A fluorescence probe study of the phosphorylation reaction of the calcium ATPase of sarcoplasmic reticulum.
The fluorescent thiol reagent N-(1-anilinonaphthyl-4)maleimide (ANM) reacts covalently with the Ca2+ ATPase moiety of fragmented sarcoplasmic reticulum in two phases as determined by the increase of fluorescence intensity and optical density at 350 nm. In the rapid phase, 5.5 nmol of ANM reacts with 1 mg of fragmented sarcoplasmic reticulum protein. Assuming that 55% of the total membrane protein is the Ca2+ ATPase, this is equivalent to 1 mol of SH/10(5) g of ATPase, designated as SH1-ANM. ANM reacts with the second SH (SH2-ANM) at a much slower rate. Reaction of ANM with both SH1-ANM and SH2-ANM produces no inhibition of phosphoenzyme (EP) formation. Upon addition of Mg . ATP in the micromolar range, at [Ca2+] = 1 microM there is an increase in the fluorescence intensity of ANM attached to SH2-ANM, while the ANM attached to SH1-ANM does not respond to Mg . ATP. Under conditions in which there is no EP formation, there is no fluorescence change. Furthermore, the enhancement of ANM fluorescence produced by Mg . ATP is reversed by ADP as it reacts with EP to form ATP. Thus, it appears that the Mg . ATP-induced fluorescence increase reflects changes of enzyme conformation produced by EP formation.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008301:Maleimides; D009132:Muscles / Q000201:enzymology; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D007202:Indicators and Reagents; D008301:Maleimides; D000244:Adenosine Diphosphate; C010854:N-(1-anilinonaphthalyl)-4-maleimide; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium