RESUMO
Sea bass (Dicentrarchus labrax L.) liver phosphofructokinase (PFK) presents biphasic kinetics with respect to fructose-6-phosphate (F-6-P) in experiments carried out with crude extract. After the enzyme had been purified, two isozymes have been detected after chromatographic treatment. The two isozymes present different kinetic behaviour PFK-L1, the first eluted phosphofructokinase activity shows positive cooperativity with respect to fructose-6-phosphate and PFK-L2, the second activity fraction, has a Hill coefficient of 0.38 (negative cooperativity). The first isozyme shows less affinity for fructose-6-phosphate than that shown by PFK-L2. The joint kinetics of both isozymes produces a biphasic kinetics with respect to fructose-6-phosphate, similar to that observed in crude extracts.
Assuntos
Bass/metabolismo , Glicólise , Isoenzimas/metabolismo , Fígado/enzimologia , Perciformes/metabolismo , Fosfofrutoquinase-1/metabolismo , Regulação Alostérica , Animais , Frutosefosfatos/metabolismo , Isoenzimas/isolamento & purificação , Cinética , Peso Molecular , Fosfofrutoquinase-1/isolamento & purificação , TemperaturaRESUMO
White muscle pyruvate kinase from sea bass presents positive cooperativity with respect to PEP substrate. The enzyme is regulated by F-1.6-P2 and L-Phenylalanine. The activator effect of F-1.6-P2 in experiments carried out for the substrate PEP with crude extract seems to indicate that the enzyme is activated in vivo by this compound. The enzyme was not inhibited by either alanine or ATP but was inhibited by L-phenylalanine. Therefore this enzyme presents kinetic and regulatory properties similar to those of the mammalian isozyme M2.
Assuntos
Frutosedifosfatos/metabolismo , Glicólise , Hexosedifosfatos/metabolismo , Músculos/enzimologia , Fenilalanina/metabolismo , Fosfoenolpiruvato/metabolismo , Piruvato Quinase/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Alanina/metabolismo , Animais , Bass , Ativação EnzimáticaRESUMO
Pyruvate kinase of sea bass liver show a joint modulation of both pH and temperature for PEP substrate. The effect of F-1,6-P2, alanine and ATP is not fundamentally affected by a variation in pH. The kinetic constants in the presence of ATP are not affected, but the intensity of its inhibitor effect varies with temperature. A study of different buffers: Tris-HC1, Tris-maleic and phosphate, on this enzymatic activity with or without effectors has been made.
Assuntos
Fígado/enzimologia , Piruvato Quinase/metabolismo , Alanina/farmacologia , Animais , Peixes , Concentração de Íons de Hidrogênio , Cinética , TermodinâmicaRESUMO
Glycolytic enzyme phosphofructokinase (PFK) from sea-bass liver shows inhibition for ATP4- and MG-ATP2-, and ATP4- is a competitive inhibitor with respect to MG-ATP2-. Free Mg2+ behaves as a mixed inhibitor on the kinetic with respect to the true enzyme substrate Mg-ATP2-, and eliminates the inhibition effect of this substrate. The kinetics with respect to Mg-ATP2- at non-inhibiting concentrations is not visibly affected by temperature of pH variation. The inhibiting effect of Mg-ATP2- is more marked at 22 and 10 degrees C (of three assayed temperatures 22, 15 and 10 degrees C and at physiological pH 6.8) as opposed to the maximum activity pH (8.0).
