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 stress proteins of Neurospora crassa, was demonstrated previously by interprotein crosslinking and by binding of Hsp80 to Hsp70 immobilized on ATP-agarose. In the present study, [Hsp70: Hsp80] complex formation was analyzed by enzyme-linked immunosorbent assays (ELISA), using specific antibodies. One protein was fixed onto ELISA plate wells and binding of the second mobile protein was monitored by retention of its cognate IgG. Binding of Hsp70 and Hsp80 to immobilized Hsp80 and Hsp70, respectively, was readily detectable at submicrogram levels. The effect of cations and various nucleotides on [Hsp70: Hsp80] complex was examined by inclusion of KCl, MgCl2, MnCl2, and nucleotides in the interaction mixture. K+ stimulated interaction between immobilized Hsp70 and Hsp80 in solution and adenosine nucleotides exerted a stimulatory effect on complexation as well. Similarly, CTP, NAD, and NADH enhanced complex formation between plate-bound Hsp70 and Hsp80 in solution, attesting to the conformational flexibility of Hsp80. Epitope blocking revealed an overlap between protein-protein contact surfaces and antibody recognition sites. Binding to alpha-carboxymethylated lactalbumin showed that Hsp70 and Hsp80 can interact with an unfolded polypeptide, individually and in complex.
Department of Biological Sciences, University of Calgary, AB, Canada.
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Ouimet
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Kapoor
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002412:Cations / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000187:drug effects; D005656:Fungal Proteins / Q000737:chemistry*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry; D007768:Lactalbumin / Q000737:chemistry; D018832:Molecular Chaperones / Q000737:chemistry; D009492:Neurospora crassa / Q000737:chemistry; D012680:Sensitivity and Specificity
D002412:Cations; D002413:Cations, Divalent; D000939:Epitopes; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D007768:Lactalbumin