Purification and characterization of salivary kallikrein from an insectivore (Scalopus aquaticus): substrate specificities, immunoreactivity, and kinetic analyses.
We report the successful one-step separation of tissue kallikrein from the salivary glands of an insectivore, the Eastern Atlantic mole (Scalopus aquaticus) by perfusion chromatography. Purified mole salivary kallikrein was characterized as a 30-kDa serine proteinase with a pI of 5.3 and a pH optimum of 9.0. It was readily recognized by human tissue kallikrein antibody in immunoblot analyses. It preferentially hydrolyzes fluorogenic peptidyl substrates with arginyl residues, rather than lysyl residues at the P1 substrate recognition site, indicating that it is like other mammalian kallikreins. Mole kallikrein efficiently releases kinin from low molecular weight human, dog, and bovine kininogen substrates with specific activities similar to that of human tissue kallikrein. Steady state kinetics performed with the synthetic tripeptidyl substrates, Phe-Phe-Arg-, Pro-Phe-Arg, and Val-Leu-Arg-7-amino-4-methylcoumarin, gave K(m) values for mole kallikrein of 3.3, 46.1, and 2.8 microM, respectively, and specificity constants, kcat/K(m), of 3818, 165, and 8714 s-1 pM-1, respectively. Mole kallikrein, when compared with human and rat tissue kallikreins, more closely resembles human kallikrein based on immunoreactivity and kininogenase activity. Mole kallikrein appears to be a member of a single gene or small multigene family. S. aquaticus is recommended for studying the evolution of mammalian proteins and may offer advantages over rodent models for biomedical research.
Richards
G P
GP
National Marine Fisheries Service, Southeast Fisheries Science Center, Charleston Laboratory, South Carolina 29422-2607, USA.
Zintz
C
C
Chao
J
J
Chao
L
L
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003429:Cross Reactions; D004285:Dogs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007610:Kallikreins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008971:Moles / Q000378:metabolism*; D051381:Rats; D012469:Salivary Glands / Q000201:enzymology*; D013045:Species Specificity; D013379:Substrate Specificity
D007610:Kallikreins