Artificial reductant enhancement of the Lowry method for protein determination

Larson, Eric M.; Howlett, Bruce; Jagendorf, André T.

doi:10.1016/0003-2697(86)90432-x

Cited by 308 publications

(112 citation statements)

References 6 publications

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“…Mitochondria were prepared from potato tubers and purified by density-gradient centrifugation exactly as described in [20]. Mitochondria1 protein was determined as described in [23].…”

Section: Materials and Methods Preparation Of Mitochondriamentioning

confidence: 99%

Localisation of the Sites of Action of Cadmium on Oxidative Phosphorylation in Potato Tuber Mitochondria Using Top‐Down Elasticity Analysis

Kesseler

Brand

1994

European Journal of Biochemistry

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The aim of this study was to identify the significant sites of action of cadmium on oxidative phosphorylation in potato tuber mitocondria. We simplified the system to three convenient subsystems linked via the production or consumption of a common intermediate, namely protonmotive force. The three subsystems were substrate oxidation, which produces protonmotive force, and the proton leak reactions and the phosphorylation reactions, which consume protonmotive force. By measuring the effect of cadmium on the kinetic response of each subsystem to protonmotive force (top-down elasticity analysis), we found that cadmium stimulated proton leak reactions and strongly inhibited substrate oxidation, but had no measurable effect on the phosphorylation reactions. Cadmium therefore decreases the amount of ATP produced/oxygen consumed (the effective P/O ratio) not by inhibiting the phosphorylation reactions directly, but by inhibiting the production of protonmotive force and by diverting proton flux from phosphorylation reactions to the proton leak reactions.The toxicity of heavy metals on aquatic and terrestrial organisms has become a major topic of toxicological research over recent decades, in particular because heavy metal concentrations have considerably increased in the environment, predominantly due to accelerated industrial contamination and air pollution [l, 21. The various heavy metals show similar characteristics. They are easily taken up into living organisms, where they accumulate in several tissues [3-61, due to a long biological half-life [7]. Once incorporated, heavy metals cause a variety of cytotoxic reactions within living cells [3-71 and affect essential metabolic pathways in plant cells [l, 6, 81. In plants, the overall result of the heavy metal impact is a decrease in growth rate and finally death [9]. This wide range of toxicity is explained by the chemical characteristics of heavy metals ; the most characteristic feature of heavy metals like cadmium, mercury and lead is a unique high affinity for the free electron pairs of SH groups [3-61. SH groups are important in enzyme function, either oxygen consumption by the substrate oxidation reactions) ; J p , rate of oxygen consumption required to pump protons out at the rate equal to the rate of proton return through the phosphorylation system; JL, rate of oxygen consumption required to pump protons out at a rate equal to the rate of proton return through the proton leak; E+ overall elasticity coefficient of a block of reactions to the common intermediate dp; the subscripts and superscripts S, P and L refer to the following three blocks of reactions: the substrate oxidation, the phosphorylation system and the proton leak branch, respectively ; Ph,MeP+, methyltriphenylphosphonium cation.Enzyme. Hexokinase; type 111: from baker's yeast (EC 2.7.1.1.).by determining structure and conformation or by direct involvement in the catalysed metabolic reaction in the active centre of the enzyme. Therefore heavy metals, as SH group reagents, affect most metabolic pathw...

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“…Mitochondria were prepared from potato tubers and purified by density-gradient centrifugation exactly as described in [20]. Mitochondria1 protein was determined as described in [23].…”

Section: Materials and Methods Preparation Of Mitochondriamentioning

confidence: 99%

Localisation of the Sites of Action of Cadmium on Oxidative Phosphorylation in Potato Tuber Mitochondria Using Top‐Down Elasticity Analysis

Kesseler

Brand

1994

European Journal of Biochemistry

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“…The fluorescence values were converted into mol equivalents of ce6 by comparison with a calibration curve constructed with known amounts of each of the conjugates. The protein content of the entire cell extract was then determined by a modified Lowry (Larson et al, 1986) method using bovine serum albumin dissolved in 0.1 M NaOH per 1% SDS to construct calibration curves. The trypsin supernatant was also checked for the presence of fluorescence, which was negligible.…”

Section: In Vitromentioning

confidence: 99%

In vivo fluorescence imaging of the transport of charged chlorin e6 conjugates in a rat orthotopic prostate tumour

et al. 1999

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Summary Polymeric drug conjugates are used in cancer therapy and, varying their molecular size and charge, will affect their in vivo transport and extravasation in tumours. Partitioning between tumour vasculature and tumour tissue will be of particular significance in the case of photosensitizer conjugates used in photodynamic therapy, where this partitioning can lead to different therapeutic effects. Poly-l-lysine chlorin e6 conjugates (derived from polymers of average M r 5000 and 25 000) were prepared both in a cationic state and by poly-succinylation in an anionic state. A fluorescence scanning laser microscope was used to follow the pharmacokinetics of these conjugates in vivo in an orthotopic rat prostate cancer model obtained with MatLyLu cells. Fluorescence was excited with the 454-528 nm group of lines of an argon laser and a 570 nm long pass filter used to isolate the emission. Results showed that the conjugates initially bound to the walls of the vasculature, before extravasating into the tissue, and eventually increasing in fluorescence. The anionic conjugates produced tissue fluorescence faster than the cationic ones, and surprisingly, the larger M r conjugates produced tissue fluorescence faster than the smaller ones with the same charge. These results are consistent with differences in aggregation state between conjugates.

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“…The O, concentration in air-saturated H,O at 25°C was assumed to be 240 p~. Mitochondrial protein was quantified by a modified Lowry assay (Larson et al, 1986).…”

Section: Lsolation and Assay Of Leaf Mitochondriamentioning

confidence: 99%

Alternative Oxidase Activity in Tobacco Leaf Mitochondria (Dependence on Tricarboxylic Acid Cycle-Mediated Redox Regulation and Pyruvate Activation)

et al. 1995

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Transgenic Nicofiana tabacum (cv Petit Havana SR1) containing high levels of mitochondrial alternative oxidase (AOX) protein due t o the introduction of a sense transgene(s) of Aoxl, the nuclear gene encoding AOX, were used t o investigate mechanisms regulating AOX activity. After purification of leaf mitochondria, a large proportion of the AOX protein was present as the oxidized (covalently associated and less active) dimer. High AOX activity in these mitochondria was dependent on both reduction of the protein by DTT (to the noncovalently associated and more active dimer) and its subsequent activation by certain a-keto acids, particularly pyruvate. Reduction of AOX t o its more active form could also be mediated by intramitochondrial reducing power generated by the oxidation of certain tricarboxylic acid cycle substrates, most notably isocitrate and malate. Our evidence suggests that NADPH may be specifically required for AOX reduction. All of the above regulatory mechanisms applied to AOX in wild-type mitochondria as well. Transgenic leaves lacking AOX due t o the introduction of an Aoxl antisense transgene or multiple sense transgenes were used to investigate the potential physiological significance of the AOXregulatory mechanisms. Under conditions in which respiratory carbon metabolism is restricted by the capacity of mitochondrial electron transport, feed-forward activation of AOX by mitochondrial reducing power and pyruvate may act t o prevent redirection of carbon metabolism, such as to fermentative pathways.

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Artificial reductant enhancement of the Lowry method for protein determination

Cited by 308 publications

References 6 publications

Localisation of the Sites of Action of Cadmium on Oxidative Phosphorylation in Potato Tuber Mitochondria Using Top‐Down Elasticity Analysis

Localisation of the Sites of Action of Cadmium on Oxidative Phosphorylation in Potato Tuber Mitochondria Using Top‐Down Elasticity Analysis

In vivo fluorescence imaging of the transport of charged chlorin e6 conjugates in a rat orthotopic prostate tumour

Alternative Oxidase Activity in Tobacco Leaf Mitochondria (Dependence on Tricarboxylic Acid Cycle-Mediated Redox Regulation and Pyruvate Activation)

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