Ingemar Ericson

[41] Isolation of submitochondrial particles with different polarities

Publisher Summary This chapter describes the purification of mitochondria from Jerusalem artichoke tubers. The polarity of inner membrane vesicles (submitochondrial particles, SMP) can be measured. The SMP of different polarities can be generated by adjustment of the ionic conditions in the disruption medium. The RO- and IO-SMP can be partially purified from a mixed population of SMP by phase partitioning. The chapter discusses methods applied only to mitochondria from potato and Jerusalem artichoke tubers, but expects that similar results will be obtained with any type of mitochondria. The use of Jerusalem artichoke tubers has several advantages over potato tubers, including (1) polysaccharide in Jerusalem artichoke tubers, inulin, is water soluble, (2) amyloplast contamination is much less prominent, such that crude mitochondria as isolated in the chapter can be used in many experiments, and (3) for the same reason the yield of mitochondria is better with Jerusalem artichoke tubers.

Preparatiön of mitochondria from gree leaves of spinach by differential centrifugation and phase partition

Abstract Mitochondria from green leaves of spinach (Spinacia oleracea L) have been prepared by a preparation procedure using differential centrifugation and partition in an aqueous dextran-poly(ethylene glycol) two-phase system. The enrichment of mitochondria in relation to chloroplast material was 200–400 times based on NAD: isocitrate dehydrogenase (IDH) activity and chlorophyll content. The protein/chlorophyll ration in the preparation was approximately 100. The apparent intactness of both the inner and the outer membrane was about 90%, as measured by latency of enzyme activities. The mitochondria oxidized NADH (respiratory control 2.1), succinate (1.6), glycine (3.8) and malate (3.0). The mitochondrial preparation was morphologically characterized by electron microscopy.

Determination of the isoelectric point of rat liver mitochondria by cross-partition.

Abstract The isoelectric points of rat liver mitochondria and mitochondrial membrane fractions determined by two independent methods, cross-partition in sodium salts and isoelectric focusing in Ampholine gradients, are in good agreement. The determined isoelectric points are 5.2–5.4 for whole mitochondria, 5.3 for the inner membrane fraction and 4.8–4.9 for the outer membrane fracton. This work shows that cross-partition which has previously only been applied to proteins can also be used to determine the isoelectric points of cell organelles and membranes. It is suggested that cross-partition in phase systems can be used to study charge and conformational changes in membranes.

Characterisation and purification of inside-out submitochondrial particles obtained from plant mitochondria

For the study of membrane components facing the matrix side of the mitochondrion and for the study of membrane asymmetry in general, it is necessary to have inside-out vesicles. Previous studies on submitochondrial particles (SMP) from plant mitochondria have in general assumed that the SMP were 100% insideout (e.g., [1,2]). Here, we have produced SMP from Arum maculaturn mitochondria by sonication and characterised them with respect to enzyme activities and contamination by matrix enzymes and outer membrane fragments. We have also determined the polarity of the particles by measuring the fraction of the vesicles which could oxidise reduced cytochrome c. By this assay the SMP were 84% inside-out. Phase partitioning in dextran-polyethylene glycol (PEG) systems will separate material according to surface properties [3] and this method has been used to separate insideout thylakoid membranes [4]. We have applied phase partitioning to the SMP fraction and obtained SMP which were 93% inside-out.

Some properties of mitochondria, mitoplasts and submitochondrial particles of different polarities from plant tissues

SOME PROPERTIES OF MITOCHONDRIA, MITOPLASTS AND SUBMITOCHONDRIAL PARTICLES OF DIFFERENT POLARITIES FROM PLANT-TISSUES

Generation and purification of submitochondrial particles of different polarities from plant mitochondria

Mitochondria were isolated from Jerusalem artichoke (Helianthus tuberosus L.) tubers in a low‐salt medium. Submitochondrial particles (SMP) produced by sonication in a low salt medium + 20 mM MgCl2 were 18% right‐side‐out (RO) as judged by the latency of cytochrome c oxidase assayed ± Triton X‐100. SMP produced by French press treatment in a low‐salt medium + 5 mM EDTA to remove bound divalent cations were 98% RO. Less extreme treatments gave SMP of intermediate polarity. There was a positive correlation between the % RO‐SMP (produced by sonication) and the % NAD+‐malate dehydrogenase enclosed within the SMP indicating that only RO‐SMP contained trapped matrix. When a mixed population of SMP (45% RO) was applied to an aqueous polymer two‐phase system, the top phase contained 76% RO‐SMP and the bottom phase mostly inside‐out SMP (26% RO). By analogy with the models for stacking of photosynthetic membranes, we propose that crista formation in the inner mitochondrial membrane is electrostatically regulated and SMP deriving from the closely stacked crista regions are inside‐out.

A comparison of the membrane composition of mitochondria isolated from spinach leaves and leaf petioles

Abstract Very pure preparations of mitochondria were obtained from spinach leaves and leaf petioles by a preparation procedure combining differential centrifugation, phase partition and Percoll density gradient centrifugation. These preparations were used for measurements of cytochrome content, lipid content and distribution of proteins between the membranes and the matrix fraction. Leaf and petiole mitochondria contained qualitatively the same cytochromes as measured by difference spectroscopy. On a protein basis leaf mitochondria contained only half the amount of cytochromes compared to petiole mitochondria. Leaf mitochondria had also a somewhat lower lipid/protein ratio than petiole mitochondria. In leaf mitochondria a relatively higher proportion of the protein was in the matrix fraction compared to petiole mitochondria. It is concluded that leaf mitochondria have less respiratory chains in relation to other membrane components compared to petiole mitochondria. The differences in composition are discussed with relation to metabolic differences between mitochondria in photosynthetic tissue and non-photosynthetic tissue.

Mitochondrial inner membrane changes determined by cross partition.

Intact beef heart mitochondria and submitochondrial particles have different isopartition points. For intact mitochondria a cross-point is obtained at pH 5.6 and for submitochondrial particles at pH 6.6. ATP but not necessarily ITP lowers the isopartition point in submitochondrial particles, indicating conformational and/or charge changes in the particulate membrane.

The Glycine Decarboxylating System in Spinach Leaf Mitochondria

Most plants lose CO2 during photosynthesis in a process called photorespiration (Tolbert 1971). This decarboxylation takes place in the mitochondria where glycine is oxidized to serine with release of NH3 and CO2 in a 1:1 ratio. This oxidation of glycine is coupled to the respiratory chain by oxidation of produced NADH. The released ammonia is efficiently refixed in the chloroplast (Bergman et al. 1981).

[40] Separation of spinach leaf mitochondria according to surface properties: Partition in aqueous polymer two-phase systems

Publisher Summary This chapter describes phase system that in combination with differential centrifugation can be used to obtain purified, functionally intact spinach leaf mitochondria. In addition, the application of some special techniques using substituted polyethylene glycol (PEG) is exemplified. The minimum concentration of PEG and Dextran needed to obtain a two-phase system is called the “critical point.” As this depends on the molecular weight of the polymers, it can be used to calibrate different batches of polymers. Ligands covalently coupled to PEG will affect the partition. The phase system used is such that in the absence of substituted PEG little material is present in the top phase. This can be achieved by high KCl concentration or by high polymer concentrations. The negatively charged carboxyl-PEG does not increase the amount of membrane material in the top phase, whereas positively charged amino-PEG will attract material into the top phase.