The crystal structure of a dipeptide complex of bovine neurophysin H has been solved at 2.8 A resolutionsolely by using single-wavelength anomalous scattering data from a single iodinated derivative. The asymmetric unit is an elongated tetramer of dimensions 110 x 40 X 30 A, composed of two dimers related by pseudo twofold symmetry. Each monomer consists of two homologous layers, each with four antiparaflel 3-strands. The two regions are connected by a helix followed by a long loop. Monomer-monomer contacts involve antiparallel 13-sheet interactions, which form a dimer with two layers of eight P-strands. (10), one of which is isomorphous with the crystals of a porcine NP-I complex (11). These contained 4, 8, and 12 molecules per asymmetric unit, respectively, suggesting that the basic aggregates of the NP-dipeptide complex in the crystals are tetramers that can self-associate to higher oligomers (10). In view of the probable importance of NP self-association in packaging of the precursor in the NSG (1, 4) and evidence that NP complexes can be present in the NSG as the precipitated or crystalline state (12, 13), analysis of the crystal structure of these complexes offers an opportunity for providing detailed structural information not only on NP folding and protein-peptide interactions but also on how the complexes might be packaged in NSG.The crystal structure of a bovine NP-II-dipeptide complex"l reported here was determined using only the singlewavelength anomalous scattering (SAS) data from an iodinated derivative crystal. Iodine was chosen as a probe because of its relatively large anomalous scattering signal (Alf' = 6.8) and its ease of incorporation into the dipeptide.The resulting derivative crystals contained iodine atoms covalently linked to the Phe residues of the bound hormone analogs.Here we report the structure of a NP molecule, as well as aspects of the methodology used in solving the structure. METHODSCocrystaflization. Bovine NP-I was purified as described (14). The dipeptide para-iodo-L-phenylalanyltyrosine amide (I-Phe-Tyr-NH2) was custom-synthesized by Peninsula Laboratories and was demonstrated by using circular dichroism (8) to bind to the hormone-binding site with high affinity. Crystals of the NP-dipeptide complex were grown at pH 7.5 by using a modification of the procedure of Yoo et al. (9); crystals of the complex of the corresponding noniodinated peptide served as seeds. The crystals obtained were nearly isomorphous with those of the complex of the uniodinated peptide, having four molecules (chains) per asymmetric unit and space group P212121 with a = 120.0 A, b = 69.4 A, and c = 62.4 A.

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The first crystal structure of the pituitary hormone oxytocin complexed with its carrier protein neurophysin has been determined and refined to 3.0 A resolution. The hormone-binding site is located at the end of a 3(10)-helix and involves residues from both domains of each monomer. Hormone residues Tyr 2, which is buried deep in the binding pocket, and Cys 1 have been confirmed as the key residues involved in neurophysin-hormone recognition. We have compared the bound oxytocin observed in the neurophysin-oxytocin complex, the X-ray structures of unbound oxytocin analogues and the NMR-derived structure for bound oxytocin. We find that while our structure is in agreement with the previous crystallographic findings, it differs from the NMR result with regard to how Tyr 2 of the hormone is recognized by neurophysin.

Circular dichroism and proton titration studies of mixtures of native or nitrated bovine neurophysin-II and lysine vasopressin confirm that there is one principal site for lysine vasopressin of very similar properties to the single oxytocin site. A second, but markedly weaker lysine vasopressin site is allowed by the data, but two thermodynamically equivalent sites for lysine vasopressin are precluded. Binding constants to nitrated bovine neurophysin-II of oxytocin, lysine vasopressin, and a series of peptide analogs of the first two to three residues of the hormones were obtained by circular dichroism using a single-site model. The data indicate that peptides containing only the first three residues of the hormones contribute almost two-thirds of the binding free energy of the hormones and that half of the binding free energy is contributed by the cooperative binding of residues 1 and 2.

The terminal step in the biosynthesis of L-ascorbic acid in peas has been shown to be the oxidation of L-galactono-y-lactone by an enzyme present in mitochondria (Mapson, Isherwood & Chen, 1954). This enzyme has been solubilized from mitocondria obtained from freshly germinated peas, cabbage leaves and cauliflower florets. Since the enzyme preparations from the mitochondria of the cauliflower florets were found to be more active than those from either peas or cabbage, we have used this material in most of the work reported here. The present paper describes the preparation and partial purification of the enzyme, together with an account of some of its properties. EXPERIMENTAL Ghemical8 L-Galactono-y-lactone was prepared by the reduction of D-galacturonic acid by borohydride as follows. D-Galacturonic acid (10 g.) was dissolved in 40 ml. of water and neutralized with NaOH to between pH 8-5 and 9 0. Borohydride was added gradually with stirring at room temperature. Samples were removed and acidified with acetic acid to remove excess of borohydride, and galacturonic acid was tested for by boiling with Fehling's solution. After reduction was complete, the solution was acidified with acetic acid to pH 5-0, a slight excess of barium acetate added, and the precipitate filtered off. Ethanol (2 vol.) was added to the solution and the precipitate was collected. After the precipitate had been washed twice with 60% (v/v) ethanol, barium was removed by Dowex 50 resin, and to the filtrate 1-2 drops of 3 N-HCl were added. The solution was concentrated to a syrup and dried in vacuo. The lactone was recrystallized from absolute ethanol.