Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.     

Purification and characterization of neutral sphingomyelinase from Helicobacter pylori

Phospholipase activities of human gastric bacterium, Helicobacter pylori, are regarded as the pathogenic factors owing to their actions on epithelial cell membranes. In this study, we purified and characterized neutral sphingomyelinase (N-SMase) from the superficial components of H. pylori strains for the first time. N-SMase was purified 2083-fold with an overall recovery of 37%. The purification steps included acid glycine extraction, ammonium sulfate precipitation, CM-Sepharose, Mono-Q, and Sephadex G-75 column chromatography. Approximate molecular mass for the native N-SMase was around 32 kDa. When N-omega-trinitrophenylaminolauryl sphingomyelin (TNPAL-SM) was used as a substrate, the purified enzyme exhibited a K(m) of 6.7 microM and a V(max) of 15.6 nmol of TNPAL-sphingosine/h/mg of protein at 37 degrees C in 50 mM phosphate-buffered saline, pH 7.4. N-SMase reaches optimal activity at pH 7.4 and has a pI of 7.15. The enzyme activity is magnesium dependent and specifically hydrolyzed sphingomyelin and phosphatidylethanolamine. The enzyme also exhibits hemolytic activity on human erythrocytes. According to Western blot analysis, a rabbit antiserum against purified N-SMase from H. pylori cross-reacted with SMase from Bacillus cereus. Sera from individuals with H. pylori infection but not uninfected ones recognizing the purified N-SMase indicated that it was produced in vivo. In enzyme-linked immunosorbent assays, the purified N-SMase used as an antigen was as effective as crude protein antigens in detecting human antibodies to H. pylori.     

Studies on phospholipases from Streptomyces. III. Purification and properties of Streptomyces hachijoensis phospholipase C.

1. Phospholipase C [EC 3.1.4.3] found in the growth medium of Streptomyces hachijoensis was purified about sixty-fold by dialysis and column chromatography on Sephadex G-50. 2. The active fraction was separated by isoelectric focusing into two fractions, phospholipase C-I (pI 6.0) and phospholipase C-II (pI 5.6). 3. Both purified phospholipases C were homogeneous by immunodiffusion and were not differentiated as regards antigencity. 4. Phospholipase C-I had maximal activity at pH 8.0 and the optimal temperature was 50degree. Phospholipase C-I was stable at 50degrees for 30 min and was stable at neutral pH. 5. The activity of phospholipase C-I was inhibited by high concentrations of various detergents such as Triton X-100, sodium, cholate, SDS and was also inhibited by Ca2+, Ba2+, Al3+, and EDTA, but was stimulated by Mg2+, and ethyl ether. 6. The Km value of phospholipase C-I was 0.9 mM, using phosphatidylcholine as a substrate. 7. By the gel filtration procedure, the molecular weights of phospholipase C-I and -II were both determined to be 18,000. 8. Phosphatidylcholine, phosphatidylinositol, cardiolipin, sphingomyelin, and lysophosphatidylcholine were hydrolyzed by phospholipase C-I, but phosphatidylethanolamine and phosphatidylserine were hydrolyzed with difficulty under the same conditions, Phospholipase C-I also hydrolyzed phosphatidic acid.     

Acid sphingomyelinase from human urine: purification and characterization

Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human urine in the presence of 0.1% Nonidet P-40. The activity could be enriched 23,000-fold by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue Sepharose and DEAE-cellulose. The last purification step yielded an enzyme preparation with a specific activity of about 2.5 mmol sphingomyelin cleaved/h per mg protein and with a yield of about 3%. Purified sphingomyelinase appeared to be homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 70 kDa. In the presence of 0.08% (w/v) sodium taurodeoxycholate the preparation showed phosphodiesterase activity toward sphingomyelin, phosphatidylcholine and phosphatidylglycerol. These activities co-purified during the entire purification procedure, indicating that the acid sphingomyelinase hydrolyses not only sphingomyelin but also the other two phospholipids, phosphatidylcholine and phosphatidylglycerol. Addition of 100 microM tripalmitoylglycerol to the assay system (which contains 100 microM sphingomyelin) instead of detergent, stimulated the reaction about 20-fold compared to an assay which did not contain detergents, thus offering a very sensitive and efficient system for the assay of sphingomyelinase in a system free of detergents. Sphingomyelin degradation was strongly inhibited by phosphatidylinositol 4',5'-bisphosphate, adenosine 3',5'-diphosphate and adenine-9-beta-D-arabinofuranoside 5'-monophosphate (50% inhibition at inhibitor concentrations of 1-5 microM).     

Ceramide and sphingomyelinases in the regulation of stress responses

Ceramide and other sphingolipids are now recognized as novel intracellular signal mediators. One of the important and regulated steps in the metabolism of sphingolipids is the hydrolysis of sphingomyelin into ceramide by sphingomyelinases. Whereas some studies suggest a role for acid sphingomyelinase in cell regulation, several lines of investigation suggest that neutral sphingomyelinase (N-SMase) plays a critical role in stress responses including apoptosis. Recently the advanced purification of neutral membrane-bound magnesium-dependent sphingomyelinase from rat brain was reported on. The specific activity of the purified N-SMase was increased by approximately 3000-fold over the rat brain homogenate, and it is specifically activated by phosphatidylserine. In cells, N-SMase may be coupled to either the redox state and/or glutathione metabolism. The significance of N-SMase and ceramide in stress responses is discussed.     

Studies on phospholipases from Streptomyces. II. Purification and properties of Streptomyces hachijoensis phospholipase D.

1. Phospholipase D [EC 3.1.4.4] from Streptomyces hachijoensis was purified about 570-fold by column chromatography on DEAE-cellulose and Sephadex G-50 followed by isoelectric focusing. 2. The purified preparation was found to be homogeneous both by immunodiffusion and polyacrylamide disc gel electrophoresis. 3. The isoelectric point was found to be around pH 8.6 and the molecular weight was about 16,000. 4. The enzyme has maximal activity at pH 7.5 at 37 degrees. The optimal temperature is around 50 degrees at pH 7.5, using 20 min incubation. 5. The enzyme was stable at 50 degrees for 90 min. At neutral pH, between 6 and 8, the enzyme retained more than 95% of its activity on 24 hr incubation at 25 degrees. However, the enzyme lost 80% of its activity under the same conditions at pH 4.0. 6. The enzyme was stimulated slightly by Ca2+, Mn2+, and Co2+, and significantly by Triton X-100 and ethyl ether. It was inhibited by Sn2+, Fe2+, Fe3+, Al3+, EDTA, sodium dodecyl sulfate, sodium cholate, and cetylpyridinium chloride. 7. This phospholipase D hydrolyzes phosphatidylethanolamine, phosphatidylcholine, cardiolipin, sphingomyelin, phosphatidylserine, and lysophosphatidylcholine, liberating the corresponding bases. 8. The Km value was 4mM, determined with phosphatidylethanolamine as a substrate.     

Effects of gentamicin on sphingomyelinase activity in cultured human renal proximal tubular cells

We have previously shown that cultured human proximal tubular cells (PT) incubated with gentamicin contain numerous "myeloid bodies." This morphological change was accompanied by the storage of phosphatidylcholine and sphingomyelin. In order to delineate the biochemical mechanisms responsible for the accumulation of sphingomyelin in cells incubated with gentamicin, we pursued detailed studies on the activity of sphingomyelinase. Characterization studies on sphingomyelinase revealed that this enzyme has a bimodal pH optima in PT cells. Optimum activity was observed at pH 5.6 (designated as acid sphingomyelinase, A-SMase) and at pH 7.4 (designated as neutral sphingomyelinase, N-SMase). The activity of both the enzymes increased proportionately in control cells as a function of days of incubation. The activity of A-SMase was 16% lower in cells incubated with gentamicin as compared to control. The most striking observation was a gradual decline in the activity of N-SMase in cells incubated with gentamicin. Thus, following 21 days of incubation of cells with 0.3 mM gentamicin, the N-SMase was 2.7-fold lower than control cells. Mg2+ stimulated and Triton X-100 inhibited the activity of N-SMase. Whereas Mg2+ had no effects, Triton X-100 stimulated the activity of the A-SMase in PT cells. Moreover, A-SMase was relatively more heat-resistant than the N-SMase. The Km values for sphingomyelin using A-SMase in control cells and cells incubated with gentamicin were 0.07 X and 0.016 X 10(-7) M, respectively, whereas the Km values for sphingomyelin using N-SMase in control cells and cells incubated with gentamicin were 1.8 X and 1.5 X 10(-7) M, respectively. These findings suggest that gentamicin exerts a competitive inhibition of the A-SMase in PT cells. In contrast, gentamicin exerts a noncompetitive inhibition of the N-SMase in PT cells. Subcellular fractionation studies revealed that A-SMase was exclusively localized in the "lysosome-rich" fraction, whereas most, if not all, the N-SMase was localized in the microsomal fraction and "plasma-membrane"-rich fraction in cultured PT cells. Cells incubated with gentamicin for 21 days contained 25% lower activity of A-SMase associated with the lysosomal fraction as compared to control. In contrast, N-SMase activity in the microsomal and plasma membrane fraction was one-half as compared to control. We conclude that gentamicin-mediated decrease in sphingomyelinase activity may be responsible for the storage of sphingomyelin in cultured human PT cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and characterization of an extracellular lipase from the conidia of Neurospora crassa

A triacylglycerol lipase (EC 3.1.1.3) from the conidia of Neurospora crassa was purified and characterized. The enzyme was purified by Sephadex G-100 column chromatography. Homogeneity was checked by PAGE, and isoelectric focusing gave a single band corresponding to a pI of 6.4. The enzyme had an apparent Mr 54000 +/- 1000 as determined by gel filtration. SDS-PAGE gave a single band of Mr 27000, suggesting the presence of two identical subunits. This lipase preferred triglycerides with C16- and C18-fatty acyl chains. It cleaved only the primary groups of triglycerides. The lipase also exhibited a marked preference for substrates containing endogenously occurring fatty acids and so may prove useful in detailed studies on the physiological relevance of fatty acyl specificity of lipases. The enzyme was not affected by detergents, or thiol-binding agents. Modification of free amino groups caused 90% inhibition, suggesting a role of these groups in the maintenance of lipase activity.     

Phospho-N-acetylmuramoyl-pentapeptide-transferase of Escherichia coli K12. Properties of the membrane-bound and the extracted and partially purified enzyme.

Phospho-N-acetylmuramoyl-pentapeptide-transferase (UDP-N-acetyl-muramoyl-L-alanyl-D-gamma-glutamyl-L-lysyl-D-alanyl-D-alanine:undecaprenoid-alcohol-phosphate-phospho-N-acetylmuramoyl-pentapeptide-transferase, EC 2.7.8.13) was solubilized by repeated freezing and thawing of crude envelopes of Escherichia coli K12. The solubilized enzyme was partially purified by gel filtration and ion-exchange chromatography. This preparation contained small amounts of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol but no endogenous lipid substrate, C55-isoprenyl phosphate, could be detected. Some catalytic properties (exchange reaction) of the solubilized enzyme were compared to those of membrane-bound transferase. The transfer activity of the partially purified transferase was restored by the addition of an aqueous lipid dispersion. All the transferase activity was found to become incorporated into the liposomes. Preincubation of the transferase preparation with phospholipase A2 or D strongly reduce both exchange and transfer activity. This suggests that phospholipids sensitive to phospholipases are necessary for the enzymatic reaction. Different effects of some neutral detergents on the exchange activity were reported.     

Sphingomyelinase of chicken erythrocyte membranes.

Most of the chicken erythrocyte's sphingomyelin is hydrolyzed when the chicken red blood cells are incubated in hypotonie solution at 37 °C.Addition of detergents, such as Triton X-100 or Na-cholate, is essential for hydrolysis of external [³H ]sphingomyelin by the erythrocyte membranes.Pure plasma membranes show relatively high sphingomyelinase activity while no activity could be detected in the soluble fraction of the cells. Mg²⁺ and Mn²⁺ activate the enzyme while Ca²⁺ and EDTA strongly inhibit its activity. The optimal pH of the membrane-bound sphingomyelinase lies between pH 7.0–9.0. The detergents Triton X-100 and Na-cholate, at concentrations of 0.5% ($\text{w}\text{v}$) solubilize the membrane-bound enzyme. Human erythrocytes fail to exhibit sphingomyelinase activity.The correlation between the sphingomyelinase activity and its localization is discussed.     

Purification and characterization of a soluble phospholipase A2 from guinea pig lung

Guinea pig lung cytosolic phospholipase A2 was purified to near homogeneity by chromatography on a phosphocellulose column, followed by Q-Sepharose, S-Sepharose, gel filtration chromatography and reverse-phase HPLC. The purified enzyme exhibited an apparent molecular weight of 16,700 by SDS-polyacrylamide gel electrophoresis. Active enzyme eluted from the gel at an apparent molecular weight of 16,700. The purified enzyme exhibited a pH optimum of 9.0 and was calcium-dependent. Guinea pig lung phospholipase A2 hydrolyzed phosphatidylcholine and phosphatidylethanolamine equally well. Substrates containing unsaturated fatty acids in the sn-2 position were hydrolyzed preferentially to those containing saturated fatty acids. Anionic detergents stimulated enzyme activity while nonionic detergents inhibited the enzyme. Disulfide reducing agents dithiothreitol, glutathione and 2-mercaptoethanol modestly stimulated enzyme activity. The sulfhydryl aklylating agent n-ethylmaleimide had no effect on enzyme activity and only high concentrations of p-hydroxymercuribenzoic acid inhibited enzyme activity. The histidine modifying agent, bromophenacyl bromide did not inhibit guinea pig lung phospholipase A2 under conditions in which Crotalus adamanteus phospholipase A2 was inhibited 80%. Manoalide inhibited guinea pig lung phospholipase A2 in a concentration-dependent manner (IC50 = 2 microM). Antibodies prepared against porcine pancreatic phospholipase A2 specifically immunoprecipitated guinea pig lung phospholipase A2 suggesting that the major phospholipase A2 in guinea pig lung cytosol is immunologically related to pancreatic phospholipase A2 in agreement with the biochemical properties of the enzyme.     

Purification and Characterization of Neutral and Acid Sphingomyelinases from Rat Brain

Neutral and acid sphingomyelinases were copurified from a rat brain P2 fraction by extraction with 1% Triton X-100, followed by (NH4)2SO4 fractionation, acetone powdering, extraction with 1% Triton X-100, (NH4)2SO4 fractionation, Sepharose CL-6B chromatography, and chromatofocusing. The neutral sphingomyelinase was eluted with buffer containing 0.4 M NaCl after the acid sphingomyelinase had been eluted with Polybuffer at pH 5.3. The neutral sphingomyelinase exhibited specific activity of 48,300 nmol/h/mg of protein, with 254-fold purification; the corresponding value for acid sphingomyelinase was 25,300 nmol/h/mg protein, with 668-fold purification from the P2 fraction. The purified neutral sphingomyelinase had no acid sphingomyelinase activity, and vice versa. The properties of the two enzymes were examined. A single band corresponding to a molecular weight of 67,000 was obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for both enzymes. The pI was estimated to be 5.5 for both on isoelectric focusing. The native molecular weights of the neutral and acid sphingomyelinases were found to be 434,000 and 284,000, respectively, on gel filtration with Sepharose CL-6B. The single band obtained for each enzyme on SDS-PAGE was identified as an antigen with antibody raised against the purified neutral sphingomyelinase. Their amino acid compositions were very similar. The neutral and acid sphingomyelinases probably consist of common polypeptides and are immunologically cross-reactive.     

Human thymidine kinase. Purification and properties of the cytosolic enzyme of placenta

Cytosolic thymidine kinase (EC 2.7.1.21) has been purified 5200-fold to apparent homogeneity from normal human placenta. The purification includes sequential affinity chromatography on blue-Sepharose and a thymidine column. The molecular weight of the enzyme determined by gel filtration and sucrose density ultracentrifugation is 92,000. The subunit molecular weight is 44,000, suggesting that the enzyme is a dimer in its native state. With isoelectric focusing, placental thymidine kinase demonstrated a single form with an isoelectric point of 9.1. The final purified enzyme preparation exhibits no immunological cross-reactivity with human mitochondrial thymidine kinase.     

Neutral sphingomyelinase: Localization in rat liver nuclei and involvement in regeneration/proliferation

We have studied the localization of neutral sphingomyelinase (N-SMase) in rat liver nuclei. The levels of neutral sphingomyelinase in regenerating liver nuclei were also assessed.We found that rat liver nuclei contain a sphingomyelinase having a pH optima of 7.2 and a kDa of 92. In intact nuclei, neutral sphingomyelinase was associated predominantly with the nuclear envelope. In regenerating/proliferating rat liver (during DNA synthesis), neutral sphingomyelinase was translocated from the nuclear envelope to the nuclear matrix. The levels of sphingomyelin in whole nuclei decreased in reverse proportion to an increase in the levels of neutral sphingomyelinase. By contrast, there was a corresponding increase in the levels of ceramide and sphingosine during cell regeneration/proliferation. Thus, endogenous nuclear neutral sphingomyelinase may play a role in the regulation of sphingomyelin levels and in relevant signal transduction reactions involving cell regeneration/proliferation. The potential significance of ceramide generation may be aimed at programmed cell death to allow the regeneration of liver mediated via target proteins such as, ceramide activated protein kinases/phospholipases or other unknown mechanisms.Abbreviations N-SMase neutral sphingomyelinase - A-SMase acid sphingomyelinase     

Human urine deoxyribonuclease II (DNase II) isoenzymes: a novel immunoaffinity purification, biochemical multiplicity, genetic heterogeneity and broad distribution among tissues and body fluids.

Deoxyribonuclease II (DNase II) was purified from the urine of a 48-year-old male (a single individual) using a column chromatography series, including concanavalin A-agarose and an immunoaffinity column utilizing anti-human spleen DNase II antibody, and was then characterized. Based on the catalytic properties of the purified enzyme, we have devised a technique of isoelectric focusing by thin-layer polyacrylamide gel electrophoresis (IEF-PAGE) combined with a specific zymogram method, for investigating the possible molecular heterogeneity of human DNase II. DNase II in urine as well as the purified form was found to exist in multiple forms with different pI values separable by IEF-PAGE within a pH range of 5-7. Since sialidase treatment of the urine sample induced simplification of the isoenzyme patterns with diminishment of anodal bands, it was clear that the multiplicity of the enzyme was in part due to differences in the sialic acid content. On screening of DNase II isoenzyme patterns in urine samples from more than 200 Japanese individuals, only the common isoenzyme pattern was observed and no electrophoretic variations were detected. However, genetic studies of urinary enzyme activity and comparative studies on the activity in urine, semen and leukocytes from the same individuals suggest that the enzyme activity level of DNase II may be under genetic control. The enzyme was widely distributed in human tissues and showed high activities in secretory body fluids such as breast milk, saliva, semen and urine, and leukocyte lysates.     

Niemann-Pick disease, Type C: evidence for the deficiency of an activating factor stimulating sphingomyelin and glucocerebroside degradation

1) Qualitative lipid analyses by thin-layer chromatography of 4 Niemann-Pick type C spleens confirmed sphingomyelin accumulation together with increase in the amount of glucocerebroside. 2) In the presence of crude sodium taurocholate as detergent, sphingomyelin degradation rates of normal and Niemann-Pick type C-cultured fibroblasts were fairly close under standard conditions at pH 5.0. In the absence of sodium taurocholate, sphingomyelinase activity was optimal at pH 4.0. Sphingomyelinase activities of fibroblasts from two patients with Niemann-Pick disease type C measured without detergent, were about 30% of that of controls. 3) Extracts from Gaucher spleen heated to 90 degrees C and devoid of sphingomyelinase activity stimulated at the optimal pH of 4.0 sphingomyelin degradation by cultured normal fibroblasts (2--4-fold, Niemann-Pick type C fibroblasts (5--9-fold), whereas similarly treated extracts from Niemann-Pick type C spleen showed no stimulation of sphingomyelin catabolism. Heated extracts from normal human spleen exhibited a smaller stimulation than that shown by Gaucher spleen. This stimulating effect could not be observed in fibroblasts from patients suffering from Niemann-Pick type B (sphingomyelinase defect). 4) Heat-treated extracts of Gaucher spleen were fractionated by ion exchange chromatography, isoelectric focusing and gel filtration. The active fractions obtained by these procedures stimulated sphingomyelin as well as glucocerebroside degradation and were absent from the corresponding Niemann-Pick type C preparations. Enriched activator preparations of Gaucher spleen stimulated sphingomyelinase activity of Niemann-Pick type C fibroblasts 25--38-fold and that of normal cells 3-fold. 5) The activating factor had an isoelectric point of 4.0 and an apparent molecular weight, as estimated by gel filtration, of 25000. Treatment with pronase E abolished its activity.     

Phosphatidylcholine/sphingomyelin metabolism crosstalk inside the nucleus

It is known that phospholipids represent a minor component of chromatin. It has been highlighted recently that these lipids are metabolized directly inside the nucleus, thanks to the presence of enzymes related to their metabolism, such as neutral sphingomyelinase, sphingomyelin synthase, reverse sphingomyelin synthase and phosphatidylcholine-specific phospholipase C. The chromatin enzymatic activities change during cell proliferation, differentiation and/or apoptosis, independently from the enzyme activities present in nuclear membrane, microsomes or cell membranes. This present study aimed to investigate crosstalk in lipid metabolism in nuclear membrane and chromatin isolated from rat liver in vitro and in vivo. The effect of neutral sphingomyelinase activity on phosphatidylcholine-specific phospholipase C and sphingomyelin synthase, which enrich the intranuclear diacylglycerol pool, and the effect of phosphatidylcholine-specific phospholipase C activity on neutral sphingomyelinase and reverse sphingomyelin synthase, which enrich the intranuclear ceramide pool, was investigated. The results show that in chromatin, there exists a phosphatidylcholine/sphingomyelin metabolism crosstalk which regulates the intranuclear ceramide/diacylglycerol pool. The enzyme activities were inhibited by D609, which demonstrated the specificity of this crosstalk. Chromatin lipid metabolism is activated in vivo during cell proliferation, indicating that it could play a role in cell function. The possible mechanism of crosstalk is discussed here, with consideration to recent advances in the field.     

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.     

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-α-[palmitoyl-1-¹⁴C]lysophosphatidylcholine as substrate. This assay avoids the problem of substrate insolubility that occurs when sphingomyelin and other lipids are used as subtrates. 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 develop the typical dermonecrotic spider lesion 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 to 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.