Kawano M, Namba Y, Hanaoka M
Microbiol Immunol. 1982;26(3):253-69. doi: 10.1111/j.1348-0421.1982.tb00176.x.
Cell hybridization of phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocyte (PBL) with murine lymphoma (EL-4) provided three hybridomas (MHH-16, MHH-20, and MHH-22) which spontaneously produced human mitogenic factor (MF). MHH-16 was serially subcloned by limiting dilution procedures, which resulted in maintaining two subclones producing human MF spontaneously for more than one year (PQL-3 and PQL-5 subcloned lines). Human MF MHH-MF) derived from supernatants of PQL-5 line cultures had a molecular weight (m.w.) of about 26,000-30,000 daltons (the major peak) with a minor peak with an m.w. of 15,000 daltons on Sephadex G-100 chromatography, and at a high concentration of NaCl (1 M), the activity of the 26,000-30,000-m.w. fraction became weak and that of the 15,000-m.w. fraction became predominant. MHH-MF had an isoelectric point of Ph 5.0-6.5. On DEAE-cellulose chromatography, MHH-MF was eluted at a fairly low salt concentration (sodium phosphate buffer 0.02 M, pH 8.0, NaCl 10 mM). After periodate treatment of this MHH-MF, the mitogenic activity almost disappeared. MHH-MF was relatively unstable to heating at 56 c for 20 min. In the presence of tunicamycin (0.3 microgram/ml), an inhibitor of N-linked glycosylation the synthesized MHH-MF showed a decrease in m.w. as follows: the major peak shifted from 26,000-30,000 to 23,000 daltons and the minor peak from 15,000 to 10,000 daltons on Sephadex G-100 chromatography. In internal labeling experiments with [3H] leucine, the 3H-labeled MF was partially purified, with mitogenic activity as a guide. This 3H-labeled MHH-MF fraction could be absorbed by PHA blasts but not by normal PBL. On SDS-PAGE under reducing conditions, only the radioactive peak of the 15,000-dalton fraction was recovered. MHH-MF obtained from the hybridoma culture supernatants may be a dimer of the 15,000-dalton fraction and a glycoprotein.
