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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The influence of fructose 2,6-bisphosphate on the activation of purified swine kidney phosphofructokinase as a function of the concentration of fructose 6P, ATP and citrate was investigated. The purified enzyme was nearly completely inhibited in the presence of 2 mM ATP. The addition of 20 nM fructose 2,6-P2 reversed the inhibition and restored more than 80% of the activity. In the absence of fructose 2,6-P2 the reaction showed a sigmoidal dependence on fructose-6-phosphate. The addition of 10 nM fructose 2,6-bisphosphate decreased the K0.5 for fructose 6-phosphate from 3 mM to 0.4 mM in the presence of 1.5 mM ATP. These results clearly show that fructose 2,6-bisphosphate increases the affinity of the enzyme for fructose 6-phosphate and decreases the inhibitory effect of ATP. The extent of inhibition by citrate was also significantly decreased in the presence of fructose 2,6-phosphate. The influence of various effectors of phosphofructokinase on the binding of ATP and fructose 6-P to the enzyme was examined in gel filtration studies. It was found that kidney phosphofructokinase binds 5.6 moles of fructose 6-P per mole of enzyme, which corresponds to about one site per subunit of tetrameric enzyme. The KD for fructose 6-P was 13 microM and in the presence of 0.5 mM ATP it increased to 27 microM. The addition of 0.3 mM citrate also increased the KD for fructose 6-P to about 40 microM. AMP, 10 microM, decreased the KD to 5 microM and the addition of fructose 2,6-phosphate decreased the KD for fructose 6-P to 0.9 microM. The addition of these compounds did not effect the maximal amount of fructose 6-P bound to the enzyme, which indicated that the binding site for these compounds might be near, but was not identical to the fructose 6-P binding site. The enzyme bound a maximum of about 12.5 moles of ATP per mole, which corresponds to 3 moles per subunit. The KD of the site with the highest affinity for ATP was 4 microM, and it increased to 15 microM in the presence of fructose 2,6-bisphosphate. The addition of 50 microM fructose 1,6-bisphosphate increased the KD for ATP to 5.9 microM. AMP increased the KD to 5.9 microM whereas 0.3 mM citrate decreased the KD for ATP to about 2 microM.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Binding and regulatory properties of phosphofructokinase from swine kidney. 609 5

The purpose of this study was to confirm the relationship between cyclic AMP(cAMP) level in plasma and changes of hormones concentrations in blood, during and after physical exercise. The results were as follows: At rest, plasma cAMP were 23.1 p mole/ml on the average and decreased after glucose loading. The level in plasma increased in proportion to the intensity of exercises. Under the 50% condition of the maximal intensity, cAMP level in plasma was about 40 p mole/ml and the contents of both thyroxine and growth hormone in serum clearly increased. And, under the 70% of the maximal, the contents of both adrenaline and noradrenaline in serum as well as that of cAMP in plasma increased. Plasma cAMP level also increased by prolongation of exercise (ca 45 p mole/ml). And when exercise lasted over 1.5 hrs, plasma glucagon level began to rise. The effect of carbohydrate load to lower the levels of plasma cAMP were also found during physical exercise. These results suggested that the cAMP level in plasma was affected, not only by the some regulating factors of glycolytic activities such as adrenaline and glucagon, but also by the production of thyroxine and growth hormone at the onset of exercise.
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PMID:[Effect of exercise on plasma cyclic AMP]. 609 99

Somatostatin binding and the ability to inhibit cyclic AMP stimulated protein kinase were investigated utilizing isolated pancreatic islets, anterior pituitary plasma membranes, adipocytes, erythrocyte ghosts, hepatic plasma membranes, and anterior pituitary secretion vesicles. Three types of response were observed. With type I response, somatostatin bound specifically to pancreatic islets and anterior pituitary secretion vesicles and inhibited cyclic AMP stimulated protein kinase. In type II response, adipocytes and anterior pituitary plasma membranes exhibited somatostatin binding but no effect of the ligand on the kinase. In erythrocyte membrane ghosts and hepatic plasma membranes, there was neither specific somatostatin binding nor protein kinase inhibition (type III response). The absence of somatostatin binding in erythrocytes or hepatic plasma membranes cannot be explained by degradation of the ligand per se. Secretory vesicles isolated from the anterior pituitary gland bind somatostatin with an average affinity which exceeds that observed in plasma membrane (for pituitary secretory vesicles Kd1 = 8.5 X 10(-8)M, Kd2 = 5.2 X 10(-7)M; for pituitary membranes Kd1 = 1.9 X 10(-8)M, Kd2 = 8.1 X 10(-7)M). The molar concentration of high affinity binding sites (Ro) for plasma membranes was 6.9 X 10(-10)M; for secretory vesicles 3.6 X 10(-9)M. Calculated in terms of somatostatin binding per U 5'nucleotidase activity, the binding for plasma membranes becomes 8.4 X 10(-14) mole/U 5'nucleotidase; secretory vesicles 4.4 X 10(-13) mole/U 5'nucleotidase. Thus, secretory vesicles are fivefold richer in high affinity receptor sites than plasma membranes. It is suggested that in order for somatostatin to act, both a receptor and an effector unit must be present. In the case of tissues secreting polypeptide hormones by granule extrusion, the secretory vesicle may possess both the receptor and the effector units. It is postulated that during the process of fusion of the plasma and secretory vesicle membranes, a high affinity binding site for somatostatin is incorporated into the plasma membrane, thereby allowing somatostatin to act at a specific locus in the cell in inhibiting hormone release.
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PMID:The relationship between somatostatin binding and cyclic AMP-stimulated protein kinase inhibition. 610 15

A protein kinase which phosphorylates and inactivates acetyl-CoA carboxylase has been purified to apparent homogeneity from rat liver. The kinase was found to exist in two forms: bound to carboxylase in a complex or in a free form that is in different stages of aggregation over a wide range of molecular weights. The purification of the kinase involved first partial purification of acetyl-CoA carboxylase through polyethylene glycol precipitation and DEAE-cellulose chromatography. The kinase was then separated from acetyl-CoA carboxylase by Sepharose 2B chromatography. The molecular weight of the kinase subunit was 170,000 as determined by sodium dodecyl sulfate-gel electrophoresis. The incorporation of 1 mol of phosphate/mole of carboxylase subunit caused complete inactivation of the carboxylase. Acetyl-CoA carboxylase, inactivated by the kinase, can be dephosphorylated and reactivated when incubated with phosphorylase phosphatase. The Km values of the kinase for acetyl-CoA carboxylase and ATP are 90 nM and 20 microM, respectively. The kinase was found to be cyclic AMP-independent, but activated by CoA. The protein kinase can phosphorylate acetyl-CoA carboxylase, protamine, and histones, but could not act on hydroxymethylglutaryl-CoA reductase or phosphorylase b.
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PMID:Purification and properties of a kinase which phosphorylates and inactivates acetyl-CoA carboxylase. 612 Jan 70

Microtubules have been isolated from immature (3-4 weeks' old) and old (11-13 years' old) bovine brains. Quantitative studies revealed that the concentration of extractable microtubule protein per gram of wet brain decreased from 0.47 mg (immature animals) to 0.34 mg (old animals). The major components of microtubule protein (tubulin and high-molecular-weight microtubule-associated proteins) do not undergo an age-correlated change. Determination of the endogenous protein kinase activity revealed that the activity associated with "immature" calf brain microtubules was six times higher than the activity present in "old" preparations. In contrast, the stimulatory effect of cyclic AMP on protein phosphorylation in microtubules from old bovine brains exceeds nine-fold the value obtained from immature animals. After addition of casein (exogenous acceptor), the basal activities increased in both preparations without altering the age-correlated difference in the specific activity. By comparing the radioactivity pattern of sodium dodecyl sulfate polyacrylamide gels after autophosphorylation of microtubule protein with [gamma-32P]ATP, 1.5 moles of phosphate per mole of high-molecular-weight microtubule-associated protein were estimated to be incorporated in preparations from immature animals and 0.9 mole of phosphate per mole of associated protein in the experiments with "old" microtubule protein. Adenosine triphosphatase activity, associated with the high-molecular-weight microtubule-associated protein 1, was determined to be 15% reduced in preparations from old animals, compared to the activity in "young" preparations. In contrast, the guanosine triphosphatase activity increased five-fold during ageing; the higher activity of this enzyme was observed both during the initial and the steady-state phases of microtubule formation.
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PMID:Age-dependent alterations of microtubule-associated enzyme activities from bovine brain (protein kinase, adenosine triphosphatase, guanosine triphosphatase). 613 97

The regulation of glycogen phosphorylase and glycogen breakdown in human skeletal muscle has been investigated using the needle biopsy technique. Preliminary studies showed that the activity of phosphorylase in vitro was dependent upon the concentration of inorganic phosphate (Pi) used in the assay system. The Km of phosphorylase a for Pi was found to be 26.2 mmol/l, and that of (a+b) (assayed in the presence of saturating AMP) was 6.8 mmol/l. Because of the difference in Km the apparent percentage of a to (a+b) activity varies with the Pi concentration used in the assay system. Phosphorylase a and (a+b) activities were therefore adjusted to saturating Pi concentrations. The ratio of the activities in this case is independent of the Pi concentration and constitutes a minimal estimate of the fraction of phosphorylase molecules in the a form. The fraction of phosphorylase in the a form in resting muscle was as a mean 22%. Despite nearly a quarter of the phosphorylase being in the a form glycogenolytic activity is extremely low. It is proposed that the concentration of Pi at the active site of the enzyme is low compared to the Km for this of either form of the enzyme, and is limiting to activity. A Pi concentration in resting muscle of 1-3 mmol/l was calculated. During epinephrine infusion at rest 90% of the phosphorylase was transformed to the a form but only a moderate increase in the glycogenolytic rate occurred. This rate approximated to 5-10% of the maximum rate of the enzyme (Vmaxa). During prolonged epinephrine infusion the glycogenolytic rate decreased despite the continuance of 90% or more of the phosphorylase in the a form. In contrast to epinephrine infusion prolonged ischemia resulted in a decrease in the mole fraction of phosphorylase a and simultaneously in an increase of the glycogenolytic rate. During isometric and dynamic exercise there was a rapid transformation of phosphorylase b to a paralleled by pronounced increase in the rate of glycogen breakdown. The increased rate of glycogenolysis during isometric exercise was close to the Vmax of phosphorylase a in vivo. When either form of exercise was continued to fatigue/exhaustion, a re-transformation of phosphorylase a to b was observed. During dynamic exercise cAMP in the muscle increased two fold. This increase was blocked by the prior administration of propranolol.+
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PMID:The regulation of glycogen phosphorylase and glycogen breakdown in human skeletal muscle. 613 34

Simultaneous studies on the secretory response of amylase and the neurotransmitter receptors of rat parotid gland, after brief treatment with agonists, showed selective alteration in beta-adrenoceptors with specific change in amylase secretion, suggesting a regulatory role of the receptors in the secretory response. The beta-adrenergic agonist (+/-)-isoprenaline (IPR) stimulated amylase secretion from rat parotid tissues much more than did the same concentration of an alpha-adrenergic or cholinergic agonist. The stimulatory effects of IPR were studied by pre-treating rat parotid tissues with IPR for 10 min and then incubating the tissue in fresh medium for 10 min. Pre-treatment with 10 microM-IPR for 10 min resulted in increased amylase secretion during further incubation with IPR and also in a lower EC50 value of amylase secretion for IPR. This treatment also resulted in selective changes in the number and affinity of beta-adrenoceptors, assessed by measuring binding of [3H]dihydroalprenolol (DHA): the maximal binding sites increased from 286/357 f-mole to mg protein and the IC50 value (the concentration for 50% inhibition of specific [3H]DHA binding) of beta-agonists, not antagonists, decreased significantly. An increase in the period of pre-treatment with IPR to 30 min resulted in a decrease in the maximal binding sites of beta-adrenoceptors and a decrease in amylase secretion during further incubation with IPR. Experiments with other agonists showed that supersensitivity of the secretory response was induced specifically by beta-agonists. Binding studies with [3H]WB-4101 and [3H]quinuclidinyl benzilate showed that alpha-adrenoceptors and muscarinic ACh receptors in rat parotid did not change under the conditions tested. The alteration in beta-adrenoceptors was parallel with a change in amylase secretion after IPR pre-treatment, but not with a change in cyclic AMP content.
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PMID:beta-Adrenoceptor alterations coupled with secretory response in rat parotid tissue. 619 86

Using mixed anhydride of AMP and mesitylene carboxylic acid carrying a fluorescent or radioactive label, it was found that the previously established irreversible inhibition of myosin ATPase is a result of protein covalent binding to the nucleotide residue of the inhibitor. The stoichiometry of the affinity labelling of heavy meromyosin is 1 mole of nucleotide residue of mixed anhydride per 1 mole of protein, that of subfragment 1-0.5 mole per 1 mole of protein. The lack of irreversible inhibition of the ATPase activity of subfragment 1 is suggestive of an existence of a regulatory substrate-binding site in the myosin molecule.
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PMID:[Affinity modification of heavy meromyosin and subfragment 1 by mixed anhydrides of [14C] AMP, epsilon AMP and mesitylene carboxylic acid]. 621 86

Rabbit brain phosphofructokinase was purified to homogeneity by a rapid procedure involving affinity chromatography and gel filtration. The enzyme consists of hybrids of the three phosphofructokinase subunit types C, A, and B. The molecular weights of these subunits are 86,000, 84,000, and 80,000, respectively; they are present in brain phosphofructokinase in a ratio of approximately 5:4:1.5. The enzyme as isolated from rabbit brain contains 0.16-0.18 mol phosphate per mole of subunit; another 0.4-0.5 mol phosphate per mole subunit can be incorporated in vitro in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase. The initial rate of phosphorylation is increased by fructose 2,6-bisphosphate or AMP and decreased by citrate or high concentrations of ammonium sulfate. All three subunit types are phosphorylated in vitro, and the phosphorylation site on each subunit is sensitive to cleavage by trypsin at a terminal region of each subunit. However, these sites show different relative rates of phosphorylation in vitro in the presence of ammonium sulfate. In vitro phosphorylation of brain phosphofructokinase had no affect on specific activity, inhibition by ATP, or activation by fructose 2,6-bisphosphate.
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PMID:Isozyme composition and phosphorylation of brain phosphofructokinase. 623 51

Autophosphorylation of cyclic AMP-dependent pig brain protein kinase has been detected. Up to 1,5 moles of gamma-32P are transferred from [gamma-32P]ATP to the dimer of the regulatory subunit. The autophosphorylation reaction is Mg2+-dependent and occurs at a high rate: more than 50% of the radioactive label is incorporated during the first minute of incubation at 30 degrees. The pH dependence of this reaction differs from that of the phosphotransferase reaction. The phosphoholoenzyme is more sensitive to cyclic AMP than the dephosphoholoenzyme; however, both forms bind up to 2 moles of 3H-cyclic AMP per 1 mole of the holoenzyme. The activation and dissociation constants for both forms of the holoenzyme have been calculated. The autophosphorylation reaction has been shown to occur via an intramolecular mechanism; the phosphorylation of the regulatory subunit can occur only within the holoenzyme. The increase in the concentration of cyclic AMP causes the latter to produce an inhibitory effect on autophosphorylation. The regulatory action of autophosphorylation on cyclic AMP-dependent protein kinases is discussed.
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PMID:[Autophosphorylation of cyclic AMP-dependent protein kinase from pig brain]. 624 75


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