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Biomedical subjects

L Packer

Publications and source records attributed to L Packer.

At least 343 records · Page 19Linked to original sources
PubMed1986

Aggregation and proton release of purple and white membranes following cleavage of the carboxyl-terminal tail of bacteriorhodopsin.

Our results indicate that the previously reported decrease in proton release by proteolyzed purple membrane sheets was due merely to the aggregation state of these preparations and not to the loss of the carboxyl-terminal tail. Changes in H+/M412 ratios obtained for purple and white membrane preparations correlate with the measured aggregation. White membrane preparations consistently exhibit H+/M412 ratios more than twice those measured for native purple membranes under the same conditions. Quasi-elastic light scattering was used to characterize the size of isolated purple and white membrane sheets before and after proteolysis. The results clearly show that native purple membrane preparations are larger in size than would be expected and that, following trypsin treatment, they are on average more than an order of magnitude larger. Negative staining electron microscopy showed that the purple membrane became aggregated in stacked arrays. Bleaching and reconstitution with retinal also affect aggregation, but iodination or nitration of purple membrane does not affect the measured size. The average size of white membranes is smaller; this is consistent with results of electron microscopy and the size increase is much less than that of purple membranes following trypsin treatment. No size change occurs with retinal reconstitution. In aggregated purple membrane preparations, protons and other cations are unable to exchange freely with the aqueous medium, explaining why proteolysis lowers the proton release from purple membrane sheets in suspension.

Bacteriorhodopsins↗
PubMed1986

Hydroperoxide metabolism in cyanobacteria.

The enzymes involved in antioxidative activity and the cellular content of the antioxidants glutathione and ascorbate in the cyanobacteria Nostoc muscorum 7119 and Synechococcus 6311 have been examined for their roles in hydroperoxide removal. High activities of ascorbate peroxidase and catalase were found in vegetative cells of both species and in the heterocysts of N. muscorum. The affinity of ascorbate peroxidase for H2O2 was 15- to 25-fold higher than that of catalase. Increased activity of ascorbate peroxidase was observed in N. muscorum when H2O2 production was enhanced by photorespiration. Catalase activity was decreased in dilute cultures whereas ascorbate peroxidase activity increased. Ascorbate peroxidase activity also increased when the CO2 concentration was reduced. Ascorbate peroxidase appears to be a key enzyme in a cascade of reactions regenerating antioxidants. Dehydroascorbate reductase was found to regenerate ascorbate, and glutathione reductase recycled glutathione. In vegetative cells glutathione was present in high amounts (2-4 mM) whereas the ascorbate content was almost 100-fold lower (20-100 microM). Glutathione peroxidase was not detected in either cyanobacterium. It is concluded from the high activity of ascorbate peroxidase activity and the levels of antioxidants found that this enzyme can effectively remove low concentrations of peroxides. Catalase may remove H2O2 produced under photooxidative conditions where the peroxide concentration is higher.

Ascorbate Peroxidases↗
PubMed1986

A comparative study on the effects of ascorbic acid deficiency and supplementation on endurance and mitochondrial oxidative capacities in various tissues of the guinea pig.

The aim of this study was to ascertain the effects of ascorbic acid deficiency and supplementation on endurance and mitochondrial oxidative capacities in various tissues of guinea pigs. Endurance capacity was significantly reduced by ascorbic acid deficiency and supplementation. Oxidative capacities were reduced in heart, liver and brown adipose tissue but it seems as if skeletal muscle is protected against ascorbic acid deficiency and supplementation-induced oxidative damage. Skeletal muscle and liver but not heart appear to be susceptible to exercise-induced oxidative damage. To a certain extent oxidative capacities could be related to the glutathione status in the various tissues.

Animals↗
PubMed1986

Carbon-13 NMR studies of salt shock-induced carbohydrate turnover in the marine cyanobacterium Agmenellum quadruplicatum.

Carbon turnover in response to abrupt changes in salinity, including the mobilization of glycogen for use in osmoregulation was studied with pulse-chase strategies utilizing nuclear magnetic resonance (NMR)-silent and NMR-detectable 12C and 13C isotopes, respectively. Growth of Agmenellum quadruplicatum in 30%-enriched 13C bicarbonate provided sufficient NMR-detectability of intracellular organic osmoregulants for these studies. A comparison of NMR spectra of intact cells and their ethanol extracts showed that the intact cell data were suitable for quantitative work, and, when combined with ESR measurements of cell volumes, yielded intracellular glucosylglycerol concentrations without disrupting the cells. NMR pulse-chase experiments were used to show that 13C-enriched glycogen, which had previously been accumulated by the cells under nitrogen-limited growth at low salinities, could be utilized for the synthesis of glucosylglycerol when the cells were abruptly transferred to hypersaline media, but only in the light. It was also shown that the accumulation of glucosylglycerol in the light occurred on a time scale similar to that of cell doubling. Depletion of glucosylglycerol when cells abruptly transferred to lower salinities appeared to be rapid--the intracellular pool of this osmoregulant was decreased 2-fold within 2 hours of hypotonic shock.

Carbon Isotopes↗
PubMed1986

Vitamin E deficiency and vitamin C supplements: exercise and mitochondrial oxidation.

The effects of dietary antioxidant vitamins E and C on exercise endurance capacity and mitochondrial oxidation were investigated in rats. The endurance capacity of both vitamin E-deficient and vitamin C-supplemented, E-deficient rats was significantly (P less than 0.05) lower (38.1 and 33.6%, respectively) than control animals. Compared with the normal and vitamin E-deficient rats, there was a significant (P less than 0.05) increase in the concentration of vitamin C in blood and liver of the vitamin E-deficient, C-supplemented animals. Hence dietary vitamin C supplementation does not prevent the inhibition of exercise endurance capacity or increased hemolysis seen in vitamin E deficiency. The mitochondrial activities for the oxidation of palmitoyl carnitine and alpha-ketoglutarate were significantly (P less than 0.05) decreased by a single bout of exercise in brown adipose tissue but not in muscle, heart, or liver from vitamin C-supplemented, E-deficient groups of rats when compared with the activities in the tissue from the same group of rats killed at rest. Similar results were also seen in brown adipose tissue from vitamin E-deficient rats. The results suggest a tissue-specific role for vitamins E and C in substrate oxidation and show that the poor endurance capacity of vitamin E-deficient rats cannot be attributed to any changes in the mitochondrial activity in skeletal or cardiac muscles. It is also concluded that vitamin C supplementation, at least at the dose employed in the present study, cannot counteract the detrimental effects associated with vitamin E deficiency.

Adipose Tissue, Brown↗
PubMed1985

The role of glutathione and ascorbate in hydroperoxide removal in cyanobacteria.

The antioxidative potential of cyanobacteria to scavenge hydroperoxides formed as by-products of photosynthetic activity was investigated in Nostoc muscorum 7119 and Synechococcus 6311. These cells contained a high concentration of glutathione, 2-5 mM, and a low concentration of ascorbate, 20-100 uM. No glutathione peroxidase was detected while the activity of ascorbate peroxidase was high, reacting with hydrogen peroxide, t-butyl hydroperoxide, and cumene hydroperoxide. Dehydroascorbate reductase was active in recycling ascorbate and glutathione reductase regenerated glutathione from glutathione disulphide. The activity of these antioxidative enzymes in the cyanobacteria was sufficient to remove between 60-230 nmoles H2O2 .mg protein-1 min-1. It is suggested that in cyanobacteria an effective reaction sequence for removal of hydroperoxides involves ascorbate peroxidase and recycling of glutathione and ascorbate.

Ascorbate Peroxidases↗
PubMed1985

EPR signals of redox active copper in EDTA washed membranes of the cyanobacterium Synechococcus 6311.

A signal of Cu2+ (g = 2.03) was detected by electron paramagmetic resonance spectroscopy in oxidized membrane preparations of Synechococcus 6311. The membranes were prepared and washed in the presence of EDTA (10mM, pH 8.0) and, hence, were depleted of adventitious copper; the treatment also would remove any membrane-associated soluble redox proteins and other paramagnetic metal ions. 0.1% Triton X-100 facilitated detection of the Cu2+ signal which was fully reduced by dithionite or ascorbate plus N,N,N',N',-tetramethyl-p-phenylenediamine, and partially reduced NADPH and NADH, which are known to donate electrons to the terminal oxidase of cyanobacteria via the respiratory chain. Using temperature dependence and power saturation of the EPR copper signal, we conclude that copper is a firmly bound constituent of the terminal oxidase in an environment which is very similar if not identical to other cytochrome c oxidase preparations.

Copper↗
PubMed1985

Interaction of tyrosine residues with the chromophore in bacteriorhodopsin.

We previously reported that the absorption spectrum at low temperatures of iodinated bacteriorhodopsin can be separated into four components with maxima at shorter wavelengths than in native bacteriorhodopsin. In this study, the time course of the formation of each spectral component after iodination was analyzed, revealing that these four components correspond to four different iodinated states of tyrosine residues interacting with the retinal chromophore of bacteriorhodopsin. Therefore at least two tyrosine residues interact with the chromophore of bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1984

Light-induced proton gradients and internal volumes in chromatophores of Rhodopseudomonas sphaeroides.

To test the predictions of the chemiosmotic hypothesis, it is essential to have sensitive and accurate measures of the aqueous volume and pH within membrane compartments. One unique feature of the present investigation is the application of electron spin resonance probes to determine internal aqueous volume and pH changes in bacterial chromatophores under virtually identical conditions. Volumes of the chromatophores ranged from 6 to 16 microliter/mg bacteriochlorophyll among different preparations, and were sensitive to the osmolarity of the suspending buffer. pH gradients reached two units in illuminated chromatophores as determined with ESR methods, and increased when KCl and valinomycin were added to the assay. Measurements with the fluorescent dye 9-amino-acridine yielded similar pH gradients, provided that an operational vesicle volume, which corrected for the binding of the dye to the membrane, was used in the calculation. The sensitivity of the ESR method allowed the measurement of pH gradients resulting from only a few light flashes. A plot of pH gradients versus number of flashes was linear up to about 30 flashes, and intercepted the origin. This result is consistent with proton release into the bulk aqueous phase after only a single light flash. This ability to measure small pH gradients offers new opportunities for the study of energy-transducing mechanisms.

Bacterial Chromatophores↗
PubMed1984

Cross-linking of bacteriorhodopsin using specific carboxyl modifications and proteolytic cleavage.

Specific carboxyl modification of purple membrane using a water-soluble carbodiimide yielded a mixture of oligomers, revealed by gel electrophoresis. Purple membrane pre-treated with papain or trypsin, cleaving the C-terminal tail, showed the same pattern of cross-linked products. Chymotryptic cleavage released amino acids 1-72 (7kD fragment) from the cross-linked products, as it did with native membrane. The tail and helices A and B are not, therefore, involved in carbodiimide-promoted cross-linking. Similar cleavage of a hydrophobic dihydroquinoline-modified sample showed that mainly intra-molecular cross-linking occurs, with little cross-linking between the large and small chymotryptic fragments.

Bacteriorhodopsins↗
PubMed1984

The role of Na2S in anoxygenic photosynthesis and H2 production in the cyanobacterium Nostoc muscorum.

Na2S is known to support anoxygenic photosynthesis in some strains of cyanobacteria and to stimulate H2 production in N2 fixing filaments of Nostoc muscorum. We have shown electron transfer between Na2S and Photosystem I to be dependent on cytochrome b559 which was detected only in vegetative cells. An electron mediator was required to support Na2S driven nitrogenase activity in isolated heterocysts. Na2S was also found to deplete the ATP pool, probably by inhibiting electron transfer from Photosystem I.

Anaerobiosis↗
PubMed1984

Effect of specific carboxyl modifications on the blue acid species and O650 photocycle intermediate of bacteriorhodopsin.

Carboxyl groups were specifically modified in purple membranes using hydrophobic or hydrophilic activating reagents and endogenous or exogenous nucleophiles. Modification of surface residues using a negatively charged nucleophile had no effect on blue-species formation, whereas surface or internal carboxyl modification using an uncharged nucleophile prevented blue-species formation. The carboxyl-modified samples showed poor correlation between blue-species and O650 intermediate. These results indicate that formation of the blue-species requires negative surface charge and is not associated with the O650 intermediate of bacteriorhodopsin, as had been previously postulated.

Bacteriorhodopsins↗
PubMed1984

Surface charge of purple membranes measured by laser Doppler velocimetry.

Laser Doppler velocimetry measurements on purple membrane suspensions from Halobacterium halobium showed a linear correlation between electrophoretic mobility and applied electric field, electrokinetic responses could be rapidly monitored. Native membranes are less charged than white membrane preparations (from the R1mW mutant). Chemical modification of carboxyl residues reduces surface charge, and nitrotyrosine modified membranes are more or less charged than native membranes at pH greater than or less than 6.5, respectively. Changes in surface charge are found upon actinic illumination and are greatest (Ca 5 X 10(-4)/A2) under conditions where decay of the M412 intermediate of the photoreaction cycle is inhibited, such as at high pH or after chemical modification.

Bacteriorhodopsins↗
PubMed1984

Na+/H+ exchange in the cyanobacterium Synechococcus 6311.

The cyanobacterium Synechococcus 6311 adapts to grow in 0.6 M NaCl by developing an efficient system for sodium extrusion. In the present investigation cells loaded with NaC1 were subjected to a large dilution. Changes in fluorescence quenching of acridine orange as a function of transmembrane Na+ gradients provide evidence that Na+/H+ exchange activity greatly enhanced in salt-adapted cells.

Adaptation, Physiological↗
PubMed1984

Oxygen radical formation induced by gossypol in rat liver microsomes and human sperm.

Gossypol, a polyphenolic compound found in cotton plants, has many potential uses, including use as a male antifertility drug and spermicide. Gossypol affects a variety of cell processes and many of these effects may be explained by a common underlying mechanism. Here we report that gossypol promotes the formation of oxygen radicals when incubated with rat liver microsomes and human sperm suggesting that oxygen radical production may be the underlying basis of its biological activity.

Animals↗
PubMed1984

Effects of training and exhaustive exercise on the mitochondrial oxidative capacity of brown adipose tissue.

Oxidation of pyruvate, alpha-ketoglutarate, palmitoylcarnitine, succinate, and ferrocytochrome c by interscapular-brown-adipose-tissue (BAT) mitochondria of untrained and trained rats were measured at rest and after running to exhaustion. At rest, BAT mitochondria from trained rats showed significantly lower activities (less than 50%) for the oxidation of all the substrates. In untrained rats the activities of the enzymes for the oxidation of all the substrates except pyruvate and succinate were lower at exhaustion compared to the resting state when expressed on a per-gram-fresh-weight basis. In trained rats all of the enzyme activities increased as a result of exhaustive exercise. These differences between the two groups of rats in the post-exercise changes in oxidative capacities suggest that following an initial adaptation, resulting in a large decrease in mitochondrial oxidative activity, training protects the residual oxidative pathways against exercise-induced inactivation. These data show that unlike exposure to cold, or overfeeding, a physiological stimulus such as exercise reduces the oxidative capacity of BAT, and therefore may reduce the thermogenic activity of the tissue in endurance-trained rats as has been addressed in the scientific literature.

Adipose Tissue, Brown↗
PubMed1984

Exercise endurance-training alters vitamin E tissue levels and red-blood-cell hemolysis in rodents.

Muscle tissue levels of d1-alpha-tocopherol (vitamin E) were significantly lower in endurance-trained rats than in sedentary animals, whether the animals were fed on vitamin-E-deficient or control (vitamin-E-sufficient) diets. In vitamin-E-deficient rats, liver tissue levels of vitamin E were significantly lower in those that were endurance-trained than in those that were sedentary; this was not the case in control animals. In addition, for vitamin-E-deficient rats, the onset of red-blood-cell hemolysis in the sedentary animals occurred one week earlier than in the endurance-trained animals. Thus, it appears that training induces a protective effect against hemolysis despite vitamin E deficiency.

Animals↗