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

Abstract

We have generated nine monoclonal antibodies against subunits of the maize (Zea mays 1.) 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 F,-ATPase from pea cotyledon mitochondria. One of the antibodies (a-ATPaseD) cross-reacted with the pea F,-ATPase asubunit and two (8-ATPaseD and 8-ATPaseE) cross-reacted with the pea Fl-ATPase j3-subunit. This established that, of the nine antibodies, four react with the maize a-ATPase subunit and the other five react with the maize 8-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 a-subunit recognizes a different epitope. Of the five 8-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. lhe monoclonal antibodies a-ATPaseD and 8-ATPaseD were bound to epoxideglass QuantAffinity beads and incubated with a purified preparation of pea Fl-ATPase. lhe ATPase activity was not inhibited when the antibodies bound the ATPase. lhe 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 a-and 8-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 F,-ATPases.Electrogenic H+-ATPases have been found in nearly a11 physiological membrane systems investigated. Each of these membrane-bound ATPases can be placed into one of severa1 groups. The three most common types of proton ATPases are E1-E2 type, Fo-F1 type, and the microsomal type (AlAwqati, 1986). The E1-E2 type ATPases are found in yeast and funga1 plasma membranes and the gastric plasma and microsomal membranes. ATPases of the microsomal type are located in the membranes of Golgi apparatus, ER, endosomes,

Expression profiles of respiratory components associated with mitochondrial biogenesis during germination and seedling growth under normal and restricted conditions in wheat

“…Monoclonal antibodies against AOX (Elthon et al, 1989) and ATP synthase subunit A (ATPA) (Luethy et al, 1993) were gifts from T. Elthon (University of Nebraska, USA). Anti-NAD9 polyclonal antibody against the wheat protein (Lammatina et al, 1993) was a gift from J. M. Grienenberger (Strasbourg University, France).…”

Proteomic Characterization of A Triton-Insoluble Fraction from Chloroplasts Defines A Novel Group of Proteins Associated with Macromolecular Structures

“…56 The quaternary structure and organization of the plastid PDC is largely unknown because the plastid PDC, unlike the mitochondrial form, dissociates rapidly in vitro. 57 The presence of all four subunits in the membrane-depleted 30k × g fraction characterized here (bands [17][18][19][20][21][25][26][27] indicates this complex can be isolated under rapid extraction conditions for further structural analyses (Table 2). Since this complex sedimented at relatively low centrifugal forces association with other enzymes is possible and may signal the presence of a larger, metabolic enzyme assembly in plastids including the heteromeric acetyl-CoA carboxylase ( Table 2).…”

Arabidopsis Photorespiratory Serine Hydroxymethyltransferase Activity Requires the Mitochondrial Accumulation of Ferredoxin-Dependent Glutamate Synthase

“…Membranes were blocked for 2 h at room temperature with blocking buffer (5% powdered milk, 1% PBS, pH 7.4, and 0.05% Tween 20). Membranes were incubated for at least 4 h with primary rabbit polyclonal antibodies against SHMT (Agrisera), Fd-GOGAT (Agrisera), or PSBA (Agrisera) or mouse monoclonal antibodies against a-tubulin (SigmaAldrich) or the b-subunit of maize (Zea mays) F1-ATPase (Luethy et al, 1993). Excess antibodies were removed by washing the blot twice for 15 min in 1% milk, 13 PBS, and 0.1% Tween 20 and twice for 15 min in 1% PBST.…”