Characterisation of a salt-stimulated ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Ion stimulation and some other properties of an ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) have been determined. The ATPase had a specific requirement for Mg²⁺ and in the presence of Mg²⁺ it was stimulated by salts of monovalent cations. The degree of stimulation by monovalent salts was influenced mainly by the anion and the order of effectiveness of the anions tested was Cl^->HCO ₃ ^- >Br^->malate>acetate>SO ₄ ^2- . For any given series of anions the magnitude of the stimulation obtained was influenced by the accompanying cation (NH ₄ ⁺ Na⁺>K⁺). This cation effect was abolished by 0.01% (v/v) Triton X-100 and it is suggested that it is the result of different permeabilities of membrane vesicles to the cations. There was no evidence of synergistic stimulation of the ATPase by mixtures of Na⁺ and K⁺. KCl- and NaCl-stimulation was maximal with salt concentrations in the range 60–150 mM. The true substrate of the enzyme was shown to be MgATP. It was shown that KCl stimulation was the result of an increase in V_max rather than a change in the affinity of the enzyme for MgATP. The ATPase was inhibited by N,N-dicyclohexylcarbodiimide, diethylstilbestrol, mersalyl and KNO₃ but other inhibitors tested (azide, oligomycin, orthovanadate, K₃[Cr(oxalate)₆] and ethyl-3-[3-dimethylaminopropyl]carbodiimide) were without effect or caused only partial inhibition at the highest concentration tested. The ATPase activity was equally distributed between pellet and supernatant fractions obtained after the subfractionation of vacuoles but the properties of the ATPase in each fraction were the same. It is suggested that beet vacuoles possess only one ATPase. The properties of the ATPase are compared with those of ATPases associated with other plant membranes and organelles and its possible role in transport at the tonoplast is discussed.Abbreviations ATP_F free ATP - ATP_T total ATP - BSA bovine serum albumen - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DNP 2,4-dinitrophenol - EDAC ethyl-3-(3-dimethylaminopropyl)carbodiimide - K_m apparent Michaelis constant - MgATP complex of Mg²⁺ and ATP - Mg _F ²⁺ free Mg²⁺ - Mg _T ² total Mg²⁺ - MES 2-(N-Morpholino)ethanesulphonic acid - Na₂EDTA disodium ethylenediaminetetraacetic acid - NEM N-ethylmaleimide - P_i inorganic phosphate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)methylamine - V_max maximum velocity     

Partial characterization of the plasma membrane ATPase from arho 0 petite strain ofSaccharomyces cerevisiae

Crude membrane preparations of arho ⁰ mutant ofSaccharomyces cerevisiae exhibit Mg²⁺-dependent ATPase activity. Over the optimal pH range, 5.0–6.75, the apparentV _max of the enzyme equals 590 nmoles of ATP hydrolyzed per minute per milligram protein, with an apparentK _m for ATP of 1.3 mM. ATP hydrolysis is insensitive to ouabain, venturicidin, aurovertin, and the protein inhibitor described by Pullman and Monroy; inhibited by oligomycin (at high concentrations) and sodium orthovandate, and it is sensitive to dicyclohexylcarbodiimide,p-hydroxymercuribenzoate, hydroxylamine, sodium fluoride, and sodium iodoacetate. The pH optimum and the inhibitor pattern distinguish the plasma membrane enzyme from the mitochondrial F₁ ATPase still present in these cells (this activity is sensitive to efrapeptin, aurovertin, and the protein inhibitor, but resistant to DCCD). In addition, the activity of the plasma membrane enzyme and its affinity for ATP are responsive to changes in the composition of the growth medium, with the highest activity observed in cells grown on methyl--d-glucoside, a sugar which results not only in partial release from catabolite repression but also requires the induction of an active transport system for growth.Author to whom correspondence should be addressed; recipient of a Research Career Award No. K06 05060 from the Institute of General Medical Sciences.     

The effect of hormones on metal and metal-ATP interactions with fat cell adenylate cyclase.

1-adrenaline, ACTH and glucagon activate the adenylate cyclase of rat adipocytes by decreasing its S_0.5(Mg²⁺) (concentration yielding 0.5 V_max) from its basal value of 11.5 to 1.2, 0.3 and 1.8 mM and by increasing its K_i(ATP^4?) from 0.03 to 0.25; 0.62 and 0.16 mM respectively. The kinetic properties of the enzyme are regulated by its state of saturation with ATP^4? or Mg²⁺; its saturation with ATP^4? and citrate^3? suppressed its basal and hormone-dependent activities. The hormone-dependent decrease in K_m and increase in V_max of the enzyme occur when shifting from suboptimal low concentrations of hormone and Mg²⁺ to optimal conditions, i.e., high concentration of hormone and low concentration of Mg²⁺. The increase in the state of saturation of the enzyme with Mg²⁺ decreases the hormone-dependent effects on V_max and results in identical values of K_m (0.14 mM) for its basal and 1-adrenaline dependent activities. CaCl₂ saturation curves at 5 mM ATP with either 5, 10 or 20 mM MgCl₂ show that the substitution of 5 mM MgCl₂ by 10 mM and 20 mM MgCl₂ increased the K_i(Ca²⁺) of the enzyme from 0.19 to 0.49 and 0.94 mM but decreased its K_i(CaATP) from 0.42 to 0.19 and 0.14 mM respectively. Only when the concentration of MgCl₂ exceeded that of ATP did 1-adrenaline and ACTH activate the enzyme by increasing its K_i(Ca²⁺), although only ACTH increased its K_i(CaATP). An increase in energy charge would decrease the intracellular concentrations of Mg²⁺ and Ca²⁺ because ATP^4? has stronger binding constants for Mg²⁺ and Ca²⁺ than ADP^3? and AMP^2?. Hence, the reported properties of the enzyme suggests that changes in energy charge may allow for metabolic feedback control of the hormonal responsiveness of the Mg²⁺, Ca²⁺, ATP^4? -sensitive adenylate cyclase.     

Effects of anisosmotic medium on cell volume,transmembrane potential and intracellular K+ activity in mouse hepatocytes

Summary Mouse hepatocytes in primary monolayer culture (4 hr) were exposed for 10 min at 37°C to anisosmotic medium of altered NaCl concentration. Hepatocytes maintained constant relative cell volume (experimental volume/control volume) as a function of external medium relative osmolality (control mOsm/experimental mOsm), ranging from 0.8 to 1.5. In contrast, the relative cell volume fit a predicted Boyle-Van't Hoff plot when the experiment was done at 4°C. Mouse liver slices were used for electrophysiologic studies, in which hepatocyte transmembrane potential (V _m ) and intracellular K⁺ activity (a _K ⁱ ) were recorded continuously by open-tip and liquid ion-exchanger ion-sensitive glass microelectrodes, respectively. Liver slices were superfused with control and then with anisosmotic medium of altered NaCl concentration.V _m increased (hyperpolarized) with hypoosmotic medium and decreased (depolarized) with hyperosmotic medium, and ln [10(experimentalV _m /controlV _m )] was a linear function of relative osmolality (control mOsm/experimental mOsm) in the range 0.8–1.5. Thea _K ⁱ did not change when medium osmolality was decreased 40–70 mOsm from control of 280 mOsm. Similar hypoosmotic stress in the presence of either 60mm K⁺ or 1mm quinine HCl or at 27°C resulted in no change inV _m compared with a 20-mV increase inV _m without the added agents or at 37°C. We conclude that mouse hepatocytes maintain their volume anda _K ⁱ in response to anisosmotic medium; however,V _m behaves as an osmometer under these conditions. Also, increases inV _m by hypoosmotic stress were abolished by conditions or agents that inhibit K⁺ conductance.     

Calcium-stimulated myofibrillar ATPase activity correlates with shortening velocity of muscle fibres in Xenopus laevis

Summary The iliofibularis muscle ofXenopus laevis is reported to contain five types of fibres which have different force—velocity relationships. Ten fibres of each type were selected on the basis of succinate dehydrogenase activity, cross-sectional area and location in the muscle, in order to assess the validity of the fibre type classification.Maximum calcium-stimulated myofibrillar ATPase activity (V _max) and apparent Michaelis constant (K _m) for ATP were determined for these 50 fibres from serial sections. The values obtained varied according to the type of fibre. Type 1 had the highest and type 5 the lowest values forK _m andV _max.In a separate experiment, single freeze-dried fibres were used to determine the relationship between their ATP content and apparentK _m for ATP. There was a tendency for high ATP concentrations in fibres with highK _m values.When myofibrillar ATPase activity was related to the maximum velocity of shortening of the five fibre types, a significant correlation was found. It is concluded that calcium-stimulated myofibrillar ATPase histochemistry allows an estimate of the maximum shortening velocity of muscle fibres fromXenopus laevis.     

Response of mitochondrial ATPase activity to uncouplers in isolated organelles and whole cells of Zajdela hepatoma

ATPase activity of coupled Zajdela hepatoma mitochondria was rendered uncoupler-sensitive by decreasing free fatty acids content in mitochondria or by preincubation of mitochondria with ATP prior to the addition of an uncoupler. The latter treatment resulted in an accelerated transport of ATP into the organelles. The effect of carbonylcyanide-m-chlorophenylhydrazone and oligomycin on the decrease of the ATP content in whole Zajdela hepatoma cells indicated that the hepatoma mitochondrial ATPase is stimulated by uncouplers $\text{in}\text{vivo}$. The conclusion is that the uncoupler-insensitive ATPase activity of coupled Zajdela hepatoma mitochondria is exhibited only by isolated organelles and results from a reduced $\text{ATP}\text{ADP}$ translocase activity.     

Kinetics of phosphate absorption by mycorrhizal and non-mycorrhizal Scots pine seedlings

Kinetics of net phosphate (P_i) uptake was measured on intact ectomycorrhizal and non‐mycorrhizal Pinus sylvestris seedlings using a semihydroponic cultivation method. The depletion of P_i in a nutrient solution was assessed over a 160–0.2 μM P_i gradient. Growth of the pine seedlings was P limited and measurements were performed 7 and 9 weeks after inoculation. Three ectomycorrhizal fungi were studied: Paxillus involutus, Suillus bovinus and Thelephoraterrestris. P_i uptake was extremely fast in plants colonised by P. involutus. The P_i concentration dropped below 0.2 μM within 4–5 h. In plants colonised with S. bovinus this occurred in 5–6 h and in plants associated with T. terrestris 8 h were needed to run through the whole concentration range. Non‐mycorrhizal plants of similar size and nutrient status decreased P_i to a concentration between 1 and 2 μM in 18 h. Data were curve fitted to a two‐phase Michaelis‐Menten equation. The apparent kinetic constants, K_m and V_max, for the high affinity P_i uptake system of the pine roots could be estimated accurately. V_max of this system was up to 7 times higher in pines associated with P. involutus than in non‐mycorrhizal seedlings. The intact extraradical mycelium greatly increased the absorption surface area of the roots (V_max). Non‐mycorrhizal plants had a K_m between 7.8 and 16.4 μM P_i. Plants mycorrhizal with P. involutus had K_m values between 2.4 and 7.2, plants colonised with S. bovinus had a K_m between 5.1 and 12.3, and seedlings associated with T. terrestris had a K_m from 4.6 to 10.1 μM P_i. All 3 ectomycorrhizal fungi had a strong impact on the P_i absorption capacity of the pine seedlings. The results also demonstrated that there is substantial heterogeneity in kinetic parameters among the different mycorrhizal root systems.     

Effect of adherence,cell morphology,and lipopolysaccharide on potassium conductance and passive membrane properties of murine macrophage J774.1 cells

Summary The effects of adherence, cell morphology, and lipopolysaccharide on electrical membrane properties and on the expression of the inwardly rectifying K conductance in J774.1 cells were investigated. Whole-cell inwardly rectifying K currents (K _i), membrane capacitance (C _m), and membrane potential (V _m) were measured using the patch-clamp technique. SpecificK _i conductance (G _K i, whole-cell Ki conductance corrected for leak and normalized to membrane capacitance) was measured as a function of time after adherence, and was found to increase almost twofold one day after plating. Membrane potential (V _m) also increased from –42±4 mV (n=32) to –58±2 mV (n=47) over the same time period.G _K i andV _m were correlated with each other;G _L (leak conductance normalized to membrane capacitance) andV _m were not. The magnitudes ofG _K i andV _m 15 min to 2 hr after adherence were unaffected by the presence of 100 m cycloheximide, but the increase inG _K iandV _m that normally occurred between 2 and 8 hr after adherence was abolished by cycloheximide treatment. Membrane properties were analyzed as a function of cell morphology, by dividing cells into three categories ranging from small round cells to large, extremely spread cells. The capacitance of spread cells increased more than twofold within one day after adherence, which indicates that spread cells inserted new membrane. Spread cells had more negative resting membrane potentials than round cells, butG _K i andG _L were not significantly different. Lipopolysaccharide-(LPS; 1 or 10 g/ml) treated cells showed increasedC _m compared to control cells plated for comparable times. In contrast to the effect of adherence, LPS-treated cells exhibited a significantly lowerG _K i than control cells, indicating that the additional membrane did not have as high a density of functionalG _K i channels. We conclude that both adherence and LPS treatment increase the total surface membrane area of J774 cells and change the density of Ki channels. In addition, this study demonstrates that membrane area and density of Ki channels can vary independently of one another.     

Regulation of mitochondrial energy production in cardiac cells of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

In skinned rat cardiac fibres, mitochondrial affinity for endogenous ADP generated by creatine kinase and Ca²⁺-activated ATPases is higher than for exogenous ADP added to the surrounding medium, suggesting that mitochondria are functionally coupled to creatine kinase and ATPases. Such a coupling may be weaker or absent in ectothermic vertebrate cardiac cells, because they typically have less elaborate intracellular membrane structures, higher glycolytic capacity and lower working temperature. Therefore, we examined skinned cardiac fibres from rainbow trout at 10 °C. The apparent mitochondrial affinity for endogenous ADP was obtained by stimulation with ATP and recording of the release of ADP into the surrounding medium. The apparent affinity for endogenous ADP was much higher than for exogenous ADP suggesting a functional coupling between mitochondria and ATPases. The apparent affinity for exogenous ADP and ATP was increased by creatine or an increase in Ca²⁺-activity, which should increase intrafibrillar turnover of ATP to ADP. In conclusion, ADP seems to be channelled from creatine kinase and ATPases to mitochondria without being released to the surrounding medium. Thus, despite difference in structure, temperature and metabolic capacity, trout myocardium resembles that of rat with regard to the regulation of mitochondrial respiration.Abbreviations ACR acceptor control ratio - ANT adenine nucleotide translocase - K_{M ADP} apparent mitochondrial affinity for ADP - K_{M ATP} apparent mitochondrial affinity for ATP - LDH lactate dehydrogenase - V_ADP ADP-stimulated respiration rate - V_{ADP max} maximal ADP-stimulated respiration rate - V_ATP ATP-stimulated respiration rate - V_{ATP max} maximal ATP-stimulated respiration rate - V₀ basal respiration rate in the absence of ADPCommunicated by G. Heldmaier     

ATP-dependent calcium transport and its correlation with Ca2+-ATPase activity in basolateral plasma membranes of rat duodenum

Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca²⁺-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH₂)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent K_m of 0.2 μM Ca²⁺ and a V_max of 5.3 nmol Ca²⁺/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca²⁺-ATPase are: K_m = 0.25 μM Ca²⁺ and V_max = 19.2 nmol P_i/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the V_max of ATP-dependent calcium uptake amounts to 25 nmol Ca²⁺/min per mg protein and of the Ca²⁺-ATPase to 23 nmol P_i/min per mg protein. The close correlation between kinetic parameters of Ca²⁺-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca²⁺-pump located in duodenal basolateral plasma membranes.     

Influence of 8-azido-ATP and other anions on the activity of cytochromec oxidase

The effect of ATP and other anions on the kinetics of cytochromec oxidation by reconstituted bovine heart cytochromec oxidase was investigated. The following results were obtained: (1) ATP and other polyvalent anions increase theK _m for cytochromec and theV _max (if assayed by the photometric method). The magnitude of the effect is proportional to the charge of the anion as follows from the series of increasing effectiveness: P_iii. (2) The kinetic effects are obtained in the millimolar physiological concentration range. (3) The kinetic changes are not saturated at high concentrations. (4) A specific interaction site for ATP at the cytosolic domain of the enzyme is concluded from the increase ofK _m for cytochromec after photolabelling of proteoliposomes with 8-azido-[-³²P]-ATP, which is protected by ATP but not by ADP. (5) No specific binding site for ATP could be identified by photolabelling with 8-azido-[-³²P]-ATP. The labelling is only partly protected by ATP or ADP.Abbreviations CCP carbonylcyanide-m-chlorophenylhydrazone - TMPD N,N,N,N-tetramethyl-1,4-phenylenediamine dihydrochloride - 8-N₃-ATP 8-azido-adenosine-5-triphosphate Dedicated to Professor Dr. Friedhelm Schneider on the occasion of his 60th birthday.     

The influence of phosphate concentration on the kinetics of uptake by maize roots

In an experiment with native maize roots depending on different phosphorus concentration in the external solution (0.001 … 50 mM P), the multiphasic character of the kinetics of phosphate uptake has been stated. The single phases are characterized by the different values of K_m and V_max. In the wide range of concentrations the isotherm of the phosphate uptake has five evident phases. The character of kinetics for the uptake of phosphate is analogical to the kinetics of the enzymatic reactions described by the Michaelis-Menten equation. On the other hand the linear dependence for the inactivated root was determined,i.e. the uptake of phosphate versus different phosphorus concentration in the external solution. The graphic representation of the logarithmic values for the phosphorus taken up versus the different phosphorus concentration in the external solution gives the biphasic course including concentration less than 1.0 mM P and more than 1.0 mM P. Within the framework of the concentration range the following values of V_max, K_m and ϕ_in were calculated under the conditions if the concentration of phosphorus is less than 1.0mMP: V_max = 1.705 μmol P × g^-1h^-1, K_m = 0.057 mM P and ϕ_in = 0.83,i.e. if the concentration of phosphorus is more than 1.0mM P: V_max = 40 μmol P × g^-1 h^-1, K_m = 16.66 mM and ϕ_in = 20. According to these results, the phosphate concentration in the external solution influences the activity of the transport mechanisms concerning their conformative changes which discretely change their working regime of membrane transport. This is also demonstrated in the change of values V_max, K_m and ϕ_in.     

Differentiation between Several Types of Phosphohydrolases in Light Microsomes of Corn Roots

The phosphohydrolase activity of a light microsomal fraction isolated from corn roots (Zea mays L. cv LG 55) was investigated. The fraction, which appears to be enriched in endoplasmic reticulum and Golgi membranes, has ATPase and pyrophosphatase activities that hydrolyze ATP and pyrophosphate at an optimum pH of 7.0, with K_m values of about 160 and 240 micromolar and with V_max values of about 200 and 50 nanomoles substrate hydrolyzed per milligram protein per minute, respectively. These enzymes differ in their sensitivity to anions and inhibitors. The ATPase is stimulated by sulfate anions, whereas pyrophosphatase is inhibited by molybdate. Furthermore, the simultaneous addition of ATP and pyrophosphate to the reaction medium increases phosphohydrolysis, suggesting that separate enzymes are operating in the membranes. We also observed that pyrophosphate competitively inhibits the ATPase, whereas ATP has no significant effect on the pyrophosphatase. On the other hand, we observed a detergent-stimulated, molybdate-insensitive inosine diphosphatase activity which, in the native state, hydrolyzes inosine diphosphate with a K_m of about 700 micromolar and a V_max of about 450 nanomoles inosine diphosphate hydrolyzed per milligram protein per minute. In the solubilized form, the enzyme appears to be fully active, exhibiting lower K_m values to hydrolyze inosine diphosphate. Furthermore, we found that native inosine diphosphatase is inhibited either by ATP or pyrophosphate, whereas inosine diphosphate inhibits the ATPase, but has no significant effect on the pyrophosphatase. It appears that inosine diphosphate is a positive modulator of the inosine diphosphatase, whereas ATP and pyrophosphate act as negative modulators of this enzyme.     

Hepatocyte water volume and potassium activity during hypotonic stress

Hepatocytes exhibit a regulatory volume decrease (RVD) during hypotonic shock, which comprises loss of intracellular K⁺ and Cl^– accompanied by hyperpolarization of transmembrane potential (V _m ) due to an increase in membrane K⁺ conductance, (G _K). To examine hepatocyte K⁺ homeostasis during RVD, double-barrel, K⁺-selective microelectrodes were used to measure changes in steady-state intracellular K⁺ activity (a _K ⁱ ) and V _m during hyposmotic stress. Cell water volume change was evaluated by measuring changes in intracellular tetramethylammonium (TMA⁺). Liver slices were superfused with modified Krebs physiological salt solution. Hyposmolality (0.8×300 mosm) was created by a 50 mm step-decrease of external sucrose concentration. Hepatocyte V _m hyperpolarized by 19 mV from –27 ± 1 to –46 ± 1 mV and a _K ⁱ decreased by 14% from 91 ± 4 to 78 ± 4 mm when slices were exposed to hyposmotic stress for 4–5 min. Both V _m and a _K ⁱ returned to control level after restoring isosmotic solution. In paired measurements, hypotonic stress induced similar changes in V _m and a _K ⁱ both control and added ouabain (1 mm) conditions, and these values returned to their control level after the osmotic stress. In another paired measurement, hypotonic shock first induced an 18-mV increase in V _m and a 15% decrease in a _K ⁱ in control condition. After loading hepatocytes with TMA⁺, the same hypotonic shock induced a 14-mV increase in V _m and a 14% decrease in a _TMA ⁱ . This accounted for a 17% increase of intracellular water volume, which was identical to the cell water volume change obtained when a _K ⁱ was used as the marker. Nonetheless, hyposmotic stress-induced changes in V _m and a _K ⁱ were blocked partly by Ba²⁺ (2 mm). We conclude that (i) hepatocyte V _m increases and a _K ⁱ decreases during hypotonic shock; (ii) the changes in hepatocyte V _m and a _K ⁱ during and after hypotonic shock are independent of the Na⁺-K⁺ pump; (iii) the decrease in a _K ⁱ during hypotonic stress results principally from hepatocyte swelling.This work was supported by grant AA-08867 from the Alcohol, Drug Abuse, and Mental Health Association.     

Role of energy in oxidative phosphorylation

This article reviews the current status of information regarding the role of energy in the process of oxidative phosphorylation by mitochondria. The available data suggest that in submitochondrial particles (SMP) energy is utilized for the binding of ADP and P_i and for the release of ATP bound at the catalytic sites of F₁-ATPase. The process of ATP synthesis on the surface of F₁ from F₁-bound ADP and P_i appears to be associated with negligible free energy change. The rate of energy production by the respiratory chain modulates the kinetics of ATP synthesis between a lowK _m (for ADP and P_i)-lowV _max mode and a highK _m -highV _max mode. TheK _m extremes for ADP are 2–3 µM and 120–150 µM, andV _max for ATP synthesis at high rates of energy production by bovine-heart SMP is about 440 s^–1 (mole F₁)^–1 at 30°C, which corresponds to 11 µmol ATP (min · mg of protein)^–1. The interaction of dicyclohexylcarbodiimide (DCCD) or oligomycin at the proteolipid (subunitc) of the membrane sector (F₀) of the ATP synthase complex alters the mode of ATP binding at the catalytic sites of F₁, probably to one of lower affinity. It has been suggested that protonic energy might be conveyed to the catalytic sites of F₁ in an analogous manner, i.e., via conformation changes in the ATP synthase complex initiated by proton-induced alterations in the structure of the DCCD-binding proteolipid. Finally, the relationship between the steady-state membrane potential () and the rates of electron transfer and ATP synthesis has been discussed. It has been shown, in agreement with the delocalized chemiosmotic mechanism, that under appropriate conditions is exquisitely sensitive to changes in the rates of energy production and consumption.     

The plasma membrane ATPase of Kloeckera apiculata: purification,characterization and effect of ethanol on activity

Partially (6-fold) purified plasma membrane ATPase from an ethanol-sensitive yeast, Kloeckera apiculata, had an optimum pH of 6.0, an optimum temperature of 35°C, a K _m of 3.6 mm ATP and a V _max of 11 mol P_i/min.mg protein. SDS-PAGE of the semi-purified plasma membrane showed a major band of 106 kDa. No in vivo activation of the ATPase by glucose was observed. Although 4% (v/v) ethanol decreased the growth rate by 50% it did not affect the ATPase. Concentrations of ethanol 2% (v/v) did, however, inhibit the enzyme in vitro. The characteristics of the enzyme did not change during growth in the presence of ethanol.     

CARRIER MEDIATED BLOOD-BRAIN BARRIER TRANSPORT OF CHOLINE AND CERTAIN CHOLINE ANALOGS

Blood-brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a K_m= 442 ± 60 μM and V_max= 10.0 ± 0.6 nmol min^-1 g^-1. In 14–15-day-old suckling forebrains a similar K_m (= 404 ± 88 μM) but higher V_max (= 12.5 ± 1.5 nmol min^-1 g^-1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis-Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex K_m= 310 ± 103 μM and V_max= 12.6 ± 2.8 nmol min^-1g^-1; paleocortex K_m= 217 ± 76 μM and V_max= 7.2 ± 1.5 nmol min^-1 g^-1). Administration of radiolabelled choline at low (5 μM) and high (100 μM) concentrations, followed by microwave fixation 60 s later and chloroform-methanol-water separations of the homogenized brain did not suggest a relationship between concentration and the appearance of label in lipid or aqueous fractions as observed in another in-vitro study elaborating two-component kinetics of choline uptake. It was observed that 60s after carotid injection 12–14% of the radiolabel in the ipsilateral cortex was found in the chloroform-soluble fraction. Hemicholinium-3 (K_i= 111 μM), dimethylaminoethanol (K_i= 42 μM), tetraethyl ammonium chloride, tetramethyl ammonium chloride, 2-hydroxyethyl triethylammonium iodide, carnitine, normal rat serum, and to a lesser extent lithium and spermidine all inhibited choline uptake in the BBB. Unsubstituted ammonium chloride and imipramine did not inhibit choline uptake. No difference was observed in blood-brain barrier choline uptake of unanesthetised, carotid artery-catheterized animals, and comparable sodium pentobarbital-anesthetized controls.     

Temperature sensitivities of extracellular enzyme Vmax and Km across thermal environments

The magnitude and direction of carbon cycle feedbacks under climate warming remain uncertain due to insufficient knowledge about the temperature sensitivities of soil microbial processes. Enzymatic rates could increase at higher temperatures, but this response could change over time if soil microbes adapt to warming. We used the Arrhenius relationship, biochemical transition state theory, and thermal physiology theory to predict the responses of extracellular enzyme V_max and K_m to temperature. Based on these concepts, we hypothesized that V_max and K_m would correlate positively with each other and show positive temperature sensitivities. For enzymes from warmer environments, we expected to find lower V_max, K_m, and K_m temperature sensitivity but higher V_max temperature sensitivity. We tested these hypotheses with isolates of the filamentous fungus Neurospora discreta collected from around the globe and with decomposing leaf litter from a warming experiment in Alaskan boreal forest. For Neurospora extracellular enzymes, V_max Q₁₀ ranged from 1.48 to 2.25, and K_m Q₁₀ ranged from 0.71 to 2.80. In agreement with theory, V_max and K_m were positively correlated for some enzymes, and V_max declined under experimental warming in Alaskan litter. However, the temperature sensitivities of V_max and K_m did not vary as expected with warming. We also found no relationship between temperature sensitivity of V_max or K_m and mean annual temperature of the isolation site for Neurospora strains. Declining V_max in the Alaskan warming treatment implies a short‐term negative feedback to climate change, but the Neurospora results suggest that climate‐driven changes in plant inputs and soil properties are important controls on enzyme kinetics in the long term. Our empirical data on enzyme V_max, K_m, and temperature sensitivities should be useful for parameterizing existing biogeochemical models, but they reveal a need to develop new theory on thermal adaptation mechanisms.     

The growth of soybean under free air [CO<Subscript>2</Subscript>] enrichment (FACE) stimulates photosynthesis while decreasing in vivo Rubisco capacity

Down-regulation of light-saturated photosynthesis (A_sat) at elevated atmospheric CO₂ concentration, [CO₂], has been demonstrated for many C₃ species and is often associated with inability to utilize additional photosynthate and/or nitrogen limitation. In soybean, a nitrogen-fixing species, both limitations are less likely than in crops lacking an N-fixing symbiont. Prior studies have used controlled environment or field enclosures where the artificial environment can modify responses to [CO₂]. A soybean free air [CO₂] enrichment (FACE) facility has provided the first opportunity to analyze the effects of elevated [CO₂] on photosynthesis under fully open-air conditions. Potential ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation (V_c,max) and electron transport through photosystem II (J_max) were determined from the responses of A_sat to intercellular [CO₂] (C_i) throughout two growing seasons. Mesophyll conductance to CO₂ (g_m) was determined from the responses of A_sat and whole chain electron transport (J) to light. Elevated [CO₂] increased A_sat by 15–20% even though there was a small, statistically significant, decrease in V_c,max. This differs from previous studies in that V_c,max/J_max decreased, inferring a shift in resource investment away from Rubisco. This raised the C_i at which the transition from Rubisco-limited to ribulose-1,5-bisphosphate regeneration-limited photosynthesis occurred. The decrease in V_c,max was not the result of a change in g_m, which was unchanged by elevated [CO₂]. This first analysis of limitations to soybean photosynthesis under fully open-air conditions reveals important differences to prior studies that have used enclosures to elevate [CO₂], most significantly a smaller response of A_sat and an apparent shift in resources away from Rubisco relative to capacity for electron transport.Abbreviations FACE Free air [CO₂] enrichment - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP Ribulose-1,5-bisphosphate - SoyFACE Soybean free air [CO₂] enrichment - VPD Vapor pressure deficit     

Regulation of the basolateral potassium conductance of theNecturus proximal tubule

Summary Two methods, the measurement of the response of the basolateral membrane potential (V _bl) of proximal tubule cells ofNecturus to step changes in basolateral K⁺ concentration, and cellular cable analysis, were used to assess the changes in basolateral potassium conductance (G _K) caused by a variety of maneuvers. The effects of some of these maneuvers on intracellular K⁺ activity (a _K ⁱ ) were also evaluated using double-barreled ion-selective electrodes. Perfusion with 0mm K⁺ basolateral solution for 15 min followed by 45 min of 1mm K⁺ solution resulted in a fall in basolateral potassium (apparent) transference number (t _K),V _bl anda _K ⁱ . Results of cable analysis showed that total basolateral resistance,R _b , rose. The electrophysiological effects of additional manipulations, known to inhibit net sodium reabsorption across the proximal tubular epithelium ofNecturus, were also investigated. Ouabain caused a fall int _K accompanied by large decreases ina _K ⁱ andV _bl. Lowering luminal sodium caused a fall int _K and a small reduction inV _bl. Selective reduction of peritubular sodium, a maneuver that has been shown to block sodium transport from lumen to peritubular fluid, also resulted in a significant decrease int _K. These results suggest thatG _K varies directly with rate of transport of the sodium pump, irrespective of the mechanism of change in pump turnover.Part of this material has been presented at the 10th International Conference on Biological Membranes (Cohen & Giebisch, 1984).