Thesis (M.S.)--University of Wisconsin--Madison, 1980. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 64-66).

Antimalarial drug discovery remains a challenging endeavour as malaria parasites continue to develop resistance to drugs, including those which are currently the last line of defence against the disease. Plasmodium falciparum is the most virulent of the malaria parasites and it delivers its deadliest impact during the erythrocytic stages of the parasite’s life cycle; a stage characterised by elevated catabolism of haemoglobin and anabolism of parasite proteins. The present study investigates the use of nanotechnology in the form of metallic silver nanoparticles (AgNPs) against P. falciparum leucine aminopeptidase (PfLAP), a validated biomedical target involved in haemoglobin metabolism. AgNPs were also tested against the human homolog cytosolic Homo sapiens leucine aminopeptidase (HsLAP) to ascertain their selective abilities. PfLAP and HsLAP were successfully expressed in Escherichia coli BL21(DE3) cells. PfLAP showed optimal thermal stability at 25 °C and optimal pH stability at pH 8.0 with a Km of 42.7 mM towards leucine-p-nitroanilide (LpNA) and a Vmax of 59.9 μmol.ml⁻¹.min⁻¹. HsLAP was optimally stable at 37 °C and at pH 7.0 with a Km of 16.7 mM and a Vmax of 17.2 μmol.ml⁻¹.min⁻¹. Both enzymes exhibited optimal activity in the presence of 2 mM Mn²⁺. On interaction with polyvinylpyrrolidone (PVP) stabilised AgNPs, both enzymes were inhibited to differing extents with PfLAP losing three fold of its catalytic efficiency relative to HsLAP. These results show the ability of AgNPs to selectively inhibit PfLAP whilst having much lesser effects on its human homolog. With the use of available targeting techniques, the present study shows the potential use of nanotechnology based approaches as “silver bullets” that can target PfLAP without adversely affecting the host. However further research needs to be conducted to better understand the mechanisms of AgNP action, drug targeting and the health and safety issues associated with nanotechnology use.

Unlike most other host-specific mycoplasmas, Mycoplasma gallinarum exists as a commensal with a host range including most poultry as well as some mammals. This property of M. gallinarum may reflect unique mechanisms for its colonization and persistence in hosts. Whereas M. gallinarum shows leucine and arginine aminopeptidase activity, the genes encoding the enzymes had not been cloned and characterized. We identified an aminopeptidase gene (APN) by oligonucleotide hybridization to a genomic library of M. gallinarum in lambda ZAPII bacteriophage. Nucleotide sequence analysis of overlapping phage clones identified a 1,362 bp open reading frame (ORF) encoding a putative leucine aminopeptidase gene. Database searches indicate that this ORF has 68% nucleotide identity and 51% amino acid identity with the M. salivarium leucine aminopeptidase gene. The active sites of the leucine aminopeptidases in other eukaryotes and prokaryotes were conserved in the cloned aminopeptidase gene. Northern-blot hybridization analysis showed that this ORF is expressed as a 1.5 kb transcript. Southern-blot hybridization analysis demonstrated this gene was present as a single copy in M. gallinarum. Characterization of the leucine aminopeptidase demonstrated that it is a metallo-aminopeptidase (EC 3.4.11.1) and is located in the cytoplasm with a weak interaction with the cell membrane. The subcellular location was further confirmed by immunoblotting with polyclonal anti-recombinant APN serum and M. gallinarum Triton-114 partitions. Immunoblotting results with sera from three chickens experimentally infected with M. gallinarum showed that there were very few proteins in M. gallinarum exposed to the host immune responses and that leucine aminopeptidase was not able to stimulate production of specific humoral antibody. Our results suggest that this leucine aminopeptidase play a role in nutrition supply for the host adaptation of M. gallinarum and that the enzyme was not strongly immunogenic.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(論文) 学位授与年月日:平成4年7月20日 淺田義正氏の博士論文として提出された

The respective kinetics of bacterial leucine aminopeptidase and [beta]-glucosidase activities were investigated to improve understanding of factors controlling activity and hydrolytic capacity in estuarine organic-poor sands. Depth distributions of enzyme activity and bulk organic matter content were determined in sediments of Aransas Bay and Copano Bay Texas, to investigate enzyme dynamics as related to the geochemical properties of the sediment. Vertical profiles of activity in sediment showed that the enzymes were more active at the surface and that the potential hydrolysis rate of leucine aminopeptidase was higher than that of [beta]-glucosidase. Vertical patterns of enzyme activity correlated (weakly) with variations in sediment organic matter (TOC, TN, and carbohydrates) content. Enrichments of sediment samples with monomeric organic compounds and inorganic nutrients did not affect leucine aminopeptidase and [beta]-glucosidase activities in short- and long-term incubations. Enzyme activity was independent of nutrient availability and suggested that microbial communities were not nutrient-limited. Time-course assays of bacterial hydrolysis of TOC, TN, and carbohydrates provided information about how substrate limitation may affect enzyme activity. Positive correlations between bulk TOC and TN content and enzyme activity indicated enzyme dependence on polymeric substrate content. Induction of enzyme activity after sediment enrichments with specific labile compounds confirmed the importance of available organic substrate to enzyme hydrolysis efficiency. A kinetic approach established the occurrence of enzyme inhibition and its effects on enzyme hydrolytic capacity. The addition of a specific-enzyme substrate to sediment samples modified enzyme parameters and indicated that a substrate-reversible type of inhibitor could reduce enzyme hydrolytic capacity. The addition of polyphenol, as a natural inhibitor of enzyme activity, to the sediment resulted in a concomitant reduction of leucine aminopeptidase activity and ammonium regeneration rate, and thus demonstrated a close coupling between enzyme activity and sediment ammonium regeneration. These research results demonstrate the dynamic nature of the hydrolytic enzymes, provide information about the mechanisms of induction and inhibition of activity, and demonstrate some implications of reducing the hydrolytic capacity to organic matter decomposition and nutrient regeneration rates. / text