A Borohydride Reduction Method for Characterization of the Acyl Phosphate Linkage in Proteins and Its Application to Sarcoplasmic Reticulum Adenosine Triphosphatase

• Published in: The Journal of biological chemistry Vol. 248; no. 23; pp. 8222 - 8226
• Main Authors: Degani, Chemda, Boyer, Paul D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.12.1973; American Society for Biochemistry and Molecular Biology
• Subjects: Acetates - analysis; Adenosine Triphosphatases - analysis; Amino Alcohols; Aspartic Acid; Binding Sites; Borohydrides; Calcium; Carboxylic Acids - analysis; Chemical Phenomena; Chemistry; Crystallization; Electrophoresis, Paper; Homoserine; Hydrolysis; Lactones; Magnesium; Membranes - analysis; Methods; Oxidation-Reduction; Phosphoproteins - analysis; Phosphoric Acids - analysis; Phosphorus Radioisotopes; Sarcoplasmic Reticulum - enzymology; Sodium; Tritium
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)43217-1

A new method for identification and characterization of an acyl phosphate linkage in phosphorylated proteins is presented. The method involves reductive cleavage of the acyl phosphate bond with sodium [3H]borohydride to form a labeled aminohydroxy acid residue. [3H]Borohydride reduction of the phosphorylated (Ca2+, Mg2+)-adenosine triphosphatase of sarcoplasmic reticulum, followed by analysis of the acid hydrolysate of the reduced enzyme, showed the formation of labeled homoserine. The results demonstrate that the phosphoryl group of sarcoplasmic reticulum ATPase is attached to the β-carboxyl group of an aspartyl residue at the active site.