Inhibition of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase by glyoxylate

The overall reaction catalyzed by the pyruvate dehydrogenase complex from rat epididymal fat tissue is inhibited by glyoxylate at concentrations greater than 10 μm. The inhibition is competitive with respect to pyruvate; K_i was found to be 80 μm. Qualitatively similar results were observed using pyruvate dehydrogenase from rat liver, kidney, and heart. Glyoxylate also inhibits the pyruvate dehydrogenase phosphate phosphatase from rat epididymal fat, with the inhibition being readily detectable using 50 μm glyoxylate. These effects of glyoxylate are largely reversed by millimolar concentrations of thiols (especially cysteine) because such compounds form relatively stable adducts with glyoxylate. Presumably these inhibitions by low levels of glyoxylate had not been previously observed, because others have used high concentrations of thiols in pyruvate dehydrogenase assays. Since the inhibitory effects are seen with suspected physiological concentrations, it seems likely that glyoxylate partially controls the activity of pyruvate dehydrogenase in vivo.     

Relative importance of pyruvate dehydrogenase interconversion and feed-back inhibition in the effect of fatty acids on pyruvate oxidation by rat heart mitochondria.

Rat heart mitochondria have been incubated with concentrations of pyruvate from 50 to 500 μm as substrate in the presence or absence of an optimal concentration of palmitoylcarnitine and with respiration limited by phosphate acceptor. The rate of pyruvate utilization has been determined and compared with the amount of active (dephosphorylated) pyruvate dehydrogenase measured in samples of mitochondria taken throughout the experiments and assayed under V_max conditions. At a given pyruvate concentration, differences in pyruvate utilization as a proportion of the content of active pyruvate dehydrogenase are attributed to differences in feed-back inhibition and interpreted in terms of the ratios of mitochondrial $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ and CoA/acetyl-CoA. Under most conditions, differences in inhibition can be attributed to differences in the CoA/acetyl-CoA ratio. Feed-back inhibition is very pronounced in the presence of palmitoylcarnitine. These parameters are also examined in the presence of dichloroacetate, used to raise the steady-state levels of active pyruvate dehydrogenase in the absence of changing the pyruvate concentration, and in the presence of various ratios of carnitine/acetylcarnitine, which predominantly change the mitochondrial CoA/acetyl-CoA ratio. The latter experiment provides evidence that a decrease in mitochondrial $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio from 3.5 to 2.2 essentially balances an increase in CoA/acetyl-CoA ratio from 0.67 to 12 in modulating enzyme interconversion, whereas the change in CoA/acetyl-CoA ratio is preponderant in effecting feed-back inhibition. Increasing the free Ca²⁺ concentration of incubation media from 10^?7 to 10^?6m using CaCl₂-ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid buffers is shown to increase the steady-state level of active pyruvate dehydrogenase in intact mitochondria, in the absence of added ionophores. Moreover, this activation is reversible. Effects of Ca²⁺ ions are dependent upon the poise of the enzyme interconversion system and were only seen in these experiments in the presence of palmitoylcarnitine.     

Changes of pyruvate dehydrogenase in brain during alloxan diabetes

Alloxan diabetes causes a decrease in the active form of pyruvate dehydrogenase in rat brain. The effect is severe in the cerebellum and brain stem compared to cerebral hemispheres. The changes observed in the total form are not as significant as those found in the active (dephosphorylated) form. The effects are reversed after administration of insulin to diabetic animals. The severity of diabetes was also found to affect the activity of pyruvate dehydrogenase with inverse correlation. There was a gradual increase in the proportion of active (dephosphorylated) form with increase of time after the onset of diabetes.     

Inactivation of alcohol dehydrogenase by 3-butyn-1-ol

Horse liver and yeast alcohol dehydrogenases are rapidly inactivated during their catalysis of the oxidation of 3-butyn-1-ol. In the case of the horse liver enzyme, the inactivation is secondary to covalent modification of the apoenzyme by an electrophilic product that accumulates in the reaction solution and that can also react with water, glutathione, and other enzymes. The modified protein exhibits enhanced ultraviolet absorbance, which is not bleached upon dialysis of the denatured enzyme at pH 7.4 for 24 h. The inactivation by 3-butyn-1-ol is more rapid than that which is afforded by the related alcohols 2-propyn-1-ol and 2-propen-1-ol under identical conditions and no inactivation is seen upon incubation with 3-hydroxypropanoic nitrile plus nicotinamide-adenine dinucleotide.     

Regulation of C4 photosynthesis: inactivation of pyruvate, Pi dikinase by ADP-dependent phosphorylation and activation by phosphorolysis

These studies provide information about the mechanism of the light/dark-mediated regulation of pyruvate, Pi dikinase (EC 2.7.9.1) in leaves. It is shown that inactivation is due to a phosphorylation of the enzyme from the beta-phosphate of ADP, and that activation occurs by phosphorolysis to remove the enzyme phosphate group. During ADP plus ATP-dependent inactivation of pyruvate, Pi dikinase in chloroplast extracts, 32P was incorporated into the enzyme from [beta-32P]ADP. Approximately 1 mol of phosphate was incorporated per mol of monomeric enzyme subunit inactivated. There was very little incorporation of label from ADP or ATP labeled variously in other positions with 32P or from the nucleotides labeled with 3H in the purine ring. Purified pyruvate, Pi dikinase was also labeled from [beta-32P]ADP during inactivation. In this system, phosphorylation of the enzyme required the addition of the "regulatory protein" shown previously to be essential for catalyzing inactivation and activation. During orthophosphate-dependent reactivation of pyruvate, Pi dikinase, it was shown that the enzyme loses 32P label and that pyrophosphate is produced. The significance of these findings in relation to regulation of the enzyme in vivo is discussed.     

Inactivation of Salmonella phosphoribosylpyrophosphate synthetase by specific chemical modification of a lysine residue.

Phosphoribosylpyrophosphate synthetase from Salmonella typhimurium contains nine lysine residues per subunit and can be inactivated by reagents specific for this amino acid. Pyridoxal-P reversibly inhibited the enzyme by about 70% by forming a Schiff base derivative with lysine. Reduction with NaBH₄ made this inactivation irreversible. Kinetic experiments indicated that the failure to inactivate the enzyme completely in a single treatment with pyridoxal-P reflects a reversible equilibrium between inactive Schiff base and a noncovalent complex. Modification of one lysine residue per subunit correlated with apparently total loss of activity. The rate of inactivation of the enzyme was decreased fourfold by saturating concentrations of ATP and was decreased at least 20-fold by formation of a quaternary complex of the enzyme with Mg²⁺, α,β-methylene ATP, and ribose-5-P. Trinitrobenzenesulfonate also irreversibly inactivated the enzyme, but this reagent was less specific in that the loss of activity corresponded to the modification of four to five lysine residues. These results suggest that an essential lysine is near the active site of Phosphoribosylpyrophosphate synthetase.     

Decarboxylation and carboxylation of pyruvate in the living mice.

Mice received intravenously [1- or 2-14C]acetate, [1-, 2- or 3-14C] or [2-14C]pyruvate and were killed 1, 3, 5 or 15 min later. The radioactivity of CO2 or HCO3- of liver or carcass as well as the radioactivity of blood glucose were measured. The ratio of the radioactivity found in these compounds after [3-14C] or [2-14C-A1pyruvate injection suggests that in the fed aminals: 1. the decarboxylation of the pyruvate was more rapid than its carboxylation, 2. most of the neosynthesized glucose was derived from pyruvate molecules which had undergone a decarboxylation followed by a condensation to citrate, 3. 1/4 to 1/3 of the pyruvate was carboxylated and 2/3 to 3/4 was decarboxylated in animals receiving a diet poor in fats.     

Letter: Studies on histidinol dehydrogenase preliminary crystallographic data

Crystals of histidinol dehydrogenase (EC 1.1.1.23) from Salmonella typhimurium have been obtained in large size suitable for single crystal X-ray studies. The following crystal data were obtained on examination of a number of X-ray precession photographs; crystal system monoclinic, $\text{a = 149.6}\text{A}\text{?}$, $\text{b = 88.9}\text{A}\text{?}$, $\text{c = 105.6}\text{A}\text{?}$, β = 124.5 °, space group C2. The density of the crystal measured by flotation in bromobenzene/xylene mixture is 1.186 g/cm³. There is one dimer molecule of molecular weight 80,000 in one crystallographically asymmetric unit.     

Defective phosphorylation and processing of beta-hexosaminidase by intact cultured fibroblasts from patients with mucolipidosis III

Previous studies of the synthesis, phosphorylation, and processing of β-hexosaminidase in cultured fibroblasts from normal individuals and from patients with mucolipidosis II (I-cell disease) (A. Hasilik and E. F. Neufeld, 1980, J. Biol. Chem.225, 4937–4946) have been extended to fibroblasts derived from patients with a related genetic disorder, mucolipidosis III (pseudo-Hurler polydystrophy). The enzyme was biosynthetically labeled in pulse-chase experiments with [³H]leucine and ³³P_i, and isolated from cells and medium by immunoprecipitation. The constitutent α and β chains of the enzyme were separated by polyacrylamide gel electrophoresis under reducing and denaturing conditions, visualized by autoradiography and fluorography, extracted from the gel, and quantitated by liquid scintillation spectrometry. Enzyme produced by fibroblasts from mucolipidosis III patients had a very low but detectable phosphate content; a high proportion of newly made enzyme was secreted, though some remained within the cells and was processed to mature enzyme; the presence of NH₄Cl during the labeling and chase did not significantly increase the amount of enzyme secreted. The β-hexosaminidase produced by mucolipidosis III fibroblasts thus resembled more closely that produced by fibroblasts from patients with mucolipidosis II than the normal enzyme. β-Hexosaminidase made by fibroblasts from mucolipidosis II heterozygotes was similar to the normal enzyme with respect to phosphorylation, processing, and secretion. Mucolipidosis II and III fibroblasts could endocytose normal precursor β-hexosaminidase and process it to the mature form. The deficiency of mature enzyme in the patients' cells may therefore be attributed to failure of the unphosphorylated enzyme to be incorporated into lysosomes, where processing would normally occur.     

The reversible phosphorylation of isocitrate dehydrogenase of Salmonella typhimurium

The 46,000 dalton phosphoprotein in Salmonella typhimurium is isocitrate dehydrogenase, an enzyme at the branch point between the glyoxylate and Krebs cycle pathways. The enzyme is phosphorylated by a kinase which is controlled by growth conditions; and it is dephosphorylated by a phosphatase. Acetate, ethanol, α-methylglucoside, and deoxyglucose cause an activation of the phosphorylation reaction in intact cells. A number of other compounds are found to affect the kinase and phosphatase activities. The reversible phosphorylation of isocitrate dehydrogenase plays a major role in the control of the Krebs cycle and glyoxylate pathways.     

UDP-glucose dehydrogenase: substrate binding stoichiometry and affinity

Precise structural parameters of polyribonucleotides single stranded helices are determined as well as those of double stranded helices of poly 2′-O-methyl A and of poly A at neutral and acid pH. Infrared linear dichroism investigations indicate the similarity of the conformation of the sugar-phosphate backbone of these single and double stranded helices. The angles of the phosphate group for single stranded helix at neutral pH is found to be oriented at 48° for the 02P02 bisector and at about 65° for the 02–03 line to the helix axis. Similar values were found for double stranded poly A helix at acid pH. These structural parameters obtained for the first time on single stranded polynucleotide helices are proposed to be valid for other similar helical chains such as poly A segments of nuclear or messenger RNA and single stranded CCA acceptor end of transfer RNA.     

A study of protein phosphorylation in shape change and Ca++-dependent serotonin release by blood platelets

Upon treatment with agents such as thrombin, collagen or concanavalin A, blood platelets change shape, secrete serotonin and phosphorylate two proteins having molecular weights of approximately 20,000 and 40,000. We have analyzed the relationship of this protein phosphorylation to shape change and release aided by the fact that while shape change occurs independently of extracellular calcium, release of serotonin displays a rather strict calcium requirement. Under limited calcium conditions, where virtually no serotonin release occurs, (Con A)-stimulated phosphorylation is uninhibited. Divalent cations (Mg⁺⁺, Co⁺⁺ and Zn⁺⁺) also inhibit release but not phosphorylation. The microtubule effectors colchicine and D₂O show concomitant effects on release and phosphorylation, indicating a microtubule involvement prior to phosphorylation. Papaverine inhibits release and phosphorylation while not strongly influencing shape change, suggesting that shape change does not require phosphorylation. We therefore conclude that phosphorylation of these proteins takes place after shape change but prior to release, and although it may be required for secretion to occur, the two processes are easily separated. Thus phosphorylation of these proteins is not likely to be an integral component of the release mechanism.     

Inhibition of d(-)-3-hydroxybutyrate dehydrogenase by malonate analoges

(1) d(-)-3-Hydroxybutyrate dehydrogenase activity from guinea pig, rat, and bovine heart and from guinea pig liver is inhibited by malonate and tartronate, and more potently by the analogs methylmalonate, bromomalonate, chloromalonate, and mesoxalate. Little or no inhibitory effect was found for aminomalonate, ethylmalonate, dimethylmalonate, succinate, glutarate, oxaloacetate, malate, propionate, pyruvate, d- and l-lactate, n-butyrate, isobutyrate, and cyclopropanecarboxylate. (2) In initial velocity kinetics at pH 8.1 with a soluble enzyme preparation from bovine heart, the inhibition by the active malonate derivatives is competitive with respect to 3-hydroxybutyrate and uncompetitive with respect to acetoacetate, NAD⁺ or NADH. With d-3-hydroxybutyrate as the variable reactant (K_{m app} = 0.26 mM) the inhibition constant of methylmalonate (K_is) was 0.09 mm. (3) The rate of utilization of d-3-hydroxybutyrate (78 μm) by coupled rat heart mitochondria in the presence of ADP was inhibited 50% by 150 μm methylmalonate. (4) With coupled guinea pig liver mitochondria oxidizing n-octanoate in the absence of added ADP, methylmalonate (1–3 mm) depressed 3-hydroxybutyrate formation substantially more than total ketone production. However, the intramitochondrial NADH (or NADPH) levels were unchanged by the addition of methylmalonate, indicating that the changes in ratios of accumulated 3-hydroxybutyrate and acetoacetate were caused by direct inhibition of 3-hydroxybutyrate dehydrogenase. Methylmalonate had the same effect on 3-hydroxybutyrate/acetoacetate ratios and ketone body formation with pyruvate or acetate as the source of acetyl groups. Similar results were obtained with malonate (10 mm) although the inhibition of total ketone formation from octanoate was more severe.     

Inhibition of amino acid transport in Bacillus subtilis by uncouplers of oxidative phosphorylation.

The effect of three uncouplers of oxidative phosphorylation, trifluoromethoxycarbon-ylcyanidephenylhydrazone (FCCP), 3,3′,4′,5-tetrachlorosalicylanilide (TCSA), and pentachlorophenol (PCP), on transport of glycine and proline by Bacillus subtilis were examined. FCCP inhibited proline uptake uncompetitively, but glycine uptake competitively. TCSA inhibited proline uptake noncompetitively, but glycine uptake competitively. PCP inhibited proline uptake noncompetitively, but glycine uptake uncompetitively. The results indicate that these uncouplers inhibit amino acid transport by interacting at specific sites rather than by reducing any central supply of energy used to fuel metabolic processes.     

Anion binding to oxidized type 2 depleted and native laccase: a spectroscopically effective model for exogenous ligand binding to the type 3--type 2 active site

Xanthine oxidase, a mammalian nitroreductase, catalyzed the binding of [³H]1-nitropyrene to DNA. The binding was dependent on the presence of hypoxanthine and was inhibited by allopurinol, a specific xanthine oxidase inhibitor. These data support the hypothesis that nitroreduction is a necessary step in the metabolic activation of 1-nitropyrene to a bacterial mutagen.     

The tryptophan synthase alpha 2 beta 2 complex: a comparison of the reactivity of amino groups in the alpha and beta 2 subunits and in the complex by differential labeling studies

The interaction of the alpha and beta 2 subunits of tryptophan synthase of Escherichia coli to form an alpha 2 beta 2 complex has been probed by differential labeling studies. In the first step the separate alpha or beta 2 subunit or the alpha 2 beta 2 complex was labeled by reductive methylation with trace amounts of [3H]HCHO in the presence of NaCNBH3. In the second step the 3H-labeled preparation was fully labeled under denaturing conditions with [14C]HCHO and NaCNBH3. Peptides containing labeled monomethyl or dimethyl amino groups were isolated after thermolytic digestion or after cyanogen bromide treatment. The 3H/14C ratio of each peptide is a measure of the relative reactivity of the amino group or groups in each peptide. The most reactive amino group in the alpha subunit, lysine-109, is strongly shielded from modification in the alpha 2 beta 2 complex. The most reactive amino group in the beta 2 subunit, the amino-terminal threonine, is not shielded from modification in the alpha 2 beta 2 complex.     

Preparation and characterization of a stable half met derivative of type 2 depleted Rhus laccase: Exogenous ligand binding to the type 3 site

We report the preparation and characterization of a stable half met (Cu(II)Cu(I)) type 2 copper depleted derivative of $\text{Rhus}$ laccase. Anion binding studies to this mixed valent type 3 protein form indicate no tight binding of anions nor group 1 - group 2 ligand behavior. This suggests that, in contrast to the well-characterized hemocyanins and tyrosinase coupled binuclear sites, exogenous ligands do $\text{not}$ appear to bridge the type 3 binuclear copper ions in laccase.     

Human granulocyte 6 phosphogluconate dehydrogenase. Purification by elective elution with NADP+, immunological and kinetic properties.

Human granulocyte 6 phosphogluconate dehydrogenase has been totally purified from a single patient with chronic granulocytic leukaemia. 48 mg of protein, of specific activity 20 IU per mg of protein, have been obtained in the course of three different steps only. The overall yield was 30 p. cent and the purification was 100 folds. Purified 6 phosphogluconate dehydrogenase was homogeneous when tested in acrylamide and acrylamide SDS gel electrophoresis or in immunodiffusion. The enzyme was immunologically identical in red blood cells, blood platelets and normal leukocytes. The fixation of both substrates, NADP-+ and 6 phosphogluconate, seemed to proceed through a non ordered mechanism. NADPH was an inhibitor strictly competitive with respect to NADP-+ and non competitive with respect to 6 phosphogluconate. 2-3 Diphosphoglycerate seemed to be able to bind on both the fixation sites of NADP-+ and 6 phosphogluconate. The inhibition by ATP was competitive with 6 phosphogluconate and non competitive with NADP-+. 6 phosphogluconate dehydrogenase was inactivated by SH reagents and was partially protected against this inactivation by both substrates. Both substrates protected the enzyme against thermal inactivation. The influence of ionic strength, pH and ions have been studied, and the results have been compared to those reported by other authors for erythrocyte enzyme.     

Characterization of calmodulin-mediated phosphorylation of cardiac muscle sarcoplasmic reticulum

Sarcoplasmic reticulum, isolated from canine cardiac muscle, was phosphorylated in the presence of exogenous cAMP-dependent protein kinase or calmodulin. This phosphorylation has been shown previously to activate sarcoplasmic reticulum calcium uptake (LePeuch et al. (1979) Biochemistry18, 5150–5157). Calmodulin appeared to activate an endogenous protein kinase present in sarcoplasmic reticulum membranes. The incorporation of phosphate increased with time. However, once all the ATP was consumed, the level of phosphorylated protein started to decrease due to the action of an endogenous protein phosphatase. Dephosphorylation occurred even when the level of phosphorylated sarcoplasmic reticulum remained constant at high ATP concentrations. The phosphorylation of sarcoplasmic reticulum in the presence of calmodulin, increased as the pH was increased from pH 5.5 to 8.5. This phosphorylation was only inhibited by KCl concentrations greater than 100 mm. The apparent K_m of cAMP-dependent protein kinase for ATP was 5.2 ± 0.2 × 10^?5m, and of the calmodulin-dependent protein kinase for ATP was 3.67 ± 0.29 × 10^?5m. Phosphorylation was maximally activated by 5–10 mm MgCl₂; higher MgCl₂ concentrations inhibited this phosphorylation. Thus the calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum could be maximally activated at sarcoplasmic concentrations of K⁺, Mg²⁺, and ATP. The calmodulindependent phosphorylation was half-maximally activated at Ca²⁺ concentrations that were significantly greater than those required to promote the formation of the sarcoplasmic reticulum Ca-activated ATPase phosphoprotein intermediate. Thus at sarcoplasmic Ca²⁺ concentrations that might be expected during systole, the sarcoplasmic reticulum calcium pump would be fully activated before any significant calmodul-independent sarcoplasmic reticulum phosphorylation occurred. However, under certain pathological conditions when the sarcoplasmic Ca²⁺ becomes elevated (e.g., in ischemia) the kinase could be activated so that the sarcoplasmic reticulum would be phosphorylated and calcium uptake augmented. Thus, the calmodulin-dependent protein kinase may only function when the heart needs to rescue itself from a possibly fatal calcium overload.