Biomedical subjects

G A Grabowski

Publications and source records attributed to G A Grabowski.

At least 91 records · Page 5Linked to original sources

PubMed1990

Acid beta-glucosidase: enzymology and molecular biology of Gaucher disease.

Human lysosomal beta-glucosidase (D-glucosyl-acylsphingosine glucohydrolase, EC 3.2.1.45) is a membrane-associated enzyme that cleaves the beta-glucosidic linkage of glucosylceramide (glucocerebroside), its natural substrate, as well as synthetic beta-glucosides. Experiments with cultured cells suggest that in vivo this glycoprotein requires interaction with negatively charged lipids and a small acidic protein, SAP-2, for optimal glucosylceramide hydrolytic rates. In vitro, detergents (Triton X-100 or bile acids) or negatively charged ganglioside or phospholipids and one of several "activator proteins" increase hydrolytic rate of lipid and water-soluble substrates. Using such in vitro assay systems and active site-directed covalent inhibitors, kinetic and structural properties of the active site have been elucidated. The defective activity of this enzyme leads to the variants of Gaucher disease, the most prevalent lysosomal storage disease. The nonneuronopathic (type 1) and neuronopathic (types 2 and 3) variants of this inherited (autosomal recessive) disease but panethnic, but type 1 is most prevalent in the Ashkenazi Jewish population. Several missense mutations, identified in the structural gene for lysosomal beta-glucosidase from Gaucher disease patients, are presumably casual to the specifically altered posttranslational oligosaccharide processing or stability of the enzyme as well as the altered in vitro kinetic properties of the residual enzyme from patient tissues.

Amino Acid Sequence↗

PubMed1990

Complex alleles of the acid beta-glucosidase gene in Gaucher disease.

Gaucher disease is inherited in an autosomal recessive manner and is the most prevalent lysosomal storage disease. Gaucher disease has marked phenotypic variation and molecular heterogeneity, and seven point mutations in the acid beta-glucosidase (beta-Glc) gene have been identified. By means of sequence-specific oligonucleotides (SSO), mutation 6433C has been detected homozygously in neuronopathic type 2 (acute) and type 3 (subacute) patients, as well as in children with severe visceral involvement who are apparently free of neuronopathic disease. To investigate the molecular basis for this puzzling finding, amplified beta-Glc cDNAs from 6433C homozygous type 2 and type 3 Gaucher disease patients were cloned and sequenced. The Swedish type 3 Gaucher disease patient was truly homozygous for alleles only containing the 6433C mutation. In comparison, the type 2 patient contained a singly mutated 6433C allele and a "complex" allele with multiple discrete point mutations (6433C, 6468C, and 6482C). Each of the mutations in the complex allele also was present in the beta-Glc pseudogene. SSO hybridization of 6433C homozygotes revealed that both type 2 patients contained additional mutations in one allele, whereas the 6433C alone was detected in both type 3 and in young severe type 1 Gaucher disease patients. These results suggest that the presence of the complex allele influences the severity of neuronopathic disease in 6433C homozygotes and reveal the central role played by the pseudogene in the formation of mutant alleles of the beta-Glc gene. Analysis of additional cDNA clones also identified two new alleles in a type 3 patient, emphasizing the molecular heterogeneity of neuronopathic Gaucher disease.

Alleles↗

PubMed1989

Comparison of RNase A, a chemical cleavage and GC-clamped denaturing gradient gel electrophoresis for the detection of mutations in exon 9 of the human acid beta-glucosidase gene.

Gaucher disease (GD), which results from mutations in the human acid beta-glucosidase (beta-Glc) gene, was used as a model system to compare the utility of three methods capable of detecting single base substitutions. PCR-amplified beta-Glc exon 9 sequences of GD patients were screened for single base mutations by GC-clamped denaturing gradient gel electrophoresis (DGGE) and RNase A cleavage of RNA-DNA heteroduplexes, and by chemical (hydroxylamine/osmium tetroxide) cleavage of dsDNA heteroduplexes. PCR products showing abnormal behaviour were cloned and sequenced. Three new point mutations were detected by this strategy. A G to C (Asp409 to His409) substitution was present in two Type 1 and one Type 3 GD patients; an A to T transversion (Asp409 to Val409) was detected in only a single Type 3 individual, and a G to T mutation (Val394 to Leu394) was present in one Type 1 and one Type 3 patient. GD thus exhibits extensive molecular heterogeneity, with at least five single base mutations in beta-Glc exon 9. In every case verified by ASO hybridization, DGGE had correctly identified the presence of the three new mutations, as well as the two previously described exon 9 mutations. In comparison, although RNase A and the chemical method were both able to detect some of these mutations, neither method reproducibly detected all of them. Additionally, DGGE was the only method that was able to reliably determine whether a given mutation was present homozygously or heterozygously. These results suggest that GC-clamped DGGE may be a more reliable and informative screening method for point mutation detection.

Base Composition↗

PubMed1989

Resting energy expenditure in Gaucher's disease type 1: effect of Gaucher's cell burden on energy requirements.

The resting energy expenditure (REE; kcal/d) of 25 patients with Gaucher's disease type 1 was determined by indirect calorimetry. The average observed REE for the group was approximately 44% greater (P less than .01) than that predicted (predicted REE) for these patient's age, sex, height, and weight. The increased caloric requirements of these patients was manifested by a height-for-age less than or equal to the fifth percentile in seven of nine growing children and a muscle mass of less than the fifth percentile in 15 of 19 patients studied. The excess REE (observed REE--predicted REE) for individual Gaucher's disease type 1 patients was directly related to their liver volume as estimated from radionuclide scans and to the mass of the spleen as measured at splenectomy. The relationship between spleen mass and excess REE was demonstrated by an average 22.0% decrease in REE following splenectomy in five patients. Based on these data, the metabolic rate of the splenic tissue removed from the patients was calculated to be 96.8 kcal/d/kg, about twofold to threefold less than that of normal splenic tissue. These findings indicate that the elevated REE observed in these patients resulted from the large mass of Gaucher's cells, which although individually hypometabolic, were cumulatively an excessive metabolic burden. Furthermore, they suggest that indirect calorimetry may be a quantitative tool for measuring disease progression and the effect of therapeutic intervention in Gaucher's disease type 1.

Adolescent↗

PubMed1989

Molecular cloning of a human co-beta-glucosidase cDNA: evidence that four sphingolipid hydrolase activator proteins are encoded by single genes in humans and rats.

Authentic cDNAs encoding the activator protein for acid beta-glucosidase (EC3.2.1.45), co-beta-glucosidase, were cloned from the pCD and lambda gt11 human cDNA libraries. Initial screening with oligonucleotide mixtures encoding amino acid sequences of co-beta-glucosidase identified partial cDNAs which were used to obtain a potentially full-length cDNA from the lambda gt11 library. This clone (2767 bp), EGTISI, contained 5' (38 bp) and 3' (1157 bp) noncoding sequences, a translation initiation site, and an open reading frame encoding 524 amino acids which included a typical hydrophobic signal sequence (16 amino acids). Computer analyses identified three regions of high similarity to co-beta-glucosidase encoded by tandem sequences in EGTISI. Searches revealed that two of these regions encoded peptides of known function; SAP1 (sphingolipid activator protein 1) and protein C (a new sphingolipid activator protein) were encoded by EGTISI sequences 5' and 3', respectively, to those for co-beta-glucosidase. The third region of similarity, encoding a theoretical peptide (undefined function), was located most 5' in the cDNA. EGTISI and its encoded polypeptide had high similarity (77% nucleotide identity and about 80% amino acid similarity) to a rat Sertoli cell cDNA and its encoded sulfated glycoprotein-1. These results indicate that a single highly conserved gene encodes the precursor for four potential sphingolipid activator proteins in rat and man.

Amino Acid Sequence↗

PubMed1989

Gaucher disease: fate of the splenic remnant after partial splenectomy--a case of rapid enlargement.

In Gaucher disease, partial splenectomy has been suggested for alleviating the complications of splenomegaly as well as for avoiding the immunologic compromise and potential acceleration of bony and hepatic involvement that may follow total splenic resection. However, the fate of the splenic remnant has been reported rarely. A subtotal splenectomy (85%) was performed in a 19-month-old girl with rapidly progressing Gaucher disease and massive splenomegaly (12% of body weight). Within 3 months, the splenic remnant had increased four-fold in size. Previous reports indicated only three Gaucher patients had significant enlargement of the splenic remnant after partial splenectomy. These findings indicate that splenomegaly may recur rapidly in Gaucher disease following partial splenectomy.

Female↗

PubMed1989

Expression of functional human acid beta-glucosidase in COS-1 and Spodoptera frugiperda cells.

A cDNA encoding human acid beta-glucosidase (N-acylsphingosyl-1-O-beta-D-glucoside: glucohydrolase, EC 3.2.1.45) expressed catalytically active enzyme in transfected COS-1 or infected Spodoptera frugiperda (Sf9) cells. The expression plasmid p91023(B) (p91023B/Glc) and a Baculovirus (AcMNPV/Glc) containing the cDNA were constructed and used with the respective cells. By immunoblotting a glycosylated, 63-kilodalton human acid-beta-glucosidase was detected in the transfected or infected cells. A 56-kilodalton human polypeptide was obtained after complete deglycosylation with N-Glycanase. The expressed human enzymes also had partial endoglycosidase H sensitivity. The human enzyme expressed at high levels in Sf9 cells and had normal immunologic properties. With the partially purified enzyme from Sf9 cells, intact function of active site was indicated by normal kcat and Kmapp or Kiapp values for alternative substrates or potent inhibitors, respectively. The expressed enzyme was also activated normally by the negatively charged lipid, taurocholate. The results of these studies indicate that the Baculovirus expression system could provide a convenient source of normal human enzyme for structure/function investigations. In addition, this expression system should prove useful for the identification and evaluation of putative etiologic point mutations in Gaucher disease variants with kinetically altered residual enzymes.

Animals↗

PubMed1989

Posttranslational processing of human lysosomal acid beta-glucosidase: a continuum of defects in Gaucher disease type 1 and type 2 fibroblasts.

The major processing steps in the maturation of the lysosomal hydrolase, acid beta-glucosidase, were examined in fibroblasts from normal individuals and from patients with types 1 and 2 Gaucher disease. In pulse-chase studies with normal fibroblasts, remodeling of N-linked oligosaccharides resulted in the temporal appearance of three molecular-weight forms of acid beta-glucosidase. An initial 64-kDa form, containing high mannose-type oligosaccharide side chains, was processed quantitatively, within 24 h, to a sialylated 69-kDa form. During the subsequent 96 h, some of the 69-kDa form is processed to 59 kDa. Glycosidase digestion studies revealed that the increase in the apparent molecular weight of the normal enzyme from 64 kDa to 69 kDa resulted primarily from the addition to sialic acid residues in the Golgi apparatus. The polypeptide backbone of both the 64-kDa and 69-kDa forms was 55.3 kDa. Processing of acid beta-glucosidase in fibroblasts from three of four type 1 (nonneuronopathic) Ashkenazi Jewish Gaucher disease patients was nearly normal. With fibroblasts from one Ashkenazi Jewish and three non-Jewish type 1 as well as from two type 2 (acute neuronopathic) Gaucher disease patients, only a 64-kDa form of acid beta-glucosidase was detected. Inefficient and incomplete processing to the 69-kDa form was found in one type 2 cell line (GM2627). These results indicate that no firm correlation exists between the type or degree of abnormal processing of acid beta-glucosidase in fibroblasts and the phenotype of Gaucher disease.

Electrophoresis, Polyacrylamide Gel↗

PubMed1989

Gaucher disease: molecular heterogeneity and phenotype-genotype correlations.

Gaucher disease (GD) is the most prevalent lysosomal storage disease. This autosomal recessive trait results from the defective activity of acid beta-glucosidase (beta-Glc). Four different exonic point mutations have been identified as causal alleles for GD. To facilitate screening for these alleles, assays were developed using allele-specific oligonucleotide hybridization to amplified genomic DNA sequences. Specifically, intron bases flanking exons 5, 9, and 10 were determined, and conditions for PCR amplification of these exons were obtained. Two different procedures were developed to distinguish signals obtained from the structural beta-Glc gene exons and those from the pseudogene. These procedures were used to determine the distribution of all known GD alleles in a population of 44 affected patients of varying phenotypes and ethnicity. The high frequency of one of the exon 9 mutations in Ashkenazi Jewish GD type 1 patients was confirmed, and, in addition, this mutation was present in ethnically diverse non-Jewish type 1 GD patients. Homozygotes (N = 5) for this allele were midly affected older individuals, and this mutant allele was not found in any patient with neuronopathic disease. The exon 10 mutation was confirmed as the predominant allele in types 2 and 3 GD. However, several type 1 GD patients, including one of Ashkenazi-Jewish heritage, also were heterozygous for this allele. The presence of this allele in type 1 patients did not correlate with the severity of clinical symptoms. The second exon 9 mutation and the exon 5 mutation were rare, since they occurred only heterozygously either in one type 2 GD patient or in two related Ashkenazi-Jewish GD patients, respectively. Although most GD patients (38 of 44) had at least one of the known mutant alleles, 57% were heterozygotes for only one of these mutations. Fourteen percent of patients were negative for all mutations. A total of 73% of GD patients had at least one unknown allele. The varying clinical phenotypes and ethnic origins of these incompletely characterized patients suggest that multiple other GD alleles exist.

Base Sequence↗

PubMed1988

Hexosaminidase A activity and amyotrophic lateral sclerosis.

Abnormalities of GM2 ganglioside metabolism owing to hexosaminidase A (Hex A) deficiency have been associated with ALS phenotypes. The clinical features described in these ALS patients with Hex A deficiency include early onset, positive family history, and/or long disease duration. In an attempt to determine prospectively the incidence of Hex A deficiency within an ALS population, the records of The Mount Sinai Medical Center ALS Clinic were reviewed to select those patients with "atypical" ALS (total N = 52), i.e. onset before age 35, positive family history, and/or disease duration greater than 90 months. The control group (total N = 50), "typical" ALS patients, did not fulfill any of these historical criteria. Hex A activity determined in isolated peripheral blood leukocytes was normal in all typical ALS patients (mean 67.3%). Hex A deficiency was not found in any atypical ALS patients. Thus, Hex A deficiency apparently is an unusual etiology of typical or atypical ALS but is of medical and genetic importance in individual families.

Age Factors↗

PubMed1988

Abnormalities in lipoprotein metabolism in Gaucher type 1 disease.

We have previously described an association between Gaucher type 1 disease and reduced levels of total, low density lipoprotein (LDL), and high density lipoprotein (HDL) cholesterol. Plasma concentrations of apolipoprotein B and apolipoprotein AI were reduced in these subjects, while plasma apolipoprotein E (apoE), which can be synthesized and secreted by macrophages, was increased. To study the pathophysiologic basis for these changes in lipoprotein and apolipoprotein levels, we studied very low density lipoprotein (VLDL), LDL, and HDL metabolism in-depth in four subjects with Gaucher disease. Gel filtration of their plasma revealed that apoE was present in essentially a single population of lipoproteins in the large HDL range. In subject no. 4, studied presplenectomy and post-splenectomy, plasma apoE levels fell after surgery in association with a redistribution of apoE among the plasma lipoproteins to a pattern seen in normal subjects. Determination of the rates of secretion and catabolism of VLDL apoB and triglyceride were within normal limits. The reduced plasma levels of LDL and HDL cholesterol, and of both plasma apoB and apoAI, were associated with increased fractional catabolic rates of these apolipoproteins in LDL and HDL. These results indicate that the hypocholesterolemia present in subjects with Gaucher type 1 disease is associated with increased fractional catabolism of LDL and HDL. These findings, together with the evidence for alternations in plasma apoE metabolism in this disorder, suggest a role for the macrophage as the basis for these abnormalities.

Adult↗

PubMed1988

Gaucher disease type 1: cloning and characterization of a cDNA encoding acid beta-glucosidase from an Ashkenazi Jewish patient.

Gaucher disease (GD) type 1 is the most prevalent lysosomal storage disease and the most prevalent genetic disease among the Ashkenazi Jews. The defective activity of acid beta-glucosidase is the enzymatic basis of GD and is inherited as an autosomal recessive trait. To investigate the genetic basis of Ashkenazi Jewish GD type 1, a cDNA encoding acid beta-glucosidase was isolated from a cDNA library constructed using splenic poly(A)+RNA from a patient. The cDNA was sequenced to identify mutations, and the presence of a single missense mutation in the patients' genome was confirmed by selective oligonucleotide hybridization and by restriction endonuclease digestion analyses of amplified genomic sequences. This G----A transition (Arg-119 to Gln-119) was present heterozygously in the index patient and his affected third cousin but was not present in normal non-Jewish individuals. This mutation is the second single base mutation found in Ashkenazi Jewish GD type 1 patients. Furthermore, results obtained with the affected third cousin suggest that at least three mutant alleles may be present in this GD subpopulation.

Child↗

PubMed1988

Human acid beta-glucosidase: inhibition studies using glucose analogues and pH variation to characterize the normal and Gaucher disease glycon binding sites.

Comparative kinetic studies with glycon inhibitors were used to investigate the properties of the active site of human acid beta-glucosidase (EC 3.2.1.45) from normal placenta and spleens of type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the pure normal enzyme, the specificity of glycon binding was assessed with 35 glucose derivatives and epimers. Most glycons were mixed type inhibitors with a predominantly competitive nature (i.e., Kis much less than Kii) and had low apparent affinity for the enzyme (Kisapp = 20-500 mmol/l). beta-Glucose-1-phosphate was unusual, since it inhibited 4-methylumbelliferyl-beta-glucoside hydrolysis in an uncompetitive pattern (Kiapp = 0.55 mmol/l) but had no effect on glucosyl ceramide hydrolysis. C-1- (1-deoxy-1-amino-beta-D-glucose) and C-3- (3-deoxy-3-amino-D-glucose) amino and C-5-imino [1-deoxynojirimycin (dNM), nojirimycin and castanospermine] substituted sugars were highly potent inhibitors with Kisapp(beta-glucose)/Kisapp approximately equal to 10(3)-10(5); an amine at C-2 did not alter Kisapp compared to beta-glucose. The variation of Kisapp with pH for the 5-imino- and 1-deoxy-1-aminoglycosides conformed to a model for the unprotonated inhibitors binding to the protonated forms (EH and EH2) of the diprotic (Vmaxapp and Vmaxapp/Kmapp) normal enzyme (pK1 = 4.7; pK2 = 6.7) with pH-independent Kisapp values of 2.9-9.0 mumol/l and 0.22 mmol/l, respectively. Several of the amine-containing inhibitors competitively protected the enzyme from inactivation by conduritol B epoxide, a covalent active site-directed inhibitor, indicating interaction with residues at that site. With the partially purified AJGD splenic enzymes, the results were the same except that Kisapp(AJGD)/Kisapp(normal) = 4-17 for dNM and 1-deoxy-1-amino-beta-glucose; this ratio was approximately equal to 1 with most other glycons, and particularly, nojirimycin and castanospermine. The results of these studies indicated that the glycon binding site of the normal acid beta-glucosidase contains important residues for interaction with the C-2, C-3 and C-4 hydroxyl groups of beta-glucose and a residue with pKa = 6.7 which was critical to the binding of amine-containing inhibitors and the hydrolysis of substrates. The findings were consistent with a specific alteration in or near the glycon binding site which results in the functional abnormalities of the mutant AJGD acid beta-glucosidase.

Adult↗

PubMed1987

Human acid beta-glucosidase: use of inhibitors, alternative substrates and amphiphiles to investigate the properties of the normal and Gaucher disease active sites.

Comparative studies with lipoidal inhibitors and alternative substrates were conducted to investigate the properties of the active site of human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) from normal placenta and spleens of Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the normal enzyme, the inhibitory potencies of series of alkyl(Cn; n = 0-18)amines, alkyl beta-glucosides and alkyl-1-deoxynojirimycins were a biphasic function of increasing chain length: i.e., large decreases in Ki,app or IC50 were found only with n greater than 4 and limiting values were approached with n = 12-14. This biphasic function of alkyl chain length was observed in the presence or absence of detergents and/or negatively charged lipids. In the presence of Triton X-100 concentrations greater than the critical micellar concentration, the relative (to deoxynojirimycin) inhibitory potencies of the N-Cn-deoxynojirimycins (n greater than 4) were decreased about 3-5-fold, due to an energy requirement to extract the inhibitors from Triton X-100 micelles. The Ki,app or IC50 of N-hexylglucosylsphingosine was inversely related to the Triton X-100 concentration and was not affected by the presence of 'co-glucosidase'. The mutual exclusion of glucon, N-Cn-deoxynojirimycin and sphingosine derivatives from the normal enzyme suggested a shared region for binding in the active site. Increasing the fatty-acid acyl chain length of glucosyl ceramide from 1 to 24 carbons had minor effects on Km,app ( = Kis,app) (8-40 microM), but increased Vmax,app up to 13-fold. With the AJGD enzyme, the inhibitor and alternative substrate findings were similar to those with the normal enzyme, except that Kis,app(AJGD)/Kis,app(normal) = 4 to 11 for the Cn-glycons and sphingosine derivatives. These results indicated that (1) the Ki,app or Km,app values for amphiphilic inhibitors or substrates reflect a balance of binding energies for two hydrophobic subsites within the enzyme's active site and Triton X-100 micelles and (2) the abnormal properties of the AJGD enzyme result from an amino-acid alteration(s) within or near a hydrophilic region which is shared by the glycon-binding site and the two hydrophobic sites of the active site.

1-Deoxynojirimycin↗

PubMed1987

Gaucher disease: genetic heterogeneity within and among the subtypes detected by immunoblotting.

The genetic heterogeneity of Gaucher disease subtypes and variants was investigated by immunoblotting of fibroblast extracts. For these studies polyclonal and monoclonal antibodies were raised to acid beta-glucosidase preparations containing a single N-terminal amino acid sequence that was colinear with that encoded by the beta-Glc cDNAs. Three forms (Mr approximately equal to 67,000, 64,000-61,000, and 58,000) of cross-reacting immunologic material (CRIM) were observed in control individuals. Decreased amounts of the same CRIM forms were detected in most type 1 Gaucher disease patients, but single CRIM forms of variable molecular weight were observed in several non-Jewish type 1 variants. One or two CRIM forms of variable molecular weight were found in neuronopathic (type 2 and type 3) patients. The amount of CRIM was severely decreased in the majority of the type 2 and type 3 patients; one American black type 2 patient was CRIM negative. With this one exception, one CRIM form was detected in the cell-free culture media from all normal or Gaucher disease fibroblasts that had an Mr approximately 2,000 greater than the highest respective intracellular molecular-weight form. All intra- or extracellular CRIM forms were reduced to a single form after deglycosylation with N-Glycanase. In addition, the radioactivity from [3H]Br-conduritol B epoxide, a specific covalent inhibitor of beta-Glc, localized to the CRIM forms of beta-Glc on immunoblots. These results indicate that all subtypes and variants of Gaucher disease result from mutations that alter the stability and/or processing of beta-Glc. Furthermore, the heterogeneity of the CRIM patterns within and among the variants of Gaucher disease cause the diagnostic usefulness of immunoblotting to be restricted to those families in which the phenotype has been well established.

Antibodies, Monoclonal↗

PubMed1986

Human acid beta-glucosidase. Use of conduritol B epoxide derivatives to investigate the catalytically active normal and Gaucher disease enzymes.

Human acid beta-glucosidase (glucosylceramidase; EC 3.2.1.45) cleaves the glycosidic bonds of glucosyl ceramide and synthetic beta-glucosides. Conduritol B epoxide (CBE) and its brominated derivative are mechanism-based inhibitors which bind covalently to the catalytic site of acid beta-glucosidase. Procedures using brominetritiated CBE and monospecific anti-human placental acid beta-glucosidase IgG were developed to determine the molar concentrations of functional acid beta-glucosidase catalytic sites in pure placental enzyme preparations from normal sources; kcat values then were calculated from Vmax = [Et]kcat using glucosyl ceramide substrates with dodecanoyl (2135 +/- 45 min-1) and hexanoyl (3200 +/- 410 min-1) fatty acid acyl chains and 4-alkyl-umbelliferyl beta-glucoside substrates with methyl (2235 +/- 197 min-1), heptyl (1972 +/- 152 min-1), nonyl (2220 +/- 247 min-1), and undecyl (773 +/- 44 min-1) alkyl chains. The respective kcat values for acid beta-glucosidase in a crude normal splenic preparation were about 60% of these values. In comparison, the kcat values of the mutant splenic acid beta-glucosidase from two Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients were about 1.5-3-fold decreased and had Km values for each substrate which were similar to those for the normal acid beta-glucosidase. The interaction of the normal and Type 1 AJGD enzymes with CBE in a 1:1 stoichiometry conformed to a model with reversible EI complexes formed prior to covalent inactivation. With CBE, the equal kmax values (maximal rate of inactivation) for the normal (0.051 +/- 0.009 min-1) and Type 1 AJGD (0.058 +/- 0.016 min-1) enzymes were consistent with the minor differences in kcat. In contrast, the Ki value (dissociation constant) (839 +/- 64 microM) for the Type 1 AJGD enzymes was about 5 times the normal Ki value (166 +/- 57 microM). These results indicated that the catalytically active Type 1 AJGD acid beta-glucosidase had nearly normal hydrolytic capacity and suggested an amino acid substitution in or near the acid beta-glucosidase active site leading to an in vivo instability of the mutant enzymatic activity.

Gaucher Disease↗

PubMed1986

Human acid beta-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site.

Human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the glucosidic bonds of glucosylceramide and synthetic beta-glucosides. The deficient activity of this hydrolase is the enzymatic defect in the subtypes and variants of Gaucher disease, the most prevalent lysosomal storage disease. To isolate and characterize the catalytic site of the normal enzyme, brominated 3H-labeled conduritol B epoxide (3H-Br-CBE), which inhibits the enzyme by binding covalently to this site, was used as an affinity label. Under optimal conditions 1 mol of 3H-Br-CBE bound to 1 mol of pure enzyme protein, indicating the presence of a single catalytic site per enzyme subunit. After V8 protease digestion of the 3H-Br-CBE-labeled homogeneous enzyme, three radiolabeled peptides, designated peptide A, B, or C, were resolved by reverse-phase HPLC. The partial amino acid sequence (37 residues) of peptide A (Mr, 5000) was determined. The sequence of this peptide, which contained the catalytic site, had exact homology to the sequence near the carboxyl terminus of the protein, as predicted from the nucleotide sequence of the full-length cDNA encoding acid beta-glucosidase.

Amino Acid Sequence↗

PubMed1986

Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose.

Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(11-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid beta-glucosidase (beta-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal beta-Glc/ml of settled gel, respectively. The purified normal enzyme (14-18% yield) had a specific activity of 1.6 X 10(6) nmol/h/mg protein and was homogeneous as evidenced by a single protein species of Mr = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the beta-Glc cDNA. Amino acid composition analyses of beta-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11%. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.

1-Deoxynojirimycin↗