The effect of high hydrostatic pressure on the modulation of regulatory enzymes from spinach chloroplasts.
High hydrostatic pressure enhanced the specific activity of regulatory enzymes of the Benson-Calvin cycle (fructose-1,6-bisphosphatase, glyceraldehyde-3-P dehydrogenase, phosphoribulokinase) which are modulated by the ferredoxin-thioredoxin system. High activity of chloroplast fructose-1,6-bisphosphatase required dithiothreitol, fructose 1,6-bisphosphate, and Ca2+. At 100 bar the A0.5 for fructose 1,6-bisphosphate (0.3 mM) was lower than that at 1 bar (1.5 mM), whereas similar variations of pressure did not alter the A0.5 for Ca2+ (55 microM). The response of chloroplast glyceraldehyde-3-P dehydrogenase exposed to 500 bar was a 4-fold increase in the NADP-linked activity; conversely, the NAD-dependent activity remained unchanged. The concerted action of high pressure and Pi (or ATP), both activators of chloroplast glyceraldehyde-3-P dehydrogenase, led to inactivation. On the other hand, the activity of phosphoribulokinase increased 10-fold when the enzyme was incubated at 1500 bar; the activation process was strictly dependent on the presence of dithiothreitol. At variance with these enzymes, bovine liver fructose-1,6-bisphosphatase, yeast glyceraldehyde-3-P dehydrogenase, and chloroplast ribulose 1,5-bisphosphate carboxylase, whose activities are not modulated by reduced thioredoxin, were inactivated by high pressure. The comparison of oligomeric enzymes revealed that the stimulation of specific activity by high pressure correlated with thioredoxin-mediated activation, and it did not depend on a particular subunit composition. Present results show that high pressure resembled thioredoxin, cosolvents, and chaotropic anions in its action on regulatory enzymes of the Benson-Calvin cycle. The comparison of physiological and non-physiological modulators suggested that thioredoxin-mediated modifications of noncovalent interactions is an important event in light-dependent regulation of chloroplast enzymes.
Prat-Gay
G
G
Instituto de Investigaciones Bioquimicas Fundacion Campomar, Buenos Aires, Argentina.
Paladini
A
A
Stein
M
M
Wolosiuk
R A
RA
D000818:Animals; D002417:Cattle; D002736:Chloroplasts / Q000201:enzymology*; D004789:Enzyme Activation; D005288:Ferredoxins / Q000378:metabolism; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006874:Hydrostatic Pressure; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D010788:Photosynthesis; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D013379:Substrate Specificity; D013879:Thioredoxins / Q000378:metabolism
D005288:Ferredoxins; D013879:Thioredoxins; D009249:NADP; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D006597:Fructose-Bisphosphatase; D012273:Ribulose-Bisphosphate Carboxylase