The active site of porcine microsomal aminopeptidase was probed by studying the inhibition of the enzyme using derivatives of ethylenediamine and diaminopropionic acid. In addition, some amino acids, substituted hydroxamates and phosphates were also tested. In order to synthesize diaminopropionic acid derivatives, CBZ-amino acid p-nitrophenyl esters were reduced to the corresponding aldehydes by lithium tri-t-butoxy-aluminohydride. Through the Strecker synthesis, the aldehyde intermediates were converted to diaminopropionitrile analogues which were then hydrolysed in acid to the desired products. Unfortunately, these compounds were not potent inhibitors for this enzyme. α-Amino acids were found to be better inhibitors than their β-amino counterparts and the Kᵢ of α-leucine was about 7-fold lower than its B-analogue. The amino group position of the amino acids is therefore important for enzyme recognition. On the other hand, N-alkylation of ethylenediamine was observed to abolish its inhibition potential. Furthermore, another unexpected finding in this work is that N- or 0-methylation of the hydroxamate group hinders the ability of these inhibitors to act as a bidentate zinc ligand. Although some phosphate derivatives that we tested showed poor inhibitory potency, phosphonamidate, a potential transition state analogue, might serve as a powerful inhibitor. In summary, the relationship between the structure and inhibitory potency of some inhibitors was demonstrated. / Thesis / Master of Science (MSc)

Recent research suggests that amino acids can significantly increase skeletal muscle glucose uptake. However, the mechanism(s) have not been fully elucidated and it is also not clear if the beneficial impact amino acids have on healthy tissue translates to insulin resistant skeletal muscle. Therefore, in this series of studies, the effects of an amino acid mixture on glucose tolerance and insulin resistance were investigated. Study 1 Experiment-1 (Exp-1) demonstrated that an amino acid mixture significantly reduced the blood glucose response to an oral glucose challenge in Sprague Dawley rats. In Study 1 Exp-2, it was found that the improved glucose tolerance was due to an increase in skeletal muscle glucose uptake. The enhanced amino acid induced muscle glucose uptake was associated with improved cellular signaling. In Study 1 we could not determine the combined and/or individual effects of insulin and amino acids on glucose uptake, so in Study 2, the hindlimb of Sprague Dawley rats were perfused with glucose with or without amino acids in the presence and absence of insulin. Study 2, confirmed our previous findings that an amino acid mixture increased skeletal muscle glucose uptake compared to a carbohydrate supplement in the presence of insulin. The enhanced amino acid-stimulated glucose uptake was not due to increased phosphatidylinositol 3-kinase (PI 3-kinase) activity, although it was related to an increase in Akt substrate of 160 kDa (AS160) phosphorylation and a greater number of glucose transporters at the plasma membrane. In the final experiment, Study 3 investigated whether amino acids could improve glucose tolerance in an insulin resistant model. Study 3 Exp-1, demonstrated that an amino acid mixture significantly lowered the blood glucose response to an oral glucose challenge in obese Zucker rats. Study 3 Exp-2 showed that the improved glucose tolerance was due to enhanced amino acid induced skeletal muscle glucose uptake. Taken together, the results of this research suggests that adding an amino acid mixture to a carbohydrate supplement improves the blood glucose response to an oral glucose challenge, acutely lowers insulin resistance and this appears due to increased skeletal muscle glucose clearance and enhanced cellular signaling. / text

Master of Science / Department of Animal Sciences and Industry / Evan C. Titgemeyer / Two experiments were conducted to determine effects of postruminal amino acid (AA) supplementation on protein deposition and signaling of the mammalian target of rapamycin (mTOR) pathway. For both experiments, 7 ruminally cannulated Holstein steers (172.7 ± 3.7 and 201.7 ± 3.8 kg initial BW in Exp. 1 and 2, respectively) were utilized in 6  6 Latin square designs with 7 d periods. A basal AA solution containing all essential AA, with the exception of lysine, were provided to all steers in each study in order to meet growth requirements, while making lysine the only limiting AA. Steers were fed 2.8 kg/d of a pelleted soyhull diet designed to be low in ruminally undegradable protein. Glucose was infused abomasally and volatile fatty acids were infused ruminally to prevent energy from being limiting. Steers were housed in metabolism crates to obtain total collection of both urine and feces. Blood and muscle biopsies of the longissimus lumborum were collected on the last day of each period. In experiment 1, treatments consisted of 2 levels of lysine (0 or 6 g/d) and 3 levels of leucine (0, 15, or 30 g/d) infused abomasally. Nitrogen retention increased with supplemental lysine. Leucine linearly decreased plasma concentrations of total AA. Plasma urea N (PUN) decreased with supplemental lysine. Total, phosphorylated, and the percent phosphorylated Akt were unaffected by treatments. The percentage of 4E-BP1 phosphorylated decreased linearly when leucine was supplemented. A tendency for a lysine x quadratic leucine effect was observed for the ratio of phosphorylated RPS6²⁴⁰/²⁴⁴ in which the intermediate level of leucine led to a decrease in the percent of RPS6²⁴⁰/²⁴⁴ phosphorylated when no lysine was supplied but increased when 6 g lysine/d was supplied. No differences were observed in the abundance of total, phosphorylated, or percent phosphorylated mTOR or in total abundance of E3 ubiquitin ligase proteins, MuRF1 or MAFbx. Experiment 2 was conducted similarly as experiment 1. Treatments consisted of 2 levels of lysine (0 or 6 g/d) and 3 mixtures of supplemental essential AA [none (control), 103 g/d essential AA (EAA), or EAA plus 30 g/d leucine (EL)] abomasally infused. Supplementation with essential AA, with or without leucine, increased the percentage of RPS6 phosphorylated, with a greater increase when leucine was included as part of the supplement. A lysine x (control vs. EAA+EL) interaction was observed for N retention in which the EAA and EL treatments did not improve N retention when no lysine was supplemented, but they increased it when 6 g lysine/d was provided. PUN increased above control when EAA or EL was provided, but PUN decreased when lysine was supplied. Supplementation of EAA or EL increased plasma total AA concentrations, but EL led to lower total plasma AA than EAA; however, concentrations were greater for EL than for control. In summary, leucine supplementation alone did not yield effects on whole-body protein deposition or on regulatory factors known to affect muscle protein synthesis, whereas a mixture of excess essential AA improved both lysine utilization and phosphorylation of RPS6²⁴⁰/²⁴⁴. These studies demonstrate the effects of essential AA, both limiting and nonlimiting, on protein deposition in growing cattle.

No description available.

The hsFLJ22386 gene, an unknown gene, was located on chromosome 16p13.3. Its protein product contains 287 amino acids with the molecular weight of 32.2 kDa approximately. Predicted by bioinformatics, hsFLJ22386 might be a protein containing a leucine zipper domain. Based on the results of reverse transcriptase polymerase chain reaction, it revealed that FLJ22386 was expressed in several nervous system tissues and several organ tissues (liver, spleen, small intestine and kidney). In human cancer cell lines, the RT-PCR results showed that FLJ22386 was expressed in brain tumor cell lines (T98G, U87MG, U251, GBM8401), nasopharyngeal epithelial cell line (NNE-3)and carcinoma cell lines (NPCTW01, NPCTW04), hepatoma cell lines (J5, Hep3B, SK-Hep-1) and lymphoma cell lines (RPMI, P3HR1, Raji, U937). Human FLJ22386 coding sequence was inserted into pEGFP-C2 plasmid, and the tag-fused gene was transfected into NIH3T3 cells to see if it has the ability to promote cell proliferation. To further investigate the protein level expression and biological functions of the gene, glutathione S-transferase-hsFLJ22386 fusion protein was expressed and used to generate anti-FLJ22386 polyclonal antibody. According to the results of RT-PCR and anchorage dependent growth assay, it is presumed that FLJ22386 may play a role in cell proliferation.

α-Isopropylmalate synthase (α-IPMS) is responsible for catalysing the first committed step in leucine biosynthesis. This pathway is found in plants and microorganisms, including pathogenic bacteria such as Mycobacterium tuberculosis and Neisseria meningitidis. α-IPMS catalyses a Claisen condensation reaction between α-ketoisovalerate (KIV) and acetyl coenzyme A (AcCoA) to form the product α-isopropylmalate (IPM). This enzyme undergoes feedback inhibition by the end product of the pathway, leucine. This regulation allows the control of the rate leucine biosynthesis. This project focuses on the α-IPMS enzymes from M. tuberculosis and N. meningitidis (MtuIPMS and NmeIPMS). These α-IPMS enzymes are homodimeric in structure. Each monomer consists of a catalytic domain which comprises of a (β/α)8 barrel fold, two subdomains and a regulatory domain, to which the allosteric binding of the natural inhibitor leucine occurs. The mechanism by which the allosteric binding of leucine leads to a decrease in enzymatic activity is not yet fully understood. Citramalate synthase (CMS) is responsible for catalysing the first committed step of threonine-independent isoleucine biosynthesis. This enzyme is extremely similar to α-IPMS in both the reaction which it catalyses and the catalytic and regulatory domain structure. CMS catalyses a Claisen condensation reaction between pyruvate and AcCoA to produce citramalate (CM). CMS is also feedback inhibited by the end product of its pathway, isoleucine. The similarity between α-IPMS and CMS enzymes resulted in and examination of the inhibitor selectivity of MtuIPMS. Amino acids in the leucine binding site were altered to their counterparts in the isoleucine binding site of the CMS enzyme to see if the selectivity of the leucine binding site could be interchanged. Results from this study show that it is possible to change inhibitor selectivity with a single amino acid substitution. However, changing the selectivity from leucine to isoleucine was unsuccessful. Instead, one of the MtuIPMS variants displayed significantly increased sensitivity to an alternative amino acid, norvaline. The MtuIPMS variants were expressed and purified using immobilised metal affinity chromatography and size-exclusion chromatography. These variants were then kinetically characterised and displayed similar binding affinities and turnover rates for the natural substrates to the wild-type enzyme. As expected changes to the leucine binding pocket had drastic effects on the sensitivity of the enzyme to its natural inhibitor. This work is described in Chapter 2 of this thesis. The mechanism by which the regulatory signal is transferred from the allosteric leucine binding site to the catalytic site in α-IPMS is not fully understood. NmeIPMS variants were created based on preliminary molecular dynamic simulations which indicated that significant changes in residue contacts were associated with leucine binding. Chapter 3 describes studies that explore the effect of single amino acid substitutions of NmeIPMS. The NmeIPMS variants were expressed and purified similarly to MtuIPMS, using immobilised metal affinity chromatography and size-exclusion chromatography. Variants were subsequently characterised via mass spectrometry, differential scanning fluorimetry and kinetic assays. It was found that each variant generated retained sensitivity to leucine but displayed significant differences in the catalytic efficiencies with AcCoA. One of the generated variants also displayed a significant increase in thermal stability. Results are drawn together in Chapter 4 along with future directions of this research. This chapter details knowledge gained into protein structure and allosteric mechanisms in this thesis.

The aim of this study was to develop an <I>in vitro</I> mammary model, based on rat mammary explants, which could be used to examine the effects of amino acid profile and concentration in the media on protein synthesis. Secondly, to ascertain whether these responses to amino acids, in particular leucine and α ketoisocaproic acid, were transmitted through the mTOR/p70 S6 kinase signalling pathway. Mammary explant protein synthesis was found to be stimulated up to twofold in response to graded levels of a complete mixture of amino acids (2 x and 4 x, normal rat plasma concentrations). The acute (1 h) stimulation of protein synthesis was at the level of translation. Inhibition of mTOR by rapamycin did not block the stimulation of protein synthesis by amino acids. In fact, when total amino acid concentrations were increased 0.5 to 4-fold, p70 S6 kinase activity decreased, despite the fact that protein synthesis was elevated up to 2.5 fold. When explants were incubated with either leucine or its transamination product α ketoisocaproic acid at 4 x normal levels in the presence of other amino acids (1 x), p70 S6 kinase activity was increased. There was a tendency for p70 S6 kinase activity to be blocked when transamination was inhibited. The failure to decrease protein synthesis by inhibition of transamination, despite the fact that p70 S6 kinase activity was inhibited, suggests that other translation factors may be more important in regulating mammary protein synthesis. This Phd thesis demonstrates a novel role for amino acids in mammary protein synthesis, whereby amino acids modulate the activity of the translation regulator p70 S6 kinase. In particular leucine and its transamination are important in the regulation of p70 S6 kinase activity. This provides the starting point for future studies exploring the role of translation factors in the regulation of mammary protein synthesis.

No description available.

Thesis (M.S.)--California Polytechnic State University, 2009. / Mode of access: Internet. Title from PDF title page; viewed on January 20, 2009. Major professor: Scott Reaves, Ph.D. "Presented to the faculty of California Polytechnic State University." "In partial fulfillment of the requirement of the degree [of] Master of Science in Agriculture with a specialization in Food Science and Nutrition." "December 2008." Includes bibliographical references (p. 81-90). Also available on microfiche.

No description available.