An analysis of the binding of fluorescence probes in mitochondrial systems

Williams, W.P.; Layton, Derek; Johnston, Carol S.

doi:10.1007/bf01869510

Cited by 15 publications

(4 citation statements)

References 26 publications

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“…178 ability to conserve energy derived from electron transport as measured by the ANS-fluorescence response (Table 1). This response is purported to monitor the energization of the mitochondrial inner membrane, which occurs during substrate oxidation (Azzi, & Montecucco, 1976;Gains & Dawson, 1976;Williams et al, 1977). In our experiment it can there-mitochondria maintain the energy-linked fluorescence response for a longer time than they maintained respiratory control.…”

Section: Resultsmentioning

confidence: 67%

Effect of chronic ethanol administration on energy metabolism and phospholipase A2 activity in rat liver

Spach¹,

Parce²,

Cunningham³

1979

Biochemical Journal

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1. For a period of 31 days male rats were given a liquid diet containing 36% of its energy as ethanol. Liver mitochondria from these animals demonstrated lowered respiratory control with succinate as substrate, a diminished energy-linked anilinonaphthalene-sulphonic acid fluorescence response, and lowered endogenous ATP concentrations. The phospholipid/protein ratio in mitochondria from these animals was unchanged; only minor alterations in the phospholipid fatty acid composition were observed. 2. In experiments where mitochondria were incubated at 18 degrees C in iso-osmotic sucrose (aging experiments), the above energy-linked properties were lost at an earlier time in organelles from ethanol-fed animals. Phospholipase A2 acitivty was depressed in mitochondria from control animals until respiratory control was lost and ATP was depleted. In contrast, no lag in the expression of phospholipase activity was observed in mitochondria from ethanol-fed rats. This loss of control of the phospholipase resulted in an earlier degradation of membrane phospholipids under the conditions of the aging experiments. 3. The ATPase (adenosine triphosphatase) activities, measured in freshly prepared tightly coupled mitochondria and in organelles uncoupled with carbonyl cyanide p-trifluoromethoxyphenylhydrazone, were not significantly different in ethanol-fed and liquid-diet control animals. When the mitochondria were aged at 18 degrees C, the activity increased with time of incubation in organelles from both groups of animals. A lag was observed, however, as the ATPase activity increased in control preparations. This lag was not present as APTase activity increased in mitochondria from ethanol-fed animals. 4. The significantly lowered values observed for energy-linked functions with succinate as an energy source demonstrate that ethanol elicits an alteration in liver mitochondria that affects the site II-site III regions of the oxidative-phosphorylation system. The apparent lack of control of the phospholipase A2 and ATPase activities in mitochondria from ethanol-fed animals suggests that the membrane microenvironment of these enzymes has been altered such that they can exert their catabolic effects more readily under conditions of mild perturbation. The fatty acid analyses demonstrate that the observed alterations both in the energy-linked functions and in control of the phospholipase and ATPase are not mediated through changes in the acyl chain composition of bulk-phase phospholipids.

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Section: Resultsmentioning

confidence: 67%

Effect of chronic ethanol administration on energy metabolism and phospholipase A2 activity in rat liver

Spach¹,

Parce²,

Cunningham³

1979

Biochemical Journal

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“…It is likely that the phospholipase, once expressed, contributes to the irreversible loss in mitochondrial metabolic functions by hydrolyzing membrane phospholipids. Figure 1 suggests that the phospholipase may contribute to the loss in the energy-linked Ans response, a property which is purported to monitor the energization of the mitochondrial inner membrane (Azzi & Montecucco, 1976;Gains & Dawson, 1976;Williams et al, 1977). Indeed, in carrying out this study we have noticed by comparing mitochondrial preparations that the rate of loss of the Ans fluorescence response is proportional to the rate of emergence of the phospholipase A activity.…”

Section: Discussionmentioning

confidence: 74%

Mitochondrial phospholipase A₂ activity and mitochondrial aging

Parce¹,

Cc²,

Waite³

1978

Biochemistry

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The changes in mitochondrial phospholipid metabolism and energy-linked functions have been followed as coupled mitochondria are allowed to age in isotonic sucrose at 18 degrees C. Analysis of the aging process has provided an approach for studying the structure--function relationships within the mitochondrion without adding external agents to perturb the membrane structure. The initial event observed in this process of deterioration is a loss of respiratory control which is paralleled by diminishing levels of ATP. As ATP levels decline, so do the rates of reacylation of monoacyglycerophosphorylethanolamine and fatty acid oxidation. In most cases the previously inactive phospholipase A2 (EC 3.1.1.4, phosphatide-2-acyl-hydrolase) begins rapid hydrolysis of membrane phosphatidylethanolamine as ATP levels approach zero. The final energy-linked phenomenon observed to decline is the anilinonaphthalenesulfonic acid fluorescence response. Evidence is presented which suggests strongly that the activity of the mitochondrial phospholipase A2 on endogenous phospholipids is suppressed in tightly coupled mitochondria. This suppression is temporally linked to ATP levels in the mitochondria. Furthermore, this study demonstrates that mitochondria which are only slightly damaged have the potential to effect membrane repair through reacylation of monoacyl phospholipids.

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“…Moreover, in the case of Ans a sharp pH optimum was found at 6.1 in contrast to the transport of H+ or Rb+. Because Ans responses are highly pH dependent and certain ambiguities exist in the interpretation of the Ans signal (Aiuchi et al, 1977;Williams et al, 1977), carbocyanine dyes (Waggoner, 1976) were used in this study. Essentially identical data were obtained using either diethyl oxadicarbocyanine (DOCC) or Di S-C3-( 5); hence we will only describe the results using DOCC.…”

Section: B Probes Of Potentialmentioning

confidence: 99%

Quantitation of hydrogen ion and potential gradients in gastric plasma membrane vesicles

Rabon¹,

Chang²,

Sachs³

1978

Biochemistry

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The ATP-dependent uptake of H+ by hog gastric parietal cell vesicles was quantitated by using the pH indicator dyes bromcresol green and malachite green, the weak bases, aminopyrine and 9-aminoacridine, and the pH electrode. A K+-dependent H+ uptake was found, with a significant difference between the quantity of H+ disappearing from the medium (deltaHo) and the quantity appearing inside the vesicle (deltaHi). 9-Aminoacridine gave a lower value for the deltaHi than any of the other probes. Probes of potential such as diethyloxadicarbocyanine or oxonol dyes showed that only secondary diffusion potentials occurred during H+ uptake and that the cationic dyes in the presence of protonophores could also be used to quantitate H+ uptake. The potential in the presence of protonophore indicated a deltaHi greater than that found with the other probes. Binding sites for acridine orange were generated either by ATP or an artificial pH gradient and corresponded to the deltaHi indicated by aminopyrine. SCN- (30mM) only partially inhibited the H+ gradient, and this, coupled with the failure to detect the physiological deltapH of 6.6, indicated that these vesicles may be an incomplete model of gastric acid secretion.

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An analysis of the binding of fluorescence probes in mitochondrial systems

Cited by 15 publications

References 26 publications

Effect of chronic ethanol administration on energy metabolism and phospholipase A2 activity in rat liver

Effect of chronic ethanol administration on energy metabolism and phospholipase A2 activity in rat liver

Mitochondrial phospholipase A₂ activity and mitochondrial aging

Quantitation of hydrogen ion and potential gradients in gastric plasma membrane vesicles

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An analysis of the binding of fluorescence probes in mitochondrial systems

Cited by 15 publications

References 26 publications

Effect of chronic ethanol administration on energy metabolism and phospholipase A2 activity in rat liver

Effect of chronic ethanol administration on energy metabolism and phospholipase A2 activity in rat liver

Mitochondrial phospholipase A2 activity and mitochondrial aging

Quantitation of hydrogen ion and potential gradients in gastric plasma membrane vesicles

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Mitochondrial phospholipase A₂ activity and mitochondrial aging