Purification of enzymatically active kallikrein hK2 from human seminal plasma.
Kallikrein hK2 is a member of the human glandular kallikrein family which includes prostate-specific antigen (PSA) and pancreatic-renal kallikrein. The purpose of this work was to isolate and characterize for the first time the enzymatically active form of the hK2 protein starting from the PCI-hK2 complex isolated from human seminal plasma (Deperthes, D., Chapdelaine, P., Tremblay, R.R., Brunet, C., Berton, J., Hébert, J., Lazure, C. and Dubé, J.Y. (1995) Biochim. Biophys. Acta 1245, 311-316). That complex was dissociated by an incubation at alkaline pH and final purification was achieved by C-18 reverse phase HPLC. The purified material contained a 27 kDa band by SDS gel electrophoresis and had the expected NH2-terminal amino acid sequence of hK2. It hydrolyzed synthetic chromogenic substrates containing esters of lysine and arginine but not of phenylalanine. Furthermore, hK2 formed molecular complexes with alpha 2 -antiplasmin, alpha 1-antichymotrypsin, antithrombin III and alpha 2-macroglobulin but not with alpha 1-antitrypsin. In conlusion, the new findings of the present paper are that the PCI-hK2 complex can be dissociated by mild procedures, that the free hK2 protein can be purified thereafter by standard HPLC procedures, that the recovered free hK2 is a trypsin-like enzyme and that it can form molecular complexes with many of the major serum proteinase inhibitors.
Frenette
G
G
Hormonal Bioregulation Laboratory, CHUL Research Center, Sainte-Foy, Quebec, Canada.
Deperthes
D
D
Tremblay
R R
RR
Lazure
C
C
Dubé
J Y
JY
D000595:Amino Acid Sequence; D000990:Antithrombin III / Q000494:pharmacology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D018046:Protein C Inhibitor / Q000737:chemistry; D012661:Semen / Q000201:enzymology*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D020840:Tissue Kallikreins; D000979:alpha-2-Antiplasmin / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000494:pharmacology
D018046:Protein C Inhibitor; D015842:Serine Proteinase Inhibitors; D000979:alpha-2-Antiplasmin; D000511:alpha-Macroglobulins; D000990:Antithrombin III; D007610:Kallikreins; D020840:Tissue Kallikreins