Graduation date: 1991

Growth hormone binding protein (GHBP) may be an important factor in the regulation of growth as well as an indirect, less invasive way of predicting the status of growth hormone receptors. Several factors (age, nutritional status, sex, and glucocorticoid administration) have been reported to influence circulating growth hormone (GH) levels, growth hormone receptor (GHR) activity and/or GHBP in mammalian species. Therefore, the studies conducted in this research were designed to determine if these factors have any affect on serum GHBP in the young broiler chicken. Serum GHBP activity was expressed as a percent specifically bound ¹²⁵IhGH (%SB), as measured by a dextran-coated charcoal assay. Serum GHBP activity was highest (mean %SB= 14.6 ± 1.2) at hatch aniedecreased linearly (r= -.9516) to 4 wk of age (mean %SB= 4.1 ± 0.6). Sex had no significant affect on serum GHBP activity from hatch to 4 wk of age. Short term nutrient deprivation (24 h fast) of 4 wk old birds had no significant affect on serum GHBP activity, nor did refeeding. Feeding birds nutrient poor diets (low energy, low protein or low energy and low protein) did not significantly affect serum GHBP activity when compared to birds fed a commercial broiler diet. Pulsatile delivery of cortisone acetate (1, 5 and 10 mg/d/b) had no affect on serum GHBP activity at any dose. These results suggest that serum GHBP activity in the chicken is not affected by many factors which do influence GHBP in mammalian species. The lack of response to nutrient deprivation and cortisone acetate may be a factor related to the age of the birds used in these studies. / Graduation date: 1993

The crystal structure of ATP-dependent phosphoenolpyruvate carboxykinase (ATP-oxaloacetate carboxy-lyase, (transphosphorylating), E.C. 4.1.1.49; PCK) from Escherichia coli K12 has been determined using a combination of multiple isomorphous replacement and density modification, and refined to a R-index of 0.202 (R-free = 0.244) at 1.9 A resolution. PCK catalyses the decarboxylation and ATP-dependent phosphorylation of oxaloacetate to form phosphoenolpyruvate, the first committed step of gluconeogenesis in E. coli. Each PCK molecule consists of a 275 residue N-terminal and 265 residue C-terminal ar mononucleotide-binding domain, with the active site located within a cleft between the two domains. PCK is an open-faced, mixed $\alpha/\beta$ protein with a unique overall tertiary structure. The putative phosphate-binding region of the ATP-binding site adopts the P-loop motif common to many ATP- and GTP-binding proteins. However, the â-sheet topology of the mononucleotide-binding fold of PCK differs from all other families within the P-loop containing nucleoside triphosphate hydrolase superfamily, suggesting PCK represents the first member of a new family of such proteins. The mononucleotide-binding domain also differs in structure from the classical mononucelotide-binding fold (CMBF), common to adenylate kinase, RecA, p21$\sp{{Ha}-ras}$, and elongation factor-Tu. Several highly-conserved amino acid residues among the ATP-dependent PCK family, including R65, Y207, K212, K213, H232, K254, T255, D269, K288 and R333 appear to make up the active site of the enzyme. A cysteine residue, C233, is located near the active site, and in the E. coli enzyme this residue is buried and is probably not involved in substrate binding or catalysis. Previous chemical modification studies, on several ATP- and GTP-dependent PCKs, have been assessed in view of these structural results. A mechanism of catalysis based on these and additional results is proposed. The structure of E. coli PCK complexed with the calcium-analogue Tb$\sp{3+}$ has been refined to an R-index of 0.205 (R-free = 0.259) at 2.5 A. Two binding sites for Tb$\sp{3+}$ have been determined, one within the active site coordinating to the side chains of K213, H232, and D269, and the other within the C-terminal domain, coordinating to the side chains of E508 and E511. No large structural movements are observed in PCK as a result of Tb$\sp{3+}$ binding, even though Ca$\sp{2+}$ is a known activator.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 129-140).

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 114-129). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 172-200).

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "December 2001. Typescript. Vita. Includes bibliographical references (leaves 326-347). Also available on the Internet.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 173-189). Also available on the World Wide Web.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 148-173). Available also in a digital version from Dissertation Abstracts.

As a ubiquitous protein-folding helper in bacterial cytosol, SlyD is a peptidylprolyl isomerase (PPIase) of the FK506-binding protein (FKBP) family. It has two important functional domains, the IF (insert-in-flap) domain with chaperone activity and the FKBP domain with PPIase activity. It also possesses a histidine- and cysteine-rich C-terminal metal-binding domain, which binds to selected divalent metal ions (e.g. Ni2+, Zn2+) and is critical for participation in metal trafficking for metalloenzymes. SlyD from Helicobacter pylori was investigated both structurally and functionally by a variety of biophysical, biochemical and molecular biology techniques. HpSlyD was cloned, expressed and purified. It binds to Ni2+ and Zn2+ with dissociation constants (Kd) of 2.74 and 3.79 μM, respectively. Both Ni2+ and Zn2+ can competitively bind to HpSlyD. The C-terminus was demonstrated to convey nickel resistance in vivo. It also binds to Bi3+ with Kd of 4.4 × 10-24 M. Furthermore, Zn2+, Cu2+ and Bi3+ can induce the dimerization or oligomerization of HpSlyD. The solution structure of the C-terminus-truncated SlyD from Helicobacter pylori (HpSlyDΔC) was determined by NMR, which demonstrates that HpSlyDΔC folds into two well-separated, orientation-independent domains. Both the FKBP and IF domains fold into a structure consisting of a four-stranded antiparallel β-sheet and an α-helix. Binding of Ni2+ instead of Zn2+ induced the conformational changes in FKBP domain, where the active sites are positioned, suggesting a regulatory role of nickel on the function of HpSlyD. It was also confirmed that HpSlyD can associate with the Tat (twin-arginine translocation) signal peptide from small subunit of [NiFe] hydrogenase (HydA), an accessory protein HpHypB for [NiFe] hydrogenase mainly by the IF domain. Surprisingly HpSlyD was found to form a complex with HpUreE, a urease chaperone, indicative of the “cross-talk” between [NiFe] hydrogenase and urease. The possible mechanism of HpSlyD for the cooperation with HpHypB was also explored. In the presence of different metal ions, HpSlyD was shown to regulate the GTPase activity of HpHypB, implicating the possible metal transfer induced by HpSlyD. It was suggested that HpSlyD modulates the nickel insertion of [NiFe] hydrogenase by controlling the GTPase activity of HpHypB. In this thesis, the SlyD protein from H. pylori was shown as an important regulator for the activation of both [NiFe] hydrogenase and urease. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy