The two Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase isozymes form heterotetramers

Two isozymes of the purine salvage enzyme hypoxanthine-guanine phosphoribos yltransferase (HGPRT) of the apicomplexan protozoan Toxoplasma gondii are e ncoded by the single HGPRT gene as a result of differential splicing. Weste rn blotting of total T. gondii protein shows that both isozymes I and II, w hich differ by 49 amino acids, are expressed. Both form enzymatically activ e homotetramers when overexpressed in Escherichia coil. The specific activi ty of HGPRT-I is five times that of HGPRT-II. When both isozymes are co-exp ressed in E. coli, HGPRT-I HGPRT-II heterotetramers form. The predominant h eterotetramer has enzymatic activity similar to HGPRT-II, and gel filtratio n chromatography demonstrates that its size is intermediate between the siz es of HGPRT-I and HGPRT-II. Mass spectrometric analysis of cross-linked hom o- and heterotetramers reveals species of distinct molecular mass for HGPRT -I, HGPRT-II, and HGPRT-I.HGPRT-II and suggests that the predominant hetero tetramer consists of one HGPRT-I subunit and three HGPRT-II subunits. The i mplications of this finding are discussed.