<i>N</i>-Polymethylenecarboxymaleimides: probes for thiol groups associated with peripheral and integral membrane proteins

Griffiths, David G.; Partis, Michael D.; Moore, Anthony L.; Proudlove, Michael O.; Beechey, R. B.

doi:10.1042/bst0110753

Cited by 5 publications

(3 citation statements)

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“…Standard deviations from the mean for each point, which were generally within 5-10 % of the means, were not included, to avoid over-crowding of the data in the Figure. Regression coefficients for each line were all greater than 0.98. phenyl-maleimide inhibit pyruvate accumulation (Papa & Paradies, 1974;Thomas & Halestrap, 1981), there is a thiol group associated with the pyruvate transporter of plant mitochondria. Inhibition by the short-chain Nethylmaleimide, but not by AM1-4, occurs because the former freely partitions into the lipid domain of the membrane, whereas the latter are prevented from doing so by their polar carboxy group (Griffiths et al, 1983). It is interesting that a direct estimation of the effect of AM 11 on pyruvate transport by S. guttatum mitochondria showed a measurable inhibition of pyruvate uptake, but it was not as marked as seen for oxidation studies.…”

Section: Resultsmentioning

confidence: 98%

“…Several of the N-polymethylenecarboxymaleimides [trivial names 'acid maleimides' (AM) followed by a number denoting the number of methylene groups in the side chain (Griffiths et al, 1981)] were found to decrease pyruvate-dependent 02 consumption in A. maculatum mitochondria (Griffiths et al, 1983). These compounds allow the maleimide residue to penetrate into the membrane, the depth of penetration being determined by the length of the polymethylene chain, and uncontrolled entry into hydrophobic regions being prevented by the ionized carboxy group (Griffiths et al, 1983). The greatest inhibition was seen with AM 11 (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Pyruvate transport by thermogenic-tissue mitochondria

Proudlove

Beechey

Moore

1987

Biochemical Journal

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1. Mitochondria isolated from the thermogenic spadices of Arum maculatum and Sauromatum guttatum plants oxidized external NADH, succinate, citrate, malate, 2-oxoglutarate and pyruvate without the need to add exogenous cofactors. 2. Oxidation of substrates was virtually all via the alternative oxidase, the cytochrome pathway constituting only 10-20% of the total activity, depending on the stage of spadix development. 3. During later stages of spadix development, pyruvate oxidation was enhanced by the addition of aspartate. This was caused by acetyl-CoA condensing with oxaloacetate, produced from pyruvate/aspartate transamination, and so decreasing feedback inhibition of pyruvate dehydrogenase. 4. Pyruvate oxidation was inhibited by the long-chain acid maleimides AM5-11, but not by those with shorter polymethylene side groups, AM1-4. 5. The alpha-cyanocinnamate derivatives UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and CHCA [alpha-cyano-4-hydroxycinnamate] inhibited pyruvate-dependent O2 consumption and the carrier-mediated uptake of pyruvate across the mitochondrial inner membrane. Characteristics of non-competitive inhibition were observed for CHCA, whereas for UK5099 the results were more complex, suggesting a very low rate of dissociation of the inhibitor-carrier complex. 6. A comparison of the values of Vmax. and Km for oxidation and transport suggested that it was the latter which controls the overall rate of pyruvate oxidation by mitochondria from both tissues.

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Pyruvate transport by thermogenic-tissue mitochondria

Proudlove

Beechey

Moore

1987

Biochemical Journal

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“…A number of reports indicate that the acid maleimides are also impermeable to mitochondrial membranes while N E M is permeable (Moore et al, 1984;Griffiths et al, 1983Griffiths et al, , 1981. Succinate dehydrogenase is accessible from the inner side of the mitochondrial membrane.…”

Section: D~s C U S S R O~mentioning

confidence: 99%

Evidence for the existence of a channel in the glucose-specific carrier EIIGlc of the Salmonella typhimurium phosphoenolpyruvate-dependent phosphotransferase system

Robillard¹,

Beechey²

1986

Biochemistry

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The effect of membrane-impermeable sulfhydryl reagents on glucose-specific enzyme I1 ( E I P ) activity has been studied in Salmonella typhimurium whole cells and in properly sealed inverted cytoplasmic membrane vesicles. Glutathione N-hexylmaleimide and N-polymethylenecarboxymaleimides inactivate methyl a-D-glucopyranoside (a-MeGlc) transport and phosphorylation in whole cell preparations at a dithiol that can be protected by oxidizing reagents, trivalent arsenicals, or phosphorylation of EIIGIC. Accessibility to this activity-linked site is restricted to small apolar reagents or to polar reagents with a hydrophobic spacer between the polar group and the reactive maleimide moiety. These same reagents inactivate a-MeGlc phosphorylation in inverted cytoplasmic membrane vesicles. Inhibition results from reaction at a dithiol that can be protected by nonpermeant mercurials, oxidants, and arsenicals as well as by phosphorylation of EII. The characteristics of this site are virtually identical with those of the activity-linked dithiol elucidated in intact cells. No evidence could be found for a second activity-linked site on the other side of the membrane when the permeable reagent A-ethylmaleimide was used. Since only one activity-linked dithiol can be detected

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