Partial purification and properties of acid sphingomyelinase from rat liver

Acid sphingomyelinase was purified approximately 5,200-fold from the mitochondria-lysosome-enriched particles of rat liver by sequential chromatography on DEAE-cellulose, octyl-Sepharose, Sephacryl S-300, Concanavalin A-Sepharose, and CM-cellulose. The specific activity of this highly purified enzyme was 3.2 mmol per hr per mg protein. The enzyme was active against 2-hexadecanoylamino-4-nitrophenylphosphorylcholine, but bis-4-methylumbelliferyl-phosphate and bis-p-nitrophenyl-phosphate were poor substrates. The preparation was free of Mg2+-dependent neutral sphingomyelinase and eight lysosomal enzymes except for the trace amount of acid phosphatase and beta-galactosidase. Apparent molecular weight of the enzyme was 200,000, estimated by Sephadex G-200 filtration in 0.1% Triton X-100. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed three major bands corresponding to molecular weights of 45,600, 44,500, and 40,000 with several minor bands. Characterization of the enzyme revealed almost the same properties as those of human tissues reported by other investigators, including pH optimum, requirement of Triton X-100, effects of metal divalent cations, phosphate ion, EDTA, some thiol blocking reagents, and amphophilic drugs.     

Complex kinetics of bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine hydrolysis by purified sphingomyelinase in the presence of Triton X-100

We have examined the hydrolysis of the synthetic phosphodiesters, bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine, by purified placental sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) in the presence of Triton X-100. Triton X-100 enhanced activity with bis(4MU)phosphate at all concentrations tested. At very low concentrations of detergent, bis(4MU)phosphate hydrolysis approached zero. Our results indicate that bis(4MU)phosphate does not form a micelle with Triton X-100. The observed enhancement of bis(4MU)phosphate activity with Triton X-100 is likely due to a direct effect of detergent on the enzyme itself. HDNP-phosphorylcholine formed its own micelle (or liposome) in the absence of Triton X-100 and, at substrate concentrations below 4 mM, hydrolysis was inhibited by Triton X-100. The extent of this inhibition varied with detergent concentrations but could be totally eliminated at substrate values above 4 mM. For theoretical reasons kinetic constants which could be obtained with the HDNP-phosphorylcholine substrate at concentrations above 4 mM are not considered to be truly representative of the real values. We conclude that neither substrate is recommended to describe the true kinetic parameters pertaining to purified sphingomyelinase. In addition, bis(4MU)phosphate may not be suitable as an aid for diagnosis of sphingomyelinase deficiency states.U     

Sphingomyelinases in human tissues. IV. Purification of sphingomyelinase from human placenta and effect of Triton X-100.

Sphingomyelinase was purified about 1700-fold from human placenta. The major steps in the procedure included chromatography on Concanavalin A-Sepharose, Sepharose 6B, and carboxymethyl-Sepharose (CM-Sepharose). The final preparation was stable for at least 3 months when stored at 4 degrees C. The enzyme was found to be heterogeneous on CM-Sepharose and isoelectric focusing. Triton X-100 which was present in most buffers used during the purification appears to be partially responsible for the heterogeneity. When Triton X-100 is removed by treatment with Bio Beads, heterogeneity was reduced. However, removal of the detergent also leads to loss of enzyme activity which could not be restored by readdition of Triton X-100. The data suggest that sphingomyelinase has a high hydrophobic character and that both its stability and electrofocusing behaviour are influenced by interaction with the nonionic detergent.     

Hydrolysis of fucosyl-GM1 ganglioside by purified pellet-associated human brain and human liver alpha-L-fucosidases without activator proteins or detergents.

Pellet-associated human brain alpha-L-fucosidase was solubilized with 0.5% (w/v) Triton X-100 and purified by affinity chromatography on agarose-6-aminohexanoyl-fucosamine resin. The procedure resulted in a 290,000-fold purification, a 58% yield and a final specific activity of 11,500 nmol/min per mg of protein. Isoelectric focusing indicated that all six major isoforms (with pI values between 4.1 and 5.3) present in crude brain pellet preparations were purified by the affinity technique. SDS/PAGE indicated the presence of one subunit (54 kDa) and a minor protein band at 67 kDa, which presumably is a contaminant since it was not immunoreactive on Western blotting. The pH optimum of the brain enzyme and its apparent Km for the synthetic substrate 4-methylumbelliferyl alpha-L-fucopyranoside were 5.5 and 0.07 mM respectively. Pellet-associated human brain and liver alpha-L-fucosidases were both capable of hydrolysing fucosyl-GM1 ganglioside without activator proteins or detergents. Linear hydrolysis rates were found only for short incubation times (1-5 min). Optimal enzymic activity at 37 degrees C was found at pH 3.4 for both alpha-L-fucosidases, with no activity at pH values above 4.0.     

Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.     

Bis-(4-methylumbelliferyl) phosphate as a substrate for the surface membrane-associated phosphodiesterase activity of pig platelets.

It was found that the newly-available compound, bis-(4-methylumbelliferyl) phosphate, could be used as a substrate for the pig platelet surface membrane-associated phosphodiesterase activity, usually assayed with bis-(p-nitrophenyl) phosphate. This enzyme activity is distinct from the phosphodiesterase activity towards 5'-dTMP-P-nitrophenyl ester, which is probably associated with intracellular membrane structures in platelets. Consequently, the use of the 4-methylumbelliferyl derivative as substrate for the phosphodiesterase activity provides a sensitive, fluorimetric assay for this marker enzyme of the platelet surface membrane.     

Release of sphingomyelin phosphodiesterase (acid sphingomyelinase) by ammonium chloride from CL 1D mouse L-cells and human fibroblasts. Partial purification and characterization of the exported enzymes

In cultured human fibroblasts and mouse L-cells the lysosomotropic agent, ammonium chloride, caused release of acid sphingomyelinase into the culture medium. The water-soluble enzymes were partially purified by sequential chromatography on ConA-Sepharose, octyl-Sepharose and Sepharose CL-4B. Mouse sphingomyelinase was purified up to 64-fold and human sphingomyelinase 134-fold from the culture medium. Specific activities were 925 nmol/(h X mg) and 1 434 nmol/(h X mg), respectively. The final enzyme preparations obtained were free of other lysosomal enzyme activities tested and had very similar properties: optimal activity at pH 4.8 (mouse enzyme) and pH 4.4 (human enzyme), Km values of 6.2 X 10(-5)M and 2.4 X 10(-5)M, respectively, and an apparent molecular mass of 68 kDa. In isoelectric focusing the enzymes peaked at pH 4.78 (mouse enzyme) and pH 4.75 (human enzyme).     

Acid Sphingomyelinase of Human Brain: Purification to Homogeneity

Abstract: Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human brain by extraction with 0.1% Triton X-100, followed by sequential chromatography on Concanavalin A-Sepharose, octyl-Sepharose, hydroxylapatite, DEAE-cellulose, red A-agarose, Sephadex G-200, and DEAE-cellulose with ampholyte elution. Sphingomyelinase activity was purified more than 20,000-fold from the starting homogenate with a 1% yield. Specific activity of up to 800 μmol/h/mg protein could be achieved. Gel electrophoresis with 6% polyacrylamide containing sodium dodecyl sulfate gave a single protein band with a molecular weight of 70,000, in good agreement with the molecular weight previously estimated from sucrose density gradient centrifugation in 0.1% Triton X-100. Triton X-100 could be readily removed from the enzyme by sucrose density gradient centrifugation. The Triton-free enzyme showed the same K _m and pH optimum. Heat stability of the enzyme was reversibly affected by Triton X-100, in that removal of the detergent made the enzyme more heat labile. The K _m of purified enzyme for sphingomyelin was 36 μ M . It was unaffected by sulfhydryl reagents, but was inhibited by dithiothreitol at high concentrations. The preparation was free of all lysosomal hydrolase activities tested, including galactosylceramidase and α-mannosidase, which tended to copurify in our previous procedure. The enzyme was inactive toward sphingosylphosphorylcholine. It was active with bis[ p -nitrophenyll- and bis[4-methylumbelliferyl]phosphate and the chromogenic and fluorogenic sphingomyelin analogues.     

Studies on sphingomyelinase and beta-glucosidase activities in Niemann-Pick disease variants. Phosphodiesterase activities measured with natural and artificial substrates

Cultured fibroblasts were studied from 12 cases of Niemann-Pick disease group C. In 11, sphingomyelinase and glucocerebrosidase (and beta-glucosidase) activities were reduced to around 50% of those of controls. On isoelectric focusing, all 12 strains lacked sphingomyelinase activity in the major cathodic region (pI 8.0). The defect was also demonstrated with the artificial phosphodiester substrates bis(4-methylumbelliferyl) phosphate and 4-methylumbelliferyl pyrophosphate diester. In control fibroblasts and those heterozygous for types A or B or group C Niemann-Pick disease, the major sphingomyelinase peak electrofocused at pI 8.0. No direct interaction could be demonstrated by mixing experiments between group C Niemann-Pick extracts and those of type A disease or Gaucher disease. Profiles for beta-glucosidase activity appeared normal in Niemann-Pick group C fibroblasts. No reduction of sphingomyelinase or glucocerebrosidase activities was found in Niemann-Pick group C liver, nor any attenuation of cathodic sphingomyelinase activity in the affected tissue. Results suggest that sphingomyelinase expression differs in fibroblasts and liver. Enzyme defects associated with Niemann-Pick disease group C were only observed in cultured cells.     

Bis-(4-methylumbelliferyl) phosphate as a substrate for the surface membrane-associated phosphodiesterase activity of pig platelets

It was found that the newly-available compound, bis-(4-methylumbelliferyl) phosphate, could be used as a substrate for the pig platelet surface membrane-associated phosphodiesterase activity, usually assayed with $\text{bis-(}\text{p-}\text{nitrophenyl)}$ phosphate. This enzyme activity is distinct from the phosphodiesterase activity towards $\text{5′ -}\text{dTMP}\text{-p-}\text{nitrophenyl}$ ester, which is probably associated with intracellular membrane structure in platelets. Consequently, the use of the 4-methylumbelliferyl derivative as substrate for the phosphodiesterase activity provides a sensitive, fluorimetric assay for this marker enzyme of the platelet surface membrane.     

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.     

Purification of particulate malate dehydrogenase and phosphoenolpyruvate carboxykinase from Leishmania mexicana mexicana

The particulate activities of Leishmania mexicana mexicana amastigote malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) have been purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5'AMP-Sepharose 4B, and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. Phosphoenolpyruvate carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SDS-gel electrophoresis were 39 800 and 33 300 for malate dehydrogenase and 63 100 and 65 100 for phosphoenolpyruvate carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km(NADH) of 41 microM and a Km(oxaloacetic acid) of 39 microM. For malate oxidation there was a Km(malate) of 3.6 mM and a Km(NAD) of 0.79 mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than 0.83 mM and malate was found to be a product inhibitor at high concentrations. However, there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors, suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts; nevertheless, their apparent importance and unique subcellular organization in the parasite make them potential targets for chemotherapeutic attack.     

Isolation and characterization of a tartrate-sensitive splenic acid phosphatase in Gaucher's disease

The acid phosphatase activity that is increased in the spleens of patients with Gaucher's disease can be separated into two principal isoenzymes by chromatography on sulphopropyl-Sephadex. The acid phosphatase species that is resistant to inhibition by l-(+)-tartrate is retained by the cation-exchange resin while the tartrate-sensitive species passes through. We have isolated and characterized the tartrate-sensitive acid phosphatase (designated SP_I) from the spleen of a patient with the adult (type 1) form of Gaucher's disease. SP_I acid phosphatase, representing approximately 30 to 50% of the total acid phosphatase activity in a detergent (Triton X-100) extract of spleen tissue, has been purified approximately 400-fold to a specific activity of 48 units/mg of protein (substrate, 4-methylumbelliferyl phosphate). The final preparation of acid phosphatase contains at least two protein components—each with phosphatase activity—when analyzed by polyacrylamide gel electrophoresis at pH 8.9 or isoelectric focusing. SP_I acid phosphatase exhibits a broad substrate specificity and catalyzes the hydrolysis of a variety of artificial and natural phosphate-containing compounds including p-nitrophenyl phosphate, α-naphthyl phosphate, phosphoenolpyruvate, and CMP. The enzyme is inhibited by l-(+)-tartrate, sodium fluoride, and ammonium molybdate and has the following properties: pH optimum, 4.5; K_m on 4-methylumbelliferyl phosphate, 44 μm; pI, 3.8–4.1; M_r, 177,400; s_20,w, 6.8.     

Human placental steroid sulfatase: purification and properties

Steroid sulfatase is recovered quantitatively from the 105,000 g h supernatant of human placental microsomes extracted with Triton X-100. The solubilized enzyme has been purified using conventional techniques. Throughout the purification procedure, steroid sulfatase appears to be heterogeneous as evidenced by certain, but not all, criteria. Following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the final preparation exhibits a major component and varying amounts of two minor ones. Antibodies raised in rabbits with the heterogeneous immunogen give rise to a single precipitation line when the native enzyme is analyzed by double immunodiffusion or by immunoelectrophoresis. In addition, using aged preparations of microsomes and immunoaffinity techniques, steroid sulfatase activity was found to be associated with the fastest migrating minor component. This finding would suggest that the apparent heterogeneity of purified steroid sulfatase is linked to degradation processes occurring within the microsomal preparations. Steroid sulfatase has a Stokes radius of 56 A, a sedimentation coefficient of 4.85 +/- 0.15S (in Triton-containing buffers) and binds 1.3 g of Triton X-100-per g of protein. The molecular weight of the Triton-protein complex was calculated to be 166,000 in which the glycoprotein portion contribution is about 43% (72,000). In contrast, the apparent molecular weight of the major polypeptide determined on calibrated SDS-gels is 62,000. The purified enzyme exhibits two pH optima with cholesterol sulfate as substrate, an acidic one at pH 5.0 and a second one at pH 7.5. The Km values for cholesterol sulfate, dehydroandrosterone sulfate and p-nitrophenylsulfate were 5.26, 14 and 1,320 microM, respectively.     

Purification and properties of human placental acid lipase

Two peaks of lysosomal acid lipase activity were purified from normal human placenta. Acid lipase I, with an estimated molecular weight of 102 500, was purified 1016-fold while acid lipase II, with an estimated molecular weight of 30 600, was purified 3031-fold. The final yields of enzyme activity for acid lipase I and II were 0.9% and 2.2% respectively. The purity of the final preparations was documented by demonstration of a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both preparations of the purified enzyme demonstrated activity towards triolein, cholesteryl oleate and the artificial substrate 4-methylumbelliferyl oleate. Examination of Km values, thermal stability, pH optima, and electrophoretic mobility revealed similar properties for the two enzyme peaks. The response of the two enzyme preparations to inhibitors was similar with both being significantly inhibited by 0.2 M NaCl, 0.2 M KCl, 5 mM HgCl2 and 5 mM p-chloromercuribenzoate. The activity of the two preparations as assayed with either triolein or cholesterol oleate was not significantly affected by the addition of bovine serum albumin. In contrast, the 4-methylumbelliferyl oleate activity of both preparations was significantly inhibitred by albumin. These findings support the hypothesis that the same enzyme or enzymes are responsible for the intralysosomal hydrolysis of triacylglycerols and cholesterol esters in human tissues.     

Purification and characterization of phytase from cotyledons of germinating soybean seeds

Soybean phytase (myo-inositol-hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from 10-day-old germinating cotyledons using a four-step purification scheme. Phytase was separable from the major acid phosphatase present, and stained as a minor band of the three acid phosphatases detectable by activity staining after gel electrophoresis. The purified enzyme exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 59 and 60 KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.5 X 10(4) M-1 cm-1. The isoelectric point of phytase, as judged by the elution profile on chromatofocusing, was about 5.5. The enzyme was totally absorbed to a Procion Red HE3B column and eluted as a single protein component at a salt concentration of 250-300 mM. The enzyme possessed a high affinity for phytic acid (apparent Km = 48 microM), and was strongly inhibited by phosphate (apparent Ki = 18 microM), vanadate, and fluoride. Characteristic of other plant phytases, the pH and temperature optima were 4.5-4.8 and 55 degrees C, respectively.     

Studies on the hydrophobic properties of sphingomyelinase.

Crude liver lysosomal sphingomyelinase (EC 3.1.4.12) displays a heterogeneous electrofocusing profile. The majority of the enzyme resolves into two major components with acidic pI values near pH 4.6 and 4.8. Several additional minor peaks of activity are seen at more basic pH values (up to pH 8.0). In the presence of 0.1% Triton X-100 (or Cutscum), the location of sphingomyelinase is shifted by about 1 pH unit to more basic pH values. Triton X-100 also increases the apparent heterogeneity of sphingomyelinase. Removal of detergent by treatment with Bio Beads SM-2 restores the acidic pI profile. This behaviour appears to be specific, since it was not shared by six glycosidases several of which hydrolyse sphingolipids. The electrofocusing profile of 3H-labelled Triton X-100 was distinct and separate from sphingomyelinase, suggesting that only a small fraction of detergent interacted directly with the enzyme. To study this behaviour in more detail we examined the effect of detergents on elution of sphingomyelinase from sphingosylphosphocholine-Sepharose. Sphingosylphosphocholine is a competitive inhibitor of sphingomyelinase (Ki 0.5 mM). Binding of enzyme was pH-dependent. Triton X-100, Cutscum and Tween 20 eluted significant amounts of enzyme at 0.01-0.02%. Total elution was achieved with up to 0.1% detergent. These data suggest that sphingomyelinase binds to neutral detergent monomers with a high degree of affinity. In excess detergent (5-7 times the critical micellar concentration) the surface charge on the protein is changed, leading to a pI shift. This behaviour probably does not occur at the active site of the enzyme, since there is no appreciable effect on substrate hydrolysis and substrate analogues were ineffective in eluting the enzyme.     

Microsomal NADH-cytochrome b5 reductase of bovine brain: purification and properties

Bovine brain microsomal NADH-cytochrome b5 (cyt. b5) reductase [EC 1.6.2.2] was solubilized by digestion with lysosomes, and purified 8,500-fold with a 20% recovery by procedures including affinity chromatography on 5'-AMP-Sepharose 4B. The purified enzyme showed one band of a molecular weight of 31,000 on polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). Polyacrylamide gel electrophoresis of the purified enzyme without SDS revealed a major band with a faint minor band, both of which exhibited NADH-cyt. b5 reductase activity. The isoelectric points of these components were 6.0 (major) and 6.3 (minor). The apparent Km values of the purified enzyme for NADH and ferricyanide were 1.1 and 4.2 microM, respectively. The apparent Km value for cyt. b5 was 14.3 microM in 10 mM potassium phosphate buffer (pH 7.5). The apparent Vmax value was 1,190 mumol cyt. b5 reduced/min/mg of protein. The NADH-cyt. b5 reductase activity of the purified enzyme was inhibited by sulfhydryl inhibitors and flavin analogues. Inhibition by phosphate buffer or other inorganic salts of the enzyme activity of the purified enzyme was proved to be of the competitive type. These properties were similar to those of NADH-cyt. b5 reductase from bovine liver microsomes or rabbit erythrocytes, although the estimated enzyme content in brain was about one-twentieth of that in liver (per g wet tissue). An immunochemical study using an antibody to purified NADH-cyt. b5 reductase bovine liver microsomes indicated that NADH-cyt. b5 reductase from brain microsomes is immunologically identical to the liver microsomal enzyme.     

Synthesis of 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and detection of skin fibroblast neuraminidase in normal humans and in sialidosis.

A procedure for the synthesis of the fluorogenic substrate analogue 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid for the human acid neuraminidase has been developed. The substrate was employed for the characterization of the enzyme in sonicates of cultured human skin fibroblasts and for enzymatic detection of the neuraminidase deficiency in the neurological storage disorder, sialidosis. Synthesis was accomplished by reacting 2-deoxy-2-chloro-4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid methyl ester with the sodium salt of 4-methylumbelliferone in acetonitrile at room temperature. The coupled product was purified on silicic acid chromatography, followed by base-catalyzed removal of the O-acetyl and methoxy blocking groups, and with additional purification of the hydrolyzed product on silicic acid. The overall yield, based on N-acetylneuraminic acid, was 37%. Under linear assay conditions, at pH 4.3, the apparent maximal velocities (nmol (mg of protein)-1 h-1) for normal fibroblasts were 58--115, 0.2--1.8 for sialidosis fibroblasts, and 28--38 for obligate heterozygotes. The apparent Km for normals was 0.13 mM.     

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.