Isolation, characterization and immunological determination of basement membrane-associated heparan sulfate proteoglycan

Basement membrane-associated heparan sulfate proteoglycan (HSPG) was extracted from isolated porcine glomerular basement membranes and purified by ion-exchange chromatography. The proteogycan was characterized by specific enzymatic digestions, by amino-acid analysis, by SDS-polyacrylamide gel electrophoresis and by density gradient centrifugation. Polyclonal antibodies were raised against the purified HSPG in rabbits. Antibodies were characterized by enzyme immunoassays, immunoprecipitation and immunohistological methods. They were shown to recognize specifically the core protein of HSPG from porcine, human and rat glomerular basement membrane but did not recognize HSPG from guinea pig or rabbit kidney. The affinity-purified antibodies did not cross-react with other basement membrane proteins like laminin, fibronectin or collagen type IV nor with chondroitin sulfate-rich or keratan sulfate-rich proteoglycans from human or bovine tissue. Using these antibodies an enzyme immunoassay was developed for determination of HSPG in the range of 1-100 ng/ml. Studies with cultured porcine endothelial cells showed that subendothelial basement membrane-associated HSPG may be determined with the enzyme immunoassay.     

Immunological and biochemical evidence for atrial-like isomyosin in thyrotoxic rabbit ventricle

Immunological, structural and enzymatic characteristics of atrial and ventricular myosin from euthyroid rabbits were analyzed and compared with ventricular myosin from hyperthyroid animals. (1) Specific antibodies against bovine atrial myosin were found to react selectively in double-immunodiffusion and enzyme immunoassay with both rabbit atrial myosin and ventricular myosin from thyroxine-treated animals. These specific anti-bovine atrial myosin antibodies reacted with the heavy chains of thyrotoxic ventricular myosin when examined by enzyme immunoassay combined with SDS-polyacrylamide gel electrophoresis. (2) In one-dimensional SDS-polyacrylamide gel electrophoresis, no difference could be demonstrated in the light chain pattern of ventricular myosin from euthyroid and hyperthyroid rabbit hearts. One-dimensional analysis of myosin heavy chains after chymotryptic digestion in the presence of SDS showed significant differences between the two ventricular myosins. Also, the peptide maps from atrial myosin resembled the pattern of peptides found with ventricular heavy chains from hyperthyroid rabbits. The steady state rate, the alkali stability and the pH sensitivity of Ca2+-ATPase activity of thyrotoxic ventricular myosin were very similar to those of atrial myosin. (3) These results provide direct immunochemical and biochemical evidence for the existence of an atrial-like isomyosin in thyrotoxic rabbit ventricles.     

Myosin types and fiber types in cardiac muscle. I. Ventricular myocardium

Antisera against bovine atrial myosin were raised in rabbits, purified by affinity chromatography, and absorbed with insolubilized ventricular myosin. Specific anti-bovine atrial myosin (anti-bAm) antibodies reacted selectively with atrial myosin heavy chains, as determined by enzyme immunoassay combined with SDS-gel electrophoresis. In direct and indirect immunofluorescence assay, anti-bAm was found to stain all atrial muscle fibers and a minor proportion of ventricular muscle fibers in the right ventricle of the bovine heart. In contrast, almost all muscle fibers in the left ventricle were unreactive. Purkinje fibers showed variable reactivity. In the rabbit heart, all atrial muscle fibers were stained by anti-bAm, whereas ventricular fibers showed a variable response in both the right and left ventricle, with a tendency for reactive fibers to be more numerous in the right ventricle and in subepicardial regions. Diversification of fiber types with respect to anti-bAm reactivity was found to occur during late stages of postnatal development in the rabbit heart and to be influenced by thyroid hormone. All ventricular muscle fibers became strongly reactive after thyroxine treatment, whereas they became unreactive or poorly reactive after propylthiouracil treatment. These findings are consistent with the existence of different ventricular isomyosins whose relative proportions can vary according to the thyroid state. Variations in ventricular isomyosin composition can account for the changes in myosin Ca2+-activated ATPase activity previously observed in cardiac muscle from hyper- and hypothyroid animals and may be responsible for the changes in the velocity of contraction of ventricular myocardium that occur under these conditions. The differential distribution of ventricular isomyosins in the normal heart suggests that fiber types with different contractile properties may coexist in the ventricular myocardium.     

Identification of two calcineurin alpha isoforms in bovine brain by two different monoclonal antibodies.

Calcineurin (calcium- and calmodulin-stimulated phosphatase) alpha subunit purified from bovine brain was found to be composed of two polypeptides, 61 KDa (alpha 1) and 59 KDa (alpha 2). The two peptides were separated and extracted from polyacrylamide gel. The immuno-peptide mapping of the purified peptides by partial proteolysis showed that the 59-KDa polypeptide was not a degradative product of the 61-KDa polypeptide. The interaction of the enzyme with two monoclonal antibodies, Vj6 and Vd3, raised against bovine brain calcineurin revealed that the 61-KDa polypeptide was recognized by both Vj6 and Vd3, whereas the 59-KDa one was recognized only by Vj6. These results indicate that there are at least two isoforms of calcineurin alpha subunits in bovine brain.     

The role of cytokinins in relation to flower-bud blasting in Iris cv. Ideal: Cytokinin determination by an improved enzyme-linked immunosorbent assay

The effect of dark and light treatment on endogenous cytokinins in internodes and buds of Iris was determined. Plant material was purified by chromatographic methods and cytokinins were assayed by an immunoassay.An indirect competitive enzyme immunoassay for the determination of zeatin- and isopentenyl-adenine cytokinins was developed. This assay, which is not dependent on the titre of the antibodies raised against zeatin riboside and isopentenyl-adenosine appeared to be specific, highly sensitive and more reproducible compared to a direct competitive enzyme immunoassay for cytokinins.Isopentenyl-adenosine was the most abundant cytokinin found, followed by zeatin: the latter counteracts bud blast when injected into dark-treated plants. Smaller amounts of isopentenyl-adenine and zeatin riboside were found. Results are in agreement with the hypothesis that deficiency of growth substances like cytokinins plays an important role in the occurrence of flower-bud blasting.A possible role for the major endogenous cytokinin, isopentenyl-adenosine, which earlier was found not to be effective in counteracting bud blast when injected into buds of dark-treated plants, is discussed.     

The enzyme defect in bovine protoporphyria. Studies with purified ferrochelatase

Ferrochelatase was purified from the livers of normal and protoporphyria cattle by chromatography on Blue Sepharose CL-6B in order to investigate the enzyme defect in this disorder. The increase in specific activity (up to 2900-fold) indicated that the normal and protoporphyria enzymes were purified to a similar degree. The mutant enzyme had catalytic activity which was 10 to 15% of normal ferrochelatase, although the Michaelis constants for protoporphyrin and iron were similar. The molecular mass of the normal and protoporphyria enzyme protein was 40 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the presence of 15 mM sodium cholate, gel filtration demonstrated a similar size. However, at a lower concentration of sodium cholate (4 mM) the molecular mass was about 240 kDa, suggesting that the purified enzymes aggregate under this condition. Polyvalent antibodies were raised in rabbits using as antigens purified normal native enzyme and normal 40-kDa protein which had been further purified by preparative SDS-PAGE. In Western blots these antibodies complexed with both the normal and mutant 40-kDa proteins. The amount of 40-kDa protein in normal and protoporphyria mitochondrial fractions was also similar as evaluated by Western blots. These studies indicate that the ferrochelatase defect in bovine protoporphyria probably results from a point gene mutation that causes a minor change in enzyme structure.     

The type 5, acid phosphatase from spleen of humans with hairy cell leukemia. Purification, properties, immunological characterization, and comparison with porcine uteroferrin

The spleens of patients with hairy cell leukemia contain high levels of a tartrate-insensitive, cationic, acid phosphatase (the human Type 5 isozyme). This phosphatase has been purified by a procedure which involves only two chromatographic steps: CM-cellulose chromatography and immunoaffinity chromatography on sheep antibodies generated against porcine uteroferrin. Uteroferrin is an abundant iron-containing acid phosphatase that can be recovered readily from porcine uterine secretions. Like uteroferrin, the purified human Type 5 phosphatase is a glycoprotein of molecular weight about 34,000. It contains two atoms of iron/molecule. The human phosphatase and uteroferrin also resemble each other closely in electrophoretic mobility, substrate specificity, and response to a variety of activators and inhibitors. Mouse monoclonal antibodies have been raised to uteroferrin and to the human Type 5 phosphatase. Three monoclonal antibodies which bind with high affinities to distinct sites on the uteroferrin molecule also recognize the human spleen enzyme, but bind to it with much lower affinity. These antibodies also recognize cationic acid phosphatases purified from bovine and rat spleens. A monoclonal antibody raised against the human enzyme, but selected for binding to uteroferrin, appears to recognize a relatively conserved site on all four phosphatases. We conclude that the human Type 5 isozyme belongs to a growing class of structurally related, iron-containing acid phosphatases which includes the iron-transport protein, uteroferrin.     

Identification of T cell recognition sites in S-antigen: dissociation of proliferative and pathogenic sites

Experimental autoimmune uveoretinitis (EAU) is a predominantly CD4+ T cell-mediated autoimmune inflammatory disease of the retina and uveal tract of the eye and the pineal gland. S-antigen, a protein found in retinal photoreceptor cells and pinealocytes, is a potent agent for the induction of EAU in susceptible species and strains. In order to identify the T cell recognition sites of S-antigen responsible for its uveitogenicity and proliferative responses, cyanogen bromide (CB) fragments as well as synthetic peptides were used to test the proliferative responses of two uveitogenic T cell lines, R9 and R17, prepared against native bovine and human S-antigen, respectively. Two nonoverlapping synthetic peptides which are known to actively induce EAU, amino acid residues 286-297 and 303-314 of the bovine sequence, were unable to induce proliferative responses in either S-antigen-specific T cell line. However, both of these sites were adjacent to synthetic peptides, residues 273-292 and 317-328, respectively, which were unable to actively induce EAU, but elicited strong proliferative responses from T cell lines raised to bovine and human S-antigen. Repeated in vitro selection of the R9 T cell line with a synthetic peptide containing one of these proliferative sites, residues 317-328, gave rise to a transiently uveitogenic T cell line. Several species-specific T cell epitopes were identified, but none of these were found to be involved in a uveitogenic response. Our results indicate that spatially separated and distinct T cell epitopes are present in S-antigen which are responsible for the active induction of EAU, lymphocyte proliferation, and the ability to adoptively transfer EAU.     

Purification and characterization of a calcium-dependent ATPase from Paramecium tetraurelia

We report the purification of a CaATPase of high specific activity from Paramecium tetraurelia. The enzyme is preferentially released into solution upon deciliation of cells by a Ca2+ shock procedure. Purification by ion exchange and gel filtration chromatography yields major peptides of 68 and 53 kDa and a minor peptide of 58 kDa, as determined by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. These three peptides yield similar proteolytic peptide maps. Rabbit antisera to the purified enzyme inhibit enzyme activity and specifically label 68- and 53-kDa bands on nitrocellulose blots of the deciliation supernatant from which the enzyme is isolated. Concanavalin A-Sepharose precipitates about 60% of ATPase activity; only the 53-kDa band binds concanavalin A on nitrocellulose blots. The purified enzyme has a specific activity of 620 +/- 70 mumol/min/mg with ATP as substrate in the presence of Ca2+, which is required for enzyme activity. As substrates, ATP and GTP are strongly preferred to UTP and CTP. The Km for ATP in the presence of 3 mM Ca2+ is approximately 20 microM. Enzyme activity is strongly inhibited by the calmodulin antagonists trifluoperazine, fluphenazine, W7, and calmidazolium. However, calmodulin is not associated with the purified enzyme, based on the enzyme's inability to bind anti-calmodulin antibodies or to stimulate brain phosphodiesterase. The intracellular origin of this ATPase, its possible function, and its relationship to several other ATPases of Paramecium are discussed.     

Purification and characterization of glucosidase II involved in N-linked glycoprotein processing in bovine mammary gland.

Glucosidase II is an endoplasmic-reticulum-localized enzyme that cleaves the two internally alpha-1,3-linked glucosyl residues of the oligosaccharide Glc alpha 1----2Glc alpha 1----3Glc alpha 1----3Man5-9GlcNAc2 during the biosynthesis of asparagine-linked glycoproteins. We have purified this enzyme to homogeneity from the lactating bovine mammary gland. The enzyme is a high-mannose-type asparagine-linked glycoprotein with a molecular mass of approx. 290 kDa. Upon SDS/polyacrylamide-gel electrophoresis under reducing conditions, the purified enzyme shows two subunits of 62 and 64 kDa, both of which are glycosylated. The pH optimum is between 6.6 and 7.0. Specific polyclonal antibodies raised against the bovine mammary enzyme also recognize a similar antigen in heart, liver and the mammary gland of bovine, guinea pig, rat and mouse. These antibodies were used to develop a sensitive enzyme-linked immunosorbent assay for glucosidase II.     

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.     

Physicochemical property of bovine brain 73-kDa stress protein

• 1.1. An approximately 70-kDa protein was purified from bovine brain using an ATP-Sepharose column.
• 2.2. The protein sample was found to contain two proteins (major 73 kDa and minor 72 kDa) on two-dimensional gel electrophoresis.
• 3.3. Antibodies raised against the 73- and 72-kDa proteins cross-reacted with stress-induced HSP73 and HSP72 from HeLa cells, respectively.
• 4.4. Heparin-binding peptides were obtained from trypsin digests of HSP73.

     

Isolation of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase from Human Brain

Abstract: The enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37) has been isolated from an acetone powder of human subcortical white matter. The yield was about 11 mg from 28 g of powder and a specific activity of 213 unitdmg protein was obtained using 2',3'-cyclic CMP as the substrate. A major protein band of molecular weight approx. 96,000 was found by gel electrophoresis under nonreducing conditions. However, two distinct protein bands of molecular weight 46,000 ± 1400 and 48,000 ± 1400 were observed when the protein sample was reduced with 10 mM-dithiothreitol and subjected to electrophoresis in more restrictive 12-15% polyacrylamide-SDS gels. This molecular weight is lower than that previously reported for the bovine enzyme. Antibodies against the purified human enzyme have been raised in New Zealand white rabbits.     

Cation-sensitive neutral endopeptidase: isolation and specificity of the bovine pituitary enzyme

A highly purified preparation of a cation-sensitive neutral endopeptidase was obtained from bovine pituitaries. The enzyme constitutes almost 0.1% of the protein in bovine pituitary homogenates. Polyacrylamide gel electrophoresis of the enzyme showed a single protein band, and in gel filtration experiments on calibrated Sepharose 6B columns the enzyme eluted slightly ahead of thyroglobulin, suggesting an apparent molecular weight of about 700,000. Polyacrylamide gel electrophoresis in SDS-containing buffers indicated the presence of three major components with molecular weights ranging from about 24,000 to 28,000. The enzyme hydrolyzes bonds between hydrophobic and small neutral amino acids in both model synthetic substrates and biologically active peptides such as substance P, LH-RH, and bradykinin. Peptide bonds in which the carbonyl group is contributed by a glutamyl or arginyl residue are also hydrolyzed, especially if they are preceded in the sequence by hydrophobic amino acids. Leupeptin exclusively inhibited enzymatic activity toward the arginine-containing substrates. This observation, together with the high molecular weight and broad specificity of the enzyme, raised the possibility that the isolated enzyme represents a proteolytic complex composed of units with distinctly different activities. Preliminary attempts to dissociate the enzyme into catalytic units of lower molecular weight were not successful and led to loss of activity.     

Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle.

Immunoreactive alpha B crystallin and a 28-kDa protein in an extract of human pectoral muscle were precipitated by (NH4)2SO4 at 40% saturation, and coeluted during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. The two proteins were separated on a column of S-Sepharose HP in the presence of 7 M urea. Further chromatography of each of the two resultant fractions on a column of Superdex 75 pg and on a TSK-SP 5PW column in the presence of urea yielded preparations of alpha B crystallin and the 28-kDa protein each of which gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final preparation of 28-kDa protein contained at least two subtypes, which were separable on the TSK-SP column. However, fragmentation patterns of the two major 28-kDa proteins after digestion with endoproteinase Asp-N were identical. Amino acid sequences of peptides formed by cleavage of the purified 28-kDa protein and alpha B crystallin were identical to those of particular regions of the deduced amino acid sequences of human small heat shock protein (HSP28) and lens alpha B crystallin, respectively. Using an immunoassay method, with antibodies raised in rabbits, we found that HSP28 was present in all human tissues tested and at high levels (greater than 1 micrograms/mg protein) in the heart and other tissues composed of striated and smooth muscles. HSP28, found with alpha B crystallin, in extracts of several human and bovine tissues was trapped on and coeluted with alpha B crystallin from an affinity column prepared with antibodies against alpha B crystallin. This result suggests that the two proteins are associated in cells.     

Regional distribution of prostaglandin endoperoxide synthase studied by enzyme-linked immunoassay using monoclonal antibodies

Prostaglandin endoperoxide synthase transforms arachidonic acid to prostaglandin H2 via prostaglandin G2. The enzyme purified from bovine vesicular gland was given to mice as antigen, and monoclonal antibodies were raised by the hybridoma technique. Two species of the monoclonal antibody recognizing different sites of the enzyme were utilized to establish a peroxidase-linked immunoassay of prostaglandin endoperoxide synthase. Fab' fragment of one of the antibodies was prepared and conjugated to horseradish peroxidase. The conjugate was then bound to prostaglandin endoperoxide synthase, and the labeled enzyme was precipitated by the addition of the other antibody. The peroxidase activity of the immunoprecipitate correlated linearly with the amount of prostaglandin endoperoxide synthase. This sensitive and convenient method to determine the enzyme amount rather than the enzyme activity was utilized to extensively screen the amount of prostaglandin endoperoxide synthase in various bovine tissues. In addition to vesicular gland, platelets and kidney medulla previously known as rich enzyme sources, the immunoenzymometric assay demonstrated a high content of the enzyme in various parts of alimentary tract and a low but significant amount of enzyme in some parts of brain.     

A major part of the polypeptide chain of tobacco mosaic virus protein is antigenic

Eighteen synthetic peptides representing virtually the entire length of the polypeptide chain of tobacco mosaic virus coat protein (TMVP) have been analyzed for their ability to bind in an enzyme immunoassay to 30 monoclonal antibodies raised against the dissociated viral subunits. Only five of the monoclonal antibodies were able to bind a number of peptides while the other 25 antibodies recognized only the complete molecule and seemed to be specific for conformational features that are absent in the peptide fragments. The 18 peptides were also tested for their ability to bind to several antisera to TMVP. Virtually the entire sequence of TMVP possessed antigenic activity. Four new epitopes were identified in the vicinity of residues 19–32, 90–95, 115–134 and 134–146. These results bring to 11 the number of continuous epitopes that have been identified in the TMVP molecule and show that the entire surface of the molecule is antigenic. When peptides of TMVP of a length of 6–8 residues were tested for antigenic activity previously a correlation was found between the location of short continuous epitopes and mobile segments of the protein. In the present study, in which longer peptides as well as monoclonal antibodies were used to probe the antigenicity of TMVP, additional conformation-dependent epitopes were shown to be present. Our results illustrate the operational nature of any definition of antigenicity and caution against the use of any single criterion for distinguishing between antigenic and non-antigenic regions of a protein.     

S-antigen-specific rat T cell lines recognize peptide fragments of S-antigen and mediate experimental autoimmune uveoretinitis and pinealitis

Two S-antigen-specific rat T cell lines expressing the T helper cell surface phenotype (W 3/25+, OX 8-) have been isolated from the spleen and lymph node cells of retinal S-antigen-immunized Lewis rats, one of which displayed neither clinical nor histopathologic signs of experimental autoimmune uveoretinitis. The other rat had recovered from severe experimental autoimmune uveoretinitis for 2 mo before isolation of the cell line. Both lines are specific for S-antigen presented by histocompatible antigen-presenting cells, and also respond in vitro to several of the peptides produced by cyanogen bromide cleavage of bovine retinal S-antigen. The lesions induced by the i.v. transfer of from 1 to 10 X 10(6) viable line cells involve the retina and pineal gland, as is found when Lewis rats are immunized with immunopathogenic doses of S-antigen. Histologic examination of the eyes and pineal glands revealed pathologic lesions typical of experimental autoimmune uveoretinitis, and consisted of marked infiltration of the retina and surrounding tissues and the pineal gland by lymphocytes and inflammatory cells. T cells capable of mediating autoimmune disease are clearly present and readily isolated from both asymptomatic and convalescent animals. No significant differences in specificity for the cyanogen bromide peptides of S-antigen or cell surface phenotype were found in the T cell lines isolated from these two rats, nor was any difference found in the specificity or titer of serum antibodies taken from the original rats for the cyanogen bromide peptides of S-antigen.     

Peptides mimicking Vibrio cholerae O139 capsular polysaccharide elicit protective antibody response

Vibrio cholerae is the etiological agent of cholera. V. cholerae serogroup O1 had been, until 1992, the only serogroup responsible for large epidemics and pandemics of cholera. In 1992, a new serotype of V. cholerae emerged in South-East Asia that caused a massive outbreak of cholera in India and neighboring countries. The new serotype was named V. cholerae O139. The main differences between V. cholerae O139 and O1 are that the former possesses a capsular polysaccharide and different lipopolysaccharide. Capsular polysaccharides are, in general, T-independent antigens giving rise to poor immune responses lacking immunological memory. In order to overcome this, monoclonal antibodies against the capsular polysaccharide of V. cholerae O139 were used to screen different phage-displayed random peptide libraries. Eight different phage clones were selected and characterized using enzyme immunoassay with the monoclonal antibodies, and then tested for specificity by competition with V. cholerae O139 capsular polysaccharide. Selected peptides were sequenced, synthesized and conjugated to bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). The conjugated peptides were used to immunize mice. It is evident that the anti-peptide mouse antibodies bind to the V. cholerae O139 capsular polysaccharide. In addition, the anti-peptide antibodies are protective in a suckling mouse model. The protective efficacy is both specific and dose-dependent. A PCT (PCT/IT2003/000489) with the publication number WO 2004/056851 has been filed for the sequences of the eight peptides.     

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.