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PubMed1973

Iodoacetamide-induced sensitization of the pancreatic beta-cells to glucose stimulation.

At a glucose concentration of 3mm or less, iodoacetamide had no effect on the release of insulin from microdissected pancreatic islets of ob/ob-mice. At higher glucose concentrations, iodoacetamide exerted both an initial stimulatory and a subsequent inhibitory action. When islets were perifused with 1mm-iodoacetamide and 17mm-glucose the inhibitory action predominated after about 15min of transient stimulation. With decreasing concentrations of iodoacetamide the stimulatory phase was gradually prolonged, and with 0.003-0.1mm-iodoacetamide stimulation only was observed for 75min. Prolonged stimulation was also noted after a short pulse of iodoacetamide. Similar responses to 0.1mm-iodoacetamide were observed with islets from normal mice. With islets from ob/ob-mice the effect of 0.1mm-iodoacetamide was reproduced with 0.1mm-iodoacetate, whereas 0.1mm-acetamide had no apparent effect. Iodoacetamide increased the V(max.) of glucose-stimulated insulin release without altering the apparent K(m) for glucose. Leucine, glibenclamide or theophylline could not replace glucose in this synergistic action with iodoacetamide. Iodoacetamide rather inhibited the insulin-releasing action of theophylline. Iodoacetamide-induced potentiation of the glucose-stimulated insulin release was rapidly and reversibly inhibited by mannoheptulose, adrenaline, or calcium deficiency. The potentiating effect on insulin release was not paralleled by effects on glucose oxidation or on islet fructose 1,6-diphosphate. However, the inhibitory action of iodoacetamide might be explained by inhibition of glycolysis as evidenced by an inhibition of glucose oxidation and a rise of fructose 1,6-diphosphate. The results support our previous hypothesis that thiol reagents can stimulate insulin release by acting on relatively superficial thiol groups in the beta-cell plasma membrane. Glycolysis seems to be necessary in order for iodoacetamide to stimulate in this way.

Amides↗
PubMed1973

The acute action of ammonia on rat brain metabolism in vivo.

1. Acute NH(4) (+) toxicity was studied by using a new apparatus that removes and freezes the brains of conscious rats within 1s. 2. Brains were removed and frozen 5min after intraperitoneal injection of ammonium acetate (2-3min before the onset of convulsions). Arterial [NH(4) (+)] rose from less than 0.01 to 1.74mm at 4-5min. The concentrations of all glycolytic intermediates measured, except glucose 6-phosphate, were increased by the indicated percentage above the control value as follows: glucose (by 41%), fructose 1,6-diphosphate (by 133%), dihydroxyacetone phosphate (by 164%), alpha-glycerophosphate (by 45%), phosphoenolpyruvate (by 67%) and pyruvate (by 26%). 4. Citrate and alpha-oxoglutarate concentrations were unchanged and that of malate was increased (by 17%). 5. Adenine nucleotides and P(i) concentrations were unchanged but the concentration of creatine phosphate decreased slightly (by 6%). 6. Brain [NH(4) (+)] increased from 0.2 to 1.53mm. Net glutamine synthesis occurred at an average rate of 0.33mumol/min per g. 7. The rate of brain glucose utilization was measured in vivo as 0.62mumol/min per g in controls and 0.81mumol/min per g after NH(4) (+) injection. 8. The arteriovenous difference of glucose and O(2) increased by 35%. 9. No significant arteriovenous differences of glutamate or glutamine were detected. Thus, although much NH(4) (+) was incorporated into glutamine the latter was not rapidly released from the brain to the circulation. 10. Plasma [K(+)] increased from 3.3 to 5.4mm. 11. The results indicate that NH(4) (+) stimulates oxidative metabolism but does not interfere with brain energy balance. The increased rate of oxidative metabolism could not be accounted for only on the basis of glutamine synthesis. We suggest that increased extracellular [NH(4) (+)] and [K(+)] decreased the resting transmembrane potential and stimulated Na(+),K(+)-stimulated adenosine triphosphatase activity thus accounting for the increased metabolic rate.

Ammonia↗
PubMed1974

The pancreatic beta-cell recognition of insulin secretagogues. Effects of calcium and sodium on glucose metabolism and insulin release.

The transport and oxidation of glucose, the content of fructose 1,6-diphosphate, and the release of insulin were studied in microdissected pancreatic islets of ob/ob mice incubated in Krebs-Ringer bicarbonate medium. Under control conditions glucose oxidation and insulin release showed a similar dependence on glucose concentration with the steepest slope in the range 5-12mm. The omission of Ca(2+), or the substitution of choline ions for Na(+), or the addition of diazoxide had little if any effect on glucose transport. However, Ca(2+) or Na(+) deficiency as well as diazoxide (7-chloro-3-methyl-1,2,4-benzothiadiazine 1,1-dioxide) or ouabain partially inhibited glucose oxidation. These alterations of medium composition also increased the islet content of fructose 1,6-diphosphate, as did the addition of adrenaline. Phentolamine [2-N-(3-hydroxyphenyl)-p-toluidinomethyl-2-imidazoline] counteracted the effects of adrenaline and Ca(2+) deficiency on islet fructose 1,6-diphosphate. After equilibration in Na(+)-deficient medium, the islets exhibited an increase in basal insulin release whereas the secretory response to glucose was inhibited. The inhibitory effects of Na(+) deficiency on the secretory responses to different concentrations of glucose correlated with those on (14)CO(2) production. When islets were incubated with 17mm-glucose, the sudden replacement of Na(+) by choline ions resulted in a marked but transient stimulation of insulin release that was not accompanied by a demonstrable increase of glucose oxidation. Galactose and 3-O-methylglucose had no effect on glucose oxidation or on insulin release. The results are consistent with a metabolic model of the beta-cell recognition of glucose as insulin secretagogue and with the assumption that Ca(2+) or Na(+) deficiency, or the addition of adrenaline or diazoxide, inhibit insulin release at some step distal to stimulus recognition. In addition the results suggest that these conditions create a partial metabolic block of glycolysis in the beta-cells. Hence the interrelationship between the processes of stimulus recognition and insulin discharge may involve a positive feedback of secretion on glucose metabolism.

Animals↗
PubMed1974

The inhibition of mitochondrial dicarboxylate transport by inorganic phosphate, some phosphate esters and some phosphonate compounds.

1. P(i) competitively inhibited succinate oxidation by intact uncoupled mitochondria in the presence of sufficient N-ethylmaleimide to block the phosphate carrier, with a K(i) of 2.5mm. 2. Of a large number of phosphate esters and phosphonate compounds, phenyl phosphate and phenylphosphonate were found to inhibit competitively uncoupled succinate oxidation by intact but not broken mitochondria. By comparison, benzoate was a relatively weak competitive inhibitor of succinate oxidation by intact mitochondria but a relatively potent inhibitor of succinate dehydrogenase. 3. Phenyl phosphate and phenylphosphonate were non-penetrant, and inhibited P(i)-dependent swelling of mitochondria suspended in isosmolar ammonium malate in a manner non-competitive with P(i). The inhibitors did not affect mitochondrial swelling when tested with P(i) alone. 4. It is concluded that: (i) phenyl phosphate and phenylphosphonate behaved as non-penetrant analogues of P(i), since their inhibitory properties were in strict contrast with those of benzoate; (ii) phenyl phosphate and phenylphosphonate interacted with the dicarboxylate carrier but not with the phosphate carrier; (iii) P(i) was effective as a competitive inhibitor of succinate oxidation because of its being either an alternative substrate for the dicarboxylate carrier or competitive with succinate for the intramitochondrial cations as proposed by Harris & Manger (1968).

Adenosine Monophosphate↗
PubMed1974

Simulation of the pentose cycle in lactating rat mammary gland.

A computer model representing the pentose cycle, the tricarboxylic acid cycle and glycolysis in slices of lactating rat mammary glands has been constructed. This model is based primarily on the studies, with radioactive chemicals, of Abraham & Chaikoff (1959) [although some of the discrepant data of Katz & Wals (1972) could be accommodated by changing one enzyme activity]. Data obtained by using [1-(14)C]-, [6-(14)C]- and [3,4-(14)C]-glucose were simulated, as well as data obtained by using unlabelled glucose (for which some new experimental data are presented). Much past work on the pentose cycle has been mainly concerned with the division of glucose flow between the pentose cycle and glycolysis, and has relied on the assumption that the system is in steady state (both labelled and unlabelled). This assumption may not apply to lactating rat mammary glands, since the model shows that the percentage flow through the shunt progressively decreased for the first 2h of a 3h experiment, and we were unable to construct a completely steady-state model. The model allows examination of many quantitative features of the system, especially the amount of material passing through key enzymes, some of which appear to be regulated by NADP(+) concentrations as proposed by McLean (1960). Supplementary information for this paper has been deposited as Supplementary Publication SUP 50023 at the British Museum (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.

Animals↗
PubMed1974

A kinetic study of pig liver pyruvate kinase activated by fructose diphosphate.

The paper reports a study of the reaction between phosphoenolpyruvate, ADP and Mg(2+) catalysed by pig liver pyruvate kinase when activated by fructose diphosphate and K(+). The experimental results are consistent with two non-sequential mechanisms in which the substrates and products of the reaction are phosphoenolpyruvate, ADP, Mg(2+), pyruvate and MgATP. Pyruvate release occurs before ADP binding. Two Mg(2+) ions are involved, though the two Mg(2+)-binding sites cannot be occupied simultaneously. An isomerized enzyme complex forms before release of MgATP. Values were determined for the Michaelis constants of the reaction. Apparent MgATP inhibition constants are also given.

Adenosine Diphosphate↗
PubMed1974

The effect of starvation on insulin secretion and glucose metabolism in mouse pancreatic islets.

1. Rates of insulin secretion, glucose utilization, lactate output, incorporation of glucose into glycogen, contents of glucose 6-phosphate, fructose 1,6-diphosphate and ATP, and maximally extractable enzyme activities of hexokinase, high-K(m) glucose-phosphorylating activity (;glucokinase'), glucose 6-phosphatase and unspecific acid phosphatase were measured in isolated pancreatic islets from fed and 48-h-starved mice. 2. In the fed state insulin secretion from isolated islets was increased five- to six-fold when the extracellular glucose concentration was raised from 2.5mm to 16.7mm; 5mm-caffeine potentiated this effect. The secretory response to glucose of islets from mice starved for 48h was diminished at all glucose concentrations from 2.5mm up to approx. 40mm. Very high glucose concentrations (60mm and above) restored the secretory response to that found in the fed state, suggesting that the K(m) value for the overall secretory process had been increased (approx. fourfold) by starvation. Addition of 5mm-caffeine to islets from starved mice also restored the insulin secretory response to 2.5-16.7mm-glucose to normal values. 3. Extractable hexokinase, ;glucokinase', glucose 6-phosphatase and unspecific phosphatase activities were not changed by starvation. 4. Glucose utilization and glycolysis (measured as the rate of formation of (3)H(2)O from [5-(3)H]glucose over a 2h period) was decreased in islets from starved mice at all glucose concentrations up to approx. 55mm. At still higher glucose concentrations up to approx. 100mm, there was no difference between the fed and starved state, suggesting that the K(m) value for the rate-limiting glucose phosphorylation had been increased (approx. twofold) by starvation. Preparation of islets omitting substrates (glucose, pyruvate, fumarate and glutamate) from the medium during collagenase treatment lowered the glucose utilization measured subsequently at 16.7mm-glucose by 38 and 30% in islets from fed and starved mice respectively. Also the 2h lactate output by the islets at 16.7mm extracellular glucose was diminished by starvation. Incorporation of glucose into glycogen was extremely low, but the rate of incorporation was more than doubled by starvation. 5. After incubation for 30min at 16.7mm-glucose the content of glucose 6-phosphate was unchanged by starvation, that of ATP was increased and the concentration of (fructose 1,6-diphosphate plus triose phosphates) was decreased. 6. Possible mechanisms behind the correlated impairment in insulin secretion and islet glucose metabolism during starvation are discussed.

Acid Phosphatase↗
PubMed1974

Kinetics of the activation of rat liver pyruvate kinase by fructose 1,6-diphosphate and methods for characterizing hysteretic transitions.

1. Kinetics of fructose 1,6-diphosphate activation of liver pyruvate kinase type I inhibited with MgATP and l-alanine are described as a function of enzyme and fructose 1,6-diphosphate concentrations. These results can be explained by a single pseudo-first-order transition of the enzyme into an active form, independent of the enzyme concentration. This rate constant, k(app.)=0.24s(-1) with 0.02mm-fructose 1,6-diphosphate (t(0.9) approximately 10s where t(0.9) is the time for 90% conversion), is an increasing function of fructose 1,6-diphosphate concentration far beyond that needed to maximally activate enzyme equilibrated with fructose 1,6-diphosphate (about 20mum). 2. The model equations are best analysed with numerical techniques which are described. These techniques are useful in studying similar slow transients frequently observed in stopped-flow studies of enzymes. 3. Shorter transients (t(0.9)=0.5-1.5s) were observed in the kinetic response of the enzyme to the addition of MgATP or phosphoenolpyruvate, but were not further characterized.

Adenosine Triphosphate↗
PubMed1974

Kinetic properties of rat liver pyruvate kinase at cellular concentrations of enzyme, substrates and modifiers.

Kinetic properties of rat liver pyruvate kinase type I at pH7.5 and 6.5 were studied with physiological ranges of substrates, modifiers and Mg(2+) concentrations at increasing enzyme concentrations, including the estimated cellular concentrations (approx. 0.1mg/ml). Enzyme properties appear unaffected by increased enzyme concentration if phosphoenolpyruvate, fructose 1,6-diphosphate and inhibitors are incubated with enzyme before starting the reaction with ADP. Our data suggest that minimum cellular concentrations of MgATP and l-alanine provide virtually complete inhibition of pyruvate kinase I at pH7.5. The most likely cellular control of existing pyruvate kinase I results from the strong restoration of enzyme activity by the small physiological amounts of fructose 1,6-diphosphate. Decreasing the pH to 6.5 also restores pyruvate kinase activity, but to only about one-third of its activity in the presence of fructose 1,6-diphosphate. Neither pyruvate nor 2-phosphoglycerate at cellular concentrations inhibit the enzyme significantly.

Adenosine Diphosphate↗
PubMed1974

Kinetic properties of cerebral pyruvate kinase.

Partly purified guinea-pig brain pyruvate kinase is not activated by fructose 1,6-diphosphate and gives hyperbolic substrate-saturation curves with phosphoenolpyruvate. It is therefore different from the L-type pyruvate kinase of mammalian liver. Inhibition by MgATP(2-) was competitive for MgADP(-) but not for phosphoenolpyruvate, and the enzyme is therefore different from the M-type pyruvate kinase, which is said to be competitively inhibited by MgATP(2-) with respect to both substrates. The K(i)(MgATP(2-)) value of approx. 8mm for the brain enzyme is higher than the values (about 2mm) reported for the muscle enzyme. Stimulation of enzymic activity was observed at low (1-2mm) concentrations of MgATP(2-). Substrate kinetic constants were K(m) (MgADP(-))=0.47mm, K(m) (phosphoenolpyruvate)=0.08mm. Free Mg(2+) at very high concentrations (over 10mm) was inhibitory (K(i)=20-32mm). Neither ADP(3-) nor 5'-AMP(2-) inhibited the activity. The brain enzyme was concluded to be different from both the M-type and the L-type of other mammalian organs such as muscle and liver.

Adenosine Diphosphate↗
PubMed1974

Properties of the phosphonomethyl isosteres of two phosphate ester glycolytic intermediates.

The enzymic synthesis of the 1-phosphonomethyl isostere of fructose 1,6-diphosphate in which the 1-phosphate (-OPO(3)H(2)) is replaced by the phosphonomethyl group (-CH(2)PO(3)H(2)) is described. The kinetic properties of this fructose diphosphate isostere and of 4-hydroxy-3-oxobutylphosphonic acid, an isostere of dihydroxyacetone phosphate, with aldolase (EC 4.1.2.13), fructose diphosphatase (EC 3.1.3.11) and glycerol phosphate dehydrogenase (EC 1.1.1.8) are described (see Table 1).

Alkaline Phosphatase↗
PubMed1974

Studies with a reconstituted muscle glycolytic system. The rate and extent of glycolysis in simulated post-mortem conditions.

The reconstituted glycolytic system described previously (Scopes, 1973) was used to simulate post-mortem glycolytic metabolism in muscle. The effects of the following factors have been investigated: ATPase (adenosine triphosphatase) amount, AMP deaminase amount, percentage of the phosphorylase in the a form and the effect of diluting the glycolytic enzyme complex as a whole. It was confirmed that the rate of metabolism was solely dependent on the amount of ATPase present and that various concentrations of the glycolytic enzymes had no effect over a wide range encompassing the variation found in anatomically different muscles. The extent of metabolism, represented by the value of the ;ultimate' pH, depended markedly on the amount of phosphorylase in the a form; as little as 1% of the a form resulted in a considerably lower pH than in its absence. To a lesser extent the amount of AMP deaminase also affected the ultimate pH, but this was probably only significant for comparisons of genetically distinct muscles with widely differing amounts of AMP deaminase. The reconstituted system behaved almost identically with regard to post-mortem glycolytic metabolism compared with intact muscle tissue. It is concluded that the controlling effectors found with the reconstituted system apply to intact muscle also.

Adenosine Monophosphate↗
PubMed1974

The carbon assimilation pathways of Methylococcus capsulatus, Pseudomonas methanica and Methylosinus trichosporium (OB3B) during growth on methane.

d-arabino-3-Hexulose 6-phosphate was prepared by condensation of formaldehyde with ribulose 5-phosphate in the presence of 3-hexulose phosphate synthase from methane-grown Methylococcus capsulatus. The 3-hexulose phosphate was unstable in solutions of pH greater than 3, giving a mixture of products in which, after dephosphorylation, allulose and fructose were detected. A complete conversion of d-ribulose 5-phosphate and formaldehyde into d-fructose 6-phosphate was demonstrated in the presence of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase (prepared from methane-grown M. capsulatus). d-Allulose 6-phosphate was prepared from d-allose by way of d-allose 6-phosphate. No evidence was found for its metabolism by extracts of M. capsulatus, thus eliminating it as an intermediate in the carbon assimilation process of this organism. A survey was made of the enzymes involved in the regeneration of pentose phosphate during C(1) assimilation via a modified pentose phosphate cycle. On the basis of the presence of the necessary enzymes, two alternative routes for cleavage of fructose 6-phosphate are suggested, one route involves fructose diphosphate aldolase and the other 6-phospho-2-keto-3-deoxygluconate aldolase. A detailed formulation of the complete ribulose monophosphate cycle of formaldehyde fixation is presented. The energy requirements for carbon assimilation by this cycle are compared with those for the serine pathway and the ribulose diphosphate cycle of carbon dioxide fixation. A cyclic scheme for oxidation of formaldehyde via 6-phosphogluconate is suggested.

Aldehyde-Lyases↗
PubMed1976

Development and properties of fructose 1,6-bisphosphatase in the endosperm of castor-bean seedlings.

1. The activity of fructose 1,6-bisphosphatase (EC 3.1.3.11) in the fatty endosperm of castor bean (Ricinus communis) increases 25-fold during germination and then declines. The developmental pattern follows that of catalase, a marker enzyme for gluconeogenesis in this tissue. 2. The enzyme at its peak of development was partially purified, and its properties were studied. It has an optimal activity at neutral pH (7.0-8.0). The apparent Km value for fructose 1,6-bisphosphate is 3.8 X 10(-5) M. The activity is inhibited by AMP allosterically with an apparent Ki value of 2.2 X 10(-4) M. The enzyme hydrolyses fructose 1,6-bisphosphate and not ribulose 1,5-bisphosphate or sedoehptulose 1,7-bisphosphate. 3. Treatment of the partially purified enzyme with acid leads to an 80% decrease in activity. The remaining activity is insensitive to AMP and has optimal activity at pH 6.7 and a high apparent Km value (2.5 X 10(-4) M) for fructose 1.6-bisphosphate. Enzyme extracted from the tissue with water instead of buffer has a similar modification. The effect of acid explains the discrepancies between this report and previous ones on the properties of the enzyme in this tissue. 4. The storage tissues of various fatty seedlings all contain a 'neutral' fructose 1,6-bisphosphatase. The activities of the enzyme from some of the tissues are inhibited by AMP. 5. The properties of the enzyme in fatty seedlings and in green leaves are discussed in comparison with that in animal tissues.

Adenosine Monophosphate↗
PubMed1976

Inosine-stimulated insulin release and metabolism of inosine in isolated mouse pancreatic islets.

Inosine is a potent primary stimulus of insulin secretion from isolated mouse islets. The inosine-induced insulin secretion was totally depressed during starvation, but was completely restored by the addition of 5 mM-caffeine to the medium and partially restored by the addition of 5 mM-glucose. Mannoheptulose (3 mg/ml) potentiated the effect of 10 mM-inosine in islets from fed mice. The mechanism of the stimulatory effect of inosine was further investigated, and it was demonstrated that pancreatic islets contain a nucleoside phosphorylase capable of converting inosine into hypoxanthine and ribose 1-phosphate. Inosine at 10 mM concentration increased the lactate production and the content of ATP, glucose 6-phosphate (fructose 1,6-diphosphate + triose phosphates) and cyclic AMP in islets from fed mice. In islets from starved mice inosine-induced lactate production was decreased and no change in the concentration of cyclic AMP could be demonstrated, whereas the concentration of ATP and glucose 6-phosphate rose. Inosine (10 mM) induced a higher concentration of (fructose 1,6-diphosphate + triose phosphates) in islets from starved mice than in islets from fed mice suggesting that in starvation the activities of glyceraldehyde 3-phosphate dehydrogenase or other enzymes below this step in glycolysis are decreased. Formation of glucose from inosine was negligible. Inosine had no direct effect on adenylate cyclase activity in islet homogenates. The observed changes in insulin secretion and islet metabolism mimic what is seen when glucose and glyceraldehyde stimulate insulin secretion, and as neither ribose nor hypoxanthine-stimulated insulin release, the results are interpreted as supporting the substrate-site hypothesis for glucose-induced insulin secretion according to which glucose has to be metabolized in the beta-cells before secretion is initiated.

Adenosine Triphosphate↗
PubMed1977

Intramolecular ionic interactions of lysine residues and a possible folding domain in fructose diphosphate aldolase.

1. Treatment with methyl acetimidate was used to probe the topography of the tetrameric fructose 1,6-diphosphate aldolase from ox liver. A single treatment with imido ester in the presence or absence of 20mM-fructose 1,6-diphosphate caused the number of amino groups in the enzyme to fall to approx. 30% of the starting number (assumed to be 30 per subunit). The catalytic activity of the aldolase modified in the presence of fructose 1,6-diphosphate was unaffected, whereas that of the enzyme modified in the absence of substrate fell by about 20%. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that the amino group of lysine-27 (the numbering is that of the highly homologous rabbit muscle enzyme) is essentially unavailable for amidination in the native enzyme and is therefore predicted to be buried in a hydrophobic environment, probably in the form of an ion-pair with a negatively charged side-chain carboxyl group. All the other lysine residues that reacted poorly with methyl acetimidate in the native enzyme (a total of 7) were found to be within the primary structure bounded by lysine-107 and lysine-227. An important member of this group of lysine residues displaying aberrant reactivity is lysine-227, which is known to form an imine with the substrate as part of the catalytic mechanism of the enzyme. 3. The results of the amidination experiments can be correlated in an interesting way with previous studies of thiol-group modification in the aldolases. Taken together, and arguing in part by analogy with the results of identical experiments with glyceraldehyde 3-phosphate dehydrogenases where the three-dimensional structure is known [Lambert & Perham (1977) Biochem. 4. 161. 49-62], they suggest that the region of primary structure from residues 107-227 may form the whole or part of a three-dimensional structural feature, perhaps a folding domain. A three-dimensional structure deduced from X-ray-crystallographic analysis will be needed to interpret these findings more closely. 4. The amino groups of lysine residues are commonly thought to reside at the 'surface' of protein structures. The patterns of specific lysine residues in glyceraldehyde 3-phosphate dehydrogenases and in aldolases that have been found to react poorly with methyl acetimidate in the native enzymes can be attributed to intramolecular ionic interactions deep in hydrophobic pockets and at the protein 'surface'. Such ionic interactions may contribute significantly to the stability of a given protein.

Amino Acid Sequence↗
PubMed1977

The mechanism of adenosine triphosphate depletion in the liver after a load of fructose. A kinetic study of liver adenylate deaminase.

1. The hepatic concentration of several nucleotides and metabolites was measured during the first few minutes after an intravenous load of fructose to mice. The first changes, observed at 30s, were a decrease in the concentration of Pi and a simultaneous accumulation of fructose 1-phosphate. The decrease in the concentrations of ATP and GTP proceeded more slowly. An increase in the concentration of IMP was detected only after 1 min and could therefore not be considered to be the cause of the accumulation of fructose 1-phosphate. 2. To explain the temporary burst of adenine nucleotide breakdown that occurs after a load of fructose, the kinetics of AMP deaminase (EC 3.5.4.6) from rat liver were reinvestigated at physiological (0.2 mM) concentration of substrate. For this purpose, a new radiochemical-assay procedure was developed. At 0.2mM-AMP a low activity could be measured, which was more than 90% inhibited by 5mM-Pi. ATP (3MM) increased the enzyme activity over 200-fold. Pi alone did not influence the ATP-activated enzyme, but 0.5mM-GTP caused a 60% inhibition. The combined effect of both inhibitors at their physiological concentrations reached 95%. 3. It is proposed that the rapid degradation of adenine nucleotides that occurs after a load of fructose is caused by a decrease in the concentration of both inhibitors, Pi and GTP, soon counteracted by the decrease in the concentration of ATP. 4. Some of the kinetic parameters of liver AMP deaminase were computed in terms of the concerted transition theory of Monod, Wyman & Changeux (1965) (J. Mol. Biol. 12, 88-118).

AMP Deaminase↗
PubMed1977

An investigation of the interactions of the allosteric modifiers of pyruvate kinase with the enzyme from Carcinus maenas hepatopancreas.

1. Pyruvate kinase purified from the hepatopancrease of Carcinus maenas exhibited sigmoidal saturation kinetics with respect to the substrate phosphoenolpyruvate in the absence of the allosteric activator fructose 1,6-bisphosphate, but normal hyperbolic saturation was seen in the presence of this activator. The activation appears to be the result of a decrease in the s0.5 (phosphoenolpyruvate) and not to a change in Vmax. 2. In the presence of ADP and ATP at a constant nucleotide-pool size the results indicate that phosphoenolpyruvate co-operativity is lost on increasing the [ATP]/[ADP] ratio. 3. Paralleling this change is the observation that the fructose 1,6-bisphosphate activation became less at the [ATP]/[ATP] ratio was increased. This was due to the enzyme exhibiting a near-maximal activity in the absence of activator. 4. L-Alanine inhibited the enzyme, but homotropic co-operative interactions were only seen with a cruder (1000000g supernatant) enzyme preparation. The inhibition by alanine could be overcome by increasing the concentration of either phosphoenolpyruvate or fructose 1,6-bisphosphate, although increasing the L-alanine concentration did not appear to be able to reverse the activation by fructose 1,6-bisphosphate. 5. In the presence of a low concentration of phosphoenolpyruvate, increasing the concentration of the product, ATP, caused an initial increase in enzyme activity, followed by an inhibitory phase. In the presence of either fructose 1,6-bisphosphate or L-alanine only inhibition was seen. 6. The inhibition by ATP could not be completely reversed by fructose 1,6-bisphosphate.

Adenosine Triphosphate↗