The two toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase isozymes form heterotetramers.
Two isozymes of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of the apicomplexan protozoan Toxoplasma gondii are encoded by the single HGPRT gene as a result of differential splicing. Western blotting of total T. gondii protein shows that both isozymes I and II, which differ by 49 amino acids, are expressed. Both form enzymatically active homotetramers when overexpressed in Escherichia coli. The specific activity of HGPRT-I is five times that of HGPRT-II. When both isozymes are co-expressed in E. coli, HGPRT-I.HGPRT-II heterotetramers form. The predominant heterotetramer has enzymatic activity similar to HGPRT-II, and gel filtration chromatography demonstrates that its size is intermediate between the sizes of HGPRT-I and HGPRT-II. Mass spectrometric analysis of cross-linked homo- and heterotetramers reveals species of distinct molecular mass for HGPRT-I, HGPRT-II, and HGPRT-I.HGPRT-II and suggests that the predominant heterotetramer consists of one HGPRT-I subunit and three HGPRT-II subunits. The implications of this finding are discussed.
White
E L
EL
Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35205, USA.
Ross
L J
LJ
Davis
R L
RL
Zywno-Van Ginkel
S
S
Vasanthakumar
G
G
Borhani
D W
DW
D000818:Animals; D001704:Biopolymers; D015153:Blotting, Western; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014122:Toxoplasma / Q000201:enzymology*
D001704:Biopolymers; D007527:Isoenzymes; D007041:Hypoxanthine Phosphoribosyltransferase