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H A Krebs

Publications and source records attributed to H A Krebs.

At least 19 recordsLinked to original sources
PubMed1990

Determination of a new anti-allergenic agent, 1-[4-[3-[4-[bis-(4-fluorophenyl)hydroxymethyl]-1-piperidinyl] propoxy]-3-methoxyphenyl]ethanone, and its active acidic metabolite in plasma by high-performance liquid chromatography.

High-performance liquid chromatographic (HPLC) methods were developed for the analysis of two compounds in a series of new antiallergenic agents, 1-[4-[3-[4-[bis(4-fluorophenyl)hydroxymethyl]-1-piperidinyl] propoxy]-3-methoxyphenyl]ethanone and its active acidic metabolite in plasma. The methods utilize ultraviolet or fluorescence detection, liquid-liquid extraction or solid-phase extraction and reversed-phase HPLC. The drugs were quantitated in samples from bioavailability studies performed in dogs. Calibrations were in the ng/ml concentration range for both compounds in plasma.

Animals↗
PubMed1983

Studies on the mechanism of the antifungal action of benzoate.

A method is described for the determination of the pH of intracellular water based on the distribution of [14C]benzoate (0.01 mM) between intra- and extra-cellular water. Benzoate at higher concentrations (2-10mM) enters the yeast cell in the undissociated form, and its neutralization within the cell can cause a shift of the pH of the intracellular water by more than 1 pH unit. Benzoate causes an accumulation of the two hexose monophosphates of yeast glucose fermentation and a decrease in intermediates beyond phosphofructokinase, suggesting inhibition at this stage. Benzoate also causes a concomitant fall in [ATP]. Phosphofructokinase is inhibited to a greater extent than hexokinase at acid pH. There is a relationship between intracellular pH, phosphofructokinase inhibition and CO2 production, suggesting that the antifungal action of benzoate is caused by an accumulation of benzoate at low external pH, which lowers the intracellular pH into the range where phosphofructokinase is sensitive. The subsequent inhibition of glycolysis causes a fall in [ATP] and thus restricts growth.

Adenine Nucleotides↗
PubMed1980

Development of narcotic drug metabolizing enzymes in the newborn rat.

Liver 9000 x g supernatant of rats from 1 day of age to adult was used as the enzyme source to study glucuronidation of morphine, O-demethylation of norcodeine and N-demtheylation and ester hydrolysis of meperidine. Uridine diphosphate glucuronyl transferase activity in 1-day-old rats was about one-fifth of that of the adult, increased to the adult level between 3 and 7 days of age and exceeded that of the adult when the rats were 28 days old. The activities of O- and N-demethylase were first observed in the liver of 3- and 7-day-old rats, respectively, and increased to the adult level at 35 days of age. Esterase activity was first observed in 14-day-old rats and increased rapidly to the adult level at 35 days of age. Drug metabolizing capacity for newborn rats, in terms of percentage of that of adults (calculated based on the specific enzyme activity, protein content of the liver and liver weight), was quite low for the first 2 weeks of life, ranging from undetectable to less than 5% of the adult level. The capacity increased during the next 3 weeks, reaching about 50 to 75% of adult activities by the 35th day.

Aging↗
PubMed1979

Cytosolic phosphorylation potential.

The tissue contents of the reactants of the myokinase (EC 2.7.4.3) and the combined glyceraldehyde-3-phophate dehydrogenase (EC 1.1.1.29)-3-phosphoglycerate kinase (EC 2.7.2.3) reactions were measured in rapidly inactivated samples of human blood and rat brain, muscle, and liver. The tissue contents of the reactants of the creatine kinase (EC 2.7.3.2) reaction were measured in rat brain and muscle. In vitro the value of the expression: KG+G = [sigma3PG] . [sigmaATP] . [sigmalactate] KLDH = [sigmaHAP]/22] . [sigmaADP][sigmaPi] . [sigmaRUVATE] (1) was found to be 0.725 x 10(7) M-1 at I = 0.25, T = 38 degrees C, and free [Mg2+] = 0.15 mM and the value measured in vivo in red cell was 0.699 x 10(7) M-1. The value of the expression KMYK = ([sigma ATP] [sigma AMP]/[ADP2]) measured under the above conditions and at pH 7.2 was found to be 0.744 while the value found in red cell was 0.784 +/- 0.037. These reactions, therefore, appear to be in a state of near-equilibrium in the red cell and the measured tissue contents of ATP and ADP, which are common reactants in both reactions, approximate closely the activity of these reactants in vivo. In brain and muscle, the value of KG + G/KLDH calculated from the measured tissue contents of the reactants was a factor of 20 or more lower than that expected at equilibrium as was the measured value of the expression: KCK = [sigma ATP] [sigma creatine] divided by [sigma ADP] [sigma creatine-P] [H+] (2) Substitution of calculated free [sigma ADP] values in the expression of KG + G/KLDH gave values of 0.83 +/- 0.19 x 10(7) M-1 for brain and muscle, respectively, which agreed well with the value of 1.65 x 10(7) M-1 measured in vitro at I = 0.25, free [Mg2+] = 1 mM, T = 38 degrees C. This agreement between two highly active enzyme systems in the same compartment is taken as evidence of the existence of near-equilibrium in both these systems and suggests that free cytosolic [sigma ADP] is probably 20-fold lower than measured cell ADP content in mitochondrial-containing tissues.

Adenine Nucleotides↗
PubMed1979

Loss of cell constituents from hepatocytes on centrifugation.

In studies of the metabolism of isolated hepatocytes, it is often necessary to measure the concentrations of cell constituents both in cells and medium. When hepatocytes are separated in the special tubes of Hems, Lund & Krebs (1975) (Biochem. J. 150, 47--50), they lose much glucose, urea and Na+, whereas there is no loss of K+, glutamate, aspartate and adenine nucleotides. Cell water is also lost, as measured by the distribution of 3H2O. This loss is mainly due to an exchange of cell water with the aqueous solution in the stems of the tubes through which the cells pass on centrifugation. In general, substances are lost only when the intracellular concentration is equal to, or lower than, the extracellular concentration. Probably solutes are lost because they travel with the water unidirectionally out of the cell. A loss of solute does not occur when the cells are centrifuged in conical tubes with a layer of silicone oil between the cell suspension and the deproteinizing layer. The reasons for the loss occurring in the special separation tubes are discussed.

Animals↗
PubMed1979

Isolation and metabolic characteristics of rat and chicken enterocytes.

1. The recent recognition of the metabolic, as opposed to absorptive, functions of the small intestine prompted efforts the improve the preparation of metabolically competent columnar absorptive cells ('enterocytes') and to study their metabolic properties. 2. With this preparation, linear rates of O2 consumption are obtained for 40 min at 37 degrees C that are more than 50% higher than rates reported by other authors. 3. Among added substrates, glucose, glutamine and glutamate are the preferred fuels of respiration. The main nitrogenous products of glutamine metabolism are NH3, alanine and glutamate. Glutamine carbon was not detectable in citrulline or proline, in contrast with the findings of Windmueller & Spaeth [(1974) J. Biol. Chem. 249, 5070-5079] in the vascularly perfused small intestine. 4. The rates of O2 uptake in the presence of glutamine or glutamate are sufficient to account for the formation of the carbon skeleton of alanine from the amino acid substrate, i.e. the ratio of O2 used/alanine formed is greater than 1.5. 5. Added ADP and ATP are rapidly degraded to AMP and IMP to a large extent by release of hydrolytic enzymes from the enterocytes into the medium. 6. Chicken enterocytes isolated by the same method are more stable; linear rates of O2 uptake are maintained for 60-70 min.

Adenine Nucleotides↗
PubMed1979

Isolation and identification of morphine n-oxide alpha- and beta-dihydromorphines, beta- or gamma-isomorphine, and hydroxylated morphine as morphine metabolites in several mammalian species.

New morphine metabolites in the urine of guinea pigs, rats, rabbits, cats, monkeys, and humans were isolated with column chromatography, solvent extraction, and TLC and identified with TLC, GLC, and GLC-mass spectrometry. In addition to the known morphine metabolites, morphine N-oxide was isolated from the urine of guinea pigs, and alpha- and beta-dihydromorphines were isolated or detected in the urine of guinea pigs, rats, and rabbits. Monohydroxymorphine was identified tentatively in the urine of guinea pigs, rats, rabbits, and cats. Dihydroxymorphine was identified tentatively in the urine of guinea pigs, rats, and possibly, rabbits. Finally, beta- or gamma-isomorphine was identified tentatively in the urine of guinea pigs. The newly described morphine metabolites may be involved in some long lasting pharmacological effects of morphine.

Animals↗
PubMed1978

Sources of ammonia for mammalian urea synthesis.

The initial rate of incorporation of [15N]alanine into the 6-amino group of the adenine nucleotides in rat hepatocytes was about one-eighteenth of the rate of incorporation into urea. Thus the purine nucleotide cycle cannot provide most of the ammonia needed in urea synthesis for the carbamoyl phosphate synthase reaction (EC 2.7.2.5). On the other hand, contrary to the view expressed by McGivan & Chappell [(1975) FEBS Lett. 52, 1--7], the experiments support the view that hepatic glutamate dehydrogenase can supply the required ammonia.

Adenine Nucleotides↗
PubMed1978

Phosphorylation of adenosine monophosphate in the mitochondrial matrix.

The origin of the GTP needed for th phosphorylation of AMP in the mitochondrial matrix was investigated. When short-chain fatty acids are metabolized by hepatocytes, AMP is readily formed within the matrix by the butyryl-CoA ligase (AMP-forming) reaction (EC 6.2.1.2). The rate of matrix AMP formation in rat hepatocytes was calculated from the rate of ketone-body formation. The rate of the reconversion of matrix AMP into ADP by GTP-AMP transphosphorylase is limited by the rate of supply of GTP. GTP can be formed either by succinic thiokinase (EC 6.2.1.4) or by nucleoside diphosphokinase (EC 2.7.4.6). The rate of the succinic thiokinase reaction was calculated from turnover of the tricarboxylic acid cycle and this was calculated from the rate of O2 consumption and ketone-body formation. The results show that nucleoside diphosphokinase can make a major contribution (up to 80%) to the supply of GTP under the test conditions.

Adenosine Monophosphate↗
PubMed1978

Rate-limiting factors in urate synthesis and gluconeogenesis in avian liver.

1. Urate synthesis and other metabolic characteristics of isolated chicken hepatocytes were studied. 2. The distinction is made between immediate precursors of the purine ring (glycine, glutamine, aspartate, formyltetrahydrofolate, bicarbonate) and ultimate precursors from which the immediate precursors are formed in the liver. 3. In hepatocytes from well-fed chickens the rate of urate synthesis was not greatly increased by the addition of amino acids or NH(4)Cl, but in hepatocytes from 72h-starved chickens the rate was much increased when alanine or asparagine was added as the only substrate. Other amino acids, when added alone, did not affect the rate. The exceptional effect of alanine and asparagine is due to the ready formation of the immediate precursors. 4. Conditions are described under which glutamine, serine, glycine plus formate, ribose and glucose increased the rate of urate synthesis. 5. At 1mm-NH(4)Cl (a concentration not much higher than that of blood plasma) the rate of urate synthesis in the presence of lactate was increased, but higher concentrations inhibited urate synthesis in the presence of lactate or alanine; with alanine even 1mm-NH(4)Cl was inhibitory. 6. Glucose synthesis from lactate, alanine or dihydroxyacetone was also inhibited by 1mm-NH(4)Cl. 7. NH(4)Cl inhibition of urate and glucose synthesis was paralleled by an increased rate of glutamine synthesis. Thus in the presence of NH(4)Cl the gluconeogenic precursors are diverted from the pathway of gluconeogenesis to that of glutamate and glutamine synthesis. This implies that the synthesis of these amino acids is the primary process in the detoxication of ammonia in the avian liver. 8. Urate synthesis, like urea synthesis, can be looked on as a cyclic process with either phosphoribosyl pyrophosphate or ribose acting as the carrier on which the purine ring is assembled. 9. The energy requirements of urate synthesis depend on whether phosphoribosyl pyrophosphate is regenerated from IMP by pyrophosphorylase or by phosphorylation and pyrophosphorylation of ribose. It is 6 or 9 pyrophosphate bonds of ATP respectively.

Acetates↗
PubMed1978

Is the adenine nucleotide translocator rate-limiting for oxidative phosphorylation?

1. The effects of atractyloside and carboxyatractyloside (between 5 and 40mum) on O(2) uptake, glucose synthesis, urea synthesis, the adenine nucleotide content and the intracellular K(+) concentration were measured in isolated hepatocytes. 2. Urea synthesis was much less inhibited than glucose synthesis by both atractylosides. Measurements of intermediary metabolites of carbohydrate metabolism in freeze-clamped liver after injection of atractyloside into rats indicate that inhibition of gluconeogenesis is due to interference at the cytosolic reactions requiring ATP (phosphoenolpyruvate carboxykinase and 3-phosphoglycerate kinase). 3. The decrease in [ATP]/[ADP]x[P(i)] after addition of atractyloside or carboxyatractyloside was restricted to the cytosol. 4. Dihydroxyacetone can be converted either into glucose with the consumption of 2mol of ATP (per mol of glucose) or into lactate with the production of 2mol of ATP. In the presence of high concentrations of atractyloside and carboxyatractyloside more ATP was produced than was used for the synthesis of glucose from dihydroxyacetone, probably for the maintenance of intracellular [K(+)]. 5. When the rates of respiration were altered by changing substrates, the degrees of inhibition of respiration and translocation by a given concentration of the atractylosides were the same, whereas at a given concentration of HCN the degree of inhibition was high at higher initial rates, and low at lower initial rates. 6. Inhibition of a complex series of reactions by atractyloside does not necessarily indicate that the translocator is a rate-limiting step in that sequence as Th. P. M. Akerboom, H. Bookelman & J. M. Tager [(1977) FEBS. Lett.74, 50-54] assume. This point is discussed.

Adenine Nucleotides↗
PubMed1978

Maintenance of glutathione content is isolated hepatocyctes.

1. During the standard procedure for the preparation of rat hepatocytes, about half of the cellular GSH (reduced glutathione) is lost. 2. This loss is prevented by the addition of 0.1 mM-EGTA (but no EDTA) to the perfusion medium. 3. On incubation with and without EGTA, isolated hepatocytes prepared in the presence of EGTA lose GSH. This loss is prevented by near-physiological concentrations of methionine or homocysteine, but not of cysteine. 4. Cysteine, at concentrations above 0.2 mM, causes a loss of GSH probably by non-enzymic formation of a mixed disulphide. 5. Serine together with methionine or homocystein increases GSH above the value in cells from starved rats in vivo. This is taken to suggest that cystathionine may be a cysteine donor in the synthesis of gamma-glutamylcysteine, the precursor of GSH.

Animals↗
PubMed1978

Reaction of formiminoglutamate with liver glutamate dehydrogenase.

1. Kinetic aspects of the reaction between crystalline bovine liver glutamate dehydrogenase and formiminoglutamate were investigated to establish the conditions under which the latter may interfere with the assay of glutamate by using glutamate dehydrogenase and to explain why formiminoglutamate accumulates in vivo after histidine loading, although it can react with glutamate dehydrogenase. The Km and Vmax. values were compared with those of the enzyme reacting with glutamate. At pH 7.4 Km for formiminoglutamate was much higher and Vmax. much lower than the values for glutamate. 2. The equilibrium constant at pH 7.0 was 0.017 micrometer with formiminoglutamate, i.e. about one two-hundredths that with glutamate. 3. In vivo the interaction between glutamate dehydrogenase and formiminoglutamate is minimal even when the concentration of the latter in the liver is greatly raised, as in cobalamine or folate deficiency after histidine loading. 4. At pH 9.3, i.e. under the conditions for the assay of glutamate by glutamate dehydrogenase, formiminoglutamate reacts readily with the enzyme.

Animals↗
PubMed1978

Metabolism and excretion of normorphine in dogs.

Normorphine metabolism was studied in dogs given 20 mg of normorphine hydrochloride/kg sc. Free and (onjugated normorphine excreted in the urine over 144 hr represented 32 and 32%, respectively, of the administered dose. Eighty percent of the urinary excretion of the drug occurred within 9 hr. One percent of the administered dose was excreted as free normorphine in the feces. The urine was chromatographed on a column. Evaporation of the washing and methanolic effluent yielded a residue, which was purified by crystallization from aqueous methanol. Results of UV and IR studies, elemental analysis, and determination of normorphine and glucuronic acid content established the identity of this metabolite as normorphine 3-glucuronide. Dihydronormorphine and dehydronormorphine were detected with GLC-mass spectrometry as minor metabolites.

Animals↗