Respiratory chain-linked nicotinamide adenine dinucleotide dehydrogenase. XVIII. Action of sulfhydryl inhibitors on different forms of the respiratory chain-linked reduced nicotinamide-adenine dinucleotide dehydrogenase

Gutman, Menachem; Mersmann, Harry J.; Luthy, James A.; Singer, Thomas P.

doi:10.1021/bi00815a016

Cited by 67 publications

(14 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Both methods were not affected by nonspecific binding effects and gave consistent and reliable results. We found no influence on our FQT measurements by a number of treatments including activation of complex I (46) and addition of bovine serum albumin or the thiol reagent N-ethylmaleimide, which were claimed to affect inhibitor binding (48,50).…”

Section: Discussionmentioning

confidence: 94%

See 1 more Smart Citation

Three Classes of Inhibitors Share a Common Binding Domain in Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase)

Okun¹,

Lümmen²,

Brandt³

1999

Journal of Biological Chemistry

306

259

View full text Add to dashboard Cite

We have developed two independent methods to measure equilibrium binding of inhibitors to membranebound and partially purified NADH:ubiquinone oxidoreductase (complex I) to characterize the binding sites for the great variety of hydrophobic compounds acting on this large and complicated enzyme. Taking Competition experiments consistently demonstrated that all tested hydrophobic inhibitors of complex I share a common binding domain with partially overlapping sites. Although the rotenone site overlaps with both the piericidin A and the capsaicin site, the latter two sites do not overlap. This is in contrast to the interpretation of enzyme kinetics that have previously been used to define three classes of complex I inhibitors. The existence of only one large inhibitor binding pocket in the hydrophobic part of complex I is discussed in the light of possible mechanisms of proton translocation.

show abstract

Section: Discussionmentioning

confidence: 94%

“…50 Values from Steady State Kinetics-To test for the activity of complex I in our SMP preparation, we determined the Michaelis-Menten parameters for NADH and NBQ. The K m values were 3.9 Ϯ 0.5 M for NADH and 2.3 Ϯ 0.2 M for NBQ.…”

mentioning

confidence: 99%

Three Classes of Inhibitors Share a Common Binding Domain in Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase)

Okun¹,

Lümmen²,

Brandt³

1999

Journal of Biological Chemistry

306

259

View full text Add to dashboard Cite

show abstract

“…Sulfhydryl group modification has been shown to profoundly alter the biological properties of membranes including the permeability characteristics of heart mitochondria, fat cells and erythrocyte membranes [7,15,17,23,27,30] as well as the activity of membrane bound ATPases [31] and a respiratory-chain linked NADH dehydrogenase [10]. Despite the number and variety of such studies, the mechanism by which sulfhydryl reagents disrupt these membrane functions has thus far remained elusive, although the implicit conclusion has been that sulfhydryl group integrity is somehow required for biological function [11].…”

mentioning

confidence: 99%

Modification of the erythrocyte membrane by sulfhydryl group reagents

Godin

Schrier

1972

J. Membrain Biol.

View full text Add to dashboard Cite

In this study, the consequences of modification of human erythrocyte membrane sulfhydryl groups by N-ethyl maleimide (NEM), 5,5'dithiobis-(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuriphenyl sulfonate (PHMPS) were investigated. These reagents differ in chemical reactivity, membrane penetrability and charge characteristics.Results of sulfhydryl modification were analyzed in terms of inhibitory effects on activities of five membrane enzymes; Mg(++)- and Na(+), K(+)-ATPase, K(+)-dependent and independentp-nitrophenyl phosphatase (NPPase) and DPNase. Structural considerations involved in the sulfhydryl-mediated inhibition were evaluated by studying the changes in susceptibility to sulfhydryl alteration produced by shearing membranes into microvesicles and by the addition of the membrane modifiers, Mg(++) and ATP.Conclusions from the data suggest that the effects of NEM appeared to result from modification of a single class of sulfhydryls; DTNB interacted with two different sulfhydryl classes. Increasing concentrations of PHMPS resulted in the sequential modification of many types of sulfhydryls, presumably as a result of increasing membrane structural disruption. DTNB and PHMPS caused solubilization of about 15% of membrane protein at concentrations giving maximal enzyme inhibition.In contrast to the usually observed parallels between Na(+), K(+)-ATPase and K(+)-dependent NPPase, activities of Mg(++)-ATPase, Na(+), K(+)-ATPase and K(+)-dependent NPPase varied independently as a result of sulfhydryl modification. We suggest complex structural and functional relationships exist among these components of the membrane ATP-hydrolyzing system.Our studies indicate that the effects of sulfhydryl group reagents on these membrane systems should not be ascribed to sulfhydryl modificationper se, but rather to the resulting structural perturbations. These effects depend upon the structural characteristics of the particular membrane preparation studied and on the chemical characteristics of the sulfhydryl group reagent used.

show abstract

“…This concept may be helpful in elucidating the terminal electron transfer step in complex I and seems to be consistent with the existence of two EPR-detectable species of complex I-associated ubisemiquinones (11,12). Some experimental results with ordinary complex I inhibitors (13)(14)(15)(16) can be explained by assuming the existence of more than one inhibitor (or ubiquinone) binding site.…”

mentioning

confidence: 62%

Specificity of Pyridinium Inhibitors of the Ubiquinone Reduction Sites in Mitochondrial Complex I

Miyoshi

Iwata

Sakamoto

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

I) 1 is a large enzyme that catalyzes the oxidation of NADH by ubiquinone coupled to proton translocation across the inner membrane (1, 2). There are a variety of inhibitors of mitochondrial complex I and with the exception of a few inhibitors which inhibit electron input into the enzyme (3, 4), all inhibitors act at or close to the ubiquinone reduction site (5). Among the inhibitors, positively charged neurotoxic N-methyl-4-phenylpyridinium (MPP ϩ ) and its alkyl analogues exhibit unique inhibitory behavior with bovine heart mitochondrial complex I (6). A series of studies of the inhibition mechanism of MPP ϩ analogues by Singer and colleagues (6 -10) have suggested that MPP ϩ analogues are bound at two sites in the enzyme, one accessible to relatively hydrophilic inhibitors (termed the "hydrophilic site") and one shielded by a hydrophobic barrier on the enzyme (the "hydrophobic site"), and that occupation of both sites is required for complete inhibition. This concept may be helpful in elucidating the terminal electron transfer step in complex I and seems to be consistent with the existence of two EPR-detectable species of complex I-associated ubisemiquinones (11,12). Some experimental results with ordinary complex I inhibitors (13-16) can be explained by assuming the existence of more than one inhibitor (or ubiquinone) binding site.In the previous study (17), we synthesized a series of MPP ϩ analogues which are much more potent than the original MPP ϩ and demonstrated that the presence of hydrophobic counteranion tetraphenylboron (TPB Ϫ ) potentiates the inhibition by MPP ϩ analogues differently depending upon the molar ratio of TPB Ϫ to the inhibitors. In the presence of a catalytic amount of TPB Ϫ , the inhibitory potency of MPP ϩ analogues was markedly enhanced, and the extent of inhibition was almost complete. The presence of an excess amount of TPB Ϫ partially reactivated the enzyme activity, and the inhibition was partly saturated (ϳ50%). This complicated inhibitory behavior could be explained by the dual binding sites model mentioned above (6), which supposes quite different hydrophobic natures of the two sites and/or their environments.If there are indeed two distinct binding sites of MPP ϩ analogues in bovine complex I, there should be specific inhibitors which act selectively at one of the two proposed binding sites since it is unlikely that the structural properties of the two sites are completely identical. We have synthesized such a selective inhibitor, MP-6 (N-methyl-4-[2-(p-tert-butylbenzyl)-propyl]pyridinium, Fig. 1) (17). In the absence of TPB Ϫ , this inhibitor showed approximately 50% inhibition at 5 M in NADH-Q 1 oxidoreductase assay, but the inhibition reached a plateau at this level over a wide range of concentrations. Weak inhibition was again observed above ϳ80 M, and maximum inhibition (Ͼ90%) was obtained only when the concentration of the inhibitor was increased to ϳ250 M. Such a marked biphasic nature of the does-response curve has not been reported previously for usual complex I i...

show abstract

Respiratory chain-linked nicotinamide adenine dinucleotide dehydrogenase. XVIII. Action of sulfhydryl inhibitors on different forms of the respiratory chain-linked reduced nicotinamide-adenine dinucleotide dehydrogenase

Cited by 67 publications

References 27 publications

Three Classes of Inhibitors Share a Common Binding Domain in Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase)

Three Classes of Inhibitors Share a Common Binding Domain in Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase)

Modification of the erythrocyte membrane by sulfhydryl group reagents

Specificity of Pyridinium Inhibitors of the Ubiquinone Reduction Sites in Mitochondrial Complex I

Contact Info

Product

Resources

About