Isolation of Carbonic Anhydrase from the Higher Plant Pisum sativum

Sequence analysis of the carboxysomal carbonic anhydrase (CcaA) from Synechocystis PCC6803, Synechococcus PCC7942 and Nostoc ATCC29133, indicated high sequence identity to the β class of plant and bacterial carbonic anhydrases (CA), and conservation of the active site region. However, the cyanobacterial enzyme has a C-terminal extension of about 75 amino acids (aa) not found in the plant enzymes, and largely absent from other bacterial enzymes. Using recombinant DNA technology, genes encoding C-terminal truncation products of up to 127 aa were overexpressed in E. coli, and partially purified lysates were analysed for CA-mediated exchange of 18O between 13C18O2and H216O. Recombinant CcaA proteins with up to 60 aa removed (CcaAΔ60) were catalytically competent, but beyond this there was an abrupt loss of activity. CcaAΔ0, along with CcaAΔ40 and CcaAΔ60, also catalysed the hydrolysis of carbon oxysulfide (COS; an isoelectronic structural analogue of CO2), but CcaAΔ63 and CcaAΔ127 did not, indicating that truncations greater than 62 aa resulted in a general loss of catalytic competency. Analysis of protein-protein interaction using the yeast two-hybrid system revealed that CcaA did not interact with the large or small Rubisco subunits (RbcL and RbcS, respectively) of Synechocystis, but there was strong CcaA-CcaA interaction. This protein interaction also ceased with C-terminal truncations in CcaA greater than 60 aa. The correlation between loss of CcaA-CcaA interaction and CcaA catalytic activity suggests that the proximal portion of the C-terminal extension is required for oligomerization, and that this oligomerization is essential for catalysis by the cyanobacterial enzyme. Thus, the C-terminal extension may play an important role in the function of CA within cyanobacterial carboxysomes, which is not required by the higher plant enzymes.

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DNA fiber replication in chromosomes of a higher plant (Pisum sativum)

Experimental Cell Research ◽

10.1016/0014-4827(75)90427-9 ◽

1975 ◽

Vol 93 (1) ◽

pp. 95-104 ◽

Cited By ~ 51

Author(s):

J. Van't Hof

Keyword(s):

Pisum Sativum ◽

Higher Plant

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The Rubisco large subunit binding protein

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1986.0048 ◽

1986 ◽

Vol 313 (1162) ◽

pp. 419-428 ◽

Cited By ~ 38

Keyword(s):

Pisum Sativum ◽

Molecular Mass ◽

Binding Protein ◽

Large Subunit ◽

Sodium Dodecyl ◽

Relative Molecular Mass ◽

Beta Subunits ◽

Higher Plant ◽

Amino Terminal ◽

Intact Chloroplasts

Newly synthesized Rubisco large subunits made by isolated intact chloroplasts from Pisum sativum are bound non-covalently to another protein, termed the Rubisco large subunit binding protein. This protein is implicated in the assembly of Rubisco in higher plant chloroplasts. The binding protein has been purified from Pisum sativum in the form of an oligomer of relative molecular mass ( M r ) about 720000. Analysis on polyacrylamide gels containing sodium dodecyl sulphate reveals equal amounts of two different types of subunit, termed alpha ( M r about 61000) and beta ( M r about 60000); thus the oligomer has the composition a α 6 β 6 . The alpha and beta subunits have been separated; their amino-terminal sequences are different, and antibodies raised against one subunit do not cross-react with the other subunit. Antibodies raised against the binding protein do not cross-react with the Rubisco large subunit, but do cross-react with polypeptides of M r about 60000 in extracts of chloroplasts from wheat, barley and tobacco, and in extracts of leucoplasts from castor-bean endosperm. The binding protein is made as a higher-molecular-mass precursor whan leaf polysomes are translated in a wheatgerm extract containing chloramphenicol, but is not synthesized by isolated intact chloroplasts. Thus the binding protein subunits are synthesized by cytoplasmic ribosomes and hence are likely to be encoded by nuclear genes. Etiolated Pisum plants contain binding protein, but exposure to light does not cause the same dramatic increase in amount that is seen in the case of Rubisco. Treatment of stromal extracts with Mg-ATP in the range 0.1-5.0 mM causes dissociation of the binding protein oligomer into monomeric subunits; CTP, GTP, UTP, AMP and cyclic AMP do not have this effect. Mg 2+ is required for dissociation but can be replaced by Ca 2+ . Newly synthesized large subunits are released when the binding protein oligomer is dissociated, but re-attach when the dissociation is reversed by removal of ATP.

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