Purification of enzymes of the kallikrein gene family (rK8 and rK9) from the rat prostate

Abstract

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.

Altered expression of extracellular matrix and proteinases in noble rat prostate gland after long‐term treatment with sex steroids

“…These proteinases were characterized as serine proteinases since their activities were inhibited by serine proteinase inhibitors. The 30-kDa serine proteinase could represent the major rat prostatic kallikrein, rK9 or rK8, based on its similarity in molecular size (25±30 kDa) and inhibition by aprotinin [59,60]. However, the identity of the 100-kDa serine proteinase is still uncertain.…”

Expression of a kallikrein-like protein in prostatic intraepithelial neoplasia in ventral prostate of the noble rat

“…Identification of kallikrein-like proteins can be difficult because of their similarities in substrate and inhibitor specificity, antigenic epitopes, amino acid sequence, and molecular mass [22]. In the present study, after SDS-PAGE analysis and blotting, the kallikreinlike nature of the prostate protein (p18) was verified by partial amino acid sequencing.…”

Effect of Boerhaavia diffusa in experimental prostatic hyperplasia in rats

“…Although the incidence of hypertrophy of the canine prostate increase with age and closely resembles that in humans, BPH have been experimentally induced in rats following prolonged treatment with testosterone. [131415] As androgen exhibited stimulating action on the growth of prostate cell, Testosterone and related hormones are demonstrated to play an important role in disease. [3] Data from large scale clinical study have demonstrated that treatment with 5α-reductase inhibitors not only significantly ameliorates symptoms, but also reduces the long-term risk of progression in patients treated surgically.…”

“…Homogenates were centrifuged at 13,000× g for 20 min and supernant collected. [15] Supernant was used as source of proteins and concentration was determined by modified biuret end point assay method.…”