Proc. Nat. Acad. Sci. USA Vol. 71, No. 8, pp. 3031-3035, August 1974

Hereditary : Evidence for a Structural Gene Mutation (6-azauridine/biochemical genetics)

THOMAS E. WORTHY, WOLFGANG GROBNER, AND WILLIAM N. KELLEY* Division of Rheumatic and Genetic Diseases, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 Communicated by J. Edwin Seegmiller, May 6, 1974

ABSTRACT Orotic aciduria is a rare autosomal re- in man (5). This proposal was based on several observations. cessive disease in man due to a deficiency of orotate phos- (i) The double enzyme defect was difficult to explain as a phoribosyltransferase (EC 2.4.2.10; -5'-phos- phate: pyrophosphate phosphoribosyltransferase) and oro- structural gene mutation. Alternatively, some mutations in tidine-5'-phosphate decarboxylase (EC 4.1.1.23; orotidine- bacteria that affected the regulation of enzyme expression 5'-phosphate carbo.Ny-lyase). We have compared certain were known to reduce the levels of two or more enzymes in a physicochemical properties of orotidine-5'-phosphate pathway (6). In addition, OPRT and ODC levels were known decarboxylase from normal and mutant fibroblasts grown to under identical conditions. Orotidine-5'-phosphate de- be subject to genetic control in bacteria (7); more recently carboxylase from homozygous mutant cells was more this type of control has been demonstrated in yeast (8). thermolabile and exhibited at different electrophoretic (ii) In erythrocytes from heterozygotes, OPRT and ODC mobility when compared to the enzyme from normal cells; levels were often substantially less than 50% of normal. This orotidine-5'-phosphate decarboxylase from one hetero- was consistent with a trans dominant effect characteristic of zygous cell strain exhibited an intermediate thermplabil- ity while the other heterozygote displayed a thermal in- some regulatory mutations in prokaryotic cells (6). More activation curve indistinguishable from normal. The recently, Pinsky and Krooth noted that the addition of 6- enzyme from both normal and mutant cells exhibited bi. azauridine to normal cells in culture resulted in a modest in- phasic kinetics with the same apparent Michaelis con- crease in the activity of OPRT and ODC, whereas a marked stants. These data suggest that the molecular defect in the enzyme of this patient with orotic aciduria is due to a mu- increase in the activity of both enzymes was observed after the tation in a gene that affects the structure of either orotate addition of this analog to cells cultured from a phosphoribosyltransferase or orotidine-5'-phosphate de- patient with orotic aciduria (9). Although the mechanism carboxylase and cannot be Attributed to a mutation in responsible for this effect in either cell type was not clearly a regulatory gene, as previously suggested. elucidated, the findings were consistent with the hypothesis Enzymatic defects of pyrimidine in eukaryotic that 6-azauridine, or a accumulating organisms are exceedingly rare and, to date, are found only as the result of the presence of this analog, was capable of in- in a group of disorders that have in common the excretion of activating the product of a regulator gene that was presum- large quantities of . In man the primary disorder ably preventing the synthesis of these enzymes in the mutant in this group is hereditary orotic aciduria, an autosomal reces- cells (10). sive genetic disease, characterized by , Although these observations in hereditary -orotic aciduria leukopenia, retarded growth and development, and an in- were consistent with a regulatory defect, they could also be creased urinary excretion of orotic acid (1, 2). Biochemically, accounted for by a mutation in a structural gene(s) coding for this disorder is characterized by either of two phenotypes: one of the involved enzymes, and definitive data were not type I is most prevalent and exhibits deficient or decreased available to differentiate between these possibilities. In the activity of orotate phosphoribosyltransferase (OPRT; EC present study, we have used the 6-azauridine-augmented 2.4.2.10; orotidine-5'-phosphate:pyrophosphate phosphoribo- activity of ODC in fibroblasts in order to compare the syltransferase) and orotidine-5'-phosphate decarboxylase physicochemical properties of this enzyme from normal sub- (ODC; EC 4.1.1.23; orotidine-'-phosphate carboxy-lyase), jects and from a patient with hereditary orotic aciduria. The the enzymes that catalyze the conversion of orotic acid to results of these experiments suggest that the double enzyme -5'-phosphate (3); type II is -characterized by a de- defect in hereditary orotic aciduria is due to a mutation in ficiency only in orotidine-5'-phosphate decarboxylase (4). either one or both of the structural genes coding for the two The genetic defect in hereditary orotic aciduria was pro- affected enzymes. A preliminary report of these findings has posed to be a consequence of a mutation in a regulator genet been presented (11). and has been cited as one of the few examples of such a defect MATERIALS AND METHODS Abbreviations: OPRT, orotate phosphoribosyltransferase; ODC. Materials. [7-'4C]Orotic acid (10.2 mCi/mmole) and [7- orotidine-5'-phosphate decarboxylase; OMP, orotidine-5'-mono- 14C]orotidine-5'-monophosphate (21 mCi/mmole) were from phosphate. New England Nuclear Corp.; 6-azauridine and tetrasodium 5- * To whom reprint requests should be addressed. phosphoribosyl-l-pyrophosphate were from Sigma Chemical t Defined by us, but not by the authors cited, as a gene that con- Co.; 6-azauridine-5'-monophosphate was from Calbiochem; trols the synthesis of an enzyme but has no effect on its structure. Sephadex G-25 was from Pharmacia; acrylamide, N,N'- 3031 Downloaded by guest on October 1, 2021 3032 Medical Sciences: Worthy et al. Proc. Nat. Acad. Sci. USA 71 (1974) methylenebisacrylamide, and N,N,N',N'-tetramethylethyl- mg/ml). The resultant solution was degassed for 15 min with enediamine were from Eastman Kodak Co.; tissue culture stirring. N,N,N',N'-Tetramethylethylene diamine (15 ul) materials were from Grand Island Biological Co.; and 10% was added, and 1.1 ml of the acrylamide solution was gently agarose was from Bio-rad. All other materials were the layered into gel tubes (70 mm X 5 mm inner diameter); the highest quality available and were purchased commercially. gels were then polymerized at room temperature for 60 min. of skin fibroblasts were ob- The gels were first subjected to electrophoresis in separation Cell Culture. Primary cultures gel buffer for 90 min at 170 at a constant current of 2.5 mA tained from explant cultures of punch biopsies from the inner of a for heredi- per gel. A stacking gel was added by layering 0.25 ml 4% forearm of normal subjects, a subject homozygous acrylamide solution, made with stacking gel buffer (45 mM tary orotic aciduria (strain 237) (12), and her heterozygous 7.1), on top of the separation gel and The cultures Tris, 32 mM\ H3PO4, pH parents (father, strain 241; mother, strain 242). polymerizing for 60 min. The gels were placed in the electro- were routinely maintained in a tissue culture medium de- mM Tris, 50 mM HCl, pH to contain 20% phoresis chamber, and anode (62 scribed by Silagi et al. (13) and modified 7.6) and cathode (42 mM Tris, 46 mM glycine, pH 9.0) (v/v) fetal-calf serum. Only cells between passages 2 and 15 buffers were added. Lysates (50-100 Al) containing brom- were used in the experiments. Fibroblasts were routinely tracking dye and sucrose (20%) before use in experiments; phenol blue (2 ,ug/ml) as the grown for 6-8 days after passage to increase the lysate density were gently layered on top of control experiments indicated that OPRT and ODC activities at were the gels, and the gels were subjected to electrophoresis 170 were stable between 6 and 9 days after passage. Cultures with a constant current of 2.5 mA per gel. Electrophoresis was fed every 3-4 days, and both normal and mutant cells were migrated to within 5 mm of the Medium containing stopped when the tracking dye refed, with Eagle's Minimal Essential end of the gels (about 90 min). The gels were removed from 10% fetal-calf serum, 2 days before use. the tubes; dye fronts and gel lengths were measured, and the Preparation of Cell Lysates. Cell lysates from confluent gels were frozen at -70°. The frozen gels were cut into 1.2- monolayers were prepared as reported (14). After centrifuga- mm slices, placed in assay tubes containing 100 M1A of 10 mM tion at 600 X g to remove cellular debris, the lysates were Trise HCl pH 7.4, and assayed for ODC activity. passed through individual Sephadex G-25 columns with a Determination of the Stokes Radius. The Stokes radius of bed volume of 1.5 ml and a void volume of 0.6 ml. Samples ODC was determined from the chromatographic behavior on (200 Il) of lysates were layered onto the top of the columns a 10% agarose column. The partition coefficient (Kay) of and allowed to enter the Sephadex; 600 ul of 10 mM Trise HCl ODC was determined by the method of Laurent and Killander (pH 7.4) was added and the column was allowed to drain. (18), with ovalbumin, alcohol dehydrogenase, and catalase as The desalted lysates were then collected by elution with 300 standards. Values of the Stokes radius of the standards were M1 of the same buffer. The desalting procedure had no ap- taken from published reports (19). The inverse error function parent effect on the specific activity of either enzyme. complement of the column partition coefficient (erfc -l) was Enzyme Assays. ODC was assayed according to Kelley and derived from the Kav as described by Ackers (20). to Beardmore (15). The assay for OPRT (14) was modified in measure 14CO2 instead of the formation of orotidine-5'- Sucrose Gradient Centrifugation was performed a Spinco addi- SW41 rotor in a Beckman L5-50 ultracentrifuge. Isokinetic monophosphate (OMP). The reaction was initiated by the method and the tubes were incubated at gradients (10-28.1%) of 11.8 ml were prepared by tion of [7-14C]orotic acid, al. and standards of 200 were for 60 min. OPRT activity was stopped by addition of 100 of McCarty et (21). Samples Mu 370 to the and at 50 for 40 hr at of a applied gradients centrifuged ,ul of 0.1 M EDTA (pH 7.4), containing 50 ,ul partially coefficient was calculated purified preparation of ODC from human erythrocytes. The 40,000 rpm. Sedimentation (520,w) formed in the on the basis of the linear relationship between the 820,t and addition of excess ODC converted all the OMP isokinetic Ovalbu- initial reaction mixture to uridylic acid and 14CO2 during an the distance migrated in the gradient (21). terminated min (10 mg/ml) and alcohol dehydrogenase (3 mg/ml) were additional 15-min incubation; the reaction was by these calculations. values of the injection of 200 MAl of 3.5 M perchloric acid. This modification used as standards for S20,w of residual OPRT in standards were taken from published reports (19). eliminated the problem activity present Molecular weights and frictional ratios of ODC were the partially purified ODC preparation. Protein was deter- radii the method of et al. calculated from the 820,w and Stokes by mined by the method of Lowry (16). Siegel and Monty (22). Enzyme Kinetics. Lysates used in the kinetic studies of ODC were prepared in 10 mM Tris HC1 (pH 7.4) with 50 mM RESULTS EDTA and assayed at a final concentration of 25 mM EDTA. Orotate Phosphoribosyltransferase and Orotidine-5'-Phosphate Under these conditions there was no change in initial velocity Decarboxylase Activities From Normal and Mutant Fibroblast and less than 0.5% of the OMP in the reaction mixture was Lysates. The activity of OPRT and ODC in desalted extracts degraded by 5'-nucleotidase to orotidine. The release of 14CO2 from 8 normal cell strains ranged from 1.03 to 4.5 and 1.32 to was proportional to time and protein concentration. 3.77 nmoles/mg of protein per hr, with mean activities of Polyacrylamide Gel Electrophoresis of ODC was performed 2.40 and 2.18, respectively (Table 1). In extracts of fibroblasts in a modified multiphasic buffer system (system B) described cultured from the parents of the patient with hereditary orotic by Rodbard and Chrambach (17). Gels of various concentra- aciduria, the activity of OPRT was 0.58 and 0.51 nmole/mg tions of acrvlamide, but with a constant 5% crosslinking, of protein per hr. ODC activity was decreased in strain 241 were made by mixing 4.5 ml of acrylamide, 5.0 ml of separa- (father), with a mean activity of 0.58 nmoles/mg of protein tion gel buffer (0.375 M Tris, 0.16 M HCl, pH 8.55), and 0.5 per hr, while in strain 242 (mother), ODC activity was normal ml of a freshly made solution of ammonium persulfate (3 (1.87 nmoles/mg of protein per hr). The activity of both Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Hereditary Orotic Aciduria 3033

OPRT and ODC was markedly decreased in fibroblast ex- tracts from the patient with hereditary orotic aciduria, with mean values of 0.03 and 0.09 nmoles/mg of protein per hr, respectively. Mixing of extracts from the mutant strain and from a normal strain led to values of enzyme activity inter- mediate between the values observed with each extract alone. The effect of substrates for other known phosphoribosyl- transferases on OPRT activity from normal fibroblast extracts was examined in the presence of a subsaturating concentration of ['4C]orotic acid (0.05 mM). None of the compounds tested to as a aoc appeared compete successfully with orotic acid sub- - sF 1.0 strate for OPRT (Table 2). Similar results were found for OPRT from the mutant fibroblasts (Table 2). Thus, the low 0 m 6 level of activity in mutant fibroblasts does not appear to be co Q4- due to a low level of affinity for orotic acid by another phos- E phoribosyltransferase. In addition, the low levels of OPRT 0 0. E were not due to an occult infection since there was neither E ir(37 visible infection nor mycoplasma, as judged by agar cultures ZD 2 E and autoradiography (23). Ic Effect of 6-Azauridine on Orotate Phosphoribosyltransferase RR Rr(241) Rr(242) and Orotidine-'-Phosphate Decarboxylase. Pinsky and Krooth (9) observed that the addition of 6-azauridine to cultured FIG. 1. Effect of 6-azauridine on OPRT and ODC activities in cultured fibroblasts. Eight normal cell strains were grown to fibroblasts led to increased activity of both OPRT and ODC. confluency and 6-azauridine was added to 0.1 mM. The cells The effect of 6-azauridine on the levels of OPRT and ODC were incubated for 16 hr, harvested, and lysed, and the extracts activity in desalted fibroblast lysates from normal and mutant were assayed. Heterozygous and mutant cell strains grown to subjects is shown in Fig 1. confluency were assayed on six different occasions. Top, OPRT; The cultivation of normal cells in 0.1 mM 6-azauridine for bottom, ODC. (Symbols) control enzyme activity, open; enzyme 16-18 hr led to a 2.5- and 3-fold increase in the activity of activity in extracts after growth in 6-azauridine, cross-hatching. OPRT and ODC, respectively. Under identical conditions, Vertical lines indicate 4± 1 SD. strain 241 exhibited a 3-fold increase in OPRT and an 8-fold increase in ODC activity, whereas strain 242 showed a 3- desalted extracts of fibroblasts grown for 16 hr in medium fold increase in OPRT but no increase in ODC. ODC from containing 0.1 mM 6-azauridine. ODC from extracts of homozygous mutant cells exhibited an increase in activity of homozygous mutant cells was more thermolabile than the about 9-fold to a mean activity of 0.85, while no apparent in- enzyme in extracts of normal cells grown under identical con- crease was seen in OPRT activity in response to 6-azauridine. ditions. Mixtures of extracts from normal and homozygous This increase in ODC activity observed in normal and mutant mutant cells with equal activities exhibited an intermediate in- cells enabled us to investigate some of the properties of this activation curve that was the approximate mean of the normal enzyme from each cell strain. Because of the low activity of and mutant curves. The enzyme in extracts from one heterozy- OPRT in fibroblasts from the patient with hereditary orotic gous parent exhibited a thermolability intermediate between aciduria, the majority of subsequent experiments deal only those of the normal and mutant homozygotes, while the with ODC. TABLE 2. Effect of substrates for other phosphoribosyltransfer Thermal Inactivation of Orotidine-5'-Phosphate Decarbox- reactions on orotate phosphoribosyltransferase activity in extracts ylase. Fig 2 illustrates the thermal inactivation of ODC from from normal and mutant fibroblasts OPRT TABLE 1. Orotate phosphoribosyltransferase and orotidine-5'- Concentra- activity* phosphate decarboxylase activities in fibroblast extracts Substrates tion (mM) Normal Mutant None 1.39 0.12 Cell Geno- Enzyme activities* Adenine 1.0 1.40 0.13 strain type Passage OPRT ODC L-Glutamine 1.0 1.31 0.15 1.0 1.35 0.06 Normal RR 5-15 2.40 0. 97 2.18 :0. 67 Nicotinic acid 1.0 1.24 0.10 241 Rr 2- 6 0.58:1:0.24 0.5840.39 Niacinamide 1.0 1.40 0.10 242 Rr 2- 6 0.51 4±0.12 1.87±0.09 Anthranilic acid 1.0 1.23 0.08 237 rr 4-15 0.03 0.03 0.09 0.02 Quinolinic acid 1.0 1.24 0.09 1.0 1.31 0.10 Eight normal cell strains (RR) were grown to confluence, harvested, and lysed. The extracts were assayed as described in A subsaturating concentration of [14C]orotic acid (0.05 mM) Materials and Methods. Heterozygous (Rr) and homozygous (rr) was added to the reaction mixture with phosphoribosylpyrophos- mutant cell strains grown to confluency were assayed on six phate immediately after the addition of the potentially com- different occasions. peting substrates. * nmoles/mg of protein per hr; mean 4 SD. * nmoles/mg of protein per hr. Downloaded by guest on October 1, 2021 3034 Medical Sciences: Worthy et al. Proc. Nat. Acad. Sci. USA 71 (1974)

2.0r

8 x 5 s~~~~-tls[4l~I.5 & 1.0 800 T -5-ea[ 11 rr 0 24680 0.2 0.4 0 0.2 0.4 -j o M RF RF mM60azaur e for 16 were eated at 57°.ARr rr z 015 15 o 0 I I eo40 Rr(7 -0 - 4 8 12 16 20 <10- 10- GEL CONCENTRATION (%) FIG. 4. Ferguson plot of ODC from normal and mutant U2 5 5 cells grown in the presence of 6-azauridine. Lysates were pre- pared as described in Materials and Methods. About 100 1Ag of 0 2 4 6 8 0 0.2 0.4 0 0.2 0.4 each lysate was loaded onto the top of polyacrylamide gels (5% TIME (min) RF RF crosslink) ranging in concentration from 4-16%. The gels were subjected to electrophoresis at 2.5 mA per gel, fractionated, and FIG. 2(left). Thermalinactivation curvesof ODC from lysates assayed. Each point is the mean of four determinations (i SD). of normal, heterozygous, and mutant fibroblasts.2))Samples (600 Recoveries were similar to those in Fig. 3. Normal, *; mutant, A. of desalted lysates from fibroblasts grown in the presence of 0.1 mM 6-azauridine for 16 hr were heated at57o1 At appropriate times, 50-iAl samples (in duplicate) were removed and placed in Ml1olecular Weight of Orotidine-5'-Phosphate Decarboxylase. tubes in an ice bath. Immediately after they were heated for 8 The Stokes radius of ODC from both normal and mutant min, the samples were assayed at 370 for 15 mm.Each point is fibroblasts grown in 6-azauridine was 58 A. Sucrose gradient the mean of five to six experiments (+ 1 SD). See Fig. 1 for centrifugation yielded 820o,w values of 5.2 for ODC from both mean control activities. Normal, A; orotic aciduria, U; hetero- zygotes (strain 241), @; heterozygote (strain 242), a o normal and mutant cells cultured in 6-azauridine. Calculation FIG. 3 (right). ODC activity profiles from polyacrylamide gels. of the molecular weight from the 820,w and Stokes radius gave Aboutrsg100 of desalted lysates from cells grown in the presence estimates of 124,500 for the enzyme from both cell strains. of 0.1 mM 6-azauridine was layered on top of 12% polyacrylamide This value is in close agreement with independent estimates of gels (5% crosslink) and subjected to electrophoresis at 2.5 mA molecular weight by polyacrylamide gel electrophoresis, per gel. The gels were frozen, fractionated into 1.2-mm slices, and which gave a value of 130,000. Calculation of the frictional placed in tubes containing 100 IdI of 10 mM Tris.HCI, pH 7.4. ratio gave an estimate of 1.76. ODC was assayed as described in Materials and Methods, except that the assay time was increased to 1 hr. Recoveries of enzyme Kinetic Properties of Orotidine-5' Phosphate Decarboxylase. activity from the gels were: normal, 50%; mutant homozygote, ODC from both cell types exhibited biphasic kinetics. At 22%; mutant heterozygotes, 50%. Top left, normal (RR); top concentrations of OMP below 1 MuM the apparent Michaelis right, strain 241 (Rr); bottom left, strain 242 (Rr); and bottom constants (Kn) were 0.33 MM and 0.25 MAM for the enzyme right, strain 237 (rr). from normal and mutant cells, respectively. Above 2 4M OMP, a second apparent Km was seen, with values of 4.5 ,uM and 2.8 MAM for the enzyme from normal and mutant cells, enzyme from the other parent exhibited a rate of thermal in- respectively. These values are similar to those reported by activation essentially indistinguishable from normal (Fig 2). Fyfe et al. in yeast (25). Electrophoretic Mobilnities of Orotidine-5'-Phosphate De- DISCUSSION carboxyta~se in Potlyacrytamide Gets. Polyacrylamide gel electro- In the present study, we have presented evidence that sug- phoresis of ODC in lysates of fibroblasts grown in the presence gests that, in at least one patient with hereditary orotic of 0.1 mM 6-azauridine is shown in Fig 3. ODC in extracts aciduria due to a deficiency of OPRT and ODC, the genetic from normal cells migrated with an RF of 0.23, whereas the defect is due to a mutation in a gene(s) that affects the struc- enzyme from mutant cells migrated with an RF Of 0-11- ture of one or both of the enzymes involved. The evidence for Extracts from the heterozygous cell strains exhibited several such a mutation is based on three observations: (i) ODC from peaks of activity, which included those peaks observed with mutant cells grown in the presence of 6-azauridine is more extracts from the normal and mutant homozygous cells. sensitive to thermal inactivation than the ODC from normal Hfendricks and Smith (24) have described a procedure for cells grown under identical conditions. (ii) ODC from mutant determining whether the electrophoretic mobility in poly- cells exhibits an electrophoretic mobility in polyacrylamide acrylamide gels is due to a difference in molecular size or gels different from that seen in normal cells when both cell charge. Nonparallel lines that intersect near 0% acrylamide strains are grown in the presence of 6-azauridine. This dif- are indicative of a difference in molecular weight, while parallel ference in the enzyme from normal and mutant cells is due to lines on a Ferguson plot indicate a difference in tbe net a difference in the net molecular charge. (iii) Electrophoresis molecular charge. Construction of a Ferguson plot comparing of lysates from both heterozygous cell strains grown in the ODC from normal and mutant cells incubated with 6-aza- presence of 6-azauridine indicates the presence of at least two uridine resulted in a set of parallel lines (Fig 4). Thus, ODC molecular forms of ODC in each cell strain. One form migrates from the mutant cells appeared to differ from the normal with an RF identical to that of the mutant enzyme, while enzyme in net charge. another form has an RF identical to the normal enzyme. Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Hereditary Orotic Aciduria 3035

Although the differences in heat stability and electropho- J. B., Wyngaarden, J. B. & Fredrickson, D. S. (McGraw- retic migration have been shown for this difference Hill, New York), 3rd ed., pp. 1003-1029. only ODC, 3. Smith, L. H., Jr., Sullivan, M. & Huguley, C. M., Jr. (1961) could reflect an alteration in the primary structure of either J. Clin. Invest. 40, 656-664. ODC or OPRT. Partial purification of OPRT and ODC 4. Fox, R. M., O'Sullivan, W. J. & Firkin, B. G. (1969) Amer. from human tissue (Grobner and Kelley, unpublished data) J. Med. 47, 332-336. suggests that the two enzyme activities copurify and that they 5. Smith, L. H. (1965) Amer. J. Med. 38, 1-6. 6. Jacob, F. & Monod, J. (1961) J. Mol. Biol. 3, 318-356. may exist in a complex, as has been reported for the enzymes 7. Beckwith, J. R., Pardee, A. B., Austrian, R. & Jacob, F, isolated from beef erythrocytes (26) and calf brain (27). (1962) J. Mol. Biol. 5, 618-634. Presumably, alteration in the structure of either enzyme could 8. Lacroute, F. (1968) J. Bacteri.l 95, 824-832. affect the net charge and stability of the complex. Therefore, 9. Pinsky, L. & Krooth, R. S. (1967) Proc. Nat. Acad. Sci. it is possible that the altered properties of ODC are due to a USA 57, 925-932. 10. Pinsky, L. & Krooth, R. S. (1967) Proc. Nat. Acad. Sci. USA mutation in the structural gene coding for OPRT. Our ob- 57, 1267-1274. servation that OPRT activity remains at control levels in 11. Worthy, T. E. & Kelley, W. N. (1973) Amer. J. Human mutant fibroblasts grown in 6-azauridine while ODC activity Genet. 25, 88A. increased 9-fold provides meager evidence for this interpreta- 12. Rogers, L. E., Warford, L. R., Patterson, R. B. & Porter, tion. The failure of ODC activity in mutant cells to attain F. S. (1968) Pediatrics 42, 415-422. 13. Silagi, S., Darlington, G. & Bruce, S. A. (1969) Proc. Nat. normal control activity, as reported by Pinsky and Krooth Acad. Sci. USA 62, 1085-1092. (9), would be explicable in terms of a destabilization of the 14. Kelley, W. N., Beardmore, T. D., Fox, I. H. & Meade, complex. J. C. (1971) Biochem. Pharmacol. 20, 1471-1478. A comparison of our data with those reported earlier by 15. Kelley, W. N. & Beardmore, T. D. (1970) Science 169, 388- 390. Pinsky and Krooth in cells from another unrelated subject 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, with hereditary orotic aciduria (9) reveals a substantial dif- R. J. (1951) J. Biol. Chem. 193, 265-275. ference in the level of both OPRT and ODC activity attained 17. Rodbard, D. & Chrambach, A. (1971) Anal. Biochem. 40, after incubation with azauridine. This finding raises the pos- 95-134. sibility that genetic heterogeneity may exist within the group 18. Laurent, T. C. & Killander, J. (1964) J. Chromatogr. 14, 317-330. of subjects with type I orotic aciduria. Perhaps, the differences 19. Smith, M. H. (1968) in Handbook of Biochemistry, ed. Sober, observed in the two heterozygous cell strains with regard to H. A. (The Chemical Rubber Co., Cleveland, Ohio), pp. the effect of azauridine and thermal inactivation of OPRT C10-17. and ODC are also a reflection of genetic heterogeneity. 20. Ackers, G. K. (1967) J. Biol. Chem. 242, 3237-3238. 21. McCarty, K. S., Stafford, D. & Brown, 0. (1968) Anal. Biochem. 24, 314-329. We thank Drs. R. B. Patterson and F. S. Porter for their 22. Siegel, L. M. & Monty, K. J. (1966) Biochim. Biophys. Acta cooperation in allowing us to study this patient. We also thank 112, 346-362. Pam Watkins for technical assistance. This research was sup- 23. Studzinski, G. P., Gierthy, J. F. & Cholon, J. J. (1973) In ported in part by USPHS Grant no. AM 14362. T.E.W. is the Vitro 8, 466-472. recipient of a USPHS Postdoctoral Fellowship Grant no. GM 24. Hendricks, J. L. & Smith, A. J. (1968) Arch. Biochem. 55365. Biophys. 126, 155-164. 25. Fyfe, J. A., Miller, R. L. & Krenitsky, T. A. (1973) J. Biol. 1. Huguley, C. M., Jr., Bain, J. A., Rivers, S. L. & Scoggins, Chem. 248, 3801-3809. R. B. (1959) Blood 14, 615-634. 26. Hatfield, D. & Wyngaarden, J. B. (1964) J. Biol. Chem. 2. Smith, L. H., Jr., Huguley, C. M., Jr. & Bain, J. R. (1972) 239, 2580-2586. in The Metabolic Basis of Inherited Disease, eds. Stanbury, 27. Appel, S. H. (1968) J. Biol. Chem. 243, 3924-3929. Downloaded by guest on October 1, 2021