Mitochondria labelling

Diazido ethidium bromide Yeast mitochondria Labeling of 1 protein, probably subunit 9 of membrane ATPase 72... 

When trihydroxycoprostanic acid labeled in the 4 position (prepared in the same manner as the 26-labeled acid) is incubated with rat liver mitochondria, labeled cholic acid is obtained. Danielsson had been carrying out parallel experiments in mouse liver mitochondria, and his work yielded valuable information concerning the pattern of hydroxylation of cholesterol. Cholesterol can be converted to 26 hydroxycholesterol and also to the 3/5, 7a, 26-triol. Danielsson also showed that liver mitochondrial preparations could convert 3 a, 7 a, 12 a trihydroxy coprostane to 3a, 7 a, 12 a, 26-tetrahydroxycoprostane, 3 a, 7 a, 12a-tri-... 

Hattori, F. Fukuda, K. Method for selecting myocardial cells using intracellular mitochondria labeled with fluorescent indicator. PCT Int. Appl. WO 2006022377, 2006 Chem. Abstr. 2006, 144, 270175. 

FIGURE 21.1 (a) All electron micrograph of a mitochondrion, (b) A drawing of a mitochondrion with components labelled, (a, B. King/BPS)... 

Fig. 4 Transmission electron microscopy of a longitudinal section of the posterior end of a Cryptosporidium parvum sporozoite showing immunogold localization of pyruvate NADP+ oxidoreductase (CpPNO). The mitochondrion-like organelle ( ) is posterior to the nucleus, and lies between the nucleus and the CB. It is labeled by mitochondrion-specific 15-nm gold anti- particles. Small -nm gold goat anti-CpPFO particles (arrows) show the localization of CpPNO. There are no 6-nm particles localized within the mitochondrion-like organelle (reprinted from Fig. 12 of Ctrnacta et al. 2006 with permission of the publishers)...

Answer The transport of fatty acid molecules into mitochondria requires a shuttle system involving a fatty acyl-carnitine intermediate. Fatty acids are first converted to fatty acyl-CoA molecules in the cytosol (by the action of acyl-CoA synthetases) then, at the outer mitochondrial membrane, the fatty acyl group is transferred to carnitine (by the action of carnitine acyl-transferase I). After transport of fatty acyl-carnitine through the inner membrane, the fatty acyl group is transferred to mitochondrial CoA. The cytosolic and mitochondrial pools of CoA are thus kept separate, and no labeled CoA from the cytosolic pool enters the mitochondrion. 

Answer The malate-aspartate shuttle transfers electrons and protons from the cytoplasm into the mitochondrion. Neither NAD+ nor NADH passes through the inner membrane, thus the labeled NAD moiety of [7-14C]NADH remains in the cytosol. The 3H on [4-3H]NADH enters the mitochondrion via the malate-aspartate shuttle (see Fig. 19-29). In the cytosol, [4-3H]NADH transfers its 3H to oxaloacetate to form [3H]malate, which enters the mitochondrion via the malate-a-ketoglutarate transporter, then donates the 3H to NAD+ to form [4-3H]NADH in the matrix. 

Figure 3. (Upper Panel). Scanning electron micrograph of an H. rufescens spermatozoon. The sperm head, from mitochondrion (M) to tip of the acrosome vesicle (granule AV) is 7 pm. The width of the nucleus (N) is 1 pm. (Lower Panel). Transmission electron micrograph of the acrosomal vesicle showing it attached to the nucleus (NF) by the rod of actin filaments (AF). The darker material labelled 1 shows the location of the 18K protein and 2 shows the location of lysin (from Lewis et al., 1980).

Draw a schematic diagram of a mitochondrion, and label the parts of this ] Question 23 ... 

For example, the study (29) of gluconeogenesls by rat hepatocytes from a [3- C] alanine precursor revealed that the label appeared not only in glucose but also In C-3 and C-2 of aspartate and glutamate. The C-3 C-2 intensity ratios In aspartate was consistently 2 1, whereas the equivalent C-2 C-3 ratio for glutamate was 1 1. The flow of C label Into glutamate and aspartate occurs via a sequence of reactions involving transamination of [3- C] alanine to fom [3- C] pyruvate which enters the mitochondrion and is carboxylated to [3- C] oxaloacetate. [3- C]... 

Fig. 5. Transfer of radioactive lipids from liposomes to cauliflower mitochondria. Radioactively labeled liposomes were incubated with cauliflower mitochondria, and after various incubation periods the radioactivity in the mitochondria per milligram of protein was determined. Two experiments were carried out in the first (lower curve) 0.56 x 10 dpm in the liposomes was offered to 3.9 mg of mitochondrial protein, while in the second (upper curve) 3.6 X 10 dpm in the liposomes was offered to 2.1 mg of mitochondrial protein. The inset gives the plot of the percentage of the initial radioactivity recovered in the mitochondrion versus time, in the same two experiments (from Douady and Mazliak, 1975).

Experiments with specifically labeled synthetic fatty acids have given conclusive evidence that no biohydrogenation of mono- and polyunsaturated fatty acids occurs in the mammalian liver (Stoffel et al. 1964). Feeding experiments with doubly-labeled linoleic, arachidonic and stearic acid disprove the often suggested hypothesis of a partial degradation and elongation of the carboxyl end of fatty acids. From these experiments, the conclusion has been drawn that a fatty acid molecule is completely degraded on contact with the 8-oxidation multienzyme of the mitochondrion. 

The different incubation times were used to find how much of the incorporating-activity is endogenous for the mitochondrion, assuming that the labeled protein that is found in the mitochondrion early would be predominantly intrinsically synthesized while that manufactured elsewhere would accumulate more slowly. However, on this basis, the results suggest not only that the labeled protein in the mitochondrion has been synthesized locally, but also that the mitochondrion may be exporting newly synthesized protein to the surroundings. 

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