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Cunningham, Lee Anna - One of the best experts on this subject based on the ideXlab platform.
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The Effects of Vasoactive Intestinal Peptide on Adrenal Steroid Hormone Secretion
1Co-Authors: Cunningham, Lee AnnaAbstract:126 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.Vasoactive intestinal Peptide (VIP)-immunoreactive nerve fibers have been demonstrated in the rat adrenal cortex in close association with zona glomerulosa cells (Holzwarth, 1984). We have studied the effects of VIP on steroid hormone secretion from the outer zones of the normal rat adrenal cortex. Intact capsule-glomerulosa preparations, consisting of the capsule, zona glomerulosa, and a small portion of the zona fasciculata were perifused in vitro. The secretory responsiveness was assessed by measuring aldosterone and corticosterone release following stimulation with the physiological secretagogues ACTH and angiotensin II. ACTH (10$\sp{-12}$ to 10$\sp{-8}$ M) stimulated dose-dependent increases in aldosterone secretion (1.9 to 36.9 fold over basal secretion) and corticosterone secretion (1.4 to 14.0 fold over basal secretion). VIP (10$\sp{-6}$ to 10$\sp{-4}$ M) stimulated dose-dependent increases of both aldosterone (1.7 to 41.0 fold) and corticosterone secretion (1.8 to 5.3 fold). However, glucagon and (N-Ac-Tyr$\sp1$-D-Phe$\sp2$)-GRF(1-29)-NH$\sb2$, Peptides structurally related to VIP, stimulated neither aldosterone nor corticosterone secretion indicating that VIP effects were specific for this Peptide. In addition, VIP (10$\sp{-5}$ M) potentiated the aldosterone but not the corticosterone secretory response to subthreshold concentrations of ACTH (10$\sp{-13}$ and 10$\sp{-12}$ M) by approximately 65-70% whereas it had no effect on the secretory response to angiotensin II. The distribution of adrenal VIP receptors was assessed by in vitro autoradiography of $\sp{125}$I-VIP binding. $\sp{125}$I-VIP (0.75 and 2.0 nM) binding was concentrated in the capsule and zona glomerulosa, coincident with the distribution of VIP nerve fibers which aborize extensively in this region. The specificity of this binding was demonstrated using unlabelled VIP, ACTH and angiotensin II.These studies demonstrate the effects of VIP on zona glomerulosa cell steroid hormone secretion indicating that the VIP innervation of the adrenal cortex provides an important regulation of adrenal steroidogenesis. The potentiated aldosterone secretory response observed with concurrent VIP and ACTH stimulation suggests that this innervation may regulate zona glomerulosa cell responsiveness to hormonal secretagogues. Further evidence for a functional interaction between the VIP innervation and zona glomerulosa cell secretion is the anatomical correlation between VIP-containing nerve fibers and specific $\sp{125}$I-VIP binding to adrenocortical cells in the capsule-zona glomerulosa.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD
Lassy, Rachel Anne Larse - One of the best experts on this subject based on the ideXlab platform.
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A Characterization of Aspartyl Peptidases in Salmonella Typhimurium
1Co-Authors: Lassy, Rachel Anne LarseAbstract:111 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2000.Few of the peptidases previously characterized from Salmonella typhimurium are capable of hydrolyzing aspartyl Peptides. Peptidase E, an aspartyl specific dipeptidase, was the first member of a new family of Peptide hydrolases that now includes peptidases from other proteobacteria and from two eukaryotes. It is shown that Peptidase E from Xenopus laevis has the same substrate specificity as Peptidase E from S. typhimurium, indicating that in this family of enzymes both the catalytic and the substrate specificity have been conserved. An alignment of the amino acid sequences of the Peptidase E family allowed for potentially important residues to be identified. Through site-directed mutagenesis of the S. typhimurium pepE, it was proposed that this enzyme is a serine hydrolase that utilizes a catalytic triad of serine, aspartate, and histidine. In extracts of a pepE strain, two additional Asp-X hydrolases were detected. The identity of each of these enzymes as well as evidence of a third Asp-X hydrolase are presented in this thesis. Two of these enzymes are shown most rapidly hydrolyze isoaspartyl Peptides (also called beta-aspartyl Peptides). Of the two isoaspartyl peptidases, one is a homolog of IadA from E. coli and the other is described for the first time in this work and is encoded by a previously unknown open reading frame called ybiK in E. coli. The ybiK gene product is a threonine hydrolase that is a member of the Ntn (N-terminal nucleophile) family of enzymes. It is synthesized as a 32 kD polyPeptide that dimerizes and undergoes processing, presumably autocatalytic, into an active form, which is a 60 kD heterotetramer. A strain of S. typhimurium lacking all of the broad specificity peptidases, as well as the aspartyl peptidases described here is still capable of growth on Asp-Leu as a leucine source. This growth is attributed to the remaining peptidase. This peptidase is specific for alpha-aspartyl Peptides, has a native molecular mass of 70 kD, and has the unusual property of requiring manganese for activity.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD
G. Aldini - One of the best experts on this subject based on the ideXlab platform.
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Development of a direct ESI-MS method for measuring the tannin precipitation effect of proline-rich Peptides and in silico studies on the proline role in tannin-protein interactions
'Elsevier BV', 2019Co-Authors: G. Aro, A. Altomare, L. Fumagalli, C. Rumio, M. Carini, G. Vistoli, G. AldiniAbstract:Tannins are a heterogeneous class of polyphenols that are present in several plants and foods. Their ability to interact and precipitate proline-rich proteins leads to different effects such as astringency or antidiarrheal activity. Thus, evaluation of the tannin content in plant extracts plays a key role in understanding their potential use as pharmaceuticals and nutraceuticals. Several methods have been proposed to study tannin-protein interactions but few of them are focused on quantification. The purpose of the present work is to set up a suitable and time efficient method able to quantify the extent of tannin protein precipitation. Bradykinin, chosen as a model, was incubated with increasing concentrations of 1,2,3,4,6-penta-O-galloyl-β-D-glucose and tannic acid selected as reference of tannic compounds. Bradykinin not precipitated was determined by a mass spectrometer TSQ Quantum Ultra Triple Quadrupole (direct infusion analysis). The results were expressed as PC 50 , which is the concentration able to precipitate 50% of the protein. The type of tannin-protein interaction was evaluated also after precipitate solubilisation. The involvement of proline residues in tannin-protein interactions was confirmed by repeating the experiment using a synthesized Peptide (RR-9) characterized by the same bradykinin sequence, but having proline residues replaced by glycine residues: no interaction occurred between the Peptide and the tannins. Moreover, modelling studies on PGG-BK and PGG-RR-9 were performed to deeply investigate the involvement of prolines: a balance of hydrophobic and H-bond contacts stabilizes the PGG-BK cluster and the proline residues exert a crucial role thus allowing the PGG molecules to elicit a sticking effect
Schrag, Joseph D. - One of the best experts on this subject based on the ideXlab platform.
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Structure and Function of Macromolecular Antifreezes (Peptide, GlycoPeptide, Fish)
1Co-Authors: Schrag, Joseph D.Abstract:100 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1984.Many polar fishes are protected from freezing by Peptide or glycoPeptide antifreezes which inhibit ice growth. The relationship between the structures of these molecules and the mechanism by which they work in investigated in this thesis.GlycoPeptide antifreezes range in molecular weight from 2600-33,000 daltons and most are polymers of the triPeptide ala-ala-thr. Each threonine is glycosidically linked to the disaccharide galactose-N-acetylgalactosamine. The glycoPeptide antifreezes from Microgadus tomcod and Eleginus gracilis are unique, however, in that they contain arginine. Determination of the amino acid sequences of these antifreezes indicates that glycoPeptide antifreeze structure is highly conserved. Arginine occasionally replaces the glycosidically linked threonine in these sequences, but the basic triPeptide repeat is otherwise maintained.The large glycoPeptides are more efficient inhibitors of ice growth than are the small proline containing glycoPeptides. Although the secondary structures of the glycoPeptides are uncertain, some investigators have proposed some ordered structures and have suggested that the proline residues of the small glycoPeptides may disrupt this structure and reduce their effectiveness. The results presented in this study, however, indicate that the observed differences in effectiveness between different sizes of glycoPeptide antifreezes can be attributed entirely to differences in molecular size rather than differences in conformation.A model of Peptide antifreeze function is based on the structure of the winter flounder Peptide. Polar amino acids are periodically spaced in this Peptide and the spacing of the polar residues in a helical conformation is presumed to allow efficient hydrogen bonding of the antifreeze of the ice surface. Some investigators predicted that all Peptide antifreezes would be similarly arranged. Peptide antifreezes were isolated from the zoarcids Rhigophila dearborni and Lycodes polaris. No periodic placement of polar groups is evident in the amino acid sequences and CD indicates little or no helical structure in these Peptides. The secondary structure of these Peptides remains uncertain, but a large amount of (beta)-structure is predicted from the amino acid sequences. Peptide antifreeze structures clearly show substantial variation.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD
Harlow, Kenneth William - One of the best experts on this subject based on the ideXlab platform.
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Chemical modification and active site studies of Salmonella typhimurium phosphoribosylpyrophosphate synthetase
1Co-Authors: Harlow, Kenneth WilliamAbstract:Comparatively little is known about the amino acid residues present at the active site of Salmonella typhimurium phosphoribosylpyrophosphate synthetase (E.C. 2.7.6.1, PRPP synthetase). Chemical modification studies using group specific and affinity labelling reagents were used to study the active site residues of the enzyme. Peptide mapping of the enzyme was performed to provide a method for rapidly locating and identifying the sites of reaction. CNBr and tryptic maps were prepared using reverse phase HPLC. Peptides from these maps were isolated and sequenced. About 85% of the primary amino acid sequence was covered. Previous work (Roberts et al., J. Biol. Chem, (1975) 250:5364-5369) suggested the presence of an essential cysteinyl residue in the active site. The present results do not support this conclusion. PRPP synthetase contains four cysteinyl residues. A single, reactive cysteinyl residue, Cys$\sb{229}$, was identified using radioactive iodoacetamide. The other 3 cysteinyl residues were equivalent in reactivity, and were labeled with iodoacetamide only under denaturing conditions. With 5,5$\sp\prime$-dithiobis(nitrobenzoic acid) reactivity is similar, except that the 3 less reactive residues react slowly under nondenaturing conditions. The extent of reaction with DTNB is dependent on the concentration of inorganic phosphate. When all the cysteinyl residues have reacted with DTNB the enzyme still possessed about 20% of the control enzymatic activity.The ATP affinity analog 5$\sp\prime$-($p$-fluorosulfonylbenzoyl)adenosine (FSBA) was used to covalently label the enzyme. It reacted with the enzyme in a limited, active site directed manner, with an apparent K$\sb{\rm D}$ of 1.7 mM. The modification was labile at alkaline and acidic pH, and no acid-stable products of reaction of FSBA with amino acid residues in proteins were identified. The site of reaction was localized to a 9 residue Peptide (residues 124-132) with histidine (His$\sb{130}$) as the only potential site of reaction with FSBA. His$\sb{130}$ is invariably conserved in PRPP synthetases from diverse origins. These results are consistent with His$\sb{130}$ being the site of reaction of FSBA with PRPP synthetase, and with His$\sb{130}$ being important to the functioning of the enzyme.U of I OnlyETDs are only available to UIUC Users without author permissio
