Kenneth L. Polakoski

Amidino-substituted aromatic heterocycles as probes of the specificity pocket of trypsin-like proteases

Abstract A number of amidino-substituted heterocycles were synthesized and examined for their inhibitory effect against arginine-directed esteroproteases, including human and bovine thrombin (EC 3.4.21.5), human factor Xa and plasmin (EC 3.4.21.7), bovine trypsin (EC 3.4.21.4), porcine pancreatic Kallikrein (EC 3.4.21.8), and boar acrosin (EC 3.4.21.10). Inhibition was competitive and reversible in all cases, and the K i values were taken to reflect binding conditions in the specificity pockets of the enzymes. A remarkably potent blocking agent was found in 5-amidinoindole. Compared to the established inhibitor benzamidine, it proved one to two orders of magnitude more effective against the majority of the proteases, the exceptions being trypsin and plasmin. Improved hydrophobic interaction, fortuitous hydrogen bonding, and charge transfer complex formation were considered in accounting for the considerable activity of the compound. Placement of the amidino moiety in the 6-position rather than in the 5-position on the indole ring resulted in a striking loss of inhibitory potency against human and bovine thrombin, factor Xa, and Kallikrein, yet slightly improved the affinity with respect to acrosin. Tightness of binding of the inhibitors to the enzymes was pH dependent and was significantly reduced below pH 7.0.

Benzamidine as an inhibitor of proacrosin activation in bull sperm

Epididymal and ejaculated sperm contain a zymogen form of acrosin (acrosomal proteinase, EC 3.4.21.10) which is converted to active enzyme prior to fertilization. Benzamidine at concentrations greater than 10 mM has been shown to inhibit the conversion of proacrosin to acrosin. Based on this inhibition, a procedure was developed for extracting and quantitating the proacrosin content of bull sperm. Sperm were isolated from semen and washed by centrifugation through 1.3 M sucrose and the outer acrosomal membrane removed by homogenization. When 25 mM benzamidine was added to the semen and wash solutions, 98% or more of the acrosin activity in the sperm homogenate was present as proacrosin. Proacrosin can be extracted from the sperm homogenate by dialysis at pH 3, which solubilized the proenzyme and removed benzamidine. Benzamidine has been useful in isolating proacrosin and provides a new method for studying the activation of proacrosin in intact sperm. Neutralization of sperm extracts, after removal of benzamidine, resulted in rapid activation of proacrosin with a pH optimum of 8.5, and activation was complete within 15 min over a pH range of 7.0 to 9.5. Rapid activation also occurred during the washing of sperm in the absence of benzamidine, and this activation correlated with a swelling of the acrosomal membrane. This rapid activation appears to result from a small amount of acrosin activity consistently present in the sperm extract. These results indicate an autocatalytic conversion of proacrosin to acrosin and suggest that disruption of the acrosomal membrane may trigger this activation.

The release of 15-hydroxyeicosatetraenoic acid by human placental trophoblast is increased in preeclampsia

OBJECTIVE We tested the hypothesis that trophoblast produces 15-hydroxyeicosatetraenoic acid, and its level is elevated in trophoblast from preeclamptic women compared with normal. We also used selective enzymatic inhibitors to determine the relative contributions of 15-lipoxygenase and the two isozymes of prostaglandin H synthase to 15-hydroxyeicosatetraenoic acid levels. STUDY DESIGN Cytotrophoblasts isolated from placentas of normal or preeclamptic women were cultured in the presence or absence of enzyme inhibitors. Media levels of 15-hydroxyeicosatetraenoic acid were measured by radioimmunoassay. RESULTS When compared with normal pregnancies, cytotrophoblasts from preeclamptic pregnancies released up to fivefold higher levels of 15-hydroxyeicosatetraenoic acid. Aspirin, an inhibitor of both the prostaglandin H synthase-1 and prostaglandin H synthase-2 isozymes, and nordihydroguaiaretic acid, a selective inhibitor of lipoxygenases, both significantly inhibited 15-hydroxyeicosatetraenoic acid production. In contrast, the selective prostaglandin H synthase-2 inhibitor NS-398 had no effect on 15-hydroxyeicosatetraenoic acid release in the absence of aspirin, but NS-398 reduced 15-hydroxyeicosatetraenoic acid levels in normal trophoblast pretreated with aspirin. CONCLUSIONS The data indicate that 15-hydroxyeicosatetraenoic acid is produced in trophoblasts and its release by cytotrophoblasts is higher in preeclamptic pregnancies compared with normal controls. Both lipoxygenase and prostaglandin H synthase contribute to 15-hydroxyeicosatetraenoic acid production, and aspirin reduces 15-hydroxyeicosatetraenoic acid secretion. We suggest 15-hydroxyeicosatetraenoic acid plays a role in the oxidation of lipoproteins and the endothelial damage characteristic of preeclampsia.

Cocaine inhibits alanine uptake by human placental microvillous membrane vesicles

OBJECTIVE The aim of this study was to determine the effects of cocaine on alanine uptake by human placental microvillous membrane vesicles and to characterize cocaine binding to the microvillous membrane. STUDY DESIGN Microvillous vesicles were isolated from the placentas of 10 human pregnancies with no history of cocaine use. The binding of tritiated cocaine to microvillous vesicle membrane and uptake of tritiated cocaine and tritiated alanine were determined with the use of filtration assays. Scatchard analyses were used to characterize cocaine binding. Sodium-independent and sodium-dependent uptake of tritiated alanine was measured in the presence and absence of (-)cocaine and its stereoisomer (+)cocaine. Uptakes were compared with the use of Student t tests. RESULTS Specific tritiated cocaine binding accounted for approximately 96% of total binding at a single-component high-affinity site in the microvillous membrane. The mediated sodium-dependent component of alanine uptake was significantly (p < 0.01) reduced in the presence of (-)cocaine but was unaffected by (+)cocaine. CONCLUSION Cocaine may contribute to fetal growth restriction by interfering with the normal activity of placental amino acid transporters necessary to maintain the nutrient gradients associated with normal fetal growth.

The rapid purification and partial characterization of human sperm proacrosin using an automated fast protein liquid chromatography (FPLC) system

A rapid and efficient procedure was developed for obtaining highly purified human proacrosin. Ejaculated spermatozoa were washed via centrifugation through 1 M sucrose containing 50 mM benzamidine and acid-extracted in the presence of benzamidine. The solubilized material was dialyzed then lyophilized. The sample was resuspended in 8 M guanidine hydrochloride in acetic acid (0.5 M) pH 2.5 and then subjected to gel permeation chromatography with an automated fast protein liquid chromatography system utilizing two Pharmacia Superose 12 columns set in tandem that were equilibrated in the same buffer. The proacrosin eluted as an individual peak that was well separated from another proteinase zymogen referred to as sperminogen. The proacrosin preparation was determined to be highly purified when observed on silver-stained SDS-polyacrylamide gels as well as on gelatin-SDS-polyacrylamide gels. The proacrosin appeared as a doublet (Mr = 55,000 and 53,000) on both of these systems. The autoconversion of proacrosin to acrosin at pH 8 resulted in a typical sigmoidal autoactivation curve. Following protein staining of SDS-polyacrylamide gels, it was shown that upon activation of purified proacrosin preparations the 55,000 and 53,000 molecular weight proteins were initially degraded to a 49,000 form and then to several lower molecular weight forms (Mr = 40,000-34,000). Similar findings with regard to proteolytic digestion were observed following gelatin-SDS-polyacrylamide zymography except that an increase with time in proteinase intensity between 58,000 and 53,000 was also observed. Cobalt and calcium were found to be potent inhibitors of the conversion of proacrosin into acrosin, while sodium resulted in much less inhibition of this process. Calcium was found to markedly enhance the proteolytic activity of human acrosin, while it had no observable influence on the acrosin hydrolysis of benzoylarginine ethyl ester. Thus, the described purification procedure resulted in a highly purified proacrosin preparation in sufficient yields to allow for its partial characterization.

An apparent high molecular weight form of boar proacrosin resulting from the presence of a protein that binds to proacrosin

Abstract Gel chromatography at pH 3.0 demonstrated that a partially purified extract of ejaculated boar spermatozoa apparently contained two proacrosins with approximate molecular weights of 88.000 and 55.000. Disc gel electrophoretic experiments indicated that the high molecular weight form was actually a complex between the low molecular weight form and a protein with a molecular weight of 29,000. The fact that this complex did not dissociate at pH 3.0 indicates the need for caution in interpretation of data obtained by acidic chromatography of proacrosin preparations.

The effects of cocaine on neutral amino acid uptake by human placental basal membrane vesicles

OBJECTIVE Prior studies have demonstrated that cocaine binds to human placental microvillous membrane vesicles at a single high-affinity site and that both 10 and 500 nmol/L cocaine inhibit sodium-dependent alanine uptake. The purpose of this study was to characterize cocaine binding to human placental basal plasma membrane and to determine the effects of cocaine on basal vesicle uptake of alanine and leucine. STUDY DESIGN Basal vesicles were isolated from the placentas of uncomplicated human pregnancies with no history of cocaine use. The binding of tritiated cocaine to basal vesicle membrane and the uptakes of tritiated cocaine, alanine, and leucine were determined with filtration assays. Alanine and leucine uptakes were measured in the presence and absence of sodium and 10 and 500 nmol/L cocaine. Cocaine binding was characterized with Scatchard analyses, and uptakes were compared by means of Student t tests. RESULTS Tritiated cocaine bound to basal membrane at two separate high-affinity sites. Sodium-dependent alanine uptake was significantly inhibited only by 500 nmol/L cocaine. Sodium-independent amino acid uptake was unaffected by cocaine. CONCLUSION Cocaine may interfere with fetal growth by impairing the activity of sodium-dependent amino acid transporters in both the microvillous and basal membrane. These membranes may be differentially sensitive to the effects of cocaine on such transporters.

Proacrosin binding protein: immuno-comparative studies in boar, bovine, hamster, human and ram

The cross reactivities of acidic extracts from boar, bovine, hamster, human and ram spermatozoa to a polyclonal antibody of the boar proacrosin binding protein has been investigated. The pH 3.0 extracts of the washed spermatozoa from each species were subjected to Western blot analysis using a polyclonal antibody developed against the 28 kDa boar proacrosin binding protein. The boar sperm extracts had a major 28 kD and a minor 29 kDa proacrosin binding protein. A similar protein of 29 kDa was present in the bovine and ram samples, while the hamster had bands at 22 and 25 kDa. Extracts of human sperm yielded a diffuse but distinct area of cross reactivity of about 25-30 kDa. These results demonstrate that proacrosin binding proteins are present in sperm of different mammalian species and they have similar molecular weights.

Identification of the in vivo sperm proacrosin into acrosin conversion sequence

Abstract 1. 1. Proacrosin and acrosin were quantitated in boar sperm which were incubated in ligated segments of gilt uteri and in buffer. 2. 2. Most of the proacrosin (70–80%) was converted into acrosin in sperm incubated in vivo for 120 min while less than 3% of the proacrosin was converted into acrosin in the control sperm. 3. 3. Electrophoretic comparison of the enzymatic activities in the sperm extracts to homogeneous m(inχ)-acrosin and m μ -acrosin suggested that the in vivo conversion sequence was: proacrosin → min(χ)-acrosin → m β acrosin.

Aspirin induces increased expression of both prostaglandin H synthase-1 and prostaglandin H synthase-2 in cultured human placental trophoblast☆☆☆★★★

OBJECTIVE We tested the hypothesis that aspirin affects trophoblast like other epithelial cells do, by inhibiting prostanoid production, inducing prostaglandin H synthase-2 expression, and enhancing secretion of 15-hydroxyeicosatetraenoic acid. STUDY DESIGN Cytotrophoblast from placentas (n = 15) of uncomplicated singleton pregnancies were cultured in medium 199 for 4 to 72 hours in the presence or absence of aspirin. RESULTS Aspirin (10(-4) M) inhibited (p < 0.01) average trophoblast prostaglandin E2 release by 60% and thromboxane B2 by 86%. Western immunoblotting showed the prostaglandin H synthase-1 was constitutively expressed in cytotrophoblast, and aspirin treatment caused a twofold increase in prostaglandin H synthase-1 expression. Prostaglandin H synthase-2 was also constitutively expressed in untreated cytotrophoblast but at lower levels than prostaglandin H synthase-1. Aspirin enhanced prostaglandin H synthase-2 expression in trophoblast cultures, but prostaglandin H synthase-2 contributed a range of only 10% to 33% (n = 4) of the total cellular prostaglandin H synthase protein pool even after aspirin induction. The increased prostaglandin H synthase expression depended on both transcription and translation because actinomycin D and cycloheximide each inhibited the increased prostaglandin H synthase protein expression after aspirin treatment. The aspirin induction of prostaglandin H synthase was accompanied by decreased release of 15-hydroxyeicosatetraenoic acid. CONCLUSIONS Trophoblast differs from other cells studied because aspirin enhances expression of both prostaglandin H synthase-1 and prostaglandin H synthase-2 isozymes while decreasing, instead of increasing, the secretion of 15-hydroxyeicosatetraenoic acid. The aspirin effects on prostaglandin H synthase synthesis and 15-hydroxyeicosatetraenoic acid release in trophoblast suggest that the mechanisms of action for aspirin in the prophylaxis of preeclampsia may be more diverse than simply altering platelet thromboxane production.