ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 247, No. 2, June, pp. 393-402,1986 Purification and Properties of the Cytoplasmic Glucose-6-Phosphate Dehydrogenase from Pea Leaves K. FICKENSCHER AND R. SCHEIBE’ Lehrstuhl fiir Pjeanzenphysiologie, lJniversit(it Bayreuth, Universitiitsstrasse SO, D-8580 Bayreuth, Federal Republic of Gemnany Received September 30,1985, and in revised form January 22,1986 A method involving affinity chromatography on the yellow dye Remazol Brilliant Gelb GL to highly purify the cytoplasmic isoenzyme of glucose-&phosphate dehydrogenase from pea shoots is described. Purification is at least 6000-fold. The specific activity of the purified enzyme is 185 pmol NADP reduced/min per mg protein. The preparation was free from any contamination of chloroplastic isoenzyme. The purified enzyme retains its activity in the presence of reducing agents which, in contrast, inactivate the chloroplast enzyme. The state of activity of the cytoplasmic and the chloroplastic isoenzyme in illuminated or darkened pea leaves was investigated using specific antibodies. While upon illumination the chloroplastic isoenzyme was in- activated by 80 to 90%, we could not find any change in activity of the cytoplasmic glucose-&phosphate dehydrogenase. ATP, ADP, NAD, NADH, and various sugar phos- phates do not inhibit the enzyme activity. Only NADPH is a strong competitive inhibitor with respect to NADP, suggesting that the enzyme is regulated by feedback inhibition by one of its products. Me ions have no influence on the activity of the enzyme. The molecular weight has found to be 240,000 for the native enzyme and 60,000 for the subunit. Throughout the purification procedure the enzyme was very unstable unless NADP was present in the buffer. o 1966 Academic press. I,,c. Glucose-6-P’ dehydrogenase (EC 1.1.1.49) is one of the key enzymes of the oxidative pentose-phosphate cycle, two isoenzymes of which have been separated from spinach leaves (1). One isoenzyme is localized in the chloroplast, the other in the cytosol. The chloroplastic isoenzyme from spinach and from pea has already been partially char- acterized (2-4), and a purification from pea chloroplasts has been reported (5). How- ever, as far as we are aware, no attempts have been made to isolate the cytoplasmic form of glucose-6-P dehydrogenase. There is not doubt that the chloroplastic glucose- 1 To whom correspondence should be addressed. ’ Abbreviations used: glucose-6-P, glucose-6-phos- phate; SDS, sodium dodecyl sulfate. 6-P dehydrogenase is light- and dithio- threitol-inactivated. But, since such a modulation of enzyme activity mediated by photosynthetic electron transport has also been postulated to occur on the cytoplasmic isoenzyme (63, we were prompted to fur- ther investigate the regulatory properties of that isoenzyme. This work describes the purification of cytoplasmic glucose-6-P dehydrogenase from pea leaves by means of affinity chro- matography and some of the enzyme’s properties. Furthermore, antibodies were raised against the purified enzyme which were used to distinguish between the ac- tivity of the chloroplastic and the cyto- plasmic isoenzyme in a rapidly prepared crude extract from illuminated or darkened leaves. 393 0003-9861/86 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.