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C Hidalgo

Publications and source records attributed to C Hidalgo.

At least 91 records · Page 5Linked to original sources
PubMed1982

Lipid-protein interactions in sarcoplasmic reticulum are not perturbed by ionophore A23187. An EPR and fluorescence study.

The divalent-cation ionophore A23187 at micromolar concentrations prevents the ATP-dependent accumulation of calcium into sarcoplasmic reticulum vesicles. Under the same conditions and throughout the temperature range of 4 degrees-37 degrees C, A23187 has no effect on either the rotational motion of the Ca2+ -ATPase in the membrane, or on the mobility of the lipid acyl chains. The steady-state fluorescence polarization of a polyene fluorescent probe incorporated into the membrane lipids was similarly unaffected by the ionophore. These results show conclusively that the mechanism of action of the ionophore does not involve significant of lipid-protein interactions.

Animals↗
PubMed1981

Immunological and biochemical properties of transverse tubule membranes isolated from rabbit skeletal muscle.

A new method for isolating transverse tubule membranes from rabbit skeletal muscle has been developed. This procedure has the advantage of being mild, fast, and producing with good yields a purified membrane fraction. The transverse tubule membranes are purified by a discontinuous sucrose density centrifugation after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. Immunofluorescence staining of cryostat sections of rabbit psoas muscle with purified goat antibodies directed against the purified membranes shows that the reacting antigens are distributed at the boundary of the A and I bands of the myofibrils where transverse tubules are localized in mammalian muscle. The purified antibodies showed no cross-reactivity with sarcoplasmic reticulum, nor did they show any fluorescence staining of the muscle plasma membrane, indicating that the isolated membranes indeed originate from the transverse tubules. The transverse tubule fraction has a characteristic protein composition distinguishable from that of sarcoplasmic reticulum, a much higher cholesterol content than that of the crude microsomes, plasma membrane, and sarcoplasmic reticulum, and a phospholipid content about twice as high as that of sarcoplasmic reticulum and plasma membrane. The purified transverse tubule membrane has a distinct phospholipid composition with high contents of sphingomyelin and phosphatidylserine. A Mg2+-activated ATPase characteristic of the transverse tubule fraction undergoes a 20-30-fold increase in specific activity during purification. The levels of Ca2+-ATPase activity present in the purified transverse tubule fraction remain comparable to those of sarcoplasmic reticulum even after extensive removal of the latter.

Adenosine Triphosphatases↗
PubMed1980

Highly purified sarcoplasmic reticulum vesicles are devoid of Ca2+-independent ('basal') ATPase activity.

On solubilization with Triton X-100 of sarcoplasmic reticulum vesicles isolated by differential centrifugation, the Ca2+-ATPase is selectively extracted while approximately half of the initial Mg2+-, or 'basal', ATPase remain in the Triton X-100 insoluble residue. The insoluble fraction, which does not contain the 100 000 dalton polypeptide of the Ca2+-ATPase, contains high levels of cytochrome c oxidase. Furthermore, its Mg2+-ATPase activity is inhibited by specific inhibitors of mitochondrial ATPase, indicating that the 'basal' ATPase separated from the Ca2+-ATPase by detergent extraction originates from mitochondrial contaminants. To minimize mitochondrial contamination, sarcoplasmic reticulum vesicles were fractionated by sedimentation in discontinuous sucrose density gradients into four fractions: heavy, intermediate and light, comprising among them 90-95% of the initial sarcoplasmic reticulum protein, and a very light fraction, which contains high levels of Mg2+-ATPase. Only the heavy, intermediate and light fractions originate from sarcoplasmic reticulum; the very light fraction is of surface membrane origin. Each fraction of sarcoplasmic reticulum origin was incubated with calcium phosphate in the presence of ATP and the loaded fractions were separated from the unloaded fractions by sedimentation in discontinuous sucrose density gradients. It was found that vesicles from the intermediate fraction had, after loading, minimal amounts of mitochondrial and surface membrane contamination, and displayed little or no Ca2+-independent basal ATPase activity. This shows conclusively that the basal ATPase is not an intrinsic enzymatic activity of the sarcoplasmic reticulum membrane, but probably originates from variable amounts of mitochondrial and surface membrane contamination in sarcoplasmic reticulum preparations isolated by conventional procedures.

Adenosine Triphosphatases↗
PubMed1978

Effect of the lipid environment on protein motion and enzymatic activity of sarcoplasmic reticulum calcium ATPase.

In order to investigate the roles of the physical states of phospholipid and protein in the enzymatic behavior of the Ca2+ -ATPase from sarcoplasmic reticulum, we have modified the lipid phase of the enzyme, observed the effects on the enzymatic activity at low temperatures, and correlated these effects with spectroscopic measurements of the rotational motions of both the lipid and protein components. Replacement of the native lipids with dipalmitoyl phosphatidylcholine inhibits ATPase activity and decreases both lipid fluidity, as monitored by EPR spectroscopy on a stearic acid spin label, and protein rotational mobility, as monitored by saturation transfer EPR spectroscopy on the covalently spin-labeled enzyme. Solubilization of the lipid-replaced enzyme with Triton X-100 reverses all three of these effects. Ten millimolar CaCl2 added either to the enzyme associated with the endogenous lipids or to the Triton X-100 soulbilized enzyme inhibits both ATPase activity and protein rotational mobility but has no detectable effect on the lipid mobility. These results are consistent with the proposal that both lipid fluidity and protein rotational mobility are essential for enzymatic activity.

Animals↗
PubMed1978

Rotational motion of the sarcoplasmic reticulum Ca2+-ATPase.

Using saturation transfer electron paramagnetic resonance, we have detected the rotational motion of a spin label rigidly attached to the sarcoplasmic reticulum Ca2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3). At 4 degrees C, the spectrum indicates an effective rotational correlation time of 60 microsec, determined by comparison with reference spectra obtained from theoretical calculations and from experiments on model systems. This motion appears to correspond to rotation of the enzyme with respect to the membrane, because the motion persists when the membrane fragments are immobilized by sedimentation and the motion stops when the polypeptide chains, but not the membrane vesicles, are crosslinked by glutaraldehyde. The rotational mobility of the enzyme increases with increasing temperature, and this increase becomes more gradual when the temperature exceeds 20 degrees C; the same kind of temperature dependence has been observed previously for lipid fluidity and enzymatic activity.

Animals↗
PubMed1977

Induction and general properties of beta-galactosidase and beta-galactoside permease in Pseudomonas BAL-31.

The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside (ONPG) by BAL-31, a marine Pseudomonas that acts as a host for bacteriophage PM2, was studied with intact cells and with cell-free extracts. A transport system for ONPG in whole cells and a beta-galactosidase activity in extracts were evident for cells grown on lactose minimal medium. It was found that the addition of isopropylthio-beta-D-galactopyranoside (IPTG) to cells growing in rich medium induced an ONPG hydrolytic activity detectable in cell extracts but cryptic in whole cells. The existence of a transport system for IPTG, which remained cryptic for ONPG, became apparent from studies of the rates of induction of beta-galactosidase as a function of cell mass at different concentrations of IPTG. The main properties of beta-galactosidase and the lactose transport system of BAL-31 were studied in terms of how they were affected by pH, temperature, or by the presence of several sugars. IPTG competitively inhibits the hydrolysis of ONPG by cell extracts. In cells pregrown on lactose, IPTG slightly inhibits the transport of ONPG. Glucose, and with less efficiency lactose, also inhibits the hydrolysis of ONPG in cell extracts. The growth of cells on lactose minimal medium was inhibited by the addition of IPTG. A mechanism for this inhibition and for the inhibition of ONPG transport by IPTG is discussed.

Carbohydrate Metabolism↗
PubMed1976

Role of phospholipids in the calcium-dependent ATPase of the sarcoplasmic reticulum. Enzymatic and ESR studies with phospholipid-replaced membranes.

Three types of partially purified ATPase enzymes having different phospholipid contents and compositions have been prepared: (a) an enzyme whose phospholipid moiety has been replaced predominantly by dioleoyl lecithin (DOL-enzyme), with about the same phospholipid content as the original sarcoplasmic reticulum, (b) dipalmitoyl lecithin-replaced enzyme whose phospholipid content is 30% of that of DOL-enzyme (DPL-enzyme), and (c) a partially delipidated enzyme with about the same phospholipid content as DPL-enzyme but with the original sarcoplasmic reticulum phospholipid composition (del-enzyme). The temperature dependence of Ca2+-activated ATPase activity of these preparations showed clearcut differences; with DOL-enzyme there was no appreciable break in the Arrhenius plot in the 3-40 degrees range; DPL-enzyme showed a break at 29 degrees, and del-enzyme and sarcoplasmic reticulum one at 18 degrees. Transition temperatures obtained from ESR studies with the use of spin-labeled stearic acid incorporated into the membranes agreed with those derived from ATPase assays. Thermo-dynamic analysis of the ATP hydrolysis rates shows that DPL-enzyme has considerably larger values of activation enthalpy and activation entropy below the transition temperature (29 degrees) than those of the other preparations, while all enzyme preparations show similar free energies of activation. The ESR data show that below their transition temperatures DPL-enzyme, and to a lesser degree del-enzyme, have a strongly restricted motion of their phospholipid molecules as compared with either DOL-enzyme or sarcoplasmic reticulum. Studies on the formation and decomposition of phosphoenzyme have been carried out with the three types of ATPase preparations. At 0 degrees, the rate of inorganic phosphate liberation is 8 times lower in DPL-enzyme than in del-enzyme with little difference in the steady state level of phosphoenzyme. In DOL-enzyme, the level of phosphoenzyme and the rate of inorganic phosphate liberation are 1.8 and 3.5 times higher than the corresponding values obtained with del-enzyme. Addition of ADP to the phosphorylated intermediate of DPL-enzyme induces a fast reversal of the phosphorylation reaction. These results indicate that the physical state of the phospholipid molecules associated with the enzyme affects the decomposition of phosphoenzyme, with little effect on the phosphorylation reaction and its reversal.

Adenosine Triphosphatases↗
PubMed1972

Methylation pattern of lambda deoxyribonucleic acid.

Deoxyribonucleic acid (DNA) extracted from phage lambda grown on Escherichia coli K-12 strain W4032 had 113 +/- 10 5-methylcytosine residues and 215 +/- 20 6-methyl adenine residues per genome, as determined by three independent methods. These methylated nucleotides were distributed equally among the two strands of lambda DNA. Shearing of double-stranded DNA to half-length fragments revealed a slight deficiency of 5-methyl cytosine in the 55% guanine plus cytosine half. Shearing the DNA to fragments of smaller length showed that the distribution of methylated nucleotides along the double helix was uniform with the exception of an undermethylated fragment arising from the center of the lambda DNA molecule. The implication of these results for the function of methylated nucleotides in the lambda DNA molecule is discussed.

Adenine↗
PubMed1970

Temperature dependence of non-electrolyte and sodium permeability in giant axon of squid.

1. The efflux of [(14)C]urea was measured in micro-injected axons at 18 degrees C. A permeability constant for urea of (0.55 +/- 0.18) x 10(-6) cm/sec was calculated from these experiments.2. The influxes of urea, thiourea, ethylene glycol, urethane and toluene were measured in perfused axons at 18 +/- 1 degrees C. The permeability constants obtained from these determinations increased in the order listed, from (0.76 +/- 0.19) x 10(-6) cm/sec for urea to 0.80 x 10(-4) cm/sec for toluene.3. The influxes of tritiated water and sodium ions at 18 degrees C were measured in perfused axons. An average permeability of (0.78 +/- 0.22) x 10(-4) cm/sec for titriated water and an average influx of 23 +/- 6 p-mole/cm(2) sec for sodium were obtained.4. Lowering the temperature of the external sea-water bathing the axon from 18 to 5 degrees C produced a decrease of 12% in the permeability of toluene, 30% for tritiated water and urethane, 55% for ethylene glycol and urea and 60% for thiourea. There was a 50% reduction in the influx of sodium for this same temperature change.5. The results obtained with the effect of temperature on permeabilities suggest that the axonal membrane has a non-homogeneous composition. A model based on the assumption of structured aqueous channels in the membrane is postulated.

Axons↗
PubMed1970

Effect of stimulation and hyperpolarization on non-electrolyte and sodium permeability in perfused axons of squid.

1. The permeability for micro-injected [(3)H]ethylene glycol was measured in resting state and during stimulation at 100/sec in squid giant axons. No detectable changes during electrical activity were observed.2. The influxes of urethane, tritiated water, ethylene glycol, urea and sodium were measured in internally perfused squid axons. Ethylene glycol and urea influxes were determined simultaneously with sodium influxes. The electrical stimulation of the fibre produced an increase in the influx of sodium but did not alter the influxes of the non-electrolytes listed above.3. Experiments were done with the combined voltage clamp-perfusion technique. The influxes of ethylene glycol and sodium were simultaneously measured in resting state and during maximum sodium current under stimulation at 10/sec. The influx of sodium increased in these conditions but the influx of ethylene glycol remained constant. In some experiments, the fibre was hyperpolarized to 10 or 20 mV, above the resting potential and the influxes of ethylene glycol and sodium were measured. The sodium influx decreased to 60% at 20 mV above the resting potential whereas the influx of ethylene glycol remained constant.4. These results indicate that in the giant axons of the squid Dosidicus gigas, sodium and non-electrolytes fluxes are not coupled.

Animals↗