Spinach Carbonic Anhydrase: Primary Sequence, Transport Into Chloroplasts and Expression in Escherichia Coli.
Date of Award
Doctor of Philosophy (PhD)
Sue G. Bartlett
Carbonic anhydrase (CA) catalyzes the reversible hydration of CO$\sb2$. Plant carbonic anhydrases are structurally distinct from those found in animals. In C$\sb3$ plants, the enzyme is located in chloroplasts. To date, little structural information on the enzyme from plants has been available. In this study the full-length cDNA for the spinach chloroplast CA was sequenced. The cDNA contained 1,156 base pairs. The open reading frame encoded a protein of 34,569 daltons. Comparison of the N-terminal sequence of this protein with that of other chloroplast precursors indicated that CA is synthesized as a precursor. The transit peptide likely contains about 60 amino acids. Indirect immunoprecipitation of translation products synthesized using pea poly A RNA indicated that the pea precursor is approximately 36,000 daltons. Incubation of either the pea or spinach precursor with isolated intact chloroplasts resulted in import of the precursor and cleavage to a polypeptide of about 30,000 daltons. Various spinach CAs containing deletions ranging from 34 to 78 amino acids were expressed in E. coli, under the control of the trc promotor. Each of the CAs assembled to yield the enzymatically active hexameric enzyme. Immunological techniques performed in E. coli expressed spinach CA as well as total plant extracts demonstrated the susceptibility of CA towards proteolysis at the N-terminal end. Furthermore, a cross-reacting protein, which is distinct from the well characterized periplasmic CA, was demonstrated to be present in extracts of Chlamydomonas reinhardti. Western blotting of CA extracts from leaves, chloroplasts or E. coli expressing CA indicated that CA is susceptible to proteolysis. N-terminal sequencing data obtained by others suggests that the degradation is from the N-terminus. Also, extracts from pea stems and leaves contained a polypeptide which cross-reacted with antibodies raised against spinach CA. Root extracts contained no cross-reacting polypeptides. These results suggest that, like other chloroplast proteins, expression of CA is regulated in a tissue specific manner.
Fawcett, Timothy William, "Spinach Carbonic Anhydrase: Primary Sequence, Transport Into Chloroplasts and Expression in Escherichia Coli." (1990). LSU Historical Dissertations and Theses. 4983.