Biomedical subjects

K B Storey

Publications and source records attributed to K B Storey.

At least 145 records · Page 8Linked to original sources

PubMed1991

Where is the glycolytic complex? A critical evaluation of present data from muscle tissue.

Associations between glycolytic enzymes and subcellular structures have been interpreted as presenting a novel mechanism of glycolytic control; reversible enzyme binding to subcellular structural components is believed to regulate enzyme activity in vivo through the formation of a multi-enzyme complex. However, three lines of evidence suggest that enzyme binding to cellular structures is not involved in the control of glycolysis. (i) Calculations of the distribution of glycolytic enzymes under the physiological cellular conditions of higher ionic strength and higher enzyme concentrations indicate that a large multi-enzyme complex would not exist. (ii) In many cases, binding to subcellular structures is accompanied by changes in enzyme kinetic parameters brought about by allosteric modification, but these changes often inhibit enzyme activity. (iii) In the case where formation of binary enzyme/enzyme complexes activates enzymes, the overall increase in flux through the enzyme reaction is negligible.

Actins↗

PubMed1991

Glucose and caffeine regulation of liver glycogen phosphorylase activity in the freeze-tolerant wood frog Rana sylvatica.

We have examined the effect of glucose and caffeine inhibition on the activity of liver glycogen phosphorylase a from the freeze-tolerant frog Rana sylvatica. Kinetic studies indicate that this enzyme exhibits similar sensitivity to glucose inhibition (glucose dissociation constant = 12.5 mM) as the mammalian enzyme. Little inhibition (less than 25%) was observed at normal glucose concentrations (1-5 mM), while significant inhibition (60-95%) occurred at glucose concentrations (50-500 mM) present in freezing-exposed animals. These results favour the hypothesis that in the normal state glucose regulates phosphorylase activity primarily through the promotion of dephosphorylation of phosphorylase a, whereas during freezing regulation is achieved through phosphorylase a inactivation. The caffeine dissociation constant (0.93 mM) and the degree of synergism between caffeine and glucose (interaction factor, alpha = 0.14) were also similar to that observed for the mammalian enzyme. Hence, if a caffeine-like ligand exists in vivo, it must be in low enough amounts during freezing to allow sufficient phosphorylase a activity for high glucose production.

Animals↗

PubMed1991

Inactivation of 6-phosphofructo-2-kinase during anaerobiosis in the marine whelk Busycon canaliculatum.

6-Phosphofructo-2-kinase (PFK-2) was analyzed in four organs of the anoxia-tolerant marine gastropod mollusk Busycon canaliculatum. Whelk PFK-2 resembled the nonhepatic enzyme from mammals with highest activity occurring in gill (22 pmol.min-1.g-1). Hepatopancreas PFK-2 was purified over 8,000-fold to a final specific activity of 11 mU/mg protein (at 20 degrees C) and gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a dimer with a native molecular mass of 142 kDa and a subunit molecular mass of 67 kDa. The purified enzyme showed negligible fructose-2,6-bisphosphatase (FBPase-2) activity, although the activity ratio of PFK-2 to FBPase-2 was 0.625 in crude extracts. In response to environmental anoxia, the activity of PFK-2 dropped in all organs to 34-56% of the corresponding aerobic value (half-time was 2 h in gill), and the Michaelis constant for fructose 6-phosphate increased by 50% (to 92 microM in gill). These changes paralleled decreases in organ fructose 2,6-bisphosphate concentration and pyruvate kinase activity and contribute to the overall glycolytic rate depression induced by anoxia in this facultative anaerobe. In vitro treatment of the anoxic form of hepatopancreas PFK-2 with alkaline phosphatase increased enzyme activity, suggesting that the aerobic and anoxic enzyme forms are interconverted by reversible protein phosphorylation. However, the protein kinase involved in this process is not yet known; incubation of aerobic PFK-2 with Mg-ATP plus adenosine 3',5'-cyclic monophosphate-dependent protein kinase or protein kinase C did not alter enzyme activity.

Anaerobiosis↗

PubMed1991

The effect of enzyme-enzyme complexes on the overall glycolytic rate in vivo.

Recent studies have demonstrated that most glycolytic enzymes can reversibly associate to form heterogeneous enzyme-enzyme (binary) complexes in vitro. However, kinetic analysis of these complexes has shown that the individual enzymes have a varied response to complex formation: some enzymes are inhibited, some are activated and some are unaffected. In order to determine the potential role of binary complexes in regulating glycolytic flux, we have mathematically calculated enzyme distributions and activities using data from in vitro binding and kinetic studies. These calculations suggest that, overall, formation of binary complexes would lower flux through phosphofructokinase and aldolase, would increase flux through glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase, and would not affect flux through triosephosphate isomerase, phosphoglycerate kinase and pyruvate kinase. The implications of these results are discussed with respect to the effect of complex formation on overall glycolytic flux and on the flux through individual enzyme loci.

Enzymes↗

PubMed1990

Phosphofructokinase from a vertebrate facultative anaerobe: effects of temperature and anoxia on the kinetic parameters of the purified enzyme from turtle white muscle.

The effects of low temperature and anoxia were determined on phosphofructokinase (PFK) purified from white skeletal muscle of the freshwater turtle, Pseudemys scripta. These effects were assayed by comparing PFK kinetic constants measured at a high (20 degrees C) and low (6 degrees C) temperature using enzyme obtained from animals held under normoxic and anoxic conditions. When assayed at 20 degrees C, PFK from anoxic animals had a lower Ka for phosphate, a lower Ka for AMP and showed no inhibition with increasing concentrations of ATP (up to 10 mM) when compared to enzyme from normoxic animals. At 6 degrees C, anoxic enzyme had a higher Km for fructose 6-phosphate and a higher I50 value for citrate with respect to normoxic enzyme. Decreasing temperature also had a differential effect on PFK kinetic parameters depending on the source of the enzyme. When normoxic enzymes were compared at 20 and 6 degrees C, the enzyme measured at 6 degrees C showed a lower Km for ATP and a lower Ka for AMP. Comparison of anoxic enzymes at these two temperatures showed that anoxic PFK at 6 degrees C had a higher Ka for phosphate, a higher Ka for AMP, and a larger Hill coefficient. A comparison of maximal velocities at varying temperature showed that normoxic enzyme (Q10 = 2.22) was more temperature sensitive than the anoxic enzyme (Q10 = 1.80). It is possible to interconvert the normoxic and anoxic forms of PFK by incubating normoxic enzyme with the active subunit of protein kinase, suggesting that the kinetic changes observed during anoxia resulted from enzyme phosphorylation. These data are discussed with respect to the mechanisms underlying white muscle function during diving and hibernation in red-eared turtles.

Adenosine Triphosphate↗

PubMed1990

Immobilization of amyloglucosidase using two forms of polyurethane polymer.

Amyloglucosidase was covalently immobilized using two hydrophilic prepolymers: Hypol FHP 2002 (creates foams) and Hypol FHP 8190H (creates gels). The foamable prepolymer was superior as a support for enzyme immobilization. The percent activity immobilized in the polyurethane foams was 25 +/- 1.5%. Large substrates (greater than 200,000 daltons in mol wt) were hydrolyzed as effectively as smaller ones by the immobilized enzyme. The Km value of the foam-immobilized enzyme increased from 0.76 mg/mL (free) to 0.86 mg/mL (immobilized), whereas the Vmax dropped from 90.9 (free) to 12.4 nmol glucose/min/mL (immobilized). The long-term (2 mo) storage stability of amyloglucosidase was enhanced by immobilization in foams (70% activity retained; free enzyme only retained 50%). Immobilization also improved the enzyme stability to various denaturing agents (sodium chloride, urea, and ethanol). The immobilized enzyme exhibited increased stability compared to the free enzyme at high temperatures (95 degrees C). Both glycogen and starch could be utilized by the immobilized enzyme, indicating that this technique could prove useful for starch hydrolysis.

Enzyme Stability↗

PubMed1990

Ice nucleating activity in the blood of the freeze-tolerant frog, Rana sylvatica.

Although the presence of antifreeze and ice nucleating agents in the hemolymph of insects has been well documented, there have been no reports of either of these types of agent in vertebrates. The technique of differential scanning calorimetry was used to examine the blood, serum, and plasma of a freeze-tolerant frog, Rana sylvatica, for the presence of antifreeze protein activity. Results demonstrate the absence of antifreeze protein but the presence of an ice nucleating agent that may serve as a functional component of the overwintering strategy of this species. Ice nucleating activity was detected in samples of cell-free blood, serum, and plasma, suggesting that the agent is a soluble component and possibly plasma protein. To our knowledge, the identification of ice nucleating activity in this freeze-tolerant vertebrate is novel.

Adaptation, Physiological↗

PubMed1990

Regulation of coenzyme utilization by mitochondrial NAD(P)-dependent malic enzyme.

1. Skeletal muscle mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1. 39, L-malate:NAD+ oxidoreductase (decarboxylating)] from herring could use both coenzymes, NAD and NADP, in a similar manner. 2. The coenzyme preference of mitochondrial NAD(P)-dependent malic enzyme was probed using dual wavelength spectroscopy and pairing the natural coenzymes, NAD or NADP with their respective thionicotinamide analogues, s-NADP or s-NAD, that have absorbance maxima in reduced forms at 400 nm. 3. s-NAD and s-NADP were found to be good alternate substrates for NAD(P)-dependent malic enzyme, the apparent Km values for the thioderivatives were similar to those of the corresponding natural coenzymes. 4. ATP produced greater inhibition of the NAD or s-NAD linked reactions than of the NADP or s-NADP-linked reactions of skeletal muscle mitochondrial NAD(P)-dependent malic enzyme. 5. At 5 mM malate concentration and in the presence of 2 mM ATP the NADP-linked reaction is favoured and the activity ratios, V(s-NADP)/V(NAD) or V(NADP)/V(s-NAD), are 6 and 26, respectively.

Adenosine Triphosphate↗

PubMed1990

Frozen and alive.

A variety of animals freeze solid during the winter months and thaw in the spring. This natural ability to survive freezing may yield clues to the cryopreservation of human tissue.

Adenosine Triphosphate↗

PubMed1990

Metabolic rate depression and biochemical adaptation in anaerobiosis, hibernation and estivation.

For many animals, the best defense against harsh environmental conditions is an escape to a hypometabolic or dormant state. Facultative metabolic rate depression is the common adaptive strategy of anaerobiosis, hibernation, and estivation, as well as a number of other arrested states. By reducing metabolic rate by a factor ranging from 5 to 100 fold or more, animals gain a comparable extension of survival time that can support months or even years of dormancy. The present review focuses on the molecular control mechanisms that regulate and coordinate cellular metabolism for the transition into dormancy. These include reversible control over the activity state of enzymes via protein phosphorylation or dephosphorylation reactions, pathway regulation via the association or dissociation of particle-bound enzyme complexes, and fructose-2,6-bisphosphate regulation of the use of carbohydrate reserves for biosynthetic purposes. These mechanisms, their interactions, and the regulatory signals (e.g., second messenger molecules, pH) that coordinate them form a common molecular basis for metabolic depression in anoxia-tolerant vertebrates (goldfish, turtles) and invertebrates (marine molluscs), hibernation in small mammals, and estivation in land snails and terrestrial toads.

Adaptation, Physiological↗

PubMed1990

Biochemistry of natural freeze tolerance in animals: molecular adaptations and applications to cryopreservation.

For a wide variety of animals, winter survival in cold climates includes the ability to tolerate ice formation in extracellular body fluids. Among terrestrially hibernating vertebrates, freeze tolerance has been documented for five amphibian and two reptile species. These species may survive for days or weeks in a frozen state with no breathing and no heart beat, and with up to 65% of total body water as extracellular ice. The biochemical mechanisms involved in natural freeze tolerance include (i) the regulation of extracellular ice formation by proteinaceous ice nucleators in body fluids, (ii) the accumulation of high concentrations of low molecular weight carbohydrates as cryoprotectants to regulate cell volume reduction during freezing and stabilize macromolecular structure, and (iii) a well-developed ischemia tolerance that supports the survival of individual organs while frozen. The present article focuses on recent advances in our understanding of the biochemistry of natural freeze tolerance in lower vertebrates and the application of these studies to the improvement of cryopreservation technology for transplantable mammalian organs.

Adaptation, Physiological↗

PubMed1990

Life in a frozen state: adaptive strategies for natural freeze tolerance in amphibians and reptiles.

Winter survival for various species of amphibians and reptiles that hibernate on land depends on freeze tolerance, the ability to survive for long periods of time with up to 65% of total body water as extracellular ice. Freeze tolerance has been described for four species of frogs, one salamander, and hatchlings of the painted turtle. A very limited tolerance also occurs in garter snakes. Studies of freeze tolerance in vertebrates have primarily focused on the wood frog Rana sylvatica and have assessed the regulation of cryoprotectant synthesis, cryoprotectant action in freezing preservation of isolated cells and tissues, metabolism and energetics under the ischemic conditions imposed by freezing, and the role of ice-nucleating agents in blood. The adaptations that preserve life at subzero temperatures for these animals illustrate the principles of vertebrate organ cryopreservation and may have important applications in the development of technology for the freezing preservation of transplantable human organs.

Adaptation, Physiological↗

PubMed1989

The regulation of 6-phosphofructo-1-kinase by insulin and glucagon in isolated hepatocytes of the American eel.

Kinetic characteristics of American eel liver 6-phosphofructo-1-kinase (PFK-1) and the effects of porcine insulin, bovine glucagon, and dibutyryl-cAMP were studied. At 0.1 mM ATP, kinetics were sigmoidal with respect to fructose-6-phosphate (F-6-P) concentrations and the S0.5 (F-6-P) increased with higher ATP concentrations. At 2 mM F-6-P, optimal ATP concentrations were 0.1 mM, with maximal inhibition at 0.5 mM. Fructose 2,6-bisphosphate (Fru-2,6-P2) offset ATP inhibition and activated the enzyme, changing F-6-P kinetic curves from sigmoidal to hyperbolic. At 2 mM F-6-P and 0.1 mM ATP the Fru-2,6-P2 activation curve was hyperbolic with a Ka of approximately 1 microM. In isolated hepatocytes, porcine insulin decreased the sensitivity of PFK-1 to ATP, an effect that was offset when bovine glucagon was also present. Insulin, alone and with glucagon, increased the Fru-2,6-P2 activation ratio. In the presence of glucagon, insulin increased Fru-2,6-P2 concentrations in hepatocytes. These effects suggest that PFK-1 is a potential regulatory point for hormones in the control of carbohydrate metabolism in the American eel liver.

Anguilla↗

PubMed1989

Regulation of glycolytic enzymes during anoxia in the turtle Pseudemys scripta.

The glycolytic enzymes glycogen phosphorylase, phosphofructokinase (PFK), and pyruvate kinase (PK) were assessed in liver, heart, red muscle, and white muscle of aerobic and 5-h anoxic turtles (Pseudemys scripta) for changes in total activity and kinetic parameters. Anoxia induced statistically significant changes in these glycolytic enzymes in each of the four organs assayed. Compared with normoxic controls, anoxic liver showed a 3.3-fold increase in glycogen phosphorylase activity, a 1.5-fold increase in the PFK I50 value for citrate (concentration that inhibits initial activity by 50%), a 1.5-fold increase in the PFK Michaelis constant (Km) value for fructose 6-phosphate (P), and an increased maximal activity of PK. Anoxic heart muscle showed a 2.6-fold decrease in glycogen phosphorylase activity and, for PFK, a 1.7-fold decrease in the Km value for ATP and a twofold increase in the I50 value for citrate. In anoxic white muscle, PFK showed a fivefold lower Km value for fructose-6-P and a threefold lower activator concentration producing half-maximal activation (A50) for potassium phosphate than the aerobic enzyme form. Changes in anoxic white muscle PK included a twofold increase in the Km value for ADP and a 1.7-fold decrease in the I50 value for alanine. In red muscle, anoxia affected only the Km value for ATP, which was 50% higher than the value for the aerobic enzyme form. Fructose 2,6-diphosphate (P2) levels also decreased in heart muscle and increased in red and white muscle during anoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Aerobiosis↗

PubMed1988

Purification of phosphofructokinase using transition-state analogue affinity chromatography.

A novel purification of phosphofructokinase has been achieved in a two step process using ion-exchange affinity chromatography and a transition-state analogue affinity column matrix. The procedure can be performed in one day, and gives a 25% yield of the starting material. The transition-state analogue chromatography is carried out using an ADP-agarose column in the presence of fructose 6-phosphate, magnesium ions and nitrate ions. In the presence of nitrate ion plus substrate, phosphofructokinase binds immobilized ADP while other proteins pass through the column. Previous studies with creatine kinase have shown that the nitrate ion mimics the planar phosphate in the transition state resulting in a complex which is stable under the relatively high ionic strength of the column buffer. This permits the elution of phosphofructokinase in a single peak of high specific activity. This column typically results in a 20-30 fold increase in specific activity with only a small loss of activity.

Animals↗

PubMed1988

Reevaluation of the "glycolytic complex" in muscle: a multitechnique approach using trout white muscle.

Preliminary characterization of the "glycolytic complex," formed in trout white muscle, revealed that phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are bound to particulate matter largely by ionic interactions; increasing neutral salt or charged metabolite concentrations released bound PFK and GAPDH. GAPDH was consistently solubilized at lower salt concentrations, indicating that it is not bound as tightly as PFK, but both enzymes were readily solubilized at physiological concentrations of salts and metabolites. pH titrations indicated that PFK binding is dependent on group(s) with a pKa of 7.3 in 30 mM imidazole. PFK binding increased at lower pH values; at 150 mM KCl the apparent pKa value is 6.5. Experiments with polyethylene glycol 8000 (PEG), which is used to mimic the high in vivo protein concentrations under in vitro conditions, showed that the binding of PFK and GAPDH increased with increasing PEG concentrations. Interestingly, at 5% PEG, only the PFK binding response depended on the ionic composition of the medium--with increased binding occurring at the pH of the exhausted muscle and decreased binding at control pH values. These results suggested that only PFK reversibly bound to cellular structures in response to changing conditions and disagrees with previous studies showing binding of several glycolytic enzymes as measured using the dilution method (F. M. Clarke, F.D. Shaw, and D.J. Morton (1980) Biochem. J. 186, 105-109). In order to determine whether artifactual binding was measured by the dilution method, two new methodologies were employed to measure enzyme binding in vivo: (a) whole muscle slices were pressed to quickly extrude cellular juice, and (b) muscle strips were finely minced and centrifuged to liberate cytoplasmic contents. Both methods indicated that, under physiological conditions, up to 70% of the total cellular phosphofructokinase may be bound, but other glycolytic enzymes are bound to a lesser extent (10-30%). This result contrasts those obtained with the dilution method, and suggests that dilution of cellular contents may result in an overestimation of the percentage of enzyme associated with cellular structures; this is dramatically shown for glyceraldehyde-3-phosphate dehydrogenase. The viability of the glycolytic complex in trout white muscle is discussed in light of the decreased binding measured using these new methodologies.

Actomyosin↗

PubMed1988

Electrophoretic analysis of liver glycogen phosphorylase activation in the freeze-tolerant wood frog.

As an adaptation for overwinter survival, the wood frog, Rana sylvatica is able to tolerate the freezing of extracellular body fluids. Tolerance is made possible by the production of very high amounts of glucose in liver which is then sent to other organs where it acts as a cryoprotectant. Cryoprotectant synthesis is under the control of glycogen phosphorylase which in turn is activated in response to ice formation. To determine the mechanism of phosphorylase activation, a quantitative analysis of phosphorylase protein concentration and enzymatic activity in liver was carried out following separation of the phosphorylated a and nonphosphorylated b forms of the enzyme on native polyacrylamide gels. The results suggest that in gels, the b form is completely inactive, even in the presence of AMP and sodium sulfate, whereas the a form is active and stimulated 3-fold by these substances. Further, phosphorylase activation appears to arise solely from conversion of the b to a form of the enzyme without an increase in phosphorylase concentration or activation of a second isozyme. The quantitative analysis presented here should prove generally useful as a simple and rapid method for examining the physiological and genetic regulation of phosphorylase in animal cells.

Animals↗

PubMed1988

Anoxic brain function: molecular mechanisms of metabolic depression.

An examination of the kinetic parameters of phosphofructokinase, pyruvate kinase and glycogen phosphorylase, and the cellular concentration of fructose 2,6-bisphosphate during anoxia in the turtle Pseudemys scripta showed that the total activity of glycogen phosphorylase, and the phosphofructokinase inhibition constants for citrate and ATP were decreased in anoxic turtle brain. These results suggest that the ability of turtle brain to survive extended periods of anoxia is the result of metabolic rate depression regulated, at the molecular level, by enzyme inactivation through anoxia-induced covalent modification.

Adenosine Triphosphate↗