Purification and some properties of inorganic pyrophosphatase from Streptococcus faecalis.

Abstract

Inorganic pyrophosphatase [EC 3.6.1.1] from Streptococcus faecalis ATCC 8043 was purified to homogeneity as judged by slab gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The purification consisted of the following steps: streptomycin sulfate precipitation, (NH4)2SO4 treatment in a Sepharose CL-4B column, DEAE-Sepharose CL-6B chromatography, gel filtration on Ultrogel AcA 34, and preparative slab gel electrophoresis. Gel filtration on Ultrogel AcA 34 was used to determine the molecular weight and slab gel electrophoresis in the presence of sodium dodecyl sulfate to study the subunit molecular weight. The enzyme appeared to be composed of four subunits with molecular weights of approximately 32,500. The molecular weight of the native enzyme was about 128,000. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, and no activity was found with a variety of other phosphate esters. The cation Mg2+ was required for maximum activity; Co2+ and Ca2+ supported 24% and 5.6% of the activity observed with Mg2+, respectively. 2,4,6-Trinitrobenzene sulfonic acid inhibited the reductants containing SH-groups activated the enzyme suggesting that lysine and cysteine have essential roles in the enzyme.