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Regional distribution of isozymes of D-amino-acid oxidase and acetylesterase in developing primate brain
EXPERIMENTAL
NEUROLOGY
Regional
28, 76-87
Distribution
Oxidase
(1970)
of
and
lsozymes
Acetylesterase Primate
JOSEPH
J.
VOLPE,
GEORGE
LEE,
of
D-Amino-Acid
in Developing
Brain
LEONARD
LASTER,
AND
J. C.
Sectiolt on Developmental Er~.qwology. Laboratory of Biomedical National Imtitute of Child Health arid Hlrrrlar~ Develop~lent, Section on Gastrootterology. Metabolic Diseases Brawk. National Imtitutc of .4vtkritis arid Metabolic Diseases, Natiortal Institutes of Health, Bethesda, Maryland Received
February
ROBINSON
l
Sciemes, and
26,197O
Isozymes of n-amino-acid oxidase and acetylesterase in different regions of the brain of the developing rhesus monkey were separated by starch gel electrophoresis. Activity of n-amino-acid oxidase was identified by the use of o-phenylalanine as substrate in a coupled peroxidase system, and acetylesterase activity was identified by using alpha-naphthyl acetate as substrate in the presence of Diazo Blue. n-amino-acid oxidase isozymes in the brain of the juvenile (2 to 3-year-old) monkey were localized primarily in brain stem, cord, and midline and lateral cerebellum but were also demonstrable in zymograms of subcortical white matter, corpus callosum, thalamus, hypothalamus, pituitary gland, and globus pallidus. Although isozymes of acetylesterase were more generally distributed throughout all brain areas, one band (band 1) was much more prominent in specimens from caudate nucleus and putamen than in specimens from any other area of brain. Isozymes of n-amino-acid oxidase were not generally detected until after birth, but then all major bands appeared fairly rapidly. By 20 weeks of postnatal age patterns of zymograms were similar to those of the adult brain. Although zymograms of the isozymes of acetylesterase in the brain of the first trimester fetus were very similar to those in regions of brain of the 2- to S-year-old animal, one band (band 6) was much more pronounced in the zymograms of fetal brain than in those from brains at any later age. The principal contribution of these studies is the demonstration of (a) an enormously higher concentration, relative to other brain regions, of one of the acetylesterase isozymes (band 1) in extracts of caudate nucleus and putamen, (b) the progressive decline of one of the acetylesterase isozymes (band 6) during fetal development, and (c) the occurrence of two different patterns of distribution and development of isozymes in the primate central nervous system. 1 Present address General Hospital, reprints to him.
of Dr Volpe Fruit Street,
is : Department of Pediatric Neurology. Boston, Massachusetts 02114. Address
76
Massachusetts requests for
ISOZYMES
IS
I’RIJIBTE
BRAIN
77
Introduction
Although there is much information available on the changes during development in total activities of various enzymes in mammalian brain, considerably less is known about the changes in the different molecular components of specific enzymes, Bonnvitn (7) demonstrated changes in the developing rat brain in the relative proportions of isozymes of lactate dehydrogenase and attempted to correlate these changes with certain physiological features of the developing nervous system. Moreover, although total activity of ,8-n-N-acetyll~exosannnidase and of ,8-galactosidase is normal or only slightly diminished in Tay-Sach’s disease (24) and in Hurler’s syndrome ( 13 ), respectively, drastic reductions in specific isozyme forms of these enzymes were noted and presumably represent the basic metabolic errors. n-Amino-acid osidase [o-amino-acid : 0, osidoreductase (deaminating) ; EC l.-l.3.3.] activity was tirst noted in mammalian liver and kidney by Krebs ( 17) and in brain by Edelbacher and Wiss (11 j. Quantitative assessment of the activity of n-amino-acid osidase in a few regions of brains of several mammals has been reported (9, 10. 12. 22 j , We recently described a method for demonstrating the electrophoretic components of several peroxide-producing enzymes. including n-amino-acid osidase (26 I. In the present paper we describe the distribution of isozymes of n-amino-acid oxidase in different regions of the brain of the developing primate. The development of isozymes of acetylesterase (acetic-ester acetylhydrolyase; EC 3.1.1.6.) in a few regions of human brain from 35 weeks of gestation to adult life has been reported by Barron and Bernsohn ( 1 ). In view of the relatively large number of esterase bands observed in samples from the adult primate brain and of the marked anatomical and biochemical changes that occur prior to 35 weeks of gestation in primate brain, we have studied acetylesterase activities in many different regions of primate brain from very early fetal life to adulthood. 1f.e prepared zymograms of n-amino-acid osidase (,substrate : D-phellylalanine 1 and xcetylesterase ( substrate : alpha-naphthyl acetate) by use of starch gel electrophoresis of specimens from 16-22 regions of the brain of the rhesus monkey at various stages of development, from the first trimester Of gestation to 2-3 years of postnatal life. Methods
Anir~r~ls. Rhesus monkeys (,d1aracn nl~claftn) were used. The fetal and infant animals were bred and maintained in a colony at the National Institutes of Health. The juvenile animals were obtained commercially but were maintained at the National Institutes of Health for at least 3 moiiths before use.
78
VOLPE
ET.
AL.
Materials. Horseradish peroxidase (,Worthington Biochemical Corp., Freehold, New Jersey), flavin-adenine dinucleotide (FAD) (, Mann Research Laboratories, Xew York, New York), D-phenylalanine (Calbiochem, Los Angeles, California), 3-amino-9-ethylcarbazole (Aldrich Biochemical Co., Milwaukee, Wisconsin ) , Diazo Blue-B ( Nutritional Biochemicals, Cleveland, Ohio), alpha-naphthyl acetate (Dajac Laboratories, Philadelphia, Pennsylvania), and starch-hydrolyzed (Connaught Medical Research Laboratories, Toronto, Canada) were obtained commercially. Preparation of Tissues. The preparation of the supernatant solutions from the dissected specimens has been described elsewhere (28). All supernatant solutions were prepared in an identical manner (tissue homogenized in 0.03 M potassium phosphate buffer, pH 6.9, in the ratio of 1 g of tissue to 3 ml of buffer, and centrifuged at 12,000 g at 0 C for 30 min ) and were stored at -20 C for 1-6 months before use. Protein concentrations were determined according to the method of Lowry, Rosebrough, Farr, and Randall (20). Zymogram patterns similar to those presented in this paper were obtained when fresh tissue was examined, and repeated freezing and thawing of supernatant solutions also did not alter their zymogram patterns. Preparation of Zylnograms. The concentration of starch gel in all studies described in this paper was 1.25 times that recommended by the supplier (23). For the studies on D-amino-acid oxidase, vertical starch gel electrophoresis (180 v, 20 hr) was performed at 4 C (27) with 0.023 M boric acid and 0.0092 M NaOH (ph 8.4) as the gel buffer, and 0.3 M boric acid and 0.06 M NaOH (ph 8.4) as the bridge buffer. With the vertical system, a larger quantity of sample can be subjected to electrophoresis. and better separation is obtained than with the horizontal system as previously described (25, 26). We found that sampleswith only two observable bands in the horizontal system could he resolved into five components by vertical electrophoresis. D-Amino-acid oxidase was detected on the sliced gels according to the method of Robinson and Lee (26)) except that n-phenylalanine was used as the substrate since it gave greater intensity of bands than n-alanine. The lower half of the gel was incubated at 37 C for 15-20 hr in a solution containing 100 ml of 1 M phosphate buffer (ph 7.5), 20 mg of 3-amino-gethylcarbazole in 5 ml of hr,N-dimethylformamide. 400 mg of D-phenylallanine, 0.9 ml of peroxidase (4400 U/ml) ; and 4 ml of 1O-J M FAD. The intensity of the staining of each band was estimated and graded according to an arbitrary scale of zero, trace, and +l through -l-4. For the studies of acetylesterase, vertical electrophoresis (27) was carried out with a discontinuous buffer system (25) (140 v, room temperature) until the brown “borate” line had migrated 15 cm from the insertion
ISOZYMES
IN
PRIM~\TE
79
RRAIA-
sites. The gel was then cooled in a 4 C cold room for 15 min before slicing. Acetylesterase was stained according to a modification of the method of Lawrence, Jlelnick, and M’eimer (19). The lower half of the gel was incubatei at 37 C for 1 hr in a solution containing 100 ml of 1 M phosphate buffer. ph 7.0), 50 mg of Diazo Blue-B, and 50 mg of alpha-naphthyl acetate which had heen previously dissolved in 0.5 ml of acetone. The intensity of of the staining of each band was estimated and graded as for n-amino-acid osidase. The lower limits of detectability were not determined for either assay .qbsence of bands on the zymograms did not, therefore, necessarily mean absenceof activity in the supernatant solutions isolated from tissue samples. Limited quantities of supernatant solutions precluded methods for concentrating the extracts in order to enhance the sensitivity of the method. These circumstances do not seriously detract from the main purpose of the study which was to compare rrlatizv activities of the isozymes as functions of development. Moreover, the data presented are consistent with quantitative data reported in previous studies (see below in Discussion) and appear to demonstrate valid regional and developmental patterns. Results
Since all supernatant solutions were prepared from tissues in an identical manner, concentrations of protein were very similar for a given region of brain from animals of the same age. Moreover, there were only minor changes with development in concentrations of protein in the supernatant solutions. 14-e could not determine any consistent relationship between the concentration of protein in the supernatant solutions (‘or of the whole homogenates) and the zymogram patterns obtained. Rcgianal
Dist~ibufion
of Iso~yr~tcs
of D-Z4?uim-,~cid
Oxidasc
in fizc Brain
of tke Jmwilc Mo~zkq~. The intensities of bands of n-amino-acid oxidase from each area of brain are summarized in Table 1. Zymograms of midbrain, pans, medulla, cord, and midline and lateral cerebellum demonstrated 5 bands ( Fig. 1 for the numbering of n-amino-acid osidase isozymes). Bands l-1 were present on the zymograms of thalamus, hypothalamus, globus pallidus, occipital white matter, corpus callosum, and superior and inferior colliculi. Bands 1, 2, and 1 were present on zymograms of pituitary gland (,anterior plus posterior) , and band 1 was present on those of frontal and parietal white matter. Samples from the remaining regions, frontal, temporal, parietal and occipital gray matter, hippocampus, caudate nucleus, ancl putamen showed no demonstrable activity. In summary, band 1 was consistently the most intensely stained of all, followed by bands 2 and 1; band 3 was graded more than + 1 only in the z!-mogram of pans. and band 5 was the least intenselv stained of all.
80
VOLPE
ET.
TABLE ISOZYMES
OF D-AMINO-ACID
OXIDASE
AL. 1
IN BRAIN
OF JUVENLE
Bands Region Frontal gray Frontal white Temporal gray Hippocampus Parietal gray Parietal white Occipital gray Occipital white Corpus callosum Caudate nucleus Putamen Globus pallidus Thalamus Hypothalamus Pituitary Sup. & Inf. colliculi Midbrain Pons Medulla Midline cerebellum Lateral cerebellum Cord
1
2
0 tr 0 0 0 fl 0
+2 +1 0 0 fl
+2 +1 +1 f3 +3 +4 +4 $4 +4 +4
0 0 0 0 0 0 0 tr tr 0 0 tr tr tr +Y +1
+2 t-2 $2 $2 +2
3 0 0 0 0 0 0 0 tr tr 0 0 tr tr tr 0 tr tr
+2 +1 +1 $1 +1
RHESUS
MONKEYS
4 0 0 0 0 0 0 0 tr tr 0 0 tr tr tr tr +1 +1 f3
+2 +1 +1 $1
5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 tr tr tr tr tr tr
a Zymograms were prepared as described in Methods and intensities of bands were graded on an arbitrary scale of from 0 to +4. tr = trace. Results of a study of samples from a single animal are presented here. Similar results were obtained with a second juvenile animal (see text).
Analysis of the brain of a second juvenile animal gave essentially identical results. The only differences in isozyme patterns were the presence of trace amounts of bands 2-4 in zymograms of parietal white matter, the absence of these bands in zymograms of occipital white matter, and the absenceof band 5 in zymograms of midbrain. Development
of Isoaymes
of D-Amino-Acid
Oxidnse
in Different
Regions
of the B&n of the Monkey. With increasing age of the animal, there was an increase in the intensity of the bands produced on the zymograms, as illustrated by the schematic representation of the zymograms of the pons from animals of various ages from 45-50 days of gestational age (first trimester ends at 56 days) to 2-3 years of postnatal life (Fig. 1). No activity was demonstrable in the whole cerebrum and brain stem (with cerebellar anlage) of the 45- to SO-day-old fetus or in the cortical gray matter areas (frontal, parietal, occipital), subcortical white matter
SI
/ i
C
0
E
F
G
FIG. 1. Zymogram patterns of isozymes of u-amino-acid osidase in samples oi pans from monkeys of the following ages: (A) 45-50 days of gestational age (whole brain stem). (B) 120-125 days of gestational age (midbrain plus pans ). (CJ 4 weeks of postnatal age, (U) 10 weeks, (E) 1-i weeks, (F) 20 weeks, and (G) juvenile (2-3 years) The positions and relative intensities of each band observed in the original gels are shown schematically. Bands xvere numbered as shown. Strip H is a photograph of the original gel schematicized in strip G. -4lthough band 5 was clearly observable in the original gel, it is scarcely discernible in the photograph. In this and subsequent figures, 0 indicates the origin, and a vertical arrow indicates that electrophoretic migration was toward the anode.
areas (frontal, pxietal), midbrain plus pans. and whole cerel)ellum of the 120- to 1.25~day-old fetus. Band 1 of trace intensity in the zymogram of medulla plus cord of the 120- to 125day-old fetus was the only manifestation of D-amino-acid oxidase activity in the fetal primate brain. Ey 3 weeks of postnatal age, the first four bands were detectable in zymograms of brain stem, cord, and cerebellum. In addition, band 5 of trace intensity had appeared in the zymogram of pons. Only band 1 in trace intensity had appeared in the zymogram of thalamus and parietal white matter. During the nest 1 months of postnatal life. there was a gradual increase of intensity of the bands in zymograms of brain stem. cord, and cerebeilum, so that by 20 weeks after birth zymograms of these areas were essentially similar to those of the juvenile i2- to 3-year-old) animal. In frontal white matter band 1 of even trace intensity was not obesrved until the specimen from the juvenile animal was studied. Similarly, in thalamus and parietal white matter, bands 2-4 were not seen until analysis of specimens from the juvenile animal. In occipital white matter, band 1 appeared at 10 weeks postnatal age and did not change significantly thereafter.
82
VOLPE
ET.
AL.
Regio.xal Distribution of Isosymes of Acetylestcrase in the Brain of the Juvedr Monkey. Eleven bands were seen on the zymograms of acetyl-
esterase in all areas of brain except frontal, parietal, and occipital white matter and cord. Band 10 was not seen in the zymograms from those areas. (Figure 3 shows the schemefor numbering the isozymes of acetylesterase.) Bands on zymograms of cortical gray matter were slightly more intense than bands on those of subcortical white matter. Bands on zymograms of caudate nucleus and putamen were generally among the most intensely stained of all. The only striking regional difference observed was the presence in zymograms of caudate nucleus and, especially, putamen of very heavily staining material migrating in the region of band 1 (Fig. 2). The present studies did not establish whether this represented a large band 1 or two or more closely migrating bands. In other areas of brain, bands 5 and 11 were the individual bands most intensely stained, as seen for frontal gray matter (Fig. 3). Bands 3, 4, and 7 were the next most intensely stained, while the bands stained most weakly were bands 9 and 10. Studies of two additional juvenile monkey brains revealed essentially similar patterns. In one, the major qualitative difference from the first animal was the presence in pons of an additional, lightly stained band migrating between bands 9 and 10. In the other, this latter band was present in all areas, except subcortical white matter and cerebellum, and a 13th, lightly stained
FIG. 2. Zymogram patterns of isozymes of acetylesterase (photograph of original gel) in samples of (A) caudate nucleus, (B) putamen, and (C) thalamus from a juvenile animal. Note the very heavily staining material in the position of band 1 (see
arrow)
in caudate nucleus and putamen in comparison
to thalamus.
ISOZYMES
IN
PRIMATE
BR.‘JTi
s3
bantl migrating between bantls 7 and S was present in all areas. The finding of 11 to 13 anodal bantls and the relative intensity of bands from region to region correspond to the isozymal and quantitative tlatx reportetl for ntlult human brain (2, 6 ) . Dcvclo~lrLcrzt of ISOZJ~~IICS of e4~~c~tylcstrr.nsl~ iu Difcrcklt Iic~giorls of the Bw;~z oj the Mo~i;cy. \t-ith but one exception ( discusseci below), the bands on the zymograms of ncetylestcrase in the primate brain increased in number and intensity with tlevelopment of the animals. ,4 representative clevelopmental pattern is illustrntetl in Fig. 3 for zymograms of frontal grnv matter. In the zymogram of the whole cerebrum of the fetus of 45-50 days gestational age. all bands escept 1, 2. and 4 were present. These latter bands were appnrent in the zymograms of the frontal xntl parietal gray and white matter of the 120- to 12j-&y-old fetus, whereas only band 4 was visiljle in the zymogram of occipital gray matter of the 120- to 125-clay-old fetus. In the zymogram of the whole brain stem ( with cerebellar nnlage ) of the 45- to SO-day-old fetus. all bnncls were present. Therefore, in contrast to the development of isozymes of D-amino-acid oxiclase, there were few qualitative differences in isozymal patterns of acetylesterase between the earliest fetal In-ains studied and the juvenile brain. By lo-20 weeks of 1:ostnatal age, occasionally as early as 4 weeks. the bantls in the zymogrnm CJf each brain
‘3
A
8
C
D
E
F
FIG. 3. Zymogram patterns of isozymes of acetylesterase (photograph gel) in samples of frontal gray matter from monkeys of the follo\ving 120-125 days of gestational age; (B) 4 weeks of postnatal age; (C) 10 14 weeks ; (E) 20 weeks; and (F) juvenile (2-3 year>). Several bands lightly in the original gels are not discernible in the photograph.
of original ages: (A) weeks; (D) that stained
84
VOLPE
ET.
AL.
region reached the levels of intensity demonstrable for those of juvenile brain regions. The most striking change in the development of isozymes of acetylesterase was in band 6 (Figs. 3 and 4). In the zymograms of whole cerebrum and brain stem of the 45 to 50-day-old fetus, band 6 stained much more intensely than any other band in zymograms of any other cortical or brain stem area at later ages. The changes of this band with maturation were essentially similar for all brain areas, and comparisons of the zymogram of whole cerebrum of the 45 to 50-day-old fetus with zymograms of frontal gray matter of the fetus at 120-125 days gestational age and of the animal at 4 weeks postnatal age illustrated the developmental change (Fig. 4). The band at 120-125 days of gestation still was greater than $4 in intensity, but by 4 weeks postnatal age it was no greater than + 1 in intensity ; it exhibited no further postnatal changes. Discussion
Isozymes of D-Amino-Acid Oxidase. Quantitative studies of a few regions of brain of several mammals (9, 12, 22), including man ( 10, 22)) revealed the highest activity of D-amino-acid oxidase to be in hindbrain regions, cor-
FIG. 4. Zymogrampatternsof isozymesof acetylesterase (photographof original gel) in samples:(A) of whole cerebrumfrom a fetus of 45-50 days gestationalage. (B) of frontal gray matter from monkeys (C) 4 weekspostnatalage. Note the deeply samples. By 4 weeks of postnatal age the juvenile animal. Several bands that stained cernible in the photograph
of
120-125
days
of
gestational
age,
and
stainedband6 (seearrow) in the fetal band is similar lightly in the
to that observed original gels are
in the not dis-
ISOZU1\IES
IN
PRIhlATE
BR.\IS
s.5
responding to the regions in which we found the most complex z\-mograms. Activity of D-amino-acid oxidase has also been detected in canine diencephalon (29), rabbit retina (14), and rat whole brain (S, 11). Our findings indicated that isozymes of n-amino-acid oxidase did not generally appear mitil after birth, but then all major bands appeared fairly rapidly and reached the intensity of the relatively mature juvenile animal by about 20 weeks of postnatal age. The most prominent feature of this period of tlevelopment in man is active myelination ( 15, 1S. 21‘1, which is, however, not restricted to the regions abundant in isozymes of o-amino-acid oxidase. The role of D-amino-acid osidase in the primate central nervous system remains to be elucidated. Isoqwcs of AflcctyltWrrasc. Zymograms of acetylesterase isozymes in brain of the developing chick and rat increase in complexity with maturation (3-5). In the rat, the most dramatic changes occur between 10 and 20 days of postnatal age coincident \vith active myelination ( 16). Zymograms prepared from a few regions of developing human brain ( 1) revealed relatively simple patterns from cerebral gray matter of a premature infant of 3S iveeks gestational age and of a 6-hr-old term infant. Patterns in the specimens from 5 and l-3-day-cld human infants were qualitatively similar to those of the adult. Patterns from cerebral and cerebellar white matter and brain stem of the S- and 12-day-old infants were also qualitatively similar to those prepared from adult material. In all of the zymograms from the .%-week-old premature, the 6-hr term infant, and the 5- and la-day-old neonatal infants there was a band that was not seen in the specimensfrom adult subjects, a finding of particular interest because in all zymograms from our first trimester (45-50 days) and third trimester (120-125 days) fetal animals, one band stained much more intensely than in more mature animals and was only lightly stained after -C weeks of postnatal age. Thus, the early development of isozymes of acetylesterase begins during periods characterized in the human by cell proliferation and then cell growth with elaboration of asonal and dendritic ramifications (21) In view of the diffuse distribution of acetylesterase isozymes throughout the nervous system. these enzymes may relate to some rather basic but still midefined function of nerve cells. Our principal contribution is the demonstration of (a) an enormously high concentration. relative to other brain areas, of one of the acetylesterase isozymes (band 1) in extracts of caudate nucleus and putamen, (b) the progressive decline of one of the acetylesterase isozymes (band 6) during fetal development, and (c) the occurrence of two different patterns of distribution and development of isozymes in the primate central nervous system. On the one hand, isozymes of acetylesterase were distributed rather diffusely throughout the brain, whereas isozymes of n-amino-acid oxidase were more
86
VOLPE
ET.
AL.
clearly localized. Whereas in early fetal life isozymes of acetylesterase were present in complexity similar to that in adult life, isozymes of D-amino-acid oxidase were absent until about the time of birth and underwent more definite developmental changes after birth. The regional differences in the molecular forms of these enzymes emphasize a still more refined aspect of the distinctive nature of each brain region. The developmental patterns demonstrate that not only individual enzyme systems, but the several molecular forms of these systems mature at different rates in different brain areas. Such regional and developmental features of isozymes are basic to the understanding of not only normal brain metabolism but those certain metabolic errors that affect the nervous system. References K. D., and J. BERNSOHN. 1968. Esterases of developing human brain. J. Networkem. 15 : 273-384. 2. BARRON, K. D., J. BERNSOHN, and A. R. HESS. 1963. Separation and properties of human brain esterases. J. H&o&em. Cytochenz. 11: 139-156. 3. BERNSOHN, J., and K. D. BARRON. 1964. Multiple molecular forms of brain hydrolases. Int. Rev. Neuvobiol. 7 : 297-344. 4. BERNSOHN, J., K. D. BARRON, and A. R. HESS. 1964. Esterase activity and zymogram patterns in developing rat brain. Progv. Brain Res. 9: 161-164. 5. BERNSOHN, J., K. D. BARRON, A. R. HESS, and M. T. HEDRICP. 1963. Alterations in properties and isoenzyme patterns of esterases in developing primate brain. J. Neurockem. 10 : 783-794. 1.
BARRON,
6. BERNSOHN, J.. L. POSSLEY, and E. LIEBERT. 1959. Study of esterase activity in human brain and serum. J. Neztrochcm. 4 : 191-201. 7. BONAVITA, V. 1964. Molecular evolution of lactate dehydrogenase in the developing nervous tissue. Progr. Brain Res. 4 : 254-272. 8. BURCH, H. B., 0. H. LOWRY, A. M. PADILLA, and A. M. COOMBS. 1956. Effects of riboflavin deficiency and realimentation on flavin enzymes of tissues J. Biol. Ckenl. 223 : 29-36. 9. DE MARCHI. W. J., and G. A. R. JO’HNSTON. 1969. The oxidation of glycine by n-amino acid oxidase in extracts of mammalian nervous tissue. J. Nezwockern. 16 : 355-362. 10. DUNN, J. T., and G. T. PERICOFF. 1963. n-amino acid oxidase activity in human tissues. Biorkirrr. Biopkys. Acta 73 : 327-331. 11. EDELBACHER, S., and 0. WISS. 1944. Zur Kenntnis des Abbaues der Aminosauren im tierischen Organismus. Heh. Ckim Acfa 27 : 1060-1073. 12. GOLDSTEIX, D. B. 1966. n-amino acid oxidase in brain: distribution in several species and inhibition by pentobarbitone. J. Ncwockenz. 13 : 101 l-1016. 13. Ho, M. W.. and J. S. O’BRIEN. 1969. Hurler’s syndrome: deficiency of a specific beta galactosidase isoenzyme. Scierkce 165 : 611-613. 14. HOTTA. K.. Y. SUGIURA, and M. TAKENO. 1959. n-amino acid oxidase in rabbit retina. ~~;fak,, Kyoto 16 : 90-97. 1.5. KEEXE, M. F. L., and E. HEWER. 1931. Some observations on myelination in the human nervous system. I. ‘gnat. 66: I-12.
ISOZYMES
Ih-
PRIM.kTE
RRr\IN
s7
16. KOC.H. W., and M. L. KOCH. 1913. Contributions to the chemical differentiation of the central nervous system. III, The chemical differentiation of the brain of the albino rat during growth. J. Rio/. C&r. 15 : 423-448. 17. KREBS, H. A. 1935. Metabolism of amino acids. III. Deamination of amino acids. BinrArnr. J. 29: 160-164. 18. L.~SGWORTHY, 0. R. 1933. Development of behavior patterns and myelination of the nervous system in the human fetus and infant. Crrr.rlci/ir Inst. Il.ash. Contrib. &hyol. 24 : 1-57. 19. J.AM.RESCE, S. H.. P. J. MEI.XICK, and H. E. WEIMER. 1960. A species comparison of serum proteins and enzymes by starch gel electrophoresis. Proc. Sot. hp. Riol. Mrtl. 105 : 572-573. 20. LOWRY. 0. H.. N. J. ROS~BRCXGH. A. L. FARR, and Ii. J. RANDALI.. 1951. Protein measurement with the Folin phenol reagent. J. Bid. Chcm 193: 265-27.5. 21. MACARTHL.R. C. G.. and E. A. DOISY. 1918-1919. Quantitative chemical changes in the human brain during growth. J. Cn~p. h:rur-ol. 30 : 445-486. 22. NEIXIS. .-\. H., LV. D. ZIEBERIXE;. and J. D. SMILACI~. 1966. Distribution of D-amino acid oxidase in bovine and human nervous tissue. J. .\-crfrorltcr~. 13: 163-168. 23. NERENRERG, S. T. 1966. “Electrophoresis.” F. A. Davis, Philadelphia. 23. OKADA, S., and J. S. O’BRIEX. 1969. Tay-Sach’s disease: generalized absence of a beta-n-x-acetylhexosaminidase component. Scicrlrc 165 : 698-700. 25. POUIXI;, M. D. 1957. Starch gel electrophoresis in a discontinuous system of buffers. Nature London 180 : 1477-1479. 26. R~BIXSON. J. C., and G. LEE. 1967. Preparation of starch gel zymograms : peroxide producing enzymes and ceruloplasmin. -4rch. Biorhou. Riopkys. 120 : 428333. 27. SMITHIES, 0. 1959. An improved procedure for starch-gel electrophoresis : further variations in the serum proteins of normal individuals. Biochrw. J. 71 : 585-587. 28. VOLPE, J .J., and L. LASTER. 1970. Trans-sulphuration in primate brain : regional distribution of methionine-activating enzyme in the brain of the Rhesus monkey at various stages of development. J. A’r~r~orlzr~. (in press). 29. YOGI, K., T. NAGATSC. and T. OZAW.4. 1956. Inhibitory action of Chlorpromazine on the oxidation of n-amino acid in the diencephalon part of the brain. Natlrrc LOlldOJl 177 : 891-891.
NEUROLOGY
Regional
28, 76-87
Distribution
Oxidase
(1970)
of
and
lsozymes
Acetylesterase Primate
JOSEPH
J.
VOLPE,
GEORGE
LEE,
of
D-Amino-Acid
in Developing
Brain
LEONARD
LASTER,
AND
J. C.
Sectiolt on Developmental Er~.qwology. Laboratory of Biomedical National Imtitute of Child Health arid Hlrrrlar~ Develop~lent, Section on Gastrootterology. Metabolic Diseases Brawk. National Imtitutc of .4vtkritis arid Metabolic Diseases, Natiortal Institutes of Health, Bethesda, Maryland Received
February
ROBINSON
l
Sciemes, and
26,197O
Isozymes of n-amino-acid oxidase and acetylesterase in different regions of the brain of the developing rhesus monkey were separated by starch gel electrophoresis. Activity of n-amino-acid oxidase was identified by the use of o-phenylalanine as substrate in a coupled peroxidase system, and acetylesterase activity was identified by using alpha-naphthyl acetate as substrate in the presence of Diazo Blue. n-amino-acid oxidase isozymes in the brain of the juvenile (2 to 3-year-old) monkey were localized primarily in brain stem, cord, and midline and lateral cerebellum but were also demonstrable in zymograms of subcortical white matter, corpus callosum, thalamus, hypothalamus, pituitary gland, and globus pallidus. Although isozymes of acetylesterase were more generally distributed throughout all brain areas, one band (band 1) was much more prominent in specimens from caudate nucleus and putamen than in specimens from any other area of brain. Isozymes of n-amino-acid oxidase were not generally detected until after birth, but then all major bands appeared fairly rapidly. By 20 weeks of postnatal age patterns of zymograms were similar to those of the adult brain. Although zymograms of the isozymes of acetylesterase in the brain of the first trimester fetus were very similar to those in regions of brain of the 2- to S-year-old animal, one band (band 6) was much more pronounced in the zymograms of fetal brain than in those from brains at any later age. The principal contribution of these studies is the demonstration of (a) an enormously higher concentration, relative to other brain regions, of one of the acetylesterase isozymes (band 1) in extracts of caudate nucleus and putamen, (b) the progressive decline of one of the acetylesterase isozymes (band 6) during fetal development, and (c) the occurrence of two different patterns of distribution and development of isozymes in the primate central nervous system. 1 Present address General Hospital, reprints to him.
of Dr Volpe Fruit Street,
is : Department of Pediatric Neurology. Boston, Massachusetts 02114. Address
76
Massachusetts requests for
ISOZYMES
IS
I’RIJIBTE
BRAIN
77
Introduction
Although there is much information available on the changes during development in total activities of various enzymes in mammalian brain, considerably less is known about the changes in the different molecular components of specific enzymes, Bonnvitn (7) demonstrated changes in the developing rat brain in the relative proportions of isozymes of lactate dehydrogenase and attempted to correlate these changes with certain physiological features of the developing nervous system. Moreover, although total activity of ,8-n-N-acetyll~exosannnidase and of ,8-galactosidase is normal or only slightly diminished in Tay-Sach’s disease (24) and in Hurler’s syndrome ( 13 ), respectively, drastic reductions in specific isozyme forms of these enzymes were noted and presumably represent the basic metabolic errors. n-Amino-acid osidase [o-amino-acid : 0, osidoreductase (deaminating) ; EC l.-l.3.3.] activity was tirst noted in mammalian liver and kidney by Krebs ( 17) and in brain by Edelbacher and Wiss (11 j. Quantitative assessment of the activity of n-amino-acid osidase in a few regions of brains of several mammals has been reported (9, 10. 12. 22 j , We recently described a method for demonstrating the electrophoretic components of several peroxide-producing enzymes. including n-amino-acid osidase (26 I. In the present paper we describe the distribution of isozymes of n-amino-acid oxidase in different regions of the brain of the developing primate. The development of isozymes of acetylesterase (acetic-ester acetylhydrolyase; EC 3.1.1.6.) in a few regions of human brain from 35 weeks of gestation to adult life has been reported by Barron and Bernsohn ( 1 ). In view of the relatively large number of esterase bands observed in samples from the adult primate brain and of the marked anatomical and biochemical changes that occur prior to 35 weeks of gestation in primate brain, we have studied acetylesterase activities in many different regions of primate brain from very early fetal life to adulthood. 1f.e prepared zymograms of n-amino-acid osidase (,substrate : D-phellylalanine 1 and xcetylesterase ( substrate : alpha-naphthyl acetate) by use of starch gel electrophoresis of specimens from 16-22 regions of the brain of the rhesus monkey at various stages of development, from the first trimester Of gestation to 2-3 years of postnatal life. Methods
Anir~r~ls. Rhesus monkeys (,d1aracn nl~claftn) were used. The fetal and infant animals were bred and maintained in a colony at the National Institutes of Health. The juvenile animals were obtained commercially but were maintained at the National Institutes of Health for at least 3 moiiths before use.
78
VOLPE
ET.
AL.
Materials. Horseradish peroxidase (,Worthington Biochemical Corp., Freehold, New Jersey), flavin-adenine dinucleotide (FAD) (, Mann Research Laboratories, Xew York, New York), D-phenylalanine (Calbiochem, Los Angeles, California), 3-amino-9-ethylcarbazole (Aldrich Biochemical Co., Milwaukee, Wisconsin ) , Diazo Blue-B ( Nutritional Biochemicals, Cleveland, Ohio), alpha-naphthyl acetate (Dajac Laboratories, Philadelphia, Pennsylvania), and starch-hydrolyzed (Connaught Medical Research Laboratories, Toronto, Canada) were obtained commercially. Preparation of Tissues. The preparation of the supernatant solutions from the dissected specimens has been described elsewhere (28). All supernatant solutions were prepared in an identical manner (tissue homogenized in 0.03 M potassium phosphate buffer, pH 6.9, in the ratio of 1 g of tissue to 3 ml of buffer, and centrifuged at 12,000 g at 0 C for 30 min ) and were stored at -20 C for 1-6 months before use. Protein concentrations were determined according to the method of Lowry, Rosebrough, Farr, and Randall (20). Zymogram patterns similar to those presented in this paper were obtained when fresh tissue was examined, and repeated freezing and thawing of supernatant solutions also did not alter their zymogram patterns. Preparation of Zylnograms. The concentration of starch gel in all studies described in this paper was 1.25 times that recommended by the supplier (23). For the studies on D-amino-acid oxidase, vertical starch gel electrophoresis (180 v, 20 hr) was performed at 4 C (27) with 0.023 M boric acid and 0.0092 M NaOH (ph 8.4) as the gel buffer, and 0.3 M boric acid and 0.06 M NaOH (ph 8.4) as the bridge buffer. With the vertical system, a larger quantity of sample can be subjected to electrophoresis. and better separation is obtained than with the horizontal system as previously described (25, 26). We found that sampleswith only two observable bands in the horizontal system could he resolved into five components by vertical electrophoresis. D-Amino-acid oxidase was detected on the sliced gels according to the method of Robinson and Lee (26)) except that n-phenylalanine was used as the substrate since it gave greater intensity of bands than n-alanine. The lower half of the gel was incubated at 37 C for 15-20 hr in a solution containing 100 ml of 1 M phosphate buffer (ph 7.5), 20 mg of 3-amino-gethylcarbazole in 5 ml of hr,N-dimethylformamide. 400 mg of D-phenylallanine, 0.9 ml of peroxidase (4400 U/ml) ; and 4 ml of 1O-J M FAD. The intensity of the staining of each band was estimated and graded according to an arbitrary scale of zero, trace, and +l through -l-4. For the studies of acetylesterase, vertical electrophoresis (27) was carried out with a discontinuous buffer system (25) (140 v, room temperature) until the brown “borate” line had migrated 15 cm from the insertion
ISOZYMES
IN
PRIM~\TE
79
RRAIA-
sites. The gel was then cooled in a 4 C cold room for 15 min before slicing. Acetylesterase was stained according to a modification of the method of Lawrence, Jlelnick, and M’eimer (19). The lower half of the gel was incubatei at 37 C for 1 hr in a solution containing 100 ml of 1 M phosphate buffer. ph 7.0), 50 mg of Diazo Blue-B, and 50 mg of alpha-naphthyl acetate which had heen previously dissolved in 0.5 ml of acetone. The intensity of of the staining of each band was estimated and graded as for n-amino-acid osidase. The lower limits of detectability were not determined for either assay .qbsence of bands on the zymograms did not, therefore, necessarily mean absenceof activity in the supernatant solutions isolated from tissue samples. Limited quantities of supernatant solutions precluded methods for concentrating the extracts in order to enhance the sensitivity of the method. These circumstances do not seriously detract from the main purpose of the study which was to compare rrlatizv activities of the isozymes as functions of development. Moreover, the data presented are consistent with quantitative data reported in previous studies (see below in Discussion) and appear to demonstrate valid regional and developmental patterns. Results
Since all supernatant solutions were prepared from tissues in an identical manner, concentrations of protein were very similar for a given region of brain from animals of the same age. Moreover, there were only minor changes with development in concentrations of protein in the supernatant solutions. 14-e could not determine any consistent relationship between the concentration of protein in the supernatant solutions (‘or of the whole homogenates) and the zymogram patterns obtained. Rcgianal
Dist~ibufion
of Iso~yr~tcs
of D-Z4?uim-,~cid
Oxidasc
in fizc Brain
of tke Jmwilc Mo~zkq~. The intensities of bands of n-amino-acid oxidase from each area of brain are summarized in Table 1. Zymograms of midbrain, pans, medulla, cord, and midline and lateral cerebellum demonstrated 5 bands ( Fig. 1 for the numbering of n-amino-acid osidase isozymes). Bands l-1 were present on the zymograms of thalamus, hypothalamus, globus pallidus, occipital white matter, corpus callosum, and superior and inferior colliculi. Bands 1, 2, and 1 were present on zymograms of pituitary gland (,anterior plus posterior) , and band 1 was present on those of frontal and parietal white matter. Samples from the remaining regions, frontal, temporal, parietal and occipital gray matter, hippocampus, caudate nucleus, ancl putamen showed no demonstrable activity. In summary, band 1 was consistently the most intensely stained of all, followed by bands 2 and 1; band 3 was graded more than + 1 only in the z!-mogram of pans. and band 5 was the least intenselv stained of all.
80
VOLPE
ET.
TABLE ISOZYMES
OF D-AMINO-ACID
OXIDASE
AL. 1
IN BRAIN
OF JUVENLE
Bands Region Frontal gray Frontal white Temporal gray Hippocampus Parietal gray Parietal white Occipital gray Occipital white Corpus callosum Caudate nucleus Putamen Globus pallidus Thalamus Hypothalamus Pituitary Sup. & Inf. colliculi Midbrain Pons Medulla Midline cerebellum Lateral cerebellum Cord
1
2
0 tr 0 0 0 fl 0
+2 +1 0 0 fl
+2 +1 +1 f3 +3 +4 +4 $4 +4 +4
0 0 0 0 0 0 0 tr tr 0 0 tr tr tr +Y +1
+2 t-2 $2 $2 +2
3 0 0 0 0 0 0 0 tr tr 0 0 tr tr tr 0 tr tr
+2 +1 +1 $1 +1
RHESUS
MONKEYS
4 0 0 0 0 0 0 0 tr tr 0 0 tr tr tr tr +1 +1 f3
+2 +1 +1 $1
5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 tr tr tr tr tr tr
a Zymograms were prepared as described in Methods and intensities of bands were graded on an arbitrary scale of from 0 to +4. tr = trace. Results of a study of samples from a single animal are presented here. Similar results were obtained with a second juvenile animal (see text).
Analysis of the brain of a second juvenile animal gave essentially identical results. The only differences in isozyme patterns were the presence of trace amounts of bands 2-4 in zymograms of parietal white matter, the absence of these bands in zymograms of occipital white matter, and the absenceof band 5 in zymograms of midbrain. Development
of Isoaymes
of D-Amino-Acid
Oxidnse
in Different
Regions
of the B&n of the Monkey. With increasing age of the animal, there was an increase in the intensity of the bands produced on the zymograms, as illustrated by the schematic representation of the zymograms of the pons from animals of various ages from 45-50 days of gestational age (first trimester ends at 56 days) to 2-3 years of postnatal life (Fig. 1). No activity was demonstrable in the whole cerebrum and brain stem (with cerebellar anlage) of the 45- to SO-day-old fetus or in the cortical gray matter areas (frontal, parietal, occipital), subcortical white matter
SI
/ i
C
0
E
F
G
FIG. 1. Zymogram patterns of isozymes of u-amino-acid osidase in samples oi pans from monkeys of the following ages: (A) 45-50 days of gestational age (whole brain stem). (B) 120-125 days of gestational age (midbrain plus pans ). (CJ 4 weeks of postnatal age, (U) 10 weeks, (E) 1-i weeks, (F) 20 weeks, and (G) juvenile (2-3 years) The positions and relative intensities of each band observed in the original gels are shown schematically. Bands xvere numbered as shown. Strip H is a photograph of the original gel schematicized in strip G. -4lthough band 5 was clearly observable in the original gel, it is scarcely discernible in the photograph. In this and subsequent figures, 0 indicates the origin, and a vertical arrow indicates that electrophoretic migration was toward the anode.
areas (frontal, pxietal), midbrain plus pans. and whole cerel)ellum of the 120- to 1.25~day-old fetus. Band 1 of trace intensity in the zymogram of medulla plus cord of the 120- to 125day-old fetus was the only manifestation of D-amino-acid oxidase activity in the fetal primate brain. Ey 3 weeks of postnatal age, the first four bands were detectable in zymograms of brain stem, cord, and cerebellum. In addition, band 5 of trace intensity had appeared in the zymogram of pons. Only band 1 in trace intensity had appeared in the zymogram of thalamus and parietal white matter. During the nest 1 months of postnatal life. there was a gradual increase of intensity of the bands in zymograms of brain stem. cord, and cerebeilum, so that by 20 weeks after birth zymograms of these areas were essentially similar to those of the juvenile i2- to 3-year-old) animal. In frontal white matter band 1 of even trace intensity was not obesrved until the specimen from the juvenile animal was studied. Similarly, in thalamus and parietal white matter, bands 2-4 were not seen until analysis of specimens from the juvenile animal. In occipital white matter, band 1 appeared at 10 weeks postnatal age and did not change significantly thereafter.
82
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AL.
Regio.xal Distribution of Isosymes of Acetylestcrase in the Brain of the Juvedr Monkey. Eleven bands were seen on the zymograms of acetyl-
esterase in all areas of brain except frontal, parietal, and occipital white matter and cord. Band 10 was not seen in the zymograms from those areas. (Figure 3 shows the schemefor numbering the isozymes of acetylesterase.) Bands on zymograms of cortical gray matter were slightly more intense than bands on those of subcortical white matter. Bands on zymograms of caudate nucleus and putamen were generally among the most intensely stained of all. The only striking regional difference observed was the presence in zymograms of caudate nucleus and, especially, putamen of very heavily staining material migrating in the region of band 1 (Fig. 2). The present studies did not establish whether this represented a large band 1 or two or more closely migrating bands. In other areas of brain, bands 5 and 11 were the individual bands most intensely stained, as seen for frontal gray matter (Fig. 3). Bands 3, 4, and 7 were the next most intensely stained, while the bands stained most weakly were bands 9 and 10. Studies of two additional juvenile monkey brains revealed essentially similar patterns. In one, the major qualitative difference from the first animal was the presence in pons of an additional, lightly stained band migrating between bands 9 and 10. In the other, this latter band was present in all areas, except subcortical white matter and cerebellum, and a 13th, lightly stained
FIG. 2. Zymogram patterns of isozymes of acetylesterase (photograph of original gel) in samples of (A) caudate nucleus, (B) putamen, and (C) thalamus from a juvenile animal. Note the very heavily staining material in the position of band 1 (see
arrow)
in caudate nucleus and putamen in comparison
to thalamus.
ISOZYMES
IN
PRIMATE
BR.‘JTi
s3
bantl migrating between bantls 7 and S was present in all areas. The finding of 11 to 13 anodal bantls and the relative intensity of bands from region to region correspond to the isozymal and quantitative tlatx reportetl for ntlult human brain (2, 6 ) . Dcvclo~lrLcrzt of ISOZJ~~IICS of e4~~c~tylcstrr.nsl~ iu Difcrcklt Iic~giorls of the Bw;~z oj the Mo~i;cy. \t-ith but one exception ( discusseci below), the bands on the zymograms of ncetylestcrase in the primate brain increased in number and intensity with tlevelopment of the animals. ,4 representative clevelopmental pattern is illustrntetl in Fig. 3 for zymograms of frontal grnv matter. In the zymogram of the whole cerebrum of the fetus of 45-50 days gestational age. all bands escept 1, 2. and 4 were present. These latter bands were appnrent in the zymograms of the frontal xntl parietal gray and white matter of the 120- to 12j-&y-old fetus, whereas only band 4 was visiljle in the zymogram of occipital gray matter of the 120- to 125-clay-old fetus. In the zymogram of the whole brain stem ( with cerebellar nnlage ) of the 45- to SO-day-old fetus. all bnncls were present. Therefore, in contrast to the development of isozymes of D-amino-acid oxiclase, there were few qualitative differences in isozymal patterns of acetylesterase between the earliest fetal In-ains studied and the juvenile brain. By lo-20 weeks of 1:ostnatal age, occasionally as early as 4 weeks. the bantls in the zymogrnm CJf each brain
‘3
A
8
C
D
E
F
FIG. 3. Zymogram patterns of isozymes of acetylesterase (photograph gel) in samples of frontal gray matter from monkeys of the follo\ving 120-125 days of gestational age; (B) 4 weeks of postnatal age; (C) 10 14 weeks ; (E) 20 weeks; and (F) juvenile (2-3 year>). Several bands lightly in the original gels are not discernible in the photograph.
of original ages: (A) weeks; (D) that stained
84
VOLPE
ET.
AL.
region reached the levels of intensity demonstrable for those of juvenile brain regions. The most striking change in the development of isozymes of acetylesterase was in band 6 (Figs. 3 and 4). In the zymograms of whole cerebrum and brain stem of the 45 to 50-day-old fetus, band 6 stained much more intensely than any other band in zymograms of any other cortical or brain stem area at later ages. The changes of this band with maturation were essentially similar for all brain areas, and comparisons of the zymogram of whole cerebrum of the 45 to 50-day-old fetus with zymograms of frontal gray matter of the fetus at 120-125 days gestational age and of the animal at 4 weeks postnatal age illustrated the developmental change (Fig. 4). The band at 120-125 days of gestation still was greater than $4 in intensity, but by 4 weeks postnatal age it was no greater than + 1 in intensity ; it exhibited no further postnatal changes. Discussion
Isozymes of D-Amino-Acid Oxidase. Quantitative studies of a few regions of brain of several mammals (9, 12, 22), including man ( 10, 22)) revealed the highest activity of D-amino-acid oxidase to be in hindbrain regions, cor-
FIG. 4. Zymogrampatternsof isozymesof acetylesterase (photographof original gel) in samples:(A) of whole cerebrumfrom a fetus of 45-50 days gestationalage. (B) of frontal gray matter from monkeys (C) 4 weekspostnatalage. Note the deeply samples. By 4 weeks of postnatal age the juvenile animal. Several bands that stained cernible in the photograph
of
120-125
days
of
gestational
age,
and
stainedband6 (seearrow) in the fetal band is similar lightly in the
to that observed original gels are
in the not dis-
ISOZU1\IES
IN
PRIhlATE
BR.\IS
s.5
responding to the regions in which we found the most complex z\-mograms. Activity of D-amino-acid oxidase has also been detected in canine diencephalon (29), rabbit retina (14), and rat whole brain (S, 11). Our findings indicated that isozymes of n-amino-acid oxidase did not generally appear mitil after birth, but then all major bands appeared fairly rapidly and reached the intensity of the relatively mature juvenile animal by about 20 weeks of postnatal age. The most prominent feature of this period of tlevelopment in man is active myelination ( 15, 1S. 21‘1, which is, however, not restricted to the regions abundant in isozymes of o-amino-acid oxidase. The role of D-amino-acid osidase in the primate central nervous system remains to be elucidated. Isoqwcs of AflcctyltWrrasc. Zymograms of acetylesterase isozymes in brain of the developing chick and rat increase in complexity with maturation (3-5). In the rat, the most dramatic changes occur between 10 and 20 days of postnatal age coincident \vith active myelination ( 16). Zymograms prepared from a few regions of developing human brain ( 1) revealed relatively simple patterns from cerebral gray matter of a premature infant of 3S iveeks gestational age and of a 6-hr-old term infant. Patterns in the specimens from 5 and l-3-day-cld human infants were qualitatively similar to those of the adult. Patterns from cerebral and cerebellar white matter and brain stem of the S- and 12-day-old infants were also qualitatively similar to those prepared from adult material. In all of the zymograms from the .%-week-old premature, the 6-hr term infant, and the 5- and la-day-old neonatal infants there was a band that was not seen in the specimensfrom adult subjects, a finding of particular interest because in all zymograms from our first trimester (45-50 days) and third trimester (120-125 days) fetal animals, one band stained much more intensely than in more mature animals and was only lightly stained after -C weeks of postnatal age. Thus, the early development of isozymes of acetylesterase begins during periods characterized in the human by cell proliferation and then cell growth with elaboration of asonal and dendritic ramifications (21) In view of the diffuse distribution of acetylesterase isozymes throughout the nervous system. these enzymes may relate to some rather basic but still midefined function of nerve cells. Our principal contribution is the demonstration of (a) an enormously high concentration. relative to other brain areas, of one of the acetylesterase isozymes (band 1) in extracts of caudate nucleus and putamen, (b) the progressive decline of one of the acetylesterase isozymes (band 6) during fetal development, and (c) the occurrence of two different patterns of distribution and development of isozymes in the primate central nervous system. On the one hand, isozymes of acetylesterase were distributed rather diffusely throughout the brain, whereas isozymes of n-amino-acid oxidase were more
86
VOLPE
ET.
AL.
clearly localized. Whereas in early fetal life isozymes of acetylesterase were present in complexity similar to that in adult life, isozymes of D-amino-acid oxidase were absent until about the time of birth and underwent more definite developmental changes after birth. The regional differences in the molecular forms of these enzymes emphasize a still more refined aspect of the distinctive nature of each brain region. The developmental patterns demonstrate that not only individual enzyme systems, but the several molecular forms of these systems mature at different rates in different brain areas. Such regional and developmental features of isozymes are basic to the understanding of not only normal brain metabolism but those certain metabolic errors that affect the nervous system. References K. D., and J. BERNSOHN. 1968. Esterases of developing human brain. J. Networkem. 15 : 273-384. 2. BARRON, K. D., J. BERNSOHN, and A. R. HESS. 1963. Separation and properties of human brain esterases. J. H&o&em. Cytochenz. 11: 139-156. 3. BERNSOHN, J., and K. D. BARRON. 1964. Multiple molecular forms of brain hydrolases. Int. Rev. Neuvobiol. 7 : 297-344. 4. BERNSOHN, J., K. D. BARRON, and A. R. HESS. 1964. Esterase activity and zymogram patterns in developing rat brain. Progv. Brain Res. 9: 161-164. 5. BERNSOHN, J., K. D. BARRON, A. R. HESS, and M. T. HEDRICP. 1963. Alterations in properties and isoenzyme patterns of esterases in developing primate brain. J. Neurockem. 10 : 783-794. 1.
BARRON,
6. BERNSOHN, J.. L. POSSLEY, and E. LIEBERT. 1959. Study of esterase activity in human brain and serum. J. Neztrochcm. 4 : 191-201. 7. BONAVITA, V. 1964. Molecular evolution of lactate dehydrogenase in the developing nervous tissue. Progr. Brain Res. 4 : 254-272. 8. BURCH, H. B., 0. H. LOWRY, A. M. PADILLA, and A. M. COOMBS. 1956. Effects of riboflavin deficiency and realimentation on flavin enzymes of tissues J. Biol. Ckenl. 223 : 29-36. 9. DE MARCHI. W. J., and G. A. R. JO’HNSTON. 1969. The oxidation of glycine by n-amino acid oxidase in extracts of mammalian nervous tissue. J. Nezwockern. 16 : 355-362. 10. DUNN, J. T., and G. T. PERICOFF. 1963. n-amino acid oxidase activity in human tissues. Biorkirrr. Biopkys. Acta 73 : 327-331. 11. EDELBACHER, S., and 0. WISS. 1944. Zur Kenntnis des Abbaues der Aminosauren im tierischen Organismus. Heh. Ckim Acfa 27 : 1060-1073. 12. GOLDSTEIX, D. B. 1966. n-amino acid oxidase in brain: distribution in several species and inhibition by pentobarbitone. J. Ncwockenz. 13 : 101 l-1016. 13. Ho, M. W.. and J. S. O’BRIEN. 1969. Hurler’s syndrome: deficiency of a specific beta galactosidase isoenzyme. Scierkce 165 : 611-613. 14. HOTTA. K.. Y. SUGIURA, and M. TAKENO. 1959. n-amino acid oxidase in rabbit retina. ~~;fak,, Kyoto 16 : 90-97. 1.5. KEEXE, M. F. L., and E. HEWER. 1931. Some observations on myelination in the human nervous system. I. ‘gnat. 66: I-12.
ISOZYMES
Ih-
PRIM.kTE
RRr\IN
s7
16. KOC.H. W., and M. L. KOCH. 1913. Contributions to the chemical differentiation of the central nervous system. III, The chemical differentiation of the brain of the albino rat during growth. J. Rio/. C&r. 15 : 423-448. 17. KREBS, H. A. 1935. Metabolism of amino acids. III. Deamination of amino acids. BinrArnr. J. 29: 160-164. 18. L.~SGWORTHY, 0. R. 1933. Development of behavior patterns and myelination of the nervous system in the human fetus and infant. Crrr.rlci/ir Inst. Il.ash. Contrib. &hyol. 24 : 1-57. 19. J.AM.RESCE, S. H.. P. J. MEI.XICK, and H. E. WEIMER. 1960. A species comparison of serum proteins and enzymes by starch gel electrophoresis. Proc. Sot. hp. Riol. Mrtl. 105 : 572-573. 20. LOWRY. 0. H.. N. J. ROS~BRCXGH. A. L. FARR, and Ii. J. RANDALI.. 1951. Protein measurement with the Folin phenol reagent. J. Bid. Chcm 193: 265-27.5. 21. MACARTHL.R. C. G.. and E. A. DOISY. 1918-1919. Quantitative chemical changes in the human brain during growth. J. Cn~p. h:rur-ol. 30 : 445-486. 22. NEIXIS. .-\. H., LV. D. ZIEBERIXE;. and J. D. SMILACI~. 1966. Distribution of D-amino acid oxidase in bovine and human nervous tissue. J. .\-crfrorltcr~. 13: 163-168. 23. NERENRERG, S. T. 1966. “Electrophoresis.” F. A. Davis, Philadelphia. 23. OKADA, S., and J. S. O’BRIEX. 1969. Tay-Sach’s disease: generalized absence of a beta-n-x-acetylhexosaminidase component. Scicrlrc 165 : 698-700. 25. POUIXI;, M. D. 1957. Starch gel electrophoresis in a discontinuous system of buffers. Nature London 180 : 1477-1479. 26. R~BIXSON. J. C., and G. LEE. 1967. Preparation of starch gel zymograms : peroxide producing enzymes and ceruloplasmin. -4rch. Biorhou. Riopkys. 120 : 428333. 27. SMITHIES, 0. 1959. An improved procedure for starch-gel electrophoresis : further variations in the serum proteins of normal individuals. Biochrw. J. 71 : 585-587. 28. VOLPE, J .J., and L. LASTER. 1970. Trans-sulphuration in primate brain : regional distribution of methionine-activating enzyme in the brain of the Rhesus monkey at various stages of development. J. A’r~r~orlzr~. (in press). 29. YOGI, K., T. NAGATSC. and T. OZAW.4. 1956. Inhibitory action of Chlorpromazine on the oxidation of n-amino acid in the diencephalon part of the brain. Natlrrc LOlldOJl 177 : 891-891.