Purine Nucleoside Phosphorylase from Human Erythrocytes
IV. CRYSTALLIZATION AND SOME PROPERTIES
R. P. Agarwal 1 and R. E. Parks Jr. 1
From the
1 From the Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island 02912
Purine nucleoside phosphorylase has been purified about 7,300-fold and crystallized from human erythrocytes (mol wt 81,000). The recrystallized enzyme exists in the form of needles and sometimes bundles of needles and has a specific activity of 96 µm units per mg of protein. A number of phenomena reported earlier for a less pure preparation of this enzyme are still seen with the crystalline enzyme. These include (a) substrate activation by inosine and (b) a constant ratio of reactivity with inosine and deoxyinosine. However, the ribosyl transfer reaction seems markedly decreased with the crystalline preparation. The effect of pH on maximal velocity suggests the occurrence of essential imidazole and sulfhydryl groups, whereas the effect of pH on the Michaelis constant suggests the existence of an essential ionizing group at about pH 7.2 in the free enzyme. Binding studies with hypoxanthine-8-14C indicate that there are at least three binding sites per molecule of enzyme. When the commercially available bovine spleen purine nucleoside phosphorylase was compared with human erythrocytic enzyme, the two enzymes were found to be similar in molecular weight. However, they have different crystal structures and different specific activities, and in contrast to the behavior of the human enzyme, the spleen enzyme did not display the phenomena of substrate activation at high concentrations of inosine.
Submitted on July 29, 1968
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
