Isolation and characterization of A CH2 domain fragment of human IgG

In~nlmodwmlst~y 1975

Vol 12. pp 333 337

Pergamon Press

Printed in Great Britain

ISOLATION AND CHARACTERIZATION OF A CH2 DOMAIN FRAGMENT OF HUMAN IgG* B.-K. S E O N and D. P R E S S M A N Roswell Park Memorial Instituter, Department of Immunology Research, 666 Elm Street, Buffalo, New York 14263, U.S.A. (First rece,ved 9 July 1974; tn revised form 21 October 1974) Abstract--A fragment corresponding to the CH2 domain of IgG was isolated and chemically character-

ized. This C,2 domain fragment was isolated by gel filtration of the reduced and alkylated IgG fragment which was obtained by the high-temperature peptic digestion of myeloma IgG. An amino-terminal amino acid sequence study revealed that the amino acid sequence of the Cn2 domain fragment corresponds to that of a 7-chain segment which begins with isoleucine 253. The mol. wt of the fragment was estimated by gel filtration in the presence of 6 M guanidine-HCl to be 8800.

INTRODUCTION In a previous paper (Soon and Pressman, 1974) we have shown that both myeloma and normal IgG immunoglobulins yield a distinct fragment upon peptic digestion at pH 4'5 and elevated temperatures, e.g. 66°C. This high-temperature fragment was isolated from two myeloma IgG proteins (both of the IgGl subclass) and normal IgG immunoglobulin. Chemical and immunological analyses showed that the high-temperature fragment was mainly derived from the constant portion. In the present study, we have isolated a subfragment from the reduced and alkylated high-temperature fragment derived from a myeloma IgG protein of IgG1 subclass, and have obtained evidence that shows that this subfragment corresponds to the Cn2 domain. MATERIALS AND METHODS lmmunoglobuhn The rnyeloma IgG protein Col which was isolated from the serum of a multiple myeloma patient of Roswell Park Memorial Institute was purified by repeated precipitation with 33~o ammonium sulfate followed by column chromatography on a DEAE-cellulose column (Seon and Pressman, 1974). The purified protein exhibited a single, welldefined arc when subjected to lmrnunoelectrophoretic analyses using goat antisera to whole human serum and to human IgG Miscellaneous reagents Pepsin was purchased from Worthington Biochemical Corp. (Freehold, N.J.). Guanidine hydrochloride was purified by the procedure of Nozaki and Tanford (1967) and its purity was checked by the u.v. absorption spectrum as described by Wong et al. (1971). In some experiments, guanidine hydrochloride of exceptional purity from Heico, Inc. (Delaware Water Gap, Pa.) was used. The reagents for amino acid sequence determinations were Beckman sequencer grade (Beckman Instrument Co., Fullerton, Calif.) or Pierce sequanal grade (Pierce Chemical Co., Rock-

ford, Ill.). Hydriodic acid (57%) was purchased from Fisher Scientific Co. (Fair Lawn, N.J.). o-Glucosamine and ogalactosamine were purchased from Sigma Chemical Co. (St. Louis, Mo.), The reagents for SDS acrylamide gel electrophoresis were from BIO-RAD (Richmond, Calif.). Isolation of subfragments of the high-temperature fragment The high-temperature fragment was isolated as reported previously (Seon and Pressman, 1974). Myeloma IgG (Col) was dissolved in 0'025 M sodium acetate buffer, pH 4.5, and digestion was carried out at 70°C for 50 min with a pepsin to substrate ratio of 1:100. No preincubation of IgG solution was carried out at 70°C without pepsin since preincubation resulted in a poor yield of the high-temperature fragment. The digestion was stopped by neutralization with 1 M Tri~HCl buffer (pH 8.0) and 0.1 N NaOH. Precipitate which appeared upon neutralization was removed by centrifugation. The clear supernatant was fractionated on a Sephadex G-150 column to give an elution pattern consisting of four peaks. The high-temperature fragment appeared at the second peak. The subfragments were isolated from the high-temperature fragment as follows: the high-temperature fragment was fully reduced and alkylated in the presence of 7 M guanidine as described previously (Seon et al., 1973); and the reduced materialwas subjected to gel filtration on a Sephadex G-150column (2 x 100 cm) in 6 M guanidine hydrochloride at 4°C. Determination of tool. w t of the subfragment Molecular weight of the reduced, alkylated fragment was determined from the elution positions observed during the gel filtration in 6 M guanidine hydrochloride as reported by Fish et al. (1969). The column (Sephadex G-150, 2 x 100cm, 6M guanidine-HC1) was first calibrated with bovine serum albumin, human myeloma IgG,/~-lactoglobulin and lysozyme, which were fully reduced and alkylated in the same manner as the high-temperature fragment. A standard curve for mol. wt was drawn from the elution positions of the above proteins of known mol. wt. Weight rather than volume was used to measure elution positions. Kd t/a was plotted as a function of mol. wt to the 0.555 power (Fish et al., 1969). In addition, as described below, mol. wt of proteins were also estimated by SDS acrylamide gel electrophoresis.

* This work was supported in part by Grant No. A103962 from the National Institute of Allergy and Infectious SDS acrylamide gel electrophoresis Diseases. Acrylamide gel electrophoresis in the presence of sodium t A umt of the New York State Department of Health. dodecyl sulfate was carried out according to Weber and 333

334

B-K SEONandD

Osborn 119691 Tbc posltlon~ and inteilsitles ol \be stained hands ~xere measured on
PRESSMAN PTI-a

0.6

0.4 J 0 ¢0 04

llllltlO-lt'l Illllhll
OUI.lntltatl'v'C alnlno acid sequence determinations were ~arrled out bx the manual, three-stage Edman degradation (Edman. 197(h as de,,cr~bed previously ISeon et a/. I97hl and 1972ul In this ~ttld\. the phen~lthlohydantom derl~atIVL" O] U l g l l n n c

\',as detcrlnlned

us tallow%

¢lfl.er t h e c x t l U C -

lion by ethyl acetate of phenylthu~hyduntou~ dcrlvatlxes h-am 1 \ H C l ( E d m a n , 1971}: Seon ct a[. 1972ulthe 1 \' HCI phase ~ as evaporated to dryness m a stream of mtrogen and the ~cs~due hydrolyzed with 57". HI tit 123 (" (Smithies ct al, 1971, Soon et a/. 1972a) Thc Icsultmg frcc urglnme ,aas dcterlnllled on all ;.lulonlutlc amino u c l d analyzer ~s u control, the 1 \ H('I phase of the p~euedmg step m the Edman degradation procedure x~us treated m cxaclly the ~,ulne nlunller

tllllllO a c i d ('~)1t111051[10tl

Protein was hydrolyzed m constant-boiling H C I m an evacuated sealed tube at I10 C for 24 or 72hr (Scon c t a / . 1971h) In some experiments protein xxas hydrolyzed m the presence of 4", th]oglycohc acM as described preciously ISeon e t a / , 1972h, Matsubaru and Sasukl. 1969). Q u a n t m t m c d e t e r m m a t m n of a m i n o acM was carrmd out on a Techmcon automatic a m m o acid analyzer l Scan et aL, 1971h) All the analyses werc done at least m duphcate. The elutlon posluon o f g l u c o s a m m c and galactosamlne were determined by adding these t u o hexosamlnes to the standard amino acid m~xture apphcd to the column The glucosammc peak appeared approximately S m m ahead of the ~ahne peak and the galactosaminc peak overlapped thc c~stmc peak which appeared 6 mm after the vahnc peak

0.2

0(/)

u

i

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0.1

IOO

200

3'O0 ml

Fig I klactumat]on ol thL ~duLcd and ulk~lalcd Inghtcmpcrature fragmcnl of mxcloma IgG ( ( o i l (a) Top 25rag of tngh-tempcraturc h-agmcnt ~ a s full~ reduced and alkylated m the plcscncc of 73.I guanldulc h~drochlorlde and the resulting mutclqul was filtered on a Sephadex G-150 column 12 - 150cm) m 6 ~,1 g u a m d m c hvdrochlorMe The column had been cuhbrated with bhle dextran 2000. e - D N P lysmc bovine serum albumin, heax and hght chums of lgG, fl-lactoglobuhn and 13so/ymc All the proteins were completcl 3 reduced and alkylated before gel filtratton. Vo and \'1 designate \old ~olumc and total volume accc,sslblc Io ,oh'ent, rc,~pcctl~Cl 3 ~+o corresponds to the eluhon position of bluc dextran 2t)00. Vl to that o f e - l ) P N lxsmc (by Bottom' the fractions making tip the shoulder m lhc top figure (as shown bv thc horizontal lmcl ~crc pooled and rechromatographed wltb blue dcxtran 2()t)(I and eD P N lvsme The fi'achon,~ lubcled Plla (top figure) and Pllb (bottom figure) uerc pooled as indicated b\ the horizontal hnes and used fo] furthcr studm~

RESt LTS l v>latum ol ~ul~lraqment.s T h e h i g h - t e m p e r a t u r e f r a g m c n t from the m ~ c l o m a I g G p r o t e i n Col w a s isolated as d e s c r i b e d in a p r e v i o u s p a p e r (Seon a n d P r e s s m a n , 1974) a n d m M a t e r i a l s a n d Methods. The reduced and alkylated high-temperature f r a g m e n t w a s s u b j e c t e d to gel filtration m the p r e s e n c e o f 6 M g u a n l d l n e HCI. A p e a k with a p r o n o u n c e d s h o u l d e r o f u.v. a b s o r b i n g m a t e r m l w as o b t a i n e d (Fig. lay. W h e n m a t e r m l f r o m the s h o u l d e r w a s r e c h r o m a t o g r a p h e d (Fig. lb), a single p e a k w a s o b t a i n e d t h a t w a s distinct f r o m the p e a k s h o w n in lqg. la. T h e real. wt o f the f r a g m e n t s m the two fractions d e s i g n a t e d P l l a a n d P l l b * were e s t i m a t e d from the e l u t i o n p o s i t i o n s as d e s c r i b e d in M a t e r m l s a n d M e t h o d s . M o l e c u l a r w e i g h t s of 14,000 a n d 8800, respectively, for P I I a a n d P I I b were o b t a i n e d (Fig. 2). As d e s c r i b e d below the P I I b f r a g m e n t c o r r e s p o n d s to the CH2 d o m a i n . T h e m o l a r yield o f the P l l b f r a g m e n t from p r o t e i n Col w a s a p p r o x i m a t e l > 20 per cent.

* T h c lngh-temperamre fragment v, as designated as "PII" since tlus fragment appeared as the sccond peak during gel fihratlon on a Sephade\ G-150 c o h m m (Seon and Pres,,,man, b)74) Thc tyro I'ractlons and the corresponding subfragments obtained here were, therefolc, designated "Plla" and "Pllb', as mdlcated m Fig. 1

~X p T r - b L v s o z v m e ~ PIT-o B - L ~c to~lobulin~L, L chain ~:

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(MW10555 Fig. 2 Determination of the mol, wt of the Plla and PIIb subfragments. Fully reduced and alkvlated protein, were filtered on a Sephadex G-150 c o h m m m the presence of 6 M guamdine (see Fig I). A standard curve ~clatmg real. wt to elutlon ~olumc was prepared usmg reduced and alkylated reference proteins of kno,~n mol. wt Kd ~ was plotted as a function of mol wt to the t)555 powel (see Matermls and Methods) The p o s m o n s of thc refcwnLc proteins on the curve are shown b~ ,m open c~rcle and those of Plla and P l l b by an "X' Thc real ~xt of Plla und Pllb were calculated to bc 1400() and S~()() respectwely

A CH2 Domain Fragment

335

!

Fig. 3 SDS acrylamide gel clectrophoresls of the PIIa and PIIb fragments. Approximately 10/~g each of the Plla (top) and PIIb (bottom) fractions were applied to 10°o acrylamlde gel containing 01"o SDS as described by Weber and Osborn (1969). The direction of electrophoresls is to the right (anode).

Examination oJ subJragments by sodium dodec yl sulfate gel electrophoresis The PIIa and PIIb fractions were subJected to acr~lamide gel electrophoresis in 0' 1 per cent sodmm dodecyl sulfate. After being stained, the gels were scanned on a densitometer. Both preparations exhibited more than one stained band (Fig. 3). The gel containing the PIIa fraction showed two intensely stained bands and a fast-running, faint band. The relative intensities as determined by the densitometer were 1: 12:7 in order of decreasing mobility. The tool. wt of the two intense bands were estimated to be 14,000 and 13,000, respectively These values agree well with the molecular weight of 14,000 for PIIa as estimated from gel filtration in the presence of guanidine. Two distinct molecular species in the PIIa fraction were also indicated by the quantitative aminoterminal amino acid sequence study described later. The gel containing the PIIb fraction showed one intensely stained band and one weakly stained band. The relative intensities of the two bands were 4: 1. The tool. wt of the intense band was estimated to be 14,000 which ~s much larger than the 8800 obtained for PIIb by gel filtration m guanidine. This can be easily understood in view of the fact that PIIb is a glycoprotem (see Table 1) and that tool. v~t of glycoproteins estimated by SDS gel electrophoresis are anomalously high (Segrest et al., 197t : Wilde, III and Koshland, 1973). The 9500mol. wt of the weaker, fast moving band agrees well with the PIIb mol. wt (8800) obtained by gel filtration. The structural differences between the fast moving and slow moving materials could be in the carbohydrate moiety rather than in the protein portion. Amino-terminal amino acid sequences The sequence of the first five amino acid residues at the amino-terminal portion was determined for each fraction by the manual, quantitative Edman degradation procedure which we have described previously in

full detail (Seon et al., 1971a and 1972a). The determination of the phenylthiohydantoin derivative of arginine was described in Materials and Methods. The PIIa fraction appears to be composed of two or more distinct polypeptide chains. The sequence determination showed two major amino acids and several other amino acids in relatively smaller proportions at each of the first five positions. This result is consistent with the two intensely stained bands in SDS acrylamide gel electrophoresis. For the further study of the PIIa fraction, additional purification is necessary and thus no data on PIIa sequences are reported here. The PIlb fraction, on the other hand, showed a single, major (over 50 per cent of the total recovery) amino acid at each of the first five positions. These are isoleucine (62"0) in the first position and then serlne (57°o), arginine (78°o), threonme (81"o) and proline (54°0), in succession. In the first position each of the minor components recovered represented less than 11 per cent of the total. At each of the other four positions the minor amino acids detected were present in proportions amounting to less than 23 per cent each and usually less than 16 per cent. This suggests that the PIIb fraction is composed mainly of one distinct polypeptide chain. The percentages of the various minor amino acids detected at each position are of the order of magnitude that is usually observed when the manual Edman degradation procedure is used to determine amino-terminal amino acid sequence of proteins (Hood et al., 1969: Seon et al., 1971a and 1972a).

Amino acid composition o] subJraqment P l lh The amino-terminal sequence, Ile-Ser-Arg-Thr-Pro-, determined for the PIIb fraction was found to correspond to a five-residue portion in the lgG 7-chain which starts at position 253 (see Discussion). In an attempt to determine whether the polypeptide chain of mol. wt 8800 in the PIIb fraction might m fact correspond to a portion of 7 chain beginning at the isoleucine residue number 253, the amino acid composition

336

B-K

S k O N a n d D PRESSMAN

of the PIIb fraction was detcrmmcd and compared v~ith the composition of a segment of the heax x cham of the myeloma lgG protem Eu ha\ mg approxHnalcl 3 the same calculated tool. x~l. ~e rcs~duc~ 253 332 As seen in Table l there is a close correspoladence between the two. Except for the glycmcs and lysines or the t)rosmes and phenylalanlncs (sec Discussionl the s~milarities m compos~Uon a~c sinking, especially since neither have a methionmc, bolh ]la~e a large amount of valine and t,ao halt-c\sllncs. ,tnd each carrles carbohydrate

I Pill obtamed by peptic dlgeshon of two myeloma lgG protems and normal lgG was mostly, if not c o m pletely, derived from the constant porhon of the m > munoglobuhn molecule (Seon and Pressman. 19741. h appears, therefore, that ttle constant domam of hghl challis Hlld "~OlllCconstant domains of hea~v chains are more stable to thermal denaturatum than the varmble domams of light and hoax ) chains. In the present study ~c ha,~e Isolated two suhl]'agmcnls. Plla and Plli~. of the high-temperature Pll lragment derwed ti'om the myeloma lgG protem ('ol alTllnO-tclnllnttl alrllnO acid sequence and a m m o acid composihon determinations demonstrate that one of the subfragments, PIIb. corresponds to thc ( ' , 2 domain and that the ammo-termmal poruon of lhls subffagmcnt starts w'lth the reslduc correspondmg t,, lhe isoleucme 253 in the hear\ chain of lgG (Eu} The conclu,~on that ~c ha~e ~olated lhc ('U2 domalll of the m).eloma protein ('O1 Is based on l\~o points. [-lrst. cxamlnalloll ol the xarlous pubhshed scqucm, es lot c h a o s oI IgG molecules showed that o n h the segment of 3'-chain which slarts Lit posHion 253 correspo~lds to the amlllO-lerminal ,sequence ob>cl"~,ed [or the Pllb ,~tlbl'lagment. No othcl segment o1' [g( J moleCl.lle c h a o s , ll~cludmg the variable portions IB,'u and Kabat. 197(), Smith c! ~tl. lU"l. k d c l m a n ct dl.. 1969: Press and Hogg. 1970, Pon~tmgl ~'t ~d, 197U, Wang et u/, 19v3: ( ' a p r a and Kehoe, 1974L could bc lt~und \~ ith :l correspondHlg "~eqtiellCC

DIS( t SSI()N

('areful, limited enzymatic dlgesllOnS of unmunoglobuhn molecules h a w been shm~n lo weld ti'agments consisting of one or more intacl, unmunoglobnhn domams {Porter. 1959; N~sonofl" ~'/ u/. 196t~: I ilstlnll and Karush, 1965; Solomon and McLaughhn. 1969, Karlson et td., 1969: Turner cl ,d, 1970: Plau/ and Tomasl, 1970: Seon el o/, 1972t ' ( UII and D'kustachlo, 1972: Hochman ct ~d, 1~}~3. Kaklmol~ and Onoue, 19731 Each domain consists of ,lboul 10(I amino acid residues and the backbone of each ~s ,t d> sulfide loop (Edelman and Gall. Ill691 Pre\ uxlslx x\c demonstrated thai tile ('h d o m a m \'~:1,,, milch lllOl-e Misceptible to peptic d~geshon :~t I ~ ( than lhc \b, domain. ~ h e r e a s tile relat~\.e susccpt~bdlhe, \\ctc reversed at 55 ( ' (Neon ct u/, 19721, and 1972t ) We showed further that the h~gh-tcmpcraltHe fragment [able

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a Residue number~ are calculated on the basis o f three arginines per molecule. h The amino acld composition of the 253-332 segment o f Eu heavy chain (~l chum) ~las calculated from the publlshed sequence (Eaelman, 1970), c Determlned as carboxymethylcysteine. Correction was made f o r the 9% decomposition dumng acid hydrolysls (Cole et a l . , 1958) d The values f o r these residues were o b t a i n e d ~ the 24 hr hydrolyzate~ obtained in the presence of 4,1 thloglycol~c acid. Th-, y i e l d of trv~tooh~n was poor probably due to the presence of carbohydrate (EastoE, 1966) e Determined in the 72 hr hydrolyzate. f The study of Howell et al. (1967) indlcate~ that m some human IgG, ene of the four tyrosines In HEu 253-33? i~ replaced with phenylalanlne. I t appears to be the case f o r IgG ~Col). g Observed in 24 hr hydrolyzates, but not in 72 hr hydrolyzates (s=e blaLerials and I'e=h~s)

OI

A Cn2 Domain Fragment Second, the segment 253-332 of the myeloma protern Eu, which contains the disulfide loop of the Cn2 domain, corresponds closely in a m i n o acid composition to that of the P I I b subfragment, a n d characteristic features in the composition of this segment of protein Eu, Le. the presence of a large a m o u n t of valine, two half-cystines and carbohydrate a n d the absence of methlonlne, are also features of the PIIb fragment. The differences m amino acid composition observed between the 253 332 segment of the myeloma protein Eu a n d the PIIb fragment could very well be due to the fact that different IgG myeloma proteins are being compared and that with all the similarities (or tdentities) to be expected in comparing constant p o m o n s of heavy chains from different proteins some differences, perhaps due to allotypic differences, are also to be expected. For example, Howell et al. (1967) obtained results which indicated that in some lgG proteins tyrosine 296 found in the Eu heavy chain is replaced by phenylalanlne. This may be the case for myeloma protern Col: the PIIb fragment contains three tyrosines and two phenylalamnes as compared with four tyrosines and one phenylalanine of the 253 332 Eu segment In addition, the composition shown for the 2 5 ~ 332 segment of the Eu heavy chain (Table 1) was calculated from the published amino acid sequence (Edelman, 1970: Edelman et al., 1969), while the composition for the PIIb fragment was obtained by amino acid analysis after acid hydrolysis. Some problems which emerge in such a comparison have been previously discussed (Seon et a l , 1972h). Regardless of the differences seen between the P I l b fragment a n d the 253 332 segment of Eu, the similarities are striking enough to strongly support our conclusion that we have ~solated the CH2 domain of the myeloma protein Col. This fragment is of considerable interest in view of the reports that the Cn2 domain carried the complement-fixing activity (Kehoe and Fougereau. 1969: Ellerson ct al., 19721. We are currently carrying out studies on the complement-fixing activittes of th~s a n d other fragments we have isolated from this myeloma protein and normal h u m a n IgG. Acknowledgements We wish to thank Dr. V. P Kreiter for useful dlscussmn and Mrs. H Tsal for her excellent techmcal as'qstance

REFERENCES Capra J D and Kehoe J. N (1974) Proc. natn 4cad Set U.S. 4. 71, 845 Cole R D., Stem W. H and Moore S. (1958) J b~ol. Chem. 233, 1359 Eastoe J. E. ( 19661 Gly~ oproteins IEdlted b~ A Gottschalkl, Chapter 5, p 112. Elsewer, Amsterdam. Edelman G M. (1970) Biochemistry 9, 3197 Edelman G. M., Cunningham B. A., Gall W E, Gottlieb P D., Rutlshauser U and Waxdal M. J. (1969) Proc natn Acad. Scl U S 4.63,78.

337

Edelman G M. and Gall W E. (1969) Ann. Ree. Blochem. 38, 415. Edman P. (1970) Protein Sequence Deternunation (Edited by S B. Needleman) Chapter 8, p. 211. Springer, New York. Ellerson J. R., Yasmeen D, Painter R. H and Dorrlngton K J. (1972) FEBS Letter,s 24, 318. Gall W E and D'Eustachlo P. 119721 Btochemlstry l 1, 4621 Fish W W. Mann K. G and Tanford C. (1969) J htol. Chem. 244, 4989. Hochman J., lnbar D. and Gwol D. (I 973) Bmchemtstry 12, 1130 Hood L., Lackland H., Elchmann K., Kindt T. J., Braun D. G. and Krause R M (1969) Proc. natn Acad. Scl. U.S.A. 63, 890. Howell J. W, Hood L. and Sanders B G (1967) 3 molec. Btol. 30, 555 Kaklmoto K and Onoue K. (1974)J. hnmun i!2, 1373. Karlsson F A, Peterson P. A. and Berggard I. (1969) Proc. hath. Acad. Scl. U S A. 64, 1257. Kehoe J. M. and Fougereau M. (1969) Nature, Lond. 224, 1212 Matsubara H and Sasakl R. M. t1969) B~ochem. biophys. Re~. Connn. 35, 175. NlsonoffA., Wtssler F. C, Llpman L. N. and Woernley D. L (1960) 4rchs. Btochem. Btophys. 89, 230. Nozakl Y. and Tanford C. (1967) Meth Enzymol. 11, 732. Plaut A G and Tomasl T. B. Jr. (1970) Proc mini. Acad. Sct. U.S q. 65, 318. Ponstingl H, Schwarz J., Relchel W. and Hilschmann N. (1970) Hoppe-Se yler's Z. Physml Chem. 351, 1591. Porter R. R 11959) Bumhem d 73, 119. Press E. M. and Hogg N M. (1970l Btochenr J. 117, 641. Segrest J. P, Jackson R L, Andre~s E. P. and Marchesl V. T (1971) Btochem. blophrs. Res. Commun. 44, 390. Seon B. K, Roholt O. A and Pressman D 11971a) lmmunochemistry 8, 585 Seon B K, Roholt O. A and Pressman D. [1971b) J b~ol. Chem. 246, 887 Seon B. K, Roholt O A. and Pressman D (1972a) J hnmun. 108, 86. Seon B. K., Roholt O A. and Pressman D 11972h) J hmnun. 109, 1201 Seon B. K., Roholt O A. and Pressman D. 11972c) J biol. Chem. 247, 2151 Seon B. K.. Yag~ Y. and Pressman D. 119731 d. hnmun. 110, 345 Seon B K. and Pressman D. 11974t d hnmun. 113, 1190. Smith G. P., Hood L and Fitch W M. (1971) Ann. Ret'. Biochem. 40, 969 Smithies O. Gibson D., Fanning E M , Goodfllesh R M., Gilman J G and Ballantyne D L. (1971) Biochemtstry 10, 4912. Solomon A and McLaughlin C. L. (1969) d bud. Chem 244, 3393 Turner M W, Benmch H H, Natwg J B (1970) Chn. exp. hnmunol. 7, 603 U tsumi S. and Karush F. (1965) Biochenu~try 4, 1766. WangA C., Gergely J and Fudenberg H H. (1973) B~ochemtstry 12, 528. Weber K. and Osborn M. (19691 J. htol. Chem 244, 4406 Wilde IIL C E and Koshland M E. (I 973) BtochenHstry 12, 3219 Wong K.-P, Roxby R and Tanford C (1971) 4nalyt. Biochem. 40, 459. Wu T T. and Kabat E. A 119701J. exp. Med. 132, 21 I.