Abstract
Crithidia fasciculata cells grown on complex medium with added [8-14C, 5'-3H]inosine or [8-14C,5'-3H]adenosine metabolize greater than 50% of the salvaged nucleosides through a pathway involving N-glycoside bond cleavage. Cell extracts contain a substantial nucleoside hydrolase activity but an insignificant purine nucleoside phosphorylase. The nucleoside hydrolase has been purified 1000-fold to greater than 99% homogeneity from kilogram quantities of C. fasciculata. The enzyme is a tetramer of Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km vary from 3.4 x 10(3) M-1 s-1 to 1.7 x 10(5) M-1 s-1 with the pyrimidine nucleosides giving the larger values. The turnover rate for inosine is 32 s-1 at 30 degrees C. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The hydrolytic reaction can be reversed to give an experimental Keq of 106 M with H2O taken as unity. The product dissociation constants for ribose and hypoxanthine are 0.7 and 6.2 mM, respectively. Deoxynucleosides or 5'-substituted nucleosides are poor substrates or do not react, and are poor inhibitors of the enzyme. The enzyme discriminates against methanol attack from solvent during steady-state catalysis, indicating the participation of an enzyme-directed water nucleophile. The pH profile for inosine hydrolysis gives two apparent pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.
Highlights
It is dependent on purine osine metabolize >60% of the salvaged nucleosides salvage and provides a convenient biological system for through a pathway involving N-glycoside bond cleav- understanding the enzymology of purine salvage in trypanoage
Isolated nucleic acids demonstrated an increase in the 14C/3Hratio to5.2 f 0.9 with [8-'4C,5'-3H]inosineand to 5.2 & 0.3 with [8-'4C,5'-3H]adenosine. Salvage of these nucleosides indicated greater than 2fold enrichment of base relative to ribose in thenucleic acids
Nucleoside hydrolase from C. fasciculuta is a nonspecific enzyme with a demonstrated capacity for nucleoside salvage in thetrypanosome
Summary
)I To whom correspondence should be addressed Dept. of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Colorimetric Assaysfor Nucleoside Hydrolase and Purine Nucleoside Phosphorylase-Catalytic activity for nucleoside hydrolase was ' Horenstein, B. After equal volume of 50 mM potassium phosphate, pH 7.2, containing 2 centrifugation, 250 pl of supernatant was removed and assayed for mM inosine, 0.1 mM EDTA, 0.1 mM dithiothreitol, 1mM benzamidine, reducing sugar as described above.Assays for purine nucleoside 1mM 1,lO-phenanthroline, 50 mM phenylmethylsulfonyl fluoride, 2 phosphorylase were the same as for nucleoside hydrolase except that mg/liter leupeptin, 50 mg/liter soybean trypsin inhibitor,and 50 mg/. Two volumes of ice-cold ethanol were added, dialyzed against 1mM potassium phosphate buffer,pH 7.3, and dried and the nucleic acids collected following centrifugation. Samples were analyzed in triplicate by reverse-phase chromatography on an Altex ODS analytical HPLC column eluted with 90 mM ammonium phosphate, pH 5.0, in 1%methanol. Ribose and inosine resonances were observed; hypoxanthine is efficiently removed by the procedure
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
