Kinetic Analysis of Corn Mitochondrial F₁-ATPase

Abstract

The activation and catalytic mechanism of corn mitochondrial F, were examined for the two distinct forms of the enzyme which appear upon storage in ammonium sulfate or glycerol. Apparently irreversible differences in the stability of the two active forms were found. Nucleosidetriphosphate induced activation of the enzyme was found to produce lasting effects on subsequent catalysis. These effects varied with both the nucleotide used for activation, and the hydrolyzed species. The substrate and metal specificity were examined with the ATP activated enzyme. Mg2 and Ca24 were found to be the most effective at promoting ATP hydrolysis. erties of the ATPases which catalyze the committed step in each pathway.In an accompanying study ofthis series, a method of obtaining purified corn mitochondrial F, was described and the subunit structure and immunochemical properties examined (18). In the current study, the activation and catalytic properties of the enzyme are examined. MATERIALS AND METHODSCorn mitochondrial F,, purified as described in the accompanying manuscript (18), was stored in 4 mm ATP, 1 mm EDTA (pH 7.0) either as a 50% (NH4)2SO4 slurry at 4°C or in 25% glycerol at -70C.Following a centrifugation and rinse step, the (NH4)2SO4 preparation was dissolved in an activation buffer consisting of 50 mM Tricine-NaOH, 0.1 mM EDTA (pH 8.0) and, unless otherwise specified, 5 mm ATP. The glycerol-stored enzyme was prepared by passage through a centrifuge column consisting of a l-ml plastic pipette tip packed with Bio-Rad P-6DG desalting gel and equilibrated with the activation buffer.In experiments dealing with NTP activation of the enzyme, activation time was measured relative to the time of dissolution or centrifugation into the activation buffer. All other assays were performed on enzyme that had been activated by incubation at 25°C for at least 1 h in the activation buffer with 5 mm NTP. Coupled assays of ATPase activity were performed at 30'C in a system consisting of 50 mM Tricine-NaOH (pH 8.0), 1.0 mm KCl, 2 mm phosphoenolpyruvate, 0.3 mM NADH, 12 units of pyruvate kinase, and 17 units lactate dehydrogenase in a total volume of 1 ml. Phosphate assays were performed in 50 mm Tricine-NaOH (pH 8.0 and 30C) and phosphate determined according to the method of Heinonen and Lahti (9). Protein concentrations were determined according to Bradford (3), using the Bio-Rad dye concentrate and fraction V BSA as a standard. Except as specified, all reagents were from Sigma. RESULTSA number of properties of the corn mitochondrial F,-ATPase were found to depend on whether the purified enzyme was stored as an (NH4)2S04 precipitate, or frozen in 25% glycerol. The ATPase activity of the glycerol-stored enzyme was generally higher and less subject to decay than the (NH4).SO4-stored material. While the former retained an activity of up to 8 Amol min-' mg-' (at 5 mm ATP) for 2 months, the activity of the precipitated enzyme decayed from -3 to 0.5 over the same period. Kinetic analyses ofan (NH4)2S04-stored preparation over this ...

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

“…Perhaps the 13-subunit of the Fj-ATPase is more sensitive to inhibition by NBD-Cl than the membrane-bound (native) ATPase (150 _ 50 ,gM) (Table IV). This is consistent with the idea that NDB-Cl binds to the catalytic site in the (3-subunit, and the conformational changes resulting in increased affinity for ATP may also (30), and trypsin inhibitor (21). The estimates of the molecular masses of the 6 and e subunits (not detectable on this gel) were made on 7.5 to 15% acrylamide gels (not shown).…”

“…Overall, the mitochondrial F,-ATPase from oat roots is comparable to the F,-ATPase derived from corn seedlings (21) and from mammalian sources (2). However, several discrepancies exist between the F, isolated from corn and that from oat roots.…”

“…HCO3 stimulates the mitochondrial ATPase from beef heart by decreasing Km.3 (the lowest affinity of three ATP binding sites) as well as by increasing V..x (8,30). It is likely that a similar mechanism for HCO3-stimulation operates in the plant mitochondrial ATPase (21). The small amount of chloride-stimulated (azide-sensitive) activity present in the native mitochondrial preparation may be due to collapse of membrane potential and not due to a specific interaction with the ATPase.…”

“…Also, the substrate and anion specificity of the purified F,-ATPase is consistent with that of the native membrane-bound enzyme of oat roots. In the purified mitochondrial ATPase from oat roots, ATP is clearly favored as a substrate over GTP, in contrast to the corn ATPase where GTP is preferentially hydrolyzed over ATP (by 3-to 4-fold) (21). Without knowing the substrate specificity of the native corn mitochondria, we cannot evaluate whether the oat ATPase is different from the corn enzyme or if the corn mitochondrial F,-ATPase has been altered during isolation or subsequent storage, as suggested by Partridge et al (21).…”

The plant mitochondrial F1‐ATPase

“…While the enzymes obtained from maize (Zea mays) [1,2], faba bean (Viciafava) [3], oat root (Avena sativa) [4] and cuckoo pint (Arum maculatum) [5], like that from mammalian sources, contain five subunits, those from sweet potato root (lpomoea batatas) [6,7], pea cotyledon (Pisum sativum) [8,9] and turnip (Brassica napus) [10] have six subunits. ATPase subunits in the five subunit preparations are designated on the basis of Lheir mobility in SDS polyacrylamide gel electrophoresis as o~, /3, y, 6, and e with approximate molecular masses of 55000, 52000, 30000, 20000 and 8000 with variations depending on the tissue source.…”