A peptide that inhibits hydroxyapatite growth is in an extended conformation on the crystal surface.

Proteins play an important role in the biological mechanisms controlling hard tissue development, but the details of molecular recognition at inorganic crystal interfaces remain poorly characterized. We have applied a recently developed homonuclear dipolar recoupling solid-state NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hydroxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N terminus of the salivary protein statherin. Constant-composition kinetic characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to the crystal surface. The DRAWS technique was used to measure the internuclear distance between two 13C labels at the carbonyl positions of the adjacent phosphoserine residues. Dipolar dephasing measured at short mixing times yielded a mean separation distance of 3.2 +/- 0.1 A. Data obtained by using longer mixing times suggest a broad distribution of conformations about this average distance. Using a more complex model with discrete alpha-helical and extended conformations did not yield a better fit to the data and was not consistent with chemical shift analysis. These results suggest that the peptide is predominantly in an extended conformation rather than an alpha-helical state on the HAP surface. Solid-state NMR approaches can thus be used to determine directly the conformation of biologically relevant peptides on HAP surfaces. A better understanding of peptide and protein conformation on biomineral surfaces may provide design principles useful for the modification of orthopedic and dental implants with coatings and biological growth factors that are designed to enhance biocompatibility with surrounding tissue.

Isolation, characterization, and radioimmunoassay of Atlantic halibut somatolactin and plasma levels during stress and reproduction in flatfish.

Somatolactin (SL), a recently identified teleost pituitary hormone which is a member of the growth hormone/prolactin family, was isolated from pituitary tissue of Atlantic halibut (Hippoglossus hippoglossus). Pituitary proteins were extracted in ammonium bicarbonate (pH 7.8), fractionated using gel filtration chromatography, and purified using reversed-phase high-performance liquid chromatography. Halibut SL was identified on the basis of molecular size (determined by gel electrophoresis and mass spectroscopy), cross-reactivity of the putative hormone with antisera to cod SL, and N-terminal amino acid sequence. Polyclonal antibodies to purified halibut SL were raised in rabbits, and a radioimmunoassay (RIA) was developed for measurement of plasma concentrations of SL using purified halibut SL as a standard. The RIA was tested in several flatfish species including Pacific halibut (Hippoglossus stenolepis), English sole (Pleuronectes vetulus), and rock sole (Lepidopsetta bilineata). The assay was specific for SL as indicated by absence of cross-reactivity with Atlantic halibut growth hormone, prolactin, and GTH alpha subunit. Dilutions of plasma and pituitary extracts from Pacific halibut, English sole, and rock sole were parallel to the Atlantic halibut SL standard curve, indicating that the assay is valid for a range of flatfish species. Using halibut SL antiserum, SL was localized in the pars intermedia of English sole pituitary, where it has been identified in previously examined teleost species. The RIA was used to measure plasma levels of SL in Atlantic halibut and English sole during reproductive development, and in English sole subjected to various types of environmental stressors, including handling and crowding. In both sole and halibut, plasma SL concentrations remained relatively constant throughout gonadal development, but dropped during or following ovulation. Plasma SL levels in English sole tended to increase in response to acute stress, in parallel with plasma cortisol levels.