ARCHIVES
OF
BIOCHEMISTRY
Purification,
AND
175, 270-278 (1976)
Composition, and Some Properties of Rat Liver Carbamyl Phosphate Synthetase (Ammonia)l LUISA
Department
BIOPHYSICS
ofBiochemistry,
RAWMAN School
of Medicine, Angeles,
AND MARY University California
ELLEN
ofsouthern 90033
JONES California,
2025 Zonal
Avenue,
Los
Received December 8, 1975 A procedure for the purification of rat liver carbamyl phosphate synthetase (ammonia) to homogeneity is described. The molecular weight of isolated active enzyme is 222,000 -t10,000; that of the subunits obtained by denaturation with sodium dodecyl sulfate in the presence of dithiothreitol is 120,000 + 5,000. The enzyme appears to be composed of two subunits of identical size. The amino acid composition of the rat liver enzyme is reported and shown to be very similar to that of the bovine enzyme. The concentration of carbamyl phosphate synthetase I in the matrix of rat liver mitochondria is about 5 x 10e4 M, very similar to that of acetylglutamate, on this basis, it is suggested that one-third to one-half of the total enzyme may be in the active form in the matrix. Calculations also indicate that normally, ATP is unlikely to limit the rate of carbamyl phosphate synthesis in vivo. Carbamyl phosphate synthetase I appears to constitute 22 to 26% of the total matrix protein of liver mitochondria; the significance of this fact is briefly examined.
Although the ammonia-dependent carbamyl phosphate synthetase of rat liver (carbamyl phosphate synthetase I) has been purified extensively and recently has been the object of physical studies, data on the molecular weight of the active protein vary widely (1, 2), and no information is available on the composition of the enzyme or the size of the subunits obtained by exhaustive denaturation with sodium dodecyl sulfate in the presence of dithiothreitel. In addition, little is known about the regulation of carbamyl phosphate synthetase I; the search for effecters has been unsuccessful except for the activation of this enzyme by acetylglutamate, and the kinetics under conditions approaching those in uiuo have not been studied. ‘I’herefore, the role that carbamyl phosphate synthetase I may play in the changes in the rate of urea synthesis observed in uiuo, in perfused liver, and in isolated liver cells cannot be evaluated.
We carried out this study to clarify and extend the physical and chemical characterization of rat liver carbamyl phosphate synthetase I and to begin to study its activity under conditons relevant to metabolism. MATERIALS
Materials. Male rats of the Sprague-Dawley strain, weighing approximately 150 g, were purchased from Zivic Miller Laboratories, Inc., Allison Park, Pennsylvania. Controlled pore glass was obtained from the Sigma Chemical Co. Dithiothreitol: glycylglycine, crystalline bovine plasma albumin, and ovalbumin were purchased from Calbiochem; bovine pancreas trypsin type I, urease type VI, and beef liver catalase (lO,OOO-25,000 units/mg) were from Sigma Chemical Co.; ornithine transcarbamylase (EC 2.1.3.3) was prepared as described by Nakamura and Jones (3); dilithium carbamyl phosphate, potassium or tricyclohexylammonium phosphoenolpyruvate, and rabbit muscle lactate dehydrogenase, pyruvate kinase, and adenylate kinase were from 3 Abbreviations used: Dl”l’, dithiothreitol; AGA, acetylglutamic acid; CAP, carbamyl phosphate; CTAB, cetyltrimethylammonium bromide; CPSase I, carbamyl phosphate synthetase (ammonia), EC 2.7.2.5; DEAE, diethylsminoethyl.
‘This work was supported by Grant No. HD 06538 from the National Institutes of Health. e To whom all correspondence should be addressed. 270 Copyright 8 1976 by Academic F’reas, Inc. All rights of reproduction in any form reserved.
AND METHODS
RAT LIVER
CARBAMYL
PHOSPHATE
Boehringer Mannheim; ultrapure ammonium sulfate and sodium dodecyl sulfate, bromophenol blue, and Coomassie brilliant blue were from Mann Research Laboratories; acrylamide, methylenebisacrylamide, N,h’,N’,N’-tetramethylethylenediamine, P-mercaptoethanol, and butanedione-2-monoxime were from Eastman Organic Chemicals. Other reagents were commercial products of analytical quality. Enzyme assays. For the assay of CPSase I, all acidic reagents were neutralized with KOH. A colorimetric assay (1) based on the conversion of CAP to citrulline was used unless stated otherwise. Samples of CPSase I were preincubated for 2 min at 37°C in a reaction mixture containing: glycylglycine, pH 7.6, 50 pmol; NHIHCOI, 50 pmol; MgSO,, 15 wmol; acetylglutamate, 5 pmol; L-ornithine, 5 pmol; ornithine transcarbamylase, 10 to 20 units. The reaction was started by addition of 5 pmol of ATP in 0.1 ml. The final volume was 1 ml, and the incubation was at 37°C for 15 min; the reaction was stopped with 0.2 ml of 5 M HClO,. A unit in this assay is the amount of enzyme that synthesizes 1 pmol of CAP (measured a8 citrulline) per minute at 37°C. Specific activity is the number of units per milligram of protein. A spectrophotometric assay based on the coupling of CPSase I activity to the oxidation of NADH was also used (1). One milliliter of reaction mixture contained: glycylglycine, pH 7.6, 50 pmol; KHCOI, 50 pmol; (NH&SO,, 35 kmol; MgSO,, 15 pmol; acetylglutamate, 10 pmol; ATP, 1.7 pmol; phosphoenolpyruvate, 2.5 pmol; NADH, 0.5 pmol; pyruvate kinase, 40 pg; lactate dehydrogenase, 200 pg; adenylate kinase, 25 pg. The reaction was started by addition of CPSase I. The concentration of some reagents was sometimes changed and will be indicated. Ornithine transcarbamylase activity at pH 7.5 was measured as previously described (4). Determination of protein. D’Pl interferes with measurements of protein absorbance at 260 and 280 nm (the oxidized derivative of D’IT absorbs light of those wavelengths) and by the method of Lowry et al. (5). Routine measurements were done by ultraviolet absorbance, using as blanks samples of the solvent prepared at the same time as those containing the enzyme and kept under similar conditions. Accurate measurements were carried out by the method of Lowry et al. (5) with dialyzed enzyme samples. Electrophoresis. Analytical disc gel electrophoresis was carried out as previously described (6-8) with bromophenol blue as the marker; 2 mA per tube was used. The gels were fixed in 12.5% trichloroacetic acid and stained for protein with Coomassie blue (9). Electrophoresis in the presence of sodium dodecyl sulfate was carried out by the method of Weber and Osborn (lo), using 5% polyacrylamide gels.
SYNTHETASE
(AMMONIA)
271
Glycerol and sucrose density gradient centrifigation. The method of Martin and Ames (11) was used for calculations of molecular weight. Eleven-milliliter linear gradients (lo-30% glycerol or lo-308 sucrose, in 0.01 M Tris-HCl, pH 7.25) were made with an ISCO Model 570 gradient former; centrifugation at 4°C 40,000 rpm, for 14 to 20 h was performed with a Beckman Model L2-65 B ultracentrifuge using a Beckman SW 41 rotor. Forty-five to sixty-four fractions were collected from the top of the gradient by means of an ISCO Model 183 density gradient fractionator. Amino acid analysis. Amino acid analyses were carried out by the method of Moore and Stein (12) after hydrolysis of the protein in 6 M HCl for 24,48, and 72 h. Serine and threonine values were corrected for decomposition by extrapolation to zero time. Half-cystine was measured as cysteic acid (13); tryptophan was measured by the method of Liu and Yang (14). Amide nitrogen was not determined. A Beckman Model 119 amino acid analyzer was used. Preparation of mitochondrin. Mitochondria were prepared as described by Myers and Slater (15) and used immediately. Preparation of antisera. Rabbit antisera to purified frog liver CPSase I and to crude fractions of rat liver CPSase I were obtained by subcutaneous injection of approximately 5 mg of protein in 1 ml of 0.15 M NaCl suspended in 1 ml of Freund’s adjuvant (0.5 ml injected per site). The rabbits were injected at least three more times at monthly intervals, each time using 1 mg of protein in NaCl, without Freund’s adjuvant. RESULTS
Stabilization
of CPSase I from rat liver.
A remarkable stabilization of this enzyme in solution had been achieved by the inclusion of KCN, mercaptoethanol, and 20% (v/v) glycerol into solutions of the protein (1). However, the dangerously large amounts of KCN at near-neutral pH required for medium scale purifications induced us to search for other stabilizing conditions. Glycerol (20%, v/v) in combination with D’IT stabilizes CPSase I to an extent which is a function of the amount of DTI’ added. At 4”C, 100% of the enzymatic activity is maintained after storage for 24 and 48 h in solutions containing 20% (v/v) glycerol containing 0.1 and 2 mM D!lT, respectively. At approximately 23°C 100 and 87% of the activity is recovered after storage for 24 and 48 h, respectively, in 20% (v/v> glycerol containing 2 mM DTI’. Throughout the purification procedure,
272
FLAIJMAN
AND
glycerol and various concentrations of D’IT were used as stabilizers of the enzyme. Whenever possible, the necessary amount of solid D’IT was added to all solutions immediately before use. Gel filtration and column chromatography were carried out at room temperature; all other procedures were carried out at 0-4°C. Purification: from rat liver.
Extraction
of CPSase
I
The following steps include several modifications of the method described by Guthohrlein and Knappe (1). Bat livers (150 g) were quickly washed with ice-cold distilled water, blotted, minced, and homogenized for 15 s with 450 ml of buffer I (0.02 M Tris-HCl, 20% (v/v) glycerol, and 1 mM D’IT, final pH 7.2) using a Waring Blendor at top speed. The homogenate was centrifuged at 15,OOOg, 4”C, for 20 min; the supernatant was discarded. The sediment was suspended in 900 ml of buffer I and was homogenized for 10 s and centrifuged as above; the supernatant was discarded. The sediment was suspended with a stirring rod in 900 ml of buffer I and centrifuged as above; the supernatant was discarded. The washed sediment was suspended in 375 ml of a buffer containing 0.02 M TrisHCl, 20% (v/v) glycerol, 2 mM DTI’, and 0.1% (w/v) CTAB; the suspension was homogenized with a Waring Blendor at top speed for 15 s. After centrifugation at 15,OOOg, 4”C, for 20 min, the supernatant was collected and the pellet was reextracted as described above. The combined supernatants had a volume of 890 ml. First
ammonium
sulfate
fractionation.
The CTAB extract was fractionated as previously described (l), except that only the fraction precipitating between 2.4 and 2.9 M ammonium sulfate was collected by suspending it in buffer B of Guthohrlein and Knappe (1). This suspension contained 1.43 g of protein in a volume of 62 ml; a 2ml sample was stored at this stage and the remainder was divided into three 20-ml portions which were stored at -20°C. Chromatography on DEAE-Sephadex and second ammonium sulfate fractionation. Each 20-ml portion of the ammonium
sulfate suspension (containing 460 mg of protein) was treated as follows: After stir-
JONES
ring at 0°C for 10 min, the suspension was centrifuged at 39,OOQg for 30 min; the supernatant was discarded. The precipitate was dissolved in 1 ml of a buffer containing 0.05 M Tris-HCl, 20% (v/v) glycerol, 0.2 nm D’IT, pH 7.2 (buffer II), plus 5 mM KCl. The rest of the procedure was as previously described (1) except that it was carried out at room temperature, buffer II was used instead of buffer A of Guthohrlein and Knappe (l), and the flow rate through the DEAE-Sephadex column was 18 ml/h. The fractions with specific activities between 1.13 and 1.6 (which were eluted -between 45 and 66 mM KCl) were pooled. The protein solution was made 2.4 M in ammonium sulfate and the resulting precipitate was discarded; the supernatant was made 3.1 M in ammonium sulfate and centrifuged. The precipitate was resuspended in a small volume of buffer B and stored at -20°C. This process was repeated (twice more) until all of the first ammonium sulfate fraction had been purified. The three pools of specific activity 1.13 to 1.6 were combined to yield a single enzyme pool which was stored at -20°C. Chromatography
on
controlled
pore
glass. A portion of the enzyme suspension from the previous step containing about 94 mg of protein was centrifuged and the precipitate was dissolved in 2 ml of buffer II plus 5 mM KCl. The solution was applied to a column (1.4 x 81 cm) of controlled pore glass (CPG-10, 2000 A pore size, 120-200 mesh), equilibrated at room temperature with the same buffer mixture; the flow rate was 24 ml/h. The column was eluted with buffer II plus 5 mM KC1; 4-ml fractions were collected. The protein appeared in a sharp peak, symmetrical except for a slight shoulder at the end of the descending limb; the shoulder contained less than 5% of the protein. The fractions containing CPSase I of specific activity 1.73 to 2 were pooled, solid ammonium sulfate was added to give a 3.1 M concentration, and the suspension was stored at -20°C. CPSase I binds tightly to the glass beads, if small amounts of the enzyme in dilute solutions are used, as much as 95%
RAT LIVER
CARBAMYL
PHOSPHATE
of the protein can remain attached to the beads and will not be eluted with high concentrations of salt, even at very high pH. Treatment of the glass column with bovine serum albumin prior to the addition of CPSase I did not prevent the binding of the latter; since all of the albumin added could be easily recovered by elution with buffer II plus 5 mu KCI, the high affinity of the glass beads for CPSase I appears to be a specific property. The recovery of enzyme was improved by applying the protein to the columns at high concentrations and by reusing the columns; the initial coating of the glass beads with CPSase I appeared to diminish further binding of this protein upon reusing the beads. Table I shows a summary of the puriilcation procedure. Analytical
SYNTHETASE
273
(AMMONIA)
sis of the purest fractions (50-100 pg/gel) was also done at pH 3.8 (8) and at pH 9.5 (6), using gels of various pore sizes. Using approximately 7% polyacrylamide gels, only one broad band was observed, always near the origin of the running gel, regardless of the pH used. At pH 9.5, CPSase I migrated into 4 and 2.5% polyacrylamide gels; in the latter case, the enzyme migrated to the center of the gel in a narrow band. No other bands were observed. Samples of CPSase I dissolved in 0.05 M glycylglycine at pH 7.2, with and without 0.5 mM AGA, and stored at 5°C for 70 h, showed an additional band on electrophoresis at pH 8 using 7% gels (7). The new band was observed only under these conditions, was somewhat diffuse, less intense than the major band remaining at the origin of the gel, and was located at the center of the gel.
disc gel electrophoresis. The
procedure used for the preparation of polyacrylamide gels was as described by Davis (6); the samples were layered directly over the stacking gels. Electrophoresis at pH 8 of the second ammonium sulfate fractions, using 7% polyacrylamide gels (7) and 75 pg of protein in 0.1 ml, showed two minor bands which penetrated well into the gel, and a major, very diffise band (1) near the beginning of the running gel. Electrophoresis of the fractions obtained after controlled-pore glass chromatography gave only the major band, even when 150 pg of protein was applied. Since CPSase I essentially does not migrate under these conditions, electrophore-
Electrophoresis after treatment with sodium dodecyl sulfate. Samples of CPSase I
(0.3 mg; sp act, about 1.87) alone and with 25 pg each of bovine serum albumin, ovalbumin, and bovine trypsin in a buffer containing 0.01 M sodium phosphate, pH 7.0, 1% (w/v) sodium dodecyl sulfate, and 1 ~-IM D’IT (total volume 1 ml) were incubated at 37°C for 2 and 24 h. To 50-~1 portions of each mixture, 20 ~1 of glycerol and 50 ~1 of 0.01 M sodium phosphate, pH 7.0, and bromophenol blue were then added; 0.1 ml of these mixtures was applied to the gels and was subjected to electrophoresis for 3.5 h. Gels to which only CPSase I had been applied showed a single narrow band,
TABLE
I CPSase I Total protein Specific activity (mg)
PURIFICATION
Fraction Crude homogenate CTAB extract First ammonium sulfate fraction DEAE-Sephadex fraction’ Second ammonium sulfate fraction Controlled pore glass fraction
Total activity (units)
OF
1,410 1,240 1,020
39,000 5,000 1,430
250 150
173 100
84
45
m v?l”li? 600 890 62
ReGT”0 100 87.4 72.3
1.45 1.48
105 5
17.8 10.5
1.87
25
5.9
0.036 0.247 0.713
D From 460 mg (20 ml) of the preceding fraction. To calculate the overall recoveries from this fraction on, a factor of 3 must be used.
274
RAIJMAN
whether the protein had been incubated 2 or 24 h; Fig. 1 shows a plot of the relative mobility of CPSase I and standards. The calculated molecular weight of the enzyme subunits (10) was 115,000 to 125,000. Attempts at renaturing (16) failed, no activity was detectable after incubation of the subunits alone and with different combinations of AGA, ATP, and Mg2+ at 4 and 22°C. Density gradient centrifugation. Solutions containing 5 mg of CPSase I (sp act, about 1.67), 0.5 mg of lactate dehydrogenase, 1 mg of catalase, and 3 mg of urease in 0.01 M Tris-HCl, pH 7.2, in a volume of 0.5 ml were prepared immediately before use. Of the mixture, 200 to 300 ~1 was layered onto glycerol or sucrose gradients. The calculated molecular weight was 222,000 +10,000; the szo,W values ranged between 10.3 and 10.9 (11). Amino acid composition. Table II shows the amino acid composition of CPSase I expressed as moles of residues per mole of subunit. It was assumed that the subunits are identical, with a molecular weight of 121,000. The synthetase contains a large number of acidic residues, some of which would have been in the amide form in the native protein. The partial specific volume calculated from the amino acid composition is 0.737 cm3/g; the error introduced in this value by the lack of data on the glutamine and asparagine content of CPSase I is small (17). Reaction of CPSase I with antisera. The interaction of the purest fractions of rat liver CPSase I with antiserum to the frog
\
1
0.2
CPSase
04
I
0.6
0.6
MOBILITY
FIG. 1. The electrophoretic mobility of CPSase I after denaturation with sodium dodecyl sulfate in the presence of DTT.
AND JONES TABLE
II
AMINO
ACID COMPOSITION OF RAT AND BEEF LIVER CPSase I AND OF THE Q.TALYTIG SUBUNIT OF E. coli CARBAMYL PHOSPHATE SYNTHETASE Amino
acid
T
tat live] 1Rat liver1 :PSaee 1L XSaee I
(mol/
m3logl, -
Beef 1 E. C& liver” subunit I CPSaee I I
Residues per 100 residues T
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
69.6 20.3 40.7 106.8 57.3 80.5 111.2 55.7 79.3 82.6 30.7" 81.3 29.5 65.1 96.2 26.2 43.4 10.1
Total
086.5
residues
6.41 1.87 3.75
9.83 5.28 7.41 10.23 5.13 7.30 7.60 2.83c 7.48 2.72
5.99 8.85 2.41 4.00
0.93
8.38 2.55 5.31
9.60 5.11 5.62 10.32 4.70 7.46
7.97 1.63 7.35 1.43 5.82 8.78 2.66
3.98 1.33
5.09 1.41 6.43 9.10 5.64 4.37 12.06 5.00 8.21 10.53 1.66 8.26 2.58 6.13 7.54 2.34 3.22 0.40
a From Ref. (20). b From Ref. (22). c This value was calculated using the average ratio of “molar equivalents in oxidized samples/molar equivalents in unoxidized samples” of four nonsulfur-containing amino acids. Because the sample of oxidized material used was very small, the minimum and maximum ratios obtained varied considerably; the respective mo1/121,00@ values arrived at from the extreme ratios are 37 and 24.5, while the residues per 100 residues vary from 3.41 to 2.25.
liver enzyme was tested by immunodiffision using agar plates; a single precipitation band was obtained, indicative of crossreactivity between the enzyme from both species. This was confirmed by assaying the rat liver enzyme after preincubation with the frog antiserum; complete inbibition of activity was observed, but the concentrations of antiserum required were approximately 10 times greater than those required for complete inhibition of similar amounts of the frog liver enzyme. The purest fractions of rat liver CPSase I also gave a single precipitation band with no spurs when tested by immunodiffision
RAT
LIVER
CARBAMYL
PHOSPHATE
against antiserum to crude rat liver enzyme (first ammonium sulfate fraction). Effect of ornithine and aspartate on the activity of mitochondrial and purified CPSase 1. Ammonium sulfate suspensions of purified CPSase I were centrifuged and the precipitates were dissolved in 0.05 M glycylglycine, pH 7.6, 20% glycerol, and 0.5 mu DTT to give an enzyme concentration of about 2 mglml; these solutions contained at most 40 mM ammonium sulfate. Small aliquots of these solutions (not greater than 50 ~1) were used in the following experiments. The effect of ornithine on the activity of CPSase I could only be studied with the purified enzyme because mitochondria contain ornithine transcarbamylase which would utilize ornithine stoichiometrically with the CAP produced by the CPSase I; the optical assay of CPSase I was used. At saturating substrate concentrations for CPSase I (those of the optical assay as described in Methods), 33 PM ornithine had no effect on the enzymatic activity; 0.3 ~-IM and 1.7 mM ornithine increased activity by 20 and 14%, respectively. At limit+ ing concentrations of KHCO, (16.7 mu) and AGA (0.33 and 1 mu), with 11.7 mM added (NH&SO,, a 10% activation by 0.33 and 1.7 mM ornithine was the only effect noticed. The calorimetric assay was used in most experiments designed to study the effect of aspartate on CPSase I; since this assay is coupled to ornithine transcarbamylase, the effect of aspartate on this enzyme was studied first. At several concentrations between 0.5 and 10 mM, aspartate had no effect on mitochondrial ornithine transcarbamylase, either at saturating substrate concentrations (4) or with limiting ornithine (1 and 2 mM). Aspartate had no effect on mitochondrial CAP synthesis at saturating substrate concentrations. At limiting HC03(10 mM) and AGA (0.5 mM), with 10 mM added NH+, a 20% increase in CAP synthesis was noticed with 0.5 to 2.5 mu aspartate, which decreased to 5% when aspartate was 5 mu. The optical assay was used in the experiments with purified CPSase I. The enzyme
SYNWIETASE
(AMMONIA)
275
activity with limiting KHC03 (16.7 II~M) and AGA (0.33 mu), with 11.7 mM added (NH&SO, was inhibited about 10% in the presence of 1 to 10 mu aspartate. No other effect was noted. DISCUSSION
The purification method used is essentially that of Guthohrlein and Knappe (l), but differs from it in several advantageous details: KCN and mercaptoethanol are eliminated; the first ammonium sulfate fractionation is reduced to two steps since most of the enzyme precipitates between 2.4 and 2.9 M ammonium sulfate; chromatography on controlled pore glass is simpler and much faster than on Sephadex G200 and yields a preparation of slightly higher specific activity than the preceding ammonium sulfate fraction; finally, all the chromatographic steps can be done at room temperature, which decreases the duration of the fractionation procedure significantly, especially when 20% (v/v) glycerol solutions must be used. Freshly dissolved CPSase I migrates with difficulty into 7% analytical polyacrylamide gels over the pH range 3.8 to 9.5, at either extreme of which the protein should have a net charge if its isoelectric point is similar to that of the frog liver enzyme, pH 6.5 (18); it migrates well only into the 2.5% gels. The molecular size of the native enzyme does not by itself explain this difficulty; it is possible that large aggregates of the enzyme are formed under the conditions of electrophoresis. The absence of minor bands under any of the conditions used indicates that the fi-actions with specific activity near 1.87 are essentially homogeneous. The electrophoretic patterns obtained after storage at 5°C provide further direct evidence of the existence of at least two molecular forms of CPSase I. The new band which appears after prolonged storage at 5°C may represent the more slowly sedimenting species previously observed by ultracentrifugation of frog (19) and rat liver (1) CPSase I. The molecular weight of active CPSase I determined by density gradient centrifugation, 222,000 + 10,000, is very close to
276
RAIJMAN
the value of 250,000 obtained by sedimentation and diffusion analysis (1). The recovery of CPSase I from glycerol gradients was about 60%; enzymatically inactive subunits which might have been formed in the course of the centrifugation would not have been detected. The molecular weight obtained by electrophoresis after denaturation with sodium dodecyl sulfate in the presence of D’M’ is 115,000 to 125,000; this suggests that CPSase I consists of two subunits of identical size. The inactive species formed at 10°C in the presence of AGA appears to have a similar molecular weight (1). Virden (2) has reported values of 316,000 + 42,000 and 160,000 & 10,000 for the molecular weight of the dimer and monomer of rat liver CPSase I, respectively. The reasons for the large discrepancy between Virden’s values and those reported earlier and in this paper are not apparent. The amino acid composition of rat and beef liver (20) CPSase I is shown in Table II; the enzymes appear to be remarkably similar in this respect. Also included is the composition of the catalytic subunit of E. coli carbamyl phosphate synthetase (glutamine) (21, 22). This subunit resembles the mammalian enzymes in that it utilizes only ammonia as the N-donor, and in its molecular weight; similarities in its amino acid composition, though present, are of less clear significance. The efficient utilization of CAP for urea synthesis requires that 1 mol of aspartate be synthesized per mole of CAP produced; this suggests that the mechanisms which regulate the availability of these substrates must be coordinated (23): CAP could stimulate aspartate synthesis by glutamic-oxaloacetic transaminase, or it could inhibit CPSase I; conversely, aspartate could affect CPSase I activity. CAP has been shown to be a mixed inhibitor of bovine liver CPSase I (24), with a Ki of 10 to 19 mM; if this value applies to the rat liver enzyme, it would appear to preclude a physiological inhibitory role for CAP, since the latter is about 0.1 mM in whole liver (4). As regards aspartate, its effects were studied at concentrations approaching those found in rat liver (0.8-l mM) and
AND JONES
at the higher levels that can be produced experimentally (23,25). Aspartate (1 to 10 mM) caused a minimal inhibition of the purified CPSase I, and a slight (5 to 20%) activation of CAP synthesis by isolated mitochondria which may be due to effects of aspartate on mitochondrial metabolism rather than on CPSase I. Krebs et al. (23) studied the effect of 1 mu ornithine on the fate of alanine nitrogen in perfused rat liver (normal liver contains about 0.15 pm01 of ornithinelg wet wt; 4). Their results suggested that ornithine can stimulate CPSase I, either by a direct effect on that enzyme, or indirectly, by increasing the concentration of arginine, which would result in increased synthesis of AGA (23). Our experiments show that 0.3 to 1.7 mM ornithine has a very small activating effect on purified CPSase I and suggest that if increased levels of ornithine stimulate CPSase I in uiuo, they do so indirectly. An adult rat on a normal diet produces about 10 mm01 of urea (therefore, of CAP) per day (25), or, for a liver weight of 10 g, about 8% of the maximum synthetic capacity measured in vitro. Since the synthesis of urea in uiuo does not proceed at a constant rate, the velocity of CPSase I must at times be greater and at times much smaller than 8% of V. However, other than AGA, no powerful physiological inhibitors or activators of CPSase I or ornithine transcarbamylase have been found; the mechanisms for the fast regulation of the first two steps of urea synthesis, therefore, remain obscure. The following findings may suggest some of the answers. Our work shows that CPSase I preparations with a specific activity of 1.87 are essentially homogeneous. Using a molecular weight of 222,000, a measured maximum CPSase I activity in vitro of 10 units/ g of rat liver, and assuming a specific activity of 2 for the pure enzyme, one can calculate that 1 g of liver contains 5 mg, or about 20 nmol, of CPSase I. Since the enzyme is in the matrix (26) and the volume of the latter is thought to be about onequarter of the total mitochondrial volume (27), the concentration of CPSase I in the mitochondrial matrix can be calculated to
RAT LIVER
CARBAMYL
PHOSPHATE
be about 4-5 x 1O-4 M (the assumption is also made that 1 g of liver equals 1 ml of water). This is an unusually high concentration: CPSase I must constitute about 22 to 26% of the total matrix protein of rat liver (since 60 to 70% of the mitochondrial protein is in the matrix, i.e., 19 to 22.4 mg of matrix protein/g of liver; 28). CPSase I should therefore be an excellent marker for studies on mammalian mitochondrial biogenesis, on the translocation of proteins from the cytoplasm into the matrix, on liver regeneration, and on the nature of the extensive adaptive changes the liver undergoes under certain nutritional conditions (29). Similar calculations show that the probable matrix concentration of AGA is 2 to 4 x 10e4 M (30), and that of ornithine transcarbamylase about 6 x 10m9 M, assuming that the specific activities of the rat and bovine enzymes are similar; their molecular weights appear to be identical (31; Kaijman and Jones, unpublished experiments). Furthermore, liver CPSase I, ornithine transcarbamylase, and AGA increase similarly in response to a 60% protein diet (30,321. The near-equimolarity of CPSase I and AGA in the matrix suggests certain regulatory possibilities. At body temperature, AGA shifts the position of equilibrium between the different forms of CPSase I towards the active form (1). Assuming that the dissociation constant of the enzyme for AGA is similar to itsK, for AGA, 1 x 10e4 M (18), and that the reaction CPSase I + AGA + CPSase I*AGA is at near-equilibrium (11, approximate values of [CPSase 1.AGAI and [free AGAI at various [total AGAI can be calculated and are listed in Table III. At 2 to 4 x 10e4 M AGA, from one- to two-thirds of the total enzyme would be in the active CPSase I*AGA form. Changes in [total AGAI should cause changes in CPSase I activity; these would be nonlinear and small in the direction of activation, but could be linear and large in the direction of deactivation. Similar calculations using the dissociation constant (4 x low5 M) of the CPSase I*ATP+Mg complex (18) show that at 4 x
SYNTHETASE
277
(AMMONIA) TABLE
CALCULATED CONCENTRATIONS AND FREE AGA AT DIFFERENT
III OF
CPSase I-AGA
CONCENTRATIONS
OF
TOT& AGAa [T$AzAl 0.5 1 2 4
[CPSase IeAGAl lo-* 0.379 0.735 1.35 2.31
M
[FygaAzA1 0.121 0.265 0.65 1.69
a The equation used in the calculations is WPSase 11 [AGAI = 10-4 M
[II ’ [CPSase I.AGA] The value of Eq. [ll is that of the K, of CPSase I for AGA; the concentration of CPSase I used in the cakulations was 3.8 X 10e4 M.
10e4 M total CPSase I and 1 mM total ATPmMg, CPSase I*ATP*Mg would be approximately 3.58 x 10T4 M. The ATP content of rat liver mitochondria has been estimated to be about 2 mu (33,34); therefore, although in uiuo ATP must bind to many liver mitochondrial proteins, it seems nevertheless possible that a significant fraction of ATP is bound to CPSase I, and that in normal rat liver ATP may not limit the activity of that enzyme. Our findings indicate that CPSase I is present in mitochondria at levels comparable to those of some pathway-related substrates such as AGA, ornithine, and CAP. In vitro experiments in search of activators or inhibitors of the synthetase have been carried out under the conditions of Michaelis-Menten kinetics, and the data obtained from them probably cannot be extrapolated to the conditions prevailing in uiuo (35, 36). The stimulation of urea synthesis by ornithine described by Krebs et al. (23) suggest that significant inhibitions or activations of CPSase I, perhaps owing to the combined action of multiple effecters, take place in uiuo. The manner in which CPSase I is regulated remains to be studied in vitro under conditions approaching the physiological; that is, among other variables, at high enzyme concentrations and at possibly limiting reactant concentrations. ACKNOWLEDGMENTS We are grateful to Mr. Gregory Headen for his
278
RAIJMAN
technical assistance; to Mr. Francis Stolzenbach and Ms. Sue Oxley for carrying out the amino acid analyses; and to Dr. Nathan 0. Kaplan, in whose laboratories they were done. Note added in proof. Since this paper was accepted for publication, it has been reported (37) that a polypeptide of 165,000 molecular weight, detected by disc gel electrophoresis in sodium dodecyl sulfate, makes up 15 to 20% of the total mitochondrial protein of rat liver; strong indirect evidence is presented in that paper which suggests that the peptide is CPSase I.
1. 2. 3.
4. 5.
10.
REFERENCES GUTHOHRLEIN, G., AND KNAPPE, J. (1968) Eur. J. Biochem. 7, 119-127. VIRDEN, R. (1972) &o&em. J. 12’7, 503-508. NAKAMURA, M., AND JONES, M. E. (1970) in Methods in Enzymology (Kaplan, N., and Colowick, S. P., eds.), Vol. 17A, pp. 286294, Academic Press, New York. RANMAN, L. (1974) B&hem. J. 138, 225-232. LOWRY, 0. H., RQ~EBROUGH, N. J., FAIR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. DAVIS, B. (1964)Ann. N. Y. Acad. Sci. 121,404427. WILLIAMS, D. E., AND REIBFELD, R. W. (1964) Ann. N. Y. Acod. Sci. 121, 373-381. REIEIFELD, R. A., LEWIS, V. J., AND WILLIAMS, D. E. (1962) Nature (London) 195, 281-283. CHRAMBACH, A., REI~ELD, R. A., WYCKOFF, M., AND ZACCARI, J. (1967) Anal. B&hem. 20, 150-154. WEBER, K., AND OSBORN, M. (1969) J. BioZ. Chem.
244,4406-4412. R. G., AND AMES, B. N. (1961) J. Biol. Chem. 236, 1372-1379. MOORE, S., AND STEIN, W. H. (1963) in Methods
11. MARTIN, 12.
in Enzymology (Kaplan, D., and Colowick, S. P., eds.)., Vol. 6, pp. 819-831, Academic Press, New York. 13. MOORE, S. (1963) J. Biol. 14. LIU, T. Y., AND YANG, 15. 16.
Chem. MYERS, them. WEBER, Chem.
Chem.
Y. H.
238,235-237. (1971) J. Biol.
246, 2842-2848. D. L., AND SLATER, E. C. (1957) BioJ. 67, 558-572. K., AND KUTER, D. J. (1971) J. Biol. 246, 4504-4509.
AND JONES 17. SCHACHMAN, H. K. (1957) in Methods in Enxymology (Kaplan, N., and Colowick, S. P., eds.), Vol. 4, pp. 32-103, Academic Press, New York. 18. MARSHALL, M., METZENBERG, R. L., AND COHEN, P. P. (1961) J. Biol. Chem. 236, 2229-2237. GRI~~LIA, S., AND RALJMAN, L. (1964) Adu. in lg. Chem. 44, 128-149. 20. ELLIOTT, K. R. F., AND TIPTON, K. F. (1973) FEBS Lett. 37, 79-81. 21. MATTHEWS, S. L., AND ANDEBBON, P. M. (1972) Biochemistry 11, 1176-1183. 22. TR(YTTA, P. P., PINKUS, L. M., HAFICHEMEYER, R. H., AND MEIWER, A. (1974) J. Biol. Chem. 249,492-499.
23. KREBS, H. A., HEMS, R., AND LUND, P. (1973) in Inborn Errors of Metabolism (Hommes, F. A., and Van Den Berg, C. J., eds.), pp. 201-215, Academic Press, London/New York. 24. ELLIOTT, K. R. F., AND TIPTON, K. F. (1974) Biochem. J. 141, 817-824. 25. LUND, P., BR~~NAN, J. T., AND EGGLESTON, L. V. (1970) in Essays in Cell Metabolism (Bartley, W., Kornberg, H. L., and Quayle, J. R., eds.), pp. 167-188, Wiley-Interscience, London. 26. GAMBLE, J. G., AND LEHNINGER, A. L. (1973) J. Biol. Chem. 248, 610-618. 27. PFAFF, E., KLINGENBERG, M., RITT, E., AND Ve GELL, W. (1968) Eur. J. Biochem. 5,222-232. 28. SCHNAITMAN, C., AND GBEENAWALT, J. W. (1968) J. Cell. Biol. 38, 158-175. 29. RAIJMAN, L. (1976) in The Urea Cycle: Discovery and Present Status (Grisolia, S., Mayor, F., and Baguena, R., eds.), Wiley-Interscience, New York, in press. 30. SHIGESADA, K., AND TATIBANA, M. (1971) J. Biol.
Chem.
246, 5588-5595.
M., AND COHEN, P. P. (1972) J. Biol. Chem. 247, 1641-1653. 32. SCHIMKE, R. T. (1962) J. Biol. Chem. 237, 459468. P., AND 33. ELBER~, R., HELDT, H. W., S~HMUCKER, 31. MARSHALL,
WIE~E, H. (1972) Hoppe-Seylers Z. Phys. Chem. 353, 702-703. P. F., AND TAGER, J. M. (1974) 34. ZUURENDONK, Biochim. Biophys. Acta 333,393-399. 35. FRIEDEN, C., AND COLMAN, R. F. (1967) J. Biol. Chem. 242, 1705-1715. SRERE, P. (1974) Life Sci. 15, 1695-1710. 36. ^_ 31’.CARK, S. (1976) J. Biol. Chem. 251, 950-961.
OF
BIOCHEMISTRY
Purification,
AND
175, 270-278 (1976)
Composition, and Some Properties of Rat Liver Carbamyl Phosphate Synthetase (Ammonia)l LUISA
Department
BIOPHYSICS
ofBiochemistry,
RAWMAN School
of Medicine, Angeles,
AND MARY University California
ELLEN
ofsouthern 90033
JONES California,
2025 Zonal
Avenue,
Los
Received December 8, 1975 A procedure for the purification of rat liver carbamyl phosphate synthetase (ammonia) to homogeneity is described. The molecular weight of isolated active enzyme is 222,000 -t10,000; that of the subunits obtained by denaturation with sodium dodecyl sulfate in the presence of dithiothreitol is 120,000 + 5,000. The enzyme appears to be composed of two subunits of identical size. The amino acid composition of the rat liver enzyme is reported and shown to be very similar to that of the bovine enzyme. The concentration of carbamyl phosphate synthetase I in the matrix of rat liver mitochondria is about 5 x 10e4 M, very similar to that of acetylglutamate, on this basis, it is suggested that one-third to one-half of the total enzyme may be in the active form in the matrix. Calculations also indicate that normally, ATP is unlikely to limit the rate of carbamyl phosphate synthesis in vivo. Carbamyl phosphate synthetase I appears to constitute 22 to 26% of the total matrix protein of liver mitochondria; the significance of this fact is briefly examined.
Although the ammonia-dependent carbamyl phosphate synthetase of rat liver (carbamyl phosphate synthetase I) has been purified extensively and recently has been the object of physical studies, data on the molecular weight of the active protein vary widely (1, 2), and no information is available on the composition of the enzyme or the size of the subunits obtained by exhaustive denaturation with sodium dodecyl sulfate in the presence of dithiothreitel. In addition, little is known about the regulation of carbamyl phosphate synthetase I; the search for effecters has been unsuccessful except for the activation of this enzyme by acetylglutamate, and the kinetics under conditions approaching those in uiuo have not been studied. ‘I’herefore, the role that carbamyl phosphate synthetase I may play in the changes in the rate of urea synthesis observed in uiuo, in perfused liver, and in isolated liver cells cannot be evaluated.
We carried out this study to clarify and extend the physical and chemical characterization of rat liver carbamyl phosphate synthetase I and to begin to study its activity under conditons relevant to metabolism. MATERIALS
Materials. Male rats of the Sprague-Dawley strain, weighing approximately 150 g, were purchased from Zivic Miller Laboratories, Inc., Allison Park, Pennsylvania. Controlled pore glass was obtained from the Sigma Chemical Co. Dithiothreitol: glycylglycine, crystalline bovine plasma albumin, and ovalbumin were purchased from Calbiochem; bovine pancreas trypsin type I, urease type VI, and beef liver catalase (lO,OOO-25,000 units/mg) were from Sigma Chemical Co.; ornithine transcarbamylase (EC 2.1.3.3) was prepared as described by Nakamura and Jones (3); dilithium carbamyl phosphate, potassium or tricyclohexylammonium phosphoenolpyruvate, and rabbit muscle lactate dehydrogenase, pyruvate kinase, and adenylate kinase were from 3 Abbreviations used: Dl”l’, dithiothreitol; AGA, acetylglutamic acid; CAP, carbamyl phosphate; CTAB, cetyltrimethylammonium bromide; CPSase I, carbamyl phosphate synthetase (ammonia), EC 2.7.2.5; DEAE, diethylsminoethyl.
‘This work was supported by Grant No. HD 06538 from the National Institutes of Health. e To whom all correspondence should be addressed. 270 Copyright 8 1976 by Academic F’reas, Inc. All rights of reproduction in any form reserved.
AND METHODS
RAT LIVER
CARBAMYL
PHOSPHATE
Boehringer Mannheim; ultrapure ammonium sulfate and sodium dodecyl sulfate, bromophenol blue, and Coomassie brilliant blue were from Mann Research Laboratories; acrylamide, methylenebisacrylamide, N,h’,N’,N’-tetramethylethylenediamine, P-mercaptoethanol, and butanedione-2-monoxime were from Eastman Organic Chemicals. Other reagents were commercial products of analytical quality. Enzyme assays. For the assay of CPSase I, all acidic reagents were neutralized with KOH. A colorimetric assay (1) based on the conversion of CAP to citrulline was used unless stated otherwise. Samples of CPSase I were preincubated for 2 min at 37°C in a reaction mixture containing: glycylglycine, pH 7.6, 50 pmol; NHIHCOI, 50 pmol; MgSO,, 15 wmol; acetylglutamate, 5 pmol; L-ornithine, 5 pmol; ornithine transcarbamylase, 10 to 20 units. The reaction was started by addition of 5 pmol of ATP in 0.1 ml. The final volume was 1 ml, and the incubation was at 37°C for 15 min; the reaction was stopped with 0.2 ml of 5 M HClO,. A unit in this assay is the amount of enzyme that synthesizes 1 pmol of CAP (measured a8 citrulline) per minute at 37°C. Specific activity is the number of units per milligram of protein. A spectrophotometric assay based on the coupling of CPSase I activity to the oxidation of NADH was also used (1). One milliliter of reaction mixture contained: glycylglycine, pH 7.6, 50 pmol; KHCOI, 50 pmol; (NH&SO,, 35 kmol; MgSO,, 15 pmol; acetylglutamate, 10 pmol; ATP, 1.7 pmol; phosphoenolpyruvate, 2.5 pmol; NADH, 0.5 pmol; pyruvate kinase, 40 pg; lactate dehydrogenase, 200 pg; adenylate kinase, 25 pg. The reaction was started by addition of CPSase I. The concentration of some reagents was sometimes changed and will be indicated. Ornithine transcarbamylase activity at pH 7.5 was measured as previously described (4). Determination of protein. D’Pl interferes with measurements of protein absorbance at 260 and 280 nm (the oxidized derivative of D’IT absorbs light of those wavelengths) and by the method of Lowry et al. (5). Routine measurements were done by ultraviolet absorbance, using as blanks samples of the solvent prepared at the same time as those containing the enzyme and kept under similar conditions. Accurate measurements were carried out by the method of Lowry et al. (5) with dialyzed enzyme samples. Electrophoresis. Analytical disc gel electrophoresis was carried out as previously described (6-8) with bromophenol blue as the marker; 2 mA per tube was used. The gels were fixed in 12.5% trichloroacetic acid and stained for protein with Coomassie blue (9). Electrophoresis in the presence of sodium dodecyl sulfate was carried out by the method of Weber and Osborn (lo), using 5% polyacrylamide gels.
SYNTHETASE
(AMMONIA)
271
Glycerol and sucrose density gradient centrifigation. The method of Martin and Ames (11) was used for calculations of molecular weight. Eleven-milliliter linear gradients (lo-30% glycerol or lo-308 sucrose, in 0.01 M Tris-HCl, pH 7.25) were made with an ISCO Model 570 gradient former; centrifugation at 4°C 40,000 rpm, for 14 to 20 h was performed with a Beckman Model L2-65 B ultracentrifuge using a Beckman SW 41 rotor. Forty-five to sixty-four fractions were collected from the top of the gradient by means of an ISCO Model 183 density gradient fractionator. Amino acid analysis. Amino acid analyses were carried out by the method of Moore and Stein (12) after hydrolysis of the protein in 6 M HCl for 24,48, and 72 h. Serine and threonine values were corrected for decomposition by extrapolation to zero time. Half-cystine was measured as cysteic acid (13); tryptophan was measured by the method of Liu and Yang (14). Amide nitrogen was not determined. A Beckman Model 119 amino acid analyzer was used. Preparation of mitochondrin. Mitochondria were prepared as described by Myers and Slater (15) and used immediately. Preparation of antisera. Rabbit antisera to purified frog liver CPSase I and to crude fractions of rat liver CPSase I were obtained by subcutaneous injection of approximately 5 mg of protein in 1 ml of 0.15 M NaCl suspended in 1 ml of Freund’s adjuvant (0.5 ml injected per site). The rabbits were injected at least three more times at monthly intervals, each time using 1 mg of protein in NaCl, without Freund’s adjuvant. RESULTS
Stabilization
of CPSase I from rat liver.
A remarkable stabilization of this enzyme in solution had been achieved by the inclusion of KCN, mercaptoethanol, and 20% (v/v) glycerol into solutions of the protein (1). However, the dangerously large amounts of KCN at near-neutral pH required for medium scale purifications induced us to search for other stabilizing conditions. Glycerol (20%, v/v) in combination with D’IT stabilizes CPSase I to an extent which is a function of the amount of DTI’ added. At 4”C, 100% of the enzymatic activity is maintained after storage for 24 and 48 h in solutions containing 20% (v/v) glycerol containing 0.1 and 2 mM D!lT, respectively. At approximately 23°C 100 and 87% of the activity is recovered after storage for 24 and 48 h, respectively, in 20% (v/v> glycerol containing 2 mM DTI’. Throughout the purification procedure,
272
FLAIJMAN
AND
glycerol and various concentrations of D’IT were used as stabilizers of the enzyme. Whenever possible, the necessary amount of solid D’IT was added to all solutions immediately before use. Gel filtration and column chromatography were carried out at room temperature; all other procedures were carried out at 0-4°C. Purification: from rat liver.
Extraction
of CPSase
I
The following steps include several modifications of the method described by Guthohrlein and Knappe (1). Bat livers (150 g) were quickly washed with ice-cold distilled water, blotted, minced, and homogenized for 15 s with 450 ml of buffer I (0.02 M Tris-HCl, 20% (v/v) glycerol, and 1 mM D’IT, final pH 7.2) using a Waring Blendor at top speed. The homogenate was centrifuged at 15,OOOg, 4”C, for 20 min; the supernatant was discarded. The sediment was suspended in 900 ml of buffer I and was homogenized for 10 s and centrifuged as above; the supernatant was discarded. The sediment was suspended with a stirring rod in 900 ml of buffer I and centrifuged as above; the supernatant was discarded. The washed sediment was suspended in 375 ml of a buffer containing 0.02 M TrisHCl, 20% (v/v) glycerol, 2 mM DTI’, and 0.1% (w/v) CTAB; the suspension was homogenized with a Waring Blendor at top speed for 15 s. After centrifugation at 15,OOOg, 4”C, for 20 min, the supernatant was collected and the pellet was reextracted as described above. The combined supernatants had a volume of 890 ml. First
ammonium
sulfate
fractionation.
The CTAB extract was fractionated as previously described (l), except that only the fraction precipitating between 2.4 and 2.9 M ammonium sulfate was collected by suspending it in buffer B of Guthohrlein and Knappe (1). This suspension contained 1.43 g of protein in a volume of 62 ml; a 2ml sample was stored at this stage and the remainder was divided into three 20-ml portions which were stored at -20°C. Chromatography on DEAE-Sephadex and second ammonium sulfate fractionation. Each 20-ml portion of the ammonium
sulfate suspension (containing 460 mg of protein) was treated as follows: After stir-
JONES
ring at 0°C for 10 min, the suspension was centrifuged at 39,OOQg for 30 min; the supernatant was discarded. The precipitate was dissolved in 1 ml of a buffer containing 0.05 M Tris-HCl, 20% (v/v) glycerol, 0.2 nm D’IT, pH 7.2 (buffer II), plus 5 mM KCl. The rest of the procedure was as previously described (1) except that it was carried out at room temperature, buffer II was used instead of buffer A of Guthohrlein and Knappe (l), and the flow rate through the DEAE-Sephadex column was 18 ml/h. The fractions with specific activities between 1.13 and 1.6 (which were eluted -between 45 and 66 mM KCl) were pooled. The protein solution was made 2.4 M in ammonium sulfate and the resulting precipitate was discarded; the supernatant was made 3.1 M in ammonium sulfate and centrifuged. The precipitate was resuspended in a small volume of buffer B and stored at -20°C. This process was repeated (twice more) until all of the first ammonium sulfate fraction had been purified. The three pools of specific activity 1.13 to 1.6 were combined to yield a single enzyme pool which was stored at -20°C. Chromatography
on
controlled
pore
glass. A portion of the enzyme suspension from the previous step containing about 94 mg of protein was centrifuged and the precipitate was dissolved in 2 ml of buffer II plus 5 mM KCl. The solution was applied to a column (1.4 x 81 cm) of controlled pore glass (CPG-10, 2000 A pore size, 120-200 mesh), equilibrated at room temperature with the same buffer mixture; the flow rate was 24 ml/h. The column was eluted with buffer II plus 5 mM KC1; 4-ml fractions were collected. The protein appeared in a sharp peak, symmetrical except for a slight shoulder at the end of the descending limb; the shoulder contained less than 5% of the protein. The fractions containing CPSase I of specific activity 1.73 to 2 were pooled, solid ammonium sulfate was added to give a 3.1 M concentration, and the suspension was stored at -20°C. CPSase I binds tightly to the glass beads, if small amounts of the enzyme in dilute solutions are used, as much as 95%
RAT LIVER
CARBAMYL
PHOSPHATE
of the protein can remain attached to the beads and will not be eluted with high concentrations of salt, even at very high pH. Treatment of the glass column with bovine serum albumin prior to the addition of CPSase I did not prevent the binding of the latter; since all of the albumin added could be easily recovered by elution with buffer II plus 5 mu KCI, the high affinity of the glass beads for CPSase I appears to be a specific property. The recovery of enzyme was improved by applying the protein to the columns at high concentrations and by reusing the columns; the initial coating of the glass beads with CPSase I appeared to diminish further binding of this protein upon reusing the beads. Table I shows a summary of the puriilcation procedure. Analytical
SYNTHETASE
273
(AMMONIA)
sis of the purest fractions (50-100 pg/gel) was also done at pH 3.8 (8) and at pH 9.5 (6), using gels of various pore sizes. Using approximately 7% polyacrylamide gels, only one broad band was observed, always near the origin of the running gel, regardless of the pH used. At pH 9.5, CPSase I migrated into 4 and 2.5% polyacrylamide gels; in the latter case, the enzyme migrated to the center of the gel in a narrow band. No other bands were observed. Samples of CPSase I dissolved in 0.05 M glycylglycine at pH 7.2, with and without 0.5 mM AGA, and stored at 5°C for 70 h, showed an additional band on electrophoresis at pH 8 using 7% gels (7). The new band was observed only under these conditions, was somewhat diffuse, less intense than the major band remaining at the origin of the gel, and was located at the center of the gel.
disc gel electrophoresis. The
procedure used for the preparation of polyacrylamide gels was as described by Davis (6); the samples were layered directly over the stacking gels. Electrophoresis at pH 8 of the second ammonium sulfate fractions, using 7% polyacrylamide gels (7) and 75 pg of protein in 0.1 ml, showed two minor bands which penetrated well into the gel, and a major, very diffise band (1) near the beginning of the running gel. Electrophoresis of the fractions obtained after controlled-pore glass chromatography gave only the major band, even when 150 pg of protein was applied. Since CPSase I essentially does not migrate under these conditions, electrophore-
Electrophoresis after treatment with sodium dodecyl sulfate. Samples of CPSase I
(0.3 mg; sp act, about 1.87) alone and with 25 pg each of bovine serum albumin, ovalbumin, and bovine trypsin in a buffer containing 0.01 M sodium phosphate, pH 7.0, 1% (w/v) sodium dodecyl sulfate, and 1 ~-IM D’IT (total volume 1 ml) were incubated at 37°C for 2 and 24 h. To 50-~1 portions of each mixture, 20 ~1 of glycerol and 50 ~1 of 0.01 M sodium phosphate, pH 7.0, and bromophenol blue were then added; 0.1 ml of these mixtures was applied to the gels and was subjected to electrophoresis for 3.5 h. Gels to which only CPSase I had been applied showed a single narrow band,
TABLE
I CPSase I Total protein Specific activity (mg)
PURIFICATION
Fraction Crude homogenate CTAB extract First ammonium sulfate fraction DEAE-Sephadex fraction’ Second ammonium sulfate fraction Controlled pore glass fraction
Total activity (units)
OF
1,410 1,240 1,020
39,000 5,000 1,430
250 150
173 100
84
45
m v?l”li? 600 890 62
ReGT”0 100 87.4 72.3
1.45 1.48
105 5
17.8 10.5
1.87
25
5.9
0.036 0.247 0.713
D From 460 mg (20 ml) of the preceding fraction. To calculate the overall recoveries from this fraction on, a factor of 3 must be used.
274
RAIJMAN
whether the protein had been incubated 2 or 24 h; Fig. 1 shows a plot of the relative mobility of CPSase I and standards. The calculated molecular weight of the enzyme subunits (10) was 115,000 to 125,000. Attempts at renaturing (16) failed, no activity was detectable after incubation of the subunits alone and with different combinations of AGA, ATP, and Mg2+ at 4 and 22°C. Density gradient centrifugation. Solutions containing 5 mg of CPSase I (sp act, about 1.67), 0.5 mg of lactate dehydrogenase, 1 mg of catalase, and 3 mg of urease in 0.01 M Tris-HCl, pH 7.2, in a volume of 0.5 ml were prepared immediately before use. Of the mixture, 200 to 300 ~1 was layered onto glycerol or sucrose gradients. The calculated molecular weight was 222,000 +10,000; the szo,W values ranged between 10.3 and 10.9 (11). Amino acid composition. Table II shows the amino acid composition of CPSase I expressed as moles of residues per mole of subunit. It was assumed that the subunits are identical, with a molecular weight of 121,000. The synthetase contains a large number of acidic residues, some of which would have been in the amide form in the native protein. The partial specific volume calculated from the amino acid composition is 0.737 cm3/g; the error introduced in this value by the lack of data on the glutamine and asparagine content of CPSase I is small (17). Reaction of CPSase I with antisera. The interaction of the purest fractions of rat liver CPSase I with antiserum to the frog
\
1
0.2
CPSase
04
I
0.6
0.6
MOBILITY
FIG. 1. The electrophoretic mobility of CPSase I after denaturation with sodium dodecyl sulfate in the presence of DTT.
AND JONES TABLE
II
AMINO
ACID COMPOSITION OF RAT AND BEEF LIVER CPSase I AND OF THE Q.TALYTIG SUBUNIT OF E. coli CARBAMYL PHOSPHATE SYNTHETASE Amino
acid
T
tat live] 1Rat liver1 :PSaee 1L XSaee I
(mol/
m3logl, -
Beef 1 E. C& liver” subunit I CPSaee I I
Residues per 100 residues T
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
69.6 20.3 40.7 106.8 57.3 80.5 111.2 55.7 79.3 82.6 30.7" 81.3 29.5 65.1 96.2 26.2 43.4 10.1
Total
086.5
residues
6.41 1.87 3.75
9.83 5.28 7.41 10.23 5.13 7.30 7.60 2.83c 7.48 2.72
5.99 8.85 2.41 4.00
0.93
8.38 2.55 5.31
9.60 5.11 5.62 10.32 4.70 7.46
7.97 1.63 7.35 1.43 5.82 8.78 2.66
3.98 1.33
5.09 1.41 6.43 9.10 5.64 4.37 12.06 5.00 8.21 10.53 1.66 8.26 2.58 6.13 7.54 2.34 3.22 0.40
a From Ref. (20). b From Ref. (22). c This value was calculated using the average ratio of “molar equivalents in oxidized samples/molar equivalents in unoxidized samples” of four nonsulfur-containing amino acids. Because the sample of oxidized material used was very small, the minimum and maximum ratios obtained varied considerably; the respective mo1/121,00@ values arrived at from the extreme ratios are 37 and 24.5, while the residues per 100 residues vary from 3.41 to 2.25.
liver enzyme was tested by immunodiffision using agar plates; a single precipitation band was obtained, indicative of crossreactivity between the enzyme from both species. This was confirmed by assaying the rat liver enzyme after preincubation with the frog antiserum; complete inbibition of activity was observed, but the concentrations of antiserum required were approximately 10 times greater than those required for complete inhibition of similar amounts of the frog liver enzyme. The purest fractions of rat liver CPSase I also gave a single precipitation band with no spurs when tested by immunodiffision
RAT
LIVER
CARBAMYL
PHOSPHATE
against antiserum to crude rat liver enzyme (first ammonium sulfate fraction). Effect of ornithine and aspartate on the activity of mitochondrial and purified CPSase 1. Ammonium sulfate suspensions of purified CPSase I were centrifuged and the precipitates were dissolved in 0.05 M glycylglycine, pH 7.6, 20% glycerol, and 0.5 mu DTT to give an enzyme concentration of about 2 mglml; these solutions contained at most 40 mM ammonium sulfate. Small aliquots of these solutions (not greater than 50 ~1) were used in the following experiments. The effect of ornithine on the activity of CPSase I could only be studied with the purified enzyme because mitochondria contain ornithine transcarbamylase which would utilize ornithine stoichiometrically with the CAP produced by the CPSase I; the optical assay of CPSase I was used. At saturating substrate concentrations for CPSase I (those of the optical assay as described in Methods), 33 PM ornithine had no effect on the enzymatic activity; 0.3 ~-IM and 1.7 mM ornithine increased activity by 20 and 14%, respectively. At limit+ ing concentrations of KHCO, (16.7 mu) and AGA (0.33 and 1 mu), with 11.7 mM added (NH&SO,, a 10% activation by 0.33 and 1.7 mM ornithine was the only effect noticed. The calorimetric assay was used in most experiments designed to study the effect of aspartate on CPSase I; since this assay is coupled to ornithine transcarbamylase, the effect of aspartate on this enzyme was studied first. At several concentrations between 0.5 and 10 mM, aspartate had no effect on mitochondrial ornithine transcarbamylase, either at saturating substrate concentrations (4) or with limiting ornithine (1 and 2 mM). Aspartate had no effect on mitochondrial CAP synthesis at saturating substrate concentrations. At limiting HC03(10 mM) and AGA (0.5 mM), with 10 mM added NH+, a 20% increase in CAP synthesis was noticed with 0.5 to 2.5 mu aspartate, which decreased to 5% when aspartate was 5 mu. The optical assay was used in the experiments with purified CPSase I. The enzyme
SYNWIETASE
(AMMONIA)
275
activity with limiting KHC03 (16.7 II~M) and AGA (0.33 mu), with 11.7 mM added (NH&SO, was inhibited about 10% in the presence of 1 to 10 mu aspartate. No other effect was noted. DISCUSSION
The purification method used is essentially that of Guthohrlein and Knappe (l), but differs from it in several advantageous details: KCN and mercaptoethanol are eliminated; the first ammonium sulfate fractionation is reduced to two steps since most of the enzyme precipitates between 2.4 and 2.9 M ammonium sulfate; chromatography on controlled pore glass is simpler and much faster than on Sephadex G200 and yields a preparation of slightly higher specific activity than the preceding ammonium sulfate fraction; finally, all the chromatographic steps can be done at room temperature, which decreases the duration of the fractionation procedure significantly, especially when 20% (v/v) glycerol solutions must be used. Freshly dissolved CPSase I migrates with difficulty into 7% analytical polyacrylamide gels over the pH range 3.8 to 9.5, at either extreme of which the protein should have a net charge if its isoelectric point is similar to that of the frog liver enzyme, pH 6.5 (18); it migrates well only into the 2.5% gels. The molecular size of the native enzyme does not by itself explain this difficulty; it is possible that large aggregates of the enzyme are formed under the conditions of electrophoresis. The absence of minor bands under any of the conditions used indicates that the fi-actions with specific activity near 1.87 are essentially homogeneous. The electrophoretic patterns obtained after storage at 5°C provide further direct evidence of the existence of at least two molecular forms of CPSase I. The new band which appears after prolonged storage at 5°C may represent the more slowly sedimenting species previously observed by ultracentrifugation of frog (19) and rat liver (1) CPSase I. The molecular weight of active CPSase I determined by density gradient centrifugation, 222,000 + 10,000, is very close to
276
RAIJMAN
the value of 250,000 obtained by sedimentation and diffusion analysis (1). The recovery of CPSase I from glycerol gradients was about 60%; enzymatically inactive subunits which might have been formed in the course of the centrifugation would not have been detected. The molecular weight obtained by electrophoresis after denaturation with sodium dodecyl sulfate in the presence of D’M’ is 115,000 to 125,000; this suggests that CPSase I consists of two subunits of identical size. The inactive species formed at 10°C in the presence of AGA appears to have a similar molecular weight (1). Virden (2) has reported values of 316,000 + 42,000 and 160,000 & 10,000 for the molecular weight of the dimer and monomer of rat liver CPSase I, respectively. The reasons for the large discrepancy between Virden’s values and those reported earlier and in this paper are not apparent. The amino acid composition of rat and beef liver (20) CPSase I is shown in Table II; the enzymes appear to be remarkably similar in this respect. Also included is the composition of the catalytic subunit of E. coli carbamyl phosphate synthetase (glutamine) (21, 22). This subunit resembles the mammalian enzymes in that it utilizes only ammonia as the N-donor, and in its molecular weight; similarities in its amino acid composition, though present, are of less clear significance. The efficient utilization of CAP for urea synthesis requires that 1 mol of aspartate be synthesized per mole of CAP produced; this suggests that the mechanisms which regulate the availability of these substrates must be coordinated (23): CAP could stimulate aspartate synthesis by glutamic-oxaloacetic transaminase, or it could inhibit CPSase I; conversely, aspartate could affect CPSase I activity. CAP has been shown to be a mixed inhibitor of bovine liver CPSase I (24), with a Ki of 10 to 19 mM; if this value applies to the rat liver enzyme, it would appear to preclude a physiological inhibitory role for CAP, since the latter is about 0.1 mM in whole liver (4). As regards aspartate, its effects were studied at concentrations approaching those found in rat liver (0.8-l mM) and
AND JONES
at the higher levels that can be produced experimentally (23,25). Aspartate (1 to 10 mM) caused a minimal inhibition of the purified CPSase I, and a slight (5 to 20%) activation of CAP synthesis by isolated mitochondria which may be due to effects of aspartate on mitochondrial metabolism rather than on CPSase I. Krebs et al. (23) studied the effect of 1 mu ornithine on the fate of alanine nitrogen in perfused rat liver (normal liver contains about 0.15 pm01 of ornithinelg wet wt; 4). Their results suggested that ornithine can stimulate CPSase I, either by a direct effect on that enzyme, or indirectly, by increasing the concentration of arginine, which would result in increased synthesis of AGA (23). Our experiments show that 0.3 to 1.7 mM ornithine has a very small activating effect on purified CPSase I and suggest that if increased levels of ornithine stimulate CPSase I in uiuo, they do so indirectly. An adult rat on a normal diet produces about 10 mm01 of urea (therefore, of CAP) per day (25), or, for a liver weight of 10 g, about 8% of the maximum synthetic capacity measured in vitro. Since the synthesis of urea in uiuo does not proceed at a constant rate, the velocity of CPSase I must at times be greater and at times much smaller than 8% of V. However, other than AGA, no powerful physiological inhibitors or activators of CPSase I or ornithine transcarbamylase have been found; the mechanisms for the fast regulation of the first two steps of urea synthesis, therefore, remain obscure. The following findings may suggest some of the answers. Our work shows that CPSase I preparations with a specific activity of 1.87 are essentially homogeneous. Using a molecular weight of 222,000, a measured maximum CPSase I activity in vitro of 10 units/ g of rat liver, and assuming a specific activity of 2 for the pure enzyme, one can calculate that 1 g of liver contains 5 mg, or about 20 nmol, of CPSase I. Since the enzyme is in the matrix (26) and the volume of the latter is thought to be about onequarter of the total mitochondrial volume (27), the concentration of CPSase I in the mitochondrial matrix can be calculated to
RAT LIVER
CARBAMYL
PHOSPHATE
be about 4-5 x 1O-4 M (the assumption is also made that 1 g of liver equals 1 ml of water). This is an unusually high concentration: CPSase I must constitute about 22 to 26% of the total matrix protein of rat liver (since 60 to 70% of the mitochondrial protein is in the matrix, i.e., 19 to 22.4 mg of matrix protein/g of liver; 28). CPSase I should therefore be an excellent marker for studies on mammalian mitochondrial biogenesis, on the translocation of proteins from the cytoplasm into the matrix, on liver regeneration, and on the nature of the extensive adaptive changes the liver undergoes under certain nutritional conditions (29). Similar calculations show that the probable matrix concentration of AGA is 2 to 4 x 10e4 M (30), and that of ornithine transcarbamylase about 6 x 10m9 M, assuming that the specific activities of the rat and bovine enzymes are similar; their molecular weights appear to be identical (31; Kaijman and Jones, unpublished experiments). Furthermore, liver CPSase I, ornithine transcarbamylase, and AGA increase similarly in response to a 60% protein diet (30,321. The near-equimolarity of CPSase I and AGA in the matrix suggests certain regulatory possibilities. At body temperature, AGA shifts the position of equilibrium between the different forms of CPSase I towards the active form (1). Assuming that the dissociation constant of the enzyme for AGA is similar to itsK, for AGA, 1 x 10e4 M (18), and that the reaction CPSase I + AGA + CPSase I*AGA is at near-equilibrium (11, approximate values of [CPSase 1.AGAI and [free AGAI at various [total AGAI can be calculated and are listed in Table III. At 2 to 4 x 10e4 M AGA, from one- to two-thirds of the total enzyme would be in the active CPSase I*AGA form. Changes in [total AGAI should cause changes in CPSase I activity; these would be nonlinear and small in the direction of activation, but could be linear and large in the direction of deactivation. Similar calculations using the dissociation constant (4 x low5 M) of the CPSase I*ATP+Mg complex (18) show that at 4 x
SYNTHETASE
277
(AMMONIA) TABLE
CALCULATED CONCENTRATIONS AND FREE AGA AT DIFFERENT
III OF
CPSase I-AGA
CONCENTRATIONS
OF
TOT& AGAa [T$AzAl 0.5 1 2 4
[CPSase IeAGAl lo-* 0.379 0.735 1.35 2.31
M
[FygaAzA1 0.121 0.265 0.65 1.69
a The equation used in the calculations is WPSase 11 [AGAI = 10-4 M
[II ’ [CPSase I.AGA] The value of Eq. [ll is that of the K, of CPSase I for AGA; the concentration of CPSase I used in the cakulations was 3.8 X 10e4 M.
10e4 M total CPSase I and 1 mM total ATPmMg, CPSase I*ATP*Mg would be approximately 3.58 x 10T4 M. The ATP content of rat liver mitochondria has been estimated to be about 2 mu (33,34); therefore, although in uiuo ATP must bind to many liver mitochondrial proteins, it seems nevertheless possible that a significant fraction of ATP is bound to CPSase I, and that in normal rat liver ATP may not limit the activity of that enzyme. Our findings indicate that CPSase I is present in mitochondria at levels comparable to those of some pathway-related substrates such as AGA, ornithine, and CAP. In vitro experiments in search of activators or inhibitors of the synthetase have been carried out under the conditions of Michaelis-Menten kinetics, and the data obtained from them probably cannot be extrapolated to the conditions prevailing in uiuo (35, 36). The stimulation of urea synthesis by ornithine described by Krebs et al. (23) suggest that significant inhibitions or activations of CPSase I, perhaps owing to the combined action of multiple effecters, take place in uiuo. The manner in which CPSase I is regulated remains to be studied in vitro under conditions approaching the physiological; that is, among other variables, at high enzyme concentrations and at possibly limiting reactant concentrations. ACKNOWLEDGMENTS We are grateful to Mr. Gregory Headen for his
278
RAIJMAN
technical assistance; to Mr. Francis Stolzenbach and Ms. Sue Oxley for carrying out the amino acid analyses; and to Dr. Nathan 0. Kaplan, in whose laboratories they were done. Note added in proof. Since this paper was accepted for publication, it has been reported (37) that a polypeptide of 165,000 molecular weight, detected by disc gel electrophoresis in sodium dodecyl sulfate, makes up 15 to 20% of the total mitochondrial protein of rat liver; strong indirect evidence is presented in that paper which suggests that the peptide is CPSase I.
1. 2. 3.
4. 5.
10.
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244,4406-4412. R. G., AND AMES, B. N. (1961) J. Biol. Chem. 236, 1372-1379. MOORE, S., AND STEIN, W. H. (1963) in Methods
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23. KREBS, H. A., HEMS, R., AND LUND, P. (1973) in Inborn Errors of Metabolism (Hommes, F. A., and Van Den Berg, C. J., eds.), pp. 201-215, Academic Press, London/New York. 24. ELLIOTT, K. R. F., AND TIPTON, K. F. (1974) Biochem. J. 141, 817-824. 25. LUND, P., BR~~NAN, J. T., AND EGGLESTON, L. V. (1970) in Essays in Cell Metabolism (Bartley, W., Kornberg, H. L., and Quayle, J. R., eds.), pp. 167-188, Wiley-Interscience, London. 26. GAMBLE, J. G., AND LEHNINGER, A. L. (1973) J. Biol. Chem. 248, 610-618. 27. PFAFF, E., KLINGENBERG, M., RITT, E., AND Ve GELL, W. (1968) Eur. J. Biochem. 5,222-232. 28. SCHNAITMAN, C., AND GBEENAWALT, J. W. (1968) J. Cell. Biol. 38, 158-175. 29. RAIJMAN, L. (1976) in The Urea Cycle: Discovery and Present Status (Grisolia, S., Mayor, F., and Baguena, R., eds.), Wiley-Interscience, New York, in press. 30. SHIGESADA, K., AND TATIBANA, M. (1971) J. Biol.
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WIE~E, H. (1972) Hoppe-Seylers Z. Phys. Chem. 353, 702-703. P. F., AND TAGER, J. M. (1974) 34. ZUURENDONK, Biochim. Biophys. Acta 333,393-399. 35. FRIEDEN, C., AND COLMAN, R. F. (1967) J. Biol. Chem. 242, 1705-1715. SRERE, P. (1974) Life Sci. 15, 1695-1710. 36. ^_ 31’.CARK, S. (1976) J. Biol. Chem. 251, 950-961.
