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

Abstract

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.

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

“…These results, taken together, indicated that this loss, which occurred during the final stage of purification, results from a perturbation of the PRTase site and not from the loss of a separate orotate PRTase protein. This interpretation is consistent with the findings of several workers that the orotate PRTase activity of UMP synthase is unusually unstable (Kasbekar et al, 1946;Appel, 1968; al" 1975; Grobner & Kelley, 1975; Reyes & Guganig, 1975; Kavipurapu & Jones, 1976;Brown & O'Sullivan, 1977).…”

“…It is now easier to reconcile the proposed or implied structures of the complex between orotate PRTase and OMP decarboxylase from various sources (Kasbekar et al, 1964;Appel, 1968;Brown & O'Sullivan, 1977). Brown & O'Sullivan (1977) proposed that UMP synthase from human erythrocytes was composed of two 13 000-dalton subunits of orotate PRTase and two 20 000-dalton subunits of OMP decarboxylase; the molecular weight of the intact protein was about 62000. Two major problems in their study confound the conclusion.…”

A Nonradioactive High-Performance Liquid Chromatographic Microassay for Uridine 5′-Monophosphate Synthase, Orotate Phosphoribosyltransferase, and Orotidine 5′-Monophosphate Decarboxylase

“…The partially purified UMPS enzyme has both physical and kinetic properties similar to the previously characterized enzyme from the same source (5,6). The molecular masses of human UMPS are reported to be ϳ51-62 kDa in different cell types (3,5,6,8,9). The native enzyme is a dimeric form with a molecular mass of ϳ110 kDa (9).…”

“…The K m 's for orotate and OMP of human UMPS are 36 and 12 M, respectively (6). The V max value for orotate of human UMPS is ϳ2400 pmol/ min/mg protein (6). These results indicate the application of the HPLC assays for enzyme purification and kinetic measurement.…”

Pyrimidine metabolism in schistosomes: A comparison with other parasites and the search for potential chemotherapeutic targets

“…As a result, no OMP or orotidine are detected in the cell (Janeway and Cha, 1977). Furthermore, in mammalian cells, OMP decarboxylase activity is greater than orotate phosphoribosyltransferase activity (Kavipurau and Jones, 1977; Brown and O’Sullivan, 1977; Levinson et al, 1979; McClard et al, 1980) presumably to facilitate the “channeling effect”. Iltzsch et al (1984) showed that this relationship between the two enzyme activities is also present in both S. mansoni and mouse liver.…”