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).