Human red cell purine nucleoside phosphorylase. Purification by biospecific affinity chromatography and physical properties (original) (raw)
Related papers
The Journal of biological chemistry, 1978
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 compl...
Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase
Protein Expression and Purification, 2003
Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (Pi) and continuous assay of reactions that generate Pi such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for Pi detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable Pi detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme. Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein. Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control. Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1 L cell culture, with a specific activity value of 80 U mg−1.
FEBS Letters, 1995
Calf spleen pnrine nucleoside phosphorylase was purified to homogeneity and its amino acid sequence was determined. The complex of the enzyme with an N(7)-acycloguanosine inhibitor crystallized in the cubic space group P213, with unit cell dimension a = 94.02 A and one monomer in the asymmetric crystal unit. The biologically active trimer is formed by the crystallographic three-fold axis. The structure was solved by molecular replacement methods, using the model of the human erythrocyte enzyme, and refined at a resolution of 2.9 A to an R-factor of 0.21. The orientation of the inhibitor at the active site is examined in relation to the catalytic activity of the enzyme in the phosphorolysis of N(7)-fl-o-purine nucleosides.
A rat model of purine nucleoside phosphorylase deficiency
Immunology, 1986
Purine nucleoside phosphorylase (NP; EC 2.4.2.1) deficiency is associated with selective T-cell dysfunction and normal B-cell immunity. In order to create an in vivo model of this immune deficiency, we administered 8-aminoguanosine to rats. This water-soluble nucleoside was rapidly converted by NP to the more potent inhibitor 8-aminoguanine, which has a Ki of 0.19 microM. The accumulation of inosine in plasma showed that administration of 8-aminoguanosine was effectively inhibiting NP activity. The administration of 8-aminoguanosine with deoxyguanosine produced increased levels of dGTP only in thymus cells, and increased levels of GTP in cells from thymus, spleen and lymph node and in red cells. This correlated with assays of deoxyguanosine kinase, which showed significantly higher activity in thymus cells than in cells from spleen and lymph node. The intraperitoneal injection of 8-aminoguanosine alone or with deoxyguanosine for 8 consecutive days caused significant decreases in the...
Structures of human purine nucleoside phosphorylase complexed with inosine and ddI
Biochemical and Biophysical Research Communications, 2004
Human purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effect on B-cell function. PNP is highly specific for 6-oxopurine nucleosides and exhibits negligible activity for 6-aminopurine nucleosides. The catalytic efficiency for inosine is 350,000-fold greater than for adenosine. Adenine nucleosides and nucleotides are deaminated by adenosine deaminase and AMP deaminase to their corresponding inosine derivatives which, in turn, may be further degraded. Here we report the crystal structures of human PNP in complex with inosine and 2 0 ,3 0 -dideoxyinosine, refined to 2.8 A A resolution using synchrotron radiation. The present structures provide explanation for ligand binding, refine the purine-binding site, and can be used for future inhibitor design.
Archives of Biochemistry and Biophysics, 2008
Human purine nucleoside phosphorylase (HsPNP) is a target for inhibitor development aiming at T-cell immune response modulation. In this work, we report the development of a new set of empirical scoring functions and its application to evaluate binding affinities and docking results. To test these new functions, we solved the structure of HsPNP and 2-mercapto-4(3H)-quinazolinone (HsPNP:MQU) binary complex at 2.7 Å resolution using synchrotron radiation, and used these functions to predict ligand position obtained in docking simulations. We also employed molecular dynamics simulations to analyze HsPNP in two conditions, as apoenzyme and in the binary complex form, in order to assess the structural features responsible for stability. Analysis of the structural differences between systems provides explanation for inhibitor binding. The use of these scoring functions to evaluate binding affinities and molecular docking results may be used to guide future efforts on virtual screening focused on HsPNP.
Journal of Immunological Methods, 1980
Indirect immunofluorescence was used as a rapid, sensitive and specific method for the visualization of the enzyme purine nucleoside phosphorylase in single cells. The enzyme was localized throughout cytoplasm of human lymphoblasts and fibroblasts but no~ in cell nuclei. This method is valuable for the detec~]o~ of mu'~ant enzyme protein in cellmediated immunodeficiencies caused by purine nucleoside phosphoryiase deficiency since it does not rely on enzyme activity. It requires only a limited number of cells and can therefore be used for the rapid screening for the presence of cross-reactive protein in immunodeficiency diseases.
European Journal of Biochemistry, 1997
Nicotinamide 1 -1l-D-riboside (Nir), the cationic, reducible moiety of the coenzyme NAD', has been confirmed as an unusual substrate for purified purine-nucleoside phosphorylase (PNP) from a mammalian source (calf spleen). It is also a substrate of the enzyme from Escherichia coli. The K,, values at pH 7, 1.48 mM and 0.62 mM, respectively, were 1-2 orders of magnitude higher than for the natural substrate inosine, but the V,,,,>, values were comparable, 96% and 35% that for Ino. The pseudo first-order rate constants, V,,,,,IK,,,, were 1 .I % and 2.5 o/o for the calf spleen and E. coli enzymes. The aglycon, nicotinamide, was neither a substrate nor an inhibitor of PNP.
A canine model of induced purine nucleoside phosphorylase deficiency
Clinical and experimental immunology, 1986
Purine nucleoside phosphorylase (NP EC 2.4.2.1) deficiency in man is associated with selective T cell dysfunction and normal B cell immunity. To create an in-vivo model of this immune deficiency, we administered 8-aminoguanosine to dogs. This water soluble nucleoside was rapidly converted by NP to the more potent product inhibitor 8-aminoguanine, which had a Ki of 0.52 microM. The accumulation of inosine and exogenous deoxyguanosine in plasma provided evidence that administration of 8-aminoguanosine was effectively inhibiting NP activity. Four dogs given 8-aminoguanosine and deoxyguanosine concurrently for 5 consecutive days showed mean reductions in peripheral blood lymphocytes of 65 +/- 9% range (55-75%) over the test period. Granulocytes, red blood cells, and plateletes remained within the normal range. Administration of 8-aminoguanosine to dogs provides a model of NP deficiency that will permit studies of the specific control of lymphopoiesis and in-vivo immune function.