Spectrophotometric studies on the interaction of sarcoplasmic-reticulum fragments with adenosine triphosphate and calcium.

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An interaction of sarcoplasmic reticulum of skeletal and cardiac muscle with bromeresol purple was studied in the absence and presence of ATP and/or calcium using dual‐wavelength spectrophotometry. In the absence of added calcium, addition of ATP induced four specific phases that were measured as successive changes in absorption intensity of bromeresol purple at the wavelength pair 590—600 nm: phase 1, rapid decrease; phase 2, rapid increase; phase 3, gradual decrease; and phase 4, gradual increase. The absorption intensity of the phases was entirely reversed in direction at the wavelength pair 615—640 nm. The wavelength of maximum absorption by bromeresol purple was 587 nm; the isosbestic point with respect to pH was 488 nm. At the wavelength pair 488–540 nm and in the presence of ATP, phase 2 and phase 4 were not observed. The intensity‐increasing phase was completely calcium‐independent and represented a blue shift of the absorption spectrum of bromeresol purple. The intensity‐decreasing phase was calcium‐dependent and included both a red shift and some pH changes of the medium. The blue shift (phase 2) accompanied the interaction of sarcoplasmic reticulum, ATP and magnesium. Addition of calcium caused a shift back to longer wavelengths (phase 3). The initiation of the blue shift (phase 2) coincided with removal of calcium in the medium by active accumulation. Calcium had no effect on the shift in the absence of ATP. The results suggest that the blue shift (shift to shorter wavelengths, phase 2) might reflect the formation of a sarcoplamic reticulum · ATP complex; the red shift (to longer wavelengths, phases 1 and 3) might reflect conversion of this complex to another intermediary form.