Localization of site-specific probes on the Ca-ATPase of sarcoplasmic reticulum using fluorescence energy transfer.

摘要: Highly reactive sulfhydryls, previously labeled with an iodoacetamide spin label on the Ca-ATPase of sarcoplasmic reticulum, were fluorescent probe, 5-(2-[iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (IAEDANS), without loss enzymatic activity. We have selectively measured apparent distance more site, relative to other site-specific probes at both nucleotide and high affinity calcium binding sites. Fluorescence energy transfer efficiencies from donor IAEDANS two acceptors: fluorescein 5′-isothiocyanate or 2′,3′-O-(2,4,3-trinitrophenyl)adenosine monophosphate, situated near using fluorescence lifetimes yields. polyacrylamide gels shows that labels are associated B tryptic fragment. The measurements consistent distances 56 68 A between these respective On hand, lanthanide, praseodymium (Pr3+), as acceptor indicate is located 16-18 site(s) this analog. Pr3+ shown be a good analog for sites enzyme since it competitively displaces calcium, evidenced by reversal specific calcium-dependent intrinsic signal inactivation ATPase activity, over same narrow range in concentration where observed. Our observations suggest portion fragment spanning cytoplasmic folded onto fragment, bringing close proximity domain.