Enzymes of energy metabolism from a vertebrate facultative anaerobe, Pseudemys scripta. Turtle heart phosphofructokinase.
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Adenine Nucleotides↗
Biomedical subjects
K B Storey
Publications and source records attributed to K B Storey.
Enzymes of energy metabolism in a vertebrate facultative anaerobe, Pseudemys scripta. Turtle heart pyruvate kinase.
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Adaptation, Physiological↗
Immobilization of glucose isomerase onto granular chicken bone.
Glucose isomerase was immobilized onto granular chicken bone (BIOBONE) by adsorption. The amount of activity bound relative to an equal amount of free enzyme was 32 +/- 1%, with the estimated specific activity decreasing from 11.1 +/- 0.7 to 3.9 +/- 0.5 U/mg protein with immobilization. Compared with the free enzyme, immobilized glucose isomerase showed a threefold increase in the Km for fructose and a fivefold decrease in Vmax. High operating temperatures were possible (greater than 55 degrees C), but continuous use and long-term storage studies showed gradual losses of activity. Both the binding and the activity of the bone-immobilized enzyme were highly resistant to treatments with detergent, ethanol, and KCl. Studies to determine mass transfer limitation effects on immobilized glucose isomerase showed that these were insignificant for this system.
Adhesiveness↗
Immobilization of amyloglucosidase onto granular chicken bone.
Amyloglucosidase was immobilized onto granular chicken bone (BIOBONE) by noncovalent interactions. The amount of activity bound relative to an equal amount of free enzyme was 13.6 +/- 0.4%. The estimated specific activity for amyloglucosidase decreased from 75.3 +/- 0.8 to 43.5 +/- 9.6 U/mg protein upon immobilization. The Km value of the bone-immobilized enzyme using glycogen as substrate increased from 3.04 +/- 0.38 mg/mL (free) to 9.04 +/- 1.51 mg/mL (immobilized), but Km showed no change upon immobilization when starches were used as substrates. A decrease in Vmax values occurred upon enzyme immobilization for all substrates, but this largely reflected the percentage of enzyme initially bound to the bone. Immobilization also improved enzyme stability in the presence of various additives (e.g., detergent, KCl, and ethanol) or under low or high pH reaction conditions. Bound amyloglucosidase maintained high activity (greater than 90%) following five cycles of continuous use at moderate (23 degrees C) and high (55 degrees C) temperatures. Data derived from Lineweaver-Burk and Arrhenius plots indicated that substrate and product diffusion limitation were minimal.
Adhesiveness↗
Substrate- and effector-induced conformational changes in phosphofructokinase from white muscle of rainbow trout (Oncorhynchus mykiss): a fluorescence study.
Results of activity and spectral studies using fluorescence show that AMP and fructose 2,6-bisphosphate (F2,6P2) activate muscle phosphofructokinase (PFK) from rainbow trout (Oncorhynchus mykiss) through specific and similar conformational changes. Inorganic compounds, such as ammonium and phosphate ions, also increase enzyme activity allosterically; however, the structural alterations in the enzyme caused by these effectors are quite different from those caused by AMP and F2,6P2. No effects of the inorganic compounds on the environment of tryptophan residues of the enzyme were observed. Mg-ATP, a substrate of the enzyme, acts as an allosteric inhibitor at high concentrations. Although Mg-ATP and citrate inhibit the enzyme activity in a synergistic way, the conformational effects of these negative effectors are different. Mg-ATP caused a drastic decrease in fluorescence intensity of the enzyme, whereas citrate did not.
Adenosine Monophosphate↗
Purification and molecular properties of glycogen phosphorylase b from trout white muscle.
Glycogen phosphorylase b (EC 2.4.1.1) was isolated from white skeletal muscle of rainbow trout (Oncorhynchus mykiss) and purified 214-fold to a final specific activity of 135 U/mg protein (assayed in the direction of glycogen breakdown at 21 degrees C) by using glycogen--concanavalin A, DEAE-Sephadex, and 3',5'-cAMP affinity chromatography. Purified phosphorylase b was a dimer with a native molecular weight of 193,000 and a subunit molecular weight of 87,000. Michaelis constants for glycogen, phosphate, and AMP were 128 microM, 31 mM and 142 microM, respectively, at pH 7.2; maximum activity of the enzyme was obtained at pH 7.5 and 25 degrees C. Glucose and ATP behaved as phosphorylase b inhibitors; glucose inhibition decreased at lower pH values. IMP did not affect the enzyme. The catalytic properties of trout phosphorylase b indicate that the enzyme would be virtually inactive at the physiological concentration of substrates and activators found in resting trout white muscle, but changes in cellular pH, ATP, Pi, and AMP levels during burst muscle work could allow phosphorylase b to augment phosphorylase a activity and make a substantial contribution to overall glycogenolysis in working trout white muscle.
Adenosine Monophosphate↗
Control of glycogenolysis and effects of exercise on phosphorylase kinase and cAMP-dependent protein kinase in rainbow trout organs.
To analyze the mechanisms of glycogen phosphorylase control in organs of the rainbow trout Oncorhynchus mykiss, activities of glycogen phosphorylase kinase (GPK) and cAMP-dependent protein kinase (PKA), as well as levels of cAMP, were quantified. The complete cascade for activating glycogen phosphorylase was present in trout organs and all components were activated in white skeletal muscle and liver during exhaustive swimming exercise. GPK and PKA showed the highest activities in the liver, being three- and four-fold higher than corresponding activities in white muscle. Exercise stimulated a 60% increase in GPK activity in the liver and a 40% rise in white muscle. Furthermore, the amount of active PKA rose from 12 to 21% in the liver and from 32 to 57% in white muscle after exhaustive exercise and the cellular levels of cAMP increased by 50% in the liver and 70% in white muscle of exercised fish. Other organs (heart, gill, brain, kidney) showed little or no change in these parameters as a result of exhaustive exercise. GPK activity in liver, muscle, and heart extracts was strongly stimulated by in vitro incubation with the catalytic subunit of mammalian PKA, activity rising by 6- to 7-fold in white muscle extracts and 2- to 2.6-fold in liver and heart extracts. This occurred in extracts from both control and exercised fish and suggested that even in fish exercised to exhaustion, the maximal enzymatic potential for activation of glycogenolysis was not expressed.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Xanthine oxidase and xanthine dehydrogenase from an estivating land snail.
During arousal from estivation in land snails, Otala lactea, active metabolic functions are restored within minutes and oxygen consumption increases dramatically. During the transition from the hypoxic conditions of estivation to normoxia it is possible that xanthine oxidase (XO) in hepatopancreas contributes to the observed lipid peroxidation. Using a fluorometric assay that is based on the oxidation of pterin, the activities and some properties of XO and XO+XDH (sum of XO and xanthine dehydrogenase activities) were measured in hepatopancreas extracts. Km values for pterin for XO and XO+XDH were 9 and 6 microM, respectively, and the Km of XDH for methylene blue was 5 microM. Both XO+XDH and XO activities were inhibited by allopurinol (I50 = 2 microM), pre-incubation at 40 degrees C, and by 5 min H2O2 pre-exposure. Inclusion of azide in the reaction promoted a rise of approximately 70-fold in the inactivation power of H2O2 due to inhibition of high endogenous catalase activity. The I50 for H2O2 of XO+XDH and XO activities in the presence of azide was 0.04 and 0.11 mM, respectively. Unlike the situation for mammalian XO, a previous reduction of O. lactea XO (by pterin) was not necessary to make the enzyme susceptible to H2O2 effects. Interestingly, methylene blue partially prevented both heat- and H2O2-induced inactivation of XO+XDH activity. These data indicate that the formation of an enzyme-methylene blue complex induces protection against heat and oxidative damage at the FAD-active site. Both XO and XO+XDH activites were significantly higher in snails after 35 days of estivation compared with active snails 24 h after arousal from dormancy. The ratio of XO/(XO+XDH) activities was also slightly increased in estivating O. lactea (from 0.07 to 0.09; P < 0.025). XO activity was 0.03 nmol.min-1.mg protein-1 in estivating snails. Compared with hepatopancreas catalase, XO activity is probably too low to contribute significantly to the net generation of oxyradicals, and hence to peroxidative damage. Rather, the low potential of XO to induce oxidative stress may constitute an adaptive advantage for O. lactea during arousal periods.
Acclimatization↗