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J M Tager

Publications and source records attributed to J M Tager.

At least 235 records · Page 13Linked to original sources
PubMed1977

Mitochondrial and cytosolic NADPH systems and isocitrate dehydrogenase indicator metabolites during ureogensis from ammonia in isolated rat hepatocytes.

1. Citrate isocitrate and 2-oxoglutarate levels were determined in isolated rat hepatocytes and in particulate and soluble fractions, thereof, obtained by the digitonin and silicone oil fractionation technique. 2. Caculated from isocitrate/2-oxoglutarate ratios ("indicator metabolite method"), the redox potential of mitochondrial free NADPH is -402 mV, whereas that of the extramitochondrial (cytosolic) space is about 10 mV more positive, -392 mV. 3; Addition of ammonia (either as ammonium chloride or from urea plus urease) to isolated hepatocytes causes preferential oxidation of mitochondrial NADPH, is demonstrated by spectrophotometry of the dihydro band and by the changes in the isocitrate/2-oxoglutarate ratios. The redox potential difference of free NADPH between mitochondria and cytosol is abolished or even reserved. 4. It is concluded that during urogenesis from ammonia mitochondrial isocitrate oxidation is shifted largely in favor of the NADP-linked as opposed to the NAD-linked enzyme; isocitrate concentration under these conditions is less than 10 muM, below the Km (isocitrate) of the NAD-linked enzyme but in the range of that for the NADP-linked enzyme. 5. Both in the absence and in the presence of ammonia there is a concentration gradient across the mitochondrial inner membrane (from mitochondria to cytosol) for citrate, isocitrate, and also, to a smaller extent, for 2-oxoglutarate. 6. These results and data in the literature on enzyme activity are in agreement with the assumption of near-equilibrium of NADP-dependent isocitrate dehydrogenases in the mitochondrial matrix and cytosolic spaces in the absence of ammonia; accordingly, during urea formation from added ammonia the redox potential of mitochondrial free NADPH is increased to -391 mV or possibly even higher if there exists an indicator error under this condition.

Ammonia↗
PubMed1976

Relationship between medium pH and that of the lysosomal matrix as studied by two independent methods.

1. The method of estimating the intralysosomal pH by measuring the distribution of [14C]methylamine in lysosomes isolated from the livers of Triton WR 1339-treated rats has been critically examined. 2. In lysed lysosomes, methylamine is bound to the membrane fragments, but this binding can be completely suppressed by increasing the concentration of monovalent cations in the medium. 3. In intact lysosomes, the binding of [14C]methylamine is only partly inhibited by monovalent cations at 25 degrees C. 4. THe accumulation of [14C]methylamine in intact lysosomes is progressively inhibited as the concentration of methylamine is increased. A similar inhibition of [14C]methylamine accumulation is obtained with NH4Cl. 5. Similar values for the intralysosomal pH were obtained from measurements of the distribution of methylamine, dimethylamine and trimethylamine, which are accumulated in the lysosomes, and of 5,5-dimethyloxazolidinedione-2,4, which is excluded. 6. The breakdown of endocytosed 123I-labelled bovine serum albumin by intact isolated lysosomes is much less sensitive to the pH of the medium than the breakdown of added protein by lysed lysosomes. 7. The intralysosomal pH has been estimated by comparing the rate of breakdown of endocytosed 125I-labelled albumin in intact lysosomes as a function of medium pH with that of added 125I-labelled albumin by lysed lysosomes at different pH values. The values obtained agree well with those calculated from the distribution of [14C]methylamine. 8. Methylamine and NH4Cl inhibit the breakdown of 125I-labelled albumin in intact lysosomes, particularly at high medium pH, but have no effect on the breakdown by lysed lysosomes. 9. It is concluded that a pH difference across the lysosomal membrane (more acidic inside than outside) is maintained by the presence of indiffusible negatively charged groups within the lysosomes, and by the permeation across the lysosomal membrane of protons together with permeant anions (or of OH- in exchange for anions).

Ammonium Chloride↗
PubMed1976

Effect of ionophores on intralysosomal pH.

1. The effect of ionophores on the intralysosomal pH (as estimated from the distribution of a weak acid or base), on the distribution of 42K+ across the lysosomal membrane, and on the intralysosomal degradation of 125I-labelled bovine serum albumin has been studied. 2. Nigericin and X537A equilibrate both 42K+ and H+ across the lysosomal membrane. Gramicidin equilibrates H+ across the lysosomal membrane, this equilibration being more effective in a NaCl than in a KCl medium. Thus all three ionophores exhibit the same ion specificity as in other membranes. 3. The effect of the exchange-diffusion ionophores cannot be imitated by the combination of valinomycin with an uncoupler. Valinomycin by itself also has no effect. 4. X537A and nigericin inhibit the intralysosomal degradation of 125I-labelled albumin only when potassium is present. In a sucrose-containing medium no effect is found. Similar results were obtained with gramicidin. 5. These data suggest that the lysosomal membrane is impermeable to monovalent cations at 25 or 37 degrees C, and that the transport of protons is organised in such a way that electroneutrality is maintained.

Animals↗
PubMed1976

Localization of neutral glycosphingolipids in human plasma.

1. The localization of the neutral glycosphingolipids glucosylceramide, lactosylceramide, trihexosylceramide and globoside in human plasma was investigated. Glycosphingolipids were isolated and analysed by gas-liquid chromatography. 2. After Sephadex gel chromatography of human plasma, about 75% of the glycosphingolipids were found in the fraction containing most of the lipoproteins. 3. After fractionation of the lipoproteins by ionic precipitation, 15-25% of each glycosphingolipid was found in the very low-density lipoprotein + chylomicron fraction, 30-45% in the low density lipoprotein fraction and 40-50% in the high density lipoprotein fraction. 4. After fractionation of lipoproteins by density-gradient ultracentrifugation, 15% of each glycosphingolipid was found in the very low-density lipoprotein + chylomicron fraction and 85% in the low density and high density lipoprotein fractions. No glycosphingolipids could be detected in the ultracentrifugal residue which contains the bulk of the albumin.

Ceramides↗
PubMed1976

A simple method for the perfusion of isolated liver cells.

The perfusion technique described in this paper offers a new and simple method for studying liver cell metabolism in a more dynamic way. It has potential use in hormonal response studies, pulsechase experiments and many other types of investigations. Finally, the perfusion technique may be applicable to other isolated cell preparations as well. The method combines some advantages of the flow-through technique with the great advantages of using isolated liver cells, apparently without disturbing the viability of the cells.

Animals↗
PubMed1976

B.C.P. Jansen Institute, University of Amsterdam, Amsterdam, The Netherlands.

In a patient with Fabry's disease who had undergone kidney transplantation to correct uremia, the neutral glycosphingolipids and alpha-galactosidase activity have been measured in plasma and urine and, 9 months later, after the death of the patient, in autopsy material. After transplantation, there was no significant increase in alpha-galactosidase activity in plasma; the activity found never exceeded 3% of the mean control value. A striking parallelism was found during the follow-up period in the increase and decrease of trihexosylceramide and globoside and also of glucosylceramide and dihexosylceramide. The alpha-galactosidase activity in spleen and liver was as low as that observed in untreated Fabry hemizygotes. These data and those obtained from autopsy material provide evidence that renal transplantation does not lead to a specific enzymic breakdown of trihexosylceramide in Fabry patients. However, no trihexosylceramide accumulation was observed in the transplanted kidney.

Adult↗
PubMed1975

Role of anion translocation across the mitochondrial membrane in the regulation of urea synthesis from ammonia by isolated rat hepatocytes.

The regulation of urea synthesis from ammonia was investigated using isolated hepatocytes from fasted rats. Addition of ammonia alone produced only a small increase of urea formation, which was stimulated 2-fold by ornithine in conjunction with a fall of ATP levels and an accumulation of citrulline. Further addition of oleate or beta-hydroxybutyrate produced an additional 2-fold stimulation of urea formation to approximately 200 mumol/g dry weight/hour. The presence of oleate also protected against the inhibitory effect of 2,4-dinitrophenol on urea synthesis and the cellular ATP content. The data suggest that both the rate of of energy production and the rate of generation of reducing equivalents from endogensou substrates are insufficient to meet the requirements for optimal rates of urea synthesis. Urea formation from NH3 in the presence of ornithine and oleate, but iin the absence of gluconeogenic precursors, was inhibited by butylmalonate, a known inhibitor of malate-phosphate exchange across the mitochondrial membrane, and stimulated by theaddition of malate and other dicarboxylic acids and amino acids to the cell suspension...

Acetoacetates↗
PubMed1975

Properties of the residual alpha-galactosidase activity in the tissues of a Fabry hemizygote.

The properties of the residual alpha-galactosidase activity in kidney, liver, spleen, fibroblasts and urine of a Fabry hemizygote have been studied using p-nitrophenyl-alpha-galactoside and 4-methylumbelliferyl-alpha-galactoside as substrates. In addition, alpha-galactosidase activity in urine has been determined with ceramidetrihexoside as substrate. The residual alpha-galactosidase activity of Fabry, measured with artificial substrate, is stimulated (6-35%) by myo-inositol and only slightly inhibited by melibiose (7-17%) in all the materials used. In contrast, the alpha-galactosidase of normal tissues and urine is inhibited (36-48%) by myo-inositol and inhibited to a much greater extent (40-50%) by melibiose. The KM for artificial substrate of the residual activity of Fabry is higher than that of the alpha-galactosidase in normal kidney, liver, spleen, fibroblasts and urine. The residual activity of Fabry is generally more stable to heating than the activity in the normal materials, although exceptions were noted. When these properties are compared with those of the alpha-galactosidase isoenzymes in normal tissues and body fluids, the residual activity of Fabry material seems to be very similar to the minor component of normal tissue (alpha-galactosidase B). Moreover, the pH optimum curve of this minor component and of the Fabry alpha-galactosidase in urine are similar, whereas the major isoenzyme (alpha-galactosidase A) shows a curve much more like that of normal urine. The findings with ceramidetrihexoside as substrate indicate a possible discrepancy. Alpha-Galactosidase A hydrolyses ceramidetrihexoside, Fabry urine preparation does not. However, alpha-galactosidase B of normal urine shows a slight but definite ceramidetrihexosidase activity. No contamination of the B preparation with alpha-galactosidase A could be detected. The minimum hypothesis, supported by most of the experimental evidence, is that the residual activity of Fabry and normal alpha-galactosidase B are identical.

Disaccharides↗
PubMed1975

Rate-limiting factors in the oxidation of ethanol by isolated rat liver cells.

The oxidation of ethanol by isolated liver cells from starved rats is limited by the rate of removal of reducing equivalents generated in the cytosol by alcohol dehydrogenase. Evidence is presented suggesting that, in these cells, transfer of reducing equivalents from the cytosol to the mitochondria is regulated by the intracellular concentrations of the intermediates of the malate-aspartate and glycerol 3-phosphate cycles, as well as by flux through the respiratory chain. In liver cells isolated from fed rats, the availability of substrate increased the cell content of intermediates of the hydrogen-transfer cycles, and enhanced ethanol uptake. Under these conditions, ethanol consumption is limited by the availability of ADP for oxidative phosphorylation.

Animals↗