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Sheep lung dipeptidase was released from a lung membrane preparation by digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. The total enzyme activity released into the supernatant was 4- to 5-fold greater than that measured in the intact membrane prior to solubilization. The release of the peptidase from the membrane by this treatment is typical of proteins anchored to the lipid bilayer by a covalent attachment of phosphatidylinositol via a C-terminal glycolipid extension. The solubilized lung peptidase was further purified by ammonium sulfate fractionation followed by affinity chromatography and high-pressure liquid chromatography. A linear relationship between log molecular weight and elution volume for proteins of known molecular weight was established using a Toya Soda TSK 3000 high-pressure liquid chromatography column, and the molecular weight of the lung dipeptidase was estimated at 105,000. The peptidase activity against glycyldehydrophenylalanine of the purified enzyme co-chromatographed in high-pressure liquid chromatography with the activity that converted leukotriene D4 to leukotriene E4. In kinetic studies using leukotriene D4 as substrate, the relationship between the rate of hydrolysis and enzyme concentration was shown to be linear over the range 20 ng to 98 ng enzyme. Values of Km and Vmax for the dipeptidase using leukotriene D4 as substrate were 43 +/- 6 microM and 11,200 +/- 400 nmol/min per mg, respectively. Inhibition of the conversion of leukotriene D4 to leukotriene E4 was observed with a series of inhibitory agents. Cilastatin, bestatin and chloracetyldehydrophenylalanine were all effective at the micromolar level with cilastatin proving to be the most effective inhibitor. Dithiothreitol was effective within the millimolar range.

Purified human renal dipeptidase was shown to exhibit no detectable activity against substrates that are characteristic for other known mammalian peptidases. The enzymic activities that were assayed were: aminopeptidase A, aminopeptidase B, aminopeptidase M, aminopeptidase P, and tripeptidase. A quantitative assay for renal dipeptidase was developed which measures the rate of release of glycine from glycylpeptides by pre-column derivatization of the amino acid with phenylisothiocyanate followed by high-performance liquid chromatography. The ratio of Vmax/Km for a series of dipeptides was used as an index of the enzyme's preference for substrates. According to the data obtained, the enzyme prefers that a bulky, hydrophobic group of the dipeptide be located at the N-terminal position. This suggests that the substrate-binding site of the enzyme may provide a hydrophobic pocket to accommodate the hydrophobic moiety at the N-terminus of the dipeptide. The unsaturated dipeptide substrate, glycyldehydrophenylalanine, was employed in spectrophotometric assays to provide kinetic analyses of enzymic inhibition. The inhibitory effect of dithiothreitol was immediate, and the kinetic data indicated reversible, competitive inhibition. These results suggest that the inhibitor competes with substrate for a coordination site of zinc within the active site of the enzyme. The reaction of renal dipeptidase with the transition-state peptide analog, bestatin, was time dependent, and velocity measurements were made after the inhibitor had been incubated with the enzyme until constant rates were observed. These steady-state rate measurements, made following preincubation of enzyme with inhibitor, were employed to show that bestatin caused apparent non-competitive inhibition of the enzyme. The inhibitory effect of the beta-lactam inhibitor, cilastatin, upon the oligomeric dipeptidase was shown to be competitive. Graphical analysis of this inhibition indicated that the subunits of the enzyme react independently during enzymic catalysis and that the catalytic event is not influenced by cooperativity between sites on the subunits. The conversion of leukotriene D4 to leukotriene E4 in the presence of human renal dipeptidase was demonstrated by HPLC procedures. This bioconversion reaction was quantitated by derivatizing the glycine produced by cleavage of the cysteinylglycine bond and isolating this derivative as a function of time. The relationship between the purified enzyme concentration and enzyme activity against leukotriene D4 was shown to be linear over the enzyme concentration range of 1 ng through 69 ng in this assay.(ABSTRACT TRUNCATED AT 400 WORDS)

The objective of the present study was to determine the temporal relationships among luteal adenylate cyclase activity, luteal phosphodiesterase activity, luteal progesterone concentration and plasma progesterone concentration during prostaglandin F2 alpha (PGF2 alpha)-induced luteolysis in ewes. Corpora lutea (CL) were removed from cycling ewes on d 9 (d 0 = first day of estrus) at 0, 2, 4, 6, 12 and 24 h (seven to eight ewes/group) after PGF2 alpha administration (im). Jugular blood samples were collected at the time of enucleation of CL and analyzed for progesterone. Plasma and luteal progesterone concentrations were decreased (P less than .05) by 4 and 12 h after PGF2 alpha injection, respectively. Basal adenylate cyclase, luteinizing hormone (LH)-activated adenylate cyclase, guanylylimidodiphosphate [Gpp(NH)p]-activated adenylate cyclase and LH plus Gpp(NH)p-activated adenylate cyclase activities were decreased (P less than .05) by 2 h after PGF2 alpha injection. The decrease in adenylate cyclase activity paralleled the decrease in plasma progesterone concentration over time. Luteinizing hormone stimulated (P less than .05) adenylate cyclase activity relative to basal activity at 0, 2, 12 and 24 h post-PGF2 alpha; whereas, Gpp(NH)p stimulated (P less than .01) adenylate cyclase activity relative to basal activity at each time point. In contrast to the decrease in adenylate cyclase activity, phosphodiesterase activity was increased (P less than .05) at 2 and 4 h post-PGF2 alpha. These results suggest that a decrease in adenylate cyclase activity coupled with an increase in phosphodiesterase activity may decrease the intracellular adenosine 3',5' cyclic monophosphate (cAMP) concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Ethanol and a variety of solvents are known to activate basal and Gpp(NH)p- and hormone-stimulated adenylate cyclase. We report here that ethanol and other solvents inhibit the activation of adenylate cyclase by forskolin. In the presence of 10 microM forskolin, 2% ethanol gives about 20% inhibition and 5% ethanol gives 40% inhibition of enzyme activity. Analysis of ethanol inhibition at several forskolin concentrations suggests that inhibition is competitive versus forskolin. Thus the effect of ethanol is greater at low forskolin concentrations and minimal at high concentrations. In addition to ethanol, inhibition of forskolin activation was observed with acetone, n-butanol, t-butanol, dimethyl formamide, dioxane, methanol and n-propanol. Dimethyl sulfoxide was inhibitory only at high concentrations (10%). Since some solvent is needed to prepare forskolin solutions and to maintain solubility at higher concentrations, the inhibitory effects reported here are an important consideration in studies employing forskolin activation. To minimize solvent inhibition we recommend that dimethyl sulfoxide be used to prepare forskolin solutions. At concentrations of 5% and less, dimethyl sulfoxide gives little if any inhibition of forskolin activation and causes only small increases in basal activity.

Forskolin, a potent activator of adenylate cyclase, has been proposed to activate this enzyme by a direct interaction with the catalytic subunit. To test this hypothesis, we examined the effects of forskolin on sperm cyclic AMP content and sperm adenylate cyclase activity. Forskolin or cholera toxin did not increase cyclic AMP content in either bull or boar sperm, whereas the inhibitors of phosphodiesterase, caffeine and methylisobutylxanthine, significantly increased sperm cyclic AMP content. Forskolin, NaF, and guanylimidodiphosphate did not activate the adenylate cyclase of either sperm membranes or cytosol. When homogenates of rat, guinea pig, or bull testes were centrifuged at 100,000 X g, the supernatant was found to contain a forskolin-stimulated adenylate cyclase. Further centrifugation of this 100,000 X g supernatant fraction at 250,000 X g for 3 hr quantitatively sedimented the forskolin-sensitive enzyme activity. We conclude that forskolin does not activate either the cytosolic or membrane-bound adenylate cyclase of mammalian sperm.

Adenylate cyclase and cyclic AMP (cAMP) phosphodiesterase have been identified and partially characterized in bacteroids of Bradyrhizobium japonicum 3I1b-143. Adenylate cyclase activity was found in the bacteroid membrane fraction, whereas cAMP phosphodiesterase activity was located in both the membrane and the cytosol. In contrast to other microorganisms, B. japonicum adenylate cyclase remained firmly bound to the membrane during treatment with detergents. Adenylate cyclase was activated four- to fivefold by 0.01% sodium dodecyl sulfate (SDS), whereas other detergents gave only slight activation. SDS had no effect on the membrane-bound cAMP phosphodiesterase but strongly inhibited the soluble enzyme, indicating that the two enzymes are different. All three enzymes were characterized by their kinetic constants, pH optima, and divalent metal ion requirements. With increasing nodule age, adenylate cyclase activity increased, the membrane-bound cAMP phosphodiesterase decreased, and the soluble cAMP phosphodiesterase remained largely unchanged. These results suggest that cAMP plays a role in symbiosis.

A procedure is described for subcellular fractionation of bull sperm which allows the isolation of outer acrosomal membrane without the use of detergent. After washing to remove seminal plasma contaminants, the acrosomal membrane is removed by homogenization and separated on a two-step sucrose gradient. The isolated membranes have been characterized by light and electron microscopy and enzyme analysis. While the acrosomal enzymes hyaluronidase and acrosin are bound to the isolated membranes, they represent only a small percentage of the total activity and therefore do not provide reliable marker enzymes for this fraction. Subcellular fractionation of sperm also yields information on the solubility of acrosomal enzymes. Two types of acrosomal enzymes have been identified on the basis of their distribution in gradient fractions. Both alpha-fucosidase and beta-N-acetyl glucosaminidase are concentrated in the soluble fraction of the gradient. In contrast, over 70% of the acrosin and hyaluronidase activity remains associated with the sperm pellet. These differences in solubility of these enzymes may reflect differences in their function in fertilization.

Human renal dipeptidase has been concentrated from kidneys by homogenization, 1-butanol solubilization, and (NH4)2SO4 fractionation. Final purification was achieved by high-pressure liquid chromatography followed by affinity chromatography. The enzyme appeared to be homogeneous by polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 220,000 by analytical high-pressure liquid chromatography. The molecular weight of human urinary dipeptidase was estimated by agarose gel filtration to be 218,000. Dissociation of human renal dipeptidase in sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced a single polypeptide (Mr 59,000). These results suggest that the native enzyme contains four subunits of Mr 59,000. Analysis of the peptidase for zinc content gave 3.9 g atoms of zinc/mol of enzyme which supports the suggestion of a 4-subunit structure. Carbohydrate analyses of the purified human dipeptidase demonstrated that it was not a glycoprotein, a characteristic that distinguishes it from porcine and rat renal dipeptidase. beta-Lactamase activity of the purified human enzyme was demonstrated by measuring its activity against the two beta-lactam antibiotics, imipenem and SCH 29482. Kinetic analyses indicated that both antibiotics undergo enzyme-catalyzed hydrolysis at rates which could produce inactivation of the antibiotics within the human kidney. The beta-lactamase inhibitor, cilastatin, demonstrated reversible competitive inhibition of the peptidase-catalyzed hydrolysis of both antibiotics with the same Ki of 0.7 microM.

Glycerate-3-kinase (EC 2.7.1.31) from spinach leaves shows absolute specificity for D-glycerate as phosphate acceptor, yielding 3-phosphoglycerate as a product. ATP complexed with either Mg2+ or Mn2+ is the preferred phosphate donor. The enzyme has Km (D-glycerate) = 0.25 mM, Km (Mg-ATP) = 0.21 mM, Vmax = 300 mumol min-1 mg protein-1, and a turnover number = 12,000 X min-1. The equilibrium constant for the reaction is approximately 300 at pH 7.8. Pyrophosphate, 3-phosphoglycerate and ribulose 1,5-bisphosphate are the strongest inhibitors among the phosphorylated and nonphosphorylated metabolites tested; however, their regulatory role in vivo is questioned. Substrate kinetics, as well as product and analog inhibition data, are consistent with a sequential random mechanism. The distinct characteristic of the glycerate kinase-catalyzed reaction is the formation of a dead-end complex between the enzyme, D-glycerate, and 3-phosphoglycerate.

Changes and interrelationships among the cellular mechanisms that may regulate luteal progesterone synthesis during the bovine estrous cycle were studied. Corpora lutea (CL) were enucleated from 30 cows via a transvaginal incision on d 4, 7, 10, 13, 16 and 19 following estrus (estrus = d 0). Mean corpus luteum weight, and luteal progesterone and plasma progesterone concentrations increased (P less than .05) from d 4 to 7 or 10, and declined following luteal regression (d 19). Unoccupied LH receptor (UOR) concentrations increased (P less than .05) more than fourfold from d 4 (38 fmol/mg protein) to d 16 (173 fmol/mg protein) before declining (P less than .05) by d 19 (30 fmol/mg protein). The dissociation constant for UOR concentrations increased (P less than .05) during the estrous cycle. Concentrations of occupied LH receptors (OR) were less (P less than .05) on d 7 than other days; however, the total number of OR/CL increased fourfold from d 4 (47 fmol/CL) to d 10 (221 fmol/CL) and remained similar thereafter. Basal adenylate cyclase, LH-activated adenylate cyclase, and Gpp(NH)p-activated adenylate cyclase activities were greatest (P less than .05) on d 7 to 16 as compared with d 4 and 19. Luteinizing hormone stimulated (P less than .05) adenylate cyclase activity relative to basal activity on d 7, 10, 13, and 16; whereas, Gpp(NH)p stimulated (P less than .05) adenylate cyclase activity relative to basal activity at each period. Phosphodiesterase was 46% greater on d 19 as compared with d 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of luteal adenylate cyclase has been measured as a function of forskolin concentration using 15 mM Mg2+ and 2, 5 and 15 mM Mn2+ as divalent cation. Analysis of these data demonstrates that activation is best described by a mixture of two binding sites which differ in their affinity for forskolin. The apparent dissociation constants of about 0.5 microM and 15 microM showed little change with type or concentration of metal ion. The overall increase in activation over basal activity (delta Vmax) was highest with Mg2+ at 1.17 nmol/min/mg protein, decreased about 10% with 2 mM Mn2+ to 1.02 pmol/min/mg and about 20% with 5 and 15 mM Mn2+ (approximately 0.9 nmol/min/mg). The major effect of increasing Mn2+ ion is to alter the amount of activation attributable to each binding site. At 2 mM Mn2+ 74% of the activation is associated with the low affinity site (Kd approximately 15 microM) compared to 80% with Mg2+. In contrast, only 41% of the activation is associated with the low affinity site using 5 and 15 mM Mn2+. In a separate experiment 5 and 15 mM Mn2+ were found to cause uncoupling of Gpp (NH) p activation. These results suggest that the different affinities for forskolin are related to differences in the interaction between the regulatory and catalytic subunit and indicate that forskolin activation will be a useful tool in studying the regulation of adenylate cyclase.

Acetoacetyl-CoA thiolase of Bradyrhizobium japonicum bacteroids has been purified greater than 130-fold. The enzyme has a molecular weight of 180,000 +/- 15,000 and consists of four identical subunits of 44,000 +/- 2,000. The enzyme was specific for acetoacetyl-CoA; ketodecanoyl-CoA did not serve as a substrate. Catalysis proceeds via a ping-pong mechanism. Iodoacetamide effectively inhibited the enzyme but acetoacetyl-CoA provided considerable protection against this compound. Magnesium was found to inhibit both the thiolysis reaction and the condensation reaction. Acetoacetyl-CoA thiolysis activity was not affected by potassium, ammonium, or several organic acids but was found to be inhibited by NADH. The inhibition by NADH may have an effect during the decline of the symbiosis.

The activity of bull sperm hyaluronidase (hyaluronate 3-glycanohydrolase, EC 3.2.1.36) is increased by the inclusion of polycations in the assay mixture. At pH 3.8, bovine serum albumin and histone give the greatest stimulation, while protamine sulfate, spermine, spermidine and hyamine 2389 stimulate to a lesser extent. Enzyme activity increases with serum albumin concentration to a nearly constant, high level at serum albumin concentrations greater than 1 mg/ml. Other stimulatory compounds show a similar concentration dependence except that inhibition of enzyme activity occurs at high concentrations of stimulator. The degree of stimulation depends on the pH, sample concentration and substrate concentration. Enzyme preparations with a low protein content give the greatest stimulation, while preparations with a high protein content show little stimulation. The concentration of serum albumin required for maximum stimulation increases with increased hyaluronic acid concentration. The results suggest that the stimulation of sperm hyaluronidase is nonspecific and results from an interaction of the polycation with hyaluronic acid. Since protein in the enzyme preparation substitutes for exogenous stimulator to a varying degree, serum albumin should be included in the assay mixture for sperm and testicular hyaluronidase to assure measurement of maximum enzyme activity.

Proacrosin has been isolated from human ejaculated spermatozoa and constitutes at least 90% of the total acrosin present. The total amount of acrosin in milli-international units per million spermatozoa was between 4.4 and 8.9, with a mean of 6.5. This is the first report of the demonstration of proacrosin and the quantitation of total acrosin in human spermatozoa.