The naturally occurring polyamines play an essential role in cell growth and proliferation. The enzyme spermidine N8- acetyltransferase catalyzes the acetylation of spermidine utilizing acetyl-CoA as the acetyl donor. In this study, an in vitro acetyltransferase assay was used to determine the types of compounds which can inhibit this reaction. The enzyme was partially purified from rat liver nuclei and solubilized in 0.4 M KCl. The Km for spermidine was 0.47 mM. Studies on the nature of the active site indicated that: (i) a sulfhydryl group is essential for optimal activity as shown by inhibition with parahydroxymercuribenzoate and N -ethylmaleimide, (ii) a metal ion does not appear to be necessary for catalytic activity of this enzyme since EDTA, 2,2-dipryridil, and 1,10 phenanthroline were poor inhibitors of this enzyme, (iii) a lysine or another primary amine is likely to play a crucial role in this reaction since succinate anhydride and 2,4,6-trinitrobenzene sulfonic acid were effective inhibitors of this reaction and (iv) tyrosine is not likely present at the catalytic site since N -acetylimidazole produced no inhibition.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 104-115).

Thesis (M.A.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on September 29, 2008) Includes bibliographical references.

Thesis (Ph.D.) - University of St Andrews, March 2008.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 112 pages. Includes Vita. Includes bibliographical references.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 82-89.

As an important posttranslational modification, protein acetylation plays critical roles in many biological processes such as gene transcription, DNA damage repair, apoptosis and metabolism. The acetylation occurs on the ε-amino group of specific lysine residues, and is catalyzed by histone acetyltransferases (HATs). In cellular contexts, HATs are found to target hundreds and thousands of substrates including histone and nonhistone proteins. Lysine acetylation changes the microenvironment of protein and may potentially alter protein activity and protein-protein interaction. The goal of this dissertation project is to investigate the impact of lysine acetylation on the catalysis of MYST HATs, and to establish the strategy for labeling substrates of the MYST HATs at cellular level. To understand the regulatory mechanism of MYST HATs, a detailed study was carried out to investigate the active site lysine acetylation of two MYST HATs (MOF and Tip60). Autoradiography and immunoblotting data shows that mutation of active site lysine differentially affects the enzyme autoacetylation activity and the cognate substrate acetylation activity. In addition, deacetylated MOF and Tip60 were prepared by using the nonspecific lysine deacetylase Sirt1. Kinetic study demonstrated that the acetylation of the active site lysine on MYST HATs marginally modulates the HAT catalysis. This work provides new insights into the regulatory mechanism of MYST catalysis. In the second part of my work, we designed and synthesized a series of Ac-CoA analogs conjugated with alkynyl or azido functional groups. Meanwhile, the active site of the MOF was engineered to expand the cofactor binding capability. Fluorescence screening was carried out to characterize the enzyme activity to Ac-CoA analogs. MOF-I317A with all analogs and MOF-I317A/H273A–5HYCoA were identified and further applied in the labeling of the cognate histone H4 protein and HAT substrates in 293T cell lysate. Visualizing of the labeled substrate was achieved using the alkynyl or azido-tagged fluorescent reporters through the copper-catalyzed azide−alkyne cycloaddition. As expected, the histone H4 protein was successfully labeled by the active enzyme-cofactor pairs. More intriguingly, multiple protein bands in cell lysate were labeled and observed. This work provides a new versatile strategy in exploring the substrates of MYST HATs at the proteomic level.

Melatonin is a hormone secreted by the pineal gland, which is known to modulate biological rhythms in mammals. This study investigated the effect of food access schedule and dietary composition on serum melatonin and pineal NAT activity in adult male Wistar rats. These rats were maintained on a 12:12 h light:dark schedule with lights on at 0800h. The rats were randomly assigned to two dietary groups. A group was simultaneously fed a protein-rich and carbohydrate-rich granulated diet and the other group fed granulated rat chow. Each dietary group was further divided based on dietary feeding schedules. Animals were fed between 0800--1600 h or fed ad libitum. The study revealed that protein intake of rats fed the dietary choice was lower with the restricted access than in the free access. In rats fed dietary choice, the nocturnal melatonin levels and pineal NAT activity were significantly lower under the restricted access feeding when compared to the ad libitum feeding schedule. This was not observed in rats fed single chow diet. In conclusion our data demonstrate that food composition does affect the nocturnal synthesis of melatonin as well as the activity of the enzyme NAT. This could be via dietary intake of tryptophan, which is a precursor melatonin synthesis in the pineal gland.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.