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Biomedical subjects

L Packer

Publications and source records attributed to L Packer.

At least 361 records · Page 20Linked to original sources
PubMed1984

Distinguishing effects of anemia and muscle iron deficiency on exercise bioenergetics in the rat.

Three weeks of dietary iron deficiency in weanling rats resulted in anemia (Hb, 3.9 vs. 14.2 g/dl in controls) and decreased oxidative capacities of skeletal muscle (as much as 90% below control values). Whole-animal maximal O2 consumption (VO2max), measured in a brief treadmill run of progressively increasing work load, was approximately 50% lower for iron-deficient rats than for controls, and maximal endurance capacity (time to exhaustion in a separate treadmill run at a constant, sub-Vo2max work load) was 90% lower for iron-deficient rats than for controls. Exchange transfusion, with packed erythrocytes or plasma, was used to adjust Hb to an intermediate concentration of approximately 9.5 g/dl in both iron-deficient and and control rats. This procedure corrected the Vo2max of iron-deficient rats to within 15% of control values, whereas endurance capacity showed no improvement. Our experimental dissociation of Vo2max and endurance capacity provides further evidence that Vo2max is not the sole determinant of endurance. We propose that defects in Vo2max during iron deficiency result primarily from diminished O2 delivery, whereas decreased endurance capacity reflects impaired muscle mitochondrial function.

Anemia, Hypochromic↗
PubMed1984

Spectroscopic characterization of nitrated purple membranes.

Light-adapted purple membranes were modified with tetranitromethane by a new light-dependent procedure at pH 5.5 which results in a blue-shifted chromophore absorbing at 530nm. This modification affects two aromatic residues. The modified bacteriorhodopsin's ground state chromophore structure is probed by circular dichroism and resonance raman spectroscopy while its photocycle is studied by laser-flash photolysis in the picosecond, microsecond and millisecond time scale. After nitration, the main findings are 1) Interactions between neighboring chromophores are lost, 2) Modified bacteriorhodopsin contains a conformationally changed chromophore but retains a protonated Schiff's base as evidenced by a resonance raman band at 1652 cm-1, 3) A red-shifted intermediate is formed in less than 10 ps after laser excitation, 4) The decay of the M-intermediate is not significantly affected whereas the rise time of the intermediate is enhanced about two fold. These observations are relevant to the role of aromatic acid residues of the apoprotein in the determination of the chromophoric characteristics in bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1983

Topographic studies of spin-labeled bacteriorhodopsin. Evidence for buried carboxyl residues and immobilization of the COOH-terminal tail.

Chemical modification and electron spin resonance techniques were used to study the topography of carboxyl residues in purple membranes. The results showed that buried carboxyl groups are located in hydrophobic protein domains at least 16 A from the membrane surface, and that the carboxyl-terminal tail is partially immobilized. Carboxyl groups on bacteriorhodopsin in purple membranes were covalently spin-labeled with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl using N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline as a highly specific coupling agent. Spin-labeled bacteriorhodopsin preparations containing an average of 2.1 +/- 0.5 spins/molecule retained photocycling and proton-pumping functions. Accessibility to the paramagnetic broadening agents, Fe(CN)3-6 and Ni2+, revealed a highly mobile surface group quenched at low concentrations of these agents, and a buried, immobilized group whose ESR signal remained at high quencher concentration. Treatment with denaturing agents greatly increased the mobility and quenching of these buried residues. A series of stearic acid spin labels bound to purple membranes was used to define the depth of paramagnetic interactions. Fe(CN)3-6 interactions were limited to surfaces whereas Ni2+ and Cu2+ effects extended into hydrophobic domains. A double modification procedure, which first blocked surface groups, selectively spin-labeled only buried carboxyl group(s) having a strongly immobilized signal. ESR analysis of the isolated carboxyl-terminal tail after trypsin treatment showed it had increased mobility, indicating that it is moderately immobilized in the native structure. These data provide evidence consistent with several models of bacteriorhodopsin tertiary structure which place carboxyls within hydrophobic domains of the protein.

Amino Acid Sequence↗
PubMed1983

Evidence for a carboxyl group in the vicinity of the retinal chromophore of bacteriorhodopsin.

Carboxyl groups of bacteriorhodopsin in purple membranes were activated using a hydrophobic reagent and then covalently labeled with a pH-sensitive reporter group, nitrotyrosine methyl ester. The membrane-bound reporter group had different spectral properties, and a pK 3 units higher than in solution. In purple membranes, an isosbestic point between the 428nm absorption peak of nitrotyrosine methyl ester, and the bacteriorhodopsin 570nm chromophore seen in alkaline titration, indicated interactions between the reporter group and retinal. Modification of white membranes (bacterioopsin from R1mW strain) revealed similar, unusual spectral and ionization properties. Thus, the hydrophobic environment, not retinal interactions per se, are responsible for the ionization behavior of the reporter group. These results indicate that a carboxyl group is near the retinal chromophore of bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1983

Vitamin E, physical exercise and tissue oxidative damage.

Oxidative damage and the role of antioxidants and prooxidants in aerobic metabolism is of great current interest; it spans areas of research such as carcinogenesis, ageing, toxicology and nutrition. We have used Bantin-Kingman female rats for both in vivo and in vitro studies. In these animals we have altered the levels of all-rac-alpha-tocopherol (vitamin E) by dietary means and have used physical exercise and visible light exposure to alter oxidative stress. Our results show a progressive and specific increase in the susceptibility of many subcellular membranes to oxidative damage with increasing levels of vitamin E deficiency and/or physical stress. In addition, endurance training raised the levels of antioxidative enzymic pathways in both skeletal and cardiac muscle.

Animals↗
PubMed1983

Nonionic detergent effects on spectroscopic characteristics and the photocycle of bacteriorhodopsin in purple membranes.

Treatment of purple membranes isolated from Halobacterium halobium with nonionic detergents caused the following effects in addition to solubilizing bacteriorhodopsin: a blue shift of the absorption spectrum of bacteriorhodopsin; an increase in tryptophan fluorescence of about twofold; an enhanced rate of bleaching in the presence of hydroxylamine; and the disappearance of the negative exciton band in the CD spectrum at 600 nm. Crosslinking of purple membranes by glutaraldehyde can prevent solubilization of bacteriorhodopsin by detergents. However, only the effect on the CD spectrum by detergents can be inhibited by crosslinking of the purple membranes prior to detergent treatment. Photocycle kinetics studies revealed that the apparent pK of the slow component of the M412 decay was decreased by the detergent treatment while the rise time for M412 formation is accelerated two- to threefold. Crosslinking also did not prevent these effects. These results demonstrate that nonionic detergents, apart from their action to monomerize bacteriorhodopsin, also affect retinal-apoprotein interactions.

Bacteriorhodopsins↗
PubMed1983

Light-dependent nitration of bacteriorhodopsin.

Purple membranes were treated with tetranitromethane to modify tyrosine residues of bacteriorhodopsin. At pH 8.0, nitration is shown to be affected by illumination during the modification. Amino acid analysis revealed about 0.7 residues nitrated if reaction was in the dark while about 2.0 tyrosines were modified if illumination greater than 540 nm was provided. Tryptophan was unaffected under both conditions. Light-dependent nitration caused a blue shift of the absorbance maximum of bacteriorhodopsin from 568 to 530 nm while no chromophore shift was observed for the dark-modified preparation. Both preparations show an absorption band at 360 nm indicative of the presence of nitrotyrosines. Reduction by dithionite eliminated the pH-dependent changes associated with the 360-nm nitrotyrosine band. Circular dichroism spectra indicate that interactions between neighboring chromophores are altered concomitant with the blue shift of the absorbance maximum by nitration. These studies show that light is required for the nitration of the tyrosine residue, and that Tyr 26 (H. D. Lemke and D. Oesterhelt (1981) Eur. J. Biochem. 115, 595-604) is probably responsible for the blue shift of the absorbance maximum. The intrinsic fluorescence and photocycle kinetics of the tyrosine-modified preparation and reduction of nitrotyrosine by dithionite were studied. In dark modification, only pH-dependent dithionite-reducible nitrotyrosines were produced. It is concluded that surface tyrosines probably do not directly participate in the proton-translocation events coupled to the photocycle of bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1982

Photoreaction of tyrosine-iodinated bacteriorhodopsin at low temperature.

To elucidate the role of tyrosine residues in the shift of lambda max and the light-driven proton pump of bacteriorhodopsin, the photochemical reaction of tyrosine-iodinated bacteriorhodopsin (tyr-mod-bR) was investigated by low-temperature spectrophotometry. After 4-5 of 11 tyrosine residues of bacteriorhodopsin were iodinated, the meta-intermediate of tyr-mod-bR in 75% glycerol solution became so stable that its decay could be observed even at room temperature and it was stable in the dark for several hours at -65 degrees C. Four batho-intermediates were formed by irradiation with green light (500 nm) at -170 degrees C. Like native bacteriorhodopsin, these batho-intermediates were photoreversible at -170 degrees C. Four corresponding meta-intermediates were also formed by irradiation at -60 degrees C. Using the difference spectra between meta-intermediates and tyr-mod-bR, the absorption spectra of four kinds of tyr-mod-bRs, batho-intermediates, and meta-intermediates were estimated. Each was at shorter wavelengths than that of its corresponding type in native bacteriorhodopsin. The results indicate that two or more tyrosine residues have some role in determining color in native bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1982

Protection against free radical formation by protein bound iron.

Naturally occurring protein-bound and artificially chelated iron have been evaluated for their catalytic effect in promoting hydroxyl radical (X OH) formation from H2O2 decomposition and on epinephrine autooxidation. Iron bound to ferritin and transferrin did not increase X OH formation or epinephrine autooxidation, whereas iron equivalents of Fe-EDTA considerably augmented those processes. After rigorous removal of contaminating trace iron, X OH can be detected at concentrations of 1.0 microM Fe3+ or 2-5 microM H2O2. Although other forms of iron found physiologically might cause considerable oxidative damage through mechanisms similar to that of Fe-EDTA, our studies indicate considerable mitigation of such toxicity in ferritin and transferrin, which constitute major forms of transport and storage of iron in vivo.

Catalysis↗
PubMed1982

Membrane effects of vitamin E deficiency: bioenergetic and surface charge density studies of skeletal muscle and liver mitochondria.

Vitamin E deficiency in rats increased the sensitivity of liver and muscle mitochondria to damage during incubation at various temperatures, irradiation with visible light, or steady-state respiration with substrates. In all cases, vitamin E-deficient mitochondria exhibited increased lipid peroxidation, reduced transmembrane potential, decreased respiratory coupling, and lower rates of electron transport compared to control mitochondria. Muscle mitochondria always showed greater negative inner membrane surface charge density, and were also more sensitive to damage than were liver mitochondria. Vitamin E-deficient mitochondria also showed slightly more negative inner membrane surface charge density compared to controls. The relationship observed between greater negative surface potential and increased sensitivity to damage provides for a new and sensitive method to probe further the role of surface charge in membrane structure and function. Implications of these new findings for the well-known human muscle myopathies and those experimentally induced by vitamin E deficiency in animals are discussed.

Animals↗