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J M Tager

Publications and source records attributed to J M Tager.

At least 199 records · Page 11Linked to original sources
PubMed1981

Identification of heterozygotes for glycogenosis 2 (acid maltase deficiency).

In 21 obligate and 9 possible heterozygotes for acid maltase deficiency (AMD) (glycogenosis 2, Pompe's disease), different methods of identifying heterozygotes have been studied. Heterozygosity could not be demonstrated by physical examination, serum CPK assays, morphological examination of muscle biopsy (including light-microscopy, histochemistry and electron-microscopy), or by ultrastructural examination of a skin biopsy. Heterozygotes could be identified to a large, but still limited extent, by measuring the acid alpha-glucosidase activity in urine, cultivated fibroblasts, leucocytes, or skeletal muscle. Heterozygotes for the generalized from of AMD could not be distinguished from those for the muscular form. The limitations of heterozygote identification by means of enzyme assays are discussed, and some practical aspects for genetic counselling are mentioned.

Female↗
PubMed1981

Relationship between the rate of citrulline synthesis and bulk changes in the intramitochondrial ATP/ADP ratio in rat-liver mitochondria.

1. The relationship between intramitochondrial and extramitochondrial ATP-utilizing systems and the intramitochondrial ATP/ADP ratio was studied in isolated rat-liver mitochondria. Citrulline synthesis was used as an intramitochondrial ATP-utilizing system, and glucose-6-phosphate synthesis as an extramitochondrial ATP-utilizing system. The intramitochondrial ATP/ADP ratio was manipulated in three ways: with succinate and different concentrations of malonate and/or hexokinase; with 2-oxoglutarate (plus oligomycin) and different concentrations of hexokinase; and with added ATP in uncoupled mitochondria (oligomycin present). 2. Under all conditions used, citrulline synthesis was strictly correlated with the bulk intramitochondrial ATP/ADP ratio. 3. The curve relating citrulline synthesis and intramitochondrial ATP/ADP was shifted towards lower ATP/ADP ratios when the activity of carbamoyl-phosphate synthetase was enhanced by increasing the mitochondrial content of N-acetylglutamate. 4. It is concluded that under the experimental conditions used the intramitochondrial adenine nucleotides behave as a homogeneous pool.

Adenosine Diphosphate↗
PubMed1981

Characterization of isolated rat-liver cells made permeable with filipin.

When isolated rat-liver cells were incubated for 1 min at 37 degrees C with filipin at a concentration of 50 microM, the plasma membrane became permeable to sucrose, inulin, glycerol 3-phosphate and other low-molecular-weight compounds. Upon removal of the filipin and subsequent incubation of the cells at 37 degrees C there was a gradual leakage of lactate dehydrogenase from the cells. However, the leakage of lactate dehydrogenase could be prevented for about 10 min by including glutathione and ATP in the incubation medium. The filipin-treated cells were able to metabolize phosphorylated sugars. The conversion of fructose 1,6-bisphosphate to fructose 6-phosphate, glucose-6-phosphate and glucose was inhibited by AMP but not by high concentrations of fructose 1,6-bisphosphate. The results indicate that filipin-treated cells can be used to study the kinetic parameters of enzymes in their macromolecular environment in situ.

Adenine Nucleotides↗
PubMed1981

A family with different clinical forms of acid maltase deficiency (glycogenosis type II): biochemical and genetic studies.

In the same family, the generalized or infantile form of acid maltase deficiency (glycogenosis type II, Pompe disease) and the muscular or adult-onset form affected different individuals. Autosomal-recessive inheritance for the two clinical forms was demonstrated in this family by assay of acid alpha-glucosidase in muscle, lymphocytes, cultured fibroblasts, and urine of asymptomatic relatives. Current biochemical techniques do not discriminate between persons heterozygous for the generalized form and those heterozygous for the muscular form. To explain the coexistence of both forms in the same family, the infant with the generalized form or her grandfather with the muscular form must have been a genetic compound of different mutant alleles for acid alpha-glucosidase.

Adult↗
PubMed1981

Control mechanisms of energy-dependent metabolic pathways in hepatocytes.

The control of respiration was studied in isolated rat-liver cells and isolated rat-liver mitochondria. When isolated rat-liver cells are perfused with different concentrations of lactate at a constant lactate/pyruvate ratio, the rate of mitochondrial respiration is a linear function of the affinity for the respiratory chain; the affinity term contains the intramitochondrial redox state and the extramitochondrial phosphate potential. However, when the cells are perfused with ammonia in addition to lactate (+ pyruvate), the stimulation of the oxygen uptake is not accompanied by an increase in the affinity. In order to obtain further insight into the relationship between oxygen uptake and intramitochondrial and extramitochondrial phosphate potential, experiments were also carried out with isolate rat-liver mitochondria incubated with an extramitochondrial ATP-utilizing system (glucose plus hexokinase) or an intramitochondrial ATP-utilizing system (citrulline synthesis). The results suggest that the rate of oxygen consumption is controlled by the intramitochondrial phosphate potential.

Adenosine Triphosphate↗
PubMed1980

A critical examination of the evidence for an MgATP-dependent proton pump in rat liver lysosomes.

(1) When lysosomes isolated from the livers of Triton WR 1339-treated rats were incubated for 30 min in the presence of 100 mM KCl and 14CH3NH2, a stimulation by MgATP of the calculated accumulation of the base was observed, in agreement with previous results (Schneider, D.L. (1979) Biochem. Biophys. Res. Commun. 87, 559-565). A similar stimulation was seen with MgITP. Excess EDTA had very little effect on the stimulation by MgATP. (2) There was little effect of MgATP or MgITP on the calculated accumulation of 14CH3NH2 if the base was added to the incubation medium 1, 3, 4 or 5 min before terminating the incubation instead of being present for the total incubation period of 30 min. (3) The accumulation of the basic dye, acridine orange, by a crude lysosomal preparation isolated from the livers of untreated rats was found to be stimulated by MgATP, in agreement with earlier results (Dell'Antone, P. (1979) Biochem. Biophys. Res. Commun. 86, 180-189). Similar results were obtained with a crude lysosomal preparation isolated from the livers of Triton WR 1339-treated rats. In both cases, the stimulation was partly oligomycin-sensitive. (4) There was very little or no effect of MgATP on the accumulation of acridine orange by preparations of pure lysosomes isolated from the livers of Triton WR 1339-treated rats. (5) Our data do not acquire us to postulate the existence of an MgATP-dependent proton pump in lysosomes.

Adenosine Triphosphate↗
PubMed1980

Origin of the ammonia found in protein-free extracts of rat-liver mitochondria and rat hepatocytes.

1. Protein-free extracts of isolated rat-liver mitochondria contain 5.17 +/- 0.19 nmol ammonia/mg protein [cf. Harris, E. J. and Bassett, D. J. (1971) FEBS Lett. 19, 214-217]. 2. The ammonia found in the protein-free extracts does not originate from lysosomes contaminating the mitochondrial preparation. 3. When isolated mitochondria are incubated with ornithine, 14CO2 and a source of ATP a small amount of citrulline is formed. This amount is stoichiometrically equivalent to the ammonia that disappears from the extramitochondrial space, whereas the amount of ammonia found in the protein-free extracts of the mitochondria remains unchanged. Similar results were obtained when the reductive amination of 2-oxoglutarate was used as an ammonia-consuming reaction. 4. When isolated mitochondria are incubated under conditions such that the glutamate dehydrogenase and 3-hydroxybutyrate dehydrogenase reactions reach equilibrium, the thermodynamically active concentration of ammonia is not equal to the concentration measured in the protein-free extracts. 5. About 80% of the ammonia found in protein-free extracts of rat-liver mitochondria is derived from a component or components with a molecular weight of greater than or equal to 50,000. 6. Protein-free extracts of isolated rat-liver cells contain considerable amounts of ammonia. After digitonin fractionation this ammonia is found in the protein-free extract of the particulate fraction. 7. It is concluded that the ammonia found in protein-free extracts of rat-liver tissue is derived from a component or components in the mitochondria and is released during deproteinization.

Ammonia↗
PubMed1979

Factors affecting the hydrolysis of ceramide-3 by alpha-galactosidase A from human liver.

1. The effect of detergents on the catalytic properties of alpha-galactosidase from human liver was studied using p-nitrophenyl-alpha-galactoside and galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosylceramide (ceramide-3) as substrates. 2. The hydrolysis of p-nitrophenyl-alpha-galactoside by alpha-galactosidase was inhibited by commercial preparations of sodium taurocholate and by taurocholate purified from these preparations by thin-layer chromatography. The extent of inhibition was dependent on the concentration of the detergent and on the amount of protein present. The impurities present in the preparation also inhibited the hydrolysis. 3. The inhibition of taurocholate preparations of p-nitrophenyl-alpha-galactoside hydrolysis was pH-dependent. 4. The inhibition by taurocholate of p-nitrophenyl-alpha-galactoside hydrolysis can be partly overcome by adding glycosphingolipids. 5. No significant hydrolysis of ceramide-3 occurs in the absence of detergent. Upon adding increasing concentrations of taurocholate, the rate of hydrolysis increases to a maximum value. At still higher taurocholate concentrations the activity decreases. 6. The concentrations of taurocholate giving a maximal rate of hydrolysis of ceramide-3 is dependent on the amount of protein present and independent of the ceramide-3 concentration. 7. When the pH dependence of the rate of hydrolysis of ceramide-3 was measured in the presence of a commercially available preparation of pure taurocholate or of crude taurocholate, curves with different shapes were obtained.

Ceramides↗
PubMed1979

Use of immobilized antibodies in investigating acid alpha-glucosidase in urine in relation to Pompe's disease.

(1) A simple method is described for the isolation of the lysosomal enzyme, acid alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from normal human liver. Antibodies raised against the purified enzyme were immobilized by covalent coupling to Sepharose 4B. (2) Acid alpha-glucosidase can be quantitatively removed from normal urine by incubating with an excess of immobilized antibody. With p-nitrophenyl-alpha-glucoside as substrate, acid alpha-glucosidase accounts for 91 +/- 3% of the total alpha-glucosidase activity at pH 4.0 IN Normal urine. (3) In urine from a patient with the infantile form of Pompe's disease ('acid maltase deficiency'), no alpha-glucosidase activity could be removed by the immobilized antibody, in agreement with the fact that acid alpha-glucosidase is absent in these patients. (4) In urine from patients with the late-onset form of Pompe's disease, 46 +/- 11% of the alpha-glucosidase activity at pH 4.0 can be removed by incubation with immobilized antibodies, indicating that residual acid alpha-glucosidase activity is present in urine of these patients. The residual acid alpha-glucosidase activity amounts to about 5% of that in the urine of control persons. (5) If acid alpha-glucosidase is adsorbed to immobilized antibodies, the activity can still be measured with p-nitrophenyl-alpha-glucoside as substrate. The Km for p-nitrophenyl-alpha-glucoside is not significantly changed by adsorbing purified acid alpha-glucosidase to immobilized antibodies. (6) The properties of acid alpha-glucosidase from urine of patients with late-onset Pompe's disease were compared with those of acid alpha-glucosidase from normal urine, both adsorbed to immobilized antiserum. The pH-activity profile of the enzyme from urine of patients with late-onset Pompe's disease can not be distinguished from that of the normal urinary enzyme. The Km for p-nitro-phenyl-alpha-glucoside of the two enzymes is identical, both at pH 4 and 3. The titration curves of the two enzymes with immobilized antibodies are identical.

Glucosidases↗
PubMed1979

Evidence against a MgATP-dependent proton pump in rat-liver lysosomes.

1. The effect of MgATP has been studied on the accumulation of the lipid-soluble anion thiocyanate, the accumulation of the lipid-soluble base methylamine, and the fluorescence of bound anilinonaphthalene sulphonate in rat-liver lysosomes. The lysosomes used were isolated from the livers of rats pretreated with Triton WR 1339. 2. The accumulation of thiocyanate is stimulated by the addition of valinomycin in the presence of K+ but not by the addition of MgATP. 3. The fluorescence of anilinonaphthalene sulphonate bound to lysosomes is enhanced by valinomycin in the presence of K+, the extent of the enhancement being dependent on the concentration of K+. In contrast, MgATP has no effect on the fluorescence. 4. The intralysosomal pH, as estimated from the distribution of methylamine, is not affected by the addition of MgATP in media with or without K+, Na+ or HCO3-. 5. These data strongly suggest that there is no MgATP-dependent proton pump in rat-liver lysosomes.

Adenosine Triphosphate↗
PubMed1979

Relationship between biochemical and clinical features in an English Anderson-Fabry family.

The relationship between biochemical parameters and clinical symptoms in angiokeratoma corporis diffusum universale (Anderson-Fabry's disease) has been studied in an extensive English family. The biochemical parameters measured were alpha-galactosidase activity in urine and in single hair roots and the urinary glycosphingolipid excretion per 24 h. The clinical symptoms evaluated included the occurrence of pain, the prevalence of skin lesions, an abnormal ECG, cornea verticillata and other features. Nine male patients and 13 female carriers were studied. No correlation between biochemical parameters and the severity of the clinical symptoms could be found either in the hemizygotes or in the heterozygotes. The urinary alpha-galactosidase activity in all the heterozygotes lay within the normal range. All the obligate heterozygotes (mothers and daughters of hemizygotes) could be detected by analysis of hair roots. Additional heterozygotes were recognized on the basis of clinical symptoms and hair root analysis.

Adolescent↗
PubMed1978

Properties of immobilized fig alpha-galactosidase and effect on ceramide-3 content of plasma from patients with Fabry's disease.

The possibility of lowering the level of ceramide-3 (galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosyl-beta(1 leads to 1)-ceramide) in the plasma of patients with Fabry's disease was investigated. An immobilized alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was prepared by coupling purified fig alpha-galactosidase to Sepharose 4B. The pH optimum for the hydrolysis of the artificial substrate p-nitro-phenyl-alpha-D-galactopyranoside was shifted by approx. 0.5--1.0 pH unit to higher pH values upon coupling of the enzyme to Sepharose 4B. The immobilized enzyme was more stable than the native enzyme to incubation at 60 degrees C. The immobilized enzyme was able to hydrolyse ceramide-3 either at pH 4.5 or at pH 7.4 in an artificial system in which sodium taurocholate was used to solubilize the substrate. In contrast, when the immobilized enzyme was incubated with normal plasma or plasma from a patient with Fabry's disease, in which elevated levels of ceramide-3 occur, no hydrolysis of the glycosphingo-lipid could be detected. The results suggest that lowering of level of ceramide-3 in plasma from patients with Fabry's disease by enzymic means is not feasible.

Drug Stability↗
PubMed1978

A new immunochemical method for the quantitative measurement of specific gene products in man-rodent somatic cell hybrids.

An immunochemical method has been developed for the quantitative determination of species-specific gene products, for instance alpha-galactosidase and N-acetyl-alpha-galactosaminidase, in man-rodent hybrid cells and in the parental cell lines. Antisera raised against the purified enzymes are covalently coupled to Sepharose 4B. The gene products are specifically removed from a cell lysate by incubating with the appropriate Sepharose-coupled antiserum. After centrifugation followed by washing of the precipitated Sepharose, the enzymic activity can be quantitatively measured on the Sepharose beads. With this technique it has been demonstrated that the ability of human N-acetyl-alpha-galactosaminidase (also known as alpha-galactosidase B) to hydrolyze alpha-galactosidic linkages is lost when the enzyme is expressed in man-Chinese hamster hybrid cells.

Animals↗