Mitochondrial pellet

The pelleted mitochondria are then resuspended in 1.0-1.5 ml TE in a 15-ml Corex tube. The mitochondrial pellet should be dispersed gently by swirling the tubes. 

Crude Mitochondrial Fraction (CMF). Part of the homogenate was spun down at 2,500 rpm (JA-20 rotor J-21 Beckman centrifuge) for 1 Omin. The supernatant was centrifuged at 13,000 rpm for 2 min and the mitochondrial pellet obtained was washed twice... 

The previous protocol can be interrupted at an intermediate stage after the first mitochondrial pellet (step 1 crude mitochondria pellet) or after the second one (step 3 pine mitochondria pellet). In both cases, mtDNA is extracted by SDS lysis of mitochiondria followed by phenol extraction (see below for a simplified preparation of phenol see also in section HI A the minipreparation of mtDNA with a similar principle). If mtDNA is contaminated, the optional step described above can be added or an alternative solution can be chosen DNase or digitonin treatment can be tried. These various protocols are described below. A protocol similar to the sucrose gradient but-involving Percoll density gradient, often of better efficiency, is described first in the next section. 

The mitochondrial pellet is not very hard. Consequently, pipette gently the supernatant, add 2 ml of solution of isolation, mix, centrifuge for 10 min at 10,000 g. If the pellet is still not hard, wash again. 

Discard the supernatant and resuspend the mitochondrial pellet in 10 mM Tris EDTA buffer pH 8 containing 0.15M NaQ and 10 mM EDTA. The total volume should be 50 ul. 

Wojciechowski and Zimowski (1975) found the most active fraction obtained from C. offidnalis cv. Radio seedlings was sedimented at 15,000 g. This crude mitochondrial pellet was fiuther fractionated by sucrose density... 

Fig. 2. Extraction of Opal from bovine liver mitochondria treated with increasing concentrations of digitonin. Protein that remains in the mitochondrial pellets was analyzed by SDS-PAGE and Western blotting. Hsp60, glntamate dehydrogenase, and ATP synthase were used as mitochondrial matrix markers. Tim23 is an integral membrane protein of the mitochondrial inner membrane. Cytochrome c is a peripheral membrane protein of the intermembrane space and porin is an integral membrane protein of the mitochondrial outer membrane. This experiment is representative of two independent experiments.

Figure 6. Time course of mitochondrial import/processing using control and VPA-treated mitochondria, f S]Methionine-labeled pSCAD, produced via in vitro transcription/translation, were incubated with freshly prepared mitochondria from control and VPA-treated rats at 27 °C for 30 min. The mitochondrial pellet was isolated and resuspended in HMS-buffer, and further incubated at 37 "C for varying periods of time. After trypsin treatment, the product was analyzed by SDS-PAGE and autoradiography (A). The results were quantitatively analyzed using a densitometer B). The amount of mature protein was expressed as percentages of the amount of the respective protein after 30 min of incubation. Symbols are (filled circles), mSCAD(controI) (open circles), mSCAD(+VPA) filled squares). mlVD(control) (filled triangles/up), mIVD(+VPA) (filled tri-angles/down), mOTC(control) broken lines, mOTC(+VPA).

Stephenson and co-workers (58) incubated metabolically active tobacco leaf discs with labeled amino acids and then fractionated them in the usual way. After 30 min it was again the microsomal pellet which had the highest activity (184 cpm/mg protein), although the mitochondrial pellet was also quite active (166 cpm/mg protein). 

Purification is most important when the presence of organelles having the same size and density leads to the preparation of crude pellets containing different types of intact organelles. This is often the case in non-green tissues, such as potato tubers or cauliflower buds. In these examples, the so-called mitochondrial pellet consist in fact of intact mitochondria and peroxisomes (potato tubers) or of intact... 

As an independent approach to determining if nascent polypeptide chains are released from attached ribosomes to the mitochondrial membrane, we labeled nascent chains as above and then determined the amount of radioactivity that could be recovered with mitochondria after removal of attached ribosomes. As shown in Table IV, when mitochondria were washed several times with buffer containing Mg" " in order to maintain the integrity of the ribosome-membrane interactions, 67% of the total incorporated radioactivity was recovered in the washed mitochondrial pellet. However, if the attached cytoplasmic ribosomes were removed by treatment with RNase and EDTA and then washed with PPi-citrate, there... 

Fig. 7. Sedimentation pattern of RNA from the supernatant fraction (A) and the mitochondrial pellet (B) of a homogenate of RNA-labeled unfertilized eggs of Ltjtechinus pictiis. A spawned female was injected with 120 iiC of uridine-5- H (specific activity 25,000 C/moIe) and shed 33 days later (6.7 cpm in RNA per egg). The labeled unfertilized eggs were washed several times by centrifugation in 0.55 M KCl, homogenized in 3 volumes of 0.01 M sodium acetate buffer at pH 5.0, centrifuged at 10,000 g for 10 minutes and the supernatant fraction set aside at 0°C. The pellet was washed twice by centrifugation with 20-30 volumes of homogenization buffer, all supernatant fractions were combined, and the pellet was resuspended in about 40 volumes of buffer. The preparations were phenol-extracted at 4°C and treated with DNase.

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