Purification of enzymes of the kallikrein gene family (rK8 and rK9) from the rat prostate.
The rat kallikrein family consists of multiple closely related proteins. A method for demonstration and identification of kallikrein-like proteins has been developed based on their differences in isoelectric point and their immunological similarity. The method, which involved separation in flat-bed isoelectro-focusing gels (pH range 3-9) and detection by immunoblotting using polyclonal antiserum against one of the family members, has been used in the present study to detect kallikrein-like proteins in the rat prostate. Nine immunoreactive kallikrein-like protein bands were detected with pI ranging from 5.30 to 8.35. Of these, six were completely purified and three were partially purified. Two proteins (pI 5.30 and 6.75-6.90) corresponded to protein bands in gels of rat submandibular-gland extracts, and were identified by partial amino acid sequence analysis as rK8 and rK9 respectively. In addition, sequence analysis revealed complete sequence similarity between rK9 and the immunoreactive prostate proteins with pI 7.15, 7.25, 7.50 and 8.27. On the basis of this finding and immunological and biochemical characterization, we concluded that all the kallikrein-like proteins detected, except for rK8, represented isoenzymes of rK9. The molecular masses of the prostate rK9 isoenzymes (24,600-29,300 Da) were close to that of submandibular-gland rK9 (24,600 Da), although differences were observed after reduction with mercaptoethanol. The prostate rK9 isoenzymes were, like submandibular-gland rK9, inhibited by soya-bean trypsin inhibitor but not by aprotinin, and were classified as serine proteases as they were inhibited by phenylmethanesulphonyl fluoride. rK8 (28,700 Da) showed no activity with any of the substrates tested, and its inhibitory profile could therefore not be studied. No other enzymes of the kallikrein family were found in the rat prostate.
Schøyen
H
H
Department of Physiology, Medical Faculty, University of Oslo, Norway.
Wassdal
I
I
Toft
K
K
Almendingen
M
M
Berg
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family; D011467:Prostate / Q000201:enzymology*; D051381:Rats; D017208:Rats, Wistar; D016622:Silver Staining; D013379:Substrate Specificity
D007527:Isoenzymes; D007610:Kallikreins