This thesis describes the development of a way to study the N8 - acetylspermidine deacetylase enzyme activity. The method created in this thesis emphasizes sensitivity, accuracy and safety. In this study, HeLa cells were cultured and extracted to yield a crude N8 - acetylspermidine deacetylase enzyme mixture. By measuring the decrease of N8 - acetylspermidine and the increase of spetmidine, N8 -acetylspermidine deacetylase enzyme activity can be determined using either a Varian 1200L LC/MS/MS or an API 3000 LC-ES (+)/MS/MS. An acetylation-derivatization method was developed because N8 -acetylspermidine and spermidine are hard to purify from a biological sample since they are not retained on a CIS solid phase extraction column or on a RP HPLC (high performance liquid chromatography) reverse phase column due to their small molecular weight and high polarity. The quantitation of N8 -acetylspem1indine over the range 2ng/ul to 5pg/ul was fit by linear regression as y = 1.064x + 0.218 with an R-squared value of 0.9996, where y is the peak area of the fragment-ion SRM (selected reaction monitoring: m/z: 188/114) chromatograms from N8 -acetylspermindine and x is the concentration of N8 - acetylspermindine. Acetylation of spermidine (SPD) and N8-acetylspermidine (N8AcSPD) with d6-acetic anhydride produces the d9 labeled triacetylated derivative of SPD and d6 labled triacetylated spermidine derivative of N8AcSPD. These triacetylated forms are retained on a C18 column. MS/MS gives characteristic m/z fragment ions for the derivatized species: N8AcSPD (278 to 215), NlAcSPD (278 to 218) and SPD (281 to 218). The fragment-ion SRM (selected reaction monitoring) chromatograms are used for the quantitation. A plot of peak area ratios for known mixtures of N8AcSPD and total SPD versus the molar ratios of N8AcSPD and total SPD was found to fit a linear regression line withy= 0.705x + 0.035 with an R-squared value of 0.9919. Quantitation of d6- and dg-tri-acetylspermidine by LC/MS/MS is possible at the low levels of materials found in cell extracts since the separation method results in a lower limit of quantitation. This approach enables the study of N8 - acetylspermidine deacetylase enzyme activity.

Submitted in fulfillment for the requirements for the degree of Doctor of Technology: Biotechnology, Durban University of Technology, Durban, South Africa, 2015. / Main focus of this study is to investigate the enzymatic-conversion of microalgal lipids to biodiesel. However, preceding steps before conversion such as drying of microalgal biomass and extraction of lipids were also studied. Downstream processing of microalgae has several challenges and there is very little literature available in this area. S. obliquus was grown in the pilot scale open pond cultivation system for biomass production. Different techniques were studied for biomass drying and extraction of lipids from harvested microalgal biomass. Effect of these drying and extraction techniques on lipid yield and quality was assessed. Energy consumption and economic evaluation was also studied. Enzymatic conversion of microalgal lipids by extracellular and whole cell lipase application was investigated. For both applications, free and immobilized lipases from different sources were screened and selected based on biodiesel conversion. Process parameters were optimized using chosen extracellular and whole cell lipases; also step-wise methanol addition was studied to improve the biodiesel conversion. Immobilized lipase was studied for its reuse. Final biodiesel was characterized for its fuel properties and compared with the specifications given by international standards. Enzymatic conversion of microalgal lipids was compared with the conventional homogeneous acid-catalyzed conversion. Enzymatic conversion and chemical conversion were techno-economically investigated based on process cost, energy consumption and processing steps. Freeze drying was the most efficient technique, however at large scale economical sun drying could also be selected as possible drying step. Microwave assisted lipid extraction performed better compared to sonication technique. Immobilized P. fluorescens lipase in extracellular application and A. niger lipase in whole cell application showed superior biodiesel conversion. The extracellular immobilized P. fluorescens lipase showed better biodiesel conversion and yields than the immobilized A. niger whole cell lipase. Both the enzyme catalysts showed lower biodiesel conversion compared to conventional chemical catalyst and higher processing cost. However, techno-economic analysis showed that, the reuse potential of immobilized lipases can significantly improve the economics. Fewer purification steps, less wastewater generation and minimal energy input are the benefits of enzymatic route of biodiesel conversion. Microalgae as a feedstock and lipase as a catalyst for conversion makes overall biodiesel production process environmentally-friendly. Data from this study has academic as well as industrial significance. Conclusions from this study form the basis for greener and sustainable scaling-up of microalgal biodiesel production process. / D

Thesis (M. App. Sc.)--Carleton University, 2004. / Includes bibliographical references (p. 130-133). Also available in electronic format on the Internet.

No description available.

A developmentally regulated cyclic AMP-dependent protein kinase has been recently reported in Dictyostelium discoideum. This report describes some of the physical and kinetic properties of the cAMP-dependent holoenzyme and its subunits. Gel filtration data suggests a holoenzyme Mr of 170,000-190,000, and catalytic and regulatory subunit Mrs of 40,000 and 49,000, respectively. These molecular weight determinations are compatible with an R₂C₂ subunit arrangement of the holoenzyme. Kinase activity required the presence of Mg²⁺ but cAMP binding to the enzyme was not dependent on divalent metal ions. The pH optimum for kinase activity was 7.5; the cAMP binding activity was not affected over a pH range of 5.0-10.0. The holoenzyme and isolated regulatory subunit had identical cAMP Kds of 28 nM. Cyclic AMP was able to dissociate the subunits when analyzed by density gradient centrifugation. Histone VII-S activated the subunits in the absence of cAMP but did not produce their dissociation. In contrast to the gel filtration data, sedimentation values indicated a dimeric holoenzyme structure. Reassociation of the subunits in the absence of cAMP occurred rapidly and was not dependent upon a preincubation with MgATP. High NaCl and low pH depressed both the total kinase activity and the ability of the subunits to reassociate as determined by activity ratio. MgATP did not decrease the ability of the holoenzyme to bind cAMP, neither did the holoenzyme possess a high affinity MgATP binding site. By the use of microdissection techniques holoenzyme levels were determined in individuals at each stage of development and in each cell type during development. Kinase activity was low and non-cAMP dependent in early aggregates but increased and became cAMP-dependent in later aggregates. Maximum activity and cAMP-dependency occurred during the slug and culmination stages. The only differential distribution of the kinase within a single-stage occurred during culmination when the activity in the stalks was approximately one-fourth that in the prespore mass. Preliminary evidence indicates that this difference is not due to an inhibitor. / Ph. D. / incomplete_metadata

Indiana University-Purdue University Indianapolis (IUPUI) / Eukaryotic cells rapidly modulate protein synthesis in response to environmental cues through the reversible phosphorylation of eukaryotic initiation factor 2 (eIF2α~P) by a family of eIF2α kinases. The eIF2 delivers initiator Met-tRNAiMet to the translational apparatus, and eIF2α~P transforms its function from a translation initiation factor into a competitive inhibitor of the guanine nucleotide exchange factor (GEF) eIF2B, which is responsible for the recycling of eIF2-GDP to the translationally-competent eIF2-GTP state. Reduced eIF2-GTP levels lower general protein synthesis, which allows for the conservation of energy and nutrients, and a restructuring of gene expression. Coincident with global translational control, eIF2α~P directs the preferential translation of mRNA encoding ATF4, a transcriptional activator of genes important for stress remediation. The term Integrated Stress Response (ISR) describes this pathway in which multiple stresses converge to phosphorylate eIF2α and enhance synthesis of ATF4 and its downstream effectors. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary across a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2α~P, while a notable cohort of key regulators are subject to preferential translation. From this latter group, we identify IBTKα as being subject to both translational and transcriptional induction during eIF2α~P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKα mRNA involves the stress-induced relief of two inhibitory uORFs in the 5’-leader of the transcript. Also identified as being subject to preferential translation is mRNA encoding the bifunctional aminoacyl tRNA synthetase EPRS. During eIF2α~P, translational regulation of EPRS is suggested to occur through the bypass of a non-canonical upstream ORF encoded by a CUG start codon, highlighting the diversity by which upstream translation initiation events can regulate expression of a downstream coding sequence. This body of work provides for a better understanding of how translational control during stress is modulated genome-wide and for the processes by which this mode of gene regulation in the ISR contributes to cell fate.