Enzymic iodination of isolated rat hepatocytes and of rat livers perfused in situ was used to discriminate between the nucleotide pyrophosphatases of endoplasmic reticulum and of plasma membranes. The location of the latter on the cell surface could also be substantiated by this method. The activity of the microsomal enzyme increased after phenobarbital treatment of the animals. The nucleotide pyrophosphates from both subcellular fractions were solubilized, purified to electrophoretic homogeneity, and their ' ' ' I content determined. The labelling of the enzyme obtained from piasma membranes was several-fold higher than that of the nucleotide pyrophosphatase from endoplasmic reticulum. This indicates a bimodal distribution of nucleotide pyrophosphatase in rat liver and the accessibility of the plasma membrane enzyme from the extracellular space.
Isolated rat hepatocytes preserved for several hours their contents of ATP, CTP, GTP, UTP and UDPG. Leakage of intracellular and membrane-bound enzymes remained in the range observed with the isolated perfused rat liver. D -Galactosamine led to a rapid decrease of the UTP and UDPG contents while ATP and GTP levels remained unaffected. The sum of acidsoluble uracil nucleotides increased immediately after the addition of D-galactosamine, indicating release of the UTP-mediated feed-back inhibition of pyrimidine nucleotide de novo biosynthesis. Addition of undine to a hepatocy te suspension resulted in a rapid increase in the levels of uridylate derivatives; the concentrations of UTP and UDPG, previously depleted by D-galactosamine, were restored within 1 h. Isolated hepatocytes appear suitable for studies on the pathogenic sequence of events elicited by D -galactosamine and on the regulation of pyrimidine biosynthesis.
Die Reaktion isolierter Hepatozyten auf Gabe von D-Galaktosamin und UridinZusammenfassung: In isolierten Rattenhepatozyten blieben die Gehalte von ATP, CTP, GTP, UTP und UDPG über mehrere Stunden hinweg weitgehend konstant. Der Austritt intrazellulärer und membrangebundener Enzyme bewegte sich im Bereich des an der isoliert perfundierten Rattenleber Beobachteten. D-Galaktosamin-Zugabe führte zu einer raschen Abnahme der UTP-und UDPG-Spiegel, während der Gehalt an ATP und GTP konstant blieb. Die Summe aller säurelösli-chen Uracilnucleotide nahm unmittelbar nach Gabe von D-Galaktosamin zu; dies deutet darauf hin, daß unter diesen Bedingungen auch in isolierten Hepatozyten die feed-back-Hemmung der
The site of feedback inhibition of the biosynthesis of pyrimidine nucleotides de novo was investigated in the isolated perfused rat liver. Hepatic uridine phosphate contents were specifically depleted by use of D-galactosamine. The effective activities of enzymes involved in the synthetic pathway were deduced from the rates of incorporation of labeled precursors into the acid-soluble uracil nucleotide pool and into some intermediates of the pathway. The labeling of hepatic urea was also monitored.When the uridine phosphate contents were less than 20 % of controls, the incorporation of ['"CIbicarbonate was stimulated about 20-fold. Label from [U-14C]oxaloacetate used as permeable precursor of intracellular aspartate was introduced into the uridylates to the same extent in normal and UTP-depleted livers. Similar results were obtained with labeled carbamoyl phosphate although the uptake of this compound by the liver was rather low. The lack of labeling of urea from exogenous carbamoyl phosphate does not indicate a free exchange of extra-and intramitochondrial carbamoyl phosphate.[ureid~-'~C]Ureidosuccinate produced in normal and D-galactosamine-treated livers almost identical labeling patterns of dihydroorotate, orotate and orotidine 5'-phosphate. The steady state concentrations of these intermediates were all below 15 nmol/g liver wet weight.
The influence of clofibrate on the glycolytic pathway in liver was studied. The changes in the activity of glucokinase and hexokinase were not significant. A reduction of phosphofructokinase (p less than 0.05) and pyruvate kinase activity was found (p less than 0.0005) during clofibrate feeding. An in vitro inhibition of these enzymes could not be demonstrated by clofibrate up to a concentration of 2.5 mM. Crossover plots of glycolytic intermediates indicate that the reduced pyruvate kinase activity may influence the glycolytic pathway in vivo. Clofibrate feeding induces a lower ATP:ADP ratio, a lower adenylate energy charge and elevates AMP levels in rat liver. This may possibly stimulate the hepatic glycogenolysis and the glucose utilisation by this organ.
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