PHYSIOLOGIA PLANTARUM 88: 113-122. 1W3 Copyrigk, © fhysiatog,^ Plamarum I'rmiril in Unmriri - all r,xJui reaerved Physical, immunological and kinetic properties of ribiilose-1,5- bisphosphate carboxylase/oxygenase from Or {Abks alba) and spruce (Picea abies) D. Afif, D. Gerant, G. Cavalie and P. Dizengremel Afif, D.,Gerant, D. Cavalie, G. and Dizetigremel. P. 1993 Physical, immunological and kinetic properties of ribulose-l,5-bisphosphate carboxylase/oxygetiase from fir (Abies alba) and spruce (Picea abies). - Physiol. Plant. 88: 113-122. Ribulose-1.5.^bisphosphate carboxylase/oxygenase {Rubisco; EC 4.1.1.39) from fir (Abies alba Mill.) and spruce (Picea abies [L.] Karst.) needles was purified to homogeneity. The enzyme was isolated from crude extracts through quantitative precipitation in 40-55% and 40-60% (NHJ^SOj for fir and spruce, respectively, followed by linear sucrose gradient centrifugation. Using two dimensional gel eiectro- phoresis, the isoelectric points were determined. For the large subunit (LSU) it was 6.7 for both species, and for the small subunit (SSU) it was 7.1 and 7.7 for fir and spruce, respectively. Very few differences in tt7ptic peptides and amino acid compo- sition of Rubisco LSU were observed between fir and spruce. By contrast, marked differences characterized the same analyses for the Rubisco SSU of the two species. Moreover, substitution of residues was observed in the sequenced N-terminal region when comparing fir and spruce SSU. The Ouchterlony technique showed no immu- nochemical difference between Rubisco of fir and spruce when a rabbit antiserum to spinach Rubisco was used. The Eadie-Hofstee plots of carboxyiase activity indicated that the apparent K^(CO;) were 31 and 36 uM for the fir and spruce enzymes, respectively. Key words - Abies atha. amino acid cotnposition. conifer, fir. N-terminal sequence. Picea abies, protein purification. Rubisco. spruce. D. Afif (corre.sponding author), D. Gerant and P. Dizengremel. Lahoratoire de Physiologie Vegetaie et Forestiere. Univ. de Nancy 1. B. P. 239, F-S45t)6 Vandoeuvre Cedex, France; G. Cavalte, Centre de Biologie et de Physiologie Vegetate, Univ. P. Sahatier. 118 route de Narbonne. F-3I062 Toulouse Cedex. France. Introduction '^^' '''* ^^^'"^^y f*"" CO, could heip to discriminate between the photosynthetic C, and C, groups (Yeoh et The essential roles of ribulose-l,5-hisphosphate car- aL 1981. Gutteridge and Keys 1985, Wessinger et al. boxylase/oxygenase (Rubisco, EC 4.1.1.39) in the pro- 1989). cesses of photosynthesis and photorespiration have led The structure of the enzyme of higher plants is com- to intensive studies of this enzyme in numerous plants. monly given as a hexadecameric structure composed of The structure and kinetic properties of Rubisco vary 8 large subunits (LSU) and 8 small subunits (SSU) according to species (Miziorko and Lorimer 1983, An- (Andrews and Lorimer 1987). The active site of the drews and Lorimer 1987) atid the amount of enzyme in enzyme is located on the LSU which is coded by a leaves depends on environmentat factors, especialJy chioropiastic gene (rbcL). The small subunit has a pro- lighl (Gutteridge and Keys 1985), nutrition (Machler et found influence on the catalytic mechanism (Andrews al. 1988) and CO, (Van Oosten et al. 1992). Compari- and Lorimer 1987) and is coded by a nuclear multigene son of the K^(CO2) values among plant species suggests family (rbcS). This mode of formation involves different Received 8 October, 1992; revised 7 January, 1993 PhysiDl. Plant. SS. 1993 1)3