Regulation of spinach chloroplast acetyl-CoA carboxylase.
We have investigated several factors which influence acetyl-CoA carboxylase (ACCase) activity in lysed spinach chloroplasts. (1) When assayed after rapid lysis of light-incubated chloroplasts, ACCase activity was 2-fold higher than activity from dark-incubated chloroplasts. Within 5 min after lysis, activity from dark-incubated chloroplasts increased, suggesting a transient inactivation or inhibition of ACCase in the dark. (2) When lysed chloroplast suspensions were incubated with 30 to 100 microM acetyl-CoA before starting assays, activity was 4-fold higher than if suspensions were not preincubated with acetyl-CoA. CoA, malonyl-CoA, propionyl-CoA, and butyryl-CoA also activated ACCase. Full acetyl-CoA activation required MgATP and was essentially complete after 8 min. ACCase activity decreased upon removal of acetyl-CoA by gel filtration and was partially restored by readdition of acetyl-CoA. Thus, ACCase activation by acetyl-CoA was reversible. (3) Dithiothreitol and thioredoxin stimulated ACCase activity, but only in preparations where ACCase activity was low. (4) ACCase was assayed in concentrations of ATP, ADP, NADPH, NADP+, Mg2+, and CO2/HCO-3, which are estimated to occur in the stroma of chloroplasts under illumination or darkness. ACCase activity from lysed chloroplast suspensions was 10-fold higher when illuminated conditions were used. However, this activity was still 5-fold to 10-fold lower than the rates required to sustain known in vivo rates of fatty acid synthesis and in vitro rates achieved under optimum assay conditions with saturating substrates.
Hunter
S C
SC
Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan, 48824, USA.
Ohlrogge
J B
JB
D000105:Acetyl Coenzyme A / Q000502:physiology; D000103:Acetyl-CoA Carboxylase / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000502:physiology; D002736:Chloroplasts / Q000737:chemistry / Q000201:enzymology*; D003065:Coenzyme A / Q000502:physiology; D003624:Darkness; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D006863:Hydrogen-Ion Concentration; D008027:Light; D008316:Malonyl Coenzyme A / Q000502:physiology; D018724:Spinacia oleracea / Q000201:enzymology*; D013347:Subcellular Fractions / Q000737:chemistry / Q000201:enzymology; D013879:Thioredoxins / Q000494:pharmacology
D000214:Acyl Coenzyme A; C024343:butyryl-coenzyme A; C009061:propionyl-coenzyme A; D008316:Malonyl Coenzyme A; D013879:Thioredoxins; D000105:Acetyl Coenzyme A; D000103:Acetyl-CoA Carboxylase; D003065:Coenzyme A; D004229:Dithiothreitol