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Biomedical subjects

E Lam

Publications and source records attributed to E Lam.

At least 91 records · Page 5Linked to original sources
PubMed1984

The effects of quinone analogues on cytochrome b6 reduction and oxidation in a reconstituted system.

The reconstituted system containing Photosystem I, plastocyanin and the cytochrome b6-f complex is used to study the effects of various quinone analogues on the redox behavior of cytochrome b6. The effects of DBMIB, DNP-INT and HQNO are compared in an attempt to discern the modes of action of these quinone analogues. Both DBMIB and DNP-INT are potent inhibitors of the plastocyanin reductase activity of the isolated cytochrome complex. However, while DBMIB abolished the oxidant-induced reduction of cytochrome b6, DNP-INT only inhibited about 25% of the net reduction. On the other hand, HQNO does not show any significant inhibition of plastocyanin reductase activity of the isolated cytochrome complex at concentrations up to 20 microM. An enhancement of the net amount of cytochrome b6 reduced is observed in the presence of HQNO. Both DNP-INT and HQNO inhibited the dark oxidation rate of cytochrome b6. The possible identity of the oxidant for cytochrome b6 is discussed. Plastoquinone is concluded to be the most likely candidate. DNP-INT is concluded to have at least two sites of inhibition in the cytochrome complex. The implications of these findings on quinone functions in the cytochrome b6-f complex are discussed.

Cytochrome b Group↗
PubMed1984

Isolation and Characterization of a Light-Harvesting Chlorophyll a/b Protein Complex Associated with Photosystem I.

A chlorophyll a/b protein complex has been isolated from a resolved native photosystem I complex by mildly dissociating sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chlorophyll a/b protein contains a single polypeptide of molecular weight 20 kilodaltons, and has a chlorophyll a/b ratio of 3.5 to 4.0. The visible absorbance spectrum of the chlorophyll a/b protein complex showed a maximum at 667 nanometers in the red region and a 77 K fluorescence emission maximum at 681 nanometers. Alternatively, by treatment of the native photosystem I complex with lithium dodecyl sulfate and Triton, the chlorophyll a/b protein complex could be isolated by chromatography on Sephadex G-75. Immunological assays using antibodies to the P(700)-chlorophyll a-protein and the photosystem II light-harvesting chlorophyll a/b protein show no cross-reaction between the photosystem I chlorophyll a/b protein and the other two chlorophyll-containing protein complexes.

Journal Article↗
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

Characterization of electron transfer from water to plastocyanin catalyzed by resolved electron transfer complexes from chloroplasts.

The photoreduction of soluble plastocyanin in a homologous reconstituted system requires a resolved Photosystem II preparation and the cytochrome b6-f complex from spinach chloroplasts. Oxygen evolution is linked quantitatively to plastocyanin reduction in the reconstituted system, indicating that water is the electron donor for the photoreduction. The photoreduction is sensitive to inhibitors which specifically interact with the Photosystem II preparation [DCMU: 3-(3,4-dichlorophenyl)-1,1-dimethylurea] as well as those which interact with the cytochrome complex (DNP-INT: 2-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenyl ether; and DBMIB: 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone). The plastocyanin requirement in the reconstituted system indicated a Km of 4.6 microM and a Vmax of 4.2 mumols plastocyanin reduced per nmol cytochrome f per h. Inhibition curves for DNP-INT and DBMIB show that both are effective inhibitors (50% inhibition at a ratio of inhibitor to cytochrome f of 1.0). The rate of reduction of plastocyanin was found to be stimulated by cations. This dependency was localized in the transfer of electrons from the Photosystem II preparation to the cytochrome complex. These results are considered in terms of organizational aspects of integral protein complexes in thylakoid membranes.

Catalysis↗
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

Reconstruction of the chloroplast noncyclic electron transport pathway from water to NADP with three integral protein complexes.

RECONSTRUCTION OF PHOTOSYNTHETIC NONCYCLIC ELECTRON TRANSPORT FROM WATER TO NADP HAS BEEN ACCOMPLISHED BY USING THREE INTEGRAL PROTEIN COMPLEXES ISOLATED FROM CHLOROPLAST THYLAKOID MEMBRANES: photosystems I and II and the cytochrome b(6)-f complex. This system shows an absolute dependence on the presence of all three protein complexes for NADP reduction, in addition to plastocyanin, ferredoxin, and ferredoxin-NADP reductase. The reconstructed system was found to be sensitive to low concentrations of known inhibitors of noncyclic electron transport. Depletion of the Rieske iron-sulfur center and bound plastoquinone from the cytochrome b(6)-f complex resulted in an inhibition of the photoreduction of NADP.

Journal Article↗
PubMed1981

Characterization of alpha-adrenoceptor populations. Quantitative relationships between cardiovascular effects initiated at central and peripheral alpha-adrenoceptors.

The agonist selectivities of central (medullary) and peripheral (vascular) alpha-adrenoceptors were compared in order to investigate a possible similarity among these two alpha-adrenoceptor populations. Linear regression equations were derived between the alpha-adrenergic potencies, mediated by these two types of alpha-adrenoceptors for 21 structurally dissimilar alpha-adrenoceptor agonists. Hypotensive potency after intravenous administration to anesthetized, normotensive rats was determined as a measure of central alpha-adrenergic activity and expressed as pC25, obtained from log dose-response curves. Peripheral alpha-adrenergic potency was quantified by means of the hypertensive effect elicited in pithed, normotensive rats after intravenous injections, yielding pC60 as the biological variable. A most significant linear relationship was generated between central hypotensive activity (pC25) and peripheral hypertensive potency (pC60), provided that log P' (octanol/buffer; pH 7.4, 37 degrees C) was included into the regression in a parabolic form. This result indicates that the central (medullary) alpha-adrenoceptors and the peripheral (vascular) alpha-adrenoceptor sites, which are excited by the drugs in question, make identical demands upon their agonists. The difference in accessibility to these peripheral and central alpha-adrenoceptor populations is adequately accounted for by a parabolic description in log P'. The apparent contradiction of this finding with the suggestion that central, hypotensive alpha-adrenoceptors are of the alpha 2 type and peripheral, vascular alpha-adrenoceptors belong to the alpha 1 subpopulation is discussed. The recent identification of an additional subclass of postsynaptic, vascular alpha 2-adrenoceptors and the lack of pronounced differential stimulating activity of the agonists at peripheral alpha-adrenoceptors may explain the present findings and clarify the paradox.

Animals↗
PubMed1981

Inhibition of the links between electron transfer and proton translocation in mitochondria.

The mechanism by which proton extrusion is linked to electron transfer in mitochondria was investigated by means of the primary amine-specific reagent fluorescamine, and of compounds obtained from the reaction of fluorescamine with simple amines (e.g. benzylamine) and with the mycosamine-containing antibiotic amphotericin B. The effect of these 'modifiers' (i.e. fluorescamine transfer chain were assayed separately using specific inhibitors to block the action associated with the other site. Both types of modifiers inhibited the proton extrusion across the membrane to a significantly greater extent than the electron transfer process in both sites II and III. In contrast, the lactone derivative (or cyclic form) of the amine-fluorescamine compounds had no significant inhibitory effect on the proton extrusion and its associated electron transfer. These results are consistent with the hypothesis that the link between proton extrusion and electron transfer in mitochondria is indirect in nature. The results show that: (a) the links involved in sites II and III are identical or very similar in nature; (b) a covalent modification of primary amino groups in the inner membrane is not essential for the expression of these differential inhibitory effects; (c) specific structural features in the amine-fluorescamine compounds, and in the mitochondria-fluorescamine derivatives, are crucial for the expression of the inhibitory effects. Our results contradict the 'redox loop' model of Mitchell, and are compatible with the proton pump concept for the linked proton translocation in oxidative phosphorylation.

Animals↗