Carbohydrate metabolism of hepatocytes from starved Japanese quail

Hepatocytes were isolated from livers of mature male and female starved Japanese quail (Coturnix coturnix japonica). The hepatocytes take up lactate and dihydroxyacetone extensively, and have a very high rate of glucose synthesis from these substrates. Fructose uptake and incorporation into glucose is much less. Pyruvate and alanine are taken up extensively, but form little glucose. There is negligible lipogenesis in cells of starved quail. Alanine increases up to 10-fold incorporation of ³HOH and ¹⁴C from several substrates into fatty acids, but it remains insignificant as compared to lipogenesis by cells of fed quail. There is little utilization of glucose, even in the presence of alanine, in marked contrast to hepatocytes from fed quail. However, glucose is phosphorylated at high rates, but most of the glucose 6-phosphate is recycled to glucose. There is a marked difference in the metabolism of polyols between the sexes. Glycerol, xylitol, and sorbitol are converted nearly quantitatively into glucose by hepatocytes of starved female quail. In cells of starved males, the uptake of polyols is higher, but conversion to glucose less efficient. In cells of starved male quail, alanine markedly stimulates the uptake of glycerol and xylitol and their conversion to glucose, but has no effect on sorbitol metabolism. In cells of female quail, alanine is without a significant effect on polyol metabolism.     

Effect of hypoxic cell radiosensitizers on glutathione level and related enzyme activities in isolated rat hepatocytes

A comparative study of the effect of misonidazole and novel radiosensitizers on glutathione (GSH) levels and related enzyme activities in isolated rat hepatocytes was performed. Incubation of hepatocytes with 5 mM radiosensitizers led to a decrease in the intracellular GSH level. The most pronounced decrease in cellular GSH was evoked by 2,4-dinitroimidazole-1-ethanol (DNIE); after incubation for only 15 min, GSH was hardly detected. DNIE-mediated GSH loss was dependent upon its concentration. DNIE reacted with GSH nonenzymatically as well as with diethylmaleate, while misonidazole and 1-methyl-2-methyl-sulfinyl-5-methoxycarbonylimidazole (KIH-3) did not. Addition of partially purified glutathione S-transferase (GST) did not enhance DNIE-mediated GSH loss in a cell-free system. DNIE inhibited glutathione peroxidase (GSH-Px), GST, and glutathione reductase (GSSG-R) activities in hepatocytes, while misonidazole and KIH-3 did not. GSH-Px activity assayed with H2O2 as substrate was the most inhibited. Inhibition of GSH-Px activity assayed with cumene hydroperoxide as substrate and GST was less than that of GSH-Px assayed with H2O2 as substrate. GSSG-R activity was decreased by DNIE, but not significantly. Incubation of purified GSH-Px with DNIE resulted in a little change in the activity when assayed with H2O2 as substrate.     

Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa: Purification by affinity chromatography and physicochemical properties

Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa was purified 150-fold by affinity chromatography on immobilized 2′-AMP. The binding of the enzyme is pH dependent. Elution was achieved with 2′-AMP, NADP⁺, or NADPH but not with 5′-AMP, NAD⁺, or NADH. The enzyme preparations appeared to be homogeneous in gel chromatography and ultracentrifugation, but only if these procedures were carried out in the presence of 2′-AMP or NADP⁺. With 2′-AMP a sedimentation coefficient of 34 S, a molecular weight of 1.6–1.7 million, and a Stokes' radius of 11.7 nm were determined. In the presence of NADP⁺ the sedimentation coefficient was 42 S and the molecular weight was 6.4 million. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed one kind of subunit with a molecular weight of 54,000. This was consistent with results from amino acid analyses and paper chromatography of peptides. Eight molar urea inactivated the enzyme but did not dissociate it into subunits. Full activity was restored after dialysis against urea-free buffer by mercaptoethanol and flavin-adenine dinucleotide.     

Pyridine nucleotide transhydrogenases of parasitic helminths.

Mitochondria from the parasitic helminth, Hymenolepis diminuta, catalyzed both NADPH:NAD⁺ and NADH:NADP⁺ transhydrogenase reactions which were demonstrable employing the appropriate acetylpyridine nucleotide derivative as the hydride ion acceptor. Thionicotinamide NAD⁺ would not serve as the oxidant in the former reaction. Under the assay conditions employed, neither reaction was energy linked, and the NADPH:NAD⁺ system was approximately five times more active than the NADH:NADP⁺ system. The NADH:NADP⁺ reaction was inhibited by phosphate and imidazole buffers, EDTA, and adenyl nucleotides, while the NADPH:NAD⁺ reaction was inhibited only slightly by imidazole and unaffected by EDTA and adenyl nucleotides. Enzyme coupling techniques revealed that both transhydrogenase systems functioned when the appropriate physiological pyridine nucleotide was the hydride ion acceptor. An NADH:NAD⁺ transhydrogenase system, which was unaffected by EDTA, or adenyl nucleotides, also was demonstrable in the mitochondria of H. diminuta. Saturation kinetics indicated that the NADH:NAD⁺ reaction was the product of an independent enzyme system. Mitochondria derived from another parasitic helminth, Ascaris lumbricoides, catalyzed only a single transhydrogenase reaction, i.e., the NADH:NAD⁺ activity. Transhydrogenase systems from both parasites were essentially membrane bound and localized on the inner mitochondrial membrane. Physiologically, the NADPH:NAD⁺ transhydrogenase of H. diminuta may serve to couple the intramitochondrial metabolism of malate (via an NADP linked “malic” enzyme) to the anaerobic NADH-dependent ATP-generating fumarate reductase system. In A. lumbricoides, where the intramitochondrial metabolism of malate depends on an NAD-linked “malic” enzyme which is localized primarily in the intermembrane space, the NADH:NAD⁺ transhydrogenase activity may serve physiologically in the translocation of hydride ions across the inner membrane to the anaerobic energy-generating fumarate reductase system.     

Perturbation of hepatic glutathione level and glutathione-related enzyme activities by repeated administration of aminopyrine in rats

The influence of repeated administration of aminopyrine on the tissue glutathione level and related enzyme activities was investigated in rats. Reduced glutathione level in the liver was not changed after 5 days of treatment but a significant increase was seen after 15 days of aminopyrine treatment. Oxidized glutathione level was unaltered throughout the experiment. Repeated administration of aminopyrine for 5 days caused a marked increase in gamma-glutamyl transpeptidase activities in liver whole homogenates as well as in the nuclear fraction, but not in liver microsomes. These results suggest that gamma-glutamyl transpeptidase located in plasma membrane may be induced by repeated administration of aminopyrine for 5 days. The activities of cytosolic glutathione peroxidase, which modulates glutathione level, were also significantly increased by aminopyrine treatment. Under the same conditions, glutathione peroxidase activity with H2O2 as a substrate was unaltered, while a time-dependent increase in the activity was found when cumene hydroperoxide was used as a substrate, even after a single administration of aminopyrine. The intracellular cysteine level was increased accompanying the increased gamma-glutamyl transpeptidase activities. Therefore, induced gamma-glutamyl transpeptidase may play a role in the reclamation of extracellular oxidized glutathione.     

Metabolism of monoacylglycerol and diacylglycerol by enzyme preparations from spinach leaves

Acetone powders prepared from a 20,000g participate preparation from spinach leaf catalyzed several reactions involving monoacylglycerol and diacylglycerol. When these substrates were presented as Triton X-100-mixed micelles, diacylglycerol gave rise to free fatty acids, monoacylglycerol, triacylglycerols, and steryl esters, and in the presence of ethanol, small amounts of ethyl esters of fatty acid. Monoacylglycerol gave rise to free fatty acids and diacylglycerol, and in the presence of ethanol, large amounts of ethyl esters of fatty acid. In the presence of bovine serum albumin, the conversion of monoacylglycerol to free fatty acid was retarded. In the presence of bovine serum albumin, steryl ester was an important product from diacylglycerol. The system containing Triton X-100-mixed micelles and bovine serum albumin permitted analysis of reaction products which showed diacylglycerol to be an acyl donor in steryl ester biosynthesis. All reactions observed in the mixed micelle system were transacylation reactions involving various acceptors: dipalmitoylglycerol → monopalmitoylglycerol + palmitate; monopalmitoylglycerol → glycerol + palmitate; dipalmitoylglycerol + sterol → monopalmitoylglycerol + steryl palmitate; monopalmitoylglycerol + ethanol → ethyl palmitate + glycerol; monopalmitoylglycerol → dipalmitoylglycerol (+glycerol); dipalmitoylglycerol → tripalmitoylglycerol (+monopalmitoylglycerol).     

The time course of glucagon action on the utilization of [U-14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U-¹⁴C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO₂. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and ¹⁴CO₂, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-¹⁴C]palmitate.     

Modification of pyruvate kinase isozymes in prolonged primary cultures of adult rat hepatocytes.

Pyruvate kinase isozymic changes were studied in the adult hepatocyte cultures, by electrophoretic, kinetic and immunological methods. We were able to maintain parenchymal cells from normal adult rat liver in non-proliferating monolayer cultures up to 10 days. Hepatocytes appeared to contain a dominant PK I type up to 4-5 days of culture. After day 5, PK III type was regularly present with PK I and after 7 days PK III type was always the only isozyme detected in culture. It must be pointed out that, by the Ouchterlony method and sometimes by electrophoresis, concentrated extracts from freshly isolated hepatocytes or starting hepatocyte cultures did also contain Pyruvate kinase PK III type. These results suggest that Pyruvate kinase III is present but partly repressed in the adult parenchymal cells and becomes derepressed in culture.     

The effect of choline deficiency on the activity of a phosphatidylcholine-requiring enzyme: Activity and properties of UDP-glucuronyltransferase in choline-deficient rats

The effect of choline deficiency on the kinetic properties of the microsomal enzyme UDP-glucuronyltransferase (EC2.4.1.17) was investigated in rats. Animals fed choline-deficient diets, as compared with animals fed a choline-replete diet or standard laboratory chow, showed almost a three-fold increase in enzyme activity when the enzyme was assayed at physiological concentrations of UDP-glucuronic acid (0.25 mM). The increase in activity appeared to be due to an enhanced affinity of the enzyme for UDP-glucuronic acid rather than to an increase in the amount of enzyme. These data indicate that the kinetic properties of tightly bound membrane enzymes are altered by a dietary change that is known to cause liver disease in the rat.     

Studies on NADPH-cytochrome c reductase I: Isolation and several properties of the crystalline enzyme from ale yeast.

NADPH-cytochrome c reductase has been isolated from a top-fermenting ale yeast, Saccharomyces cerevisiae (Narragansett strain), after ca. a 240-fold purification over the initial extract of an acetone powder, with a final specific activity (at pH 7.6, 30 °C) of ca. 150 μmol cytochrome c reduced min^?1mg^?1 protein. The preparation appears to be homogeneous by the criteria of: sedimentation velocity; electrophoresis on cellulose acetate in buffers above neutrality; and by polyacrylamide gel electrophoresis. Although the reductase appeared to partially separate into species “A” and “B” on DEAE-cellulose at pH 8.8, the two species have proven to be indistinguishable electrophoretically (above pH 8) and by sedimentation. By sedimentation equilibrium at 20 °C, a molecular weight of ca. 6.8 (± 0.4) × 10⁴ was obtained with use of a $\text{V}\text{?}{}_{\mathrm{20\; °}}\text{= 0.741}$ calculated from its amino acid composition. After disruption in 4 m guanidinium chloride- 10 mm dithioerythritol- 1 mm EDTA, pH 6.4 at 20 °C, an $\text{M}\text{?}{}_{\mathrm{<mtext>r</mtext>}}$ of 3.4 (± 0.1) × 10⁴ resulted, which points to a subunit structure of two polypeptide chains per mole. Confirmatory evidence of the two-subunit structure with similar, if not identical, polypeptide chains was obtained by polyacrylamide gel electrophoresis in dodecyl-sulfate, after disruption in 4 m urea and 2% sodium dodecyl sulfate, and yielded a subunit molecular weight of ca. 4 × 10⁴. Sulfhydryl group titration with 4,4′-dithiodipyridine under acidic conditions revealed one sulfhydryl group per monomer, which apparently is necessary for the catalytic reduction of cytochrome c. NADPH, as well as FAD, protects this-SH group from reaction with 5,5′-dithiobis (2-nitrobenzoate). The visible absorption spectrum of the oxidized enzyme (as prepared) has absorption maxima at 383 and 455 nm, typical of a flavoprotein. Flavin analysis (after dissociation by thermal denaturation of the “A” protein) conducted fluorometrically, revealed the presence of 2.0 mol of FAD per 70,000 g, in confirmation of the deduced subunit structure. The identity of the FAD dissociated from either “A” or “B” protein was confirmed by recombination with apo-d-amino acid oxidase and by thin-layer chromatography. A kinetic approach was used to estimate the dissociation constant for either FAD or FMN (which also yields a catalytically active enzyme) to the apoprotein reductase at 30 °C and pH 7.6 (0.05 m phosphate) and yielded values of 4.7 × 10^?8m for FAD and 4.4 × 10^?8m for FMN.     

Enzyme-induced aziridine formation by isolated hepatocytes

The metabolism of 2-bromoethylaminonaphthoquinone in hepatocytes isolated from rats was studied. This compound was chemically inert in the reaction system used. However, in buffer solution containing isolated hepatocytes, it was gradually converted into aziridinylnaphthoquinone. Under the same reaction conditions, 4-chlorobutylaminonaphthoquinone also gave the cyclization products, pyrrolidinylnaphthoquinone. Cellular GSH decreased in both reactions.     

Tissue fractionation in rat brain, kidney and liver. I. Intracellular localization of a 5-methyltetrahydrofolic acid requiring enzyme.

The intracellular distribution of N-methyl-transferase requiring 5-methyl-tetrahydrofolic acid (5 MT-NMT) was studied in brain, kidney and liver of rats. Among these different tissues, the kidney displayed the highest enzyme activity, more than 20 times the activity detected in the brain. As the striatum and, to a lesser extent the hypothalamus, were found to contain slightly higher 5 MT-NMT than other cerebral regions, they were also selected for the study of the subcellular localization. Tissue fractionation was performed by differential centrifugation yielding five different fractions which were analyzed for their enzymatic content not only of 5 MT-NMT but also of marker enzymes, such as cytochrome oxidase, acid phosphatase and inosine diphosphatase. In all the tissues studied, 5 MT-NMT was recovered in the supernatant fraction. Therefore one may consider this enzyme to belong to the cytosol. Although a neuronal localization cannot be excluded, it is beyond doubt that the enzyme is contained in other cellular types. In the brain fractionation, the five fraction procedure seems to be very useful especially when the subcellular distribution of a given enzyme is compared to that obtained in other tissues like liver or kidney. Finally 5 MT-NMT may be considered a good marker enzyme for the supernatant fraction.     

Adenine nucleotide content of liver mitochondria increases after glucagon treatment of rats or isolated hepatocytes

Glucagon injected into rats via tail vein or incubated with isolated hepatocytes in vitro caused a 22–35% increase in the intramitochondrial ATP+ADP+AMP content. There was no statistically significant change in state 3 or uncoupled respiratory rates in these mitochondria. Similar glucagon effects were noted irrespective of the medium used or time required for isolation of mitochondria. There has been recent controversy over possible artifacts in assessing the effects of glucagon on mitochondrial function. The present results suggest that the glucagon-induced increase in the matrix adenine nucleotide content is a reproducible phenomenon with probable physiological significance.     

Pig heart mitochondrial ATPase : properties of purified and membrane-bound enzyme. Effects of flavonoids.

Soluble ATPase (F1) has been purified from pig heart mitochondria. The purified enzyme had a high specific activity and was homogeneous as checked by ultracentrifugation and electrofocusing. It could be dissociated into subunits by cold-treatment or sodium dodecyl sulfate denaturation. The molecular weights of the two major and three minor subunits could be estimated by sodium dodecyl sulfate gel electrophoresis. The native enzyme had an isoelectric point of 5.2 while the cold-denatured enzyme showed three main bands focusing at pH 5.0, 5.2, and 5.4. Kinetic properties (Vm and Km (atp) have been compared for the soluble and membrane bound ATPase in presence of various anions. Inhibitory effects of Quercetin and other flavonoids have been tested in order to get an insight on the interaction between ATPase and its natural inhibitor.     

Inhibition of the oxidation of acetaldehyde and formaldehyde by hepatocytes and mitochondria by crotonaldehyde

Crotonaldehyde was oxidized by disrupted rat liver mitochondrial fractions or by intact mitochondria at rates that were only 10 to 15% that of acetaldehyde. Although a poor substrate for oxidation, crotonaldehyde is an effective inhibitor of the oxidation of acetaldehyde by mitochondrial aldehyde dehydrogenase, by intact mitochondria, and by isolated hepatocytes. Inhibition by crotonaldehyde was competitive with respect to acetaldehyde, and the Ki for crotonaldehyde was about 5 to 20 microM. Crotonaldehyde had no effect on the oxidation of glutamate or succinate. Very low levels of acetaldehyde were detected during the metabolism of ethanol. Crotonaldehyde increased the accumulation of acetaldehyde more than 10-fold, indicating that crotonaldehyde, besides inhibiting the oxidation of added acetaldehyde, also inhibited the oxidation of acetaldehyde generated by the metabolism of ethanol. Formaldehyde was a substrate for the low-Km mitochondrial aldehyde dehydrogenase, as well as for a cytosolic, glutathione-dependent formaldehyde dehydrogenase. Crotonaldehyde was a potent inhibitor of mitochondrial oxidation of formaldehyde, but had no effect on the activity of formaldehyde dehydrogenase. In hepatocytes, crotonaldehyde produced about 30 to 40% inhibition of formaldehyde oxidation, which was similar to the inhibition produced by cyanamide. This suggested that part of the formaldehyde oxidation occurred via the mitochondrial aldehyde dehydrogenase, and part via formaldehyde dehydrogenase. The fact that inhibition by crotonaldehyde is competitive may be of value since other commonly used inhibitors of aldehyde dehydrogenase are irreversible inhibitors of the enzyme.     

Hepatic heme metabolism in selenium-deficient rats: effect of phenobarbital.

Hepatic heme metabolism was examined in selenium (Se)-deficient and Se-adequate (control) rats. Administration of phenobarbital stimulated heme synthesis in the liver in both Se-deficient and Se-adequate rats. In contrast to these results, phenobarbital increased microsomal heme oxygenase (MHO) activity six- to eightfold in Se-deficient but not control rats. These data suggest that the previously reported abnormalities of cytochrome P-450 induction in Se-deficient rats are related to increased degradation of hepatic heme.     

A three-step procedure for the isolation of peptides derived from adenine nucleotide binding sites of enzymes labeled with p-fluorosulfonyl [14C]benzoyl-5'-adenosine.

A simple three-step procedure is described for the purification of a labeled peptide from a tryptic digest of the β-subunit of the F₁-ATPase after the enzyme had been inactivated with p-fluorosulfonyl-[¹⁴C]benzoyl-5′-adenosine. The procedure involves: (1) anion-exchange chromatography of a tryptic digest of the labeled β-subunit on diethylaminoethyl-Sephadex; (2) treatment of the peptides in the radioactive peak from the first step with 0.1m NaOH under conditions in which the ester bond in the label is hydrolyzed; and (3) anion-exchange chromatography of the treated peptides under conditions identical to those of the first step after removal of the NaOH by gel filtration. Cleavage of the ester bond in the second step releases adenosine and specifically introduces an additional negative charge onto the labeled peptide. Thus, it is resolved from the peptides that contaminate it in the third step.     

Nucleotide sequence of polypyrimidines from cloned mouse DNA as determined by base-specific blockage of exonuclease action

Cloned fragments of mouse DNA have been screened for the presence of long polypyrimidine/polypurine segments. The polypyrimidine portion of one such segment (about 200 nucleotides in length) has been isolated by acidic depurination of the entire cloned fragment and plasmid vector followed by selective precipitation and 5'-32P labeling. This polypyrimidine has been used to demonstrate a new procedure for sequencing. Covalent modification of thymine with a water-soluble carbodiimide, or cytosine with glutaric anhydride, at low levels blocked the action of snake venom exonuclease. After deblocking, separation of the products of digestion by polyacrylamide gel electrophoresis yields a sequence ladder which can be used to determine the position of C and T residues as in other sequencing methods. A sequence of 72 residues adjacent to the 5' end has been established, consisting principally of the repeating tetranucleotide (CCTT)n. A low ratio of endonuclease to exonuclease is essential for application of this method to sequences of this size. Accordingly, a very sensitive modification of a fluorometric endonuclease assay was developed and used to optimize pH and Mg2+ conditions to favor exonuclease activity over the accompanying endonuclease activity. The results clearly indicate that long polypyrimidine tracts can be efficiently prepared and their sequences determined with this method using commercially available exonuclease preparations without additional purification.     

Ionic dependence and transmission of epidermal action potentials in a newt embryo

The ionic dependency and transmission of epidermal action potentials have been examined from tailbud to hatching stages of newt embryos. Previously we have reported that the epidermal action potential is composed of a fast- and slow-action component; only the slow-action component, however, is transmitted to other cells. We address in this report the mechanism by which these responses are mediated. The slow-action potential is not produced in Na⁺-free saline, tricaine saline, or following the application of TTX, and thus appears to be Na⁺ dependent. The fast-action potential on the other hand is blocked by application of Co²⁺ and verapamil saline and thus appears to be Ca²⁺ dependent. The slow-action potentials appear to be chemically transmitted since they are transmitted even to those cells which are electrically uncoupled at low intracellular pH (NaHCO₃ + HCl, pH 6.2). Furthermore 1 μM curare and atropine are inhibitory to transmission of the slow potential. Epidermal cells of the newt embryo are sensitive to acetylcholine (ACh) applied by hydrostatic ejection through a micropipet. The latter observation further suggests that propagation of the slow-action potential is, in part, a chemical event.