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Double deficiencies of urea cycle enzymes in human liver
HIOCHEMI~AI
Double
MEDI(
INE
21, ??6-233 (1979)
Deficiencies
of Urea Cycle Enzymes
in Human
Liver’
LLJISA RAIJMAN~ Department
of Biochemistry. 2025 Zonal
University of Southern California. A\aenue, Los Angeles. Cal@rnia
School 90033
qf Medicine.
Received January 4, 1979
In the last 20 years, congenital deficiencies affecting each one of the urea-cycle enzymes in human liver have been described (1). A characteristic of these diseases is a tendency of patients to develop hyperammonemia; in general, the severity of the hyperammonemia is greatest when carbamyl phosphate synthetase (ammonia) or ornithine transcarbamylase is affected (1). When argininosuccinate synthetase, argininosuccinate lyase, or arginase is affected, amino acid intermediates of the urea cycle formed by reactions preceding the enzymatic block tend to be elevated in blood and excreted in urine; it is possible, therefore, to arrive at reasonably certain identification of the biochemical lesion by examination of those fluids. In carbamyl phosphate synthetase (ammonia) and ornithine transcarbamylase deficiencies, no specific intermediates accumulate in blood, and a definitive diagnosis can be achieved only by measuring the activities of these enzymes in liver. The true incidence of these diseases is not known, due to the fact that they are often fatal in the neonatal period, and that neither the measurement of ammonia in blood nor of urea-cycle enzymes in liver is a routine procedure at present. Given the devastating effects of neonatal hyperammonemia (l), it is important to characterize all the mutations that result in defects in the urea-cycle enzymes, and to complete our knowledge of their mode of transmission. This paper describes studies on the livers of two hyperammonemic infants who died in the neonatal period. In each case, the liver is shown to be deficient in two urea-cycle enzyme activities. ’ Supported by Grant AM 19417 from the United States Public Health Service, National Institutes of Health, and an award from the National Reye’s Syndrome Foundation, Inc., Bryan, Ohio. * To whom all correspondence should be addressed. 226 000&2944/79/020226-08$02.00/O Copyright All @Is
@I 1979 by Academic Press, Inc. of reproduction in any form reserved.
UREA CYCLE ENZYME
MATERIALS
DEFICIENCIES
227
AND METHODS
Reagents. Ornithine transcarbamylase from Streptococcus faecalis, with a specific activity of 2500 pmole/min/mg protein, was prepared by the method of Nakamura and Jones (2); argininosuccinate lyase from steer liver, with a specific activity of 200 pmole/hr/mg protein, was prepared by the method of Ratner (3). Pyruvate kinase and glutamate dehydrogenase in glycerol were purchased from Boehringer-Mannheim, Indianapolis, Indiana; urease (type VI) and arginase3 from Sigma Chemical Company, St. Louis, Missouri. All other reagents were purchased from Boehringer-Mannheim, Sigma Chemical Company, or other sources of analytical quality reagents. Purified CPSase from human liver. This enzyme was purified by a method described for the rat liver enzyme (4), with modifications. Fractions of approximately 95% purity catalyzed the synthesis of 1.6 pmole of carbamyl phosphate per milligram protein per minute under the assay conditions described in (4) (L. Raijman, unpublished experiments). Tissue samples. Human liver samples of two infant boys who had severe perinatal hyperammonemia were obtained at autopsies performed within 2 hr after death. The fragments were immediately frozen and stored at -80°C. Control values were obtained from autopsy liver samples from three infants aged 1 hr, 6 weeks, and 3 months, who had died of respiratory distress, circulatory collapse, and consequences of birth trauma. Autopsies were done 6, 12, and 5 hr after death; the tissues obtained were frozen and stored as described above. Fresh rat liver was obtained from male Sprague-Dawley rats weighing 125 to 150 g, purchased from Mission Laboratory and Supplies, Rosemead, California. Preparation of tissue homogenates. Frozen portions of human liver or fresh rat liver were homogenized in 10 vol of ice-cold deionized water, using a Potter-Elvehjem homogenizer with a Teflon pestle, making 10 passes at 1100 t-pm. The homogenates were kept at 0°C for 5 min and then assayed simultaneously; all incubations for enzyme assays were completed within 90 min. The assays of enzyme activities in human liver were completed within 2 weeks after obtaining the tissue samples. Enzyme assays; general conditions. The amounts of liver homogenate used in each assay were sufficient to synthesize about 0.5 pmole or less of the measured reaction products; the reactions were linear with respect to enzyme concentration within this range of synthesis and for the duration of incubation. At least three different quantitites of homogenate were assayed in duplicate together with zero-time controls. All reactions were 3 Abbreviations: carbamyl phosphate synthetase (ammonia) (EC 2.7.2.5), CPSase; ornithine transcarbamylase (EC 2.1.3.3), OTCase; argininosuccinate synthetase (EC 6.3.4.5), ASase; argininosuccinate lyase (EC 4.3.2. I), ALase; arginase (EC 3.5.3. l), ARGase.
228
LUISA
RAIJMAN
stopped with 0.5 ml of 5 M HCIO,; precipitated protein was removed by centrifugation. Human and rat liver homogenates were assayed simultaneously. Assay of CPSase. All acidic reagents were neutralized to pH 7.4 with KOH. Assay mixtures contained 50 mM NH,HCO,, 5 mM acetylglutamate. 15 mM MgC&, 5 mM ornithine, 50 mM glycylglycine buffer, pH 7.4, 50 ,ug of OTCase, 50 kg of pyruvate kinase (ammonium sulfate suspension), and liver homogenate. The mixtures were preincubated at 37°C for 3 min. The reaction was then started by addition of 0.1 ml of a solution containing enough ATP and phosphoenolpyruvate to give final concentrations of 5 mM and 12.5 mM, respectively. Final volume was 1 ml; incubation was at 37°C for 15 min. Citrulline formed was measured calorimetrically (5). Assay of OTCase. This enzyme was assayed at pH 7.6 and 8.4. Assay mixtures contained 5 mM carbamyl phosphate; 5 mM ornithine; 200 mM triethanolamine, pH 7.6 or 8.4, and aliquots of a 1: 10 (v/v) dilution of liver homogenates in ice-cold water made immediately before addition. Final volume was 1 ml; incubation was at 37°C for 15 min. Citrulline formed was measured calorimetrically (5). In addition, OTCase was assayed in the presence of 2 to 20 mM carbamyl phosphate (with 5 mM ornithine), and of 2.5 to 20 mM ornithine (with 5 mM carbamyl phosphate). ASase, ALase, and ARGase were assayed by modifications of the methods of Schimke (6); only the modifications are described below. The ASase assay contained K-phosphate buffer at pH 7.5, 10 pg of pyruvate kinase in glycerol, 250 pg of ALase, and 300 pg of ARGase (which was not activated with Mn2+); final volume was 1 ml and incubation was at 37°C for 30 min. Urea formed was measured enzymatically. One milliliter of deproteinized reaction mixture was brought to about pH 9 with KOH followed by K&O3 (a very small amount of universal indicator was used). Volume changes due to the neutralization were recorded. These mixtures were placed in a desiccator over H.&SO, for approximately 4 hr. Residual ammonia and urea were measured spectrophotometrically as described by Gutmann and Bergmeyer (7). After the addition of 20 pg of urease, the reaction reached completion in 50-60 min. Controls were run to correct for small amounts of ammonia present in some urease preparations. The recovery of urea by this assay approaches 100%. The ALase assay contained 250 pg of ARGase not activated with Mn2+; final volume was 0.25 ml, and incubation was at 37°C for 15 min. Controls were run to correct for a small amount of ALase which might be present in the ARGase preparation. Urea formed was measured calorimetrically by the same method for citrulline (5). ARGase was activated by preincubation at 55°C for 15 min in the presence of 50 mM MnCl,. The ARGase assay contained a final concentra-
UREA
CYCLE
ENZYME
229
DEFICIENCIES
tion of 1 mM MnCl,; final volume was 1 ml, and incubation was at 37°C for 5 min. Urea formed was measured calorimetrically. Enzyme activities in liver are expressed as micromoles of measured produce formed per gram tissue per hour. The results obtained with these assays are comparable with those obtained by similar reported methods (6,8). Control values for normal rat and human liver also agree with reported values (6,8,9). Electrophoresis. Samples of liver homogenates or of purified human CPSase from normal liver, in a buffer containing 1% sodium dodecyl sulfate, 0.04 M dithiothreitol, 1 mM EDTA, and 0.04 M Tris-acetate, pH 6.4, were denatured by heating them at 100°C for 5 min; a few crystals of sucrose and a trace of tracking dye (pyronin Y) were added to the samples. Aliquots containing 50 to 100 pg of protein were applied to 6% acrylamide gels containing 0.2 M Tris-acetate buffer, pH 6.4, and 0.1% sodium dodecyl sulfate. The running buffer contained 0.2 M Tris-acetate, pH 6.4, and 0.1% sodium dodecyl sulfate. Electrophoresis was done under constant current, 4 mA per tube for 30 min, then 6-8 mA per tube until the tracking dye approached the bottom of the gels. The gels were fixed and stained by the method of Neville (10). Protein was measured by the method of Lowry et al. (11). RESULTS AND DISCUSSION Enzyme activities. The activities of the urea-cycle enzymes in the two samples of human liver are shown in Table 1. CPSase was undetectable in both liver samples. OTCase was markedly lower than the control range in the first case, and somewhat decreased in the second. The OTCase of the liver of case 1 behaved like the normal human enzyme as regards its activity at various concentrations of both substrates, and at pH 8.4. ASase was slightly lower than the control range in the first case, and TABLE ACT~WY
1
OF THE UREA CYCLE ENZYMES IN Two SAMPLES OF HUMAN LIVER Activity (pmole/g liver/hr)
Source of tissue
CPSase
Case 1 Case 2 Control 1 Control 2 Control 3
P P 125 250 140
Protein
OTCase
ASase
ALase
ARGase
(mdg liver)
67ob 2O.w 1680 3500 2300
53.7 12.0 54.4 69.0 65.0
200 240 150 170 110
20,000 32,100 20,000 24,200 22,000
125 135 115 130 108
a The limit of detection of the assay of CPSase is approximately b At pH 7.6, 5 mh+ omithine and 5 mM carbamyl phosphate.
7 pmole/g/hr.
230
LUISA
RAIJMAN
markedly decreased in the second. ALase was higher than the control range in both casesi ARGase activity was within the control range. Electrophoretic patterns. In the rat, CPSase is one of the major components of liver mitochondria, amounting to about 20% of the total protein of the mitochondrial matrix (4,12); it is also the only subunit polypeptide of molecular weight 165,000 to be found in large amounts in liver mitochondrial extracts (12). Consequently, rat liver CPSase can be easily identified by disc gel electrophoresis of mitochondrial extracts after protein denaturation with sodium dodecyl sulfate in the presence of dithiothreitol. The electrophoretic pattern of similarly treated extracts of human liver mitochondria also shows a major polypeptide of molecular weight 162,000 5 1000, which is the subunit molecular weight of purified human liver CPSase (L. Raijman, unpublished experiments). Since the CPSase activity of human liver is similar to that of rat liver (6,13) and the subunit molecular weight and molecular activity of human CPSase are essentially the same as those of the rat enzyme (12,4), it can be inferred that CPSase also constitutes a major portion of the mitochondrial proteins of human liver, and that the polypeptide of molecular weight 162,000 is primarily CPSase. The electrophoretic pattern of denatured human cytoplasm shows only one barely detectable component of molecular weight similar to that of CPSase. The amount of this polypeptide is small enough that its contribution to the electrophoretic pattern of denatured samples of whole human homogenates is negligible. Consequently, the subunit of CPSase can be identified by electrophoresis of denatured mitochondrial extracts or whole homogenates of human liver. Figure 1 shows the patterns obtained from homogenates of normal human liver and of the livers of cases 1 and 2, and from partially purified CPSase from a normal human liver. The pattern obtained from case 1 is indistinguishable from that of normal human liver, and contains a major distinct band with the same migration characteristics as purified CPSase. This band is absent from the pattern obtained from the liver of case 2. The existence of mutations affecting the activity of each of the ureacycle enzymes has been documented; in all cases but one, only one enzyme was significantly decreased. Palmer et al. (14) observed that females, but not males, with partial OTCase deficiencies tended to have decreased activity of CPSase; in one case, the exception mentioned above, CPSase activity was markedly decreased. The data reported in this paper partially characterize two different mutations affecting male infants; in each instance two urea-cycle enzymes 4 ALase activity was higher than the control range in the liver of 10 hyperammonemic children we have studied. In one case, it was as high as to form 320 pmoie of arginine per gram liver per hour.
FIG. 1. Disc gel electrophoresis of homogenates of human livers and of partially purified, normal human CPSase. (A) Shows the pattern of a sample of normal human liver homogenate containing 60 pg of denatured protein. (B) Shows the pattern obtained from a liver homogenate of case 1 (CPSase and OTCase deficiencies) on the center gel, and from partially purified human CPSase on the right gel; the samples applied to the gels contained 100 f 5 pg of protein. The arrows mark the front of the CPSase band.
,r$
232
LUISA RAIJMAN
are affected. In the first case, CPSase and OTCase activities are undetectable and markedly decreased, respectively; the residual OTCase does not appear to be either a K, or a pH variant of the normal enzyme. In the second case, CPSase and ASase activities are undetectable and greatly reduced, respectively. The two mutants differ further: In the first case, a polypeptide of the same molecular weight as CPSase is present in liver extracts, but is either enzymatically inactive or very unstable; in the second case, CPSase polypeptide is absent, suggesting either that the latter was not synthesized, or that it was a variant extremely susceptible to proteolytic degradation The frequency of mutations affecting the urea cycle enzymes is not known with certainty for a number of reasons discussed in (1). The incidence of the most common of these defects (ALase deficiency) is about I in !90,000 newborns (1); OTCase, and even more so CPSase and ASase deficiencies are thought to occur less frequently. On this basis, and provided that the true incidence of these diseases is not orders of magnitude greater than is believed at present, the probability that the findings reported here are due to double mutations is very low, of the order of 1 in lOlo. It appears more probable that in each case a single mutation at a different locus may have affected two enzymes. SUMMARY
The activities of the enzymes of the urea cycle were measured in liver samples from two infants with severe hyperammonemia who had died a few days after birth. In the first case, carbamyl phosphate synthetase (ammonia) activity was undetectable in liver homogenates, and ornithine transcarbamylase activity was significantly lower than that of human controls. In the second case, carbamyl phosphate synthetase (ammonia) was undetectable, and argininosuccinate synthetase activity was greatly diminished with respect to controls. The disc gel electrophoretic pattern of dodecyl sulfate-treated liver homogenates of the first case was indistinguishable from patterns obtained from normal infant livers, and contained a major band in the position normally occupied by carbamyl phosphate synthetase (ammonia) peptide, which in this case must be presumed to have been enzymatically inactive or unstable. The electrophoretic pattern of liver homogenates of the second case did not contain a similar peptide. ACKNOWLEDGMENTS I am grateful to Mr. Shing F. Kwan for excellent technical assistance, and to Drs. K. N. F. Shaw, G. N. Donnell, and H. Neustein, of Childrens Hospital of Los Angeles, for providing the samples of tissue used in this work.
UREA CYCLE ENZYME
DEFICIENCIES
233
REFERENCES 1. Shih, V. E., in “The Urea Cycle: Discovery and Present Status” (S. Grisolia, F. Mayor, and R. Baguena, Eds.), pp. 367-414 Wiley-Interscience, New York, 1976. 2. Nakamura, M., and Jones, M. E., in “Methods in Enzymology” (N. Kaplan, and S. P. Colowick, Eds.), Vol. 17A, pp. 286-294. Academic Press, New York, 1970. 3. Ratner, S., in “Methods in Enzymology” (N. Kaplan, and S. P. Colowick, Eds.), Vol. 17A, pp. 304-309. Academic Press, New York, 1970. 4. Raijman, L., and Jones, M. E., Arch. Biochem. Biophys. 175, 270-278 (1976). 5. Guthiihrlein, G., and Knappe, J., Anal. Biochem. 26, 188-191 (1%8). 6. Schimke, R. T., J. Biol. Chem. 237, 459-468 (1%2). 7. Gutmann, I., and Bergmeyer, H. U., in “Methods of Enzymatic Analysis” (H. U. Bergmeyer, Ed.), Vol. 4, pp. 1794-1798. Academic Press, New York, 1974. 8. Nuzum, C. T., and Snodgrass, P. J., in “‘The Urea Cycle: Discovery and Present Status” (S. Grisolia, F. Mayor, and R. Baguena, Eds.), pp. 325-349. WileyInterscience, New York, 1976. 9. Glick, N. R., Snodgrass, P. J., and Schafer, I. A., Amer. J. Human. Genet. 28,22-30 (1976).
10. Neville, D. M., Jr., J. Biol. Chem. 246, 6328-6334 (1971). 11. Lowry, 0. H., Rosebrough, N. J., Fat-r, A. C., and Randall, R. J., J. Biol. Chem. 193, 265-275 (1951). 12. Clarke, S., J. Biol. Chem. 251, 950-961 (1976). 13. Nuzum, C. T., and Snodgrass, P. J., Science 172, 1042-1043 (1971). 14. Palmer, T., Oberholzer, V. G., Burgess, E. A., Butler, L. J., and Levin, B., Arch. Dis. Child. 49, 443-449 (1974).
Double
MEDI(
INE
21, ??6-233 (1979)
Deficiencies
of Urea Cycle Enzymes
in Human
Liver’
LLJISA RAIJMAN~ Department
of Biochemistry. 2025 Zonal
University of Southern California. A\aenue, Los Angeles. Cal@rnia
School 90033
qf Medicine.
Received January 4, 1979
In the last 20 years, congenital deficiencies affecting each one of the urea-cycle enzymes in human liver have been described (1). A characteristic of these diseases is a tendency of patients to develop hyperammonemia; in general, the severity of the hyperammonemia is greatest when carbamyl phosphate synthetase (ammonia) or ornithine transcarbamylase is affected (1). When argininosuccinate synthetase, argininosuccinate lyase, or arginase is affected, amino acid intermediates of the urea cycle formed by reactions preceding the enzymatic block tend to be elevated in blood and excreted in urine; it is possible, therefore, to arrive at reasonably certain identification of the biochemical lesion by examination of those fluids. In carbamyl phosphate synthetase (ammonia) and ornithine transcarbamylase deficiencies, no specific intermediates accumulate in blood, and a definitive diagnosis can be achieved only by measuring the activities of these enzymes in liver. The true incidence of these diseases is not known, due to the fact that they are often fatal in the neonatal period, and that neither the measurement of ammonia in blood nor of urea-cycle enzymes in liver is a routine procedure at present. Given the devastating effects of neonatal hyperammonemia (l), it is important to characterize all the mutations that result in defects in the urea-cycle enzymes, and to complete our knowledge of their mode of transmission. This paper describes studies on the livers of two hyperammonemic infants who died in the neonatal period. In each case, the liver is shown to be deficient in two urea-cycle enzyme activities. ’ Supported by Grant AM 19417 from the United States Public Health Service, National Institutes of Health, and an award from the National Reye’s Syndrome Foundation, Inc., Bryan, Ohio. * To whom all correspondence should be addressed. 226 000&2944/79/020226-08$02.00/O Copyright All @Is
@I 1979 by Academic Press, Inc. of reproduction in any form reserved.
UREA CYCLE ENZYME
MATERIALS
DEFICIENCIES
227
AND METHODS
Reagents. Ornithine transcarbamylase from Streptococcus faecalis, with a specific activity of 2500 pmole/min/mg protein, was prepared by the method of Nakamura and Jones (2); argininosuccinate lyase from steer liver, with a specific activity of 200 pmole/hr/mg protein, was prepared by the method of Ratner (3). Pyruvate kinase and glutamate dehydrogenase in glycerol were purchased from Boehringer-Mannheim, Indianapolis, Indiana; urease (type VI) and arginase3 from Sigma Chemical Company, St. Louis, Missouri. All other reagents were purchased from Boehringer-Mannheim, Sigma Chemical Company, or other sources of analytical quality reagents. Purified CPSase from human liver. This enzyme was purified by a method described for the rat liver enzyme (4), with modifications. Fractions of approximately 95% purity catalyzed the synthesis of 1.6 pmole of carbamyl phosphate per milligram protein per minute under the assay conditions described in (4) (L. Raijman, unpublished experiments). Tissue samples. Human liver samples of two infant boys who had severe perinatal hyperammonemia were obtained at autopsies performed within 2 hr after death. The fragments were immediately frozen and stored at -80°C. Control values were obtained from autopsy liver samples from three infants aged 1 hr, 6 weeks, and 3 months, who had died of respiratory distress, circulatory collapse, and consequences of birth trauma. Autopsies were done 6, 12, and 5 hr after death; the tissues obtained were frozen and stored as described above. Fresh rat liver was obtained from male Sprague-Dawley rats weighing 125 to 150 g, purchased from Mission Laboratory and Supplies, Rosemead, California. Preparation of tissue homogenates. Frozen portions of human liver or fresh rat liver were homogenized in 10 vol of ice-cold deionized water, using a Potter-Elvehjem homogenizer with a Teflon pestle, making 10 passes at 1100 t-pm. The homogenates were kept at 0°C for 5 min and then assayed simultaneously; all incubations for enzyme assays were completed within 90 min. The assays of enzyme activities in human liver were completed within 2 weeks after obtaining the tissue samples. Enzyme assays; general conditions. The amounts of liver homogenate used in each assay were sufficient to synthesize about 0.5 pmole or less of the measured reaction products; the reactions were linear with respect to enzyme concentration within this range of synthesis and for the duration of incubation. At least three different quantitites of homogenate were assayed in duplicate together with zero-time controls. All reactions were 3 Abbreviations: carbamyl phosphate synthetase (ammonia) (EC 2.7.2.5), CPSase; ornithine transcarbamylase (EC 2.1.3.3), OTCase; argininosuccinate synthetase (EC 6.3.4.5), ASase; argininosuccinate lyase (EC 4.3.2. I), ALase; arginase (EC 3.5.3. l), ARGase.
228
LUISA
RAIJMAN
stopped with 0.5 ml of 5 M HCIO,; precipitated protein was removed by centrifugation. Human and rat liver homogenates were assayed simultaneously. Assay of CPSase. All acidic reagents were neutralized to pH 7.4 with KOH. Assay mixtures contained 50 mM NH,HCO,, 5 mM acetylglutamate. 15 mM MgC&, 5 mM ornithine, 50 mM glycylglycine buffer, pH 7.4, 50 ,ug of OTCase, 50 kg of pyruvate kinase (ammonium sulfate suspension), and liver homogenate. The mixtures were preincubated at 37°C for 3 min. The reaction was then started by addition of 0.1 ml of a solution containing enough ATP and phosphoenolpyruvate to give final concentrations of 5 mM and 12.5 mM, respectively. Final volume was 1 ml; incubation was at 37°C for 15 min. Citrulline formed was measured calorimetrically (5). Assay of OTCase. This enzyme was assayed at pH 7.6 and 8.4. Assay mixtures contained 5 mM carbamyl phosphate; 5 mM ornithine; 200 mM triethanolamine, pH 7.6 or 8.4, and aliquots of a 1: 10 (v/v) dilution of liver homogenates in ice-cold water made immediately before addition. Final volume was 1 ml; incubation was at 37°C for 15 min. Citrulline formed was measured calorimetrically (5). In addition, OTCase was assayed in the presence of 2 to 20 mM carbamyl phosphate (with 5 mM ornithine), and of 2.5 to 20 mM ornithine (with 5 mM carbamyl phosphate). ASase, ALase, and ARGase were assayed by modifications of the methods of Schimke (6); only the modifications are described below. The ASase assay contained K-phosphate buffer at pH 7.5, 10 pg of pyruvate kinase in glycerol, 250 pg of ALase, and 300 pg of ARGase (which was not activated with Mn2+); final volume was 1 ml and incubation was at 37°C for 30 min. Urea formed was measured enzymatically. One milliliter of deproteinized reaction mixture was brought to about pH 9 with KOH followed by K&O3 (a very small amount of universal indicator was used). Volume changes due to the neutralization were recorded. These mixtures were placed in a desiccator over H.&SO, for approximately 4 hr. Residual ammonia and urea were measured spectrophotometrically as described by Gutmann and Bergmeyer (7). After the addition of 20 pg of urease, the reaction reached completion in 50-60 min. Controls were run to correct for small amounts of ammonia present in some urease preparations. The recovery of urea by this assay approaches 100%. The ALase assay contained 250 pg of ARGase not activated with Mn2+; final volume was 0.25 ml, and incubation was at 37°C for 15 min. Controls were run to correct for a small amount of ALase which might be present in the ARGase preparation. Urea formed was measured calorimetrically by the same method for citrulline (5). ARGase was activated by preincubation at 55°C for 15 min in the presence of 50 mM MnCl,. The ARGase assay contained a final concentra-
UREA
CYCLE
ENZYME
229
DEFICIENCIES
tion of 1 mM MnCl,; final volume was 1 ml, and incubation was at 37°C for 5 min. Urea formed was measured calorimetrically. Enzyme activities in liver are expressed as micromoles of measured produce formed per gram tissue per hour. The results obtained with these assays are comparable with those obtained by similar reported methods (6,8). Control values for normal rat and human liver also agree with reported values (6,8,9). Electrophoresis. Samples of liver homogenates or of purified human CPSase from normal liver, in a buffer containing 1% sodium dodecyl sulfate, 0.04 M dithiothreitol, 1 mM EDTA, and 0.04 M Tris-acetate, pH 6.4, were denatured by heating them at 100°C for 5 min; a few crystals of sucrose and a trace of tracking dye (pyronin Y) were added to the samples. Aliquots containing 50 to 100 pg of protein were applied to 6% acrylamide gels containing 0.2 M Tris-acetate buffer, pH 6.4, and 0.1% sodium dodecyl sulfate. The running buffer contained 0.2 M Tris-acetate, pH 6.4, and 0.1% sodium dodecyl sulfate. Electrophoresis was done under constant current, 4 mA per tube for 30 min, then 6-8 mA per tube until the tracking dye approached the bottom of the gels. The gels were fixed and stained by the method of Neville (10). Protein was measured by the method of Lowry et al. (11). RESULTS AND DISCUSSION Enzyme activities. The activities of the urea-cycle enzymes in the two samples of human liver are shown in Table 1. CPSase was undetectable in both liver samples. OTCase was markedly lower than the control range in the first case, and somewhat decreased in the second. The OTCase of the liver of case 1 behaved like the normal human enzyme as regards its activity at various concentrations of both substrates, and at pH 8.4. ASase was slightly lower than the control range in the first case, and TABLE ACT~WY
1
OF THE UREA CYCLE ENZYMES IN Two SAMPLES OF HUMAN LIVER Activity (pmole/g liver/hr)
Source of tissue
CPSase
Case 1 Case 2 Control 1 Control 2 Control 3
P P 125 250 140
Protein
OTCase
ASase
ALase
ARGase
(mdg liver)
67ob 2O.w 1680 3500 2300
53.7 12.0 54.4 69.0 65.0
200 240 150 170 110
20,000 32,100 20,000 24,200 22,000
125 135 115 130 108
a The limit of detection of the assay of CPSase is approximately b At pH 7.6, 5 mh+ omithine and 5 mM carbamyl phosphate.
7 pmole/g/hr.
230
LUISA
RAIJMAN
markedly decreased in the second. ALase was higher than the control range in both casesi ARGase activity was within the control range. Electrophoretic patterns. In the rat, CPSase is one of the major components of liver mitochondria, amounting to about 20% of the total protein of the mitochondrial matrix (4,12); it is also the only subunit polypeptide of molecular weight 165,000 to be found in large amounts in liver mitochondrial extracts (12). Consequently, rat liver CPSase can be easily identified by disc gel electrophoresis of mitochondrial extracts after protein denaturation with sodium dodecyl sulfate in the presence of dithiothreitol. The electrophoretic pattern of similarly treated extracts of human liver mitochondria also shows a major polypeptide of molecular weight 162,000 5 1000, which is the subunit molecular weight of purified human liver CPSase (L. Raijman, unpublished experiments). Since the CPSase activity of human liver is similar to that of rat liver (6,13) and the subunit molecular weight and molecular activity of human CPSase are essentially the same as those of the rat enzyme (12,4), it can be inferred that CPSase also constitutes a major portion of the mitochondrial proteins of human liver, and that the polypeptide of molecular weight 162,000 is primarily CPSase. The electrophoretic pattern of denatured human cytoplasm shows only one barely detectable component of molecular weight similar to that of CPSase. The amount of this polypeptide is small enough that its contribution to the electrophoretic pattern of denatured samples of whole human homogenates is negligible. Consequently, the subunit of CPSase can be identified by electrophoresis of denatured mitochondrial extracts or whole homogenates of human liver. Figure 1 shows the patterns obtained from homogenates of normal human liver and of the livers of cases 1 and 2, and from partially purified CPSase from a normal human liver. The pattern obtained from case 1 is indistinguishable from that of normal human liver, and contains a major distinct band with the same migration characteristics as purified CPSase. This band is absent from the pattern obtained from the liver of case 2. The existence of mutations affecting the activity of each of the ureacycle enzymes has been documented; in all cases but one, only one enzyme was significantly decreased. Palmer et al. (14) observed that females, but not males, with partial OTCase deficiencies tended to have decreased activity of CPSase; in one case, the exception mentioned above, CPSase activity was markedly decreased. The data reported in this paper partially characterize two different mutations affecting male infants; in each instance two urea-cycle enzymes 4 ALase activity was higher than the control range in the liver of 10 hyperammonemic children we have studied. In one case, it was as high as to form 320 pmoie of arginine per gram liver per hour.
FIG. 1. Disc gel electrophoresis of homogenates of human livers and of partially purified, normal human CPSase. (A) Shows the pattern of a sample of normal human liver homogenate containing 60 pg of denatured protein. (B) Shows the pattern obtained from a liver homogenate of case 1 (CPSase and OTCase deficiencies) on the center gel, and from partially purified human CPSase on the right gel; the samples applied to the gels contained 100 f 5 pg of protein. The arrows mark the front of the CPSase band.
,r$
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LUISA RAIJMAN
are affected. In the first case, CPSase and OTCase activities are undetectable and markedly decreased, respectively; the residual OTCase does not appear to be either a K, or a pH variant of the normal enzyme. In the second case, CPSase and ASase activities are undetectable and greatly reduced, respectively. The two mutants differ further: In the first case, a polypeptide of the same molecular weight as CPSase is present in liver extracts, but is either enzymatically inactive or very unstable; in the second case, CPSase polypeptide is absent, suggesting either that the latter was not synthesized, or that it was a variant extremely susceptible to proteolytic degradation The frequency of mutations affecting the urea cycle enzymes is not known with certainty for a number of reasons discussed in (1). The incidence of the most common of these defects (ALase deficiency) is about I in !90,000 newborns (1); OTCase, and even more so CPSase and ASase deficiencies are thought to occur less frequently. On this basis, and provided that the true incidence of these diseases is not orders of magnitude greater than is believed at present, the probability that the findings reported here are due to double mutations is very low, of the order of 1 in lOlo. It appears more probable that in each case a single mutation at a different locus may have affected two enzymes. SUMMARY
The activities of the enzymes of the urea cycle were measured in liver samples from two infants with severe hyperammonemia who had died a few days after birth. In the first case, carbamyl phosphate synthetase (ammonia) activity was undetectable in liver homogenates, and ornithine transcarbamylase activity was significantly lower than that of human controls. In the second case, carbamyl phosphate synthetase (ammonia) was undetectable, and argininosuccinate synthetase activity was greatly diminished with respect to controls. The disc gel electrophoretic pattern of dodecyl sulfate-treated liver homogenates of the first case was indistinguishable from patterns obtained from normal infant livers, and contained a major band in the position normally occupied by carbamyl phosphate synthetase (ammonia) peptide, which in this case must be presumed to have been enzymatically inactive or unstable. The electrophoretic pattern of liver homogenates of the second case did not contain a similar peptide. ACKNOWLEDGMENTS I am grateful to Mr. Shing F. Kwan for excellent technical assistance, and to Drs. K. N. F. Shaw, G. N. Donnell, and H. Neustein, of Childrens Hospital of Los Angeles, for providing the samples of tissue used in this work.
UREA CYCLE ENZYME
DEFICIENCIES
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