The oxidation of reduced nicotinamide adenine dinucleotide, malate-pyruvate, and succinate by corn mitochondria in buffered 0.2 M KCl was determined as a function of divalent cations. Ni2+, Mg2+, Co2+, Ca2+, Mn2+, Sr2+, and Ba2+ stimulated reduced nicotinamide adenine dinucleotide oxidation in the absence of inorganic phosphate, with Ca2+ and Sr2+ having the greatest effect. Malate-pyruvate and succinate oxidation was stimulated by Ca2+, Ba2+, and Sr2+, but only in the presence of inorganic phosphate. Ca2+, Sr2+, and Ba2+ produced a simulated state 4 to state 3 transition with all three substrates, but only with malate-pyruvate and succinate was there a return to state 4. The order of divalent cation effectiveness suggests that the rate of water substitution from the cation inner coordination hydration sphere may be a rate-limiting step in certain mitochondrial reactions involving electron transport and phosphorylation.Plant mitochondria readily oxidize exogenous reduced nicotinamide adenine dinucleotide by a pathway stimulated by salts in general and by divalent cations in particular (6, 9). Hackett (6) found that the divalent cation stimulation was greater than that due to the ionic strength effect of monovalent salts. Associated with the respiratory stimulation was a light absorbancy increase, indicative of structural changes. Hackett suggested that the stimulation was due to either a greater NADH permeability or a release of some limiting step in the respiratory chain which might be the breakdown of high energy intermediates.The oxidation of NADH by corn mitochondria is stimulated by Ca2+ (17), but this is not true for malate-pyruvate or succinate oxidation (7). Only in the presence of inorganic phosphate is organic acid oxidation promoted and this can be attributed to the coupling of respiration to phosphate transport (10, 16). Hence, if Ca2+ is enhancing substrate permeability, it does so only for NADH. It seems likely that Hackett's second alternative is more probable; divalent cations release some rate-limiting step of the NADH oxidation pathway.The Ca2+-stimulated oxidation of NADH is accompanied by extra Ca2+ binding (10). When respiration ceases, the Ca2+ is released. Hanson and Miller (8) METHODSIsolation of Mitochondria. Mitochondria were isolated from 3-day-old corn shoots (Zea mays L., WF9 X M14) by a procedure similar to that used by Kenefick and Hanson (10). Corn seedlings were grown on paper toweling saturated with a 0.1 mm CaCl2 solution. Approximately 100 g of corn shoots were ground in an ice-cold mortar with 200 ml of 0.5 M sucrose, 50 mM tris-HCl (pH 7.5), 50 mm KH2PO4, and 5 mm disodium EDTA (pH 7.5). The homogenate was strained through four layers of cheesecloth and cleared of cell debris by centrifuging at 1,500g for 10 min, and the mitochondria were collected by centrifugation at 28,00g for 5 min. The mitochondria were then resuspended in a total of 50 ml of 0.4 M sucrose and again centrifuged at 1 ,500g for 10 min. The supernatant was decanted into clean tubes and 10 ml o...

Abstract. Respiration-linked contraction of corn mitochondria is not noticeably reduced by low, uncoupling concentr*tions of dinitrophenol. However, if a oontraction/respiration ratio is calculated, the contraction proves to be uncoupled. Previous statements that contraction cannot be uncoupled from respiration are in error.The uncoupling of c-ontraction is oonsistent with the concept that dinitrophenol attacks a primary non.phosphoryla-ted high energy intermediate (I,.X). It is proposed thot this intermediate is linked to some oontracti-le mechanism such that the degree of contraction reflects the level of intermediate.One of the more perplexing aspects of respiration-linked contraction in swollen mitochondria is the ineffeotiveness of uncoupling agents on the process. Our observations on 'corn mitochondria (2,18,19,21) show 'that only high concentrations of uncoupler (e.g. 100-500 FM DN'P2) wi,ll accelerate spontaneous swelling and reduce contraction. In a phosphorylating medium, isutch concenftrations alimost eliminate oxidative phosphorylation and strongly inhibit the respiration. Low concentrations of uncoupler (e.g. 10-30 KM DNP) which are adequate 'to depress P/O ratios, accelerate ATPase, release acceptorless respiration, and reduce Ca24 + Pi transport, have little or no effect on respirationlinked contraction. The same in(senisitivity is fouindl with animal mitochondria (1,4, 7).We proposed that contraction is closely linked to respiration and cannot be reduced by uncoupling agents un'less they depress respiration (21). When evidence was obtained supporting our contention that contraction is associated wiith energy conservation, presumably as I-X, the hypothetical nonphosphorylated high energy intermediate, a scheme was presented relating contraction to -the level of Ir--(13).Recenitly we have re-examined the action of DNP on contraction and find our previoous statements to be in error. Dinitrophenol does uncouple contraction if expressed on a respiration b)asis comparable to the P/O ratio. Furthermore, net contraction can be reduced by DN'P w'hille the respiration rate is stilil very high. The findings 'Supported by grants from the Atomic Energy -Commi,ssion

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