Article

Isolation of Active Mitochondria From Tomato Fruit

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Abstract

An improved method for isolating mitochondria from tomato fruit (Lycopersicon esculentum Mill.) is described. The fruit is chilled, and the tissue of the fruit wall cut by hand into very thin slices with a razor blade while immersed in a buffer containing 0.4 m sucrose, 2 mm MgCl(2), 8 mm EDTA, 4 mm cysteine, 10 mm KCl, 0.5 mg per ml bovine serum albumin 50 mm tris-HCl, pH 7.6. The pH is monitored and kept within the range of 7.0 to 7.2 by dropwise addition of 1 n KOH during cutting. The tissue is strained through 8 layers of cheesecloth and centrifuged at 2000 x g for 15 minutes. The supernatant is then centrifuged at 11,000 x g for 20 minutes, and the sediment is washed once with a medium containing 0.4 m sucrose, 10 mm KCl, 1 mm MgCl(2), 10 mm tris-HCl, 10 mm KH(2)PO(4) and bovine serum albumin (0.5 mg per ml), pH 7.2. Electron microscope studies show that this method gives homogeneous, relatively intact mitochondria; they have a higher respiratory control ratio than those reported by other workers. The method was also tested successfully on fruits of cantaloupe and ;Honey Dew' melon.

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... 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. ...
... Studies made with isolated mitochondria must always be interpreted with caution, because the mitochondria may have been changed or damaged during the isolation procedure, and the preparation may be contaminated with nonmitochondrial material (11,34). Furthermore, the mitochondria isolated with one method may not show the same respiratory activities as those with other methods. ...
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... The mitochondria in the HBP pulp after 12, 54, 60, and 78 DOS were extracted according to methods described by Ku et al. (1968) and Day and Hanson (1978) with minor modifications. Exactly 1.5 g of the HBP pulp was ground in liquid nitrogen, and 3 mL of 60 mmol/L Tris-HCl (pH 7.4) was added. ...
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Full-text available
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This paper represents an attempt to develop logically the basic premises that tissue fractionation is: 1.(a) a chemical method to be conducted according to the ground rules which govern chemical fractionation in general;2.(b) potentially applicable to the separation and characterization of all elements of cellular organization, whether known or unknown, which are not irretrievably lost in the initial grinding of the cells.It is shown that the approach which best answers these prerequisites is a purely analytical one, untrammeled by any preconceived idea of the cytological composition of the isolated fractions, and allowing their biochemical properties to be expressed as continuous functions of the physical parameter which determines the behaviour of subcellular components in the fractionation system chosen. Examples are given which illustrate the application of density gradient centrifugation in this type of approach, as well as the advantages which can be derived from the use of media of different composition.The results of such experiments are expressed in the form of distribution curves of biochemical constituents, as with other fractionation methods such as chromatography or electrophoresis, but with the difference that the independent variable is related to a property, not of the constituent but of its host-particles. These curves can be taken to represent the mass distribution of the particles themselves if the constituent is assumed to be homogeneously distributed amongst them (postulate of biochemical homogeneity). This assumption has been verified for a number of enzymes, which provide valuable markers to fix the position of their host-particles on the distribution diagrams.By comparing the distribution of an unlocalized constituent against the background of known distributions, especially under a variety of experimental conditions, and by making use of all additional data which can be obtained by ancillary experiments, it is usually possible either to demonstrate the association of the constituent with a known intracellular component, or to bring to light its possible localization in an as yet unidentified type of particle. The existence, chemical properties, and structural features of the latter can be further established when enough analytical resolution has been achieved to warrant a preparative attempt. Lysosomes as well as microbodies containing urate oxidase, catalase and D-amino acid oxidase have been identified in rat liver by following an approach of this kind.In the design of tissue fractionation experiments and in the interpretation of their results, it is essential to observe a rigorous logic and to employ an appropriately accurate vocabulary. The most important requirement in this respect is to maintain a strict distinction between the intracellular organelles or structures as they occur within the cells, the populations of particulate aggregates as they are present in the homogenate and react to the fractionation procedure, and the subcellular fractions as they are isolated and analysed.
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The mitochondria isolated from dark-grown mung bean hypocotyls oxidize succinate, l-malate, and externally added reduced nicotine adenine dinucleotide (NADH) with good respiratory control. While the pattern of respiration resembles that of animal mitochondria, there are 4 basic differences between the respiratory properties of mung bean and animal mitochondria: A) the ability to oxidize NADH, B) the pattern of succinate and malate oxidation, C) the rate of oxygen uptake, and D) the adenosine-5'-diphosphate to oxygen ratios.The apparent ;Km' for malate of mung bean mitochondria is about one order higher than that expected from malic dehydrogenase in animal mitochondria, whereas the affinity for phosphate is about 5 times higher with plant mitochondria than rat-liver mitochondria. While the half-maximal stimulation of respiration by adenosine-5'-diphosphate is practically identical to that of animal mitochondria, higher concentrations of adenosine-5'-diphosphate cause some decrease in its stimulating action.
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