IN DILUTE-LETHAL MICE

Transcription

1 PHENYLALANINE METABOLISM AND PHENYLKETONURIA IN DILUTE-LETHAL MICE V. G. ZANNONI, W. W. WEBER,1 P. VAN VALEN, A. RUBIN, R. BERNSTEIN AND B. N. LA DU Department of Pharmacology, New York University Medical School, New, York, New York Received July 25, 1966 MICE homozygous for the dilute-lethal (8) gene have been reported by COLEMAN (1960) and RAUCH and YOST (1963) to have low liver phenylalanine hydroxylase activity. This deficiency in phenylalanine metabolism has been reported to be due to the presence of an inhibitor of the hydroxylase localized in a particulate fraction of liver (15,000 x g sediment). These investigators have pointed out that the enzymatic deficiency and several neurological disturbances of dilute-lethal homozygotes resemble phenylketonuria in humans. The condition is inherited as an autosomal Mendelian recessive character. The mice are normal at birth, but opisthotonic seizures appear when about 14 days old and occur with increased frequency and severity until death at about 3 weeks of age. KELTON and RAUCH (1962) report that myelin formation is initially normal but myelin degeneration occurs shortly therea ter, a few days before the onset of the abnormal neurological signs and continues until death. The hair of dilute-lethal homozygotes is also pale as in homozygotes for the nonlethal dilute (d) allele. This dilution is apparently due to the abnormal clumping of melanin granules around the nucleus rather than a reduction in the amount of pigment ( MARKERT and SILVERS 1956). Further investigations with these animals, as a model of experimental phenylketonuria, are of considerable interest in studying the relationship between the biochemical disturbances and the central nervous system pathology in phenylketonuria. This paper presents comparative biochemical studies using stocks of three independent dilute-lethal mutations. The concentration of phenylalanine in blood, phenylpyruvic acid excreted in urine and liver phenylalanine hydroxylase activity are compared. Differences in susceptibility of liver phenylalanine hydroxylase to inhibition by L-phenylalanine and phenylpyruvic acid will be described. A preliminary report of this work has been presented (WEBER, VAN VALEN, LA Du and ZANNONI 1965). MATERIALS AND METHODS Enzyme assays: Phenylalanine hydroxylase activity in homogenates of mouse liver was based on the method of UDENFRIEND and COOPER (1952.a). Mouse livers were homogenized in a Potter- Reupient of Career Scientist Award of the Health Research Councll of the City of New Pork under contract # I441 Genetics 54 : December 196G

2 1392 v. G. ZANNONI et al. Elvehjem homogenizer in cold isotonic (1.15%) KC1, ph 7.0 to yield a 20% crude homogenate. For most of the studies reported in this paper 0.25 ml of crude homogenate was used. Incubations were carried out in 25 ml Erlenmeyer flasks containing phosphate buffer, 0.2~, ph 7.0; nicotinamide, 5 pmoles; diphosphopyridine nucleotide (DPN), 0.6 pmole. The experimental flasks contained 2.0 pmoles of L-phenylalanine. The reaction was started by the addition of an equal aliquot of the homogenate to both control and experimental flasks. The total volume of the reaction mixture was 1.5 ml. The flasks were incubated at 25" for 60 minutes in a Dubnoff shaker, in air. After the incubation the reaction mixtures were deproteinized with 0.5 ml of 20% trichloroacetic acid. After centrifugation, 1.0 ml of the supernatant fraction was used to determine the amount of tyrosine formed by the colorimetric reaction with l-nitroso-2-naphthol ( UDENFIUEND and COOPER 1952a, b). Tyrosine transaminase activity was measured spectrophotometrically at 25" by following the appearance of the enol-borate complex of p-hydroxyphenylpyruvic acid at 310 mp according to the method of LIN and KNOX (1957). p-hydroxyphenylpyruvic acid oxidase activity was measured spectrophotometrically at 25" by following the disappearance of the enol-borate complex of p-hydroxyphenylpyruvic acid at 308 mp as previously described (ZANNONI and LA Du 1960). Determination of phenylalanine and phenylpyruvic acid in blood and urine: Heparinized blood samples ( ml) were deproteinized by adding an equal volume of 7% perchloric acid. The protein free supernatant fraction was neutralized with 14% KOH and after standing, perchlorate was removed by centrifugation. The clear supernatant fraction was then analyzed for phenylalanine by the enzymatic spectrophotometric method of LA Du and MICHAEL (1960) and phenylpyruvic acid was determined by the same method except that snake venom L-amino acid oxidase was omitted. Pooled urine samples from animals of each genotype were also analyzed for phenylpyruvic acid. In these analyses aliquots of urine (0.01 to 0.03 ml) were used directly. Protei= Determinations for protein were made by the colorimetric method of LOWRY, ROSE- BROUGH, FARR and RANDALL (1951) and crystalline bovine serum albumin (Fraction V) was used as the standard. Although enzyme activity values are expressed in terms of wet weight of liver, these values were all determined on a protein basis. Genetic background of dilute-iethal mice: The dilute locus is in linkage group I1 of the house mouse (Mus musculus), closely linked to the recessive gene short-ear (se) with a crossover frequency of 0.15% (GOODWINS and VINCENT 1955). Animals carrying the wild-type allele, (D), at this locus have normal coat color, and animals homozygous for the recessive allele (d) have a marked dilution of color. The dilute-lethal mutant stocks used in the experiments described are: 1. dlse/dse and dlse/dse. The mutant stock, dlse/dse was obtained from Jackson Laboratory, Bar Harbor, Maine, and was similar to that used by COLEMAN (1960). The dse genes were introduced when the stock was being expanded for experimentation. 2. 'd'se/dse. A mutant stock obtained from the Oak Ridge National Laboratory; produced by irradiation; mutant 14 FATw. 3. di5se/dse. A mutant stock obtained from the Hanvell Laboratories, England. Dilution of coat color is less and survival is about one week longer in the d15/d15 animals than in the dl/dz or the 'd/,d' animals. In each of the three lines animals homozygous for the respective dilute-lethal allele were obtained by breeding heterozygous parents. The three expected genotypes were classified using the short-ear marker and coat color (when applicable). Mice homozygous for the dilute-lethal alleles were always distinguished by repeated observation of their abnormal neurological behavior. Animals too young to show these signs were not used. The young mice were not separated from their mothers prior to the experiment. Reagents: L-phenylalanine, L-tyrosine, phenylpyruvic acid, nicotinamide and diphosphopyridine nucleotide (DPN) were obtained from Nutritional Biochemical Corporation, Cleveland, Ohio. RESULTS Blood phenylalanine concentration and urine phen ylpyruzric acid excretion in dilute-lethal mice: Previous reports have suggested that dilute-lethal homozygotes

3 PHENYLKETONURIA IN DILUTE-LETHAL MICE 1393 TABLE 1 Blood phenylalanine and urinary phenylpyruvic acid in mice days old Stock Bar Harbor Oak Ridge Harwell Genotype dl/dl* dl/d,?, I * d /d d15/d15* d15/d Blood phenylalanine (mg/lw ml) !r c k t: Urinary phenylpyruvic acid (mg/ml)t %0.20 * Dilute-lethal genotype. The number of animals is given in parentheses. + Blood phenylalanine levels were determined by the method of LA Du and MICHAEL (1960). Urinary phenylpyruvic acid was measured daily on poled samples by the same method with snake venom L-amino acid oxidase omitted. Values for blood phenylalanine are given as means i- standard deviation. have elevated levels of blood phenylalanine and increased excretion of phenylalanine metabolites such as phenylpyruvic acid but quantitative data establishing these biochemical alterations have not been supplied. These measurements have been made in the various genotypes of the three stocks of dilute-lethal mice and the results are summarized in Table 1. Determinations were made in mice days of age since it is at this time that other investigators have found liver phenylalanine hydroxylase to be inhibited (COLEMAN 1960; RAUCH and YOST 1963). However, as can be observed, there was no significant increase in blood phenylalanine concentration or urinary prenylpyruvic acid excretion in animals of any of the genotypes. The blood phenylalanine concentration and urine phenylpyruvic acid excretion in animals homozygous for the dilute-lethal alleles (d /dz, d15/p, d / d ) were not significantly different from those of the other genotypes. TABLE 2 Liuer phenylalanine hydroxylase in mice 21 to 26 days old Stock Genotype Phenylalanine hydroxylase+ (pmoles tyrosine formed/hr/g liver Bar Harbor dl/dl* dl/d Oak Ridge I t I* I Hanvell dl 5/&5 * d15/d & f f * * f f * Dilute-lethal genotype. The number of animals is given in parentheses. + Phenylalanine hydroxylase activity was assayed with 0.25 ml 20% crude homogenate as described in M.ATERIALS and XCTHOUS. Values are given as means rt standard deviation.

4 1394 v. G. ZANNONI et al. Liver phenylalanine hydroxylase activity in dilute-lethal mice: Since the blood phenylalanine concentration and phenylpyruvic acid in the urine were not significantly increased in the dilute-lethal homozygotes it was essential to determine the activity of liver phenylalanine hydroxylase in animals of these various genotypes (Table 2). The data indicate that there is no significant decrease or inhibition of liver phenylalanine hydroxylase in crude liver homogenates. The similarity in liver hydroxylase activity in all of these animals is in accord with our data on blood phenylalanine concentration and phenylpyruvic acid excretion in these animals (Table 1 ). However, these data are not in agreement with the previously reported findings of COLEMAN (1960) and RAUCH and YOST (1963) and these differences led to further investigations. COLEMAN (1960) reported the presence of an inhibitor in the 15,000 x g particulate fraction of liver to account for phenylalanine hydroxylase inhibition in crude liver homogenates. Therefore a comparison was made of hydroxylase activity in both crude homogenates and 15,000 X g supernatant fractions in liver preparations of animals of various genotypes (Table 3). Phenylalanine hydroxylase activity was found to be lower in crude liver homogenates than in the 15,000 x g supernatant fractions in all genotypes. Furthermore, this difference was also observed in liver homogenates prepared from dog, rat and guinea pig. However, there was no indication that crude liver homogenates prepared from dilute-lethal mice had a more potent particulate fraction inhibitor than was present in the animals of the other genotypes. Inhibition of liver phenylalanine hydroxylase activity by L-phenylalanine or phenylpyruuic acid: Although there was no significant difference in phenylalanine hydroxylase activity between dilute-lethal homozygotes and the other genotypes in the three stocks, the possibility was considered that a dietary factor might explain the greater inhibition in the animals homozygous for the dilute- TABLE 3 Liver phenylalanine hydroxylase in crude homogenate and 15,000 X. g supernatant fraction of mice, rat, dog and guinea pig Species Genotype.- Phenylalanine hydroxylase' (#moles tyrosine formed/hr/g liver) Crude 15,ODoXg Percent homogenate supematant inhibition+ Mice$ Homozygous dilute-lethal 'd'/,d' % Homozygous % Homozygous dilute-lethal d15/d % Heterozygous d15/d % Homozygous % Rat % Dog... 1.o % Guinea Pig % * Phenylalanine hydroxylase activity was assayed with 0.50 ml of 20% "de homogenate or 0.50 ml of the 15,OOOXg supernatant fraction as desuibed in MATERIALS and METHODS. Each value gwen for the mice was determined on a sample of 6 to 8 pooled livers. t %, Inhibition == (activity in 15,OOOxg supernatant fraction - activity in crude homogenate) X 100. activity in 15,DoOXg supernatant fraction $ Mice, 21 to 26 days old.

5 6< PHENYLKETONURIA IN DILUTE-LETHAL MICE 1395 TABLE 4 Inhibition of liver phenylalanine hydroxylase after intraperitoneal injection of L-phenylalanine* Phenylalanine hydroxylasei (pmoles tyrosine formed/hr/g liver) Genotype No I.P. L-phenylalanine I.P. L-phenylalanine Homozygous (lethal) d / d (13)$ 1.03 rt rt 0.15 (12)$ Heterozygous Id /, ( 14) 1.06 * k 0.20 (8) Homozygous ( 13) 1.15 & * 0.14 (9) * Mice, 21 to 26 days old, received an intraperitoneal (I.P.) injection of L-phenylalanine (10 mg in saline/lo g body weight) one hour before sacrifice. Blood samples were also taken at this time and analyzed for phenylalanine by the method of L.4 Du and MICHAEL (1960); the values of phenylalanine in the blood of the injected ammals ranged hom mg/100 ml. -1 Phenylalanine hydroxylase activity was assayed with 0.25 ml of 20% crude liver homogenate as described in ILIZTER14LS and METHODS. Values are given as means standard deviation. Nnmber in parenthesesxnumber of animals. lethal alleles as was observed by the other investigators. Therefore, it was of interest to determine how the animals of the different genotypes would respond to either a load of L-phenylalanine or a metabolite of the amino acid, such as phenylpyruvic acid. The results of intraperitoneal injection of L-phenylalanine are shown in Table 4. L-phenylalanine caused greater inhibition of hydroxylase activity (83%) in dilute-lethal homozygotes than in the animals of the other genotypes (44 to 49%). Even more striking differences were observed following an intraperitoneal injection of phenylpyruvic acid (Table 5). The latter metabolite of L-phenylalanine completely inhibited hydroxylase activity in the dilutelethal homozygotes; d / d ( >90% ) but this compound produced no hydroxylase inhibition when injected into heterozygotes, d /d. Blood phenylalanine concentrations were markedly elevated in both groups of animals and it is evident that they are both able to convert phenylpyruvic acid to L-phenylalanine very efficiently. The effect of supplementing the diet with extra L-phenylalanine was also determined. The diet was supplemented daily with L-phenylalanine added to the drinking water (15 to 20 mg/ml) for one week. The mice remained with their mothers during this period but were drinking the supplemented water. In addition TABLE 5 Inhibition of liver phenylalanine hydroxylase after intraperitoneal injection of phenylpyruvic acid* Phenylalanine hydroxylase Blood after I.P. phenylpyruvic acid+ phenylalaninet Genotype (pmoles tyrosine fonned/hr/g liver) (mg/lm ml) Homozygous (lethal) d / d (8) 0.12 rt Heterozygous d /d (8) Mice, 21 to 26 days old, received an intraperitoneal (1.P.) injection of phenylpyruvic acid (IO mg in saline/lo g body weight) one hour before sacrifice. t Phenylalanine hydroxylase activity was assayed with 0.5 ml of 20% liver mouse homogenate as described in MATERIALS and METHODS. Values are given as means k standard deviation. Liver phenylalanine hydroxylase activity in animals not injected with phenylpyruvic add was $ Blood samples were taken at the time of sacrifice and phenylalanine levels were determined by the method of LA Du and MICHAEL (1960).

6 ._.~ 1396 v. G. ZANNONI et al. TABLE 6 Inhibition of liver phenylalanine hydroxylase after feeding L-phenylalanine* Phenylalanine hydroxylase+ Blood Urinary phenyl- (pmoles tyrosine phenylalanine$ pyruvic acid: Genotype formed/hr/g liver) (mg/100 ml) (nig/ml) Homozygous (lethal) d15/d15 (12) Heterozygous d15/d (11) 1.69 < Homozygous (12) 1.71 < A group of ten mice (d'5/&5), 21 to 26 days old, not receiving extra L-phenylalanine had liver phenylalanine hydroxylase activity equal to 1.60 pmoles tyrosine formed/hr/g liver. * The diet was supplemented daily with L-phenylalanine added to the drinking water (15 to 20 mg/ml) for one week. The animals were sacrificed on the eighth day, +Phenylalanine hydroxylase activity was assayed with 0.50 ml of 20% crude liver homogenate as described in MATHRIAI.S and METHODS. Average values are given. $ Blood samples were taken at time of sacrifice and phenylalanine levels were determined by the method of LA DU and MICH~EL (1960). Urinary phenylpyruvic acid war; measured daily on pooled samples by the same method with the snake venom r-amino acid oxidase omitted. Average values are given. each animal was given orally 0.2 ml to 0.3 ml of the same solution of L-phenylalanine per day. As with intraperitoneal injection of L-phenylalanine the liver hydroxylase activity of the dilute-lethal homozygotes was markedly inhibited and both the blood phenylalanine concentration and urine phenylpyruvic acid excretion were elevated (Table 6). In contrast, liver phenylalanine hydroxylase activity, blood phenylalanine concentration and urinary phenylpyruvic acid excretion were not significantly different from the values obtained on the regular diet when animals of the other genotypes were fed L-phenylalanine. It is apparent from the data presented in Tables 5 and 6 that liver phenylalanine hydroxylase activity is markedly lower in homozygous dilute-lethal mice compared to animals of the other genotypes when either the L-phenylalanine intake is increased or when phenylpyruvic acid is given. It is also of interest that other enzymes in tyrosine metabolism, such as tyrosine transaminase and p-hydroxyphenylpymvic acid oxidase, were not significantly altered in either dilute-lethal homozygotes or the other Eenotypes following an increased intake of L-phenylalanine. The inhibition of phenylalanine hydroxylase activity in animals which had TABLE 7 Liuer phenylalanine hydroxylase actiuity in crud? homogenates and 15,000 x g supernatant fractions after intraperitoneal injection of L-phenylalanins* -. ~ Phenylalanine hydroxylaset (pmoles tyrosine fomled/hr/g liver) No I.P. phenylalanine - ~ After I.P. phenylalanine ~_~ Crude 15,000Xg super- Crude 15,000Xg super- Genotype homogenate natant fraction homogenate natant fraction ~ ~ Homozygous (lethal) 'd'/'d' <0.05 <0.05 Homozygous * Mice, 21 to 26 days old received an intraperitoneal (I.P.) injection of L-phenylalanine (10 mg in saline/lo g body weight) one hour before sacrifice. At this time, livers were removed, homogenized in 1.15% saline to give a 20% crude homogenate. A portion of the homogenate was centrifuged at 15,000 x g for 30 minutes at 4". The resulting supematant fraction was used in the assay, t Phenylalanine hydroxylase activity was assayed with 0.25 ml of 20% crude homogenate or with 0.25 ml of the 15,OOOXg supernatant fraction as described in MATERIALS and METHODS.

7 < L PHENYLKETONURIA IN DILUTE-LETHAL MICE 1397 received L-phenylalanine was observed using crude liver homogenate preparations. It was of interest to determine whether there existed a relationship between this inhibition and the particulate fraction inhibitor described by COLEMAN (1960). Therefore, a comparison was made of hydroxylase activity in crude homogenate5 and 15,000 x g supernatant fractions prepared from dilute-lethal homozygotes after they had received an intraperitoneal injection of L-phenylalanine (Table 7). Neither the crude homogenate nor the 15,000 x g supernatant fraction had any detectable hydroxylase activity. Furthermore, crude homogenates prepared from the homozygous () control animals had phenylalanine hydroxylase activity inhibited to approximately the same extent (50%) whether or not they had previously received L-phenylalanine. Other experiments not included in Table 7 have shown that particles (15,000 x g sediment) prepared from animals pretreated with L-phenylalanine and added to the 15,000 X g supernatant iraction did not inhibit phenylalanine hydroxylase to any greater extent than the corresponding particulate fraction prepared from animals not pretreated with L-phenylalanine. DISCUSSION Our investigations on the metabolism of phenylalanine in three stocks of mice carrying independent dilute-lethal alleles indicate that phenylalanine hydroxylase activity is not significantly reduced in animals homozygous for any of the dilute-lethal alleles. Furthermore. these animals have no apparent elevation in the concentration of phenylalanine in the blood nor in phenylpyruvic acid excreted in the urine. WOOLF (1963) also found no increase in the concentration of phenylalanine in the blood of mice homozygous for a dilute-lethal gene nor was there any phenylpyruvic acid or o-hydroxyphenylacetic acid in the urine. Nevertheless, the dilute-lethal homozygotes show the typical neurological signs and die at about three weeks of age, as previously noted. Our biochemical results differ from those reported by COLEMAN (1960) and RAUCH and YOST (1963). COLEMAN attributed the deficiency in phenylalanine hydroxylase to an inhibitor present in the particulate fraction (15,000 x g sediment) in liver and suggested that the inhibitor is present in larger amounts in livers of dilute-lethal homozygotes. RAUCH and YOST (1963) reported that serum phenylalanine levels of dilute-lethal homozygotes have blood concentrations of phenylalanine ten times higher than normal. They suggested that reduced liver phenylalanine hydroxylase activity leads to an elevation in serum phenylalanine and the latter may be responsible for the degeneration of myelin in the central nervous system and the neurological changes observed. From the biochemical data obtained in our laboratory, it is concluded that the neurological symptoms and death at the predicted age in homozygous dilute-lethal animals occur indepndently and are not associated causally with a reduction in liver phenylalanine hydroxylase activity. Further evidence for the presence of an inhibitor of phenylalanine hydroxylase in the particulate fraction of liver ( x g sediment) has been presented but the degree of inhibition of hy-

8 1398 v. G. ZANNONI et al. droxylase activity observed (comparing crude homogenate versus 15,000 x g supernatant fractions) is in the same order of magnitude in all three genotypes and for that matter, in other species such as rat, dog and guinea pig. It might appear that further investigations of phenylalanine metabolism in dilute-lethal homozygotes would not be pertinent to phenylketonuria and other biochemical explanations for the neuropathology should be sought. However, we did find that the liver phenylalanine hydroxylase in mice homozygous for the dilute-lethal alleles was more susceptible to inhibition following the administration of L-phenylalanine or phenylpyruvic acid. When phenylalanine hydroxylase activity was decreased by these agents the concentration of phenylalanine in the blood was markedly increased and phenylpyruvic acid was excreted in the urine. Thus, at least some of the biochemical features of phenylketonuria can be reproduced more completely in dilute-lethal homozygotes than in the other genotypes. The biochemical basis for the greater sensitivity of liver hydroxylase to inhibition after giving L-phenylalanine or phenylpyruvic acid to animals homozygous for dilute-lethal alleles warrants further investigation. These differences could be due to a qualitative alteration in either the structure of the hydroxylase or the accessory enzyme system reducing the pteridine cofactor or the availability of the pteridine cofactor might also be affected. Another possibility is that the metabolism of phenylalanine is qualitatively or quantitatively different in the dilutelethal homozygotes so that an inhibitory metabolite accounts for the differences in inhibition observed. However, it is evident that dietary factors such as the phenylalanine intake are important and may explain some of the differences in results obtained in other laboratories. Even though the progressive neurological symptoms can not now seem to be attributed to a disorder of phenylalanine metabolism in animals homozygous for dilute-lethal alleles further biochemical studies with these animals may be pertinent to our understanding of phenylketonuria in man. For example, if we understood why the hydroxylase is inhibited more completely in the dilute-lethal homozygotes when given L-phenylalanine it might then be possible to show that a similar mechanism accounts for the low phenylalanine hydroxylase activity inherited in some phenylketonuric families. This investigation was supported by a research grant, AM and, in part, by grant AM from the Public Health Service. We are grateful to DR. LIANE RUSSELL of Oak Ridge National Laboratory for providing us with the initial stock of the d allele, to DR. A. G. S-LE of the M.R.C. Radiobiological Research Unit, Harwell, England for the initial stock of the di5 allele, and to the Jackson Laboratory for the initial stock of the dz allele. SUMMARY Our investigations on the metabolism of phenylalanine in mice from three stocks carrying independent dilute-lethal alleles have shown that the animals homozygous for these alleles have a pronounced inhibition of liver phenylalanine hydroxylase after receiving either L-phenylalanine or phenylpyruvic acid. Inhibition of the hydroxylase is accompanied by an elevation of blood phenylalanine

9 L< PHENYLKETONURIA IN DILUTE-LETHAL MICE 1399 concentration and urine phenylpyruvic acid excretion. Thus, the biochemical changes observed are similar to those in phenylketonuria. However, if the animals were not challenged with L-phenylalanine or phenylpyruvic acid the activity of liver phenylalanine hydroxylase was normal in all animals regardless of genotype. The concentration of phenylalanine in the blood was not elevated and phenylpyruvic acid was not excreted in the urine. However, those animals homozygous for the dilute-lethal alleles nevertheless showed neurological symptoms and died at about three weeks of age. The usefulness of these animals in investigating the biochemical disturbances in phenylketonuria has been discussed. LITERATURE CITED COLEMAN, D. L., 1960 Phenylalanine hydroxylase activity in dilute and nondilute strains of mice. Arch. Biochem. Biophys. 91 : GOODWINS, I. R., and M. A. C. VINCENT, 1955 Further data on linkage between short-ear and Maltese dilution in the house mouse. Heredity 9: KELTON, D. E., and H. RAUCH, 1962 Myelination and myelin degeneration in the central nervous system of dilute-lethal mice. Exptl. Neural. 6: LA Du, B. N., and P. J. MICHAEL, 1960 An enzymatic spectrophotometric method for the determination of phenylalanine in blood. J. Lab. Clin. Med. 55: LIN, E. C. C., and W. E. KNOX, 1957 Adaptation of the rat liver tyrosine ketoglutarate transaminase. Biochim. Biophys. Acta 26: LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL, 1951 Protein measurement with folin phenol reagent. J. Biol. Chem. 193: MARKERT, C. L., and W. K. SILVERS, 1956 The effects of genotype and cell environment on melanoblast differentiation in the house mouse. Genetics 41 : RAUCH, H., and M. T. YOST, 1963 Phenylalanine metabolism in dilute-lethal mice. Genetics 48: UDENFRIEND, S., and J. R. COOPER, 1952a The enzymic conversion of phenylalanine to tyrosine. J. Biol. Chem. 194: b The chemical estimation of tyrosine and tyramine. J. Biol. Chem. 196: WEBER, W. W., P. VAN VALEN, B. N. LA Du, and V. G. ZANNONI, 1965 Phenylalanine metabolism in dilute lethal mice. (Abstr.) Federation Proc. 24: 470. WOOLF, L. I., 1963 Inherited metabolic disorders: Errors of phenylalanine and tyrosine metabolism. Advan. Clin. Chem. 6 : ZANNONI, V. G., and B. N. LA Du, 1960 Studies on the defect in tyrosine metabolism in scorbutic guinea pigs. J. Biol. Chem. 235: