Methyldopa (L-alpha-Methyl-3,4-dihydroxyphenylalanine) is a catecholamine used as an antihypertensive agent.1 Alpha-Methyldopa is not used as frequently anymore due to side effects, but it is still used especially in developing countries due to its low cost. Indications are mostly for the management of pregnancyinduced hypertension (PIH), as it is relatively safe in pregnancy compared to other antihypertensive drugs. This project is intended to increase the already-existing knowledge base of the mechanism of pharmacological action and to stimulate further investigation through research. The sympathetic nervous system is a division of the autonomic nervous system and it is responsible for the “flight-or-fight” response. It is involuntary and constantly active to maintain homeostasis in the human body. Sympathetic responses include an increase in heart rate, blood pressure and cardiac output, dilation of pupils and bronchioles, constriction of blood vessels, contraction of sphincters and inhibition of gut motility and secretions. The purpose of this study is to evaluate the activity of the sympathetic nervous system of volunteers by three different techniques (QT interval and Heart rate variability and Skin conductance) after a week of a bi-daily dosage of alphamethyldopa. All volunteers received either 250mg alpha-methyldopa orally or a placebo tablet in a randomized, double blind, placebo controlled study design. The correlation between the following techniques was also evaluated: Skin conductance as measured by the ProComp Infiniti Biofeedback apparatus, QT interval on ECG and HRV measured with Viport apparatus. A salivary sample was collected to evaluate the effect of alpha-methyldopa on salivary cortisol using an ELISA kit for analysis. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Pharmacology / unrestricted

The metabolism of tyrosine is essential for many critical biochemical events including catecholamine synthesis, melanogenesis and insect cuticle sclerotization. These pathways are highly regulated in both insects and mammals by many well-characterized enzymes including dopa decarboxylase and tyrosine hydroxylase. On the other hand, there are still many enzymes involved in these processes that we know very little about. Dopachrome tautomerase (DCT), dopachrome conversion enzyme (DCE) and α-methyldopa resistant protein (AMD) fall into the category of the less characterized enzymes. Dopachrome is a pivotal intermediate in melanogenesis. Mammalian DCT and insect DCE both use dopachrome as a substrate. DCE catalyzes a decarboxylative structural rearrangement of dopachrome to 5,6-dihydroxyindole (DHI), whereas DCT mediates the isomerization/tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). DHI is oxidized easily, leading to the production of melanin, as well as reactive oxygen species (ROS). DHICA is less reactive, relative to DHI, and consequently produces less toxic byproducts during melanogenesis; therefore DCT plays an important role in detoxification of DHI and ROS. Purification and MS analysis of DCE and DCT determined that N-glycosylation is a primary post-translational modification. Q-TOF mass spectrometry was used to determine N-glycosylation patterns from Aedes aegypti DCE and MALDI-TOF/TOF was used to determine multiple glycosylation sites in DCT. N-glycosylation is critical for the folding and trafficking of secreted proteins in the endomembrane system. The analysis of glycosylation sites in DCE and DCT therefore is essential toward achieving a comprehensive understanding of their structure and function. Like DCT, AMD also plays a protective role. The AMD protein was originally identified in Drosophila mutants hypersensitive to α-methyldopa, an inhibitor of dopa decarboxylase (DDC). Production of dopamine by DDC is critical for developing insects because dopamine conjugates are used as crosslinking agents for cuticle sclerotization. Although there has been much discussion into the function of AMD, what exactly this protein does has been unknown. AMD shares 48% sequence identity with DDC, however we have found that AMD is an enzyme, which possesses a different catalytic activity. GC-MS analysis of AMD enzymatic reaction components revealed that AMD catalyzes the oxidative decarboxylation of L-DOPA to DOPAL, and also the oxidative decarboxlation of α-methyldopa to 3,4-dihydroxyphenylacetone. In summary, multiple N-glycosylation sites were characterized in DCT and DCE, furthermore a new protein function has been demonstrated for AMD. These experiments were performed using classical biochemistry techniques in combination with mass spectrometry. / Ph. D.