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chromatograms were developed in ethyl acetate:propan-2-
ol:ammonia:water (27:23:5:3, by volume).
4.5. Enzymes assays
The nucleosidase was assayed quantitatively in a reaction
mixture (50 ll) containing 0.1 M sodium acetate buffer (pH
4.75), 2 mM CaCl2, 0.1 mM [3H]-labelled substrate (300
000 cpm), other additions (e.g. salt) and a rate-limiting quan-
tity of enzyme fraction. The reaction was carried out at
30 ꢁC. To estimate reaction rates, 3 ll aliquots were spotted
on to TLC plates, usually at four time intervals, unlabelled
standards added and the plates developed for 30–40 min to
separate the labeled nucleoside from corresponding product
base. The compounds were visualized under short-wave UV
light, spots of the products excised, immersed in scintillation
cocktail and the radioactivity measured.
During enzyme purification, the activity of guanosine
nucleosidase was monitored by TLC analysis of the conver-
sion of 0.5 mM guanosine into guanine. This qualitative
approach was sufficient for making quick decisions about
which fractions from one step of enzyme purification could
be collected and used in a following step. The same quali-
tative assay was applied for testing unlabelled guanosine
analogues as potential substrate of the lupin nucleosidase
(for example see Fig. 3).
Burch, L.R., Stuchbury, T., 1986. Purification and properties of adenosine
nucleosidase from tomato (Lycopersicon esculentum) roots and leaves.
J. Plant Physiol. 125, 267–273.
Bzowska, A., Kulikowska, E., Shugar, D., 2000. Purine nucleoside
phosphorylases: properties, functions, and clinical aspects. Pharmacol.
Ther. 88, 349–425.
Cacciapuoti, G., Bertoldo, C., Brio, A., Zappia, V., Porcelli, M., 2003.
Purification and characterization of 50-methylthioadenosine phosphor-
ylase from the hyperthermophilic archaeon Pyrococcus furiosus.
Extermophiles 7, 159–168.
Campos, A., Rijo-Johansen, M.J., Carneiro, M.F., Fevereiro, P., 2005.
Purification and characterization of adenosine nucleosidase from
Coffea arabica young leaves. Phytochemistry 66, 147–151.
Chen, Ch.-M., Kristopeit, S.M., 1981. Metabolism of cytokinin: deribo-
sylation of cytokinin ribonucleoside by adenosine nucleosidase from
wheat germ cells. Plant Physiol. 68, 1020–1023.
Davis, B.J., 1964. Disc electrophoresis. II. Method and application to
human serum protein. Ann. N.Y. Acad. Sci. 121, 404–427.
Degano, M., Almo, S.C., Sachettini, J.C., Schramm, V.L., 1998.
Trypanosomal nucleoside hydrolase. A novel mechanism from the
structure with a transition-state inhibitor. Biochemistry 37, 6277–
6285.
Dixon, M., Webb, E.C., 1964. Enzymes, second ed. Academic Press, New
York.
´
Estupinan, B., Schramm, V.L., 1994. Guanosine–inosine-preferring nucle-
˜
oside N-glycohydrolase from Crithidia fasciculata. J. Biol. Chem. 269,
23068–23073.
4.6. Kinetic parameters
Gianotti, A.J., Tower, P.A., Sheley, J.H., Conte, P.A., Spiro, C., Ferro,
A.J., Fitchen, J.H., Riscoe, M.K., 1990. Selective killing of Klebsiella
pneumoniae by 5-trifluoromethylthioribose. Chemotherapeutic exploi-
tation of the enzyme 5-methylthioribose kinase. J. Biol. Chem. 265,
831–837.
Guranowski, A., 1982. Purine catabolism in plants; purification and some
properties of inosine nucleosidase from yellow lupin (Lupinus luteus L.)
seeds. Plant Physiol. 70, 344–349.
The Km values for guanosine and inosine were estimated
in the standard reaction mixture with substrate concentra-
tions ranging between 1 and 12 lM. The values were com-
puted from the Eadie-Hofstee plot (v versus v/[S]). Ki
values were calculated according to the method of Dixon
and Webb (1964).
Guranowski, A., Barankiewicz, J., 1979. Purine salvage in cotyledons of
germinating lupin seeds. FEBS Lett. 104, 95–98.
Guranowski, A., Paszewski, A., 1982. Metabolism of 50-methylthioaden-
osine in Aspergillus nidulans; an alternative pathway for methionine
synthesis via utilization of the nucleoside methylthio group. Biochim.
Biophys. Acta 717, 289–294.
Guranowski, A., Pawełkiewicz, J., 1978. Adenosylhomocysteinase and
adenosine nucleosidase activities in Lupinus luteus cotyledons during
seed formation and germination. Planta 139, 245–247.
Guranowski, A., Schneider, Z., 1977. Purification and characterization of
adenosine nucleosidase from barley leaves. Biochim. Biophys. Acta
482, 145–158.
4.7. Protein quantification
Protein concentration was determined by the turbidi-
metric tannin method (Mejbaum-Katzenellenbogen, 1955)
using bovine serum albumin as standard. For column elu-
ates the absorbance at 280 nm was monitored.
Acknowledgement
Guranowski, A.B., Chiang, P.K., Cantoni, G.L., 1981. 50-Methylthioa-
denosine nucleosidase; purification and characterization of the enzyme
from Lupinus luteus seeds. Eur. J. Biochem. 114, 293–299.
Imagawa, H., Yamano, H., Inoue, K., Takino, Y., 1979. Purification and
properties of adenosine nucleosidases from tea leaves. Agric. Biol.
Chem. 43, 2337–2342.
Jakubowski, H., Guranowski, A., 1983. Enzymes hydrolyzing ApppA
and/or AppppA in higher plants; purification and some properties of
diadenosine triphosphatase, diadenosine tetraphosphatase, and phos-
phodiesterase from yellow lupin (Lupinus luteus) seeds. J. Biol. Chem.
258, 9982–9989.
Financial support from the Agricultural University in
´
Poznan, within grant 264/R/81/W, is gratefully
acknowledged.
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