Niemann Pick disease: presence of the magnesium-dependent sphingomyelinase in brain of the infantile form of the disease.

Abstract— Brain of a 14-month-old patient with the infantile form of Niemann Pick disease was found to be practically devoid of sphingomyelinase activity when assayed at pH 5. When assayed at pH 7.4, in the presence of magnesium ions considerable hydrolysis of sphingomyelin was obtained by brain preparations. The rate of hydrolis of a homogenate of grey matter was about 0.3 μmol. mg protein^?1. H^?1, corresponding to about 15 μmol of sphingomyelin hydrolysed perg brain in 1 h. The possibility is suggested that the presence of the extra-lysosomal, magnesium dependent sphingomyelinase in brain of Niemann Pick patients may be responsible, in part, for the lesser accumulation of sphingomyelin in this tissue relative to other organs, such as liver or spleen.     

Effects of metal ions on sphingomyelinase activity of Bacillus cereus

Some divalent metal ions were examined for their effects on sphingomyelinase activity of Bacillus cereus. The enzyme activity toward mixed micelles of sphingomyelin and Triton X-100 proved to be stimulated by Co2+ and Mn2+, as well as by Mg2+. Km's for Co2+ and Mn2+ were 7.4 and 1.7 microM, respectively, being smaller than the Km for Mg2+ (38 microM). Sr2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 1 mM. Zn2+ completely abolished the enzyme activity at concentrations above 0.5 mM. The concentration of Zn2+ causing 50% inhibition of the enzyme activity was 2.5 microM. Inhibition by Zn2+ was not restored by increasing concentrations of Mg2+ when the concentration of Zn2+ was above 10 microM. Ba2+ was without effect. When sphingomyelinase was incubated with unsealed ghosts of bovine erythrocytes at 37 degrees C, the enzyme was significantly adsorbed onto the membrane in the presence of Mn2+, Co2+, Sr2+ or Ba2+. Incubation with intact or Pronase-treated erythrocytes caused enzyme adsorption only in the presence of Mn2+. In the course of incubation, the enzyme was first adsorbed on the membranes of intact bovine erythrocytes in the presence of Mn2+; then sphingomyelin breakdown proceeded with ensuing desorption of adsorbed enzyme. Hot-cold hemolysis occurred in parallel with sphingomyelin breakdown. In this case, the hydrolysis of membranous sphingomyelin as well as the initial enzyme adsorption took place in the following order: unsealed ghosts greater than Pronase-treated erythrocytes greater than intact erythrocytes.     

Comparative Hydrolysis of Sphingomyelin and 2-N-(Hexadecanoyl)-Amino-4-Nitrophenyl-Phosphorylcholine by Normal Human Brain Homogenate at Acid and Neutral pH

Hydrolysis of sphingomyelin and 2-N-(hexade-canoyl)-amino-4-nitrophenyl-phosphorylcholine (HDA-PC), a synthetic analogue of sphingomyelin, by acid and Mg-dependent neutral sphingomyelinases was tested with a homogenate of normal human brain cortex. Results demonstrated quite different substrate specificities for these enzymes. Acid sphingomyelinase, which is neither activated by MgCl₂ nor inhibited by EDTA, hydrolyzed both substrates (the hydrolysis ratio of HDA-PC to sphingomyelin is ?2). In contrast, Mg-dependent neutral sphingomyelinase, which is inhibited by EDTA and reactivated by MgCl₂, hydrolyzed only sphingomyelin (the hydrolysis ratio of HDA-PC to sphingomyelin is ?0-0.05). This synthetic substrate seems to be useful for selective determination of acid sphingomyelinase and for avoiding interference of Mg-dependent neutral sphingomyelinase.     

Sphingomyelinase activities in neuronal cell cultures.

Human and rat brains have been previously demonstrated to contain three sphinomyelinases, one lysosomal with a pH optimum of 5.0, one with a pH optimum of 7.4 and a dependence on magnesium and another with a pH optimum of 7.0 and no divalent cation requirement. Using brain cell cultures and clonal cell lines of both neuronal and glial origin the activities of the pH 5.0 and pH 7.4 (magnesium-dependent) sphingomyelinase were examined. Sphingomyelinase activity measured at pH 5.0 was found in all the cell lines tested including G26, C6, N18 (differentiated and undifferentiated), mouse “L” cells, human skin fibroblasts, fetal mouse brain surface cultures and fetal mouse brain reaggregate cultures. However, pH 7.4 (magnesium-dependent) sphingomyelinase activity was found only in the N18 cell lines and the reaggregate cultures suggesting the probable localization of this activity in neurons. Although the pH 7.4 sphingomyelinase activity was found in the fetal mouse brain used for the surface cultures this activity was rapidly lost. This enzyme may play an important role in neuronal development and maturation.     

Enzymatic hydrolysis of sphingomyelin liposomes by normal tissues and tissues from patients with Niemann-Pick disease

Liposomes of [3H]sphingomyelin are readily hydrolyzed by extracts of human spleen, liver, cultured skin fibroblasts and purified placental sphingomyelinase in the absence of detergents. The pH optimum for hydrolysis by liver and spleen extracts was 6.5-7.0 while the fibroblast activity showed an optimum at pH 4.0-4.3. However, the pH optimum for purified placental sphingomyelinase in the presence of Triton X-100 (pH 5.0) is only slightly different from that displayed with liposomes (pH 5.3). The data clearly show that hydrolysis of liposomal sphingomyelin by sphingomyelinase is affected by the composition and purity of the enzyme source.     

A new Zn2+-stimulated sphingomyelinase in fetal bovine serum

Fetal bovine serum contains a Zn2+-dependent sphingomyelinase with optimal activity at pH 5.5 in vitro. Activity could be demonstrated with a liposomal sphingomyelin substrate suspension but was stimulated up to 15-fold by Triton X-100. Under a variety of conditions tested, phosphatidylcholine, lysophosphatidylcholine, glycerophosphocholine, and p-nitrophenyl phosphate were not substrates for this activity. Several inhibitors of serum alkaline and acid phosphatases had no effect on the activity. The enzyme resembles the acid lysosomal sphingomyelinase in pH optimum and inhibition by AMP but differs in inhibition by EDTA, stimulation by Zn2+, and heat lability at 55 degrees C. It resembles the neutral, Mg2+-stimulated enzyme in inhibition by EDTA and heat lability but differs in metal ion requirement and pH optima. Of the sera tested, activity was highest in fetal bovine serum, with fetal bovine greater than newborn bovine greater than horse greater than human; more than 95% of the activity is in the lipoprotein-free infranatant of serum (d greater than 1.21). This activity appears to be a hitherto undescribed sphingomyelinase. Its biological functions are not known but may subserve a special role in sphingomyelin catabolism in the circulation, in blood vessel walls, or in the tissue(s) of origin.     

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)     

Purification to homogeneity of human placental acid sphingomyelinase

Acid sphingomyelinase was purified to homogeneity from human placenta in the presence of a dialyzable detergent, n-octyl-beta-D-glucopyranoside. The major steps in the procedure included column chromatographies with Con A-Sepharose, sphingosylphosphorylcholine-Sepharose 4B, hexyl-agarose, and Mono P. The purified enzyme with pI 7.4 had a specific activity of approx 170,000 units/mg protein with a yield of 3.6%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single protein band of Mr 62,000. Gel filtration with a Superose 12 column gave a single peak, and the enzyme in the presence 50 mM n-octyl-beta-D-glucopyranoside was of Mr 123,000, indicating that the native enzyme occurs in a dimeric form. The optimal pH was 5.5 with both sphingomyelin and an artificial substrate, 2-N-hexadecanoylamino-4-nitrophenylphosphorylcholine. The Km values were 55 microM with sphingomyelin and 340 microM with the artificial substrate. The enzyme activity was not affected by Mg2+ (1-5 mM), confirming that the enzyme is acid sphingomyelinase. The enzyme was stable at -80 degrees C for more than 4 months. In addition to the enzyme with pI 7.4, the Mono P chromatofocusing gave two peaks (pI 7.0 and 6.7) possessing the enzymatic activity.     

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.     

Adsorption of sphingomyelinase of Bacillus cereus onto erythrocyte membranes

Sphingomyelinase of Bacillus cereus proved to be specifically adsorbed onto mammalian erythrocyte membranes in the presence of either Ca2+ or Ca2+ plus Mg2+ in the order of sphingomyelin content; i.e., sheep, bovine greater than porcine greater than rat erythrocytes. No appreciable adsorption was observed in the presence of Mg2+ alone nor in the absence of divalent metal ions. The enzyme adsorption onto bovine erythrocytes was dependent upon the incubation temperature. By shifting the temperature from 37 to 0 degrees C, sphingomyelinase once adsorbed onto the surface of bovine erythrocytes was released into the supernatant. Ca2+ proved to be an essential factor for the enzyme adsorption: The addition of 1 mM Ca2+ enhanced the adsorptive process, but inhibited sphingomyelin hydrolysis and hot or hot-cold hemolysis of erythrocytes, while the addition of 1 mM Ca2+ plus 1 mM Mg2+ enhanced sphingomyelin breakdown and hemolysis as well as the enzyme adsorption. However, when the amount of sphingomyelin fell off to 0.2-0.7 nmol/ml or less by the action of sphingomyelinase, the enzyme once adsorbed was completely released from the surface of erythrocytes. The result indicates that the major binding site for sphingomyelinase is sphingomyelin. In the presence of 1 mM Mg2+ alone, the enzymatic hydrolysis of sphingomyelin and hemolysis proceeded whereas the enzyme adsorption was not encountered during 60 min incubation at 37 degrees C. The change in the molar ratio of Ca2+ to Mg2+ affected the enzyme adsorption and sphingomyelin breakdown; the higher Ca2+ enhanced the adsorption whereas the higher Mg2+ stimulated sphingomyelin hydrolysis.     

Characterization and localization of neutral sphingomyelinase in bovine adrenal medulla

Homogenates of bovine adrenal medullae hydrolyzed exogenous sphingomyelin at 4.3 +/- 1.6 nmol X mg-1 X min-1 and 97% of this sphingomyelinase activity was sedimentable at 110,000 g. The sphingomyelinase had a broad pH optimum centered at pH 7. Enzymatic activity was maximal with 80 microM added Mn2+; Mg2+ supported less than half maximal activity and both Ca2+ and EDTA inhibited activity. No activity was detected in the absence of Triton X-100. Response to detergent was biphasic with dose-dependent stimulation from 0.02% to 0.05% Triton X-100 followed by inhibition with increasing concentrations of detergent. Activity in response to detergent was also modulated by protein concentration. Sphingomyelinase activity was associated with a plasma membrane-microsomal fraction. Phosphatidylcholine was not hydrolyzed under optimal conditions for sphingomyelin hydrolysis and a variety of other conditions. Neutral-active sphingomyelinase activity in adrenal medulla was similar in magnitude to that observed in other non-neural bovine tissues. This study demonstrates the presence of a potent neutral-active sphingomyelinase in a plasma membrane-microsomal fraction of bovine adrenal medulla. This enzyme may be involved in membrane fusion and lysis during catecholamine secretion through its ability to alter membrane composition.     

A neutral sphingomyelinase in spermatozoal plasma membranes

A highly active neutral sphingomyelinase was observed for the first time in ram spermatozoal plasma membranes. The optimal conditions for the enzyme activity are pH 7.4, 40 mM MgCl2, 40 min of incubation, and 267 nmol sphingomyelin. Ca2+ and cholesterol were found to inhibit sphingomyelinase activity.     

Interfacial regulation of bacterial sphingomyelinase activity

The objective of this study was to define how the quality of the buffer/membrane interface influences the activity of bacterial sphingomyelinase acting at the interface. The enzyme reaction was carried out in a zero-order trough using a surface barostat. This approach allowed for proper control of the physico-chemical properties of the substrate molecules. Since the molecular area of ceramide is smaller than that of sphingomyelin, the hydrolysis reaction could be followed `on-line' from the monolayer area decrease at constant surface pressure. The hydrolysis reaction could be divided into two separate phases, the first being the lag-phase (time between enzyme addition and commencement of the monolayer area change), and the second phase being the actual hydrolysis reaction (from which a maximal degradation rate could be determined). The activity of sphingomyelinase (Staphylococcus aureus) toward bovine brain sphingomyelin (bb-SM) was markedly enhanced by Mg²⁺ (maximal activation at 5 mM). Mg²⁺ also influenced the lag-phase of the reaction (the lag-time increased markedly when the Mg²⁺ concentration decreased below 1 mM). Saturated sphingomyelins (bb-SM and N-palmitoyl sphingomyelin [N-P-SM]) were more slowly degraded than the mono-unsaturated N-oleoyl sphingomyelin (N-O-SM). Both bb-SM and N-P-SM monolayers underwent a phase-transition at room temperature, whereas the N-O-SM monolayer did not. The phase-transition (liquid-expanded to liquid-condensed) was observed to greatly increase the lag-time of the hydrolysis reaction. The activity of sphingomyelinase was also sensitive to the lateral surface pressure of the monolayer membrane. Maximal degradation rate was achieved at 20 mN/m (with bb-SM, 30°C); above this pressure the lag-time of the reaction increased sharply. The inclusion of 4 mol% of cholesterol into a [³H]sphingomyelin monolayer markedly increased the extent of [³H]sphingomyelin degradation, and shortened the lag-time of the reaction. The inclusion of 10 mol% of zwitterionic or negatively charged phospholipids to the [³H]sphingomyelin monolayer did not affect the sphingomyelinase reaction significantly. In conclusion, this study has demonstrated that the physico-chemical properties of the substrate molecules have a dominating influence on the activity of a bacterial sphingomyelinase acting at the buffer/membrane interface.     

Sphingomyelinase activity associated with human plasma low density lipoprotein

Isolated human plasma low density lipoprotein (LDL) was observed to possess sphingomyelinase activity. Accordingly, the formation of ceramide was catalyzed by LDL at 37 degrees C using tertiary liposomes composed of sphingomyelin (mole fraction (x) = 0.2), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (x = 0.7), 1, 2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (x = 0.1), and either the fluorescent sphingomyelin analog Bodipy-sphingomyelin or [(14)C]sphingomyelin as substrates. However, this activity was not present in either very low density lipoprotein or the high density lipoprotein subfractions HDL(2) and HDL(3). Oxidation of LDL abrogated its sphingomyelinase activity. Aggregation of the liposomes upon incubation with LDL was evident from the light scattering measurements. Microinjection of LDL to the surface of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-d-sphingomyelin (C16:0-sphingomyelin), and Bodipy-sphingomyelin as a fluorescent tracer (0.75:- 0.20:0.05, respectively) revealed the induction of vectorial budding of vesicles, resembling endocytosis.     

A sphingomyelinase-resistant pool of sphingomyelin in the nuclear membrane of hen erythrocytes

Experiments in which hen erythrocytes were exposed to the action of exogenous sphingomyelinase (Staphylococcus aureus) or to their endogenous plasma membrane sphingomyelinase showed that about 15% of the total sphingomyelin was resistant to breakdown either in intact or lysed cells. This resistant pool of sphingomyelin seems likely to reside in the nuclear membranes of the cells, so that essentially all the plasma membrane sphingomyelin can be broken down by exogenous sphingomyelinase acting on intact cells, suggesting that plasma membrane sphingomyelin is exclusively localised in the outer lipid leaflet. Paradoxically, introduction of Ca2+ into the intact cells using A23187 causes the breakdown of up to 30% of total cell sphingomyelin inside the cells but without apparently affecting the putative nuclear pool of sphingomyelin and this suggests that Ca2+ may alter the original disposition of sphingomyelin in the membrane so that originally outer leaflet sphingomyelin becomes accessible to the endogenous sphingomyelinase inside the cells. No differences were seen in the fatty acid compositions of sphingomyelin degradable by exogenous sphingomyelinase, sphingomyelin degradable in the presence of A23187/Ca2+ or the enzyme-resistant pool of sphingomyelin.     

Acid sphingomyelinase of human placenta: purification, properties, and 125iodine labeling

Human placental acid sphingomyelinase was highly purified in the presence of Triton X-100. DEAE-Sephacel chromatography and chromatofocusing were the most effective steps in the purification procedure. Enzyme purification was 380,000 nmol/mg protein/h. Characterization and radioiodination were carried out with the chromatofocusing fraction containing highly purified enzyme. The purified enzyme contained no activity of eleven other lysosomal hydrolases but hydrolyzed bis-p-nitrophenyl phosphate slowly compared with [14C]sphingomyelin and chromogenic substrates. SDS-gel electrophoresis revealed two distinct protein bands with molecular weights of 70,500 and 39,800. This enzyme had a molecular weight of 200,000 as determined by analytical gel filtration. The pH optimum was 5.0 and Km was 52.6 x 10(-5) M for [14C]sphingomyelin. Highly purified sphingomyelinase was labeled with 125iodine by the use of Enzymobeads. Labeled sphingomyelinase preparation was rapidly cleared from blood with t1/2 of 1 min. It was absorbed mostly into the liver and presumably largely excreted from there. This labeled enzyme may be useful in metabolic studies in normal animals and animal models of genetic lysosomal storage disorders.     

Catabolism of Exogenous and Endogenous Sphingomyelin and Phosphatidylcholine by Homogenates and Subcellular Fractions of Cultured Neuroblastoma Cells. Effects of Anesthetics

Cultured murine neuroblastoma cells contain a neutral, Mg2+-stimulated sphingomyelinase and an alkaline phosphatidylcholine-hydrolyzing activity that are enriched in the plasma membrane fraction. The reaction products of sphingomyelin catabolism are phosphocholine and ceramide and those of phosphatidylcholine, glycerophosphocholine and fatty acid. These reactions were studied with endogenous as well as exogenous liposomal substrates. With both exogenous and endogenous substrates, the sphingomyelinase activity was stimulated two- to threefold by Mg2+ and a further three- to fourfold by volatile anesthetic agents. Stimulation was concentration-dependent and corresponded to anesthetic potency: methoxyflurane greater than halothane greater than enflurane. Greater than 80% of the plasma membrane sphingomyelin was hydrolyzed within 2 h in the presence of Mg2+ and anesthetic. In contrast, the activity with exogenous and endogenous phosphatidylcholine was unaffected by Mg2+ or Ca2+ and was markedly inhibited (50-80%) by anesthetic agents. The degree of inhibition was concentration-dependent and corresponded to anesthetic potency. The quantitative importance of choline-containing lipids in cell membranes, the relatively exclusive localization of the neutral Mg2+-stimulated sphingomyelinase in cells of neural origin, the totally different type of hydrolytic attack on phosphatidylcholine, and the reciprocal effects of anesthetics on the hydrolysis of these two lipids strongly suggest important roles for these activities in cell membranes in general and in the neuron in particular.     

The action of sphingomyelinase from Bacillus cereus on ATP-depleted bovine erythrocyte membranes and different lipid composition of liposomes

The presence of cholesterol or phosphatidylethanolamine in sphingomyelin liposomes enhanced 2- to 10-fold the breakdown of sphingomyelin by sphingomyelinase from Bacillus cereus. On the other hand, the presence of phosphatidylcholine was either without effect or slightly stimulative at a higher molar ratio of phosphatidylcholine to sphingomyelin (3/1). In the bovine erythrocytes and their ghosts, the increase by 40-50% or the decrease by 10-23% in membranous cholesterol brought about acceleration or deceleration of enzymatic degradation of sphingomyelin by 50 or 40-50%, respectively. The depletion of ATP (less than 0.9 mg ATP/100 ml packed erythrocytes) enhanced K+ leakage from, and hot hemolysis (lysis without cold shock) of, bovine erythrocytes but decelerated the breakdown of sphingomyelin and hot-cold hemolysis (lysis induced by ice-cold shock to sphingomyelinase-treated erythrocytes), either in the presence of 1 mM MgCl2 alone or in the presence of 1 mM MgCl2 and 1 mM CaCl2. Also, ATP depletion enhanced the adsorption of sphingomyelinase onto bovine erythrocyte membranes in the presence of 1 mM CaCl2 up to 81% of total activity, without appreciable K+ leakage and hot or hot-cold hemolysis. These results suggest that the presence of cholesterol or phosphatidylethanolamine in biomembranes makes the membranes more susceptible to the attack of sphingomyelinase from B. cereus and that the segregation of lipids and proteins in the erythrocyte membranes by ATP depletion causes the deceleration of sphingomyelin hydrolysis despite the enhanced enzyme adsorption onto the erythrocyte membranes.     

Beta-amyloid protein oligomers induced by metal ions and acid pH are distinct from those generated by slow spontaneous ageing at neutral pH.

Amyloid protein (Abeta1-40) aggregation and conformation was examined using native and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the results compared with those obtained by atomic force microscopy, and with Congo red binding, sedimentation and turbidity assays. The amount of Abeta aggregation measured was different, depending upon the method used. Incubation for 15 min at pH 5.0 or in the presence of Fe2+, Cu2+ or Zn2+ did not alter the level of Abeta oligomers observed on SDS and native gels. However, the slow aggregation of Abeta to form high molecular mass species over 5 days was inhibited. In contrast, when Abeta aggregation was monitored using a Congo red binding assay or sedimentation assay, a rapid increase in Abeta aggregation was observed after incubation for 15 min at pH 5.0, or in the presence of Fe2+, Cu2+ or Zn2+. The low pH-, Zn2+- or Cu2+-induced Abeta aggregation measured in a turbidity assay was reversible. In contrast, a considerable proportion of the Abeta aggregation measured by native and SDS/PAGE was stable. Atomic force microscopy studies showed that Abeta aged at pH 5.0 or in the presence of Zn2+ produced larger looser rod-shaped aggregates than at pH 7.4. Abeta that had been aged at pH 7.4 was more cytotoxic than Abeta aged at pH 5.0. Taken together, the results suggest that Abeta oligomerizes via two mutually exclusive mechanisms to form two different types of aggregates, which differ in their cytotoxic properties.     

Phosphatidylinositol-3,5-Bisphosphate is a potent and selective inhibitor of acid sphingomyelinase

Acid sphingomyelinase (A-SMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to phosphorylcholine and ceramide. Inherited deficiencies of acid sphingomyelinase activity result in various clinical forms of Niemann-Pick disease, which are characterised by massive lysosomal accumulation of sphingomyelin. Sphingomyelin hydrolysis by both, acid sphingomyelinase and membrane-associated neutral sphingomyelinase, plays also an important role in cellular signaling systems regulating proliferation, apoptosis and differentiation. Here, we present a potent and selective novel inhibitor of A-SMase, L-alpha-phosphatidyl-D-myo-inositol-3,5-bisphosphate (PtdIns3,5P2), a naturally occurring substance detected in mammalian, plant and yeast cells. The inhibition constant Ki for the new A-SMase inhibitor PtdIns3,5P2 is 0.53 microM as determined in a micellar assay system with radiolabeled sphingomyelin as substrate and recombinant human A-SMase purified from insect cells. Even at concentrations of up to 50 microM, PtdIns3,5P2 neither decreased plasma membrane-associated, magnesium-dependent neutral sphingomyelinase activity, nor was it an inhibitor of the lysosomal hydrolases beta-hexosaminidase A and acid ceramidase. Other phosphoinositides tested had no or a much weaker effect on acid sphingomyelinase. Different inositol-bisphosphates were studied to elucidate structure-activity relationships for A-SMase inhibition. Our investigations provide an insight into the structural features required for selective, efficient inhibition of acid sphingomyelinase and may also be used as starting point for the development of new potent A-SMase inhibitors optimised for diverse applications.