Uncoupling of Respiration-Linked Contraction in Corn Mitochondria

Self Cite

Kaempferol (3,5,7,4'tetrahvdroxyflavone) inhibited the rate of state 3 substrate oxidation, but not the state 4 rate. This, along with the kaempferol inhibition of substrate-driven calcium-phosphate deposition, provided evidence that kaempferol was acting specifically on the phosphorylation mechanism and not on electron transfer. Kaempferol, however, did not inhibit ATP-driven contraction while oligomycin did. Comparisons of kaempferol with mersalyl indicated that kaempferol did not inhibit phosphorylation by blocking phosphate transport. Both kaempferol and 2, 4-dinitrophenol inhibited calcium-phosphate transport, but kaempferol did not stimulate respiration to the extent that 2, 4-dinitrophenol did under acceptorless conditions. Kaempferol had no effect on NADHdriven contraction in a potassium chloride reaction medium. The site of kaempferol effect is thus seen to be unique from oligomycin and more like aurovertin, likely acting before the formation of the phosphorylated high energy intermediate, but not as an uncoupler in the traditional 2,4-dinitrophenol mode.Flavonoid compounds have been reported to be distributed widely throughout the plant kingdom (11). Despite this wide distribution, little evidence has been presented linking their occurrence with a role in growth or metabolism. Indirect evidence suggesting a role in dormancy and in root and shoot growth has been presented by Harborne (11). It was determined that kaempferol (3,5,7,4'-tetrahydroxyflavone) was a cofactor for in vitro IAA oxidase while quercetin (3,5,7,3',4'-pentahydroxyflavone) inhibited the activity of the enzyme (6). Stenlid (16) conducted an extensive survey of the effects of many flavonoids on ATP production by mitochondria isolated from several plant species. From this work it is evident that minor changes in the hydroxyl pattern affect the degree of inhibition of ATP formation. The work of Stenlid (16) chloroplasts (20). Kaempferol glucosides were a major component of the flavonoids quantitated.Galston (7) has hypothesized from data with quercetin and kaempferol glucosides that flavonoids could play a broadly significant role in contractile and membranous phenomena in plants. Such possibilities would involve transport mechanisms associated with the production and utilization of high energy bonds, most probably as ATP and would thus suggest a possible interaction of mitochondria and flavonoids under certain conditions in vivo. In vitro mitochondrial experiments in which these interactions are studied should be useful in the furtherance of hypotheses such as Galston's (7).When the specific effects of kaempferol on mitochondrial mechanisms for oxidative phosphorylation are being considered, several sites of action are possible as evidenced in studies with other inhibitors. 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 phosphat...

Section: Discussionsupporting

confidence: 69%

“…This is somewhat surprising since DNP has been reported to reduce the contraction of isolated corn mitochondria in KCI (10). However, in the previous experiments of Hanson et al (10), the reaction media contained phosphate and Ca2 .…”

Section: Discussionmentioning

confidence: 88%

Kaempferol Inhibitions of Corn Mitochondrial Phosphorylation

Koeppe

Miller²

1974

Self Cite

“…The buffer employed in the isolation of plant mitochondria is either phosphate (39)(40)(41)45), or tris-HCl (25,26,33,34,36), or both (17,22,31,32). Recently, Drury et al (14)(15)(16) Plant Physiol.…”

Section: Discussionmentioning

confidence: 99%

“…27,40), cysteine is often added to the grinding medium (5, 6, 13-16, 24-26, 28-30, 33-36, 40, 41). Several workers, on the other hand, do not employ cysteine in their isolation media atall (17,22,31,32,39,45). In our experiments with mung bean mitochondria to clarify this discrepancy in theliterature, we found that the deletion of cysteine from the grinding medium altogether did not damage the mitochondrial activity (unpublished results).…”

Section: Discussionmentioning

confidence: 99%

Necessary Conditions for Isolation of Tightly Coupled Higher Plant Mitochondria

Ikuma

1970

Recently, a number of workers have reported the isolation of mitochondria from various higher plant tissues, which show good respiratory and phosphorylative activities (2, 5, 6, 11-17, 21-36, 38-45). The method in general consists of tissue disruption and homogenization in an appropriate medium, isolation of the mitochondrial fraction from filtered tissue homogenate by means of differential centrifugation, and final washing of the resultant mitochondrial pellet. If the techniques of isolation are compared in detail, however, a wide variety of differences is seen in the method of tissue disruption, in the composition of isolation media, and in the patterns of differential centrifugation. In fact, the same group sometimes changes the procedure with little explanation for the modification. In addition, there are several reports indicating that the method successfully employed in one laboratory does not apply to another laboratory (cf. 34,39, 40,45). These situations may reflect the fact that all the parameters which enter into the conditions of mitochondrial isolation have not been exhaustively studied.Another point which does not appear in the literature frequently is the fact that plant mitochondria age faster than animal mitochondria (cf. 16, 28, 34). In addition, the yield of mitochondria, calculated on the basis of initial fresh weight, is usually much less from higher plant tissues than that from animal tissues. This necessitates the use of a large quantity of tissue

“…ATP synthesis was sensitive to the electron transport inhibitor KCN, the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and the coupling factor inhibitor oligomychr. Experi Studies from several laboratories have shown that oxidative phosphorylation in plant mitochondria is similar in many respects to that in animal mitochondria (12,13,15). While intact plant and animal mitochondria have been well characterized, they are not ideal systems for studying the mechanism or the control of oxidative phosphorylation.…”

Section: Introductionmentioning

confidence: 99%

Oxidative Phosphorylation in Pea Cotyledon Submitochondrial Particles

Grubmeyer

Melanson²,

Duncan³

et al. 1979

Mitochondria and submitochondrial particles (SMP) from pea cotyledoDs were shown to catalyze oxidative phosphorylation as measured by 32p; uptake into pbosphate esters. ATP synthesis was sensitive to the electron transport inhibitor KCN, the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and the coupling factor inhibitor oligomychr. Experi Studies from several laboratories have shown that oxidative phosphorylation in plant mitochondria is similar in many respects to that in animal mitochondria (12,13,15). While intact plant and animal mitochondria have been well characterized, they are not ideal systems for studying the mechanism or the control of oxidative phosphorylation. Access of all charged substrates to the inner matrix is governed by specific carriers that may, under some conditions, control apparent substrate affinities and maximal rates of oxidative phosphorylation (29).In contrast, the use of submitochondrial particles is particularly advantageous in that substrates in solution have direct access to the catalytic site on the ATPase-ATP synthetase enzyme complex. In this report, pea cotyledon SMP are shown to catalyze high rates of succinate and NADH oxidation, phosphorylate ADP, and perform an ATP-Pi exchange reaction. The kinetic parameters for ATP synthesis were determined from analysis of these data. In addition, the properties, including sensitivities to inhibitors, of the SMP are reported. MATERIALS AND METHODSPea seeds (Pisum sativum L. cv. Homesteader) were germinated at 27 C in the dark for 4 days in Vermiculite. Cotyledons (1,200 ml) were harvested and mitochondria prepared as previously reported (25). The mitochondria were resuspended in 60 ml of 0.25 M sucrose and centrifuged at 20,000g for 7 min. The dark brown pellet was cleaned of a light brown layer by suction and the mitochondria (approximately 250 mg of protein) were resuspended in 10 ml of 0.25 M sucrose.For the preparation of SMP, the mitochondria could either be used immediately or stored overnight at -20 C without change in the yield or activity of the resulting SMP preparation.The above mitochondrial suspension was diluted to 60 ml in a beaker with ice cold SMP buffer (0.25 M sucrose, 50 ims Tes, brought to pH 7.0 at 20 C with Tris). The beaker was placed in an ice bath and sonicated for two 1-min bursts, separated by a 1-min