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F Y Choy

Publications and source records attributed to F Y Choy.

32 records · Page 2Linked to original sources
PubMed1989

Purification of lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts using high-performance liquid chromatography.

Glucocerebrosidase from human skin fibroblasts was purified more than 2300-fold to apparent homogeneity with an overall yield of 39% using taurocholate extraction, ammonium sulfate fractionation, and high-performance hydrophobic interaction and gel permeation column chromatography. This relatively high yield is attributed to two modifications from previously published procedures: (i) the elimination of a butanol delipidation step that resulted in substantial loss of enzyme activity; and (ii) the use of 2% (w/v) sodium taurocholate instead of 1-2% sodium cholate that resulted in more than 90% solubilization of total membrane-bound enzyme activity. Confluent monolayers of human cultured skin fibroblasts (approximately 3.6 x 10(8) cells) were harvested from 10 roller bottles. Glucocerebrosidase in the cell pellet was solubilized with 2% (w/v) sodium taurocholate, fractionated in 14% ammonium sulfate, and applied to a high-performance hydrophobic interaction phenyl-5PW column. After an ammonium sulfate descending linear gradient step, glucocerebrosidase was eluted from the column at 4% cholate concentration using a 0-5% linear cholate gradient. There was a 36-fold purification and 80% recovery. In the subsequent step, concentrated glucocerebrosidase fractions from the phenyl column were injected into two Bio-Sil TSK-250 gel permeation columns joined in series. Glucocerebrosidase peak activity was eluted at 263 ml corresponding to Mr 76,000. There was an 18-fold purification and 78% recovery. The enzyme preparation was then recycled through the phenyl-5PW column in order to remove a remaining contaminant.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1989

Purification and characterization of activated human erythrocyte prolidase.

Prolidase (E.C. 3.4.13.9) has been purified 7500-fold to homogeneity from human erythrocytes in a Mn2+-activated form using conventional and fast protein liquid chromatography columns. The procedure includes a 1-h incubation of the crude hemolysate at 50 degrees C with 1 mM MnCl2. Following this novel step, prolidase retains full activity, obviating the requirement for preincubation of each enzyme fraction with Mn2+ prior to assay. Preincubation with MnCl2 does not change the isoelectric point of the enzyme. The molecular weight of the purified enzyme was 58,000 when measured by SDS-PAGE. Western blotting, using rabbit antibody raised to human kidney prolidase, with partially purified erythrocyte enzyme revealed a cross-reacting band at Mr 58,000.

Blood Proteins↗
PubMed1988

Intrafamilial clinical variability of type 1 Gaucher disease in a French-Canadian family.

Glucocerebroside beta-glucosidase (glucocerebrosidase) activity was determined from peripheral blood lymphocytes and cultured skin fibroblasts of eight full sibs in a French-Canadian family at risk for Gaucher disease, an autosomal recessive sphingolipidosis resulting from deficient glucocerebrosidase activity. The diagnosis of type 1, non-neuronopathic Gaucher disease was made in all of the five affected sibs on the basis of deficient (7.5 to 15.5% of control mean) glucocerebrosidase activity and absence of neurological involvement. Normal levels of enzyme activity were found in two of the three asymptomatic sibs. The third asymptomatic sib had an intermediate level (about 50% of control mean) of fibroblast and lymphocyte glucocerebrosidase activity, indicating that he is a carrier. Considerable clinical heterogeneity was noted among the five affected sibs. One patient is mildly affected and so far has not developed any orthopaedic complications associated with Gaucher disease. His haematological complications were also reversed after splenectomy 24 years ago. In contrast to this mild presentation, the patient's splenectomised sister has been very anaemic and thrombocytopenic. There have been severe orthopaedic complications associated with Gaucher disease, including vertebral compression, avascular necrosis, and pathological fracture of the long bones. The clinical picture of the other three affected sibs appeared to vary between the two extremes. Although the asymptomatic parents of the patients died many years ago, their heterozygous status with respect to Gaucher disease can be deduced by the presence of Gaucher homozygotes, normal homozygotes, and one heterozygote among their eight offspring. Present findings suggest that the clinical variability of type 1 Gaucher disease may be attributed to variable expressions of the same Gaucher mutant alleles, in addition to the presence of multiple mutant alleles that are widely disseminated in the population.

Adult↗
PubMed1987

Gaucher disease: accurate identification of asymptomatic French-Canadian carrier using nonlabeled authentic sphingolipid substrate N-palmitoyl dihydroglucocerebroside.

Gaucher disease is an autosomal recessive sphingolipidosis associated with deficient glucocerebroside beta-glucosidase activity. It is a panethnic metabolic disorder, but the carrier frequency is particularly high among Ashkenazi Jews (estimated between 1:12-1:25). In order to establish a reliable and convenient biochemical assay method for differentiating asymptomatic Gaucher carriers from normal individuals, glucocerebroside beta-glucosidase activity was determined in peripheral blood lymphocytes and cultured skin fibroblasts of 11 Gaucher obligate heterozygotes using the authentic nonlabeled sphingolipid substrate N-palmitoyl dihydroglucocerebroside and the artificial fluorogenic substrate 4-methylumbelliferyl-beta-D-glucopyranoside (4MUGP). The level of lymphocyte beta-glucosidase activity on the glucocerebroside substrate was observed to range from 42-65% of that of the control mean, and there was no overlap of enzyme activity between the Gaucher heterozygotes and controls. However, when the artificial fluorogenic substrate 4MUGP was used, the level of beta-glucosidase activity in 2 of the Gaucher obligate heterozygotes was noted to overlap with that of the control individuals. Contrary to findings in the lymphocytes, cultured skin fibroblasts appear to be a reliable enzyme source for Gaucher carrier detection even when the artificial fluorogenic 4MUGP substrate was used, as the level of beta-glucosidase activity in all of the Gaucher obligate heterozygotes tested was intermediate and distinctly separated from that of the control persons. Using the lymphocyte glucocerebroside beta-glucosidase assay and fibroblast 4MUGP beta-glucosidase assay methods, we identified the carrier status in 3 other relatives and ruled it out in 4 others. These data suggest that nonlabeled glucocerebroside is a reliable and highly specific substrate for either lymphocyte or fibroblast beta-glucosidase activity assay in identifying asymptomatic Gaucher carriers. Use of the 4MUGP substrate for differentiating Gaucher heterozygotes from control persons, on the other hand, should be restricted to the fibroblast enzyme assay method, as considerable overlap of enzyme activity was noted in lymphocytes.

Adult↗
PubMed1986

Purification of human placental glucocerebrosidase using a two-step high-performance hydrophobic and gel permeation column chromatography method.

Glucocerebrosidase was purified from human placenta approximately 10,600-fold to apparent homogeneity with an overall yield of 37% using cholate extraction, ammonium sulfate fractionation, butanol delipidation, and a two-step high-performance hydrophobic and gel permeation column chromatography method. A Phenyl-5PW (21.5 X 150 mm) column was used in the first step. Approximately one litre of delipidated and dialysed extract containing 3.7 X 10(6) units of enzyme activity from 1 kg of placental tissue was processed by the column at a flow rate of 5 ml/min. Glucocerebrosidase was eluted using a linear cholate gradient (2-3%). There was a 50-fold purification and 89% recovery. The run was completed in about 7 h. In the second step, the concentrated enzyme preparation from the phenyl column was run through two Bio-Sil TSK 250 gel permeation columns (21.5 X 600 mm) connected in series at a flow rate of 1.5 ml/min. A symmetrical peak of glucocerebrosidase activity (Ve = 253 ml) which had constant specific activity (47,000 units/h/mg protein) was noted. There was a 17-fold purification and 80% recovery in this run which was completed in 4 h. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and protein staining with silver compounds of the purified preparation revealed the presence of one band of Mr 68,000.

Chromatography, Gel↗
PubMed1986

In situ radiation-inactivation size of fibroblast membrane-bound acid beta-glucosidase in Gaucher type 1, type 2 and type 3 disease.

The radiation-inactivation size of membrane-bound acid beta-glucosidase in cultured skin fibroblasts of four normal individuals, five Gaucher type 1 (non-neuropathic), four Gaucher type 2 (acute neuropathic) and three Gaucher type 3 (sub-acute neuropathic) patients was determined using the radiation-inactivation method. The radiation-inactivation size of the enzyme in the control, Gaucher type 2 and Gaucher type 3 fibroblasts ranged from 94 000 to 128 800, and no statistical significant difference was found in the enzyme size between the normal and Gaucher cells nor among the Gaucher type 2 and type 3 cells. Contrary to the normal, Gaucher type 2 and Gaucher type 3 enzyme, the radiation-inactivation size of membrane-bound acid beta-glucosidase in all of the Gaucher type 1 fibroblasts tested is significantly higher, ranging from 158 400 to 235 300. The size of the control lysosomal enzyme, sphingomyelinase, also determined by the radiation-inactivation method in fibroblasts of normal individuals and patients with the three Gaucher subtypes, was between 70 000 and 74 500 and indistinguishable from each other. Since the molecular weight of acid beta-glucosidase subunit determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was about 60 000 (Pentchev, P.G., Brady, R.O., Hibbert, S.P., Gal, A.E. and Shapiro, C. (1973) J. Biol. Chem. 248, 5256-5261), the above data suggest that: (i) the normal fibroblast enzyme, as well as the Gaucher type 2 and type 3 mutant enzyme, in the membrane-bound form, exists as a dimer; (ii) the underlying biochemical and genetic defect in non-neuropathic (type 1) and neuropathic (type 2 and type 3) Gaucher disease is very different from each other; and (iii) subunit interaction of the mutant enzyme may be present in Gaucher type 1 fibroblasts, resulting in the formation of a higher-molecular-weight aggregate.

Adult↗
PubMed1985

Gaucher disease: comparative study of acid phosphatase and glucocerebrosidase in normal and type-1 Gaucher tissues.

Acid phosphatase activity was determined in serum, cultured fibroblasts, and peripheral blood lymphocytes of six splenectomized adult patients with non-neuropathic Gaucher disease in two Canadian families. Elevated levels of serum acid phosphatase activity (520-711% of normal) were found in four patients who also developed orthopedic complications associated with Gaucher disease, including intermittent bone pain, arthritis, collapse of femoral head, and pathological fractures. Serum acid phosphatase activity in two patients who do not have bone involvement were found to be within the normal range. Contrary to the serum enzyme, acid phosphatase activity in lymphocytes and cultured fibroblasts of all of the patients was within the normal range. Deficient glucocerebrosidase (7.5-15.5% of normal) and acid beta-glucosidase (13.8-27.8% of normal) activities were noted in all probands. Similarly, normal levels of fibroblast and lymphocyte acid phosphatase activity were found in Gaucher heterozygotes whose glucocerebrosidase activity was about 50% of normal. Acid polyacrylamide gel electrophoresis and acid phosphatase activity staining of the patients' sera showed that the elevated acid phosphatase is isozyme type 5 osteoclastic origin. The apparent Michaelis constant, Km, of fibroblast glucocerebrosidase for the natural substrate was 0.6 +/- 0.1 mM for controls and 0.6 +/- 0.2 mM for the patients. These data suggest that the assay of serum acid phosphatase activity for the presumptive diagnosis of Gaucher disease is not completely reliable and that the elevated level of serum acid phosphatase in Gaucher disease is most likely a secondary phenomenon which may be indicative of bone involvement in some patients with this disorder. It also demonstrates the clinical heterogeneity of type 1 Gaucher disease, even among full sibs of the same heterozygous parents.

Acid Phosphatase↗
PubMed1984

Gaucher disease: the effects of phosphatidylserine on glucocerebrosidase from normal and Gaucher fibroblasts.

Glucocerebroside beta-glucosidase (glucocerebrosidase) activity was assayed from cultured fibroblasts of normal individuals, and patients with type 1 (non-neuropathic), type 2 (acute neuropathic), and type 3 (subacute neuropathic) form of Gaucher disease. Residual glucocerebrosidase activity of patients was 8.9 to 17.4% of normal controls, and there was no clear correlation between the level of residual enzyme activity and the different clinical subtypes of the disease. When membrane-bound glucocerebrosidase activity was assayed in the presence of crude brain lipid extracts or purified phosphatidylserine, enzyme from both the normal and type 1 Gaucher fibroblasts was stimulated dramatically (35-60% by crude extracts, 85-90% by phosphatidylserine). This stimulation was not observed with fibroblast glucocerebrosidase of an infantile type 2 and two juvenile type 3 Gaucher patients. The presence of inhibitors of glucocerebrosidase in these type 2 and type 3 Gaucher cells was not detected. Contrary to the mutant enzyme from these Gaucher fibroblasts, glucocerebrosidase from fibroblasts of two adult type 3 Gaucher patients with cerebral involvement was stimulated substantially (72-85%) by phosphatidylserine. When membrane-bound glucocerebrosidase from fibroblasts of the infantile type 2 and juvenile type 3 patients was solubilized with sodium cholate (1% w/v) and delipidated, the phospholipid stimulation of enzyme activity was restored. These findings suggest that considerable clinical and biochemical heterogeneity exists among patients with neuropathic Gaucher disease and that phosphatidylserine activation cannot be used as a reliable indicator in predicting future onset of neurodegeneration in Gaucher patients. The possibility of an aberrant binding of mutant glucocerebrosidase to the lysosomal membrane in juvenile type 3 form of Gaucher disease is discussed.

Adult↗
PubMed1983

Survey, purification, and properties of sugar phosphate phosphohydrolase among microorganisms.

Sugar phosphate phosphohydrolase was purified approximately 500- to 600-fold to apparent homogeneity from Escherichia coli B, Escherichia coli C, Escherichia coli var. communior, Escherichia acidilactici, Enterobacter aerogenes, Neisseria meningitidis, and Saccharomyces cereviseae. The molecular weights of the enzyme as estimated by gel filtration ranged from 97 X 10(3) to 101 X 10(3). The enzyme was composed of two subunits with the same molecular weight which ranged from 50 X 10(3) to 52 X 10(3), as determined by sodium dodecyl sulfate gel electrophoresis. Homogeneous enzyme preparations hydrolyse all the tested alpha-D-aldohexose 1-phosphate, D-(keto or aldo)hexose 6-phosphate, and pentose phosphate substrates significantly. When the microorganisms were transferred from growth medium with 1% glucose to that without glucose, there were dramatic increases in both the specific and total enzyme activities. At least three isozymes appeared to be present in S. cereviseae, and two appeared to be present in E. coli B, E. coli var. communior, and N. meningitidis. Rabbit antiserum immunized against sugar phosphate phosphohydrolase purified from E. coli B cross-reacted with both the crude extracts and purified preparations of the enzyme from the other microorganisms. The presence of neither sugar phosphate phosphohydrolase activity nor immunocrossreacting material was detected in the following microorganisms: Aspergillus niger, Azotobacter chroococcum, Bacillus subtilis, Bacillus pumilis, Citrobacter freundii, Clostridium butyricum, Corynebacterium xerosis, Flavobacterium aquatile, Flavobacterium synxanthum, Lactobacillus bulgaricus, Micrococcus coralinus, Neisseria perflava, Neurospora crassa, Penicilium expansum, Penicilium notatum, Proteus mirabilis, Proteus vulgaris, Pseudomonas fluorescens, Saccharomyces fermenti, Sarcina lutea, and Streptomyces antibioticus. At present, no conclusive relationship can be established between the phosphoenolpyruvate phosphotransferase system and the enzyme sugar phosphate phosphohydrolase among microorganisms. The physiological role of sugar phosphate phosphohydrolase as a transferase and regulatory enzyme is discussed.

Bacteria↗
PubMed1980

Gaucher's disease II. Studies on the kinetics of beta-glucosidase and the effects of sodium taurocholate in normal and Gaucher tissues.

beta-Glucosidase activity from normal human cultured fibroblasts was not affected by the presence of up to 0.1% (w/v) (1.86 mM) purified sodium taurocholate. At concentrations greater than 0.1%, there was a gradual decrease in activity. Conversely, beta-glucosidase activity from fibroblasts of three patients with juvenile onset Gaucher's disease was preferentially inhibited by the detergent at concentrations as low as 0.025% (0.46 mM). A 40% decrease in activity was observed at 0.1%. Crude sodium taurocholate was more potent in inhibiting beta-glucosidase activity from both the normal and Gaucher fibroblasts. However, very high background fluorescence and inconsistent results were observed when different batches of the crude taurocholate from the same or different sources were used. Similarly, beta-glucosidase activity from Gaucher splenic tissue homogenates, supernatant fluids (40,000 x g) and residue pellets was preferentially inhibited by purified sodium taurocholate. These findings indicate that the reliability and sensitivity of the enzyme assay for Gaucher's disease can be enhanced by determining beta-glucosidase activity in both the absence and presence of purified sodium taurocholate, particularly when variant cases with relatively high residual enzyme activity are encountered. In contrast to the enzyme from fibroblasts and spleens, beta-glucosidase activity from human placenta was markedly activated (greater than 300%) by the presence of 0.08% (1.49 mM) purified sodium taurocholate or 0.1 mM phosphatidyl serine, suggesting the presence of a predominate form of beta-glucosidase, possibly glucocerebroside beta-glucosidase, which is activated by the detergent. The apparent Michaelis constant (Km) for both the soluble and membrane-bound enzyme from normal fibroblasts was 1.6 +/- 0.1 mM. Kms from a patient with severe juvenile Gaucher's disease and two other patients with milder manifestations were 0.8 +/- 0.2 and 3.3 +/- 0.3 mM, respectively.

Adolescent↗
PubMed1980

Gaucher disease. III. Substrate specificity of glucocerebrosidase and the use of nonlabeled natural substrates for the investigation of patients.

A reproducible and convenient method for assaying glucocerebrosidase activity using the natural substrates has been developed. From the insoluble pellet fraction of cultured skin fibroblast homogenates, released glucose was measured enzymically using hexokinase coupled with the glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP) system. Optimal enzyme assay conditions required both Triton X-100 and sodium taurocholate, pH 4.8. Glucocerebrosidase activities from three patients with type 1 Gaucher disease were 17.5%, 15.8%, and 11.2% of normal (normal = 198 +/- 14 nmol/hr per mg protein, n = 3). The first patient had normal beta-glucosidase activity with the artificial fluorogenic umbelliferone substrate. Interference with the accuracy of the glucose-dependent assay system by either glycolytic or gluconeogenic enzyme activites was not detected under these experimental conditions, and when substrates with long fatty-acid chain lengths (C = 22) were used, markedly decreased glucocerebrosidase activity occurred in both normal individuals and patients. The apparent Km's for the natural substrates were 0.56 +/- 0.05 mM for controls and 2.2-3.3 mM for Gaucher fibroblasts. These data further support the hypothesis that a structurally altered and catalytically deficient enzyme is synthesized in patients with type 1 Gaucher disease and illustrate the value of the natural substrate in investigating patients.

Cells, Cultured↗
PubMedDate not supplied

Novel point mutation (W184R) in neonatal type 2 Gaucher disease.

Gaucher disease is the most prevalent inherited sphingolipidosis and results from deficient glucocerebrosidase activity. Three clinical forms of Gaucher disease have been described: type 1, or non-neuronopathic; type 2, or acute neuronopathic; and type 3, or subacute neuronopathic. We have identified a novel mutation in a patient of Russian-British descent who died of type 2 Gaucher disease a few hours after birth. A heterozygous T --> C transition mutation in exon 6, cDNA nucleotide position 667, results in the substitution of tryptophan by arginine at amino acid residue 184 (W184R) of glucocerebrosidase. This mutation creates a new cleavage site for the restriction endonuclease Hinf1. We developed a method that utilizes Hinf1 restriction endonuclease analysis to confirm the presence of the mutation and test family members. The second mutation identified in the other glucocerebrosidase allele of the patient is mutation L444P, a severe mutation frequent in type 2 and 3 Gaucher disease. Since the patient died very shortly after birth, we postulate that the W184R/L444P genotype may result in little or no detectable glucocerebrosidase activity and thus a poor prognosis.

Base Sequence↗