Protein kinase and phosphatase responses to anoxia in crayfish, Orconectes virilis: purification and characterization of cAMP-dependent protein kinase

The freshwater crayfish, Orconectes virilis, shows good anoxia tolerance, enduring 20 h in N₂-bubbled water at 15°C. Metabolic responses to anoxia by tolerant species often include reversible phosphorylation control over selected enzymes. To analyze the role of serine/threonine kinases and phosphatases in signal transduction during anoxia in O. virilis, changes in the activities of cAMP-dependent protein kinase (PKA) and protein phosphatases 1, 2A, and 2C were measured in tail muscle and hepatopancreas over a time course of exposure to N₂-bubbled water. A strong increase in the percentage of PKA present as the free catalytic subunit (% PKAc) occurred between 1 and 2 h of anoxia exposure whereas phosphatase activities were strongly reduced. This suggests that PKA-mediated events are important in the initial response by tissues to declining oxygen availability. As oxygen deprivation became severe and prolonged (5–20 h) these changes reversed; the % PKAc fell to below control values and activities of phosphatases returned to or rose above control values. Subcellular fractionation also showed a decrease in PKA associated with the plasma membrane after 20 h anoxia whereas cytosolic PKA content increased. PKAc purified from tail muscle showed a molecular weight of 43.8±0.4 kDa, a pH optimum of 6.8, a high affinity for Mg ATP (K_m=131.0±14.4 μM) and Kemptide (K_m=31.6±5.2 μM). Crayfish PKAc was sensitive to temperature change; a break in the Arrhenius plot occurred at approximately 15°C with a 2.5-fold rise in activation energy at temperatures <15°C. These studies demonstrate a role for serine/threonine protein kinases and phosphatases in the metabolic adjustments to oxygen depletion by crayfish organs.     

A Novel Arginine Kinase with Substrate Specificity Towards <Emphasis Type="SmallCaps">d</Emphasis>-arginine

We determined the cDNA-derived amino acid sequences of two arginine kinases (AK1, AK2) from the annelid Sabellastarte indica, cloned the cDNAs into pMAL plasmid and expressed them in E. coli. The phylogenetic analyses suggested that Sabellastarte AKs have evolved from a CK-related gene, not from the usual AK gene. The recombinant Sabellastarte AK1 showed a broad specificity towards various guanidine compounds, while the Sabellastarte AK2 mainly showed stronger activity for both d- and l-arginine, a very unique substrate specificity not seen before in usual AKs. We isolated guanidino compounds from the body wall musculature of Sabellastarte, and found that the major compound is d-arginine with a concentration of 4.85 ± 0.51 mmol/kg. From these results, we suggest strongly that in Sabellastarte, d-arginine is the major phosphagen substrate and that the AK2 with substrate specificity towards d-arginine, catalyzes the phosphorylation of d-arginine.     

Carbon balance and energetics of cyooprotectant synthesis in a freeze-tolerant insect: responses to perturbation by anoxia

Summary The capacity for polyol synthesis by larvae of Eurosta solidaginis was evaluated under aerobic versus anoxic (N₂ gas atmosphere) conditions. Glycerol production occurred readily in aerobic larvae at 13°C. Under anoxic conditions, however, net glycerol accumulation was only 57% of the aerobic value after 18 d, but the total hydroxyl equivalents available for cryoprotection were balanced by the additional synthesis of sorbitol. The efficiency of carbon conversion to polyols was much lower in anaerobic larvae. The ATP requirement of glycerol biosynthesis necessitated a 22% greater consumption of carbohydrate, when anaerobic and resulted in the accumulation of equimolar amounts of l-lactate and l-alanine as fermentative end products. The ratio of polyols produced to glycolytic end products formed was consistent with the use of the hexose monophosphate shunt to generate the reducing equivalents needed for cryoprotectant synthesis. A comparable experiment analyzed sorbitol synthesis at 3°C under aerobic versus anoxic conditions. Sorbitol synthesis was initiated more rapidly in anaerobic larvae, and the final sorbitol levels attained after 18 d were 60% higher than in aerobic larvae. The enhanced sorbitol output under anoxia may be due to an obligate channeling of a high percentage of total carbon flow through the hexose monophosphate shunt at 3°C. Carbon processed in this way generates NADPH which, along with the NADH output of glycolysis, must be reoxidized if anaerobic ATP synthesis is to continue. Redox balance within the hexose monophosphate shunt is maintained through NADPH consumption in the synthesis of sorbitol.     

Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog,Rana sylvatica

The wood frog (Rana sylvatica) can survive the winter in a frozen state, in which the frog’s tissues are also exposed to dehydration, ischemia, and anoxia. Critical to wood frog survival under these conditions is a global metabolic rate depression, the accumulation of glucose as a cryoprotectant, and a reliance on anaerobic glycolysis for energy production. Pyruvate kinase (PK) catalyzes the final reaction of aerobic glycolysis, generating pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP. This study investigated the effect of each stress condition experienced by R. sylvatica during freezing, including dehydration and anoxia, on PK regulation. PK from muscle of frozen and dehydrated frogs exhibited a lower affinity for PEP (K_m = 0.098 ± 0.003 and K_m = 0.092 ± 0.008) than PK from control and anoxic conditions (K_m = 0.065 ± 0.003 and K_m = 0.073 ± 0.002). Immunoblotting showed greater serine phosphorylation on muscle PK from frozen and dehydrated frogs relative to control and anoxic states, suggesting a reversible phosphorylation regulatory mechanism for PK activity during freezing stress. Furthermore, PK from frozen animals exhibited greater stability under thermal and urea-induced denaturing conditions than PK from control animals. Phosphorylation of PK during freezing may contribute to mediating energy conservation and maintaining intracellular cryoprotectant levels, as well as increase enzyme stability during stress.     

cGMP-stimulated protein kinase phosphorylates pyruvate kinase in an anoxia-tolerant marine mollusc

Summary A cystosolic protein kinase that phosphorylates pyruvate kinase (PK) in vitro has been identified in crude homogenates of heart, radular retractor, and foot muscle from the anoxia-tolerant marine whelk Busycon canaliculatum. Protein kinase action was measured by following changes in PK kinetic parameters: phosphorylated PK has a higher K _m value for phosphoenolpyruvate and a lower I₅₀ value for l-alanine. The crude protein kinase readily phosphorylated PK in a Mg²⁺-and ATP-dependent manner in the absence of any added effector. This activity was not affected by the addition of either cAMP (a stimulator of protein kinase A) or Ca²⁺ plus phorbol 12-myristate 13-acetate (stimulators of protein kinase C) to the incubation medium. Addition of cGMP to the homogenate, however, increased the rate of PK phosphorylation giving a 3–4-fold increase in the rate of change in PK kinetic parameters that was readily apparent after 5h. Complete time-courses of changes in PK kinetic parameters in the presence and absence of cGMP showed that cGMP increased the rate, but not the final extent, of PK phosphorylation. These results indicate that PK inactivation by enzyme phosphorylation in response to anoxia in whelk tissues may be mediated by a cyclic GMP stimulated protein kinase in response to changing levels of cGMP. This conclusion was further supported by data indicating that the total activity of protein kinase was the same in both anoxic and aerobic animals, and that the total PK phosphatase activity was also constant. Changes in PK phosphorylation during anoxia are not, therefore, the result of changes in the total amount of protein kinase or phosphatase.Abbreviations cAMP adenosine 3:5-monophosphate - cGMP guanosine 3:5-monophosphate - PK pyruvate kinase - PMA phorbol 12-myristate, 13-acetate - PEP phosphoenolpyruvate - K _m Michaelis constant - I ₅₀ inhibitor concentration that reduces enzyme activity by 50%     

Differential survival of Venus gallina and Scapharca inaequivalvis during anoxic stress: Covalent modification of phosphofructokinase and glycogen phosphorylase during anoxia

Summary Biochemical mechanisms underlying anaerobiosis were assessed in two Mediterranean bivalve species, Scapharca inaequivalvis and Venus gallina, with widely differing tolerances for oxygen lack. These species displayed LT₅₀ values for anoxic survival at 17–18°C of 17 and 4 d, respectively. Succinate and alanine were the major products of 24 h anaerobic metabolism in both species but only S. inaequivalvis further metabolized succinate to propionate. Both species reduced metabolic rate while anoxic but metabolic arrest was more pronounced in S. inaequivalvis. Calculated ATP turnover rate (MATP) during exposure to N₂-bubbled seawater was only 4.51% of the aerobic rate in S. inaequivalvis but was 12.68% in V. gallina. To counteract a greater load of acid end products, V. gallina foot showed a significantly greater buffering capacity, 23.38±0.20 slykes, compared to 19.6±0.79 slykes in S. inaequivalvis. The two species also differed distinctly in the enzymatic regulation of anaerobiosis. In V. gallina anoxia exposure caused only a small change in PFK kinetic parameters (a decrease in K_a AMP) and had no effect on glycogen phosphorylase. By contrast, S. inaequivalvis foot showed a strong modification of enzyme properties in anoxia. The percentage of glycogen phosphorylase in the a form dropped significantly only in S. inaequivalvis. Other changes included alterations in the properties of PFK leading to a less active enzyme form in anoxia. Compared to the aerobic enzyme form, PFK from anoxic foot showed a reduced affinity for fructose-6-P (K_m increased 2.4-fold), greater inhibition by ATP (I₅₀ decreased 6.8-fold), and an increase in sensitivity to AMP activation (K_a decreased by 50%). These enzyme changes appear to be key to a glycolytic rate depression during anaerobiosis in S. inaequivalvis foot muscle.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-(2-aminoethyl)-tetraacetic acid - Fructose-2,6-P ₂ fructose-2,6-bisphosphate - Fructose-6-P fructose-6-phosphate - K _a AMP Activation constant (concentration of AMP required to increase the reaction to twice the rate it shows in the absence of AMP) - MATP ATP turnover rate - P _i inorganic phosphate - PCA Perchloric acid - PFK 6-phosphofructo-1-kinase - TCA Trichloroacetic acid     

Role of covalent modification in the control of glycolytic enzymes in response to environmental anoxia in goldfish

Summary The effects of environmental anoxia (24 h at 7°C in N₂/CO bubbled water) on the maximal activities, selected kinetic properties, and isoelectric points of phosphofructokinase and pyruvate kinase were measured in eight tissues of the goldfish,Carassius auratus, in order to evaluate the role of possible covalent modification of enzymes in glycolytic rate control and metabolic depression during facultative anaerobiosis. Both enzymes showed modified kinetic properties as a result of anoxia in liver, kidney, brain, spleen, gill, and heart. Effects of anoxia on properties of pyruvate kinase included reducedV _max, increased S_0.5 for phosphoenolpyruvate, increasedK _a for fructose-1,6-bisphosphate, and strongly reduced I₅₀ for alanine; all these effects are consistent with an anoxia-induced phosphorylation of pyruvate kinase to produce a less active enzyme form. Anoxia-induced alterations in phosphofructokinase kinetics included tissue-specific changes in S_0.5 for fructose-6-phosphate, Hill coefficient,K _a values for fructose-2,6-bisphosphate, AMP, and NH ₄ ⁺ , and I₅₀ values for ATP and citrate, the direction of changes being generally consistent with the production of a less active enzyme form in the anoxic tissue. Enzymes from aerobic versus anoxic skeletal muscle (both red and white) did not differ in kinetic properties but anoxic enzyme forms had significantly different pI values than the corresponding aerobic forms. Enzyme phosphorylation-dephosphorylation as the basis of the anoxia-induced changes in the kinetic properties of PFK and PK was further tested in liver: treatment of the aerobic forms of both enzymes with cAMP dependent protein kinase altered enzyme kinetic properties to those typical of the anoxic enzymes while alkaline phosphatase treatment of the anoxic enzyme forms had the opposite effect. The data provide strong evidence that coordinated glycolytic rate control, as part of an overall metabolic rate depression during anoxia, is mediated via anoxia-induced covalent modification of regulatory enzymes.Abbreviations cAMP cyclic 35 adenosine monophosphate - F16P ₂ fructose-1,6-bisphosphate - F26P ₂ fructose-2,6-bisphosphate - F6P fructose-6-phosphate - PEP phosphoenolpyruvate - PFK phosphofructokinase (E.C. 2.7.1.11) - PK pyruvate kinase (E.C. 2.7.1.40) - PMSF phenylmethylsulfonyl fluoride     

l-Arginine Effects on Na+ Transport in M-1 Mouse Cortical Collecting Duct Cells—A Cationic Amino Acid Absorbing Epithelium

The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using continuous short circuit current (I _SC ) measurements in HCO₃ ⁻/CO₂ buffered solution. Steady state I _SC averaged 73.8 ± 3.2 μA/cm² (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na⁺ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y⁺ and a basal amino acid transport system y⁺L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I _SC averaging 36.6 ± 5.4 μA/cm² (n= 19) and 32.0 ± 7.2 μA/cm² (n= 8), respectively. The response was preserved in the absence of bath Cl⁻ (n= 4), but was abolished either in the absence of apical Na⁺ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I _SC response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na⁺ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract passive cationic amino acid leak into the CCD in the presence of electrogenic Na⁺ transport, consistent with reports of stimulated expression of Na⁺ and cationic amino acid transporters by aldosterone. Received: 11 September 2000/Revised: 6 December 2000     

Transport-limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition

Summary The anaerobic glucose uptake (at 20°, pH 3.5) by resting cells of Saccharomyces cerevisiae followed unidirectional Michaelis-Menten kinetics and was competitively inhibited by l-sorbose; K _m and K _i were respectively 5.6×10^-4 m and 1.8×10^-1 m; V _max was 6.5×10^-8 moles mg^-1 min^-1. The aerobic uptake of glucose by resting yeast was also inhibited by l-sorbose but did not follow unidirectional Michaelis-Menten kinetics. Glucose-limited growth in the chemostat of a respiration-deficient mutant of S. cerevisiae was competitively inhibited by l-sorbose. As predicted by theory for transport-limited growth in the chemostat (van Uden, 1967) the steady state glucose concentrations were linear functions of the l-sorbose concentrations with different slopes at different dilution rates; K _m and K _i were respectively 7.2×10^-4 m and 1.8×10^-1 m. It is concluded that glucose transport was the rate-limiting step of anaerobic fermentation of S. cerevisiae and of growth of the mutant and that l-sorbose is a competitive inhibitor of active glucose transport in this yeast. The latter conclusion is accommodated in the transport model of van Steveninck and Rothstein (1965).     

Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

Kinetic and regulatory properties of pyruvate kinase isozymes from flight muscle and fat body of the cockroach,Periplaneta americana

Summary Pyruvate kinases from flight muscle and fat body of the cockroach,Periplaneta americana, were purified to homogeneity. The two tissues contained different forms of the enzyme which were separable by starch gel electrophoresis and isoelectric focusing (pI=5.75 for flight muscle and 6.15 for fat body). Both enzymes had molecular weights of 235,000±20,000.Flight muscle pyruvate kinase displayed Michaelis-Menten kinetics with respect to both ADP and P-enolpyruvate withK _m values of 0.27 and 0.04 mM, respectively.K _m for Mg²⁺ was 0.60 mM andK _a for K⁺ was 15 mM. The enzyme was weakly inhibitied by four compounds, ATP, arginine-P,l-alanine and citrate with apparentK _i values of 3.5, 15, 20 and 24 mM, respectively. Competitive inhibition by 3 mM ATP or 10 mM arginine-P raised theK _m for P-enolpyruvate to 0.067 or 0.057 mM. Fructose-1,6-P₂ did not activate the enzyme but reversed inhibitions by ATP and arginine-P.Fat body pyruvate kinase showed sigmoidal kinetics with respect to P-enolpyruvate with S_0.5=0.32 mM andn _H=1.43.K _m values for ADP and Mg²⁺ were 0.30 and 0.80 mM, respectively with aK _a for K⁺ of 10 mM. ATP andl-alanine were inhibitors of the enzyme; 2 mM ATP raised S_0.5 for P-enolpyruvate to 0.48 mM while 3 mMl-alanine increased S_0.5 to 0.84 mM. Neither citrate nor arginine-P inhibited the enzyme but citrate affected the enzyme by reversingl-alanine inhibition. Fat body pyruvate kinase was strongly activated by fructose-1,6-P₂ with an apparentK _a of 1.5 M. Fructose-1,6-P₂ at 0.1 mM reduced S_0.5 for P-enolpyruvate to 0.05 mM andn _H to 1.0.Flight muscle and fat body pyruvate kinases from the cockroach show properties analogous to those of the muscle and liver forms of mammalian pyruvate kinase. Fat body pyruvate kinase is suited for on-off function in a tissue with a gluconeogenic capacity. Strong allosteric control with a feed-forward activation by fructose-1,6-P₂ is key to coordinating enzyme function with glycolytic rate. The function of flight muscle pyruvate kinase in energy production during flight is aided by a lowK _m for P-enolpyruvate, weak inhibitor effects by high energy phosphates and deinhibition of these effects by fructose-1,6-P₂.     

Tyrosine hydroxylase activity in the central nervous system of the crayfish,Pacifastacus leniusculus (Crustacea,Decapoda)

Summary Tyrosine hydroxylase, responsible for the formation ofl-dopa froml-tyrosine, has been identified in the central nervous system of the crayfish,Pacifastacus leniusculus (Crustacea, Decapoda). It requires pterine as cofactor and is inhibited by a number of known tyrosine hydroxylase inhibitors; iron-chelators, tyrosine analogues and also by the catecholamines, dopamine and noradrenaline. Iron enhances the activity of the enzyme. It differs from the vertebrate tyrosine hydroxylase in having a more alkaline pH optimum and a higher affinity for the pterine cofactor. Kinetic studies were performed andK _m andV _max values are presented. Dopa formed was identified and quantitatively measured by high pressure liquid chromatography (HPLC) and electrochemical detection.     

Alanine dehydrogenase of the β-lactam antibiotic producer Streptomyces clavuligerus

l-Alanine dehydrogenase was found in extracts of the antibiotic producer Streptomyces clavuligerus. The enzyme was induced by ammonia, and the level of induction was dependend on the extracellular concentration. l-Alanine was the only amino acid able to induce alanine dehydrogenase. The enzyme was characterized from a 38-fold purified preparation. Pyruvate (K _m =1.1 mM), ammonia (K _m =20 mM) and NADH (K _m =0.14 mM) were required for the reductive amination, and l-alanine (K _m =9.1 mM) and NAD (K _m =0.5 mM) for the oxidative deaminating reaction. The aminating reaction was inhibited by alanine, serine and NADPH. Alanine inhibited uncompetitively with respect to NADH (K _i =1.6 mM) and noncompetitively with respect to ammonia (K _i =2.0 mM) and pyruvate (K _i =3.0 mM). In the aminating reaction 3-hydroxypyruvate, glyoxylate and 2-oxobutyrate could partially (6–7%) substitute pyruvate. Alanine dehydrogenase from S. clavuligerus differed with respect to its molecular weight (92000) and its kinetic properties from those described for other microorganisms.Abbreviation Alanine-DH l-alanine:NAD oxidoreductase     

Redistribution of hepatocyte chloride during l-alanine uptake

We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (V _m), intracellular K⁺, Cl^-, and Na⁺ activities (a ⁱ _k, a _Cl ⁱ and a _Na ⁱ ), and water volume during l-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 m l-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 mm tetramethylammonium ion (TMA⁺) for 10 min. Changes in cell water volume during l-alanine uptake were determined by changes in intracellular, steady-state TMA⁺ activity measured with the K⁺ electrode. Hepatocyte control V _m was -33±1 mV. l-alanine uptake first depolarized V _m by 2±0.2 mV and then hyperpolarized V _m by 5 mV to-38±1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, a _Na ⁱ increased by 30% from 19±2 to 25±3 mm (P < 0.01), and a _K ⁱ did not change significantly from 83±3 mm. However, with added ouabain (1 mm) l-alanine caused only a 2-mV increase in V _m, but now a _K ⁱ decreased from 61±3 to 54±5 mm (P < 0.05). Hyperpolarization of V _m by l-alanine uptake also resulted in a 38% decrease of a _Cl ⁱ from 20±2 to 12±3 mm (P < 0.001). Changes in V _m and V _Cl — V _m voltage traces were parallel during the time of l-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl^– with the V _m in hepatocytes. Added Ba²⁺ abolished the l-alanineinduced hyperpolarization, and a _Cl ⁱ remained unchanged. Hepatocyte water volume during l-alanine uptake increased by 12±3%. This swelling did not account for any changes in ion activities following l-alanine uptake. We conclude that hepatocyte a _K ⁱ is regulated by increased Na⁺-K⁺ pump activity during l-alanine uptake in spite of cell swelling and increased V _m due to increased K⁺ conductance. The hyperpolarization of V _m during l-alanine uptake provides electromotive force to decrease a _Cl ⁱ . The latter may contribute to hepatocyte volume regulation during organic solute transport.This work was supported by grant AA-08867 from the Alcohol, Drug Abuse, and Mental Health Association.     

Anoxia and freezing exposures stimulate covalent modification of enzymes of carbohydrate metabolism in Littorina littorea

The effects of anoxia (N₂ atmosphere at 5 °C) or freezing (at-8 °C) exposure in vivo on the activities of five enzymes of carbohydrate metabolism were assessed in foot muscle and hepatopancreases of the marine periwinkle Littorina littorea. Changes in glycogen phosphorylase, glycogen synthetase, pyruvate kinase and pyruvate dehydrogenase under either stress were generally consistent with covalent modification of the enzymes to decrease enzyme activity and/or convert the enzyme to a less active form. However, no evidence for a similar covalent modification of phosphofructokinase was found. The metabolic effects of freezing and anoxia were generally similar, suggesting that a primary contributor to freezing survival is the implementation of anaerobic metabolism and metabolic arrest mechanisms that also promote anoxia survival in marine molluses. However, in hepatopancreas phosphorylase was activated and pyruvate kinase remained in two enzyme forms in freezing-exposed snails, contrary to the results for anoxic animals. Ion exchange chromatography on DE-52 Sephadex revealed the presence of two forms of pyruvate kinase in both tissues of control L. littorea, eluting at 30–50 mmol·1^-1 KCl (peak I) or 90–110 mmol·1^-1 KCl (peak II). Anoxia exposure converted pyruvate kinase in both tissues to the peak I form, as did freezing for foot muscle pyruvate kinase. Kinetic analysis showed that peak I pyruvate kinase had lower affinities for substrates, phosphoenolpyruvate and ADP, and was very strongly inhibited by l-alanine compared with the peak II enzyme. Peak I pyruvate kinase had an I ₅₀ value for l-alanine of 0.38 mmol·1^-1, whereas peak II pyruvate kinase was unaffected by l-alanine evenat 40 mmol·1^-1. In vitro incubation of extracts from control foot muscle under conditions promoting phosphorylation or dephosphorylation identified the peak I and II forms as the low and high phosphate forms, respectively. This result for L. littorea pyruvate kinase was highly unusual and contrary to the typical effect of anoxia on pyruvate kinase in marine molluscs which is to stimulate the phosphorylation of pyruvate kinase and, thereby, convert the enzyme to a less active form.Abbreviations AABS p-(p-aminophenylazo)benzene sulphonic acid - F2, 6P fructose-2,6-bisphosphate - F6P fructose-6-phosphate - G6P glucose-6-phosphate - GP glycogen phosphorylase - GS glycogen synthase - I ₅₀ inhibitor concentration reducing enzyme velocity by 50% - MR metabolic rate - PDH pyruvate dehydrogenase - PEP phosphoenopyruvate - PFK phosphofructokinase - PK pyruvate kinase - SW sea water - F _a air temperature - TCA trichloroacetic acid - UDPG uridine-diphosphate glucose - WW wet weight     

Brief antecedent anoxia preserves mitochondrial function after sustained undersupply: A subcellular correlate to ischemic preconditioning?

Background: There is increasing evidence that mitochondria – owning a high degree of autonomy within the cell – might represent the target organelles of the myocardial protection afforded by ischemic preconditioning. It was the aim of the study to investigate a possible subcellular correlate to ischemic preconditioning at the mitochondrial level. In addition, we tested whether this protection depends on mitochondrial ATP-dependent potassium channels (K _ATP) and an might involve an attenuation of mitochondrial ATP hydrolysis during sustained anoxia.Methods and Results: Sustained anoxia (A, 14 min) and reoxygenation (R) completely inhibited state 3 and state 4 respiration of isolated ventricular mitochondria from Wistar rats. An antecedent brief anoxic incubation (4 min) followed by reoxygenation (2 min) prevented this loss of mitochondrial function. The protection afforded by anoxic preconditioning could be mimicked by the K _ATP opener diazoxide (30 μmol/l) and was completely inhibited by the K _ATP blocker 5-hydroxydecanoic acid (300 μmol/l). Structural mitochondrial integrity, as estimated from externalization of the mitochondrial enzymes creatine kinase and glutamateoxalacetate transaminase, remained unchanged between the groups, as did mitochondrial ATP loss during anoxia.Conclusion: For the first time, we provide direct evidence for a subcellular preconditioning-like functional mitochondrial adaptation to sustained anoxia. This effect apparently depends on opening of K_ATP but is independent of ATP preservation.     

Substrate Selectivity and pH Dependence of KAAT1 Expressed in Xenopus laevis Oocytes

When expressed in Xenopus oocytes KAAT1 increases tenfold the transport of l-leucine. Substitution of NaCl with 100 mm LiCl, RbCl or KCl allows a reduced but significant activation of l-leucine uptakes. Chloride-dependence is not strict since other pseudohalide anions such as thyocyanate are accepted. KAAT1 is highly sensitive to pH. It can transport l-leucine at pH 5.5 and 8, but the maximum uptake has been observed at pH 10, near to the physiological pH value, when amino and carboxylic groups are both deprotonated. The pH value mainly influences the V _max in Na⁺ activation curves and l-leucine kinetics. The kinetic parameters are K _mNa = 4.6 ± 2 mm, V _maxNa = 14.8 ± 1.7 pmol/oocyte/5 min for pH 8.0 and K _mNa = 2.8 ± 0.7 mm, V _maxNa = 31.3 ± 1.9 pmol/oocyte/5 min for pH 10.0. The kinetic parameters of l-leucine uptake are: K _m = 120.4 ± 24.2 μm, V _max = 23.2 ± 1.4 pmol/oocyte/5 min at pH 8.0 and K _m = 81.3 ± 24.2 μm, V _max = 65.6 ± 3.9 pmol/oocyte/5 min at pH 10.0. On the basis of inhibition experiments, the structural features required for KAAT1 substrates are: (i) a carboxylic group, (ii) an unsubstituted α-amino group, (iii) the side chain is unnecessary, if present it should be uncharged regardless of length and ramification. Received: 27 April 1999/Revised: 10 January 2000     

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996     

Effect of oligomycin on NADH oxidation and its coupled phosphorylation with the particulate fraction from dark aerobically grown Rhodospirillum rubrum

Summary NADH oxidation with the particulate fraction from dark aerobically grown Rhodospirillum rubrum is significantly stimulated by the addition of phosphate (Pi) and Mg⁺⁺, or Pi, Mg⁺⁺, ATP and the hexokinase-glucose system. K _m values for Pi in NADH oxidation and phosphorylation are 10^–3 m and 8×10^–4 m, respectively. These K _m values are almost the same as in corresponding photophosphorylation and oxidative phosphorylation catalyzed with chromatophores. As in the case of NADH oxidation with chromatophores, NADH oxidation with the particulate fraction has an optimal pH at 7.5 without additions, which is shifted to 6.9 by the addition of Pi and Mg⁺⁺, or Pi, Mg⁺⁺, ATP and the hexokinase-glucose system. The optimal pH for coupled phosphorylation is 6.9. 10 g per ml of oligomycin can suppress stimulation of NADH oxidation by Pi, or by the energy trapping system, and prevent the shift of optimal pH. The particulate fraction can catalyze Pi-incorporation into glucose-6-phosphate without externally added ATP, so that Pi-incorporation is inhibited by oligomycin. From these findings, it is concluded that NADH oxidation in the particulate fraction is tightly coupled to phosphorylation.     

Amino acid metabolism in the thermophilic phototroph,Chloroflexus aurantiacus: properties and metabolic role of two l-threonine (l-serine) dehydratases

Two l-threonine (l-serine) dehydratases (EC 4.2.1.16) of the thermophilic phototrophic bacterium Chloroflexus aurantiacus Ok-70-fl were purified to electrophoretic homogeneity by procedures involving anion exchange and hydrophobic interaction chromatography. Only one of the two enzymes was sensitive to inhibition by l-isoleucine (K _i=2 M) and activation by l-valine. The isoleucine-insensitive dehydratase was active with l-threonine (K _m=20 mM) as well as with l-serine (K _m=10 mM) whereas the other enzyme, which displayed much higher affinity to l-threonine (K _m=1.3 mM), was inactivated when acting on l-serine. Both dehydratases contained pyridoxal-5-phosphate as cofactor. When assayed by gel filtration techniques at 20 to 25° C, the molecular weights of both enzymes were found to be 106,000±6,000. In sodium dodecylsulfate-polyacrylamide gel electrophoresis, the two dehydratases yielded only one type of subunit with a molecular weight of 55,000±3,000. The isoleucine-insensitive enzyme was subject to a glucose-mediated catabolite repression.Abbreviations A absorbance - ile isoleucine - PLP pyridoxal-5-phosphate - SDS sodium dodecyl sulfate - TDH threonine dehydratase - U unit