A Candida albicans mutant impaired in the utilization of N-acetylglucosamine

Indicator plates containing eosin, methylene blue, glucosamine and proline were used to select mutants of Candida albicans impaired in the utilization of glucosamine. One such mutant, strain hOG298, grew on glucosamine at a slower rate than the parent and was severely impaired in growth on N-acetylglucosamine. The mutant was unable to express the first three steps in the N-acetylglucosamine pathway: viz the permease, N-acetylglucosamine kinase and N-acetylglucosamine-6-phosphate deacetylase. Glucosamine-6-phosphate deaminase was, however, induced by N-acetylglucosamine. The mutant still possessed a constitutive uptake system and kinase activity for glucosamine but glucosamine neither increased the glucosamine kinase activity nor induced N-acetylglucosamine kinase. These findings accounted for the decreased growth rate on glucosamine. The parent strain formed germ-tubes in N-acetylglucosamine or 4% (v/v) serum but the mutant formed germ-tubes only in serum.     

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.     

Crystallographic structure of human beta-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis

Lysosomal beta-hexosaminidase A (Hex A) is essential for the degradation of GM2 gangliosides in the central and peripheral nervous system. Accumulation of GM2 leads to severely debilitating neurodegeneration associated with Tay-Sachs disease (TSD), Sandoff disease (SD) and AB variant. Here, we present the X-ray crystallographic structure of Hex A to 2.8 A resolution and the structure of Hex A in complex with NAG-thiazoline, (NGT) to 3.25 A resolution. NGT, a mechanism-based inhibitor, has been shown to act as a chemical chaperone that, to some extent, prevents misfolding of a Hex A mutant associated with adult onset Tay Sachs disease and, as a result, increases the residual activity of Hex A to a level above the critical threshold for disease. The crystal structure of Hex A reveals an alphabeta heterodimer, with each subunit having a functional active site. Only the alpha-subunit active site can hydrolyze GM2 gangliosides due to a flexible loop structure that is removed post-translationally from beta, and to the presence of alphaAsn423 and alphaArg424. The loop structure is involved in binding the GM2 activator protein, while alphaArg424 is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. The beta-subunit lacks these key residues and has betaAsp452 and betaLeu453 in their place; the beta-subunit therefore cleaves only neutral substrates efficiently. Mutations in the alpha-subunit, associated with TSD, and those in the beta-subunit, associated with SD are discussed. The effect of NGT binding in the active site of a mutant Hex A and its effect on protein function is discussed.     

The presence of two different infantile Tay-Sachs disease mutations in a Cajun population.

A study was undertaken to characterize the mutation(s) responsible for Tay-Sachs disease (TSD) in a Cajun population in southwest Louisiana and to identify the origins of these mutations. Eleven of 12 infantile TSD alleles examined in six families had the beta-hexosaminidase A (Hex A) alpha-subunit exon 11 insertion mutation that is present in approximately 70% of Ashkenazi Jewish TSD heterozygotes. The mutation in the remaining allele was a single-base transition in the donor splice site of the alpha-subunit intron 9. To determine the origins of these two mutations in the Cajun population, the TSD carrier status was enzymatically determined for 90 members of four of the six families, and extensive pedigrees were constructed for all carriers. A single ancestral couple from France was found to be common to most of the carriers of the exon 11 insertion. Pedigree data suggest that this mutation has been in the Cajun population since its founding over 2 centuries ago and that it may be widely distributed within the population. In contrast, the intron 9 mutation apparently was introduced within the last century and probably is limited to a few Louisiana families.     

Galactose-6-sulfatase from Actinobacillus sp. IFO-13310 and its action on sulfated oligosaccharides from keratan sulfate.

A 6-sulfatase specific for sugasr of the galactose configuration was purified 81-fold from the crude extract of Actinobacillus sp. IFO-13310. This preparation contained activity towards both N-acetylgalactosamine 6-sulfate and galactose 6-sulfate (relative activity, 2.4 : 1). The enzyme also release inorganic sulfate from the non-reducing galactose 6-sulfate end group of a trisaccharide disulfate prepared from keratan sulfate by sequential degradation with endo-beta-galactosidase, N-acetylglucosamine-6-sulfatase and exo-beta-N-acetylglucosaminidase. In addition, a tetrasaccharide trisulfate bearing the non-reducing N-acetylglucosamine 6-sulfate end group, also enzymatically prepared from keratan sulfate, was degraded to give rise to inorganic sulfate, N-acetylglucosamine and galactose by the sequential action of this enzyme, N-acetylglucosamine-6-sulfatase, exo-beta-N-acetylglucosaminidase and exo-beta-galactosidase (Charonia lampas).     

Enzymatic deacetylation of N-acetylglucosamine residues in cell wall peptidoglycan

An enzyme which catalyzes the hydrolysis of acetamido groups of N-acetylglucosamine residues in cell wall peptidoglycan was found in the supernatant and 20,000 X g pellet fractions of Bacillus cereus. Autolysis of the latter fraction resulted in solubilization and activation of the deacetylase. Among various bacteria, strains of B. cereus which contain high proportions of N-unsubstituted glucosamine residues in their cell wall peptidoglycan components are particularly rich in the deacetylase. The peptidoglycan deacetylase is distinguishable from N-acetylglucosamine-6-phosphate deacetylase [EC 3.5.1.25] on the basis of their cellular distribution and chromatographic behavior. The rate of reaction of the deacetylase with (N-acetylglucosaminyl-N-acetylmuramic acid)3 [abbreviated as (GlcNAc-MurNAc)3] is less than 1/100 of that with peptidoglycan, while the enzyme is inactive towards (GlcNAc-MurNAc)2, GlcNAc-MurNAc, and monomeric N-acetylglucosamine derivatives. The enzyme also deacetylates partially O-hydroxyethylated chitin. The concentrations of peptidoglycan and partially O-hydroxyethylated chitin required for half-maximum activities were found to be 0.29 and 6.9 mg per ml (or 0.17 and 20 mM with respect to N-acetylglucosamine residues), respectively. The occurrence of this enzyme accounts for the formation of cell wall peptidoglycan N-unsubstituted at the glucosamine residues.     

Repression and induction of the nag regulon of Escherichia coll K-12: the roles of nagC and nagA in maintenance of the uninduced state

The nag regulon located at 15.5 min on the Escherichia coli chromosome consists of two divergent operons, nagE and nagBACD, encoding genes involved in the uptake and metabolism of N-acetylglucosamine. Null mutations have been created in each of the genes by insertion of antibiotic resistance cartridges. The phenotypes of the strains carrying the insertions in nagE, B and A were consistent with the previous identification of gene products: nagE, EII(Nag), the N-acetylglucosamine specific transporter of the phosphotransferase system and nagB and nagA, the two enzymes necessary for the degradation of N-acetylglucosamine. Insertions in the nagC result in derepression of the nag genes, which is consistent with earlier observations that the nagC gene encodes the repressor of the regulon. Insertions in nagA also provoke a derepression, implying that nagA has a role in the regulation of the expression of the nag regulon as well as in the degradation of the amino-sugars. N-acetylglucosamine-6-phosphate, the intracellular product of N-acetylglucosamine transport and the substrate of the nagA gene product, is shown to be an inducer of the regulon and this suggests how nagA mutations result in derepression: the absence of N-acetylglucosamine-6-phosphate deacetylase allows N-acetylglucosamine-6-phosphate to accumulate and induce the regulon.     

弧菌几丁质降解代谢及调控机理的研究进展

对近年来在弧菌中几丁质降解代谢的主要过程及调控机理方面的研究进行了综述,指出弧菌降解几丁质主要分为3个步骤,几丁质的水解、几丁寡糖和N-乙酰基葡萄糖胺的运输、几丁二糖和N-乙酰基葡萄糖胺-6-磷酸的进一步降解等,且此过程的许多环节均受到二元信号传导系统的调控。     

Mutational analyses of Tay-Sachs disease: studies on Tay-Sachs carriers of French Canadian background living in New England.

Tay-Sachs disease (TSD) results from mutations in HEXA that cause Hex A deficiency. Heterozygote-screening programs have been applied in groups with an increased TSD incidence, such as Ashkenazi Jews and French Canadians in Quebec. These programs are complicated by benign mutations that cause apparent Hex A deficiency but not TSD. Benign mutations account for only approximately 2% of Jewish and approximately 36% of non-Jewish enzyme-defined carriers. A carrier frequency of 1/53 (n = 1,434) was found in an ongoing prospective analysis of persons of French Canadian background living in New England by using an enzyme-based assay. DNA from enzyme-defined carriers from this population was analyzed to determine the molecular basis of Hex A deficiency. Samples (36) were tested for common mutations, and samples that were negative for these were screened for uncommon or novel mutations by using SSCP analysis. Exons showing mobility shifts were sequenced, and most mutations were confirmed by restriction enzyme digestion. Known disease-causing mutations were found in nine samples (four had a 7.6-kb deletion found in 80% of French Canadian TSD alleles), and known benign mutations were found in four samples. Seven novel changes were identified, including G748A in four samples. The molecular basis of Hex A deficiency in this carrier population differs from that of French Canadian TSD patients. Screening centers should be aware of the presence of benign mutations among U.S. French Canadians or Franco-Americans.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermolabile hexosaminidase (Hex) B: diverse frequencies among Jewish communities and implication for screening of sera for Hex A deficiencies

Twenty unrelated people with thermolabile beta-hexosaminidase (Hex) B were identified in random samples of 41,561 adult Israeli Jews whose sera were screened for Hex A levels. Eighteen of them originated from contiguous Middle Eastern countries (n = 1,337) and only 2 were Ashkenazi Jews (n = 38,388). None was found among the screened Moroccan Jews (n = 1,524). The commonly used screening test for detection of Tay-Sachs disease (TSD) carriers is the serum Hex heat inactivation method (HIM), which is based on the assumption that Hex A is the only thermolabile component of Hex; However, in the presence of thermolabile Hex B, HIM could lead to false-negative results. Since TSD is very rare among Mideastern Jews while thermolabile Hex B is very rare among Ashkenazi and Moroccan Jews, it is concluded that at present there is almost no risk of such false-negative results. In order to further reduce this risk it is recommended that screening of people of Mideastern or mixed ancestry be done with HIM in leukocytes rather than in serum or with the specific substrate for Hex A in serum.     

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.     

Assay and properties of N-acetylglucosamine-6-phosphate deacetylase from rat liver

A single-vial assay has been developed for N-acetylglucosamine-6-phosphate deacetylase, in which [3H]acetate released from 3H-acetyl-labeled substrate is measured in a biphasic liquid scintillation counting system after acidification of the reaction mixture. The deacetylase was partially purified from rat liver, and some of its properties were determined. Chromatography on a calibrated Sepharose CL-6B column indicated a molecular weight of 345,000. The Km for the substrate at pH 8.0 was 0.3 mM. Glucosamine 6-phosphate and glucose 6-phosphate inhibited the enzyme, whereas N-acetylgalactosamine, N-acetylglucosamine, N-acetylglucosamine 1-phosphate, and glucosamine 1-phosphate were without effect. The effects of several divalent cations were also examined. Under the conditions tested, Ca2+, Mg2+, and Ba2+ had essentially no effect, whereas Mn2+, Ni2+, and Cu2+ were inhibitory and Co2+ stimulated activity at low concentrations but inhibited above 5 mM. An increase in the ionic strength of the reaction mixture to 0.3 M decreased the activity by 40%.     

Sandhoff disease heterozygote detection: a component of population screening for Tay-Sachs disease carriers. I. Statistical methods

Serum and leukocyte hexosaminidase profiles (total activity and percent heat-labile activity levels) in obligate Sandhoff disease (SHD) heterozygotes differ from those of obligate Tay-Sachs disease (TSD) heterozygotes and noncarrier individuals. We have developed a procedure to identify, with 95% sensitivity, carriers of the allele(s) for SHD among individuals screened in a TSD heterozygote identification program. Using multivariate statistical methods of cluster analysis and discriminant analysis on serum and leukocyte hexosaminidase profiles from 102 potential SHD carriers, a linear discriminant function to classify individuals as SHD carriers or SHD noncarriers was constructed. This function classifies the serum and leukocyte profiles from all 15 obligate SHD heterozygotes studied, as those of SHD carriers. A 95% isodensity ellipse derived from only the serum hexosaminidase profiles of the 15 SHD obligate carriers has been applied to a TSD screened sample of 37,843 Jewish and non-Jewish individuals. A potential recall rate of screened individuals for serum retests and leukocyte assays of 2.01% has been estimated. These statistical methods enhance the TSD heterozygote screening program by permitting one to detect SHD heterozygotes within the screened population.     

Intralysosomal formation and metabolic fate of N-acetylglucosamine 6-sulfate from keratan sulfate

The physiological relevance of the ability of beta-N-acetylhexosaminidase A to liberate N-acetylglucosamine 6-sulfate from polymeric keratan sulfate was investigated. Upon intravenous injection into rats of [35S]sulfate-labeled proteokeratan sulfate up to 25% of the radioactivity excreted with the urine were identified as N-acetyl-glucosamine 6-sulfate. Within 24 h, however, excretion of inorganic sulfate rose at the expense of the sulfated monosaccharide. Upon incubation in vitro of liver lysosomes from rats treated with proteokeratan sulfate, inorganic sulfate and minor amounts of sulfated monosaccharide were found in the incubation fluid. Cultured rat peritoneal macrophages ingested proteokeratan sulfate with a clearance rate of 6-9 micrograms X h-1 X mg cell protein-1 and degraded it rapidly. Inorganic sulfate but not N-acetylglucosamine 6-sulfate was delivered to the culture medium. During a chase period the amount of intracellular N-acetylglucosamine 6-sulfate fell, and a corresponding amount of sulfate could be found extracellularly. Significant amount of N-acetylglucosamine 6-sulfate were only found in the culture medium when the cells were challenged with zymosan. These results suggest that N-acetylglucosamine 6-sulfate is a physiological intermediate during the degradation of keratan sulfate, but is usually hydrolyzed intralysosomally by N-acetylglucosamine-6-sulfate sulfatase. Genetic deficiency of the sulfatase in humans therefore results in excessive excretion of the sulfated amino sugar but not of keratan sulfate.     

Low levels of beta hexosaminidase A in healthy individuals with apparent deficiency of this enzyme.

Appreciable beta hexosaminidase A (hex A) activity has been detected in cultured skin fibroblasts and melanoma tissue from healthy individuals previously reported as having deficiency of hex A activity indistinguishable from that of patients with Tay-Sachs disease (TSD). Identification and quantitation of hex A, amounting to 3.5%-6.9% of total beta hexosaminidase activity, has been obtained by cellulose acetate gel electrophoresis, DEAE-cellulose ion-exchange chromatography, radial immunodiffusion, and radioimmunoassay. Previous family studies suggested that these individuals may be compound heterozygotes for the common mutant TSD gene and a rare (allelic) mutant gene. Thus, the postulated rate mutant gene appears to code for the expression of low amounts of hex A. Heterozygotes for the rare mutant may be indistinguishable from heterozygotes for the common TSD mutant. However, direct visualization and quantitation of hex A by the methods described may prevent false-positive prenatal diagnosis of TSD in fetuses having the incomplete hex A deficiency of the type described in the four healthy individuals.     

A second mutation associated with apparent beta-hexosaminidase A pseudodeficiency: identification and frequency estimation.

Deficient activity of beta-hexosaminidase A (Hex A), resulting from mutations in the HEXA gene, typically causes Tay-Sachs disease. However, healthy individuals lacking Hex A activity against synthetic substrates (i.e., individuals who are pseudodeficient) have been described. Recently, an apparently benign C739-to-T (Arg247Trp) mutation was found among individuals with Hex A levels indistinguishable from those of carriers of Tay-Sachs disease. This allele, when in compound heterozygosity with a second "disease-causing" allele, results in Hex A pseudodeficiency. We examined the HEXA gene of a healthy 42-year-old who was Hex A deficient but did not have the C739-to-T mutation. The HEXA exons were PCR amplified, and the products were analyzed for mutations by using restriction-enzyme digestion or single-strand gel electrophoresis. A G805-to-A (Gly269Ser) mutation associated with adult-onset GM2 gangliosidosis was found on one chromosome. A new mutation, C745-to-T (Arg249Trp), was identified on the second chromosome. This mutation was detected in an additional 4/63 (6%) non-Jewish and 0/218 Ashkenazi Jewish enzyme-defined carriers. Although the Arg249Trp change may result in a late-onset form of GM2 gangliosidosis, any phenotype must be very mild. This new mutation and the benign C739-to-T mutation together account for approximately 38% of non-Jewish enzyme-defined carriers. Because carriers of the C739-to-T and C745-to-T mutations cannot be differentiated from carriers of disease-causing alleles by using the classical biochemical screening approaches, DNA-based analyses for these mutations should be offered for non-Jewish enzyme-defined heterozygotes, before definitive counseling is provided.     

Rapid identification of HEXA mutations in Tay-Sachs patients

Tay-Sachs disease (TSD) is a recessively inherited neurodegenerative disorder due to mutations in the HEXA gene resulting in a β-hexosaminidase A (Hex A) deficiency. The purpose of this study was to characterize the molecular abnormalities in patients with infantile or later-onset forms of the disease. The complete sequencing of the 14 exons and flanking regions of the HEXA gene was performed with a unique technical condition in 10 unrelated TSD patients. Eleven mutations were identified, including five splice mutations, one insertion, two deletions and three single-base substitutions. Four mutations were novel: two splice mutations (IVS8+5G > A, IVS2+4delAGTA), one missense mutation in exon 6 (c.621T > G (p.D207E)) and one small deletion (c.1211-1212delTG) in exon 11 resulting in a premature stop codon at residue 429. The c.621T > G missense mutation was found in a patient presenting an infantile form. Its putative role in the pathogenesis of TSD is suspected as residue 207 is highly conserved in human, mouse and rat. Moreover, structural modelling predicted changes likely to affect substrate binding and catalytic activity of the enzyme. The time-saving procedure reported here could be useful for the characterization of Tay-Sachs-causing mutations, in particular in non-Ashkenazi patients mainly exhibiting rare mutations.     

Divergent homeobox gene hex regulates promoter of the Na(+)-dependent bile acid cotransporter

The divergent homeobox gene Hex is expressed in both developing and mature liver. A putative Hex binding site was identified in the promoter region of the liver-specific Na(+)-bile acid cotransporter gene (ntcp), and we hypothesized that Hex regulates the ntcp promoter through this site. Successive 5'-deletions of the ntcp promoter in a luciferase reporter construct transfected into Hep G2 cells confirmed a Hex response element (HRE) within the ntcp promoter (nt -733/-714). Moreover, p-CMHex transactivated a heterologous promoter construct containing HRE multimers (p4xHRELUC), whereas a 5-bp mutation of the core HRE eliminated transactivation. A dominant negative form of Hex (p-Hex-DN) suppressed basal luciferase activity of p-4xHRELUC and inhibited activation of this construct by p-CMHex. Interestingly, p-CMHex transactivated the HRE in Hep G2 cells but not in fibroblast-derived COS cells, suggesting the possibility that Hex protein requires an additional liver cell-specific factor(s) for full activity. Electrophoretic mobility shift assays confirmed that liver and Hep G2 cells contain a specific nuclear protein that binds the native HRE. We have demonstrated that the liver-specific ntcp gene promoter is the first known target of Hex and is a useful tool for evaluating function of the Hex protein.     

Sanfilippo D syndrome: estimation of N-acetylglucosamine-6-sulfatase activity with a radiolabeled monosulfated disaccharide substrate

N-Acetylglucosamine-6-sulfatase activity was assayed by incubation of the radiolabeled disaccharide O-(a-N-acetylglucosamine-6-sulfate)-(1----3)-L-[6-3H]-idonic acid (GlcNAc6S-IdOA), with homogenates of leucocytes, cultured fibroblasts, and urine from normal individuals, patients affected with N-acetylglucosamine-6-sulfatase-deficiency (Sanfilippo D syndrome, mucopolysaccharidosis type IIID), and patients affected with other mucopolysaccharidoses and lysosomal storage disorders. The assay clearly distinguished affected homozygotes from their obligate heterozygotes and normal controls and other lysosomal storage disorders. Sulfatase activity in fibroblasts, leucocytes, and urine toward GlcNAc6S-IdOA exhibited a pH optimum at 4.2, 4.5, and 5.1, respectively. Sulfatase activity in fibroblasts had an apparent Km of 7.2 microM and was significantly inhibited by both sulfate and phosphate ions. The action of fibroblast or leucocyte N-acetylglucosamine-6-sulfatase activity toward GlcNAc6S-IdOA is recommended for the routine enzymatic detection and classification of mucopolysaccharidosis type IIID patients.     

Pyrimethamine as a potential pharmacological chaperone for late-onset forms of GM2 gangliosidosis

Late-onset GM2 gangliosidosis is composed of two related, autosomal recessive, neurodegenerative diseases, both resulting from deficiency of lysosomal, heterodimeric beta-hexosaminidase A (Hex A, alphabeta). Pharmacological chaperones (PC) are small molecules that can stabilize the conformation of a mutant protein, allowing it to pass the quality control system of the endoplasmic reticulum. To date all successful PCs have also been competitive inhibitors. Screening for Hex A inhibitors in a library of 1040 Food Drug Administration-approved compounds identified pyrimethamine (PYR (2,4-diamino 5-(4-chlorophenyl)-6-ethylpyrimidine)) as the most potent inhibitor. Cell lines from 10 late-onset Tay-Sachs (11 alpha-mutations, 2 novel) and 7 Sandhoff (9 beta-mutations, 4 novel) disease patients, were cultured with PYR at concentrations corresponding to therapeutic doses. Cells carrying the most common late-onset mutation, alphaG269S, showed significant increases in residual Hex A activity, as did all 7 of the beta-mutants tested. Cells responding to PC treatment included those carrying mutants resulting in reduced Hex heat stability and partial splice junction mutations of the inherently less stable alpha-subunit. PYR, which binds to the active site in domain II, was able to function as PC even to domain I beta-mutants. We concluded that PYR functions as a mutation-specific PC, variably enhancing residual lysosomal Hex A levels in late-onset GM2 gangliosidosis patient cells.