Nature of the rate-limiting step in the supply of inorganic carbon for photosynthesis in isolated asparagus mesophyll cells.

Accumulation of acid-stable and acid-labile C has been studied in cells isolated from cladophylls of Asparagus sprengeri regel, as a function of the concentrations of the various inorganic C (C(i)) species in the external medium.The rate of CO(2) fixation was higher when C(i) was supplied as CO(2) as opposed to HCO(3) (-), at the same concentration. Participation of HCO(3) (-) was indicated when the external CO(2) and HCO(3) (-) concentrations were chosen such that, owing to interconversion between C(i) species, the same CO(2) concentrations would be reached at a certain point in time, regardless of which species was supplied initially; at this point, a higher fixation rate was observed in the case of HCO(3) (-) supply.In the presence of carbonic anhydrase, the apparent affinity for C(i) was raised. This enzyme raised fixation rate even under steady-state conditions, but only at limiting external C(i) concentrations. The decrease in external CO(2) concentration was correlated with a decreasing internal C(i) level when CO(2) was the species supplied.When 10 mum CO(2) was supplied initially fixation rate was almost independent of pH. However, when HCO(3) (-) was supplied at concentrations calculated to yield 10 mum CO(2) at equilibrium, fixation rate rose with pH. Carbonic anhydrase raised the fixation rate over the entire pH range when HCO(3) (-) was the species supplied.It is concluded that CO(2) was the major C(i) species permeating from the medium to the carboxylating site. Further, our results bring clear evidence that formation of CO(2) from HCO(3) (-) in the unstirred layer, and the diffusion of both species in this layer, rate limit CO(2) fixation by these isolated cells.