Purification and characterization of human erythrocyte purine nucleoside phosphorylase and its subunits.

V Zannis,D Doyle,D.W Martin

doi:10.1016/s0021-9258(17)38238-8

Abstract

Purine nucleoside phosphorylase (EC 2.4.2.1; purine nucleoside:orthophosphate ribosyltransferase) from fresh human erythrocytes has been purified to homogeneity in two steps with an overall yield of 56%. The purification involves DEAE-Sephadex chromatography followed by affinity chromatography on a column of Sepharose/formycin B. This scheme is suitable for purification of the phosphorylase from as little as 0.1 ml of packed erythrocytes. The native enzyme appears to be a trimer with native molecular weight of 93,800 and the subunit molecular weight of 29,700 +/- 1,100. Two-dimensional gel electrophoresis of the purified enzyme under denaturing conditions revealed four major separable subunits (numbered 1 to 4) with the same molecular weight. The apparent isoelectric points of subunits 1 to 4 in 9.5 M urea are 6.63, 6.41, 6.29, and 6.20, respectively. The different subunits are likely the result of post-translational modification of the enzyme and provide an explanation of the complex native isoelectric focusing pattern of purine nucleoside phosphorylase from erythrocytes. Three of the four subunits are detectable in two-dimensional electrophoretic gels of crude hemolysates. Knowing the location of the subunits of purine nucleoside phosphorylase in a two-dimensional electropherogram allows one to characterize the purine nucleoside phosphorylase in crude cell extracts from individuals with variant or mutant purine nucleoside phosphorylase as demonstrated in a subsequent communication. Partial purification of the phosphorylase from 1 ml of erythrocytes on DEAE-Sephadex increases the sensitivity of detection of the subunits to the 0.3% level.

Highlights

DEAE-Sephadex chromatography followed by affinity chromatography on a column of Sepharoselformycin B
The use of formycin B, a competitive inhibitor (Ki - 1OV M) of purine nucleoside phosphorylase
The purine nucleoside phosphorylase is judged pure by several criteria. (a) It exhibits a single component upon electrophoresis on acrylamide in the presence of sodium dodecyl sulfate. (bj The specific catalytic activity remained constant during the elution from the affinity chromatographic column and in a subsequent step. (c) When electrophoresed in native acrylamide gels of three different concentrations and (d) when isoelectric focused in acrylamide under native conditions, the catalytic activity of the purified purine nucleoside phosphorylase and the protein detected by staining coincide

Summary

Introduction

DEAE-Sephadex chromatography followed by affinity chromatography on a column of Sepharoselformycin B. This scheme is suitable for purification of the phosphorylase from as little as 0.1 ml of packed erythrocytes. Partial purification of the phosphorylase from 1 ml of erythrocytes on DEAE-Sephadex increases the sensitivity of the detection of the subunits to the 0.3% level. Purine nucleoside phosphorylase from mammalian sources reversibly catalyzes the phosphorolysis of naturally occurring purine ribo- or 2’.deoxyribonucleosides with the possible exception of adenosine (l-5).

Objectives

Results

Conclusion