Kinetic investigation of the hydrolysis of aminoacyl p-nitroanilides by dipeptidyl peptidase IV from human and pig kidney

Dipeptidyl peptidase IV (dipeptidyl-peptide hydrolase, EC 3.4.14.5), an enzyme that participates in the catabolism of bradykinin and Substance P as well as the post-translational processing of various other peptides, has been purified from human and pig kidney. The assay reaction involved the cleavage of p-nitroaniline (pNA) from various dipeptidyl p-nitroanilides. The specific activities of the human and pig enzyme (with Gly-Pro-pNA at pH 7.6) were 49.2 and 45.8, respectively. The dependence of initial reaction velocity on substrate concentration was determined for a variety of dipeptidyl p-nitroanilides over the concentration range 0.05 to 2.0 mM. Most of the substrates tested produced significant non-hyperbolic behavior for the function v vs. S at concentrations above 0.5 mM. As to differences between the two enzymes, the pig enzyme exhibited featureless (i.e., hyperbolic) behavior with Glu-Pro-pNA concentrations as high as 2.0 mM, whereas the human enzyme produced significant non-hyperbolic behavior for the function v vs. S, beginning at S = 0.4 mM. Thus, the human and pig dipeptidyl peptidases IV are kinetically distinct enzyme forms.     

The expression of free oligosaccharides in human seminal plasma

Human seminal plasma is a complex mixture of proteins, glycoproteins, peptides, glycopeptides, and prostaglandins secreted by organs of the male reproductive tract. The components of this fluid have been implicated in the suppression of immune response, agonistic effects on sperm-egg binding, and promotion of successful implantation of the human embryo. Fractionation followed by biophysical analyses revealed that free oligosaccharides constitute a major component of the total glycoconjugates within seminal plasma. Significant findings of our analyses include the following: (i) the concentration of free oligosaccharides is 0.3-0.4 mg/ml; (ii) mono- and difucosylated forms of the disaccharide lactose are major components; (iii) many of the remaining oligosaccharides are also rich in fucose and carry Lewis(x) and/or Lewis(y) epitopes; (iv) a subset of the oligosaccharides express the reducing end sequence (GlcNAcbeta1-3/4Glc) not reported in human milk oligosaccharides; (v) oligosaccharides in seminal plasma exclusively express type 2 (Galbeta1-4GlcNAc) but not the type 1 sequences (Galbeta1-3GlcNAc) that predominate in human milk glycans; and (vi) the structural diversity of seminal plasma oligosaccharides is far less than human milk oligosaccharides. The agonistic effect of both fucose and fucosylated glycoconjugates on human sperm-egg binding in vitro suggests that fucosylated oligosaccharides may also promote fertilization in the female reproductive tract.     

Lucigenin chemiluminescence in human seminal plasma

Seminal plasma protects spermatozoa from the detrimental effects of reactive oxygen species such as hydrogen peroxide. We investigated the lucigenin-dependent chemiluminescence in cell-free seminal plasma from andrological patients. The seminal plasma was separated from cells by centrifugation. In all seminal plasmas studied lucigenin-dependent chemiluminescence (LCL) was detected. The LCL showed a strong pH-dependence. The signal was stable if samples were stored at +4°C for up to 4 days or up to 8 days at -80°C. Filtration of the samples (0.45 and 0.22 μm pore size) did not lower their luminescence. The addition of superoxide dismutase (SOD) and ascorbic acid oxidase (AAO) lowered LCL nearly to baseline values while trolox and desferal showed moderate effect, whereas allopurinol had no effect. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radicals in seminal plasma. Physiological concentrations of ascorbic acid yielded SOD-inhibitable lucigenin-chemiluminescence. The nitroblue-tetrazolium assay showed that ascorbic acid in buffer solution produced formazan. Superoxide-anion radicals were not detected in seminal plasma by the spin-trap DEPMPO due to their low steady state concentration. It is concluded that in seminal plasma ascorbate reacts with molecular oxygen yielding ascorbyl radicals and superoxide anion. If lucigenin is added to seminal plasma, reducing substances present, such as ascorbate, reduce lucigenin to the corresponding radical; this radical reacts with molecular oxygen and also forms O₂^-2. So LCL in human seminal plasma results from the autoxidation of ascorbate and the oxidation of the reduced lucigenin. While the physiological relevance of the former mechanism is unknown, the latter is an artifact.     

Lucigenin chemiluminescence in human seminal plasma

Seminal plasma protects spermatozoa from the detrimental effects of reactive oxygen species such as hydrogen peroxide. We investigated the lucigenin-dependent chemiluminescence in cell-free seminal plasma from andrological patients. The seminal plasma was separated from cells by centrifugation. In all seminal plasmas studied lucigenin-dependent chemiluminescence (LCL) was detected. The LCL showed a strong pH-dependence. The signal was stable if samples were stored at +4°C for up to 4 days or up to 8 days at -80°C. Filtration of the samples (0.45 and 0.22 μm pore size) did not lower their luminescence. The addition of superoxide dismutase (SOD) and ascorbic acid oxidase (AAO) lowered LCL nearly to baseline values while trolox and desferal showed moderate effect, whereas allopurinol had no effect. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radicals in seminal plasma. Physiological concentrations of ascorbic acid yielded SOD-inhibitable lucigenin-chemiluminescence. The nitroblue-tetrazolium assay showed that ascorbic acid in buffer solution produced formazan. Superoxide-anion radicals were not detected in seminal plasma by the spin-trap DEPMPO due to their low steady state concentration. It is concluded that in seminal plasma ascorbate reacts with molecular oxygen yielding ascorbyl radicals and superoxide anion. If lucigenin is added to seminal plasma, reducing substances present, such as ascorbate, reduce lucigenin to the corresponding radical; this radical reacts with molecular oxygen and also forms O₂^-2.. So LCL in human seminal plasma results from the autoxidation of ascorbate and the oxidation of the reduced lucigenin. While the physiological relevance of the former mechanism is unknown, the latter is an artifact.     

Fibronectin fragments in human seminal plasma

The study has revealed the presence of fibronectin (FN) fragments and a lack of intact FN in 72 seminal plasma samples. The FN fragmentation was examined by immunoblotting with a monoclonal antibody specific to the central cellular FN domain and was confirmed with a monoclonal antibody directed to the C-terminal domain of FN. Nine FN fragments between 60 and 200 kDa and five fragments of 60-150 kDa were identified in seminal plasma samples of normozoospermic and of terato-, oligoterato-, and oligoasthenoterato-spermic groups, respectively. The relative amounts of the 60, 90 and 100 kDa FN fragments were 2-3 times higher in seminal plasmas with abnormal semen characteristics than in the normozoospermic group. The results suggest that seminal plasma FN fragments may contribute to fertilization and the analysis of FN fragmentation may have a diagnostic value in andrological investigations.     

Purification and characterization of human seminal plasma aminopeptidase

A 50.4-fold purification of aminopeptidase is achieved by alcohol precipitation, DEAE-cellulose, CM-cellulose and finally Sephadex G-200 chromatography. On polyacrylamide gel electrophoresis of the purified enzyme after molecular sieving on Sephadex G-200, only one band was obtained, suggesting that the enzyme preparation was obtained almost homogeneous by three steps of column chromatography. Aminopeptidase showed highest activity at pH 7.0, using a buffer system, of 70 mM Na-phosphate. The enzyme was found to be active at 40 degrees C, even at 60 degrees C (80% activity), suggesting that the human seminal plasma enzyme is fairly thermostable. Amongst the various aminoacyl derivatives evaluated as substrates in the present study, L-alanine beta-naphthylamide hydrochloride was found to have the highest rate of hydrolysis. Ovalbumin showed effective cleavage in comparison to that of other natural substrates. The Km value for the purified seminal plasma aminopeptidase towards L-alanine beta-naphthylamide hydrochloride was 4 x 10(-4) M. Hg+2 showed highest inhibitory effect than other metal ions tested in the present study. Concentration causing 50% inhibition of the enzyme (I50) by Hg2+ was 4.7 x 10(-6) M. Inhibition by EDTA at 1 mM concentration in the incubation system was higher than by EGTA and sodium azide, suggesting that the enzyme contains a metallo group at the active site. A 50% inhibition of the enzyme by EDTA was obtained at 5.11 x 10(-3) M. The Ackerman and Potter plot for EDTA inhibition suggests that EDTA is a reversible inhibitor of seminal plasma aminopeptidase. A single molecular form of aminopeptidase was found to be present in human seminal plasma as shown by polyacrylamide activity gel electrophoresis.     

Tissue kallikrein of human seminal plasma is secreted by the prostate gland

Samples of human seminal plasma were subjected to gel filtration, and the eluted fractions were analysed for their contents of tissue kallikrein-like antigen, arginine esterase activity and kininogenase activity. Two peaks of tissue kallikrein-like antigen were detected with apparent molecular masses of about 72 and 48 kDa. As judged by the criteria of molecular mass, immunoreactivity, kininogenase activity, identification of the released kinin as kallidin and inhibition studies, a genuine tissue kallikrein has been identified in the 48-kDa peak. In addition, this peak contains one or more species of immunoreactive tissue kallikrein which differ in molecular mass and enzymatic activities. The 72-kDa peak probably represents the complex of tissue kallikrein with alpha 1-proteinase inhibitor rather than a true high molecular mass tissue kallikrein. The prostate gland was identified as the site of origin of the tissue kallikrein in the seminal fluid by indirect methods and by demonstrating immunoreactive tissue kallikrein in prostatic tissue and secretion.     

The identity of the acid and alkaline phosphatases of human seminal plasma.

Examination of human seminal plasma showed that there was no p-nitrophenylphosphatase activity maximum at alkaline pH values. A constant ratio of enzyme activities in 8 split ejaculates, identical temperature dependence and copurification through a three-step purification procedure led us to conclude that the alkaline phosphatase in human semen is identical to acid phosphatase.     

The immunochemical identification of beta-globulin in human seminal plasma]

Using affinity chromatography there was isolated a protein from human seminal plasma, which was named beta-globulin of seminal plasma (beta-GSP). It was demonstrated, that beta-GSP possesses heterogeneity, consists of two subunits with the M. W. about 19 and 15 kD, it has electrophoretic mobility of b1-globulin and possesses affinity to immobilized 17-beta-estradiol. Relatively high concentration of beta-GSP was found in seminal plasma. Besides, this protein was determined in small amounts in saliva. One can suppose, that beta-GSP is a secretory protein.