Purification and characterization of myosin from bovine retina

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca²⁺ and a low activity in the presence of Mg²⁺. The Mg²⁺-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal mucle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

Purification, characterization and substrate specificity of calmodulin-dependent myosin light-chain kinase from bovine brain.

A substrate-specific calmodulin-dependent myosin light-chain kinase (MLCK) was purified 45,000-fold to near homogeneity from bovine brain in 12% yield. Bovine brain MLCK phosphorylates a serine residue in the isolated turkey gizzard myosin light chain (MLC), with a specific activity of 1.8 mumol/min per mg of enzyme. The regulatory MLC present in intact gizzard myosin is also phosphorylated by the enzyme. The Mr-19,000 rabbit skeletal-muscle MLC is a substrate; however, the rate of its phosphorylation is at best 30% of that obtained with turkey gizzard MLC. Phosphorylation of all other protein substrates tested is less than 1% of that observed with gizzard MLC as substrate. SDS/polyacrylamide-gel electrophoresis of purified MLCK reveals the presence of a major protein band with an apparent Mr of 152000, which is capable of binding 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of MLCK by the catalytic subunit of cyclic-AMP-dependent protein kinase results in the incorporation of phosphate into the Mr-152,000 protein band and a marked decrease in the affinity of MLCK for calmodulin. The presence of Ca2+ and calmodulin inhibits the phosphorylation of the enzyme. Bovine brain MLCK appears similar to MLCKs isolated from platelets and various forms of muscle.     

Purification and characterization of myosin from bovine retina.

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca2+ and a low activity in the presence of Mg2+. The Mg2+-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal muscle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

Purification and identification of myosin heavy chain kinase from bovine brain

A high salt extract of bovine brain was found to contain a protein kinase which catalyzed the phosphorylation of heavy chain of brain myosin. The protein kinase, designated as myosin heavy chain kinase, has been purified by column chromatography on phosphocellulose, Sephacryl S-300, and hydroxylapatite. During the purification, the myosin heavy chain kinase was found to co-purify with casein kinase II. Furthermore, upon polyacrylamide gel electrophoresis of the purified enzyme under non-denaturing conditions, both the heavy chain kinase and casein kinase activities were found to comigrate. The purified enzyme phosphorylated casein, phosvitin, troponin T, and isolated 20,000-dalton light chain of gizzard myosin, but not histone or protamine. The kinase did not require Ca2+-calmodulin, or cyclic AMP for activity. Heparin, which is known to be a specific inhibitor of casein kinase II, inhibited the heavy chain kinase activity. These results indicate that the myosin heavy chain kinase is identical to casein kinase II. The myosin heavy chain kinase catalyzed the phosphorylation of the heavy chains in intact brain myosin. The heavy chains in intact gizzard myosin were also phosphorylated, but to a much lesser extent. The heavy chains of skeletal muscle and cardiac muscle myosins were not phosphorylated to an appreciable extent. Although the light chains isolated from brain and gizzard myosins were efficiently phosphorylated by the same enzyme, the rates of phosphorylation of these light chains in the intact myosins were very small. From these results it is suggested that casein kinase II plays a role as a myosin heavy chain kinase for brain myosin rather than as a myosin light chain kinase.     

Isolation and characterization of calcineurin from bovine brain

Calcineurin was isolated from bovine cerebrum extracts by sequential chromatography on Affi-Gel blue and calmodulin affinity columns. Calcineurin so isolated was approximately 90% pure and was composed of equimolar amounts of subunit A (Mr = 61 000-63 000) and subunit B (Mr = 15 000-17 000) when examined by sodium dodecyl sulfate gel electrophoresis. A polypeptide (less than 10%) with Mr = 71 000 whose function and role remains to be investigated, was routinely detected in the calcineurin preparation. Both inhibitory activity (towards calmodulin-dependent cAMP phosphodiesterase) and phosphatase activity (with 32P-labelled myelin basic protein as substrate) were associated with calcineurin as evidenced by (i) coelution from Affi-Gel blue, Affi-Gel calmodulin, diethythaminoethyl-Sepharose, and Sephacryl S-200 chromatography columns; (ii) association with the same protein band on nondenaturing gels; (iii) similar stability upon storage at 4 degrees C and with repeated freezing and thawing; and (iv) parallel heat inactivation. Phosphatase activity of calcineurin was maximal with 32P-labelled myelin basic protein as the substrate. Using this substrate, enzyme activity was generally stimulated 5- to 10-fold in the presence of Ca2+ and calmodulin; half-maximal activation (A0.5) was observed with 25 nM calmodulin. Calmodulin increased the Vmax of the reaction without affecting the Km for the substrate. Optimum temperature and pH for the reaction were 45 degrees C and 7, respectively, in both the absence and presence of Ca2+ and calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characterization of a novel cholinergic neuron-specific ganglioside in bovine brain.

A new ganglioside antigen, termed Chol-1 alpha-b, recognized by cholinergic neuron-specific antibody, Chol-1 alpha, was isolated from bovine brain ganglioside mixture using Q-Sepharose. The yield was approximately 1.3 mg from 5 g of the total ganglioside. The chemical structure was characterized as a novel ganglioside by means of gas-liquid chromatography, a permethylation study, mild acid hydrolysis, thin layer chromatography-enzyme immunostaining, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The ganglioside has the following unique structure. [formula: see text] When examined by thin layer chromatography immunostaining and enzyme-linked immunosorbant assays, this ganglioside has the most intense immunoreactivity with Chol-1 alpha antibody among bovine brain gangliosides. As combined with our previous results (Hirabayashi, Y., Hyogo, A., Nakao, T., Tsuchiya, K., Suzuki, Y., Matsumoto, M., Kon, K., and Ando, S. (1990) J. Biol. Chem. 265, 8144-8151; Ando, S., Hirabayashi, Y., Kon, K., Inagaki, F., Tate, S., and Whittaker, X. (1992) J. Biochem. (Tokyo), 111, 287-290), the present study indicates the occurrence of a new series of gangliosides containing N-acetylneuraminic acid residue attaching to N-acetylgalactosamine as cholinergic specific antigens.     

Solubilization and characterization of muscarinic receptors from bovine brain

This study compared the capacity of different detergents to solubilize the muscarinic cholinergic receptor (mAChR) from bovine brain, evaluated various procedures for the measurement of [3H]-L-quinuclidinyl benzilate [( 3H]-L-QNB) binding to solubilized receptors, and examined some physical and chemical characteristics of the soluble material. An active form of the mAChR was solubilized using digitonin (1%), Triton X-100 (0.5%), and a digitonin-cholate mixture (1%, 0.1%). Values of maximal binding (Bmax) were 2.01, 0.47, and 0.68 pmoles/mg protein, respectively. Comparison of equilibrium dialysis, charcoal adsorption, and polyethylene glycol precipitation indicated that these methods differ in their estimation of Bmax. A decrease in [3H]-L-QNB binding to digitonin solubilized receptors occurred upon dilution or incubation at 37 degrees. The half-life at 37 degrees C was 25 min., but was increased by glycerol. Antagonist binding to digitonin solubilized receptors was saturable and of high affinity. Agonist binding had Hill coefficients less than 1 and was increased by micromolar concentrations of cupric ions.     

Isolation and characterization of cathepsin B from bovine brain

Cathepsin B (EC 3.4.22.1) was purified 746-fold with a 21% recovery from bovine brain by autolysis, fractional precipitation with acetone, carboxy-methyl-Sephadex chromatography, affinity chromatography on a cystamine containing column and gel filtration chromatography. The purified cathepsin B eluted on gel filtration with an apparent molecular weight of 27,000 but was resolved into three bands of 30,000, 25,000 and 5,000 molecular weight by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Antibodies to cathepsin B, raised against the 30,000 dalton band, were shown by immunoblots to react with both the 30,000 and 25,000 dalton proteins with results suggesting that the former predominated as the immunoreactive form in bovine brain homogenates. Isoelectric focusing demonstrated multiple bands, ranging from pH 4.75–5.2 with the major band at pH 5.1–5.2, all of which were capable of degrading N-carbobenzoxy-l-arginyl-l-arginine 4-methoxy--naphthylamide. The cathepsin B activity against N-benzoyl-dl-arginine -naphthylamide (BANA) and bovine myelin basic protein (MBP) had a pH optimum of pH 6.0. The Km for the degradation of BANA was 1.0 mM and 5.1 mM when assayed in the presence of 1% and 2.5% dimethylsulfoxide, respectively. Cathepsin B from bovine brain has many properties similar to cathepsin B isolated from other organs. The degradative effect of cathepsin B on MBP suggests a role for this proteinase in inflammatory demyelination.     

Purification and characterization of squid brain myosin.

Myosin was extracted from frozen squid brain and purified by a modification of the procedure of Pollard et al. (Pollard, T.D., Thomas, S.M., and Niederman, R. (1974) Anal. Biochem. 60, 258-266). Myosin was eluted from Bio-Gel A-15m column as a single peak of (K+-EDTA)-activated ATPase ((K+-EDTA)-ATPase) activity with an average partition coefficient (Kav) of 0.22. In sodium dodecyl sulfate-acrylamide gel electrophoresis, the purified myosin showed a predominant band with similar electrophoretic mobility as the heavy chain of rabbit skeletal muscle myosin, and two less intense bands near the bottom of the gel. No actin band was seen. The properties of the (K+-EDTA)-ATPase activity were: (a) the time course of the reaction was biphasic at 25 degrees but linear at 32 degrees; (b) the optimum rate of reaction was obtained between 0.3 and 0.8 M KCl; (c) the pH optimum was between 8.0 and 9.0; (d) the reaction was specific for ATP with an apparent Km of 0.19 mM. ATPase activity in 0.06 M KCl and 5 mM MgCl2 was increased about 1.5 times by a 10-fold excess of rabbit skeletal muscle F-actin and about 5 times by a 40-fold excess. The actin activation was inhibited slightly by the addition of 0.2 mM CaCl2 and completely by the addition of 10 mM CaCl2. Myosin formed arrowhead patterns with rabbit skeletal muscle F-actin as observed by electron microscopy of negatively stained samples. It also aggregated in bipolar filaments which attached to decorated actin filaments at different angles, as well as formed cross-connections and ladder-like patterns between actin filaments. These two forms of interactions between myosin and actin were abolished by treatment with MgATP.     

Isolation from bovine brain of 155 kDa component exhibiting myosin light chain kinase activity

1) Taking myosin light chain kinase (MLCK) activity as the index, bovine extract was fractionated by the use of anion-exchange chromatography, cation-exchange chromatography, and calmodulin affinity chromatography. The kinase activity of the fraction thus obtained was elevated up to about 12,400 times over that of the original crude extract. 2) The fraction mentioned above was subjected again to anion exchange chromatography. The kinase activities were divided into two parts, i.e., part I which contained the 155 kDa component and part II which was virtually free of 155 kDa component. The MLCK activity of part I was considerably lower than that of part II. 3) Part I was subjected to gel filtration using AcA 34 gel and the 155 kDa component was isolated. The fraction contained the 155 kDa component in a homogeneous state and showed myosin specific kinase activity, which was about 2 X 10(5) times that of the original crude extract. 4) The high kinase activity of part II seemed to be ascribable to the 130 kDa component, in accord with the report of Hathaway, Adelstein, and Klee (J. Biol. Chem. 256, 8183-8189, 1981).     

Purification and characterization of fructose-1,6-bisphosphatase from bovine brain

Fructose-1,6-bisphosphatase from bovine brain tissue has been purified to near homogeneity. This enzyme is similar to other mammalian fructose-1,6-bisphosphatases in many respects, and its properties are distinctly different from those reported for the enzyme from rat brain [A. L. Majumder and F. Eisenberg (1977) Proc. Natl. Acad. Sci. USA 74, 3222-3225; S. Chattoraj and A. L. Majumder (1986) Biochem. Biophys. Res. Commun. 139, 571-580]. The bovine enzyme (sp act 4, pH ratio (7.5/9.6) = 3.6) has a pH optimum of 7.5. The Km is 2 microM. Divalent metal ion is required for activity, and Vmax is obtained at either 4 mM Mg2+ or 0.3 mM Mn2+. Fructose 2,6-bisphosphate is a competitive inhibitor (Ki = 0.07 microM), and AMP a noncompetitive inhibitor (kis = 24 microM, Kii = 10 microM) of bovine brain fructose-1,6-bisphosphatase. The enzyme activity is enhanced by small amounts of EDTA relative to metal, and AMP inhibits fructose-1,6-bisphosphatase in either the presence or absence of the metal chelator; however, AMP is more effective in the absence of EDTA.     

Partial purification and characterization of sialate O-acetylesterase from bovine brain

From bovine brain an esterase was purified 2,600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8-6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e.g. alpha-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-4-O-acetylneuraminic acid. Other primary acetyl esters such as 6-O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, however, is a substrate for this esterase. Whereas N-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. N-Acetylated sialic acids are better substrates than the analogous N-glycoloyl derivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and partial characterization of glyoxalase I from bovine brain

Glyoxalase I was purified to homogeneity from bovine brain using affinity chromatography on S-hexylglutathione-Sepharose 6B with a yield of 22%. The enzyme was a dimer (44,000 Daltons) composed of, apparently, identical subunits (22,000 Daltons), as shown by SDS electrophoresis, and contained one mole of Zn2+/monomer. The active site metal ion, Zn2+, was removed by dialysis against EDTA, but the activity of the apoenzyme obtained was not completely restored after addition of Co2+ and Zn2+ (<25%), while a recovery of 50% was obtained after addition of Mg2+. The enzyme was inhibited by S-bromobenzylglutathione and S-p-nitrobenzylglutathione with a Ki value of 21 microM and 32 microM, respectively. The highest dissociation constant observed for the brain enzyme with respect to that reported for human erythrocytes, or other mammalian forms of enzyme could be related to a tissue-specific dependence of the glyoxalase I activity.     

Purification and characterization of thiamine triphosphatase from bovine brain.

Soluble thiamine triphosphatase (EC 3.6.1.28) of bovine brain has been purified 68,000-fold to an electrophoretically homogeneous state with an overall recovery of 5.5% by hydrophobic chromatography on Toyopearl HW-60, Sephadex G-75 gel filtration, DEAE-Toyopearl 650M chromatography and Blue Sepharose CL-4B chromatography. The enzyme has an absolute specificity among thiamine and nucleoside phosphate esters for thiamine triphosphate and shows no nonspecific phosphatase activities. Thiamine triphosphatase is composed of a single polypeptide chain with molecular mass of 33,900 kDa as estimated by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 8.7 and is dependent on divalent metal ions. Mg2+ has been found to be the most effective among cations tested. A study of the reaction kinetics over a wide range of thiamine triphosphate concentrations has revealed a biphasic saturation curve being described by higher-degree rational polynomials.     

Purification and characterization of glycolipid transfer protein from bovine brain

Bovine brain contains a lipid transfer protein that is specific for neutral glycosphingolipids and gangliosides but does not stimulate phospholipid or neutral lipid intermembrane transfer (Brown, R.E., Stephenson, F.A., Markello, T., Barenholz, Y. and Thompson, T.E. (1985) Chem. Phys. Lipids 38, 79-93). This report describes a new procedure for purifying glycolipid transfer protein from bovine brain as well as a characterization of the resulting protein. Chief among the newly introduced approaches are dye-ligand and fast protein cation-exchange liquid chromatography. Other modifications include increasing the overall scale of purification, incorporating a pH precipitation step and adding different proteinase inhibitors. The resulting procedure simplifies and accelerates the purification process while yielding a homogeneous protein. The purified protein has a molecular weight near 23 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chromatofocusing reveals that glycolipid transfer protein activity co-elutes with the 23 kDa protein and has an isoelectric point near pH 9.0. A similar isoelectric point is observed using denaturing isoelectric focusing conditions. The protein's amino acid composition reveals high levels of amino acids with non-polar side chains (48%). Based on the findings reported here and on previously published data, bovine brain glycolipid transfer protein has been compared to other lipid transfer proteins as well as lysosomal sphingolipid activator proteins.     

Structural characterization of beta-cardiac myosin subfragment 1 in solution

beta-cardiac myosin subfragment 1 (betaS1) tertiary structure and dynamics were characterized with proteolytic digestion, nucleotide analogue trapping kinetics, and intrinsic fluorescence changes accompanying nucleotide binding. Proteolysis of betaS1 produces the 25, 50, and 20 kDa fragments and a new cut within the 50-kDa fragment at Arg369. F-actin inhibits cleavage of the 50-kDa fragment and fails to inhibit cleavage at the 50/20 kDa junction, suggesting betaS1 presents an actoS1 conformation fundamentally different from skeletal S1. Time-dependent changes in Mg(2+)-ATPase accompanying proteolysis identifies cleavage points that lie within the energy transduction pathway. The nucleotide analogue trapping kinetics reveal the presence of a reversible weakly actin attached state. Comparison of nucleotide analogue induced betaS1 structures with the transient structures occurring during ATPase indicates analogue induced and transient structures are in a one-to-one correspondence. Tryptophan fluorescence enhancement accompanies the binding or trapping of nucleotide or nucleotide analogues. Isolation of Trp508 fluorescence shows it is an ATP-sensitive tryptophan and that its vicinity changes conformation sequentially with the transient intermediates accompanying ATPase. These studies elucidate energy transduction and suggest how mutations of betaS1 implicated in disease might undermine function, stability, or efficiency.     

Isolation,characterization and localization of bovine adrenal medullary myosin

Summary Myosin was isolated in high purity from the bovine adrenal medulla by gel filtration and ion exchange chromatography. The purified myosin was analyzed by electrophoresis in gels containing SDS and found to contain a 200,000 molecular weight heavy chain and major light chains of molecular weights 20,000 and 17,000 in a 111 molar ratio. At high ionic strength the myosin had high Ca-ATPase and K-EDTA-ATPase activities and low Mg-ATPase activity. At low ionic strength, the Mg-ATPase was activated to a low level by rabbit muscle actin. The myosin was found to decorate F-actin in the absence, but not the presence of ATP. In low ionic strength solutions, the myosin assembled into characteristic bipolar filaments.The distribution of this myosin in the adrenal medulla and of cross-reacting myosin in several other bovine tissues was determined with the use of antimedullary myosin immunoglobulin G as a specific stain that was detected by direct and indirect immunofluorescence. In the medulla strong staining was seen between the chords of chromaffin cells indicating the presence of a highly muscular vasculature that may perform functions analogous to those of the myoepithelium of exocrine glands. The chromaffin cells showed weak positive staining around the nuclei and in a pattern radiating toward adjacent blood vessels. Cells of the inner zone of the adrenal cortex showed strong staining in the peripheral cytoplasm while cells in the intermediate and outer zones did not stain. In a blood smear, platelets and the cytoplasm of leukocytes stained strongly while erythrocytes did not stain. In striated muscle and the gray and white matter of the cerebrum only the capillaries and larger vessels stained. In the liver the phagocytic cells bordering vascular sinuses stained strongly while the hepatocytes were separated from one another by a 2 micron trilaminar band possibly representing the microfilament web surrounding the bile canaliculi and associated with junctional complexes.The results suggest that myosin is present in several highly differentiated, non-motile tissue cells where it may play a role in secretion or other specialized functions.The author gratefully acknowledges the support and encouragement received from Francis D. Carlson (Johns Hopkins University) and Harvey B. Pollard (National Institutes of Health) in whose laboratories the majority of this work was performed, as well as additional advice and assistance from John Cebra, Richard Cone, William F. Harrington, Shin Lin, Robert Wyllie and the members of their laboratories     

Structural analysis of the carbohydrate chains of beta-N-acetylhexosaminidases from bovine brain.

The oligosaccharide structures of bovine brain beta-N-acetylhexosaminidases A and B (EC 3.2.1.30) were studied at the glycopeptide level by employing 500 MHz 1H-n.m.r. spectroscopy and methylation analysis involving g.l.c.-m.s. More than 90% of the chains were found to be of the oligomannoside type, containing, on average, five to six mannose residues. Biantennary N-acetyl-lactosamine-type chains terminated in N-acetylneuraminic acid were found to comprise the remaining 5-10% of the total carbohydrate. The isoenzyme forms A and B do not differ from each other in the structure of their carbohydrate moiety, but do deviate in carbohydrate content and, in consequence, in the number of carbohydrate chains per molecule.     

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.