Details

Autor(en) / Beteiligte

• Madamanchi, N.R
• Anderson, J.V
• Alscher, R.G
• Cramer, C.L
• Hess, J.L

Titel

Purification of multiple forms of glutathione reductase from pea (Pisum sativum L.) seedlings and enzyme levels in ozone-fumigated pea leaves

Ist Teil von

• Plant physiology (Bethesda), 1992-09, Vol.100 (1), p.138-145

Ort / Verlag

Rockville, MD: American Society of Plant Physiologists

Erscheinungsjahr

1992

Link zum Volltext

Quelle

EZB Electronic Journals Library

Beschreibungen/Notizen

• Glutathione reductase was purified from pea seedlings using a procedure that included 2',5'-ADP Sepharose, fast protein liquid chromatography (FPLC)-anion exchange, and FPLC-hydrophobic interaction chromatography. The purified glutathione reductase was resolved into six isoforms by chromatofocusing. The isoform eluting with an isoelectric point of 4.9 accounted for 18% of the total activity. The five isoforms with isoelectric points between 4.1 and 4.8 accounted for 82% of the activity. Purified glutathione reductase from isolated, intact chloroplasts also resolved into six isoforms after chromatofocusing. The isoform eluting at pH 4.9 constituted a minor fraction of the total activity. By comparing the chromatofocusing profile of the seedling extract with that of the chloroplast extract, we inferred that the least acidic isoform was extraplastidic and that the five isoforms eluting from pH 4.1 to 4.8 were plastidic. Both the plastidic (five isoforms were pooled) and extraplastidic glutathione reductases had a native molecular mass of 114 kD. The plastidic glutathione reductase is a homodimer with a subunit molecular mass of 55 kD. Both glutathione reductases had optimum activity at pH 7.8. The Km for the oxidized form of glutathione (GSSG) was 56.0 and 33.8 micromoles for plastidic and extraplastidic glutathione reductase, respectively, at 25 degrees C. The Km for NADPH was 4.8 and 4.0 micromoles for plastidic and extraplastidic isoforms, respectively. Antiserum raised against the plastidic glutathione reductase recognized a 55-kD polypeptide from purified antigen on western blots. In addition to the 55-kD polypeptide, another 36-kD polypeptide appeared on western blots of leaf crude extracts and the purified extraplastidic isoform. The lower molecular mass polypeptide might represent GSSG-independent enzyme activity observed on activity-staining gels of crude extracts or a protein that has an epitope similar to that in glutathione reductase