ANALYSIS OF COMPLEX-FORMATION BETWEEN HSP80 AND HSP70, CYTOSOLIC MOLECULAR CHAPERONES OF NEUROSPORA-CRASSA, BY ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISA)

A physical association between Hsp70 and Hsp80, the major cytosolic st ress proteins of Necrlospora crassa, was demonstrated previously by in terprotein crosslinking and by binding of Hsp80 to Hsp70 immobilized o n ATP-agarose. In the present study, [Hsp70:Hsp80] complex formation w as analyzed by enzyme-linked immunosorbent assays (ELISA), using speci fic antibodies. One protein was fixed onto ELISA plate: wells and bind ing of the second mobile protein was monitored by retention of its cog nate IgG. Binding of Hsp70 and Hsp 80 to immobilized Hsp80 and Hsp70, respectively, was readily detectable at submicrogram levels. The effec t of cations and various nucleotides on [Hsp70:Hsp80] complex was exam ined by inclusion of KCl, MgCl2, MnCl2, and nucleotides in the interac tion mixture. K+ stimulated interaction between immobilized Hsp70 and Hsp80 in solution and adenosine nucleotides exerted a stimulatory effe ct on complexation as well. Similarly, CTP, NAD, and NADH enhanced com plex formation between plate-bound Hsp70 and Hsp 80 in solution, attes ting to the conformational flexibility of Hsp80. Epitope blocking reve aled an overlap between protein-protein contact surfaces and antibody recognition sites. Binding to alpha-carbosym-thylated lactalbumin show ed that Hsp70 and Hsp 80 can interact with an unfolded polypeptide, in dividually and in complex.