Biomedical subjects

K B Storey

Publications and source records attributed to K B Storey.

At least 55 records · Page 3Linked to original sources

PubMed1999

Reversible phosphorylation control of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad, Scaphiopus couchii.

Both pyruvate kinase (PK) and phosphofructokinase (PFK) occur in two different forms, separable by isoelectric focusing (IEF), in skeletal muscle of the spadefoot toad Scaphiopus couchii. During estivation (aerobic dormancy) the proportions of the two forms changed compared with controls; in both cases the amount of enzyme in Peak I (pI = 5.3-5.4) decreased whereas activity in Peak II (isoelectric point = 6.2-6.4) increased. In vitro incubation of crude muscle extracts with 32P-ATP under conditions that promoted the activity of cAMP-dependent protein kinase led to strong radiolabeling associated with Peak I, but not Peak II, and reverse phase HPLC confirmed that 32P was associated with the subunits of both PK and PFK found in Peak I. Specific radiolabeling of Peak I PK and PFK by protein kinase A was further confirmed using immunoprecipitation. In total, this information allowed identification of the Peaks I and II enzymes as the phosphorylated and dephosphorylated forms, respectively, and the effect of estivation was to increase the proportion of dephosphorylated PK and PFK in muscle. Analysis of the kinetic properties of partially purified PK and PFK revealed significant kinetic differences between the two forms of each enzyme. For PK, the Peak II (low phosphate) enzyme showed a 1.6-fold higher Km for phosphoenolpyruvate and a 2.4-fold higher Ka for fructose-1,6-bisphosphate than did the Peak I (high phosphate) form. These kinetic properties suggest that Peak II PK is the less active form, and coupled with the shift to predominantly the Peak II form during estivation (87% Peak II vs. 13% Peak I), are consistent with a suppression of PK activity in estivating muscle, as part of the overall metabolic rate depression of the estivating state. A similar shift to predominantly the Peak II, low phosphate, form of PFK (75% Peak II, 25% Peak I) in muscle of estivating animals is also consistent with metabolic suppression since phosphorylation of vertebrate skeletal muscle PFK is typically stimulated during exercise to enhance enzyme binding to myofibrils in active muscle. Peak II PFK also showed reduced sensitivity to inhibition by Mg:ATP (I50 50% higher) compared with the Peak I form suggesting that the enzyme in estivating muscle is less tightly regulated by cellular adenylate status than in awake toads. The data indicate that reversible phosphorylation control over the activity states of enzymes of intermediary metabolism is an important mechanism for regulating transitions between dormant and active states in estivating species.

Animals↗

PubMed1999

Brain gamma-glutamyltranspeptidase: characteristics, development and thyroid hormone dependency of the enzyme in isolated microvessels and neuronal/glial cell plasma membranes.

The characteristics, cellular locus and regulation of the enzyme gamma-glutamyltranspeptidase (gammaGT) in brain were examined. In rat brain homogenates, the activity of the enzyme exhibited tissue differences--kidney>>>brain==testis>>liver>>skeletal muscle=ventricular muscle and regional differences--brain stem>hippocampus=cerebellum>cerebral cortex, with no significant species/strain differences in the select group of mammals studied. Methods were developed for the isolation from brain of microvessels (MV) and plasma membranes from neuronal/glial cells (N/G PM) utilizing morphological indicators and marker analyses. GammaGT activity was >12 higher in MV than N/G PM; however the enzyme displayed: stability, heat-activation and inhibition with maleate to the same extent in both fractions. A comparative study indicated that in the N/G PM fraction, gammaGT activity was low in all animals studied; gammaGT activity in MV however, was barely detectable in amphibians and reptiles, very low in birds and very high in mammal -mirroring the phylogenetic development of a functional blood-brain barrier. In the rat, gammaGT in both MV and N/G PM displayed a pronounced postnatal increase in activity but the extent and the patterns were different--in all cases, that of the MV greatly exceeded that of the N/G PM and in the MV, the enzyme activity the exhibited the same pattern as the postnatal development of the blood-brain barrier. The induction of congenital hypothyroidism by propylthiouracil (PTU) had no effect on gammaGT in N/G PM but effected a one third reduction in the activity of gammaGT in MV. The normalization by thyroid hormone replacement indicated that MVgammaGT is under thyroid hormone control. The induction of hypothyroidism by PTU in the adult, however, was without effect on enzyme activity in either fraction. The implications of the thyroid hormone dependency of MVgammaGT in the neonatal period and the relationship of gammaGT to the function of the blood brain-barrier is discussed.

Aging↗

PubMed1999

Phosphorylation of H,K-ATPase alpha-subunit in microsomes from rabbit gastric mucosa by cAMP-dependent protein kinase.

A 100-kDa protein that is a main component of the microsomal fraction from rabbit gastric mucosa is phosphorylated by cAMP-dependent protein kinase (PKA) in the presence of 0.2% Triton X-100. Microsomes from rabbit gastric mucosa possess activity of H,K-ATPase but not activity of Na,K-ATPase. Incubation of microsomes with 5 microM fluorescein 5'-isothiocyanate (FITC) results in both an inhibition of H,K-ATPase and labeling of a protein with an electrophoretic mobility corresponding to the mobility of the protein phosphorylated by PKA. The data suggest that the alpha-subunit of H,K-ATPase can be a potential target for PKA phosphorylation.

Animals↗

PubMed1999

Living in the cold: freeze-induced gene responses in freeze-tolerant vertebrates.

1. Winter survival for numerous cold-blooded animals includes freeze tolerance: the ability to endure the conversion of as much as 65% of total body water into extracellular ice. Selected molecular adaptations underlying freeze tolerance (e.g. cryoprotectants, ice nucleating proteins) have been widely studied, but the full range of metabolic adjustments needed for freeze endurance remains unknown. 2. Recent studies using gene screening techniques are providing a different approach to the search for biochemical responses that support freezing survival by identifying genes and proteins that are up-regulated by freezing or thawing in freeze-tolerant amphibians and reptiles. 3. Screening of a cDNA library from wood frog liver revealed the freeze-induced up-regulation of genes coding for the alpha- and gamma-subunits of fibrinogen (a plasma clotting protein), the mitochondrial ADP/ATP translocase and a novel 10 kDa protein containing a nuclear exporting sequence. 4. Northern blotting revealed that these genes were differentially responsive to two of the component stresses of freezing (dehydration and anoxia), indicating that different genes are induced by signals radiating either from cell volume change or oxygen deprivation during freezing. 5. Freeze up-regulation of fibrinogen synthesis in liver and other organs appears to be a damage repair response that anticipates a need for enhanced plasma clotting capacity to deal with ice crystal damage to capillary beds. 6. Up-regulation of ADP/ATP translocase in frog liver is linked with ischaemia resistance and studies with freeze-tolerant turtles have shown that other genes encoding proteins involved in mitochondrial energetics (NADH-ubiquinone oxido-reductase subunit 5, cytochrome C oxidase subunit 1) are also up-regulated by both anoxia and freezing exposures. 7. These studies are making major advances in our understanding of freeze tolerance as a natural phenomenon and also highlight new key areas that can be targeted by applied interventions for the optimization of medical cryopreservation techniques for cells, tissues and organs.

Amino Acid Sequence↗

PubMed1999

Metabolic adjustments during daily torpor in the Djungarian hamster.

Djungarian hamsters (Phodopus sungorus) acclimated to a short photoperiod (8:16-h light-dark cycle) display spontaneous daily torpor with ad libitum food availability. The time course of body temperature (Tb), metabolic rate, respiratory quotient (RQ), and substrate and enzyme changes was measured during entrance into torpor and in deep torpor. RQ, blood glucose, and serum lipids are high during the first hours of torpor but then gradually decline, suggesting that glucose is the primary fuel during the first hours of torpor, with a gradual change to lipid utilization. No major changes in enzyme activities were observed during torpor except for inactivation of the pyruvate dehydrogenase (PDH) complex in liver, brown adipose tissue, and heart muscle. PDH inactivation closely correlates with the reduction of total metabolic rate, whereas in brain, kidney, diaphragm, and skeletal muscle, PDH activity was maintained at the initial level. These findings suggest inhibition of carbohydrate oxidation in heart, brown adipose tissue, and liver during entrance into daily torpor.

Adaptation, Physiological↗

PubMed1999

Characteristics of the interaction of melittin with sarcoplasmic reticulum membranes.

Addition of an amphipathic bee venom peptide, melittin, to sarcoplasmic reticulum (SR) vesicles isolated from rabbit skeletal muscles resulted in a fast (<1 min) blue shift in the fluorescence maximum of the melittin--SR membrane complex. Over the following 45 min the position of the fluorescence maximum did not change, but the fluorescence intensity of the melittin--SR membrane complex decreased by approximately 35% with rate constant 0.14 min-1. Melittin rapidly quenched the isotropic signal in the EPR spectrum of spin-labeled stearic acid added to SR membranes. Further changes in the spectral parameters of the spin probe bound to SR membranes in the presence of melittin indicated an increase of the viscosity of the probe microenvironment (empiric parameter T/eta was decreased by approximately 35% with rate constant 0.11 min-1). The surface potential of SR membranes measured using a pH-sensitive dye, neutral red, decreased after melittin addition from -60 to -30 mV. It was demonstrated with the use of a cross-linking agent, cupric o-phenanthroline, that melittin induced slow aggregation of Ca-ATPase protein in SR membranes; the content of enzyme in the monomeric form decreased with rate constant 0.14 min-1. It is concluded that melittin binds rapidly to SR membranes, inducing slow changes in Ca-ATPase conformation and oligomeric state as well as structural transitions in the lipid bilayer of SR membranes.

Animals↗

PubMed1999

Comparative characteristics of sarcoplasmic reticulum preparations from skeletal muscles of the ground squirrel Spermophilus undulatus, rats, and rabbits.

A comparison of sarcoplasmic reticulum (SR) preparations from skeletal muscles of ground squirrels Spermophilus undulatus, rats, and rabbits established that on the basis of protein yield and phospholipid/protein ratio these preparations are practically the same. Nevertheless, the specific activity of Ca-ATPase, the main protein component of SR membranes, in SR preparations of the ground squirrel skeletal muscles is only about half of the activity in SR preparations of rats and rabbits. Significant differences in protein composition of the preparations were detected: ground squirrel SR differed by an unusually high content of a 205 kD protein (probably myosin) and a number of low-molecular-weight SR protein components, and the SR preparations of rabbits are characterized by a high content of the Ca-binding proteins calsequestrin and sarcalumenin. Use of the anionic carbocyanine dye Stains-All established that all preparations contained only three proteins which are stained dark blue by this dye: calsequestrin, sarcalumenin, and a histidine-rich Ca-binding protein. The electrophoretic mobility of calsequestrin was identical in all preparations (molecular mass 63 kD), whereas sarcalumenin and histidine-rich Ca-binding protein are probably present in different isoforms with molecular masses of 130, 145, and 160 and 165, 155, and 170 kD, respectively, in SR preparations of ground squirrels, rats, and rabbits. Analysis of the fluorescence parameters of the fluorescent probes 8-anilino-1-naphthalene sulfonic acid and pyrene bound to SR membranes showed that the properties of the lipid bilayer in the SR membranes of the preparations differed considerably. It is suggested that the differences in protein composition and/or structural state of the ground squirrel SR membrane lipid bilayer could be the reason for the low Ca-ATPase activity in these preparations.

Animals↗

PubMed1998

Protein kinase A from bat skeletal muscle: a kinetic study of the enzyme from a hibernating mammal.

The catalytic subunit of adenosine 3'-5'-cyclic mono-phosphate-dependent protein kinase (PKAc) was purified to homogeneity from skeletal muscle of the little brown bat, Myotis lucifugus. The purification procedure was highly reproducible, resulting in a final activity of 205 nmol phosphate transferred/min/mg protein at 22 degreesC. Identification of the enzyme as a protein kinase A was confirmed through the use of specific PKA inhibitors. The catalytic subunit had a molecular weight of 54.6 +/- 3.5 kDa.Km values for Kemptide and Mg-ATP were 9.1 +/- 0.2 and 94.1 +/- 4.5 microM at 37 degreesC, respectively. Both values decreased significantly at 5 degreesC to 37 and 52% of their values at the higher temperature. Similar temperature effects on Km values were found with the purified commercial pig heart enzyme. Neutral salts had little effect on enzyme activity (I 50 values >400 mM) but NaF had an I 50 of 38 mM; except for fluoride, ions were less inhibitory at 5 degreesC, compared with 37 degreesC. Arrhenius plots showed evidence of a temperature-dependent conformational change; a distinct break in the plot occurred at 10 degreesC giving calculated activation energies of 5.6 +/- 0. 46 kJ/ mol at temperatures above 10 degreesC++and 29.5 +/- 2.0 kJ/mol below 10 degreesC. Porcine PKAc, by contrast, showed a linear Arrhenius plot over the entire temperature range tested and an intermediate activation energy of 15.9 +/- 0.3 kJ/mol. The pH optimum of bat PKAc also changed dramatically with temperature falling from 8.5 at 37 degreesC to 5.5 at 5 degreesC, an effect that could substantially change enzyme activity in vivo at the low body temperature of the hibernating state. Overall, low temperature had both positive (increased the percentage of PKAc, reduced Km values, increased I 50 values for salts) and negative (increased activation energy, acidic shift of pH optimum) effects on PKAc but the substantial positive effects of low temperature on the enzyme suggest an important role for continued PKA action in signal transduction in the hibernating animal.

Animals↗

PubMed1998

Biophysics of freezing in liver of the freeze-tolerant wood frog, R. sylvatica.

This study investigates the water transport characteristics during freezing in the liver tissue of the freeze-tolerant wood frog Rana sylvatica. Experiments were performed using both low temperature microscopy and a differential scanning calorimeter (DSC). Tissue samples were cooled at 2 and 5 degree C/min by a "two-step" freezing technique to end temperatures of -4, -6, -8, -10, and -20 degrees C, followed by a slam cooling (> 1000 degrees C/min) step. Stereological analysis of the low temperature microscopy results leads to the conclusions that 74% of the control tissue is cellular (26% vascular), Vb (osmotically inactive cell volume) is 0.4 Vo and the Krogh cylinder dimensions are: distance between adjacent sinusoid centers, delta X = 64 microns, original sinusoid (vascular) radius, rvo = 18.4 microns and length of the Krogh cylinder, L = 0.71 microns (assuming a single isolated hepatocyte cell diameter of 16 microns). A parallel study was also done using the DSC at 2 and 5 degrees C/min, and the measured heat releases from the tissue were used to calculate water transport data. Both techniques confirmed that tissue cooled at 5 degrees C/min does not dehydrate completely, but does so when cooled at 2 degrees C/min. By curve fitting a model to 5 degrees C/min water transport data from both techniques the biophysical parameters of water transport were obtained: Lpg = 1.76 microns/min-atm and ELp = 75.5 Kcal/mol. A modified Krogh model was used to account for the fact that approximately 24% of the hepatocytes were found not to be in direct contact with the vasculature. This model was then used to explain the experimentally measured water retention in some cells on the basis of different volumetric responses to dehydration of cells directly adjacent to vascular spaces and cells at least one cell removed from the vascular spaces.

Animals↗

PubMed1998

Antioxidant defenses and lipid peroxidation damage in estivating toads, Scaphiopus couchii.

Tissue-specific changes in antioxidant defenses and lipid peroxidation damage were analyzed in spadefoot toads, Scaphiopus couchii, to determine how these responded during estivation, a state of suppressed oxygen consumption. Maximal activities of glutathione-S-transferase, glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase were measured in six organs from 2-month-estivated toads and compared with activities in animals awakened for 10 days after estivation. Activities of many enzymes, particularly the glutathione-linked enzymes, were significantly lower in tissues of estivating toads than in awake toads. This indicates that enzymatic antioxidant defenses are probably modulated in response to the rate of reactive oxygen species generation in tissues, which is proportional to oxygen consumption. Antioxidant enzyme activities were largely insensitive to high urea, which accumulates during estivation, but were inhibited by elevated KCl. Levels of reduced glutathione were also significantly lower in three organs during estivation and all organs, except skeletal muscle, exhibited a higher oxidized/reduced glutathione ratio, indicating a more oxidized state during estivation. Products of lipid peroxidation (conjugated dienes, lipid hydroperoxides) were higher in tissues of estivated than control toads, suggesting accumulated oxidative damage to lipids during dormancy. One enzymatic source of free radical generation, xanthine oxidase, appeared to have little impact because its activity was detectable only in liver and was significantly lower in estivated toads. The data indicate that both enzymatic and metabolite antioxidant defenses in toads are adaptable systems that are modulated in estivating versus awake states.

Animals↗

PubMed1998

cAMP-dependent protein kinase from brown adipose tissue: temperature effects on kinetic properties and enzyme role in hibernating ground squirrels.

Arousal from hibernation requires thermogenesis in brown adipose tissue, a process that is stimulated by beta-adrenergic signals, leading to a rise in intracellular 3',5'-cyclic adenosine monophosphate AMP (cAMP) and activating cAMP-dependent protein kinase A (PKA) to phosphorylate a suite of target proteins and activate lipolysis and uncoupled respiration. To determine whether specific adaptations (perhaps temperature-dependent) facilitate PKA kinetic properties or protein-phosphorylating ability, the catalytic subunit of PKA (PKAc) from interscapular brown adipose of the ground squirrel Spermophilus richardsonii, was purified (final specific activity = 279 nmol phosphate transferred per min per mg protein) and characterized. Physical properties of PKAc included a molecular weight of 41 kDa and an isoelectric point of 7.8 +/- 0.08. A change in assay temperature from a euthermic value (37 degrees C) to one typical of hibernating body temperature (5 degrees C) had numerous significant effects on ground squirrel PKAc including: (a) pH optimum rose from 6.8 at 37 degrees C to 8.7 at 5 degrees C, (b) K(m) values at 37 degrees C for Mg.ATP (49.2 +/- 3.4 microM) and for two phosphate acceptors, Kemptide (50.0 +/- 5.5 microM) and Histone IIA (0.41 +/- 0.05 mg/ml) decreased by 53%, 80% and 51%, respectively, at 5 degrees C, and (c) inhibition by KCl, NaCl and NH4Cl was reduced. However, temperature change had little or no effect on K(m) values of rabbit PKAc, suggesting a specific positive thermal modulation of the hibernator enzyme. Arrhenius plots also differed for the two enzymes; ground squirrel PKAc showed a break in the Arrhenius relationship at 9 degrees C and activation energies that were 29.1 +/- 1.0 kJ/mol for temperatures > 9 degrees C and 2.3-fold higher at 68.1 +/- 2.1 kJ/mol for temperatures < 9 degrees C, whereas the rabbit enzyme showed a breakpoint at 17 degrees C with a 13-fold higher activation energy over the lower temperature range. However, fluorescence analysis of PKAc in the absence of substrates, showed a linear change in fluorescence intensity and wavelength of maximal fluorescence over the entire temperature range; this suggested that the protein conformational change indicated by the break in the Arrhenius plot was substrate-related. Temperature change also affected the Hill coefficient for cAMP dissociation of the ground squirrel PKA holoenzyme which rose from 1.12 +/- 0.18 at 37 degrees C to 2.19 +/- 0.07 at 5 degrees C, making the release of catalytic subunits at low temperature much more responsive to small changes in cAMP levels. Analysis of PKAc function via in vitro incubations of extracts of ground squirrel brown adipose with 32P-ATP + cAMP in the presence versus absence of a PKA inhibitor, also revealed major differences in the patterns of phosphoproteins, both between euthermic and hibernating animals as well as between 37 and 5 degrees C incubation temperatures; this suggests that there are both different targets of PKAc phosphorylation in the hibernating animal and that temperature affects the capacity of PKAc to phosphorylate different targets. Both of these observations, plus the species-specific and temperature-dependent changes in ground squirrel PKAc kinetic properties, suggest differential control of the enzyme in vivo at euthermic versus hibernating body temperatures in a manner that would facilitate a rapid and large activation of the enzyme during arousal from torpor.

Adipose Tissue, Brown↗

PubMed1998

Antioxidant defenses and metabolic depression. The hypothesis of preparation for oxidative stress in land snails.

The roles of enzymatic antioxidant defenses in the natural tolerance of environmental stresses that impose changes in oxygen availability and oxygen consumption on animals is discussed with a particular focus on the biochemistry of estivation and metabolic depression in pulmonate land snails. Despite reduced oxygen consumption and PO2 during estivation, which should also mean reduced production of oxyradicals, the activities of antioxidant enzymes, such as superoxide dismutase and catalase, increased in 30 day-estivating snails. This appears to be an adaptation that allows the snails to deal with oxidative stress that takes place during arousal when PO2 and oxygen consumption rise rapidly. Indeed, oxidative stress was indicated by increased levels of lipid peroxidation damage products accumulating in hepatopancreas within minutes after arousal was initiated. The various metabolic sites responsible for free radical generation during arousal are still unknown but it seems unlikely that the enzyme xanthine oxidase plays any substantial role in this despite being implicated in oxidative stress in mammalian models of ischemia/reperfusion. We propose that the activation of antioxidant defenses in the organs of Otala lactea during estivation is a preparative mechanism against oxidative stress during arousal. Increased activities of antioxidant enzymes have also observed under other stress situations in which the actual production of oxyradicals should decrease. For example, antioxidant defenses are enhanced during anoxia exposure in garter snakes Thamnophis sirtalis parietalis (10 h at 5 degrees C) and leopard frogs Rana pipiens (30 h at 5 degrees C) and during freezing exposure (an ischemic condition due to plasma freezing) in T. sirtalis parietalis and wood frogs Rana sylvatica. It seems that enhancement of antioxidant enzymes during either anoxia or freezing is used as a preparatory mechanism to deal with a physiological oxidative stress that occurs rapidly within the early minutes of recovery during reoxygenation or thawing. Thus, a wide range of stress tolerant animals display coordinated changes in antioxidant defenses that allow them to deal with oxidative stress that occurs as part of natural cycles of stress/recovery that alter oxygen levels in tissues. The molecular mechanisms that trigger and regulate changes in antioxidant enzyme activities in these species are still unknown but could prove to have key relevance for the development of new intervention strategies in the treatment of cardiovascular ischemia/reperfusion injuries in humans.

Animals↗

PubMed1998

Role of antioxidant defenses in the tolerance of severe dehydration by anurans. The case of the leopard frog Rana pipiens.

Many anurans have excellent dehydration tolerance that allows endurance of the loss of up to 50-60% of total body water. One of the effects of severe dehydration is circulatory impairment due the reduced volume and increased viscosity of blood, which leads to organ hypoxia. The rehydration situation, therefore, involves a reoxygenation of tissues that may include elements of oxidative stress that resemble the injury in post-ischemic reperfusion of mammalian organs. The role of endogenous defenses against oxygen radicals in the tolerance of severe dehydration by leopard frogs, Rana pipiens, was investigated by monitoring the activities of antioxidant enzymes and glutathione levels (reduced GSH and oxidized GSSG) in leg muscle and liver of control, 50%-dehydrated, and fully rehydrated frogs. The maximal activities of muscle catalase and liver glutathione peroxidase, measured per mg soluble protein, increased significantly by 52 and 74%, respectively, after dehydration whereas muscle superoxide dismutase and glutathione reductase activities responded oppositely, decreasing by 32 and 35%, respectively. Enzyme activities returned to control levels after full rehydration. Hepatic GSH and GSSG increased early in the rehydration process (30% recovery of total body water), but returned to control levels after full recovery. A similar trend was observed for liver GSSG. The elevation of antioxidant defenses against peroxides during dehydration could provide protection against post-hypoxic oxyradical stress during rehydration. Indeed, analysis of one product of lipid peroxidation, thiobarbituric acid reactive substances, in frog tissues gave no indication of oxidative stress during the dehydration/rehydration cycle.

Animals↗

PubMed1998

Protein kinase C from rainbow trout brain: identification and characterization of three isozymes.

Free and membrane-associated fractions of protein kinase C (PKC) from rainbow trout (Onchorynchus mykiss) brain tissue were separated by hydroxylapatite chromatography and characterized kinetically. In both resting fish and in fish swum to exhaustion, approximately 40% of the total PKC activity was bound to membranes. Quantification of the three distinct hydroxylapatite chromatography peaks (PKC types gamma, beta and alpha) in cytosolic and membrane fractions revealed different isozyme distributions. The cytosolic fraction contained 21% PKC type gamma, 52% PKC type beta and 27% PKC type alpha. The membrane-associated fraction contained 23% PKC type gamma, 28% PKC type beta and 49% PKC type alpha. Kinetic characterization of the three isozymes showed that PKC type gamma was almost completely activated by Ca2+ alone whereas PKC type beta and PKC type alpha were 40% and 60% activated by Ca2+. Full activity for all enzymes was observed only in the presence of phosphatidylserine and diacylglycerol. Differences in the kinetic constants for the three isozymes were also apparent. PKC type gamma had a much lower affinity for Histone III-S when compared with PKC types beta and alpha (100 micrograms/ml as compared with 1.7 and 5.7 micrograms/ml). PKC type gamma also had a lower affinity for calcium (0.22 microM) when compared with PKC type beta (0.08 microM) and PKC type alpha (0.05 microM). PKC type alpha had a lower affinity for phosphatidylserine (8.6 micrograms/ml) when compared with PKC type gamma (0.37 microgram/ml) and PKC type beta (0.89 microgram/ml).

Animals↗

PubMed1998

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

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 that from a nonhibernator. We found that the Kd for oleate of the liver fatty acid binding protein (1.5 microM) isolated from ground squirrel (Spermophilus richardsonii) was temperature insensitive over 5-37 degrees C, while the rat liver fatty acid binding protein was affected with the Kd at 37 degrees C being about half (0.8 microM) 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 the two proteins at 5 and 37 degrees 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 matched 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 degrees C than at 37 degrees 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.

Animals↗

PubMed1998

Comparative study of free and bound glycolytic enzymes from sea scorpion brain.

Free and bound forms of hexokinase, pyruvate kinase, and lactate dehydrogenase were prepared from the brain of the sea scorpion (Scorpaena porcus) in a low ionic strength medium. Properties of the free and bound forms were compared to determine whether binding to particulate matter could influence enzyme function or stability in vivo. Changes in pH differently affected the activity of the free and bound forms of all three enzymes. Furthermore, bound forms of hexokinase and pyruvate kinase were more stable than the free enzymes to heating at 45 degrees C. Bound hexokinase showed higher affinity for substrates (ATP, glucose) than the free form and bound lactate dehydrogenase had greater affinity for pyruvate and NADH. Although the affinities of the two forms of pyruvate kinase for substrates were similar, Hill coefficients for phosphoenolpyruvate as well as inhibition by ATP differed between the two enzyme forms. Free and bound lactate dehydrogenase also showed differences in Hill coefficients and bound lactate dehydrogenase was less sensitive to substrate inhibition by high pyruvate concentrations. The possible physiological role of the binding of these glycolytic enzymes to subcellular structures is discussed.

Animals↗

PubMed1998

Effect of hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion tissues.

The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 mumol/g wet weight and in muscle led to its increase from 3.64 to 25.1 mumol/g wet weight. Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK) by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.

Animals↗

PubMed1998

Freeze tolerance in the wood frog Rana sylvatica is associated with unusual structural features in insulin but not in glucagon.

The wood frog Rana sylvatica utilises glucose, derived from hepatic glycogen, as a cryoprotectant in order to survive freezing during winter hibernation, and glycogenolysis is initiated by hormonal and/or neural stimuli. The primary structure of insulin was determined from R. sylvatica and from two species of freeze-intolerant Ranid frogs R. catesbeiana (American bullfrog) and R. ridibunda (European green frog). All three insulins contain a dipeptide (Lys-Pro) extension to the N-terminus of the A-chain. The amino acid sequences of insulins from R. catesbeiana and R. ridibunda differ by only one residue (Asp for Glu at B21) but R. sylvatica insulin differs from R. catesbeiana insulin at A12 (Thr-->Met), A23 (Asn-->Ser), B5 (Tyr-->His) and B13 (Glu-->Asp). The residue at A23 (corresponding to A21 in human insulin) has been otherwise fully conserved during evolution and the residue at B13 has been strongly conserved in tetrapods. Insulin isolated from specimens of R. sylvatica that had been frozen for 24 h and from control animals that had not been frozen had the same structure, showing that freezing did not alter the pathway of post-translational processing of proinsulin. R. sylvatica glucagon was isolated in two molecular forms. Glucagon-29 was identical to R. catesbeiana glucagon-29 and contains only one amino acid substitution (Thr-->Ser) compared with human glucagon. Glucagon-36 represents glucagon-29 extended from its C-terminus by Lys-Arg-Ser-Gly-Gly-Ile-Ser and is identical to R. catesbeiana glucagon-36. We speculate that selective changes in the structure of the insulin molecule may contribute to the anomalous regulation of glycogen phosphorylase in the wood frog.

Adaptation, Physiological↗