Uptake and metabolism of radioactively labeled sphingomyelin in cultured skin fibroblasts from controls and patients with Niemann-Pick disease and other lysosomal storage diseases

The metabolism of [stearoyl-1-14C]- and [choline-methyl-14C]sphingomyelin, [stearoyl-1-14C]ceramide-1-phospho-N,N-dimethylethanolamine (demethylsphingomyelin) and [choline-methyl-14C]phosphatidylcholine was measured 1, 3 and 5 days after uptake from the media of cultured skin fibroblasts. This was done to measure the relative contributions of lysosomal sphingomyelinase and plasma membrane phosphocholine transferase on the metabolism of sphingomyelin, a component of all cell membranes. By using cell lines from controls and from patients with Niemann-Pick disease and other lysosomal storage diseases, it was concluded that a significant portion (10-15%) of the observed degradation of sphingomyelin is due to exchange of the phosphocholine moiety producing phosphatidylcholine. Although cell lines from type A and B Niemann-Pick disease have only 0-2% of lysosomal sphingomyelinase activity measured in vitro, three cell lines from type B Niemann-Pick disease could metabolize 54.4% of the labeled sphingomyelin by day 3 while cell lines from type A Niemann-Pick disease could only metabolize 18.5% by day 3. This compares to 86.7% metabolized in control cells by day 3. Cells from one patient with juvenile Niemann-Pick disease and one with type D Niemann-Pick disease metabolized sphingomyelin normally while cells from two other patients with juvenile or type C Niemann-Pick disease could only metabolize 58.2% by day 3. Cells from patients with I-cell disease and 'lactosylceramidosis' also demonstrated decreased metabolism of sphingomyelin (55.1 and 54.9% by day 3, respectively). Cells from the patient with Farber disease accumulated [14C]stearic acid-labeled ceramide produced from [14C]sphingomyelin. Studies with choline-labeled sphingomyelin and phosphatidylcholine demonstrated that phosphocholine exchange takes place in either direction in the cells, and this is normal in Niemann-Pick disease. Studies in cells from patients with all clinical types of sphingomyelinase deficiency have led to new methods for diagnosis and prognosis and to a better understanding of sphingomyelin metabolism.     

Lysosomal involvement in cellular turnover of plasma membrane sphingomyelin

At least two isoenzymes of sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12), including lysosomal acid sphingomyelinase and nonlysosomal magnesium-dependent neutral sphingomyelinase, catalyse the degradation of sphingomyelin in cultured human skin fibroblasts. A genetically determined disorder of sphingomyelin metabolism, type A Niemann-Pick disease, is characterized by a deficiency of lysosomal acid sphingomyelinase. To investigate the involvement of lysosomes in the degradation of cellular membrane sphingomyelin, we have undertaken studies to compare the turnover of plasma membrane sphingomyelin in fibroblasts from a patient with type A Niemann-Pick disease, which completely lack acid sphingomyelinase activity but retain nonlysosomal neutral sphingomyelinase activity, with turnover in fibroblasts from normal individuals. Plasma membrane sphingomyelin was labeled by incubating cells at low temperature with phosphatidylcholine vesicles containing radioactive sphingomyelin. A fluorescent analog of sphingomyelin, N-4-nitrobenzo-2-oxa-1,3-diazoleaminocaproyl sphingosylphosphorylcholine (NBD-sphingomyelin) is seen to be readily transferred at low temperature from phosphatidylcholine liposomes to the plasma membranes of cultured human fibroblasts. Moreover, when kinetic studies were done in parallel, a constant ratio of [14C]oleoylsphingosylphosphorylcholine ( [14C]sphingomyelin) to NBD-sphingomyelin was taken up at low temperature by the fibroblast cells, suggesting that [14C]sphingomyelin undergoes a similar transfer. The comparison of sphingomyelin turnover at 37 degrees C in normal fibroblasts compared to Niemann-Pick diseased fibroblasts shows that a rapid turnover of plasma membrane-associated sphingomyelin within the first 30 min appears to be similar in both normal and Niemann-Pick diseased cells. This rapid turnover appears to be primarily due to rapid removal of the [14C]sphingomyelin from the cell surface into the incubation medium. During long-term incubation, an increase in the formation of [14C]ceramide correlating with the degradation of [14C]sphingomyelin is observed in normal fibroblasts. In contrast, the level of [14C]ceramide remains constant in Niemann-Pick diseased cells, which correlates with a higher level of intact [14C]sphingomyelin remaining in these cells compared to normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Niemann-Pick disease type B: prenatal diagnosis and enzymatic and chemical studies on fetal brain and liver.

Patients with Niemann-Pick disease type A have a severe neurovisceral disease caused by a deficiency of lysosomal sphingomyelinase activity in all tissues examined. The patients with the type B form have signs and symptoms related to storage of sphingomyelin in the spleen, liver, and lungs, while neurologically they remain normal. They also have a severe deficiency of lysosomal sphingomyelinase activity in all tissues previously examined. Here the brain and liver of a fetus with Niemann-Pick disease type B are examined for enzymatic anc chemical changes. Despite careful analysis, no measurable lysosomal sphingomyelinase could be measured in either organ. Lipid changes were comparable to those observed in fetuses aborted with Niemann-Pick disease type A. The affected child in this family is now age 3 and remains neurologically normal but continues to show organ enlargement and lung infiltration of lipids. It appears that the lack of neurological involvement in type B patients cannot be due to an obvious presence of significant lysosomal sphingomyelinase activity in brain.     

Niemann-Pick disease, Type C: evidence for the deficiency of an activating factor stimulating sphingomyelin and glucocerebroside degradation

1) Qualitative lipid analyses by thin-layer chromatography of 4 Niemann-Pick type C spleens confirmed sphingomyelin accumulation together with increase in the amount of glucocerebroside. 2) In the presence of crude sodium taurocholate as detergent, sphingomyelin degradation rates of normal and Niemann-Pick type C-cultured fibroblasts were fairly close under standard conditions at pH 5.0. In the absence of sodium taurocholate, sphingomyelinase activity was optimal at pH 4.0. Sphingomyelinase activities of fibroblasts from two patients with Niemann-Pick disease type C measured without detergent, were about 30% of that of controls. 3) Extracts from Gaucher spleen heated to 90 degrees C and devoid of sphingomyelinase activity stimulated at the optimal pH of 4.0 sphingomyelin degradation by cultured normal fibroblasts (2--4-fold, Niemann-Pick type C fibroblasts (5--9-fold), whereas similarly treated extracts from Niemann-Pick type C spleen showed no stimulation of sphingomyelin catabolism. Heated extracts from normal human spleen exhibited a smaller stimulation than that shown by Gaucher spleen. This stimulating effect could not be observed in fibroblasts from patients suffering from Niemann-Pick type B (sphingomyelinase defect). 4) Heat-treated extracts of Gaucher spleen were fractionated by ion exchange chromatography, isoelectric focusing and gel filtration. The active fractions obtained by these procedures stimulated sphingomyelin as well as glucocerebroside degradation and were absent from the corresponding Niemann-Pick type C preparations. Enriched activator preparations of Gaucher spleen stimulated sphingomyelinase activity of Niemann-Pick type C fibroblasts 25--38-fold and that of normal cells 3-fold. 5) The activating factor had an isoelectric point of 4.0 and an apparent molecular weight, as estimated by gel filtration, of 25000. Treatment with pronase E abolished its activity.     

Biosynthesis of sphingomyelinase in normal and Niemann-Pick fibroblasts

Deficient sphingomyelinase activity and massive storage of sphingomyelin are common to two clinically different forms of Niemann-Pick disease, called types A and B. Polyclonal antisera to human sphingomyelinase precipitated both enzyme activity and the polypeptide chain of purified placental sphingomyelinase. In normal fibroblasts, following a 19-h labelling period with [35S]methionine and immunoprecipitation of the labelled proteins, sphingomyelinase occurred as a single polypeptide with a mean molecular mass of 110 kilodaltons (kDa). Niemann-Pick disease type A and B fibroblasts also synthesized a sphingomyelinase polypeptide having the same molecular mass as that found in normal fibroblasts. In I-cell disease fibroblasts, a reduced amount of cross-reacting material was detected, suggesting that sphingomyelinase may be targeted to the lysosome via the phosphomannosyl receptor. Pulse-chase experiments demonstrated sphingomyelinase processing, as judged by a substantial loss of radiolabel and the appearance of an 84-kDa intermediate form of the enzyme. These results confirm and extend previous work based on autopsy specimens and urine, and show that Niemann-Pick disease fibroblasts synthesize a sphingomyelinase polypeptide. We show for the first time that an 84-kDa processed form of the enzyme is biosynthetically related to the 110-kDa polypeptide.     

Uptake and intracellular degradation of fluorescent sphingomyelin by fibroblasts from normal individuals and a patient with Niemann-Pick disease

Sphingomyelin, labelled with a fluorescent probe, pyrene, in the fatty acyl residue was associated with fetal calf serum; approx. 80% of the sphingomyelin was found in the low- and high-density lipoproteins. This was added to the growth medium of cultured human skin fibroblasts from normal individuals and a patient with Niemann-Pick disease type A, devoid of acid sphingomyelinase activity. The fluorescent sphingomyelin was taken up by both cell types, but only the former degraded it to produce fluorescent ceramide. Differences between normal and Niemann-Pick cells in sphingomyelin content or ceramide production were observed after several hours uptake. A more pronounced difference was noted when cells were incubated for 1 day with fluorescent sphingomyelin and then for two to three days in medium devoid of this compound. Under these conditions, the fluorescence intensity of the Niemann-Pick cells remained practically constant while that of their normal counterparts was almost completely eliminated from the cells. Comparison of fluorescence intensities of these two cell types could be made directly on aqueous suspensions of whole cells or, alternatively, on their lipid extracts. For evaluation of the degradation of fluorescent sphingomyelin to ceramide within the cells, several procedures were developed for the rapid isolation of the latter compound from the total lipid extract. The results suggest that when associated with the constituents of the fetal calf serum, sphingomyelin is taken up by the cells and transported into the lysosomal compartment where it is degraded to ceramide. Use of the fluorescent derivative of sphingomyelin provided a simple and rapid procedure for following the uptake by and degradation within the cultured cells. It also permitted the establishment of differences in the rates of degradation of the fluorescent sphingomyelin by cells with a normal metabolism and others lacking sphingomyelinase (i.e., Niemann-Pick disease type A cells).     

Connecting Hsp70, sphingolipid metabolism and lysosomal stability

Heat shock protein 70 (Hsp70) is an evolutionary highly conserved molecular chaperone. Upon cancer-associated translocation to the lysosomal compartment, it promotes cell survival by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced death. We have recently shown that Hsp70 stabilizes lysosomes by binding to the endo-lysosomal lipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingolipid catabolism. The Hsp70–BMP interaction enhances the activity of acid sphingomyelinase, an important enzyme that hydrolyzes sphingomyelin. Importantly, treatment with recombinant Hsp70 effectively reverts the dramatic increase in lysosomal volume and decrease in lysosomal stability in cells from patients with Niemann-Pick disease, a genetic disorder associated with reduced acid sphingomyelinase activity. These findings give new insight into the mechanisms controlling lysosomal stability and integrity, and open new exciting possibilities for the treatment of cancer as well as Niemann-Pick disease.     

Somatic cell hybridisation studies showing different gene mutations in Niemann-Pick variants

Summary Cultured skin fibroblasts from patients with different clinical types of Niemann-Pick disease were hybridized and sphingomyelinase activities were measured in the heterokaryon cell population. Both the natural substrate (³H-choline) sphingomyelin and the chromogenic analogue hexadecanoylamino-4-nitrophenylphosphorylcholine were used in the complementation analysis. In fusions between cells from type C Niemann-Pick disease with those from type A or B a clear restoration of sphingomyelinase activity occurred, whereas no complementation was found in other fusion combinations. The results indicate that at least two different genes are involved in the mutations leading to the different Niemann-Pick variants.     

Effects of dimethylsulfoxide on sphingomyelinase activities in normal and Niemann-Pick type A, B and C fibroblasts

The effects of dimethylsulfoxide (DMSO) on sphingomyelinase activity measured at pH range 3.5-8.0 were examined in normal and Niemann-Pick disease type A, B and C fibroblasts culture. In normal cells, a minor activity was observed at pH 7.5, which was 3- to 4-fold lower than a major one at pH 5.0. Both activities at pH 5.0 and 7.5 were Mg2+-independent and localized to lysosomes. Niemann-Pick type C cells had 30-50% residual sphingomyelinase activity at both pH 5.0 and 7.5, as compared to normal control cells, whereas type A and B cells exhibited virtually no activity over the entire pH range examined. Treatment with 2% DMSO caused a marked increase in sphingomyelinase activities at pH 5.0 and 7.5 in normal and Niemann-Pick disease type C cells, while in type A and B cells, both activities remained virtually unchanged after DMSO treatment. The increase in sphingomyelinase activity at pH 5.0 induced in normal cells by DMSO resulted in an increase in the Vmax without a substantial change in the Km and was inhibited by the simultaneous addition of 10 micrograms/ml of cycloheximide. By comparison, a less than 2-fold increase in other lysosomal hydrolase activities was observed after DMSO treatment in all cell lines examined.     

Sphingomyelinase and nonspecific phosphodiesterase activities in Epstein-Barr virus-transformed lymphoid cell lines from Niemann-Pick disease A, B and C

Acid sphingomyelinase activity determined using the natural substrate, [choline-methyl-14C]sphingomyelin, or the chromogenic synthetic analogue, 2-N-(hexadecanoyl)amino-4-nitrophenylphosphorylcholine, was deficient in Epstein-Barr virus-transformed lymphoid cell lines from Niemann-Pick disease types A and B. In contrast, lines from Niemann-Pick disease type C and "sea-blue histiocyte syndrome" showed a sphingomyelinase activity within the normal range. Bis(4-methylumbelliferyl)phosphate and bis(4-methylumbelliferyl)pyrophosphate phosphodiesterase activities were not deficient in any Niemann-Pick disease cell line. These results demonstrate the validity of such cell lines as an experimental model system for enzymatic studies of Niemann-Pick disease.     

Posttranslational regulation of acid sphingomyelinase in niemann-pick type C1 fibroblasts and free cholesterol-enriched chinese hamster ovary cells

Niemann-Pick type C disease is characterized by the accumulation of cholesterol and other lipids within the lysosomal compartment, a process that is often accompanied by a reduction in acid sphingomyelinase activity. These studies demonstrate that a CHO cell mutant (CT-60), which accumulates lysosomal cholesterol because of a defective NP-C1 protein, has approximately 5-10% of the acid sphingomyelinase activity of its parental cell line (25-RA) or wild type (CHO-K1) cells. The cholesterol-induced reduction in acid sphingomyelinase activity can be reproduced in CHO-K1 cells by incubation in the presence of low density lipoprotein (LDL) and progesterone, which impairs the normal egress of LDL-derived cholesterol from the lysosomal compartment. Kinetic analysis of sphingomyelin hydrolysis in cell extracts suggests that the CT60 cells have a reduced amount of functional acid sphingomyelinase as indicated by a 10-fold reduction in the apparent V(max). Western blot analysis using antibodies generated to synthetic peptides corresponding to segments within the carboxyl-terminal region of acid sphingomyelinase demonstrate that both the CT60 and the LDL/progesterone-treated CHO-K1 cells possess near normal levels of acid sphingomyelinase protein. Likewise, Niemann-Pick type C fibroblasts also displayed normal acid sphingomyelinase protein but negligible levels of acid sphingomyelinase activity. These data suggest that cholesterol-induced inhibition is a posttranslational event, perhaps involving cofactor mediated modulation of enzymatic activity or alterations in acid sphingomyelinase protein trafficking and maturation.     

Phosphatidylinositol-3,5-Bisphosphate is a potent and selective inhibitor of acid sphingomyelinase

Acid sphingomyelinase (A-SMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to phosphorylcholine and ceramide. Inherited deficiencies of acid sphingomyelinase activity result in various clinical forms of Niemann-Pick disease, which are characterised by massive lysosomal accumulation of sphingomyelin. Sphingomyelin hydrolysis by both, acid sphingomyelinase and membrane-associated neutral sphingomyelinase, plays also an important role in cellular signaling systems regulating proliferation, apoptosis and differentiation. Here, we present a potent and selective novel inhibitor of A-SMase, L-alpha-phosphatidyl-D-myo-inositol-3,5-bisphosphate (PtdIns3,5P2), a naturally occurring substance detected in mammalian, plant and yeast cells. The inhibition constant Ki for the new A-SMase inhibitor PtdIns3,5P2 is 0.53 microM as determined in a micellar assay system with radiolabeled sphingomyelin as substrate and recombinant human A-SMase purified from insect cells. Even at concentrations of up to 50 microM, PtdIns3,5P2 neither decreased plasma membrane-associated, magnesium-dependent neutral sphingomyelinase activity, nor was it an inhibitor of the lysosomal hydrolases beta-hexosaminidase A and acid ceramidase. Other phosphoinositides tested had no or a much weaker effect on acid sphingomyelinase. Different inositol-bisphosphates were studied to elucidate structure-activity relationships for A-SMase inhibition. Our investigations provide an insight into the structural features required for selective, efficient inhibition of acid sphingomyelinase and may also be used as starting point for the development of new potent A-SMase inhibitors optimised for diverse applications.     

In situ degradation of sphingomyelin by cultured normal fibroblasts and fibroblasts from patients with Niemann-Pick disease type A and C

Cultured normal human skin fibroblasts actively degraded sphingomyelin [choline-methyl-¹⁴C] introduced in ethanolic solution in the culture medium. After 17 h incubation, about 65 to 80 % of the cellular radioactivity was recovered in phosphatidylcholine. In fibroblasts from Niemann-Pick disease type A the $\text{in situ}$ degradation of sphingomyelin was less than 2 % of controls, which was in good agreement with the strong decrease of the sphingomyelinase activity measured in $\text{vitro}$ by conventional methods. In the two cases of Niemann-Pick disease type C studied, the in situ degradation of sphingomyelin was significantly but not dramatically decreased compared to controls.     

Ultrastructural investigations on two lymphoid cell lines from Niemann-Pick disease type B

Lymphoid cell lines (LCL) were established by Epstein-Barr Virus (EBV) transformation of blood B-lymphocytes from two different patients affected with Niemann-Pick disease (NPD) type B. Those lines were severely deficient in sphingomyelinase activity (8% and 10% residual activity). Ultrastructural investigations showed in both these lines the presence of numerous osmiophilic, dense and pleiomorphic inclusions characteristic of lysosomal storage (due to the accumulation of amphiphilic lipids) similar to those observed in tissues from NPD.     

Ito cells in lysosomal storage disorders. An ultrastructural study

An ultrastructural study was performed in a series of liver biopsies from patients with various lysosomal storage diseases to evaluate the extent of lysosomal hypertrophy and hyperplasia in Ito cells (ICs). In previous studies this has been considered to be absent or only rudimentary. Lysosomal storage was recognized by the presence of storage cytosomes surrounded by limiting membranes and by the appearance of their content which was identical to that in other hepatic storage lysosomes. Storage was found in sphingomyelinase deficiency (Niemann-Pick disease types A, B), in Wolman's disease, GM1 gangliosidosis, mucopolysaccharidosis and in multiple sulphatase deficiency. In type C Niemann-Pick disease it was virtually absent with the exception of cases with prominent hepatic symptomatology. Storage was of variable degree and was accompanied by a decrease in the physiological fat content (cytoplasmic lipid droplets). The degree to which ICs were affected correlated only with the extent to which nonspecific fibroblasts were involved in the specimens studied and thus seems to reflect storage in the fibroblastic population.     

Soluble sphingomyelinase from human urine as antigen for obtaining anti-sphingomyelinase antibodies

A soluble form of lysosomal sphingomyelinase was partially purified from human urine using concanavalin A-Sepharose 4B, Sephadex G-100 and octyl-Sepharose 4B chromatography. The octyl-Sepharose 4B eluate was used to immunise a rabbit. The antiserum obtained was able to precipitate about 70% of the sphingomyelinase activity present in urine from control subjects. Both the immunoprecipitable and non-precipitable activities were found to be deficient in urine from patients with Niemann-Pick disease Type A and Type B. In contrast, both activities were present in urine from patients with Niemann-Pick disease Type C. The antiserum was able to precipitate about 80% of the sphingomyelinase activity present in an aqueous extract of placenta.     

High sphingomyelin levels induce lysosomal damage and autophagy dysfunction in Niemann Pick disease type A

Niemann Pick disease type A (NPA), which is caused by loss of function mutations in the acid sphingomyelinase (ASM) gene, is a lysosomal storage disorder leading to neurodegeneration. Yet, lysosomal dysfunction and its consequences in the disease are poorly characterized. Here we show that undegraded molecules build up in neurons of acid sphingomyelinase knockout mice and in fibroblasts from NPA patients in which autophagolysosomes accumulate. The latter is not due to alterations in autophagy initiation or autophagosome–lysosome fusion but because of inefficient autophago–lysosomal clearance. This, in turn, can be explained by lysosomal membrane permeabilization leading to cytosolic release of Cathepsin B. High sphingomyelin (SM) levels account for these effects as they can be induced in control cells on addition of the lipid and reverted on SM-lowering strategies in ASM-deficient cells. These results unveil a relevant role for SM in autophagy modulation and characterize autophagy anomalies in NPA, opening new perspectives for therapeutic interventions.     

The role of sphingomyelin in phosphatidylcholine metabolism in cultured human fibroblasts from control and sphingomyelin lipidosis patients and in Chinese hamster ovary cells.

Human fibroblasts in culture take up exogenous [choline-Me-3H,32P]sphingomyelin (SM) from the medium and incorporate it into cellular SM and phosphatidylcholine [Spence, Clarke & Cook (1983) J. Biol. Chem. 258, 8595-8600]. The ratio of [3H]choline/[32P]Pi is similar in SM and phosphatidylcholine, indicating that the phosphocholine (P-Cho) moiety is transferred intact. Similar results are obtained with Niemann-Pick (NP) cells which are deficient in lysosomal sphingomyelinase activity, suggesting that the P-Cho transfer may not be mediated by the lysosomal sphingomyelinase and that alternative pathways of sphingomyelin catabolism are present in cultured cells. In this study we have shown that: (1) the P-Cho pool in control and NP cells incubated with exogenous labelled SM has a specific radioactivity intermediate between that of SM and PtdCho; (2) expansion of the intracellular P-Cho pool by incubation with exogenous choline reduces the incorporation of [3H]choline from SM into PtdCho; and (3) incorporation of P-Cho from SM into PtdCho is decreased at the non-permissive temperature in Chinese hamster ovary cells with a temperature-sensitive mutation in the cytidylyltransferase reaction. These results suggest that incorporation of P-Cho from SM into PtdCho involves a reaction sequence in which P-Cho is hydrolysed from SM by a sphingomyelinase, followed by incorporation of P-Cho into PtdCho via the cytidine pathway of biosynthesis (SM----P-Cho----CDP-Cho----PtdCho). The appreciable incorporation of P-Cho from SM into PtdCho in sphingomyelinase-deficient NP cells suggests a more substantial or effective lysosomal sphingomyelinase activity in intact cells than is measured in vitro, and/or a significant contribution by other sphingomyelinase activities in these cells.     

Molecular basis of acid sphingomyelinase deficiency in a patient with Niemann-Pick disease type A.

Niemann-Pick disease, an autosomal recessive lysosomal storage disorder, is caused by deficiency of acid sphingomyelinase. Sequence analysis of mRNA and genomic DNA of fibroblasts of a type A patient showed a single G1729 to A nucleotide transition. This mutation resulted in a substitution of serine for normal glycine at position 577 of the peptide sequence. Amplification of the genomic DNA region around the mutation and subsequent sequencing yielded exclusively the same base change found at the cDNA level. Expression studies with this abnormal cDNA in COS-1 cells revealed a complete loss of enzymatic activity of the mutated protein. These findings indicate that this mutation is responsible for the clinical disease of the patient.     

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.