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.     

Phosphatidylinositol-specific phospholipase C-gamma1 undergoes pH-induced activation and conformational change

Phospholipase C-gamma1 displayed sigmoidal kinetics with a S(0.5) value of 0.17 mole fraction PIP(2) when assayed at pH 6.8 using detergent:lipid mixed micelles. The pH optimum for hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C-gamma1 was dependent on the mole fraction of substrate in the micelle. The pH optimum was 5.5 when the enzyme was assayed below the S(0.5). The pH optima shifted to a pH range of 6.0-6.3 when the enzyme was assayed above the S(0.5). The kinetic parameters for phospholipase C-gamma1 assayed at various pH values from pH 7.0 to 5.0 yielded similar n values (n=4), but the constant, K', decreased from 1x10(-2) (mole fraction)(2) at pH 7.0 to 1x10(-5) (mole fraction)(2) at pH 5.0. Maximum enzyme specificity occurred at pH values below pH 6.0 as determined by the plot of logk(cat)/S(0.5) versus pH. Intrinsic fluorescence spectroscopy revealed that at a pH value above 7.0 or below 6.3, tryptophan quenching occurred. Fluorescence quenching experiments performed with acrylamide determined phospholipase C-gamma1 incubated at pH 5.0 had a larger collisional quenching constant than enzyme incubated at pH 7.0. Lowering the pH to 5.0 apparently resulted in interior tryptophans becoming more solvent accessible. These data suggest that pH may activate phospholipase C-gamma1 by disrupting ionizable groups leading to a conformational change.     

Extracellular Enzyme From Myxobacter AL-1 That Exhibits Both β-1,4-Glucanase and Chitosanase Activities

An enzyme that has both beta-1,4-glucanase and chitosanase activities is characterized. Evidence for homogeneity was obtained from electrophoresis and sedimentation velocity studies; only one N-terminal amino acid, valine, was found. Results of denaturation studies showed that beta-1,4-glucanase and chitosanase activities decreased at equal rates. With carboxymethylcellulose as the substrate, a K(m) of 1.68 g of carboxymethylcellulose per liter of solution and a V(max) of 2.20 x 10(-9) mol/min were found. With chitosan (the beta-1,4-polymer of glucosamine) as the substrate, a K(m) of 0.30 g of chitosan per liter of solution and a V(max) of 0.75 x 10(-9) mol/min were found. A pH optimum of 5.0 was found for beta-1,4-glucanase activity, and pH optima of 5.0 and 6.8 were found for chitosanase activity. beta-1,4-Glucanase activity had a temperature optimum of 38 C, and chitosanase activity had a temperature optimum of 70 C. Chitosan stabilized both enzyme activities at 70 C. Cellotriose was the smallest polymer capable of hydrolysis. Glucosamine was released by action of the enzyme upon cell wall preparations of several fungi.     

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

2'',3''-CYCLIC NADP AS A SUBSTRATE FOR 2'',3''-CYCLIC NUCLEOTIDE 3''-PHOSPHOHYDROLASE

Abstract– 2',3'-Cyclic NADP has been prepared by cyclization of NADP at pH 6 in the presence of l-ethyl-(3-dimethylaminopropyl)-carbodiimide. The NADP derivative is readily hydrolyzed to NADP by the enzyme in brain and nerve that hydrolyzes 2',3'-cyclic nucleotides to 2'-phospho esters. The K _m for this substrate is the same as that for 2',3'-cyclic AMP (0.22 m m ) at pH 6 and 25°C. The two substrates are hydrolyzed by the phosphohydrolase at similar maximum velocities. The nicotinamide moiety in cyclic NADP thus has little effect on the enzyme-substrate interaction. This synthetic substrate can be used in a rapid (2 min) and sensitive (10 ng of 31-fold purified enzyme) spectrophotometric coupled enzyme assay for 2',3'-cyclic nucleotide 3'-phosphohydrolase; in this assay the hydrolysis proceeds in the presence of glucose-6-phosphate dehydrogenase and its substrate and the NADPH formed is measured by the increase in absorbance at 340 nm. The assay is applicable to tissue extracts as well as to purified preparations of the enzyme. There is no interference from nucleases of the pancreatic RNase A type.     

Chick embryo liver beta-glucuronidase. Comparison of activity on natural and artificial substrates.

The beta-glucuronidase in homogenates of 12-day chick embryo livers catalyzed the release of glucuronic acid from 4-methylumbelliferyl-beta-D-glucuronide and from the nonreducing terminals of the hexasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4 at rates of 143, 114, and 108 nmol of glucuronic acid/h/mg of protein, respectively, when assayed at pH 3.5 in 0.05 M sodium acetate buffer. During a 60-fold purification of the enzyme, the ratios of the activities on these substrates did not change. When 4-methylumbelliferyl-beta-D-glucuronide was used as substrate the enzyme was active at pH values from 3.0 to 5.5, with maximal activity between pH values 4.0 and 4.5. Concentrations of NaCl from 0.15 to 0.3 M inhibited the activity at low pH values but activated the enzyme between pH 4.0 and 5.5. The enzyme was active on the chondroitin-6-SO4 hexasaccharide from pH 3.0 to 5.5, with a broad optimum between 3.0 and 4.5. NaCl inhibited the activity on the oligosaccharide substrate at all pH values. Eadie-Scatchard plots of rates of 4-methylumbelliferyl-beta-D-glucuronide hydrolysis at substrate concentrations ranging from 2 to 1000 microM showed multiple kinetic forms of the enzyme, a form with a Km of approximately 11 microM, and a second form with a Km of approximately 225 microM. The pH optimum of the low Km form was 3.5 to 4.0; that of the high Km form was pH 4.5. NaCl inhibited the activity of the low Km form, but activated the high Km form of the enzyme. Chondroitin SO4 oligosaccharides competed with 4-methylumbelliferyl-beta-D-glucuronide for the low Km form of the enzyme but had little effect on the hydrolysis of 4-methylumbelliferyl-beta-D-glucuronide by the high Km form of the enzyme. The activities of the beta-glucuronidase on tetra-, hexa-, octa-, and decasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4, measured using a new assay procedure which can detect the formation of 1 nmol of product, were similar, although rates were somewhat lower for the higher oligosaccharides. With the exception of the chondroitin-4-SO4 tetrasaccharide, all of the oligosaccharide substrates saturated the enzyme at concentrations of 20 to 30 microM, indicating Km values of less than 10 to 15 microM for the oligosaccharides. Highly purified beta-glcuronidases from human placenta and from rat preputial gland also showed multiple kinetic forms when assayed using 4-methylumbelliferyl-beta-D-glucuronide as substrate.     

Magnesium-dependent sphingomyelinase.

An enzyme which requires divalent metals and hydrolyses sphingomyelin to ceramide and phosphorylcholine is present in rat and human brain and practically absent from other organs. The greatest activity is associated with the microsomal fraction. It had an optimal pH at about 7.4, required magnesium or manganese ions and was completely inhibited by EDTA. Triton X-100 was required for optimal activity and this detergent could also be used to partly solubilize the enzyme from rat brain microsomes. Lecithin was hydrolyzed at only 2% of the corresponding rate of hydrolysis of sphingomyelin.     

Oxidation of corticosteroids to steroidal-21-oic acids by human liver enzyme.

An enzyme that oxidizes corticosteroids to acidic metabolites has been purified from postmortem human liver. The most rapidly oxidized substrate was 11-deoxycorticosterone (DOC). Other corticosteroids were oxidized at rates that were 10% or less of DOC. The products of DOC oxidation were 3, 20-dioxopregn-4-en-21-oic acid and 20-hydroxy-3-oxopregn-4-en-21-oic acid. The 20-keto acid was the predominant metabolite in all enzyme preparations. Keto acid and hydroxy acid were not interconverted. Enzyme activity was assayed by measuring the transfer of tritium from [21-3H]DOC to water. The enzyme is yellow, and has spectral maxima at 278 and 405 nm. Inhibition by o-phenanthroline suggests that it may be a metalloenzyme. Molecular weight was estimated at 74 000 +/- 8 000; a pH maximum occurred at pH 8-8.5. This enzyme may participate in the in vivo conversion of corticosteroids to the acidic metabolites that we have described previously (H.L. Bradlow et al. (1973), J. Clin. Endocrinol. Metab. 37, 811).     

Purified Rat Brain Microvessels Exhibit Both Acid and Neutral Sphingomyelinase Activities

Purified rat brain microvessels have been shown to hydrolyze radiolabeled sphingomyelin by means of two different enzyme systems. Enzymatic activity was detected at pH 7.4 and was strongly stimulated by magnesium or manganese and inhibited by calcium. Activity at pH 5.1 could also be found and was not dependent on any of these cations. At neutral pH and in the presence of magnesium, the rate of sphingomyelin hydrolysis did not exhibit a linear relationship with protein concentration. In contrast, increasing the protein concentration from 0.05 to 0.5 mg/ml resulted in a constant increase of sphingomyelin hydrolysis at pH 5.1. Kinetic parameters of both neutral and acid activities have been determined and were similar in magnitude to values reported previously for neural sphingomyelinases. This work demonstrates the occurrence of a neutral sphingomyelinase activity in purified rat brain microvessels, an observation raising the question of its role at the level of the blood-brain interface.     

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.     

Evaluation of the performance of immobilized penicillin G acylase using active-site titration

Penicillin G acylase from Escherichia coli was immobilized on Eupergit C with different enzyme loading. The activity of the immobilized preparations was assayed in the hydrolysis of penicillin G and was found to be much lower than would be expected on the basis of the residual enzyme activity in the immobilization supernatant. Active-site titration demonstrated that the immobilized enzyme molecules on average had turnover rates much lower than that of the dissolved enzyme. This was attributed to diffusion limitations of substrate and product inhibition. Indeed, when the immobilized preparations were crushed, the activity increased from 587 U g-1 to up to 974 U g-1. The immobilized preparations exhibited up to 15% lower turnover rates than the dissolved enzyme in cephalexin synthesis from 7-ADCA and D-(-)-phenylglycine amide. The synthesis over hydrolysis ratios of the immobilized preparations were also much lower than that of the dissolved enzyme. This was partly due to diffusion limitations but also to an intrinsic property of the immobilized enzyme because the synthesis over hydrolysis ratio of the crushed preparations was much lower than that of the dissolved enzyme.     

Purification and characterization of a cyclic nucleotide-regulated 5'-nucleotidase from potatoe.

A procedure is presented for the rapid purification of a 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) from potato tubers, involving ammonium sulphate fractionation and chromatography on phosphocellulose, DEAE-cellulose and Sephadex G-75. Application of this procedure results in a 6000-fold purification of the 5'-nucleotidase and the final preparations are virtually homogeneous, yielding only one protein band on electrophorsis in polyacrylamide gels in non-dissociating or dissociating conditions. The 5'-nucleotidase has a molecular weight of 50 000 from gel filtration experiments. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified 5'-nucleotidase reveals one major band of molecular weight 25 000. The 5'-nucleotidase is competitively inhibited by cyclic nucleotides, having micromolar Ki values for cyclic AMP and cyclic GMP at pH 5.0 and pH 8.0. The enzyme has a pH optimum of 5.0 with 5'-GMP as substrate. While 5'-AMP and 3'-AMP are hydrolyzed at comparable rates at pH 5.0, at pH 8.0 the rate of hydrolysis of 3'-AMP is only 4% of that with 5'-AMP. ADP, ATP and 2'-AMP are very poor substrates for the enzyme. The nucleotidase has micromolar Km values for nucleoside 5'-monophosphates other than 5'-NMP. A wide variety of divalent cations activate the 5'-nucleotidase.     

The pH dependence of breakdown of various purified brain proteins by cathepsin D preparations

In a continuing study of control processes of cerebral protein catabolism we compared the activity of cathepsin D from three sources (rat brain, bovine brain, and bovine spleen) on purified CNS proteins (tubulin, actin, calmodulin, S-100 and glial fibrillary acidic protein). The pH optimum was 5 for hydrolysis with tubulin as substrate for all three enzyme preparations, and it was pH 4 with the other substrates. The pH dependence curve was somewhat variable, with S-100 breakdown relatively more active at an acidic pH range. The formation of initial breakdown products and the further catabolism of the breakdown products was dependent on pH; hence the pattern of peptides formed from glial fibrillary acidic protein was different in incubations at different pH's. The relative activity of the enzyme preparations differed, depending on the substrate: with tubulin and S-100 as substrates, rat brain cathepsin D was the most active and the bovine spleen enzyme was the least active. With calmodulin and glial fibrillary acidic protein as substrates, rat brain and spleen cathepsin D activities were similar, and bovine brain cathepsin D showed the lowest activity. Actin breakdown fell between these two patterns.The rates of breakdown of the substrates were different; expressed as μg of substrate split per unit enzyme per h, with rat brain cathepsin D activity was 8–9 with calmodulin and S-100, 4 with glial fibrillary acidic protein, 1.8 with actin, and 0.9 with tubulin. The results show that there are differences in the properties of a protease like cathepsin D, depending on its source; furthermore, the rate of breakdown and the characteristics of breakdown are also dependent on the substrate.We recently measured the breakdown of brain tubulin by cerebral cathepsin D in a continuing study of the mechanisms and controls of cerebral protein catabolism (Bracco et al., 1982a). We found that tubulin breakdown is heterogeneous, that membrane-bound tubulin is resistant to cathepsin D but susceptible to thrombin (Bracco et al., 1982b), and that cytoplasmic tubulin was in at least two pools, one with a higher, another with a lower, rate of breakdown. The pH optimum of tubulin breakdown by cerebral cathepsin D differed significantly from the pH optimum of hemoglobin breakdown by the same enzyme.These findings showed that the properties of breakdown by a cerebral protease depend on the substrate. To further examine this dependence of properties of breakdown on the substrate, we now report measurements of pH dependence of breakdown of several purified proteins (tubulin, actin, calmodulin, S-100, glial fibrillary acidic protein [GFA]) from brain by cathepsin D preparations from three sources, rat brain, bovine brain, and bovine spleen. We also compare the rate of breakdown of the various proteins with the rate of hemoglobin breakdown.     

Hydrolysis of sphingomyelin and phosphatidylcholine by midgut homogenates of the stable fly

Qualitative and quantitative analyses were made to characterize the enzymatic degradation of sphingomyelin and phosphatidylcholine by midgut homogenates of the adult stable fly, Stomoxys calcitrans (L.). The results indicated that sphingomyelin was hydrolyzed by an enzyme with sphingomyelinase-like properties, and that phosphatidylcholine was hydrolyzed by an enzyme with properties similar to phospholipase C. The optimum pH for the sphingomyelinase was 7.6, and the rate of hydrolysis of sphingomyelin at that pH was linear from 1 to 4 nmol of substrate and 5 to 25 micrograms of enzyme preparation. Dialysis of the homogenates against Tris-HCl and imidazole buffers resulted in a decrease of sphingomyelinase activity by 59% and 98%, respectively, and the original activity was not restored with the addition of Ca++, Mg++, or Mn++.     

Sphingomyelinase activity at pH 7.4 in human brain and a comparison to activity at pH 5.0.

A hitherto undescribed sphingomyelinase (sph'ase 7.4) of human brain has been studied in crude and partially purified (3- to 4- fold) extracts of grey matter, and compared to the known sphingomyelinase with an acid pH optimum (sph'ase 5.0). Its specificity for sphingomyelin as substrate is similar to that of sph'ase 5.0, but it differs from sph'ase 5.0 in its pH optimum (7.4 vs 5.0) and in a requirement for Mg2+ for optimal activity. Other properties of sph'ase 7.4 that distinguish it from sph'ase 5.0 include (a) its lack of appreciable solubilization during dialysis of crude homogenates (b) a more marked concentrations in grey matter than in white matter (9- to 13- fold vs 1.5- to 2-fold for sph'ase 5.0); (c) inhibition by Ca2+ and Cd2+ ions, and by EDTA; (D) stimulation by dithiothreitol, and inhibition by cysteine, N-ethylmaleimide, and p-hydroxymercuribenzoate; (e) lack of inhibition by nucleotides (AMP.ADP, and ATP) and by NAD plus NADH; and (f) relative instability to storage or manipulation between -20degrees C and 40degrees C. These differences indicate the SPH'ASE 7.4 is a different enzyme protein from sph'ase 5.0. Unlike sph'ase 5.0, which is widely distributed in mammalian tissues, sph'ase 7.4 occurs predominantly in grey matter and little activity was observed is spleen, liver, or leukocytes. The high levels of this enzyme in brain suggest a role related to the specific functions of this organ or to the need for a more stringent control of sphingomyelin catabolism in brain as compared to other organs.     

A lipid analogue that inhibits sphingomyelin hydrolysis and synthesis, increases ceramide, and leads to cell death

We report the synthesis and characterization of a novel thiourea derivative of sphingomyelin (AD2765). In vitro assays using pure enzyme and/or cell extracts revealed that this compound inhibited the hydrolysis of BODIPY-conjugated or 14C-labeled sphingomyelin by acid sphingomyelinase and Mg2+-dependent neutral sphingomyelinase. Studies in normal human skin fibroblasts further revealed that AD2765 was taken up by cells and inhibited the hydrolysis of BODIPY-conjugated sphingomyelin in situ. In situ and in vitro studies also showed that this compound inhibited the synthesis of sphingomyelin from BODIPY-conjugated ceramide. The specificity of AD2765 for enzymes involved in sphingomyelin metabolism was demonstrated by the fact that it had no effect on the hydrolysis of BODIPY-conjugated ceramide by acid ceramidase or on the synthesis of BODIPY-conjugated glucosylceramide from BODIPY-conjugated ceramide. The overall effect of AD2765 on sphingomyelin metabolism was concentration-dependent, and treatment of normal human skin fibroblasts or cancer cells with this compound at concentrations > 10 microM led to an increase in cellular ceramide and cell death. Thus, AD2765 might be used to manipulate sphingomyelin metabolism in various ways, potentially to reduce substrate accumulation in cells from types A and B Niemann-Pick disease patients, and/or to affect the growth of human cancer cells.     

Hydrolysis of lipid monolayers and the substrate specificity of hepatic lipase

The substrate specificities of the phospholipase and triglyceridase activities of purified rat liver hepatic lipase were compared using lipid monolayers so that the substrates were presented to the enzyme in a controlled physical state. The rate of hydrolysis of 14C-labeled lipid at constant surface pressure in the presence of hepatic lipase and fatty acid-free bovine serum albumin at 33 degrees C was determined by monitoring the decrease of surface radioactivity. In monolayers of sphingomyelin/cholesterol (2:1, mol/mol) containing either 1 mol% triacylglycerol, 1 mol% phosphatidylethanolamine, or 10 and 20 mol% phosphatidylcholine, hepatic lipase clearly showed a preference for unsaturated over saturated lipids. In addition, with a sphingomyelin/cholesterol (2:1) monolayer containing 1 mol% of lipid substrate, hepatic lipase showed the following preference: triolein = dioleoylphosphatidylethanolamine much greater than dioleoylphosphatidylcholine; the respective rates of hydrolysis were 15.3 +/- 1.2, 14.9 +/- 0.8, and 0.5 +/- 0.1 mumol fatty acid produced/h per mg hepatic lipase. Overall, it appears that when comparing rates of hydrolysis of molecules within a given lipid class, hydrocarbon chain interactions are important. However, when comparing different lipid classes such as phosphatidylcholines and phosphatidylethanolamines, it is apparent that the polar group has a significant influence on the rate of hydrolysis. The rate of [14C]triolein hydrolysis, when mixed at surface concentrations of up to 2 mol% in a sphingomyelin/cholesterol (2:1) monolayer, was significantly faster than when triolein was present in a 1-oleyl-2-palmitylphosphatidylcholine monolayer; the rates of hydrolysis were 47.7 +/- 5.4 and 8.9 +/- 0.8 mumol fatty acid produced/h per mg hepatic lipase, respectively. The monolayer physical state and the miscibility of the substrate in the inert matrix influence the presentation of the substrate to the enzyme, thereby affecting the hydrolysis rate.     

Activity of human hepatic beta-galactosidase toward natural glycosphingolipid substrates.

1. Human hepatic "acid" beta-galactosidase preparations, which had been purified approximately 250-fold, were examined for activities toward 4-methylumbelliferyl beta-galactoside, galactosylceramide, lactosylceramide, galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosyl-glucosylceramide (GM1-Ganglioside) and galactosyl-Cacetylgalactosaminyl-galactosyl-glucosylceramide (asialo GM1-ganglioside). 2. The enzyme was active toward the synthetic substrate, GM1-ganglioside and asialo GM1-ganglioside but was inactive toward galactosylceramide. Under our assay conditions, optimized for lactosylceramidase II, the preparations were as active toward lactosylceramide as toward GM1-ganglioside or its asialo derivative. Teh apparent Km values for the three natural substrates were similar. When determined by the assay system of Wenger, D.A., Sattler, M., Clark, C. and McKelvey, H. (1974) Clin. Chim. Acta 56, 199-206, lactosylceramidecleaving activity was 0.2% of that determined by our assay system. This confirmed our previous suggestion that the Wenger assay system determines exclusively the activity of lactosylceramidase I, which is probably identical with galactosylceramide beta-galactosidase. 3. Crude sodium taurocholate was far more effective than pure taurocholate in stimualting hydrolysis of the three glycosphingolipids by the beta-galactosidase. However, crude tauroxycholate, suggesting that the unique activating capacity of the crude taurocholate might be due to taurodeoxycholate present as the major impurity. 4. Cl- was generally stimulatory for hydrolysis of the natural glycosphingolipids by our enzyme preparation. Effects of additional oleic acid and Triton X-100 Were generally minor in either direction. 5. When the enzyme preparation was diluted with water, activity toward the synthetic substrate declined rapidly while those toward the natural substrates were essentially stable. Activity toward the synthetic substrate remained much more stable when the enzyme was diluted with 0.1 M sodium citrate/phosphate buffer, pH 5.0. 6. These observations provide insight into the complex relationship among the human hepatic beta-galactosidases.     

Purification and properties of a phospholipase C that has high activity toward sphingomyelin from Aspergillus saitoi

An enzyme hydrolyzing sphingomyelin was purified from extracts of solid cultures of Aspergillus saitoi 7041 by fractionation with isopropanol followed by successive column chromatographies on DEAE-Sepharose CL-6B, butyl-Toyopearl 650 M, and phenyl-Sepharose CL-4B. The preparation of purified enzyme was homogeneous and had an activity increased 81-fold over that of the isopropanol fraction. The yield was about 65%. The molecular weight was estimated to be 54,000 by sodium dodecyl sulfate-gel electrophoresis. The enzyme solution had a violet color and contained iron atoms. The enzyme catalyzed the hydrolysis of sphingomyelin to N-acylsphingosine and phosphorylcholine. The optimum pH for hydrolytic activity was around 3.5. The Km values for sphingomyelin and 2-hexadecanoylamino-4-nitrophenylphosphorylcholine were 0.11 and 0.33 mM, respectively. The enzyme also catalyzed the hydrolysis of other phospholipids; the order of its hydrolytic activity at a substrate concentration of 2.5 mM was phosphatidylcholine greater than or equal to sphingomyelin = phosphatidylethanolamine = lysophosphatidylethanolamine greater than phosphatidyl DL-glycerol = phosphatidyl L-serine greater than phosphatidylinositol. From these results, this enzyme appears to be a new type of phospholipase C(phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3).     

Enzymatic properties of immobilized beta-galactosidase from Curvularia inaequalis]

beta-Galactosidase (EC 3.2.1.23) from fungus Curvularia inaequalis was modified by active brilliant orange KH and adsorbed on DEAE-Sephadex A-50. The lactose hydrolysis was studied in a continous flow on the column packed with the immobilized enzyme. The pH and temperatures optima for the substrate hydrolysis by the immobilized enzyme were shown to remain unchanged. A certain destabilizing effect of the matrix on the enzyme resistance to hear denaturation was observed. The activation parameters of denaturation of the native enzyme as well as those of the dye-modified and immobilized preparations were determined.