Fig. 4. (A) Superose 6 gel filtration of soluble fractions from L.mexicana wild type (WT) and ΔGDPMP promastigote lysates. The enzymatic activities of GDPMP and UDPGP were determined in each fraction. The elution positions of size standards are indicated by arrows. (B) High-pH anion-exchange HPLC of 2 M TFA hydrolysates of L.mexicana WT and ΔGDPMP promastigote membranes. The hexose fraction of equal amounts of cells (∼1 × 10⁸) was loaded onto a Carbo-Pac PA10 column. (C) SDS–PAGE/fluorography of delipidated total promastigote lysates from [³H]myo-inositol-labeled L.mexicana WT (lane 1), L.mexicana ΔGDPMP (lane 2) and L.mexicana ΔGDPMP + cRIBLmxGDPMP (lane 3). Each lane was loaded with 2.5 × 10⁷ delipidated promastigotes labeled overnight with [³H]myo-inositol. The positions of LPG and the major GPI-anchored surface metalloproteinase gp63 are indicated by a bar and an arrow, respectively. (D–G) SDS–PAGE/immunoblot of L.mexicana culture supernatants. Loading was normalized to SAP activity (2 mU). (D and E) Lane 1, WT; lane 2, ΔGDPMP. (F) Lane 1, WT; lane 2, ΔGDPMP; lane 3, ΔGDPM + cRIBLmxGDPMP. (G) Lane 1, WT; lane 2, ΔPMI; lane 3, ΔPMI + 20 µM Man; lane 4, ΔPMI + 200 µM Man; lane 5, ΔGDPMP; lane 6, ΔGDPMP + 200 µM Man; lane 7, ΔGDPMP + 2 mM Man; lane 8, WT. (H) Growth curves of L.mexicana WT and of L.mexicana ΔGDPMP in the presence of various Man concentrations. (D), (F) and (G) were probed with affinity-purified rabbit anti-L.mexicana GDPMP antibodies, while (E) was probed with mAb L7.25. The molecular masses and relative positions of standard proteins are indicated on the gels and blots in (C–G).