RIOCHEMICAL
MEDICINE.
17, I?x-I-t() 11977,
Multiple Molecular Decarboxylase MARY
T. CAMPBELL,’ AND WILLIAM
Departmenr
of Medicine, Ro.val
Forms of Orotidylate from Human Liver NEIL
D. GALLAGHER,
J. O’SULLIVAN’
Universitp of Sydney Prince Alfred Hospital.
and Deportment of Gasrroenreroiogy, Sydne!. Alrstraliu
Received July 14, 1976
INTRODUCTION
Uridine-5’-monophosphate (UMP)3 is synthesized from erotic acid by two enzymic steps in de ~OVO pyrimidine biosynthesis: the conversion of erotic acid to orotidine-5’-monophosphate (OMP) by orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10) and decarboxylation of OMP to form UMP by orotidylate decarboxylase (ODCase; EC 4.1.1.23). These enzymes appear to be very closely associated in mammalian systems. Their activities remain linked during a variety of purification procedures (l-3) and also following alteration in their activity as a result of therapy with purine and pyrimidine antimetabolites (4-6). In addition, they are both grossly deficient in hereditary erotic aciduria Type I (7), a potentially lethal inborn error of metabolism in man. Investigation of these two enzymes in man has been effectively limited to erythrocytes, where the de no~o pyrimidine pathway is residual (7), and to diploid fibroblast cultures (8-10). In the present paper, we report some preliminary findings on OPRTase and ODCase in human liver, a tissue in which both enzymes remain functional. In particular, the decarboxylase, which is the more stable of the two enzymes, was found to occur in several oligomeric forms, interconversion of which was affected by ionic strength, the presence of thiols, and UMP. Some comparisons ’ Present address: Laboratory of Liver Physiopathology, Rega Institute, Minderbroedersstraat 10, 3000-Leuven, Belgium. * Present address: School of Biochemistry. University of New South Wales, Kensington, N.S.W. 2033, Australia. 3 Abbreviations used: UMP, uridine-5’-monophosphate: OMP, orotidine-S’-monophosphate; OPRTase, orotate phosphoribosyltransferase; ODCase, orotidylate decarboxylase; XMP, xanthosine monophosphate; PRPP, phosphoribosylpyrophosphate: HEPES, N-2hydroxyethylpiperazine-N’-2-ethane sulfonic acid; MOPS, morpholinopropanesulfonic acid; EGTA, ethyleneglycol-bis(&aminoethyl ether)-N.N’-tetraacetic acid. I28 ISSN 0OW?YJ4
HUMAN
LIVER
OROTIDYLATE
are made with similar oligomeric (11.12). MATERIALS
DECARBOXYLASE
129
forms derived from human erythrocytes AND METHODS
Materials
Orotic acid, UMP, XMP, AMP, CMP, and tetrasodium phosphoribosylpyrophosphate (PRPP) were purchased from Sigma Chemical Company; 6-azaUMP, OMP, HEPES (N-2-hydroxyethylpiperazine - N’ 2 - ethanesulfonic acid) and MOPS (morpholinopropane-sulfonic acid) were from Calbiochem Pty. Ltd. [Carboxy - ‘*C]orotic acid and [carboxy‘*ClOMP were obtained from New England Nuclear Corporation. Oxipurinol [4,6 - dihydroxypyrazolo(3,4 - d)pyrimidine] was a gift from Welicome (Australasia) Pty. Ltd. while Sephadex G-25 and G-150 were obtained from Pharmacia Fine Chemicals. Bovine serum albumin (Fraction V) was from Armour Pharmaceutical Company, pepsin and cytochrome c were from Sigma, catalase was from Calbiochem, and soybean trypsin inhibitor was from Worthington Biochemical Corporation. Creatine kinase was prepared from rabbit muscle by the method of Kuby et al. (13) and yeast ODCase was prepared by a modification of the method of Umezu et al. (14). All other materials were of analytical reagent grade. Enzyme
Preparation
During the early stages of this work, human liver was obtained from Royal Prince Alfred Hospital, Sydney, as soon as possible after death (within 2 hr), immediately frozen, and stored at -20” until required. At a later stage, liver was obtained from kidney donors immediately after death, rapidly frozen, and stored in liquid air. Portions of liver were homogenized in 4 or 6 vol of a buffer containing 20 mM HEPES-KOH, 0.15 M KCl, 0.2 mM EDTA, and 0.1 mM dithiothreitol, pH 7.2. Homogenization was carried out in a Sorvall Omnimix for 5 mm at 0” and the homogenate was centrifuged at 25,OOOg for 1 hr. Protamine sulfate in the same buffer solution was added dropwise to the supernatant to give a final concentration of O.l%, equilibrated for 5 min, and centrifuged. The clear supernatant was brought to 35% saturation with finely ground ammonium sulfate and centrifuged, and the resulting supernatant was brought to 65% saturation. The pellet obtained after centrifugation was dissolved in 5 mM HEPES-KOH, 0.15 M KCl, 0.2 mM dithiothreitol, pH 7.2, and the volume was adjusted to 5% of the original supernatant volume. The fraction was either dialyzed overnight or desalted on Sephadex G-25. All fractions were immediately assayed for OPRTase and ODCase activities, then stored at -20” or in liquid air for further analysis.
130
CAMPBELL,
GALLAGHER
AND
O’SULLIVAN
Enzyme Assuys ODCase was assayed by the collection of ‘“CO, from [ccrrbo.r.v14C]OMP as described by Fox et al. (4), except that the assay buffer was 25 mM MOPS, pH 6.8. When assaying dilute fractions from gel filtration experiments, 150 pg of bovine serum albumin was included in the assay. Unless otherwise stated, all assays were carried out at 10 PM OMP concentration (specific activity, 2000-3000 dpm per nmole). OPRTase was assayed by the same method using [carboxy-i4C]orotic acid, with the OMP formed being immediately decarboxylated by endogenous or added yeast decarboxylase. The assay buffer was 50 mM Tris-HCl, pH 7.4, containing 3 mM MgC&, with an erotic acid concentration of 100 PM (specific activity, 950 dpm per nmole) and a PRPP concentration of 250 PM. For some gel filtration experiments purified yeast ODCase was included in the assay mixture at a level sufficient to decarboxylate 120 nmoles of OMP in 1 hr. In most other experiments the level of liver ODCase was sufficiently high to make further additions of yeast enzyme unnecessary. Protein concentrations were determined by absorbance at 280 and 260 nm (15). Gel Filtration
Experiments
Changes in the aggregation state of the enzyme were assessed by chromatography on Sephadex G-150 columns (1.5 x 70 cm) at 4”. The sample volume was l-2 ml and the flow rate was 8-10 ml per hr. The columns were calibrated with catalase (240,000 MW), creatine kinase (83,000), pepsin (35,000), soybean trypsin inhibitor (21,500), and cytochrome c (12,200). In all chromatography experiments, a trace amount of blue dextran was included in the sample in order to accurately locate the void volume. Rechromatography experiments, which generally involved very low concentrations of protein, were carried out in the presence of bovine serum albumin (0.5 mg/ml). RESULTS
Enzyme Preparation ODCase and OPRTase were present in both particulate and soluble fractions of human liver. Approximately 45% of both enzyme activities was present in the 25,OOOg supernatant after the livers had been frozen at -20”. This value was increased only slightly (to 55%) by including 1% Triton X-100 in the homogenizing medium. However, rapid freezing of liver in liquid air released 80-85% of both enzyme activities into the supernatant fraction. The subcellular location of the particulate activity
HUMAN
LIVER
OROTIDYLATE
131
DECARBOXYLASE
was not determined since fresh livers suitable for cell fractionation studies were not available. The ODCase was found to be stable in crude supernatants for up to 3 weeks when stored at 0” but lost 20-30% of its activity when stored at -20”. The OPRTase was much more unstable at 0” and had lost 50% of its activity after 1 week at -20”. The conditions under which the liver was originally stored also considerably affected the level of enzyme activity. For livers frozen in liquid air immediately after death, the soluble decarboxylase activity was 17-21 nmoles of UMP formed per hour per milligram of protein, while the activity in livers frozen at -20” was l-5 nmoles per hour per milligram of protein. Only part of this difference could be attributed to the greater solubilization of enzyme from liver stored in liquid air. The two enzymes showed parallel behavior during purification, until dialysis (Table 1). At this stage, 40% of the OPRTase activity was lost when dialyzed against buffered 0.15 M KCl. If the extracts were dialyzed or desalted in low ionic strength buffer (25 mM KCl), 85-90% of the OPRTase activity was lost immediately and the remaining activity was lost within the first few days of storage at -20”. Under similar conditions of low ionic strength, only 20% of the decarboxylase was lost. In the final preparations made under optimum conditions (0.15 M KCl, 0.2 mM dithiothreitol) both enzyme activities remained stable when stored in liquid air for several weeks or at -20” for several days. TABLE PREPARATION
OF OROTIDYLATE
PHOSPHORJBOSYLTRANSFERASE
FROM
Protein Fraction Cell supematant Protamine supernatant Ammonium sulfate (35-65%) Dialysis
1
DECARBOXYLASE
AND
HUMAN
OROTATE
LIVERY
ODCase
OPRTase
mg/ml
Total mg
Units/ m
Total units
Units/ mg
Total units
20 10
510 260
17 34
8670 8910
7 14
3510 3540
46 29
116 105
55 54
6414 5740
23 16
2650 1640
a ODCase and OPRTase were prepared as described in Materials and Methods from fresh human liver rapidly frozen in liquid air. All fractions were assayed for both enzyme activities immediately after preparation. ODCase was assayed at 25 PM OMP ([carboxy-W]OMP: specific activity, 2000 dpm/nmole), and OPRTase was assayed at 100 PM erotic acid ([carboxy-i4C]orotic acid; specific activity, 950 dpm/nmole). One unit of enzyme activity is defined as the formation of 1 nmole product in 1 hr at 37”.
132
('AMPHELL.GALLAGHER
AND O'SULLIVAN
The activity of ODCase was unaffected over the pH range 6.0-7.4 when preincubated at these values for 30 min at 37”, but was rapidly inactivated at pH values above 8.0. The optimum pH for enzyme activity was found to be 6.7 at 37” using a Tris-maleate-glycine buffer over a pH range of 5.0-9.0. The following compounds were without significant effect on the decarboxylase activity when included in the incubation medium: EDTA, EGTA. MgCl, (all at 1 and 10 mM); chloride salts of Ca2+, Co*+. and Mn”+: erotic acid. PRPP, dithiothreitol, N-ethylmaleimide (all at 0.1 and 1.0 mM): and oxipurinol (0.1 mM). The sulfate salts of Cu2+ and Zn’+ produced 90% inhibition at 0.1 mM concentration and inorganic phosphate, UMP, XMP, and 6-azaUMP were also inhibitory (see below). Gel Filtration
on Sephadex G-150
Chromatography of the partially purified ODCase on Sephadex G-150 gave rise to more than one peak of activity. The relative amounts of the various molecular weight forms varied according to the history of the stored liver. Liverfrozen in liquid air. Elution profiles of ODCase and OPRTase on Sephadex G-150 are shown in Fig. 1A. Both enzyme activities eluted as single symmetrical peaks with the same elution volume, corresponding to a MW of 63,000. Recovery of activity was 60-70% for the decarboxylase and 25% for the transferase. Upon rechromatography under the same buffer conditions, 80% of the decarboxylase activity was recovered and the elution profile remained unchanged even after storage for at least 6 weeks in liquid air. It was not possible to confirm this for the OPRTase, as sufficient activity did not survive rechromatography. While a similar elution profile was also obtained for ODCase if the initial chromatography was carried out in low ionic strength buffer (25 mM KC1 instead of 0.15 M KCl) with or without dithiothreitol present, the elution profile of the enzyme isolated under these conditions was found to change considerably during subsequent storage and rechromatography. First, after storage for 24 hr in buffered 25 mM KC1 without dithiothreitol, both aggregation and dissociation were observed. Half the ODCase eluted with a MW of approximately 35,000 and the rest eluted at the void volume, with only a trace of the original 63,000 MW form (Fig. 1B). This changed profile was accompanied by a loss of more than 70% of the ODCase activity, while OPRTase activity was completely lost. Second, if dithiothreitol was included in the low ionic strength buffer, dissociation was still observed but aggregation was not. Under these conditions, a variable amount of activity (40-N%) was recovered in the 35,000 MW form, while the rest remained in the 63,000 MW form. In this case, the dissociation was accompanied by a loss of half the total enzyme activity
HUMAN
LIVER OROTIDYLATE
DECARBOXYLASE
133
FIG. 1. Sephadex G-150 chromatography of ODCase and OPRTase from human liver frozen in liquid air. Ammonium sulfate extracts were prepared as described in Materials and Methods. (A) Elution protile on a Sephadex G-150 column (1.5 x 70 cm) of ODCase (04) and OPRTase (O---O) in 5 mM HEPESKOH, pH 7.2, containing 0.15 M KC1 and 0.2 mh4 dithiothreitol. The protein profile (monitored at 280 nm) is indicated by open circles (B) Elution profile of the peak Sephadex fractions after dialysis and rechromatog(0 -0). raphy in 5 mM HEPES-KOH, pH 7.2, containing 25 mM KC1 and no dithiothreitol. Only ODCase activity was detected. The sample volumes were 1 ml and the flow rate was 8-10 ml/hr. Fractions of 1.1 ml were collected and points are plotted for each two or three fractions. Recovery of activity was 60% for ODCase and 25% for OPRTase in experiment A and 27% for ODCase in experiment B. Elution was carried out at 0”. The void volume was determined with blue dextran. Inset: Plot of molecular weight as a function of elution volume for standard proteins (see Materials and Methods). The elution volumes of the 35,000 and 63,000 molecular weight forms of ODCase are indicated by arrows.
applied to the column. Increasing the ionic strength did not reverse this inactivation. Both the void volume form and the 35,000 MW form of the ODCase could be rechromatographed independently with no further change in the elution profiles. The void volume enzyme was stable to prolonged storage at -2O”, but the 35,000 MW form rapidly lost all activity unless stored in bovine serum albumin (0.5 mg/ml). Liver stored af -20”. With ODCase prepared from liver which had been stored at -2O”, half the activity eluted at the 35,000 MW position while
134
CAMPBELL>.
GAL.LAGHER
AND O’SULLIVAN
the rest eluted in a broad peak, the fractions containing maximum activity eluting with a MW of approximately 105,000 (Fig. 2). If these peak fractions were rechromatographed immediately. the elution volume was unaltered. However, following storage at -20” (in the absence of dithiothreitol). the 105,000 form gradually aggregated to the void volume enzyme over a period of 1-2 weeks. In contrast to the 63,000 form, there was no detectable dissociation during storage in low ionic strength buffer. The aggregation appeared to be greatly accelerated when the enzyme was prepared in 50 mM phosphate buffer, an inhibitor of the erythrocyte enzyme (II), instead of HEPES-KOH. Although the 105,000 MW form was predominant on the day of preparation, chromatography of the ammonium sulfate fractions on subsequent days showed an increasing aggregation to the void volume form until only a trace of the 105,000 MW form remained. Incubation of the 105,000 MW form with other inhibitors, GazaUMP, XMP, or oxipurinol (all at 0.1 mM) for 2 hr at 37”. also caused an accelerated aggregation to the void volume form. Behavior qf Various Forms of ODCase Heat stability. The void volume enzyme and the 105,000 MW enzyme were both relatively stable to mild heat treatment at 45” (Fig. 3). The 35,000 MW form was extremely unstable but could be stabilized by strong inhibitors. After incubation of aliquots of this form for 2 hr at 37” with XMP (0.1 mM) or 6-azaUMP (0.1 mM) and chromatography to remove the I '
40
60 Elutlon
80 Volume
100
-6
120
(ml)
FIG. 2. Sephadex G-150 chromatography of OMP decarboxylase from human liver stored at -20”. Ammonium sulfate extracts were prepared from liver stored at -20” and chromatographed on Sephadex G-150 as described in the legend to Fig. 1, except that a different column was used and the sample size was 2.5 ml. Fractions of 1.4 ml were collected and assayed for ODCase. Every alternate fraction is indicated (04). The protein profile is shown by the fine line (O-O). Recovery of activity was 35%. The void volume is indicated by k’,,.
HUMAN
? .Z -c ‘i ::L t
LIVER OROTIDYLATE
DECARBOXYLASE
135
40
20..
\ 0
: 10
20
;I 30
lime tmin) FIG. 3. Stability of OMP decarboxylase ohgomers at 45”. Preparations of the void volume enzyme (0) and the 105,000 (A) and 35,090 (0) molecular weight forms were obtained by gel filtration in 10 mM HEPES-KOH, pH 7.2, and 25 mM KCl. One-milliliter samples were incubated in a 45” water bath; aliquots were removed at the times indicated, rapidly cooled in ice, and kept at 0” until assayed. For assay of ODCase, the enzyme ahquots were brought to 37” in the incubation medium (3 min) before addition of substrate. The activity is expressed as a percentage of the initial activity of unincubated samples. Initial specific activities and protein concentrations, respectively, were: (i) void volume enzyme, 5.1 nmoles/mg of protein/hr (9 mg of protein/ml); (ii) 105,000 MW enzyme, 4.0 nmoles/mg of proteinihr (4.6 mg of protein/ml); (iii) 35,000 MW enzyme, 6.8 nmoles/mg of proteinihr (1 mg of protein/ml).
inhibitors, no loss of activity was observed and the molecular weight remained unaltered. In contrast, virtually all activity was lost after incubation in the absence of inhibitors (Fig. 3). Aggregation of the 35,000 MW enzyme. Incubation of the low molecular weight form of ODCase with UMP (0.5 mM), in the presence of 0.15 M KC1 and 10 mM dithiothreitol for 3 hr at 37”, caused aggregation to a 67,000 MW form with traces of higher aggregates (Fig. 4). No effect was observed with other inhibitors, such as 6-azaUMP (0.1 mM), XMP (0.1 mM), and inorganic phosphate (50 mM). In general, these experiments were hampered by the low activity of the 35,000 MW form, and bovine serum albumin (0.5 m&ml) was included in elution buffers in order to recover maximum activity. To attain reproducible aggregation of the decarboxylase, it was necessary to include both a high ionic strength and a thiol and to use freshly prepared enzyme. There was no detectable recovery of OPRTase in reaggregated ODCase preparations. Dissociation of the void volume enzyme. The void volume form of ODCase could be almost completely dissociated by mild treatment with guanidine-HCl. After incubation with 0.5 M guanidineHC1 for 5 min at O”, followed by immediate gel filtration, most of the recovered activity
136
CAMPBELL,,
GALLAGHER
Elution
volume
AND O’SULLIVAN
(ml)
FIG. 4. Aggregation of the 35,000 molecular weight form of ODCase by UMP. The 35,000 molecular weight fraction obtained by gel filtration was incubated with 0.5 mM UMP for 3 hr at 37’ in a buffer containing 10 mM HEPES-KOH, pH 7.2, 0.15 M KCI. 10 mM dithiothreitol, and 0.5 mg/ml of serum albumin. The sample, with a trace of blue dextran, was chromatographed on Sephadex G-150 using the same buffer, except that 1.0 mM dithiothreitol was used. Fractions were assayed for ODCase (04). The elution profile of control enzyme is included for comparison (O---e).
was present as the 35,000 MW form (Fig. 5). Only small amounts of activity remained at the void volume and also at positions corresponding to molecular weights of 110,000 and 63,000. The conditions used for this dissociation caused no significant loss of enzyme activity in the sample before chromatography. 12 -. ‘:
1
vo 1
35,000 I
0vI $
a--
r .Z .? 6 d i!
.‘:
4--
J
[
ri 0
30
50 Elution
70 Volume
90
110
(ml)
FIG. 5. Dissociation of the void volume form of ODCase by guanidineHC1. A fresh sample of the high molecular weight void volume enzyme was incubated in 0.5 M guanidine-HCI for 5 min at 0” and immediately applied to a Sephadex G-150 column. Fractions were assayed for OMP decarboxylase activity. Experimental conditions were as for Fig. 1. Recovery of activity was 42%.
HUMAN
LIVER
OROTIDYLATE
137
DECARBOXYLASE
A small amount of activity (lo-20%) was also consistently obtained in the 35,000 MW form after incubation of the void volume enzyme with 10 or 20 mM dithiothreitol for 2 hr at 37”. However, no change in the elution profile could be obtained by heat treatment alone or in the presence of phosphate or 6-azaUMP or by variation of the ionic strength or protein concentration. Kinetic properties. The individual high molecular weight aggregates of ODCase gave linear Lineweaver-Burk plots with K, values of 0.12 PM. Enzyme preparations containing a mixture of different aggregates gave a single linear response when tested over a wide range of OMP concentrations (0.07-10 PM), with K, values varying from 0.2 to 0.5 PM in different preparations. The 35,000 MW form gave nonlinear Lineweaver-Burk plots concave upward, in which enzyme activity fell rapidly at substrate concentrations lower than 0.5 PM. However, when bovine serum albumin (300 pg) was included during the assay, the plots approached linearity. This suggested that the concave plots were due to instability of the low MW enzyme at low substrate concentrations, as has been found by other workers (2,3,8). An accurate K, value could not be obtained for this form but it was estimated as approximately 0.3 PM. A number of compounds which have been shown to competitively inhibit erythrocyte ODCase was tested as inhibitors of the 105,000 MW form of the liver enzyme. Ki values are shown in Table 2 compared to values reported for the rat liver enzyme (16) and also with results for the 250,000 MW form of human erythrocyte ODCase (11,17).
TABLE INHIBITION
Inhibitor 6-azaUMP XMP UMP CMP AMP Phosphate
CONSTANTS
Human liver co.01 0.3 16 40
80 12,000
FOR
2 HUMAN LIVER ODCase” Rat livep 0.1
Human erythrocytesc 0.06 1.9 11
700
800
>50 22,000
a Inhibition constants obtained for the 105,000 MW form of the human liver enzyme are compared to values reported for rat liver (16) and for the 250,000 MW form of the human erythrocyte enzyme (11, 17). K, values for the human liver enzyme were determined from Dixon plots using varying concentrations of inhibitors at a fixed substrate concentration of 0.36 ~1M. K, for OMP in the same experiments was 0.12 FM. All Ki values are expressed as micromolar concentrations. b Creasey and Handschumacher (16). c Brown (17).
138
CAMPBELL,. GALLAGHER ANDO‘SULLIVAN DISCUSSION
Apart from the problem of obtaining sufficient suitable material, experiments with human liver ODCase and OPRTase were severely hampered by the high lability of both enzymes, in particular the OPRTase, difficulties which were only partially overcome during the course of the investigation. Thus, many of the conclusions are based on results with fairly crude preparations and relatively low yields. However, the results do provide some insight into the nature of ODCase, in particular, from this source. As was found for the erythrocyte enzyme (1 l), ODCase activity was associated with a number of species. In the case of the liver enzyme, these species corresponded to molecular weights of approximately 35,000, 63,000, and 105,000 and to a species eluting in the void volume from Sephadex G- 150. This correlates with an oligomeric series based on an enzymatically active ‘~monomer” of 35,000 molecular weight. The proportion of the different forms observed experimentally appeared to depend on a number of factors, including the mode of storage of the tissue, the ionic strength, and the presence of sulfhydryl agents and of UMP, the product of the reaction. Extrapolating from our experimental findings, the most probable in viva form would be that of MW 63,000. This was the form obtained in highest yield when purification was carried out on liver frozen in liquid air immediately after death. It was also the form in which optimal activities of OPRTase were observed. The 105,000 MW form, which was associated with much lower yields of activity, was always observed in the presence of monomer, suggesting that some molecular weight changes had already started in the liver during storage at -20”. Dissociation of the 63,000 MW form, which resulted in significant loss of activity, occurred in conditions of low ionic strength. Reaggregation could not be produced by increasing the ionic strength alone. However, it did occur after incubation with UMP, the product of the enzymic reaction, provided a thiol was present, implying the presence of free sulfhydry1 groups as a prerequisite for aggregation. Other inhibitors of the enzyme did not produce aggregation of the low molecular weight form but were able to stabilize it against heat inactivation. The possibility that the enzyme substrate, OMP, could also cause aggregation could not be tested unequivocally. Aggregation above the 63,000 MW form appeared to involve several factors, one of which could be polymerization due to disulfide bond formation, since spontaneous aggregation occurred in preparations not containing a thiol. The higher aggregates showed much greater stability to cold storage and heat inactivation than did the monomer. The kinetic and stability properties of the enzyme eluting at the void volume and of the
HUMAN
LIVER
OROTIDYLATE
DECARBOXYLASE
139
105,000 MW forms appeared identical to each other, in contrast to the similar forms observed in hemolysates where the K, was found to decrease with increasing molecular weight (11). We cannot eliminate the possibility that interconversion between the two high molecular weight forms from liver occurred during incubation in the assay medium. The overall pattern of interconversion of the enzyme forms observed for human liver decarboxylase shows many similarities to that found for the erythrocyte decarboxylase. The latter enzyme was found to exist in at least three forms, associated with OPRTase activity, of molecular weights 62,000, Il5,000, and 250,000 (corresponding to monomer, dimer, and tetramer) (11). In this case, aggregation of the 62,000 MW form was promoted by a wide range of compounds, including erotic acid and a number of competitive inhibitors of the decarboxylase. Recently, evidence has been obtained for ODCase, free of OPRTase, with a MW of 35,000-40,000.4 It seems reasonable to suppose that this form of the enzyme corresponds to the 35,000 MW form from human liver. OPRTase from human liver eluted on Sephadex G-150 with the same elution volume as the 63,000 MW form of ODCase. However, unlike the decarboxylase, the transferase lost large amounts of activity during chromatography, even in this molecular weight form. Furthermore, any conditions which led to an alteration in the molecular weight of the ODCase also caused a rapid, and almost total, loss of the OPRTase activity. The observations show a close physical relationship between the two enzyme activities but do not distinguish the possibilities of one or two separate proteins being involved. The two activities are associated with separable enzymes in calf thymus (1) and mouse leukemia cells (18), but separation of the two enzymes invariably led to large losses of the transferase activity. For the human liver, it might be suggested that a twoenzyme complex exists in which the stability and activity of the transferase is intimately dependent on the integrity of the decarboxylase dimer. Thus, UMP (or perhaps OMP) would have a critical role in maintaining the stability of the complex. Factors which alter the binding of UMP to the complex, or otherwise interfere with the stability of this complex, may have considerable physiological significance in view of recent suggestions that OPRTase represents a locus of control of pyrimidine biosynthesis in mammals (3,191. SUMMARY
Orotidylate decarboxylase activity from human liver has been demonstrated to be associated with a number of molecular weight species. 4 G. K. Brown
and W. J. O’Sullivan,
unpublished
observations.
I 40
CAMPBELL.
GALL.4GHER
.4ND
O‘SUI.LIV.4N
Depending on ionic strength. the presence of thiols and storage condition of the liver, variable proportions of forms with molecular weights of 35,000,63,000, and 105,000 could be separated on Sephadex G-150. Fresh liver, which had been frozen at -7o”, gave predominantly the 63.000 form. which was considered to be the most likely form to occur itI \silvj. This form could be dissociated to the 35,000 form by low ionic strength and aggregated to a much higher MW form in the absence of thiols. The dissociation could be reversed by incubation with UMP. Orotate phosphoribosyltransferase co-eluted from Sephadex G- 150 with the 63,000 form of the decarboxylase. Its activity was much more labile than the decarboxylase and was completely lost during any interconversions. ACKNOWLEDGMENTS We thank Mr. C. S. Lee for his assistance with some of these experiments. The study was supported by the Bushells Trust Fund of the A. W. Morrow Gastroenterology Unit. Royal Prince Alfred Hospital, Sydney and by the National Health and Medical Research Council of Australia.
REFERENCES I. Kasbekar. D. K., Nagabhushanam. A., and Greenberg, D. M..J. Biol. Chem. 239,4245 (1964). 2. Appel, S. H., J. Bid. Chem. 243, 3924 (1968). 3. Shoaf, W. T., and Jones, M. E.. Biochemistry 12, 4039 (1973). 4. Fox, R. M., Wood, M. H., and O’Sullivan. W. J., J. C/in. Inr~rsr. 50, 1050 (1971). 5. Brown, G. K.. Fox, R. M.. and O’Sullivan, W. J.. Biochem. Pharmcrcol. 21. 2469 (1972). 6. Cornell, R. C., Milstein, H. G.. and Fox. R. M., Arch. Dernmrol.. December 1976. 7. Smith, L. H.. Jr., Huguley, C. M., Jr., and Bain, J. A., irr “The Metabolic Basis of Inherited Disease” (J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, Eds.). 3rd ed.. p. 1003. McGraw-Hill, New York, 1972. 8. Pinsky. L., and Krooth, R. S.. Proc. Nut. Acad. Sci. USA 57, 925 (1967). 9. Pinsky, L., and Krooth. R. S.. Proc. Not. Acud. Sci. USA 57. 1267 (1967). IO. Krooth, R. S., Lam. G. F. M., and Chen-Kiang, S. Y., Cell 3, 55 (1974). 1I. Brown, G. K.. Fox, R. M.. and O’Sullivan, W. J.. J. Bid. Chem. 250, 7352 (1975). 12. Grobner, W.. and Kelley, W. N., Biochem. Pharmncd. 24, 379 (1975). 13. Kuby, S. A., Noda, L., and Lardy, H. A., J. Bid. Chem. 209, 191 (1954). 14. Umezu, K.. Amaya. T.. Yoshimoto. A., and Tomita, K., J. Biochem. (Japan) 70, 249 (1971). 15. Dawson, R. M. C., Elliott, D. C., Elliott. W. H., and Jones, K. M., Eds., “Data for Biochemical Research,” 2nd ed.. p. 625. Clarendon Press. Oxford. 1Y6Y. 16. Creasey. W. A., and Handschumacher, R. E.. J. Biol. Chem. 236, 2058 (1961). 17. Brown, G. K.. TheOrotate Phosphoribosyltransferase-Orotidylate Decarboxytase Complex from Human Erythrocytes. Ph.D. thesis. Sydney University. 1976. 18. Reyes, P., and Guganig, M. E.. J. Bid. Chem. 250, 5097 (1975). 19. Hoogenraad, N. J.. and Lee. D. C.. J. Biol. Chem. 249, 2763 (1974).
MEDICINE.
17, I?x-I-t() 11977,
Multiple Molecular Decarboxylase MARY
T. CAMPBELL,’ AND WILLIAM
Departmenr
of Medicine, Ro.val
Forms of Orotidylate from Human Liver NEIL
D. GALLAGHER,
J. O’SULLIVAN’
Universitp of Sydney Prince Alfred Hospital.
and Deportment of Gasrroenreroiogy, Sydne!. Alrstraliu
Received July 14, 1976
INTRODUCTION
Uridine-5’-monophosphate (UMP)3 is synthesized from erotic acid by two enzymic steps in de ~OVO pyrimidine biosynthesis: the conversion of erotic acid to orotidine-5’-monophosphate (OMP) by orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10) and decarboxylation of OMP to form UMP by orotidylate decarboxylase (ODCase; EC 4.1.1.23). These enzymes appear to be very closely associated in mammalian systems. Their activities remain linked during a variety of purification procedures (l-3) and also following alteration in their activity as a result of therapy with purine and pyrimidine antimetabolites (4-6). In addition, they are both grossly deficient in hereditary erotic aciduria Type I (7), a potentially lethal inborn error of metabolism in man. Investigation of these two enzymes in man has been effectively limited to erythrocytes, where the de no~o pyrimidine pathway is residual (7), and to diploid fibroblast cultures (8-10). In the present paper, we report some preliminary findings on OPRTase and ODCase in human liver, a tissue in which both enzymes remain functional. In particular, the decarboxylase, which is the more stable of the two enzymes, was found to occur in several oligomeric forms, interconversion of which was affected by ionic strength, the presence of thiols, and UMP. Some comparisons ’ Present address: Laboratory of Liver Physiopathology, Rega Institute, Minderbroedersstraat 10, 3000-Leuven, Belgium. * Present address: School of Biochemistry. University of New South Wales, Kensington, N.S.W. 2033, Australia. 3 Abbreviations used: UMP, uridine-5’-monophosphate: OMP, orotidine-S’-monophosphate; OPRTase, orotate phosphoribosyltransferase; ODCase, orotidylate decarboxylase; XMP, xanthosine monophosphate; PRPP, phosphoribosylpyrophosphate: HEPES, N-2hydroxyethylpiperazine-N’-2-ethane sulfonic acid; MOPS, morpholinopropanesulfonic acid; EGTA, ethyleneglycol-bis(&aminoethyl ether)-N.N’-tetraacetic acid. I28 ISSN 0OW?YJ4
HUMAN
LIVER
OROTIDYLATE
are made with similar oligomeric (11.12). MATERIALS
DECARBOXYLASE
129
forms derived from human erythrocytes AND METHODS
Materials
Orotic acid, UMP, XMP, AMP, CMP, and tetrasodium phosphoribosylpyrophosphate (PRPP) were purchased from Sigma Chemical Company; 6-azaUMP, OMP, HEPES (N-2-hydroxyethylpiperazine - N’ 2 - ethanesulfonic acid) and MOPS (morpholinopropane-sulfonic acid) were from Calbiochem Pty. Ltd. [Carboxy - ‘*C]orotic acid and [carboxy‘*ClOMP were obtained from New England Nuclear Corporation. Oxipurinol [4,6 - dihydroxypyrazolo(3,4 - d)pyrimidine] was a gift from Welicome (Australasia) Pty. Ltd. while Sephadex G-25 and G-150 were obtained from Pharmacia Fine Chemicals. Bovine serum albumin (Fraction V) was from Armour Pharmaceutical Company, pepsin and cytochrome c were from Sigma, catalase was from Calbiochem, and soybean trypsin inhibitor was from Worthington Biochemical Corporation. Creatine kinase was prepared from rabbit muscle by the method of Kuby et al. (13) and yeast ODCase was prepared by a modification of the method of Umezu et al. (14). All other materials were of analytical reagent grade. Enzyme
Preparation
During the early stages of this work, human liver was obtained from Royal Prince Alfred Hospital, Sydney, as soon as possible after death (within 2 hr), immediately frozen, and stored at -20” until required. At a later stage, liver was obtained from kidney donors immediately after death, rapidly frozen, and stored in liquid air. Portions of liver were homogenized in 4 or 6 vol of a buffer containing 20 mM HEPES-KOH, 0.15 M KCl, 0.2 mM EDTA, and 0.1 mM dithiothreitol, pH 7.2. Homogenization was carried out in a Sorvall Omnimix for 5 mm at 0” and the homogenate was centrifuged at 25,OOOg for 1 hr. Protamine sulfate in the same buffer solution was added dropwise to the supernatant to give a final concentration of O.l%, equilibrated for 5 min, and centrifuged. The clear supernatant was brought to 35% saturation with finely ground ammonium sulfate and centrifuged, and the resulting supernatant was brought to 65% saturation. The pellet obtained after centrifugation was dissolved in 5 mM HEPES-KOH, 0.15 M KCl, 0.2 mM dithiothreitol, pH 7.2, and the volume was adjusted to 5% of the original supernatant volume. The fraction was either dialyzed overnight or desalted on Sephadex G-25. All fractions were immediately assayed for OPRTase and ODCase activities, then stored at -20” or in liquid air for further analysis.
130
CAMPBELL,
GALLAGHER
AND
O’SULLIVAN
Enzyme Assuys ODCase was assayed by the collection of ‘“CO, from [ccrrbo.r.v14C]OMP as described by Fox et al. (4), except that the assay buffer was 25 mM MOPS, pH 6.8. When assaying dilute fractions from gel filtration experiments, 150 pg of bovine serum albumin was included in the assay. Unless otherwise stated, all assays were carried out at 10 PM OMP concentration (specific activity, 2000-3000 dpm per nmole). OPRTase was assayed by the same method using [carboxy-i4C]orotic acid, with the OMP formed being immediately decarboxylated by endogenous or added yeast decarboxylase. The assay buffer was 50 mM Tris-HCl, pH 7.4, containing 3 mM MgC&, with an erotic acid concentration of 100 PM (specific activity, 950 dpm per nmole) and a PRPP concentration of 250 PM. For some gel filtration experiments purified yeast ODCase was included in the assay mixture at a level sufficient to decarboxylate 120 nmoles of OMP in 1 hr. In most other experiments the level of liver ODCase was sufficiently high to make further additions of yeast enzyme unnecessary. Protein concentrations were determined by absorbance at 280 and 260 nm (15). Gel Filtration
Experiments
Changes in the aggregation state of the enzyme were assessed by chromatography on Sephadex G-150 columns (1.5 x 70 cm) at 4”. The sample volume was l-2 ml and the flow rate was 8-10 ml per hr. The columns were calibrated with catalase (240,000 MW), creatine kinase (83,000), pepsin (35,000), soybean trypsin inhibitor (21,500), and cytochrome c (12,200). In all chromatography experiments, a trace amount of blue dextran was included in the sample in order to accurately locate the void volume. Rechromatography experiments, which generally involved very low concentrations of protein, were carried out in the presence of bovine serum albumin (0.5 mg/ml). RESULTS
Enzyme Preparation ODCase and OPRTase were present in both particulate and soluble fractions of human liver. Approximately 45% of both enzyme activities was present in the 25,OOOg supernatant after the livers had been frozen at -20”. This value was increased only slightly (to 55%) by including 1% Triton X-100 in the homogenizing medium. However, rapid freezing of liver in liquid air released 80-85% of both enzyme activities into the supernatant fraction. The subcellular location of the particulate activity
HUMAN
LIVER
OROTIDYLATE
131
DECARBOXYLASE
was not determined since fresh livers suitable for cell fractionation studies were not available. The ODCase was found to be stable in crude supernatants for up to 3 weeks when stored at 0” but lost 20-30% of its activity when stored at -20”. The OPRTase was much more unstable at 0” and had lost 50% of its activity after 1 week at -20”. The conditions under which the liver was originally stored also considerably affected the level of enzyme activity. For livers frozen in liquid air immediately after death, the soluble decarboxylase activity was 17-21 nmoles of UMP formed per hour per milligram of protein, while the activity in livers frozen at -20” was l-5 nmoles per hour per milligram of protein. Only part of this difference could be attributed to the greater solubilization of enzyme from liver stored in liquid air. The two enzymes showed parallel behavior during purification, until dialysis (Table 1). At this stage, 40% of the OPRTase activity was lost when dialyzed against buffered 0.15 M KCl. If the extracts were dialyzed or desalted in low ionic strength buffer (25 mM KCl), 85-90% of the OPRTase activity was lost immediately and the remaining activity was lost within the first few days of storage at -20”. Under similar conditions of low ionic strength, only 20% of the decarboxylase was lost. In the final preparations made under optimum conditions (0.15 M KCl, 0.2 mM dithiothreitol) both enzyme activities remained stable when stored in liquid air for several weeks or at -20” for several days. TABLE PREPARATION
OF OROTIDYLATE
PHOSPHORJBOSYLTRANSFERASE
FROM
Protein Fraction Cell supematant Protamine supernatant Ammonium sulfate (35-65%) Dialysis
1
DECARBOXYLASE
AND
HUMAN
OROTATE
LIVERY
ODCase
OPRTase
mg/ml
Total mg
Units/ m
Total units
Units/ mg
Total units
20 10
510 260
17 34
8670 8910
7 14
3510 3540
46 29
116 105
55 54
6414 5740
23 16
2650 1640
a ODCase and OPRTase were prepared as described in Materials and Methods from fresh human liver rapidly frozen in liquid air. All fractions were assayed for both enzyme activities immediately after preparation. ODCase was assayed at 25 PM OMP ([carboxy-W]OMP: specific activity, 2000 dpm/nmole), and OPRTase was assayed at 100 PM erotic acid ([carboxy-i4C]orotic acid; specific activity, 950 dpm/nmole). One unit of enzyme activity is defined as the formation of 1 nmole product in 1 hr at 37”.
132
('AMPHELL.GALLAGHER
AND O'SULLIVAN
The activity of ODCase was unaffected over the pH range 6.0-7.4 when preincubated at these values for 30 min at 37”, but was rapidly inactivated at pH values above 8.0. The optimum pH for enzyme activity was found to be 6.7 at 37” using a Tris-maleate-glycine buffer over a pH range of 5.0-9.0. The following compounds were without significant effect on the decarboxylase activity when included in the incubation medium: EDTA, EGTA. MgCl, (all at 1 and 10 mM); chloride salts of Ca2+, Co*+. and Mn”+: erotic acid. PRPP, dithiothreitol, N-ethylmaleimide (all at 0.1 and 1.0 mM): and oxipurinol (0.1 mM). The sulfate salts of Cu2+ and Zn’+ produced 90% inhibition at 0.1 mM concentration and inorganic phosphate, UMP, XMP, and 6-azaUMP were also inhibitory (see below). Gel Filtration
on Sephadex G-150
Chromatography of the partially purified ODCase on Sephadex G-150 gave rise to more than one peak of activity. The relative amounts of the various molecular weight forms varied according to the history of the stored liver. Liverfrozen in liquid air. Elution profiles of ODCase and OPRTase on Sephadex G-150 are shown in Fig. 1A. Both enzyme activities eluted as single symmetrical peaks with the same elution volume, corresponding to a MW of 63,000. Recovery of activity was 60-70% for the decarboxylase and 25% for the transferase. Upon rechromatography under the same buffer conditions, 80% of the decarboxylase activity was recovered and the elution profile remained unchanged even after storage for at least 6 weeks in liquid air. It was not possible to confirm this for the OPRTase, as sufficient activity did not survive rechromatography. While a similar elution profile was also obtained for ODCase if the initial chromatography was carried out in low ionic strength buffer (25 mM KC1 instead of 0.15 M KCl) with or without dithiothreitol present, the elution profile of the enzyme isolated under these conditions was found to change considerably during subsequent storage and rechromatography. First, after storage for 24 hr in buffered 25 mM KC1 without dithiothreitol, both aggregation and dissociation were observed. Half the ODCase eluted with a MW of approximately 35,000 and the rest eluted at the void volume, with only a trace of the original 63,000 MW form (Fig. 1B). This changed profile was accompanied by a loss of more than 70% of the ODCase activity, while OPRTase activity was completely lost. Second, if dithiothreitol was included in the low ionic strength buffer, dissociation was still observed but aggregation was not. Under these conditions, a variable amount of activity (40-N%) was recovered in the 35,000 MW form, while the rest remained in the 63,000 MW form. In this case, the dissociation was accompanied by a loss of half the total enzyme activity
HUMAN
LIVER OROTIDYLATE
DECARBOXYLASE
133
FIG. 1. Sephadex G-150 chromatography of ODCase and OPRTase from human liver frozen in liquid air. Ammonium sulfate extracts were prepared as described in Materials and Methods. (A) Elution protile on a Sephadex G-150 column (1.5 x 70 cm) of ODCase (04) and OPRTase (O---O) in 5 mM HEPESKOH, pH 7.2, containing 0.15 M KC1 and 0.2 mh4 dithiothreitol. The protein profile (monitored at 280 nm) is indicated by open circles (B) Elution profile of the peak Sephadex fractions after dialysis and rechromatog(0 -0). raphy in 5 mM HEPES-KOH, pH 7.2, containing 25 mM KC1 and no dithiothreitol. Only ODCase activity was detected. The sample volumes were 1 ml and the flow rate was 8-10 ml/hr. Fractions of 1.1 ml were collected and points are plotted for each two or three fractions. Recovery of activity was 60% for ODCase and 25% for OPRTase in experiment A and 27% for ODCase in experiment B. Elution was carried out at 0”. The void volume was determined with blue dextran. Inset: Plot of molecular weight as a function of elution volume for standard proteins (see Materials and Methods). The elution volumes of the 35,000 and 63,000 molecular weight forms of ODCase are indicated by arrows.
applied to the column. Increasing the ionic strength did not reverse this inactivation. Both the void volume form and the 35,000 MW form of the ODCase could be rechromatographed independently with no further change in the elution profiles. The void volume enzyme was stable to prolonged storage at -2O”, but the 35,000 MW form rapidly lost all activity unless stored in bovine serum albumin (0.5 mg/ml). Liver stored af -20”. With ODCase prepared from liver which had been stored at -2O”, half the activity eluted at the 35,000 MW position while
134
CAMPBELL>.
GAL.LAGHER
AND O’SULLIVAN
the rest eluted in a broad peak, the fractions containing maximum activity eluting with a MW of approximately 105,000 (Fig. 2). If these peak fractions were rechromatographed immediately. the elution volume was unaltered. However, following storage at -20” (in the absence of dithiothreitol). the 105,000 form gradually aggregated to the void volume enzyme over a period of 1-2 weeks. In contrast to the 63,000 form, there was no detectable dissociation during storage in low ionic strength buffer. The aggregation appeared to be greatly accelerated when the enzyme was prepared in 50 mM phosphate buffer, an inhibitor of the erythrocyte enzyme (II), instead of HEPES-KOH. Although the 105,000 MW form was predominant on the day of preparation, chromatography of the ammonium sulfate fractions on subsequent days showed an increasing aggregation to the void volume form until only a trace of the 105,000 MW form remained. Incubation of the 105,000 MW form with other inhibitors, GazaUMP, XMP, or oxipurinol (all at 0.1 mM) for 2 hr at 37”. also caused an accelerated aggregation to the void volume form. Behavior qf Various Forms of ODCase Heat stability. The void volume enzyme and the 105,000 MW enzyme were both relatively stable to mild heat treatment at 45” (Fig. 3). The 35,000 MW form was extremely unstable but could be stabilized by strong inhibitors. After incubation of aliquots of this form for 2 hr at 37” with XMP (0.1 mM) or 6-azaUMP (0.1 mM) and chromatography to remove the I '
40
60 Elutlon
80 Volume
100
-6
120
(ml)
FIG. 2. Sephadex G-150 chromatography of OMP decarboxylase from human liver stored at -20”. Ammonium sulfate extracts were prepared from liver stored at -20” and chromatographed on Sephadex G-150 as described in the legend to Fig. 1, except that a different column was used and the sample size was 2.5 ml. Fractions of 1.4 ml were collected and assayed for ODCase. Every alternate fraction is indicated (04). The protein profile is shown by the fine line (O-O). Recovery of activity was 35%. The void volume is indicated by k’,,.
HUMAN
? .Z -c ‘i ::L t
LIVER OROTIDYLATE
DECARBOXYLASE
135
40
20..
\ 0
: 10
20
;I 30
lime tmin) FIG. 3. Stability of OMP decarboxylase ohgomers at 45”. Preparations of the void volume enzyme (0) and the 105,000 (A) and 35,090 (0) molecular weight forms were obtained by gel filtration in 10 mM HEPES-KOH, pH 7.2, and 25 mM KCl. One-milliliter samples were incubated in a 45” water bath; aliquots were removed at the times indicated, rapidly cooled in ice, and kept at 0” until assayed. For assay of ODCase, the enzyme ahquots were brought to 37” in the incubation medium (3 min) before addition of substrate. The activity is expressed as a percentage of the initial activity of unincubated samples. Initial specific activities and protein concentrations, respectively, were: (i) void volume enzyme, 5.1 nmoles/mg of protein/hr (9 mg of protein/ml); (ii) 105,000 MW enzyme, 4.0 nmoles/mg of proteinihr (4.6 mg of protein/ml); (iii) 35,000 MW enzyme, 6.8 nmoles/mg of proteinihr (1 mg of protein/ml).
inhibitors, no loss of activity was observed and the molecular weight remained unaltered. In contrast, virtually all activity was lost after incubation in the absence of inhibitors (Fig. 3). Aggregation of the 35,000 MW enzyme. Incubation of the low molecular weight form of ODCase with UMP (0.5 mM), in the presence of 0.15 M KC1 and 10 mM dithiothreitol for 3 hr at 37”, caused aggregation to a 67,000 MW form with traces of higher aggregates (Fig. 4). No effect was observed with other inhibitors, such as 6-azaUMP (0.1 mM), XMP (0.1 mM), and inorganic phosphate (50 mM). In general, these experiments were hampered by the low activity of the 35,000 MW form, and bovine serum albumin (0.5 m&ml) was included in elution buffers in order to recover maximum activity. To attain reproducible aggregation of the decarboxylase, it was necessary to include both a high ionic strength and a thiol and to use freshly prepared enzyme. There was no detectable recovery of OPRTase in reaggregated ODCase preparations. Dissociation of the void volume enzyme. The void volume form of ODCase could be almost completely dissociated by mild treatment with guanidine-HCl. After incubation with 0.5 M guanidineHC1 for 5 min at O”, followed by immediate gel filtration, most of the recovered activity
136
CAMPBELL,,
GALLAGHER
Elution
volume
AND O’SULLIVAN
(ml)
FIG. 4. Aggregation of the 35,000 molecular weight form of ODCase by UMP. The 35,000 molecular weight fraction obtained by gel filtration was incubated with 0.5 mM UMP for 3 hr at 37’ in a buffer containing 10 mM HEPES-KOH, pH 7.2, 0.15 M KCI. 10 mM dithiothreitol, and 0.5 mg/ml of serum albumin. The sample, with a trace of blue dextran, was chromatographed on Sephadex G-150 using the same buffer, except that 1.0 mM dithiothreitol was used. Fractions were assayed for ODCase (04). The elution profile of control enzyme is included for comparison (O---e).
was present as the 35,000 MW form (Fig. 5). Only small amounts of activity remained at the void volume and also at positions corresponding to molecular weights of 110,000 and 63,000. The conditions used for this dissociation caused no significant loss of enzyme activity in the sample before chromatography. 12 -. ‘:
1
vo 1
35,000 I
0vI $
a--
r .Z .? 6 d i!
.‘:
4--
J
[
ri 0
30
50 Elution
70 Volume
90
110
(ml)
FIG. 5. Dissociation of the void volume form of ODCase by guanidineHC1. A fresh sample of the high molecular weight void volume enzyme was incubated in 0.5 M guanidine-HCI for 5 min at 0” and immediately applied to a Sephadex G-150 column. Fractions were assayed for OMP decarboxylase activity. Experimental conditions were as for Fig. 1. Recovery of activity was 42%.
HUMAN
LIVER
OROTIDYLATE
137
DECARBOXYLASE
A small amount of activity (lo-20%) was also consistently obtained in the 35,000 MW form after incubation of the void volume enzyme with 10 or 20 mM dithiothreitol for 2 hr at 37”. However, no change in the elution profile could be obtained by heat treatment alone or in the presence of phosphate or 6-azaUMP or by variation of the ionic strength or protein concentration. Kinetic properties. The individual high molecular weight aggregates of ODCase gave linear Lineweaver-Burk plots with K, values of 0.12 PM. Enzyme preparations containing a mixture of different aggregates gave a single linear response when tested over a wide range of OMP concentrations (0.07-10 PM), with K, values varying from 0.2 to 0.5 PM in different preparations. The 35,000 MW form gave nonlinear Lineweaver-Burk plots concave upward, in which enzyme activity fell rapidly at substrate concentrations lower than 0.5 PM. However, when bovine serum albumin (300 pg) was included during the assay, the plots approached linearity. This suggested that the concave plots were due to instability of the low MW enzyme at low substrate concentrations, as has been found by other workers (2,3,8). An accurate K, value could not be obtained for this form but it was estimated as approximately 0.3 PM. A number of compounds which have been shown to competitively inhibit erythrocyte ODCase was tested as inhibitors of the 105,000 MW form of the liver enzyme. Ki values are shown in Table 2 compared to values reported for the rat liver enzyme (16) and also with results for the 250,000 MW form of human erythrocyte ODCase (11,17).
TABLE INHIBITION
Inhibitor 6-azaUMP XMP UMP CMP AMP Phosphate
CONSTANTS
Human liver co.01 0.3 16 40
80 12,000
FOR
2 HUMAN LIVER ODCase” Rat livep 0.1
Human erythrocytesc 0.06 1.9 11
700
800
>50 22,000
a Inhibition constants obtained for the 105,000 MW form of the human liver enzyme are compared to values reported for rat liver (16) and for the 250,000 MW form of the human erythrocyte enzyme (11, 17). K, values for the human liver enzyme were determined from Dixon plots using varying concentrations of inhibitors at a fixed substrate concentration of 0.36 ~1M. K, for OMP in the same experiments was 0.12 FM. All Ki values are expressed as micromolar concentrations. b Creasey and Handschumacher (16). c Brown (17).
138
CAMPBELL,. GALLAGHER ANDO‘SULLIVAN DISCUSSION
Apart from the problem of obtaining sufficient suitable material, experiments with human liver ODCase and OPRTase were severely hampered by the high lability of both enzymes, in particular the OPRTase, difficulties which were only partially overcome during the course of the investigation. Thus, many of the conclusions are based on results with fairly crude preparations and relatively low yields. However, the results do provide some insight into the nature of ODCase, in particular, from this source. As was found for the erythrocyte enzyme (1 l), ODCase activity was associated with a number of species. In the case of the liver enzyme, these species corresponded to molecular weights of approximately 35,000, 63,000, and 105,000 and to a species eluting in the void volume from Sephadex G- 150. This correlates with an oligomeric series based on an enzymatically active ‘~monomer” of 35,000 molecular weight. The proportion of the different forms observed experimentally appeared to depend on a number of factors, including the mode of storage of the tissue, the ionic strength, and the presence of sulfhydryl agents and of UMP, the product of the reaction. Extrapolating from our experimental findings, the most probable in viva form would be that of MW 63,000. This was the form obtained in highest yield when purification was carried out on liver frozen in liquid air immediately after death. It was also the form in which optimal activities of OPRTase were observed. The 105,000 MW form, which was associated with much lower yields of activity, was always observed in the presence of monomer, suggesting that some molecular weight changes had already started in the liver during storage at -20”. Dissociation of the 63,000 MW form, which resulted in significant loss of activity, occurred in conditions of low ionic strength. Reaggregation could not be produced by increasing the ionic strength alone. However, it did occur after incubation with UMP, the product of the enzymic reaction, provided a thiol was present, implying the presence of free sulfhydry1 groups as a prerequisite for aggregation. Other inhibitors of the enzyme did not produce aggregation of the low molecular weight form but were able to stabilize it against heat inactivation. The possibility that the enzyme substrate, OMP, could also cause aggregation could not be tested unequivocally. Aggregation above the 63,000 MW form appeared to involve several factors, one of which could be polymerization due to disulfide bond formation, since spontaneous aggregation occurred in preparations not containing a thiol. The higher aggregates showed much greater stability to cold storage and heat inactivation than did the monomer. The kinetic and stability properties of the enzyme eluting at the void volume and of the
HUMAN
LIVER
OROTIDYLATE
DECARBOXYLASE
139
105,000 MW forms appeared identical to each other, in contrast to the similar forms observed in hemolysates where the K, was found to decrease with increasing molecular weight (11). We cannot eliminate the possibility that interconversion between the two high molecular weight forms from liver occurred during incubation in the assay medium. The overall pattern of interconversion of the enzyme forms observed for human liver decarboxylase shows many similarities to that found for the erythrocyte decarboxylase. The latter enzyme was found to exist in at least three forms, associated with OPRTase activity, of molecular weights 62,000, Il5,000, and 250,000 (corresponding to monomer, dimer, and tetramer) (11). In this case, aggregation of the 62,000 MW form was promoted by a wide range of compounds, including erotic acid and a number of competitive inhibitors of the decarboxylase. Recently, evidence has been obtained for ODCase, free of OPRTase, with a MW of 35,000-40,000.4 It seems reasonable to suppose that this form of the enzyme corresponds to the 35,000 MW form from human liver. OPRTase from human liver eluted on Sephadex G-150 with the same elution volume as the 63,000 MW form of ODCase. However, unlike the decarboxylase, the transferase lost large amounts of activity during chromatography, even in this molecular weight form. Furthermore, any conditions which led to an alteration in the molecular weight of the ODCase also caused a rapid, and almost total, loss of the OPRTase activity. The observations show a close physical relationship between the two enzyme activities but do not distinguish the possibilities of one or two separate proteins being involved. The two activities are associated with separable enzymes in calf thymus (1) and mouse leukemia cells (18), but separation of the two enzymes invariably led to large losses of the transferase activity. For the human liver, it might be suggested that a twoenzyme complex exists in which the stability and activity of the transferase is intimately dependent on the integrity of the decarboxylase dimer. Thus, UMP (or perhaps OMP) would have a critical role in maintaining the stability of the complex. Factors which alter the binding of UMP to the complex, or otherwise interfere with the stability of this complex, may have considerable physiological significance in view of recent suggestions that OPRTase represents a locus of control of pyrimidine biosynthesis in mammals (3,191. SUMMARY
Orotidylate decarboxylase activity from human liver has been demonstrated to be associated with a number of molecular weight species. 4 G. K. Brown
and W. J. O’Sullivan,
unpublished
observations.
I 40
CAMPBELL.
GALL.4GHER
.4ND
O‘SUI.LIV.4N
Depending on ionic strength. the presence of thiols and storage condition of the liver, variable proportions of forms with molecular weights of 35,000,63,000, and 105,000 could be separated on Sephadex G-150. Fresh liver, which had been frozen at -7o”, gave predominantly the 63.000 form. which was considered to be the most likely form to occur itI \silvj. This form could be dissociated to the 35,000 form by low ionic strength and aggregated to a much higher MW form in the absence of thiols. The dissociation could be reversed by incubation with UMP. Orotate phosphoribosyltransferase co-eluted from Sephadex G- 150 with the 63,000 form of the decarboxylase. Its activity was much more labile than the decarboxylase and was completely lost during any interconversions. ACKNOWLEDGMENTS We thank Mr. C. S. Lee for his assistance with some of these experiments. The study was supported by the Bushells Trust Fund of the A. W. Morrow Gastroenterology Unit. Royal Prince Alfred Hospital, Sydney and by the National Health and Medical Research Council of Australia.
REFERENCES I. Kasbekar. D. K., Nagabhushanam. A., and Greenberg, D. M..J. Biol. Chem. 239,4245 (1964). 2. Appel, S. H., J. Bid. Chem. 243, 3924 (1968). 3. Shoaf, W. T., and Jones, M. E.. Biochemistry 12, 4039 (1973). 4. Fox, R. M., Wood, M. H., and O’Sullivan. W. J., J. C/in. Inr~rsr. 50, 1050 (1971). 5. Brown, G. K.. Fox, R. M.. and O’Sullivan, W. J.. Biochem. Pharmcrcol. 21. 2469 (1972). 6. Cornell, R. C., Milstein, H. G.. and Fox. R. M., Arch. Dernmrol.. December 1976. 7. Smith, L. H.. Jr., Huguley, C. M., Jr., and Bain, J. A., irr “The Metabolic Basis of Inherited Disease” (J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, Eds.). 3rd ed.. p. 1003. McGraw-Hill, New York, 1972. 8. Pinsky. L., and Krooth, R. S.. Proc. Nut. Acad. Sci. USA 57, 925 (1967). 9. Pinsky, L., and Krooth. R. S.. Proc. Not. Acud. Sci. USA 57. 1267 (1967). IO. Krooth, R. S., Lam. G. F. M., and Chen-Kiang, S. Y., Cell 3, 55 (1974). 1I. Brown, G. K.. Fox, R. M.. and O’Sullivan, W. J.. J. Bid. Chem. 250, 7352 (1975). 12. Grobner, W.. and Kelley, W. N., Biochem. Pharmncd. 24, 379 (1975). 13. Kuby, S. A., Noda, L., and Lardy, H. A., J. Bid. Chem. 209, 191 (1954). 14. Umezu, K.. Amaya. T.. Yoshimoto. A., and Tomita, K., J. Biochem. (Japan) 70, 249 (1971). 15. Dawson, R. M. C., Elliott, D. C., Elliott. W. H., and Jones, K. M., Eds., “Data for Biochemical Research,” 2nd ed.. p. 625. Clarendon Press. Oxford. 1Y6Y. 16. Creasey. W. A., and Handschumacher, R. E.. J. Biol. Chem. 236, 2058 (1961). 17. Brown, G. K.. TheOrotate Phosphoribosyltransferase-Orotidylate Decarboxytase Complex from Human Erythrocytes. Ph.D. thesis. Sydney University. 1976. 18. Reyes, P., and Guganig, M. E.. J. Bid. Chem. 250, 5097 (1975). 19. Hoogenraad, N. J.. and Lee. D. C.. J. Biol. Chem. 249, 2763 (1974).
