Purine nucleoside phosphorylase: Isolation and characterization

1990 ◽  
Vol 55 (12) ◽  
pp. 2987-2999 ◽  
Author(s):  
Katarina Šedivá ◽  
Ivan Votruba ◽  
Antonín Holý ◽  
Ivan Rosenberg
Keyword(s):  
Molecular Weight ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Leukemia Cells ◽  
Ph Optimum ◽  
Nucleoside Phosphorylase ◽  
Isolation And Characterization ◽  
Reaction Proceeds ◽  
The Matrix ◽  
Sepharose 4B

Purine nucleoside phosphorylase (PNP) from mouse leukemia cells L1210 was purified to homogeneity by a combination of ion exchange and affinity chromatography using AE-Sepharose 4B and 9-(p-succinylaminobenzyl)hypoxanthine as the matrix and the ligand, respectively. The native enzyme has a molecular weight of 104 000 and consists of three subunits of equal molecular weight of 34 000. The results of isoelectric focusing showed that the enzyme is considerably microheterogeneous over the pI-range 4.0-5.8 and most likely consists of eight isozymes. The temperature and pH-optimum of phosphorolysis, purine nucleoside synthesis and also of transribosylation is identical, namely 55 °C and pH 7.4. The transribosylation reaction proceeds in the presence of phosphate only. The following Km-values (μmol l-1) were determined for phosphorolysis: inosine 40, 2'-deoxyinosine 47, guanosine 27, 2'-deoxyguanosine 32. The Km-values (μmol l-1) of purine riboside and deoxyriboside synthesis are lower than the values for phosphorolysis (hypoxanthine 18 and 34, resp., guanine 8 and 11, resp.). An affinity lower by one order shows PNP for (-D-ribose-1-phosphate, (-D-2-deoxyribose-1-phosphate (Km = 200 μmol l-1 in both cases) and phosphate (Km = 805 μmol l-1). The substrate specificity of the enzyme was also studied: positions N(1), C(2) and C(8) are decisive for the binding of the substrate (purine nucleoside).

Adenosine binding to low-molecular-weight purine nucleoside phosphorylase: the structural basis for recognition based on its complex with the enzyme fromSchistosoma mansoni

2009 ◽  
Vol 66 (1) ◽  
pp. 73-79 ◽  
Author(s):  
Humberto M. Pereira ◽  
Martha M. Rezende ◽  
Marcelo Santos Castilho ◽  
Glaucius Oliva ◽  
Richard C. Garratt
Keyword(s):  
Molecular Weight ◽  
Purine Nucleoside Phosphorylase ◽  
De Novo ◽  
Purine Nucleoside ◽  
Structural Basis ◽  
Low Molecular Weight ◽  
Salvage Pathway ◽  
Structural Explanation ◽  
Nucleoside Phosphorylase ◽  
Transition State Analogues

Schistosomes are unable to synthesize purinesde novoand depend exclusively on the salvage pathway for their purine requirements. It has been suggested that blockage of this pathway could lead to parasite death. The enzyme purine nucleoside phosphorylase (PNP) is one of its key components and molecules designed to inhibit the low-molecular-weight (LMW) PNPs, which include both the human and schistosome enzymes, are typically analogues of the natural substrates inosine and guanosine. Here, it is shown that adenosine both binds toSchistosoma mansoniPNP and behaves as a weak micromolar inhibitor of inosine phosphorolysis. Furthermore, the first crystal structures of complexes of an LMW PNP with adenosine and adenine are reported, together with those with inosine and hypoxanthine. These are used to propose a structural explanation for the selective binding of adenosine to some LMW PNPs but not to others. The results indicate that transition-state analogues based on adenosine or other 6-amino nucleosides should not be discounted as potential starting points for alternative inhibitors.

Purification and characterization of purine nucleoside phosphorylase from developing embryos of Hyalomma dromedarii

1991 ◽  
Vol 69 (4) ◽  
pp. 223-231 ◽  
Author(s):  
Mamdouh Y. Kamel ◽  
Afaf S. Fahmy ◽  
Abdel H. Ghazy ◽  
Magda A. Mohamed
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Catalytic Mechanism ◽  
Kinetic Studies ◽  
Purine Nucleoside ◽  
Hyalomma Dromedarii ◽  
Ph Optimum ◽  
Nucleoside Phosphorylase ◽  
Apparent Homogeneity ◽  
Camel Tick

Purine nucleoside phosphorylase from Hyalomma dromedarii, the camel tick, was purified to apparent homogeneity. A molecular weight of 56 000 – 58 000 was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. Unlike purine nucleoside phosphorylase preparations from other tissues, the H. dromedarii enzyme was unstable in the presence of β-mercaptoethanol. The enzyme had a sharp pH optimum at pH 6.5. It catalyzed the phosphorolysis and arsenolysis of ribo- and deoxyribo-nucleosides of hypoxanthine and guanine, but not of adenine or pyrimidine nucleosides. The Km values of the enzyme at the optimal pH for inosine, deoxyinosine, guanosine, and deoxyguanosine were 0.31, 0.67, 0.55, and 0.33 mM, respectively. Inactivation and kinetic studies suggested that histidine and cysteine residues were essential for activity. The pKa values determined for catalytic ionizable groups were 6–7 and 8–9. The enzyme was completely inactivated by thiol reagents and reactivated by excess β-mercaptoethanol. The enzyme was also susceptible to pH-dependent photooxidation in the presence of methylene blue, implicating histidine. Initial velocity studies showed an intersecting pattern of double-reciprocal plots of the data, consistent with a sequential mechanism.Key words: Acarina, Hyalomma dromedarii, purine nucleoside phosphorylase, kinetics, active site, catalytic mechanism.

scholarly journals β-deuterium kinetic isotope effects in the purine nucleoside phosphorylase reaction

1991 ◽  
Vol 278 (2) ◽  
pp. 487-491 ◽  
Author(s):  
X M Guo ◽  
M Ashwell ◽  
M L Sinnott ◽  
T A Krenitsky
Keyword(s):  
Transition State ◽  
Purine Nucleoside Phosphorylase ◽  
Perturbation Technique ◽  
Isotope Effects ◽  
Purine Nucleoside ◽  
Ph Optimum ◽  
Nucleoside Phosphorylase ◽  
Ph Variation ◽  
Kinetic Isotope ◽  
And Inversion

1. [2′-2H]Inosine was made from inosine by tetraisopropyldisiloxanyl protection of the 3′- and 5′-positions, oxidation with dimethyl sulphoxide and acetic anhydride, immediate NaB2H4 reduction of the oxo sugar product and inversion at C-2′ of the resultant protected [2′-2H]arabino-inosine by trifluoromethanesulphonylation and reaction with caesium propionate, followed by deprotection. 2. The equilibrium-perturbation technique was used to measure beta 2H(V/K) for phosphorolysis of this compound by the purine nucleoside phosphorylase of Escherichia coli as a function of pH. 3. The pH variation indicates an intrinsic effect of 1.068 masked by isotopically silent steps near the pH optimum. 4. The similar pH variation of these beta-deuterium effects and the alpha-deuterium effects measured previously [Stein & Cordes (1981) J. Biol. Chem. 256, 767-772; Lehikoinen, Sinnott & Krenitsky (1989) Biochem. J. 257, 355-359] for this reaction provides the first experimental reassurance for the common assumption that pH changes merely mask and unmask the chemical steps in an enzyme-catalysed reaction, and do not detectably alter transition-state structure. 5. The dihedral angle between the C-H-2′ bond and the electron-deficient p-orbital at the transition state is in the range 32-48 degrees, in accord with an essentially planar furanose ring.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

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