Properties and prenatal ontogeny of beta-D-mannosidase in selected goat tissues.

beta-D-Mannosidase activity in selected normal adult, neonatal and foetal goat tissues and in tissues from animals affected with caprine beta-mannosidosis was examined with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. The enzyme in normal adult thyroid, kidney and brain exhibited a sharp unimodal pH optimum at pH 5.0, whereas the enzyme in both normal adult and mutant liver exhibited broad pH ranges of activity (pH 4.5-8.0). No residual enzyme was detectable in mutant kidney or brain; in contrast, residual activity in mutant liver was 52% of that in a neonatal control. Concanavalin A-Sepharose 4B (Con A-Sepharose) fractionation of normal adult liver beta-D-mannosidase resolved the enzyme into an unbound (non-lysosomal) from (52%) with a broad pH range of activity (pH 4.5-8.0) and a bound (lysosomal) form (48%) with a sharp pH optimum of 5.5. The enzyme in mutant liver consisted entirely of the unbound (non-lysosomal) form. Beta-D-Mannosidase activity in normal adult thyroid, kidney and brain was resolved by chromatofocusing into two major isoenzymes, with pI 5.5 and 5.9, and traces of a minor isoenzyme, with pI 5.0. In normal adult liver the enzyme was also resolved into three isoenzymes with similar pI values; however, that with pI 5.0 predominated. The predominant form of the enzyme in 60-day-foetal liver was bound by Con A, exhibited a unimodal pH optimum (5.0) and was resolved into two isoenzymes, with pI 5.4 and 5.8; only traces of an isoenzyme with pI 5.0 were detectable. Total hepatic beta-D-mannosidase activity increased progressively towards adult values during the last 90 days of gestation as a result of increasing non-lysosomal isoenzyme activity (pI 5.0). Lysosomal beta-D-mannosidase was shown to occur in all normal goat tissues studied as multiple isoenzymes, which are genetically and developmentally distinct from the non-lysosomal isoenzyme occurring predominantly, if not exclusively, in liver.     

The separation and characterization of marmoset kidney beta-D-galactosidase and beta-D-glucosidase.

beta-D-Galactosidase and beta-D-glucosidase activities were determined in homogenates of marmoset kidney by using the appropriate 4-methylumbelliferyl glycoside, beta-D-Galactosidase activity was separated into two main components by ion-exchange chromatography on DEAE-cellulose, starch-gel electrophoresis, isoelectric focusing and gel filtration on Sephadex G-200. One form designated A had a pI of 5.1, was loosely bound to DEAE-cellulose at pH7.0, remained near the origin on starch-gel electrophoresis at pH 7.0 and had an apparent molecular weight of 160000. The second beta-D-galactosidase component, designated B, was associated with the total beta-D-glucosidase activity, had a pI of 4.3, was firmly bound to DEAE-cellulose, migrated rapidly towards the anode on starch-gel electrophoresis and had an apparent molecular weight of 50000. The optimum pH values of beta-D-galactosidase A and B were 4.5 and 6.0 respectively. beta-D-Galactosidase A was activated by 0.1 M-NaC1 but the activity of the B form was inhibited by 1 M-NaC1 at pH 4.5. beta-D-galactosidase had a bimodal distribution, the A form being recovered in the lysosomal fraction whereas the B form was present in the soluble fraction, as was the major portion of the beta-D-glucosidase activity. The lysosomal and soluble forms were further characterized by DEAE-cellulose chromatography.     

N-acetyl-beta-d-glucosaminidase in marmoset kidney, serum and urine.

N-Acetyl-beta-D-glucosaminidase activities were determined in homogenates of marmoset kidney, in serum and in urine by using the 4-methylumbelliferyl substrate. The enzyme activity was separated into several components by DEAE-cellulose ion-exchange chromatography, starch-gel electrophoresis and isoelectric focusing. The kidney contained two major forms of the enzyme, A and B, which had similar pH optima and Km values. The A-form bound to DEAE-cellulose at pH 6.8, migrated towards the anode on starch-gel electrophoresis and had a pI of 5.0. The B-form did not bind to DEAE-cellulose at pH 6.8, remained near the origin on starch-gel electrophoresis and had a pI of 7.64. The isoenzymes also differed in heat stability, the B-form being the more stable. Serum contained B-form activity and, in addition, two intermediate forms (I1 and I2) were loosely bound to DEAE-cellulose. The serum A-form activity was less firmly bound to DEAE-cellulose than was the tissue A-form and was designated As. Serum from a pregnant marmoset contained a form which may be analogous to the human P-isoenzyme. Urine contained only a small amount of B-form activity, the majority being present in the A-form. The kidney A- and B-forms both had mol.wts. of 96000--100000 and the activity was predominantly lysosomal. Partial purification of the kidney A isoenzyme was undertaken. Immunoprecipitation studies indicated a relationship between marmoset kidney A-form and human liver A-form activity.     

Cellular localization and substrate specificity of isoelectric forms of human liver neuraminidase activity.

The four major isoelectric forms of human liver neuraminidase (with pI values between 3.4 and 4.8) have been isolated by preparative isoelectric focusing and characterized with regard to their substrate specificity using glycoprotein, glycopeptide, oligosaccharide and ganglioside natural substrates. All forms exhibited a rather broad linkage specificity and were capable of hydrolyzing sialic acid glycosidically linked alpha 2-3, alpha 2-6 and alpha 2-8, although differential rates of hydrolysis of the substrates were found for each form. The most acidic form 1 (pI 3.4) was most active on sialyl-lactose, whereas form 2 (pI 3.9) and 3 (pI 4.4) were most active on the more hydrophobic ganglioside substrates. Form 4 (pI 4.8) was most active on the low-Mr hydrophilic substrates (fetuin glycopeptide, sialyl-lactose). Each form was less active on the glycoprotein fetuin than on a glycopeptide derived from fetuin. Organelle-enriched fractions were prepared from fresh human liver tissue and neuraminidase activity on 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid was recovered in plasma membrane, microsomal, lysosomal and cytosolic preparations. Isoelectric focusing of the neuraminidase activity recovered in each of these preparations resulted in significantly different isoelectric profiles (number, relative amounts and pI values of forms) for each preparation. The differential substrate specificity of the isoelectric forms and the different isoelectric focusing profiles of neuraminidase activity recovered in subcellular-enriched fractions suggest that specific isoelectric forms with broad but defined substrate specificity are enriched at separate sites within the cell.     

PROPERTIES OF ACID β-d-GALACTOSIDASE ISOLATED FROM I-CELL DISEASE BRAIN AND SPLEEN

Abstract— Acid 4-methylumbelliferyl β- d -galactosidase activity from autopsied I-cell disease brain and spleen tissues was 28% and 35% respectively of normal activity. Acid β- d -gatactosidase (β- d -galactoside galactohydrolase, EC 3.2.1.23) from two I-cell disease brains demonstrated a 5-fold increase over normal for the proportion of enzyme activity which did not adsorb to Concanavalin A-Sepharose 4B, while acid β- d -galactosidase from two I-cell disease spleens demonstrated a 21–35-fold increase in the proportion of unadsorbed enzyme activity. Normal and I-cell disease acid β- d -galactosidase present in crude brain and spleen supernatant fluids and in preparations partially purified on Concanavalin A-Sepharose 4B had similar apparent K _m values with respect to 4-methylumbelliferyl β- d -galactopyranoside and G_M1-ganglioside. Isoelectric focusing profiles of normal and I-cell disease acid β- d -galactosidase from crude brain and spleen-supernatant fluids and partially purified preparations were similar. Neuraminidase treatment and subsequent isoelectric focusing of the partially purified normal and I-cell disease enzyme preparations from brain and spleen revealed increases in the proportion of I-cell β- d -galactosidases found at neutral pH values, suggesting that the electrophoretic variations observed for the I-cell enzymes may not be attributed solely to changes in sialic acid composition.     

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.     

Isoelectric focusing patterns of staphylococcal exfoliative toxin

Multiple components in two antigenic variants of staphylococcal exfoliative toxin were demonstrated by isoelectric focusing in polyacrylamide gel. The main components had an isoionic point of pH=7.0 while other components ranged from pI=4.5 to pI=8.5. Each individual component was shown to possess full biologic and serologic activity after focusing. Isoelectric focusing in the presence of 8 M urea also showed microheterogeneity in exfoliative toxin preparations. The various components appear to be reversible conformers, possibly raised by exposure to the ph gradient of the ampholines.     

Microheterogeneity of human kininogen by isoelectric focusing and crossed immunoelectrophoresis.

LMW kininogen was isolated from whole human plasma by gel filtration on Sephadex G-200 (Kav 0.34) followed by DEAE-chromatography according to earlier established methods. Further purification was performed with specific Sepharose-antibody columns to remove protein contaminants, avoiding procedures which may denature kininogen. The microheterogeneity was investigated by isoelectric focusing in column in the pH-gradients 3.5-10, 4-6 and 3.5-5. Kininogen components were determined by single radial immunodiffusion against monospecific anti-human kininogen serum, in comparison with focusing of whole plasma. 40% of isolated as well as whole plasma kininogen focused at pI 4.5; the respective focusing ranges were pI 4.4-4.7 (60--80%) and pI 4.3-4.6 (92%). The results were verified by crossed immunoelectrophoresis. The pI 4.5 component is apparently the main native form of human kininogen as shown by focusing of whole human blood bank plasma. Earlier described difficulty of separating kininogen and alpha2HS-glycoprotein was verified by crossed immunoelectrophoresis which showed approximately seven kininogen components after focusing in polyacrylamide gel electrophoresis at pI 4.5-5.0 and four alpha 2HS components at pI 4.2-4.6.     

Sphingomyelinase activities in cultured skin fibroblasts from patients with Niemann-Pick disease

Summary Sphingomyelinase activity in cultured skin fibroblasts from a fetus affected with infantile-type Niemann-Pick disease was 0.5% of control activity; the activities in cells from two patients with adult-type disease (Cases 2 and 3) were 5.0% and 59.0%.Sphingomyelinase activity was separated into three peaks (I–III) by isoelectric focusing. The isoelectric points were 4.5, 4.9, and 5.2 for peaks I, II, and III, respectively. The three peaks in the Case 2 cells were drastically reduced; only a very small peak could be distinguished (pI of 4.7). On the other hand, three peaks were observed in the Case 3 cells. Peak I had a pI of 4.4, peak II a pI of 4.7, and peak III a pI of 5.2. Peak I was found at near normal level, but both peaks II and III were markedly reduced.Sphingomyelinase in the peak I fraction obtained from isoelectric focusing in Case 3 cells was found to have the same Km value as that in control cells.     

The N-acetylhexosaminidase components of the ram testis and epididymis

Three and four N-acetylhexosaminidase components, from ram testis and epididymis respectively, have been separated by ion-exchange chromatography on DEAE-cellulose. Although they all have the same molecular weight (approx. 140000) and very similar catalytic properties towards the synthetic substrates, 4-methylumbelliferyl N-acetyl-beta-glucosaminide and N-acetyl-beta-galactosaminide, isoelectric focusing of the individual components showed that each had a distinct pI value. Isoelectric focusing has also been used to demonstrate the occurrence of multiple forms in ejaculated ram semen.     

Purification and characterization of NADPH-dependent methemoglobin reductase from the nucleated erythrocytes of bullfrog, Rana catesbeiana

Two protein components having a NADPH-dependent methemoglobin reductase activity were purified to electrophoretic homogeneity from the erythrocytes of the bullfrog, Rana catesbeiana. Their molecular properties were investigated. The components were separated by isoelectric focusing, having discrete bands of pI 5.0 and 7.5, respectively. The pI 5.0 component, designated F-5.0, was faint yellow, with a broad absorption in the range of 400-450 nm, while the pI 7.5 component, designated F-7.5, was colorless and did not absorb in that range. The molecular weight was estimated to be 22,000 for both components by gel filtration and SDS-PAGE. When F-5.0 was subjected to isoelectric focusing repeatedly, the protein part of that component gradually moved to and refocused at pH 7.5, leaving a yellow color at acidic pH. Both F-5.0 and F-7.5 were highly specific for NADPH and had the same kinetic properties in catalyzing the reduction of MB, DCPIP, FMN, or FAD, and that of methemoglobin or cytochrome c in the presence of a certain dye. They were also indistinguishable from one another in their amino acid compositions and were completely identical in the N-terminal sequence of 24 amino acid residues. These findings strongly suggest that the two components can be attributed to the same enzyme molecule, carrying an identical protein moiety but interacting differently with some unidentified biological pigments, and that they are equivalent in their molecular and kinetic properties to the NADPH-dependent enzyme(s) occurring in human erythrocytes.     

Use of preparative isoelectric focusing in a Sephadex gel slab to separate immunizing and growth facilitating moieties in crude 3 M KCl extracts of a murine fibrosarcoma

Summary Crude 3 M KCl extracts of the methylcholanthrene-induced fibrosarcoma of C3H/HeJ mice, MCA-F, were demonstrated to contain two fractions, one inducing tumor resistance and the other facilitating the outgrowth of neoplastic cell challenge. In immunoprotection tests in syngeneic C3H/HeJ mice, optimal doses of crude solubilized tumor antigen afforded only a 28% reduction in growth compared with saline-treated controls. When crude extracts were fractionated by preparative isoelectric focusing (pIEF) in a slab of superfine Sephadex G-75, significant biologic activity was demonstrated in two fractions. Fraction (Fr) 1, pI 2.5–3.6, induced potent tumor facilitation, increasing the tumor size by more than 100%, while Fr 15, pI 5.8–6.0, engendered resistance that reduced their respective biological effects to MCA-F, but not the antigenically unrelated MCA-D tumor. Thus 3 M KCl extracts contain at least two biologically active components, one immunoprotective and one tumor-facilitating. Since the weak immunoprotective activity of crude materials may represent the vectorial effect of these antagonistic components, subsequent molecular characterization of both moieties may afford insight into the complex response of hosts toward tumors. Furthermore, TSTA purified by the rapid method of isoelectric focusing may be a more suitable reagent for immunotherapy than the parent crude 3 M KCl extracts by virtue of the absence of facilitating antigens.Abbreviations CE crude 3 M KCl extract - pIEF preparative isoelectric focusing - Fr fraction from pIEF - MCA-F and MCA-D antigenically different methylcholanthrene-induced fibrosarcomas of C3H/HeJ mice - TSTA tumor specific transplantation antigens     

Galactosyl (alpha 1--4)galactosylceramide: galactosyl hydrolase activity in normal and Fabry plasma

An α-galactosidase fraction which hydrolyzes galactosyl(α1→4)galactosylceramide and 4-methylumbelliferyl-α-galactoside has been isolated from normal human plasma by affinity chromaography. It was partially separated into two enzymatically active proteins by isoelectric focusing or cellulose acetate electrophoresis. The protein fraction obtained by affinity chromatography of Fabry plasma also was divided into two proteins, but only the protein of slower electrophoretic mobility had detectable enzymatic activity. These results indicate that the accumulation of galactosyl(α1→4)galactosylceramide in certain organs in Fabry's disease is due to an alteration of a specific α-galactosidase.     

Lipoprotein substrates of lipoprotein lipase and hepatic triacylglycerol lipase from human post-heparin plasma.

The number and the substrate specificities of glutathione thiol esterases of human red blood cells have been investigated by gel electrophoresis and isoelectric focusing and staining methods devised for the location of these enzymes on gels. Several glutathione thiol esterase forms, both unspecific (with respect to the S-acyl group of the substrate) and specific were found. Electrophoresis on both polyacrylamide and agarose gels resolved three enzyme components with apparently similar substrate specificity. Isoelectric focusing in liquid column separated two unspecific thiol esterase components with S-lactoylglutathione (pI = 8.4) and S-propionylglutathione (pI = 8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (pI = 5.2) and S-succinylglutathione hydrolase (pI = 9.0). Isoelectric focusing on polyacrylamide gel resolved nine unspecific glutathione thiol esterase bands (between pH values 7.0 and 8.4). Partially purified glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) from erythrocytes or liver still gave three components on electrophoresis and several activity bands on gel electrofocusing. These results indicate that human red cells contain at least four separate glutathione thiol esterases. Glyoxalase II, one of these enzymes, apparently occurs in multiple forms. These were neither influenced by preptreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.     

Microheterogeneity of biliverdin reductase in rat liver and spleen: selective suppression of enzyme variants in liver by bromobenzene

Recently we have reported the detection of multiple net-charge and molecular mass variants of biliverdin reductase in the rat liver. We now report an apparent selective change in the electrophoretic profile of the reductase variants in the liver by in vivo bromobenzene treatment (2 mmol/kg, sc, 24 h). Using two-dimensional electrophoresis and isoelectric focusing, one molecular mass species of the reductase (Mr 30,400) appeared to be selectively suppressed by bromobenzene treatment. This molecular mass species was the main component of two isoelectric focusing bands with pI6.23 and 5.91. The effect in vivo of bromobenzene could not be duplicated by in vitro experiments involving treatment of purified enzyme with bromobenzene in the presence of a NADPH-dependent microsomal drug metabolizing system. The phenomenon of multiplicity of the reductase was not limited to the liver. Multiplicity of the enzyme was detected also in the spleen; however, the pattern of composition of the reductase variants vastly differed from that of the liver. In the spleen, variants with pI 5.76, 5.61, and 5.48 were the prevalent forms; the variant with pI 6.23 was absent, and pI 5.91 was present in a minute amount. Further, bromobenzene did not affect the composition pattern of net-charge variants in this organ. Also, the splenic biliverdin reductase activity was refractory to in vivo bromobenzene treatment, whereas the liver reductase activity with both NADH and NADPH was altered by the treatment. The possible significance of the presence of multiple variants of biliverdin reductase and the change in their composition caused by bromobenzene is discussed.     

Isolation and properties of multiple forms of histidine decarboxylase from rat gastric mucosa.

The heterogeneity of histidine decarboxylase from rat gastric mucosa was studied. The partially purified enzyme was fractionated by preparative isoelectric focusing on a flat-gel bed by using narrow pH-range carrier ampholytes and a short focusing time. The activity was resolved, with about 95% recovery, into three forms, designated I, II and III, with pI values of 5.90, 5.60 and 5.35 respectively. These three forms exhibited similar molecular weights, indicating that the forms were not the result of different degrees of polymerization. By preparative refocusing each form refocused as a single peak of enzyme activity with reproducible pI, but a high loss of activity occurred with repeated focusing. Forms I, II and III were purified by the combined use of preparative isoelectric focusing and gel chromatography and other fractionation methods. The active forms could be distinguished by electrophoresis and isoelectric focusing on polyacrylamide gels and displayed protein heterogeneity. These forms were found in the crude extract and in the partially purified preparations in the presence or absence of proteinase inhibitors. Form II had the highest specific activity, but all three forms had the same optimum pH and Km value for histidine.     

Partial purification and characterization of dipeptidyl peptidase II (DPP II) from guinea pig testes

Depeptidyl peptidase (DPP II) was partially purified from guinea pig testes by (NH₄)₂SO₄ precipitation, Con A-Sepharose 4B chromatography, and Sephadex G-200 chromatography to a specific activity of 27.4 μmol Ala₃ hydrolyzed min^?1 mg^?1 protein. Chromatography on a calibrated G-200 column yielded a molecular weight of 135,000 daltons for the enzyme. Sodium dodecyl sulfate polyacrylamide electrophoresis showed an enrichment of a broad doublet at 64–66,000 daltons. The enzyme had optimal activity toward hydrolysis of L-alanyl-alanyl-alanine at pH 4.5 and showed sensitivity to cations of increasing size with Tris producing the most inhibition of those tested. The enzyme was moderately inhibited by serine proteinase inhibitors. Thin-layer chromatography revealed the dipeptidase nature of the enzyme's activity on tripeptides and dipeptidyl arylamides. A doublet of activity occurred when nitrocellulose electroblots of nondenaturing gel electrophoresis of the (NH₄)₂SO₄ fraction were reacted with the specific DPP II substrate, lysyl-alanyl-4-methoxy-2-napthylamide. Analytical isoelectric focusing of the G-200 fraction followed by fluorescent enzyme activity detection that used cellulose triacetate overlay membranes impregnated with the specific DPP II substrate, lysyl-alanyl-7-amino-4-trifluoromethylcou-marin, revealed multiple isoforms focusing at pI = 4.8–5.6. Two prominent bands focused at pI = 4.9 and pI = 5.1. The properties of guinea pig testicular DPP II are compared and contrasted with similar dipeptidyl peptidases from other sources.     

Isoelectric focusing of glutathione S-transferases from rat liver and kidney

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.     

Neutral sphingomyelinase from human urine. Purification and preparation of monospecific antibodies

A neutral sphingomyelinase which cleaves phosphorylcholine from sphingomyelin at a pH optima of 7.4 was purified 440-fold to apparent homogeneity from normal human urine concentrate employing Sephadex G-75 column chromatography, preparative isoelectric focusing, and sphingosylphospholcholine CH-Sepharose column chromatography. The enzyme is composed of a single polypeptide whose apparent molecular weight is 92,000. Analytical isoelectric focusing revealed that the pI of this enzyme is 6.5. Purified neutral sphingomyelinase was devoid of beta-galactosidase and beta-N-acetylglucosaminidase activity originally present in the urine concentrate. The purified neutral sphingomyelinase (N-SMase) had low levels of phospholipase A1 and A2 activity when phosphatidylcholine was used as a substrate and detergents were included in the assay mixture. However, it had no phospholipase activity toward phosphatidylglycerol and sphingomyelin at pH 4.5 irrespective of the presence or absence of detergents. Monospecific polyclonal antibodies raised against N-SMase immunoprecipitated approximately 70% of N-SMase activity from urine, human kidney proximal tubular cells, and partially purified membrane-bound N-SMase from these cells. Western immunoblot assays revealed that the monospecific polyclonal antibody against urinary N-SMase recognized both the urinary N-SMase and the membrane-bound N-SMase. Because this enzyme is distinct biochemically and immunologically as compared to acid sphingomyelinase (EC 3.1.4.12), we would like to assign it an enzyme catalog number of EC 3.1.4.13. The availability of N-SMase and corresponding antibody will be useful in studying various aspects of this enzyme in biological systems.