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J T Russell

Publications and source records attributed to J T Russell.

11 recordsLinked to original sources
PubMed1979

Optical probe responses on sarcoplasmic reticulum. Oxacarbocyanines.

Absorbance and fluorescence changes of oxacarbocyanine dyes during ATP-induced Ca2+ transport in rabbit sarcoplasmic reticulum were analyzed. The response of the probes is complex and contains contributions from the binding of Ca2+ and ATP to the membrane. In a medium of 0.12 M KCl and 5 mM MgCl2, the fluorescence of Di-O-C5(3) is decreased by Ca2+ or ATP with apparent dissociation constants of 0.2 and 5 micron, respectively. This suggests that oxacarbocyanines respond to binding of Ca2+ and ATP at the active site of Ca2+ transport ATPase. The effect of ATP is observed in the absence of divalent cations. Further changes in the fluorescence or absorbance of cyanine dyes occur at millimolar concentrations of Ca2+ or during ATP-induced Ca2+ uptake, which can be related to Ca2+ binding to low affinity, relatively nonspecific binding sites on the membrane, that can also bind K+ and Mg2+. The optical changes due to Ca2+ accumulation are most pronounced in media of 0.25 M sucrose and much reduced in 0.12 M KCl and 5 mM MgCl2, in accord with competition by K+ and Mg2+ for the low affinity Ca2+ binding sites. These effects must be taken into account in the evaluation of the magnitude and direction of membrane potential in sarcoplasmic reticulum vesicles during Ca2+ uptake and release.

Adenosine Triphosphate↗
PubMed1979

Optical probe responses on sarcoplasmic reticulum. Merocyanine and oxonol dyes.

The fluorescence and absorbance of merocyanine 540 in suspensions of skeletal muscle microsomes is altered by the binding of Ca2+ and other cations to the membrane. The order of effectiveness of various cations in causing this effect is La greater than Ca congruent to Mg greater than K. Competition between Ca2+, Mg2+, and K+ suggests the involvement of low affinity, relatively nonspecific cation binding sites in the process. Changes in the fluorescence and absorbance of merocyanine were also observed during ATP-dependent accumulation of calcium into sarcoplasmic reticulum. These changes are satisfactorily explained by the binding of accumulated calcium to binding sites on the interior of sarcoplasmic reticulum membrane. The small absorbance response of the oxonol dye bis[1,3-dibutylbarbituric acid-(5)]trimethinoxonol to Ca2+ and ATP is qualitatively similar to that of merocyanine 540 and can be readily explained by the same mechanism. We have found no clear evidence that any of the observed dye responses are due to changes in the diffusion potential across the sarcoplasmic reticulum membrane generated by an electrogenic transport mechanism. The possibility is considered that merocyanine and oxonol dyes respond to changes in electrostatic surface potential caused by the binding of cations.

Adenosine Triphosphate↗
PubMed1979

Trypsin liberates an arginine vasopressin-like peptide and neurophysin from a Mr 20,000 putative common precursor.

Although the hypothesis that vasopressin and its associated neurophysin are synthesized together in one macromolecular common precursor was put forward more than a decade ago, direct conformation of this hypothesis has been lacking. A [35S]cysteine-labeled putative precursor for vasopressin-related neurophysin (Mr 20,000, pI 6.1) has been isolated from the supraoptic nuclei of rats. This precursor was subjected to limited proteolysis with trypsin which produced a Mr 10,000 protein and peptide products. The former was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and its behavior in isoelectric focusing systems (pI 4.6-4.8). The tryptic peptides proved to be vasopressin-like because they: (i) were rich in cysteine, (ii) comigrated with vasopressin on gel filtration columns in 6 M guanidine HCl, (iii) bound to a neurophysin-Sepharose affinity column at pH 5.7, and (iv) were recognized by antibodies against vasopressin. These data on the Mr 20,000, pI 6.1 protein represent direct experimental evidence for a candidate for the common precursor of vasopressin and neurophysin. We propose that this common precursor be called "propressophysin."

Animals↗
PubMed1979

Optical probe responses on sarcoplasmic reticulum: oxacarbocyanines as probes of membrane potential.

The relationship between Ca2+ fluxes and the ion diffusion potential was analyzed on sarcoplasmic reticulum membranes using oxacarbocyanine dyes as optical probes for membrane potential. 3.3'-Diethyloxodicarbocyanine responds to ATP-induced Ca2+ uptake by isolated sarcoplasmic reticulum vesicles with a decrease in absorbance at 600 nm. The optical change is reversed during Ca2+ release from sarcoplasmic reticulum induced by KCl or by ADP and inorganic phosphate. The absorbance changes are largely attributable to the binding of accumulated Ca2+ to the membrane. There is no indication that sustained changes in membrane diffusion potential would accompany pump-mediated Ca2+ fluxes. A large change in the absorbance of 3,3'-diethyloxodicarbocyanine was observed on sarcoplasmic reticulum vesicles under the influence of membrane potential generated by valinomycin in the presence of a K+ gradient or by ionophore A23187 in the presence of a Ca2+ gradient. The maximum of the potential-dependent absorbance change is at 575--580 nm. The potentials generated by valinomycin or ionophore A23187 are short-lived due to the high permeability of sarcoplasmic reticulum membranes for cations and anions. There is no correlation between the direction and magnitude of the artifically imposed membrane potential and the rate of Ca2+ uptake or release by isolated sarcoplasmic reticulum vesicles.

Adenosine Triphosphate↗
PubMed1978

The influence of pH on the absorption spectrum of arsenazo III.

The absorption spectrum of arsenazo III in media containing K+, Mg2+ and Ca2+ is sharply influenced by pH in the range of 7.5--5.0. The effect of pH is particularly pronounced in the wavelength range 532--602 nm due to the large pH dependence of the dissociation constant of Mg-arsenazo III complex. Therefore absorption changes at these wavelengths during muscle contraction cannot be used as reliable indicators of free ionized Ca2+ concentration in the cell. The effect of pH is less pronounced, but still noticeable at the wavelength pairs 575--650 or 660--685 nm. Multiple layers of muscle cells grown on polystyrene coils permit measurement of absorption changes of arsenazo III, introduced into the cells, by equilibration with 0.5 mM arsenazo III under routine culture conditions. The absorbance changes recorded at 660--685 nm are probably related to changes in intracellular free Ca2+ concentration.

Animals↗
PubMed1978

ATP-induced release of vasopressin associated with phosphorylation of isolated bovine neurohypophyseal secretory granule membranes.

Secretory granules isolated from ox neurohypophyses released their content of vasopressin in the presence of ATP and Mg2+. A half maximal ATP concentration of 0.25 mM was found. Ca2+ was not necessary for the effect. High concentrations of ADP, AMP and ITP were shown to mimic the effect of ATP. Utilizing this effect of ATP combined with iodonitrotetrazolium treatment to make mitochondria heavier, a method is described to obtain granule "ghosts" in a purified form. They were shown to be phosphorylated when granules were incubated with [gamma-32P] ATP.

Adenosine Triphosphate↗
PubMed1977

Fusion of neurohypophyseal membranes in vitro.

Freeze cleaving electron microscopy has shown that fusion of isolated secretory vesicles from bovine neurohypophyses was induced by Ca2+ in micromolar concentrations. Mg2+ and Sr2+ were ineffective. Mg2+ inhibited Ca2+-induced fusion. In suspensions containing secretory vesicles as well as sheets of cell membrane, release of vasopressin parallel to intervesicular fusion and fusion of secretory vesicles with sheets of cell membrane was observed after exposure to Ca2+. Mg2+ and Sr2+ were ineffective in replacing Ca2+ as trigger for fusion or vasopressin release. Intervesicular fusion and exocytotic profiles were observed when isolated neurohypophyses or neurosecretosomes were exposed to cold.

Animals↗
PubMed1977

Isolation and purification of calcium-binding proteins from bovine neurohypophyses.

An acidic calcium-binding protein was isolated from the soluble fraction of the homogenate of ox neurohypophyses. The protein has a molecular weight of 35 000 and a subunit weight of 15 000. The purification procedure involved ammonium sulphate fractionation, DEAE-cellulose chromatography and gel filtration on Sephadex G-100 and Sephadex G-50. Conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated it to be a protein distinct from the S-100 protein and the soluble hormone-binding proteins (neurophysins) abundant in the neurohypophysis. This appears to be the only Ca2+-binding protein in the soluble part of the homogenate, with an apparent Kdiss for Ca2+ of 1.1 X 10(-5) M (at 22 degrees C) and a binding capacity of 2 mol of calcium per mol of protein. Two different Ca2+-binding proteins of molecular weights 16 500 and 68 000, respectively, were identified in the sodium-deoxycholate-soluble proteins from an ox neurohypophysial microsome fraction. One of them (the former) has been isolated in high purity by DEAE-cellulose chromatography and gel filtration on Sephadex G-200. This protein binds 4 mol of calcium per mol of protein with an apparent Kdiss of 1.0 X 10(-5) M (at 22 degrees C). The sodium-deoxycholate-insoluble proteins from the microsomal fraction also have Ca2+-binding components. The soluble Ca2+-binding protein has properties similar to and may be identical to Ca2+-binding proteins which have been isolated from bovine brain and have been demonstrated to be modulators of brain cyclic nucleotide phosphodiesterase and of actinomyosin ATPase. It also resembles Ca2+-binding proteins isolated from bovine adrenals and the electroplax from electrophorus electricus.

Animals↗
PubMed1976

Calcium and stimulus secretion coupling in the neurohypophysis. V. The effects of the Ca2+ ionophores A23187 and X537 A on vasopressin release and 45Ca2+ efflux; interactions with sodium and a verapamil analogue (D600).

Slices from ox neurohypophyses were incubated in a calcium-free medium with the ionophores A23187 or X537A. X537A (5 X 10(-5) mol/l) caused a marked release of vasopressin, neurophysin and protein to the medium. A23187 (2 X 10(-5) mol/l) did not cause any release by itself, but when Ca2+ was added to the medium in the presence of the ionophore, an increase in the release of vasopressin, neuorphysin and protein occurred. Release of lactate dehydrogenase and peptidase were not affected by the ionophores. The secretion caused by A23187 was abolished by D600 (a verapamil analogue) (2 X 10(-5) mol/l) whereas the effect of X537A was unchanged. The effects of X537A were strongly inhibited by removal of sodium from the medium. Re-addition of sodium to the medium caused a marked release. Gramicidin (10(-6) or 5 X 10(-5) mol/l) had no effect on secretion. Efflux of 45Ca2+ from pre-loaded slices was drastically reduced in a sodium-free medium. X537A caused an increase in the efflux rate of 45Ca2+ both in medium with a normal concentration of sodium and when slices had been incubated in a sodium-free medium. A23187 and X537A both released 45Ca2+ from a neurohypophyseal mitochondrial fraction. When sodium in a concentration of 20 mmol/l was added to this fraction, the Ca2+ accumulation was inhibited. This effect was reduced by inorganic phosphate up to a concentration of 2 mmol/l.

Animals↗
PubMed1975

Adenosine triphosphate dependent calcium uptake by subcellular fractions from bovine neurohypophyses.

Bovine neurohypophyses were fractionated by differential and density gradient ultracentrifugation and the Ca-2+ uptake and ATPase activities in the microsomal, mitochondrial and secretory granule fractions were studied. The microsomal and mitochondrial fractions accumulated Ca-2+ in the presence of ATP. The accumulation by the latter per mg protein was at least twice as large as by the former. This Ca2+ accumulation was accompanied by liberation of inorganic phosphate (Pi). In the presence of sodium azide (2 mM) Ca-2+ uptake and Pi liberation were inhibited in the mitochondrial, but not in the microsomal fraction. Further studies of the microsomal fractions revealed that the ATP-dependent Ca-2+ uptake and Pi liberation activities were temperature and pH-dependent and required Mg-2+. Both activities were stimulated by very low concentrations of Ca-2+ (1-10 muM) and were inhibited by EGTA (2 mM). N-ethylmaleimide (2 mM) inhibited both the Ca-2+ uptake and ATPase activities of the microsomal fraction. These results suggest the presence of a membrane ATPase that is stimulated by both Ca-2+ and Mg-2+. It is suggested that the observed Ca-2+ uptake activities are involved in maintaining a low axoplasmic free Ca-2+ concentration, thus playing an important role in the release mechanism of vasopressin by the neurosecretory terminals.

Adenosine Triphosphatases↗