Daniel Miller, M.Sc. Biology, 1983

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



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.



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



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



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



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



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



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.


Thomas D. Pfister, M.Sc. Chemistry 1999

Phosphorylation regulation in two species of cold-hardy goldenrod gall insects.



The larvae of two cold-hardy insects, the freeze avoiding gall moth Epiblema scudderiana and freeze tolerant gall fly Eurosta solidaginis, endure prolonged exposures to subzero temperatures while overwintering. Both rely on a reduction in metabolic rate and the production of polyols for survival. The present study analyzed the role of several signal transduction enzymes in cold hardiness. Changes in cAMP-dependent protein kinase (PKA), protein phosphatases-1 (PP1), 2A, 2C and protein tyrosine phosphatase activities were monitored over the course of the winter season and also in insects exposed to -4C, -20C or anoxic conditions. The catalytic subunit of PKA (PKAc) and PP1 were purified to homogeneity from both species. Effects of low temperature on the enzymes included increased affinity of PKAc for the Kemptide substrate, and increased inhibition by okadaic acid of PP1. Differential regulation of kinases and phosphatases by low temperature appears to be key to regulating polyol production and, hence, cold survival.


Estatira Sepehr, M.Sc. Chemistry 2002

Effect of fermentation of folate bioavailability.



The objective of this study was to assess the association between dietary fibre (DF) and fermentable substrates (FS) intake, colonic bacterial fermentation, and folate bioavailability. Folate is important in human nutrition because folate deficiencies are associated with neural tube defects and anaemia and excess folate intake may mask other disease states. Folate may also modulate hyperhomocysteynemia, an independent risk factor for cardiovascular disease. Current recommended intakes (400 ug/d) are based solely on folacin intake but it has been suggested that folacin may also be obtained indirectly from colonic bacteria, stimulated by fermentation of DF and FS. In order to study this possibility, male weanling Sprague Dawley rats were folate depleted by feeding a low folacin AIN93G formulated basal diet for 28 d and then fed diets differing in DF or FS and folate (0.25-1.0 mg/kg diet) to replete liver stories. During the repletion phase, the increase in liver folate was proportional only to the dietary folate content and did not vary with changing dietary DF or FS. An antibiotic (succinylsulfathiazole) was added to inhibit gut bacterial populations and bacterial fermentation. Addition of the antibiotic lowered total liver folate in controls and test diets equally suggesting an effect of succinylsulfathiazole on folate absorption or metabolism. Changes in cecal volatile fatty acids, increases in fecal diaminopimelic acid excretion, DF or FS balance as well as increases in fecal folate content provided evidence for an increased bacterial fermentation, an increased bacterial excretion, and an increased production of folate in the colon. However, colonic folate produced by bacterial fermentation was not bioavailable.

Zhouli (Julie) Ni, M.Sc. Biology 2004

Antioxidant defense in the hibernating thirteen-lined ground squirrel, Spermophilus tridecemlineatus.



Dramatic changes in blood flow and oxygen consumption during torpor-arousal cycles could increase the risk of oxidative stress to tissues of mammalian hibernators. Changes to antioxidant defense systems during hibernation were assessed in thirteen-lined ground squirrels, Spermophilus tridecemlineatus, focusing on heme oxygenase 1 (HO1), superoxide dismutase (SOD) and the thioredoxin (Trx) system. Compared with euthermic control squirrels, inducible HO1 was elevated at both the transcriptional and translational level in multiple tissues of hibernating squirrels. The Nrf2/ARE signal transduction pathway commonly regulates HO1 and protein levels of both the transcription factor Nrf2 and its cofactor MafG were elevated in concert with HO1 implicating this pathway in HO1 expression in hibernation. Both cytosolic Cu/Zn SOD and mitochondrial Mn SOD increased significantly in squirrel muscle during hibernation, probably contributing to antioxidant defense during arousal from torpor when oxygen consumption by muscle increases rapidly to support shivering thermogenesis. Proteins of the thioredoxin system (Trx1, Trx2, TrxR2) also rose in multiple tissues during hibernation, perhaps to remedy a glutathione redox imbalance during hibernation. Amino acid sequencing of HO1 and SOD showed strong conservation of sequence compared with nonhibernating mammals but selected substitutions were found that could aid enzyme function at low body temperatures. The data support the idea that hibernators experience oxidative stress over torpor-arousal cycles and in response activate antioxidant defenses to minimize damage. A better understanding of antioxidant defense systems in hibernation may guide the use of antioxidants in medical treatments for ischemia/reperfusion damage and technology for cold preservation of organs.