PubMed Health⌕ Search

Biomedical subjects

J M Tager

Publications and source records attributed to J M Tager.

At least 181 records · Page 10Linked to original sources
PubMed1983

Bicarbonate and the pathway of glutamate oxidation in isolated rat-liver mitochondria.

1. The factors affecting the pathway of glutamate oxidation were studied in isolated rat-liver mitochondria in incubations of 2-3 min. 2. It was found that bicarbonate at a physiological concentration has a profound effect on the pathway of glutamate oxidation. Ammonia formation via glutamate dehydrogenase is stimulated by bicarbonate [from 5.48 +/- 0.29 (n = 10) to 9.57 +/- 0.73 (n = 8) nmol X min-1 X mg protein-1], whereas aspartate formation via the transamination pathway is inhibited [from 38.41 +/- 2.24 (n = 9) to 24.56 +/- 3.28 (n = 6) nmol X min-1 X mg protein-1]. 3. Bicarbonate has no effect on the rate of transport of glutamate via the glutamate-hydroxyl translocator. 4. The interaction of bicarbonate with the pathway of glutamate oxidation occurs primarily at the level of succinate dehydrogenase, due to competitive inhibition of the enzyme by bicarbonate. 5. Inhibition by bicarbonate of the transamination pathway leads to a decrease in intramitochondrial 2-oxoglutarate, so that the deamination pathway is stimulated. 6. Using an equation which describes flux through glutamate dehydrogenase kinetically, it could be shown that the bicarbonate-induced decrease in intramitochondrial 2-oxoglutarate quantitatively accounts for the enhanced rate of deamination. 7. It is concluded that in the intact liver flux through glutamate dehydrogenase is sufficient to account for the ammonia formation required for urea synthesis from substrates such as alanine.

Adenosine Diphosphate↗
PubMed1983

Measurement of binding of adenine nucleotides and phosphate to cytosolic proteins in permeabilized rat-liver cells.

1. A method is described for measuring the binding of metabolites to cytosolic proteins in situ in isolated rat-liver cells treated with filipin to render the plasma membrane permeable to compounds of low molecular weight. 2. There is no binding of ATP or inorganic phosphate to cytosolic proteins, either in the presence or in the absence of Mg2+. 3. Binding of ADP to cytosolic proteins occurs both in the absence and in the presence of Mg2+. The concentration of binding sites was 0.68 and 0.52 mumol/g dry weight of cells (n = 3-4) in the absence and presence of Mg2+, respectively. The corresponding Kd values were 320 microM and 235 microM.

Adenosine Diphosphate↗
PubMed1983

Control of mitochondrial respiration.

The control theory of Kacser and Burns [in: Rate Control of Biological Processes (Davies, D.D. ed) pp. 65-104, Cambridge University Press, London, 1973] and Heinrich and Rapoport [Eur. J. Biochem. (1974) 42, 97-105] has been used to quantify the amount of control exerted by different steps on mitochondrial oxidative phosphorylation in rat-liver mitochondria. Inhibitors were used to manipulate the amount of active enzyme. The control strength of the adenine nucleotide translocator was measured by carrying out titrations with carboxyatractyloside. In state 4, the control strength of the translocator was found to be zero. As the rate of respiration was increased by adding hexokinase, the control strength of the translocator increased to a maximum value of approximately 30% at approximately 80% of state 3 respiration. In state 3, control of respiration is distributed between a number of steps, including the adenine nucleotide translocator, the dicarboxylate carrier and cytochrome c oxidase. The measured values for the distribution of control agree very well with those calculated with the aid of a model for mitochondrial oxidative phosphorylation developed by Bohnensack et al. [Biochim. Biophys. Acta (1982) 680, 271-280].

Adenosine Diphosphate↗
PubMed1983

Assignment of the gene coding for human beta-glucocerebrosidase to the region q21-q31 of chromosome 1 using monoclonal antibodies.

A series of man-Chinese hamster somatic cell hybrids with a variable content of human chromosomes was used to study the localization of the human gene coding for the lysosomal enzyme beta-glucocerebrosidase (EC 3.2.1.45). In lysates made from hybrid cells, the human enzyme was specifically recognized by a mouse monoclonal antibody raised against human placental beta-glucocerebrosidase. This monoclonal antibody did not cross-react with Chinese hamster beta-glucocerebrosidase. After reaction of the antibody with the enzyme, beta-glucocerebrosidase was precipitated by addition of Protein A-Sepharose beads, and was detected on the beads by its enzymatic activity. From the analysis of a series of man-Chinese hamster hybrids, among which were hybrids with specific segments of chromosome 1, we conclude that the gene coding for human beta-glucocerebrosidase is localized in the region q21-q31 of chromosome 1.

Animals↗
PubMed1982

Biosynthesis of acid alpha-glucosidase in late-onset forms of glycogenosis type II (Pompe's disease).

Cultured human skin fibroblasts from control persons and from patients with the generalized and late-onset forms of Pompe's disease were labelled with radioactive leucine and the incorporation of radioactivity into acid alpha-glucosidase and cathepsin D was analysed by immunoprecipitation, gel electrophoresis and fluorography. When the labelling was carried out for 6-12 h in the presence of NH4Cl, the labelling of secreted alpha-glucosidase relative to that of secreted cathepsin D in fibroblasts from patients with the late-onset form of Pompe's disease was less than 15% of that in fibroblasts from control persons. However, when the fibroblasts were labelled for less than 1 h, the relative rate of incorporation of radioactivity into acid alpha-glucosidase was rather similar in the two types of fibroblasts. In fibroblasts from patients with the generalized form of Pompe's disease no incorporation of radioactivity into acid alpha-glucosidase could be detected.

Cathepsin D↗
PubMed1982

An evaluation of the metabolite indicator method for determining the cytosolic phosphate potential in rat liver cells.

The cytosolic phosphate potential was estimated in isolated rat liver parenchymal cells incubated with various gluconeogenic substrates. The value of the cytosolic [ATP]/[ADP][Pi] ratio was either estimated directly from measurements of ATP, ADP and Pi after digitonin fractionation of the cells, or calculated by the metabolite indicator method. When cells were incubated with lactate, pyruvate or alanine so that net flux through the indicator enzymes was in gluconeogenic direction, there was excellent agreement between the values obtained by the two methods over a wide range of fluxes. However, when the cells were incubated with substrates that could be converted both to glucose and to lactate so that net flux through the indicator enzymes was in the glycolytic direction, a large difference in the values of the cytosolic [ATP]/([ATP][Pi]) ratio as derived by the two methods was observed. It is concluded that the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase plus 3-phosphoglycerate kinase is out of equilibrium when flux through the reaction is the glycolytic direction, and that use of the metabolite indicator method for the calculation of the cytosolic phosphate potential under these conditions leads to erroneous values.

Adenosine Diphosphate↗
PubMed1982

Quantification of the contribution of various steps to the control of mitochondrial respiration.

Using principles developed by Kacser and Burns ((1973) in Rate Control of Biological Processes (Davies, D. D., ed) pp. 65-104, Cambridge University Press, London) and Heinrich and Rapoport ((1974) Eur. J. Biochem. 42, 97-105), inhibitor titration studies were carried out in order to quantify the amount of control (control strength) exerted by different steps in oxidative phosphorylation on the rate of mitochondrial oxygen uptake. In the resting state of respiration, nearly all control is exerted by the passive permeability of the mitochondrial inner membrane to protons. In the intermediate states and even in the active state of respiration, control is distributed among different steps including the adenine nucleotide translocator and cytochrome c oxidase. It is therefore not possible to speak of the rate-limiting step in oxidative phosphorylation. Since both the adenine nucleotide translocator and cytochrome c oxidase appear to be rate-controlling, hypotheses suggesting that respiration is fully controlled by either of these two steps alone need to be modified.

Animals↗
PubMed1981

Monoclonal antibodies against human acid alpha-glucosidase.

Acid alpha-glucosidase purified from human placenta was used to immunize a mouse (strain Balb/cHeA) according to a procedure described earlier (Stähli, C., Staehlin, T., Miggiano, V., Schmidt, J. and Häring, P. (1980) J. Immunol. Methods 32, 297-304). After fusion of spleen cells with myeloma cells, about 10% of the hybrid clones obtained produced antibodies against acid alpha-glucosidase. Finally, eight stable clones producing antibodies against the enzyme were obtained. When purified acid alpha-glucosidase is analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, two major protein bands (mol. wt. 76000 and 70000,) a minor band of mol. wt. 9600 and several minor bands with a mol. wt. of 67000 or lower are seen. Since all these components react with the monoclonal antibodies, they must have at least one antigenic determinant in common.

Animals↗
PubMed1981

Accumulation of weak bases in relation to intralysosomal pH in cultured human skin fibroblasts.

The volume of the lysosomal compartment in cultured human skin fibroblasts was estimated from the distribution between the cells and the medium of tracer amounts of labelled methylamine and chloroquine, which accumulate in the lysosomes, 2,2-dimethyloxazolidine-2,4-dione, which accumulates in the soluble cytoplasmic compartment relative to the lysosomes, and sucrose, which is excluded by the cells. In a foetal fibroblast line, the fractional volume of the lysosomal compartment was 0.044 +/- 0.007 (n = 8). In fibroblasts from a patient with the I-cell disease, the fractional volume was 0.15. The fractional volume of the lysosomal compartment was used to calculate the intralysosomal pH from the accumulation of the weak bases in the cells. The mean value obtained was 5.29 +/- 0.04 (n = 8). In fibroblasts incubated with various concentrations of chloroquine, the fractional volume of the lysosomal compartment and the accumulation of chloroquine in the cells were used to calculate the concentration of chloroquine in the lysosomes. The intralysosomal concentration increased from 3 to 114 mM as the extracellular concentration increased from 1 to 100 microM. Concomitantly, the intralysosomal pH increased from 5.3 in the absence of chloroquine to 5.9 in the presence of 100 microM chloroquine. A similar increase in intralysosomal pH could be calculated in fibroblasts incubated with different concentrations of ammonia.

Cell Line↗