Purification and Properties of Orotic Acid-decarboxylating Enzymes from Calf Thymus

Kasbekar, D. K.; Nagabhushanam, A.; Greenberg, David M.

doi:10.1016/s0021-9258(18)91164-6

Cited by 69 publications

(6 citation statements)

References 7 publications

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“…The results presented here account for the copurification of orotate phosphoribosyltransferase and orotidylate decarboxylase and also the greater lability of the phosphoribosyltransferase as previously described for the enzymes from mammalian tissues (Kasbekar et al, 1964;Appel, 1968;Brown et al, 1975;Grobner and Kelley, 1975). The difficulty in separating the two enzyme activities can be compared to the situation in yeast, where the two enzymes are readily separable (Umezu et al, 1971).…”

Section: Discussionmentioning

confidence: 68%

“…Phosphoribosyltransferase activity was not associated with this form, though it was detected in association with the decarboxylase in a species with a molecular weight of approximately 63 000 (Campbell et al, 1977). Kasbekar et al (1964) have claimed separation of the two enzyme activities by starch gel electrophoresis of the partially purified enzymes from calf thymus. Reyes and Guganig (1975) also obtained an apparent separation of the two enzyme activities on a sucrose gradient following treatment of the phosphoribosyltransferase-decarboxylase complex from murine leukemia P1534J with elastase.2…”

Section: Discussionmentioning

confidence: 99%

“…The two enzymes appear to behave in a coordinated fashion in all mammalian tissues in which they have been studied. Copurification of the enzymes has been observed by a number of investigators, subject to the limitations imposed by the greater instability of the phosphoribosyltransferase (Kasbekar et al, 1964;Appel, 1968;Brown et al, 1975; Grobner and Kelley, 1975). Also, the ratio of the two activities was found to be constant over a wide range in fresh preparations of human hemolysate (Fox et al, 1971).…”

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confidence: 99%

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Subunit structure of the orotate phosphoribosyltransferase-orotidylate decarboxylase complex from human erythrocytes

Brown¹,

O'Sullivan²

1977

Biochemistry

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A complex of orotate phosphoribosyltransferase and orotidylate decarboxylase has been shown to exist in three molecular weight forms (Brown, G. K., Fox, R. M., and O'Sullivan, W. J. (1975), J. Biol. Chem. 250, 7352). The smallest of these, of molecular weight 62 000, was subjected to further study. On the basis of the inactivation of the enzyme activities, carried out in the presence of low concentrations of guanidine hydrochloride, and of changes in molecular weight of preparations during aging, it was inferred that the enzyme complex contained more than one type of subunit. This was confirmed by chromatography on Sephadex G-75 after preincubation in guanidine hydrochloride or with guanidine hydrochloride in the elution buffer. It was concluded that the enzyme complex consisted of two types of subunits, two decarboxylase units of molecular weight approximately 20 000 and two further subunits of approximately 13 000. The subunits could be separated and reassociated with partial recovery of both activities. A 40 000 molecular weight form had full decarboxylase activity but no phosphoribosyltransferase activity. Restoration of the 62 000 molecular weight form resulted in restoration of both enzymatic activities. An intermediate species of molecular weight 50 000 representing a combination of the decarboxylase dimer with one of the 13 000 subunits was also demonstrated. This form required the presence of dithiothreitol in order to manifest phosphoribosyltransferase activity. A model of the system has been proposed that accounts for both the different molecular weight forms and also for the deficiency of both activities in the rare inborn error of metabolism, hereditary orotic aciduria.

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Section: Discussionmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Subunit structure of the orotate phosphoribosyltransferase-orotidylate decarboxylase complex from human erythrocytes

Brown¹,

O'Sullivan²

1977

Biochemistry

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“…This loss of orotate PRTase is not surprising, despite efforts to provide maximal stablizing conditions (Table II). Several workers have demonstrated that the orotate PRTase activity of UMP synthase is far more labile than the OMP decarboxylase activity (Kasbekar et al, 1964;Appel, 1968;Brown et al, 1975;Grobner & Kelley, 1975;Reyes & Guganig, 1975; Kavipurapu & Jones, 1976;Brown & O'-Sullivan, 1977). We feel that the partial loss of orotate PRTase activity results from an abrupt change, induced by the extensive purification of the protein, in the structure of the PRTase domain of UMP synthase.…”

Section: Discussionmentioning

confidence: 99%

“…However, occasional reports have indicated that the two enzyme activities could be on separate polypeptides. Kasbekar et al (1964) observed that the two activities separated during electrophoresis in a starch gel, although the orotate PRTase activity was frequently lost; Appel (1968) could not recover the transferase activity after electrophoresis. Brown & O '-Sullivan (1977) inferred from experiments on a partially purified protein from human erythrocytes that the orotate PRTase:OMP decarboxylase complex was composed of two subunits (approximately 13 000 daltons) responsible for the orotate PRTase activity plus two subunits (approximately 20 000 daltons) responsible for the OMP decarboxylase activity.…”

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confidence: 99%

Isolation and initial characterization of the single polypeptide that ssynthesizes uridine 5'-monophosphate from orotate in Ehrlich ascites carcinoma. Purification by tandem affinity chromatography of uridine-5'-monophosphate synthase

McClard¹,

Black²,

Livingstone³

et al. 1980

Biochemistry

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UMP synthase, or multienzyme pyr-5,6 (orotate phosphoribosyltransferase:orotidine monophosphate decarboxylase), has been purified from Ehrlich ascites carcinoma to apparent homogeneity. The purification was achieved by the use of 5-[2-[N-(2-aminoethyl)carbamyl]ethyl]-6-azauridine 5'-monophosphate-agarose and phosphocellulose affinity columns linked in tandem by a flow dialysis system. The purified protein has amolecular weight of approximately 51500 as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Both enzyme activities cosediment with an S20,w value of 3.7 S, which corresponds to a molecular weight of about 50000. Two-dimensional electrophoresis of UMP synthase shows that the protein exists as two isomeric forms with isoelectric points of 5.85 (major form) and 5.65 (minor form). Both forms have the same molecular weight of 51500 and contain both active centers. These results clearly show that the last two enzyme activities of de novo UMP biosynthesis occur on a single polypeptide chain of approximately 51500 daltons and that this polypeptide exists in at least two isomeric forms.

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Thymidylate synthetase activity and fluorouracil sensitivity of human colonic cancer and normal mucosal tissue preparations

Wolberg

Morin

1981

Cancer

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This study compares thymidylate synthetase activity and certain aspects of fluorinated pyrimidine metabolism in tissue preparations derived from human colonic adenocarcinoma and from normal colonic mucosa. The purpose was to define differences that might be sufficient to explain the selective antitumor response of some colonic adenocarcinomas to 5-fluorouracil (FU) chemotherapy. Some carcinomas had much greater thymidylate synthetase activity than did other carcinomas and all normal mucosal preparations. Such carcinomas may be highly reliant on thymidylate synthetase and hence susceptible to its inhibition. Similarly, the smaller amount of FdUMP dephosphorylation found in all carcinomas compared with autologous normal mucosal preparations might result in prolonged thymidylate synthetase inhibition. In addition, greater thymidylate synthetase inhibition was produced by 1 X 10(-4) M FU in an occasional tumor than was found in normal mucosal preparations.

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Purification and Properties of Orotic Acid-decarboxylating Enzymes from Calf Thymus

Cited by 69 publications

References 7 publications

Subunit structure of the orotate phosphoribosyltransferase-orotidylate decarboxylase complex from human erythrocytes

Subunit structure of the orotate phosphoribosyltransferase-orotidylate decarboxylase complex from human erythrocytes

Isolation and initial characterization of the single polypeptide that ssynthesizes uridine 5'-monophosphate from orotate in Ehrlich ascites carcinoma. Purification by tandem affinity chromatography of uridine-5'-monophosphate synthase

Thymidylate synthetase activity and fluorouracil sensitivity of human colonic cancer and normal mucosal tissue preparations

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