Trapping of transition metal-nucleotide complexes in myosin subfragment 1 by cross-linking thiols; divalent transition metal probes of the active site.
Recent experiments [Wells, J., & Yount, R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966] have shown it is possible to trap MgADP and other nucleotides stably at the active site of myosin by cross-linking two thiol groups. A variety of cross-linking reagents including chelation of the two thiols by cobalt (III) phenanthroline or covalent reaction with N,N'-p-phenylenedimaleimide (pPDM) are effective trapping agents. No trapping of nucleotides occurs in the absence of divalent metals. Thus far Mg2+, Mn2+, Co2+, Ni2+, and Ca2+ but not Zn2+ all function to promote trapping of the 1:1 divalent metal-ADP complex and to enhance the rate of ATPase inactivation. Substitution-inert Cr(III) complexes of ADP, ATP, or pyrophosphate that bind very weakly or not at all to the active site are not trapped by cross-linking. While the stability of the trapped divalent metals varies, e.g., t1/2 of 0.5-7 days at 0 degree C, they are stable enough to permit accurate spectral measurements of the Mn2+ and Co2+ trapped complexes. Electron paramagnetic resonance (EPR) measurements of Mn2+ bound to 5'-adenylyl imidodiphosphate or complexed to myosin chymotryptic subfragment 1 indicate that the metal is bound at the active site. Circular dichroism (CD) and visible absorption studies of the Co2+ . ADP trapped complex indicate the metal ion is in an asymmetric octahedral environment. EPR and CD measurements show that the environment of the metal nucleotide is the same whether bound reversibly or stably trapped at the active site.
D000227:Adenine Nucleotides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000818:Animals; D001665:Binding Sites; D002118:Calcium; D002413:Cations, Divalent; D055598:Chemical Phenomena; D002621:Chemistry; D002857:Chromium; D003035:Cobalt; D003432:Cross-Linking Reagents; D008301:Maleimides; D008345:Manganese; D008670:Metals; D015879:Myosin Subfragments; D009218:Myosins; D010446:Peptide Fragments; D010618:Phenanthrolines; D011817:Rabbits; D013057:Spectrum Analysis; D013438:Sulfhydryl Compounds
D000227:Adenine Nucleotides; D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D008301:Maleimides; D008670:Metals; D015879:Myosin Subfragments; D010446:Peptide Fragments; D010618:Phenanthrolines; D013438:Sulfhydryl Compounds; D002857:Chromium; D003035:Cobalt; D008345:Manganese; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C011328:N,N'-4-phenylenedimaleimide; D009218:Myosins; D002118:Calcium; C025205:1,10-phenanthroline