Association of Carbonic Anhydrase Activity with Carboxysomes Isolated from the Cyanobacterium Synechococcus PCC7942.
The development of a simple method for the isolation of purified carboxysomes from the cyanobacterium Synechococcus PCC7942 has made it possible to identify a specific and inducible, intracellular carbonic anhydrase (CA) activity that is strongly associated with carboxysomes. This was shown, in part, through enzyme recovery experiments that indicated that a clear majority of a CA activity that is sensitive to the CA inhibitor ethoxyzolamide (I(50) = 4 mum) copurifies with a majority of the cell's ribulose-1,5-bisphosphate carboxylase/oxygenase activity in a highly purified pelletable fraction. Electron microscopy of this pelletable fraction revealed the presence of carboxysomes that were physically intact. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of carboxysome proteins showed that the large and small subunits of ribulose-1,5-bisphosphate carbosylase/oxygenase were clearly prominent and that several other minor proteins could be distinguished. The specific location of this carboxysomal CA activity is further reinforced by the finding that a previously isolated high CO(2)-requiring mutant, Type II/No. 68 (G.D. Price, M.R. Badger [1989] Plant Physiol 91: 514-525), displayed a 30-fold reduction in carboxysome-associated CA activity when tested under optimal conditions. Carboxysomal CA has the unusual property of being inactivated by dithiothreitol. The enzyme also requires 20 mm Mg(2+) (as MgSO(4)) for near maximum activity; other divalent cations, such as Ca(2+) and Mn(2+), also stimulate carboxysomal CA activity, but to a lesser extent than Mg(2+). Results are discussed in relation to the role of carboxysomes in the CO(2)-concentrating mechanism in cyanobacteria and the role that carboxysomal CA activity appears to play in this process.
Price
G D
GD
Plant Environmental Biology Group, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra, 2601, Australia.
Coleman
J R
JR
Badger
M R
MR