Arnost Horak

Monoclonal Antibodies to the [alpha]- and [beta]-Subunits of the Plant Mitochondrial F1-ATPase

We have generated nine monoclonal antibodies against subunits of the maize (Zea mays L.) mitochondrial F1-ATPase. These monoclonal antibodies were generated by immunizing mice against maize mitochondrial fractions and randomly collecting useful hybridomas. To prove that these monoclonal antibodies were directed against ATPase subunits, we tested their cross-reactivity with purified F1-ATPase from pea cotyledon mitochondria. One of the antibodies ([alpha]-ATPaseD) cross-reacted with the pea F1-ATPase [alpha]-subunit and two ([beta]-ATPaseD and [beta]-ATPaseE) cross-reacted with the pea F1-ATPase [beta]-subunit. This established that, of the nine antibodies, four react with the maize [alpha]-ATPase subunit and the other five react with the maize [beta]-ATPase subunit. Most of the monoclonal antibodies cross-react with the F1-ATPase from a wide range of plant species. Each of the four monoclonal antibodies raised against the [alpha]-subunit recognizes a different epitope. Of the five [beta]-subunit antibodies, at least three different epitopes are recognized. Direct incubation of the monoclonal antibodies with the F1-ATPase failed to inhibit the ATPase activity. The monoclonal antibodies [alpha]-ATPaseD and [beta]-ATPaseD were bound to epoxide-glass QuantAffinity beads and incubated with a purified preparation of pea F1-ATPase. The ATPase activity was not inhibited when the antibodies bound the ATPase. The antibodies were used to help map the pea F1-ATPase subunits on a two-dimensional map of whole pea cotyledon mitochondrial protein. In addition, the antibodies have revealed antigenic similarities between various isoforms observed for the [alpha]- and [beta]-subunits of the purified F1-ATPase. The specificity of these monoclonal antibodies, along with their cross-species recognition and their ability to bind the F1-ATPase without inhibiting enzymic function, makes these antibodies useful and invaluable tools for the further purification and characterization of plant mitochondrial F1-ATPases.

Coupling factor activity of the purified pea mitochondrial F1-ATPase

The pea cotyledon mitochondrial F1-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose/2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF1-ATPase. The molecular weights of pea mitochondrial F1-ATPase and pea chloroplast CF1-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F1-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF1-ATPase. The purified mitochondrial F1-ATPase exhibited coupling factor activity. In spite of the observed differences between CF1 and F1, the mitochondrial enzyme stimulated ATP formation in CF1-depleted pea chloroplast membranes. Thus, the mitochondrial F1 was able to substitute functionally for the chloroplast CF1 in reconstituting photophosphorylation.

Subunit composition and cold stability of the pea cotyledon mitochondrial F1-ATPase

Abstract The F 1 -ATPase isolated from pea cotyledon submitochondrial particles contained six types of subunit with molecular weights of 57 000 (α), 55 000 (β), 36 500 (γ), 26 500 (δ), 22 500 (δ′) and 8000 (e). The same polypeptide composition was observed even when the purification was carried out at 4°C in the presence of proteolytic inhibitors, suggesting that the sixth subunit was not a proteolytic product formed during the isolation procedure. The six-subunit F 1 -ATPase exhibited considerable cold stability: it retained 65% of its activity after 24 h of incubation at 0°C and was more than 90% active after 48 h of incubation at 4°C. The 26.5 kDa protein could be dissociated from the remaining F 1 -ATPase by centrifugation in a linear sucrose gradient containing (NH 4 ) 2 SO 4 and deoxycholate. The resulting five-subunit F 1 -ATPase was considerably less stable at 0°C than the six-subunit enzyme. Several features suggest the possibility of a functional and structural relationship between the 26.5 kDa protein of the pea cotyledon mitochondria and the mammalian oligomycin-sensitivity-conferring protein.

Restoration of ATP synthesis in urea-treated membranes prepared from pea cotyledon mitochondria

Submitochondrial particles were prepared from pea cotyledon mitochondria by sonication in a medium containing 5 mM MgCl2. The resulting particles (Mg2+-submitochondrial particles) catalyzed oxidative phosphorylation at the rate of 100–200 nmol ATP formed / min per mg protein. Treatment of Mg2+-submitochondrial particles with 3.0 M urea resulted in a preparation of highly resolved particles with low ATPase activity and no capacity for oxidative phosphorylation. However, the resulting membranes were not capable of reconstitution of oxidative posphorylation with the purified mitochondrial F1-ATPase. Urea particles capable of reconstitution of oxidative phosphorylation could be prepared by extracting Mg2+-submitochondrial particles with concentrations of urea ranging from 1.7 to 2.0 M. We have used 1.9 M urea for large-scale preparation of urea particles that could be stored in liquid nitrogen without any loss of reconstitution capacity. The residual oxidative phosphorylation rate of these particles was 6–8 nmol ATP / min per mg protein and this rate could increase to 60–70 nmol ATP / min per mg protein on incubation with saturating amounts of purified mitochondrial F1-ATPase. In contrast to the mitochondrial F1, purified activated pea chloroplast CF1 was unable to stimulate ATP synthesis in 1.9 M urea particles.

Interaction of a methylene diphosphonate analog of ADP with photosynthetic membranes of chloroplasts

Summary The ADP analog α,β-methylene adenosine 5′-diphosphate was photophosphorylated to α,β-methylene adenosine 5′-triphosphate by spinach chloroplasts. The phosphorylation of the analog was not due to nucleotide diphosphokinase activity. The ADP analog was binding to the nucleotide binding sites on the photosynthetic membranes as well as to the solubilized coupling factor protein. Binding of ADP to the photosynthetic membranes was decreased in the presence of the ADP analog indicating a direct interaction of the ADP analog with the ADP binding sites on the thylakoids.

Purification of the subunits of pea mitochondrial F1-ATPase by high-performance liquid chromatography.

Abstract The subunits of the F1-ATPase from pea cotyledon mitochondria were purified by reversed-phase chromatography. The resolution of the subunits was affected by several chromatographic parameters: a reversed-phase C8 column was superior to the less hydrophobic Bio-Gel TSK Phenyl-5-PW column for the resolution of the subunits, acetonitrile was more suitable for good separation of the subunits than 2-propanol and the flow-rate had a significant effect on peak height but little effect on the column resolving power. Tandem chromatography on two reversed-phase chromatography columns with different hydrophobicities was used in an attempt to isolate F1-ATPase subunits directly from soluble proteins extracted from submitochondrial particles.

Reconstitution of Oxidative Phosphorylation in Pea Cotyledon Submitochondrial Particles

We have described the purification and subunit composition of the mitochondrial F1-ATPase from pea cotyledons1. The purified F1-ATPase stimulated ATP formation in CF1-depleted pea chloroplast membranes1. In this report, we describe the preparation of mitochondrial membranes which have low rates of residual oxidative phosphorylation and show that the ATP formation in these membranes is stimulated on incubation with the purified mitochondrial F1-ATPase.