Publication Date


Document Type


First Advisor

Holbrook, Gabriel P.

Degree Name

M.S. (Master of Science)

Legacy Department

Department of Biological Sciences


Plants; Effect of light on; Botanical chemistry


Studies of CA1-P metabolism in whole leaves and intact chloroplasts have indicated that CAl-Pase may be regulated by irradiance levels. This study examined the regulation of CAl-Pase in vitro by thioredoxin and phosphorylated metabolites. Its interaction with reducing agents and changes in ionic strength was also addressed. FPLC gel filtration was the most effective step in the purification of CAl-Pase, giving specific activity values similar to previous purifications from tobacco leaves. CAl-Pase from Phaseolus and Nicotiana, which produce drastically different amounts of CA1-P, gave similar responses to the potential activator compounds DTT, FBP, and thioredoxin. There was no activating response of CAl-Pase with thioredoxin f or m from spinach or from the bacteria Spirulina platensis and Escherichia coli. This suggests that CAl-Pase may not be regulated by this system, whereas E. coli thioredoxin was capable of stimulating the activity of purified spinach fructose bisphosphatase and malate dehydrogenase. Oxidation of CAl-Pase by glutathione inhibited catalysis, which could not be restored by a 20-fold excess concentration of DTT or NADH. Four millimolar DTT alone did not activate CAl-Pase. However, in the presence of 10 mM FBP, DTT at this concentration increased the activity of CAl-Pase 2-fold over 10 mM FBP alone. The increased ionic strength caused by 0- 25 mM KCl increased CAl-Pase activity. This response was further enhanced in the presence of FBP and DTT after a short preincubation time, suggesting a synergistic effect of this combination of metabolites and reducing agents. The lack of any positive response of CAl-Pase to DTT or thioredoxin, and the irreversibility of oxidation treatments, indicate that CAl-Pase may not be directly light activated. However, CAl-Pase may be regulated indirectly by changes occurring in the chloroplast stroma during illumination, such as increased ionic strength and increased concentrations of certain phosphorylated metabolites.


Includes bibliographical references (pages [95]-101)


viii, 101 pages




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