Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota.
The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated Mr of 124,000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.
Cella
R
R
Dipartimento di Genetica e Microbiologia A. Buzzati Traverso, Pavia, Italy.
Carbonera
D
D
Orsi
R
R
Ferri
G
G
Iadarola
P
P
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D007700:Kinetics; D007895:Leishmania tropica / Q000201:enzymology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000378:metabolism
D046911:Macromolecular Substances; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase