Memory and imprinting effects in multienzyme complexes--I. Isolation, dissociation, and reassociation of a phosphoribulokinase-glyceraldehyde-3-phosphate dehydrogenase complex from Chlamydomonas reinhardtii chloroplasts.
A bienzyme complex made up of phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase has been isolated and purified from chloroplasts of Chlamydomonas reinhardtii. The complex contains four phosphoribulokinase and eight glyceraldehyde-3-phosphate dehydrogenase polypeptide chains. As phosphoribulokinase is dimeric and glyceraldehyde-3-phosphate dehydrogenase tetrameric, it is concluded that the complex comprises two phosphoribulokinase and two glyceraldehyde-3-phosphate dehydrogenase molecules. Its overall molecular mass is 460 kDa, which is in excellent agreement with its stoichiometry. Moreover, owing to the nature of the two enzymes, this complex must catalyse two nonconsecutive reactions. The bienzyme complex tended to spontaneously dissociate into the free enzymes upon dilution. This dissociation process was considerably promoted by reducing agents such as dithiothreitol or reduced thioredoxin. The kinetics of the dissociation process induced by dithiothreitol or reduced thioredoxin were paralleled by an increase of activity of phosphoribulokinase. The dissociation of the complex was reversible. If oxidized phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase were mixed, a certain amount of the complex was formed. The reconstituted complex displayed properties that were indistinguishable from those of the native complex extracted from chloroplasts of Chlamydomonas reinhardtii. These results suggest that the concentration of the complex in vivo must vary depending on the light intensity.
Avilan
L
L
Institut Jacques Monod, Université Paris VII, France.
Gontero
B
B
Lebreton
S
S
Ricard
J
J
D000818:Animals; D015153:Blotting, Western; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D002736:Chloroplasts / Q000201:enzymology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000494:pharmacology; D017886:Durapatite; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D013879:Thioredoxins / Q000494:pharmacology
D009097:Multienzyme Complexes; D009243:NAD; D013879:Thioredoxins; D009249:NADP; D017886:Durapatite; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D004229:Dithiothreitol