M. Sc. Thesis Abstracts

Yulia Maistrovski, M.Sc. Biology 2013

Regulation of anti-apoptotic pathways in skeletal muscle and liver of an estivating species, Xenopus laevis

 

Abstract:

This study investigated the anti-apoptotic pathways activated during dehydration and estivation of the African clawed frog, Xenopus laevis. Staying immobile for a significant period of time can cause serious tissue atrophy in non-adapted animals. To preserve skeletal muscle and liver mass, certain metabolic pathways with functions in cell preservation and protection must be activated. This study focused on two families of transcription factors, NF-κB and STAT whose target genes include those with anti-apoptotic functions. The data indicated an upregulation of the NF-ᴋB pathway in liver of dehydrated frogs along with downstream targets Bcl-xL and c-IAP. STAT3 appeared to be active in liver whereas STAT5 was active in skeletal muscle. Relative levels of the NF-κB and STAT downstream target Bcl-2 were elevated in response to dehydration. Analysis of pro- and anti-apoptotic microRNAs indicated that these contribute to post-transcriptional regulation of mRNA transcripts that encode proteins with roles in cell survival.

Jean Abboud, M.Sc. Biology 2015

Regulation of pyruvate kinase and glycerol-3-phosphate dehydrogenase in the freeze tolerant goldenrod gall fly: Role in polyol cryoprotectant production

 

Abstract:

Larvae of the goldenrod gall fly, Eurosta solidaginis, can survive winter temperatures as cold as –55oC by using the freeze tolerance strategy of cold hardiness. This study examines pyruvate kinase (PK) and glycerol-3-phosphate dehydrogenase (G3PDH) that were purified from gall fly larvae acclimated to 5oC (control) and −15oC (freeze-exposed). The roles that these enzymes play in polyol cryoprotectant synthesis and metabolic regulation were investigated. A primary focus was the potential for reversible post-translational modifications (PTMs) to regulate enzyme activity. Several PTMs were analyzed for PK and G3PDH from control and freeze-exposed gall fly larvae using dot-blot analysis. Results showed that low and high phosphate forms of each enzyme exist and these differ in kinetic properties, G3PDH also showed different levels of ubiquitination. Changes in substrate affinities and different interactions with endogenous sugars suggest that PK activity is maintained whereas G3PDH is inhibited during freezing.

 

Tamara English, M.Sc. Biology, 1995

Comparisons of the effects of temperature on the liver fatty acid binding proteins from hibernator and nonhibernator mammals

 

Abstract:

Hibernating mammals rely heavily on lipid metabolism to supply energy during hibernation. We wondered if the fatty acid binding protein from a hibernator responded to temperature differently than a non-hibernator. We found that the Kd for oleate of the liver fatty acid binding protein (1.5 mM) isolated from ground squirrel (Spermophilus richardsonii) was temperature insensitive over 5-37°C, while the rat liver fatty acid binding protein was affected with the Kd at 37°C being about half (0.8 mM) that found at lower temperatures. This same trend was observed when comparing the specificity of various fatty acids of differing chain length and degree of unsaturation for both proteins at 5°C and 37°C. At the lower temperature, ground squirrel protein bound long chain unsaturated fatty acids, particularly linoleate and linolenate, at least as well as at the higher temperature and matches requirements for these fatty acids in the diet. The most common long chain fatty acid, palmitate, was a more effective ligand for ground squirrel liver fatty acid binding protein at 5°C than at 37°C with the opposite occurring in the eutherm. Rat protein was clearly not adapted to function optimally at temperatures lower than the animal’s body temperature.

Daniel Miller, M.Sc. Biology, 1983

Gas-Liquid Chromatography and Enzymatic Determination of Alanopine and Strombine in Tissues of Marine Invertebrates

 

Abstract:

Gas-liquid chromatography (GLC) and enzymatic assays were developed for quantitating the imino acids, alanopine and strombine, alternate products of anaerobic glycolysis (replacing lactate) in the tissues of many marine invertebrates. For GLC analysis, t -strombine (2-methyliminodiacetic acid) and meso-alanopine (2,2’ iminodipropionic acid) were chromatographed as N-trifluoroacetyl isobutyl esters. Modifications of techniques used for GLC analysis of amino acids were required to overcome steric hindrance in the acylation reaction caused by the presence of imino, rather than amino, groups. Both imino acids were separated from each other and from all amino acids by GLC. Detection limit of the technique was 0.05 microg imino acid. Enzymatic determination of imino acids made use of the alanopine-specific alanopine dehydrogenase (ADH) purified from the periwinkle, Litterina littorea, and the strombine/ alanopine utilizing strombine dehydrogenase (SDH) from the clam, Mercenaria mercenaria, with assay conditions: 300 mM hydrazine buffer, pH 9.0, 5 mM NAD, and 0.3 unit ADH or 1.0 unit SDH. Enzymatic determinations of mixtures of alanopine and strombine in tissue samples required a dual analysis using both enzymes. Production of alanopine and strombine during anoxic stress in two species of marine molluscs was quantitated.

Roy D. Cole, M.Sc. Biology, 1984

Temperature effect on bivalve anaerobic metabolism.

 

Abstract:

The response of anaerobic metabolism in Mercenaria mercenaria to long term and acute temperature stress revealed qualitative and quantitative changes in metabolic flux. (A) The rate of anaerobic metabolism was sensitive to both long term and acute temperature change. (B) The choice of anaerobic end product was affected by environmental temperature. (C)Long term exposure to high temperature results in acclimation of anaerobic metabolism not seen during acute exposure to high temperature.

 

David Kelly, M.Sc. Biology 1988

Organ-specific control of glycolysis in anoxic turtles

 

Abstract:

Control of glycolysis during anoxia was investigated in five organs (heart, brain, liver, and red and white skeletal muscles) of the freshwater turtle, Pseudemys scripta, after 1 or 5 h of submergence in N2-bubbled water. Lactate was produced as the metabolic end product, with distinct organ differences in the amount (net lactate accumulation was 2.4-fold higher in brain than white muscle) and rate (lactate production in liver dropped 16-fold after the 1st h) of lactate accumulation. ATP and total adenylate contents of all organs were reduced (by 15-32%) after 1 h of submergence, but energy charge was maintained, after 5 h, adenylate contents had fully recovered. Changes in the levels of hexose and triose phosphate intermediates of glycolysis indicated an activation of glycolysis within the 1st h of anoxia exposure in brain, heart, and skeletal muscles. By 5 h, however, these were reversed, and a glycolytic rate depression was indicated, consistent with the overall metabolic rate depression accompanying long-term anaerobiosis in the turtle. Crossover analysis indicated glycolytic control at the pyruvate kinase reaction in all organs during both glycolytic activation and metabolic depression; regulatory control at the phosphofructokinase locus was primarily important only during glycolytic activation in heart and red muscle. The same analysis indicated a very rapid glycolytic inhibition in liver occurring within the 1st h of anoxia exposure; this allows glycogenolysis to be directed toward glucose export yielding the fermentative fuel used by other organs during anoxia.

Ross Whitwam, M.Sc. Biology 1988

Pyruvate kinase from the land snail Otala lactea: regulation by reversible phosphorylation during estivation and anoxia

 

Abstract:

Pyruvate kinase (PK) from tissues of the desert snail Otala lactea (Muller) undergoes a stable modification of its kinetic properties during estivation or in response to anoxia stress. In foot muscle and mantle, the kinetic changes induced by either state were virtually identical and were consistent with a less active enzyme form in estivation or anoxia: S0.5 PEP increased, and I50 values for Mg-ATP and L-alanine decreased, compared to the enzyme in control (aroused) snails. Estivation and anoxia also changed the properties of PK from hepatopancreas; some changes were consistent with a more active enzyme form (S0.5 PEP decreased, I50 values for Mg-ATP and L-alanine increased) but the enzyme lost all sensitivity to the potent activator fructose-1,6-bisphosphate. A time course of changes in I50 Mg-ATP for foot PK and S0.5 PEP for hepatopancreas PK revealed that estivation-induced changes in enzyme properties occurred between 12 and 48 h after snails were deprived of access to food and water, whereas the reversal of these changes occurred within as little as 10 min in foot muscle after arousal was initiated. The molecular basis of the stable modification of PK kinetics appears to be reversible protein phosphorylation. The action of added cyclic-AMP-dependent protein kinase on foot or hepatopancreas PK from control (aroused) snails changed PK kinetic parameters to those characteristic of the enzyme form in estivating animals; the addition of stimulators of endogenous cyclic-GMP-dependent protein kinase or protein kinase C had the same effect. Conversely, treatment with added phosphatases reconverted the properties of foot muscle PK from estivating snails to those characteristic of the control enzyme. The data suggest that reversible phosphorylation control over the activity state of regulatory enzymes of glycolysis is one mechanism contributing to the overall metabolic rate depression of the estivating state.

Clark Holden, M.Sc. Biology 1991

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

 

Abstract:

The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE+ ion exchange and 3′-5′-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3+0.74 and 2.7+0.87 micromol glucose-1-P/min/g wet weight, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units/mg protein, respectively. Both enzymes were dimers with native molecular weights of 2l5 000 + l8 000 for glycogen phosphorylase a and 209 000+15 000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87 000+2 000. Both enzymes showed pH optima of 7.5 at 22 °C and a break in the Arrhenius relationship with a two- to fourfold increase in activation energy below 10 °C. Michaelis constant values for glycogen at 22 °C were 0.12+0.004 mg/ml for glycogen phosphorylase a and 0.87+0.034 mg/ml for glycogen phosphorylase b, the Michaelis constant for inorganic phosphate was 6.5 + 0.07 mmol/L for glycogen phosphorylase a and 23.6 mmol/L for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a Ka of 0.176+0.004 mmol/L. Michaelis constant and Ka values decreased by two- to fivefold at 5°C compared with 22 °C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol/L) but was inhibitory to both enzyme forms at high concentrations (2 mol/L). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glyeogen phosphorylase b.

Aleixo Muise, M.Sc. Chemistry 1993

Reversible phosphorylation of fructose 1,6-bisphosphatase mediates enzyme role in glycerol metabolism in the freeze-avoiding gall moth Epiblema scudderiana

 

Abstract:

Fructose-1,6-bisphosphatse (FBPase) from larvae of the freeze-avoiding gall moth Epiblema scudderiana occurs in two forms which are interconverted by reversible phosphorylation and separable by CM-cellulose column chromatography. The phosphoenzyme has properties that would make it the more active form in vivo. Compared with the dephosphorylated form, the phosphoenzyme had 3-fold lower values for Km fructose-1,6-bisphosphate and Ka Mg2+ and lower sensitivities to allosteric inhibitors (I50 values for fructose-2,6-bisphosphate and AMP were 50 % and 10-fold higher, respectively). The proportions of the two enzyme forms in the larvae changed with the seasons and with acclimation to warm (15°C) versus cold (4°C) temperatures. The phosphorylated enzyme predominated (70% of total activity) in early autumn and during the spring, as well as in warm acclimated larvae, all situations where gluconeogenesis via FBPase would be favoured. During the autumn cold-hardening period when the larvae are actively synthesizing the antifreeze, glycerol, the ratio of the two enzyme forms changed to about 50:50. This, plus allosteric inhibition and low temperature effects on enzyme kinetics, would effectively suppress FBPase activity and prevent futile recycling of glycerol carbon back into glycogen during the winter months when the 2 M pool of polyol must be sustained for antifreeze protection. Acclimation studies suggested that low temperature itself might be the signal that triggers enzyme dephosphorylation and this could integrate control over FBPase with the well known phosphorylation-mediated activation of glycogen phosphorylase by low temperature in cold-hardy insects.

Peggy L. Schade, M.Sc. Chemistry, 1996

Glycogen degradation by alpha-glucosidase from a hibernating ground squirrel, Spermophilus lateralis, and a freeze tolerant frog, Rana sylvatica

 

Abstract:

Glycogen degradation in cells can proceed via two routes, the well-known phosphorolytic pathway via glycogen phosphorylase (GP) and the glucosidic pathway involving alpha-glucosidase. The present study analyzes alpha-glucosidase and its potential for net contributions to organ glucose production in two species that exhibit unusual glucose requirements while overwintering at low body temperatures: the hibernating golden-mantled ground squirrel, Spermophilus lateralis, and the freeze-tolerant wood frog, Rana sylvatica. Mammalian liver (ground squirrel, rat) possessed two neutral isozymes of alpha-glucosidase and one acidic form (pH optima 7.0 and 4.0) whereas amphibian liver (wood frog, leopard frog) had one neutral and one acidic enzyme. Tissue distribution of acidic and neutral forms revealed highest activities in liver and kidney and significant changes in activity during hibernation or freezing in selected tissues. Compared with the amount of active GP, acid alpha-glucosidase could make a substantial contribution to net glucose production in several wood frog organs, notably heart. Liver isozymes were separated by isoelectrofocusing prior to characterization of substrate specificity and affinity, inhibitor effects, and thermal denaturation. Neutral isozymes of all species were similar showing limited use of glycogen, strong thermal denaturation at 45°C and above, and generally reduced substrate affinity at the low temperatures characteristic of the hibernating state. Acidic isozymes used glycogen, showed good thermal stability and substrate affinities were largely temperature-independent. Inhibition of acidic alpha-glucosidase by salts and carbohydrates was reduced for the wood frog enzyme, compared with the leopard frog, a factor that may improve enzyme function in the frozen state.