Conformational comparison of stored and secreted ovine pituitary prolactin

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 225, No. 2, September, pp. 482-489,1983

Conformational

Comparison of Stored and Secreted Ovine Pituitary Prolactin’~*

THOMAS A. BEWLEY;

PETER COLOSI,

AND

FRANK TALAMANTES

Hormone Research Laboratoq, University of Cal$brnia, San Francisco, California 941.&9;and Thimann Laboratories, University of Ca&fmnia, Santa Cruz, California 95064 Received March 23, 1983

Highly purified samples of stored and secreted ovine pituitary prolactin have been compared with regard to those conformational properties evidenced by ultraviolet absorption and circular dichroism measurements. No significant differences were found in any of the optical properties measured. The previously reported absence of tryptophanyl circular dichroism in the secreted forms of rat and mouse prolactins may be typical only of rodent hormones and not a general phenomenon.

A previous study (1) indicated a significant conformational difference between so-called “stored” rat prolactin (PRL), isolated from homogenates of anterior pituitary tissue, and so-called “secreted” prolactin, isolated from cultured pituitary cells in the absence of hypothalamic inhibition to secretion. This difference consisted of a complete absence of any CD signal from the two tryptophan residues in the secreted form (1). The tryptophanyl CD of the stored form was readily apparent (1). More recently, secreted mPRL was also shown to lack any tryptophanyl CD (2). Unfortunately, stored mPRL has not been available for comparison. ’ Dedicated to Dr. Choh Hao Li on the occasion of his 70th birthday. a This work was supported in part by grants from the National Institutes of Health; AM-18677 and AM6097 (T.A.B.), along with HRR-08132 and HD-14966 (F.T.). ’ To whom all correspondence should be addressed. ’ Abbreviations used: PRL, pituitary prolactin; oPRL, ovine prolactin; mPRL, mouse prolactin; rPRL, rat prolactin; SDS, sodium dodecyl sulfate; Em, molar extinction coefficient; aEn, change in EM observed in difference spectra; Atg,,, absorptivity maximum of a l.O-mg/ml solution with a l.O-cm pathlength at wavelength X; [ejmuv, mean residue molecular ellipticity; [@In, molar elliptic@; E/S, enzyme to substrate ratio (weight/weight). 0003-9861/83 $3.00 Copyright All rights

0 1983 by Academic Press. Inc. of reproduction in any form reserved.

From the physicochemical point of view, stored oPRL is certainly the most thoroughly studied of all the prolactins (3). It was of special interest therefore to compare the secreted form of oPRL with stored oPRL. As part of this study, we have also extended and refined certain optical parameters of stored oPRL. MATERIALS

AND

METHODS

Secreted oPRL was prepared from tissue culture media as described elsewhere for rodent hormones (4-6). The highly purified product was characterized by amino acid analyses (7), exclusion chromatography on Sephadex G-100, and disk electrophoresis on 10% polyacrylamide gels (alkalin pH) in the presence and absence of SDS as previously described (4,5). A preliminary bioassay was performed in the pigeon cropsac assay as modified by Nicoll(8). Stored oPRL was prepared as described by Li et al (9). All uv absorption measurements were made on a Perkin-Elmer Model 552 double-beam spectrophotometer equipped with temperature control, background correction, and second derivative accessories. Molar extinction coefficients (Eu) were evaluated from thermolysin digests as described by Bewley (10). Difference absorption spectra, obtained during thermolysin digestion, were performed as described previously (2). CD measurements were carried out on a Cary Model 60 spectropolarimeter equipped with a Model 6002 circular dichroism attachment. A mean residue weight of 115 (MI = 22,674) was assumed for both forms of oPRL, and a-helix contents were es-

482

CONFORMATION

OF OVINE

PITUITARY

PROLACTIN

483

WAVELENGTH (nm)

FIG. 1. Zero-order absorption spectra of stored oPRL. Native, pH 8.2 pH 1.5 (- - -). Tyrosyl difference absorption obtained between 2 aliquots used to determine EM as described in (10). The pHL5andpH13(...), were essentially identical. The zero position of the EM scale is placed at plot) light scattering position for 279 nm (21).

(-), digest titrated to of the digest titrated to spectra of secreted oPRL the extrapolated (log-log

imum presents a broad, indefinite region between 280 and 274 nm rather than a sharp peak. Fine structure, due to phenRESULTS ylalanine can be seen as a series of shoulAmino acid analyses of native and per- ders between 270 and 255 nm. In all reformic acid oxidized samples of secreted spects, the spectrum of secreted oPRL is oPRL (data not shown) were not signifiindistinguishable from that of the stored cantly different from parallel runs, or pre- protein (data not shown). EM values have viously published (12) values for stored been determined to be 20,770 f 160 (M-’ oPRL. Exclusion chromatography on Se- cm-‘) at 279.6 f 0.1 nm for 3 different prepphadex G-100, and electrophoresis on arations (3 digests each) of the stored horpolyacrylamide gels (with and without mone, and 20,670 + 500 (M-’ cm-‘) also at SDS) also failed to distinguish between the 279.6 f 0.1 mm, for 3 digests of the secreted secreted and stored forms (data not form. These EM values correspond to shown). A preliminary bioassay in the pi- Ao.‘% 1 cm.2795 = 0.916 + 0.007 for stored oPRL, geon crop-sac indicated dried mucosal and A’.‘% 1 em379.6 = 0.912 f 0.024 for secreted weight of 23.0 + 5 mg for an 8-pg dose of oPRL. secreted oPRL, 21.7 + 1.9 mg for 8 pg of The second-order spectrum of native, stored oPRL, and 10.3 + 0.3 mg for saline stored oPRL is presented in Fig. 2A (solid injected controls. These results suggest line), with the corrected second-order minthat the secreted hormone is certainly po- ima being listed in Table I. Again, the sectent, although more thorough bioassays ond-order spectrum of the secreted protein will be required to firmly establish the rel- (Fig. 2A, dashed line) is indistinguishable ative potencies. from the stored form. Figure 1 presents the zero-order uv abExtensive digestion of either stored or sorption spectrum of native, stored oPRL. secreted oPRL with the enzyme thermoA distinct shoulder, arising from absorp- lysin produces the hypochromic and bluetion by the two tryptophan residues, can shift effects shown for the zero-order specbe seen near 290 nm. The absorption max- trum of stored oPRL in Fig. 1. The extent timated according to Chen et aL (11). All optical measurements were made in 0.1 M Tris-Cl buffer (pH 8.2).

BEWLEY,

COLOSI,

AND

TALAMANTES

d2 Abs. dX*

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-0.04

-0.06 I,

240

I,

I

260

I

11

11

11

200

I1

300

I

I

I

320

WAVELENGTH (nm)

FIG. 2. Second-order absorption spectra of stored oPRL (-) and secreted oPRL (- - -), with native proteins at pH 8.2 in (A), and digests titrated to pH 1.5 in (B). The spectrum ( + . .) of a model mixture containing N-acetylamino acid amide forms of Trp, Tyr, and Phe, plus free cystine in molar ratios of 2:7:6:3, respectively, is also shown in (B). The insets and circled areas show regions where digestion produces large changes. Bands labeled with roman numerals are described in the text. The various spectra have been offset on the vertical axis for clarity.

of blue-shifting after digestion and titration to pH 1.5 is even more apparent from the second-order spectra shown in Fig. 2B (corrected second-order minima for the stored form are listed in Table I). Again, both stored and secreted proteins display precisely equivalent spectra after digestion. Figure 2B also contains the second-

order spectrum of a model mixture of Nacetylamino acid amides of Trp, Tyr, Phe, and Cys prepared with the same molar ratios as found in oPRL. The importance of the nearly complete congruency of the model mixture spectrum with the digest spectra will be found under the Discussion section.

CONFORMATION

OF OVINE

PITUITARY

TABLE

485

PROLACTIN

I

SECOND-ORDER SPECTRAL MINIMA FOR STORED oPRL”

Native, pH 8.2 (nm)

Chromophore assignment in native

299.5 (w)

Trp

289.7 282.9 b 278-279 (s)

Tw Trp + Tyr Tyr

274.4 268.3 * 264.7 261.5 (w) 258.7 252.8 248.1

Trp Phe + slight Tyr Phe Phe Phe Phe Phe

Digest (nm) pH 8.2

pH 1.5

-

-

288.0 281.5 -

287.6 281.4 -

275.2 ~272 (s) 267.1 263.7 ~260 (VW) 257.6 252.0 247.3

275.0 =272 (s) 267.0 263.6 -260 (VW) 257.3 251.3 247.0

Model mix, pH 1.5 (nm)

Chromophore assignment in digest and model mix

-

-

287.6 231.5 -

Tw Trp + Tyr

274.6 272.2 (w) 266.9 263.5 260.1 (VW) 257.3 251.3 246.7

5r Trp Phe + slight Tyr Phe Phe Phe Phe Phe

-

a Corrected as described in (10). The values for secreted oPRL are all within 0.1 nm of those for stored oPRL. *Unresolvable, composite band containing contributions from at least two types of chromophore. c Molar ratios of: 2 Trp; 7 Tyr; 6 Phe, and 3 Cys. (w) = weak. (VW) = very weak. (s) = shoulder.

The difference between the zero-order spectra of the intact and digested forms of these proteins can be compared more precisely from difference spectra taken during the digestion, In these experiments both sample and reference cells contain the protein solution. A small amount of enzyme (E/S = l/1000) is added to the sample cell. As the sample digests, increasingly intense difference spectra are produced as a function of time as shown in Figs. 3A and B. Both the rate of development of the difference spectra (Fig. 4), and their overall final forms and intensities (Figs. 3A and B) are identical in the two oPRLs. Figure 5 shows the CD spectra of stored and secreted oPRL. Below 250 nm, both spectra contain equivalently intense negative bands at 223 and 209 nm which are characteristic of a-helical proteins. Identical a-helix contents of 65 + 5% are estimated for both proteins. In the region of side-chain absorption, both secreted and stored oPRL display identical, moderately intense, positive CD bands at 297 and weaker, negative bands at 291 nm, both of which may be assigned to tryptophan residues (13). The remainder of the side-chain CD spectra are nearly identical although

the secreted form shows slightly more negative values between 293 and 258 nm which are just beyond the usual range of experimental error. DISCUSSION

Previous studies of stored and secreted rPRL (1) were unable to detect significant differences in molecular weight, amino acid composition, or electrophoretic behavior, with only minor differences in biological and immunological potencies. However, a significant conformational difference involving local environments around the tryptophan residues in the two forms was noted (1). In the present study, we have also been unable to detect any differences in molecular size, composition, or electrical charge for stored and secreted oPRL. Both proteins also appear to be approximately equipotent in a preliminary bioassay. In view of the results with rPRL, a more thorough conformational comparison was undertaken with the ovine hormone, both to better define the optical properties of the ovine protein, and to more precisely compare the two forms.

486

BEWLEY,

COLOSI,

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AND

TALAMANTES

which is due only to conformation. The magnitude of this purely conformational contribution may be seen by comparing the zero-order spectra of the native and digested forms of stored oPRL shown in Fig. 1. The digested protein has also been employed to determine the EM, and thereby the concentrations, of the native proteins by a procedure described elsewhere (10). The tyrosyl ionization difference spectrum, central to the Ena determination, is also shown in Fig. 1. The near equivalence of the native spectra and the EM values of the stored and secreted proteins strongly suggests very similar conformations for the two molecules. Second-O&r Spectra Recent studies (2,10,16,1’7) have shown that second-order spectra (the second deTime (mid

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FIG. 3. Difference absorption spectra of secreted oPRL (A) and stored oPRL (B), taken during digestion at pH 8.2 with thermolysin (E/S = l/1000). The time (min), after adding the enzyme, at which each spectral scan was begun is shown. The position of the 2’74.0nm isosbestic point is also indicated.

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Spectra

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Local environmental effects such as refractive index, proximity of charged groups, and hydrogen bonds, produce significant alterations in the absorption spectra of the chromophoric side-chains of Trp, Tyr, Phe, and to a lesser extent Cys residues (14, 15). Thus, the absorption spectrum of a globular protein is significantly altered from that of a model mixture of its chromophoric amino acids (or of a completely denatured form of the protein) by an amount that is highly characteristic of the folded form of the native molecule. In the present study we have used exhaustive digestion with the proteolytic enzyme thermolysin to completely destroy the conformation of the protein, thus revealing that portion of the absorption spectrum

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log Time (min) FIG. 4. The rate of change in Al& at 290.8 nm for digesting samples of secreted oPRL (0) and stored oPRL (o), taken from difference spectra as shown in Fig. 3.

CONFORMATION

t -0

6-

"E 0

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OF OVINE

PITUITARY

487

PROLACTIN

r --2 7

r

--4

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210

230

250

255

260 265

270

275

280

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290

295

300

305

310

WAVELENGTH (nm) FIG. 5. Circular 8.2.

dichroism

spectra of stored oPRL (-)

rivative of the zero-order spectrum with respect to wavelength) contain far more precise data with regard to wavelength positions and fine structure details than do the more familiar zero-order spectra. Moreover, second-order spectra are relatively free of light scattering artifacts (16, 1’7). The essentially perfect congruence of the second-order spectra of intact, stored and secreted oPRL (Fig. ‘2A) indicates very similar, if not equivalent, local environments for all chromophoric residues in both proteins. Exact wavelength positions and chromophore assignments in the various native and digested forms are listed in Table I. The congruence in the second-order spectra of the two digests titrated to pH 1.5, with the spectrum of a model mixture at the same pH (Fig. 2B), clearly demonstrates that digestion and titration can remove all conformational contributions. This point, which is crucial to the EMdeterminations, is further discussed elsewhere (10). The congruency of both digest spectra with the model mixture spectrum also unequivocally indicates the presence of two tryptophan residues in secreted oPRL (2). Tryptophan was the only residue

and secreted oPRL (0). taken at pH

not determined in amino acid analyses of the two proteins. The second-order spectra also display conformationally dependent spectral details to much greater precision than zeroorder spectra. For example, the weak Trp band at 299.5 nm (labeled I in Fig. 2A), is completely absent in the digested forms. This band, which is only barely discernable in the native protein of Fig. 1, is completely dependent on the folded form’s ability to produce a particular microenvironment around the Trp residues. At present we do not know which Trp (or if both) are involved, or what details of the microenvironment produce this particular feature. By reference to model compound spectra, the intense band at 298-287 nm, and the weak shoulder at 274.4 nm (labeled III and III’ in Fig. 2) may also be assigned to Trp residues (10,14-17). Similarly, most of the intense band near 282-281 nm, and the weaker band near 279-2’78 nm (labeled II and II’ in Fig. 2) may be assigned to Tyr absorption. The very complex patterns of these bands, including changes in relative intensity (circled areas in Fig. 2), reflect unequal blue-shifting of the two chromophore types. These patterns are extremely

488

BEWLEY,

COLOSI,

sensitive to, and characteristic of, the folded form of the protein. Again, the apparent equivalence of these spectral details, which are not readily apparent in zero-order spectra, strongly argues for nearly equivalent conformations in stored and secreted oPRL. fiflerence

Absorption

Spectra

AND

TALAMANTES

digestion presumably begins, until the difference spectra begin to develop. This suggests that one or more of the initial proteolytic “nicks” do not result in gross unfolding of the native conformation. Circular

Dichroism

Spectra

The CD spectra of Fig. 5 confirm the conclusions derived from absorption spectra. Difference absorption spectra provide The basic folding of the peptide backbone another approach for direct visualization of that portion of the absorption which is is completely identical in the two forms of due only to conformational effects. As oPRL. The a-helix contents reported herein are slightly higher than reported previshown in Fig. 3, digestion of the protein in the sample cell is accompanied by in- ously for stored oPRL (13). This is due creasingly intense blue-shift peaks at 290.8 to use of an improved calculation procedure (11). Virtually identical helical connm, due to exposure of previously buried Trp residues, and at 284.9 nm, due to ex- tents (65 + 5%) have been reported for secreted mPRL (2) and found for stored posure of buried Tyr residues. The shoulder near 300 nm arises from Trp, while the forms of human, porcine, and equine PRL shoulder near 278 nm comes from Tyr (18). (Bewley and Li, unpublished results). Most noteworthy is the appearance of Exposure of buried Phe shows up as fine structure peaks between 270 and 250 nm. distinct CD bands at 297 and 291 nm arisThe final difference spectra (24 hr) of stored ing from Trp residues in the secreted oPRL. and secreted oPRL are essentially equiv- This is in contrast to the results reported alent, within experimental error. More- for secreted rPRL (1) and mPRL (2). The over, both forms display a sharp isosbestic slight difference in the CD spectra between point at 274.0 nm. This point can also be 295 and 260 nm is just beyond the realm seen in the zero-order spectra of Fig. 1, of experimental error. If real, it might be and is of considerable utility since it rep- assignable to a slight alteration in the CD resents a unique wavelength at which the signal from one or more disulfide bonds (19,20), but this will require more detailed EM of the protein is independent of the investigation. conformational state of the molecule. The EM at this wavelength can therefore be CONCLUSION used to accurately determine the concentration of the protein, even in the absence The type of conformational differences of information with regard to its con- previously observed between stored and formation. The isosbestic point EM val- secreted rPRL, and which may exist beues were found to be 20,200 M-’ cm-’ tween stored and secreted mPRL, do not ~&%%m = 0.891) for stored oPRL, and extend to oPRL. 20,100 M-’ cm-’ (AT:&,, = 0.886) for the ACKNOWLEDGMENTS secreted form. The essentially equivalent rates at which The authors thank Professor C. H. Li for his interest the major Trp difference peaks at 290.8 nm in this study, and for the gift of stored oPRL. We form during digestion (Fig. 4) suggests that also thank Professor J. Ramachandran and Dr. Koji not only is the local conformation around Muramoto for making a sample of their stored oPRL these residues virtually identical in the two available to us. oPRLs, but that the stability of this conREFERENCES formation is also quite similar in the two molecules. It is also apparent from Fig. 4 1. FARMER, S. W., BEWLEY, T. A., RUSSEL, S. M., AND that there is a lag period of lo-15 min beNICOLL, C. S. (1976) Biochim Biophys A& 437, tween the time the enzyme is added and 562-570.

CONFORMATION

OF OVINE

2. BEWLEY, T. A., COLOSI, P., AND TALAMANTES, F. (1982) Biochemistry 21,4238-4243. 3. LI, C. H. (1980) in Hormonal Proteins and Peptides-Prolactin (C. H. Li, ed.), Vol. 8, pp. 2-34, Academic Press, New York. 4. COLOSI, P., MARKOFF, E., LEVY, A., OGREN, L., SHINE, N., AND TALAMANTES, F. (1981) ETW?P crirwlogy 108,850-854. 5. SHOER, L. F., SHINE, N. R., AND TALAMANTES, F. (1978) B&him Biophys. Acta, 527, 336-34’7. 6. MARKOFF, E., COLOSI, P., AND TALAMANTES, F. (1981) Life Sci. 28, 203-211. 7. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S. (1958) And Chem. 30,1190-1206. 8. NICOLL, C. S. (1967) Endocrinology 80. 641-655. 9. LI, C. H., DIXON, J. S., Lo. T.-B., SCHMIDT, K. D., AND PANKOV, Y. A. (1970) Arch. Biochem Bie phys. 141, ‘705-737. 10. BEWLEY, T. A. (1982) And Biochem 123, 55-65. 11. CHEN, Y.-H., YANG, J.-T., AND MARTINEZ, H. M. (1972) Biochemistry 11,412&4131. 12. HOUGHTEN, R. A., AND LI, C. H. (1976) B&him Biophys. A& 439,240-249.

PITUITARY

489

PROLACTIN

13. BEWLEY, T. A., AND LI, C. H. (1972) Biochemistry 11, 884-888. 14. WETLAUFER, D. B. (1962) A&v. Protein 303-390.

Chem. 17,

15. DONOVAN, J. W. (1969) In Physical Principles and Techniques of Protein Chemistry (S. J. Leach, ed.), Part A, pp. 102-170, Academic Press, New York. 16. BALESTRIERI, C., COLONNA, G., GIOVANE, A., IRACE, G., AND SERVILLO, L. (1978) Eur. J B&hem 90,433-440. 17. BALESTRERI, C., COLONNA, G., GIOVANE, A., IRACE, G., AND SERVILLO, L. (1980) And Biochem. 106, 49-54. 18. HERSKOVITS, T. T., AND SORENSEN, M. (1968) Bie chemistry 7,2533-2542. 19. BEWLEY, T. A. (1977) Biochemistry

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20. DONEEN, B. A., BEWLEY, T. A., ANDLI, C. H. (1979) Biochemistry 18,4851-4860. 21. LEACH, S. J., AND SCHERAGA, H. A. (1960) J. Am Chem Sex. 82, 4790-4792.