Summary
The plasma membrane (PM) of higher plants contains a major ascorbate-reducible, high-potentialb-type cytochrome, named cytochromeb 561 (cytb 561). In this paper a rapid purification protocol for the cytb 561 of bean hypocotyls PM is described. An almost 200-fold increase of cytb 561 specific concentration was achieved with respect to the PM fraction, which contained about 0.2 nmol of ascorbate-reducible heme per mg protein. The procedure can be performed in one day starting from purified PMs obtained by the phase-partitioning procedure. However, cytb 561 proved to be unstable during chromatographic purification and the amount of protein finally recovered was low. Purified cytb 561 eluted as a 130,000 Da protein-detergent complex from gel-filtration columns. It was completely reduced by ascorbate and reduced-minus-oxidized spectra showed α-, β- and γ-bands at 561, 530, and 429 nm respectively, not unlike the spectra of whole PMs. This work represents an initial approach to the biochemical characterization of the cytb 561 of higher plants, formerly suggested to be related to cytb 561 of animal chromaffin granules.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- cytb 561 :
-
cytochromeb 561
- PM:
-
plasma membrane
- UPV:
-
upper-phase vesicles
- GSII:
-
glucan synthase II
- CCR:
-
NADH-dependent cytochromec reductase
- CCO:
-
cytochromec oxidase
- TX-100R:
-
reduced Triton X-100
References
Asard H, Venken M, Caubergs RJ, Reijnders W, Oltmann FL, De Greef JA (1989)b-Type cytochromes in higher plant plasma membranes. Plant Physiol 90: 1077–1083
—, Horemans N, Caubergs RJ (1992) Transmembrane electron transport in ascorbate-loaded plasma membrane vesicles from higher plants involves ab-type cytochrome. FEBS Lett 306: 143–146
— —, Mertens J, Caubergs RJ (1994) The plant plasma membrane b-type cytochrome: an overview. Bel J Bot 127: 171–183
—, Caubergs RJ (1995) Involvement of ascorbic acid and a b-type cytochrome in plant plasma membrane redox reactions. Protoplasma 184: 36–41
— —, Preger V, Trost P (1998) Plasma membraneb-type cytochromes. In: Asard H, Bérczi A, Caubergs RJ (eds) Plasma membrane redox systems and their role in biological stress and disease. Kluwer, Dordrecht, pp 1–32
Askerlund P, Larsson C, Widell S (1989) Cytochromes of plant plasma membranes: characterization by absorbance difference spectroscopy and redox titration. Physiol Plant 76: 123–134
Barr R, Pan RS, Crane FL, Brightman AO, Morré DJ (1992) Destruction of vitamin K1 of cultured carrot cells by ultraviolet radiation and its effect on plasma membrane electron transport reactions. Biochem Int 27: 449–456
Bérczi A, Møller IM (1998) NADH-monodehydroascorbate oxidore-ductase is one of the redox enzymes in spinach leaf plasma membranes. Plant Physiol 116: 1029–1036
Bensadoun A, Weinstein D (1976) Assay of proteins in the presence of interfering materials. Anal Biochem 70: 241–250
Crane FL, Sun IL, Sun EE, Crowe RA (1995) Plasma membrane redox and regulation of cell growth. Protoplasma 184: 3–7
Guerinot ML, Yi Y (1994) Iron: nutritious, noxious, and not readily available. Plant Physiol 104: 815–820
Horemans N, Asard H, Caubergs RJ (1994) The role of the ascorbate free radical as an electron acceptor to cytochromeb-mediated transplasma membrane electron transport in higher plants. Plant Physiol 104: 1455–1458
Kent UM, Fleming PJ (1987) Purified cytochromeb 561 catalyzes transmembrane electron transfer for dopamine beta hydroxylase and peptidyl glycine alpha-amidating monooxygenase activities in reconstituted systems. J Biol Chem 262: 8174–8178
Lamb C, Dixon RA (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 48: 251–275
Larsson C, Sommarin M, Widell S (1994) Isolation of highly purified plant plasma membranes and separation of inside-out and right-side out vesicles. Methods Enzymol 228: 451–469
Luster DG, Buckhout TJ (1989) Purification and identification of a plasma membrane associated electron transport protein from maize (Zea mays) roots. Plant Physiol 91: 1014–1019
Lüthje S, Döring O, Heuer S, Lüthen H, Böttger M (1997) Oxidore-ductases in plant plasma membranes. Biochim Biophys Acta 1331: 81–102
Moog PR, Brüggemann W (1995) Iron reductase systems on the plant plasma membrane: a review. In: Abadia J (ed) Iron nutrition in soils and plants. Kluwer, Dordrecht, pp 343–362
Perin MS, Fried VA, Slaughter CA, Sudhof TC (1988) The structure of cytochromeb 561, a secretory vesicle-specific electron transport protein. EMBO J 7: 2697–2703
Rubinstein B, Stern A (1991) The role of plasma membrane redox activity in light effects in plants. J Bioenerg Biomembr 23: 393–408
Segal AW (1995) The NADPH oxidase of phagocytic cells is an electron pump that alkalinises the phagocytic vacuole. Protoplasma 184: 86–103
Serrano A, Cordoba F, Gonzales-Reyes JA, Navas P, Villalba JM (1994) Purification and characterization of two distinct NAD(P)H dehydrogenases from onion (Allium cepa L.) root plasma membrane. Plant Physiol 106: 87–96
Sparia F, Bagnaresi P, Scagliarini S, Trost P (1997) NADH Fe(III)-chelate reductase of maize roots is an active cytochrome b5 reductase. FEBS Lett 414: 571–575
Trost P, Foscarini S, Preger V, Honora P, Vitale L, Pupillo P (1997) Dissecting the diphenylene iodonium-sensitive NAD(P)H:quinone oxidoreductase ofCucurbita plasma membrane. Plant Physiol 114: 737–746
Tsubaki M, Nakayama M, Okuyama E, Ichikawa Y, Hori H (1997) Existence of two hemeb centers in cytochromeb 561 from bovine adrenal chromaffin vesicles as revealed by a new purification procedure and EPR spectroscopy. J Biol Chem 272: 23206–23210
Valenti V, Minardi P, Guerrini F, Mazzucchi U, Pupillo P (1989) Increase of plasma membrane NADH-duroquinone reductase in tobacco leaves treated with protein-lipopolysaccharide complexes. Plant Physiol Biochem 27: 569–575
Van Gestelen P, Asard H, Caubergs RJ (1997) Solubilization and separation of a plant plasma membrane NADPH-O2 −-synthase from other NAD(P)H-oxidoreductases. Plant Physiol 115: 1–8
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Scagliarini, S., Rotino, L., Bäurle, I. et al. Initial purification study of the cytochromeb 561 of bean hypocotyl plasma membrane. Protoplasma 205, 66–73 (1998). https://doi.org/10.1007/BF01279295
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01279295