4',6-Diamidino-2-phenylindole, a novel conformational probe of the sarcoplasmic reticulum Ca2+ pump, and its effect on Ca2+ release.
Sarcoplasmic reticulum vesicles were noncovalently labeled at micromolar concentrations with the polycationic fluorescent reagent 4',6-diamidino-2-phenylindole (DAPI), and changes in the fluorescence intensity of the membrane-bound dye associated with functions of the Ca2+ pump and Ca2+ release were investigated. It was found that 1) DAPI fluorescence changed in the [Ca2+] range in which high affinity Ca2+ binding to the Ca2+-ATPase takes place. The time course of the Ca2+-induced changes of DAPI fluorescence was essentially the mirror image of that of tryptophan fluorescence. 2) The fluorescence intensity of bound DAPI decreased upon increase of the intravesicular [Ca2+] by either ATP-dependent Ca2+ accumulation or incubation with millimolar Ca2+ in the presence of a calcium ionophore. 3) Upon induction of Ca2+ release by adding caffeine after the completion of Ca2+ uptake, DAPI fluorescence showed transient changes. Two classes of binding sites of the sarcoplasmic reticulum membrane for DAPI were clearly distinguishable: a high affinity site (Ka = 3.0 X 10(5) M-1) with a capacity of about 1 mol/mol of Ca2+-ATPase (8.0 nmol/mg of protein) and low affinity sites with about 20-fold lower affinity and 10-fold larger capacity. The partially purified Ca2+-ATPase showed similar characteristics of high affinity DAPI binding, suggesting that DAPI bound to its high affinity site on the Ca2+-ATPase monitors the enzyme conformational changes coupled with the events described above. The high affinity binding of DAPI to the enzyme led to an increase of the initial rate of Ca2+ uptake and the inhibition of Ca2+ release induced by caffeine or ionic replacement. These results suggest that the Ca2+-ATPase is involved in some steps of the Ca2+ release mechanism.
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007211:Indoles / Q000378:metabolism*; D008832:Microchemistry; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D007211:Indoles; D000001:Calcimycin; D002110:Caffeine; C007293:DAPI; D000252:Calcium-Transporting ATPases; D002118:Calcium