Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 114-124).

Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 121-133).

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Nov 30

Proefschrift Rijksuniversiteit Groningen. / Met lit. opg. - Met samenvattingen in het Nederlands en Engels.

The enzymes phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) were studied in the yeast Sporobolomyces roseus (Kluyver and van Neil). Cells grown on a glucose-salts medium were ground with alumina, and the cell-free buffer extract was fractionated with ammonium sulfate. Enzyme activity was assayed by measuring spectrophotometrically the cinnamic acid and p-coumaric acid produced from phenylalanine and tyrosine respectively. Further attempts at purification resulted in the inactivation of the TAL. Although the two enzymes were not separated by the purification procedures used, there is some evidence that the deamination of phenylalanine and tyrosine are catalyzed by different proteins, and not by a single enzyme with wide specificity. TAL appears to be precipitated by lower concentrations of ammonium sulfate than is PAL. The pH curves of the two enzymes are different. The specific activities of the two enzymes can be changed relative to one another in the cell by changing the medium upon which the cells were grown. The rates of production of the two enzymes vary independently during the growth of the cells. While the proteins are probably distinct, the production and activity of each enzyme seem to be under common control. Peak production of both enzymes occurs during late logarithmic-early stationary phase in the growth of a batch culture. Replacement media containing either phenylalanine or tyrosine stimulate the production of both PAL and TAL. Similarly, media containing cinnamic acid or p-coumaric acid repress the formation of the enzymes. Studies using labelled substrate show that both products inhibit the action of both enzymes. / Science, Faculty of / Botany, Department of / Graduate

Interest in brain catecholamines has grown considerably in the last few years in view of their possible role as neurotransmitters. This investigation deals primarily with the enzyme tyrosine hydroxylase which is thought to be the rate limiting step in catecholamine synthesis. Using tyrosine hydroxylase measurements and catecholamine depletion techniques,attempts were made to determine the site of increased synthesis of catecholamines in animals exposed to cold. Brain, heart and spleen do not appear to be the organs involved in this change. Adrenals may be of significance but the results were suggestive rather than conclusive. Tyrosine hydroxylase distribution in brain was determined in various regions of rat, rabbit and cat brain, and activity was shown to be highest in the caudate, septal area, nucleus accombens, and anterior perforating substance, with much lower activities in other regions such as hippocampus, amygdala, hypothalamus, thalamus, cortex, cerebellum and brain stem. Using these distribution studies as indications of normal tyrosine hydroxylase activity in areas of rat brain, and electrolytic lesion techniques, studies were carried out to determine noradrenergic and dopanergic pathways in brain. Catecholamine fibers from their origin in the midbrain were traced in the midbrain and diencephalon to the caudate and septal area, and the relative positions of each group of fibers determined along their course. / Medicine, Faculty of / Graduate

No description available.

No description available.

Protein phosphorylation is utilized universally as a mechanism of signal transduction. However, the use of tyrosine phosphorylation by bacteria has been a matter of dispute. Conventional wisdom dictated that "prokaryotic phosphorylation" was typified by phosphorylation of histidine and aspartate residues of proteins, while "eukaryotic phosphorylation" was characterized by modification of serine, threonine, or tyrosine residues. Increasing numbers of reports have emerged challenging the traditional view of "prokaryotic" and "eukaryotic" phosphorlyation. One of the strongest links unifying prokaryotic and eukaryotic protein phosphorylation to date is IphP, a genomically-encoded dual-specificity protein phosphatase from the cyanobacterium Nostoc commune UTEX 584 bearing the active-site signature sequence of eukaryotic tyrosine-specific and dual-specificity protein phosphatases. The catalytic properties and substrate specificity of IphP were examined in detail. The enzyme was able to discriminate among a variety of exogenous peptides and proteins. Kinetic studies revealed that IphP favors protein / peptide substrates over low molecular weight compounds. Heparin effected IphP activity in a substrate-dependent manner. Enzyme activity toward casein (P-Ser) and MAP kinase (P-Thr/P-Tyr) was stimulated in the presence of the polyanion, whereas activity was inhibited by heparin toward other protein substrates. Both stimulation and inhibition by heparin were dose-dependent. The ability to stimulate IphP activity toward select substrates was attributed to the ability of heparin to recruit the enzyme and substrate to the same microenvironment. To facilitate future genetic studies examining the role of tyrosine phosphorylation in cyanobacteria, we searched for evidence of protein tyrosine phosphorylation in Anabaena PCC 7120. In a collaborative effort with the laboratory of Dr. Potts, tyrosine phosphorylated proteins were identified in Anabaena utilizing several approaches, including comparative labelling with alpha- vs gamma-32P-ATP, phosphoamino acid analysis, and selective hydrolysis with a tyrosine specific protein phosphatase. Together, these data unequivocally demonstrate the presence of tyrosine-phosphorylated proteins in Anabaena PCC 7120. Extracts of Anabaena PCC 7120 were examined for protein tyrosine phosphatase activity. An apparent PTP activity was detected, partially purified, and characterized. The protein phosphatase was ~38kDa by SDS-PAGE and sucrose density gradient centrifugation and displayed dual-specificity protein phosphatase (DSP) activity in vitro. The enzyme was localized to the periplasm and was thus assigned the title PAD, for Periplasmic Anabaena DSP. Periplasmic phosphoproteins of ~120 and 55 kDa that had been radiolabelled in vitro were dephosphorylated by partially purified PAD. PAD activity varied in vivo ~5-fold in a rhthymic, seemingly diurnal manner. Periplasmic proteins, including the 55kDa protein, were labelled in vivo and the degree of radiolabel incorporated into these proteins varied inversely with PAD activity. / Ph. D.

Tyrosine is involved in many biological processes including protein synthesis. This dissertation is focused on two different aspects: tyrosine catabolism and tyrosine derivative metabolism. Tyrosine undergoes degradation via tyrosine aminotransferase (TAT). Deficiency of TAT leads to some disease conditions or tyrosinemia type II. TAT has been characterized in several species, including humans. Mouse tyrosine aminotransferase was used as a model protein for the tyrosine catabolism portion of this study. Characterization of TAT included its expression in a bacterial expression system, purification using various chromatographic techniques, crystallization under different conditions, and its kinetic analysis, and molecular dynamics simulations. Based on sequence, structure, and kinetic data we have shown that mouse TAT behaves like human TAT. Our crystallization studies added new insights into the mechanism of TAT by shedding light on involvement of a disulfide bond in the regulation of mTAT. Molecular dynamics analysis provided perspective on the differences (preferences) in the substrate specificities of mouse and Trypanosome cruzi TAT. Tyrosine is a precursor of several key neurotransmitters. These neurotransmitters must be regulated in order to function properly. The hypothetical N-acetyltransferases from Aedes aegypti were used as model proteins for investigation of tyrosine derivative metabolism. We found nine potential arylalkylamine N-acetyltransferase (AANAT) genes in Ae. aegypti. Phylogenetic analysis suggests that these Ae. aegypti AANATs (AeAANATs) can be further divided into three clusters. Phylogenetic analysis suggests that insect AANATs may have different functions as compared with the mammalian AANATs, for which function is specific to circadian rhythm regulation. PCR amplification indicates that eight of the nine putative AeAANATs are expressed in the mosquito. Expression of the eight putative AeAANATs and substrate screening of their recombinant proteins against dopamine, octopamine, tyramine, epinephrine, tryptamine, 5-hydroxytryptamine, and methoxytryptamine established that five of the eight putative AeAANATs are true AANATs. The discontinuous expression profiles of AeAANAT genes were studied in detail. Six of the AeAANATs were expressed in the head before and after blood feeding, suggesting their potential role in neurotransmission inactivation. Down-regulation of these genes after blood feeding suggests that blood feeding or factors related to blood feeding impact on the regulation of these genes. Kinetic studies determined that two AeAANAT proteins are highly efficient in mediating the acetylation of dopamine and 5-hydroxytryptamine. Substrate analysis of AeAANATs supports the notion that acetylation of arylalkylamines is vital to the biology of mosquito species, and that these genes emerged in response to specific pressures related to necessities for biogenic amine acetylation. / Ph. D.