RESUMO
6-phosphofructo-1-kinase (PFK) is a calmodulin (CaM)-binding protein that plays a key role on the regulation of glycolysis. Each PFK monomer binds two CaM molecules inducing the dissociation of the active tetrameric conformation of the enzyme into dimers, thus inhibiting it. Recently, we have reported that the binding of one CaM per PFK monomer promotes the dimerization of the enzyme although maintaining its full catalytic activity. The present work aims to understand the regulatory role of these active PFK dimers induced by CaM. We show that the inhibition of PFK activity by ATP (>1 mM) is abolished in the presence of CaM. CaM decreases the affinity of PFK for its substrates, fructose-6-phophate and ATP. Moreover, CaM activates PFK in the presence of citrate and lactate, two inhibitory metabolites that induce the dimerization of PFK tetramers, as well as potentiate the stimulatory action of ADP and fructose-2,6-bisphosphate. Under all the conditions tested CaM induces the formation of PFK dimers, supporting that these CaM-bound dimers are active and less susceptible to inhibition by allosteric ligands. In the end, we suggest that CaM binding to PFK, which is stimulated by Ca(2+), represents an important way to increase the glycolytic pathway in cells.
Assuntos
Calmodulina/farmacologia , Fosfofrutoquinase-1/metabolismo , Trifosfato de Adenosina/antagonistas & inibidores , Regulação Alostérica , Concentração de Íons de Hidrogênio , Fosfofrutoquinase-1/efeitos dos fármacos , Multimerização Proteica , Estrutura Quaternária de Proteína , Regulação para CimaRESUMO
Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. Clotrimazole is an anti-fungal azole derivative recently recognized as a calmodulin antagonist with promising anti-cancer effect. Here, we show that clotrimazole induced morphological and functional alterations on human breast cancer derived cell line, MCF-7. The drug decreased cell viability in a dose- and time-dependent manner, exhibiting an IC50 of 88.6+/-5.3 microM and a t0.5 of 89.7+/-7.2 min, with 50 microM clotrimazole. Morphological changes were evident as observed by scanning electron microscopy, which revealed the completely loss of protrusion responsible for cell adhesion after a 180 min of treatment with 50 microM clotrimazole. Giemsa stained cells observed by optical microscopy show morphological alterations and a marked nuclear condensation. These changes occurred in parallel to the detachment of the glycolytic enzymes, 6-phosphofructo-1-kinase and aldolase, from cytoskeleton. After a 45 min treatment with 50 microM clotrimazole, the remaining activities in a cytoskeleton enriched fraction was 16.4+/-3.6% and 41.0+/-15.6% of control for 6-phosphofructo-1-kinase and aldolase, respectively. Immunocytochemistry experiments revealed a decrease in the co-localization of 6-phosphofructo-1-kinase and F-actin after clotrimazole treatment, suggesting the site of detachment of the enzymes. Altogether, our results support evidence for apoptotic events that might be started by clotrimazole involving inhibition of glycolytic flux in MCF-7 cells and makes this drug a promising agent in the fight against human breast cancer.
Assuntos
Neoplasias da Mama/enzimologia , Carcinoma Ductal de Mama/enzimologia , Clotrimazol/farmacologia , Citoesqueleto/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Actinas/efeitos dos fármacos , Actinas/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Frutose-Bifosfato Aldolase/efeitos dos fármacos , Frutose-Bifosfato Aldolase/metabolismo , Glucose/metabolismo , Humanos , Ácido Láctico/metabolismo , Faloidina/química , Faloidina/metabolismo , Fosfofrutoquinase-1/efeitos dos fármacos , Fosfofrutoquinase-1/metabolismo , Rodaminas/química , Rodaminas/metabolismo , Estreptavidina/química , Estreptavidina/metabolismoRESUMO
The contamination of water by metal compounds is a worldwide environmental problem. This study was undertaken to evaluate the impact of short-term cadmium exposure on metabolic patterns of the freshwater fish Oreochromis niloticus. The fish were exposed to 320, 640, 1,280 and 2,560 microg/l sublethal concentrations of Cd++ (CdCl2) in water for 7 days. The specific activities of the enzymes phosphofructo kinase (PFK-E.C.2.7.1.11.), lactate dehydrogenase (LDH-E.C.1.1.1.27.) and creatine kinase (CKE.C.2.7.3.2.) were decreased in white muscle after cadmium treatments, indicating decreases in the capacity of glycolysis in this tissue. Cadmium exposure induced increased glucose concentration in white muscle of fish. On the other hand, cadmium exposure at sublethal concentrations increased phosphofructo kinase and LDH in red muscle of fish. Cadmium significantly decreased total protein concentrations in liver and white muscle regardless of tissue glycogen levels. The data suggest that cadmium acts as a stressor, leading to metabolic alterations similar to those observed in starvation.
Assuntos
Cádmio/efeitos adversos , Creatina Quinase/metabolismo , L-Lactato Desidrogenase/metabolismo , Fosfofrutoquinase-1/metabolismo , Tilápia/fisiologia , Poluentes Químicos da Água/efeitos adversos , Animais , Creatina Quinase/efeitos dos fármacos , Relação Dose-Resposta a Droga , Exposição Ambiental , Glicogênio/análise , L-Lactato Desidrogenase/efeitos dos fármacos , Fígado/química , Músculo Esquelético/química , Fosfofrutoquinase-1/efeitos dos fármacosRESUMO
La sialadenosis en animales de laboratorio puede ser provocada por la administración de un agonista betadrenérgico conocido como isoproterenol. Su efecto produce hipertrofia e hiperplasia de las glándulas salivales, especialmente de la glándula parótida. La 6-fosfofructo-2-quinasa (PKF-2) es una enzima bifuncional que cataliza tanto la síntesis como la degradación de la fructosa-2,6-difosfato. este metabolito es un potente activador de la 6-fosfofructo-1-quinasa (PKF-1) enzima clave en el proceso glucolítico. En este trabajo determinamos la actividad de la PKF-2 y el contenido de fructosa-2,6-bifosfato en glándulas parótidas de ratas, al ser estimuladas con varias dosis de isoproterenol