Purification, characterization and cDNA cloning of soluble carbonic anhydrase from Chlorella sorokiniana grown under ordinary air.
Soluble carbonic anhydrase (CA, EC 4.2.1.1) inducible by low levels of CO2 was purified from the unicellular green alga Chlorella sorokiniana grown at alkaline pH. The purified CA had a specific activity of 2,300 units (mg protein)-1. The molecular mass of the CA was found to be 100 kDa by non-dissociating (native)-polyacrylamide gel electrophoresis and 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 50-kDa subunit was recognized by concanavalin A. These results suggest that the protein has a dimeric form with two 50-kDa subunits that are glycosylated in an asparagine-linked manner. The native CA was revealed by isoelectric focusing to be a very acidic protein with an isoelectric point of 4.2. About 60% of the CA activity was inhibited by 0.5 M NaCl. The enzyme was inactivated over 95% by preincubation with 50 mM dithiothreitol but not with 1 mM dithiothreitol. After partial amino acid sequence analysis, a cDNA clone of the CA was isolated and characterized. The cloned cDNA fragment encoded a 348-amino-acid polypeptide (36,709 Da) including an NH2-terminal hydrophobic signal peptide composed of 35 amino acids (3,725 Da). Conserved regions of sequences found in animal CAs, in the periplasmic (pCA) and the intracellular CAs of Chlamydomonas, and in the plasma-membrane-bound CA of Dunaliella (Dca) were also found in this Chlorella CA. The signal sequence was significantly homologous to the pCA and the Dca. The internal signal sequence between the large and the small subunits reported for pCA was not found in this Chlorella CA. The soluble CA of this alga was an alpha-type CA with salt-sensitive, periplasm-locating and acidic properties and very different from pCA and Dca with their salt-sensitive/neutral and salt-resistant/acidic properties, respectively.
Satoh
A
A
Marine Biotechnology Institute, Kamaishi Laboratories, Iwate, Japan.
Iwasaki
T
T
Odani
S
S
Shiraiwa
Y
Y
D000388:Air; D000595:Amino Acid Sequence; D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002708:Chlorella / Q000201:enzymology* / Q000254:growth & development; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D014461:Ultracentrifugation
D018076:DNA, Complementary; D002256:Carbonic Anhydrases