PubMed Health⌕ Search

Biomedical subjects

K B Storey

Publications and source records attributed to K B Storey.

At least 127 records · Page 7Linked to original sources
PubMed1993

Characterization of alpha-glucosidases from rainbow trout liver.

Three forms of alpha-glucosidase were separable in trout liver by DE-52 chromatography, isoelectrofocusing, and gel filtration. Two forms showed acid pH optima, hydrolyzed glycogen, maltose, and 4-methylumbelliferyl alpha-glucoside, and were associated with the lysosomes. The third enzyme form was largely associated with microsomes and was present in the highest activity; it showed a neutral pH optimum and did not hydrolyze glycogen. Molecular weights were 181 +/- 2, 130 +/- 1.5, and 365 +/- 3 kDa for the acid types I and II and the neutral enzyme, respectively. Maximal activities and kinetic and physical properties of the three enzymes were compared in liver samples from control, resting fish versus fish that underwent exhaustive swimming exercise. The properties of liver acid alpha-glucosidase type I changed significantly in response to exercise; maximal activity increased by 80% and Km values for both glycogen and maltose dropped by 50% in exercised, versus control, fish. Under the same exercise condition, liver glycogen phosphorylase a activity also increased 4.4-fold. These changes in alpha-glucosidase type I are consistent with an activation of the enzyme, in parallel with phosphorylase activation, under physiological stress conditions that promote glycogenolysis and glucose export from liver. These results are, we believe, the first demonstration of the activation of the glucosidic route of glycogenolysis in response to a physiological stress and suggest that the glucosidic route has a significant role to play in complementing the phosphorolytic pathway in the metabolic response by liver to the fuel demands of working muscle.

Animals↗
PubMed1993

Antioxidant defenses in the tolerance of freezing and anoxia by garter snakes.

The garter snake Thamnophis sirtalis parietalis can readily tolerate several hours of freezing or anoxia exposure. Both stresses halt oxygen availability to tissues and to endure these stresses snakes must cope with potential oxidative stress arising as a result of the ischemic/anoxic condition followed by reperfusion of aerated blood during recovery. To determine whether antioxidant defenses are important for freezing and anoxia survival, we monitored the activities of antioxidant enzymes and the levels of glutathione (GSH and GSSG) during freezing (5 h at -2.5 degrees C) and anoxia (10 h under N2 gas at 5 degrees C) exposures in three organs (muscle, liver, and lung) of snakes. Freezing resulted in a significant rise in the activity of muscle and lung catalase (by 183 and 63%) and in muscle glutathione peroxidase (52%). Anoxia enhanced muscle and liver superoxide dismutase activities (by 59 and 118%) and also caused a 57% increase in muscle GSH levels. The increase in muscle GSH concentration in anoxia (from 0.45 to 0.71 mM) could also stimulate muscle glutathione peroxidase activity in vivo by 1.5-fold because of its low affinity for GSH (Km = 11 mM). The ratio of GSSG/GSH was not affected by experimental state in any tissue, suggesting that oxidative stress did not occur during the freezing or anoxic exposure. Rather, H2O2- and O2(-)-detoxification systems may be activated in preparation for possible oxygen free radical overgeneration during thawing or reoxygenation. Antioxidant defenses appear to be part of the adaptive machinery for reptilian tolerance of freezing and anoxia.

Adaptation, Physiological↗
PubMed1993

Adaptations of plasma membrane glucose transport facilitate cryoprotectant distribution in freeze-tolerant frogs.

Natural freeze tolerance in several anuran species involves the accumulation of high concentrations of glucose as a cryoprotectant in body fluids and tissues. The present study identifies an important new molecular mechanism supporting freeze tolerance, an adaptive increase in the capacity for facilitated transport of cryoprotectant across plasma membranes by increasing the numbers and/or activity of plasma membrane glucose transporters. Glucose transport by membranes isolated from liver and skeletal muscle was analyzed in two species, the freeze-tolerant wood frog Rana sylvatica and the freeze-intolerant leopard frog Rana pipiens. Membranes from both liver and muscle of R. sylvatica displayed much higher rates of carrier-mediated glucose transport, measured by a rapid filtration technique, compared with corresponding rates for R. pipiens membranes. For the liver Vmax values for glucose transport by membrane vesicles were 69 +/- 18 and 8.4 +/- 2.3 nmol.mg protein-1.s-1 at 10 degrees C for R. sylvatica and R. pipiens, respectively. This difference was due primarily to a greater number of glucose transporters in R. sylvatica liver membranes; the total number of transporter sites, determined by cytochalasin B binding, was 4.7-fold higher in the freeze-tolerant species. For muscle membranes, the Vmax for glucose transport was 4.9 +/- 1 and 0.6 +/- 0.16 nmol.mg-1 x s-1 at 22 degrees C for R. sylvatica and R. pipiens, respectively. However, in muscle there were no differences in the number of membrane transporters between species.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Nitrophenylphosphatase↗
PubMed1993

Dehydration tolerance in wood frogs: a new perspective on development of amphibian freeze tolerance.

Wood frogs, Rana sylvatica, tolerate the loss of 50-60% of total body water during experimental dehydration. The rate of water loss for unprotected frogs is the same whether animals are frozen (at -2 degrees C) or unfrozen (at 1 degrees C) but is greatly reduced when frogs are frozen under a protective layer of moss. Dehydrational death could occur in as little as 7-9 days for unprotected animals; this indicates the importance for winter survival of selecting well-protected and damp hibernation sites. Prior dehydration affected the cooling and freezing properties of frogs, reducing supercooling point and the amount of ice formed after 24 h at -2 degrees C and acting synergistically with freezing exposure in stimulating cryoprotectant synthesis. Analysis of the effects of controlled dehydration at 5 degrees C showed that changes in body water content alone (without freezing) stimulated liver glycogenolysis and the export of high concentrations of glucose into blood and other organs. Autumn-collected frogs dehydrated to 50% of total body water lost showed glucose levels of 165-1,409 nmol/mg protein in different organs, increases of 9- to 313-fold compared with control values and reaching final levels very similar to those induced by freezing exposure. The data support the proposal that various adaptations for natural freeze tolerance may have been derived from preexisting mechanisms for dealing with water stress in amphibians and that cell volume change may be one of the signals involved in triggering and sustaining molecular adaptations (e.g., cryoprotectant output) that support freezing survival.

Adaptation, Physiological↗
PubMed1993

De novo protein synthesis and protein phosphorylation during anoxia and recovery in the red-eared turtle.

Changes in de novo protein synthesis and protein phosphorylation were monitored during anoxia and recovery in the red-eared slider Trachemys (= Pseudemys) scripta elegans. Time courses of 35S-radiolabeled methionine incorporation into acid-precipitable material showed an increase up to 5 h postinjection and remained constant after this time. Comparison of the total and acid-precipitable 35S label incorporation into tissues from 20-h control, anoxic, and recovering animals showed differences between these groups: total radioactivity in brain was 2.9-fold lower in recovering turtles, whereas protein-associated radioactivity was 2.4-fold higher in anoxic liver, 2.3-fold lower in recovering skeletal muscle, and 3.7-fold lower in recovering brain tissue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled proteins showed the existence of a newly synthesized protein band (relative molecular mass = 72 kDa) that was apparent only in 20-h recovering liver and skeletal muscle. Use of 32P labeling to monitor changes in protein phosphorylation patterns during anoxia revealed 1.6-, 1.4-, and 1.5-fold increases in 32P incorporation in anoxic brain, heart, and liver, respectively. Changes in protein phosphorylation were localized to the plasma membrane and cytosolic fractions in brain and to the cytosolic fraction in liver.

Animals↗
PubMed1992

Phosphofructokinase from white muscle of the rainbow trout, Oncorhynchus mykiss: purification and properties.

Phosphofructokinase was purified and characterized from the white skeletal muscle of rainbow trout Oncorhynchus mykiss. Purification involved three steps: ion-exchange chromatography on hydroxyapatite and affinity chromatography on phosphocellulose and ATP-agarose. A final specific activity of 75 units per mg of protein at 22 degrees C and pH 7.2 with 40% recovery was obtained. The purified enzyme gave a single band on SDS-PAGE with a subunit molecular mass of 76.5 +/- 0.6 kDa. Based on gel filtration analysis, the active form of the enzyme was found to be composed of six identical subunits. A high isoelectric point (7.1) was found for this enzyme. Arrhenius plots of the enzyme activity showed a sharp transition at 15-16 degrees C. The pH optimum of the enzyme was 8.0-8.5 at physiological level of ATP and positive modulators shifted the optimum to lower pH values. Amino-acid analysis revealed a lower content of the aromatic residues Phe, Tyr and Trp and higher level of Ser residue than in the rabbit muscle enzyme.

Amino Acids↗
PubMed1992

A kinetic description of sequential, reversible, Michaelis-Menten reactions: practical application of theory to metabolic pathways.

Equations are presented which describe a linear coupled system of reactions that utilize a single substrate and convert it to product by way of several intermediate enzyme catalysed steps. The present analysis extends previous results by assuming that the enzymes obey reversible Michaelis-Menten kinetics. In order for the system to reach steady state one must assume that the initial substrate concentration and the final product concentration are buffered to a constant value. Using the present analysis it can be shown that the system will not enter a steady state if the maximal velocity of any forward reaction is less than the steady state flux through the system. This condition represents a practical test for determining if a system will enter steady state but is valid only when the rate of the primary enzyme is not affected allosterically be intermediates in the pathway. The equations are used to analyse a portion of the rat liver glycogenic pathway that catalyses the conversion of glucose to fructose 1,6-bisphosphate.

Animals↗
PubMed1992

Bound and determined: a computer program for making buffers of defined ion concentrations.

A computer program that allows the preparation of buffers containing known concentrations of metal-ligand complexes at defined pH values and temperatures is described. Ligands are defined as compounds that bind metals and may include AMP, ADP, ATP, GMP, GDP, GTP, EGTA, EDTA, BAPTA, phosphate, sulfate, chloride, monocarboxylic acids, dicarboxylic acids, organophosphates, and/or citric acid. Metals may include sodium, potassium, magnesium, calcium, and/or manganese. The program uses association constants corrected for temperature and ionic strength so that solutions between 0 and 40 degrees C and between pH values of 4 and 10 can be defined. The program can perform the following: (i) calculate the concentration of all metal-ligand complexes when total metal and total ligand concentrations are known, (ii) calculate the concentration of metal ion required to make a solution of known free metal ion concentration when total ligand concentrations are known, (iii) calculate the concentration of ligand required to make a solution of known free metal ion concentration when total metal concentrations are known, and (iv) calculate the total concentrations of metal and ligand required to make a buffer of known metal-ligand concentration. Options i-iii are useful for making buffers of defined free metal ion concentrations; option iv is useful for making buffers of defined metal-nucleotide concentrations.

Adenosine Triphosphate↗
PubMed1992

Biochemical modification of plasma ice nucleating activity in a freeze-tolerant frog.

Recently, we reported the presence of ice nucleating activity, apparently proteinaceous, in the plasma of a freeze-tolerant frog, Rana sylvatica, collected in autumn and spring. Although this protein has not been purified, its ice nucleating behavior can act as an internal reference for tests that attempt to modify its ability to nucleate ice formation. If the addition of a chemical reagent alters the temperature of ice crystallization compared with the control, it can be assumed that protein modification may have occurred. The ice nucleating protein in R. sylvatica showed resistance to proteolysis with four different proteases although there was a significant reduction in the temperatures of nucleation with these treatments (ANOVA P less than 0.001). However, ice nucleating activity was lost when plasma was treated with the addition of urea or N-bromosuccinimide. Modification of protein sulphydryl groups with iodoacetamide did not affect the crystallization temperature (Tc) but treatment with iodoacetic acid resulted in a significant increase in Tc of plasma. An abrupt loss of ice nucleating ability was observed in plasma samples after heating above 87 degrees C. Anomalous potentiation of ice nucleating activity occurred when the plasma was heated to and held at temperatures between 67-75 degrees C.

Acclimatization↗
PubMed1992

Hormonal effects on glycogen metabolism in isolated hepatocytes of a freeze-tolerant frog.

To determine whether specific hormonal responses were involved in the production of cryoprotectant (glucose) by liver of the freeze tolerant wood frog, Rana sylvatica, metabolically active hepatocytes were isolated in reasonable yields (mean 20.1 +/- 1.30% SEM, n = 29) by in situ liver perfusion with collagenase. Freshly isolated cells from autumn-collected frogs contained large amounts of glycogen (650 mumol glucosyl units/g packed cells) and produced glucose from this endogenous reserve at a rate of 10 mumol g-1 hr-1 at 0 degrees. Glucose output from cells was highly responsive to the addition of hormones; rates of glucose release increased 2.1-, 1.7-, and 1.7-fold with the addition of 10(-7) M bovine glucagon, 10(-7) M epinephrine, and 5 x 10(-6) M dibutyryl-cyclic AMP, respectively. Norepinephrine, 5-hydroxytryptamine, and bovine insulin were without effect at 0.1 microM/l. Hormone stimulation of glucose release was correlated with an increase in both the total activity and the percentage a of glycogen phosphorylase in hepatocytes. However, none of the hormones tested affected the kinetic properties of hepatocyte pyruvate kinase, suggesting the absence of covalent modification control of the enzyme. The data indicate that the freezing-stimulated production of large quantities of glucose as a cryoprotectant by R. sylvatica liver does not involve qualitative differences in the hormonal control of liver glycogenolysis, compared with other lower vertebrates. However, quantitative differences were seen, such as the much greater phosphorylase activity, 4.38 +/- 0.33 mumol min-1 g-1 packed cells, in freshly isolated R. sylvatica hepatocytes compared with 0.36 +/- 0.06 mumol min-1 g-1 in Rana pipiens hepatocytes.

Animals↗
PubMed1992

Subcellular enzyme binding and the regulation of glycolysis in anoxic turtle brain.

The association of glycolytic enzymes with the particulate fraction of the cell was assessed in the brain of the freshwater turtle, Pseudemys scripta elegans, using three different methodologies. Each method showed that a large percentage of each of eight enzymes was bound in brain. The effect of environmental anoxia (5 or 20 h submergence in N2-bubbled water at 7 degrees C) on the distribution of enzymes between free and bound states was analyzed. All three techniques showed a significant increase in the percentages of brain aldolase and glyceraldehyde-3-phosphate dehydrogenase bound during anoxia and no change in lactate dehydrogenase or creatine kinase binding. Two methodologies also showed an increase in the percent bound during anoxia for hexokinase, phosphofructokinase, and phosphoglycerate kinase. An increased association of glycolytic enzymes with structural elements of the cell during anoxia may physically position the glycolytic pathway to facilitate coupling between this ATP-generating pathway and ATP-utilizing processes, such as membrane ion pumps.

Animals↗
PubMed1992

Natural freezing survival by painted turtles Chrysemys picta marginata and C. picta bellii.

Hatchlings of both the Midland (Chrysemys picta marginata) and Western (C. picta bellii) subspecies of the painted turtle tolerate the freezing of extracellular body fluids while overwintering in terrestrial nests. Fall-collected hatchlings survived 3 days of continuous freezing at -2.5 degrees C, with ice contents of 43.5 +/- 1.0% of total body water (SE; n = 24) for C. picta marginata and 46.5 +/- 0.8% (n = 32) for C. picta bellii. Survival times dropped to 4-5 h when temperature was lowered to -4 degrees C, correlated with ice contents of greater than or equal to 50%. However, C. picta marginata tested immediately after excavation from nests in the spring showed greater freeze tolerance, with survival extending to 11 days at -2.5 degrees C and a higher mean ice content of 50.2 +/- 1.2% (n = 6). Spring hatchlings also had high supercooling points, -1.07 +/- 0.13 degrees C (n = 8), that dropped within 3 days to -4.83 +/- 0.83 degrees C (n = 4), suggesting a breakdown of endogenous ice-nucleating agents when hibernation ended. A search for possible cryoprotectants showed that both subspecies accumulated glucose and lactate in liver during freezing (net increase = 3-13 mumols/g wet wt); both also maintained large free amino acid pools in organs, with taurine making up 21-47% of the total.

Acclimatization↗
PubMed1992

Cryomicroscopic analysis of freezing in liver of the freeze-tolerant wood frog.

The technique of directional solidification coupled with low-temperature scanning electron microscopy was applied to analyze the freezing of liver slices from the freeze-tolerant frog Rana sylvatica. Micrographs of liver slices from 5 degrees C-acclimated frogs frozen on the directional stage to -7 degrees C showed continuous ice formed along an expanded vasculature with hepatocytes that were shrunken and virtually dehydrated. However, when frogs were given a survivable freezing exposure at -4 degrees C for 24 h, liver slices subsequently frozen in vitro at -7 degrees C were much less shrunken and the presence of intracellular ice crystals (formed when samples were plunged into liquid N2 before microscopy) demonstrated that ample free water remained in these hepatocytes at -7 degrees C. This reduced level of cell dehydration was correlated with the buildup of 280 +/- 61 mumol/g wet wt glucose as a cryoprotectant in liver during the -4 degrees C exposure in vivo. The study provides the first direct cytological analysis of the freezing process in an organ of a freeze-tolerant vertebrate and the first confirmation of the relationship between maintenance of a critical minimum cell volume and freezing survival by these animals.

Acclimatization↗
PubMed1992

Responses to freezing exposure of hatchling turtles Trachemys scripta elegans: factors influencing the development of freeze tolerance by reptiles.

Hatchling red-eared turtles Trachemys (= Pseudemys) scripta elegans (Wied) from a Louisiana population display a significant ability to withstand the freezing of extracellular body fluids. All animals survived at least 2 h of freezing at -2.5 or -4 degrees C. At -2.5 degrees C, survival declined to 50% after 6 h of freezing and no animals recovered after 24 h or longer, when mean ice content reached 54.7 +/- 1.4% of total body water. At -4 degrees C, all turtles recovered from 4 h of freezing exposure with a mean ice content of 49.6 +/- 2.4%, but survival dropped sharply thereafter with no animals recovering after 8 h, when ice content had reached 64.5 +/- 0.7%. Survival times were substantially shorter and percentage ice values greater than comparable values for hatchling painted turtles (Chrysemys picta (Schneider)) from northern populations subjected to identical freezing exposures. The ability to synthesize cryoprotectants in response to freezing was poorly developed in T. s. elegans; maximal accumulation of glucose was only 3.2 mumol g-1 wet mass in liver. Lactate content increased two- to threefold in oxygen-sensitive organs (heart and brain) during freezing, but levels of lactate and other putative cryoprotectants were unchanged in other organs. Total free amino acid content rose significantly in liver, muscle and blood during freezing; increased taurine concentration was primarily responsible for the changes in liver and blood. The capacity for freezing survival by T. s. elegans hatchlings from southern populations would be of limited use for hibernation in a cold climate, but the metabolic responses to freezing displayed by these animals might be enhanced by northern populations to increase their freeze tolerance.

Acclimatization↗
PubMed1991

Re-evaluation of the glycerol-3-phosphate dehydrogenase/L-lactate dehydrogenase enzyme system. Evidence against the direct transfer of NADH between active sites.

An investigation of the direct transfer of metabolites from rabbit muscle L-lactate dehydrogenase (LDH, EC 1.1.1.27) to glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.1.8) revealed discrepancies between theoretical predictions and experimental results. Measurements of the GPDH reaction rate at a fixed NADH concentration and in the presence of increasing LDH concentrations gave experimental results similar to those previously obtained by Srivastava, Smolen, Betts, Fukushima, Spivey & Bernhard [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6464-6468]. However, a mathematical solution of the direct-transfer-mechanism equations as described by Srivastava et al. (1989) showed that the direct-transfer model did not adequately describe the experimental behaviour of the reaction rate at increasing LDH concentrations. In addition, experiments designed to measure the formation of an LDH4.NADH.GPDH2 complex, predicted by the direct-transfer model, indicated that no significant formation of tertiary complex occurred. An examination of other kinetic models, developed to describe the LDH/GPDH/NADH system better, revealed that the experimental results may be best explained by assuming that free NADH, and not E1.NADH, is the sole substrate for GPDH. These results suggest that direct transfer of NADH between rabbit muscle LDH and GPDH does not occur in vitro.

Animals↗
PubMed1991

A quantitative evaluation of the effect of enzyme complexes on the glycolytic rate in vivo: mathematical modeling of the glycolytic complex.

The cellular distribution of free and bound glycolytic enzymes in vivo was estimated by means of a model based on previously determined association constants for individual binding interactions and in vivo protein concentrations. The calculations revealed that a significant proportion of the enzymes would be either associated with F-actin, or bound in binary enzyme-enzyme complexes in vivo. An analysis of the relative concentration, and relative activity, of F-actin-bound enzymes suggested that a complete glycolytic complex, composed of all enzymatic steps from phosphofructokinase (PFK) to lactate dehydrogenase (LDH) does not exist. This was indicated by a very low concentration of F-actin-associated phosphoglycerate kinase (PGK) and by a very low activity of F-actin bound aldolase and PGK; this model showed that aldolase and PGK would be absent from any F-actin bound complex. An analysis of soluble enzyme-enzyme associations indicated that formation of binary enzyme complexes may lead to an increased overall flux through glyceraldehyde 3-phosphate dehydrogenase and LDH, but would serve to decrease flux through PFK and aldolase. A 1.4-fold activation of PFK, which occurs when the soluble enzyme binds to F-actin, suggested that reversible binding of PFK to F-actin may represent a novel cellular mechanism for controlling glycolytic flux during periods of increased metabolic demand by controlling the key regulatory enzyme of glycolysis.

Actins↗
PubMed1991

The role of protein kinases in anoxia tolerance in facultative anaerobes: purification and characterization of a protein kinase that phosphorylates pyruvate kinase.

A protein kinase which phosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia-tolerant gastropod mollusc Busycon canaliculatum. Purification involved four steps: poly(ethylene glycol) fractionation, affinity chromatography on Blue agarose, ion-exchange chromatography on phosphocellulose and preparative isoelectric focusing (pI = 5.5). The activity was monitored by following changes in pyruvate kinase I50 values for L-alanine which have previously been linked to changes in the degree of enzyme phosphorylation. The correlation between enzyme phosphorylation and changes in the L-alanine inhibition constant was also directly demonstrated in the present paper by radioactively labelling PK with [tau-32P]ATP. The final purified protein kinase solution gave a single band on SDS-gel electrophoresis with a molecular weight of 37,000 +/- 2000. Kinetic analysis of the purified protein kinase (PK-kinase) showed a pH optimum of 7.0, an absolute requirement for magnesium ions (Km = 1.29 mM), a relatively high affinity for MgATP (Km = 57 microM), and inhibition by increasing salt concentrations (I50 = 55 mM KCl). The protein kinase activity was not affected by either spermine, heparin, cAMP, cGMP or concentrations of CaCl2 less than 10 mM. The enzyme did not phosphorylate either phosphofructokinase or glycogen phosphorylase, two enzymes that are also phosphorylated during anoxia in whelks. The purified enzyme is different from the catalytic subunit of cAMP-dependent protein kinase as shown by the inability of cAMP to stimulate the protein kinase at all stages of the preparation; cAMP did not activate either crude enzyme, the 7% poly(ethylene glycol) supernatant, or any of the column eluant peak fractions when measured by changes in pyruvate kinase kinetic parameters.

Adenosine Triphosphate↗