Phosphate-induced Stimulation of Acceptorless Respiration in Corn Mitochondria

Abstract

In a recent paper (11) we suggested that high state 4 respiration rates in corn mitochondria are linked to energy expenditure in phosphate transport. State 4 respiration was considerably augmented by polycations which served to increase phosphate transport, producing swelling. It seems to be widely accepted that energy-linked ion transport in mitochondria is a process capable of releasing coupled respiration (3,20,21), although it is sometimes pointed out that the stoichiometry is disturbing (3), and that permeant anions are required (20 MATERIALS AND METHODS Mitochondria. Corn mitochondria (Zea mays L., WF9(Tms) X M14) were isolated and assayed as previously described (11). Final suspension of the mitochondria was in the same medium as used for the basic reaction mixture: 200 mm sucrose, 10 mm TES buffer, 1 mm MgSO,, 1 mg/ml bovine serum albumin, adjusted to pH 7.6 with KOH. Mitochondria concentrations were adjusted to approximately 10 mg protein/ml, with protein determinations corrected for the BSA of the suspending medium. Reaction vessels contained 4.4 ml of the basic medium plus 0.1 ml of mitochondria suspension (about 1 mg protein), with additives as indicated in figures and tables. Temperature was 28 or 29 C. Recordings of 02 consumption and percentage of transmission were as previously described (1 1). ATP Measurements. Two-ml samples were withdrawn from the reaction vessel in the spectrophotometer before and 1 min after adding 300 nmoles ADP. In determination of adenylate kinase blanks the same procedure was followed, but initial additions were made of 20 /M rotenone, 3 tM antimycin A, and 20 ,ug oligomycin, and addition of NADH was omitted. The samples were added to 3 ml of 20 mm glycine buffer, pH 7.7, in a boiling water bath. After 15 min the tubes were cooled in an ice bath, and the denaturated mitochondria were centrifuged down at 5000g for 10 min. Aliquots of the extract were assayed for ATP by a luciferase assay (15) using an

Potassium and Phosphate Uptake in Corn Roots

“…If the main driving forces, A I and ApH generated by electrogenic proton pumps (10) are used for K+ and Pi uptake, respectively, it should be possible to differentiate the energycoupling requirements for these two ions either by changing the proton gradient across the membrane or by chemically modifying the transport carrier. Accordingly, several chemical probes such as the ATPase stimulator, FC2 (18), the inhibitor, DES (2), the OH-/Pi antiporter inhibitor, mersalyl (8,16) 4 h, or more, are "stable" in their physiological activities (7,20), most of the experiments were conducted using washed tissues.…”

Kaempferol Inhibitions of Corn Mitochondrial Phosphorylation

“…If the site of DNP inhibition is associated with the dissipation of I X, then it seems unlikely that the pumping of K+, which results in contraction, in a KCI medium is involved directly with I~X. Hanson et al (8) suggest this schematically where they show the outpumping of K+ to be associated with the proton motive force generated from respiration and not from the dissipation of I -X.…”

“…This transport is inhibited by mersalyl and can be followed via percentage transmittance changes (8). When comparative studies of the effects of mersalyl and kaempferol were conducted, kaempferol was found to have no effect on the transport of phosphate.…”

“…The uptake of phosphate to produce the high energy phosphorylated intermediate is a coupled event that is inhibited by mersalyl (4, 5, 19) and can be followed spectrophotometrically as mitochondrial swelling in a sucrose-KCl reaction medium (8). This swelling was inhibited by mersalyl (8, Fig.…”

“…These include the oligomycinsensitive site in some way associated with X P (3), the DNPtsensitive site most likely associated with a proton-motive force or the nonphosphorylated intermediate (2,8,15), or the mersalyl-sensitive site associated with phosphate transport (4,5,8,19). Additional studies of the effects of kaempferol on calcium transport and the substrate, or ATP-induced contraction of mitochondria swollen in KCI are also possible.…”