Contracted State as an Energy Source for Ca Binding and Ca + Inorganic Phosphate Accumulation by Corn Mitochondria

Abstract

Swnmary. An investigation has been made of the possibility of tutilizing the potential energy of the contracted state of corn mitochondria to drive Ca + inorganic phosphate accumulation. Contraction was obtained with succinate or NADH oxidation. In the succinate experiments the mitochondria were contracted in buffered KCl layered over sucrose in centrifuge tubes and centrifuged down through distinct wash, reactive and isotope exchange layers. In the NADH experiments, ion accumulation was initiated upon exhaustion of the substrate. The results show that mitochondria in the contracted state will actively bind some 45Ca, but no real accumtulation occurs until inorganic phosphate is available. Substrate powered contraction in the presence of inorganic phosphate also provides a potential for accumulation upon subsequent reaction of the mitochondria with Ca. It is deducted that contraction is due to X-I formation, to which Ca will bind. Subsequent reaction with inorganic phosphate produces CaX-P, which is the transport moiety. When X-P is formed first, Ca also reacts to produce CaX-P. Hence it is immaterial which ion reacts first with the contracted state. Contraction is believed to result from the action of a mechanoenzyme, presumably I-. The stability of CaX-I must be low for the mitochondria swell very rapidly tupon exhatustion of NADH or blocking of stuccinate oxidation by cyanide.Previotus papers from this laboratory have reported the characteristics of corn mitochondria in Ca + Pi accumulation and in swelling-contraction. Substrate-powered calcium accumulation is dependent upon phosphate, and phosphate acctumulation is dependent tupon calcium, or the related strontium ion which is about half as effective (10,25). The mitochondria show no pronounced tuncotupling responses to Ca; the small decline in P /O ratio with 1 myN Ca is due to diversion of phosphate from ATP formation into Pi acctumtulation (7).Swelling is spontaneous in buffered KCl, with rapid contraction not only with ATP and Mg btut also with the simple addition of an oxidizable substrate (23). The ATP-powered contraction is oligomycin sensitive and releases Pi, accounting for the bulk of the ATPase activity (23). The substrate powered contraction is very closely linked to respiration and is inhibited about 40 % by 1 mm phosphate (23,25). Calciuim releases the phosphate inhibition

Inhibition of Postoxidative Calcium Release in Corn Mitochondria by Inorganic Phosphate

“…A postenergized addition of phosphate leads to phosphate uptake and enhaced calcium retention. Oligomycin, KCN,2, or (4,6). As previously reported (4, 6), there is no discernible respiration after the point of reswelling.…”

“…Mitochondria were isolated from 3-day-old etiolated corn shoots (Zea mays L., WF9 X M14) in 400 mm sucrose, 5 mm EDTA, 50 mM KH2PO4, adjusted to pH 7.6 with tris as previously described (6), except that ADP was omitted from the washing medium. The Pi of the isolation medium was labeled with 2 mc of carrier-free "P in experiments involving endogenous Pi determination.…”

“…Some years ago Kenefick and Hanson (6) proposed that the contracted state of corn mitochondria produced by respiration in a KCI medium was associated with a conservation of energy which could be used in ion transport even after respiration ceased. Mitochondria contracted in the presence of Ca2`or Pi and centrifuged down through successive wash, reaction, and isotope exchange layers were shown to have accumulated 'Ca2+.…”

Effect of Ozone on Swelling of Tobacco Mitochondria

“…Swel,ling and contraction of mitochondria are known to be associated with phosphorylation and respiration (1,6,9,12,19,20,22). On this basis it i-s reasonable to assuime that the high 03 sensitivity of the phosphorylative system in tobacco leaf mitochondria which resulted in severe inhibition in oxidaitive phosphorylation (11) The positive coirrelation between the increasing loss of proteins, as wel!l as other substances absorbing at 260 and 280 imn, after removal of proteins, an>(l the length of expo-sure to 0. provides evidence for 03-'induce,d permeability of mitochondrial membrane (fig 3,4).…”

“…Since respiration and phosphorylation were reparted to be associaited wiith swelling and contraction of aniimal and plant mitochondria (1,2,6,9,12,19,20,22), kthe present study was undertaken 'to determine the effect of 03 on the contractili'ty and permeab.ility oif mitochondria isolated from tobacco plants. Although swelling and contracbion of animal mitoehondria have been extensively studied, charadterization of swelling and contraction 'in plant mitochondria has not been reported until recently (7,9,14,16,17,22). In this paper I report changes in swelilling and permeability of tobacco mitochondria as a resul!t of exposture to ozone.…”