M. Sc. Thesis Abstracts

Regulation of glucose-6-phosphate dehydrogenase and hexokinase in anoxia-tolerant mollusks: role of reversible phosphorylation

Abstract:

Metabolic rate depression is key to animal survival without oxygen and requires coordinated suppression of ATP-generating and ATP-consuming cellular functions by stable regulatory mechanisms. This thesis examined the possible role of reversible protein phosphorylation in metabolic suppression in organs of the anoxia-tolerant intertidal marine mollusc, Littorina littorea. Studies focused on glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme of the pentose phosphate pathway (PPP), and hexokinase (HK), an important enzyme at the forefront of carbohydrate metabolism. The data show that hepatopancreas G6PDH is regulated by phosphorylation and entry into anoxia leads to a more dephosphorylated form, whose properties suggest a more active enzyme. This would favour enhanced carbon flow through the PPP to sustain NADPH production for antioxidant defense. Furthermore, the study provides one of the first demonstrations of coordinated regulation of the PPP between active and hypometabolic states and implicates specific protein kinases and phosphatases in G6PDH regulation. Studies of HK showed that it is also regulated by reversible protein phosphorylation, in a tissue-specific manner. In the hepatopancreas, it appears that HK affinity for one of its substrates is enhanced during anoxia whereas in foot muscle, HK activity is suppressed. Differences between control and anoxic HK were also observed in their susceptibility to urea denaturation, response to specific protein kinase and protein phosphatase incubations as well as their elution profiles from an ion-exchange column. Overall, these studies confirm an integral role of reversible protein phosphorylation in the suppression and reorganization of L.littorea metabolism for anoxia survival.

Regulation of NF-κB and p53 in the liver and skeletal muscle of the freeze tolerant wood frog, Rana sylvatica

Abstract:   

The wood frog, Rana sylvatica, is the primary model animal used for studying vertebrate freeze tolerance.  During Canadian winters, wood frogs can endure the freezing of about 70% of their total body water and then thaw and resume life in the spring.  Frogs have multiple ways to protect themselves against potential freezing injuries including adaptive changes to intermediary metabolism and gene expression.  One way that wood frogs deal with freezing stress is via upregulation of several freeze-responsive genes.  Previous studies provided excellent presumptive evidence for the involvement of the NF-κB and p53 transcription factors in freeze tolerance.  The studies in this thesis used Western blotting to quantify levels of NF-κB subunits p50 and p65, its inhibitor, p-IκB, and downstream targets (ferritin heavy chain, manganese superoxide dismutase) as well as protein levels of p53, post-translationally modified p53, and some p53 downstream genes in the muscle and liver of control versus frozen wood frogs. Nuclear distributions of NF-κB and p53 were also assessed. RT-PCR was used to quantify transcript levels of select targets of NF-κB and p53.  Significant increases in the expression levels of NF-κBand its downstream targets as well as in levels of p53, post-translationally modified p53, and its downstream targets were observed during freezing.  These findings suggest the activation of NF-κB antioxidant defenses in the wood frog during freezing in anticipation of reperfusion during thawing and the activation of p53 in the wood frog which would lead to cell cycle arrest in the frozen state.

Regulation of the Tb-E2F pathway in the freeze tolerant wood frog, Rana sylvatica

Abstract:   

The North American wood frog, Rana sylvatica, is a primary model animal used in the study of freeze tolerance in vertebrates. During Canadian winters, wood frogs endure the freezing of about 65-70% of their total body water. Anoxia, ischemia, oxidative stress and many other consequences are a result of freeze/thaw cycles. A variety of adaptations are known that protect the frogs against potential freezing injuries as well as regulate their intermediary metabolism and gene expression to support survival. Selected transcription factors have critical roles to play in freezing survival by regulating the expression of genes that control the adaptations needed to handle freezing stress. In my present study, the retinoblastoma (Rb) protein coupled with the E2F transcription factor family were demonstrated to have roles in controlling the cell cycle in wood frog liver and skeletal muscle during freezing and associated stresses (anoxia, dehydration). Western blotting was used to quantify total Rb, phosphorylation or acetylation at different sites on Rb, E2F members and selected downstream targets under E2F control. Other central regulators of the cell cycle were also quantified including Cyclins, Cyclin dependent kinases (Cdks), and checkpoint proteins. Nuclear distributions of Rb-E2F and Cdk:Cyclins were also assessed. RT-PCR was used to quantify mRNA transcript levels of Cyclin D1 which decreased significant during freezing as well as c-Myc, a downstream target of E2F. The data indicate that the cell cycle is under regulation during freezing through E2F upregulation and Rb phosphorylation via Cdk:Cyclin activity.

Phosphorylation of glycolytic enzymes in hibernation.

Abstract:

Glycerol-3-phosphate dehydrogenase (G3PDH) and lactate dehydrogenase (LDH) were examined for differential phosphorylation, accompanying kinetics and stability inSpermophilus richardsonii liver and skeletal muscle. Hibernator G3PDH had a higher phosphate content and differential kinetics in both tissues which could be manipulated by kinase and phosphatase incubations in the G3P utilizing direction. Arrhenius plots and activation energies (Ea) showed that the hibernator form had a lower Ea in both tissues, especially in the liver.Euthermic LDH had a higher phosphate content and differential kinetics which could be manipulated by kinase and phosphatase incubations in both directions. Euthermic forms had lower Ea values in both tissues. G3PDH and LDH had differential urea I50 values with the hibernator more susceptible to urea denaturation in both tissues. Urea I50 values could be manipulated in LDH by kinase and phosphatase incubations in the liver but not in muscle.

Expression pattern of the novel freeze-responsive genes li16, fr10 and fr47 in the wood frog, Rana sylvatica

Abstract:

The ability of the wood frog (Rana sylvatica) to freeze up to 65% of its total body water allows it to endure subzero temperatures encountered during winter.  To survive the stresses associated with freezing, frogs have evolved multiple molecular adaptations including expression of three novel genes: li16, fr10 and fr47.  All three genes were found to be freeze responsive in a tissue-dependent manner.  They also all respond to anoxia and dehydration stresses, suggesting that the transcription of these genes is triggered by the low oxygen conditions common to all three stresses.  Protein levels were elevated after freezing for Li16; however FR10 and FR47 generally showed no change or a decrease in protein after freezing.  Transcripts of all three genes were also detected during tadpole development, indicating a possible additional role for metamorphosis.  Finally,bioinformatic analysis of their sequences provided insight into functional motifs and potential regulatory sites on the proteins.

Organ-specific metabolism during anoxia and recovery from anoxia in the cherrystone clam, Mercenaria mercenaria

Abstract:

The levels of intermediary metabolites and end products were quantified in the tissues of the cherrystone clam, Mercenaria mercenaria, over a time course of 96 h of anoxia followed by 48 h of aerobic recovery. Succinate and alanine accumulated as anaerobic products while glycogen and aspartate were utilized as substrates. Succinate accumulation ranged from 12-14 micromol/g wet weight in muscle (phasic and catch adductor, foot) to 25 micromol/g in gill and mantle with 32 micromol/mL released into the mantle cavity fluid. Lesser amounts of alanine were produced, the ratio succinate:alanine varying from 1.4:1 in phasic adductor to 3.2:1 in mantle at 96 h. Aspartate reserves apparently supply the carbon for succinate synthesis over the first 6-12 h of anoxia; subsequent succinate and alanine production probably results from glycogen fermentation. The imino acids alanopine and strombine were not produced in appreciable amounts (less than 1 micromol/g) during anoxia. When returned to aerated seawater, control levels of alanine and aspartate were reestablished within 24 h; accumulated succinate was catabolized within 48 h. Glycogen content of all tissues showed a sharp decline after 6 h of recovery, perhaps due to enhanced energy demands, but levels increased later in recovery. Tissue ATP levels, which were depressed during anoxia, were restored by 24 h.

Immobilization of Cellulase Using Polyurethane Foam

Abstract: 

Cellulase was covalently immobilized using a hydrophilic polyurethane foam (Hypol FHP 2002). Compared to the free enzyme immobilized cellulase showed a dramatic decrease (7.5-fold) in the Michaelis constant for carboxymethylcellulose. The immobilized enzyme also had a broader and more basic pH optimum (pH 5.5-6.0) a greater stability under heat-denaturing or liquid nitrogen-freezing conditions and was relatively more efficient in utilizing insoluble cellulose substrates. High molecular weight compounds (Blue Dextran) could move throughout the foam matrix indicating permeability to insoluble celluloses; activity could be further improved 2.4-fold after powdering foams under liquid nitrogen. The improved kinetic and stability features of the immobilized cellulase combined with advantageous properties of the polyurethane foam (resistance to enzymatic degradation plasticity of shape and size) suggest that this mechanism of cellulase immobilization has high potential for application in the industrial degradation of celluloses.

Role of enzyme binding in muscle metabolism of the goldfish

Abstract:

Regulation of glycolytic metabolism in muscle by the reversible association of enzymes with the particulate fraction of the cell was assessed in heart and white skeletal muscle of the goldfish Carassius auratus. Three physiological states were compared: rested aerobic controls, 21 h of anoxia exposure in water bubbled with N2 and CO, and exhaustive swimming. Heart muscle responded to the anoxia exposure with an increase in the percentage bound for phosphofructokinase (PFK), aldolase, and pyruvate kinase, the greatest increase being for PFK (from 35 to 48%). When fish swam to exhaustion, however, no changes in the percentage bound occurred for the eight enzymes assayed in heart. In white muscle neither anoxia nor exhaustive swimming altered the binding of the eight enzymes to the particulate fraction of the cell, except for a significant decrease in the percentage of alcohol dehydrogenase bound in anoxic muscle. PFK binding was particularly high in white muscle (63-72% bound, assessed by two methods). PFK binding was sensitive to pH in both organs, with binding increasing as pH decreased. This suggests that binding of PFK could be enhanced when metabolism is highly dependent on glycolytic ATP production due to the fall in cellular pH as a result of metabolic acidosis.

Immobilization of Amyloglucosidase onto Granular Chicken Bone

Abstract:

Amyloglucosidase was immobilized onto granular chicken bone (BIOBONE TM) by noncovalent interactions. The amount of activity bound relative to an equal amount of free enzyme was 13.6+0.4%. The estimated specific activity for amyloglucosidase decreased from 75.3+0.8 to 43.5+9.6 U/mg protein upon immobilization. The Km value of the bone-immobilized enzyme using glycogen as substrate increased from 3.04+0.38 mg/mL (free) to 9.04+l.51 mg/mL (immobilized), but Km showed no change upon immobilization when starches were used as substrates. A decrease in Vmax values occurred upon enzyme immobilization for all substrates, but this largely reflected the percentage of enzyme initially bound to the bone. Immobilization also improved enzyme stability in the presence of various additives (e.g., detergent, KCl, and ethanol) or under low or high pH reaction conditions. Bound amyloglucosidase maintained high activity (greater than 90%) following five cycles of continuous use at moderate (23°C) and high (55°C) temperatures. Data derived from Lineweaver-Burk and Arrhenius plots indicated that substrate and product diffusion limitation were minimal.

Regulation of enzymes of carbohydrate metabolism during anoxia in the salt marsh bivalve Geukensia demissus

Abstract:

The effects of anoxia exposure (2 or 12 h at 5°C) on the tissue-specific responses by enzymes of carbohydrate metabolism were analyzed in mantle, gill, hepatopancreas and adductor muscle of the intertidal bivalve Geukensia demissus demissus. Reversible enzyme phosphorylation has been identified in other species of marine molluscs as a mechanism for coordinating the suppression of metabolic rate and the redirection of carbon into fermentative pathways under anoxic conditions. The present study shows patterns of response to anoxia by five enzymes of carbohydrate metabolism including glycogen synthetase (GS) and pyruvate dehydrogenase (PDH) whose responses to anoxia have not before been analyzed in marine molluscs. Anoxia-induced changes in properties, consistent with reversible phosphorylation modification of the enzymes, were found for pyruvate kinase (PK) and pyruvate dehydrogenase (PDH) in all tissues and in selected tissues for glycogen phosphorylase (GP) and GS. However, phosphofructokinase did not appear to be modified in any tissue during anoxia. Within 2 h of anoxia exposure, PK showed a sharp drop in the activity ratio (at subsaturating vs. saturating PEP concentrations) that indicated a stable modification of enzyme Km for PEP; for example, in gill the ratio (determined at 0.75 & 7.5 mM PEP) fell from 0.39 + 0.08 for aerobic controls to 0.08 + 0.01 after 2 h anoxia. The percentage of PDH in the active a form also dropped significantly in anoxia from 80-84 % a in controls to 65-75% a in anoxic tissues. Changes in both of these enzymes are consistent with anoxia-induced metabolic rate suppression. By contrast, anoxia exposure increased GP activity in gill and adductor muscle indicating a need for increased glycogenolysis during anoxia in these tissues. Total phosphorylase (a + b) activity increased in both tissues as also did the %a in adductor; for example, in adductor, active GPa content rose from 0.09 + 0.02 U/g wet weight in controls to 0.24 + 0.01 U/gww after 2 h and 0.16 + 0.02 U/gww after 12 h anoxia exposure. GS behaved oppositely in hepatopancreas showing a significant decrease in total activity in 12 h anoxic tissue but GS was unaffected by anoxia in gill or adductor muscle. In mantle, oppositely directed changes in total GS activity and the % active resulted in no change in the activity of the active I form during anoxia.