TY - JOUR
T1 - Studies on guinea-pig macrophage migration inhibitory factor (MIF). II. Purification of MIF after treatment with reducing and denaturing agents
AU - Kotkes, P.
AU - Pick, E.
PY - 1979
Y1 - 1979
N2 - Treatment of guinea-pig macrophage migration inhibitory factor (MIF) containing culture supernatants with the denaturing agents guanidine hydrochloride (Gu HCl) or urea, in the presence or absence of the reducing agent 2-mercaptoethanol (2-ME), or with sodium dodecyl sulphate (SDS), does not destroy biological activity. Alkylation of reduced MIF preparations results in a considerable decrease or total loss of MIF activity. Treatment of supernatants with the combinations, Gu HCl and 2-ME or urea and 2-ME results in the recovery of MIF activity in association with molecules less than 30,000 in molecular weight (mol. wt). After removal of the agents by dialysis, MIF activity is found associated with molecules larger than 30,000. The reduction in mol. wt. is dependent on the presence of 2-ME. When MIF-containing supernatants are treated with urea and SDS and fractionated by preparative polyacrylamide gel electrophoresis (PAGE) in the presence of the same agents, MIF activity is recovered in the mol. wt range of 42,000-80,000. When supernatants are treated with 2-ME, in addition to urea and SDS, and preparative PAGE is performed in their presence, MIF activity is found associated with material having a mol. wt of 15,000-18,000. Analytical SDS-PAGE of this fraction reveals 2 or 3 closely grouped bands corresponding to the above mol. wt range.
AB - Treatment of guinea-pig macrophage migration inhibitory factor (MIF) containing culture supernatants with the denaturing agents guanidine hydrochloride (Gu HCl) or urea, in the presence or absence of the reducing agent 2-mercaptoethanol (2-ME), or with sodium dodecyl sulphate (SDS), does not destroy biological activity. Alkylation of reduced MIF preparations results in a considerable decrease or total loss of MIF activity. Treatment of supernatants with the combinations, Gu HCl and 2-ME or urea and 2-ME results in the recovery of MIF activity in association with molecules less than 30,000 in molecular weight (mol. wt). After removal of the agents by dialysis, MIF activity is found associated with molecules larger than 30,000. The reduction in mol. wt. is dependent on the presence of 2-ME. When MIF-containing supernatants are treated with urea and SDS and fractionated by preparative polyacrylamide gel electrophoresis (PAGE) in the presence of the same agents, MIF activity is recovered in the mol. wt range of 42,000-80,000. When supernatants are treated with 2-ME, in addition to urea and SDS, and preparative PAGE is performed in their presence, MIF activity is found associated with material having a mol. wt of 15,000-18,000. Analytical SDS-PAGE of this fraction reveals 2 or 3 closely grouped bands corresponding to the above mol. wt range.
UR - http://www.scopus.com/inward/record.url?scp=0018296388&partnerID=8YFLogxK
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AN - SCOPUS:0018296388
SN - 0009-9104
VL - 37
SP - 540
EP - 550
JO - Clinical and Experimental Immunology
JF - Clinical and Experimental Immunology
IS - 3
ER -