Proc. Natl. Acad. Sci. USAVol. 75, No. 12, pp. 5778-5782, December 1978Biochemistry

Localization of the site of adenylylation of glutamine synthetase byelectron microscopy of an enzyme-antibody complex

(immune electron microscopy/ethenoadenosine/antibodies against nucleosides/regulation)

RAYMOND J. FRINK*, DAVID EISENBERGtt, AND DOHN G. GLITZ*t*Department of Biological Chemistry, School of Medicine, tChemistry Department, and Molecular Biology Institute, University of California,Los Angeles, California 90024

Communicated by Paul D. Boyer, August 7,1978

ABSTRACT Antibodiesto'thenucleosidelN6-ethenoaden-osine have 'been used',to 'localize the site of Iadenylylation ofthe glutamine synthetase [L-lutamate:ammonia ligase (ADP-forming), EC 6.3.1.21 of Escherichia coli. Antibodies were in-duced in rabbits by injection of a bovine albumin-ethenoaden-osine conjugate. The resulting antisera strongly bound ethe-noadenosine, its 5'-nucleotide, or protein conjugates of the nu-cleoside; little or no crossreaction was seen to adenosine, AMP,or the protein carrie Etenoadenylylated glutamine synthetasewas prepared by modification of the enzyme by the E. coliadenylyltransferase, using etheno-ATP as a substrate. Theethenoadenylylated glutamine synthetase was precipitated byantibodies to ethenoadenosine in conjunction with goat anti-rabbit gamma globulin. Electron micrographs of reactionmixtures of ethenoadenylylated glutamine synthetase andanti-ethenoadenosine showed individual enzyme moleculescomplexed with one or more antibodies and pairs of enzymemolecules crosslinked by a single antibody. The approximatesite of adenylylation was located from the apparent area ofcontact between enzyme and antibody. We conclude that theadenylylation sites are on the priphery of the bilayered hex-agonal disc, offset by 15 ± 10° from the 2-fold axis of symmetrythrough a vertex of the hexagon and 20 + 10 A from the planebetween the layers of the disc.

Stadtman and Ginsburg (1) described the glutamine synthetase[L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] ofEscherichia coli as an enzyme of singular metabolic impor-tance, and Tyler (2) characterized it in addition as a generalcontrol element of nitrogen assimilation in enteric bacteria. Theenzyme permits the utilization of ammonia as a source of ni-trogen, and with glutamate synthase and various transaminasesit links the metabolism of proteins and amino acids to that ofcarbohydrates. Glutamine synthetase thus provides a particu-larly good point for the regulation of cellular processes.

Regulation of glutamine synthetase occurs by severalmechanisms (1). One is enzyme adenylylation; AMP is bound,via a phosphate ester bond, to the phenolic oxygen of a singletyrosyl residue in each of the twelve identical polypeptidechains of the enzyme (3). Adenylylation results in a lessenedbiosynthetic activity and increased sensitivity to feedback in-hibition (1), and it also has been reported to affect the autoge-nous control of transcription of the gene for glutamine syn-thetase (4-6).The glutamine synthetase of E. colh is composed of twelve

identical protein subunits, each with a molecular weight of50,000 (1). Electron microscopy (1, 7) shows it to be a bilayereddisc or hexagon, each layer being composed of six subunits. Inthe presence ofmM quantities of divalent cations (1, 8), enzymemolecules stack to form cables. This work is concerned with thelocalization of the adenylylation site in the glutamine synthetasedodecamer. Immunoelectron microscopy-the visualization

in electron micrographs of antibody-linked biological struc-tures-has been particularly valuable in the localization of theindividual proteins of the E. coil ribosomal subunits (9-14). Thesame approach has been used to localize a nucleoside, N6-dimethyladenosine, in the small ribosomal subunit (15). Wereasoned that anti-nucleoside antibodies could be similarlyuseful in localizing the adenylylated sites of glutamine syn-thetase.

Anti-nucleoside antibodies can be induced by nucleosidesor nucleotides linked to carrier proteins or synthetic polypep-tides (16). We have attempted to use antibodies to adenosineor AMP to study the adenylylation of glutamine synthetase, butthe affinity of our antibodies was insufficient to permit for-mation of stable complexes. However, such an approach hasbeen used by others (17), and during the course of this work webecame aware of further related research by R. Hohman andE. R. Stadtman. Instead, we have produced antibodies to amodified nucleoside, 1,N6-ethenoadenosine (18). These anti-bodies were then used to localize the modified nucleoside onglutamine synthetase that had been ethenoadenylylated byusing etheno-ATP and the E. colh adenylyltransferase respon-sible for enzyme modification (19, 20).

MATERIALS AND METHODSGlutamine synthetase in a low state of adenylylation (GS% toGS%: two to three AMP residues per enzyme dodecamer) wasisolated from E. coil W grown to late logarithmic phase (21, 22).The enzyme was purified according to Woolfolk et al. (23) asmodified by Shapiro and Stadtman (21). The state of adenyl-ylation was estimated in the y-glutamyltransferase procedure(21).

ATP:glutamine synthetase adenylyltransferase was preparedby the method of Caban and Ginsburg (24) through the secondstep of DEAE-cellulose chromatography (step 5 of their pro-cedure). Ethenoadenylylated glutamine synthetase was thenprepared by a variation (20) in the technique described byGinsburg (19, 25); one to two molecules of ethenoadenosinewere incorporated per dodecamer, for a total preparation ofGS% to GS1.

Procedures for the synthesis and characterization of nucle-oside-protein conjugates, immunization, blood collection, andantibody characterization have been described (5, 26, 27).Binding of radioactive nucleoside to antibody was measuredby using a membrane filter assay as previously described (15,28). Tritiated 1,N6-ethenoadenosine was prepared by MoravekBiochemicals, City of Industry, CA. Unlabeled nucleosides andnucleotides were purchased from Sigma Chemical Co. Goatanti-rabbit gamma globulin was purchased from Calbio-chem.

For electron microscopy, 100-120 ,ug of ethenoadenylylatedglutamine synthetase plus 0.7-3.0 equivalents of antibodycombining site (calculated as binding capacity for [3H]ethe-

5778

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

Proc. Natl. Acad. Sci. USA 75 (1978) 5779

noadenosine) per enzyme dodecamer were incubated for 15min at 370C in 25 yd of a buffer containipg 0.15 M Nah , 0.01M MnCl2, and 0.05 M Tris-HCI, pH 7.2. Reaction mixtures werefreed of excess globulins by passage at 40C through a 0.9 X 30cm column of Bio-Gel A-0.5 m (Bio-Rad Laboratories) equili-brated with the same buffer. In some experiments, short sin-gle-stranded glutamine synthetase cables were then generatedby incubation at room temperature for 30-45 min in 2.5 mMCoCl2. Samples were prepared for microscopy immediatelyafter dilution and negative staining with 1% uranyl acetate, bythe method of Valentine et al. (7) as modified by Lake andKahan (11). Electron micrographs were recorded with a PhillipsEM 400 microscope operated at 80 kV and a magnification ofX64,500.

RESULTSAntibody Formation and Characterization. Antibodies to

1,N6-ethenoadenosine and its 5'-nucleotide were induced byinjection into rabbits of a nucleoside- (or nucleotide-) bovinealbumin complex. In each instance a strong response to thehapten was seen; immunodiffusion showed heavy precipitinlines with either bovine or rabbit albumin complexes of ethe-noadenosine or its nucleotide. Little or no precipitation of un-modified bovine albumin, rabbit albumin, or rabbit albumincomplexes of adenosine or AMP could be detected in any serum.In all subsequent experiments, serum from one rabbit (no. 87)was used. 'Measurement of [3H]ethenoadenosine bindingshowed 15 nmol of ligand bound per ml of serum. The globulinfraction of the serum was purified by ammonium sulfatefractionation and gel filtration; its [3H]ethenoadenosine bindingcapacity was 32 nmol/ml.

Binding specificity was examined by measurement of inhi-bition of [3Hlethenoadenosine binding by a variety of nucleo-sides and nucleotides, as shown in Fig. 1. The specificity forethenoadenosine is seen to be very great, and the affinity foreither ethenoadenosine or ethenoadenylate is sufficient to ex-pect ,significant' interaction with the nucleotide in etheno-adenylylated glutaminel synthetase. Some crossreactivityl isshown by hydrophobic adenosine derivatives modified at po-

90

70

.650 f

30-

1001-9 -8 -7 -6 -5 -4 -3 -2 -1

log inhibitor concentration

FIG. 1. Effect of inhibitors on [3H]ethenoadenosine binding byantibodies. Reaction mixtures (250 gl) contained radioactive ethe-noadenosine (50 nM, 0.1 MCi/mi) (1 Ci = 3.7 X 1010 becquerels), un-labeled nucleoside as indicated, and antibody (equivalent to 0.125 glof the globulin preparation) in 0.14 M NaClI/0.02 M Tris-HCI buffer,pH 7.2. Samples were incubated 30 min at 37°C, followed by 60 minat 0°C, and 200-Ml portions were then rapidly passed through Milli-pore 25-mm type HA filters (previously wetted with buffer). Eachfilter was immediately washed with three 2-ml portions of cold buffer,and 3H bound to the filters was measured by scintillation counting.Inhibitors used were: 0, lN6-ethenoadenosine; *, lN6-etheno-5'-adenylic acid; A, N6-isopentenyladenosine; A, 1-methyladenosine;0, adenine; *, 5'-adenylic acid; o, adenosine.

Table 1. Antibody precipitation of ethenoadenylylatedglutamine synthetase

Glutamine synthetaseactivity, %

Antibody preparation Precipitate Supernatant

Nonimmune serum 4 97Anti-adenosine 6 94Anti-5'-adenylic acid 5 93

Anti-ethenoadenosine 42 16Anti-5'-ethenoadenylic acid 24 67

Reaction mixtures (150 ,l) included 5 Mg of ethenoadenylylatedglutamine synthetase and 10,ul of the appropriate serum in 0.14 MNaCl/0.02 M Tris-HCl, pH 7.2. After 15 min at either 37°C or 0°C,1 equivalent of goat anti-rabbit gamma globulin was added (100 ,M),and the samples were incubated a further 15 min. The samples werethen centrifuged (5 min at 500 X g), the precipitates were washed with250 Ml of buffer, and all fractions were assayed (22). All values areexpressed as the percentage of activity added and have an estimatederror of ±5%.

sition 1 of the purine ring at N6. However, these moieties arenot present in glutamine synthetase, and would not interferein the experiments below.

Interaction of Antibodies with Glutamine Synthetase.Antibodies to adenosine, AMP, ethenoadenosine, or etheno-AMP were permitted to interact with adenylylated or ethe-noadenylylated glutamine synthetase, and any complexesformed were then precipitated with a second antibody (goatanti-rabbit gamma globulin). Adenylylated glutamine syn-thetase was never found to be precipitated in such experiments.In contrast, ethenoadenylylated glutamine synthetase wasprecipitated to a significant-extent by antibodies to etheno-adenosine'or to ethenoadenylate.These results aresummarizedin Table 1.

Electron Microscopy of Antibody-Glutamine SynthetaseComplexes. Ethenoadenylylated glutamine synthetase andantibodies to ethenoadenosine were permitted to interact, andexcess globulins were removed by gel filtration. Electron mi-croscopy of the reaction mixtures showed several forms ofglutamine synthetase-antibody complexes. Images were in-terpreted in the terms of the position of the contact region ofthe antibody on the surface of the enzyme. As shown in Fig. 2,this position is defined by three coordinates: r, 0, and z. Theradial coordinate r gives the distance of the contact point fromthe central 6-fold axis of the oligomer; it can vary from 0 A toabout 70 A (half the molecular diameter). The polar coordinate0 gives the extent of rotation of the radius vector around the6-fold axis; 0 can assume values from 0° to 30'C. The coordinate

r~~~~~~~~

A B

FIG. 2. Structure of glutamine synthetase defining the coordi-nates used in this paper. (A) Face-on view, defining the radial coor-dinate r and the polar coordinate 0. (B) Side view defining the coor-dinate z, which is parallel to the molecular 6-fold axis.

Biochemistry: Frink etA

Proc. Natl. Acad. Sci. USA 75 (1978)

FIG. 3. Electron micrograph of ethenoadenylylated glutamine synthetase allowed to react with antibodies to ethenoadenosine. The arrowsindicate enzyme-antibody complexes. Bar length: 1000 A.

along the 6-fold axis, z, can assume values between 0 A (froma contact point on the plane bisecting the two hexagonal layers)to about 45 A (for a contact point on one of the outer hexagonalfaces).A general field such as that in Fig. 3 shows characteristic

glutamine synthetase structures in either hexagonal face-on(6-fold) or side (2-fold) views, glutamine synthetase moleculescomplexed with one or more IgG molecules, and pairs of glu-tamine synthetase molecules joined by a single IgG. Localizationof the site of adenylylation of glutamine synthetase was at-tempted by examination of ca. 250 individual enzyme-antibody

complexes. Fig. 4 shows a gallery of complexes in which theenzyme is seen in its face-on (6-fold) view. Twenty glutaminesynthetase dimers, each crosslinked by a single IgG moleculeas in Fig. 4A, were found to be separated by as little as 70 A andas much as 115 A (± 5 A), with a mean separation of 95 A. Thisrange may be compared with the results of Valentine and Green(29); they reported a mean separation of 120 A for the com-bining sites of an IgG molecule with the Fab fragment arms180° apart. With smaller inter-arm angles the separation is less,and they reported no separation greater than 150 A. From theirmicrographs we estimate the most common separation to be

--K

.-.#-, -C.

3C t0c..0 -tN-f

i)

.:

.- .4. ;,.

,S

FIG. 4. Antibody-glutamine synthetase complexes showing the enzyme in a face-on (6-fold) view. (A) Enzyme molecules crosslinked byIgG molecules. (B) Single glutamine synthetase molecules with one or more bound antibody molecules. Below each frame is an interpretivedrawing. Bar length: 1000 A.

5780 Biochemistry: Frink et al.

11) 0I-)1) 40 C)i

,., - %% J OV"'I-, - I.

Proc. Nati. Acad. Sci. USA 75 (1978) 5781

A

/3

B~~~~~~~~~~~~~~~~i

-IFIG. 5. Gallery of antibody-glutamine synthetase complexes showing the enzyme in a side (2-fold) view. (A) Individual enzyme molecules

with attached antibody and antibody-linked enzyme molecules. Below each frame is an interpretive drawing. (B) Antibody complexes involvingshort single-stranded cables of glutamine synthetase. Bar length: 1000 A.

about 100 A. Because the separation of contact points of cross-linked glutamine synthetase molecules is about the same as thereported separation of IgG binding sites, the ethenoadenylateresidues must lie near the outer periphery of the enzyme doubledisc, far from the molecular 6-fold axis. We estimate the radialcoordinate r to be 70 i 10 A, roughly the radius of the gluta-mine synthetase molecule. This value is also most consistent withthe interpretation of side views or of glutamine synthetase ca-bles discussed below.Many complexes of single glutamine synthetase dodecamers

plus one or more antibody molecules were also found (Fig. 4B).In these images the site of attachment of antibody was analyzedwith respect to. the intersection of a 2-fold axis with the mo-lecular envelope of the enzyme. Many enzyme molecules ap-pear as regular hexagons; in these cases antibody attachmentis usually seen near but not directly on a point of the hexagon.Some enzyme molecules appear as six roughly circular units;in these cases the site of antibody attachment often appearsslightly displaced from the 2-fold axis extending through thecenttrs of both the dodecamer and the circular unit. Becausethe width of a Fab arm of an IgG molecule is roughly 40 A, andthe glutamine synthetase subunit is roughly 45 A in diameter,we cannot expect fine mapping of the etheno-AMP site. Nev-ertheless, in most images the center of the antibody combiningsite appears to point at the molecular envelope between the twotypes of molecular 2-fold axes. Accordingly, we estimate thepolar coordinate 0 to be 15 + 100.

Antibody-glutamine synthetase complexes that show side(2-fold) views of the enzyme are presented in Fig. 5. Attach-ment sites are generally less clear in side views (Fig. 5A) or inglutamine synthetase cables (Fig. 5B) but consistently showantibody meeting the molecular envelope at the outer edge ofthe double disc, near the center of a subunit layer or slightlydisplaced toward the plane of the 2-fold axis between the layers.We estimate the value of the z coordinate to be 20 i 10 A.

DISCUSSIONAttempts to demonstrate an interaction of adenylylated glu-tamine synthetase with antibodies to adenosine or AMP havenot been successful in our hands. We presume this to be due tothe relatively low affinity of our anti-adenosine and anti-AMPpreparations; 50% inhibition of adenosine binding was seen at

inhibitor concentrations of about 10 MM. Although otherworkers (30-32) have described anti-adenosine antibodies thatsometimes show a greater affinity than those we produced, wefelt that use of the more hydrophobic ethenoadenosine as ahapten could give antibodies with significantly greater affinityand specificity for the derivative. Fig. 1 shows this to be true;50% inhibition values for ethenoadenosine or its nucleotide are0.1 uM or less, and far removed from those found for othernucleosides.

Ethenoadenylylation of glutamine synthetase by the ade-nylyltransferase occurs at the same site as normal adenylylation,and ethenoadenylylated and normally adenylylated enzymeare functionally identical in experiments in vitro (20). Exam-ination of glutamine synthetase-antibody complexes fromseveral preparations has allowed the approximate localizationof the adenylylation site indicated in Fig. 6. We place the AMPresidue on the outer surface of the envelope of the double disc(r = 70 + 10 A) and roughly midway between the plane of thesix 2-fold axes at the interface of the two discs and the plane atthe top (or bottom) of the double disc (z = 20 ± 10 A). Theposition on the outer edge seems to lie between the intersectionsof adjacent 2-fold axes with the molecular envelope (0 = 15 i100).

FIG. 6. Model of glutamine synthetase showing the proposedsite of adenylylation.

Andrew . .e -"I .;"-A

. .&-

Biochemistry: Frink et al.

1.;-Ii. .0"1'1.

Ali.-1I ..

,y.r ... .

I.-

?It

3

VW,I

4.

4'. ,.

. '.

Proc. Natl. Acad. Sci. USA 75 (1978)

Several facts indicate that the AMP must be near to the sur-face of the glutamine synthetase molecular envelope. First, inphysiological regulation of glutamine synthetase the nucleotideis added and removed by the regulatory enzymes (1), and it canalso be removed with venom phosphodiesterase (33). Adeny-lylated and unadenylylated enzyme have similar quaternarystructures as judged by chemical and physical properties(summarized in ref. 1) and by x-ray crystallography (34). Thisis not easily reconcilable with a deeply buried AMP residue.Finally, the measurements of separations of pairs of enzymemolecules bound to a single IgG suggest that the binding siteis not deeply buried beneath the enzyme surface.

Given that the AMP residue is near the molecular surface,several lines of evidence show that it lies on the cylindrical edge,rather than the top or bottom faces of the double disc: (i) at-tached antibodies do not hinder enzyme molecules frompolymerizing into strands along their 6-fold faces; (ii) IgGmolecules bound to strands or to molecules that display sideviews invariably meet the enzyme molecule at the cylindricalsurface rather than the top or bottom surfaces; (iii) the observedseparation of antibody-linked enzyme molecules is most con-sistent with a binding site on the periphery of the glutaminesynthetase molecule.

Localization of the site of adenylylation on the outer edgeof the disc leads to predictions about other aspects of glutaminesynthetase structure and function. If, as has been suggested (2,4-4), glutamine synthetase participates in regulation of tran-sciion in a way dependent upon its state of adenylylation,then we predict that it is the edge of the double disc that mustinteract with other molecules (perhaps including DNA) thatare involved in regulation. Using magnetic resonance and en-ergy transfer techniques, Villafranca et al. (35) have localizedthe regulatory and catalytic metal-binding sites of the enzymewithin a distance of 10 A of the adenylyl phosphorus. Thus ourresult would seem to indicate that the catalytic site of the en-zyme is within the outer edge of the disc.We thank Drs. J. Langer, J. Lake, and F. Eiserling for assistance and

advice in electron microscopy and R. Stone and R. Ito for help in im-munological procedures. Support was provided through NationalScience Foundation Grant PCM 77 14872 (to D.G.G.) and U.S. PublicHealth Service Grant GM 16925 (to D.E.) and Training Grant GM00364.

1. Stadtman, E. R. & Ginsburg, A. (1974) in The Enzymes, ed.Boyer, P. D. (Academic, New York), 3rd Ed., Vol. 10, pp.755-807.

2. Tyler, B. (1978) Annul Rev. Biochem. 47, 1127-1162.3. Shapiro, B. M. & Stadtman, E. R. (1968) J. Biol. Chem. 243,

3769-3771.4. Foor, F., Janssen, K. A. & Magasanik, B. (1975) Proc. Natl. Acad.

Sci. USA 72,4844-4848.5. Janssen, K. A. & Magasanik, B. (1977) J. Bacteriol. 129, 993-

1000.6. Gaillardin, C. M. & Magasanik, B. (1978) J. Bacteriol. 133,

1329-1338.

7. Valentine, R. C., Shapiro, B. M. & Stadtman, E. R. (1968) Bio-chemistry 7, 2143-2152.

8. Frey, T. G., Eisenberg, D. & Eiserling, F. A. (1975) Proc. Natl.Aced. Sci. USA 72, 3402-3406.

9. Lake, J. A., Pendergast, M., Kahan, L. & Nomura, M. (1974) J.Supramol. Struct. 2, 189-195.

10. Lake, J. A., Pendergast, M., Kahan, L. & Nomura, M. (1974) Proc.Nati. Acad. Sci. USA 71,4688-4692.

11. Lake, J. A. & Kahan, L. (1975) J. Mol. Biol. 99, 631-644.12. Tischendorf, G. W., Zeichhardt, H. & Stoffler, G. (1974) Mol.

Gen. Genet. 134, 187-208.13. Tischendorf, G. W., Zeichhardt, H. & Stoffler, G. (1974) Mol.

Gen. Genet. 134,209-223.14. Tischendorf, G. W., Zeichhardt, H. & Stoffler, G. (1975) Proc.

Natl. Acad. Sci. USA 72,4820-4824.15. Politz, S. M. & Glitz, D. G. (1977) Proc. Natl. Acad. Sci. USA 74,

1468-1472.16. Stollar, B. D. (1973) in The Antigens, ed. Sela, M. (Academic,

New York), Vol. 1, pp. 1-85.17. Hohman, R. J. & Stadtman, E. R. (1978) Biochem. Biophys. Res.

Commun. 82, 865-870.18. Secrist, J. A., Barrio, J. R., Leonard, N. J. & Weber, G. (1972)

Biochemistry 11,3499-3506.19. Ginsburg, A. (1970) Methods Enzymol. 17,927-941.20. Timmons, R. B., Huang, C. Y., Stadtman, E. R. & Chock, P. B.

(1973) in Metabolic Interconversion of Enzymes, eds. Fischer,E. H., Krebs, E. G., Neurath, H. & Stadtman, E. R. (SpringerVerlag, New York), pp. 209-220.

21. Shapiro, B. M. & Stadtman, E. R. (1970) Methods Enzymol. 17,910-922.

22. Phares, E. F. (1971) Methods Enzymol. 22,441-476.23. Woolfolk, C. A., Shapiro, B. M. & Stadtman, E. R. (1966) Arch.

Biochem. Biophys. 116, 177-192.24. Caban, C. E. & Ginsburg, A. (1976) Biochemistry 15, 1569-

1580.25. Hennig, S. B. & Ginsburg, A. (1971) Arch. Biochem. Biophys. 144,

611-627.26. Eichler, D. C. & Glitz, D. G. (1974) Biochim. Biophys. Acta 335,

303-317.27. Erlanger, B. F. & Beiser, S. M. (1964) Proc. Natl. Acad. Sci. USA

52,68-74.28. Humayun, M. Z. & Jacob, T. M. (1973) Biochim. Biophys. Acta

331,41-53.29. Valentine, R. C. & Green, N. M. (1967) J. Mol. Biol. 27, 615-

617.30. Rosenberg, B. J., Erlanger, B. F. & Beiser, S. M. (1973) Bio-

chemistry 12, 2191-2197.31. Cameron, B. J. & Erlanger, B. F. (1976) Immunochemistry 13,

263-269.32. Drocourt, J. L. & Leng, M. (1975) Eur. J. Biochem. 56, 149-

155.33. Shapiro, B. M., Kingdon, H. S. & Stadtman, E. R. (1967) Proc.

Natl. Acad. Sci. USA 58,642-649.34. Heidner, E. J., Frey, T. G., Held, J., Weissman, L. J., Fenna, R.

E., Lei, M., Harel, M., Kabsch, H., Sweet, R. M. & Eisenberg, D.(1978) J. Mol. Biol. 122, 163-173.

35. Villafranca, J. J., Rhee, S. G. & Chock, P. G. (1978) Proc. Natl.Acad. Sci. USA 75, 1255-1259.

5782 Biochemistry: Frink et al.