The goal of the present study is to understand the discoursive negotiation of the claims upon the use of public space. Specifically is to focused on the analysis of purification of public space from homeless persons in middle-sized city. On the basis of my research of homeless persons that included de semi-structured interviews with selected actors (policemen, politicians, employees of social services and of the non-profit organizations), analysis of documents (especially journalistic and legal) and active participant observation.
Malate dehydrogenase (EC 1.1,1,37.) (MDH) was purified to apparent homogeneity from the cytosolic fraction of the protozoan Trichomonas vaginalis Donné. The four step purification included ion-exchange chromatography (DEAE-Sephacel and Q-Sepharose, elution with NaCl) and affinity chromatography (Reactive Red Agarose, elution with NADH and NaCl). The enzyme was purified about 132-fold (30.6% yield) to a specific activity of 352 units mg~'. The Km values determined at pH 7.8 (pH optimum from 7.5 to 8.3) for oxaloacetate and NADH were 16.2 pM and 10.6 μΜ, respectively. The MDH activity was inhibited by the substrate, decreasing to 50% at about 1 mM concentration of oxaloacetate. The reverse reaction from malate to oxaloacetate showed a pH optimum around pH 9.5. The Km for malate and NAD* (determined at pH 7.8) were 1220 pM and 69.9 pM, respectively. SDS-PAGE analysis of the purified MDH revealed a single band with an apparent size of 34.5 kDa. The native molecular weight was estimated by HPLC gel filtration to be 60 kDa, which indicates that the T. vaginalis MDH exists as a dimer.
Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme in the metabolism of vitamin B6 (VB6) in Bombyx mori. A fusion expressional vector pET-22b-BPLK-His was constructed using a sub-cloning technique, the recombinant B. mori PLK was then expressed in Escherichia coli, purified and characterized. Bioinformatics were used to deduce the protein structure and genomic organization of this enzyme. Using Ni Sepharose affinity column chromatography, the recombinant protein was purified to very high degree (approximately 90%). The recombinant PLK exhibits a high specific enzymatic activity (1800 nmol/min/mg of protein). The maximum catalytic activity of this enzyme was recorded over a narrow pH range (5.5-6.0) and Zn2+ is the most effective cation for catalysis under saturating substrate concentrations. When only triethanolamine is present as the cation, K+ is an activator of PLK. A double reciprocal plot of initial velocity suggests that the enzyme catalyses the reaction by means of a sequential catalytic mechanism. Under optimal conditions, the Km value for the substrates of ATP and pyridoxal are 57.9 ± 5.1 and 44.1 ± 3.9 µM. B. mori's genome contains a single copy of the PLK gene, which is 7.73 kb long and contains five exons and four introns, and is located on the eighth chromosome. The PLK may be a dimer with two identical subunits under native conditions, and it is hypothesized that each monomer contains eight α-helices (α1-8), nine β-strands (β1-9) and two segments of 310 helices. and Shuo-Hao HUANG, Wang MA, Ping-Ping ZHANG, Jian-Yun ZHANG, Yan-Feng XIE, Long-Quan HUANG.