Figure 1.

Molecular mass determination of native precursor and mature API under steady-state conditions. (A) Glycerol gradient analysis of precursor and mature API under steady-state conditions. Wild-type (SEY6210) cells were grown to midlog phase, lysed with glass beads, and the resulting cell extracts were separated on 20–50% glycerol gradients. Collected fractions were subjected to Western blotting with antiserum to API. The reaction of a chemifluorescent substrate (ECF) with an alkaline phosphatase– conjugated secondary antibody allowed for the quantitation of the Western blots by a chemifluorescence scanner (Molecular Dynamics) as shown in the graph corresponding to the Western blot signals. Molecular mass standards indicated are hen egg albumin (45 kD), aldolase (158 kD), catalase (240 kD), ferritin (450 kD), and thyroglobulin (669 kD). Both mature (mAPI) and precursor API (prAPI) peaks appeared in fraction 7, cofractionating with the thyroglobulin molecular mass standard. (B) Hedrick-Smith calculation of the molecular masses of precursor and mature API. Relative mobilities were measured for protein standards resolved on native gels of 4.0 to 5.5% acrylamide and the negative slopes were determined by plotting the relative mobilities as a function of gel percentage. A standard curve was then generated by plotting the negative slopes of the protein standards as a function of molecular mass. Extracts from wild-type and pep4Δ strains were run on the same gels, and their molecular masses were calculated with reference to the standard curve. Molecular masses of mAPI and precursor API were determined to be 592 and 752 kD, respectively.