- Email: [email protected]
Looking at lens proteins
TIBS -
June 1979
137 References 1 Habermann,
E. (1972)
S&we
177, 314-321
2 Habermann, E. and Jentach, J. (1967) Seyler’s Z. Physiol. Chem. 348, 37-50
Hoppe-
3 Kreil. G. and Bachmayer, B&hem. 20. 344-350
f:w.
4 Suchanek. Schreicr, 309-315
G., Kind&Mtigge, M. (1975) Eur.
5 Aviv. H. and Leder, P. (1972) Sci. U.S.A. 69, 1408-1412
H. (1971)
J.
I., Kreil. G. and Rioc,hem 60.
Proc. Nut. Awd.
6 Bishop, J., Rosbach, M. and Evans, 1. Mol. Biol. 85, 75-86
-r-M5 by 7M Lwe and let live, girls!
cell, since the prepeptide is cleaved even before the polypeptide chain is completed. Only the translation of a mRNA in a cell-free system free of membranes and associated proteases yields these presecretory polypeptides. Prepromelittin obviously falls into this category of precursors and it is therefore not surprising that only promelittin, Bnd not prepromelittin has been detected in the venom glands. We have recently tried
Looking Hans Bloemendal
to develop an in vitro system for the cleavage of the presequence. In these experiments labelled prepromelittin has been incubated with subcellular fractions from rat liver in the presence of DOC. A peptide is generated which behaves like promelittin in our fractionation procedure and which has the same N-terminal sequence [ 141. Using this test we are now attempting to isolate and characterize this important intracellular protease.
at lens proteins
Hans Bloemendal and Wilfried W. de Jong are at the Department of Biochemistry, University of Nijmegen, ‘Heyendael’ Geert Grootplein Noord 21) Nijmegen, The Netherlands.
resemble plasma.
those
in the dialysate
(I 974) G.
8 Kindas-Mtiggc, I., Frasel, L. and Diggelmann. (1976) J. Mol. Rio/. 105, 177
H.
9 Suchanek, G.. Krcil, G. and Hermodson. M. (1978) Proc. Nut. Acad. Sri. U.S.A. 75. 701704 10 Kreil, G., Suchanek, G. and Kind&Mtigge. (1977) Fed. Pmt. 36, 2081-2086 I1 Kreil. G., Suchanek, G., Kaschnitz, Kind&+Mugge, I. (1978) Febs llth Copenhagen, 1977. 47. 79-88 G. and Krein, G. (1977) 12 Suchanek. Acad. Sci. U.S.A. 74, 975-978 13 Blobel. G. and Dobberstein. Biol. 67, 835-85 1
I.
R. and Mwting Proc Nat.
B. (1975)
J. (‘~11.
14 Kaschnitz, R. and Kreil, G. (1978) Biwhrm. Biophy~. Res. Commun. 83, 901-907
which
gradually
enlarges
upon
The crystallins
The eye lens is highly suitable for studies on differentiation and aging since it is derived from a single pure cell line and does not show the phenomenon ofcell death. Yet the major lenticular proteins (crystallins) undergo changes in their structure upon aging. The evolution of these proteins has been very conservative. The eye lens is an excellent tool for the study of a variety of biological mechanisms whose understanding is also relevant for the elucidation of similar processes in other tissues [l]. Unlike all other organs the lens is an ‘isolated’ entity, completely devoid of blood supply and innervation. Since, however, it does need nourishment for its maintenance during the life span of the animal, the supply of nutrients has to come by a pathway other than the blood stream. It is thought that the so-called aqueous humor, which is interposed between the lens and blood capillaries, provides the nutritional requirements. Actually the nature and concentration of many constituents in the aqueous humor
D.
7 Kind%-Miigge, I., Lane. C. D. and Kreil. (1974) J. Mol. Biol. 87, 45 1-462
nucleus aging.
and Wilfried W. de Jong
J.
of blood
The organ The lens is completely enclosed within a membrane, called the lens capsule. Underneath the capsule at the anterior side of the lens a single layer of epithelial cells is situated that differentiate into fiber-like cells. This process is accompanied by a considerable increase in the volume of the cells and the synthesis of protein. As new fibers are produced the older ones are displaced toward the center of the lens. Hence the central region of an adult lens, called the nucleus or core, represents the fiber cells that have been synthesized at the embryonic and fetal stage. As lens cells are never shed, the concentration of closely packed cells increases in the lens center, resulting in a
In all mammalian lenses there are three major classes of water-soluble proteins, designated (Y-, /3- and y-crystallins. This classification suggests that the protein composition is simpler than it really is. For instance, cY-crystallin can be subdivided into a group of very high molecular weight and another one with an average molecuz lar weight of about 800,000. @Crystallin exists as a group of proteins of higher (PH) and lower weights. (PL) molecular Whereas the a-crystallins differ only in size but have qualitatively the same subunit composition, the P-crystallins are composed of different polypeptides albeit they share the major P-subunit PBp. Finally the y-crystallins are a population of monomeric water-soluble lens proteins with a molecular weight lower than 28,000 (cf. Fig. 1). The crystallins account for almost 90% of the total lens protein. The biochemistry of these proteins has been studied in great detail with particular emphasis on subunit composition [2] and biosynthesis [3]. A satisfactory separation of the major crystallin classes can be obtained by a single passage of the 10,000 x g super@Elsevier/North-Holland
Biomedical
Press
1979
138 natant fraction of a lens homogenate through a gel filtration column packed with Sephadex G200, Biogcl P300 or Ultrogel AcA34 (Fig. IA). Analysis of the individual fractions from such a column is achicvcd upon gel elcctrophoresis in polyacrylamide containing either 6-8 M urea (separation on basis of charge) (Fig. 1B) or 0.10/o sodium dodecylsulfate (separation on basis of size) (Fig. IC). Some crystallin subunits are direct gene products, which means that they arise by translation of a specific messenger RNA on ribosomes. However, quite a number of crystallin polypeptide chains arise as a result of post-translational modification of pre-existing direct gent products. Firm evidence for the deamidation of the primary gent products has only been provided for the major cu-crystallin polypeptidc. CYAZ.It could be shown that newly synthesized Lu-crystallin contains only aAp and CYB~, while cram and aBI originate from post-translational modification. Also. in fetal lenses cvAl is still lacking. The crystallins arc structural proteins which undergo changes in their architecture upon aging. The nature of these changes is two-fold. First, the molecular size and conformation alters gradually. Secondly, defined breakdown, starting from the C-terminal end. and the deamidation process already mentioned occur. The latter phenomena have been observed for cr-crystallin [4], but may also be true for a number of @crystallin polypeptide chains. Aging of proteins in
the lens nucleus is also associated with racemization of aspartyl residues at a rate of 0.14% per year [S].
Non-crystallins When discussing lens protein in general. almost automatically the crystallins come to mind. However, one should realize that numerous other proteins exist in the lens, albeit in most cases neither their structure nor their function has been elucidated. Very recently we were able to show that at least one protein is an important constituent of the lens, despite its low concentration (at most l-2% of the total soluble crystallin fraction). This component appeared to be identical to actin as judged by chemical, physical and electron microscopical criteria [6]. Although the precise function of this non-muscle actin has not been elucidated, it may have a distinct role in accommodation, the phenomenon whereby the lenses of certain animals change their shapes temporarily. WC have also found some evidence that part of the lens polyribosomes are bound to the cytoskeleton (and possibly also to plasma membrane fragments) and that actin is in\,olved in the binding [7]. Another minor protein in lens is the natural ribonuclease inhibitor which can be separated relatively easily from the bulk of structural proteins [8]. Of the numerous enzymes existing in lenticular tissue, leucine amino peptidase has to be mentioned. This enzyme, which occurs in relatively high concentration. has been studied rather extensively [9].
Evolution of crystallins The comparatively simple structure and function of the lens makes it an attractive object for comparative and evolutionary studies. The vertebrate-type of Icns must have come into existence some 500 million years ago. Since that time it has diverged into different shapes, ranging from perfectly spherical in most aquatic and nocturnal animals to flattened and ovoid forms in some other animals. ‘I‘hc refractive propertics of the lens :tre dctermincd not only by its curvature, but also by its consistency. which varies from solid and unpliable in some Icnscs to almost liquid in others. The Icns nucleus is often more solid than the outer layers. A rather soft and flexible lens is obviously required in those species which accommodatc by altering the lens shape. Large differences in protein content and composition are found among lenses of different species [IO]. Basically the same types of proteins. (Y-, p- and y-cryctallins. are found in all species, with &crystallin occurring in addition in birds and reptiles. The proportions in which these crystallins occur vary greatly. Likewise, the oligomcric aggregates of cy-. /3- and &crystallin vary in size and charge. and the stoichiometry of the component monomers differs from species to species. as do the isoelectric points of these monomers. To a lesser extent such changes also occur during development and aging of the lens, between different layers of the lens. All these differences are not primarily due to large sequence differences in the various primary gene products. All tho
A280
p la
_____-_I^_
fjlb
______
6Bp ___..__
20
Fig. I.
30
40
(A) Gel Jiltrnlion pattcm
conruining conruining
50 60 70 Fraction number A of ~lre strtrc/urnl
aAl
lens proteins
6 .\I urea. The patiern .~IIOM.Sthe uthrcnir composition sodium dodecyl wlfutc.
(crysrrdlins).
(B) Elecrrophoretic
oftherrystallins.
separation
(C) Electrophoretic
ofpeok
separation
from
Fig. I A on polyac~rylrmlidc
gc1.s
o frhe 4 crysrollin
fiiarfions
cla.~ses on polyacrylrrmidr~
gr1.s
TIBS -
June 1970 Ser Leu 20 Val Thr 10 Ala ac-Met-Asp-lle-Ala-Ile-Gln-His-Pro-Trp-Phe-Lys-Arg-Thr-Leu-Gly-Pro-Phe-Ty~-Pro-Ser-Arg-Leu-Phe-AspThr Leu Ile Asn Ala 40 30 -Gln-Phe-Phe-Gly-Glu-Gly-Leu-Phe-Glu-Tyr-Asp-Leu-Leu-Pro-Phe-Leu-Ser-Ser-Thr-lle-Ser-Pro-Tyr-Tyr-ArgMet Leu Asp Val Phe Leu Phe
70 Ala
Gln Ser Glu 50 60 Val Arg -Gln-Ser-Leu-Phe-Arg-Thr-Val-Leu-Asp-Ser-Gly-ile-Ser-Glu-Val-Arg-Ser-Asp-Arg-Asp-Lys-Phe-Vai-lle-PhePhe Ser Phe Glu Arg
Leu
Tyr Leu
90 Leu Asp 80 Tyr -Leu-Asp-Val-Lys-His-Phe-Ser-Pro-Glu-Asp-Leu-Thr-Val-Lys-Vai-Gln-Glu-Asp-Phe-Val-Glu-lle-His-Gly-LysSer Ile Leu Asp Ile 110 100 Ser 120 -His-Asn-Glu-Arg-Gln-Asp-Asp-His-Gly-Tyr-ile-Ser-Arg-Glu-Phe-His-Arg-Arg-Tyr-Arg-Leu-Pro-Ser-Asn-VaiSer
Leu Ala Ala His Asp Val Gln Thr Asn
Thr Ile 130 Val 140 CYS -Asp-GIn-Ser-Ala-Leu-Ser-Cys-Ser-Leu-Ser-Ala-Asp-Gly-Met-Leu-Thr-Phe-Ser-Gly-Pro-Lys-lle-Pro-Ser-GlyThr Ser Ile Ser Leu Met Val 150 Leu Del GUY 160 Ala Ser Thr Leu 170 Met Glu Ser Ser Pro Thr Asp -Val-Asp-Ala-Gly-His-Ser-Glu-Arg-Ala-lle-Pro-Val-Ser-Arg-Glu-Glu-Lys-Pro-Ser-Ser-Ala-Pro-Ser-Ser-COOH Pro Thr Ser Ser Leu G/Y Pro Met
Asp Ser
Thr
Fig. 2. The amino acid sequence ofthr bovine cr-crystallin A chain is shown as the continuous. c,entral line. DijJerences from this sequence observed in the a4 chains of 16 other mammalian species are placed above the line, and differences observed in chicken and frog WI chains below the line.
evidence indicates that most crystallin chains evolve at a rather slow rate. Considerable immunological crossreactivity exists between the crystallins of the most diverse vertebrates. Detailed comparative sequence data are only available for the a-crystallin A chain, but these reveal that it is one of the slowest-evolving proteins [ 111. Limited sequence data of cr-crystallin B chain and of y-crystallin chains also indicate a similarly slow rate of evolution. The very tight packing of proteins of the lens cells, necessitating extensive contacts between amino acid side chains, may be related to this phenomenon. Duplication of crystallin genes The differences in overall protein patterns are mainly due to differential gene expression and to different patterns of post-translational modification. Such a differential gene expression is made easier by the fact that gene duplications in the course of evolution have led to the existence of several related genes for each of the crystallins. The calf cY-crystallin A and B chains show 57% sequence homology and are coded by genes which must have originated by duplication of an ancestral gene before the last common
ancestor of present-day vertebrates existed. At least four genes code for the monomeric calf y-crystallin chains [ 121. These chains show fewer sequence differences than between the aA and aB chains, indicating that the gene duplications responsible for these differences have occurred more recently. Whereas all vertebrates appear to have (YA and aB chains, albeit in differing proportions. the number of y-chains varies considerably, even in related species. Also, the P-crystallins are built up by a number of related chains coded by different genes. By modulating the expression of gents of the different crystallins, the organism has an effective means of regulating aggregate size and charge properties. Unfortunately little is known about the relation between the differences in protein pattern and the functional and structural properties of the lens. Sensitive immunological techniques have shown that there are no crystallins outside the ocular lens. i.e. they are strictly organ-specific. Also the primary structures of cr- and y-crystallins have no detectable relationships to any other known protein sequence; this places each of them in a protein superfamily of its own [131.
Although the (YA and (YB genes probably originated by an ancient event of gene duplication. no evidence has been found for further, more recent, duplications of either the aA or cuB gene. In fact the rat ‘possesses both a normal cuA chain and a small amount of another aA-like chain which only differs by the presence of an internal insertion of 22 residues in the latter chain. Although this cYA-like chain could be due to a gene duplication followed by an unknown and peculiar insertion of 66 base pairs, we suspect that the internally elongated (YA chain is rather the result of defective splicing during the processing of the aA mRNA precursor. leaving a stretch of 66 nucleotidcs which would normally be removed [ 141. Sequence changes in aA chain The cu-crystallin A chains of 17 mammalian species, the chicken and a species of frog have been sequenced so far. Their comparisons should provide a picture of the evolutionary changes of this protein [ll]. Most changes in the sequence of crA chains are amino acid substitutions due to single base replacements in the codons of the aA gene. Only one case of deletion has been observed: a single residue in position 153 of the aA
140 chain of higher primates. The observed amino acid replacements (Fig. 2) demonstrate some of the characteristics of protein evolution. With few exceptions the replacements are of a chemicallyconservative character, i.c. properties like hydrophobicity, polarity and charge are hardly changed by most substitutions. Replacements by chemically similar residues are the most frequent ones, e.g. Ser *Thr, Val tf Leu and Asp c) Glu. The distribution of substitutions over the chain is remarkably uneven, which may be related to structural requirements of the a-crystallin aggregate, allowing more substitutions to be maintained in some regions than in others. The highest density of substitutions is found in the region 146-158, which is known to be located at the surface of the cr-crystallin aggregate [ 151. The mammalian aA chain sequences, together with those of chicken and frog, have been analysed by a computer program to construct a phylogenetic tree Ill]. The procedure is such that the final tree is the most parsimonious one, i.e. requiring the smallest number of substitutions to connect all sequences in the tree. The result (Fig. 3) mostly conforms nicely to the known relationships between species and gives indications of the phylogenetic position of species for which fossil or
TIBS morphologic data are scarce or contradictory. Although the (YA chain generally evolves slowly, at a rate of one amino replacement per 100 residues in about 35 million years, this rate varies considerably in different lineages. In the last 70 million years the crA chain apparently has undergone some seven substitutions in the line leading to man, against none in the lines to whale, rat and rabbit. The comparative study of eye lens crystallins thus reveals a picture of slow but steady changes. It is still too early to decide to what extent these changes reflect adaptations to changing functional requirements of the lens or are due to gradual, functionally neutral, so-called non-Darwinian, divergence in the course of millions of years. Biosynthesis
of lens protein
The lens is also attractive for the study of protein synthesis since, as we have seen, the organ is composed mainly of crystallins that represent a rather small number of specific structural proteins. When studied in organ culture it appears that about 50% of the total protein synthesis is directed towards the production of cr-crystallin [ 161. Interesting results have been obtained recently upon investigation of protein biosynthesis in cultured lens epithelium [17]. It appears that during
Fig. 3. Computer analysis of the sequences of the aA chains of 17 mammalian species, chicken and frog produced this ‘phylogenetic tree’. The calculated numbers of amino acid substitutions are indicated for each branch. The estimated times of divergence are based on palaeontological data. (From [I I].)
June 1979
“Oh Darling, your eyes, they’re fascinating! You certainly must have radical substitution in your crystallins!”
differentiation a qualitative change in the synthesis of crystallins occurs. The transition from epithelial cell to the fiber is accompanied by a marked increase in CYA chains and several P-crystallin polypeptides, while the synthesis of non-crystallin protein slows down. Strikingly, different regions in the epithelial monolayer have different protein biosynthetic activities. Lens mRNA [ 181 or lens polyribosomes [19] can be injected into living oocytes. In this system translation of the heterologous messenger proceeds for at least two days. It has been estimated that 20-30 crystallin polypeptide chains are produced per hour and per added mRNA molecule. De no\‘0 synthesis of lens protein can also he studied in a variety of heterologous cell-free systems by adding either isolated mRNA or lens polyribosomes. The newly synthesized products are analysed by SDS-gel electrophoresis followed by scintillation autoradiography. In order to detect the synthesis of a specific protein, immunoprecipitation with the antiserum directed against that protein has to precede the electrophoretic analysis. Detection of in vitro synthesis of the specific lens plasma membrane protein MP34 has been achieved in this way [3]. Cell-free incubation of a reticulocyte lysate with 14s rat lens mRNA revealed that the synthesis of the rat Lu-crystallin chain with the insertion [~YA’“~],is directed by the same messenger fraction which also codes for the synthesis of the normal aA chain [20]. Moreover antiserum raised against calf lens a+crystallin in which no CYA”‘~is present, precipitated the rat drains chain. This observation illustrates not only the high degree of homology between a-crystallins of two different species, but also confirms the very close relationship between normal rat CXAZ,and aAl”\, the polypeptide with the insertion of 22 amino acids. In conclusion we want to state that it is
TIBS -
141
June 1979
advantageous to use the lens for the study of the regulation of the synthesis of tissue-specific proteins. It offers a population of well-defined cells which show distinct stages of differentiation and biosynthetic display characteristic activities of highly specialized proteins. References Bloemcndal, H. (1977) Science 197. 127-138 Harding, J. J. and Dilley, K. J. (1976) Ex[>. Eyr Res. 22, 1 Bloemendal, H. (1977) in International Review of Biochemistry, Biochemistry of Cell Differmtiation II (Paul, J.. ed.). University Park Press, Baltimore. Vol. 15. pp. 195-225 Van Klcef. F. S. M., De Jong. W. W. and Hoenders. H. J. (1975) Nnrurt, (London) 258, 264 Masters. P. M., Bada, J. L. and Zigler, J. S. (1977) Narure (London) 268. 71-73
Tadpoles,
Kibbelaar.
M. A., Selten-Vcrsteegen,
,4
15
Bloemendal, H., Zweers, A., Koopmans, M. and van den Broek, W. (1977) Biochem. Biophys. Res. Commun. 77. 41&425
16
Hanson, H.,GIasser, D. and Kirschke, Z. Physiol. Chem. 340. 107
l7
H. (1965)
Biochemical D. C.. Vol. 5.
Cohen, L. H., Westerhuis, L. W., de Jong, W. W. and Bloemendal, H. (1978) Eur. J. Eiochem. 89, 259-266 Siczen, R. J. and Hoenders, Len. 80, 75-80 Spector, A. and Kinoshita, Opthalmol. 3, 5 17-522
H. J. (1977)
FtBS
J. H. (1964)
fflresr.
Vermorken, A. J. M. and Bloemendal. Nature (London) 271, 779-181
H.
(I Y78)
10 Clayton, R. M. (1974) in The Eye (Davson, H. and Graham, L. T. Jr. eds), Academic Press, New York and London, Vol. 5, 399
M.. Bloemen18 Berns, A. J. M., van Kraaikamp, dal, H. and Lane, C. D. (1972) Proc. Nar. Acad. Sci. U.S.A. 69, 1606-1609
11 De Jong, W. W., Gleaves, J. T. and Boultcr, (1977) J. Mol. Evol. 10, 123-135
19 Asselbergs, Bloemcndal, 5 17-524
12 Slingsby, C. and Croft. Res. 26, 291-304
L. R. (1978)
D.
Exp. Eye
13 Dayhoff. M. O., Barker, W. C. and Hunt, L. T. (1976) in Atlas of Protein Sequence and Structure
drugs and toxicities W. Robert Jondorf
The theory of Brodie and his collaborators that the non-specific drug metabolizing enzymes were developed in terrestrial species of animals essentially to enable them to convert lipid-soluble exogenous compounds to more readily excretable polar derivatives has withstood the test of time. The theory has come into focus again because of our better understanding of hormonal influences on evolutionary drives, and also because of the foreseeable consequences of contaminating the global environment with more and more unmetabolizable lipid-soluble compounds. In the course of studies on age-related factors in drug metabolism in mice, guinea-pigs [I] and chicks, and in reviewing the whole subject of drug metabolism in terms of development and evolution [3-51 certain new facts connected with the regulatory importance of thyroid hormones and prolactin have come to light [5] which ought to be more fully integrated with concepts of drug metabolism.
for like other juvenile aquatic vertebrates lipid-soluble they could eliminate exogenous compounds by dialysis through the lipoidal membranes of gills or skin. This may no longer be strictly true since various halogenated environmental contaminants persist in most ecosystems [6] and at best, aquatic vertebrates lacking drug metabolizing enzymes can do no more than equilibrate with some of these contaminants. After adaptation of the aquatic tadpoles and toadpoles to life on Amphibian beginnings land by metamorphosis they develop the The story really began about 20 years ability to oxidatively metabolize and ago when it was established, partly on the conjugate test drugs [3,4]. This illustration basis of experiments with amphibia [3,41, of a biochemical metamorphosis was as that the ability to metabolize relatively vivid as that of the sequential changes lipid-soluble drugs and foreign comoccurring in nitrogen excretion patterns pounds to more water-soluble entities was and the developmental changes in plasma a characteristic of biochemical evolution proteins, visual pigments and haemoaccompanying the transition from aquatic globin [7]. to terrestrial life. Tadpoles of toads and of The ‘terrestrial drive’ of amphibian certain kinds of frogs could not metabolize metamorphosis appears to be controlled drugs either by oxidation or conjugation, by thyroid hormones and can be manipulated towards precocious development by W. Robert JondorA lives at 3 Cough Way, thyroxine 171. AltematiGely it can be Cambridge, U.K. and is currently associated with inhibited by antithyroid compounds such laboratories in Newmarket working on the detection of drugs in horses.
(Dayhoff, M. O., cd.), National Research Foundation, Washington Suppl. 2, Ch. 2
A. M. E.,
Dunia, 1.. Benedetti, E. L. and Bloemendal, H. (1979) Eur. J. Biochem. 95, S43. Bloemendal, H., Kibbelaar, M. A., Ramaekers, F. C. S., Selten-Versteegen, A. M. E., Dunia, 1. and Benedetti, E. L. (1978) in Protides of the Biological Fluids 26 Colloq. (Peeters, H., cd.), Pergamon Press, Oxford (in press)
as Z-thiouracil and thionrea.
These and
F. A. M., van Venrooij, W. J. and H. (1978) Eur. 1. Eiochem. 87,
20 Cohen, L. H., Westerhuis, L. W., Smits. D. P. and Bloemendal, H. (1978) Eur. J. Biochem. 89, 251-258
other antithyroid compounds have been shown to interfere with thyroid peroxidase activity [8], a model precursor reaction for the (mammalian) synthesis of thyroxine in vivo (Table I). The antithyroid compounds also inhibit key enzymes required for urea or pyrimidine synthesis [9] such as carbamyl phosphate synthetase (also shown in Table I). The terrestrial drive of tadpole development is opposed in nature by a juvenilizing ‘aquatic drive’, regulated by pituitary prolactin release [ 101. The effect of prolactin on tadpoles is to promote growth without metamorphosis, while adult newts return to the water to mate and spawn under the influence of this hormone [lo]. It can be readily recognized that antithyroid compounds and compounds that interfered with prolactin release or feedback control would have profound toxicological effects on orderly synchronized development in higher animals subject to similar hormonal controls. The story continues with mammals. Drugs, the new-born and induction Drug-metabolizing enzyme activity in placental mammals is altogether absent or deficient in the foetus and the new-born [l]. This was confirmed particularly elegantly when the various components of the mixed function oxidase system in rabbit liver were measured during preg nancy and for two weeks after parturition correlated with the [I 11, and were electron-microscopic appearance of the liver and with measurements of micro. somal drug-metabolizing activities a’ various stages of development. Drui metabolism in new-born mammals can bt induced very readily by administerinl 0
ElseviedNorth-Holland
Biomedii
Press 197’
June 1979
137 References 1 Habermann,
E. (1972)
S&we
177, 314-321
2 Habermann, E. and Jentach, J. (1967) Seyler’s Z. Physiol. Chem. 348, 37-50
Hoppe-
3 Kreil. G. and Bachmayer, B&hem. 20. 344-350
f:w.
4 Suchanek. Schreicr, 309-315
G., Kind&Mtigge, M. (1975) Eur.
5 Aviv. H. and Leder, P. (1972) Sci. U.S.A. 69, 1408-1412
H. (1971)
J.
I., Kreil. G. and Rioc,hem 60.
Proc. Nut. Awd.
6 Bishop, J., Rosbach, M. and Evans, 1. Mol. Biol. 85, 75-86
-r-M5 by 7M Lwe and let live, girls!
cell, since the prepeptide is cleaved even before the polypeptide chain is completed. Only the translation of a mRNA in a cell-free system free of membranes and associated proteases yields these presecretory polypeptides. Prepromelittin obviously falls into this category of precursors and it is therefore not surprising that only promelittin, Bnd not prepromelittin has been detected in the venom glands. We have recently tried
Looking Hans Bloemendal
to develop an in vitro system for the cleavage of the presequence. In these experiments labelled prepromelittin has been incubated with subcellular fractions from rat liver in the presence of DOC. A peptide is generated which behaves like promelittin in our fractionation procedure and which has the same N-terminal sequence [ 141. Using this test we are now attempting to isolate and characterize this important intracellular protease.
at lens proteins
Hans Bloemendal and Wilfried W. de Jong are at the Department of Biochemistry, University of Nijmegen, ‘Heyendael’ Geert Grootplein Noord 21) Nijmegen, The Netherlands.
resemble plasma.
those
in the dialysate
(I 974) G.
8 Kindas-Mtiggc, I., Frasel, L. and Diggelmann. (1976) J. Mol. Rio/. 105, 177
H.
9 Suchanek, G.. Krcil, G. and Hermodson. M. (1978) Proc. Nut. Acad. Sri. U.S.A. 75. 701704 10 Kreil, G., Suchanek, G. and Kind&Mtigge. (1977) Fed. Pmt. 36, 2081-2086 I1 Kreil. G., Suchanek, G., Kaschnitz, Kind&+Mugge, I. (1978) Febs llth Copenhagen, 1977. 47. 79-88 G. and Krein, G. (1977) 12 Suchanek. Acad. Sci. U.S.A. 74, 975-978 13 Blobel. G. and Dobberstein. Biol. 67, 835-85 1
I.
R. and Mwting Proc Nat.
B. (1975)
J. (‘~11.
14 Kaschnitz, R. and Kreil, G. (1978) Biwhrm. Biophy~. Res. Commun. 83, 901-907
which
gradually
enlarges
upon
The crystallins
The eye lens is highly suitable for studies on differentiation and aging since it is derived from a single pure cell line and does not show the phenomenon ofcell death. Yet the major lenticular proteins (crystallins) undergo changes in their structure upon aging. The evolution of these proteins has been very conservative. The eye lens is an excellent tool for the study of a variety of biological mechanisms whose understanding is also relevant for the elucidation of similar processes in other tissues [l]. Unlike all other organs the lens is an ‘isolated’ entity, completely devoid of blood supply and innervation. Since, however, it does need nourishment for its maintenance during the life span of the animal, the supply of nutrients has to come by a pathway other than the blood stream. It is thought that the so-called aqueous humor, which is interposed between the lens and blood capillaries, provides the nutritional requirements. Actually the nature and concentration of many constituents in the aqueous humor
D.
7 Kind%-Miigge, I., Lane. C. D. and Kreil. (1974) J. Mol. Biol. 87, 45 1-462
nucleus aging.
and Wilfried W. de Jong
J.
of blood
The organ The lens is completely enclosed within a membrane, called the lens capsule. Underneath the capsule at the anterior side of the lens a single layer of epithelial cells is situated that differentiate into fiber-like cells. This process is accompanied by a considerable increase in the volume of the cells and the synthesis of protein. As new fibers are produced the older ones are displaced toward the center of the lens. Hence the central region of an adult lens, called the nucleus or core, represents the fiber cells that have been synthesized at the embryonic and fetal stage. As lens cells are never shed, the concentration of closely packed cells increases in the lens center, resulting in a
In all mammalian lenses there are three major classes of water-soluble proteins, designated (Y-, /3- and y-crystallins. This classification suggests that the protein composition is simpler than it really is. For instance, cY-crystallin can be subdivided into a group of very high molecular weight and another one with an average molecuz lar weight of about 800,000. @Crystallin exists as a group of proteins of higher (PH) and lower weights. (PL) molecular Whereas the a-crystallins differ only in size but have qualitatively the same subunit composition, the P-crystallins are composed of different polypeptides albeit they share the major P-subunit PBp. Finally the y-crystallins are a population of monomeric water-soluble lens proteins with a molecular weight lower than 28,000 (cf. Fig. 1). The crystallins account for almost 90% of the total lens protein. The biochemistry of these proteins has been studied in great detail with particular emphasis on subunit composition [2] and biosynthesis [3]. A satisfactory separation of the major crystallin classes can be obtained by a single passage of the 10,000 x g super@Elsevier/North-Holland
Biomedical
Press
1979
138 natant fraction of a lens homogenate through a gel filtration column packed with Sephadex G200, Biogcl P300 or Ultrogel AcA34 (Fig. IA). Analysis of the individual fractions from such a column is achicvcd upon gel elcctrophoresis in polyacrylamide containing either 6-8 M urea (separation on basis of charge) (Fig. 1B) or 0.10/o sodium dodecylsulfate (separation on basis of size) (Fig. IC). Some crystallin subunits are direct gene products, which means that they arise by translation of a specific messenger RNA on ribosomes. However, quite a number of crystallin polypeptide chains arise as a result of post-translational modification of pre-existing direct gent products. Firm evidence for the deamidation of the primary gent products has only been provided for the major cu-crystallin polypeptidc. CYAZ.It could be shown that newly synthesized Lu-crystallin contains only aAp and CYB~, while cram and aBI originate from post-translational modification. Also. in fetal lenses cvAl is still lacking. The crystallins arc structural proteins which undergo changes in their architecture upon aging. The nature of these changes is two-fold. First, the molecular size and conformation alters gradually. Secondly, defined breakdown, starting from the C-terminal end. and the deamidation process already mentioned occur. The latter phenomena have been observed for cr-crystallin [4], but may also be true for a number of @crystallin polypeptide chains. Aging of proteins in
the lens nucleus is also associated with racemization of aspartyl residues at a rate of 0.14% per year [S].
Non-crystallins When discussing lens protein in general. almost automatically the crystallins come to mind. However, one should realize that numerous other proteins exist in the lens, albeit in most cases neither their structure nor their function has been elucidated. Very recently we were able to show that at least one protein is an important constituent of the lens, despite its low concentration (at most l-2% of the total soluble crystallin fraction). This component appeared to be identical to actin as judged by chemical, physical and electron microscopical criteria [6]. Although the precise function of this non-muscle actin has not been elucidated, it may have a distinct role in accommodation, the phenomenon whereby the lenses of certain animals change their shapes temporarily. WC have also found some evidence that part of the lens polyribosomes are bound to the cytoskeleton (and possibly also to plasma membrane fragments) and that actin is in\,olved in the binding [7]. Another minor protein in lens is the natural ribonuclease inhibitor which can be separated relatively easily from the bulk of structural proteins [8]. Of the numerous enzymes existing in lenticular tissue, leucine amino peptidase has to be mentioned. This enzyme, which occurs in relatively high concentration. has been studied rather extensively [9].
Evolution of crystallins The comparatively simple structure and function of the lens makes it an attractive object for comparative and evolutionary studies. The vertebrate-type of Icns must have come into existence some 500 million years ago. Since that time it has diverged into different shapes, ranging from perfectly spherical in most aquatic and nocturnal animals to flattened and ovoid forms in some other animals. ‘I‘hc refractive propertics of the lens :tre dctermincd not only by its curvature, but also by its consistency. which varies from solid and unpliable in some Icnscs to almost liquid in others. The Icns nucleus is often more solid than the outer layers. A rather soft and flexible lens is obviously required in those species which accommodatc by altering the lens shape. Large differences in protein content and composition are found among lenses of different species [IO]. Basically the same types of proteins. (Y-, p- and y-cryctallins. are found in all species, with &crystallin occurring in addition in birds and reptiles. The proportions in which these crystallins occur vary greatly. Likewise, the oligomcric aggregates of cy-. /3- and &crystallin vary in size and charge. and the stoichiometry of the component monomers differs from species to species. as do the isoelectric points of these monomers. To a lesser extent such changes also occur during development and aging of the lens, between different layers of the lens. All these differences are not primarily due to large sequence differences in the various primary gene products. All tho
A280
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(B) Elecrrophoretic
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TIBS -
June 1970 Ser Leu 20 Val Thr 10 Ala ac-Met-Asp-lle-Ala-Ile-Gln-His-Pro-Trp-Phe-Lys-Arg-Thr-Leu-Gly-Pro-Phe-Ty~-Pro-Ser-Arg-Leu-Phe-AspThr Leu Ile Asn Ala 40 30 -Gln-Phe-Phe-Gly-Glu-Gly-Leu-Phe-Glu-Tyr-Asp-Leu-Leu-Pro-Phe-Leu-Ser-Ser-Thr-lle-Ser-Pro-Tyr-Tyr-ArgMet Leu Asp Val Phe Leu Phe
70 Ala
Gln Ser Glu 50 60 Val Arg -Gln-Ser-Leu-Phe-Arg-Thr-Val-Leu-Asp-Ser-Gly-ile-Ser-Glu-Val-Arg-Ser-Asp-Arg-Asp-Lys-Phe-Vai-lle-PhePhe Ser Phe Glu Arg
Leu
Tyr Leu
90 Leu Asp 80 Tyr -Leu-Asp-Val-Lys-His-Phe-Ser-Pro-Glu-Asp-Leu-Thr-Val-Lys-Vai-Gln-Glu-Asp-Phe-Val-Glu-lle-His-Gly-LysSer Ile Leu Asp Ile 110 100 Ser 120 -His-Asn-Glu-Arg-Gln-Asp-Asp-His-Gly-Tyr-ile-Ser-Arg-Glu-Phe-His-Arg-Arg-Tyr-Arg-Leu-Pro-Ser-Asn-VaiSer
Leu Ala Ala His Asp Val Gln Thr Asn
Thr Ile 130 Val 140 CYS -Asp-GIn-Ser-Ala-Leu-Ser-Cys-Ser-Leu-Ser-Ala-Asp-Gly-Met-Leu-Thr-Phe-Ser-Gly-Pro-Lys-lle-Pro-Ser-GlyThr Ser Ile Ser Leu Met Val 150 Leu Del GUY 160 Ala Ser Thr Leu 170 Met Glu Ser Ser Pro Thr Asp -Val-Asp-Ala-Gly-His-Ser-Glu-Arg-Ala-lle-Pro-Val-Ser-Arg-Glu-Glu-Lys-Pro-Ser-Ser-Ala-Pro-Ser-Ser-COOH Pro Thr Ser Ser Leu G/Y Pro Met
Asp Ser
Thr
Fig. 2. The amino acid sequence ofthr bovine cr-crystallin A chain is shown as the continuous. c,entral line. DijJerences from this sequence observed in the a4 chains of 16 other mammalian species are placed above the line, and differences observed in chicken and frog WI chains below the line.
evidence indicates that most crystallin chains evolve at a rather slow rate. Considerable immunological crossreactivity exists between the crystallins of the most diverse vertebrates. Detailed comparative sequence data are only available for the a-crystallin A chain, but these reveal that it is one of the slowest-evolving proteins [ 111. Limited sequence data of cr-crystallin B chain and of y-crystallin chains also indicate a similarly slow rate of evolution. The very tight packing of proteins of the lens cells, necessitating extensive contacts between amino acid side chains, may be related to this phenomenon. Duplication of crystallin genes The differences in overall protein patterns are mainly due to differential gene expression and to different patterns of post-translational modification. Such a differential gene expression is made easier by the fact that gene duplications in the course of evolution have led to the existence of several related genes for each of the crystallins. The calf cY-crystallin A and B chains show 57% sequence homology and are coded by genes which must have originated by duplication of an ancestral gene before the last common
ancestor of present-day vertebrates existed. At least four genes code for the monomeric calf y-crystallin chains [ 121. These chains show fewer sequence differences than between the aA and aB chains, indicating that the gene duplications responsible for these differences have occurred more recently. Whereas all vertebrates appear to have (YA and aB chains, albeit in differing proportions. the number of y-chains varies considerably, even in related species. Also, the P-crystallins are built up by a number of related chains coded by different genes. By modulating the expression of gents of the different crystallins, the organism has an effective means of regulating aggregate size and charge properties. Unfortunately little is known about the relation between the differences in protein pattern and the functional and structural properties of the lens. Sensitive immunological techniques have shown that there are no crystallins outside the ocular lens. i.e. they are strictly organ-specific. Also the primary structures of cr- and y-crystallins have no detectable relationships to any other known protein sequence; this places each of them in a protein superfamily of its own [131.
Although the (YA and (YB genes probably originated by an ancient event of gene duplication. no evidence has been found for further, more recent, duplications of either the aA or cuB gene. In fact the rat ‘possesses both a normal cuA chain and a small amount of another aA-like chain which only differs by the presence of an internal insertion of 22 residues in the latter chain. Although this cYA-like chain could be due to a gene duplication followed by an unknown and peculiar insertion of 66 base pairs, we suspect that the internally elongated (YA chain is rather the result of defective splicing during the processing of the aA mRNA precursor. leaving a stretch of 66 nucleotidcs which would normally be removed [ 141. Sequence changes in aA chain The cu-crystallin A chains of 17 mammalian species, the chicken and a species of frog have been sequenced so far. Their comparisons should provide a picture of the evolutionary changes of this protein [ll]. Most changes in the sequence of crA chains are amino acid substitutions due to single base replacements in the codons of the aA gene. Only one case of deletion has been observed: a single residue in position 153 of the aA
140 chain of higher primates. The observed amino acid replacements (Fig. 2) demonstrate some of the characteristics of protein evolution. With few exceptions the replacements are of a chemicallyconservative character, i.c. properties like hydrophobicity, polarity and charge are hardly changed by most substitutions. Replacements by chemically similar residues are the most frequent ones, e.g. Ser *Thr, Val tf Leu and Asp c) Glu. The distribution of substitutions over the chain is remarkably uneven, which may be related to structural requirements of the a-crystallin aggregate, allowing more substitutions to be maintained in some regions than in others. The highest density of substitutions is found in the region 146-158, which is known to be located at the surface of the cr-crystallin aggregate [ 151. The mammalian aA chain sequences, together with those of chicken and frog, have been analysed by a computer program to construct a phylogenetic tree Ill]. The procedure is such that the final tree is the most parsimonious one, i.e. requiring the smallest number of substitutions to connect all sequences in the tree. The result (Fig. 3) mostly conforms nicely to the known relationships between species and gives indications of the phylogenetic position of species for which fossil or
TIBS morphologic data are scarce or contradictory. Although the (YA chain generally evolves slowly, at a rate of one amino replacement per 100 residues in about 35 million years, this rate varies considerably in different lineages. In the last 70 million years the crA chain apparently has undergone some seven substitutions in the line leading to man, against none in the lines to whale, rat and rabbit. The comparative study of eye lens crystallins thus reveals a picture of slow but steady changes. It is still too early to decide to what extent these changes reflect adaptations to changing functional requirements of the lens or are due to gradual, functionally neutral, so-called non-Darwinian, divergence in the course of millions of years. Biosynthesis
of lens protein
The lens is also attractive for the study of protein synthesis since, as we have seen, the organ is composed mainly of crystallins that represent a rather small number of specific structural proteins. When studied in organ culture it appears that about 50% of the total protein synthesis is directed towards the production of cr-crystallin [ 161. Interesting results have been obtained recently upon investigation of protein biosynthesis in cultured lens epithelium [17]. It appears that during
Fig. 3. Computer analysis of the sequences of the aA chains of 17 mammalian species, chicken and frog produced this ‘phylogenetic tree’. The calculated numbers of amino acid substitutions are indicated for each branch. The estimated times of divergence are based on palaeontological data. (From [I I].)
June 1979
“Oh Darling, your eyes, they’re fascinating! You certainly must have radical substitution in your crystallins!”
differentiation a qualitative change in the synthesis of crystallins occurs. The transition from epithelial cell to the fiber is accompanied by a marked increase in CYA chains and several P-crystallin polypeptides, while the synthesis of non-crystallin protein slows down. Strikingly, different regions in the epithelial monolayer have different protein biosynthetic activities. Lens mRNA [ 181 or lens polyribosomes [19] can be injected into living oocytes. In this system translation of the heterologous messenger proceeds for at least two days. It has been estimated that 20-30 crystallin polypeptide chains are produced per hour and per added mRNA molecule. De no\‘0 synthesis of lens protein can also he studied in a variety of heterologous cell-free systems by adding either isolated mRNA or lens polyribosomes. The newly synthesized products are analysed by SDS-gel electrophoresis followed by scintillation autoradiography. In order to detect the synthesis of a specific protein, immunoprecipitation with the antiserum directed against that protein has to precede the electrophoretic analysis. Detection of in vitro synthesis of the specific lens plasma membrane protein MP34 has been achieved in this way [3]. Cell-free incubation of a reticulocyte lysate with 14s rat lens mRNA revealed that the synthesis of the rat Lu-crystallin chain with the insertion [~YA’“~],is directed by the same messenger fraction which also codes for the synthesis of the normal aA chain [20]. Moreover antiserum raised against calf lens a+crystallin in which no CYA”‘~is present, precipitated the rat drains chain. This observation illustrates not only the high degree of homology between a-crystallins of two different species, but also confirms the very close relationship between normal rat CXAZ,and aAl”\, the polypeptide with the insertion of 22 amino acids. In conclusion we want to state that it is
TIBS -
141
June 1979
advantageous to use the lens for the study of the regulation of the synthesis of tissue-specific proteins. It offers a population of well-defined cells which show distinct stages of differentiation and biosynthetic display characteristic activities of highly specialized proteins. References Bloemcndal, H. (1977) Science 197. 127-138 Harding, J. J. and Dilley, K. J. (1976) Ex[>. Eyr Res. 22, 1 Bloemendal, H. (1977) in International Review of Biochemistry, Biochemistry of Cell Differmtiation II (Paul, J.. ed.). University Park Press, Baltimore. Vol. 15. pp. 195-225 Van Klcef. F. S. M., De Jong. W. W. and Hoenders. H. J. (1975) Nnrurt, (London) 258, 264 Masters. P. M., Bada, J. L. and Zigler, J. S. (1977) Narure (London) 268. 71-73
Tadpoles,
Kibbelaar.
M. A., Selten-Vcrsteegen,
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Bloemendal, H., Zweers, A., Koopmans, M. and van den Broek, W. (1977) Biochem. Biophys. Res. Commun. 77. 41&425
16
Hanson, H.,GIasser, D. and Kirschke, Z. Physiol. Chem. 340. 107
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Biochemical D. C.. Vol. 5.
Cohen, L. H., Westerhuis, L. W., de Jong, W. W. and Bloemendal, H. (1978) Eur. J. Eiochem. 89, 259-266 Siczen, R. J. and Hoenders, Len. 80, 75-80 Spector, A. and Kinoshita, Opthalmol. 3, 5 17-522
H. J. (1977)
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Vermorken, A. J. M. and Bloemendal. Nature (London) 271, 779-181
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10 Clayton, R. M. (1974) in The Eye (Davson, H. and Graham, L. T. Jr. eds), Academic Press, New York and London, Vol. 5, 399
M.. Bloemen18 Berns, A. J. M., van Kraaikamp, dal, H. and Lane, C. D. (1972) Proc. Nar. Acad. Sci. U.S.A. 69, 1606-1609
11 De Jong, W. W., Gleaves, J. T. and Boultcr, (1977) J. Mol. Evol. 10, 123-135
19 Asselbergs, Bloemcndal, 5 17-524
12 Slingsby, C. and Croft. Res. 26, 291-304
L. R. (1978)
D.
Exp. Eye
13 Dayhoff. M. O., Barker, W. C. and Hunt, L. T. (1976) in Atlas of Protein Sequence and Structure
drugs and toxicities W. Robert Jondorf
The theory of Brodie and his collaborators that the non-specific drug metabolizing enzymes were developed in terrestrial species of animals essentially to enable them to convert lipid-soluble exogenous compounds to more readily excretable polar derivatives has withstood the test of time. The theory has come into focus again because of our better understanding of hormonal influences on evolutionary drives, and also because of the foreseeable consequences of contaminating the global environment with more and more unmetabolizable lipid-soluble compounds. In the course of studies on age-related factors in drug metabolism in mice, guinea-pigs [I] and chicks, and in reviewing the whole subject of drug metabolism in terms of development and evolution [3-51 certain new facts connected with the regulatory importance of thyroid hormones and prolactin have come to light [5] which ought to be more fully integrated with concepts of drug metabolism.
for like other juvenile aquatic vertebrates lipid-soluble they could eliminate exogenous compounds by dialysis through the lipoidal membranes of gills or skin. This may no longer be strictly true since various halogenated environmental contaminants persist in most ecosystems [6] and at best, aquatic vertebrates lacking drug metabolizing enzymes can do no more than equilibrate with some of these contaminants. After adaptation of the aquatic tadpoles and toadpoles to life on Amphibian beginnings land by metamorphosis they develop the The story really began about 20 years ability to oxidatively metabolize and ago when it was established, partly on the conjugate test drugs [3,4]. This illustration basis of experiments with amphibia [3,41, of a biochemical metamorphosis was as that the ability to metabolize relatively vivid as that of the sequential changes lipid-soluble drugs and foreign comoccurring in nitrogen excretion patterns pounds to more water-soluble entities was and the developmental changes in plasma a characteristic of biochemical evolution proteins, visual pigments and haemoaccompanying the transition from aquatic globin [7]. to terrestrial life. Tadpoles of toads and of The ‘terrestrial drive’ of amphibian certain kinds of frogs could not metabolize metamorphosis appears to be controlled drugs either by oxidation or conjugation, by thyroid hormones and can be manipulated towards precocious development by W. Robert JondorA lives at 3 Cough Way, thyroxine 171. AltematiGely it can be Cambridge, U.K. and is currently associated with inhibited by antithyroid compounds such laboratories in Newmarket working on the detection of drugs in horses.
(Dayhoff, M. O., cd.), National Research Foundation, Washington Suppl. 2, Ch. 2
A. M. E.,
Dunia, 1.. Benedetti, E. L. and Bloemendal, H. (1979) Eur. J. Biochem. 95, S43. Bloemendal, H., Kibbelaar, M. A., Ramaekers, F. C. S., Selten-Versteegen, A. M. E., Dunia, 1. and Benedetti, E. L. (1978) in Protides of the Biological Fluids 26 Colloq. (Peeters, H., cd.), Pergamon Press, Oxford (in press)
as Z-thiouracil and thionrea.
These and
F. A. M., van Venrooij, W. J. and H. (1978) Eur. 1. Eiochem. 87,
20 Cohen, L. H., Westerhuis, L. W., Smits. D. P. and Bloemendal, H. (1978) Eur. J. Biochem. 89, 251-258
other antithyroid compounds have been shown to interfere with thyroid peroxidase activity [8], a model precursor reaction for the (mammalian) synthesis of thyroxine in vivo (Table I). The antithyroid compounds also inhibit key enzymes required for urea or pyrimidine synthesis [9] such as carbamyl phosphate synthetase (also shown in Table I). The terrestrial drive of tadpole development is opposed in nature by a juvenilizing ‘aquatic drive’, regulated by pituitary prolactin release [ 101. The effect of prolactin on tadpoles is to promote growth without metamorphosis, while adult newts return to the water to mate and spawn under the influence of this hormone [lo]. It can be readily recognized that antithyroid compounds and compounds that interfered with prolactin release or feedback control would have profound toxicological effects on orderly synchronized development in higher animals subject to similar hormonal controls. The story continues with mammals. Drugs, the new-born and induction Drug-metabolizing enzyme activity in placental mammals is altogether absent or deficient in the foetus and the new-born [l]. This was confirmed particularly elegantly when the various components of the mixed function oxidase system in rabbit liver were measured during preg nancy and for two weeks after parturition correlated with the [I 11, and were electron-microscopic appearance of the liver and with measurements of micro. somal drug-metabolizing activities a’ various stages of development. Drui metabolism in new-born mammals can bt induced very readily by administerinl 0
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