The presence of a short redox chain in the membrane of intact potato tuber peroxisomes and the association of malate dehydrogenase with the peroxisomal membrane
(1993) In Physiologia Plantarum 88(1). p.19-28- Abstract
- Peroxisomes and mitochondria were purified from potato tubers (Solanum tuberosum L. cv. Bintje) by differential centrifugation followed by separation on a continuous Percoll gradient containing 0.3 M sucrose in the lower half and 0.3 M mannitol in the upper half. The peroxisomes band at the bottom and the mitochondria in the middle of this type of gradient. Mitochondrial contamination of the peroxisomes was only 2% [as judged by cytochrome c oxidase (EC 1.3.9.1) activity]. Contamination by amyloplasts, plasma membrane and endoplasmic reticulum was also minimal. The peroxisomes were 80% intact as judged by malate dehydrogenase (MDH, NAD−-dependent; EC 1.1.1.37) latency.The specific activity of NADH-ferricyanide reductase and NADH-Cyt c... (More)
- Peroxisomes and mitochondria were purified from potato tubers (Solanum tuberosum L. cv. Bintje) by differential centrifugation followed by separation on a continuous Percoll gradient containing 0.3 M sucrose in the lower half and 0.3 M mannitol in the upper half. The peroxisomes band at the bottom and the mitochondria in the middle of this type of gradient. Mitochondrial contamination of the peroxisomes was only 2% [as judged by cytochrome c oxidase (EC 1.3.9.1) activity]. Contamination by amyloplasts, plasma membrane and endoplasmic reticulum was also minimal. The peroxisomes were 80% intact as judged by malate dehydrogenase (MDH, NAD−-dependent; EC 1.1.1.37) latency.The specific activity of NADH-ferricyanide reductase and NADH-Cyt c reductase was 0.22 and 0.051 μmol (mg protein)−1 min−1 in freshly isolated peroxisomes, respectively. The active site of the reductase appeared to be on the inner surface of the membrane. The peroxisomes also contained a b-type cytochrome. Frozen peroxisomes were subfractionated by osmotic rupture followed by centrifugation to separate the soluble proteins from the peroxisomal membrane. About half the MDH and 30% of the NADH-ferricyanide reductase activity was associated with the membrane but only 6% of the catalase (EC 1.11.1.6) activity. A further wash removed 75% of the residual catalase with only a small loss of MDH or NADH-ferricyanide reductase activity. MDH appears to be closely associated with the peroxisomal membrane.When the purified peroxisomal membrane was analyzed by SDS-PAGE followed by silver staining, prominent bands at 22, 40, 41, 48, 53 and 74 kDa were observed. After immunoblotting the purified peroxisomal membrane, a band at 53 kDa showed strong cross-reactivity with antibodies raised against NADH-ferricyanide reductase. Since the NADH-ferricyanide reductase activity in the peroxisomal membrane could be shown to be specific for the β-hydrogen of NADH, the activity could not be due to contamination by endoplasmic reticulum where the reductase is α-specific. We conclude that the peroxisomal membrane contains a short redox chain, consisting of a NADH-ferricyanide reductase and a b-type cytochrome, similar to that of e.g. the plasma membrane. The role of this redox chain has yet to be elucidated. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/30036b98-0aa5-430e-b3f8-df3b543229a9
- author
- Struglics, André LU ; Fredlund, Kenneth M ; Rasmusson, Allan LU and Möller, Ian M
- organization
- publishing date
- 1993
- type
- Contribution to journal
- publication status
- published
- keywords
- Cytochrome b, malate dehydrogenase, membrane integrity, NADH‐ferricyanide reductase, peroxisome, potato, redox chain, Solanum tuberosum.
- in
- Physiologia Plantarum
- volume
- 88
- issue
- 1
- pages
- 19 - 28
- publisher
- John Wiley & Sons Inc.
- external identifiers
-
- scopus:84989741846
- ISSN
- 0031-9317
- DOI
- 10.1111/j.1399-3054.1993.tb01755.x
- language
- English
- LU publication?
- yes
- id
- 30036b98-0aa5-430e-b3f8-df3b543229a9
- date added to LUP
- 2016-11-03 15:15:06
- date last changed
- 2024-01-04 15:32:43
@article{30036b98-0aa5-430e-b3f8-df3b543229a9, abstract = {{Peroxisomes and mitochondria were purified from potato tubers (Solanum tuberosum L. cv. Bintje) by differential centrifugation followed by separation on a continuous Percoll gradient containing 0.3 M sucrose in the lower half and 0.3 M mannitol in the upper half. The peroxisomes band at the bottom and the mitochondria in the middle of this type of gradient. Mitochondrial contamination of the peroxisomes was only 2% [as judged by cytochrome c oxidase (EC 1.3.9.1) activity]. Contamination by amyloplasts, plasma membrane and endoplasmic reticulum was also minimal. The peroxisomes were 80% intact as judged by malate dehydrogenase (MDH, NAD−-dependent; EC 1.1.1.37) latency.The specific activity of NADH-ferricyanide reductase and NADH-Cyt c reductase was 0.22 and 0.051 μmol (mg protein)−1 min−1 in freshly isolated peroxisomes, respectively. The active site of the reductase appeared to be on the inner surface of the membrane. The peroxisomes also contained a b-type cytochrome. Frozen peroxisomes were subfractionated by osmotic rupture followed by centrifugation to separate the soluble proteins from the peroxisomal membrane. About half the MDH and 30% of the NADH-ferricyanide reductase activity was associated with the membrane but only 6% of the catalase (EC 1.11.1.6) activity. A further wash removed 75% of the residual catalase with only a small loss of MDH or NADH-ferricyanide reductase activity. MDH appears to be closely associated with the peroxisomal membrane.When the purified peroxisomal membrane was analyzed by SDS-PAGE followed by silver staining, prominent bands at 22, 40, 41, 48, 53 and 74 kDa were observed. After immunoblotting the purified peroxisomal membrane, a band at 53 kDa showed strong cross-reactivity with antibodies raised against NADH-ferricyanide reductase. Since the NADH-ferricyanide reductase activity in the peroxisomal membrane could be shown to be specific for the β-hydrogen of NADH, the activity could not be due to contamination by endoplasmic reticulum where the reductase is α-specific. We conclude that the peroxisomal membrane contains a short redox chain, consisting of a NADH-ferricyanide reductase and a b-type cytochrome, similar to that of e.g. the plasma membrane. The role of this redox chain has yet to be elucidated.}}, author = {{Struglics, André and Fredlund, Kenneth M and Rasmusson, Allan and Möller, Ian M}}, issn = {{0031-9317}}, keywords = {{Cytochrome b; malate dehydrogenase; membrane integrity; NADH‐ferricyanide reductase; peroxisome; potato; redox chain; Solanum tuberosum.}}, language = {{eng}}, number = {{1}}, pages = {{19--28}}, publisher = {{John Wiley & Sons Inc.}}, series = {{Physiologia Plantarum}}, title = {{The presence of a short redox chain in the membrane of intact potato tuber peroxisomes and the association of malate dehydrogenase with the peroxisomal membrane}}, url = {{http://dx.doi.org/10.1111/j.1399-3054.1993.tb01755.x}}, doi = {{10.1111/j.1399-3054.1993.tb01755.x}}, volume = {{88}}, year = {{1993}}, }