On Methods of Isolation of Active, Tightly Coupled Mitochondria of Wheat Seedlings

Sarkissian, Igor V.; Srivastava, H. K.

doi:10.1104/pp.43.9.1406

Cited by 39 publications

(21 citation statements)

References 13 publications

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“…The respiration data, presented as Arrhenius plots in Figure 1 1970 (22) for mitochondria from pears, the RC ratios shown in Table I fell within the range usually observed for succinate oxidation by plant mitochondria (1,3,5,23,26,27,30) and by rat liver mitochondria (6). Similarly, the ADP:O ratios, although exhibiting some variability, show no consistent pattern which can be related to temperature effects and approach the theoretical value of 2 for succinate oxidation.…”

Section: Resultsmentioning

confidence: 51%

Oxidative Activity of Mitochondria Isolated from Plant Tissues Sensitive and Resistant to Chilling Injury

1970

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Arrhenius plots of the respiration rates of nmitochondria isolated from chilling sensitive plant tissuies (tonmato and cucumber fruit, and sweet potato roots) showed a linear decrease from 23 C to about 9 to 12 C (with Qio values of 1.3 to 1.6), at which point there was a marked deviation with an increased slope as temperatures were reduced to 1.5 C (Qio of 2.2 to 6.3). The phorylative activities (assayed at 25 C) of mitochondria, isolated from chilling sensitive sweet potato roots, were impaired as a result of chilling treatment. However, many factors, such as the polyphenol content of the parent tissue (4, 11), can markedly influence both oxidative rates and apparent phosphorylative ability of isolated plant mitochondria. Thus, where plant material is given a differential pretreatment, it is most difficult to ascertain whether the observed differences in activity represent a direct effect of the chilling treatment on the mitochondria or an indirect effect due to the release or activation of some components which might be operational during isolation. It has also been noted that the method of tissue disruption (8), the amount of mitochondrial protein in the assay mixture (20), and the time involved between tissue disruption and assay (18) influence the activity of isolated mitochondria but which may be of little significance in vivo.The experiments reported here were carried out with mitochondria from plant tissue not subjected to chilling treatment, with the aim of evaluating the influence of temperature on oxidation and phosphorylation of these isolated mitochondria. The results show that in mitochondria from chilling sensitive tissues there is a marked depression in the respiratory rates below the critical temperature for chilling injury (10 C) which is not observed with mitochondria from resistant tissues. It was also established that temperatures between 1.5 and 25 C do not influence the phosphorylative efficiency of mitochondria from either sensitive or resistant plant tissues.Mitochondria isolated from chilling resistant tissues show the capacity to swell to greater extent than the mitochondria from chilling sensitive tissues (14), indicating that the membranes of mitochondria from chilling sensitive tissues are less flexible than those from chilling resistant tissues. Furthermore, this difference in flexibility was related to the relative proportion of saturated and unsaturated fatty acids of the membrane lipids. A study of the temperature at which mixtures of the predominant saturated and unsaturated fatty acids, found in the membranes of plant mitochondria, solidify showed that a small increase in the proportion of unsaturated fatty acids causes a large decrease in the solidification temperature (13). Thus (Beta vulgaris L.).The mitochondria were prepared and respiratory activity was determined by the methods described in the preceding paper (20). Since it was shown (20) that the respiration of isolated mitochondria was greatly reduced and the mitochondria lost respiratory control when th...

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Section: Resultsmentioning

confidence: 51%

Oxidative Activity of Mitochondria Isolated from Plant Tissues Sensitive and Resistant to Chilling Injury

1970

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“…Mitochondria were isolated from shoot tips by a modification of the method of Sarkissian and Srivastava (21). The shoots were homogenized in a mortar using approximately 20 ml (per g fresh weight tissue) of a grinding medium composed of 0.5 M sucrose, 1 mM Na2EDTA, 0.067 M KH,PO,, and 0.75 mg of bovine serum albumin per ml.…”

Section: Methodsmentioning

confidence: 99%

Structural and Functional Responses of Wheat Mitochondrial Membranes to Growth at Low Temperatures

Miller

Pomeroy²

1974

Plant Physiol.

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The responses in membrane lipid composition, structure, and function of four cultivars of wheat (Triticum aestivum L.) to growth at Differences in cold tolerance between hardy and nonhardy plant species may reside partially in the ability of the intracellular membranes to adapt to low temperatures (7,10,11,22,23). Mitochondria were selected for a comparison of the structural and functional characteristics of membrane components of four wheat varieties which exhibit quantitatively different degrees of cold hardiness (6,16,19). Since the oxidative and phosphorylative activities of the respiratory membrane may be assessed after growth of seedlings at various temperatures, these activities may be used as intrinsic probes of membrane function. Lyons and Raison (15) indicated that oxidative activity of sweet potato mitochondria declines during storage at 0 C, and that certain phospholipid fractions of the mitochondrial membranes also decrease during cold storage.Structural alterations in the mitochondrial membrane may be inferred from differences in the fatty acid composition of the isolated mitochondrial phospholipids (13). The involvement of membrane lipids in the coupling of the oxidation of substrates with energy conservation and utilization (24) is not completely understood and this fact necessitates further examination of the relationships between lipid composition and membrane structure and function.A more direct physical probe of intact membrane structure has been developed recently (8,17,20). The insertion into the hydrophobic membrane regions of a lipid-soluble probe molecule bearing a nitroxyl-free radical moiety allows assessment of the conditions under which structural changes occur. By recording the electron spin resonance spectra of the nitroxide label over a temperature range, a relative motion parameter, the correlation time, T,, may be determined at the known absolute temperatures. Changes in the slope of Arrhenius plots of T, have been interpreted as indicating phase changes in membrane structure (5). The motion parameter provides a relative measure of molecular restraint within the fluid lipid matrix of the membrane. Correlation of abrupt changes in the temperature dependence of rates of mitochondrial oxidations with changes in the temperature dependence of the molecular motion parameter have been made for plant and animal species (14,15).Studies on the respiratory properties of mitochondria isolated from developing wheat seedlings have indicated that mitochondria with high respiratory rates, and excellent respiratory control and ADP :0 ratios can be obtained from dark-grown 2-to 3-day-old winter wheat seedlings. It has also been shown that these seedlings will develop a high degree of freezing resistance when germinated and grown in the dark at 2 C (4). In winter wheat cultivars, cold resistance is genetically controlled within a species and can be quantitatively measured (6,16,19). Hence, comparisons of the properties of well defined isolated membrane components from tissues grown a...

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“…Especially rapid methods of isolation hav_e blen shown to favor tightly coupled and stable (3 hr at 0 C) mitochondria (5,6,11). However, the maceration procedures employed with fruit tissues, designed to affect maximal control of pH (10), are particularly slow.…”

Section: A0¢ -Tmentioning

confidence: 99%

“Survival” of Mitochondria in Vitro

Romani

Ozelkok²

1973

Plant Physiol.

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Isolated mitochondria have been maintained active and coupled for 72 hours at 25 C. Survival (retention of respiratory control) is a function of incubation temperature and dependent upon aeration and substrate. ATP does not entirely substitute for substrate, indicating a need for products of active metabolism other than energy. An improvement in respiratory control is often observed during the first several hours of incubation. Sedimentation and resuspension at 24-hour intervals prolonged survival. As revealed by electron microscopy, mitochondria maintained their basic structure during a 72-hour period at 25 C.Survival is a dynamic, energy-requiring process and must be distinguished from so-called "aging" of organelles at ice temperatures. As a manifestation of partial autonomy, survival may prove useful in assessing aspects of mitochondrial function and the mitochondrial-cellular interrelationship.The prospects of prolonged incubation of isolated organelles were heightened by the reports of Ridley and Leech (7) and Giles and Sarafis (2) demonstrating the survival of isolated chloroplasts for several days. In earlier experiments (8) we have shown that mitochondria will retain respiratory control for 20 hr at 25 C when supplied with substrate and appropriate cofactors. The present paper describes the influence of physical and energy parameters that further extend the retention of respiratory control, referred to hereafter as survival, to at least 72 hr at 25 C. A preliminary report of this work has appeared (9).MATERIALS AND METHODS Avocados (Persea americana "Fuerte" Mill) were obtained from orchards in southern California or from local wholesale markets and used as a source of mitochondria.Isolation Techniques. The isolation procedure used throughout these experiments was essentially that described by Lance et al. (3) with minor modifications. Peeled and pitted pieces of avocado were grated into isolation medium (1:3, w/v) composed of 0.25 M sucrose, 0.05 M potassium phosphate buffer (pH 7.2), 5 mm EDTA, 0.2% polyvinylpyrollidone (40,000 mol wt), 0.1 % bovine serum albumin, and 5 mM /3-mercaptoethanol. Grating was done below the liquid surface (10). Cellular debris was removed by centrifugation at 1,200g for 10 min. After additional centrifugation at 14,000g for 15 min the mitochondrial pellet was resuspended in "wash" medium (0.25 M sucrose, 0.05 M phosphate buffer (pH 7.2), 0.1% bovine serum albumin, and S mm /3-mercaptoethanol). The suspension was centrifuged at 1,000g for 5 min and the resultant supernatant fraction at 8,000g for 10 min. The final pellet was resuspended in a few tenths of a milliliter of wash medium and held at 0 C, for a period seldom exceeding 1 hr, until the mitochondria were assayed or incubated at 25 C.Incubation and Assay. As a standard incubation procedure based on earlier experiments (8), the mitochondria were suspended in 3 ml of reagent mixture (see legend, Fig. 1) contained in a 20-ml beaker. At the start of the incubation 0.4 ,umole of ADP was also added, and the beake...

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On Methods of Isolation of Active, Tightly Coupled Mitochondria of Wheat Seedlings

Cited by 39 publications

References 13 publications

Oxidative Activity of Mitochondria Isolated from Plant Tissues Sensitive and Resistant to Chilling Injury

Oxidative Activity of Mitochondria Isolated from Plant Tissues Sensitive and Resistant to Chilling Injury

Structural and Functional Responses of Wheat Mitochondrial Membranes to Growth at Low Temperatures

“Survival” of Mitochondria in Vitro

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