Search PubMed⌕ Search

Biomedical subjects

R R Sharp

Publications and source records attributed to R R Sharp.

At least 37 records · Page 2Linked to original sources
PubMed1983

Evidence that the H+ electrochemical gradient across membranes of chromaffin granules is not involved in exocytosis.

The possibility that the large H+ electrochemical potential of chromaffin granules, the secretory granules of adrenal medullary chromaffin cells, plays an important role in exocytosis was investigated in cultures of chromaffin cells from bovine adrenal medulla. Methylamine uptake into the cells, [gamma-31P]phosphate nmr of ATP within intracellular chromaffin granules, O2 consumption of intracellular mitochondria, and MgATP-stimulated catecholamine uptake into chromaffin granules isolated from cultured chromaffin cells were assessed to determine whether various manipulations altered the H+ electrochemical gradients of intracellular chromaffin granules or mitochondria. Catecholamine secretion was not significantly altered by ammonium, methylamine, nigericin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or dicyclohexylcarbodiimide under conditions when the pH of intracellular chromaffin granules was reduced or when granular or mitochondrial processes were uncoupled from H+ electrochemical gradients. The data indicate that the H+ electrochemical gradient across the chromaffin granule membrane does not play a role in exocytosis.

Adrenal Medulla↗
PubMed1982

Molecular mobilities and the lowered osmolality of the chromaffin granule aqueous phase.

Carbon-13 spin-lattice relaxation times, T1, have been measured in whole adrenal medullary tissue slices, in suspensions of isolated chromaffin granules, in the reconcentrated chromaffin granule lysate, and in various model solutions containing catecholamines. ATP, chromogranins and Ca2+. Reorientational correlation times have been calculated at 10 degrees C using T1 data and nuclear Overhauser enhancements for protonated carbons on both catecholamines and nucleotides. Correlation times in all media are relatively short and characteristic of highly fluid aqueous phases. Adrenalin and ATP exhibit substantial differences in correlation times in all media, however, the ratio tau R (ATP): tau R(catecholamine) ranging from 2.4 in simple 3:1 adrenalin-ATP solutions to 4 in intact chromaffin granules. This difference, as well as the relatively high absolute reorientational mobilities of both components, confirms the importance of labile ionic interactions between ATP and catecholamines, but rules out the presence of high concentrations of base-stacked structures. Participation of the chromogranins in ternary complexes with catecholamines and ATP appears to be of minor importance. Ionic interactions to the protein are not reflected in either 13C T1 values or chemical shifts of arginine or glutamate sidechain resonances, or in the 13C chemical shifts of ATP or catecholamines. Very labile protein-ATP binding appears to be reflected in the correlation time measurements, however, which show selective immobilization of ATP relative to catecholamine in the presence of soluble protein. Osmotic measurements indicate that solutions containing adrenaline, ATP and Ca2+ are highly nonideal, but probably not sufficiently so to account fully for the osmotic stabilization of the chromaffin through their polyelectrolyte properties, exert a significant influence on the intragranular osmolality. The osmotic lowering due to polyion-counterion interactions has been estimated semiquantitatively using a theory developed by Oosawa.

Adenosine Triphosphate↗
PubMed1982

Water permeability of the chromaffin granule membrane.

NMR spin-lattice relaxation rates of solvent protons have been used to measure the water permeability coefficient of the chromaffin granule membrane. The technique involves labeling the chromaffin granule interior with Mn+2, which provides an efficient relaxation pathway for intravesicular solvent protons. Added Mn+2 spontaneously accumulates in the chromaffin granule matrix in the presence of the divalent cation-specific ionophore A23187 and is maintained against a large concentration gradient. In this way, the internal proton relaxation rate is readily augmented to values some 10(2)-10(3) times greater than that in the extravesicular water space. Transmembranal water transport permits solvent protons in the extravesicular water space, in which most of the observed NMR signal orginates, to sample the highly relaxive environment of the chromaffin granule matrix. By this process, water permeation shortens the observed relaxation rate. The diffusive water permeability coefficient of the chromaffin granule membrane has been measured over the temperature range 0-38 degrees C. The permeability coefficient measured at 25 degrees C is comparable to a previously reported value for planar lipid bilayers composed of ox brain lipids and cholesterol (Pd approximately equal to 0.37-0.53 10(-3)) cm X s-1 at 25 degrees C) but is substantially less than values for the plasma membranes of erythrocytes and Chlorella. Hypothesized hydrophilic "pores," thought to provide parallel permeation pathways in the latter membranes, appear to be absent in chromaffin granule membranes. The water permeation rate exhibits Arrhenius temperature behavior and does not reflect a phase transition at 32 degrees-34 degrees C observed previously in ESR spin-label studies of chromaffin granule ghosts.

Adrenal Medulla↗
PubMed1981

Factors influencing hydroxylamine inactivation of photosynthetic water oxidation.

The kinetics of Mn release during NH2OH inactivation of the water oxidizing reaction is largely insensitive to the S-state present during addition of NH2OH. This appears to reflect reduction by NH2OH of higher S-states to a common more reduced state (S0 or S-1) which alone is susceptible to NH2OH inactivation. Sequences of saturating flashes with dark intervals in the range 0.2--5 S-1 effectively prevent NH2OH inactivation and the associated liberation of manganese. This light-induced protection disappears rapidly when the dark interval is longer than about 5 S. Under continuous illumination, protection against NH2OH inactivation is maximally effective at intensities in the range 10(3)--10(4) erg . cm-2 . S-1. This behavior differs from that of NH2OH-induced Mn release, which is strongly inhibited at all intensities greater than 10(3) erg . cm-2 . S-1. This indicates that two distinct processes are responsible for inactivation of water oxidation at high and low intensities. Higher S-states appear to be immune to the reaction by which NH2OH liberates manganese, although the overall process of water oxidation is inactivated by NH2OH in the presence of intense light. The light-induced protection phenomenon is abolished by 50 microM DCMU, but not by high concentrations of carbonyl cyanide m-chlorophenylhydrazone, which accelerates inactivation reactions of the water-splitting enzyme, Y (an ADRY reagent). The latter compound accelerates both inactivation of water oxidation and manganese extraction in the dark.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1981

Stoichiometry, inhibitor sensitivity, and organization of manganese associated with photosynthetic oxygen evolution.

Chloroplast thylakoid membranes isolated in the presence of EDTA retain high rates of O(2) evolution (>/=340 mumol.h(-1).mg chlorophyll(-1)) but contain no Mn(2+) that is detectable by electron paramagnetic resonance (EPR) at room temperature. The total Mn(2+) content of these preparations is 4.6 per 400 chlorophylls; 0.6 Mn(2+) can be released by addition of Ca(2+), a treatment that does not affect O(2) evolution. The remaining Mn(2+) (4 per 400 chlorophylls) appears to be functionally associated with O(2) evolution activity. Inhibition by Tris, NH(2)OH, or heat will release a small fraction of Mn(2+) from these membranes ( approximately 25% with Tris, for example). Addition of Ca(2+) further enhances Mn(2+) release so that for Tris and for NH(2)OH, 2 and 3, respectively, Mn(2+) per 400 chlorophylls are extracted from the O(2)-evolving complex. Based on the microwave power-saturation properties of the EPR signal IIf, which arises from an intermediate electron carrier in the water splitting process, it appears that one of the four Mn(2+) associated with photosystem II is uniquely sensitive to Tris. A new model is proposed for the organization and inhibitor sensitivity of manganese in the O(2)-evolving complex.

Journal Article↗
PubMed1980

NMR relaxivity changes in chloroplast suspensions. Effects of NH2OH and of treatments altering the redox state of the photosynthetic electron transport chain.

Treatments (illumination, chemical oxidation or reduction) which are potentially capable of producing paramagnetic centers in chloroplast thylakoid membranes do not produce enhancements of the proton magnetic relaxivities of these preparations. However, exposure of thylakoid membranes to varying concentrations of hydroxylamine induces a time-dependent increase in relaxivity for which the steady-state magnitude is dependent on hydroxylamine concentration. The appearance of relaxivity is correlated kinetically with inactivation of oxygen-evolving centers; in addition both processes show a threshold effect with respect to hydroxylamine concentration. Kinetic analyses of these hydroxylamine-induced effects suggest that at low (less than or equal to 100 microM) and at intermediate (200--500 microM) concentrations, hydroxylamine extraction is partially counteracted by a reverse process that reactivates oxygen-evolving centers in the dark.

Chloroplasts↗
PubMed1980

The kinetics of water exchange across the chloroplast membrane.

The kinetics of water exchange across the membrane of class II chloroplasts has been studied by two NMR methods. Both methods utilize Dy(en)3+ (en = ethylenediamine) to induce a transmembranal chemical shift the order of 40 Hz in the water proton resonance. The shift reagent is impermeant to the chloroplast membrane, inert as a redox reagent, soluble at millimolar concentrations at neutral pH, and associated with a large, virtually temperature independent molar shift (0.10-0.12 ppm/mM). Water exchange across the membrane is monitored by two independent experiments. In the first, chemical exchange causes line broadening in the water proton resonance in the high-resolution spectrum. Measurement of the incremental linewidth as a function of transmembranal chemical shift determines the exchange kinetics as well as the fractions of water protons in internal and external media. In the second experiment, chemical exchange causes the transverse relaxation time, as measured by the Carr-Purcell-Gill-Meiboom technique, to be dependent on the 180 degree pulse spacing. The two experiments, while independent of each other, depend on the same set of theoretical parameters. These parameters are overdetermined by simultaneous analysis of both experiments. The mean lifetime of a water proton in the inner thylakoid space is found to be 1.1 +/- 0.8 ms at 25 degrees C and 2.75 +/- 0.4 ms at 3 degrees C in NH2OH/EDTA-treated chloroplasts. Values derived from dark-adapted chloroplasts that are active with respect to oxygen evolution are 1.1 +/- 0.3 ms (25 degrees C) and 1.75 +/- 0.4 ms (3 degrees C). The internal thylakoid volume is also determined in principle by the data, but uncertainties in the membrane volume and the transmembranal chemical shift severely limits the accuracy of this measurement.

Chloroplasts↗
PubMed1980

Field-dispersion profiles of the proton spin-lattice relaxation rate in chloroplast suspensions. Effect of manganese extraction by EDTA, Tris, and hydroxylamine.

Proton spin-lattice relaxation rates (R1) have been measured in a variety of dark-adapted chloroplast suspensions over a range of field stengths between 1 and 15 kG (4-65 MHz). When the effects of EDTA or Tris washing on chloroplast relaxivities are compared, the pool of Mn associated with oxygen evolution is seen not to contribute significantly to relaxivity. Instead, nearly all of the observed relaxivity, which is characterized by a paramagnetic maximum near 20.7 MHz in the field dispersion profile of R1, appears to arise from contaminating non-functional Mn(II) that can be removed by EDTA during the isolation procedure. These observations, which contradict previous reports ascribing chloroplast relaxivity to the water-oxidizing system, require a reevaluation of proposed models, derived from NMR studies, of the state of Mn in the water-splitting reaction. Chloroplasts from which loosely bound non-functional Mn has been removed by EDTA washing do show an enhancement of relaxivity when exposed to NH2OH at concentrations known to inactivate water oxidation. This NH2OH-induced relaxivity is comprised of Mn(II) in two distinct paramagnetic sites. One site is chelatable by EDTA, whereas the other site is not. This finding suggests that some Mn(II) tightly bound to thylakoid membranes can contribute to relaxivity after inactivation of the oxygen-evolving reaction.

Binding Sites↗
PubMed1980

The soluble components of chromaffin granules. A carbon-13 NMR survey.

Carbon-13 NMR spectra of the reconcentrated chromaffin granule lysate have been obtained at 50 MHz and 62.9 MHz. The spectrum contains a number of assignable resonances in addition to those of the main soluble components (catecholamines, adenine nucleotides and chromogranin). Guanine and uridine nucleotides are present at levels of 0.13 and 0.08 mol/mol adenine nucleotides, respectively. Concentrations of cytidine nucleotides and NAD+ are below the detection limit (0.02 mol/mol adenine nucleotides). An unidentified low molecular weight species, thought to be an adenine-containing oligonucleotide, is also present. Ascorbic acid was observed at a concentration of 0.14 mol/mol adenine nucleotides, but both dopamine and dehydroascorbic acid were below the detection limit. Protein resonances agree well with the reported amino acid composition of chromogranin A, with the exception of tryptophan and glutamine which have not previously been measured. The concentrations of these residues are estimated to be 12 +/- 3 and 39 +/- 5 residues per 77 000 dalton unit of chromogranin A. Substantial intensity due to unsaturated fatty acid side-chains in solubilized lipid is seen in the olefinic carbon region and in the methylene region, suggesting the presence of lipoprotein. Unassigned carbohydrate resonances are also present, but are largely obscured by sucrose in the isolation medium.

Adenine Nucleotides↗
PubMed1979

Composition of the aqueous phase of chromaffin granules.

Nuclear magnetic resonance spectroscopy has been used to determine the composition of the aqueous phase of bovine chromaffin granules. Relative concentrations of catecholamines (epinephrine plus norepinephrine), ATP and chromogranins have been measured from integrated intensities in the proton spectra using computer simulation techniques. Most or all of the catecholamines (97 +/- 8%) are present in the aqueous phase and contribute to the high resolution spectrum. The catecholamine:ATP molar ratio (4.41 +/- 0.45) determined by NMR is close to the value (4.45) derived from biochemical assay indicating that most or all of the ATP is present with catecholamine in the aqueous phase. Catecholamine:protein ratios show that approximately 45% of the soluble protein freed by lysis is not NMR visible. Intensity from this fraction does not appear under highly denaturing conditions (8 M urea) but reappears after hydrolysis. This behavior is similar to that of recently isolated soluble lipoprotein complexes. Variations in the NMR spectra associated with (1) different preparative procedures; (2) different suspension media, and (3) increasing osmolality are described. The fact that high concentrations of epinephrine and ATP (approximately 700 mM total) are dissolved in the aqueous phase implies that solution phase interactions at least partially ionic in nature are responsible for the low internal osmolality of chromaffin granules in vivo. Ordered phases containing a substantial fraction of the total catecholamine in an osmotically inactive form are not present.

Adenosine Triphosphate↗
PubMed1977

Analysis of the carbon-13 and proton NMR spectra of bovine chromaffin granules.

Natural abundance carbon-13 and proton NMR spectra of bovine chromaffin granules have been obtained and analyzed using computer simulation techniques. High resolution spectra show the presence of a fluid aqueous phase containing epinephrine, ATP and a random coil protein. The protein spectrum contains unusually intense resonances due to glutamic acid and proline and has been simulated satisfactorily using the known amino acid composition of chromogranin A. The lipid phase of chromaffin granules gives rise to intense, but very broad, resonances in the carbon-13 spectrum. Protons in the lipid phase are also observable as a very rapid component of the proton-free induction decay (T2 approximately equal to 15 microns). Linewidths of the carbon-13 spectra have been used to set upper limits on rotational correlation times and on the motional anisotropy in the aqueous phase. These limits show that the aqueous phase is a simple solution (not a gel) that is isotropic over regions much larger than solute dimensions. No gel transition is observed between -3 and 25 degrees C. The carbon-13 spectra are definitely inconsistent with a lipoprotein matrix model and chromaffin granules previously proposed by Helle and Serck-Hanssen ((1975) Mol. Cell, Biochem. 6, 127-146). Relative carbon-13 intensities of ATP and epinephrine are not consistent with the known 1 : 4 mol ratio of these components. This fact suggests that epinephrine and ATP are not directly complexed in intact chromaffin granules.

Adenosine Triphosphate↗
PubMed1977

Molecular mobilities of soluble components in the aqueous phase of chromaffin granules.

NMR relaxation times have been used to characterize molecular motion and intermolecular complexes in the aqueous phase of bovine chromaffin granules. Partially relaxed 13C and proton spectra have been obtained at 3 and 25 degrees C. T1 measurements of five protonated carbons on epinephrine (C2, C5, C6, CHOH and NCH3) give a correlation time of 0.15 (10(-9)) s at 25 degrees C for the catechol ring and methine carbon, while the effective correlation time for the NCH3 group is somewhat shorter due to its internal degree of rotational freedom. Resonances of protonated carbons on the soluble protein chromogranin give very similar correlation times: 0.20 (10(-9)) s for the peptide alpha-carbon and 0.2 (10(-9)) s for the methylene sidechain carbons of glutamic acid. The correlation time (tauR) of ATP was not measured directly using 13C T1 data due to the weakness of its spectrum, but its reorientation appears to be substantially slower than that of epinephrine or chromogranin. This conclusion is based on three observations: (1) the qualitative temperature dependence of T1 for H2 and H8 on the adenine ring places tauR for ATP to the right of the T1 minimum, or tauR greater than or equal to 1.0 (10(-9)) s; (2) 13C-resonances of ATP have anomalously low amplitudes compared with epinephrine resonances, a fact that is readily explained only if ATP undergoes substantially slower reorientation; and (3) a comparison of the T1 data of H8 in chromaffin granules and in a dilute aqueous solution, where tauR for ATP can be measured directly indicates that tauR approximately 1.0 (10(-9)) s at 25 degrees C in the granules. The relaxation data are consistent with the concept of a storage complex based on electrostatic interactions between a polyion (chromogranin) and its counterions (ATP and epinephrine), in which ATP cross-links cationic sidechains of the protein.

Adenosine Triphosphate↗