Precocious axons and improved survival of rat hippocampal neurons on lysine-alanine polymer substrates.

We tested the hypothesis that other polymers of lysine would be better substrates for culture of CNS neurons than polylysine itself. In a serum-free medium optimized for survival of hippocampal neurons grown on substrates of poly-D-lysine, 13% more neurons survived on substrates to which a sequential copolymer of lysine and alanine (LAS) was applied (P = 0.006). The effect was specific for the sequential polymer, in contrast to the random copolymer of lysine and alanine. This suggests that average cationic charge density is not as important as the spacing of these charges. More dramatically, immunostaining for the axon-associated microtubule-associated protein, tau, indicated a 2-fold higher rate of fiber growth on LAS. The somatodendritic cytoskeletal component MAP2 also appeared to be increased in cells cultured on LAS. This suggests that cytoskeletal differentiation in general and axon formation in particular are stimulated by the LAS substrate. Scanning electron microscopy supported this conclusion. By circular dichroism, the conformation of LAS in phosphate-buffered saline appeared to be a random coil, indistinguishable from poly-D-lysine. These results indicate that LAS is a superior substrate to polylysine for growth of CNS neurons. LAS may be useful for regeneration of damaged circuits in the CNS as well as a substrate for connections to a neuroprosthesis.

17O, 14N and 15N n.m.r. studies of the Co2+ complexes of cyclo(Pro17O-Gly15N) and cyclo(Gly17O-Pro) in aqueous solution.

The complexation of cyclo(Pro17O-Gly15N) and cyclo(Gly17O-Pro) with Co2+ ions has been studied by 17O, 14N and 15N n.m.r. spectroscopy in aqueous solution. 17O, 14N and 15N transverse relaxation times and chemical shifts were measured as a function of temperature. The 17O n.m.r. studies unequivocally demonstrate that the cobaltous ion binds to the peptide oxygen of both compounds. The hyperfine coupling constant and the peptide residence times were found to be A = -0.165 MHz and -0.145 MHz, tau m = 16, and 92 microseconds for cyclo(Pro17O-Gly15N) and cyclo(Gly17O-Pro), respectively. The 14N and 15N studies of labeled cyclo(Pro17O-Gly15N) do not indicate binding at either the Gly15N or the Pro14N site.

Synthesis of 17O isotope labeled Leu-enkephalin and 17O n.m.r.

Oxygen-17 isotope was introduced into the alpha-carboxyl group of glycine, 1-phenylalanine, 1-leucine and 1-tyrosine by acid catalyzed exchange of 17O from H2O(17) or by acid hydrolysis of respective amino acid methyl esters in H2O(17). Quantitative enrichment of glycine was achieved by acid hydrolysis of amino acetonitrile in H2O(17). For alpha-amino protection in amino acids t-butoxycarbonyl (Boc) group was employed for 17O labeled enkephalin synthesis. Five analogues of Leu-enkephalins (I-V) labeled with 17O at different amino acid residues were synthesized by solid phase method. 17O n.m.r. spectra were measured at 24.4 and 67.8 MHz for Leu-enkephalins 17O labeled at Gly2 and Phe4 positions. A downfield shift was observed for 17O labeled Gly2 Leu-enkephalin upon heating. This shift is indicative of the rupture of intramolecular hydrogen bonds. The preliminary results confirm the hypothesis that an intramolecular hydrogen bond exists between the carbonyl group of Gly2 and NH group of Leu5.

17O n.m.r. studies of amino acids in the solid state, in single- and polycrystalline forms.

A single crystal of 17O enriched alpha-glycine was grown by slow evaporation of aqueous solution. 17O n.m.r. studies of the alpha-glycine molecule in single crystalline form revealed five 17O transitions each consisting of two lines due to inequivalency of the oxygen atoms in the unit cell, with each of these lines revealing a dipolar interaction between the 17O and the nearest hydrogen atom. The spectral width was found to be of the order of magnitude of MHz and the linewidths of the order of magnitude of 2 kHz.

17O and 14N n.m.r. studies of the Co (II) interaction with cyclo(Ala*-Ala) in aqueous solution.

The complexation of cyclo(Ala*-Ala) with the cobaltous ions in aqueous solution was investigated by 17O and 14N n.m.r. spectroscopy. The 17O and 14N transverse relaxation time (T2p) and chemical shift (delta omega a) of cyclo(Ala*-Ala) were measured as a function of the temperature at pH = 7.03 +/- 0.02, and pH = 6.45 +/- 0.02, and as a function of pH at room temperature. No effects of pH on the transverse relaxation time and chemical shift were observed. Complementary 17O studies of the solvent water molecules were also carried out. The hyperfine coupling constant and the entropy and enthalpy of activation for the exchange of cyclo(Ala*-Ala) and water molecules between the coordinated and noncoordinated states were determined by least-square fit of theoretical equation for the chemical shift delta omega a to experimental data. The hyperfine coupling constant of the peptide bound oxygen was determined to be (-1.6 +/- 0.1) X 10(5) Hz and the entropy and enthalpy (32.0 +/- 3.0) kJ/mol and (-12.0 +/- 1.0) e.u, respectively. Information obtained from 17O n.m.r. study allows some inferences concerning the probable coordination sphere of the cobaltous ion. There are three types of complexes: Co(H2O)6(2+), CoL X 5H2O and CoL2 X 4H2O, with relative concentrations 19.9%, 2.9%, and 77.2%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen-18 isotope labeling and its effect on carbon-13 chemical shifts of the peptide bond.

The effects of 18O isotopes on 13C NMR chemical shifts of peptide carbonyl carbons were found to be 0.028 ppm for glyclyglycine and 0.029 ppm for glycylproline at 50.31 MHz.