Modulation of the binding characteristics of a fluorescent nucleotide derivative to the sarcoplasmic reticulum adenosinetriphosphatase.
Trinitrophenyladenosine monophosphate (TNP-AMP) binding to the phosphorylated Ca2+-ATPase of sarcoplasmic reticulum results in manyfold higher fluorescence intensity and longer lifetimes of the nucleotide analogue, as compared to TNP-AMP binding to the nonphosphorylated enzyme. This is observed when the phosphoenzyme intermediate is formed either from ATP or from inorganic phosphate (Pi). An important question is whether the TNP-AMP fluorescence properties can also reflect the kinetically defined interconversions of different phosphoenzyme species during catalysis. We have approached this question by manipulating the phosphorylation conditions in a manner which is known to result in accumulation of different species of the phosphoenzyme, i.e., by variations in pH, substrates, and K+ and Ca2+ concentrations. Decreasing pH or increasing [K+] caused large decreases in fluorescence intensity at a given concentration of TNP-AMP under conditions of phosphorylation with either ATP or Pi. In contrast, low to high intravesicular Ca2+ concentrations had no effect on fluorescence during steady-state turnover. TNP-AMP titrations of the phosphorylated enzyme stabilized in different states revealed that H+ and K+ caused a shift in TNP-AMP binding affinity to the site responsible for high fluorescence enhancement, while maintaining approximately the same maximal fluorescence yield at saturation. The fluorescence lifetimes of TNP-AMP bound to phosphoenzyme did not change with variations in pH, [K+], and substrates. We conclude that the environment of that part of the TNP-AMP binding site which binds the trinitrophenyl moiety undergoes a change upon enzyme phosphorylation resulting in enhanced fluorescence yield; this change is invariant between different phosphoenzyme species.(ABSTRACT TRUNCATED AT 250 WORDS)
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D000249:Adenosine Monophosphate; C036744:2',3'-O (2,4,6-trinitrocyclohexadienylidine)adenosine 5'-monophosphate; D000252:Calcium-Transporting ATPases