Purification and Properties of Soluble and Bound γ-Glutamyltransferases from Radish Cotyledon

Cloning and molecular analysis of radish (Raphanus sativus L.) cDNAs encoding heterodimeric .GAMMA.-glutamyltransferases

“…NP177140) revealed a putative protein that apparently has a complete small subunit but lacks approximately 70% of the GGT large subunit. The N-terminal amino acid sequences of radish GGT small subunits (Nakano et al 2006) were highly identical to those of the small subunits deduced from Arabidopsis AtGGT (accession no. Z49240), At4g39650 (accession no.…”

“…In accord with this, it was reported that AtGGT overexpressed in tobacco plants was a membrane-bound protein that might be associated with the plasma membrane, as in the case of mammalian GGTs (Storozhenko et al 2002). In contrast, radish heterodimeric and monomeric GGTs were purified and characterized as symplastic soluble enzymes and cell wall bound enzymes, respectively (Nakano et al 2004;Nakano et al 2006). These results strongly suggest that the N-terminal hydrophobic domain of at least some of the radish heterodimeric GGT isoforms may be cleaved after protein sorting.…”

“…After protein processing, this threonine becomes the N-terminal residue of E. coli GGT (Suzuki and Kumagai 2002) and it is also necessary for enzyme catalysis (Inoue et al 2000). The same case applies to the heterodimeric type of radish GGTs (Nakano et al 2006).…”

“…In order to isolate a possible At4g29210 orthologue in radish, we followed an RT-PCR strategy based on the use of degenerate primers against the highly conserved Nterminal amino acid sequence (TSHFSIVDSD) of the small subunits (Nakano et al 2006) and At4g29210 cDNA specific primers. Primary PCR was performed on 5-day-old radish cotyledon cDNA using the degenerate primer 1 (5Ј-ACRAGYCATTTYTCSATMG-5Ј) and the At4g29210 primer 1 (5Ј-CTCCACCTGATAGC-TCCT-3Ј).…”

“…However, at present, we cannot disregard the possibility that RsGGT3 will encode a protein without GGT activity. The fact that heterodimeric GGTs were purified from the radish soluble fraction (Nakano et al 2006) indicates that RsGGT3 and/or yet to be identified radish cDNAs encode heterodimeric soluble GGT isoforms. The biological significance of the different heterodimeric RsGGT isoforms should be determined in further studies.…”