inosinic-acid and glucose-1-phosphate

In this study we have revised our original procedure of yeast metabolites extraction. We showed that: (a) less than 5% of intracellular metabolites leaks out during the step of rapid arrest of cellular metabolism by quenching yeast cells into a 60% methanol solution kept at -40 degrees C; and (b) with a few exception, the stability of metabolites were not altered during the 3 min boiling procedure in a buffered ethanol solution. However, there was a loss of external added metabolites of 5-30%, depending on the type of metabolites. This was mainly attributable to their retention on cellular debris after ethanol treatment, which prevented centrifugation of the cellular extracts before evaporation of ethanol. We further simplified our previous high-performance ionic chromatography (HPIC) techniques for easier, more reliable and robust quantitative measurements of organic acids, sugar phosphates and sugar nucleotides, and extended these techniques to purine and pyrimidine bases, using a variable wavelength detector set at 220 and 260 nm in tandem with a pulsed electrochemical or suppressed conductivity detector. These protocols were successfully applied to a glucose pulse to carbon-limited yeast cultures on purines metabolism. This study showed that glucose induced a fast activation of the purine salvage pathway, as indicated by a transient drop of ATP and ADP with a concomitant rise of IMP and inosine. This metabolic perturbation was accompanied by a rapid increase in the activity of the ISN1-encoded specific IMP-5'-nucleotidase. The mechanism of this activation remains to be determined.

Topics: Adenine Nucleotides; Analytic Sample Preparation Methods; Fructosephosphates; Fumarates; Glucose; Glucose-6-Phosphate; Glucosephosphates; Inosine; Inosine Monophosphate; Pyruvic Acid; Saccharomyces cerevisiae; Sugar Phosphates; Trehalose

The mechanism of activation of glycogen phosphorylase is incompletely understood, although adenosine and inosine nucleotides are known to be important allosteric activators. In this study the activation of glycogen phosphorylases a and b from bovine liver by adenosine 5'-monophosphate (AMP) and inosine 5'-monophosphate (IMP) has been investigated and the results compared with the activation of the muscle isozyme by the same nucleotides. Enzyme activity was determined by spectrophotometric measurement of inorganic phosphate produced in the phosphorylase-catalysed reaction of glycogen synthesis. Liver phosphorylase b binds both nucleotides non-co-operatively (Hill coefficients of 1.0 +/- 0.1), with changes in the maximum velocity to 75 or 80 mumol min-1 mg-1 in the presence of adenosine 5'-monophosphate or inosine 5'-monophosphate, respectively, but no change in the enzyme affinity towards the substrate, glucose-1-phosphate. Binding of glucose-1-phosphate is co-operative and the kinetic data have been fitted with the Monod-Wyman-Changeux model. Liver phosphorylase a has a maximum velocity similar to that of the b form in the presence of nucleotides. Binding of glucose-1-phosphate to the enzyme is non-co-operative (Hill coefficient of 1.0 +/- 0.1) and the affinities in the presence of the nucleotides (Michaelis constants of 28 +/- 0.2 mM or 27 +/- 0.2 mM for adenosine 5'-monophosphate or inosine 5'-monophosphate) are stronger than those of the b form. It is concluded that the activity of bovine liver phosphorylase a and b is similarly influenced by adenosine 5'-monophosphate or inosine 5'-monophosphate. The b form seems to behave like muscle phosphorylase b in response to inosine 5'-phosphate; however, the binding of adenosine 5'-phosphate does not induce the conformational change necessary to activate the liver enzyme, as occurs with the muscle isozyme.

Topics: Adenosine Monophosphate; Animals; Cattle; Enzyme Activation; Glucosephosphates; Inosine Monophosphate; Liver; Models, Chemical; Phosphorylase a; Phosphorylase b

The relationship between muscle glycogen concentration and the rate of glycogen breakdown during short, intense contraction has been investigated in man. Prior to the experiment, muscle glycogen content was manipulated by a combination of exercise and diet, and varied from 155 +/- 19 to 350 +/- 25 mmol kg-1 dry muscle (36-81 mmol kg-1 wet wt). The quadriceps femoris muscle was stimulated electrically at a frequency of 20 Hz for 1 min. The blood flow to the leg was occluded during the experiment and muscle biopsies were taken before and after 10, 30 and 60 s stimulation. Force development and glycogenolytic rate were maintained constant during electrical stimulation and similar in all conditions, irrespective of the initial glycogen concentration. The phosphorylase a fraction was increased after 10 s stimulation, but returned to the initial values at the end of the stimulation. Muscle ATP was unaltered during the first 30 s stimulation, but decreased thereafter. The decrease in ATP was accompanied by a stoichiometric increase in inosine monophosphate. Phosphocreatine decreased during stimulation and was almost depleted at the end of stimulation. Muscle lactate and glucose 6-phosphate (Glu 6-P) increased during stimulation. None of these changes was significantly affected by the reduced glycogen contents. It is concluded that the rate of muscle glycogen breakdown is not affected by the initial glycogen level in the range of 155 +/- 19 to 350 +/- 25 mmol kg-1 dry muscle.

Topics: Adenosine Triphosphate; Adolescent; Adult; Electric Stimulation; Fructosephosphates; Glucose-6-Phosphate; Glucosephosphates; Glycogen; Humans; Inosine Monophosphate; Lactates; Lactic Acid; Male; Muscles; Phosphocreatine; Phosphorylase a

Phosphorylase ab was prepared in vitro by partial phosphorylation of rabbit skeletal muscle phosphorylase b and was isolated by DEAE-Sephacel chromatography. Its phosphorylated and non-phosphorylated subunits could not be distinguished by different affinity to substrates, activators or inhibitors, indicating their coordinated function. In the absence of nucleotide activators, the Km values for Pi and glucose-1-P were 28 mM and 18 mM, respectively. Activity in the presence of 16 mM glucose-1-P was doubled by 10(-4) M AMP or 10(-3) M IMP, mainly by lowering the Km for glucose-1-P. Half-maximum activation was exerted by 2 microM AMP or 0.1 mM IMP. Activation by these nucleotides showed no cooperativity. Glucose exerted competitive inhibition with respect to glucose-1-P, while for the inhibition by glucose-6-P an allosteric mechanism is suggested; the appropriate Ki values were 4.5 mM and 1.5 mM, respectively. The Hill coefficient for glucose-1-P binding was about 1.0, even in the presence of glucose (up to 10 mM), but 10 mM glucose-6-P lowered it to 0.47, indicating a negative heterotropic cooperativity. Effective regulation of the activity of phosphorylase ab by physiological concentrations of Pi, AMP, IMP and glucose-6-P suggests its metabolic control under in vivo condition.

Topics: Adenosine Monophosphate; Allosteric Regulation; Animals; Binding, Competitive; Caffeine; Enzyme Activation; Glucose; Glucose-6-Phosphate; Glucosephosphates; Inosine Monophosphate; Kinetics; Muscles; Phosphorylase a; Phosphorylase b; Phosphorylases; Phosphorylation; Rabbits