RESUMO
The structural gene ldhL for a thermostable L-(+)-lactate dehydrogenase was cloned from Thermoanaerobacter ethanolicus JW200. The nucleotide sequence of the ldhL gene was determined and shown to have the capacity to encode a protein of 311 amino acids (33.5 kDa). By 5'-RACE analysis, the ldhL transcription start point was confirmed to be derived from the -10 region closest to the initiation codon. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by nickel-affinity chromatography. It was shown to be allosteric in the presence of fructose-1,6-bisphosphate. The optimum pH and temperature for the enzyme were 5.8 and 60 degrees capital ES, Cyrillic in the pyruvate reduction and 7.0 and 70 degrees capital ES, Cyrillic in the lactate oxidation reaction, respectively. The kinetic parameters K(m,app) and k(cat,app) for pyruvate were 0.18 mM and 520 U/mg, respectively, and in the absence of fructose-1,6-bisphosphate, a 1.2-fold increase in K(m,app) and a 16-fold decrease in k(cat,app) were determined. The K(m,app) and k(cat,app) values for lactate were 60 mM and 0.58 U/mg, respectively, and they were not affected by fructose-1,6-bisphosphate. The enzyme was greatly inhibited by Zn2+, Ag(+), Cu2+, Fe3+, and Pb(3+). The extreme thermostability of the enzyme was reflected in its unaltered activity over 5 h at 70 degrees C.
Assuntos
L-Lactato Desidrogenase/metabolismo , Thermoanaerobacter/enzimologia , Regulação Alostérica , Sequência de Bases , Frutose-Bifosfatase/metabolismo , Cinética , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/genética , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
The author of this article visited China for the purpose of helping the faculty of the School of Nursing learn research skills by participating in research--a kind of learn by doing. Both investigators had conducted quantitative studies in the US--one with children and one with adults--that were adapted for use in China. Seeking to also include a qualitative study, the investigators explored several possible research areas. Because pain is a universal phenomena, it was chosen as the subject for the study using the qualitative methodology reported in the first part of this article.
