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Introduction: The increase in demand for post-bariatric plastic surgery has revealed a high prevalence of psychopathologies in patients undergoing the procedure, requiring the need to diagnose these diseases in the preoperative period. The use of specific psychological screening tools has been promoted; however, a gold-standard method has not yet been fully established. Objective: To carry out a review of the literature for alternatives available for the preliminary psychological evaluation of patients who are candidates for post-bariatric plastic surgery, presenting the method recommended in the Post-Bariatric Plastic Surgery outpatient clinic of the Federal University of Mato Grosso do Sul (UFMS). Methods: We reviewed the clinical trials which employed psychological screening tools preoperatively in patients who were candidates for post-bariatric plastic surgery; the MEDLINE/PubMed database was searched using keywords such as “bariatric surgery”, “body image”, “quality of life”, “obesity”, “plastic surgery” and “psychiatry”, for clinical trials published in the last 20 years. Herein, we discuss the findings and analyze the most common methodologies used. Results: Only four clinical trials used psychopathology screening tools in postbariatric plastic surgeries preoperatively, and one method could not be identified. Conclusion: The use of appropriate strategies to screen for psychopathologies helps prevent significant losses in the postoperative period, but the tools still lack validation in the post-bariatric population. Besides possessing extensive clinical-surgical technical knowledge, the plastic surgeon must remain attentive to the signs and psychopathological symptoms in these patients, referring them for psychological and psychiatric evaluation when indicated.

DELLA proteins restrain the cell proliferation and enlargement that characterizes the growth of plant organs. Gibberellin stimulates growth via 26S proteasome-dependent destruction of DELLAs, thus relieving DELLA-mediated growth restraint. Here, we show that the Arabidopsis thaliana sleepy1gar2-1 (sly1gar2-1) mutant allele encodes a mutant subunit (sly1gar2-1) of an SCF(SLY1) E3 ubiquitin ligase complex. SLY1 (the wild-type form) and sly1gar2-1 both confer substrate specificity on this complex via specific binding to the DELLA proteins. However, sly1gar2-1 interacts more strongly with the DELLA target than does SLY1. In addition, the strength of the SCFSLY1-DELLA interaction is increased by target phosphorylation. Growth-promoting DELLA destruction is dependent on SLY1 availability, on the strength of the interaction between SLY1 and the DELLA target, and on promotion of the SCFSLY1-DELLA interaction by DELLA phosphorylation., (Copyright 2004 American Society of Plant Biologists)

The phytohormone gibberellin (GA) regulates the development and fertility of Arabidopsis flowers. The mature flowers of GA-deficient mutant plants typically exhibit reduced elongation growth of petals and stamens. In addition, GA-deficiency blocks anther development, resulting in male sterility. Previous analyses have shown that GA promotes the elongation of plant organs by opposing the function of the DELLA proteins, a family of nuclear growth repressors. However, it was not clear that the DELLA proteins are involved in the GA-regulation of stamen and anther development. We show that GA regulates cell elongation rather than cell division during Arabidopsis stamen filament elongation. In addition, GA regulates the cellular developmental pathway of anthers leading from microspore to mature pollen grain. Genetic analysis shows that the Arabidopsis DELLA proteins RGA and RGL2 jointly repress petal, stamen and anther development in GA-deficient plants, and that this function is enhanced by RGL1 activity. GA thus promotes Arabidopsis petal, stamen and anther development by opposing the function of the DELLA proteins RGA, RGL1 and RGL2.

DELLA proteins are nuclear repressors of plant gibberellin (GA) responses. Here, we investigate the properties of SLN1, a DELLA protein from barley that is destabilized by GA treatment. Using specific inhibitors of proteasome function, we show that proteasome-mediated protein degradation is necessary for GA-mediated destabilization of SLN1. We also show that GA responses, such as the aleurone alpha-amylase response and seedling leaf extension growth, require proteasome-dependent GA-mediated SLN1 destabilization. In further experiments with protein kinase and protein phosphatase inhibitors, we identify two additional signaling steps that are necessary for GA response and for GA-mediated destabilization of SLN1. Thus, GA signaling involves protein phosphorylation and dephosphorylation steps and promotes the derepression of GA responses via proteasome-dependent destabilization of DELLA repressors.

Bioactive gibberellin (GA) is an essential regulator of vascular plant development. The GAI gene of Arabidopsis thaliana (L.) Heynh. encodes a product (GAI) that is involved in GA signalling. The dominant mutant gai allele encodes an altered product (gai) that confers reduced GA responses, dwarfism, and elevated endogenous GA levels. Recessive, presumed loss-of-function alleles of GAI confer normal height and resistance to the GA biosynthesis inhibitor paclobutrazol. One explanation for these observations is that GAI is a growth repressor whose activity is opposed by GA, whilst gai retains a constitutive repressor activity that is less affected by GA. Previously, we described gai-t6, a mutant allele which contains an insertion of a maize Ds transposable element into gai. Here we describe the molecular and physiological characterization of two further alleles (gai-t5, gai-t7) identified during the Ds mutagenesis experiment. These alleles confer paclobutrazol resistance and normal endogenous GA levels. Thus the phenotype conferred by gai-t5, gai-t6 and gai-t7 is not due to elevated GA levels, but is due to loss of gai, a constitutively active plant growth repressor.

Bioactive gibberellins (GAs) are essential endogenous regulators of plant growth. GA signaling is mediated via GAI, a nuclear member of the GRAS family of plant transcription factors. Previous experiments have suggested that GAI is a GA-derepressible repressor of plant growth. Here we test this hypothesis by examining the effects of the expression of Arabidopsis GAI in transgenic Basmati rice. High-level expression of GAI caused dwarfism and reduced GA responses, and the strength of this effect was correlated with the level of transgene expression. In particular, the expression of GAI abolished the GA-mediated induction of rice aleurone alpha-amylase activity, thus implicating GAI orthologs in the well-characterized cereal aleurone GA response. The GA derepressible repressor model predicts that high-level expression of GAI should confer dwarfism, and these observations are consistent with this prediction.

World wheat grain yields increased substantially in the 1960s and 1970s because farmers rapidly adopted the new varieties and cultivation methods of the so-called 'green revolution'. The new varieties are shorter, increase grain yield at the expense of straw biomass, and are more resistant to damage by wind and rain. These wheats are short because they respond abnormally to the plant growth hormone gibberellin. This reduced response to gibberellin is conferred by mutant dwarfing alleles at one of two Reduced height-1 (Rht-B1 and Rht-D1) loci. Here we show that Rht-B1/Rht-D1 and maize dwarf-8 (d8) are orthologues of the Arabidopsis Gibberellin Insensitive (GAI) gene. These genes encode proteins that resemble nuclear transcription factors and contain an SH2-like domain, indicating that phosphotyrosine may participate in gibberellin signalling. Six different orthologous dwarfing mutant alleles encode proteins that are altered in a conserved amino-terminal gibberellin signalling domain. Transgenic rice plants containing a mutant GAI allele give reduced responses to gibberellin and are dwarfed, indicating that mutant GAI orthologues could be used to increase yield in a wide range of crop species.

Active gibberellins (GAs) are endogenous factors that regulate plant growth and development in a dose-dependent fashion. Mutant plants that are GA deficient, or exhibit reduced GA responses, display a characteristic dwarf phenotype. Extragenic suppressor analysis has resulted in the isolation of Arabidopsis mutations, which partially suppress the dwarf phenotype conferred by GA deficiency and reduced GA-response mutations. Here we describe detailed studies of the effects of two of these suppressors, spy-7 and gar2-1, on several different GA-responsive growth processes (seed germination, vegetative growth, stem elongation, chlorophyll accumulation, and flowering) and on the in planta amounts of active and inactive GA species. The results of these experiments show that spy-7 and gar2-1 affect the GA dose-response relationship for a wide range of GA responses and suggest that all GA-regulated processes are controlled through a negatively acting GA-signaling pathway.

The Arabidopsis gai mutant allele confers a reduction in gibberellin (GA) responsiveness. Here we report the molecular cloning of GAI and a closely related gene GRS. The predicted GAI (wild-type) and gai (mutant) proteins differ only by the deletion of a 17-amino-acid segment from within the amino-terminal region. GAI and GRS contain nuclear localization signals, a region of homology to a putative transcription factor, and motifs characteristic of transcriptional coactivators. Genetic analysis indicates that GAI is a repressor of GA responses, that GA can release this repression, and that gai is a mutant repressor that is relatively resistant to the effects of GA. Mutations at SPY and GAR2 suppress the gai phenotype, indicating the involvement of GAI, SPY, and GAR2 in a signaling pathway that regulates GA responses negatively. The existence of this pathway suggests that GA modulates plant growth through derepression rather than through simple stimulation.

Ouabain-sensitive uncoupled Na+ efflux has been studied in human, pig, and rat red cells and in vesicles containing reconstituted kidney Na+/K+ pumps obtained from these same species. The red cells from the different species gave qualitatively similar results; the uncoupled Na+ efflux was 15-30% of the Na+/K+ exchange rate, and this flux was inhibited at 5 mM extracellular Na+ (Na+o). At higher levels of Na+o there was a monotonic increase in the Na+ efflux. As has previously been observed in human red cells, the uncoupled efflux from pig red cells consists of Na+ and anion cotransport, suggesting that anion cotransport may be a general characteristic of uncoupled Na+ efflux in red cells. The uncoupled Na+ efflux carried out by pig and rat kidney Na+/K+ pumps differs from the red cell activity in that it represents no more than 2-4% of the Na+/K+ exchange rate and that 5 mM Na+o does not inhibit this efflux. Furthermore, the efflux does not appear to be dependent on anion cotransport. Vesicles containing human kidney Na+/K+ pumps differ from vesicles derived from pig or rat kidneys in that the Na+ efflux is not inhibited or stimulated by Na+ present on the opposite side; it thus appears that the Na+,K(+)-ATPase in these vesicles may be incapable of Na+/Na+ exchange. These results indicate that the ligand and kinetic properties of the uncoupled Na+ efflux mode of red cells are markedly different from kidney-derived Na+/K+ pumps reconstituted into proteoliposomes. The basis for these differences may be inherent in the Na+/K+ pumps themselves or represent differences between the two types of preparations studied.

Calretinin is a member of the EF-hand calcium-binding protein family, with a high similarity with calbindin D28k. The chick calretinin cDNA sequence was reconstructed in a M13 vector and transferred into an expression plasmid derived from the pET series. The calretinin gene was expressed in Escherichia coli and produced immunoreactive calretinin of the expected size. Bacterially expressed calretinin was purified with successive ammonium-sulfate precipitation, DEAE chromatography, hydroxyapatite chromatography, Sephadex G-75 chromatography and Mono-Q chromatography. Normally, 1.0-1.5 mg calretinin was obtained from 1 l bacterial culture with a protein recovery of 0.5-1.5%. Calbindin D28k was purified similarly from bacteria using an expression plasmid provided by W. Hunziker. Calcium-binding activity of purified proteins was measured by equilibrium dialysis in calcium/EGTA mixtures with 45Ca as tracer. Both calretinin and calbindin D28k bound 3-4 Ca2+/molecule (calretinin, 4.0 +/- 0.5; calbindin D28k, 3.5 +/- 0.4), implying that at least one of the canonical EF-hand domains does not bind calcium. The Kd was 0.3-0.5 microM with little difference between the values for the two proteins.

We have used renal (Na,K)-ATPase, covalently labeled with fluorescein, and phospholipid vesicles reconstituted with labeled enzyme, to detect conformational transitions induced by acetyl phosphate in the presence of Mg2+ and Na+ ions. Equilibrium fluorescence measurements show quenching of the fluorescein fluorescence, which is thought to reflect conversion of the initial E1 form to the phosphorylated E2P form. These fluorescence changes occur on inside-out-oriented pumps. The rates of acetyl phosphate-induced fluorescence changes have been measured using a stopped-flow fluorimeter. The rate of fluorescence quenching (1.5-3 s-1) is a measure of the rate of the E1P(Na)----E2P transition. The quenching is preceded by a fast fluorescence increase (12.3 +/- 4 s-1) associated with phosphorylation of E1 to E1P(Na), shown clearly in experiments with enzyme treated with oligomycin. Oligomycin greatly reduces the rate of the fluorescence quenching (0.044 +/- 0.01 s-1). Using potassium-loaded vesicles treated with valinomycin or lithium-loaded vesicles treated with Li+ ionophore N,N'-diheptyl-N,N'-didiethyl ether, 5,5-dimethyl-3,7-dioxanonanediamide in order to induce electrical diffusion potentials, negative inside, the rates of the fluorescence quenching are accelerated by up to 4-fold. The experiments demonstrate that the conformational transition E1P(Na)----E2P, associated with transport of 3 Na+ ions, is a voltage-sensitive reaction, carrying a net positive charge. This confirms a prediction based on transport experiments. In experiments with fluorescein-labeled (Na,K)-ATPase, the use of acetyl phosphate rather than ATP, which does not bind, provides a valuable tool to detect fluorescence signals accompanying steps in the turnover cycle.

1. Ouabain-sensitive K or Rb influx was measure into ghosts resealed to contain ATP concentrations of 1 micrometers-3 mM and no K. 2. Increasing ATP from 1 to 100 micro M, at saturation external K, increased K influx about twentyfold while have no effect on the ratio of ouabain-sensitive K influx to ouabain-sensitive ATPase activity. 3. Increasing external K decreased the apparent affinity for ATP. Similarly increasing ATP decreased the apparent affinity for external K. 4. The K influx can be empirically described as: influx = VmaxK2/(K + Kapp)2. Increasing ATP increased Vmax and (Kapp)2 by the same amount. 5. These results are consistent with a consecutive model for the Na pump in which an ATP-dependent reaction follows a K-activated dephosphorylation.

1. When magnesium and orthophosphate are added to Na+,K+-ATPase containing occluded rubidium ions, and suspended in a medium containing free rubidium ions, only 50% of the occluded rubidium is released rapidly. This is because the release of occluded rubidium is ordered, and the replacement (by rubidium ions from the medium) of the first occluded rubidium ions to leave slows the departure of the remaining occluded ions. 2. Since the Na+,K+-ATPase probably exists in the membrane as a structural dimer, the ordered release might represent either the ordered emptying of the two halves of the dimer, or the ordered release of the two rubidium ions thought to be contained in each promoter. 3. The present experiments were designed to decide between these possibilities by examining the behaviour of Na+,K+-ATPase in which about half of the protomers had been randomly inactivated by pre-treatment either with fluorescein isothiocyanate or with alpha-chymotrypsin. 4. The results show that the release of rubidium ions from each protomer is ordered.

1. A phosphodiesterase that cleaves glycerophosphoinositol into glycerophosphate and inositol has been detected in rat tissues. 2. The enzyme requires Mg2+ (Mn2+) and has a pH optimum of 7.7. 3. The richest sources of the enzyme are kidney and intestinal mucosa. In pancreas subcellular fractions it occurs largely in the microsomal fraction. 4. The enzyme is inhibited by excess substrate and by the reaction product glycerophosphate. 5. Temperature-stability studies and other observations distinguish the enzyme from other membrane-bound phosphodiesterases active at an alkaline pH e.g. glycerophosphoinositol inositophosphohydrolase, glycerophosphocholine diesterase, inositol cyclic phosphate phosphodiesterase and phosphodiesterase I.

The routes of 14C excretion following the administration of a single oral 230 mg/kg body weight dose of 2-ethoxyethanol [ethanol-1,2-14C] or 2-ethoxyethanol [ethoxy-1-14C] to male Sprague-Dawley rats were investigated. Elimination of the 14C by the urinary route accounted for 76 to 80% of the dose within 96 hr. The main pathway of biotransformation is oxidation to the corresponding acid, with some subsequent conjugation of the acid metabolite with glycine. The major metabolites, ethoxyacetic acid and N-ethoxy-acetyl glycine, representing 73 to 76% of the administered dose, were eliminated in the urine. The major difference in the metabolic profiles of the two radiochemicals was in the rate and amount of 14CO2 expired via the lung. Of the administered 14C, 11.7% of the ethoxy-labeled and 4.6% of the ethanol-labeled compounds were eliminated as CO2. The biological half-time was 9.9 +/- 1.5 hr for the ethoxy-labeled compound and 12.5 +/- 1.9 hr for the ethanol label. After administration of the ethanol-labeled compound, the only radiolabeled component found in the rat testes was identified as ethoxyacetic acid. Results of this study suggest that the reported testicular effects in the rat may be a result of tissue levels of ethoxyacetic acid.

(1) The hydrolysis of (32)P- or myo-[2-(3)H]inositol-labelled rat liver microsomal phospholipids by rat liver lysosomal enzymes has been studied. (2) The relative rates of hydrolysis of phospholipids at pH4.5 are: sphingomyelin>phosphatidylethanolamine>phosphatidylcholine> phosphatidylinositol. (3) The predominant products of phosphatidylcholine and phosphatidylethanolamine hydrolysis are their corresponding lyso-compounds, indicating a slow rate of total deacylation. (4) Ca(2+) inhibits the hydrolysis of all phospholipids, though only appreciably at high (>5mm) concentration. The hydrolysis of sphingomyelin is considerably less sensitive to Ca(2+) than that of glycerophospholipids. (5) Analysis of the water-soluble products of phosphatidylinositol hydrolysis (by using myo-[(3)H]inositol-labelled microsomal fraction as a substrate) produced evidence that more than 95% of the product is phosphoinositol, which was derived by direct cleavage from phosphatidylinositol, rather than by hydrolysis of glycerophosphoinositol. (6) This production of phosphoinositol, allied with negligible lysophosphatidylinositol formation and a detectable accumulation of diacylglycerol, indicates that lysosomes hydrolyse membrane phosphatidylinositol almost exclusively in a phospholipase C-like manner. (7) Comparisons are drawn between the hydrolysis by lysosomal enzymes of membrane substrates and that of pure phospholipid substrates, and also the possible role of phosphatidylinositol-specific lysosomal phospholipase C in cellular phosphatidylinositol catabolism is discussed.

1. Co2+ ions can replace Mg2+ ions as co-factors for the Na+-K+ pump purified from dog kidney outer medulla. The evidence comes from (a) measurement of ouabain-sensitive Na+,K+-ATPase activity, (b) measurement of ATP-dependent 22Na uptake catalysed by the Na+-K+ pump reconstituted into phospholipid vesicles, (c) measurements of phosphorylation of the Na+-K+ pump either in the presence of ATP and sodium ions or in the presence of inorganic phosphate, and (d) measurement of occlusion of rubidium ions through the route involving phosphorylation and dephosphorylation. 2. Purified Na+,K+-ATPase incubated in the presence of ATP, Na+ ions and [60Co]CoCl2, can carry occluded Co2+ ions through a cation-exchange resin. The enzyme fails to occlude the divalent cation (i) if ADP replaces ATP, (ii) if the enzyme is heat-inactivated, (iii) if the enzyme is inactivated by treatment with fluorescein isothiocyanate, (iv) if K+ replaces Na+ in the incubation medium, (v) if Na+ ions are omitted, and (vi) if Mg2+ ions are added in a sufficient concentration. 3. The amount of occluded Co2+ ions is unaffected by pre-treatment of the Na+,K+-ATPase with oligomycin, which stabilizes the phosphoenzyme in the E1P form. 4. The addition of K+ ions to Na+,K+-ATPase that has been phosphorylated in the presence of ATP, Na+ ions and [60Co]CoCl2 releases the occluded Co2+ ions from the enzyme. Under those conditions, K+ ions accelerate the hydrolysis of the phosphoenzyme, and become occluded in the resulting dephosphoenzyme. 5. The stoichiometry of Co2+ ion occlusion is about one occluded Co2+ ion per phosphorylation site. 6. These results support the hypothesis that, in the normal working of the Na+-K+ pump, Mg2+ ions are trapped in the phosphorylated forms of the enzyme, and are released by a K+-dependent dephosphorylation reaction.

The hypothesis that the ADP-sensitive form of phosphorylated Na+, K+-ATPase contains occluded sodium ions has been tested by a procedure which involves (i) modifying the enzyme with alpha-chymotrypsin or N-ethylmaleimide (NEM) so that the ADP-sensitive form is more stable than it is in the native enzyme, (ii) phosphorylating the modified enzyme with ATP in the presence of labelled sodium ions, and (iii) forcing the phosphorylated enzyme rapidly through a cation-exchange column and measuring the labelled sodium in the effluent. The results show that ADP-sensitive phosphoenzyme prepared from alpha-chymotrypsin- or NEM-modified Na+, K+-ATPase is able to carry labelled sodium ions through a cation-exchange resin. This behaviour was not seen with native Na+, K+-ATPase or when phosphorylation was prevented by the omission of magnesium ions or by the substitution of adenylyl(beta, gamma-methylene)diphosphonate (AMP-PCP) for ATP. The occluded sodium ions were rapidly released when the phosphoenzyme was dephosphorylated by ADP. When alpha-chymotrypsin-modified enzyme was phosphorylated by ATP with 1 mM-sodium in the medium, close to three sodium ions were occluded per phospho group. The stoicheiometry at much lower sodium concentrations could not be determined satisfactorily. A consideration of the rate constants of the reactions thought to be involved in the occlusion of sodium and in the release of sodium from the occluded state shows that, so far as they are known, these constants are compatible with the hypothesis that the occluded-sodium form of the phosphoenzyme plays a central role in sodium transport through the pump.

When Na,K-ATPase containing occluded rubidium ions is exposed to orthophosphate, in the presence of magnesium ions, there is a rapid release of half or all of the occluded ions. This behaviour is observed irrespective of whether the occluded-rubidium form of the enzyme is generated by putting the unphosphorylated enzyme in a sodium-free medium containing rubidium ions, or by allowing rubidium ions to catalyse the hydrolysis of phosphoenzyme made by adding ATP to enzyme suspended in a medium containing sodium and magnesium ions. The release of occluded rubidium ions by orthophosphate requires the presence of magnesium, presumably because phosphorylation is necessary. Whether the addition of orthophosphate causes the rapid release of all or of half of the occluded rubidium depends on whether free rubidium (or potassium, thallium or (probably) caesium ions) are present in the medium at the time the orthophosphate is added. In the absence of free ions of these species, all of the occluded rubidium is released. In their presence (in adequate concentration), only half of the occluded rubidium is released. The relative effectiveness of the different potassium congeners in preventing the rapid release of 50% of the occluded rubidium when orthophosphate is added is: thallium greater than rubidium greater than potassium greater than caesium. Lithium and sodium are ineffective even at high concentrations, and sodium ions strongly antagonize the effect of free rubidium ions. In a sodium-free, Tris medium, the concentration of free rubidium ions necessary for a half-maximal effect is about 30 microM. In a medium containing 250 microM-free rubidium, the concentration of sodium necessary to reduce the effect of free rubidium by 50% is about 500 microM. These figures are compatible with the hypothesis that the free rubidium or other ions act at the potassium-loading sites at the extracellular face of the pump. By starting with enzyme occluding unlabelled rubidium, and using 86Rb-labelled free rubidium, it is possible to show that the free ions that prevent the rapid release of half of the occluded ions themselves become occluded. These experiments are significant in two ways. First, they provide direct evidence for the existence of a second route for the release of occluded rubidium (and therefore presumably of occluded potassium) ions. Secondly, they seem to require that the release of occluded ions by this route occurs in an ordered fashion.

1. It is now widely believed that the main rate-limiting step in the sodium-potassium pump (Na, K-ATPase) cycle is a conformational change between two forms of the dephosphoenzyme (E2 and E1) and that this change releases to the cell interior potassium ions occluded within the E2 form. 2. If this hypothesis is correct, and if occluded ions cannot be released directly from dephosphoenzyme in the E2 conformation, we should expect that, under any given conditions, the rate of release of the occluded ions would be identical with the rate of the conformational change. 3. Using the potassium congeners 86Rb, 137Cs and 204Tl, the rates of release of the occluded ions can be measured by a rapid ion-exchange technique. Using the fluorescence probes fluorescein isothiocyanate (FITC), eosin or 5-iodoacetamido-fluorescein (5-IAF), the rates of the conformational change can be measured by stopped-flow fluorimetry. 4. A comparison of the two rates in the absence of ATP showed that the rate of release of the occluded ions was usually somewhat faster than the rate of the fluorescence change. The discrepancy was probably caused by a very slow direct release of occluded ions from enzyme in the E2 form, but we cannot exclude the possibility that it is the result of systematic errors. In the presence of 5 microM-ATP, both rates were increased and there was no significant difference between them. 5. The results are compatible with the hypothesis that the same conformational change alters the fluorescence of the fluorescent probes and releases the occluded potassium congener ions.

The effects of DPI 201-106 were examined on contractions of papillary muscles from the right ventricle of feline heart, on [3H]-ouabain binding and Na+, K+-ATPase activity in feline ventricular membrane particles and on ouabain-sensitive 86Rb uptake in human erythrocytes. DPI 201-106 partially inhibited the Na+ pump at concentrations that caused pronounced positive inotropic effects.

The potassium:potassium (K-K) exchange through the sodium pump has been measured as the ouabain-sensitive 86Rb uptake by Na-free ghosts resealed to contain various concentrations of ATP, orthophosphate and K. The exchange is activated by increasing either internal or external K+ (Rb+) ion concentration. The activation curves can be described by simple Michaelis kinetics as: exchange = Vmax [K]/(Kapp + [K]). Increasing ATP concentration increases the apparent affinity for external K ions but decreases the apparent affinity for internal K (Ki+). Increasing [ATP] from 1 microM to 1 mM typically increases the Kapp for Ki+ from less than 1 mM to about 30 mM. Increasing ATP first activates the exchange but, after an optimal concentration is reached, further increase of ATP inhibits. The value of ATP concentration which gives the maximum flux depends on the internal and external K+ concentrations. The higher [Ki], the greater the optimal ATP concentration. Increasing external K (Rb) decreases the optimal ATP concentration. Increasing the concentration of orthophosphate (Pi) activates the exchange at high ATP but inhibits at low ATP concentration. A concentration of Pi which stimulates the exchange at high external K (Rb) can inhibit at low external K (Rb). These findings are in agreement with a consecutive or ping-pong model of the K-K exchange. We suggest that previous experiments have not shown the inhibitory effects of ATP and Pi because of the particular range of concentrations investigated.

1. Ouabain-sensitive Rb influx was measured into K-free resealed red cell ghosts. The effects of inorganic phosphate (Pi) were examined. 2. Phosphate decreased the magnitude of the influx. Increasing Pi lowered the apparent affinity for both ATP and external rubidium ions. The effects of Pi on the affinity for external Rb were greatest at low ATP concentrations. 3. In the nominal absence of phosphate, increasing ATP from 1 to 100 microM had little effect on the Rb influx from solutions of low (1 microM) rubidium concentrations. In the presence of Pi, ATP increased Rb uptake markedly, even from solutions of low Rb concentration. 4. The above interactions between Pi, ATP and external Rb are consistent with a consecutive scheme for the Na pump in which phosphate is released after potassium binds at the external surface and before ATP binds to release potassium ions to the internal solution. 5. Previous failures to find an effect of phosphate on either the affinity for ATP or that for external potassium (rubidium) ions are shown to be equally consistent with the model. The lack of change of apparent affinity is shown to result from the restricted range of concentrations used in these previous experiments.

1. The occlusion of rubidium ions by Na, K-ATPase has been investigated by suspending enzyme prepared from pig kidney outer medulla in media containing low concentrations of (86)Rb, forcing the suspensions rapidly through small columns of cation-exchange resin, and measuring the amounts of radioactivity emerging from the columns.2. When the suspension media contained 2 mM-ATP or ADP, or 15 mM-NaCl, the amounts of radioactivity emerging from the columns were greatly (and similarly) reduced, presumably because both nucleotides and sodium ions stabilized the enzyme in the E(1) form. (See p. 19 for definition of E(1) and E(2)). The extra radioactivity carried through the columns when nucleotides and sodium were absent was taken as a measure of the amount of rubidium occluded within the enzyme (in the E(2) form) when it emerged from the resin.3. By varying the flow rate, and therefore the time spent by the enzyme on the resin, and relating this to the amount of radioactivity emerging from the columns, we have been able to estimate the rate constant for the conformational change (E(2) --> E(1)) that allows the occluded rubidium ions to escape. At 20 degrees C, and in the absence of nucleotides, it is about 0.1 S(-1).4. The rate constant for rubidium release was the same in a sodium-containing as in a potassium-containing medium. The opposite effects of sodium and potassium ions on the poise of the equilibrium between the E(1) and the E(2) forms of the enzyme must, therefore, be due solely to opposite effects of these ions on the rate of conversion of E(1) to E(2).5. The rate constant for rubidium release was greatly increased by ATP and by ADP. Both nucleotides appeared to act at low-affinity sites and without phosphorylating the enzyme.6. Orthovanadate, in the presence of magnesium ions, stabilized the enzyme in the occluded-rubidium (E(2)Rb) form.7. Ouabain, in the presence of magnesium ions, prevented the occlusion of rubidium ions.8. We have measured the amount of rubidium occluded by the enzyme as a function of rubidium concentration, and estimate that at saturating rubidium concentrations about three rubidium ions can be occluded per phosphorylation site (or per ouabain-binding site).9. We have found that the occluded-rubidium form of the enzyme can also be formed by allowing rubidium ions to catalyse the hydrolysis of phosphoenzyme generated by the addition of ATP to enzyme suspended in a high-sodium medium.10. The properties of the occluded-rubidium form of the enzyme, and of the two routes that can lead to its formation, suggest that an analagous occluded-potassium form plays a central role in the transport of potassium ions through the sodium-potassium pump. This hypothesis is supported by a detailed consideration of the probable magnitudes of the rate constants of the individual reactions making up the two routes.

In the presence of ATP and of Mg(2+), human erythrocyte membranes show a phosphatase activity towards p-nitrophenyl phosphate which is activated by low concentrations of Ca(2+). The effect of Ca(2+) is strongly enhanced if either K(+) or Na(+) is also present. Activation of the p-nitrophenyl phosphate phosphatase by Ca(2+) reaches a half-maximum at about 8mum-Ca(2+) and is apparent only when the ion has access to the inner surface of the cell membrane. Ca(2+)-dependent phosphatase activity can only be observed if ATP is at the inner surface of the cell membrane, and the presence of ATP seems to be absolutely necessary, since either its removal or its replacement by other nucleoside triphosphates abolishes the activating effect of Ca(2+). The properties of the (ATP+Ca(2+))-dependent phosphatase are very similar to those of the Ca(2+)-dependent ATPase (adenosine triphosphatase), also present in erythrocyte membranes, which probably is involved in Ca(2+) transport in erythrocytes. The similarities suggest that both activities may be properties of the same molecular system. This view is further supported by the fact that p-nitrophenyl phosphate inhibits to a similar extent Ca(2+)-dependent ATPase activity and ATP-dependent Ca(2+) extrusion from erythrocytes.