Characterization of Hex S, the major residual beta hexosaminidase activity in type O Gm2 gangliosidosis (Sandhoff-Jatzkewitz disease).

Hex S, the major residual beta hexosaminidase activity present in tissues, fluids, and cultured skin fibroblasts of patients with type 0 GM2 gangliosidosis, was isolated and characterized biochemically and immunologically. when appropriate tissue homogenates were tested by electrophoresis on cellulose acetate gels, hex S as well as hex C, the corresponding minor beta hexosaminidase component found in normal visceral tissues, migrated with greater anodic mobilities than hex A. However, a small but reproducible electrophoretic difference was observed between partially purified hex S and hex C components. Hex S and hex C had slightly higher apparent molecular weights than those of hex A or hex G; no major differences were found between hex S and hex A in thermostability, pH optimum, or kinetic properties. Hex S, like hex C from placenta, reacted with an antiserum directed towards the unique antigenic determinants alpha of hex A, indicating that hex S, hex C, and hex A share a common antigenic determinant. No reactivity of hex S was detected with an antiserum directed toward the common antigenic determinant beta of hex A and hex B. These results suggest that further biochemical and immunologic characterization of hex S and elucidation of its relationships with hex A, hex B, and hex C may significantly contribute to the understanding of the molecular defects in the GM2 gangliosidoses.     

The human GM2 activator protein. A substrate specific cofactor of beta-hexosaminidase A.

Ganglioside GD1a-GalNAc was isolated from Tay-Sachs brain, tritium-labeled in its sphingosine moiety, and its enzymic degradation studied in vitro and in cultured fibroblasts. When offered as micelles, GD1a-GalNAc was almost not hydrolyzed by Hex A or Hex B, while after incorporation of the ganglioside into the outer leaflet of liposomes, the terminal GalNAc residue was rapidly split off by Hex a. In striking contrast to ganglioside GM2, the major glycolipid substrate of Hex A, the enzymic hydrolysis of GD1a-GalNAc was not promoted by the GM2 activator protein, although the activator protein did bind GD1a-GalNAc to form a water-soluble complex. Pathobiochemical studies corroborate these results. After incorporation of [3H]GD1a-GalNAc into cultured skin fibroblasts from healthy subjects and from patients with different variants of GM2 gangliosidosis, its degradation was found to be strongly attenuated in mutant cells with Hex A deficiencies such as variant B (Tay-Sachs disease), variant B1 and variant 0 (Sandhoff disease), while in cells with variant AB (GM2 activator deficiency), its catabolism was blocked only at the level of GM2. In line with these metabolic studies, a normal content of GD1a-GalNAc was found in brains of patients who had succumbed to variant AB of GM2 gangliosidosis whereas in brains from variants B, B1, and 0, its concentration was considerably elevated (up to 19-fold). Together with studies on the enzymic degradation of GM2 derivatives with modifications in the ceramide portion, these results indicate that mainly steric hindrance by adjacent lipid molecules impedes the access of Hex A to membrane-bound GM2 (whose degradation therefore depends on solubilization by the GM2 activator) and in addition that the interaction between the GM2. GM2 activator complex and the enzyme must be highly specific.     

Juvenile Sandhoff disease: some properties of the residual hexosaminidase in cultured fibroblasts.

The residual hexosaminidase isoenzymes in juvenile Sandhoff and infantile Sandhoff disease fibroblasts, have been determined by starch gel electrophoresis and column isoelectric focusing. Hex A and hex S are the major residual isozymes in fibroblasts from the juvenile patient, while hex B is barely detectable. Only hex S could be detected in fibroblasts from infantile Sandhoff patients. These results suggest that the defects in juvenile and infantile Sandhoff disease may be different allelic modifications of the beta subunit common to hex A and hex B.     

Validity of lymphoid cell line for enzymatic studies of GM2-gangliosidosis variant 0 (Sandhoff disease)

A lymphoid cell line established by Epstein-Barr virus (EBV)-transformation of peripheral blood B-lymphocytes from a patient with Sandhoff disease showed a severe deficiency of beta-N-acetylhexosaminidase activity (residual activity around 10% of that in lymphoid cell lines from normals or other lipidotic patients). This residual beta-N-acetylhexosaminidase was completely heat-labile in contrast to that of normals. The molecular forms of residual beta-N-acetylhexosaminidase from Sandhoff lymphoid cell line were separated by Con A-sepharose and electrofocusing. Their properties and electrofocusing profiles were compared to those from Sandhoff fibroblasts and from fetal brain: this comparison permitted to identify the residual molecular forms with Hex S and Hex C. The microheterogeneity of Hex S and Hex C, demonstrated by electrofocusing, was discussed. 2-Acetamido-2-deoxy-D-galactonolactone (GalNAcLone) showed a strong inhibitory effect on lysosomal Hex A, B and S, but only a very slight effect on Hex C. Studies of the inhibition type (competitive on Hex A, B and S and mixed on Hex C) gave some informations about the enzymatic site. Elsewhere, differences in affinity of GalNAcLone for the various isoenzymes could be utilized to define optimal assay conditions for specifically determining Hex C (standard assay containing 400 mumol/l of GalNAcLone). These results demonstrated that EBV-transformed lymphoid cell lines represent an accurate model system for enzymatic studies of Sandhoff disease.     

Purification, biochemical properties and active sites of N-acetyl-beta-D-hexosaminidases from human seminal plasma.

Two isoenzymes of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) (Hex A and Hex B) from human seminal plasma were purified to homogeneity with specific activities of 26 and 60 units/mg of protein respectively. N-Acetyl-beta-D-glucosaminidase activity was inseparable from N-acetyl-beta-D-galactosaminidase activity in both Hex A and Hex B by various conventional chromatographic procedures. Although Km values of N-acetyl-beta-glucosaminidase activity of Hex A and Hex B were similar (1.33 mM), those of N-acetyl-beta-galactosaminidase activity were 0.14 mM for Hex A and 0.40 mM for Hex B. However, pH optima and temperature optima were identical for N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities of both isoenzymes; Hex A was far more heat-sensitive than Hex B. Thiol-reactive compounds such as silver salts, mercuric salts, p-chloromercuribenzoate and thimerosal strongly inhibited the N-acetyl-beta-glucosaminidase activities of both isoenzymes. GSH protected the enzyme activities from inactivation caused by these reagents, confirming the presence of thiol groups at the active centres. Inhibitions of N-acetyl-beta-glucosaminidase activities of both isoenzymes by metal salts and organic anions were comparable; acetate and arsenite were effective inhibitors for both isoenzymes. In contrast, inhibitions of N-acetyl-beta-glucosaminidase activities of the two isoenzymes by iodoacetic acid, iodoacetamide and ethylmaleimide were not comparable; Hex B was more susceptible to inhibition by these agents at 20 mM concentration. The N-acetyl-beta-glucosaminidase activities of both isoenzymes are strongly inhibited, in decreasing order, by N-acetyl-galactosamine, mannosamine, disaccharic acid lactone, N-acetylglucosamine and gluconolactone. The Ki values of the N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities for N-acetylhexosamines and results from mixed-substrate kinetics indicated that the activities for the two substrates are located at different sites in Hex A and at the same site in Hex B. The Mr values of Hex A and Hex B were determined to be 195,000 and 210,000 respectively by gel filtration through Sephadex G-200. SDS/polyacrylamide-gel electrophoresis revealed that Hex A and Hex B are each composed of four subunits corresponding to Mr about 50,000 each. No further polypeptide chain was obtained after reduction and alkylation of Hex A and Hex B with 10 mM-dithiothreitol and 10 mM-iodoacetamide.     

Purification, characterization and kinetics of beta-N-acetylhexosaminidases A and B from the slug Arion rufus L

Two beta-N-acetylhexosaminidases have been purified to homogeneity and characterized, from the digestive gland of the slug A. rufus L., showing very high specific activities. Hexosaminidase A (Hex A) was purified 1300-fold with a yield of 12%, and hexosaminidase B (Hex B) was purified 1400-fold with a yield of 20%. Purified Hex A or Hex B run as a single protein band in polyacrylamide gel disc electrophoresis, showing different mobilities. The purified preparations do not show any of the other glycosidase activities present in the crude extract. beta-N-acetylglucosaminidase (GlcNAc-ase) and beta-N-acetylgalactosaminidase (GalNAc-ase) activities are always associated in a single peak for each enzyme form, with constant activity ratio, in all the purification steps, since they are catalyzed by the same enzyme (Hex A or Hex B). The optimal pH for both forms are 4.5 for GlcNAc-ase and 4.0 for GalNAc-ase activity. Hex B shows thermal and pH-stability higher than Hex A. The isoelectric points are 4.5 and 5.5 for A and B forms, respectively. The molecular weight is 150 000 for Hex A and 320 000 for Hex B. The amino acid composition of purified Hex A and B presents some differences concerning particularly Cys, Thr, Ser, Glu and Ile. The ratios Vmax/Km show that GlcNAc-ase is the main activity of both enzyme forms. beta-N-acetylglucosides and beta-N-acetylgalactosides completely compete for a common active site in mixed-substrates experiments. The Ki values are always coincident for GlcNAc-ase and GalNAc-ase activities, using competitive inhibitors (the corresponding lactones). These results strongly suggest that both activities are catalyzed by the same active site in both Hex A and B. Inhibition of the enzyme activities was found with the corresponding lactones, N-acetyl hexosamines, mannose, mannosides, HgCl2 and lead acetate; activation, with ribose, and with some chlorides and sulphates of divalent cations.     

Impaired proteolytic processing of lysosomal N-acetyl-beta-hexosaminidase in cultured fibroblasts from patients with infantile generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from patients suffering with infantile generalized N-acetylneuraminic acid (NeuAc) storage disease accumulate free NeuAc in a population of lysosomes less dense than those observed in normal fibroblasts (1.035 vs. greater than 1.07 mean density), as assessed by the distribution of lysosomal enzyme activities and NeuAc on Percoll gradients after subcellular fractionation. In the present study, normal and affected fibroblasts were labeled with [35S]methionine, and cell homogenates or subcellular fractions from Percoll gradients were immunoprecipitated with polyclonal antibodies to lysosomal N-acetyl-beta-hexosaminidase (Hex); immunoprecipitated polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis. The synthesis and initial processing of Hex polypeptides were comparable in normal and affected fibroblasts, but mature polypeptides were quantitatively localized in "buoyant" lysosomes of affected cells, along with Hex activity; moreover, mature alpha-chain of Hex was approximately 2 kDa larger than that observed in normal cells. The molecular weight difference was apparently due to impaired proteolytic processing of alpha-chain in affected fibroblasts, since treatment of immunoprecipitated alpha-chain from normal and affected cells with neuraminidase and endo-beta-N-acetylglucosaminidase H failed to resolve the molecular weight difference. The impaired processing was observed to be persistent (after a chase of up to 200 h), but had no apparent effect on the turnover or activity of Hex in affected fibroblasts. The observed proteolytic processing defect may be primary or secondary in infantile NeuAc storage disease.     

Prenatal diagnosis of Tay-Sachs disease. Reflectometry of hexosaminidase A,B, and C/S bands on zymograms

Summary Hexosaminidase (Hex) A, B, and C/S were electrophoretically separated from cultured amniotic fluid cells, fetal brain, and white blood cells. Photographs of cellulose acetate zymograms were evaluated by reflectometric scanning. The usefulness and limitations of this rapid method were shown. Hex A was completely absent in the amniotic fluid cells of one out of three pregnancies at risk for Tay-Sachs disease, but Hex C/S was present in this case. The prenatal diagnosis of Tay-Sachs disease was made, and confirmed with the fetal material after abortion. Hex C/S was distinguishable from a residual or heterozygous Hex A activity. In the two other risk pregnancies, reflectometric Hex A activities were found to be 50 and 34% of control; the heterozygous stage was presumed for the fetuses.     

Biosynthesis and maturation of arylsulfatase B in normal and mutant cultured human fibroblasts

The biosynthesis of arylsulfatase B in normal and mutant human skin fibroblasts was studied by metabolic labeling with radioactive amino acids, monosaccharides, or 32Pi and by specific immunoprecipitation followed by polyacrylamide gel electrophoresis and fluorography. Three major polypeptides with apparent molecular weights of 47,000, 40,000, and 31,000 were found intracellularly and one of 64,000 in the medium. Pulse-chase labeling and uptake experiments showed that arylsulfatase B synthesized and secreted as a 64,000 precursor was intracellularly processed within less than 24 h via short lived intermediates to two different forms. Form I (chains of 47,000 and 11,500) was labeled earlier and was about twice as stable as form II (chains of 40,000 and 31,000). The secreted 64,000 precursor and the 40,000 chain of form II contained oligosaccharides resistant to endo-beta-N-acetylglucosaminidase H. In the other chains mainly cleavable and phosphorylated oligosaccharides were found. Arylsulfatase B activity was associated with the 64,000 precursor and with form I, but not with form II. Fibroblasts of four patients with the severe form of mucopolysaccharidosis type VI, which were deficient in arylsulfatase B activity, synthesized and secreted the 64,000 precursor at a normal rate. This precursor, however, had little if any catalytic activity and one of its mature forms (I) was rapidly degraded.     

Epstein-Barr virus transformed lymphoid cell lines as a new model system in culture for the study of GM2-gangliosidoses: Tay-Sachs and Sandhoff diseases

Epstein-Barr Virus transformed cell lines (LCL) were established from blood B-lymphocytes of patients affected with GM2-gangliosidoses variant O (Sandhoff disease, SD) and variant B (Tay-Sachs disease, TSD). LCL from SD showed a severe deficiency of activity of the major lysosomal beta-N-acetylhexosaminidase isoenzymes, Hex A and B; the residual activity was due to Hex S and Hex C. In LCL from TSD, the whole Hex activity was not deficient but isoenzyme composition was completely abnormal. Ultrastructural investigations showed the presence of pleiomorphic enlarged lysosomes appearing as clear vacuoles containing a finely fibrillo-granular material characteristic of the visceral lysosomal storage of gangliosidoses.     

Presence of arylsulfatase A (ARS A) in multiple sulfatase deficiency disorder fibroblasts.

Multiple deficiency disorder fibroblasts cultured in MEM-CO2 showed deficiencies of arylsulfatase A(ARS A) comparable to the deficiency in metachromatic leukodystrophy fibroblasts. However, the MSDD fibroblasts cultured in MEM-HEPES contained near normal levels of ARS A. Moreover, the enzyme from the latter fibroblasts was indistinguishable from ARS A of control fibroblasts on DEAE-cellulose chromatography, ratio of activity with several substrates, thermal inactivation, sensitivity to inhibitors, and precipitation by antiserum to human ARS A. These data support the conclusion that the ARS A genome is intact in MSDD fibroblasts and, by extension, in MSDD patients. Other sulfatases were present at levels ranging from mildly deficient to near normal but never as low as seen in the corresponding specific sulfatase deficient disorders.     

Expression of Hex homeobox gene during skin development: Increase in epidermal cell proliferation by transfecting the Hex to the dermis

A number of homeobox genes have been found to be expressed in skin and its appendages, such as scale and feather, and appear to be candidates for the regulation of the development of these tissues. We report that the proline-rich divergent homeobox gene Hex is expressed during development of chick embryonic skin and its appendages (scale and feather). In situ hybridization analysis revealed that, during development of the skin, a transient expression of the Hex gene was observed. While the expression of Hex in the dermis was closely correlated with proliferation activity of epidermal basal cells, that in the epidermis was related to a suppression of epidermal differentiation. When dermal fibroblasts were transfected with Hex, stimulation of both DNA synthesis and proliferation of the epidermal cells followed by two-fold scale ridge elongation and increase in epidermal area was observed during culture of the skin, whereas epidemal keratinization was not affected. This is the first study to demonstrate that Hex is expressed during development of the skin and its appendages and that its expression in the dermal cells regulates epidermal cell proliferation through epithelial mesenchymal interaction.     

Processing of human beta-galactosidase in GM1-gangliosidosis and Morquio B syndrome

The nature of the molecular defect resulting in the beta-galactosidase deficiency in different forms of GM1-gangliosidosis and mucopolysaccharidosis IV B (Morquio B syndrome) was investigated. Normal and mutant cultured skin fibroblasts were labeled in vivo with [3H]leucine and immunoprecipitation studies with human anti-beta-galactosidase antiserum were performed, followed by polyacrylamide gel electrophoresis and fluorography. In Morquio B syndrome, the mutation does not interfere with the normal processing and intralysosomal aggregation of beta-galactosidase. In cells from infantile and adult GM1-gangliosidosis, 85-kDa precursor beta-galactosidase was found to be synthesized normally but more than 90% of the enzyme was subsequently degraded at one of the early steps in posttranslational processing. The residual 5-10% beta-galactosidase activity in adult GM1-gangliosidosis is 64-kDa mature lysosomal enzyme with normal catalytic properties but with a reduced ability of the monomeric form to aggregate into high molecular weight multimers. Knowledge of the exact nature of the molecular defect underlying beta-galactosidase deficiency in man may lead to a better understanding of the clinical and pathological heterogeneity among patients with different types of GM1-gangliosidosis and Morquio B syndrome.     

Hereditary heat-labile hexosaminidase B: a variant whose homozygotes synthesize a functional HEX A.

Homozygosity for a mutant allele at the beta-chain locus of hexosaminidase (HEX), resulting in a variant of heat-labile HEX B, is reported for the first time in two healthy children. HEX activity in their sera, leukocytes, and cultured skin fibroblasts is severely deficient when measured on the synthetic substrate 4-MU-GLcNAc. However, their cultured skin fibroblasts synthesize and process both alpha and beta chains of HEX, and their lymphoid cells hydrolyze normally the natural ganglioside GM2. This mutation is, therefore, different from at least one of the beta-chain mutations found in previously published families with heat-labile HEX B.     

Heterogeneity for adenosine deaminase deficiency: Expression of the enzyme in cultured skin fibroblasts and amniotic fluid cells.

Adenosine deaminase (ADA) could be quantitated and the isozyme pattern characterized in cultured amniotic fluid cells. In 20 amniotic fluid cell cultures the mean specific activity was 14.3 U/g protein +/- 6.7 (SD) and compared favorably with values of 14.6 U/g protein +/- 6.8 (SD) observed in 26 cultures of skin fibroblasts. In cultures of skin fibroblasts established from two obligate heterozygotes for ADA deficiency, the specific activity of ADA was 7.0 and 7.7 U/g protein. The ADA isozyme pattern that existed in cultures of amniotic fluid cells was the same as that observed in cultured skin fibroblasts. This identification of the same apparent enzyme may permit the prenatal diagnosis of that form of combined immunodeficiency disease caused by ADA deficiency. Residual enzyme activity of less than 1% and 10% of the mean of normal fibroblasts could be measured in cultured fibroblasts from two unrelated children with ADA deficiency and combined immunodeficiency disease. The tissue-specific enzyme from cultured skin fibroblasts from the child with 10% residual activity had a faster electrophoretic mobility and greater heat stability than normal ADA. This enzymatic evidence indicates that at least two mutant alleles exist at the locus for ADA which predispose to combined immunodeficiency disease when present in the homozygous state.     

Pathochemistry, pathogenesis and enzyme replacement in multiple-sulfatase deficiency

Multiple-sulfatase deficiency (MSD) is now considered to be heterogeneous and could be classified into three or four clinical phenotypes according to the onset of the disease: neonatal, late infantile, juvenile and possibly adult type. Neonatal-type MSD shows severe clinical involvement and practically no arylsulfatase A, B and C activities in cultured skin fibroblasts. Furthermore, arylsulfatase A activity in neonatal-type MSD was not enhanced by the addition of thiosulfate. Therefore, it is distinct from late infantile-type MSD. The degradation of 14C-sulfatide can occur in MSD-cultured skin fibroblasts and was much higher than in late infantile-type MLD. The addition of thiol protease such as leupeptin to cultured MSD skin fibroblasts enhanced arylsulfatase A activity as well as the degradation of 14C-sulfatide. This suggests that the decreased activities of arylsulfatase A is due to an acceleration of the enzyme degradation. Enzyme replacement by the addition of arylsulfatases of different sources (human liver, brain, fungus) was carried out in cultured MSD skin fibroblasts. Human enzymes of arylsulfatase A and B were mostly taken up by MSD cells rather than those of fungus origin. By the exposure to leukocytes to cultured skin fibroblasts, MSD cells corrected arylsulfatase A and B activities.     

Molecular analysis of the acid sphingomyelinase deficiency in a family with an intermediate form of Niemann-Pick disease.

A novel point mutation in the lysosomal acid sphingomyelinase gene has been identified in the recently reported Serbian family with a clinically and biochemically atypical intermediate form of Niemann-Pick disease. The mutation was a T1171-->G transversion resulting in substitution of glycine for normal tryptophan at amino acid residue 391. The coding sequence was otherwise normal. All of the five affected individuals were almost certainly homoallelic, and both of the two obligate heterozygotes studied also carried the same mutation. This mutation is therefore likely to be directly associated with the atypical phenotype of these patients. Expression in COS-1 cells suggested a higher residual activity than that in cultured fibroblasts. A recently developed high-affinity rabbit antihuman sphingomyelinase antibody allowed us to study for the first time the biosynthesis, processing, and targeting of a mutant sphingomyelinase by metabolic labeling of cultured fibroblasts. The mutant enzyme protein was normally synthesized, processed, and routed to the lysosome but was apparently unstable and degraded rapidly once it reached the lysosome. Together with the finding of the relatively high residual activity in COS-1 cells, we interpret our observations to mean that instability and rapid breakdown of the mature mutant enzyme protein, due to the mutation rather than direct inactivation of the catalytic activity, is the primary mechanism for the deficiency of sphingomyelinase activity in these patients. A high prevalence of this mutation in the Serbian population is likely, since the family pedigree indicates that members from four reportedly unrelated families must have contributed the same mutation.     

The molecular defect in an autosomal dominant form of osteogenesis imperfecta. Synthesis of type I procollagen containing cysteine in the triple-helical domain of pro-alpha 1(I) chains

Synthesis of procollagen was examined in skin fibroblasts from a patient with a moderately severe autosomal dominant form of osteogenesis imperfecta. Proteolytic removal of the propeptide regions of newly synthesized procollagen, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, revealed the presence of type I collagen in which two alpha 1(I) chains were linked through interchain disulfide bonds. Fragmentation of the disulfide-bonded alpha 1(I) dimers with vertebrate collagenase and cyanogen bromide demonstrated the presence of a cysteine residue in alpha 1(I)CB8, a fragment containing amino acid residues 124-402 of the alpha 1(I) collagen chain. Cysteine residues are not normally found in the triple-helical domain of type I collagen chains. The heterozygous nature of the molecular defect resulted in the formation of three kinds of type I trimers: a normal type with normal pro-alpha(I) chains, a type I trimer with one mutant pro-alpha 1(I) chain and two normal chains, and a type I trimer containing two mutant pro-alpha 1(I) chains and one normal pro-alpha 2(I) chain. The presence of one or two mutant pro-alpha 1(I) chains in trimers of type I procollagen was found to reduce the thermal stability of the protein by 2.5 and 1 degree C, respectively. In addition to post-translational overmodification, procollagen containing one mutant pro-alpha 1(I) chain was also cleared more slowly from cultured fibroblasts. The most likely explanation for these disruptive changes in the physical stability and secretion of the mutant procollagen is that a cysteine residue is substituted for a glycine in half of the pro-alpha 1(I) chains synthesized by the patient's fibroblasts.     

Properties of α-l-iduronidase in cultured skin fibroblasts from α-l-iduronidase-deficient patients

Summary On DEAE cellulose column chromatography, -l-iduronidase in cultured skin fibroblasts was resolved into two distinct components, forms A and B. They had similar K_m values for 4-methylumbelliferyl--l-iduronide, but differed in pH optima and thermal stability. Form B was more heat-stable than form A.Residual -l-iduronidase activity in Hurler fibroblasts was heat-stable, while that in Scheie fibroblasts was heat-labile, and moreover, that in Hurler-Scheie compound fibroblasts lay intermediate between Hurler and Scheie syndromes. These findings demonstrated that Hurler syndrome, Scheie syndrome and Hurler-Scheie compound were enzymatically distinguishable.     

Glutamic Acid Decarboxylase and γ-Aminobutyric Acid in Huntington''s Disease Fibroblasts and Other Cultured Cells, Determined by a [3H]Muscimol Radioreceptor Assay

A sensitive and reproducible [3H]muscimol radioreceptor assay was developed for measuring low levels of both glutamic acid decarboxylase activity and gamma-aminobutyric acid. By using this technique, endogenous gamma-aminobutyric acid and glutamic acid decarboxylase activity were detected in two rat neuroblastomas, B35 and B50, a human medulloblastoma cell line, TE671, and cultured human skin fibroblasts. Glutamic acid decarboxylase activities and gamma-aminobutyric acid levels were compared for human skin fibroblasts obtained from patients with Huntington's disease and their controls in a well-controlled, blind study. However, no significant difference was found to either measure between Huntington and control cells. Glutamic acid decarboxylase activity was relatively low in all cell types examined except for the TE671 cells, which had more than four times the activity found in the other cells. This human medulloblastoma cell line appears to be a good model for studying gamma-aminobutyric acid metabolism and the control of glutamic acid decarboxylase expression.