91] Inorganic pyrophosphatase from yeast: [formula omitted

Abstract

This chapter discusses the determination of inorganic pyrophosphatase from yeast. The assay method is based on the formation of inorganic orthophosphate from inorganic pyrophosphate under suitable conditions of pH, temperature, concentration of substrate, and concentration of activating ion. One unit of enzyme is defined as that amount liberating 1 mg. of orthopbosphate P per minute at 30° and pH 7.2. Specific activity is expressed as milligrams of P liberated per minute per milligram of protein. The steps described in the purification procedure are: (1) plasmolysis of yeast with toluene at 38 to 40°, followed by extraction with water at 5°, (2) fractionation with ammonium sulfate, (3) autolysis at 5° followed by refractionation with ammonium sulfate, (4) adsorption of impurities on Ca3(PO4)2 gel, (5) dialysis, (6) crystallization in 22% ethyl alcohol, (7) first recrystallization, (8) second recrystallization, and (9) drying and storing. The purified enzyme is quite specific for inorganic pyrophosphate. It does not hydrolyze adenosine diphosphate, adenosine triphosphate, or thiamine pyrophosphate. The chapter also describes an alternative purification procedure is because it involves fewer steps and is simpler when only small quantities of enzyme are required. It is based on the work of Heppel and Hilmoe.