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Biomedical subjects

D M Jameson

Publications and source records attributed to D M Jameson.

68 records · Page 4Linked to original sources
PubMed1985

Mechanisms of inhibition of (Na,K)-ATPase by hydrostatic pressure studied with fluorescent probes.

To investigate the mechanisms by which hydrostatic pressure inhibits (Na,K)-ATPase, we measured enzyme activity, as a function of pressure and temperature, of purified (Na,K)-ATPase from dog kidney and eel electroplax, and we monitored protein conformation, possible subunit interactions, and the fluidity of the membrane with fluorescent probes. The (Na,K)-ATPase and p-nitrophenylphosphatase activities were inhibited reversibly by pressures below 1.5 kilobars (eel enzyme) and 2.5 kilobars (dog kidney enzyme). Above these pressures, the enzymes were inactivated irreversibly. The plots of 1n(activity) versus pressure were curvilinear; this indicates that the reversible inhibition by pressure involves two or more rate-limiting steps. The calculated activation volumes varied with temperature and pressure and were larger for the (Na,K)-ATPase activity compared to the p-nitrophenylphosphatase activity. The fluorescence polarization of three hydrophobic probes decreased with increasing temperature (10-36 degrees C) and increased with increasing pressure (10(-3)-1.5 kilobars), reversibly, without any evidence of a lipid phase transition. Plots of enzyme activity versus fluorescence polarization of the lipid probes showed an inverse relationship; this indicates that enzyme activity was directly related to the fluidity of the membrane as measured by the lipid probes. Pressure had no effect on the fluorescence polarization of two cardiac glycoside probes nor on the efficiency of resonance energy transfer between either donor and acceptor cardiac glycosides specifically bound to the ouabain sites of different alpha-subunits, or tryptophan and the bound cardiac glycoside probe. These results suggest that dissociation of dimeric alpha-subunits is not related to the inhibition by pressure, and that the cardiac glycoside-complexed enzyme conformation is stabilized by pressure. It is concluded that increased pressure decreases the membrane fluidity which hinders conformational transitions associated with rate-limiting steps of the (Na,K)-ATPase reaction. It is proposed that ion-bound or -occluded forms of (Na,K)-ATPase are stabilized by pressure because they occupy a smaller volume.

Animals↗
PubMed1984

A model of dynamic quenching of fluorescence in globular proteins.

A model is presented for the quenching of a fluorophore in a protein interior. At low quencher concentration the quenching process is determined by the acquisition rate of quencher by the protein, the migration rate of quencher in the protein interior, and the exit rate of quencher from the protein. In cases where the fluorescence emission observed in the absence of quencher could be described by a single exponential decay, the presence of quencher led to doubly exponential decay times, and the aforementioned exit rates of the quencher could be determined from experimental data. At high quencher concentration, the processes became more complex, and the deterministic rate equations used at low quencher concentration had to be modified to take into account the Poisson distribution of quencher molecules throughout the protein ensemble and also by using a migration rate for quencher in the protein interior that is a function of the quencher concentration. Simulations performed for typical fluorescent probes in proteins showed good agreement with experiments.

Kinetics↗
PubMed1984

Oxygen distribution and migration within Mbdes Fe and Hbdes Fe. Multifrequency phase and modulation fluorometry study.

Quenching of the intensity and lifetime of porphyrin fluorescence from Mbdes Fe and Hbdes Fe (iron-free myoglobin and hemoglobin) by oxygen was investigated using a multifrequency cross-correlation phase fluorometer. The single exponential decay characteristic of porphyrin emission of Mbdes Fe and Hbdes Fe became doubly exponential upon application of oxygen pressure. The results were interpreted in terms of a general model of dynamic quenching of fluorescence in globular proteins. The model accounted for the rate k+ of acquisition of quencher by the protein, the exit rate k- of quencher from the protein, and the migration rate chi of quencher in the protein interior. The values of k+, k-, and chi were different for Mbdes Fe and Hbdes Fe. The addition of 40% sucrose, which increased the bulk viscosity sixfold, modified these rates. These results are discussed and compared with previous quenching studies on proteins. The significance of these results and the model for the interpretation of protein quenching studies is emphasized.

Animals↗
PubMed1982

Acrylamide and oxygen fluorescence quenching studies with liver alcohol dehydrogenase using steady-state and phase fluorometry.

The fluorescence lifetime of liver alcohol dehydrogenase (LADH) has been determined by phase fluorometry at various emission wavelengths and as a function of the concentration of the quencher acrylamide. Acrylamide selectively quenches the fluorescence of the surface tryptophanyl residue Trp-15, thus allowing the fluorescence lifetime of this residue and the buried residue Trp-314 to be evaluated. Values of tau15 = 6.9 ns and tau314 = 3.6 ns are obtained, in qualitative agreement with lifetimes of these residues determined from fluorescence decay studies [Ross, J.B.A., Schmidt, C.J., & Brand, L. (1981) Biochemistry 20, 4369-4377]. The quenching of the fluorescence of LADH by oxygen has also been studied. Quenching by oxygen results in a blue shift in the fluorescence of the protein and a downward-curving Stern-Volmer plot. These data, along with oxygen quenching studies in the presence of 1 M acrylamide, are consistent with a model in which oxygen quenches the fluorescence of Trp-314 and -15 with quenching constants of 3.5 and 25 M-1, respectively. Thus, as in studies with other quenchers, Trp-314 is found to be less accessible to the quencher oxygen than is Trp-15. A lifetime Stern-Volmer plot has also been obtained for the oxygen quenching of LADH. Such a plot deviates somewhat from the intensity Stern-Volmer plot as predicted by simulations of the quenching of two-component systems.

Acrylamides↗
PubMed1978

The use of the fluorescent probe alpha-parinaric acid to determine the physical state of the intracytoplasmic membranes of the photosynthetic bacterium, Rhodopseudomonas sphaeroides.

alpha-Parinaric acid has been used to determine the degree of ordering of the hydrocarbon region of purified intracytoplasmic membranes of Rhodopseudomonas sphaeroides. The usefulness of alpha-parinaric acid as a probe of membrane fluidity was established by comparison of its fluorescent properties in phosphatidylcholine vesicles with those of the more commonly used fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene. Both fluorescent probes were shown to monitor similar environments in the phosphatidylcholine vesicles when the phospholipids were maintained at temperatures above their phase transition temperature. The rotational mobility of alpha-parinaric acid in the intracytoplasmic membranes was determined from 0 to 50 degrees C, a region where no phase transitions were detectable. The rotational mobility of alpha-parinaric acid dissolved in vesicles formed from total extracted intracytoplasmic membrane phospholipids, was 2--3-fold greater than that measured in the intact intracytoplasmic membranes; demonstrating that the presence of protein greatly reduces the mobility of the phospholipid acyl chains of the intracytoplasmic membranes. Due to the high protein content of these membranes, the perturbing effect of protein on acyl chain mobility may extend to virtually all the intracytoplasmic membrane phospholipid.

Cell Membrane↗