Structural Studies on Avian Pancreatic Polypeptide and STEPHEN P. WOOD. Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College,.
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curves for radioimmunoassay that exactly coincided with the curve obtained with native tubulin. This may constitute an important advantage of our test in view of its application to the measurement of the tubulin content of cells in culture in vitro. The manipulations needed for homogenization of the cells and extraction of the tubulin undoubtedly favour denaturation of the latter. In addition, whereas colchicine binds only to the dimer form of tubulin, antibodies are expected to react equally well with dimers and polymers. Also, the cytoplasm may contain biosynthetic precursor forms of tubulin that may not bind colchicine, but most probably would extensively cross-react ant igenically . Finally, using immunoprecipitationand radioimmunoassay in parallel, we were able to show an extensive antigenic cross-reactivity of rat brain tubulin with tubulins isolated from rabbit and pig brains by the same procedure. This would extend the use of our standard assay based on rat brain tubulin to substrates isolated from a variety of mammalian cell cultures, either normal or transformed. Brinkley, B. R., Fuller, G. M. & Highfield, D. P. (1975)Proc.Natl. Acad. Sci. U S A . 72,49814985
Catt, K. & Treager, G. W. (1967) Science 158, 1570-1572 De Mey, J., Hoebeke, J., De Brabander, M., Geuens, G. & Joniau, M. (1976) Nature (London) 264,273-275
Edelman, G. M. (1976) Science 192,218-226 Edelman, G. M. & Yahara, I. (1 976) Proc. Natl. Acad. Sci. U.S.A. 73,2047-205 1 Fuller, G. M., Brinkley, B. R. & Boughter, J. M. (1975) Science 187, 948-950 Hunter, W. M. & Greenwood, F. C. (1962) Nature (London) 194,495-496 Shelanski, M. L., Gaskin, F. & Cantor, C. R. (1973) Proc. Natl. Acad. Sci. U.S.A. 70,765-768 Sherline, P., Bodwin, C. K. & Kipnis, D. M. (1974) Anal. Biochem. 62,400-407 Tucker, R. W., Frankel, F. R.& Sanford, K. K. (1976) J . Cell Biol. 70,107a
Structural Studies on Avian Pancreatic Polypeptide JAMES E. PITTS, JOHN A. JENKINS, IAN J. TICKLE, THOMAS L. BLUNDELL and STEPHEN P. WOOD Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, University of London, Malet Street, London WC1E 7HX, U.K. A 36-residue pancreatic polypeptide with some hormonal properties has been purified from chicken and turkey pancreas by using acid/ethanol extraction, gel filtration on Sephadex G-50 with 10% (v/v) acetic acid and anion-exchange chromatography at pH8.3 on DEAE-Sephadex A25 in 7 ~ - u r e aThe . methods used are very similar to those used in the preparation of insulin and glucagon. The initial characterization of this material was reported by Kimmel et al. (1968). In subsequent studies they have chemically characterized the material and shown that it is present in the pancreas, is secreted into the circulation of chicks after feeding (Langslow et al., 1973) and gives rise to a decrease in hepatic glycogen without concomitant hyperglycaemia. Decreases in plasma glycerol and large increases in gastric secretion of acid and pepsin have also been noted (Hazelwood et al., 1973; Kimmel et al., 1975). The material that we have prepared has been crystallized by exploiting its positive temperature coefficient of solubility between 20" and 50°C at neutral pH. The crystals are monoclinic with space group C2. Preliminary isomorphous-replacement experiments have so far provided a single-site derivative with Hg(NO&. A low-resolution electrondensity map phased with this derivative by using anomalous scattering, considered together with Patterson-function calculations and study of the sequence, suggest that the molecules are partly helical and are arranged as a compact dimer about the crystallographic 2-fold axis. The sequence of the polypeptide (Fig. 1) shows two distinct regions. The N-terminal part of the sequence has a high proline content, and the C-terminal half (residues 17-34) shows alternating regions of hydrophobic and hydrophilic residues. If this region of the
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BIOCHEMICAL SOCIETY TRANSACTIONS
Gly-Pro-Ser-Gln-Pro-Thr-Tyr-Pro-Gly-Asp-Asp-Ala-Pro-Val-Glu-ASp-~eu-I~e-
1
18
Arg-Phe-Tyr-Asp-Asn-Leu-Gln-Gln-Tyr-Leu-Asn-Val-Val-Thr-Arg-His-Arg-~r-NH~ 19
36
Fig. 1. Amino acid sequence of avian pancreatic polypeptide (Kimmel et a/., 1975)
molecule were helical, then the helix would have well-defined hydrophilic and hydrophobic faces. This can be clearly demonstrated by placing the residues on a n Edmundson-Schiffer wheel (Schiffer & Edmundson, 1967; Wood et al., 1977). Such a n arrangement might be important in the association of the molecules. Homologous polypeptides from various mammalian sources have been characterized (Lin & Chance, 1972). The hydrophobic nature of the proposed helical segment is conserved. The structure and association properties of these molecules offer an interesting comparison with insulin and glucagon, which are pancreatic protein and polypeptide hormones respectively, which have been studied in great detail (Pullen et a/., 1976; Sasaki et al., 1975). Hazelwood, R. L., Turner, S. D., Kimmel, J. R. &Pollock, H. G. (1973) Gen. Comp. Endocrinol. 21,485-497 Kimmel, J. R., Pollock, H. G. & Hazelwood, R. L. (1968) Endocrinology 83,1323-1330 Kimmel, J . R., Hayden, L. J. &Pollock, H. G. (1975)J. Biol. Chem. 250,9369-9376 Langslow, D. R., Kimmel, J. R. & Pollock, H. G. (1973)Endocrinology 93,558-565 Lin, T . M. & Chance, R. E. (1972) Gastroenterology 62, 852 (Abstr.) Pullen, R. A., Lindsay, D. G., Wood, S. P., Tickle, 1. J., Blundell, T. L., Wollmer, A., Krail, G., Brandenburg, D., Zahn, H., Gliemann, J. & Gammeltoft, S. (1976) Nature (London) 259, 369-373 Sasaki, K., Dockerill, S., Adamiak, D. A., Tickle, I. J. & Blundell, T. L. (1975) Nature (London) 251,751-757 Schiffer, N . & Edmundson, A. B. (1967) Biopbys. J . 7,121-134 Wood, S. P., Pitts, J. E., Blundell, T. L., Tickle, I. J. &Jenkins, J. A. (1977) Eur. J. Biochem. in the press
The Structure at 0.3nm ( 3 i ) Resolution of the Acid Proteinase from Endothia parasitica JOHN A. JENKINS, IAN J. TICKLE, TREVOR SEWELL and THOMAS L. BLUNDELL Laboratory of Molecular Biology, Department of Crystallography, Birkbeck College, University of London, Malet Street, London WCI E 7HX, U.K.
The acid proteinase from Endothia parasitica was extracted and crystallized as described previously (Jenkins et al., 1975). X-ray-diffraction data were collected for crystals of native enzyme and six heavy-atom derivatives by using a Hilger and Watts four-circle diffractometer. An electron-density map was computed at 0.3 nm (3A) resolution by using phases calculated from isomorphous data from all the derivatives. The electron-density map allows a clear identification of the molecular boundary, and there are large regions of solvent. Within the boundary the polypeptide chain can be followed for about 305 residues and many large and well-defined side chains are apparent. The molecule is organized into two lobes, with a deep cleft about 2.5nm (25A) in length between them (see Fig. 1). The left lobe of Fig. 1 contains an extensive sheet structure which at one end folds over to form a barrel-like structure. There are many anti-parallel loops, but only one well1977
