KINETIC AND THERMODYNAMIC ASPECTS OF ATP-DEPENDENT CA2+ TRANSPORT IN SARCOPLASMIC RETICULUM USING ATPßS DIASTEREOMERS AS SUBSTRATES

Abstract

Publisher Summary This chapter discusses the use of ATPβS dia-stereomers, the SR ion-transporting system, and the hydrolysis of adenosine triphosphate (ATP) and ATPβS; it also discusses the kinetics of phosoho-enzyme formation after rapid mixing of [γ-32P]ATP or [γ- 32P]ATPβS(S) followed by rapid quenching. The chapter discusses a study to illustrate the HPLC elution profile of AMP, adenosine diphosphate (ADP), ADPβS, ATP, and ATPβS after single injection. The 10 μl injection consisted of an aqueous standard mixture containing 20 μM each of AMP, ADP, ATP, and ATPsS, and 10 uM of ADPsS. The chromatographic separation of ATP, ATPβS, ADP, ADPβS, and AMP was achieved at 20°C on a Partisil 10 SAX, 20 μ particle size ionic exchange column, 250 mm long, 4.6 mm i.d., supplemented with a small precolumn (30 X 2.9 mm i.d.) filled with similar but coarser particulate material, using a Waters 600 solvent delivery system with a model U6K sample injector and a model 441 absorbance detector. The mobile phase, consisting of 500 mM ammonium phosphate (pH 4.4), was pumped at a flow rate of 2 ml/min (1800 lb/square inch). Nucleotide absorbance was recorded at 214 nm (0.01 absorbance full scale).