Regulation of glycolysis in the land snail Oreohelix during estivation and artificial hypercapnia

Summary Respiration rates, hemolymph acid-base variables, and metabolite levels were measured in the land snail Oreohelix during a brief period of estivation (4 days) and during exposure of non-estivating snails to elevated levels of ambient CO₂ (34 and 58 mmHg). Respiration rate dropped during entry into estivation reflecting decreased glycolytic flux. Analyses of metabolite levels in foot muscle and digestive gland tissues demonstrated that glycolytic control during early estivation was primarily vested in pyruvate kinase. Relative to control values, the mass action ratio (MAR) of this reaction decreased by a factor of 4 in the foot muscle and by a factor of 2.7 in the digestive gland, reflecting inhibition of this enzyme in both tissues. No other glycolytic reaction appeared to be inhibited. Exposure of non-estivating snails to artificial hypercapnia resulted in extracellular acid-base conditions similar to those seen during estivation and also promoted a reduction in respiration rate. However, the points of glycolytic control were different: artificial hypercapnia resulted in inhibition of phosphofructokinase in foot muscle and hexokinase in digestive gland. Furthermore, elevated ambient CO₂ had no effect on the MAR of the pyruvate kinase reaction in non-estivating snails. These results suggest that the action of extracellular acid-base variables alone cannot fully explain the down-regulation of glycolysis which occurs during estivation in this land snail.Abbreviations ADP adenosine 5'-diphosphate - AEC adenylate energy charge - AMP adenosine 5'-monophosphate - ANOVA analysis of variance - ATP adenosine 5'-triphosphate - CCO₂ total content of carbon dioxide - EDTA ethylenediamine tetraacetic acid (disodium salt) - AG ^o change in molar standard Gibbs energy - IMP inosine 5'-monophosphate - K _a activation constant - K _eq equilibrium constant - K _m Michaelis constant - MAR mass action ratio - P probability - PCA perchloric acid - PCO₂ partial pressure of carbon dioxide - pH _i intracellular pH - P _i inorganic phosphate - r Spearman's coefficient of rank correlation - SD standard deviation     

Urea and salt effects on enzymes from estivating and non-estivating amphibians

The effects of urea, cations (K⁺, NH₄, Na⁺, Cs⁺, Li⁺), and trimethylamines on the maximal activities and kinetic properties of pyruvate kinase (PK) and phosphofructokinase (PFK) from skeletal muscle, were analyzed in two anuran amphibians, an estivating species, the spadefoot toadScaphiopus couchii, and a semi-aquatic species, the leopard frogRana pipiens. Urea, which accumulates naturally to levels of 200–300 mM during estivation in toads, had only minor effects on the V_max, kinetic constants and pH curves of PK from either species and no effects on PFK V_max or kinetic constants. Trimethylamine oxide neither affected enzyme activity directly or changed enzyme response to urea. By contrast, high KCl (200 mM) lowered the V_max of toad PFK and of PK from both species and altered the K_m values for both substrates of frog PFK. Other cations were even more inhibitory; for example, the V_max of PK from either species was reduced by more than 80% by the addition of 200 mM NH₄Cl, NaCl, CsCi, or LiCl. High KCl also significantly changed the K_m values for substrates of toad lactate dehydrogenase and strongly reduced the V_max of glutamate dehydrogenase and NAD-dependent isocitrate dehydrogenase in both species whereas 300 mM urea had relatively little effect on these enzymes. The perturbing effect of urea on enzymes and the counteracting effect of trimethylamines that has been reported for elasmobranch fishes (that maintain high concentrations of both solutes naturally) does not appear to apply to amphibian enzymes. Rather, we found that urea is largely a non-perturbing solute for anuran enzymes (I₅₀ values were>1 M for both PK and PFK in both species) and we propose that its accumulation in high concentrations during estivation helps to minimize the increase in cellular ionic strength that would otherwise occur during desiccation and to alleviate the accompanying negative effects of high salt on individual enzyme activities and overall metabolic regulation.Abbreviations PFK 6-phosphofructo-1-kinase - PK pyruvate kinase     

Metabolic responses of the South American ornate horned frog (Ceratophrys ornata) to estivation

We examined the metabolic responses of the South American frog, Ceratophrys ornata, to laboratory-induced estivation. Whole-animal and mass-specific oxygen consumption rates (VO₂) did not change during fasting or 56 days of estivation, despite observing significant decreases in body mass. The maintenance of mass-specific metabolic rate at routine levels during estivation suggests that metabolic rate suppression is not a major response to estivation in this species. There was a significant decline in liver glycogen and a loss of adipose tissue mass during estivation, suggesting that both carbohydrate and lipid pathways are used to fuel metabolism during estivation. The activity of pyruvate dehydrogenase, an important regulator of carbohydrate oxidation, and carnitine palmitoyltransferase and 3-hydroxyacyl-CoA dehydrogenase, regulators of lipid oxidation, showed no significant change in activity in liver, heart, and muscle between estivating and active frogs. There was an increase in plasma osmolality, which is characteristic of estivating animals. Overall, our metabolic analysis of estivation in C. ornata indicates that this species does not employ a dramatic suppression metabolic rate to survive dehydration stress and that both endogenous carbohydrates and lipids are used as metabolic fuels.     

Seasonal periodicity of enteric nitric oxide synthesis and its regulation in the snail, Helix lucorum

Abstract. The snail Helix lucorum has been used as a model to study the adaptation of a nitric oxide (NO)‐forming enteric neural network to the long‐term resting period of summer estivation or winter hibernation. Quantification of the NO‐derived nitrite established that NO formation is confined to the nitric oxide synthase (NOS)‐containing myenteric network of the mid‐intestine. In active snails but not in resting snails, NO production could be enhanced by the NOS substrate l ‐arginine (l ‐ARG, 1 mM). We followed the enteric NO synthesis in a snail population kept at natural conditions for 1 year. Our findings indicate that NO synthesis was depressed in July during entry to the estivation, had a peak in autumn before hibernation, and finally was reduced during hibernation. Monoamines (histamine, serotonin, and adrenalin) could inhibit the NO liberation in active snails. Cofactors of NOS (β‐NADPH, β‐NAD, FAD, FMN, Ca²⁺, TH4) did not alter the low nitrite production in hibernating snails. We conclude that enteric NO synthesis in H. lucorum has a regular seasonal periodicity following the annual physiological cycles of terrestrial snails. During estivation or hibernation, NOS activity is blocked. Monoamines, the levels of which are elevated during hibernation, can trigger decreased NOS activity. The reduced activity of NOS cannot be restored by the administration of NOS cofactors; therefore, their absence cannot be the cause of the temporarily blocked L‐ARG/NO conversion ability of NOS.     

The cystathionine‐β‐synthase domains on the guanosine 5′’‐monophosphate reductase and inosine 5′‐monophosphate dehydrogenase enzymes from Leishmania regulate enzymatic activity in response to guanylate and adenylate nucleotide levels

The Leishmania guanosine 5′‐monophosphate reductase (GMPR) and inosine 5′‐monophosphate dehydrogenase (IMPDH) are purine metabolic enzymes that function maintaining the cellular adenylate and guanylate nucleotide. Interestingly, both enzymes contain a cystathionine‐β‐synthase domain (CBS). To investigate this metabolic regulation, the Leishmania GMPR was cloned and shown to be sufficient to complement the guaC (GMPR), but not the guaB (IMPDH), mutation in Escherichia coli. Kinetic studies confirmed that the Leishmania GMPR catalyzed a strict NADPH‐dependent reductive deamination of GMP to produce IMP. Addition of GTP or high levels of GMP induced a marked increase in activity without altering the K_m values for the substrates. In contrast, the binding of ATP decreased the GMPR activity and increased the GMP K_m value 10‐fold. These kinetic changes were correlated with changes in the GMPR quaternary structure, induced by the binding of GMP, GTP, or ATP to the GMPR CBS domain. The capacity of these CBS domains to mediate the catalytic activity of the IMPDH and GMPR provides a regulatory mechanism for balancing the intracellular adenylate and guanylate pools.     

Purification and Characterization of Lactate Dehydrogenase in the Foot Muscle and Hepatopancreas of <Emphasis Type="Italic">Otala lactea</Emphasis>

Lactate dehydrogenase (LDH) has a crucial role in maintaining ATP production as the terminal enzyme in anaerobic glycolysis. This study will determine the effect of posttranslational modifications (PTMs) on the activity of LDH in the foot muscle and hepatopancreas of an estivating snail, Otala lactea. LDH in foot muscle of O. lactea was purified to homogeneity and partially purified in hepatopancreas in a two-step and three-step process, respectively. The kinetic properties and stability of these isoforms were determined where there was a significant difference in K_m and I₅₀ values with pyruvate and urea separately in foot muscle; however, hepatopancreas exhibited significant differences in K_m and I₅₀ in salt between control and stress. Interestingly, hepatopancreas has a higher affinity for pyruvate in the control state whereas foot muscle has a higher affinity for its substrate in the estivated state. PTMs of each isoform were identified using immunoblotting and dot blots, which prove to be significantly higher in the control state. Overall, foot muscle LDH enters a low phosphorylation state during estivation allowing more efficiency in consuming pyruvate with higher thermal stability but less structural stability. Hepatopancreas LDH becomes dephosphorylated in the estivating snail that decreases the efficiency of the enzyme in the forward direction; however, the snail has an increased tolerance to the presence of salt when water becomes scarce. Such tissue-specific regulations indicate the organism’s ability to reduce energy consumption when undergoing metabolic depression.     

Studies on the extra- and intracellular acid-base status and its role on metabolic depression in the land snail <Emphasis Type="Italic">Helix lucorum</Emphasis> (L.) during estivation

The aim of the present study was to examine the acid-base status of extra- and intracellular fluids and its possible role on the regulation of the metabolic rate of Helix lucorum during prolonged estivation. For this purpose, the rate of oxygen consumption for active and estivating snails was determined. The acid-base status was also examined in the hemolymph and tissues from active and estivating snails acclimated at 25 degrees C. In addition, the buffer values of hemolymph and tissues were determined in order to examine whether there is a change in the snails during estivation. The rate of oxygen consumption decreased significantly within the 1st 10 days of estivation from 122.51+/-10 microl.g(-1).h(-1) to 25.86+/-5.2 microl.g(-1).h(-1), indicating a marked decrease in metabolic rate. P(CO2)increased within the 1st 20 days of estivation from 13.52+/-0.68 mmHg to 25.09+/-2.05 mmHg, while the pH of hemolymph (pH(e)) decreased from 7.72+/-0.04 to 7.44+/-0.06. The level of bicarbonates decreased in the hemolymph of estivating snails, indicating a metabolic acidosis, which was moderate in extracellular fluids. In contrast to pH(e), the intracellular pH (pH(i)) was maintained in the tissues of estivating H. lucorum, indicating a regulation of pH(i) despite the developed hypercapnia. According to the results presented here, it seems that the timing of pH(e) changes does not correlate with the timing of metabolic rate reduction in estivating H. lucorum.     

Enhanced [<Superscript>3</Superscript>H] Glutamate Binding in the Cerebellum of Insulin-Induced Hypoglycaemic and Streptozotocin-Induced Diabetic Rats

Aim Energy deprivation causes neuronal death affecting the cognitive and memory ability of an individual. The kinetic parameters of glutamate dehydrogenase (GDH), the enzyme involved in the production of glutamate, was studied in the cerebellum and liver and the binding parameters of glutamate receptors in the cerebellum of insulin-induced hypoglycaemic and streptozotocin-induced diabetic rats were studied to reveal the role of glutamate excitotoxicity. Methods A single intrafemoral dose of streptozotocin was administered to induce diabetes. Hypoglycaemia was induced by appropriate doses of insulin subcutaneously in control and diabetic rats. The kinetic parameters V _max and K _m of GDH were studied spectrophotometrically at different substrate concentrations of α-ketoglutarate. Glutamate receptor binding assay was done with different concentrations of [³H] Glutamate. Results The GDH enzyme assay showed a significant increase (P < 0.001) in the V _max of the enzyme in the cerebellum of hypoglycaemic and diabetic rat groups when compared to control. The V _max of hypoglycaemic groups was significantly increased (P < 0.001) when compared to diabetic group. In the liver, the V _max of GDH was significantly increased (P < 0.001) in the diabetic and diabetic hypoglycaemia group when compared to control. The V _max of GDH increased significantly (P < 0.001) in the diabetic hypoglycaemic rats compared to diabetic group, whereas the control hypoglycaemic rats showed a significant decrease in V _max (P < 0.001) when compared to diabetic and diabetic hypoglycaemic rats. The K _m showed no significant change amongst the groups in cerebellum and liver. Scatchard analysis showed a significant increase (P < 0.001) in B _max in the cerebellum of hypoglycaemic and diabetic rats when compared to control. The B _max of hypoglycaemic rats significantly increased (P < 0.001) when compared to diabetic group. In hypoglycaemic groups, B _max of the control hypoglycaemic rats showed a significant increase (P < 0.001) compared to diabetic hypoglycaemic rats. The K _d of the diabetic group decreased significantly (P < 0.01) when compared to control and control hypoglycaemic rats. There was a significant decrease (P < 0.05) in the K _d of diabetic hypoglycaemic group when compared to the control hypoglycaemic rats. Conclusion Our studies demonstrated the increased enzyme activity in the hypoglycaemic rats with increased production of extracellular glutamate. The present study also revealed increased binding parameters of glutamate receptors reflecting an increased receptor number with increase in the affinity. This increased number of receptors and the increased glutamate production will lead to glutamate excitotoxicity and neuronal degeneration which has an impact on the cognitive and memory ability. This has immense clinical significance in the management of diabetes and insulin therapy.     

Functional analysis of the <Emphasis Type="Italic">Burkholderia cenocepacia</Emphasis> J2315 BceA<Subscript>J</Subscript> protein with phosphomannose isomerase and GDP-<Emphasis Type="SmallCaps">d</Emphasis>-mannose pyrophosphorylase activities

The bceA _J gene from the cystic fibrosis isolate Burkholderia cenocepacia J2315 encodes a 56-kDa bifunctional protein, with phosphomannose isomerase (PMI) and guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMP) activities, a new member of the poorly characterised type II PMI class of proteins. Due to the lack of homology between the type II PMIs and the human PMI, this class of proteins are being regarded as interesting potential targets to develop new antimicrobials. The BceA_J protein conserves the four typical motifs of type II PMIs: the pyrophosphorylase signature, the GMP active site, the PMI active site and the zinc-binding motif. After overproduction of BceA_J by Escherichia coli as a histidine tag derivative, the protein was purified to homogeneity by affinity chromatography. The GMP activity is dependent on the presence of Mg²⁺ or Ca²⁺ as cofactors, while the PMI activity uses a broader range of divalent ions, in the order of activation Mg²⁺ > Ca²⁺ > Mn²⁺ > Co²⁺ > Ni²⁺. The kinetic parameters K _m, V _max and K _cat/K _m for the PMI and GMP activities were determined. Results suggest that the enzyme favours the formation of GDP-mannose instead of mannose catabolism, thus channelling precursors to the formation of glycoconjugates.     

Effectors of metabolic depression in an estivating pulmonate snail (Helix aspersa): whole animal and in vitro tissue studies

 We have examined metabolic depression in the land snail (Helix aspersa) during estivation, and have developed a tissue model of metabolic depression using an in vitro mantle preparation. The metabolic rate of H. aspersa is depressed by 84% in vivo within 4 weeks of onset of estivation, and this metabolic depression is accompanied by a decrease in haemolymph PO₂ and pH, and an increase in haemolymph PCO₂. The in vitro mantle preparation has a stable O₂ consumption and energy charge, and an energy charge similar to that of mantle in vivo. The in vitro mantle is an O₂-conforming tissue, with VO₂ varying curvilinearly with PO₂. Consequently, we have developed a mathematical method of calculating tissue VO₂ at any PO₂. These calculations show that under appropriate incubation conditions of pH and PO₂, the mantle from estivating animals shows a stable in vitro metabolic depression of 48% compared to mantle from control snails. The extrinsic effects of PO₂ and pH account for 70% of the total in vitro metabolic depression of mantle tissue; intrinsic effectors contribute a further 30%. Accepted April 26, 1996     

Kinetic properties of microsomal 3β hydroxysteroid dehydrogenase-isomerase from the testis of Bufo arenarum H

3β-hydroxysteroid dehydrogenase 5-ene isomerase (3βHSD/I) activity is necessary for the biosynthesis of hormonally active steroids. A dual distribution of the enzyme was described in toad testes. The present study demonstrates that in testicular tissue of Bufo arenarum H., microsomal 3βHSD/I has more affinity for dehydroepiandrosterone (DHEA) than for pregnenolone (K_m=0.17±0.03 and 1.02 μM, respectively). The Hill coefficient for the conversion of DHEA and pregnenolone were 1.04 and 1.01, respectively. The inclusion of DHEA in the kinetic analysis of pregnenolone conversion affected V_max while K_m was not modified, suggesting a non-competitive inhibition of the conversion of pregnenolone. K_i was calculated from replot of Dixon's slope for each substrate concentration. K_i from the intercept and the slope of this replot were similar (0.276±0.01 and 0.263±0.02 μM) and higher than the K_m for DHEA. The K_m and K_i values suggest the presence of two different binding sites. When pregnenolone was present in the assays with DHEA as substrate, no effect was observed on the V_max while K_m values slightly increased with pregnenolone concentration. Consequently, pregnenolone inhibited the transformation of DHEA in a competitive fashion. These studies suggest that, in this species, the microsomal biosyntheses of androgens and progesterone are catalysed by different active sites.     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.     

Isovaleric Acid Reduces Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity in Synaptic Membranes from Cerebral Cortex of Young Rats

1. Patients affected by isovaleric acidemia (IVAcidemia) suffer from acute episodes of encephalopathy. However, the mechanisms underlying the neuropathology of this disease are poorly known. The objective of the present study was to investigate the in vitro effects of the metabolites that predominantly accumulate in IVAcidemia, namely isovaleric acid (IVA), 3-hydroxyisovaleric acid (3-OHIVA) and isovalerylglycine (IVG), on important parameters of energy metabolism, such as ¹⁴CO₂ production from acetate and the activities of the respiratory chain complexes I–IV, creatine kinase and Na⁺, K⁺-ATPase in synaptic plasma membranes from cerebral cortex homogenates of 30-day-old rats. 2. We observed that 3-OHIVA acid and IVG did not affect all the parameters analyzed. Similarly, ¹⁴CO₂ production from acetate (Krebs cycle activity), the activities of creatine kinase, and of the respiratory chain complexes was not modified by IVA. In contrast, IVA exposition to cortical homogenates provoked a marked inhibition of Na⁺, K⁺-ATPase activity. However, this activity was not changed when IVA was directly exposed to purified synaptic plasma membranes, suggesting an indirect effect of this organic acid on the enzyme. Furthermore, pretreatment of cortical homogenates with α-tocopherol and creatine totally prevented IVA-induced inhibition on Na⁺, K⁺-ATPase activity from synaptic plasma membranes, whereas glutathione (GSH) and the NO synthase inhibitor N^ω-nitro-l-arginine methyl ester (L-NAME) did not alter this inhibition. 3. These data indicate that peroxide radicals were probably involved in this inhibitory effect. Since Na⁺, K⁺-ATPase is a critical enzyme for normal brain development and functioning and necessary to maintain neuronal excitability, it is presumed that the inhibitory effect of IVA on this activity may be involved in the pathophysiology of the neurological dysfunction of isovaleric acidemic patients.     

Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Activity in the Posterior Gills of the Blue Crab, <Emphasis Type="Italic">Callinectes ornatus</Emphasis> (Decapoda,Brachyura): Modulation of ATP Hydrolysis by the Biogenic Amines Spermidine and Spermine

We investigated the effect of the exogenous polyamines spermine, spermidine and putrescine on modulation by ATP, K⁺, Na⁺, NH₄ ⁺ and Mg²⁺ and on inhibition by ouabain of posterior gill microsomal Na⁺,K⁺-ATPase activity in the blue crab, Callinectes ornatus, acclimated to a dilute medium (21‰ salinity). This is the first kinetic demonstration of competition between spermine and spermidine for the cation sites of a crustacean Na⁺,K⁺-ATPase. Polyamine inhibition is enhanced at low cation concentrations: spermidine almost completely inhibited total ATPase activity, while spermine inhibition attained 58%; putrescine had a negligible effect on Na⁺,K⁺-ATPase activity. Spermine and spermidine affected both V and K for ATP hydrolysis but did not affect ouabain-insensitive ATPase activity. ATP hydrolysis in the absence of spermine and spermidine obeyed Michaelis–Menten behavior, in contrast to the cooperative kinetics seen for both polyamines. Modulation of V and K by K⁺, Na⁺, NH₄ ⁺ and Mg²⁺ varied considerably in the presence of spermine and spermidine. These findings suggest that polyamine inhibition of Na⁺,K⁺-ATPase activity may be of physiological relevance to crustaceans that occupy habitats of variable salinity.     

Studies of Ca<Superscript>2+</Superscript> ATPase (SERCA) Inhibition

The Ca²⁺ transport ATPase of intracellular membranes (SERCA) can be inhibited by a series of chemical compounds such as Thapsigargin (TG), 2,5-di(tert-butyl)hydroquinone (DBHQ) and 1,3-dibromo-2,4,6-tris (methyl-isothio-uronium) benzene (Br₂-TITU). These compounds have specific binding sites in the ATPase protein, and different mechanisms of inhibition. On the other hand, SERCA gene silencing offers a convenient and specific method for suppression of SERCA activity in cells. The physiological and pharmacological implications of SERCA inhibition are discussed.     

Cloning and functional characterization of the high-affinity K<Superscript>+</Superscript> transporter HAK1 of pepper

High-affinity K⁺ uptake in plants plays a crucial role in K⁺ nutrition and different systems have been postulated to contribute to the high-affinity K⁺ uptake. The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K⁺ uptake observed in roots. Pepper plants starved of K⁺ for 3 d showed high-affinity K⁺ uptake (K _m of 6 M K⁺) that was very sensitive to NH and their roots expressed a high-affinity K⁺ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters. When expressed in yeast (Saccharomyces cerevisiae), CaHAK1 mediated high-affinity K⁺ and Rb⁺ uptake with K _m values of 3.3 and 1.9 M, respectively. Rb⁺ uptake was competitively inhibited by micromolar concentrations of NH and Cs⁺, and by millimolar concentrations of Na⁺.     

Mechanisms Underlying Regulation of a Barium -sensitive K<Superscript>+ </Superscript>Conductance by ATP in Single Proximal Tubule Cells Isolated from Frog Kidney

K⁺ channels play an important role in pump-leak coupling and volume regulation in the renal proximal tubule. Previous experiments have identified a barium-sensitive K⁺ conductance (G_Ba) in proximal tubule cells isolated from frog kidneys. In this paper we examine the regulation of G_Ba by ATP. G_Ba was measured in single cells isolated from frog kidney using the whole-cell patch-clamp technique. G_Ba was activated by 2 mM intracellular ATP. This activation was enhanced by inhibition of protein kinase C and attenuated by inhibition of protein kinase A, indicating reciprocal regulation by these kinases. Activation by ATP was reduced in the presence of a hypertonic bath solution, suggesting that cell swelling was required. However, after activation to steady-state, G_Ba was not sensitive to cell-volume changes. Hypotonic shock-induced volume regulation was inhibited by barium and quinidine, inhibitors of G_Ba. The effect of maximal inhibitory concentrations of barium and quinidine on volume regulation was similar and addition of both blockers together did not augment the inhibitory response. G_Ba was also activated by ADP, via a mechanism dependent on the presence of Mg²⁺. However, the responses to ADP and ATP were not additive, suggesting that these nucleotides may share a common mechanism of activation. The regulation of G_Ba by ATP was biphasic, with a half-maximal activating concentration of 0.89 mM and a half maximal inhibitory concentration of 6.71 mM. The sensitivity to nucleotides suggests that G_Ba may be regulated by the metabolic state of the cell. Furthermore, the sensitivity to solution osmolality, coupled with the blocker profile of inhibition of volume regulation, suggests that G_Ba could play a role in volume regulation.     

Regulation of glucose-6-phosphate dehydrogenase by reversible phosphorylation in liver of a freeze tolerant frog

Glucose-6-phosphate dehydrogenase (G6PDH) and the pentose phosphate pathway play a key role in reductive biosynthesis and antioxidant defense, while diverting glucose from other cellular functions. G6PDH was isolated from liver of the wood frog, Rana sylvatica, a freeze tolerant species that uses glucose as a cryoprotectant. Analysis of kinetic parameters (K _m and V _max) of G6PDH showed a significant increase in K _m G6P (from 98.2 ± 3.8 to 121 ± 5.3 μM) and K _m NADP⁺ (from 65.5 ± 2.3 to 89.1 ± 4.8 μM) in frogs following freezing exposure, indicating lower affinity for G6PDH substrates in this state. Subsequent analyses indicated that differential phosphorylation of G6PDH between the two states was responsible for the altered kinetic properties. Thus, two differentially charged forms of G6PDH were resolved by DEAE ion-exchange chromatography and, compared with controls, the proportion of G6PDH activity in peak I decreased and in peak II increased in liver from frozen frogs. G6PDH in peak I had a K _m G6P of 94.1 ± 1.1 μM and K _m NADP⁺ of 61.2 ± 3.5 μM, whereas Peak II G6PDH showed higher values (K _m G6P was 172 ± 4.3 μM, K _m NADP⁺ was 98.2 ± 3.3 μM). G6PDH from each peak was incubated with ions and second messengers to stimulate the actions of protein kinases with results indicating that G6PDH can be phosphorylated by protein kinase G, protein kinase C, AMP-activated protein kinase, or calmodulin-dependent protein kinase. The data indicate that in control frogs, G6PDH is in a high phosphate form and displays a high substrate affinity, whereas in frozen frogs G6PDH is less phosphorylated, with lower substrate affinity.     

Structural stability and functional analysis of L-asparaginase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>

We report studies on an L-asparaginase from Pyrococcus furiosus, cloned and expressed in Escherichia coli and purified to homogeneity. Protein stability and enzyme kinetic parameters were determined. The enzyme was found to be thermostable, natively dimeric, and glutaminase-free, with optimum activity at pH 9.0. It showed a K _m of 12 mM and a substrate inhibition profile above 20 mM L-asparagine. Urea could not induce unfolding and enzyme inactivation; however, with guanidine hydrochloride (GdnCl) a two-state unfolding pattern was observed. Reduced activity and an altered near-UV-CD signal for protein at low GdnCl concentration (1 M) suggested tertiary structural changes at the enzyme active site. A homology three-dimensional model was developed and the structural information was combined with activity and stability data to give functional clues about the asparaginase.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange activity in the alkaliphile halotolerant cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

The activity of Na⁺/H⁺ exchanger to remove toxic Na⁺ is important for growth of organisms under high salinity. In this study, the halotolerant cyanobacterium Aphanothece halophytica was shown to possess Na⁺/H⁺ exchange activity since exogenously added Na⁺ could dissipate a pre-formed pH gradient, and decrease extracellular pH. Kinetic analysis yielded apparent K _m (Na⁺) and V _max of 20.7 ± 3.1 mM and 3,333 ± 370 nmol H⁺ min⁻¹ mg⁻¹, respectively. For cells grown under salt-stress condition, the apparent K _m (Na⁺) and V _max was 18.3 ± 3.5 mM and 3,703 ± 350 nmol H⁺ min⁻¹ mg⁻¹, respectively. Three cations with decreasing efficiency namely Li⁺, Ca²⁺, and K⁺ were also able to dissipate pH gradient. Only marginal exchange activity was observed for Mg²⁺. The exchange activity was strongly inhibited by Na⁺-gradient dissipators, monensin, and sodium ionophore as well as by CCCP, a protonophore. A. halophytica showed high Na⁺/H⁺ exchange activity at neutral and alkaline pH up to pH 10. Cells grown at pH 7.6 under high salinity exhibited higher Na⁺/H⁺ exchange activity than those grown under low salinity during 15 days of growth suggesting a role of Na⁺/H⁺ exchanger for salt tolerance in A. halophytica. Cells grown at alkaline pH of 9.0 also exhibited a progressive increase of Na⁺/H⁺ exchange activity during 15 days of growth.