Ph. D. Thesis Abstracts

Thomas A. Churchill, Ph.D. Biology 1992

Metabolic biochemistry of freeze tolerance in vertebrates

 

Abstract:

A unique group of vertebrate animals has developed complex metabolic adaptations that enable them to survive freezing. This thesis investigates: i) cryoprotectant synthesis in freeze tolerant frogs, ii) freeze tolerance in a newly identified freeze tolerant vertebrate, the garter snake, and iii) metabolic responses elicited by other stresses (anoxia and dehydration) in the garter snake and two frog species. Investigation of cryoprotectant synthesis in spring frogs,Pseudacris crucifer, revealed large amounts of glucose produced during freezing; approximately 0.1 molar. Changes in the levels of glycolytic intermediates indicated that an activation of glycogen phosphorylase and phosphofructokinase (PFK) directed glycolytic flux to cryoprotectant synthesis. Tissue glucose distribution was much lower than in fall animals. These results suggested seasonal variation in glucose transport mechanisms. A similar investigation of cryoprotectant synthesis in spring Hyla versicolorshowed a maintenance of regulatory enzyme controls at glycogen phosphorylase and PFK directing glycogen carbon to cryoprotectant. Only glucose was synthesized as cryoprotectant; quantities of glycerol (the major cryoprotectant of winter H. versicolor) showed no increase. The amount of cryoprotectant produced was directed correlated to glycogen content in the liver. Investigation of freeze tolerance in garter snakes revealed that these snakes were only partially freeze tolerant. Survival of brief freezing exposures (5-10 h at -2.5°C; 30-50% ice) was possible. Two amino acids, glutamate and taurine, were implicated as possible cryoprotective agents. Comparison of the metabolic responses (adenylate levels, anaerobic end products, glycolytic flux) to freezing in garter snakes were similar to those elicited by anoxia. Dehydration timecourses were investigated in two freeze tolerant frog species, Rana sylvatica andPseudacris crucifer. Even though whole body water contents dropped by 50-60 %, individual tissues exhibited little or no change in water content. There were many similarities between metabolic responses to dehydration and those to freezing. The most remarkable similarity between freezing and dehydration was the accumulation of glucose, presumably acting as a cellular protectant; quantities in liver rose to 127 and 220 micromole per gram in R. sylvatica and P. crucifer, respectively.

Yanjing Su, Ph.D. Chemistry, 1992

Phosphofructokinase from white skeletal muscle and liver of rainbow trout (Oncorhynchus mykiss): isolation, characterization and study of enzymatic regulation

 

Abstract:

Phosphofructokinase (PFK) isozymes from white skeletal muscle and liver of rainbow trout Oncorhynchus mykiss were purified to electrophoretic homogeneity. Muscle PFK was purified 175-fold using phosphocellulose, hydroxylapatite, and ATP-agarose affinity chromatography whereas liver PFK was purified 13,400-fold using acetone precipitation, heat treatment, ammonium sulfate fractionation, and ATP-agarose chromatography. Muscle PFK was a homohexamer having a native molecular mass of 478,000. The enzyme was regulated by the levels of fructose-6-phosphate (F6P), ATP, pH, and allosteric effectors including activators (NH4+, inorganic phosphate, AMP, ADP, and fructose-2,6-bisphosphate [F2,6P2]) and inhibitors (citrate, phosphoenolpyruvate [PEP], and ATP). Activators increased the enzyme affinity for F6P and released the inhibition by ATP or citrate. Citrate inhibited the enzyme synergistically with ATP. Arrhenius plots of the enzyme activity showed discontinuity at 15 to 16°C, presumably due to conformational alterations in the enzyme. The kinetic behavior of muscle PFK was significantly altered by protein kinase – mediated phosphorylation. The high-phosphate form of the enzyme showed higher activity with increased affinity for F6P and less inhibition by ATP. Protein concentration affected enzyme activity as assessed by two different methods. PFK showed a higher Vmax, lower S0.5 F6P and higher I50 values for ATP as enzyme concentration increased. The association of PFK with myofibrils of trout muscle was affected by pH, ionic strength, protein concentration, and the levels of metabolites or the effectors of the enzyme with binding favored by lower pH values and increased protein concentration. During exercise, muscle PFK is probably activated by increases in the levels of enzyme activators and enzyme phosphorylation state, and enhanced PFK association with myofibrils. Trout liver PFK was also regulated by the levels of F6P, ATP, NH4+, inorganic phosphate, AMP, and F2,6P2. However, the liver enzyme was not sensitive to citrate inhibition. Contrary to its muscle counterpart, liver PFK was inhibited by protein phosphorylation catalyzed by the catalytic subunit of cAMP-dependent protein kinase and activated by the removal of phosphate through acid phosphatase. The high-phosphate form of liver PFK exhibited a lower Vmax, an increased S0.5 F6P, and higher I50 values for ATP.

 

Hossein Mehrani, Ph.D. Chemistry, 1994

Regulation of glycogen metabolism by protein phosphorylation during environmental stress

 

Abstract:

The enzymes involved in the phosphorylation controlled glycogen catabolic pathway were studied in two different model systems involving anoxia: functional anoxia in exercised fish and environmental anoxia in turtle. Glycogen phosphorylase b from rainbow trout,Oncorhynchus mykiss, white skeletal muscle was purified to near homogeneity. Glucose and ATP inhibited the enzyme; glucose inhibition decreased at lower pH values. Michaelis constants for glycogen, phosphate, and AMP were 128 micromolar, 31 millimolar, and 142 micromolar respectively, at pH 7.2; maximum enzyme activity was obtained at pH 7.5 and 25°C Exhaustive swimming exercise altered tissue glycogen phosphorylase kinase (GPK) and protein kinase A (PKA), GPK activity increasing by 60% in liver and 40% in white muscle of exercised fish. The amount of active PKA rose from 12% to 21% in liver and from 32% to 57% in white muscle after exhaustive swimming coupled with 50% and 70% increases in cellular cyclic AMP levels, respectively. Three forms of alpha-glucosidase were identified in trout liver. Two forms showed acid pH optima, hydrolyzed glycogen, maltose and 4-methylumbelliferyl alpha-glucoside (MUalphaG), and were associated with lysosomes whereas the third was microsomal, had a neutral pH optimum and did not hydrolyze glycogen. Properties of acid alpha-glucosidase type I changed significantly during exercise; maximal activity increased by 80% and Km values for glycogen and maltose dropped by 50% in exercised, versus control, fish. Exposure of turtles, Trachemys scripta elegans, to submergence anoxia at 7°C, elevated activities of phosphorolytic and glucosidic enzymes in some organs. Phosphorylase a in liver and heart increased significantly after 5 h of anoxia. PKA activity increased 2.3-fold in liver within 1 h of anoxia accompanied by a 60% increase in cAMP levels; however, with longer anoxia active PKA was suppressed to 2.1-3.7% of the total. Protein phosphatase-1 (PP-1) activity in liver decreased to 63% of controls within 1 h and remained suppressed over the subsequent 20 h of anoxia. PP-1 activity also fell in anoxic red muscle and decreased transiently in brain. Within one hour of anoxia, 40% of protein kinase C beta isomer (PKC-beta) and over 80% of PKC-alpha were translocated from cytosol to the membrane fraction. Activity of acid alpha-glucosidase also increased in liver of anoxic turtles. PKA, PP-1, PKC-alpha, and PKC-beta from control turtle liver were purified to homogeneity; physical and kinetic properties of these are presented.

 

Clark P. Holden, Ph.D. Biology, 1995

Signal transduction and the function of second messengers and protein kinases in the control of glycogenolysis and cryoprotectant production in freeze tolerant vertebrates

 

Abstract:

A unique group of lower vertebrates, termed freeze tolerant, have developed complex biochemical mechanisms that allow them to survive whole body freezing. Perhaps the most important of these mechanisms is the production of cryoprotectant, usually in the form of glucose, via an activation of glycogenolysis as a response to freezing. This thesis investigates: (1) whether protein kinase enzymes are responsible for the early activation of glycolysis that stimulates cryoprotectant production in freeze tolerant vertebrates and/or the subsequent inhibition of the pathway in the later stages of a freezing episode, (2) the comparative aspects of the responses of protein kinases elicited by the environmental stresses of dehydration and anoxia, to those of freezing, and (3) the role of the free catalytic subunit of cyclic AMP-dependent protein kinase (PKAc) and protein kinase C (PKC) in cryoprotectant production in Rana sylvatica liver. Short freezing episodes caused a dramatic increase in the percentage of free catalytic PKA (PKAc). This response diminished with increased time frozen in tissues of freeze tolerant vertebrates. The measurement of second messengers over a freezing time course revealed significant increases in adenosine 3′,5′- cyclic monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) in liver and muscle of 4 freeze tolerant animals. An activation of PKA and PKC by these molecules, in response to freezing, is likely. Dehydration stress. not anoxia stress, elicited a remarkably similar protein kinase response to that seen during freezing. Levels of PKAc and its second messenger cAMP, as well as the second messenger for PKC, IP3, rose in dehydrated liver tissue whereas anoxia did not activate PKAc and only increased liver IP3. Purified R. sylvatica liver PKAc was not inhibited by high concentrations of salt or glucose and showed an increased affinity for substrates at low temperatures. Additional evidence showed that a freezing stress did not initiate a translocation of PKC to the plasma membrane in R. sylvatica liver and brain tissues. Freeze tolerant vertebrates possess an adaptive strategy to combat freezing which links second messengers and protein kinases to the post-translational modification of glycolytic enzymes conducting the biosynthesis of cryoprotectant.

 

 

Denis R. Joanisse, Ph.D. Biology, 1995

Metabolism during overwintering in two species of cold-hardy goldenrod gall insects

 

Abstract:

Metabolism was examined in the larvae of two cold-hardy goldenrod gall insects, the freeze tolerant fly Eurosta solidaginis (Fitch) (Diptera, Tephritidae) and the freeze avoiding moth Epiblema scudderiana (Clemens) (Lepidoptera, Olethreutidae) during overwintering. Acclimatization resulted in specific changes in enzyme activities and cellular metabolites. Enzyme activity data suggested metabolic shifts concurrent with the requirements of both species. Larvae shifted metabolism towards cryoprotectant synthesis in the fall, as witnessed by increased activities of glycerol and sorbitol producing enzymes, and towards the removal of these polyols in the spring with increased activities of catabolic enzymes. Measured increases in lipid unsaturation in the fall helped to maintain membrane fluidity during cold-exposure. Decreased activities of enzymes of the tricarboxylic acid cycle paralleled the reduced demands on oxidative metabolism over the winter. In Eurosta solidaginis, decreased oxidative metabolism and the anoxic state of the animal when frozen further resulted in the lack of lipid reserve depletion over the winter, whereas freeze avoiding Epiblema scudderiana larvae appeared to use lipid reserves to support basal metabolism over the winter. Decreased activities of antioxidant enzymes in Eurosta solidaginisover the winter suggested that these larvae do not experience increased reactive oxygen species formation during freezing and thawing. Increasing antioxidant enzyme activities in Epiblema scudderianaover the winter suggested that these larvae may be subject to oxidative stress, perhaps due to the maintenance of aerobic metabolism during cold exposure.

 

Jean E. Grundy, Ph.D. Chemistry, 1996

Antioxidant defenses during estivation in the spadefoot toad, Scaphiopus couchii

 

Abstract:

Antioxidant enzymes and lipid peroxidation damage were analyzed in an estivating species, to determine whether estivation (a dormancy induced by hot, dry environmental conditions) represented an oxidative stress and to determine whether changes in organ antioxidant systems were made in response to estivation. The model animal used was Scaphiopus couchii, the spadefoot toad, that estivates for about 10 months per year in the Arizona desert during which it loses about 50% of its body water and builds up high levels of electrolytes and osmolytes in body fluids. Activities of six antioxidant enzymes (AOE) and xanthine oxidase/xanthine dehydrogenase as well as the small molecular weight free radical scavenger glutathione were measured in liver, lung, heart, kidney, gut and leg muscle of control and dormant toads. Damage to membranes was assessed by measurement of extent of lipid peroxidation by three different methods. The glutathione ratio, (GSSG) /(GSH), rose significantly in several tissues during estivation. In addition, activities of key AOE were reduced in several tissues, perhaps indicating that the metabolic cost of keeping antioxidant defenses high during estivation was greater than the cost of maintaining AOE at their previous level. Three antioxidant enzymes were further examined: Catalase (CAT) and glutathione reductase (GR) were purified and characterized from liver and glutathione S-transferase (GST) from liver and muscle of awake and estivated toads. CAT activity was lower in liver during estivation but the kinetic properties of catalase were the same in estivated and awake toads. Catalase from toad liver was an active dimer, which has not been demonstrated previously in a eukaryote, to our knowledge. GR and GST of toad liver had altered kinetic properties during estivation, indicating pre- or post-translational modification of the enzyme in the dormant animal. However, GST in muscle was the same for awake and estivated toads. GST is a multiclass family of enzymes which generally render lipid-soluble material soluble in the cytosol, via conjugation to glutathione. Three class of GST, Mu Pi and Alpha, have different substrate specificities; these parameters were used to classify frog and toad GSTs. GST, GR and CAT were relatively insensitive to physiological urea concentrations but were strongly modulated by KCl, suggesting that urea buildup during estivation may serve to modulate passive KCl accumulation as a result of dehydration.

William G. Willmore, Ph.D. Biology 1997

Molecular adaptation to anoxia and recovery from anoxia in the freshwater turtle Trachemys scripta elegans

 

Abstract:

The responses to stresses associated with oxygen extremes, namely anoxia and oxidative stress associated with recovery from anoxia, were studied in an organism that must tolerate both extremes. The model organism used was the turtle Trachemys scripta elegans that can survive complete anoxia for up to 18 weeks at lower temperatures. Metabolic adaptations that support survival of oxygen extremes in this organism that were examined included: (a) tissue-specific changes in the maximal activities of enzymes of intermediary metabolism, (b) an assessment of the metabolite and enzymatic antioxidant defenses utilized to combat the oxidative stress encountered upon recovery from anoxia, (c) adaptations in specific enzymatic antioxidant defenses and (d) adaptations to anoxia and recovery from anoxia of a specific protease. Of the key enzymes of intermediary metabolism, 14 instances of reduced and only 3 instances of increased enzyme activities were observed in tissues during anoxia as compared to controls. Brain had changes related to both metabolic rate depression and neurotransmitter release. Maximal activities which increased during anoxia included G6PDH in heart, MDH in white muscle and CPT in kidney. Anoxia stress and aerobic recovery produced relatively few changes in organ antioxidant enzyme activities and levels of lipid peroxidation products. Changes included tissue specific decreases in antioxidant enzymatic activities during anoxia, particularly SOD and CAT. The levels of enzymatic antioxidant activities were high in turtles in comparison with other vertebrates and non-vertebrates. Turtles maintained high levels of total glutathione in tissues in comparison to other ectotherms. Tissue-specific changes in the maximal activities of the glutathione-related enzymes occurred, the most dramatic being a decrease in gamma-GTPase during anoxia to 2% of control values. Turtle liver contained one homodimeric alpha class and a unique heterodimeric alpha-like GST. Turtle liver GR revealed a high affinity for GSSG. No new isoforms of either GST or GR were formed during anoxia. MPC displayed an increase in peptidylglutamyl-peptide bond hydrolyzing activity during recovery from anoxia, possibly as a result of oxidative damage to proteins in this tissue. Overall, T. S. elegans maintains continually high defenses against the stresses of anoxia and oxidative stress upon recovery from anoxia.

Bradley J. Thatcher, Ph.D. Biology, 1997

Properties of enzymes from mammalian hibernators: Structure function relationships

 

Abstract:

The examination of four enzymes, glutathione S-transferase (GST), glutathione reductase (GR), aspartate aminotransferase (AspAT), and glutamate dehydrogenase (GDH), from two hibernators Cynomys leucurus andSpermophilus richardsonii were used to show three different responses to a hibernating life style. The production of a new (isozyme) of GST, alpha-class, in the liver and muscle tissues of Cynomys leucurus. N-terminal and amino acid compositions show strong homology alpha-class GSTs from a variety of species. The kinetic profile and structural studies suggest that this isozyme is similar to other alpha-class GSTs. PDM 18 showed a broader temperature optima and lower thermal stability compared to a comparable rabbit enzyme. A different form of GDH was found in the liver tissue of Spermophilus richardsonii in the hibernating animal. This new isozyme of GDH differed from the nonhibernating and bovine enzyme in both physical and kinetic parameters. There is a shift in the directional preference between the two ground squirrel forms of the enzyme in the presence of GTP, ATP, AMP. Further kinetic differences are also reflected in I50, Km, and Ka values between the two ground squirrel isozymes. The N-terminal sequence of the two isozymes is identical over the first 12 residues; there are differences in the molecular weight by size exclusion chromatography, and retention time on reverse phase HPLC suggesting small differences in the polypeptide chain. The final observed case was that there were no substantial kinetic and minor structural changes between the enzyme from the hibernator and from a nonhibernator. This was seen with two enzymes from the study GR (from prairie dog) and AspAT (from ground squirrel).

 

Kyra J. Cowan, Ph.D. Chemistry, 1998

Evidence for the reversible phosphorylation control of metabolism during prolonged environmental stress

 

Abstract:

I hypothesized that the environmental conditions endured during estivation in the spadefoot toad, Scaphiopus couchii, would dramatically affect metabolism and metabolic regulation in toad organs, and that reversible protein phosphorylation is important in the control of the necessary metabolic adaptations. Enzyme activities in tissues of control versus 2 month estivated toads revealed that glycolysis and ketone body metabolism dropped in liver, and that amino acid catabolism and NADPH production increased in brain and muscle. The influence of urea, which can build to 300 mM in estivating toads to aid desiccation resistance, was assessed on liver and muscle enzymes. Although known as a protein denaturant, urea had little effect on toad enzymes, being less disruptive of enzyme activities than 300 mM KCl. Estivation led to significant changes in the kinetic properties of both pyruvate kinase and phosphofructokinase that were mediated by reversible phosphorylation. To explore this mode of metabolic regulation, estivation effects on the activities of multiple protein kinases and protein phosphatases were studied. Protein kinase A (PKA) activities dropped in all tissues, and dormancy strongly affected type-1 and type-2 protein phosphatases. Estivation effects on the isozyme types, activities and distribution of protein tyrosine kinases (PTKs) and phosphatases (PTPs) revealed three major forms of PTKs and four PTPs in muscle and overall activities of PTKs decreased during estivation in the soluble fractions of liver, lung, and leg muscle, with the opposite pattern in heart. PTP activity in soluble fractions of gut, heart, and leg muscle also increased in estivation. Tissue specific changes in the enzyme activities and protein levels of mitogen-activated protein kinases (including Raf-1, ERK 1 & 2, MAPKAPK-1, JNK, p38) also occurred during estivation as did levels of various transcription factors (ATF-2, Elk-1, Egr-1, CREB). Involvement of these kinases and transcription factors implicates tissue-specific changes in gene expression during estivation and together with estivation-induced reversible phosphorylation of enzymes indicates that widespread, coordinated controls are used to readjust metabolism for the needs of the estivating state including metabolic rate depression, desiccation tolerance and long term starvation.

 

Justin A. MacDonald, Ph.D. Biology, 1998

Enzyme thermal adaptations and signal transduction involvement in ground squirrel hibernation

 

Abstract:

Regulatory enzymes are integral to the regulation of metabolism, and reversible protein phosphorylation by kinases and phosphatases controls most aspects of cell life. Brown adipose (BAT) PKA, liver PP-1 and muscle phosphofructokinase (PFK) from the ground squirrel were purified to homogeneity; physical and kinetic properties of these were assessed with respect to function at low temperature. An assessment of PKA catalytic subunit function via in vitro incubations of ground squirrel BAT extracts with 32P-ATP revealed differences in the patterns of phosphorylated proteins between euthermic and hibernating animals and between 37° and 5°C. The addition of 10% (w/v) polyethylene glycol reversed all negative effects of cold temperature, low pH and urea on PFK stability. A reduction in assay temperature from 37° to 5°C had numerous effects on ground squirrel enzymes including changes in pH optima, decreases in Km values for substrates, and reduced inhibition by salts. The percentage of membrane associated PKC increased during hibernation in liver, but the % active PKA was unaffected by hibernation in any tissue. Changes in PP-1 and -2 activities in tissues of euthermic and hibernating animals showed that phosphatases are regulated in hibernation. The subcellular localization of PP-1 in liver and muscle was affected by hibernation. The mitogen-activated protein kinase (MAPKs) MAPK-activated protein kinase (MAPKAPK-1 and -2) activities were affected by hibernation. p70S6Kkinase activity decreased in kidney during hibernation. c-jun kinase activity increased in 4 hibernating tissues but decreased slightly in brain. p38 was activated in hibernating muscle and heart. The responses of immediate-early gene products c-jun, egr-1, and c-myc were tissue specific with no apparent overall pattern. Maintenance of muscle energy status at the expense of the total adenylate pool was accompanied by a 60% decrease in the Na+K+-ATPase activity. Enzyme activity was reduced in vitro when incubated with protein kinase activators and was relieved by addition of alkaline phosphatase. A change in the ATP dependency of the enzyme also occurred in hibernation. Overall, protein phosphorylation is key to the regulation of hibernating metabolism in ground squirrels, and hibernator enzymes demonstrate functional adaptations to cold temperature.