Absence Of Adenosine Triphosphate Research Articles

Facftors affecting the relative magnitudes of the sodium:potassium and sodium:sodium exchanges catalysed by the sodium pump.

1. The effects of external potassium on sodium: potassium exchange and sodium: sodium exchange in human red cells have been estimated from measurements of ouabain-sensitive potassium influx and ouabain-sensitive sodium influx in media containing different concentrations of potassium.2. As the external potassium concentration is increased from zero to 5 mM, sodium:sodium exchange-as judged by ouabain-sensitive sodium influx-is progressively suppressed, and sodium:potassium exchange-as judged by ouabain-sensitive potassium influx-is progressively increased. Both exchanges are half-maximal between 1 and 2 mM-K, and at 5 mM-K sodium: sodium exchange becomes very small as sodium: potassium exchange approaches a maximum.3. Experiments have been carried out, mainly on resealed ghosts, to determine what factors affect the magnitude of the sodium:sodium exchange in potassium-free solutions.4. Sodium:sodium exchange does not occur in the absence of adenosine triphosphate (ATP).5. Ghosts containing high concentrations of sodium, no potassium and high concentrations of ATP show no ouabain-sensitive loss of sodium into potassium-free solutions. The ability to carry out sodium:sodium exchange can be restored by replacing most of the internal sodium with potassium or by preparing the cells so that they contain much more orthophosphate (P(i)) than ATP.6. Ghosts containing sodium in low concentration, potassium in high concentration and with a low [ATP]/([ADP].[P(i)]) ratio show a greater ouabain-sensitive loss of sodium into potassium-free media than into media containing potassium; i.e. external potassium reduces ouabain-sensitive sodium efflux.7. The effect of P(i) is not the result of competitive inhibition of the transport ATPase since P(i) at the concentrations used does not inhibit ATPase activity in fragmented ghosts.

Respiration and phosphorylation of mitochondria isolated from the skeletal muscle of diabetic and normal subjects.

A group of diabetics, well controlled as far as blood sugar, free fatty acids and acetoacetate were concerned but with a high frequency of diabetic angiopathy in different organs, was compared with a group of controls in terms of the respiration and phosphorylation reactions in mitochondria isolated from skeletal muscle. Two keto-acidotic patients were also studied. The reactions studied were: respiration with glutamate or pyruvate plus malate as substrates, and also ratios of esterified inorganic orthophosphate to oxygen consumed and respiratory control with these substrates. The respiratory control in the absence of adenosine triphosphate was measured for pyruvate plus malate, and also the influence of serum albumin on this. Adenosine triphosphatase was measured under basal conditions and after maximal activation with 2,4-dinitrophenol. No certain differences were found between the non-ketotic diabetics and controls. Caution must be exercised in the interpretation of certain of the results because of difficulties in standardization of the mitochondrial isolation procedure in a patient material with wide ranges of age, infiltration of fibrous material in muscles, atrophy of muscles and other factors.

Studies on the Transfer of Incomplete Polypeptide Chains across Rat Liver Microsomal Membranes in Vitro

Abstract A rat liver microsomal system in vitro was used to study the transport of protein from the attached ribosomes to the contents of the microsomal vesicle. Puromycin caused a release of the protein from the ribosomes and the released protein was transferred to the contents of the microsomal vesicle. The transfer of nascent protein across the microsomal membrane was not accompanied by any changes in phospholipid metabolism and occurred in the absence of adenosine triphosphate and of any added enzyme and also at 0°. The only requirement needed for transport of the nascent protein across the microsomal membrane is that it be released from the ribosomes.

Interaction of Actin and Myosin during Clearing

SUPERPRECIPITATION of actomyosin has long been thought to involve both binding of the actin to the myosin and activation of the myosin adenosine triphosphatase (ATPase) by actin in the presence of magnesium1. The clearing response, on the other hand, has been reported to result from dissociation of the actomyosin with a concomitant loss of this ATPase activation2. In both situations, the physically observable binding and the activation of the myosin ATPase by actin were thought to change in parallel3, but some recent observations have been difficult to reconcile with this conclusion. In investigating the effect of tropomyosin B on actomyosin, A. M. Katz4 noted that actin can activate the myosin ATPase without superprecipitation occurring. In other investigations, Leadbeater and Perry5, and Perry and Cotterill6, found that actin activates the ATPase of heavy meromyosin under conditions where viscosity measurements indicated that the two proteins might be dissociated. Extrapolating these data to the actomyosin system, Perry et al. argued that the interaction of actin and myosin which results in activation of the myosin ATPase is not accompanied by physical interaction as strong as that which occurs between the two proteins in the absence of adenosine triphosphate (ATP). Maruyama and Gergely2, however, noted that actomyosin undergoes a brief viscosity increase simultaneously with the rise in ATPase just before superprecipitation, which suggests that strong physical interaction does accompany the activation of the myosin ATPase by actin.

Brain Mitochondria: IV. THE ACTIVATION OF FATTY ACIDS IN BOVINE BRAIN MITOCHONDRIA

Abstract Fatty acid oxidation was studied in the presence or absence of adenosine triphosphate with the use of sodium octanoate-1-14C as substrate. In the absence of ATP, fatty acid oxidation proceeded at a rate 5 to 30% of that obtained in the presence of optimal ATP. The rate of oxidation was partially inhibited by the uncoupling agents dinitrophenol, Dicumarol, or arsenate, completely inhibited by cyanide or antimycin A, and stimulated by oligomycin or azide. The dinitrophenol inhibition was not reversed by oligomycin, although the arsenate inhibition was reversed by oligomycin. Fatty acid oxidation was only slightly inhibited by arsenite, suggesting that substrate level phosphorylation makes only a small contribution to fatty acid activation. In the presence of ATP or ADP, fatty acid oxidation was inhibited both by uncoupling agents and by oligomycin. The oligomycin inhibition was reversed by increasing concentrations of ATP. After preincubation with glucose and hexokinase, the rate of fatty acid oxidation in the absence of ATP was only slightly reduced. Addition of oligomycin reversed this inhibition. Fluoride ion inhibited fatty acid oxidation in the presence of ATP or ADP but had no effect on the oxidative rate in the absence of nucleotides. These results indicate that fatty acids are activated in brain mitochondria by two different mechanisms: (a) an ATP-linked reaction which used primarily the ATP produced during respiration and (b) a system involving high energy intermediates of oxidative phosphorylation.

Niacin Metabolism in Mycobacteria1,2

Human tubercle bacilli, as compared with other mycobacteria, produce a large amount of in the culture medium (1). The niacin (2), which distinguishes human tubercle bacilli biochemically from other mycobacteria, is based on this difference in production. Albertson and Moat (3) reported that niacin-14C was formed from aspartic acid-14C and suggested that was formed by the condensation of a four-carbon dicarboxylic acid with a three-carbon compound. Konno and co-workers (4, 5), using cellfree extracts of mycobacteria, reported that quinolinic acid was a precursor of ribonucleotide (deamido-NMN). The enzyme activity that converts quinolinic acid to deamidoNMN was found to parallel production. Nakamura, Nishizuka, and Hayaishi (6), using a purified beef liver extract system, demonstrated the regulation of nicotinamide adenine dinucleotide (NAD) biosynthesis by adenosine triphosphate (ATP). In the absence of ATP, deamido-NMN is converted to ribonucleoside and eventually to niacin. However, when a sufficient amount of ATP is available, and ribonucleoside are utilized for the synthesis of deamido-NMN, and no accumulates. The production of excess by human strains of mycobacteria suggested that a similar mechanism might be operative, and the studies reported here were designed to test this hypothesis.

HISTOCHEMICAL DEMONSTRATION OF UPTAKE OF EXOGENOUS NOREPINEPHRINE BY ADRENERGIC FIBERS IN VITRO.

Adrenergic fibers in isolated iris preparations were demonstrated histochemically by means of the formaldehyde condensation technique. In the presence of adenosine triphosphate (ATP) there was a net uptake of norepinephrine added in vitro by the adrenergic fibers producing a striking increase in fluorescence. In the absence of ATP only a slight increase in fluorescence was observed when the tissue was exposed to high concentrations of norepinephrine.

ATP-stimulated adenylic acid deaminase activity at site of aqueous humor production

A specific 5-adenylic acid (5-AMP) deaminase was studied in preparations of hog ciliary process tissue. It was determined that while the activity of this deaminase is enhanced by adenosinetriphosphate (ATP), this activity is not completely blocked in the absence of ATP. Deamination proceeds only at the expense of 5-AMP, but a myokinase-like-active system may be prepared, such that 5-AMP is added to the substrate pool. In this latter case total levels of deamination are generally reflective of added 5-AMP and myokinase-like reaction accumulated 5-AMP. The general acceptance of ciliary process tissue as the site of aqueous humor production leads one to a reasonable conclusion that the very active 5-AMP deaminase studied plays some as yet unexplained role in the dynamics of aqueous secretion. Possibilities concerning the specific role(s) played are discussed. Functional analogy between aqueous humor and cerebrospinal fluid production is again suggested by the fact that an adenine nucleotide deaminase is found also in choroid plexus tissue.

Peptide-bond synthesis in higher plants. II. Studies on the mechanism of synthesis of γ-glutamylcysteine

An enzyme has been purified some 50-fold from wheat germ extracts which will catalyze the synthesis of γ-glutamylcysteine from glutamic acid and cysteine. The system has an absolute requirement for adenosine triphosphate (ATP) and for magnesium and potassium ions. No other univalent or bivalent metals could be found to effectively replace magnesium and potassium. The enzyme alone does not catalyze any signifycant exchange of orthophosphate-P 32 with ATP, but rapidly catalyzes such an exchange in the presence of glutamate. Cysteine reduces this glutamate-induced exchange. Conversely, ADP 32 exchanges with ATP in the presence of the enzyme alone, with the amino acids having only a slight inhibitory effect on this exchange. The enzyme also catalyzes the exchange of cysteine-S 35 with γ-glutamylcysteine in the absence of ATP. The nature of the phosphate exchange would seem to preclude the formation of a glutamyl phosphate as an intermediate in the peptide synthesis, but is consistent with the formation of enzyme-bound intermediates.

Thermal enzymes. III. Apyrase from a thermophilic bacterium

An enzyme, apyrase, obtained from thermophile No. 2184 possesses heat stability at 65 °C., the temperature preferred by the organism for growth. Apyrase from the thermophile has an activation energy of 9600 cal./ mole. Its Michaelis constant is 0.00065 at 60 °C. The enzyme is inhibited by fluoride, citrate, and copper, but is activated by magnesium. Apyrase from a mesophilic bacterium, although possessing a fair amount of stability in the presence of substrate, does not have the resistance to heat possessed by the thermophile enzyme. The apyrase from potato is inactivated readily on heating in the absence of adenosine triphosphate.