Energy interconversion in sarcoplasmic reticulum vesicles in the presence of Ca2+ and Sr2+ gradients

Sarcoplasmic reticulum vesicles of rabbit skeletal muscle are able to accumulate Ca2+ or Sr2+ at the expense of ATP hydrolysis. Depending on the conditions used, vesicles loaded with Ca2+ can catalyze either an ATP in equilibrium Pi exchange or the synthesis of ATP from ADP and Pi. Both reactions are impaired in vesicles loaded with Sr2+. The Sr2+ concentration required for half-maximal ATPase activity increases from 2 microM to 60-70 microM when the Mg2+ concentration is raised from 0.5 to 50 mM. The enzyme is phosphorylated by ATP in the presence of Sr2+. The steady state level of phosphoenzyme varies depending on both the Sr2+ and Mg2+ concentrations in the medium. Phosphorylation of the enzyme by Pi is inhibited by both Ca2+ and Sr2+. In the presence of 2 and 20 mM Mg2+, half-maximal inhibition is attained in the presence of 4 and 8 microM Ca2+ or in the presence of 0.24 mM and more than 2 mM Sr2+, respectively. After the addition of Sr2+, the phosphoenzyme is cleaved with two different rate constants, 0.5-1.5 s-1 and 10-18 s-1. The fraction of phosphoenzyme cleaved at a slow rate is smaller the higher the Sr2+ concentration in the medium. Ca2+ inhibition of enzyme phosphorylation by Pi is overcome by the addition of ITP. This is not observed when Ca2+ is replaced by Sr2+.     

Ca2+ translocation and catalytic activity of the sarcoplasmic reticulum ATPase. Modulation by ATP, Ca2+, and Pi

The ratio between Ca2+ uptake and Ca(2+)-dependent ATP hydrolysis measured in sarcoplasmic reticulum vesicles of rabbit skeletal muscle was found to vary greatly depending on the concentrations of oxalate or Pi used. In the presence of 5 mM oxalate, 20 mM Pi, and 1 mM Pi, the ratios found were in the range of 1.4-2.3, 0.6-0.8, and 0.01-0.10, respectively. The rates of Ca2+ exchange and ATP synthesis were measured at the steady state by adding trace amounts of 45Ca and 32Pi, after the vesicles had been loaded with Ca2+. In the presence of 1 mM Pi, 10 mM MgCl2, and 0.2 mM CaCl2, the ratio between Ca2+ exchange and ATP synthesis varied from 9 to 14. This ratio approached two when Ca2+ in the medium was reduced to a very low level, or when in the presence of Ca2+, dimethyl sulfoxide was added to the assay medium, or when the Pi concentration was raised from 1 to 20 mM. A ratio of two was also measured when the steady state was attained using ITP instead of ATP. In all the conditions that led to a ratio close to two, there was an increase in the fraction of enzyme phosphorylated by Pi. It is proposed that the coupling between Ca2+ translocation and ATP hydrolysis or synthesis is modulated by the phosphorylation of the ATPase by Pi.     

Ca2+-dependent inhibitory effects of Na+ and K− on Ca2+ transport in sarcoplasmic reticulum vesicles

Effects of Na⁺ and K⁺ on Ca²⁺ transport by sarcoplasmic reticulum vesicles were studied in a medium containing high Mg²⁺ and ATP (2mM) and low Ca²⁺ (0.44μM) concentrations. Under these conditions, Na⁺ and K⁺ inhibit Ca²⁺ uptake. ATPase activity and membrane phosphorylation by ATP. Since the concentrations of ATP and Ca²⁺ used are consistent with relaxation in vivo, the results suggest that under physiological resting conditions the Ca²⁺ pump of the sarcoplasmic reticulum operates below its maximal capacity.     

Regulation of steady state level of phosphoenzyme and ATP synthesis in sarcoplasmic reticulum vesicles during reversal of the Ca2+ pump.

The role of the Ca2+ concentration gradient in ATP synthesis and membrane phosphorylation by Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. The Pi concentration required to attain 50% of the maximal membrane phosphorylation varies significantly in the pH range of 5.5 to 4.5, the optimal being at pH 6.0. In the pH range of 6.0 to 7.0, this concentration of Pi was 4- to 10-fold higher in empty vesicles than in vesicles loaded with calcium phosphate, i.e. having transmembrane Ca2+ concentration gradient. ATP, ADP, and Ca2+ inhibit the membrane phosphorylation by Pi, the inhibition being greater at pH 7.0 than at pH 6.0. The pH profile for ATP synthesis shows a higher optimum than for membrane phosphorylation. The optimum pH for synthesis, but not for phosphorylation depends on whether the vesicles were previously loaded with calcium phosphate or with calcium oxalate. Addition of Ca2+ to the assay medium inhibits the extent of membrane phosphorylation and the rate of ATP synthesis to different extents. Evidence is presented that the rate of membrane phosphorylation by Pi is higher than the rate by which the phosphoprotein transfers its pohsphate to ADP for the ATP synthesis.     

Transient state kinetic studies of phosphorylation by ATP and Pi of the calcium-dependent ATPase from sarcoplasmic reticulum.

The ATPase of the sarcoplasmic reticulum is phosphorylated by ATP in the presence of Ca2+. A rapid phosphorylation was observed when the enzyme was preincubated with Ca2+ prior to the addition of 0.1 or 1 mM ATP. The rate of phosphorylation was decreased when Ca2+ was omitted from the preincubation medium and added with ATP when the reaction was started. The rate of phosphorylation by ATP was further decreased when Pi was included in the preincubation medium without Ca2+. In this case, the enzyme was phosphorylated by Pi during the preincubation. When Ca2+ and ATP were added, a burst of phosphorylation by ATP was observed in the initial 16 ms. In the subsequent incubation intervals, the phosphorylation by ATP was synchronous with the hydrolysis of the phosphoenzyme formed by Pi. The rate of hydrolysis of the phosphoenzyme formed by Pi was measured when either the Pi concentration was decreased 10 fold, or when Ca2+, ATP or ATP plus Ca2+ was added to the medium. Upon the single addition of Ca2+, the time for half-maximal decay was in the range 500--1000 ms. In all other conditions it was in the range 70--90 ms.     

Heat production by skeletal muscles of rats and rabbits and utilization of glucose 6-phosphate as ATP regenerative system by rats and rabbits heart Ca2+-ATPase

This report is divided in two parts. The first section shows that vesicles derived from the sarcoplasmic reticulum of rats skeletal muscle can cleave ATP at a faster rate and produce more heat that the vesicles derived from rabbit skeletal muscle. In the second part, we compared the rates of Ca²⁺ transport and ATP hydrolysis by rats and rabbits heart sarcoplasmic reticulum. It is shown that the two vesicles preparations are able to use glucose 6-phosphate and hexokinase as an ATP regenerative system. The rates of Ca²⁺-uptake and ATP hydrolysis measured with glucose 6-phosphate and hexokinase is four to six times slower than that measured with phosphoenolpyruvate and pyruvate kinase as ATP regenerative system.     

Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes

DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene-operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing the transposition have to be introduced into the cell to be studied (microbial systems and applications are reviewed in ref. 1). However, all in vivo systems have a number of technical limitations. For instance, the transposase must be expressed in the target host, the transposon must be introduced into the host on a suicide vector, and the transposase usually is expressed in subsequent generations, resulting in potential genetic instability. A number of in vitro transposition systems (for Tn5, Tn7, Mu, Himar1, and Ty1) have been described, which bypass many limitations of in vivo systems. For this purpose, we have developed a technique for transposition that involves the formation in vitro of released Tn5 transposition complexes (TransposomesTM) followed by introduction of the complexes into the target cell of choice by electroporation. In this report, we show that this simple, robust technology can generate high-efficiency transposition in all tested bacterial species (Escherichia coli, Salmonella typhimurium, and Proteus vulgaris) We also isolated transposition events in the yeast Saccharomyces cerevisiae.     

Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.     

Proton transport in maize tonoplasts supported by fructose-1,6-bisphosphate cleavage. Pyrophosphate-dependent phosphofructokinase as a pyrophosphate-regenerating system

The energy derived from pyrophosphate (PPi) hydrolysis is used to pump protons across the tonoplast membrane, thus forming a proton gradient. In a plant's cytosol, the concentration of PPi varies between 10 and 800 microm, and the PPi concentration needed for one-half maximal activity of the maize (Zea mays) root tonoplast H+-pyrophosphatase is 30 microm. In this report, we show that the H+-pyrophosphatase of maize root vacuoles is able to hydrolyze PPi (Reaction 2) formed by Reaction 1, which is catalyzed by PPi-dependent phosphofructokinase (PFP): Fructose-1,6-bisphosphate (F1,6BP) + Pi <--> PPi +Fructose-6-phosphate (F6 P) (reaction 1) PPi --> 2 Pi (reaction 2) H+cyt --> H+vac (reaction 3) F1,6BP + H+cyt <--> H+vac + F6P + Pi (reaction 4) During the steady state, one-half of the inorganic phosphate released (Reaction 4) is ultimately derived from F1,6BP, whereas PFP continuously regenerates the pyrophosphate (PPi) hydrolyzed. A proton gradient (DeltapH) can be built up in tonoplast vesicles using PFP as a PPi-regenerating system. The Delta pH formed by the H+-pyrophosphatase can be dissipated by addition of 20 mm F6P, which drives Reaction 1 to the left and decreases the PPi available for the H+-pyrophosphatase. The maximal Delta pH attained by the pyrophosphatase coupled to the PFP reaction can be maintained by PFP activities far below those found in higher plants tissues.