ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 244, No. 1, January, pp. l-15,1986 Enzyme Regulation in C4 Photosynthesis: Purification, Properties, and Activities of Thioredoxins from C4 and C3 Plants’ NANCY A. CRAWFORD, BOIHON C. YEE, STEVEN W. HUTCHESON,’ RICARDO A. WOLOSIUK,3 AND BOB B. BUCHANAN Lhisim of MoLwular Plant Biology, Hilgard Hall, University of Califmia, Berkeley, California 94720 Received June 20,1985, and in revised form September 13, 1985 Procedures are described for the purification to homogeneity of chloroplast thiore- doxins f and m from leaves of corn (Zea muys, a C4 plant) and spinach (Spinacea oleracea, a C3 plant). The Cs and CJthioredoxins were similar immunologically and biochemically, but differed in certain of their physiochemical properties. The f thioredoxins from the two species were capable of activating both NADP-malate dehydrogenase (EC 1.1.1.37) and fructose-1,6-bisphosphatase (EC 3.1.3.11) when tested in standard thioredoxin assays. Relative to its spinach counterpart, corn thioredoxin f showed a greater molecular mass (15.0-16.0 kDa vs 10.5 kDa), lower isoelectric point (ca. 5.2 vs 6.0), and lower ability to form a stable noncovalent complex with its target fructose bisphosphatase enzyme. The C3 and Cq m thioredoxins were similar in their specificity (ability to activate NADP- malate dehydrogenase, and not fructose-1,6-bisphosphatase) and isoelectric points (ea. 4.8), but differed slightly in molecular mass (13.0 kDa for spinach vs 13.5 kDa for corn) and substantially in their immunological properties. Results’obtained in con- junction with these studies demonstrated that the thioredoxin m-linked activation of NADP-malate dehydrogenase in selectively enhanced by the presence of halide ions (e.g., chloride) and by an organic solvent (e.g., 2-propanol). The results suggest that in viva NADP-malate dehydrogenase interacts with thylakoid membranes and is regulated to a greater extent by thioredoxin m than thioredoxin $ o 1~66 Academic PRESS, I~C. The ferredoxin/thioredoxin system, consisting of ferredoxin, ferredoxin-thio- redoxin reductase, and two thioredoxins, functions in enzyme regulation in different types of oxygenic photosynthesis, including cyanobacteria, Ca, C4 and crassulacean acid i Sixth paper of a series. The first five papers are identified in Ref. (34). This work was supported by grants from the Competitive Research Grants Office of the U. S. Department of Agriculture (Photosyn- thesis Program) and the National Science Foundation (Metabolic Biology Program and, in the initial stages, the U. S.-Latin America Program). ‘Present address: Department of Botany, Univer- sity of Maryland, College Park, Md. 20742. a Permanent address: Fundacion Campomar, An- tonio Machado 151/Av. Patricias Argentina 435,1405- Buenos Aires, Argentina. metabolism (CAM)4 plants (l-3). The thio- redoxin components of this system (thio- redoxins f and m) have been purified and studied from C3 (4, 5) and CAM plants (6, 7), but, aside from their presence (8,9), lit- tle is known about these proteins in C4 plants. We have, therefore, isolated thio- redoxins f and m from leaves of corn, a classical C4 plant, and determined certain physiochemical and biochemical proper- ties. The results demonstrate that these C4 4 Abbreviations used: CAM, crassulacean acid me- tabolism; PMSF, phenylmethylsulfonylfluoride; FPLC, fast protein liquid chromatography; ELISA, enzyme- linked immunosorption assay; Mops, 3-(N-morphol- ino)propanesulfonic acid; TEMED, N,N,N’,N’-tetra- methylethylenediamine; Mes, 2-(N-morphoIino)eth- anesulfonic acid. 1 0003-9861/86 $3.00 Copyright 0 1986 by Academic Press. Inc. All rigbta of reproduction in any form reserved.