Michael A. Lea

Synchronous behavior pattern of key glycolytic enzymes: Glucokinase, phosphofructokinase, and pyruvate kinase

Abstract In thyroid hormone deficient rats, the hepatic concentration of FAD was significantly reduced. This reduction appeared to be due to decreased activity of hepatic flavokinase, which was also observed under these conditions. Administration of large doses of riboflavin to hypothyroid rats restored the concentration of FAD to normal, probably by producing greater saturation of the enzyme with its substrate. Riboflavin also had a slightly stimulatory effect upon flavokinase activity. Similarities between riboflavin deficiency and thyroid hormone deficiency were noted with respect to the hepatic levels of FAD, the activity of several flavoprotein enzymes, and the response to correction of the deficiency. The feature common to both conditions appears to be reduced formation of FAD from riboflavin. In hyperthyroid animals, hepatic FAD levels were not increased above normal, despite a substantial increase in flavokinase activity. The results provide further evidence for an apparent upper limit in the hepatic concentration of FAD. The increase in flavokinase produced by thyroxine, like increases in activity of certain flavoprotein enzymes, was not blocked by treatment with actinomycin-D.

Feedback Inhibition of Key Glycolytic Enzymes in Liver: Action of Free Fatty Acids

Increasing concentrations of sodium octanoate were progressively inhibitory to the activities of glucokinase, hexokinase, phosphofructokinase, and pyruvate kinase. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases were also markedly inhibited. Other enzymes of carbohydrate metabolism such as lactate dehydrogenase, phosphohexose isomerase, and fructose-1,6-diphosphatase were not decreased. Among the key glycolytic enzymes, the inhibition of pyruvate kinase by the fatty acid was most marked. The biological significance of the inhibition of the key glycolytic enzymes is interpreted as a feedback inhibitory mechanism in regulation of fatty acid biosynthesis. The mechanism may function for rapid adaptation by which the organism can use the fatty acid level as a metabolic directional switch in decreasing glycolysis and turning on gluconeogenesis.

Regulation of gluconeogenesis and glycolysis: Studies of mechanisms controlling enzyme activity

Abstract Various regulatory mechanisms functioning at the enzyme activity and enzyme biosynthetic level in carbohydrate metabolism were explored. The regulatory influences operate on receptor sites on the enzyme molecules and on the enzyme-forming systems. These enzymatic control mechanisms were examined as the basis for rapid and chronic regulation of gluconeogenesis and glycolysis by hormones and regulatory signal molecules. This work presented the effects of NADH, free fatty acids, and other compounds on strategic enzymes of liver carbohydrate metabolism. The selective action of free fatty acids was characterized in terms of inhibition of key enzymes of glycolysis, and the direct oxidative pathway, and of certain enzymes in the Krebs cycle. Since liver pyruvate kinase is pivotal in the regulation of gluconeogenesis and glycolysis, detailed attention was given to the effects of fructose 1,6-diphosphate, ATP, ADP, NADH, magnesium, free fatty acids, and other regulatory molecules on this enzyme.

Inhibitory effects of tea extracts and (−)-epigallocatechin gallate on DNA synthesis and proliferation of hepatoma and erythroleukemia cells

Polyphenols extracted from green or black tea with ethyl acetate were strongly inhibitory for DNA synthesis in HTC rat hepatoma cells and DS19 mouse erythroleukemia cells at concentrations of 0.1-0.2 mg/ml. There was less inhibition with a subsequent black tea fraction extracted with butanol and with the residual water-soluble fraction. Although cell proliferation was inhibited by (-)-epigallocatechin gallate and the tea extracts, there were only marginal effects on differentiation of DS19 cells as judged by hemoglobin synthesis.

The effect of mannoheptulose on the phosphorylation of glucose and the secretion of insulin by islets of Langerhans

Abstract Insulin secretion induced in vitro by glucose in pancreatic tissue of the rat is unaffected by addition of phloridzin and 3-0-methylglucose to the incubation media, suggesting that the beta-cells are freely permeable to glucose. D-glucosamine and mannoheptulose markedly inhibit the stimulant effect of glucose upon insulin secretion. The inhibitory effect of mannoheptulose is reversible and of the competitive type. Mannoheptulose also competitively inhibits the phosphorylation of glucose by isolated islet homogenates. Phosphorylation of glucose thus appears as a necessary step in the stimulation of insulin secretion by this sugar.

Induction of histone acetylation and inhibition of growth of mouse erythroleukemia cells by S-allylmercaptocysteine

Growth-inhibitory effects on DS19 mouse erythroleukemia cells were seen in the micromolar concentration range with allicin and S-allylmercaptocysteine and in the millimolar range with allyl butyrate, allyl phenyl sulfone, and S-allyl cysteine. Increased acetylation of histones was induced by incubation of cells with the allyl compounds at concentrations similar to those that resulted in the inhibition of cell proliferation. The induction of histone acetylation by S-allylmercaptocysteine was also observed in Caco-2 human colon cancer cells and T47D human breast cancer cells. In contrast to the effect on histone acetylation, there was a decrease in the incorporation of phosphate into histones when DS19 cells were incubated with 25 μM S-allylmercaptocysteine. Histone deacetylase activity was inhibited by allyl butyrate, but there was little or no effect with the allyl sulfur compounds examined in this study. A similar degree of downregulation of histone deacetylase and histone acetyltransferase was observed when DS19 cells were incubated with S-allylmercaptocysteine or allyl isothiocyanate. The induction of histone acetylation by S-allylmercaptocysteine was not blocked by a proteasome inhibitor. The mechanism by which S-allylmercaptocysteine induces histone acetylation remains to be characterized. It may be related in part to metabolism to allyl mercaptan, which is a more effective inhibitor of histone deacetylase.

The molecular correlation concept of neoplasia

Abstract We examined the intermediary metabolism and enzymology of a spectrum of liver tumors of different growth rates. The new results we presented along with those assembled from the literature indicate that a definite pattern of intermediary metabolism does exist. Special stress was laid on the identification of the biochemical parameters which correlate positively or negatively with the growth rate in the hepatomas. With this concept in mind a pattern was identified showing that the synthetic pathways of carbohydrate and lipid metabolism, and the breakdown pathways of protein and of nucleic acid metabolism decrease parallel with the increase in hepatoma growth rate. On the other hand, the catabolic pathway of carbohydrate metabolism increases progressively, whereas in protein and nucleic acid metabolism the anabolic pathways increase gradually parallel with the increase in hepatoma growth rate. An application of our concepts developed for carbohydrate metabolism interprets these findings as examples of antagonism between synthetic and breakdown pathways centering on opposing, key, one-way, rate-limiting reactions. The metabolic parameters and enzyme activities which do not correlate with the growth rate are usually identifiable as non-rate-limiting reactions or enzymes present in an excess. The metabolic pattern identified in the spectrum of liver tumors of different growth rates offers a framework for experimental testing and application of these concepts to other types of tumors and to chemotherapy.

Sequential feedback inhibition and regulation of liver carbohydrate metabolism through control of enzyme activity

Abstract 1. (1)|The free fatty acids, octanoic, lauric, myristic, palmitic, and elaidic acids, were examined for their ability to inhibit the key glycolytic enzymes. The effective inhibitory concentrations of the FFA were in the physiological range of rat or human systems. 2. (2)|Acetyl-CoA was observed to exert a dose- and preincubation time-dependent inhibitory action on hepatic glucokinase and pyruvate kinase. When acetyl-CoA was added to the reaction mixture of liver pyruvate kinase assay system a K i = 5.1 m m was found, but when it was added to the supernantant fluid for a 10-min preincubation a K i = 30 μ m was observed. The inhibition was not of a competitive type. Acetyl-CoA required only a few seconds for the interaction resulting in the inhibition of liver pyruvate kinase. 3. (3)|Acetyl-CoA also inhibited rat muscle pyruvate kinase with a K i = 2.0 × 10 −4 m , and the inhibition was of a non-competitive type. 4. (4)|Acetyl-CoA inhibited glucokinase, in a preincubation system, yielding a K i = 75 μ m . The inhibition of this enzyme was through a non-competitive mechanism. 5. (5)|There was no inhibition observed with acetyl-CoA against liver hexokinase, phosphofructokinase, lactate dehydrogenase, glucose 6-phosphatase or fructose 1,6-diphosphatase. Yeast glucose 6-phosphate dehydrogenase and rabbit muscle lactate dehydrogenase were also unaffected by acetyl-CoA. 6. (6)|FFA which is an end product of glucose catabolism acts as a feedback inhibitor by inhibiting hexokinase, glucokinase, phosphofructokinase, and pyruvate kinase. Acetyl-CoA which is an end product of the degradation of FFA reconfirms the feedback inhibition by inhibiting glucokinase and pyruvate kinase activities. This phenomenon was called sequential feedback inhibition. 7. (7)|Phosphoenolpyruvate when preincubated for 10 min with liver supernatant fraction caused a progressive inhibition of glucokinase activity with a K i = 2.1 m m . Kinetic experiments showed that the Km for glucokinase was not affected and the inhibition was not of a competitive type. Liver hexokinase was not inhibited by phosphoenolpyruvate. 8. (8)| l -alanine exerted a concentration-dependent inhibition of liver pyruvate kinase activity which was detectable at low concentrations of PEP. At the usual PEP concentration employed in the routine pyruvate kinase assay system l -alanine was not inhibitory with or without preincubation. At low concentrations of PEP l -alanine acted as a competitive inhibitor against PEP. Using a preincubation mixture it was observed that alanine protected pyruvate kinase from thermal inactivation. d -alanine and beta-alanine were not inhibitory to liver pyruvate kinase and neither these amino acids nor l -alanine inhibited muscle pyruvate kinase. 9. (9)| l -alanine exerted no effect on hepatic glucokinase, hexokinase, glucose 6-phosphatase or fructose 1,6-diphosphatase when added to the incubation mixture, or with preincubation, or at different levels of the respective substrates. 10. (10)|The factors regulating activity and biosynthesis of liver pyruvate kinase were reviewed and discussed.

Induction of histone acetylation in mouse erythroleukemia cells by some organosulfur compounds including allyl isothiocyanate

In previous studies we observed that some allyl sulfides can cause increased acetylation of histones and differentiation in DS19 mouse erythroleukemia cells. In the present work we observed increased acetylation of histones with allyl isothiocyanate and butanethiol but not with butyl sulfide or butyl disulfide. Increased acetylation of histones was established by change in electrophoretic mobility, incorporation of [3H]acetate or immunoblotting. Histone deacetylase in nuclei of DS19 cells was inhibited 74% by 0.5 mM allyl mercaptan and 43% by 0.5 mM butanethiol but was not significantly affected by 0.5 mM allyl isothiocyanate. There was some degree of reversibility in the effect of allyl isothiocyanate when the cells were incubated for 15 hr in fresh medium. The data suggested that allyl isothiocyanate may stimulate histone acetylation rather than inhibit histone deacetylation. Addition of allyl isothiocyanate, however, had very little or no additional effect on the induction of histone acetylation caused by trichostatin A. Histone acetyltransferase activity determined in cell homogenates was not increased by preincubation of cells with allyl isothiocyanate or inclusion of allyl isothiocyanate in the assay medium. It was concluded that treatment of mouse erythroleukemia cells with allyl isothiocyanate can cause increased acetylation of histones but the mechanism for this effect requires further elucidation.

Acid soluble nuclear proteins of rat liver: Differential absorbance of bound dyes and changes in neoplasia

Summary Evidence was obtained for the presence in several hepatomas of a minor histone previously reported to be absent in tumors. The concentration of this protein was inversely related to hepatoma growth rate. Comparison of light absorbance by bound dyes showed that the protein appeared to be a minor component of acid soluble nuclear proteins when stained with Amido Black or Fast Green but a major component when stained with Coomassie blue.