Insulin and glucagon's reciprocal mediation of a dual regulation mechanism for pyruvate kinase

The addition of glucagon to hepatocytes in primary culture produced a rapid and sustained increase in the K_m ($\text{1.27 m}\text{M}$ phosphoenol pyruvate) of pyruvate kinase. The low K_m ($\text{0.4 m}\text{M}$) form of the enzyme was seen when cells were retreated with insulin, demonstrating a short-term regulation mechanism. Injections of insulin, glucagon or glucagon followed by insulin demonstrated that a similar mechanism occurs $\text{in}\text{vivo}$. Results from longer times after injection indicated that another mechanism occurs when altered activity was the result of changes in V_max and not K_m. Thus, a dual mechanism for regulation of pyruvate kinase occurs. A rapid responding system functions by modification of the enzyme, while a long-term system functions by altering the rate of synthesis, thus changing the amount of enzyme present.     

Kinetic properties of pyruvate kinase isolated from rat hepatic tumours.

The results demonstrate the existence of L and M forms of pyruvate kinase in rat hepatomas. Tumours were induced by feeding N-Nitrosodiethylamine. The kinetic properties of the L-type tumour enzyme was markedly different from the L-enzyme form found in normal liver. The L-form of tumour enzyme was purified by DEAE cellulose-Sephadex G200 chromatography (Sp. activity 41 units/mg). $\text{MgADP}{}^{\mathrm{?}}\text{ADP}{}^{\mathrm{2?}}$ of $\text{20}\text{1}$ gave optimum activity for both the intrinsic and F1,6di-P stimulated reactions. ATP did not inhibit the enzyme. Alanine (2.5 nM) caused 60% inhibition at low PEP concentrations (0.25 mM). The homotropic effector (PEP) exhibited a complex allosteric pattern and saturation kinetics were not observed for either the intrinsic or F1,6di-P stimulated reactions with PEP concentrations as high as 10 mM.     

Magnetic interaction of nickel(III) and the iron-sulphur cluster in hydrogenase from Chromatium vinosum

Upon partial reduction of hydrogenase from Chromatium vinosum with ascorbate plus phenazine methosulphate, EPR signals due to Ni(III) and a [3Fe-xS] cluster appear simultaneously and with equal intensities. Since the intact enzyme shows no $\text{S =}\text{1}\text{2}$ signals, it is concluded that Ni(III) and a [4Fe-4S]³⁺ cluster interact magnetically in such a way as to prevent the detection of the two paramagnets as individual $\text{S =}\text{1}\text{2}$ systems. This interaction is thought to be the origin of a signal in which Fe is involved and which is not due to an $\text{S =}\text{1}\text{2}$ system (Albracht, S.P.J., Albrecht-Ellmer, K.J., Schmedding, D.J.M. and Slater, E.C. (1982) Biochim. Biophys. Acta 681, 330–334). A variable fraction of the enzyme preparation shows signals due to Ni(III) and a [3Fe-xS] cluster with equal intensities without any further treatment. These are thought to be derived from irreversibly inactivated enzyme molecules. The enzyme contains no selenium.     

Cyclic AMP stimulated phosphorylation of liver pyruvate kinase in hepatocytes.

The addition of glucagon (10^?6 M) to an incubation mixture containing ³²Pi and hepatocytes isolated from livers of rats fed $\text{ad libitum}$ results in both a 3-fold increased incorporation of ³²P into L-type pyruvate kinase and a decreased catalytic activity. The ³²P incorporated into pyruvate kinase was covalently bound to the enzyme as evidenced by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In addition, exogenous cyclic AMP (10^?3 M) stimulated the phosphorylation and the suppression of catalytic activity to a similar extent. On the other hand, insulin (10^?7 M) had essentially no effect on the incorporation of ³²P into pyruvate kinase or on its catalytic activity under the conditions used in this study. These results suggest that phosphorylation of pyruvate kinase $\text{invivo}$ is stimulated by glucagon via cyclic AMP and cyclic AMP-dependent protein kinase and that the activity of the enzyme is, at least in part, regulated by a phosphorylation-dephosphorylation mechanism.     

Evidence for existence and a tentative identification of coenzyme in yeast thiamine pyrophosphokinase.

Thiamine pyrophosphokinase (E.C. 2.7.6.2.) from $\text{Saccharomyces cerevisiae}$ was found to require the presence of a non-protein, non-metal compound for its activity. $\text{myo}$-Inositol was found capable of stimulating the kinase activity in the presumably resolved but otherwise crude sample of the enzyme. The hexytol was also found capable of inducing the enzyme in growing yeast cells. The cultured yeast cells, in which the kinase had been induced, were used as source of the enzyme for its purification. The compound that had been left adsorbed to the final column of DEAE-Sephadex was proved to have a coenzyme activity towards the enzyme and tentatively identified with $\text{myo}$-inositol 1-pyrophosphate. A sample of synthetic $\text{myo}$-inositol 1-pyrophosphate was made and its coenzyme activity was observed.     

Modulation of 25-hydroxyvitamin D3-24-hydroxylase by aminophylline: a cytochrome P-450 monooxygenase system.

The enzymatic activity of human erythrocyte pyruvate kinase was found to decrease on incubation of the purified enzyme with red blood cell ghosts, ATP and cAMP. If $\text{[}{}^{32}\text{P]γATP}$ was used radioactivity was found associated with the protein after gel electrophoresis. Various effectors protected the enzyme against phosphorylation. Treatment of the modified enzyme with a protein phosphatase restored enzymatic activity and also caused the loss of the radioactive label. Modification of the pyruvate kinase in this way altered the affinity of the enzyme for one of its substrates (phosphoenolpyruvate), but the binding of the other substrate (ADP) was unaffected.     

Detection of two forms of glutamine synthetase in Bacillus brevis (A. G. 4)

A laboratory isolate of $\text{Bacillus}\text{brevis}$ could grow and sporulate on an amino acid, viz., alanine or glutamate or aspartate as single source of carbon and nitrogen. It failed to sporulate if the amino acid was replaced by the corresponding keto acid and ammonium sulphate in the medium, although, normal growth was observed. One of the key enzymes in nitrogen assimilation, the glutamine synthetase, has been purified by DE-52 and affinity column chromatography from both alanine and pyruvate grown cells. The kinetic and other properties of both of these enzymes were studied. The enzyme isolated from alanine grown cells differed significantly from that isolated from pyruvate grown cells (viz.,pH optima, response to Mg⁺⁺ and other effectors). A possible role of glutamine synthetase in the initiation of bacterial sporulation is discussed.     

Phosphorylation of calf thymus H1 histone by calcium-activated,phospholipid-dependent protein kinase

Ca²⁺-activated, phospholipid-dependent protein kinase recently found in mammalian tissues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) $\text{J.}$$\text{Biol.}$$\text{Chem.}$$\text{254}$, 3692–3695) is able to phosphorylate five fractions of calf thymus histone. H1 histone serves as a preferential substrate, and approximately two moles of phosphate are incorporated into every mole of this histone. Analysis on the N-bromosuccinimide-bisected fragments of this radioactive histone has revealed that the enzyme phosphorylates preferentially seryl and threonyl residues located in the carboxyl-terminal half of this histone molecule.     

Adrenergic regulation of pyruvate kinase and gluconeogenesis in hepatocytes from phosphorylase kinase-deficient (gsdgsd) rats

Hepatocytes were prepared from a strain of rats deficient in hepatic phosphorylase $\text{b}$ kinase and were used to assess the role of this enzyme in the adrenergic regulation of pyruvate kinase and gluconeogenesis. Epinephrine (10 μM) stimulated glucose output and gluconeogenesis from 1.8 mM lactate but did not significantly affect the concentration of hepatocyte glycogen. In addition epinephrine treatment led to an inhibition of pyruvate kinase. The stimulation of gluconeogenesis and the inhibition of pyruvate kinase by epinephrine were blocked by both α- and β-antagonists: similar effects with epinephrine were observed in cells from control animals. It is concluded that mechanisms for the adrenergic regulation of pyruvate kinase and gluconeogenesis are similar in hepatocytes from both phosphorylase kinase-deficient and normal rats.     

Observation of carbon labelling in cell metabolites using proton spin echo NMR

A heteronuclear spin echo experiment is described which allows detection of both ¹²C and ¹³C labelled species in a ¹H spectrum. Fractional labelling of ¹³C labelled metabolites can thus be observed. The method is illustrated with a study of the exchange of ¹³C label between the methyl groups of alanine and pyruvate catalysed by the enzyme alanine aminotransferase (E.C. 2.6.1.2) both in the human erythrocyte and $\text{in}\text{,}\text{vitro}$.     

The regulation of pyruvate dehydrogenase by fatty acids in isolated rabbit heart mitochondria.

The rate of pyruvate oxidation by isolated rabbit heart mitochondria was inhibited by fatty acylcarnitine derivatives. The extent of inhibition by pyruvate oxidation in State 3 was greatest with palmitylcarnitine and only a minimal inhibition was observed with acetylcarnitine, while octanoylcarnitine or octanoate caused an intermediate extent of inhibition. Analyses of the intramitochondrial $\text{ATP}\text{ADP}$ and $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratios under the different conditions of incubation indicated that it is unlikely that changes in either or both of these parameters were the primary negative effectors of the rate of pyruvate oxidation. A positive correlation between the decrease in the rate of pyruvate oxidation and the decrease in the level of free CoASH in the mitochondria was observed. Extraction and assay of the pyruvate dehydrogenase from rabbit heart mitochondria during the time course of the fatty acid-mediated inhibition of pyruvate oxidation indicated that pyruvate dehydrogenase was strongly inactivated when palmitylcarnitine was the fatty acid, while incubation with octanoate and acetylcarnitine resulted in less extensive inactivation of pyruvate dehydrogenase. Measurement of the effects of NADH, NAD⁺, acetyl-CoA, and CoASH on the inactivation of pyruvate dehydrogenase extracted from rabbit heart mitochondria indicated that NADH and acetyl-CoA activated the pyruvate dehydrogenasee kinase while CoASH strongly inhibited the kinase and NAD⁺ was without effect. In addition, palmityl-CoA and octanoyl-CoA had little, if any, effect on the pyruvate dehydrogenase kinase activity. It was observed that palmityl-CoA but not octanoyl-CoA strongly inhibited the activity of the extracted pyruvate dehydrogenase. Hence, it is concluded that (a) decreased mitochondrial CoASH levels, which essentially remove a potent inhibitor of the pyruvate dehydrogenase kinase, (b) possibly a diminished free CoASH supply, which may be utilized as a substrate for the active complex, and (c) direct inhibitory effects of palmityl-CoA on the active form of the pyruvate dehydrogenase complex combine to make palmitylcarnitine a much more potent inhibitor of mitochondrial pyruvate oxidation than shorter chain length acylcarnitine derivatives.     

Letter: Electron microscopy of pyruvate kinase crystals from Saccharomyces carlsbergensis

Crystals of pyruvate kinase have been analysed by electron microscopy, optical diffraction and filtering, and the following parameters were obtained: $\text{2a = 93}\text{A}\text{?}$, $\text{2b = 126}\text{A}\text{?}$, $\text{l = 35·2}\text{A}\text{?}$. A comparison of these data with the parameters obtained from small-angle X-ray scattering measurements indicates that two molecules of the tetrameric enzyme are arranged in one packing unit.     

Unmasking of an essential thiol during function of the membrane-bound enzyme II of the phosphoenolpyruvate β-glucoside phosphotransferase system of Escherichia coli

β-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents. This inactivation is strongly enhanced by the presence of transported substrates. In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme II^bgl appeared as the target of N-ethylmaleimide inactivation. The same feature was found in the case of methyl-α-d-glucoside uptake via enzyme II^glc.It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-α-d-glucoside only sensitizes enzyme II^bglc and $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ only sensitizes enzyme II^bgl towards N-ethylmaleimide inactivation.The inactivation of enzyme II^bgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis. In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ phosphorylation. Both results suggest that the inactivator resistent form of enzyme II^bgl is an energized form of the enzyme.     

A cyclic AMP dependent protein kinase in Dictyostelium discoideum

A cyclic AMP-dependent protein kinase was found to appear during the time course of development of $\text{Dictyostelium}\text{discoideum}$. No cyclic AMP dependency was observed at any stage of development in crude 110,000 X G soluble extracts. After partial purification, however, extracts from post-aggregation stages contained enzyme that was activated up to 6-fold by cyclic AMP, whereas protein kinase from earlier stages was not affected by cyclic AMP. Likewise, cyclic AMP binding activity increased from the aggregation to the slug stage of development. Approximately one-half of the total cyclic AMP binding activity co-purified with the cyclic AMP dependent protein kinase. The enzyme from $\text{Dictyostelium}$ showed similarities to mammalian protein kinases with respect to its kinetic properties but differed in its behavior on ion-exchange chromatography.     

Glucose transport and metabolism in cultured human skin fibroblasts

Human skin fibroblast cultures, seeded at $\text{10}{}^5\text{cells}\text{5}\text{cm}$ plate and allowed to grow to confluence at approx. $\text{10}{}^6\text{cells}\text{5}\text{cm}$ plate, utilized a glycolytic mode of metabolism where the ratio of glucose utilized to lactate produced wa 0.62±0.05 (Zielke, R.H., Ozand, P.T., Tyldon, J.I., Sevdalian, D.A. and Cornblath, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4110–4114) (mean±S.E.). When the glucose in the medium was exhausted, the lactate produced during the highly glycolytic phase was then reutilized. In monolayer cultures that had been washed with phosphate-buffered saline, rates of glucose utilization were measured at 0.25 and 2 mM glucose by monitoring the appearance of ³H₂O from [5-³H]glucose. Rate of utilization for each concentration of glucose decreased markedly as the cultures became more confluent. This decrease also correlated with a reduced ability to transport glucose as measured by 2-deoxy-[³H]glucose uptake in washed monolayer cultures. In washed confluent culture of fibroblasts, glucose utilization was markedly decreased by the presence of pyruvate and lactate but not by glutamine. The respiratory inhibitors, rotenone and antimycin, did not increase the rate of glucose utilization except when added in combination with pyruvate. We conclude that cultured skin fibroblasts posses a highly glycolytic mode of metabolism but that this mode can become more oxidative in the presence of sufficient quantities of pyruvate and lactate.     

Cooperative lipid activation of (Na+ + K+)-ATPase as a consequence of non-cooperative lipid-protein interactions

Lipid activation data for $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (Ottolenghi, P. (1979) Eur. J. Biochem. 99, 113–131) have been subjected to a regression and fitting analysis based on a recent kinetic model (Sandermann, H. (1982) Eur. J. Biochem, 127, 123–128). The observed kinetic cooperativity could be generated from strictly non-cooperative binding events involving the known number of 30 boundary lipid-binding sites per ATPase monomer. Apparent lipid dissociation equilibrium constants of between 0.3 and 5 μM were obtained, enzyme activity being associated only with the fully lipid-substituted enzyme and enzyme-lipid complexes with less than six unoccupied lipid-binding sites. The enzyme appeared to operate close to a maximum of cooperativity.     

On the defect of a dCMP hydroxymethylase mutant of bacteriophage T4 showing enzyme activity in extracts

Infection by $\text{L}\text{13}$, a temperature-sensitive mutant of gene 42 of phage T4, the structural gene for dCMP hydroxymethylase, previously was shown not to form T4 DNA at nonpermissive temperatures. Yet the enzyme activity was found in extracts. Since inactivation of the enzyme was not reversible, we have examined acid-soluble extracts of cells infected at nonpermissive temperature by $\text{tsL}\text{13}$ for 5-hydroxymethyldCMP in order to determine whether the enzyme functioned $\text{in vivo}$. A double mutant of $\text{tsL13}$ and $\text{amB}\text{24}$ (5-hydroxymethyldCMP kinase) did not form the nucleotide at nonpermissive temperature, but the control, $\text{amB}\text{24}$, formed large quantities. From these results and previous temperature-shift studies it is suggested that the enzyme is normally activated to function $\text{in vivo}$ between 5 and 8 minutes after infection.