Inactivation of aminated horseradish peroxidase by interaction with S-Sepharose.

Horseradish peroxidase which had been aminated by periodate oxidation and reductive amination was purified by cation-exchange chromatography on S-Sepharose. Instead of the expected single peak of aminated enzyme, two distinct peaks of protein were eluted from the column. Evaluation of the protein in each of the two distributions showed that peak number 1 had spectral properties and specific activity similar to those of native enzyme. Distribution number 2 had a threefold reduction in the extinction in the Soret region at 404 nm and was completely devoid of enzymatic activity. This inactivation was caused by a specific interaction between the aminated peroxidase and the S-Sepharose matrix, resulting in a displacement of the heme prosthetic group out of its native orientation. The inactivation of the aminated peroxidase was found to be dependent on time, pH, and the support matrix itself. These results indicate that the S-Sepharose and Mono-S resins are not interchangeable, despite the chemical similarities of the two resins.

An internal clock reaction used in a one-step enzyme immunochromatographic assay of theophylline in whole blood.

We describe the development and performance of a second-generation enzyme immunochromatography method for visually quantifying theophylline in whole blood without the use of instrumentation. We have developed the novel concept of an internal chemical clock reaction to combine the capillary-migration and color-generation protocol of the two-step immunochromatographic assay into a single-step, simultaneous protocol. The two assay components are (a) chromatographic paper to which glucose oxidase (EC 1.1.3.4) and monoclonal antibody to theophylline have been immobilized, and (b) an enzyme reagent consisting of glucose, dicarboxidine, ascorbate, and theophylline-labeled horseradish peroxidase (EC 1.11.1.7). The ascorbate acts as an internal clock by inhibiting premature color formation until the ascorbate has been completely consumed in the peroxidase-mediated reaction. Color is then generated rapidly, producing a clearly visible front on the paper. Performance evaluations of the 20-min one-step assay show very good precision, analytical recovery, specificity, and accuracy. This simplified protocol is reliable and convenient for therapeutic drug monitoring in the physician's office.

Vasectomy and vasovasostomy have no effect on seminal plasma zinc concentrations.

Mean zinc concentrations were determined for human seminal plasma obtained from 110 nonvasectomized men (139 micrograms. per ml.), 43 recently vasectomized men (144 micrograms. per ml.), 25 long-term vasectomized men (139 micrograms. per ml.) and 25 men who had undergone vasovasostomy (129 micrograms. per ml.). The results indicate that there is no significant short-term or long-term effect of these surgical procedures on prostatic secretory function as measured by seminal plasma zinc concentration.

PIP: Zinc levels in seminal plasma were compared in vasectomized men 3-6 wks after surgery, men before and after vasovasostomy, and men consulting for infertility. Semen denatured with perchloric acid, frozen, thawed, and centrifuged, was analyzed by atomic absorption spectroscopy. There were no differences in mean zinc concentrations: 110 infertility patients averaged 139 mcg/ml; 43 vasectomized men averaged 144; 25 vasectomized 2 to 19 yrs ago averaged 139; these men after vasovasostomy averaged 129 mcg/ml. There was no correlation between zinc content in seminal plasma and sperm count. The results indicate that there is no significant effect of vasectomy or of vasovasostomy on secretory function of the prostate.

Mitogenic factors in prostatic tissue and expressed prostatic secretion.

Expressed prostatic secretions and extracts of benign prostatic hyperplasia tissue contain a polypeptide growth factor(s) that stimulates the uptake of tritium-labeled thymidine by cultured 3T3 fibroblasts. Mitogenic activity was present in expressed prostatic secretions and extracts of benign prostatic hyperplasia tissue. The apparent molecular weights of the mitogenic fractions were estimated to be 300,000, 150,000 and 60,000 daltons for prostatic tissue extracts, and 30,000 daltons for expressed prostatic secretions. Bioassays yielded a mean of 27 units of mitogenic activity per mg. protein in expressed prostatic secretions obtained from men with normal and enlarged prostate glands. There was no difference in bioassayable mitogenic activity in the expressed prostatic secretions from normal and benign prostatic hyperplasia samples but gel filtration studies revealed a high molecular weight component present only in samples from men with prostatic enlargement. A dialyzable low molecular weight inhibitor of fibroblast growth was found in the prostatic tissues and expressed prostatic secretions. We report the characterization studies and discuss the possible roles of growth factors in the pathogenesis of benign prostatic hyperplasia.

Evidence against a zinc binding peptide in pilocarpine-stimulated canine prostatic secretions.

Pilocarpine-stimulated canine prostatic secretions were subjected to gel filtration chromatography over Sephadex G-100 and Bio Gel P-4. Free zinc and the zinc in pilocarpine-stimulated canine prostatic secretions were observed to elute in the same fractions from the Sephadex G-100 column. In addition, Sephadex G-100 chromatography could not resolve free zinc from the small peptide bacitracin. However, a column packed with Bio Gel P-4 could resolve free zinc and bacitracin. When pilocarpine-stimulated canine prostatic secretions were chromatographed over the Bio Gel P-4 column, the major portion of the zinc eluted in the same fractions as did free zinc. No zinc was observed in the fractions where the small peptide bacitracin was found to elute. These results indicate that contrary to a previous report, zinc in pilocarpine-stimulated canine prostatic secretions was not bound to an eight amino acid peptide, but, rather, behaved chromatographically like free zinc.

Proacrosin conversion inhibitor. Purification and initial characterization of a boar sperm protein which prevents the conversion of proacrosin into acrosin.

A proacrosin conversion inhibitor present in boar spermatozoa has been purified and initially characterized. Purification methods included sequential acid extractions of washed spermatozoa at pH 4.0, pH 3.5, and pH 2.5 followed by successive gel filtrations of the pH 2.5 sperm extract supernatant over Sephadex G-75 and G-50. The resulting 8.8-fold purified materials were judged to be homogeneous by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, had an estimated molecular weight of 12,800, and a constant specific activity of 65 units/mg. Treatment with the proteinases acrosin, trypsin, or chymotrypsin destroyed the highly purified proacrosin conversion inhibitor, indicating that it is a protein. Additional properties of the inhibitor included stability to long periods of storage at pH 3.0 and 4 degrees C, stability to boiling and lyophilization, and an absolute requirement for divalent cations to maintain activity. The highly purified proacrosin conversion inhibitor does not inhibit acrosin. Therefore, it apparently acts to prevent proacrosin conversion by selectively inhibiting the zymogen's self-catalyzed conversion mechanism.

Stimulation of proteolytic activity of boar sperm acrosin by divalent metal ions.

The in-vitro proteolytic activity of boar sperm acrosin was stimulated by a series of monovalent and divalent metal ions. Equivalent concentrations of monovalent cations resulted in nearly identical increases in proteolytic activity, probably related to the increased ionic strength of the incubation medium. However, at concentrations of monovalent cations that resulted in a 2- to 3-fold stimulation of proteolysis of Azocoll, divalent metal ions caused a 24-(magnesium) to 46-(calcium) fold increase in proteolytic activity. We suggest that the divalent metal ion binds to acrosin and thus increases the proteolytic activity of acrosin to an extent greater than that due to the increased ionic strength of the incubation medium.

Boar acrosin. Association of an endogenous membrane proteinase with phospholipid membranes.

Acrosin, an enzyme required for fertilization, is an endogenous proteinase associated with membranes of the sperm acrosome. Liposomes were utilized as a model system to evaluate the mode of association between highly purified boar acrosin and phospholipid bilayer membranes. Acrosin was observed to bind to liposomes containing acidic phospholipids such as phosphatidylglycerol, cardiolipin, and phosphatidylserine. There was no apparent binding of acrosin to liposomes which consisted of nonacidic phospholipids, thus indicating that an ionic phospholipid constituent was required for binding. Increased ionic strength caused a significant reduction in acrosin-liposome association with an inverse effect on enzyme-membrane dissociation. Acrosin-liposome association and dissociation were similarly effected by increasing concentrations of divalent cations at constant ionic strength, and by reductions in pH. Equilibrium binding experiments, with anionic liposomes, suggest the presence of either multiple classes of independent binding sites, or apparent negative cooperativity, with a range in the apparent affinity constant (Ka) from 2 x 10(11) M-1 at low acrosin concentrations to 3 x 10(8) M-1 at high acrosin concentrations. The membrane-associated enzyme was accessible to concanavalin A, and to high molecular weight substrates, demonstrating that a portion of the acrosin molecule is exposed at the membrane surface. In addition, acrosin binding to liposomes had no apparent effect of hydrolysis of soluble protein or synthetic substrates. The results demonstrate that acrosin-liposome binding is due in part to electrostatic charge interactions and indicate that the enzyme has properties of an extrinsic membrane protein.

Selective binding of zinc ions to heparin rather than to other glycosaminoglycans.

The relative binding affinity of Zn2+ to several glycosaminoglycans was determined by gel-filtration chromatography. Binding was observed only between Zn2+ and heparin. No binding was observed between Zn2+ and chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate of hyaluronic acid. All of the glycosaminoglycans contained carboxy groups, but only heparin bound Zn2+. This observation suggests that, contrary to a previously proposed hypothesis, simple electrostatic interactions between the negatively charged carboxy groups of the glycosaminoglycans and the positively charged Zn2+ cannot explain the observed binding.

Antibacterial substances in prostatic fluid.

We have identified the "prostatic antibacterial factor," responsible for the antibacterial activity of normal prostatic fluid as free zinc. It appears as if the bactericidal activity of the EPS is related to the amount of zinc present in the fluid and may play a role in the natural resistance of the male urinary tract to infection. In addition, the determination of the zinc content of the expressed prostatic secretion may be a useful test in diagnosing patients with chronic bacterial prostatitis or those who are likely to be susceptible to prostatitis. The factors responsible for the marked decrease of zinc in the prostatic fluid of patients with bacterial prostatitis and methods of altering the zinc level in the fluid as a possible means of eradicating chronic bacterial prostatitis or increasing the resistance of the patient to the disease are important questions requiring further study.

Polyamine inhibition of the conversion of human proacrosin to acrosin.

The conversion of human proacrosin to acrosin was inhibited by polyamines. The order of effectiveness was spermine greater than spermidine greater than cadaverine greater than putrescine greater than 1,3,-diaminopropane. These results are similar to those obtained for the conversion of boar proacrosin to acrosin. Unlike the effects on boar acrosin, however, polyamines did not affect the esterolytic activity of human acrosin but had a slight stimulatory effect on the proteolytic activity of human acrosin.

Fertilization: a uterine glycosaminoglycan stimulates the conversion of sperm proacrosin to acrosin.

Acrosin is a proteinase required for mammalian fertilization, and in freshly ejaculated spermatozoa exists as an inactive zymogen, proacrosin. A factor prsent in uterine flushings of gilts stimulates the conversion of highly pruified boar proacrosin to acrosin. Characterization of this factor indicates that its active component is a glycosaminoglycan.