Transport of adenine, hypoxanthine and uracil into Escherichia coli.

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.     

Adrenal glucocorticoids, adenine nucleotide translocation, and mitochondrial calcium accumulation.

The administration of dexamethasone to rats markedly diminished the initial rate and maximal extent of substrate-dependent calcium uptake in subsequently isolated liver mitochondria, and enhanced the release of calcium. The apparent Km for calcium transport was not altered by dexamethasone treatment and it ranged from 50 to 80 muM when an EDTA/Ca buffer system was used in the presence of magnesium, and 20 muM when an NTA/Ca buffer system without magnesium was employed. In contrast, when ATP was employed as the energy source, there was no significant difference in initial rate, Km, or the extent of calcium accumulation between mitochondria from control and dexamethasone-treated animals. Although mitochondria from dexamethasone-treated animal showed 15% less cytochrome c oxidase activity/mg of protein, overall respiratory capacity and ATP production from ADP were the same as in control mitochondria. However, mitochondria from dexamethasone-treated animals translocated ATP from inside to outside faster than those from control animals. When the ATP in the medium was depleted by glucose and hexokinase, both types of mitochondria retained essentially all the preloaded calcium until total ATP reached a critical level (7 approximately 5 mumol of ATP/mg of protein). When ATP content fell below this critical level, mitochondria released all the calcium quickly. Dexamethasone treatment increased the susceptibility of mitochondria to the depletion of ATP. These data indicate that the dexamethasone-induced decrease in maximal calcium transport and in calcium retention carrier system per se, but o an altered ability of the mitochondria to regulate intramitochondrial ATP content.     

Interrelationships of copper, cytochrome oxidase, phospholipid synthesis and adenine nucleotide binding.

Studies are reported on the interrelationships in liver mitochondria of copper status, cytochrome oxidase activity, adenine nucleotide binding capacity and phospholipid synthesis. Direct exposure of mitochondria to cyanide or diethyldithiocarbamate depressed cytochrome oxidase activity; ADP-binding and phospholipid synthesis. Fractionation of mitochondria to increase the specific activity of cytochrome oxidase about 10-fold did not increase the affinity to bind ADP. Ageing of mitochondria or dialysis of mitochondria or mitochondrial membrane preparations against water or diethyldithiocarbamate at 0--2 degrees for 18 h did not decrease cytochrome oxidase activity or copper content of reisolated and resuspended mitochondria or mitochondrial membrane preparations, but considerably reduced the affinity to bind ADP. The respiratory inhibitors, fluoride and azide, at concentrations inhibitory to cytochrome oxidase did not reduce ADP-binding or phospholipid synthesis. Atractyloside did not inhibit cytochrome oxidase activity but did inhibit ADP-binding and phospholipid synthesis. Pre-incubation of mitochondrial membrane preparations with Cu++ increased the copper content and ADP-binding affinity. The results indicate that cytochrome oxidase is not the ADP-binding site of the mitochondrial membrane system and that reduced cytochrome oxidase activity per se does not depress binding affinity. Copper appears to be a component of the adenine nucleotide binding sites of mitochondrial membranes because the copper-complexing agents, cyanide and diethyldithiocarbamate, depressed ADP-binding, while increased mitochondrial membrane copper content increased ADP-binding.     

Reduced S-adenosylhomocysteine hydrolase. Kinetics and thermodynamics for binding of 3'-ketoadenosine, adenosine, and adenine.

S-Adenosylhomocysteine hydrolase (SAHase) was resolved into apoenzyme and NAD+ by acidic ammonium sulfate treatment. The apoenzyme was catalytically inactive, but could be reconstituted to active enzyme with NAD+. Reduced SAHase (ENADH) that was prepared by reconstitution of the apoenzyme with NADH was catalytically inactive. ENADH was oxidized by 3'-ketoadenosine to active SAHase. The recovery of activity paralleled the oxidation of enzyme-bound NADH. The association rate constant for ENADH and 3'-ketoadenosine was 6.1 x 10(2) M-1 s-1, and the dissociation rate constant was calculated to be 4 x 10(-7) s-1. This association rate constant was considerably smaller than the association rate constant for adenosine and SAHase (greater than 10(7) M-1 s-1). However, the observed pseudo first-order rate constant for reaction of 3'-ketoadenosine with ENADH (0.6 s-1 with 1 mM 3'-ketoadenosine) approached kcat for the hydrolytic reaction (1.2 s-1). Thus, bound 3'-ketoadenosine probably reacted sufficiently rapidly with ENADH to be considered a kinetically competent intermediate. The dissociation constants of SAHase for adenosine and 4',5'-dehydroadenosine, substrates for the enzyme, were 9 and 14 microM, respectively. In contrast, the dissociation constants of ENADH for 3'-ketoadenosine and 4',5'-dehydro-3'-ketoadenosine, intermediates of the catalytic reaction, were significantly lower with values of 600 and 300 pM, respectively. The equilibrium constant for reduction of enzyme-bound NAD+ in the absence of an adenosine analogue, as estimated from cyanide binding studies, was 10-fold more favorable than that for free NAD+. ENADH was highly fluorescent (emission maximum 428 nm, excitation 340 nm) with a quantum yield that was six times that of free NADH. Since SAHase reduced by adenosine was not highly fluorescent, enzyme-bound intermediates quenched the fluorescence of enzyme-bound NADH. Adenosine and adenine quenched the fluorescence of ENADH. Cyanide formed a complex with SAHase that was analogous to ENADH. Adenine stabilized this complex sufficiently that addition of 65 microM adenine and 25 mM cyanide to SAHase caused total complex formation with loss of over 95% of the catalytic activity.     

Metabolic modulation of neurotransmitter release--adenosine, adenine nucleotides, potassium, hyperosmolarity, and hydrogen ion.

Evidence has accumulated that several factors, which have been proposed as mediators of exercise hyperemia, can modulate adrenergic neurotransmission in blood vessels. Adenosine and the adenine nucleotides depress the response of isolated blood vessels of the dog to nerve stimulation more than that to exogenous norepinephrine; this difference is explained by a decreased release of the neurotransmitter. Potassium, hyperosmolarity, and acidosis also depress adrenergic neurotransmission in isolated veins. These results are consistent with the hypothesis that metabolic changes in the vicinity of the adrenergic neuroeffector junction are capable of decreasing the output of neurotransmitter to the blood vessels in the exercising muscle.     

Unusual sensitivity of Escherichia coli to adenine or adenine plus histidine.

The W3110 strain of Escherichia coli K-12 is unusually sensitive to adenine. Inhibition of growth is relieved by a combination of thiamine and uridine (or cytidine). In the presence of histidine, inhibition is more severe and is relieved by a combination of thiamine, glycine, uridine (or cytidine), and inosine (or guanosine).     

Nicotinamide adenine dinucleotide phosphate-malic enzyme of rat liver. Purification, properties, and immunochemical studies.

Rat liver malic enzyme (EC 1.1.1.40) was purified from livers of rats fasted and refed a high sucrose diet containing 1% desiccated thyroid powder. The purification was accomplished by a six-step procedure. The specific activity of the purified enzyme was increased 181-fold above that of the initial high speed supernatant of liver extracts. Slight additional purification of malic enzyme was achieved with preparative disc electrophoresis. The specific activities of the purified rat liver malic enzyme from the least two steps were between 28.0 and 30.5 units per mg of protein. Homogeneity of the purified enzyme was determined by disc and starch gel electrophoresis as well as sedimentation velocity and sedimentation equilibrium studies. The molecular weight and S20, w values of rat liver malic enzyme are 268,000 and 10.2, respectively. Amino acid analysis based on milligram of protein hydrolyzed yielded higher amounts of leucine and glutamic acid but lower quantities of alanine and voline per subunit than the corresponding Escherichia coli enzyme...     

Bull semen nicotinamide adenine dinucleotide nucleosidase. VI. Fluorescence studies of the enzyme with reduced nicotinamide adenine dinucleotide

The binding of NADH to bull semen NAD nucleosidase was observed to be accompanied by a considerable enhancement of the fluorescence of NADH. The fluorescence enhancement observed in the binding of NADH to the enzyme was utilized to study the stoichiometry of binding of this compound to the enzyme. Results obtained from the fluorescence titration of the enzyme with NADH indicated the binding of one mole of NADH per mole of enzyme (36,000 g). The dissociation constant for the enzyme-NADH complex was determined to be 2.52 × 10^?6m. NADH was also found to be a very effective competitive inhibitor of the NADase-catalyzed hydrolysis of NAD, and the inhibitor dissociation constant (K_I) for the enzyme-NADH complex was determined to be 2.99 × 10^?6m which was in good agreement with the value obtained from the fluorescence titration experiments.     

Human adenine phosphoribosyltransferase. Affinity purification, subunit structure, amino acid composition, and peptide mapping.

Adenine phosphoribosyltransferase (EC 2.4.2.7) has been purified 55,000-fold from normal human erythrocytes. The native molecular weight of the enzyme is 38,200 as determined by sedimentation equilibrium centrifugation. The subunit molecular weight is 18,000 as determined by sodium dodecyl sulfate gel electrophoresis and 17,000 as determined by gel filtration in guanidine hydrochloride, suggesting that the enzyme is a dimer in its native state. Cross-linking the enzyme with dimethylsuberimidate confirms the dimeric structure and peptide mapping data suggested that the subunits are quite similar if not identical. The amino acid composition reveals that 33% of the residues are hydrophobic.     

Sensitivity of the response of cytosolic calcium in Quin-2-loaded rat hepatocytes to glucagon, adenine nucleosides, and adenine nucleotides

Hepatocytes from fasted rats were used to study the effect of glucagon on intracellular free cytosolic Ca2+ ([Ca2+]i) indicated by the use of Quin-2-calcium fluorescence. It was found that, in both male and female rats, glucagon increased [Ca2+]i at a half-maximally effective concentration (Kact) of 0.3 nM, a concentration known to be half-maximal for affecting several hepatic functions. Acute chelation of extracellular Ca2+ did not obliterate the hormone effect but shortened its duration. Cyclic AMP, 5'-AMP, ADP, and ATP also increased [Ca2+]i, while adenosine 2':3'-monophosphate and 3'-AMP did not. The rise in [Ca2+]i brought about by glucagon at near physiological concentrations may be responsible for the stimulation of glutamate metabolism produced acutely by glucagon.