FIGURE 2.

Purification and identification of MCT-1. The substances that induced β-hexosaminidase release from HL-60-derived neutrophilic/granulocytic cells were fractionated and purified by gel filtration chromatography using a Sephadex G-25 column (A), preparative RP-HPLC using an ODS column (B), ion-exchange HPLC using a cation-exchange column (C), and analytical RP-HPLC using a C₁₈ column (D). The ability of each fraction to induce β-hexosaminidase release from the differentiated HL-60 cells was tested, and the activity in each fraction is denoted with a gray column. Fractions containing the activity were combined as indicated in each panel. In E, the active 12th fraction in D was further purified and analyzed by micro-RP-HPLC using a C₂/C₁₈ column, and each peak fraction was collected separately. Only one of the fractions was able to induce enzyme release. The substances in this peak fraction (PAG1-BII(F12) peak) were subjected to fast atom bombardment mass spectrometry, which demonstrated a [M + H]⁺ of 2569.2 (F).