Benedict J. Campbell

Platelet aggregation and sphingomyelinase D activity of a purified toxin from the venom of Loxosceles reclusa.

A facile and quantitative assay for measuring the activity of sphingomyelinase D in recluse spider venom has been developed using L-alpha-[palmitoyl-1-14C]lysophosphatidylcholine as substrate. This assay avoids the problem of substrate insolubility that occurs when sphingomyelin and other insoluble lipids are used as substrates. This assay has been employed in gel filtration and isoelectric focusing isolation techniques to purify sphingomyelinase D from spider venom. The purified sphingomyelinase exhibits four active enzyme forms in isoelectric focusing with pI values of 8.7, 8.4, 8.2, and 7.8. Each active form when examined in SDS-polyacrylamide gel electrophoresis gave an estimated molecular weight of 32 000. The four active enzyme forms were immunologically cross-reactive with each other as demonstrated with radioimmune assays using an antiserum developed to one of the active forms. Each active form hydrolysed sphingomyelin to release choline and produce N-acylsphingosine phosphate. One of the active enzyme forms was characterized further in dermonecrosis and platelet aggregation measurements. This purified sphingomyelinase D was identified as a poisonous toxin that can developed typical dermonecrotic spider lesions when injected into experimental animals at levels expected to be delivered in a normal bite. Furthermore, the purified toxin acts to aggregate human blood platelets. The toxin-induced platelet aggregation has been related to serotonin release as aggregation occurs, and it has been shown to be inhibited by EDTA over the range of 0.6 yo 3.0 mM EDTA. It is suggested that spider-induced dermonecrosis could result in part from platelet aggregation at and near the site of envenomation.

Red blood cell lysis induced by the venom of the brown recluse spider: The role of sphingomyelinase D

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A2-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.

Hyaluronidase and esterase activities of the venom of the poisonous brown recluse spider

Abstract Venom of Loxosceles reclusa free from impurities was expressed from venom glands collected by microdissection. Polyacrylamide gel electrophoresis of the venom at pH 8.3 demonstrated 7 or 8 major plus 3 or 4 minor components. Upon electrophoresis at pH 4.9 two major components plus 3 or 4 minor components were noted. Monophoretic hyaluronidase prepared by Sephadex gel filtration and electrophoresis at pH 8.3 exhibited optimum activity from pH 5.0 to 6.6. Sodium dodecyl sulfate gel electrophoresis of purified hyaluronidase revealed two components with estimated molecular weights of 33,000 and 63,000. The purified hyaluronidase exhibited activity against chondroitin sulfate, types A, B, and C at approximately 20–30% of that upon hyaluronic acid. The enzyme was inhibited 10–20% by the heavy metal ions, Fe+3 and Cu+2. Rabbit antivenom inhibited the spreading effect of whole venom in vivo and completely inhibited hyaluronidase in vitro. Incorporation of [14C]leucine into the spider venom led to the separation of hyaluronidase from the dermonecrotic activity of the venom. The venom demonstrated activity against carbobenzoxy- l -tyrosine-p-nitrophenyl ester and β-naphthylacetate which was inhibited approximately 65% by 2.5 × 10−3 m levels of EDTA and EGTA but not by 2.5 × 10−4 m o-phenanthroline. The esterase activity resisted concentrations of p-chloromercuribenzoate which totally inactivated papain. The venom appeared devoid of collagenase, dipeptidase, acetylcholinesterase, phosphodiesterase, ribonuclease A, and deoxyribonuclease.

The purification and properties of a particulate renal dipeptidase.

Summary By the use of glycyldehydrophenylalanine as assay substrate the peptidase was isolated from hog-kidney particulate material following solubilization with n -butanol. Purification techniques employed included ammonium sulfate fractionation, diethylaminoethyl-cellulose and carboxymethyl-cellulose chromatography, followed by Sephadex-gel filtration. The purified enzyme exhibited homogeneity in ultra-centrifugation and acrylamide-gel electrophoresis experiments. The molecular weight of the enzyme estimated by the approach-to-equilibrium method was 47 200. The zinc content of the purified enzyme measured colorimetrically and by atomic absorption spectroscopy was 1.36 μg Zn per mg protein indicating that the enzyme contains one atom of zinc per mole enzyme. Peptidase activity could be reduced by removal of zinc using o -phenanthroline dialysis and could be restored by dialysis against zinc-containing buffers. The enzyme catalyzed the hydrolysis of a variety of dipeptides including glycylglycine, L -leucylglycine, L -alanylglycine, and L -seryl-glycine; however, it did not promote the cleavage of L -leucylglycylglycine, L -alanyl-glycylglycine, L -serylglycylglycine, L -leucinamide or D -leucylglycine.

Bull sperm adenylate cyclase: Localization and partial characterization

Abstract Adenylate cyclase activity has been studied in subcellular fractions isolated from bull sperm. The plasma membrane fraction had the highest specific activity for adenylate cyclase and contained approximately 15% of the total activity. However, 30% of the adenylate cyclase activity was present in the soluble fraction and did not sediment after centrifugation at 200,000g for 2 h. Low levels of guanylate cyclase were present in the soluble fraction, but cannot account for the soluble adenylate cyclase activity. These results indicate that bull sperm contain both soluble and membrane-bound forms of adenylate cyclase. Plasma membrane-bound adenylate cyclase had a pH optimum between pH 7.0 and 9.0 with maximum activity near pH 8.5. Variation of ATP concentration gave normal saturation kinetics in the presence of excess Mg2+, with a Km of approximately 4 m m . When Mg2+ concentration is limiting, inhibition is observed at high ATP concentrations. Membrane-bound adenylate cyclase is not stimulated by 10 m m F− and has much higher rates with 25 m m Mn2+ than with Mg2+. At pH 7.6, with 3 m m ATP and 15 m m Mg2+, specific activities of 2–3 and 1.0–1.5 nmol/10 min/mg of protein were obtained for plasma and acrosomal membranes, respectively. Comparable activities with 25 m m Mn2+ are 20–30 and 10–15 nmol/min/mg of protein, respectively. Plasma membrane-bound adenylate cyclase was not activated by thyroxine, luteinizing hormone, or prostaglandins A1, E1, E2, F1α, and F2α.

Thiol-disulfide interchange reactions between serum albumin and disulfides.

Abstract Thiol-disulfide interchange between serum albumins and l -cystine or lysine vasopressin was studied by p- chloromercuribenzoate (PCMB) titration and by incubation with radioactive disulfides followed by separation of reaction products using Sephadex G-15 gel filtration. Good agreement between these two techniques was obtained. Serum albumins were purified by Sephadex G-150 gel filtration, and lysine vasopressin was purified by CM-cellulose ion-exchange chromatography and Sephadex G-25 gel filtration. [3H]Lysine vasopressin was prepared by reduction of the iodinated hormone with 3H and purified by ion-exchange chromatography and gel filtration. The PCMB technique was primarily used to confirm the gel filtration technique. The gel filtration technique was used to measure the kinetic and equilibrium parameters of reactions between l -[14C]cystine and human serum albumin and between [3H]lysine vasopressin and bovine mercaptalbumin. The pH profiles for these reactions were also determined. Kinetic experiments indicated that these thiol-disulfide interchange reactions proceeded at two distinct rates, and equilibrium experiments were measured only with respect to the fast-reacting component. Mg2+ was demonstrated to inhibit the rate and extent of reaction between [3H]lysine vasopressin and bovine serum albumin, while Ca2+ had no significant effect at similar concentrations.

Bioconversion of leukotriene D4 by lung dipeptidase

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.

Uptake of glycine froml-alanylglycine into renal brush border vesicles

SummaryIsolated renal brush border microvilli vesicles were employed to study the uptake of radiolabel froml-Ala · [3H]Gly andd-Ala · [3H]Gly as well as to determine the presence of dipeptidase activity. Microvilli vesicles were prepared from porcine kidney cortex by differential centrifugation through hypotonic Tris buffer containing Mg2+. The microvilli vesicles transiently accumulated radiolabel froml-Ala · [3H]Gly to higher levels than were initially present in the incubation medium (overshoot phenomenon). This accumulation was dependent on the presence of an inward-directed (extravesicular > intravesicular) Na+ gradient and was osmotically sensitive and linear with respect to microvilli protein concentration. Analysis of intravesicular contents revealed that all3H uptake froml-Ala · [3H]Gly appeared as free glycine. Hydrolysis studies demonstrated the rate ofl-ala · [3H]Gly hydrolysis to free alanine and [3H] glycine by the microvilli to be greatly in excess of their rate of radiolabel uptake from this dipeptide. In addition, the uptake profiles and kinetic constants for vesicular uptake of radiolabel froml-Ala · [3H]Gly and free glycine were demonstrated to be identical when measured by double-labeling techniques in the same experiments. These results indicate thatl-Ala · [3H]Gly is hydrolyzed at the external surface of the microvilli with the [3H]glycine released being transported into the vesicles by a Na+ gradient-dependent system identical to that employed for free glycine.Microvilli vesicle uptake of radiolabel fromd-Ala · [3H]Gly exhibited no Na+ dependent “overshoot” effect.d-Ala · [3H]Gly was completely resistant to microvilli-catalyzed hydrolysis.Analysis of the microvilli for renal dipeptidase, an enzyme with hydrolytic activity against a wide range ofl-dipeptides, revealed this enzyme to be enriched in the microvilli vesicles to a degree equivalent to that observed for marker enzymes for renal microvilli.Renal dipeptidase catalyzed hydrolysis ofl-Ala · Gly but notd-Ala · Gly, as was the case with microvilli-catalyzed hydrolysis of these dipeptides.With its location in the renal brush border microvilli and its hydrolytic action againstl-dipeptides, renal dipeptidase may act at the luminal surface of the proximal tubule cell to hydrolyzel-dipeptides present in the glomerular filtrate, with the resultant free amino acids transported across the brush border microvilli by Na+ gradient-dependent processes.

β-lactamase activity of renal dipeptidase against N-formimidoyl-thienamycin

Kinetic analyses of the rate of enzyme — catalyzed hydrolysis of the antibiotic, N-formimidoyl-thienamycin, by purified renal dipeptidase demonstrated that the purified mammalian enzyme is responsible for the breakdown of the β-lactam antibiotic. The effect of z-2-(2,2-dimethyl-cyclopropanecarboxamido)-2-butenoic acid upon the rate of enzyme — catalyzed hydrolysis of N-formimidoylthienamycin and glycyldehydrophenylalanine was characterized as reversible, competitive inhibition for both substrates. Comparable Kis in the range of 1 μM for both substrates indicate that the enzyme acts upon the dipeptide and the antibiotic within the same catalytic site and that dipeptidase and β-lactamase activity are functional properties of the same enzyme. These results demonstrate for the first time β-lactamase activity exhibited by a specific mammalian enzyme.

Specificity and inhibition studies of human renal dipeptidase

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 enzymes 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)