg-pNA and DL-val-leu-arg-pNA, and plasmin substrate D-val-leu-lys-pNA. RSMG enzyme is more reactive towards Bz-val-gly-arg-pNA and tosyl-gly-pro-arg-pNA, preferential chromogenic substrates for trypsin-like proteases and thrombin, respectively. Both leupeptin and aprotinin inhibit RUK strongly, but soy bean trypsin inhibitor has no effect on this enzyme. RSMG enzyme is poorly inhibited by any of these inhibitors. The data suggest that although both enzymes are members of tissue kallikrein multigene family, urinary enzyme is a true kallikrein and RSMG enzyme is a kallikrein-like serine protease with different substrate specificity.
Bedi
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Department of Oral Biology, State University of New York, Buffalo 14214.
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D007610:Kallikreins / Q000187:drug effects / Q000302:isolation & purification* / Q000652:urine; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012697:Serine Endopeptidases / Q000302:isolation & purification*; D013363:Submandibular Gland / Q000201:enzymology*; D013379:Substrate Specificity
D011480:Protease Inhibitors; D007610:Kallikreins; D012697:Serine Endopeptidases