Isolation and Characterization of a cDNA Coding for Pea Chloroplastic Carbonic Anhydrase

Abstract

Using a polyclonal antibody generated against the purified pea (Pisum sativum) carbonic anhydrase (CA) monomeric species, we have isolated and characterized a cDNA coding for this enzyme. Protein sequence analysis was used to confirm the identity of the clone. The presence of a large transit peptide suggests that CA is transported into the chloroplast and then processed to the mature size of approximately 26 kilodaltons. Northern hybridization, using the CA cDNA as a probe of total leaf RNA, revealed a single transcript of 1.45 kilobase pairs. This transcript was not detected in RNA extracted from root or etiolated leaf tissue. Comparison of the deduced amino acid sequence with that of spinach CA showed approximately 68% identity over the length of the nascent protein but with greater similarity observed within the mature protein sequences. In addition, regions of the pea and spinach CA proteins were found to be significantly similar to the Escherichia coli cyanate permease.One of the more abundant soluble proteins in the leaves of higher plants is the

Kinetic studies of pea carbonic anhydrase

“…In a previous study (Johansson and Forsman, 1992), we showed this doublet to represent subunits of two sizes present within pea chloroplasts. Calculation of the corresponding molecular masses from the published cDNA sequences (Roeske and Ogren, 1990;Majeau and Coleman, 1991) and N-terminal amino acid sequences from isolated subunits gives masses of 24.2 kDa and 28.2 kDa. Non-denaturing gel electrophoresis also gives a doublet but with a mass around 230 kDa (data not shown).…”

“…Today we know the amino acid sequences for plant CAs from spinach (Fawcett et al, 1990;Burnell et al, 1990), pea (Roeske and Ogren, 1990;Majeau and Coleman, 1991), tobacco (Majeau and Coleman, 1992) and Arubidopsis thuliuna (Raines et al, 1992) and they are highly similar with 54% identity and 80% similarity. CAs from the two procaryotes Synechococcus (Fukuzawa et al, 1992) and Escherichia coZi (Sung and Fuchs, 1988;Guilloton et al, 1992) have also been sequenced and found to belong to the plant CA group.…”

Characterization and Expression of Two cDNAs Encoding Carbonic Anhydrase in Arabidopsis thaliana

“…Initially, a total of 106 recombinants were screened with a 617-bp SacI-BamHI intemal restriction fragment of the pea CA cDNA (Majeau and Coleman, 1991). The hybridization was done at 65OC and the final washing at 55OC.…”

“…After the sample was centrifuged (10 min a t l2,OOOg), the supematant was made 0.1 M Na2C03/DT1', and the soluble protein samples were analyzed by SDS-I'AGE. Acrylamide gels were blotted onto nitrocellulose according to the manufacturer's specifications (Pharmacia Multiphor I1 Systems Handbook 18-1013-42), and westem analysis was performed by incubation with a polyclonal antibody against pea CA (Majeau and Coleman, 1991).…”

Processing of the chloroplast transit peptide of pea carbonic anhydrase in chloroplasts and in Escherichia coli Identification of two cleavage sites

“…cDNA sequences coding for spinach CA [4,5] and pea CA [6,7] have been published and the derived amino Abbreviufiorrs: CA, Carbonic anhydrase; RCA 11, human carbonic anhydrase 11; Kubisco, ribulosc-I,5-bisphosphate carboxylase; cTP, chloroplast transit peptide; TPTG, isopropyl-I&o-thiogalactopyranoside.…”