Human ornithine transcarbamylase. Purification and characterization of the enzyme from normal liver and the liver of a Reye's syndrome patient.

Ornithine transcarbamylase was purified and characterized from normal human liver. The properties of this enzyme were compared to those of ornithine transcarbamylase purified from the liver of a patient with Reye's syndrome. The enzyme isolated from both sources appeared virtually identical for a variety of biochemical characteristics. The native molecular weight of ornithine transcarbamylase is 110,000 as determined by gel filtration. Electrophoresis of the enzyme, dissociated by sodium dodecyl sulfate, indicated that the enzyme exists as a trimer of identical or similar subunits of 36,500 daltons. Ornithine transcarbamylase from normal liver has an isoelectric point of 7.95, and the value for the enzyme from the Reye's syndrome liver was 8.05. No evidence of multiple species was found during the purification or subsequent characterization of the enzyme. The enzyme exhibited normal Michaelia-Menten kinetics, and the apparent Michaelis constants for L-ornithine and carbamyl phosphate are 0.20 mM and 0.09 mM, respectively. Inhibitor studies established the structural requirements for L-ornithine antagonists. L-Norvaline is the best competitive inhibitor of the enzyme with respect to L-ornithine. This study indicated that the reduced level of ornithine transcarbamylase activity commonly observed in Reye's syndrome is not necessarily due to structural or functional alterations of the enzyme.

Ornithine transcarbamylase was purified and characterized from normal human liver. The properties of this enzyme were compared to those of ornithine transcarbamylase purified from the liver of a patient with Reye's syndrome. The enzyme isolated from both sources appeared virtually identical for a variety of biochemical characteristics. The native molecular weight of ornithine transcarbamylase is 110,000 as determined by gel filtration. Electrophoresis of the enzyme, dissociated by sodium dodecyl sulfate, indicated that the enzyme exists as a trimer of identical or similar subunits of 36,500 daltons. Ornithine transcarbamylase from normal liver has an isoelectric point of 7.95, and the value for the enzyme from the Reye's syndrome liver was 8.05. No evidence of multiple species was found during the purification or subsequent characterization of the enzyme. The enzyme exhibited normal Michaelis-Menten kinetics, and the apparent Michaelis constants for L-ornithine and carbamyl phosphate are 0.20 mM and 0.09 mM, respectively. Inhibitor studies established the structural requirements for L-ornithine antagonists. L-Norvaline is the best competitive inhibitor of the enzyme with respect to L-ornithine. This study indicated that the reduced level of ornlthine transcarbamylase activity commonly observed in Reye's syndrome is not necessarily due to structural or functional alterations of the enzyme.
The urea cycle consists of five enzymes, three of which are located in the cell's cytoplasm and two in the matrix of the mitochondrion (l-5

Measurement ofornithine Transcarbamylase
Ornithine transcarbamylase activity was measured by a modification of the method of Ceriotti (29). Unless specified otherwise, reaction mixtures contained 0.5 pmol of carbamyl phosphate; 0.5 pmol of L-ornithine; 10 wmol of triethanolamine, pH 8.0; and enzyme in a final volume of 0.2 ml. Triethanolamine buffer was used in the assay mixture because consistently higher specific activities were obtained with this buffer compared to Tris or glycylglycine buffers. Following incubation for 10 min at 37", the reaction was stopped by the addition of 3.0 ml of the chromogenic reagent described by Ceriotti (291. The composition of the chromogenic reagent is as follows: Solution A consists of 4 g of antipyrine, 50 mg of Fe,(SO& .9H,O, and 100 ml of H,SO, diluted to 1 liter; Solution B consists'of 5 g of diacetyl monoxime, 3 g of Brij 35, and 50 ml of acetic acid diluted to 1 liter. Equal volumes of Solutions A and B are mixed before use. The reaction tubes were covered, heated at 90" for 15 min, and cooled at room temperature.
The absorbance at 460 nm was determined in a Gilford 240 spectrophotometer.
All assays were conducted under conditions such that citrulline production was directly proportional to the enzyme concentration for the time interval of the assay. Specific activity is expressed as micromoles of citrulline produced per min per mg of protein.
Protein concentration was estimated as described by Lowry et al. (30) using bovine serum albumin (Fraction Vl as the standard.

Isolation of Ornithine Transcarbamylase
Step 1: Homogenate-Five grams of liver were minced and handhomogenized (Ten-Broeck tissue grinder) in 15 ml of 10 rnM triethanolamine buffer, pH 8.0, containing 0.3% Triton X-100. This and all subsequent procedures were carried out at 4".
Step 2: Extract-Particulate debris was removed from the homogenate by centrifugation at 45,000 x g for 15 min. The pellet was washed with 5 ml of the homogenization buffer and centrifuged as before. The two supernatant fractions were combined. Step 3; DEAE-chromatography-The extract was applied to a column of DEAE (1.8 x 25 cm) equilibrated in 10 mM triethanolamine buffer, pH 8.0, according to Ref. 31. The ornithine transcarbamylase activity did not bind to the DEAE, and was completely eluted by washing the column with 30 ml of the equilibration buffer.
Step 4: Hydroxylapatite Chromatography -Fractions containing the ornithine transcarbamylase activity from the DEAE-column (35 ml) were combined and applied to a column containing Bio-Gel HTP (1.5 x 15 cm) equilibrated with 10 rnrvr potassium phosphate buffer, pH 7.0. The column was washed with 50 ml of 0.2 M potassium phosphate buffer, pH 7.0. Ornithine transcarbamylase activity was eluted with a 300.ml linear gradient of potassium phosphate, pH 7.0 (0.2 to 0.5 M), and 2.0-ml fractions were collected. The fractions containing ornithine transcarbamylase activity were pooled, concentrated, and dialyzed against 10 mM potassium phosphate buffer, pH 7.0, in an Amicon Diaflo cell (PM-10 membrane).
The final volume was 4 ml.
Step 5; Gel Filtration-The dialyzed preparation from hydroxylapatite was applied to a Sephadex G-200 column (2.5 x 90 cm) equilibrated in 10 rnM triethanolamine buffer, pH 8.0. The column was eluted (ascending) with the equilibration buffer, and 2.0-ml fractions were collected. Fractions containing ornithine transcarbamylase activity were pooled and concentrated in an Amicon Diaflo cell (PM-10 membrane).

Molecular
Weight Determination Initially the molecular weights were determined by calibration of the Sephadex G-200 column used for the final step in the purification scheme (Step 5). However, limiting amounts of available liver from the Reye's syndrome patient made multiple determinations impractical with enzyme of this purity.
Thus, extracts were prepared from 200 mg of liver in 10 mM triethanolamine buffer, pH 8.0, containing 0.3% Triton X-100. The extracts were applied to a Sephadex G-200 column (1.5 x 55 cm) equilibrated in 10 mM triethanolamine buffer, pH 8.0. The column was eluted (ascending) with the equilibration buffer, and 2.0-ml fractions were collected. more accurately the p1 of the enzyme. The results of this latter experiment are shown in Fig. 3. Only one peak of enzyme activity from each source is evident. The p1 values for ornithine transcarbamylase from normal and Reye's syndrome livers were 7.95 and 8.05, respectively. Similar results were found when crude extracts of human liver were subjected to electrofocusing.

Molecular Weight and Subunit
Composition -The molecular weight of human ornithine transcarbamylase from both normal and Reye's syndrome livers was determined by gel filtration.
No evidence was found in the present study for multiple species of the enzyme. The molecular weight of ornithine transcarbamylase from both sources was 110,000. The subunit composition was determined by polyacrylamide gel electrophoresis under dissociating conditions. Purified enzyme (from DEAF, hydroxylapatite, and electrofocusing) was dissociated as described under "Experimental Procedures" and subsequently electrophoresed.
The subunit molecular weight of ornithine transcarbamylase from both normal and Reye's syndrome livers was found to be 36,500 (Fig. 4) the native enzyme is comprised of three similar or identical subunits of 36,500 daltons.

DISCUSSION
The present study is concerned with the purification and characterization of ornithine transcarbamylase from human liver, and a comparison of the properties of the enzyme from normal human liver and from a liver of a patient with Reye's syndrome. The purification of ornithine transcarbamylase from both normal and Reye's syndrome livers was achieved by column chromatography.
While previous investigators have relied mainly on the solubility and heat stability properties of the enzyme (10, 13, 141, the present procedure utilized DEAEcellulose, hydroxylapatite, and Sephadex G-200 chromatography to achieve a simple and rapid purification.
The enzyme was purified approximately 150-fold from intact liver within 2 days.
Abnormal kinetic properties of ornithine transcarbamylase have been reported for two diseases characterized by hyperammonemia, Reye's syndrome and ornithine transcarbamylase deficiency disease. Investigators have speculated that in the latter disease a decreased affinity of the enzyme for L-ornithine or carbamyl phosphate results in the accumulation of toxic levels of ammonia (36,37). Similar abnormal kinetic data have been reported for ornithine transcarbamylase from one Reye's syndrome patient with an B-fold increase in the K,,, for L-ornithine (21). No kinetic abnormalities of purified ornithine transcarbamylase from Reye's syndrome liver were discerned in the present study ( Figs. 1 and 2). The apparent K,,, values for L-ornithine and carbamyl phosphate were 0.20 and 0.09 mM, respectively. These values are similar to previously reported values (26,27,36,38). There was no indication of any inhibition by either substrate as had been reported (15,27). The present study does not support the hypothesis of kinetic alterations in ornithine transcarbamylase which could result in the hyperammonemia characteristic of Reye's syndrome; however, since it is based on the kinetic evaluation of the enzyme from only one patient, the data presented do not categorically eliminate the possibility that kinetic variations could play a role in some cases of the disease. Various compounds were tested for possible modulating effects on ornithine transcarbamylase activity. I,-Norvaline (2aminopentanoic acid) is structurally similar to L-ornithine (2,5-diaminopentanoic acid) and serves as a competitive inhibitor (data not shown) of the human enzyme (Table III). Similar inhibition of the bovine enzyme has been reported (11). Interestingly, 2,4-diaminobutyrate is a relatively poor inhibitor, while L-lysine (2,6-diaminohexanoic acid) was noninhibitory at 20 mM. These results, coupled with the potent inhibition afforded by L-norvaline, strongly suggest that the length of the side chain is more important than the presence of the amino group on the side chain. This is further illustrated by the lack of inhibition by L-norleucine (2-aminohexanoic acid). The side chain of this aliphatic amino acid is only one methylene group longer than L-norvaline. The necessity for the oc-carboxyl and a-amino groups is demonstrated by the noninhibitory nature of 2,4-diaminobutane and 4-pentenoic acid, respectively.

Human Ornithine Transcarbamylase
Amino-4-pentenoic acid is structurally similar to L-norvaline, and is likewise a very potent inhibitor of human ornithine transcarbamylase activity (Table III). This compound also inhibits the enzyme in intact mitochondria.' Methylenecyclopropylacetic acid is a metabolite of hypoglytin and has been reported to be responsible for at least some of the symptoms of Jamaican vomiting disease (39). 4-Pentenoic acid is an analog of this compound, and when administered to rats, will produce most of the symptoms exhibited in Jamaican vomiting disease (42). 4-Pentenoic acid did not directly inhibit ornithine transcarbamylase from rat liver (25). Similarly, in the present study, no inhibitory effect was found with the purified human enzyme at concentrations up to 50 mM (data not shown).
Previous reports on the biophysical characteristics of ornithine transcarbamylase have demonstrated the presence of more than one species of the enzyme in the human liver. However, in the present study only one peak of enzyme activity was found by electrofocusing in either the normal or the diseased liver. The isoelectric points of ornithine transcarbamylase from normal and Reye's syndrome liver were 7.95 and 8.05, respectively. These values for purified human enzyme are in agreement with that reported by Reichard, who found a p1 of 8.0 for the enzyme isolated from rat liver (14). Arashima et al. (17) have demonstrated two major species of ornithine transcarbamylase in a crude human liver homogenate by electrofocusing. The isoelectric points of the two species were 3.2 and 4.4. The reason for the discrepancies in isoelectric points is unclear.
The native molecular weight of human ornithine transcarbamylase from both normal and Reye's syndrome livers was determined by gel filtration to be 110,000. This is in good agreement with molecular weight determinations of the bovine and rat enzymes measured by centrifugation methods (10, 13). Previously, it had been reported that human ornithine transcarbamylase can be resolved into two molecular weight species by gel filtration (16). Throughout the purification procedure of the present study as well as subsequent characterization, no evidence of multiple forms was found. The results of disc gel electrophoresis indicated that the enzyme from both normal and Reye's syndrome livers consists of three identical or similar subunits of 36,500 daltons. This unusual trimeric subunit arrangement was also reported for the rat and bovine ornithine transcarbamylase (10, 13). The purification and characterization of the human enzyme revealed many similarities to ornithine transcarbamylase isolated from two other mammalian liver sources, rat and bovine. The most striking of these properties are the native molecular weight, the apparent subunit structure, and the pattern of inhibition by L-ornithine-related compounds. In addition, ornithine transcarbamylase isolated from normal and Reye's syndrome livers was found to be identical for a variety of properties. Table IV summarizes the findings and demonstrates that no significant differences exist between the normal enzyme and that isolated from this particular case of Reye's syndrome. The similarities in enzyme activity and biophysical properties studied here suggest that the biochemical lesion associated with Reye's syndrome does not involve a functional alteration of ornithine transcarbamylase.
There are two possible alternatives: (a) an alteration in the first enzyme of the urea cycle, carbamyl phosphate synthetase, which is also located in the mitochondrion, and (6)