Human kallikrein hK2 has low kininogenase activity while prostate-specific antigen (hK3) has none

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Abstract

In the present paper, we determined the kinin-releasing activity of human prostatic kallikrein hK2 and compared it to one of the kallikreins hK1 and prostate specific antigen (hK3). Kinin-like substances active on the rabbit jugular vein were progressively produced when nanomolar concentrations of hK2 were incubated with heated plasma. However in these experiments, hK1 appeared much more potent than hK2 while hK3 was totally inactive. When hK2 was incubated with purified high molecular weight kininogen, several peptides were generated as shown by the analysis on C18 reverse-phase HPLC. Kinin activity was localized exclusively in a small peak having an elution time identical to that of bradykinin while the only important peak obtained with hK1 corresponded to Lys-bradykinin. Finally, the rate of kinin production of hK2 was found to be more than a thousandfold lower than that of hK1. These experiments show that kallikreins hK2 has only a low kininogenase activity. However, it is not excluded that some of the peptides produced by hK2 action could have other types of biological activity.

Introduction

Glandular kallikreins were originally discovered because of their ability to generate kinins. With the widespread use of the recombinant DNA technology in the eighties, several new kallikreins have been added to that family in rat, mouse and man 1, 2, 3. Some of the new kallikreins turned out to have significant kininogenase activity. Therefore, it has become customary to test the enzymatic activity of novel kallikreins using blood serum kininogens as substrates. In our laboratory, we have recently isolated from seminal plasma the third human kallikrein gene product 4, 5which is presently known as hK2. In view of the demonstration that hK2 is a trypsin-like enzyme 5, 6, 7, it appeared important to determine whether it had kinin-releasing activity. The kinins produced in seminal plasma are thought to enhance sperm motility through their interaction with a B2 type receptor in the membrane of human spermatozoa [8]. The kinin producing activity in human seminal plasma has previously been attributed to immunoreactive kallikrein hK1 whose presence has been detected in low amounts by immunoassay procedures in that fluid 9, 10. Furthermore, prostate-specific antigen (hK3) has recently been proposed to be able to release a kinin-like substance on proteolysis of seminal vesicle fluid [11]. Therefore, this paper is aimed not only at assaying the kinin-releasing activity of hK2 but also at determining the relative importance of each human kallikrein hK1, hK2 and hK3 in the kinin production of seminal plasma.

Section snippets

Kallikreins hK1, hK2 and hK3

The purified kallikrein hK1 preparation used in these studies was a generous gift from Dr. Lee Chao of the Medical University of South Carolina, Charleston, South Carolina.

The purification of hK2 was done as described in previous publications 4, 5. hK3 was obtained by sequential chromatography on DEAE-Sepharose, Sephadex G-100 and CM-Sepharose as recently reported [6]. That hK3 preparation had chymotrypsin-like activity on protein substrates but no trypsin-like activity using carbobenzoxylysine

Results

In the first series of experiments, we examined the in vitro kinin-releasing activity of the three members of the human kallikrein gene family, hK1, hK2 and hK3 using heated citrated plasma as a source of kininogens and the rabbit jugular vein contraction as the kinin detection system. Fig. 1 shows that 3 nM concentrations of kallikrein hK1 very rapidly produced kinins able to generate maximal contraction of the rabbit jugular vein. The production of kinin-like substances was much slower with a

Discussion

These results show that kallikrein hK2 has significant kininogenase activity. However, that activity is low when compared to that of kallikrein hK1. Furthermore in our experimental conditions, hK3 was found to be completely devoid of kininogenase activity. That observation is not in accord with the recent report suggesting that hK3 was able to generate kinin-like substances when incubated with seminal vesicle fluid [11]. The difference between the conclusions of the two studies must be ascribed

Acknowledgements

We are grateful to Mrs. Lucie Turcotte for typing the original manuscript. This study was supported by a grant from the Medical Research Council of Canada.

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