Isolation and Antigenic Characterization of Corn Mitochondrial F₁-ATPase

Abstract

Corn mitochondrial F1-ATPase was purified from submitochondrial particles by chloroform extraction. Enzyme stored in ammonium sulfate at 40C was substantially activated by ATP, while enzyme stored at -70'C in 25% glycerol was not. Enzyme in glycerol remained fully active (8)(9) micromoles Pi released per minute per milligram), while the ammonium sulfate preparations steadily lost activity over a 2-month storage period. The enzyme was cold labile, and inactived by 4 minutes at 60'C. Treatment with octylglucoside resulted in complete loss of activity, while vanadate had no effect on activity. The apparent subunit molecular weights of corn mitochondrial F,-ATPase were determined by SDSpolyacrylamide gel electrophoresis to be 58,000 (a), 55,000 (8), 35,000 (-y), 22,000 (a), and 12,000 (e). Monoclonal (9). No detailed comparisons of mitochondrial F,-ATPase and chloroplastic CF,-ATPase from the same plant species has as yet been accomplished, and the similarities or differences between these enzymes can only be inferred by studies ofanimal mitochondrial Fr-ATPases.The fact that plant cells contain two unique organelles capable of energy production by oxidative phosphorylation in one case, or photosynthetic phosphorylation in the other, makes them an attractive model for comparative studies. Detailed studies of plant mitochondrial F,-ATPase will provide the basis for comparison to chloroplastic CF,-ATPase. Comparisons of these two enzymes from the same cell may provide valuable information on aspects of structure, regulation, biosynthesis, and evolution of the enzyme within these two organelles. This paper reports the isolation, purification, physical and enzymic properties of the F,-ATPase from corn mitochondria. In addition, the antigenic properties ofthis enzyme are compared to those from CF,-ATPases and mamallian and E. coli F,-ATPases using both polyclonal and monoclonal antibodies. MATERIALS AND METHODS Purification of

Purification and Characterization of the Soluble F1-ATPase of Oat Root Mitochondria

“…Occasionally, we have detected a polypeptide slightly larger than 6 on SDS-PAGE which may correlate to 6', as described by Iwasaki and Ashai (14). We estimate e to be 14 kD, in agreement with that obtained for the esubunit of corn F1-ATPase (26).…”

“…The structural characteristics are similar to those described for other higher plants (14,26). We find that the inhibitor and modulator sensitivities of the purified F,-ATPase from oat roots are similar to the native mitochondrial enzyme.…”

Nucleotide Sequence of the F1-ATPase α Subunit Gene from Maize Mitochondria

“…The F, is the best characterized segment of the ATPase complex, consisting of five subunits located in the matrix of mitochondria. These F, subunits exist in a 3 a:3 (l:1 y:2 6:1 e stoichiometric ratio (26). Between the membrane-bound Fo and the matrix F, is the oligomycin-sensitivity-conferring protein.…”

Purification and Characterization of Mitochondrial F1-ATPase fromLilium longiflorumBulbs

“…Hinomoto were stored in a refrigerator until use. Submitochondrial particles (SMP) were prepared from the bulbs by the method of Spitsberg et al 5 ) with the exception of the addition of insoluble polyvinylpyrrolidone (5%, w/w) to the homogenization. All these procedures were done at 4°C.…”

“…F cATPase was dissolved from SMP at room temperature by CHCl 3 treatment. 5) The solubilized F 1-ATPase was further purified by gel filtration and anion exchange column chromatography4) using Fractogel HW-55F and Fractogel DEAE-650M at room temperature. ATPase activity was assayed by colorimetric measurement of Pi released during the 10 min incubation at 30°C using Fiske-Subbarow reagent.…”