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Foi estudado em pastagem diferida de Brachiaria decumbens, o efeito de quatro tipos de suplementos na terminação de bovinos Nelore no período da seca. Utilizaram-se 64 animais da raça Nelore, machos castrados, com idade aproximada de 34 meses e peso corporal (PC) inicial variando de 360 a 380 kg, distribuídos inteiramente ao acaso em quatro tratamentos. Os tratamentos foram quatro formas de suplementação da pastagem: pasto diferido de Brachiaria decumbens + sal proteico energético mineral (SuPEM), considerado controle; pasto diferido de Brachiaria decumbens + ureia + farelo de algodão (28% PB) + milho grão moído (SuMi); pasto diferido de Brachiaria decumbens + ureia + farelo de algodão (28% PB) + polpa cítrica (SuPC); pasto diferido de Brachiaria decumbens + ureia + farelo de algodão (28% PB) + casca de soja (SuCS). A pastagem foi diferida por 170 dias e apresentou 3.482 kg MS/ha de forragem, permitindo 1,56 UA/ha (ingestão de MS de 2,25% do PC e 50% de eficiência de pastejo). Os animais receberam o suplemento ad libitum no tratamento SuPEM e em % PC nos demais tratamentos, de julho a outubro. Os animais foram abatidos com PC mínimo de 457 kg. Foram determinadas as variáveis: peso final (PF), ganho de peso no período (GPP), ganho de peso diário (GPD), peso da carcaça quente (PCQ) e rendimento da carcaça quente (RCQ). Na variável PF, o tipo de suplemento dos tratamentos SuMi, SuPC e SuCS, permitiu maior aporte de nutrientes, e assim apresentaram melhor desempenho (P<0,05) comparados ao tratamento SuPEM (média de 478,68 vs 412,62 kg). Esse mesmo efeito foi observado nos outros parâmetros estudados. Quando foi analisado o GPP e GPD, o tratamento SuCS foi superior (P<0,05) aos tratamentos SuMi e SuPC, em razão do aumento na oferta de concentrado, e o tratamento SuPEM foi inferior aos demais. O PCQ e RCQ foi maior no tratamento SuCS em decorrência do melhor desempenho, com 260,05 kg e 53,92%, respectivamente. A suplementação de bovinos no período da seca...(AU)

This study evaluated the effect of four types of supplement on the finishing of Nellore cattle on deferred Brachiaria decumbens pasture during the dry season. Sixty-four castrated Nellore males with an age of approximately 34 months and initial body weight (BW) ranging from 360 to 380 kg were divided into 16 animals per treatment in a completely randomized design. The treatments consisted of four types of pasture supplement: deferred Brachiaria decumbens pasture + energy protein mineral salt (SuEPM) used as control; deferred Brachiaria decumbens pasture + urea + cottonseed meal (28% CP) + ground corn grain (SuCo); deferred Brachiaria decumbens pasture + urea + cottonseed meal (28% CP) + citrus pulp (SuCPu); deferred Brachiaria decumbens pasture + urea + cottonseed meal (28% CP) + soy hull (SuSH). The pasture was deferred for 170 days and provided 3,482 kg DM/ha of forage, permitting a stocking rate of 1.56 AU/ha (DM intake of 2.25% BW and 50% pasture efficiency). The animals received the supplement ad libitum in the SuEPM treatment and as % BW in the other treatments from July to October. The animals were slaughtered at a minimum BW of 457 kg. The following variables were evaluated: final weight, weight gain during the period (WG), average daily gain (ADG), hot carcass weight (HCW), and hot carcass yield (HCY). With respect to final weight, the supplement in the SuCo, SuCPu and SuSH treatments permitted a greater supply of nutrients and the animals therefore exhibited better performance (P<0.05) compared to the SuEPM treatment (mean of 478.68 vs 412.62 kg). The same effect was observed for the other parameters studied. Analysis of WG and ADG showed that SuSH was superior to the SuCo and SuCPu treatments (P<0.05) due to the increased offer of concentrate and SuEPM was inferior to the other treatments. Higher HCW (260.05 kg) and HCY (53.92%) were obtained with treatment SuSH as a result of greater performance. Supplementation of cattle during the dry ...(AU)

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Foi estudado em pastagem diferida de Brachiaria decumbens, o efeito de quatro tipos de suplementos na terminação de bovinos Nelore no período da seca. Utilizaram-se 64 animais da raça Nelore, machos castrados, com idade aproximada de 34 meses e peso corporal (PC) inicial variando de 360 a 380 kg, distribuídos inteiramente ao acaso em quatro tratamentos. Os tratamentos foram quatro formas de suplementação da pastagem: pasto diferido de Brachiaria decumbens + sal proteico energético mineral (SuPEM), considerado controle; pasto diferido de Brachiaria decumbens + ureia + farelo de algodão (28% PB) + milho grão moído (SuMi); pasto diferido de Brachiaria decumbens + ureia + farelo de algodão (28% PB) + polpa cítrica (SuPC); pasto diferido de Brachiaria decumbens + ureia + farelo de algodão (28% PB) + casca de soja (SuCS). A pastagem foi diferida por 170 dias e apresentou 3.482 kg MS/ha de forragem, permitindo 1,56 UA/ha (ingestão de MS de 2,25% do PC e 50% de eficiência de pastejo). Os animais receberam o suplemento ad libitum no tratamento SuPEM e em % PC nos demais tratamentos, de julho a outubro. Os animais foram abatidos com PC mínimo de 457 kg. Foram determinadas as variáveis: peso final (PF), ganho de peso no período (GPP), ganho de peso diário (GPD), peso da carcaça quente (PCQ) e rendimento da carcaça quente (RCQ). Na variável PF, o tipo de suplemento dos tratamentos SuMi, SuPC e SuCS, permitiu maior aporte de nutrientes, e assim apresentaram melhor desempenho (P<0,05) comparados ao tratamento SuPEM (média de 478,68 vs 412,62 kg). Esse mesmo efeito foi observado nos outros parâmetros estudados. Quando foi analisado o GPP e GPD, o tratamento SuCS foi superior (P<0,05) aos tratamentos SuMi e SuPC, em razão do aumento na oferta de concentrado, e o tratamento SuPEM foi inferior aos demais. O PCQ e RCQ foi maior no tratamento SuCS em decorrência do melhor desempenho, com 260,05 kg e 53,92%, respectivamente. A suplementação de bovinos no período da seca...

This study evaluated the effect of four types of supplement on the finishing of Nellore cattle on deferred Brachiaria decumbens pasture during the dry season. Sixty-four castrated Nellore males with an age of approximately 34 months and initial body weight (BW) ranging from 360 to 380 kg were divided into 16 animals per treatment in a completely randomized design. The treatments consisted of four types of pasture supplement: deferred Brachiaria decumbens pasture + energy protein mineral salt (SuEPM) used as control; deferred Brachiaria decumbens pasture + urea + cottonseed meal (28% CP) + ground corn grain (SuCo); deferred Brachiaria decumbens pasture + urea + cottonseed meal (28% CP) + citrus pulp (SuCPu); deferred Brachiaria decumbens pasture + urea + cottonseed meal (28% CP) + soy hull (SuSH). The pasture was deferred for 170 days and provided 3,482 kg DM/ha of forage, permitting a stocking rate of 1.56 AU/ha (DM intake of 2.25% BW and 50% pasture efficiency). The animals received the supplement ad libitum in the SuEPM treatment and as % BW in the other treatments from July to October. The animals were slaughtered at a minimum BW of 457 kg. The following variables were evaluated: final weight, weight gain during the period (WG), average daily gain (ADG), hot carcass weight (HCW), and hot carcass yield (HCY). With respect to final weight, the supplement in the SuCo, SuCPu and SuSH treatments permitted a greater supply of nutrients and the animals therefore exhibited better performance (P<0.05) compared to the SuEPM treatment (mean of 478.68 vs 412.62 kg). The same effect was observed for the other parameters studied. Analysis of WG and ADG showed that SuSH was superior to the SuCo and SuCPu treatments (P<0.05) due to the increased offer of concentrate and SuEPM was inferior to the other treatments. Higher HCW (260.05 kg) and HCY (53.92%) were obtained with treatment SuSH as a result of greater performance. Supplementation of cattle during the dry ...

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In this study we use a theoretical approach to study the volumetric response of goldfish hepatocytes challenged by osmotic gradients and compared it with that of hepatocytes from another teleost (the trout) and a mammal (the rat). Particular focus was given to the multiple non-linear interactions of transport systems enabling hypotonically challenged cells to trigger a compensatory response known as volume regulatory decrease or RVD. For this purpose we employed a mathematical model which describes the rates of change of the intracellular concentrations of main diffusible ions, of the cell volume, and of the membrane potential. The model was fitted to experimental data on the kinetics of volume change of hepatocytes challenged by anisotonic media. In trout and rat hepatocytes, experimental results had shown that hypotonic cell swelling was followed by RVD, whereas goldfish cells swelled with no concomitant RVD (M.V. Espelt et al., 2003, J. Exp. Biol. 206, 513-522). A comparison between data predicted by the model and that obtained experimentally suggests that in trout and rat hepatocytes hypotonicity activates a sensor element and this, in turn, activates an otherwise silent efflux of KCl - whose kinetics could be successfully predicted - thereby leading to volume down-regulation. In contrast, with regard to the absence of RVD in goldfish hepatocytes the model proposed suggests that either a sensor element triggering RVD is absent or that the effector mechanism (the loss of KCl) remains inactive under the conditions employed. In line with this, we recently found that extracellular nucleotides may be required to induce RVD in these cells, indicating that our model could indeed lead to useful predictions.

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The synthesis of 6,6-dibromo-3alpha-(diphenylphosphate)oxymethyl-2,2-dimethyl penam sulfone (3a), 6alpha-chloro-3alpha-(diphenylphosphate)oxymethyl-2,2-dimethyl penam sulfone (3b), benzyl 6alpha-(diphenyl-phosphate)oxypenicillanate sulfone (4) and 6,6-dibromo-3alpha-(methylphosphate)carbonyl-2,2-dimethylpenam sulfone (12) are reported. When tested as inhibitors of human leukocyte elastase, the compound 4 proved to be the most active.

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P-ATPases are characterized by the formation of acid-stable phosphorylated intermediates (EP) during their reaction cycle. We have developed a microscale method to determine EP that involves the phosphorylation of the enzyme using [gamma-(32)P]ATP and precipitation with TCA; separation of the sample by SDS-PAGE, and measurement of the enzyme protein and (32)P-labeled EP by digital analysis of both the stained gel and its autoradiogram, respectively. The principal advantages of this method over typical procedures (filtration and centrifugation) are the low amount of enzyme required and the substantial decrease in the blank values and data scattering produced by unspecific phosphorylation and nonquantitative recovering of the enzyme. Application of this new method to a purified preparation of the plasma membrane calcium ATPase (PMCA) results in overcoming the difficulties of measuring EP at high ATP concentrations. A biphasic behavior of the substrate curve for EP was observed when the study was extended to ATP levels within the physiological range. Since, in principle, the method does not require the use of highly purified preparations, it could be helpful for the study of phosphorylated intermediates especially under conditions in which small amounts of protein are available, e.g., mutated variants of P-ATPases.

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In the Albers-Post model, occlusion of K(+) in the E(2) conformer of the enzyme (E) is an obligatory step of Na(+)/K(+)-ATPase reaction. If this were so the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) should be equal to the rate constant for deocclusion. We tested this prediction in a partially purified Na(+)/K(+)-ATPase from pig kidney by means of rapid filtration to measure the occlusion using the K(+) congener Rb(+). Assuming that always two Rb(+) are occluded per enzyme, the steady-state levels of occluded forms and the kinetics of deocclusion were adequately described by the Albers-Post model over a very wide range of [ATP] and [Rb(+)]. The same happened with the kinetics of ATP hydrolysis. However, the value of the parameters that gave best fit differed from those for occlusion in such a way that the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) became much larger than the rate constant for deocclusion when [Rb(+)] <10 mM. This points to the presence of an extra ATP hydrolysis that is not Na(+)-ATPase activity and that does not involve occlusion. A possible way of explaining this is to posit that the binding of a single Rb(+) increases ATP hydrolysis without occlusion.

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The present paper describes a quenching-and-washing chamber (QWC) to be used with a rapid-mixing apparatus (RMA) for the study of processes in the millisecond time scale. The QWC enables fast, nondestructive quenching by cooling and dilution of reactants in particulate systems that can be trapped on a filter. The reaction mixture (e.g., at 25 degrees C) is injected from the RMA into the QWC where it is immediately mixed with a stream of ice-cold solution flowing at a rate of 15-40 ml s-1. Quenching requires that the process studied is slowed considerably by cooling to 0-2 degrees C and/or by removal of reactants by dilution. The equipment was characterized through a study of the tight binding (occlusion) of 86Rb+ to purified, membrane-bound Na+/K+-ATPase. Millipore filters of 0.22-0.80 microm pore size trapped close to 100% of the enzyme protein. Enzyme with occluded 86Rb+ was formed in the RMA under conditions where the rate constant for release of Rb+ at 25 degrees C is up to 25 s-1 and then injected into the QWC. The high off-rate constant is due to the presence of 2.5 mM ATP, which accelerates release of Rb+. The recovery of occluded 86Rb+ on the filter was at least 90%, indicating that both cooling of the reactants and dilution of ATP are fast enough to stop the reaction. The quenching time was 3-4 ms.

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Extracellular [gamma-32P]ATP added to a suspension of goldfish hepatocytes can be hydrolyzed to ADP plus gamma-32Pi due to the presence of an ecto-ATPase located in the plasma membrane. Ecto-ATPase activity was a hyperbolic function of ATP concentration ([ATP]), with apparent maximal activity of 8.3 +/- 0.4 nmol P(i).(10(6) cells)-1.min-1 and substrate concentration at which a half-maximal hydrolysis rate is obtained of 667 +/- 123 microM. Ecto-ATPase activity was inhibited 70% by suramin but was insensitive to inhibitors of transport ATPases. Addition of 5 microM [alpha-32P]ATP to the hepatocyte suspension induced the extracellular release of alpha-32P(i) [8.2 pmol.(10(6) cells)-1.min-1] and adenosine, suggesting the presence of other ectonucleotidase(s). Exposure of cell suspensions to 5 microM [2,8-3H]ATP resulted in uptake of [2,8-3H]adenosine at 7.9 pmol.(10(6) cells)-1.min-1. Addition of low micromolar [ATP] strongly increased cytosolic free Ca2+ (Ca2+i). This effect could be partially mimicked by adenosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable analog of ATP. The blockage of both glycolysis and oxidative phosphorylation led to a sixfold increase of Ca2+i and an 80% decrease of intracellular ATP, but ecto-ATPase activity was insensitive to these metabolic changes. Ecto-ATPase activity represents the first step leading to the complete hydrolysis of extracellular ATP, which allows 1) termination of the action of ATP on specific purinoceptors and 2) the resulting adenosine to be taken up by the cells.

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According to the Albers-Post model the hydrolysis of ATP catalyzed by the Na+/K(+)-ATPase requires the sequential formation of at least two conformers of a phosphoenzyme (E1P and E2P), followed by the K(+)-stimulated hydrolysis of E2P. In this paper we show that this model is a particular case of a more general class of models in all of which the ratio between ATPase activity (v) and total phosphoenzyme level (EP) in steady state is determined solely by the rate constants of interconversion between phosphoconformers and of dephosphorylation. Since these are thought to be unaffected by ATP, the substrate curves for ATPase activity and EP should be identical in shape so that the ratio v/EP ought to be independent of the concentration of ATP. We tested this prediction by parallel measurements of v and EP as a function of [ATP] in the absence or presence of non-limiting concentrations of K+, Rb+ or NH+4. In the absence of K+ or its congeners, both curves followed Michaelis-Menten kinetics, with almost identical Km values (0.16 microM) so that v/EP remained independent of [ATP]. In the presence of either K+, Rb+ or NH+4, v and EP increased with [ATP] along the sum of two Michaelis-Menten equations. The biphasic response of v is well known but, to the best of our knowledge, our results are the first demonstration that the response of EP to [ATP] is also biphasic. Under these conditions, the ratio v/EP increased with [ATP] from 19.8 to 40.1 s-1 along a hyperbola that was half-maximal at 9.5 microM. To preserve the validity of the current model it seems necessary to assume that ATP acts on the E1P <--> E2P transition and/or on the rate of hydrolysis of E2P. The latter possibility was ruled out. We also found that to fit the Albers-Post model to our data, the rate constant for K+ deocclussion from E2 has to be about 10-times higher than that reported from measurements of partial reactions. The results indicate that the Albers-Post model quantitatively predicts the experimental behavior of the Na(+)-ATPase activity but is unable to do this for the Na+/K(+)-ATPase activity, unless additional and yet unproved hypothesis are included.

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The maximum rate of phosphorylation (rm) of a highly purified Na,K-ATPase from red outer medulla of pig kidney was measured at 25 degrees C as a function of ATP concentration in media with Mg2+, Na+, and no K+. When rm was plotted as a function of the concentration of ATP a biphasic response was observed with a hyperbolic component of high affinity (Km = 15.7 +/- 2.6 microM) and low velocity ((rm)max = 460 +/- 40 nmol of Pi/(mg of protein.s)) plus a parabolic component which showed no saturation up to 1000 microM ATP, concentration at which rm was 1768.1 +/- 429.6 nmol Pi/(mg protein.s) (mean +/- S.E.; n = 3). This low affinity effect of ATP on the rate of phosphorylation disappeared when the Na,K-ATPase underwent turnover in medium without K+ suggesting that, like superphosphorylation (Peluffo, R. D., Garrahan, P. J., and Rega, A. F. (1992) J. Biol. Chem. 267, 6596-6601), it required the enzyme to be at rest. This property of the Na,K-ATPase was not predicted by the Albers-Post reaction scheme. The observed behavior of the enzyme could be simulated by a scheme that involves a resting enzyme (Er) functionally different from E1 or E2, which is able to bind three molecules of ATP, one with high and two with low affinity, and that after phosphorylation is converted into the phosphointermediates that are generally considered to participate in the reaction cycle described by Albers and Post.

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Using the K+ congener Tl+ and rapid mixing combined with special quench techniques, we have investigated (at 20 degrees C) what is usually assumed to be the enzymatic correlate of active transport of K+ by Na,K-ATPase: the Tl(+)-catalyzed dephosphorylation of the K(+)-sensitive phosphointermediate(s), EP, and the resulting occlusion of Tl+ in the enzyme protein. We measured [EP] and [occluded Tl] as a function of time in phosphorylation, as well as dephosphorylation experiments with the following results. First, we found that with 150 mM Na+ and 600 mM Na(+)--NO3- was the anion--[Tl+] = 0.1-1 mM was without influence on the phosphorylation rate. Tl(+)-catalyzed dephosphorylation and Tl+ occlusion appeared to be simultaneous, and the stoichiometry was always 2 Tl+ occluded/EP dephosphorylated. Second, we tried computer simulations of the transient kinetic experiments, using an Albers-Post-type reaction scheme. This produced satisfactory curve-fits only in the case of 150 mM Na+, and although we could arrange that calculated [EP]steady-state was equal to that measured, the calculated steady-state Na,Tl-ATPase hydrolysis rates were always two to four times the rates measured directly. Third, we propose, as one (possibly of several) solution to these discrepancies between model and data, an expanded kinetic model consisting of an initiation reaction sequence followed by a propagation (or steady-state) reaction cycle. In this alternative model the first turnover of the enzyme is kinetically different from subsequent reaction cycles, and this allowed us to obtain both satisfactory curve-fits and accordance between calculated and measured values of hydrolysis-rate and [EP]steady-state.

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