G L I C K S 0 N,
Egyed, A. (1973), Biochim. Biophys. Acta 304,805. Englander, S. W.. and Crowe, D. (1965),Ana/. Biochem. 12,579. Fletcher, J., and Huehns, E. R. (1967), Nature (London)215, 584. Fletcher, J., and Huehns, E. R. (1968), Nature (London) 218, 1211. Greene, F. C.. and Feeney, R. E. (1968), Biochernistrj 7,1366. Lanczos, C . (1956), Applied Analysis, Engelwood Cliffs, N. J.. Prentice-Hall, pp 272-280. Mann, K . G., Fish, W. W., COY,A. C . , and Tanford, C . (1970), Biochemistry 9, 1348.
C U N N I N GH AM,
AND
MAR S HA LL
Morgan, E. H . (1971), Biochim. Biophj's. Acta244,103. Price, E. M., and Gibson, J. F. (1972), Biochem. Biophys. Res. Commun. 46,646. Schade, A . L.: and Reinhart, R. W. (1966), Protides Biol. Nuids, Proc. Colloq. 14,75. van der Waerden, B. L. (1969), Mathematical Statistics, Heidelberg, Germany, Springer-Verlag, pp 127-133. Williams, S. C.?and Woodworth. R . C . (1973), J . B i d . Chem. (in press). Young, J. W., and Perkins, D. J. (1968), Eur. J . Bioclrem. 4, 385.
Proton Magnetic Resonance Study of Angiotensin I1 (Asn'Val') in Aqueous Solutionf Jerry D. Glickson,*,$ William D. Cunningham, and Garland R . Marshall
All the resolved resonances in the 220-MHz proton magnetic resonance (prnr) spectrum of angiotensin [I (AsnIVaI;) (AII') in D 2 0 have been assigned to specific hydrogens. In H,O additional assignments were made of resotlances originating from peptide N H hydrogens of Phe and Arg. primary amide N H hydrogens of Asn: and the four cquicalent guanidino NH hydrogens of Arg. Conformational transitions associated with titration of the a-amino and/or the imidazole group(s) (pK, = 6.6 i 0.2) and with titration of the phenol group ( p K , = 10.2 i 0.2) have been confirmed. Pmr determined pKa values in H,O at 23' and in D n O at 4' (shown in parentheses) are all normal: carboxyl 3.07 + 0.03, imidazole 6.26 0.04 (6.82 = 0.02), a-amino (6.98
ABSTRZCT:
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A
ngiotensin I1 (AH)' is a potent natural pressor agent derived from an a2-macroglobulin by renin hydrolysis. The present study deals with a more readily available synthetic congener, angiotensinamide (AII)' (Asn1-Arg2-Va13-Tyr4Val'-His"Pro'-Phe*), which elicits similar biological responses to AI1 but dif'fers chemically from it only in the replacement of the N-terminal Asp residue of AI1 by Asn. It has long been recognized that the conformation of the hormone may be related to its biological activity (Bumpus et d.. 1961). Even though it is the conformation of the hormone at the receptor site which is of primary significance (Marshall and Bosshard, 1972), the free solution orientation of this hormone has received the most attention because . ~. -.
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From the Division of Molecular Biophysics, Laboratory of Molecular Biology, University of Alabama in Birmingham School of Mediciiic (J. D . G . and W. D. c.),Birmingham, Alabama, and from the Dcp,irtment of Physiology and Biophysics, Washington University School of Medicinc, St. Louis, Missouri 63110 (G. R . M,). Receiaed A p r t i 17, 1973. This research was supported by Public Health Service Grtints A M - 1 3025 a n d HE-19509 and an Established Investigator '1w;ircI f r o m the American Heart Association to G. R . M . $ P r c x n t acldrcss: Division of Hematology-Oncology of the Departtiictit of Vcdicinc and The Cancer Research and Training Program, UiiiLcrsity of Alabama i n Birmingham, School of Medicine, Birmingliiim, Ala. 35294. 1 Abbreviations used : angiotensin I1 ( M I ) , and angiotensin I1 (Asti'V;il5) ( A H ' ) . i-
3684
I ~ I O C H E M I S T R Y ,V O L .
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2 0.04), and phenol 10.2 i 0.2 (10.5 I 0.5). The peptide NH-aCH coupling constants in acidic solution are: 6.5 = 0.3 (Arg), 6.0 = 0.5, 7.2 0.5, 7.3 0.3 (Phe), 7.0 i 0.3, and 8.0 = 0.4. Various classes of labile hydrogens were defined on the basis of their exchange rates as determined from broadening of their respective resonances. The pmr data are consistent either with a rapid equilibrium between various conformations or with a unique conformation of the hormone, but additional evidence is required to definitively determine the structure(s) significantly contributing to the equilibrium. Previously proposed structures excluded by these data include the a helix, the conventional p turn, the y turn, and a structure stabilized by a salt bridge.
*
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it is more easily monitored by available methods. In free solution AI1 has been variously described as an a helix (Smeby et a/., 1962), random-coil (Paiva et a/., 1963), antiparallel pleated sheet (Fermandjian et u l . , 1972a, 1972b; Printz et a/., 1972a), and a number of more complex conformations (Weinkam and Jorgensen, 1971a; Glauser et a/., 1970). The effect of p H on the conformation of the hormone in free solution has also been debated. Thus, Paiva ef al. (1963) report no conformational transitions between pH 2.5 and 8.5, but others have associated conformational perturbations with titration of the carboxyl group (Weinkam and Jorgensen, 1971a, 1971b), the imidazole group (Craig et nl., 1964), and the phenol group (De Fernandez et a/., 1968). Clarification of this controversy is desirable not only in order to delineate the structure-activity relationship of AII, but also in order to define the capabilities and limitations of various methods employed in studying the conformation of peptides in solution. Toward this end we have undertaken a detailed proton magnetic resonance (prnr) study of the conformation of AII'. A preliminary report of our observations has been presented (Glickson et af., 1972). Here we present a more detailed description of the method of resonance assignment, the effects of p H on conformation, analysis of peptide NH-aCH coupling constants, and estimation of proton exchange rates from line-broadening data.
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J . Amer. Chem. Soc. 91,1513. Paiva, T. B., Paiva, A . C . M.. and Scheraga, H . A. (1963), Biocheniistrj, 2, 1327. Pople, J. A,, Schneider. W. G . , and Bernstein, H . J . (1959), in High-resolution Nuclear Magnetic Resonance, New York, N . Y.: McGraw-Hill, Chapter 10. Printz, M. P., Nemethy, G., and Bleich, H . (1972a)? Nuture (London),New Biol. 237. 135. Printz, M. P.. Williams, H . D., and Craig, L. C . (1972b), Proc. Nut. Acad. Sci. L'. S . 69, 378. Ramachandran, G. N., Chandrasekaran, R . , and Kopple, K . D. (1971), Biopo/j,mers 10,2113.
Smeby, R . R., Arakawa, K., Bumpus, F. M., and Marsh, M. M. (1962): Biochim.Biophys. Acta 58,550. Tonelli, A. E., and Bovey, F. A. (1970), Macromo/eculrs 3, 410. Torchia. D. A,. and Bovey, F. A. (1971): Macromolecules 4 , 246. Urry, D. W.: and Ohnishi, M. (1970), in Spectroscopic Approaches to Biomolecular Conformation, Urry, D. W.. Ed.. Chicago, Ill., American Medical Association, Chapter VII. Walter, R.: Glickson, J . D., Schwartz, I. L., Havran, R . 'T'.. Meienhofer, J., and Urry, D. W. (1972). Proc. Nut. Acud. Sci. U . S . 69,1920. Weinkam, R . J , , and Jorgensen, E. C . (1971a). J . An7er. C/iem. Soc. 93,7038. Weinkam, R. J., and Jorgensen, E. C. (1971b). J . Amer. Chen7. Soc. 93,7033. Zimmer. S., Haar, W.? Maurer, W., Ruterjans, H., Fermandjian, S., and Fromageot, P. (1972), Eur. J . Biochem. 29, 80.
Purification and Characterization of a Steroid Receptor from Chick Embryo Livert Alice S. Tu and Evangelos N . Moudrianakis*
,USTRACT: The high-speed supernatant fraction of the 16day-old chick embryo liver homogenate was found to contain a macromolecular species that could form tight complexes with steroid hormones. This hormone receptor was purified by isoelectric precipitation, high-speed centrifugation, DEAEcellulose column chromatography. and preparative disc electrophoresis. The purified receptor migrated as one diffuse band on analytical acrylamide gel electrophoresis and had an isoelectric point of 5.3, as shown by electrofocusing. The hormone-receptor complex sedimented as a single 4 s species in sticrosr gradient centrifugation in several concentrations of KCI. The binding of hydrocortisone to the receptor has a temperature optimum at 37": and saturation is reached within 10 min of incubation. The binding was insensitive to changes in pH within the range of 4.5-9.4. By the use of equilibrium dialysis it was estimated that the association constant for the
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everal approaches have been used for the elucidation of the mechanism of hormone action. Injection of radioactive hormone into animals showed concentration of radioactivity in specific target organs (Bellamy er d,, 1962; Litwack and Baierga, 1967). Induction of RNA and protein synthesis after in riro injection of hormone into an animal has also been demonstrated (Kenney, 1962; Schimke et ul,, 1965; Teng and Hamilton, 1968). The specificity of action manifested by the hormone is incompatible with its being a low molecular weight substance. I t has heen hypothesized (Jensen and Jacobson, 1960) that the ~
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C:oiiirihutioii No. 728 from t h c Dcpnrtmcnt of Biology, Johns H o p k i n s Uiiivcrsit!, Baltimorc, Mnr>lancl 21218. Receiced Janirar). 9, iY7.7. Supportstl b! Grant HD-326 from the National Institutes of H C d I ill.
3692
BIOCHEMISTRY V O, L .
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binding of hydrocortisone was 7.5 X lo8 hi-' at 4:) while that for the binding of corticosterone was 3.6 x los hi-l at 4'. The protein nature of the receptor was suggested by its susceptibility to proteolytic enzymes. Estradiol and testosterone were found to bind to the crude receptor preparation (the high-speed supernate). This binding, however, would not reach saturation even when high concentrations of hormones were used. In addition, neither estradiol nor testosterone would compete with the binding of hydrocortisone or corticosterone by the receptor. After the receptor was puritied by preparative disc electrophoresis, the binding of hydrocortisone and corticosterone was at least 3000-fold higher than that of estradiol and testosterone, when compared in the range where the binding of hydrocortisone was linear with respect to hormone concentrations.
target tissue may play an important role in the final physiological manifestation of hormone action. More recently, receptor molecules for specific hormones have been isolated from their target organs. Such a receptor was initially isolated from the rat or calf uterus, which bound estrogen hormones (Toft and Gorski, 1966; Jensen et a/., 1967; Erdos, 1968; Puca et a/,, 1971). The hormone-receptor complex was isolated from the soluble supernate of the uterine homogenate and was found to sediment as an 8s molecule on sucrose gradient. This 8s molecule dissociated into a 4-5s species upon treatment with 0.3 M KCI. Similar receptor molecules have been reported for other steroid hormones (Edelman and Fimognari, 1968 ; Gardner and Tomkins, 1969; Sherman et a/., 1970). There is evidence that these receptor molecules enter the nucleus of the target tissue in the presence of hor-
