Enzymatic characterization of arginine esterase from dog seminal plasma.

Previously purified arginine esterase from dog seminal plasma was characterized enzymatically. The enzyme was found to have a rather narrow specificity for arginine esters, much less for lysine esters and was practically devoid of activity towards tyrosine esters, casein, albumin and azocoll. It had a broad optimum pH between 8 and 9. It presented no kallikrein-like activities either in the blood pressure test in dog or in the rat uterus contraction test. It was inhibited by bovine pancreas trypsin inhibitor, aprotinin, phenylalanylprolyl arginine chloromethyl ketone, diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, sodium dodecyl sulfate and leupeptin, but not by soybean trypsin inhibitor, tosyllysine chloromethyl ketone, tosylamide-2-phenylethyl chloromethyl ketone, iodoacetamide, Triton X-100 and EDTA. Experiments involving incubation of prostatic cytosol with purified arginine esterase showed that actin was the only important prostatic protein that was extensively hydrolyzed by this enzyme. It is not known presently whether the hydrolysis of actin is related to a true physiological function of the enzyme and whether actin and arginine esterase ever come into contact with each other in vivo. These properties indicate that arginine esterase from dog seminal plasma is different from other known proteinases including classical kallikreins, although it presents many similarities with this class of enzyme.

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.

Characterization of rhesus monkey prostate specific antigen cDNA.

Using a RT-PCR approach, we obtained two overlapping cDNA clones containing the entire 1.5 kb sequence of rhesus monkey prostate specific antigen (rmPSA). The sequence obtained revealed an open reading frame of 261 amino acids. One potential N-glycosylation site was identified at Asn-78. The calculated molecular mass for the unglycosylated mature protein was 26,147 Da. Extensive amino acid homology (89%) was observed between rmPSA and its human counterpart. These results demonstrate that rhesus monkey and man prostate share a major biochemical component, and suggest that this animal species might be useful to answer specific questions related to human prostatic function and pathology.

Characterization of canine pancreas kallikrein cDNA.

Using a combination of primer extension and RT-PCR, the cDNA encoding a canine tissue kallikrein expressed in the pancreas was cloned and sequenced. The cloned 0.85 kbp cDNA contained a complete open reading frame encoding a polypeptide of 261 amino acids. The calculated molecular mass of the processed, unglycosylated, 237 amino acid protein was 26,428 Da. Its mRNA was expressed at high levels in the pancreas, kidney and submaxillary gland. The sequence of the encoded protein was highly homologous with canine prostatic arginine esterase (66%) and human renal/pancreatic kallikrein (74%). Therefore, the cloned cDNA encoded a previously uncharacterized canine kallikrein enzyme which was named dog renal/pancreatic kallikrein or dK2 according to the new nomenclature for kallikrein gene family members. Because of its specific pattern of tissue expression and the presence of all the amino acid residues necessary for kininogenase activity, we suggest that dK2 is the canine true tissue kallikrein.

Identification of glandular kallikrein in dog pancreas and determination of its tissue distribution.

In order to establish a formal link between previously purified canine urinary kallikrein and dog pancreatic kallikrein whose cDNA sequence has recently been published, we have isolated the pancreatic kallikrein from that animal species. Pancreatic cytosol proteins were sequentially subjected to chromatography on DEAE-Sepharose CL-6B and Concanavalin A-Sepharose, to an autolysis step and finally to two-dimensional gel electrophoresis. Kallikrein immunoreactive spots were identified with an antibody directed against canine urinary kallikrein. These proteins were isolated after electroblotting and the amino acid sequence of their NH2-terminal portion was determined by microsequencing. The sequence was found to be identical to the one deduced from pancreatic kallikrein cDNA. Using the same antibody and immunohistochemical procedures, kallikrein was found to be present in the pancreas, the salivary glands, the kidney, the colon, the lungs and the testis. These results thus confirm the molecular nature of a glandular kallikrein in the canine species.

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

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.

Isolation of prostatic kallikrein hK2, also known as hGK-1, in human seminal plasma.

To demonstrate the presence of kallikrein hK2 in the human prostate and seminal plasma, we used mouse monoclonal antibodies (MAb) against a recombinant hK2-fusion protein. Using one of these MAb 9D5, we detected the presence of several major immunoreactive spots of 22 kDa and minor ones of 31 and 55 kDa in prostate cytosol and seminal plasma. After ion exchange and immunoaffinity chromatography of seminal plasma proteins, the 22-kDa immunoreactive proteins were isolated along with 55- and 75-kDa proteins. The NH2-terminal amino acid sequencing permitted identification of fragments of hK2 and protein C inhibitor, respectively, in the 22- ad 55-kDa bands. Furthermore, immunoblotting experiments in one and two-D gels with two different anti-hK2 MAbs and one polyclonal anti-PCI antibody suggested that the major 55- and 75-kDa bands were covalent hK2-PCI complexes containing either the full-length hK2 chain or only its carboxyterminal fragment in the presence of mercaptoethanol. These results demonstrate for the first time the existence of kallikrein hK2 and suggest that PCI may regulate its activity in seminal plasma.

Specific insulin binding sites in rat testis: characterization and variation.

We have found high affinity binding of insulin not only in rat liver and kidney, but also in testis and male sex accessory tissues, prostate, seminal vesicle, and epididymis. We have studied particularly the characteristics of insulin binding in the testis. Membranes sedimenting at 100,000 X g showed the highest binding after 6-20 h of incubation at 0 C. Higher temperatures (15 and 25 C) resulted in lower binding. More than 90% of membrane-bound radioactivity after long incubations at 0 C was eluted at the same position as insulin by Sephadex G-50 chromatography. Membranes could be stored at -80 C for several weeks without loss of activity. Studies on binding specificity showed the following order of competition relative to insulin (100): desalanine insulin (84), proinsulin (2), and desoctapeptide insulin (1). Other peptidic hormones, LH, FSH, PRL, GH, glucagon, and ACTH-(1-24) were totally ineffective. Scatchard representation of the binding data could be resolved into two components with respective affinity constant (Ka) of 1.6 X 10(9) M-1 and 3 X 10(6) M-1. Testicular high affinity binding in adult rats did not vary after 3 days of starvation. However, it increased with age from 1-6 months. By contrast, in rat liver, this type of binding increased after starvation but decreased slightly at 6 months of age. These results show that testicular insulin receptors are similar to those of the liver but may have a different physiological control.

In vivo effects of steroid hormones on the testosterone 5alpha-reductase in skin.

We have studied the hormonal regulation of the levels of testosterone 5alpha-reductase in rat skin by performing surgical manipulations (gonadectomy and/or adrenalectomy) of the rats and by steroid administration to ovariectomized- adrenalectomized rats. Castration significantly increases (P smaller than 0.01) the enzyme activity in the sole of the foot but not in the thoracic skin of male rats. In female rats, significant elevations of the enzyme activity are observed in the thoracic skin (P smaller than 0.01) and in the sole of the foot (P smaller than 0.05) after ovariectomy. Estrogen administration decreases the enzyme activity in thoracic skin, tail skin and vulvar skin of ovariectomized-adrenalectomized rats. Testosterone and dihydrotestosterone decrease the activity in the sole of the foot and increase the activity in vulvar skin. Progesterone and corticosterone administration produce no significant effects in the four skin types studied.

Binding of methyltrienolone to glucocorticoid receptors in rat muscle cytosol.

Androgen receptor measurements with [3H]-methyltrienolone [17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881)] or [3H]testosterone in vastus lateralis muscle cytosol of intact male rats yielded similar numbers of binding sites. Gonadectomy significantly increased the androgen receptor values, and both steroids gave similar results. The binding of [3H]-testosterone was not affected by adrenalectomy. In contrast, [3H]R1881 binding increased 2-fold after adrenalectomy. Competition experiments as well as sucrose density gradient analysis indicated that R1881, in addition to its binding of the androgen receptor, was also bound to glucocorticoid receptor. These results show that the synthetic androgen R1881 must be used with caution in tissues suspected to contain glucocorticoid receptors.

Testosterone 5alpha-reductase in rat skin homogenates: measurement in the skin at various anatomical sites in males and females.

Experiments were disigned to find suitable conditions to determine the 5alpha-reducatase activity in rat skin homogenates. The dihydrotestosterone formation was linear until 15 min. The 5alpha- reductase was very heat labile at 37-c in absence of testosterone and NADPH since complete loss of activity occurred after one hour of preincubation. At OC, there was a 20% decrease after 5 hr. The enzyme activiity was measured in the skin at various anatomical sites. It was found to vary with each region in the following order in the male; tail,greater than scrotum greater than ear greater than genital skin greater than dorsum smaller than or equal to thorax greater than sole of the foot. In a few tissues, the enzyme activity was higher than in the prostate. Corresponding female tissues exhibited lower reductase activity except the sole of the foot in which the contrary was found. This significant sex difference indicates that sex hormones may be involved in the control of the levels of that enzyme in rat skin.

Effect of combination treatment with zanoterone (WIN 49596), a steroidal androgen receptor antagonist, and finasteride (MK-906), a steroidal 5 alpha-reductase inhibitor, on the prostate and testes of beagle dogs.

The effects of the steroidal androgen receptor antagonist zanoterone (WIN 49596) and the steroidal 5 alpha-reductase inhibitor finasteride (MK-906) either alone or in combination on prostatic size, histomorphology, and biochemistry were determined in the intact male dog. Additionally, the effects of treatment with zanoterone and/or finasteride on testicular size, serum testosterone and LH levels, and spermatogenesis were determined in the same dogs. Daily oral treatment for 16 weeks with either zanoterone alone at 10 mg/kg.day or finasteride alone at 1.0 mg/kg.day reduced (P < 0.05) the size of the prostate, resulted in mild to moderate diffuse glandular atrophy of the prostate, and decreased prostatic DNA and prostatic arginine esterase (the primary canine prostatic protein) levels compared to those in intact controls. These changes occurred with no effect on testicular weight, testicular histomorphology, daily sperm production, or serum LH levels. Serum testosterone concentrations were increased (P < 0.05) approximately 3-fold in the 10 mg/kg.day zanoterone treatment group compared to those in intact controls. Combination treatment of male dogs for 16 weeks with zanoterone (10 mg/kg.day) plus finasteride (1.0 mg/kg.day) orally also reduced (P < 0.05) prostate size, resulted in moderate to marked diffuse prostatic glandular atrophy, and decreased prostatic DNA and arginine esterase levels more than either drug alone, without affecting testicular size, testicular histomorphology, serum LH concentrations, or serum testosterone concentrations compared to those in intact controls. The effects of combination treatment with zanoterone and finasteride on prostatic size; histomorphology; and DNA, arginine esterase protein, and arginine esterase mRNA levels were similar to those observed in castrate controls. In addition, in situ estimates of prostatic size using transrectal ultrasonography indicated that the median time to 70% prostatic regression in dogs administered combination zanoterone plus finasteride was similar to that in castrate controls (9.6 and 9.3 weeks, respectively), indicating that the combination was more effective in causing prostatic regression than either drug alone. Finally, at the dosages used, no adverse effects of combination treatment with zanoterone plus finasteride on testicular or other major body organ weights were observed. Based on these results, combination therapy using zanoterone and finasteride for the treatment of human androgen-dependent disorders such as benign prostatic hyperplasia and prostate cancer has potential utility.

Progestin binding in testes from three siblings with the syndrome of male pseudohermaphroditism with testicular feminization.

We have found a specific binding protein for synthetic progestins 6,7-[3H]methyltrienolone (R1881) and 17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione (R5020) and in the testis cytosol from three "sisters" with the complete form of the testicular feminization syndrome. The binding component sediments in the 8S region of sucrose gradients. It is saturable. The apparent affinity constant (Ka) for R5020 was determined in two cases and found to be 1.8 and 0.6 X 10(8) M-1. The number of binding sites calculated from Scatchard plots is relatively high: 572 and 826 fmol/mg protein. Competition studies indicate that this putative receptor is specific for natural and synthetic progestins but not for 5 alpha-dihydrotestosterone and cortisol. Similar progestin binding could not be found in normal human and rat testes.

High level of expression in the prostate of a human glandular kallikrein mRNA related to prostate-specific antigen.

Using a synthetic oligonucleotide primer complementary to human prostate-specific antigen mRNA, we found that an additional sequence possibly similar to human glandular kallikrein-1 could be read by a primer-extension sequencing technique. We were able to confirm the identity of that additional sequence with another oligonucleotide primer complementary to a specific region of the human glandular kallikrein-1 mRNA sequence. Northern blot analysis with 2 oligonucleotide probes respectively specific for prostate-specific antigen and human glandular kallikrein-1 mRNAs showed that the length of both mRNAs was similar at 1.5 kb. The level of human glandular kallikrein-1 mRNA relative to that of prostate-specific antigen could be estimated as approx. 10-20%. This study constitutes the first evidence that the human glandular kallikrein-1 gene is expressed at a high level in a human tissue.

Demonstration of DNA binding factors interacting with a fragment of the canine prostate arginine esterase gene promoter.

We have studied, by the gel mobility shift assay, the interaction of DNA binding proteins with a fragment of the proximal promoter (from nucleotides -177 to -47) of the androgen-regulated canine prostate arginine esterase gene. Several shifted bands were obtained using nuclear extracts from various tissues. In the case of the prostate, the intensity of some of the shifted bands was decreased or increased when the extracts were prepared from animals that had been castrated 12 days earlier. Several of the DNA-protein complexes could be assigned to an interaction with part or all of the sequence GGGGGTGGGGG from-124 to -114. We also obtained evidence for the presence of protein(s) interacting with an Sp1 motif present in the same fragment. These results suggest that some ubiquitous factors different from the androgen receptors could be involved in the regulation of the arginine esterase gene.

Nucleotide sequence of the androgen-dependent arginine esterase mRNA of canine prostate.

The nucleotide sequence of canine prostate arginine esterase mRNA was determined using a 400 bp cDNA clone and primer-extended cDNA transcripts for the 5'-coding and noncoding regions. The mRNA contains 864 nucleotides encoding a protein of 236 amino acids preceded by 24 amino acids which constitutes both the signal and the zymogen peptides. The sequence indicates the presence of one potential glycosylation site. A high degree of homology was found between the canine enzyme and other members of the kallikrein family including human prostate specific antigen. The protein appears to be specified by a single gene.

The major androgen-dependent protease in dog prostate belongs to the kallikrein family: confirmation by partial amino acid sequencing.

Canine prostate fluids and seminal plasma contain a major androgen-dependent protein which was identified as a proteolytic enzyme exhibiting an Arg-esterase activity. This protease, as characterized, is shown to be present as a two-chain structure held together by at least one disulfide bridge and composed of approximately 220 amino acids. Amino acid sequence determination of both chains has revealed a clear homology to other known amino acid sequences of serine proteases. Furthermore, the comparison of the presented 58 amino acids of the Arg-esterase with the other sequences revealed a very strong homology (larger than 50%) to members of the kallikrein family. The two chain structure could thus result from autolysis of a single chain enzyme in the 'kallikrein autolysis loop'. Amino acid composition of the canine prostatic enzyme suggests that it is related, but not identical, to pancreatic canine kallikrein.

Search for progesterone receptors in testes from various animal species.

Using [3H]R5020 as binding probe, we have looked for the presence of progesterone receptors in testis cytosol from a few vertebrate species ranging from the turtles to the humans. In addition, we have tested in the rat several experimental conditions potentially susceptible to induce progesterone receptors. With the exception of chicken, tfm patients and possibly the frogs, progesterone receptors could not be conclusively demonstrated in any of the other animal species tested nor could they be induced in the rat. Surprisingly, they were not present in Stanley-Grumbeck pseudohermaphrodite rats. In chicken testes, the levels of progesterone receptor were variable (3--45 fmol per mg prot.) in the adults and high (87 fmol per mg prot.) in one-day-old animals. The presence of progesterone receptor, a marker of estradiol action in many estrogen-target tissues, seems to be parallel to the capacity of chicken testes for sex reversal under the influence of estrogens. In human tfm, the presence of these receptors could also indicate that the gonads are estrogen-sensitive or that they were so during embryonic life although the nature of estrogenic action is not known.

Transcriptional regulation of dog prostate arginine esterase gene by androgens.

These studies were designed to define the molecular events involved in the modulation of dog prostate arginine esterase gene expression following short castration intervals and androgen treatment. Arginine esterase enzymatic activity and protein levels decreased about 50% 24 h after castration. Thereafter, a more progressive decrease was observed, resulting in 2-4-fold lower levels in 12-day castrates than in the intact controls. Total prostatic arginine esterase mRNA levels slowly decreased during the first five days after castration but more abruptly thereafter and were about 150-fold lower in 12-day castrated animals. By contrast, in isolated prostatic nuclei, levels of arginine esterase RNA precursors and mature transcripts rapidly fell following orchiectomy, with a 50-70% decrease 24 h after castration. Nuclear run-on experiments confirmed that the latter effects were the result of decreased arginine esterase gene transcription. All these changes could be at least partially reversed by administration of testosterone cypionate. Furthermore, no striking modifications in the proportion of epithelial/stromal cells in the prostatic tissue were observed following orchiectomy. These results show that castration and androgens exert very rapid effects on the gene expression of arginine esterase, and that the regulation occurs at the transcriptional level.

Androgen regulation of canine prostatic arginine esterase mRNA using cloned cDNA.

Canine prostatic arginine esterase complementary DNA has been cloned in pPBS27, a new cloning vector. The relative abundance of androgen-regulated mRNA in intact dog prostate was reflected by the finding that a high proportion of the clones in the cDNA library hybridized strongly by plaque or colony hybridization with a poly(A)+ RNA probe from intact dog prostate but not with a poly(A)+ RNA probe from castrated dog prostate. One clone carrying a 400 base pairs cDNA insert was selected for further studies. Translation of the hybrid-selected RNA in a cell-free system resulted in the production of a 31 kDa peptide immunoprecipitable by antibodies against arginine esterase. This identification was confirmed by partial sequence analysis of the cDNA revealing an encoding protein with high homology to known kallikreins. Northern blot analysis of poly(A)+ and total RNA showed that arginine esterase mRNA had an approximate size of 1.0 kb which corresponded to a major androgen-regulated RNA species that could be observed after denaturing agarose gel electrophoresis of prostatic poly(A)+ RNA from intact dogs. Dot-blot analysis showed that dogs which had been castrated 3 weeks before had more than 100-fold lower arginine esterase mRNA level than intact dogs or castrated dogs treated with Depo-testosterone.