RESUMO
Lactate dehydrogenase was purified 21-fold from liver of Varanus bengalensis using colchicine-sepharose column chromatography. The crude enzyme showed two isoenzymes (LDH-5 and LDH-4) by agarose gel electrophoresis (AGE). The purified enzyme showed a single band after SDS-PAGE corresponding to molecular mass of 35 kDa. The molecular mass of native enzyme was about 140 kDa. The optimum pH for the forward reaction was 7.5 while that for the reverse reaction was pH 9.5. The K-m values for pyruvate, NADH, lactate and NAD(+) were 0.17 +/- 0.037, 0.02 +/- 0.004, 12.4 +/- 3.05 and 0.38 +/- 0.032 mM, respectively. Pre-heating of enzyme showed that its t(50) was 40-50 degrees C. Oxalate and n-hexanediol were inhibitors for both forward and reverse reactions. Among divalent ions, Cu++ was shown to be more effective inhibitor for the forward reaction.
Assuntos
Cromatografia , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Íons , Isoenzimas , L-Lactato Desidrogenase , Ácido Láctico , Fígado , NAD , Ácido PirúvicoRESUMO
The present study is designed to establish a xenograft model of human promyelocytic leukemia cell mutant (HL-60-AR) deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT) in nude mice. A solid leukemia sarcoma developed after subcutaneous inoculation with HL-60-AR cells. Comparative studies of HL-60-AR/Nu tumor cells in nude mice and cultured HL-60-AR cells in vitro revealed virtual identity as shown by light microscopic morphology, ultrastructure of cell, cytochemistry, chromosome analysis, LDH isoenzyme pattern, genetic markers and differentiated characters assay. Up to now, twelve generations haw been transmitted in rive by inoculating with the solid tumor Cells developed in nude mice. This nude mice model in which human leukemia cells grew could be considered as a useful model for in rive studies of human leukemic cells proliferation, differentiation and the screening for anti-leukemia drugs.