| 1. |
- Møller, Ian Max, et al.
(författare)
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Isolation of Highly Purified, Intact, and Functional Mitochondria from Potato Tubers Using a Two-in-One Percoll Density Gradient
- 2022
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Ingår i: Plant Mitochondria : Methods and Protocols - Methods and Protocols. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. - 9781071616529 - 9781071616536 ; 2363, s. 39-50
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Bokkapitel (refereegranskat)abstract
- The isolation of mitochondria from potato tubers (Solanum tuberosum L.) is described, but the methodology can easily be adapted to other storage tissues. After homogenization of the tissue, filtration and differential centrifugation, the key step is a Percoll density gradient centrifugation. The Percoll gradient contains two parts: a bottom part containing Percoll in 0.3 M sucrose, and a slightly less dense top part containing Percoll in 0.3 M mannitol. After centrifugation, a density gradient is formed that is almost linear in the central part, and this is where the band containing the purified intact mitochondria is formed. This method makes it possible to process large amounts of plant material (2–6 kg) and saves at least 1.5 h on the preparation time compared to methods where two consecutive purification methods are used. Nonetheless, it yields large amounts of mitochondria (50–125 mg protein) of very high purity, intactness and functionality.
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| 2. |
- Rasmusson, Allan G., et al.
(författare)
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Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin
- 2022
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Ingår i: Plant Mitochondria : Methods and Protocols - Methods and Protocols. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. - 9781071616529 - 9781071616536 ; 2363, s. 77-84
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Bokkapitel (refereegranskat)abstract
- We here describe measurements of respiratory enzymes in situ, which can be done on very small cell samples and make mitochondrial isolation unnecessary. The method is based on the ability of the fungal peptide alamethicin to permeate biological membranes from the net positively charged side, and form nonspecific ion channels. These channels allow rapid transport of substrates and products across the plasma membrane, the inner mitochondrial membrane, and the inner plastid envelope. In this way, mitochondrial enzyme activities can be studied without disrupting the cells. The enzymes can be investigated in their natural proteinaceous environment and the activity of enzymes, also those sensitive to detergents or to dilution, can be quantified on a whole cell basis. We here present protocols for in situ measurement of two mitochondrial enzymatic activities: malate oxidation measured as oxygen consumption by the electron transport chain, which is sensitive to detergents, and NAD+-isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme that dissociates upon dilution.
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| 3. |
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| 4. |
- Agius, Stephanie C, et al.
(författare)
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NAD(P) turnover in plant mitochondria
- 2001
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Ingår i: Australian Journal of Plant Physiology. - 0310-7841. ; 28:6, s. 461-470
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Tidskriftsartikel (refereegranskat)abstract
- An analytical procedure based on alkaline extraction and HPLC analysis was adapted for quantification of pyridine nucleotides in plant mitochondria. The amounts of NAD and NADP extracted from seven different species varied from 1.0 to 3.7 and 0 to 0.5 nmol (mg protein) –1 , respectively. Although NADP was found in four species, its reduced form was in all cases below the detection limit of 0.1 nmol (mg protein) –1 . The NAD pool was mainly oxidized in the absence of substrates. However, oxidation of substrates followed by anaerobiosis caused 50–92% NAD pool reduction, indicating that the majority of the NAD+ was metabolically active. The NAD reduction level in potato tuber mitochondria oxidizing malate varied with assay conditions. The highest level of reduction (>80%) was reached at anaerobiosis, at pH 6.5 and 7.2, conditions favouring malic enzyme (ME), whereas the lowest reduction level (0%) was observed at pH 7.5, conditions favouring malate dehydrogenase (MDH). Mitochondria incubated at 0°C without respiratory substrate showed a loss of endogenous NAD + which correlated with a decline in the rate of oxidation of NAD+ -linked substrates. The lost NAD+ was mainly recovered as breakdown products in both the surrounding medium and the mitochondria. When submitochondrial fractions were incubated with NAD + or NADP + , the highest rate of NAD(P)+metabolism was detected in the outer membrane fraction. The metabolites detected, adenosine monophosphate (AMP), nicotinamide mononucleotide (NMN) and adenosine, imply that several enzymes involved in pyridine nucleotide degradation, including an NAD pyrophosphatase, are localized to the outer membrane.
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| 5. |
- Aidemark, Mari, et al.
(författare)
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Regulation of callose synthase activity in situ in alamethicin-permeabilized Arabidopsis and tobacco suspension cells
- 2009
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Ingår i: BMC Plant Biology. - : Springer Science and Business Media LLC. - 1471-2229. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Background: The cell wall component callose is mainly synthesized at certain developmental stages and after wounding or pathogen attack. Callose synthases are membrane-bound enzymes that have been relatively well characterized in vitro using isolated membrane fractions or purified enzyme. However, little is known about their functional properties in situ, under conditions when the cell wall is intact. To allow in situ investigations of the regulation of callose synthesis, cell suspensions of Arabidopsis thaliana (Col-0), and tobacco (BY-2), were permeabilized with the channel-forming peptide alamethicin. Results: Nucleic acid-binding dyes and marker enzymes demonstrated alamethicin permeabilization of plasma membrane, mitochondria and plastids, also allowing callose synthase measurements. In the presence of alamethicin, Ca2+ addition was required for callose synthase activity, and the activity was further stimulated by Mg2+ Cells pretreated with oryzalin to destabilize the microtubules prior to alamethicin permeabilization showed significantly lower callose synthase activity as compared to non-treated cells. As judged by aniline blue staining, the callose formed was deposited both at the cell walls joining adjacent cells and at discrete punctate locations earlier described as half plasmodesmata on the outer walls. This pattern was unaffected by oryzalin pretreatment, showing a quantitative rather than a qualitative effect of polymerized tubulin on callose synthase activity. No callose was deposited unless alamethicin, Ca2+ and UDP-glucose were present. Tubulin and callose synthase were furthermore part of the same plasma membrane protein complex, as judged by two-dimensional blue native SDS-PAGE. Conclusion: Alamethicin permeabilization allowed determination of callose synthase regulation and tubulin interaction in the natural crowded cellular environment and under conditions where contacts between the cell wall, the plasma membrane and cytoskeletal macromolecules remained. The results also suggest that alamethicin permeabilization induces a defense response mimicking the natural physical separation of cells (for example when intercellulars are formed), during which plasmodesmata are transiently left open.
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| 6. |
- Aidemark, Mari, et al.
(författare)
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Trichoderma viride cellulase induces resistance to the antibiotic pore-forming peptide alamethicin associated with changes in the plasma membrane lipid composition of tobacco BY-2 cells
- 2010
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Ingår i: Bmc Plant Biology. - : Springer Science and Business Media LLC. - 1471-2229. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background: Alamethicin is a membrane-active peptide isolated from the beneficial root-colonising fungus Trichoderma viride. This peptide can insert into membranes to form voltage-dependent pores. We have previously shown that alamethicin efficiently permeabilises the plasma membrane, mitochondria and plastids of cultured plant cells. In the present investigation, tobacco cells (Nicotiana tabacum L. cv Bright Yellow-2) were pre-treated with elicitors of defence responses to study whether this would affect permeabilisation. Results: Oxygen consumption experiments showed that added cellulase, already upon a limited cell wall digestion, induced a cellular resistance to alamethicin permeabilisation. This effect could not be elicited by xylanase or bacterial elicitors such as flg22 or elf18. The induction of alamethicin resistance was independent of novel protein synthesis. Also, the permeabilisation was unaffected by the membrane-depolarising agent FCCP. As judged by lipid analyses, isolated plasma membranes from cellulase-pretreated tobacco cells contained less negatively charged phospholipids ( PS and PI), yet higher ratios of membrane lipid fatty acid to sterol and to protein, as compared to control membranes. Conclusion: We suggest that altered membrane lipid composition as induced by cellulase activity may render the cells resistant to alamethicin. This induced resistance could reflect a natural process where the plant cells alter their sensitivity to membrane pore-forming agents secreted by Trichoderma spp. to attack other microorganisms, and thus adding to the beneficial effect that Trichoderma has for plant root growth. Furthermore, our data extends previous reports on artificial membranes on the importance of lipid packing and charge for alamethicin permeabilisation to in vivo conditions.
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| 7. |
- Björn, Lars Olof, et al.
(författare)
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Photosensitivity in sponge due to cytochrome c oxidase?
- 2009
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Ingår i: Photochemical and Photobiological Sciences. - : Springer Science and Business Media LLC. - 1474-9092 .- 1474-905X. ; 8, s. 755-757
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Tidskriftsartikel (refereegranskat)abstract
- An action spectrum for photosensitivity in sponge larvae published by Leys et al. [J. Comp. Physiol., A, 2002, 188, 199–202] was interpreted by the authors as being due to a combination of light absorption by flavin or carotenoid in the blue region, and another pigment such as pterin in the long-wavelength region. Here we show here that their action spectrum closely matches the absorption spectrum of reduced cytochrome c oxidase that is present in sponges, and compare with other photoreactions which are thought to be due to this chromoprotein.
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| 8. |
- Browse, John A., et al.
(författare)
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Respiration and lipid metabolism
- 2010
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Ingår i: Plant Physiology (5th edition). - 9780878938667 - 0878938664 ; , s. 305-342
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Bokkapitel (refereegranskat)abstract
- Abstract is not available
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| 9. |
- Browse, John A., et al.
(författare)
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Respiration and Lipid Metabolism
- 2014. - 6th ed.
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Ingår i: Plant Physiology and Development. - 9781605352558 - 9781605354354 ; , s. 317-352
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Bokkapitel (refereegranskat)
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| 10. |
- Browse, John, et al.
(författare)
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Respiration and lipid metabolism
- 2006
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Ingår i: Plant Physiology, 4th Ed.. ; , s. 253-288
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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