Data from ¹H nuclear magnetic resonance studies on the Fv fragment of protein 315, a Dnp-binding BALB/c mouse lgA(λ₂) myeloma protein, have been used to refine a predicted structure of the combining site of the protein. The Dnp-binding subsite in the modified structure is composed of the side chains of three aromatic amino acids Trp 93_L, Tyr 3⁴_L and 3⁴_H. A fourth aromatic amino acid residue is close to the side chain-NH-CH 2 -group, this is Tyr 33 H - The antibody-hapten binding is a simple encounter process, which causes no extensive conformation change in the Fv fragment. A method for paramagnetic structural studies has been devised using Dnp derivatives with cllgophosphate side chains, which create a specific manganese binding site on the Fv fragment-hapten complex. The distances from the bound metal ion to the imidazole side chains of two of the three hlstldine residues of protein 315 have been determined. ¹H nuclear magnetic resonance has been used to study the histidine residues of the Fv fragment of protein 315. It has been shown that one of the three histidine residues (102_H) is close to the combining site, but that this residue does not participate directly in binding haptens. ³¹ P nuclear magnetic resonance studies have shown the presence of a positively charged amino acid side chain near the entrance of the combining site of the Fv fragment. This residue has been identified as Arg 95_L. The mode of binding of trini trophenyl derivatives to the Fv fragment has been studied by ¹H nuclear magnetic resonance. It is concluded that these haptens, when bound to the Kv fragment, make contacts with the same amino acid side chains as Dnp derivatives.

Upon starvation, individual amoebae of D. discoideum cooperate to produce a cell aggregate. The aggregate then elongates into a slug which moves chemotactically towards food sources. Culmination of the developmental cycle results in the formation of a fruiting body containing differentiated stalk and spore cells. Ammonia is produced naturally in the aggregates during starvation and is implicated in the regulation of the developmental cycle. Addition of excess ammonia leads to inhibition of aggregation, increased slug migration, delay of culmination and the promotion of spore formation. This thesis examines the possible intracellular mechanism(s) through which ammonia might affect developmental processes. It has been postulated that ammonia might act by directly inhibiting a particular enzymatic activity, alternatively it could act as a weak base to raise the pH of the cytosol or an intracellular compartment. To investigate these possibilities, the effects of a variety of weak bases were tested on aggregation and culmination. All of the weak bases tested inhibited development in a dose-dependent way, which argues against the notion that ammonia acts by direct enzyme inhibition since the bases are structurally distinct. In addition, the inhibition potencies of the bases varied according to their protonophoric activity: the most effective protonophores were also the most effective developmental inhibitors. This suggested that the bases were likely to be acting to neutralize an intracellular compartment in these experiments,a result which implicates acidic compartments in the control of development. That weak bases can indeed dissipate the pH of intracellular vesicles was confirmed in vitro by monitoring acidification of isolated vesicles in the presence of the bases. In vivo experiments using ³¹P-NMR probes of the vesicular system confirmed this and showed that cytosolic pH was stable in the presence of bases. Genetic evidence for the role of acidic compartments in development was also gathered. Both the acidification-defective (Hgr) mutants and a mutant created by antisense expression of a fraction of the B subunit cDNA of the vacuolar H⁺- ATPase were defective in development, displaying developmental behaviour reminiscent of weak base-treated wild-type strains. This shows that defective vacuolar acidification can mimic the effect of addition of ammonia and indicates that an acidic vesicle is the target of ammonia In vivo.

Lateral diffusion of phospholipids is a process essential to membrane function, and can be measured by nuclear magnetic resonance (NMR). This project will use CODEX (Centerband-Only Detection of Exchange), the most highly-evolved NMR experiment combining magic angle spinning with exchange sensitivity, to measure lateral diffusion. 31P CODEX NMR measurements were performed on phospholipids in small unilamellar vesicles (SUV) in a high viscosity solution to slow the rotational tumbling of the SUV and minimize its influence on the CODEX decay. For SUV composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), alone or mixed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) or cholesterol (CHOL), 31P CODEX spectra showed well-resolved resonances for POPC and POPG, with monoexponential decays for both, from which the correlation time for molecular motion could be extracted. The lateral diffusion coefficients were determined, and fell in the range of 1.0 - 3.3 x 10-12 m2s-1 at 10°C, which agree with established literature values for POPC and POPG.

Lateral diffusion of phospholipids is a process essential to membrane function, and can be measured by nuclear magnetic resonance (NMR). This project will use CODEX (Centerband-Only Detection of Exchange), the most highly-evolved NMR experiment combining magic angle spinning with exchange sensitivity, to measure lateral diffusion. 31P CODEX NMR measurements were performed on phospholipids in small unilamellar vesicles (SUV) in a high viscosity solution to slow the rotational tumbling of the SUV and minimize its influence on the CODEX decay. For SUV composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), alone or mixed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) or cholesterol (CHOL), 31P CODEX spectra showed well-resolved resonances for POPC and POPG, with monoexponential decays for both, from which the correlation time for molecular motion could be extracted. The lateral diffusion coefficients were determined, and fell in the range of 1.0 - 3.3 x 10-12 m2s-1 at 10°C, which agree with established literature values for POPC and POPG.

No description available.

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1978. / Bibliography: leaves 160-164. / Microfiche. / xiv, 164 leaves ill

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1984. / Bibliography: leaves 181-186. / Microfiche. / lMaster negative: Microfiche MS33173. / xiii, 186 leaves, bound ill. 29 cm

Typescript. / Thesis (Ph. D.)--University of Hawaii, 1970. / Includes bibliographies. / 2 pt. in 1 v illus., tables

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1976. / Bibliography: leaves [312]-320. / Microfiche. / xxx, 320 leaves ill

Thesis (Ph. D.)--University of Adelaide, 1986. / Includes bibliographical references.