Characterization of the subunit structure of human placental nucleoside phosphorylase by immunochemistry

Ghangas, G.; Reem, G.H.

Journal of Biological Chemistry 254(10): 4233-4237


ISSN/ISBN: 0021-9258
PMID: 108272
Accession: 068520885

Download citation:  

Article/Abstract emailed within 1 workday
Payments are secure & encrypted
Powered by Stripe
Powered by PayPal

Purine nucleoside phosphorylase (EC, purine-nucleoside:orthophosphate ribosyltransferase) was purified to homogeneity from human placenta and an antibody specifically directed against the human enzyme was prepared. The antibody was characterized by crossed immunoelectrophoresis. It cross-reacts with the human enzyme derived from placenta and from erythrocytes but does not recognize bovine spleen purine nucleoside phosphorylase. The subunit structure of the human placental enzyme was characterized by immunoprecipitation of crude placental extracts and by 2-dimensional analysis of the purified enzyme. This technique is suitable for the characterization of the subunit structure of the enzyme protein without requiring prior purification which could affect the protein structure. It is also applicable to the study of variant forms of human purine nucleoside phosphorylase which are deficient in enzymatic activity. The placental enzyme is simpler in structure than the partially purified erythrocyte enzyme. It consists of 2 major subunits of the same MW (31,000 .+-. 500) but different isoelectric points. The native MW of the purified placental enzyme is 93,000 .+-. 1000 and is trimeric in structure. The trimeric structure of the purified placental enzyme was demonstrated by electrophoresis following cross-linking with dimethylsuberimidate. Comparison of the subunit structure of human erythrocyte purine nucleoside phosphorylase immunoprecipitated from crude hemolysates showed the structure of the enzyme of erythrocytes to be more complex than that of the placenta, a tissue which synthesizes enzymes de novo. The enzyme immunoprecipitated from hemolysates consists of 4 subunits of the same MW but different isoelectric points. The subunit structure of erythrocyte purine nucleoside phosphorylase prior to purification is apparently the same as that described for the partially purified erythrocyte enzyme, but is more complex in structure than the placental enzyme. Three isozymes of the placental enzyme can be detected by native isoelectric focusing, while 7 bands of the erythrocyte enzyme are apparent; the simpler isozyme pattern of the placental enzyme is consistent with its less complex subunit structure when compared with the erythrocyte enzyme.