Date of Award:

5-1-1992

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Department name when degree awarded

Biology

Committee Chair(s)

Keith A. Mott

Committee

Keith A. Mott

Committee

Bruce Bugbee

Committee

Richard J. Mueller

Abstract

A spectrophotometric assay for the activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase was developed. This assay was used to examine the effects of CO2 and the inactive-Rubisco:ribulose 1,5-bisphosphate complex (ER) concentrations on the rate of activase-catalyzed Rubisco activation reaction in a purified preparation. ER and CO2 in this reaction were consistent with two-substrate Michaelis Menton kinetics. The implications of these data and their relation to previously published data on the effects of ER and CO2 on activase are discussed. The spectrophotometric activase assay was also used to measure activase activity in extracts from spinach (Spinacea oleracea L.) leaves undergoing steady- and nonsteady-state photosynthesis following an increase from darkness to a high PFD. Analyses of these samples showed that steady-state activase activity increased with PFD and saturated below 300 µmol m-2 s-1. Following an increase in PFD from darkness to a high PFD, activase activity increased over a period of 5 to 6 minutes, after which it was constant. The light-dependence of Rubisco activase is consistent with previous gas-exchange data showing two interdependent processes in the activation of Rubisco following an increase in PFD. Phosphorylation of serine and threonine residues of 41 and 45 KDa polypeptides was observed in activase preparations and in lysed chloroplasts. When unbound ATP was removed from the preparations by gel filtration, activase activity declined and free Pi increased over a similar time period. A phosphatase inhibitor, okadaic acid, prevented both of these processes. The data suggest that ATP is necessary for phosphorylation of activase to produce a catalytically active form and that there is an ATP requirement during catalysis. Phosphorylation of activase in lysed chloroplasts was found to be stimulated by light up to PFD values of approximately 200 µmol m-2 s-1. The data also suggest the involvement of a light-dependent protein kinase and a role for photosynthetic electron transport in regulating the process. Phosphorylation under illuminated conditions was also found to be dependent on ATP and free Mg2+. The results are discussed in the context of light-regulation of Rubisco activity during steady- and nonsteady-state photosynthesis.

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