No description available.

No description available.

No description available.

Thesis (doctoral)--Köln, 1987.

Bielefeld, Universiẗat, Diss., 2000.

Kynureninase (EC.3.7.1.3) is an unusual pyridoxal 5'-phosphate (PLP) dependent enzyme which catalyses the beta-gamma hydrolytic cleavage of kynurenine to give anthranilic acid and alanine. The enzyme is a potential therapeutic target for controlling the levels of metabolites, quinolinic acid and kynurenic acid, that are important in a variety of neurodegenerative and inflammatory disorders, such as Alzheimer's disease, AIDS, Lyme disease and poliovirus. Studies have been carried out to elucidate the mechanism of the enzyme. A phosphinic acid analogue of kynurenine, designed as a putative transition state mimic for the reaction, has been prepared. This was found to be a competitive inhibitor, with a KI of 4.19 mM when its interaction with kynureninase, isolated from Pseudomonas fluorescens, was examined. The corresponding methyl phosphinate was also prepared. This was found to be a more potent inhibitor, with a KI of 0.88 mM. It is proposed that methylation of the acid removes a destabilising interaction between the negative charge of the ionised phosphinic acid and some group of the active site. A number of analogues of kynurenine were prepared in order to obtain information on the specificity of the enzyme and the interactions at the active site. A racemic mixture of desaminokynurenine was found to have a KI of 23.2 μM. Cyclohexyl and naphthyl amino acid derivatives prepared by the same method were found to be weak competitive inhibitors of the enzyme with KI values of 844 μM and 207 μM repectively. Desaminokynurenine was found to be a substrate with a rate of reaction twenty times slower than that of kynurenine. The naphthyl amino acid derivative was also found to be a substrate. The results suggested that interactions with the aromatic ring are important and that the naphthyl amino acid derivative may be too large for the active site. The synthesis of 25-[2-2H]-kynurenine is described using two different routes. Diacetylation / racemisation of racemic kynurenine in deuterium oxide followed by acylase catalysed resolution is the most direct route. The alternative is to prepare 2S-[2-2H]-tryptophan by a similar procedure and then to convert this to 2S-[2-2H]-kynurenine via ozonolysis. This compound has been used in isotope studies to gain an understanding of the mechanism of the enzyme catalysed reaction. The primary deuterium isotope effect, DV= 0.98 and D(V/K) = 3.6, showed that for the kynureninase catalysed reaction, alpha-H abstraction is partially rate limiting. A solvent isotope effect, DV= 4.4 and D(V/K) = 4.6 was also determined, showing that a proton transfer is also partially rate limiting.

No description available.

Ostrich organs/tissue/fluids were screened for plasma kallikrein-like, tissue kallikrein-like and tonin-like activity in a continuous-fluorogenic-assay system using Pro-Phe-Arg-7-amino-4-methylcoumarine, Phe- Arg-7-amino-4-methylcoumarine and Val-Leu-Arg--7-amino-4-trifluoro-methylcoumarine as substrates. Ostrich liver and kidney showed the highest specific plasma kallikrein-like activity. Ostrich adrenal glands and kidney showed the highest specific tissue kallikrein-like and tonin-like activity. Ostrich high molecular weight kininogen was purified from plasma and low molecular weight kininogen was partially purified. The N-terminal amino acid sequences of both high- and low molecular weight kininogens from ostrich plasma were determined. Ostrich plasma high molecular weight kininogen was purified as a 118 kD protein. The purified high molecular weight kininogen inhibits the cysteine proteinase papain at a ratio of one molecule HKG to two molecules of papain. Ornitho kinin-like molecules were detected in ostrich urine using reverse phase HPLC.

The little studied plasma kallikrein-kinin system of poikilotherm vertebrates was investigated in several species offish, amphibians, and reptiles, and compared to the well-known mammalian enzyme system. It was found that the plasmas of all fish and amphibians tested differed from reptilian and mammalian plasma in their inability to release a kinin-like factor when reacted with trypsin or glass, and no evidence was obtained to suggest that these plasmas contain enzymic machinery which can produce a kinin. However, it was shown that heat-denatured plasma from these animals did develop biological activity when treated with hog pancreas kallikrein, an enzyme specific for releasing kinins. Thus, the equivalent of a kininogen might exist in these plasmas. Since turtle plasma produced a kinin by endogenous enzymes, detailed studies of this system were conducted. By a variety of criteria, enzymic mechanisms for kinin production in this plasma were closely similar to those of mammalian plasma. However, purification of the turtle kinin released by endogenous enzymes, followed by pharmacological and chemical tests showed that this kinin was chemically different from bradykinin, its mammalian counterpart. Data obtained from amino acid analysis of the peptide, and from certain pharmacological tests, strongly suggested that the structure of turtle kininis 6-thr-bradykinin; i.e., that a threonine residue has been substituted for a serine . The possible significance of this finding is discussed. Preliminary studies of the pharmacological effects of bradykinin on aspects of blood pressure and flow in the turtle itself are described. Intra-arterial injections of bradykinin over a wide range of doses always produced a press or response which could be greatly reduced by adrenergic blockade. This is in contrast with the effect of, the peptide in mammals, where there is typically a hypotensive response which cannot be reduced by adrenergic blockade. The significance of this difference is discussed, and approaches to future investigations are suggested. / Science, Faculty of / Zoology, Department of / Graduate

Plasma kallikrein (PK) is a coagulation factor that activates Factor XII in the contact pathway and generates vasoactive bradykinin from kininogen. C1-esterase inhibitor (C1INH) regulates PK levels. C1INH deficiency manifests as potentially life-threatening hereditary angioedema (HAE). Several drugs are licensed for HAE treatment and/or prophylaxis, including C1INH concentrates and recombinant Ecallantide (rEcall). rEcall is a small protein comprised of the first Kunitz domain of Tissue Factor Pathway Inhibitor and was substituted at seven residues making it a PK-specific inhibitor. rEcall is licensed for HAE treatment, but its short half-life prevents prophylactic application. This project focused on comparing the inhibition of PK and the in vivo clearance of hexahistidine-tagged rEcall (H6-rEcall) and H6-rEcall genetically fused to human serum albumin (H6-rEcall-HSA), or an albumin binding domain (H6-rEcall-ABD). To determine if the orientation of the constructs contributed to activity and half-life, proteins were reoriented, and HSA was moved from the C- to N-terminus. Proteins produced were H6-rEcall (64 amino acids), H6-rEcall-ABD (110 amino acids), H6-rEcall-HSA (666 amino acids), H6-rEcall-HSA, rEcall-H6 (64 amino acids), and HSA-rEcall- H6 (666 amino acids). All proteins were expressed in Pichia pastoris, secreted, and purified from conditioned media via nickel-chelate chromatography. PK activity was assessed via amidolysis of S2302, and the inhibitory constant (Ki) was determined. Purified proteins were radiolabeled with 125I and injected intravenously into CD1 mice. Acid-precipitable radioactivity in timed blood samples was expressed as a percentage of the peak post-injection value. Values were means ± SD, n = 6. Next, the role of Lipoprotein related receptor protein 1 (LRPI) or neonatal Fc receptor (FcRn) in rEcall proteins’ clearance was examined by performing a competition experiment. In this experiment, rEcall associated with albumin was co-administered with a competitor ligand that blocked FcRn or LRP1. To further investigate the role of these receptors and the organs responsible for rEcall proteins’ clearance, organ distribution was conducted by cannulating mice via the right jugular vein and delivering 125I-rEcall or 125I-HSA-rEcall. The organs analyzed were the kidneys, liver, spleen, and heart. In vitro, rEcall and its modified proteins had a Ki of ~ 2 – 3 nM. In vivo, fusion proteins behaved similarly and presented some pharmacokinetic enhancements. Two hours post-injection, 50 ± 10% of H6-rEcall-HSA and 36 ± 6 % of HSA-rEcall-H6 remained in the circulation vs. 6 ± 2 % of H6-rEcall and 7 ± 1% of H6-rEcall-ABD (p <0.0001), while 75 ± 5% of HSA-H6 was recovered in the circulation. In the competition experiment, the presence of GST-RAP elevated the recovery of 125I- H6-rEcall-HSA 2.2-fold, from 37 ± 3 % for GST + H6-rEcall-HSA to 81 ± 3 % for GST-RAP + H6-rEcall-HSA. Competition with IVIg also significantly increased the recovery of 125I- H6- rEcall-HSA, but to a lesser degree, by 1.13-fold (to 42 ± 5% for IVIg + H6-rEcall-HSA). Similar significant changes were also observed 30 mins after injection, of 2.9- and 1.9-fold, respectively. Finally, results indicate that albumin changed rEcall’s organ distribution. Of the four organs extracted 30 minutes after injection, rEcall-H6 was predominantly found in the kidneys, and fusion to albumin largely redirected rEcall to the liver. 3.6-fold more HSA-rEcall-H6 was found in the liver in comparison to rEcall (p<0.0001). In contrast, 3.3-fold more rEcall was observed in the kidneys vs. rEcall-HSA (p<0.0001). In conclusion, fusing HSA to either the N- or C-terminus of rEcall extended its plasma residency time and did not impact its function towards PK. / Thesis / Master of Health Sciences (MSc)