The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.     

Some effects of benomyl and carbendazim on Aphis fabae and Acyrthosiphon pisum on field bean (Vicia faba)

Field bean plants were treated with benomyl in a glasshouse, then nymphs of Aphis fabae and Acyrthosiphon pisum were caged on the second pinnate leaf. Soil drenches at concentrations of 150 and 75 μg benomyl/ml or above increased mortality of A. fabae and A. pisum respectively; 250 μg a.i./ml increased mortality and decreased progeny production of alate A. fabae. The effect on mortality persisted for at least 16 days after treatment. Foliar sprays increased mortality at concentrations of 75 μg a.i./ml and above (A. fabae). Field populations of A. pisum were reduced when bean plants were drenched or sprayed at a concentration of 250 μg a.i./ml and A. fabae populations were reduced by drenches but not by foliar sprays. A commercial formulation of carbendazim (Bavistin) increased aphid mortality whereas the formulation medium did not. Under the experimental conditions, benomyl affected the distribution of both species on young bean plants but did not induce a repellent effect; aphids preferred untreated leaves. Mortality and preference tests, and a field experiment, indicated that A. pisum was affected more than A. fabae.     

Dibutylchloromethyltin chloride, a covalent inhibitor of the adenosine triphosphate synthase complex

1. The synthesis of dibutylchloromethyltin chloride, a new covalent inhibitor of the mitochondrial ATP synthase [oligomycin-sensitive ATPase (adenosine triphosphatase)] complex is described, together with a method for preparing dibutylchloro[(3)H]methyltin chloride. 2. Studies with the yeast mitochondrial oligomycin-sensitive ATPase complex show that dibutylchloromethyltin chloride inhibits both the membrane-bound enzyme and also the purified Triton X-100-dispersed preparation. 3. F(1)-ATPase is not inhibited even at 500nmol of dibutylchloromethyltin chloride/mg of protein, and the general inhibitory properties are similar to those of triethyltin, oligomycin and dicyclohexylcarbodi-imide, known energy-transfer inhibitors of oxidative phosphorylation. 4. Binding studies with yeast submitochondrial particles show that dibutylchloromethyltin chloride antagonizes the binding of triethyl[(113)Sn]tin, indicating that there is an interaction between the two inhibitor-binding sites. 5. Unlike triethyltin, inhibition by dibutylchloromethyltin chloride is due to a covalent interaction which titrates a component of the inner mitochondrial membrane present at a concentration of 8-9nmol/mg of protein. 6. All of the labelled component can be extracted with chloroform/methanol (2:1, v/v), and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the chloroform/methanol extract indicates that the labelled component has an apparent mol.wt. of 6000-8000. However, t.l.c. reveals the presence of only one labelled component which is lipophilic and non-protein and is distinct from the free inhibitor, mitochondrial phospholipids and the dicyclohexylcarbodi-imide-binding protein (subunit 9). 7. Inhibition of mitochondrial ATPase and oxidative phosphorylation is correlated with specific interaction with a non-protein lipophilic component of the mitochondrial inner membrane which is proposed to be a co-factor or intermediate of oxidative phosphorylation.     

Cross-linking of protein by ω-maleimido alkanoylN-hydroxysuccinimido esters

A series of chemical bifunctional cross-linking reagents, the succinimidyl maleimides, has been synthesized. Using hemoglobin as a model protein, it has been shown that these molecules react rapidly with sulfhydryl groups and more slowly with amino groups. The result is a high degree of specific cross-linking between the intramolecular subunits.     

Interaction of the dibutylchloromethyltin chloride binding site with the carbodiimide binding site in mitochondria

An examination of the effect of dibutylchloromethyltin/chloride on the carbodiimide binding proteolipid of mitrochondrial ATPase has revealed that in the presence of the alkyltin, (1) binding of dicyclohexycarbodiimide is decreased (2) the electron spin resonance spectrum of a nitroxide analogue of dicyclohexylcarbodiimide exhibits line broadening characteristic of either an increase of polarity or a decrease in viscosity of the carbodiimide binding site (3) the rate of reduction of the nitroxide probe by ascorbate is increased threefold. These phenomena suggest a possible mode of action for the inhibition of ATP synthesis by alkyltins.