Two types of sarcoplasmic reticulum-orthophosphate interactions observed with dye probe.
The interaction of Pi with sarcoplasmic reticulum (SR) isolated from rabbit skeletal muscle was studied using bromocresol purple (BCP) as a probe and a dual-wavelength spectrophotometer. Two kinds of absorption-intensity changes controlled by a low concentration of Ca2+ (greater than 10(-6)M) were observed after addition of Pi; an increase phase (in the presence of Ca2+), and a decrease phase (in the presence of EGTA). The increase phase was rapid, Ca2+-dependent, Mg2+-enhanced (depressed by high Mg2+ concentration) and not inhibited by PCMB and was suggested to reflect the formation of an SR-Pi complex. The decrease phase was slower than the increase phase, and was strongly inhibited by the low concentration of Ca2+. It required Mg2+, and was completely inhibited by p-chloromercuribenzoate or deoxycholate. It was suggested to reflect the formation of SR-Pi (phosphorylated protein). ATP inhibited this phase by converting it completely to an SR-MgATP phase. PPi was effective for inducing the decrease phase but PPPi was not. From measurements of these phases, the association constants of the SR-Pi complex and SR-Pi at pH 8.8 in the reaction scheme, SR + Pi in equilibrium SR-Pi in equilibrium SR-Pi, were calculated as 5.4 X 10 M-1 and 1.8 X 10(3) M-1, respectively. From the completely different responses of SR-Pi and SR-Pi observed with BCP a marked difference in the conformations of these enzyme states was suggested.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002729:Chloromercuribenzoates / Q000494:pharmacology; D004396:Coloring Agents / Q000378:metabolism*; D003840:Deoxycholic Acid / Q000494:pharmacology; D011756:Diphosphates / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles; D010710:Phosphates / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013053:Spectrophotometry
D002729:Chloromercuribenzoates; D004396:Coloring Agents; D011756:Diphosphates; D010710:Phosphates; D003840:Deoxycholic Acid; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium