Purification and Properties of Orotidine-5′-Phosphate Pyrophosphorylase and Orotidine-5′-Phosphate Decarboxylase from Bakers' Yeast

Umezu, Kohei; Amaya, T.; Yoshimoto, Akio; Tomita, K.

doi:10.1093/oxfordjournals.jbchem.a129637

Cited by 92 publications

(48 citation statements)

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“…S. cerevisiae BY2 was described previously (5 (7,32,35). The clarified lysates were further centrifuged in a Beckman Ti6O rotor at 50,000 rpm for 3 h at0°C, and the supernatants were adjusted to a concentration of 10 mg of protein per ml.…”

Section: Methodsmentioning

confidence: 99%

Interaction of GAL4 and GAL80 gene regulatory proteins in vitro.

Lue¹,

Chasman²,

Buchman³

et al. 1987

Mol. Cell. Biol.

163

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The GAL80 protein of Saccharomyces cerevisiae, synthesized in vitro, bound tightly to GAL4 protein and to a GAL4 protein-upstream activation sequence DNA complex, as shown by (i) coimmunoprecipitation of GAL4 and GAL80 proteins with anti-GAL4 antiserum, (ii) an electrophoretic mobility shift of a GAL4 protein-upstream activation sequence DNA complex upon the addition of GAL80 protein, and (iii) GAL4-dependent binding of GAL80 protein to upstream activation sequence DNA immobilized on Sepharose beads. Anti-GAL4 antisera were raised against a GAL4-URA3 fusion protein, which could be purified to homogeneity in a single step with the use of an affinity chromatographic procedure for the URA3 gene product.

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Section: Methodsmentioning

confidence: 99%

Interaction of GAL4 and GAL80 gene regulatory proteins in vitro.

Lue¹,

Chasman²,

Buchman³

et al. 1987

Mol. Cell. Biol.

163

View full text Add to dashboard Cite

show abstract

“…Ali and Sloan (2) . However, the effects of these metal ions on OPRTase have not been investigated because the presence of these ions will influence the UV absorption of orotate and make detection difficult (30). Here we determined the extinction coefficient of orotate in the presence of 2 mM divalent metal ions at 298 nm.…”

Section: Resultsmentioning

confidence: 99%

“…Measurements of the initial velocities of the phosphoribosyl transfer reaction were carried out according to the method of Umezu et al (30). The ⌬ε value for the conversion of orotate to OMP was determined to be 3,780 M Ϫ1 cm Ϫ1 at 298 nm under our experimental conditions.…”

mentioning

confidence: 99%

Orotate Phosphoribosyltransferase fromCorynebacterium ammoniagenesLacking a Conserved Lysine

Wang

et al. 2007

J Bacteriol

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The pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase), was cloned by nested PCR and colony blotting from Corynebacterium ammoniagenes ATCC 6872, which is widely used in nucleotide production. Sequence analysis shows that there is a lack of an important conserved lysine (Lys 73 in Salmonella enterica serovar Typhimurium OPRTase) in the C. ammoniagenes OPRTase. This lysine has been considered to contribute to the initiation of catalysis. The enzyme was overexpressed and purified from a recombinant Escherichia coli strain. The molecular mass of the purified OPRTase was determined to be 45.4 ؎ 1.5 kDa by gel filtration. Since the molecular mass for the subunit of the enzyme was 21.3 ؎ 0.6 kDa, the native enzyme exists as a dimer. Divalent magnesium was necessary for the activity of the enzyme and can be substituted for by Mn 2؉ and Co 2؉ . The optimal pH for the forward (phosphoribosyl transfer) reaction is 10.5 to 11.5, which is higher than that of other reported OPRTases, and the optimal pH for the reverse (pyrophosphorolysis) reaction is 5.5 to 6.5. The K m values for the four substrates were determined to be 33 M for orotate, 64 M for 5-phosphoribosyl-1-pyrophosphate (PRPP), 45 M for orotidine-5-phosphate (OMP), and 36 M for pyrophosphate. The K m value for OMP is much larger than those of other organisms. These differences may be due to the absence of Lys 73, which is present in the active sites of other OPRTases and is known to interact with OMP and PRPP.

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“…Since OMP, an enzymatic product of OPRTase reaction, was unstable, we excluded the possibility of unexpected labile products from OMP by using exogenous purified yeast ODCase added into the OPRTase reaction in order to convert all OMP into UMP (12,13). The individual OPRTase and ODCase activities in the bifunctional UMPS of human red cells, human mononuclear cells, and mouse red cells were measured separately in their crude enzyme preparations by using the HPLC assays as described under Materials and Methods (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…Two highperformance liquid chromatographic (HPLC) assays using nonradiochemical method have been used for measurement of the very low UMPS activities of human chorionic villus and red cell (10,11). Most of the monofunctional OPRTase and ODCase assays are based on spectrophotometric quantitation of orotate conversion to OMP and OMP conversion to UMP at 295 and 285 nm, respectively (12)(13)(14)(15)(16)(17).…”

Section: Mg 2ϩmentioning

confidence: 99%