Saroj Joshi

Activation of potassium-dependent H+ efflux from mitochondria by cadmium and phenylarsine oxide.

Addition of Cd2+ or phenylarsine oxide (PhAsO) to respiring rat liver mitochondria results first in acidification of the medium (H+ efflux) followed by disappearance of H+ (discharge of the pH gradient or uncoupling). The first phase of H+ efflux is dependent upon the presence of K+ in the medium, and is not seen in the presence of valinomycin, which is consistent with the conclusion that H+ efflux is linked to membrane potential-dependent uptake of K+. These effects are abolished by low levels of 2,3-dimercaptopropanol but potentiated by excess of 2-mercaptoethanol, showing involvement of a dithiol type of group in the response. Mersalyl produces only the H+ efflux, and subsequent addition of Cd2+ or PhAsO produces collapse of the ΔpH.

Isolation of a highly active H+-ATPase from beef heart mitochondria.

The lysolecithin extraction procedure originally described by Sadleret al. (1974) has been modified to yield a H+-ATPase with high levels of Pi-ATP exchange activity (400–600 nmol × min−1 × mg−1). This activity is further enhanced (1400–1600 nmol × min−1 × mg−1) following sucrose density gradient centrifugation in the presence of asolectin. This enhancement results in part from a lipid-dependent activation and in part from removal of inactive complexes. The H+ translocating activity of the complex has been determined spectrophotometrically using binding of oxonol VI as an indicator of membrane potential. Pi-ATP exchange, ATP hydrolysis, and oxonol binding are sensitive to energy-transfer inhibitors (oligomycin, rutamycin) and/or uncouplers (DNP, FCCP).

Elimination of thiol reagent interference during Lowry protein determination

Abstract Pretreatment of protein samples containing mono- or dithiol reagents with N -ethylmaleimide significantly reduces interference during subsequent protein determinations by the Lowry method. When the Lowry procedure as modified by M. A. K. Markwell et al. ( Anal. Biochem. 87 , 206) is utilized, the effectiveness of N -ethylmaleimide pretreatment is not influenced by a variety of substances including detergents, lipids, EDTA, sucrose, and salts. The combination of the modified procedure of Markwell et al. with N -ethylmaleimide pretreatment is rapid, easily applied to a large number of samples, and equally applicable to both soluble and membranous protein samples.

[48] Purification of coupling factor B

Publisher Summary This chapter describes the coupling factor B activity as assayed by measuring the stimulation of anaerobic ATP-driven NAD reduction by succinate in ammonia ethylenediaminetetraacetic acid (AE) particles at 38 ° C. The particles exposed to ethylenediaminetetraacetic acid (EDTA) at pH 8.5–8.8 are selectively deficient in factor B, unlike particles depleted at higher pH. Factor B can be isolated equally satisfactorily from heavy, light, or mixed mitochondria. Isolation of mitochondria from beef heart tissue has been revised to give better yields and better coupled preparations. Respiratory control ratios approaching 3.0 are obtained using glutamate plus malate as substrate. The steps of isolation of factor B isolation are preparation of mitochondrial acetone powder, crude extract, diethylaminoethyl-cellulose (DEAE-C) chromatography, purification on CM-cellulose, and molecular sieving, on Sephadex G-75-40. Factor B purified also appears to be homogeneous, showing a single weakly stained band with Coomassie blue in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) corresponding to a molecular weight of approximately 13,000–15,000 daltons.

Formation of an intramolecular disulfide bond in the mitochondrial adenine nucleotide translocase

Adenine nucleotide translocase in electron transport particles or in H+‐ATPase preparation from bovine heart mitochondria is capable of forming both inter‐ and intramolecular disulfide bridges upon reaction with copper‐o‐phenanthroline. We have examined the localisation of the intramolecular disulfide bridge in the protein chain by peptide fragmentation methods. The most likely position of the disulfide bridge is between cysteine 159 and 256, but the possibility of the presence of a second disulfide bridge formed between 129 and 256 cannot be ruled out. Our experimental results support the theoretical model proposed [(1982) FEBS Lett. 144, 250‐254] for the topography of the translocase and provide a more accurate description of the arrangement of some of the hydrophilic segments in the molecule.

Monoclonal antibodies to mitochondrial coupling factor B

Two monoclonal antibodies (MAb 1 and IV) have been prepared which showed high and specific reactions towards bovine heart mitochondrial coupling factor B (FB). Both have been identified as sub‐type IgG1 of mouse immunoglobulins. MAb I reacts with purified and functionally active fB, alkylated or oxidized forms of FB and even with peptides formed on digestion of FB with trypsin. When used together, MAb I and IV reacted with FB in immunoblots of normal and urea treated samples of mitochondria, submitochondrial particles, ammonia‐EDTA extracted particles, and H+‐ATPase. Both MAbs inhibited FB‐stimulated ATP‐dependent reverse electron flow activity when fB was incubated with the antibody either before or after its addition to FB‐deficient AE‐particles. Reactivity of MAb I towards fB declined upon exposure of fb to guanidine HCl while reactivity of MAb IV remained unaltered.

Occurrence of proteins immunoreactive with anti-coupling factor B in phosphorylating membrane preparations

Coupling factor B has been isolated from beef heart mitochondria, apparently in multiple forms which differ in molecular weight and specific activity. Since it has no known intrinsic catalytic activity, detection and quantitation have been based upon the factor B-dependent stimulation of ATP-linked activities in factor B-deficient sub-mitochondrial particles. This communication reports the development of a reliable and more universally applicable enzyme-linked immunosorbent assay (ELISA) for detection and quantitation of factor B in soluble or membranous preparations. The assay requires nanoliter volumes of rabbit antiserum raised against purified factor B and will detect nanogram amounts of the coupling factor. Analysis of beef heart submitochondrial particles using a competitive binding ELISA indicated a factor B content of 0.27 nmol/mg protein, making factor B stoichiometric with F1 (0.3--0.6 nmol/mg). Furthermore, application of the factor B ELISA has indicated the presence of material cross-reacting with the beef heart factor B-antiserum in phosphorylating membranes from chloroplasts, Escherichia coli, Paracoccus denitrificans and the thermophilic bacterium, PS3. Negative results were obtained with mitochondria and microsomes from rat liver, purple membranes from Halobium halobacterium and sarcoplasmic reticulum from rabbit skeletal muscle.

ATP synthase complex from bovine heart mitochondria: The oligomycin sensitivity conferring protein is essential for dicyclohexyl carbodiimide-sensitive ATPase

The requirement of bovine heart mitochondrial oligomycin sensitivity conferring protein (OSCP) in conferring dicyclohexylcarbodiimide (DCCD)-sensitivity to membrane-bound F1 was investigated by using OSCP-depleted membrane fraction (UF0) of ATP synthase. The ATPase activity of UF0-F1 was completely insensitive to DCCD while that of UF0-F1-OSCP was inhibited 95% by 16 microM DCCD. Both UF0-F1 and UF0-F1-OSCP complexes bound 5 nmol [14C]DCCD/mg UF0, and all the radioactivity was found to be associated with the DCCD-binding proteolipid. The data suggest that OSCP may be necessary for transmitting not only energy-linked signals, but also signals induced by F0 inhibitory ligands in mitochondrial energy transduction.

Further observations on coupling factor A

The isolation, purification and characterization of coupling factor A from beef heart mitochondria has been reported [l-3]. Its subunit composition is similar to that of Fr-ATPase [4]. These studies revealed that factor A is a different form of mitochondrial Fr-ATPase [5]. It stimulates the activity of urea-depleted sub~tochond~al particles in oxidative phospho~lation coupled to NADH or succinate oxidation, ATP-supported reversed electron flow, and Pi-ATP exchange. It differs from Fi-ATPase in that it is cold-stable, and the ATPase specific activity is latent, although it can be enhanced to match Fr -ATPase activity by exposing it to elevated temperatures. It has been suggested in the past that the lower ATPase activity of factor A may result from the presence of mitochondrial ATPase inhibitor [6] in amounts sufficient to mask most of the enzyme activity [7,8]. Analysis of factor A and Fr-ATPase for in~bitor content showed that both had roughly the same ~~bitor content (0.4 mol1340 000 g ATPase protein) but only Fr had high ATPase activity [3]. However, there remained the possibility that the inhibitor was bound to a non-specific, non-functional site in Fr , but it was located at its proper site in factor A leading to masking of the ATPase activity. The only way to overcome this criticism is to prepare factor A in a form that had zero inhibitor content. It was found [9] that membrane bound, as well as soluble Fr and Fi-X [IO] preparations form a fluorescent complex with the antibiotic inhibitor, aurove~in. They reported that a factor A preparation did not form such a complex with aurovertin and further proposed that it was either dissociated into

Restoration of Pi-ATP exchange in the oligomycin-sensitive ATPase: effect of a coupling factor.

Summary P i -ATP exchange activity has been restored to the bovine heart mitochondrial ATPase. The exchange rates can be doubled by addition of coupling factor 1 (F 1 and the oligomycin-sensitivity conferring protein (OSCP), and further stimulated 2·5-fold by the addition of a coupling factor which has Factor B as a major component. The activity is sensitive to oligomycin, uncoupler or valinomycin + K + .