NATURE VOL. 224 DECEMBER 27 1969
`
`1307
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`Institutes of Health, US National Science Foundat.ion a nu
`the Cooley's Anemia Foundat,ion.
`
`J . FUHR
`C. NATTA
`P.A. MARKS
`A.BANK
`
`Department e,f Medicine,
`Columbia University College of Physicians and Surgeons,
`New York.
`Rec~ivcd Aw;ust 11 ; re\'ised October 2, 1969.
`
`' Weatherall, D. J'.. The Thalaa.,aemia S 1/1ldronie8 (Blackwell, Oxford, JY65).
`' Hurka., E., and Marks, P. A., Nature, 199, 706 (1963).
`• B ank, A., and ~£arks, P. A. , .T. Clin. Invest., 45, 330 (1966).
`• Clegg, J. B. Weatherall , D. J., Na·Nakorn , S., and W.isi, P., !Yat1tre, 220,
`604 (1068).
`~ :Fuhr, J. E., London, I. ;r., and Grayzel , A. I., Proc. US 1Ya.t. Acad. S ci.,
`63, 129 (1969).
`• Nomura, M., and Lowry, C., P>·oc. US Nat . A cad. Sri., 58, 9 ,16 (1967).
`'Bishop, J., Biochim. Biophys. Acta.,119, 130 (1966).
`
`cule arc not reveale u in the electron m icrographs, t his
`part has been omitted from the model.
`Fig. 3a represents a single I-'- + L chain protomeric unit,
`with three numbered cysteino residues available for d i•
`sulphide bridge formation. The numbering, which nm;;
`from the N-tenninus, is that used previously•, but ,
`because the model represents t h o folded polypep tidf•
`chains , the linear sequence of bridges shown h er e need
`not correspond to that in tho extended ch a in. In Fig. :3b
`a pair of these units hav e been assembled through bridges
`2 and 4 to form a dirneric subunit, leaving two cyst eine
`residues 3 available for bridging t o identical n eighbouring
`units, generating the cyclic structure of the I gM molemtle,
`Fig. 3c. It is this latter view of t h e rnolec11le which w,•
`believe corresponds t o images of t he type seen in Fig. l b.
`The brancrung referred to in Fig. le occurs at a flexibk(cid:173)
`"hinge" region a ssociated with bridge 2, as representc-d
`in the model in F ig. 3d.
`Each of the ten I-'-+ L chain pro tornor uni ts carries a
`single antigen-binding site, situated at the out ermost tip
`of the units ananged radially in the I gM molecule. This
`positioning follows from electron micrographs of Ig:M
`antibody molecules cross-linking two bacterial flagella,
`as seen, for example , in F ig. 2a . When, however, IgM
`molecules are seen in profile attached along a s ingl<"
`bacterial flagellum, they a re "staple" -like in appearanc,i•
`(Fig. 2b). Similar forrns of IgM ,m tibo<lies have oe.ell
`seen at t a ched to fragments of erythronyte membran e 18.
`
`Conformation of the Free and Antigen(cid:173)
`bound lgM Antibody Molecules
`THE two major classes of antibody molecule, I g G and
`IgM 1
`• have muleuul~r w,:,igh~1:; of Hi0,000 a ml ll00,000,
`respectively. They are composed of heavy and lig ht
`polypeptide chains. In IgiVI each heav y (1-1-)
`chain, molecular weight 70,000, is linked by
`a disu lphide bridge to a light (L) chain,
`molecular weight 23,000, and is proba bly
`linke<l by three further bridges to other p.
`chains•-10.
`A model accounting for the flexible Y •
`s haped structure of IgG seen in the electron
`mieroseope has already been d escribed 11 , 12,
`and we now propose a m od el for the Ig:M
`antibody consistent both wit h the appeara n ce
`of free and antigen-bound molecules in elec(cid:173)
`tron micrographs and with
`t,ho av ailable
`chemical data . W o ha ve exa mined I g:M p re-
`parations from a variety of mammals and
`from the chicken a nd dogfish, and complexes
`o f Salmon ella flagella with antiflagella IgM
`from several of these species 13 • 14. All speci(cid:173)
`m e ns were negatively s t ained with col.cl 2
`per cent sodium phosphotungstatc (pH 7·3)
`on holey carbon films'• and dried at 4° C.
`The ~tellate appearance of the free IgM
`molecule (Fig. la) is somewhat varia b le.
`SomC't-imos we find molecules display ing the
`sim ple symmetry of that s hown in I<'ig . l b.
`Svehag and h is collea.gues16 •1 7 have publish ed
`g ood pictures of this t y pe, which they in(cid:173)
`t,erpret as arising from five cyclically linked
`s ubunits. \Ve a grne with t his interpretation ,
`but believe that the Ig:M molecule consists of
`two similar halves which in this view are
`s uperimposed. The arms are about 25 A wide 1'
`a nd 100 A lon g ; the Jess well defined central
`region of the molec ule is a pproxima tely 100 A
`acr oss. Fig. le sh ows examples display ing a
`rnor e commonlv encountered feat ure :
`in
`ea ch , one or m o~e of the radial units appea rs
`branch ed .into t wo, an d in on e ease all five
`a rms a re branched. The length of the
`branched sections of t,ho arms is 55-70 A.
`"\Ve suggest t hat the points a t which the
`a rms branch are flexible regions of the
`molecule wher e
`t wo heavy polypcpt;ide
`ch ains in each subunit are joined. This
`feature is embodied
`in the m odel sh own
`i n Fig. a.
`Because the details of t he
`material present, at, the centre of the m ole-
`
`(t
`
`c
`
`1.:r.-11.i"'-·~~·.11
`
`( iv)
`(ii)
`(iii)
`( i)
`F ig. l. Electron micrographs of free Jgjl,I molecules. a . IgM preparation isola te<! from
`the scrum of mice carry ing plasma cell tumour MOPC 104E (generously provider! Jn·
`Dr A. J . Munro). b and c, Selected individual IgM molecules; CiHiv) as a ; (v) isolated
`from dogfish scrum. The har represents 250 A.
`
`© 1969 Nature Publishing Group
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`

`

`1308
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`a
`
`NATURE VOL. 224 DECEMBER 27 1969
`
`constmctPd by linking each dimeric subun_it (F ig. :]b) to
`the next but one on either side.
`The Y-shaped appeara nce and variable confonnation
`of the IgM subunit seen
`in the electron mirrosr ope
`
`(I)
`
`(Ill)
`
`(ii)
`
`(i\')
`
`b
`Fig. 2. Electron micrographl! of IgM antibody molecules bound to
`Salmon,,Ua flagella.
`a, J)og6sll antibody cross-linking two flagella;
`b: (I) and (II) dogllah, (ill) sheep and (iv) rabbit antlbodiee, each
`attached to a single flagellum. The bar l'epresents 250 )..
`
`The reduced maximum dimension of molecules in the
`staple form is evident when Figs. 2a and b are compared.
`This appearance can be explained if several of the radial
`units have folded at a flexible hinge region to resemble
`the legs of a table, thus making possible the attachment
`of a ll , or most of, the terminal binding sites to antigenic
`determinants on the surface of a single flagellum. This
`folded conformation, represented by the model in ):<'ig. 3d,
`is confirmed by the fact that the perimeters of the staples
`have the approximate length, 300 A, of the extended
`IgM molecule. In Fig. 3d the legs are folded only at the
`hinge region near bridge 2 already postulated as their
`branch point. In some staples, however, the cross-bar is
`only 100 A long, with uprights of comparable length.
`These uprights would be formed from entire 100 A arms,
`with the central 100 A disk in pro.file forming tho cross(cid:173)
`bar. Thus, in addition to the flexible regions at the
`branched points of the arms, hinging can also occur in
`those regions of the heavy chains linked to neighbours
`by bridge 3.
`In addition to those already mentioned, the following
`known features of the I gM molecule are consistent w-ith
`our model: (1) The interhea.vy-ehain disulphide bridges
`a.re symmetrical•.
`(2) Reductive cleavage of a single
`bridge to each heavy chain yields dimeric subunits with
`a structure corresponding to t hat seen in Fig. 3b9 •19 •
`(:1) Enzymatic cleavage of IgM preparations yields••-22
`(a) an Fab fragment, with a molecular weight of 45,000,
`having a single antigen-binding site, and composed of an
`L chain and part of a heavy chain carrying no inter(cid:173)
`heavy-cha.in bridge; (b) an (Fab"), fragment, of molecular
`weight 120,000, carrying two antigen-binding sites; bridgo
`2 links the halves of this dimeric fragment, which arises
`from cleavage between bridges 2 and 3; (o) a fragment,
`Fe, of molecular weight 300,000, carrying no L chains 0r
`antigen-binding sites;
`this is the central disk of tho
`IgM molecule, composed of the ten C terminal ends of
`the µ chains linked cyclically by bridges 3 and 4.
`An alternative symmetrical IgM model fulfilling the
`chemical requirements but not immediately reconcilable
`with tho structure seen in the electron microscope can bo
`
`Fig. 3. Schematic mod els of the IgM molecule and il s snlJunils. "· A
`single µ+ 1.. chain protomeric unit ; 2, 3 and 4 are cystcinc re~idues
`available for interchain dll!ttlphide bridge formation: the L chain and
`disulphide bridge 1 linking it to the heavy (µ ) chain lie between the
`antigen b inding site (B) and cystelne residue 2. h, A dimer fonned by
`linki1;g two protomers through bridges 2 and 4.
`r , Model showing
`how firn of the dimerlc units shown In b linked cyc!icall~' through
`disulph ide bridges 3 are lU!!!Cmbled in the lgl\I molecule. d, l\Iodel
`inoorporating the features shown In c bu t with the subunits opened to
`demonstrate the proposed fl exibility about a " hinge" region asso(cid:173)
`ciated with bridge 2. The resull,ing branched appearance matches t hr.t
`in r crl.nln eled -ron mlcrograph.s.
`
`Fii,. -!.
`lllodcl of IgM a ntibody houud to a particul:\lc antigen. p hoto(cid:173)
`graphed in profile so as w simulate the "staple" appp,ararn:e in miC'ro~
`gr-,.phs of the t ype seen in F ig. 2b.
`
`© 1969 Nature Publishing Group
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`

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`NATURE VOL. 224 DECEMBER 27 1969
`
`resemble those of IgG11 •12. Such flexibility in antibodies
`must facilitate multipoint attachment.
`The altered
`stabilized conformation in the bound IgM molecule may
`be that w hich is recognized by, and activates, the first
`component of complement, leading to phagocytosis and
`mernbrnne lysis.
`Finally, the similarity of mammalian and dogfish IgM
`an1,ibodies is of interest, suggesting that t he features
`described were present in the common ancestor of the
`dogfish and onrselves.
`
`A. FEINSTEIN
`E.A.MUNN
`
`ARC Institute of Animal Phys iology ,
`Babra ham,
`Cambridge.
`ltcc·cive<l Sept.ember l 5; revised October 29. 1060.
`' I.lull. Wo,ld H ealth Omo.nization, 30, 447 (1964).
`• Pain. ii. Fl. , Biochim. Bioph11s. Acta, 94, 183 (1965).
`• Rmall, l'. A .. a11<l La.nun, M. E ., Bfr,r.hemistrv, 6, 259 (1966).
`• ~1ontgomer.v, 1-'. C., Dorrington, K. ;r., and Rockey, J . H ., Biochemistry, 8.
`1247 (1969).
`• Miller, I<'., and Metzger, II., J. Biol. Chem., 240, 3325 (1965).
`• Lamm, M. E., and Small, P . A., BiocltFmistry, 5, 267 (1966).
`' Suzuki, T., and Dontsch, H.F., J. Rio!. Chem., 242, 2725 (1967).
`• ~farchalonis, J., am! Edelman, G. J.I., Science, 154, 1567 (1966).
`• lleale, D., and F einstein, A., Biochem. J ., 112, 187 (1969).
`10 :\lillcr, F. , and Metzger, H., J. B iol. Chem., 240, 4740 (1965).
`11 Feinstein , A., and Rowe, A. J ., Nature, 205, 147 (1965).
`"Valentine, R. C., and Green, N. l\f., J. Mol. Biol., 27, 615 (1967).
`"I-'cinsteln, A., and Munn, E. A., J. Physiol., 186, 64P (]966).
`"Feinstein, A., a nd Munn, E. A., P roc. Seve,uh Int. Cong. Biochmn., Tokyo,
`982 (rn67J.
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`"Chcsubro, ]3., llloth, B., and Svehag, S.-K, J. Ezp. M ed., 127, 399 (1968).
`" H umphrey, .T. H ., and Do urmashkin, R. R., in Comple,nent (ed it. by
`Wo lstenholme. (I.E. W., :i.mt Knight, J.), 175 (Churcl,il!, J,ondon, 1965) .
`... J\lorrls, J . E ., 1.mtl Inman, 11·. P ., [111,1m,inochemi8lry, 7 , 285'1 (1968).
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`"Dorrin;.ton, K. J., an d )iihncscu, C., Immunochemistry (in t he prC$S).
`
`Multiple Forms of Monoamine Oxidase
`in the Developing Brain
`MuoH a ttention has recently been centred on the biosyn(cid:173)
`thosis and m etabolism of the catecholamines and indole(cid:173)
`a lky lamines. Because the enzyme mono1:1.mine oxidase
`(deaminating), EC
`(monoarnine : 0 2 oxidoreductase
`1.4.:l.4) is involved in the m etabolism of both these classes
`o f biogenic amines, and because of our interest in develop(cid:173)
`m ent'•' we were prompted to investigate the nature of
`this enzyme and especially its activity in developing
`brain and other tissues of the maturing organism.
`Several investigators have suggested that there might
`be multiple forms (isoenzymes) of monoamine oxidase
`(J.VIAO), based on indirect evidence derived from multiple
`substrate and inhibitor studies 3 - 7• There are even some
`r eports of attempts to isolate the pure enzyme which
`also suggest the possibility of the existence of one or more
`multi ple forms of MAO (refs. 8-13). As a means of
`approaching this problem, we attempted to look at any
`differences in MAO(s ) there m ight be between embryonic
`and adult bro.in.
`We used 16 da.y old embryo and adult New Hampshire
`red hens. After the appropriate tissue h ad been homo(cid:173)
`genized, the monoamine oxidase was solubili:r.cd by sonic
`oscillation14 for 30, 60 or 120 min, partially purified by
`ammonium s ulphate precipitat ion and then subjected t o
`polyacry lamide gel electrophoresis. In some oases, the
`enzyme was solubilized by h omogenization of the tissue
`with 15 per cent 'Lubrol'. The general methods and
`techniriues of gel electrophoresis used were those d escribed
`
`1309
`
`by Davis 15. The gel concentration wus 7·25 p er cont and a
`Tris glycine buffer at pH 8·3 was used. The rwming time
`of the electrophoresis was 2 h at 4° C with current at 4·5
`mA per tube. Monoamine oxidase activity was revealed
`by the appearance of plll'ple coloured bands after incuba(cid:173)
`tion with a nitroblun tetrazolium s ubstrate staining solu(cid:173)
`tion. Tyro.mine, kynuramine, benzylamine• or trypt(cid:173)
`amine 1 • was used as substrate. All substrates were used
`in excess. Monoamine oxidase activity was a lso d eterm(cid:173)
`ined by the spectr ophotometric method o f Weissbach et al.
`with ky nurami ne as substrate".
`
`C
`R
`A
`J<' ig. 1. Gel electrophoretic patterns or chick brain munoamine oxid11se.
`A, 16 day embryo; B, 1 day old chick ; C. :,dnlt. Arrows point to
`bands discUBSed in text. Arrow labelled rn ind icates the dye marker.
`The anode is at the bottom of the tube wh ile t-h• cathode is at the top.
`
`The results of acrylamide gel electrophoresis of em (cid:173)
`bryonic, newborn a nd adult chick brains stained for
`monoaminc oxidase activity, with benzylamine as sub(cid:173)
`strate, are sh own in Fig. l. The slow m oving MAO band
`which is always obtained from embryonic brain is com(cid:173)
`p letely absent from the pattern d erived from the adult
`brain. lt is also apparent t hat the newborn chick brain
`has a pattern similar to that of the embryonic brain. At.
`least five bands with monoarnino oxidase activity a re
`common to both adult and infant bra in. Our preliminary
`r esults indicate that t hese same MAO hand patterns a re
`obtained whether the tissue is disrupted by homogen iza(cid:173)
`t ion followed by sonication or d isrupted by homogeniza(cid:173)
`t ion with a non-ionic d etergent. The data obtained after
`sonication for 120 min were the same a s those a fter 30 min.
`These result s arc in accord with the statement by Kim
`and D 'Iorio that either method of solubilizing the enzyme
`results in similar p atterns of MAO active bands.
`Similar patterns of the multiple forms of MAO arc
`obtained if tyramine or k ynuramine is used as substrate
`of the enzyme, alth ough there seem to be quantitative
`differences in t he relative intensities of t he various bands.
`It should be pointed out, however, that when chick brain
`MAO patterns, from embryo as well as adult, were d eterm(cid:173)
`ined on acrylamide gel using tryptamino as substrate,
`no bands could be observed. This is in contrast t,o rat
`brain, where we and others 18 liavn obser ved that trypt(cid:173)
`amine as well as the other substrates mentioned earlier
`can readily serve as substrate for the multiple forms of
`the em:ymc .
`
`© 1969 Nature Publishing Group
`
`