No CAS activity could be detected, indicating that the CAS/CS specific activity ratio of this protein might be low

No CAS activity could be detected, indicating that the CAS/CS specific activity ratio of this protein might be low. purified CAS exhibits detectable CS activity (Hendrickson and Conn, 1969; Ikegami et al., 1988a, 1988b; Maruyama et al., 1998), according to the reaction (Eq. 2): 2 On the other hand, purified CSs from various plants species display some CAS activity (Ikegami et al., 1993). In spinach, CAS activity is usually predominantly present in the mitochondrial fraction but can also be detected in chloroplast and cytosol fractions (Warrilow and Hawkesford, 1998). In cocklebur seed cotyledon, cytosolic CAS activity has been attributed to CS (Maruyama et al., 1998). Compartment-specific CS isoforms have been purified from spinach and cauliflower cytosol, chloroplast, and mitochondrialfractions (Lunn et al., 1990; Rolland et al., 1992). In spinach,cDNAs encoding cytosolic and mitochondrial CS isoforms have Detomidine hydrochloride been isolated (Saito et al., 1992, 1993, 1994) (for a proposal of a new nomenclature for CS-like genes, (-substituted Ala synthase) family according to the rules recommended by the Commission rate on Herb Gene Nomenclature (http://mbclserver.rutgers.edu/CPGN/Guide.html). The extensive structural and functional similarities between CS and CAS prompted us to examine the identity of a CS isoform with CAS. In spinach mitochondria extracts, it was not possible to separate CS from CAS protein (Warrilow and Hawkesford, 1998), raising the possibility that the mitochondrial Bsas3 isoform of CS could actually be a CAS and not a CS, as was thought previously. To test this hypothesis, Detomidine hydrochloride we searched for homolog cDNAs in Arabidopsis and decided the kinetic characteristics of CS and CAS activities of spinach and Arabidopsis CS-like recombinant proteins. We conclude that this mitochondrial Bsas3 isoform is the CAS in spinach and Arabidopsis. This is the first report of the identification of CAS-encoding cDNA in living organisms. RESULTS Identification of New Members of the CS-Like Protein Family in Arabidopsis Among the 45,752 expressed sequence tags Detomidine hydrochloride (ESTs) of Arabidopsis present in the dbEST database (release November 26, 1999), 52 were assigned as encoding a CS. They could be classified into eight families, four of which did not match any previously reported CS sequence. ESTs representative of Detomidine hydrochloride these families were sequenced and named and (-contained an intron at position 260 to 351 and an in-frame stop codon at position 764 to 766. This imperfectly processed mRNA was corrected manually for the Detomidine hydrochloride purpose of establishing the phylogeny of herb CS-like proteins. The corrected protein was 325 amino acids long, with a calculated molecular mass of 34 kD, and was predicted to be cytosolic by the PSORT program (Nakai and Kanehisa, 1992). contained a full-length open reading frame (ORF) encoding a 324-amino acid polypeptide of 34.3 kD, which was predicted to be cytosolic. contained a partial ORF, comprising only the last 174 amino acids of the protein. contained a full-length ORF encoding a 368-amino acid polypeptide of 39.9 kD, which was predicted to be mitochondrial. Phylogenetic analysis indicated that herb CS-like proteins CPB2 could be separated into six groups, defined as Bsas1, Bsas2, Bsas3, Bsas4, Bsas5, and Bsas6 classes (Fig. ?(Fig.1).1). The ARAth;Bsas1;1 putative protein belonged to the Bsas1 class, whereas ARAth;Bsas4;1 and ARAth;Bsas4;2, together with BRAju;Bsas4;1 from Indian mustard (and appeared to be identical to and respectively, the sequences that were released in the directories recently. Open in another window Shape 1 Phylogeny of Bsas family members proteins in vegetation. Complete proteins sequences had been aligned using Clustal X (Thompson et al., 1997). Unrooted phylogenetic tree was determined from the neighbor-joining technique and then attracted using Drawtree through the Phylip bundle (Phylogeny Inference Bundle, edition 3.57c, Division of Genetics, College or university of Washington, Seattle). The real numbers indicate the bootstrap values for 100 replicates. Accession amounts are summarized in Desk ?TableI.We. *, Sequence manually corrected; #, partial series only. Sequences whose actual CAS or CS actions have already been determined are indicated in daring. Sequences whose real subcellular localization have already been established are underlined. Functional Characterization of CS-Like Recombinant Protein Expressed inside a Cys Auxotroph NK3 mutant, that have been lacking in CS activity (Desk ?(TableII).II). The NK3.