RESUMO
Phosphofructokinase-1 plays a key role in the regulation of carbohydrate metabolism. Its activity can be used as an indicator of the glycolytic flux in a tissue sample. The method most commonly employed to determine phosphofructokinase-1 activity is based on oxidation of NADH by the use of aldolase, triosephosphate isomerase, and alpha-glycerophosphate dehydrogenase. This method suffers from several disadvantages, including interactions of the auxiliary enzymes with phosphofructokinase-1. Other methods that have been used also require auxiliary enzymes or are less sensitive than a coupled assay. Here, we propose a direct method to determine phosphofructokinase-1 activity, without the use of auxiliary enzymes. This method employs fructose-6-phosphate and ATP labeled with 32P in the gamma position ([gamma-32P]ATP), and leads to the formation of ADP and fructose-1,6-bisphosphate labeled with 32P ([1-32P]fructose-1,6-bisphosphate). Activated charcoal is used to adsorb unreacted [gamma-32P]ATP, and the radioactive product in the supernatant, [1-32P]fructose-1,6-bisphosphate, is analyzed on a liquid scintillation counter. The proposed method is precise and relatively inexpensive, and can be applied to determine phosphofructokinase-1 activity in cellular extracts as well as in the purified enzyme.
Assuntos
Fosfofrutoquinase-1/análise , Trifosfato de Adenosina , Animais , Chlorocebus aethiops , Eritrócitos/enzimologia , Frutosefosfatos , Humanos , Cinética , Músculo Esquelético/enzimologia , Fosfofrutoquinase-1/sangue , Fosfofrutoquinase-1/isolamento & purificação , Fosfofrutoquinase-1 Muscular/análise , Fosfofrutoquinase-1 Muscular/isolamento & purificação , Radioisótopos de Fósforo , Coelhos , Radiometria/métodos , Contagem de Cintilação , Espectrofotometria/métodos , Especificidade por Substrato , Células VeroRESUMO
14CO2 production from [1-14C] glucose, the rate of glycolysis measured by the value of lactate production and the activities of various enzymes were determined in buffalo erythrocytes. Buffalo red cell glycolytic metabolites were estimated and used for the calculation of the mass action ratios of reactions catalyzed by the glycolytic enzymes of Bubalus bubalis. A comparison of the values of the mass action ratios with the equilibrium constants of the various glycolytic reactions indicate that hexokinase, phosphofructokinase, phosphoglycerate kinase and pyruvate kinase reactions are displaced from equilibrium, suggesting a regulatory role for each of these enzymes in buffalo erythrocyte glycolysis.
Assuntos
Glicemia/metabolismo , Búfalos/sangue , Eritrócitos/metabolismo , Animais , Dióxido de Carbono/sangue , Glicólise , Hexoquinase/sangue , Cinética , Fosfofrutoquinase-1/sangue , Fosfoglicerato Quinase/sangue , Piruvato Quinase/sangueRESUMO
Phosphofructokinase (PFK) from human polymorphonuclear leukocytes (PMN) was characterized by immunological titration with subunit specific antibodies and column chromatography on QAE-Sephadex in three different groups: control, type II diabetic, and obese individuals. It was found that PMN phosphofructokinase in the three groups consists mainly of a mixture of L4 and M4 homotetramers with possibly some hybrid forms. The predominant subunit was the L-type. A 24% decrease in the specific activity of the L-type isozyme was observed and an intermediate form (I-isozyme) having 23% of the total activity in diabetic individuals appeared. In obese individuals a 30% decrease was observed in the activity of M-type isozyme and 9% of the total activity corresponded to the intermediate form. Kinetic studies showed different regulatory properties among the isozymes from the three groups. The lower PFK activity found in diabetic and obese individuals can be associated with the decreased activity in the L-type isozyme (for diabetic individuals) and in the M-type isozyme (for obese individuals); the lower activity can also be associated with the four times lower affinity for F-6-P showed by the M-type isozyme, the decreased sensitivity to ATP inhibition (for both isozymes), and the appearance of an intermediate form with a different kinetic behaviour.
Assuntos
Resistência à Insulina/fisiologia , Isoenzimas/sangue , Neutrófilos/enzimologia , Fosfofrutoquinase-1/sangue , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Estudos de Casos e Controles , Citratos/farmacologia , Ácido Cítrico , Diabetes Mellitus Tipo 2/enzimologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Frutosedifosfatos/farmacologia , Frutosefosfatos/metabolismo , Frutosefosfatos/farmacologia , Humanos , Isoenzimas/antagonistas & inibidores , Cinética , Obesidade/enzimologia , Fosfofrutoquinase-1/antagonistas & inibidores , Especificidade por SubstratoRESUMO
Phosphofructokinase from human polymorphonuclear leukocytes has low cooperativity and high affinity for its substrate, F-6-P. It is resistant to ATP inhibition at pH 8; however, at pH 7.1 it becomes sensitive to the effect of this compound. It is activated by F-1, 6-P2; it is not very sensitive to citrate inhibition and F-2, 6-P2 has no effect on its activity. With these kinetic characteristics we assume that perhaps the predominant L-type subunit is accompanied by an F-type component.
Assuntos
Neutrófilos/enzimologia , Fosfofrutoquinase-1/sangue , Citratos/farmacologia , Ativação Enzimática , Frutosedifosfatos/farmacologia , Humanos , Técnicas In Vitro , Cinética , Fosfofrutoquinase-1/antagonistas & inibidores , Valores de ReferênciaRESUMO
Cord blood samples from ten term infants were fractionated into populations of young and old erythrocytes and compared with cells prepared in a similar fashion from eight normal adults. The old cell fraction from the newborn infants had very low phosphofructokinase activity and did not display the usual decline of activity of the enzymes phosphoglycerate kinase and enolase. In addition, the old cells from the newborn infants demonstrated impaired glucose consumption, which, upon analysis of the pattern of glycolytic intermediates, appeared to be a result of the phosphofructokinase deficiency. These findings suggest that cells produced earlier in gestation possess the developmental characteristics of fetal blood to a more significant degree and that these biochemical alterations may produce functional impairment.