Biomedical subjects

K B Storey

Publications and source records attributed to K B Storey.

At least 91 records · Page 5Linked to original sources

PubMed1996

Liver protein kinase C isozymes: properties and enzyme role in a vertebrate facultative anaerobe.

Protein kinase C was purified to homogeneity from liver of the anoxia-tolerant turtle (Trachemys scripta elegans). Two isozymes were present and were identified as PKC alpha and PKC beta by hydroxylapatite chromatography and cross-reaction with specific antibodies to the mammalian isozymes. Kinetic characterization of the isozymes showed that both required phospholipids and Ca2+ for activation and both were inhibited by low concentrations of PKC inhibitors. The PKC alpha was activated more strongly by phosphatidylinositol and lysophosphatidylinositol compared with PKC beta. Treatment with trypsin did not activate turtle PKC isozymes, but generated inactive PKC beta, whereas PKC alpha was resistant to inactivation. Anoxia exposure of turtles in vivo, via submergence in N2-gassed water at 7 degrees C, altered the activity and subcellular distribution of PKC in liver. After 1 hr of anoxic exposure at 7 degrees C, the activity of membrane-bound PKC had increased by 2.4-fold and represented a translocation of 40% of PKC beta and more than 80% of PKC alpha from the cytosol to the membrane-associated fraction. With longer submergence, however, membrane-bound PKC activity was suppressed again. This two-phase response to anoxia by PKC suggests that an activation of PKC, through its translocation to the membrane, is important in mediating the initial metabolic responses to submergence, which include an activation of glycogenolysis during the hypoxia transition period. With sustained anoxia exposure, the subsequent reduction of PKC activity may be part of the overall mechanism of metabolic rate depression that allows endurance of prolonged anoxia.

Anaerobiosis↗

PubMed1996

Oxidative damage and antioxidants in Rana sylvatica, the freeze-tolerant wood frog.

Freeze-tolerant wood frogs (Rana sylvatica) must endure prolonged ischemia on freezing. Reperfusion on thawing brings with it the potential or oxidative damage due to reactive oxygen species formation, a well-known consequence of mammalian ischemia-reperfusion. To determine whether oxidative damage occurs during thawing and how frogs deal with this, we examined oxidative damage and antioxidant and prooxidant systems in tissues of Rana sylvatica and a nonfreezing relative, Rana pipiens. Glutathione status indicated little oxidative stress in tissues during freezing or thawing; an increase of the glutathione pool in the oxidized form was observed during freezing only in Rana sylvatica kidney (by 85%) and brain (by 33%). Oxidative damage to tissue lipids, measured as the levels of thiobarbituric acid-reactive substances and/or by an Fe(III)-xylenol orange assay, did not increase above control values pver a freeze-thaw time course. Correlative data showing increased activities of some antioxidant enzymes during freezing, notably glutathione peroxidase (increasing 1.2- to 2.5-fold), as well as constitutively higher activities of antioxidant enzymes and higher levels of glutathione in the freeze-tolerant species compared with Rana pipiens, suggest that antioxidant defenses play a key role in amphibian freeze tolerance.

Adaptation, Physiological↗

PubMed1996

Relationship between anoxia exposure and antioxidant status in the frog Rana pipiens.

The biochemical adaptations of cellular antioxidant defenses that permit anoxia-tolerant animals to deal effectively with rapid and large changes in oxygen availability, and hence oxidative stress, during transitions from anoxia to normoxia provide insights into the strategies of antioxidant defense that could help to minimize reperfusion injuries to mammalian organs after anoxia/ischemia stress. The present study analyzes the effects of 30 h anoxia exposure followed by reoxygenation on the antioxidant defenses (activities of five enzymes, glutathione status) and lipid peroxidation damage to organs of the leopard frog Rana pipiens (5 degrees C-adapted autumn frogs). Exposure to 30 h anoxia resulted in significant increases in the activities of skeletal muscle and heart catalase (by 53 and 47%), heart and brain glutathione peroxidase (by 75 and 30%), and brain glutathione S-transferase (by 66%). In most cases, enzyme activities had returned to the control values after 40 h aerobic recovery. Activities of superoxide dismutase and glutathione reductase were unaltered in all of the organs, and anoxia/recovery had no effect on any of the enzymes in liver. Glutathione equivalents (GSH-eq) were maintained in four organs during anoxia but decreased by 32% in brain during anoxia. Brain GSH-eq had recovered after 90 min reoxygenation, and, in addition, hepatic GSH-eq rose by 71% after 90 min reoxygenation. The ratio of oxidized glutathione to GSH-eq was also affected by anoxia in an organ-specific way. Lipid peroxidation, assessed as the content of thiobarbituric acid-reactive substances (TBARS), was unaltered in skeletal muscle and liver after 30 h anoxia exposure or short (25 and 90 min)- or long-term (40 h) periods of reoxygenation, indicating that cycles of natural and survivable anoxia/reoxygenation occur without significant increase in TBARS in selected organs. Overall, the data demonstrate that elements of the antioxidant system of R. pipiens are induced during anoxia exposures as a possible preparation for dealing with potentially harmful oxygen reperfusion stress.

Animals↗

PubMed1996

Signal transduction, second messenger, and protein kinase responses during freezing exposures in wood frogs.

Changes in the percentage of adenosine 3', 5'-cyclic monophosphate (cAMP)-dependent protein kinase present as the active catalytic subunit (PKAc) and in the levels of the second messengers cAMP, guanosine 3',5'-cyclic monophosphate (cGMP), and D-myo-inositol 1,4,5-trisphosphate (IP3) were quantified in tissues of the freeze-tolerant wood frog Rana sylvatica over the course of freezing at -2.5 degrees C and thawing at 5 degrees C. Freezing exposure rapidly raised liver cAMP concentration and %PKAc (by 2- and 6-fold, respectively) within 2 min postnucleation; both peaked and stabilized between 5 and 60 min postnucleation but declined with longer freezing. Other organs also showed elevated PKAc during freezing, particularly skeletal muscle. By contrast, cGMP concentration was reduced in muscle and kidney after 24 h of freezing but rose after thawing in muscle. Liver also showed a twofold elevation of cGMP during thawing. The protein kinase C (PKC) second messenger, IP3, rose throughout freezing in liver, reaching levels 11-fold higher than control values after 24 h of freezing. IP3 was also elevated in brain after 4 and 8 h of freezing. The different patterns of cAMP, protein kinase A (PKA), and IP3 changes in liver suggest that, whereas cAMP and PKA clearly mediate the rapid activation of glucose output as a cryoprotectant, IP3 and PKC may be involved instead with metabolic responses that deal with the consequences of long-term freezing, such as ischemia resistance or cell volume control.

Animals↗

PubMed1996

Biochemistry below 0 degrees C: nature's frozen vertebrates.

Although alien to man, the ability to endure the freezing of extracellular body fluids during the winter has developed in several species of terrestrially hibernating frogs and turtles as well as in many species of insects and other invertebrates. Wood frogs, for example, can endure freezing for at least 2 weeks with no breathing, no heart beat or blood circulation, and with up to 65% of their total body water as ice. Our studies are providing a comprehensive view of the requirements for natural freezing survival and of the physical and metabolic protection that must be offered for effective cryopreservation of vertebrate organs. Molecular mechanisms of natural freeze tolerance in lower vertebrates include: 1) control over ice crystal growth in plasma by ice nucleating proteins, 2) the accumulation of low molecular weight cryoprotectants to minimize intracellular dehydration and stabilize macromolecular components, and 3) good ischemia tolerance by all organs that may include metabolic arrest mechanisms to reduce organ energy requirements while frozen. Cryomicroscopy of tissue slices and magnetic resonance imaging (MRI) of whole animals is revealing the natural mode of ice propagation through an organism. MRI has also revealed that thawing is non-uniform; core organs (with high cryoprotectant levels) melt first, facilitating the early resumption of heart beat and blood circulation. Studies of the production and actions of the natural cryoprotectant, glucose, in frogs have shown its importance in maintaining a critical minimum cell volume in frozen organs and new work on the metabolic effects of whole body dehydration in 3 species of frogs has indicated that adaptations supporting freeze tolerance grew out of mechanisms that deal with desiccation resistance in amphibians. Studies of the regulation of cryoprotectant glucose synthesis by wood frog liver have shown the role of protein kinases and of alpha and beta adrenergic receptors in regulating the glycemic response, and of changes in membrane glucose transporter proteins to facilitate cryoprotectant distribution.

Adenosine Triphosphate↗

PubMed1996

Purification and characterization of a protein phosphatase that dephosphorylates pyruvate kinase in an anoxia tolerant animal.

A protein phosphatase that dephosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia tolerant gastropod mollusc Busycon canaliculatum. Purification involved three steps: negative chromatography through Blue Dextran and CM Sephadex, affinity chromatography on DEAE Sephadex and gel exclusion chromatography on Sephacryl S-400. Pyruvate kinase phosphatase (PK-Pase) activity was monitored by following changes in PK I50 values for L-alanine that had previously been linked to changes in the degree of PK phosphorylation. The purified PK-Pase gave a single band on SDS-polyacrylamide gel electrophoresis with a molecular weight of 41 +/- 1 kdaltons. Isoelectric focusing analysis showed that the PK-Pase had an isoelectric point of 4.2 +/- 0.1. Kinetic analysis showed that the enzyme was a Type 2C protein phosphatase with a pH optimum of 6.5. Maximal activity required the presence of magnesium ions (KM = 7.9 +/- 0.6 microM) although high concentrations of Mg2+ were inhibitory (I50 = 2.3 +/- 0.4 mM). The protein phosphatase activity was not affected by either spermine, cAMP, cGMP, potassium phosphate, tartrate, NaF, HgCl2, citrate or concentrations of CaCl2 less than 10 mM. The enzyme could also use ATP, ADP, and GTP as substrates.

Animals↗

PubMed1996

Multicatalytic proteinase activity in turtle liver: responses to anoxia stress and recovery.

Activities of the multicatalytic proteinase complex (MPC) were detected in turtle (Trachemys scripta elegans) liver. The ratio of peptidylglutamyl-peptide bond hydrolyzing, trypsin-like, and chymotrypsin-like activities was 6:2.7:1 for the MPC partially purified by Sepharose CL-6B gel filtration. Molecular mass of the turtle liver enzyme was 940 +/- 46 kD. Nondenaturing PAGE revealed a single band containing MPC activity reacting with peptide substrate. In vivo anoxia exposure (20 h submergence in N2-bubbled water) and subsequent 24 h aerobic recovery stimulated changes in liver protease activity. Peptidylglutamyl-peptide bond hydrolyzing activity of the partially purified MPC increased by 29% during aerobic recovery. Elevated MPC activity during recovery may serve to catabolize specific stress-related proteins or to remove proteins damaged by oxygen free radicals generated upon the reintroduction of oxygen.

Aerobiosis↗

PubMed1996

Oxidative stress: animal adaptations in nature.

As a consequence of aerobic life, an organism must deal with the continuous generation of reactive oxygen species (O2-, H202, .OH) as byproducts of metabolism and defend itself against the harm that these can do to cellular macromolecules. Organisms protect themselves from such damage with both enzymatic and nonenzymatic antioxidant defenses. However, the reperfusion injuries noted after ischemic insult in mammalian organs and ascribed to a burst of reactive oxygen species produced when oxygenated blood is reintroduced demonstrate that the antioxidant defenses of many organisms can be overwhelmed, Although unusual among most mammals, many organisms routinely experience wide variation in oxygen availability to their tissues due to factors such as environmental oxygen lack, breath-hold diving, extracellular freezing, or apnoeic breathing patterns in arrested metabolic states. In recent studies using various animal models (anoxia-tolerant turtles, freeze-tolerant snakes and frogs, estivating snails) our laboratory has explored the adaptations of antioxidant defenses that allow such organisms to deal with rapid changes in tissue oxygenation with little or no accumulation of damage products. The key to successful transitions in several systems is the induction, during the oxygen-limited state, of elevated activities of antioxidant and associated enzymes, such as catalase, superoxide dismutase, glutathione-S-transferase, and glutathione peroxidase, so that damage during the reintroduction of oxygen (such as lipid peroxidation) is minimized. However, animals that are excellent facultative anaerobes, such as freshwater turtles, appear to deal with potential of oxidative stress during the anoxic-aerobic transition by maintaining constitutively high antioxidant defenses (e.g. enzyme activities similar to those of mammals and much higher than those of anoxia-intolerant lower vertebrates) that can readily accommodate the burst of reactive oxygen species generation when breathing is renewed.

Adaptation, Physiological↗

PubMed1995

cAMP-dependent protein kinase and anoxia survival in turtles: purification and properties of liver PKA.

The catalytic subunit of turtle (Trachemys scripta elegans) liver cyclic AMP-dependent protein kinase (PKAc) was purified to homogeneity with a final specific activity of 65,783 pmol phosphate transferred.min-1.mg protein-1. Subunit molecular weight was 42-43 kDa as determined by SDS-PAGE and Sephacryl S-300 chromatography. The isolectric point was pH 6.41 +/- 0.02. Turtle liver PKAc showed highest activity with kemptide as its substrate; activity with other artificial substrates, histone IIA and protamine, was only 21 and 11%, respectively, of the activity with kemptide. Km values were 83 +/- 6.5 microM for Mg.ATP and 11.7 +/- 0.5 microM for kemptide and enzyme activity was strongly reduced by inhibitors of mammalian PKA (H-89, PKA-1) but not by inhibitors of other protein kinases. The enzyme was also inhibited by salts, especially fluoride salts (I50 about 30 mM), and showed a sharp break in the Arrhenius plot (0-45 degrees C) with activation energy increasing by 4-fold from 27.9 +/- 1.85 to 115 +/- 2.5 kJ/mol for temperatures above versus below 15 degrees C. Temperature effects may be important in suppressing PKA function, and therefore PKA-mediated responses, in vivo to enhance anoxic survival time during winter hibernation under water. Analysis of the effects of in vivo anoxia exposure at 7 degrees C on PKA in turtle organs showed a rapid 2.3-fold increase in the amount of active enzyme in liver within 1 h of anoxic submergence accompanied by a 60% increase cAMP levels; with longer anoxia (5 or 20 h) the percentage of active PKA was suppressed to 2.1-3.7% of the total.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Protein phosphorylation patterns during aestivation in the land snail Otala lactea.

Protein phosphorylation patterns were investigated in whole tissues and subcellular fractions of active and aestivating Otala lactea (Müller) (Pulmonata, Helicidae). Measurement of overall protein phosphorylation showed that incorporation of 32P increased until the second day after injection and remained constant for the remaining 4 days of the time course. Comparison of tissues from aestivating and active snails on day 3 showed a decreased protein phosphorylation in aestivating snails (44% of active). No differences in total and protein-associated radioactivity for foot, mantle or haemolymph were observed. Subcellular fractionation of the hepatopancreas localized the changes to plasma membrane, microsomal, and cytosolic fractions: values for aestivating animals were reduced to 71, 37 and 58% of the corresponding active values. Separation of the individual subcellular fractions on isoelectric focusing columns revealed differences in the phosphate incorporation patterns. Plasma membrane from aestivating animal hepatopancreas had a lower overall level of incorporation and fewer radioactive peaks in the pH 7-10 region than did the plasma membrane fraction from active animals. SDS-PAGE analysis of plasma membrane fractions from active and aestivating snails showed a relative decrease in phosphorylation between 60-80 kDa and 30-40 kDa. IEF analysis of cytosolic proteins from aestivating snail hepatopancreas also showed peaks of radioactivity that were apparently shifted by 0.3 pH units toward higher pI values. Increased phosphate incorporation was observed at a peak that corresponded to the pI value for pyruvate kinase in aestivating snails but definite assignment of peaks was not possible. SDS-PAGE analysis of cytosolic proteins showed an aestivation-related decrease in relative protein phosphorylation between 30-35 kDa and 40-45 kDa. A relative increase in phosphorylation during aestivation was observed for proteins between 16-22 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Evidence for aestivation specific proteins in Otala lactea.

Changes in [35S]methionine protein labeling patterns were examined by following incorporation into the acid precipitate protein fraction of land snails, Otala lactea (Müller) (Pulmonata, Helicidae). Labeled proteins were analyzed by SDS polyacrylamide gel electrophoresis and isoelectric focusing columns. Snails in four different physiological states were compared: active controls, short term aestivating snails (injected and allowed to enter aestivation), long term aestivating snails (aestivated for 14 days, injected, and maintained in the aestivating state), and snails aroused after aestivation (aestivated, injected, and aroused). Protein associated radioactivity was measured over a 7 day time course post injection. Autoradiographic analysis of SDS-polyacrylamide gels showed increases in the radioactivity of four proteins: 91 kDa (hepatopancreas, day 1 in long term aestivating animals), 50 kDa (hepatopancreas, day 2 in short term aestivating snails), 70 kDa and 30 kDa (foot, day 2 in short term aestivating animals). Hepatopancreas and foot from day 1 long term aestivating and day 2 short term aestivating animals were also analyzed by isoelectric focusing columns. Several pH-specific differences were apparent when controls and aestivating animals were analyzed. In particular a peak of radioactivity was observed at pH 5.05 in 1 d long term aestivating hepatopancreas and at pH 4.30 in 2d short term aestivating animals. Several differences were noted in foot with no specific pattern emerging. SDS-polyacrylamide gel electrophoresis analysis of the hepatopancreas peaks showed the appearance of several bands with increased radioactivity, including the 91 kDa and 50 kDa proteins described above. These results suggest that O. lactea aestivation specific proteins may be involved in the transition to a depressed metabolic state.

Animals↗

PubMed1995

Effects of anoxia on protein phosphatase in turtle organs: purification and properties of protein phosphatase type-1 from turtle liver.

Protein phosphatase type 1 (PP-1) was analyzed in organs of the red-eared slider turtle, Trachemys scripta elegans, a species capable of long-term anoxia survival. During anoxic submergence at 7 degrees C, PP-1 activity in liver rapidly decreased to 63% of the control value within the first hour and remained suppressed over the subsequent 20 h of anoxia. PP-1 activity was also suppressed in red skeletal muscle during anoxia and dropped transiently (after 1 h) in brain but did not change in heart or white muscle. PP-1 was purified from turtle liver using polyethylene glycol fractionation and chromatography on DEAE-cellulose, blue dextran, Sephacryl S-200, and ADP-agarose. A 3000-fold purification was achieved with a final specific activity of 3156 nmol released min-1 mg protein-1 using 32P-labeled phosphorylase a as the substrate. Turtle liver PP-1 was a monomer of molecular mass 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or 38 +/- 2 kDa by Sephacryl S-200 gel filtration. The enzyme was inhibited by okadaic acid (Ki 12.6 +/- 1.4 nM) and AMP (Ki 23 +/- 2 microM) as well as by ADP, ATP, and IMP. Regulation of liver PP-1 appears to be an integral part of anoxia-induced changes in liver glycogenolysis and metabolic rate suppression.

Adenosine Monophosphate↗

PubMed1995

The optimal depot fat composition for hibernation by golden-mantled ground squirrels (Spermophilus lateralis).

Golden-mantled ground squirrels (Spermophilus lateralis) are herbivores that hibernate during winter. Although little is known about the nutritional/physiological constraints on hibernation, numerous studies have demonstrated that increasing the amount of linoleic acid (a polyunsaturated fatty acid) in the diet enhances hibernation. This is probably because high linoleic acid diets reduce the melting points of the depot fats produced for hibernation which makes them more metabolizable at low body temperatures. This suggests that a major limitation on hibernation may be obtaining enough linoleic acid in the diet for proper hibernation. In all previous studies, however, the amount of linoleic acid in the diets of free-ranging animals was either not considered, or the range of dietary linoleic acid contents in the experiments was less than that of natural diets. It is thus not known whether the amount of linoleic acid available to hibernators under natural conditions actually limits their torpor patterns. A series of laboratory feeding and hibernation experiments were conducted with S. lateralis and artificial diets with different linoleic acid contents that were either below or above the linoleic acid content of the natural diet. The results demonstrated that when dietary linoleic acid contents are either below or above natural levels, hibernation ability is greatly reduced. Hibernation ability was reduced when the squirrels were maintained on a high linoleic acid diet probably by the production of toxic lipid peroxides in brown adipose tissues. The results indicate that there is an optimal level of dietary linoleic acid for proper hibernation, and this is equal to that of the natural diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1995

Quantification of lipid peroxidation in tissue extracts based on Fe(III)xylenol orange complex formation.

Commonly used spectrophotometric methods for determining the extent of lipid peroxidation in animal tissue extracts, such as measurements of diene conjugation and thiobarbituric acid reactive substances (TBARS), have been criticized for their lack of specificity. This study shows that lipid hydroperoxides can be effectively quantified in animal tissue extracts using an assay based on the formation of a Fe(III)xylenol orange complex. Addition of H2O2, cumene hydroperoxides, or methanolic tissue extracts to an acidic reaction mixture containing 0.25 mM Fe(II) and 0.1 mM xylenol orange caused the formation of a broad Fe(III)xylenol orange complex absorbance peak at 560-580 nm with a corresponding decrease in the xylenol orange peak at 440 nm. Complex formation measured at 580 nm was saturable with both xylenol orange and Fe (II) concentration. Addition of ascorbic acid, GSH, and cysteine (0.3-5 mM) caused a saturable reduction of the Fe(III)xylenol orange complex. Formation of the Fe(III)xylenol orange complex was linear with the amount of tissue extract added. A significant correlation (r = 0.88, p < 0.005) existed between the xylenol orange method of estimating lipid peroxidation and the conventional TBARS assay in a series of animal tissues tested. The time course of increase in A580nm in tests using tissue extracts was typical of a free radical reaction; a lag phase was followed by a log phase. No increase in A580nm was observed up to 24 h when highly peroxidizable arachidonic acid was assayed. These results indicate that the formation of the Fe(III)xylenol orange complex reflects a chemical amplification of the original level of lipid hydroperoxides present in tissue extracts and that peroxidizable lipids do not influence the assay. The potential usefulness of the xylenol orange assay for comparative biochemical and toxicological studies of oxidative stress is discussed.

Animals↗

PubMed1995

Enzymatic control of glycogenolysis during anoxic submergence in the freshwater turtle Trachemys scripta.

Freshwater turtles Trachemys scripta elegans endure prolonged severe hypoxia, and even complete anoxia, while diving or hibernating underwater. Metabolic adaptations supporting survival include the activation of glycogenolysis and glucose output from liver, as well as strong metabolic rate depression. The present study analyzes the enzymes of both the phosphorolytic (glycogen phosphorylase, phosphorylase b kinase, cAMP-dependent protein kinase) and glucosidic (alpha-glucosidase) pathways of glycogenolysis in turtle organs. Turtles were subjected to 5 hr of submergence in N2-bubbled water at 7 degrees C and then activities of phosphorolytic and glucosidic enzymes were assayed in liver, heart, brain, and red and white skeletal muscle, and compared with aerobic controls. In vitro incubations also assessed protein kinase A control of phosphorolytic enzymes. A functional enzyme cascade system for the activation of glycogen phosphorylase was found in all organs, and both phosphorylase and phosphorylase kinase were stimulated by in vitro incubation with the catalytic subunit of cAMP-dependent protein kinase. Anoxic submergence led to significant increases in phosphorylase activities in liver and heart (phosphorylase a rose 2- and 2.5-fold, respectively) but phosphorylase kinase and protein kinase A activities in liver were reduced after 5 hr exposure. Both acidic (pH 4) and neutral (pH 7) forms of alpha-glucosidase were detected in all five organs with highest activities in liver. Activity of acid alpha-glucosidase, which degrades lysosomal glycogen, increased by 2-fold in liver during anoxic submergence. The data show that glycogen breakdown in turtle liver during anoxic submergence may result from coordinated activations of both the cytoplasmic phosphorolytic and the lysosomal glucosidic pathways of glycogenolysis.

Adaptation, Physiological↗

PubMed1995

Antioxidant defenses and metabolic depression in a pulmonate land snail.

During arousal from estivation oxygen consumption by land snails (Otala lactea) increases severalfold. To determine whether snails prepared for an accompanying rise in the rates of oxyradical generation by altering their antioxidant defense mechanisms, changes in the activities of antioxidant enzymes and lipid peroxidation products were quantified in foot and hepatopancreas of control, 30-day estivating, and aroused snails. Compared with controls, estivating O. lactea showed significant increases in the activities of foot muscle superoxide dismutase (SOD) (increasing by 56-67%), catalase (51-72%), and glutathione S-transferase (79-108%), whereas, in hepatopancreas, SOD (57-78%) and glutathione peroxidase (93-144%) increased. Within 40 min after arousal began, hepatopancreas glutathione peroxidase activity had returned to control values, but SOD showed a further 70% increase in activity but then returned to control levels by 80 min. Estivation had no effect on total glutathione (GSH + 2 GSSG) concentrations in tissues, but GSSG content had increased about twofold in both organs of 30-day dormant snails. Lipid peoxidation (quantified as thiobarbituric acid reactive substances) was significantly enhanced at the onset of arousal from dormancy, indicating that oxidative stress and tissue damage occurred at this time. The data suggest that antioxidant defenses in snail organs are increased while snails are in the hypometabolic state as a preparation for oxidative stress during arousal.

Animals↗

PubMed1995

Metabolic effects of dehydration on an aquatic frog, Rana pipiens.

Cellular responses to dehydration were analyzed in six organs of leopard frogs Rana pipiens. Frogs at 5 degrees C endured the loss of up to 50% of their total body water content but water contents of individual organs were strongly defended. Skeletal muscle water content was strongly affected by dehydration, dropping from 80.7% of wet mass in controls to 67.2% in frogs that had lost 50% of their total body water. However, water contents of internal organs dropped by only 3-8% of their wet masses. Water contents of all organs except skeletal muscle were fully restored by 24h of rehydration in water at 5 degrees C. Dehydration had no consistent effect on the protein content of five organs but in a sixth, the kidney, protein levels were elevated (by 60-72%) at the higher levels of dehydration and during rehydration. Dehydration led to a rapid increase in glucose concentration in the liver; compared with control values of 13 +/- 2 nmol mg-1 protein, levels were doubled by 12.2% dehydration and continued to increase to a maximum of 307 +/- 44 nmol mg-1 protein (20 mumol g-1 wet mass) in 50% dehydrated frogs. Glucose accumulation was supported by a decrease in liver glycogen content and a parallel rise in glucose 6-phosphate levels, but not in the levels of other glycolytic intermediates, confirming that glycogenolytic flux was being directed into glucose synthesis. Blood glucose levels also increased as a function of increasing dehydration, reaching values 13.8 times higher than controls, but only the kidney and brain showed a significant accumulation of glucose over the course of dehydration.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1995

Phosphofructokinase binding to myofibrils in fish muscle: influences of ionic strength and metabolite levels on enzyme complex formation.

The interaction of 6-phosphofructo-1-kinase (PFK) with myofibrils was assessed using the purified proteins from rainbow trout white skeletal muscle. More than 70% of PFK activity was bound at pH values between 6 and 7 but higher pHs dissociated the complex. Increasing salt concentrations also reduced PFK binding, with greater sensitivity to salt at pH 7.0 vs pH 6.6. Substrates and allosteric effectors also reduced enzyme binding to myofibrils; 50% of enzyme was released at 0.23, 0.24, 1.3, and 2.0 mM for fructose 6-phosphate, Mg.ATP, ATP and AMP, respectively. However, the addition of a protein crowding agent, poly(ethylene)glycol, greatly enhanced PFK binding to myofibrils, particularly at high pH (8.0) or high [KCl]. The studies suggest that reversible binding of PFK to myofibrils may be an important factor in the control of anaerobic glycolysis in vivo, especially under the cellular conditions of burst swimming exercise.

Actins↗