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J T Russell

Publications and source records attributed to J T Russell.

At least 73 records · Page 4Linked to original sources
PubMed1982

Proopiocortin-converting enzyme activity in bovine neurosecretory granules.

Neurosecretory granules (NSGs) from neural lobes of bovine pituitary glands were isolated in a highly purified form by metrizamide-sucrose gradient centrifugation. The purified NSGs were lysed and centrifuged, and the supernatants were further fractionated by gel filtration on Sephadex G-75. Proopiocortin-converting enzyme activity was assayed by incubation of [3H]arginine- or [3H]phenylalanine-labeled toad proopiocortin with NSG supernatant fractions. The processed products were identified by immunoprecipitation with ACTH and beta-endorphin antisera, followed by acid-urea gel electrophoresis. The optimum pH for the enzyme-mediated conversion was around pH 5.0. Conversion of toad proopiocortin by NSG converting enzyme activity was inhibited by leupeptin, antipain, p-chloromercuribenzoate, and pepstatin A, but not by diisopropyl fluorophosphate, EDTA, or N-alpha-p-tosyl-L-lysine-chloromethyl ketone HCl. The results suggest that the proopiocortin-converting enzyme activity in bovine neurosecretory granules is due to an acid-thiol protease which may contain secondary hydrophobic binding sites that are involved in substrate recognition.

Adrenocorticotropic Hormone↗
PubMed1981

Measurement of delta pH and membrane potential in isolated neurosecretory vesicles from bovine neurohypophyses.

The steady state concentration ratios of [3H]methylamine and [14C]SCN- were measured to determine the pH gradient and electrical potential, respectively, across the membrane of purified, intact, neurosecretory vesicles from bovine posterior pituitary. The intravesicular pH was 5.8 when the medium pH was 7. Varying the external pH had only a small effect on the intravesicular pH and resulted in a change in the pH gradient. Extrapolation to zero pH gradient yielded a value of 5.5 for the intravesicular pH. When the medium pH was 7, the pH gradient collapsed in the presence of ammonium sulfate or the combination of nigericin and K+. Measurement of the thiocyanate distribution across the vesicle membrane suggested the existence of an inside negative membrane potential. Mg2+ ATP caused a marked shift in the potential to +50 mV (inside positive) without altering the intravesicular pH. This shift was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The possible roles of the large pH gradient and membrane potential in the storage of peptide hormones and their secretion is discussed.

Adenosine Triphosphate↗
PubMed1981

Time course of appearance and release of [35S]cysteine labelled neurophysins and peptides in the neurohypophysis.

[35S]cysteine injected adjacent to the supraoptic nuclei (SON) of rats is rapidly incorporated into two macromolecular (both about 20,000 Daltons) common precursors of arginine vasopressin (AVP) and its associated neurophysin, and oxytocin (OT) and its neurophysin. Conversion of the larger precursor proteins to the smaller peptides appears to occur intragranularly during axonal transport to the neurohypophysis. The labelled products of this conversion (neurophysin, AVP, and OT) are released, in a Ca2+-dependent manner, from the posterior pituitary in response to depolarization by veratridine. Both the rates of biosynthesis and of processing of the precursors are greatly increased by increased functional activity (i.e. secretion) of the hypothalamo--neurohypophysial system.

Animals↗
PubMed1981

Multiple-rate components of axonally transported proteins in the hypothalamo-neurohypophysial system of the rat.

The transport of labeled proteins from the hypothalamus to the neurohypophysis following 35S-methionine injection into the rat supraoptic nucleus was studied using a unique approach adapted for the study of short-axon systems. Multiple-rate components to those found in other neuronal systems were demonstrated. Neurosecretory vesicle-containing proteins (e.g., neurophysins) were transported at fast rates (greater than 120 mm/day), whereas the cytoskeletal protein, actin, moved principally in the slow component of transport. Two-dimensional gel electrophoresis was used to analyze the diverse patterns of labeled proteins found in the various rate components of axonal transport in this system.

Actins↗
PubMed1980

Synthesis, transport, and release of posterior pituitary hormones.

Vasopressin and oxytocin are made and released by neurons of the hypothalamo-neurohypophysial system. Pulse labeling these neurons with radioactive amino acid indicates that the two hormones and their respective neurophysin carrier proteins are synthesized as parts of separate precursor proteins. The precursors seem to be processed into smaller, biologically active molecules while they are being transported along the axon.

Animals↗
PubMed1980

Biosynthesis of vasopressin, oxytocin, and neurophysins: isolation and characterization of two common precursors (propressophysin and prooxyphysin).

[35S]Cysteine-labeled putative precursors for vasopressin-associated neurophysin (NP-VP) and oxytocin-associated neurophysin (NP-OT) were isolated from the supraoptic nuclei (SONs) of normal rats. Homozygous Brattleboro rats were deficient in one of these precursors, the NP-VP precursor. Direct support for the hypothesis that OT and its NP- and VP and its NP are synthesized from two separate macromolecular common precursors was obtained by limited proteolysis of the precursors with trypsin and identification of the fragments as NPs, VP, or OT by a number of criteria (14). In this paper, these common precursors designated propressophysin (precursor for NP-VP and arginine VP) and prooxyphysin (precursor for NP-OT and OT) were further characterized. Propressophysin was specifically bound by Concanavalin-A-Sepharose and was eluted by 0.2 M alpha-methyl mannoside, showing that it is glycosylated. In contrast, prooxyphysin does not appear to be glycosylated. Using [3H]fucose as label injected near the SONs, two proteins (mol wt, approximately 10,000) were identified, which appear to be derived from propressophysin, that are rapidly transported to the posterior pituitary of normal rats. These two proteins were absent in homozygous Brattleboro animals. Both propressophysin and prooxyphysin were bound by a NP-Sepharose affinity support. The binding was different from that of arginine VP or OT to NP, since it was independent of pH and was hydrophobic in nature. In addition to prooxyphysin, the SONs of homozygous Brattleboro rats also contained other [35S]cysteine-labeled protein (mol wt, 20,000) composed of a NP-like protein and NP-binding peptides. The tryptic peptides derived from these proteins were very different in their chromatographic (high performance liquid chromatography) properties than the tryptic peptides derived from propressophysin and prooxyphysin. This protein ('X') was not bound by a Concanavalin-A-Sepharose affinity column, although it had chromatographic properties similar to propressophysin on Sephadex G-75.

Animals↗
PubMed1980

Biosynthesis of neuronal peptides: implications for neurobiology.

Many biologically active peptides (e.g., insulin, nerve growth factor, ACTH, endorphin, parathyroid hormone, etc.) appear to be synthesized first as prohormones, which are then converted intracellularly to the biologically active products by various post-translational modifications. Peptides of neuronal origin (e.g., vasopressin and oxytocin) are synthesized by similar mechanisms. The prominent role of post-translational processing in determining the final peptide products allows for the possibility that different peptides will by generated from identical prohormones in different cells.

Adrenocorticotropic Hormone↗
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↗