K. U. Prasad

Chemotaxis of fibroblasts toward nonapeptide of elastin

Bovine ligamentum fibroblasts, which produce elastin, migrate towards a positive chemical gradient of human platelet-derived growth factor and of the tropoelastin repeat hexapeptide Val-Gly-Val-Ala-Pro-Gly, as previously shown. They are also responsive to two permutations of a nonapeptide that repeats in tropoelastin, i.e., Ala-Gly-Val-Pro-Gly-Phe-Gly-Val-Gly and Gly-Phe-Gly-Val-Gly-Ala-Gly-Val-Pro. Concentration curves and checkerboard assays prove that the nonapeptides are chemoattractants. The component pentapeptide, Gly-Phe-Gly-Val-Gly, is chemotactic, while the component tetrapeptide Ala-Gly-Val-Pro is not. The hexapeptide competitively suppresses the nonapeptide chemotaxis suggesting the involvement of a common cell receptor. The results support the concept that elastin has multiple cell recognition sites as measured by the chemotactic response and that among the hydrophobic repeating sequences of elastin chemotacticity is selectively and multiply localized.

Cesium-133 NMR longitudinal relaxation study of ion binding to the Gramicidin transmembrane channel

Abstract Cesium ion interaction with the Gramicidin transmembrane channel is studied using cesium-133 NMR. The channel system provides unusual insight into the use of spin - 7 2 nuclei to obtain ion interaction information. In spite of the small quadrupole moment for 133Cs, the absolute magnitude of which is 1 40 that of 23Na, the change in longitudinal relaxation rate for 133Cs interaction with channels is twice that observed for 23Na under identical conditions. Also, only small positive ion resonance chemical shifts are experimentally observed which are of opposite sign to those observed for the other alkali metal ions. The possibility of the cesium ion pairing with chloride ion in the channel binding site is explored as explanation for the small positive chemical shift and large T1 changes. The large T1 changes provide values for both tight and weak binding constants, i.e., Kbt ≈ 60/M and Kbw ≈ 4/M. In the null region of the partially relaxed spectra two narrow lines (separated by 20 Hz at 13 MHz) and a broad line were observed and the longitudinal relaxation rates were faster for the narrow components than for the broad component. Evidence is presented indicating that all resolvable lines were derived from a single equilibrated pool of spin - 7 2 nuclei. The null region demonstrates that a single, intense, narrow resonance line dominates the longitudinal relaxation in accordance with the analyses of Bull, Forsen, and Turner.

Poly (VAL1-PRO2-ALA3-VAL4-GLY5): A Reversible, Inverse Thermoplastic

Initial characterization of the sequential polypentapeptide, poly (VPAVG) an analog of the polypentapeptide of elastin poly (VPGVG), is reported. It undergoes a concentration dependent inverse temperature transition, in which it is soluble in water below 25°C and aggregates on raising the temperature. When crosslinked by 20 Mrads of γ-irradiation to give X20-poly (VPAVG), an elastomeric matrix is formed. At 25°C, this material exhibits an elastic modulus of 1.5 x 105 dynes/cm2, similar to that found at that temperature for X20-poly (VPGVG). On raising the temperature to 37°C, however, the elastic modulus increases three orders of magnitude to 3 × 108 dynes/cm2 which is two orders of magnitude greater than obtained for X20-poly (VPGVG). This reversible hardening on raising the temperature defines X20-poly (VPAVG) as a reversible, inverse thermoplastic. This property makes it an interesting new material to add to the already described set of bioelastic materials. The potential applications and extensions of applications made possible by this additional bioelastic material are briefly considered.

On the mechanism of channel-length dependence of gramicidin single-channel conductance

Single-channel conductance data on four different gramicidin channel lengths demonstrate that conductance magnitude is neither inversely dependent on the square of the channel length nor on the image force arising from differences in the extent of lipid dimpling (Jordan and Vayl (1985) Biochim. Biophys. Acta 818, 416-420). Rather the conductance differences are consistent with the decreased off-rate constant for the singly occupied state as the ionic radius decreases from that of cesium ion to sodium ion coupled with the decreased probability of the doubly occupied channel due to increased ion-ion repulsion as the channel is shortened (Urry et al. (1984) Biochim. Biophys. Acta 774, 115-119).

Complete proton and carbon-13 resonance assignments of the cyclodecapeptide of elastin by combined use of multiple and selective proton-decoupled 13C and 1H spectra

Abstract Multiple and selective 1 H irradiation techniques were used to assign all the peptide 1 H and 13 C resonances to their respective residues for the cyclodecapeptide, cyclo-( l ·Val 1 - l ·Pro 2 -Gly 3 - l ·Val 4 -Gly 5 ) 2 , analog of the polypentapeptide of elastin. As the structure of interest has twofold symmetry on the NMR time scale, the primary problems are to delineate the resonances of Val, from Val, and those of Gly 3 from Gly 5 and to assign the five peptide C O resonances. Irradiation of the Pro 2 α CH while observing the carbonyl carbon spectrum allows identification of the pro 2 C O resonance, which is selectively intensified. The Gly NH which on selective irradiation also causes the Pro 2 C O to become selectively intensified is the Gly 3 N H . The GIy 3 N H can be irradiated to identify the Gly 3 αC H 2 protons in the PMR spectrum, etc. With this combined use of selective proton irradiation of a peptide N H and an αC H proton while observing the CMR spectrum and with the usual proton homonuclear decoupling, it becomes possible to assign all of the resonances. This approach obviates the expensive and time-consuming process of achieving difficult assignments by synthetic isotopic enrichments.

On the Mechanism whereby Phosphorylation Modulates Protein Folding Relevance to Protein Tangles and Plaques of Alzheimer's Disease

In the macromolecular tangles of Alzheimer’s disease there are abnormally phosphorylated proteins. These phosphorylated proteins are components of the intraneuronal paired helical filaments and extraneuronal amyloid deposits characteristic of Alzheimer’s disease,’,* and they include the microtubule-associated tau proteins3s4 and the amyloid precursor protein^.^ In the present report, the sensitivity of protein folding and assembly to phosphorylation is demonstrated in an elastic protein element model. The mechanism whereby phosphorylation modulates protein structure and function is not the often discussed electrostatic interaction, but rather is what appears to be a more efficient mechanism for the chemical modulation of protein structure. This can be demonstrated in synthetic model contractile proteins which are capable of chemomechanical transduction6 in which the amount of chemical energy to perform a given amount of mechanical work can be compared with that of mechanochemical systems based on the electrostatic repulsion m e ~ h a n i s m . ~

Polypentapeptide of Elastin as an Elastomeric Biomaterial

The polypentapeptide of elastin, (L·-Val1-L·Pro2-Gly3-L·Val4-Gly5)n, is synthesized with \(\bar n\)>200. The purity of the synthesis is verified by carbon-13 nuclear magnetic resonance spectra. Coacervate concentrations are γ-irradiation cross-linked at 2,6,10,14, 18,26 and 34 MRADs to produce elastomeric bands. The dependence of the elastic modulus on cross-linking dose is demonstrated and thermoelasticity studies indicate a dominantly entropic elastomeric force. These results are discussed in connection with the property of the polypentapeptide to self-align into fibers, in connection with a proposed entropy source for the elasticity utilizing a class of β-spiral conformations and in terms of specific analogs which had been synthesized to test concepts of conformation and entropy source. Finally, the biocompatibility of the polypentapeptide is considered.

Elastomeric Polypeptide Biomaterials: Introduction of Hexapeptide Repeats (Hard Segments) in the Polypentapeptide

A repeating sequence of elastin, the hexapeptide (Val-Ala-Pro-Gly-Val-Gly), has been variously introduced into matrices of the polypentapeptide of elastin, (Val1-Pro2-Gly3-Val4-Gly5)n, to examine a possible structural role of interlocking of hexapeptide segments of one chain with hexapeptide segments of a second chain. The aggregational properties of oligohexapeptides with n = 1,2,3 and 4, of polyhexapeptides with n = 100, of polypentapeptide with n = 200, of 1:2 mixtures of polyhexapeptide with polypentapeptide, respectively, and of cosequential polyhexapentapeptide with a ratio of hexapeptide to pentapeptide of 1:2 were determined. Samples vith 60% water by weight were prepared and γ-irradiation crosslinked at 20 MRAD to obtain elastomeric bands. The introduction of hexapeptide, under conditions where hexapeptide-hexapeptide interactions occured, was shown to be able to increase elastic modulus by as much as an order of magnitude. This demonstrated structural role, of the hexapeptide, its previously demonstrated chemotactic role and the effect of introducing occasional lysine residues in place of Val4 in the polypentapeptide sequence were considered in a general way in discussing the potentials of elastomeric polypeptide biomaterials.