Kallistatin: a novel human tissue kallikrein inhibitor. Purification, characterization, and reactive center sequence.
A novel human tissue kallikrein inhibitor designated as kallistatin has been purified from plasma to apparent homogeneity by polyethylene glycol fractionation and successive chromatography on heparin-Agarose, DEAE-Sepharose, hydroxylapatite, and phenyl-Superose columns. A purification factor of 4350 was achieved with a yield of approximately 1.35 mg per liter of plasma. The purified inhibitor migrates as a single band with an apparent molecular mass of 58 kDa when analyzed on SDS-polyacrylamide gel electrophoresis under reducing conditions. It is an acidic protein with pI values ranging from 4.6 to 5.2. No immunological cross-reactivity was found by Western blot analyses between kallistatin and other serpins. Kallistatin inhibits human tissue kallikrein's activity toward kininogen and tripeptide substrates. The second-order reaction rate constant (ka) was determined to be 2.6 x 10(4) M-1 s-1 using Pro-Phe-Arg-MCA. The inhibition is accompanied by formation of an equimolar, heat- and SDS-stable complex between tissue kallikrein and kallistatin, and by generation of a small carboxyl-terminal fragment from the inhibitor due to cleavage at the reactive site by tissue kallikrein. Heparin blocks kallistatin's complex formation with tissue kallikrein and abolishes its inhibitory effect on tissue kallikrein's activity. The amino-terminal residue of kallistatin is blocked. Sequence analysis of the carboxyl-terminal fragment generated from kallistatin reveals the reactive center sequence from P1' to P15', which shares sequence similarity with, but is different from known serpins including protein C inhibitor, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. The results show that kallistatin is a new member of the serpin superfamily that inhibits human tissue kallikrein.
Zhou
G X
GX
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425.
Chao
L
L
Chao
J
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D002845:Chromatography; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006882:Hydroxyapatites; D007525:Isoelectric Focusing; D007610:Kallikreins / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D015843:Serpins / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D013379:Substrate Specificity; D020840:Tissue Kallikreins
D002352:Carrier Proteins; D006882:Hydroxyapatites; D015843:Serpins; C051324:kallistatin; D017886:Durapatite; D007610:Kallikreins; D020840:Tissue Kallikreins