RESUMO
The interaction of pig heart AMP deaminase with different chemical species of phosphatidylcholine and with natural plasma membranes has been investigated. Phospholipids added to the system either as natural biological membranes (plasma membrane vesicles) or in the form of liposomes containing unsaturated phosphatidylcholine considerably enhanced AMP deaminase activity. The secondary structure of pig heart AMP deaminase in the absence and in the presence of dioleoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine liposomes was investigated by Fourier-transform infrared spectroscopy. Quantitative analysis of the amide I band showed that the enzyme contains 45% beta-sheets, 28% alpha-helix, 16% turns and 11% non-ordered structure. In the presence of dioleoyl phosphatidylcholine liposomes, the beta/alpha content ratio decreased; this decrease was dependent on the amount of lipid added. This phenomenon was not observed in the case of dipalmitoyl phosphatidylcholine liposomes. These data suggest a possible role for membrane phospholipids in the regulation of AMP deaminase activity.
Assuntos
AMP Desaminase/metabolismo , Bicamadas Lipídicas/metabolismo , Miocárdio/enzimologia , Fosfolipídeos/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/farmacologia , AMP Desaminase/química , Trifosfato de Adenosina/farmacologia , Adenilil Imidodifosfato/farmacologia , Animais , Análise de Fourier , Cinética , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/farmacologia , Estrutura Secundária de Proteína , Espectrofotometria Infravermelho , SuínosRESUMO
Phosphatidate bilayers composed of dilauroylphosphatidate, dimyristoylphosphatidate, dipalmitoylphosphatidate and dioleoylphosphatidate were prepared. Their interaction with AMP deaminase isolated from pig heart was investigated. Dioleoylphosphatidate bilayers were found to exert non-competitive inhibition on the AMP deaminase with a Ki of 15 x 10(-6) M. This inhibition is three orders of magnitude stronger than that exerted by orthophosphate. The phosphatidate species containing saturated fatty acids were either non-inhibitory or inhibited enzyme activity rather poorly. However, alkalinization of the medium from pH 6.5 to pH 7.9 led to the inhibition of pig heart AMP deaminase by dilauroylphosphatidate bilayers. This was accompanied by the fluidization of the saturated phosphatidate species, i.e. the lowering of their phase transition temperature in alkaline pH, as measured by light-scattering and fluorescence scans. The possible significance of these findings for the regulation of AMP deaminase activity in vivo by natural membranes is discussed.
Assuntos
AMP Desaminase/antagonistas & inibidores , Bicamadas Lipídicas/metabolismo , Miocárdio/enzimologia , Nucleotídeo Desaminases/antagonistas & inibidores , Ácidos Fosfatídicos/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Lipossomos/metabolismo , Fosfatos/farmacologia , Espectrometria de Fluorescência , Suínos , TemperaturaRESUMO
AMP-deaminase purified from pig heart has been found to be activated by liposomes prepared from phospholipids extracted from pig heart mitochondria, microsomes and cytoplasm, as well as by intact microsomes. The activation by phospholipids occurred only in the presence of ATP and after the enzyme had been preincubated with liposomes for 30 min. Liposomes prepared from cardiolipin displayed an inhibitory effect on pig heart AMP-deaminase.
Assuntos
AMP Desaminase/metabolismo , Miocárdio/enzimologia , Nucleotídeo Desaminases/metabolismo , Fosfolipídeos/farmacologia , AMP Desaminase/antagonistas & inibidores , Trifosfato de Adenosina/farmacologia , Animais , Cardiolipinas/farmacologia , Citosol/análise , Ativação Enzimática/efeitos dos fármacos , Cinética , Lipossomos/farmacologia , Lipídeos de Membrana/farmacologia , Microssomos/análise , Mitocôndrias Cardíacas/análise , Miocárdio/análise , SuínosRESUMO
Liposomes made of sphingomyelin were found to inhibit both ATP-activated and non-activated AMP deaminase from pig brain, in contrast to liposomes made of egg yolk phosphatidylcholine which exhibited an activating effect on the ATP-activated enzyme, being without effect on AMP deaminase in the absence of ATP. Dioleoylphosphatidylcholine exerted a similar effect as egg yolk phosphatidylcholine but dipalmitoylphosphatidylcholine was without effect.
Assuntos
AMP Desaminase/metabolismo , Encéfalo/enzimologia , Nucleotídeo Desaminases/metabolismo , Fosfatidilcolinas/farmacologia , Esfingomielinas/farmacologia , 1,2-Dipalmitoilfosfatidilcolina/farmacologia , AMP Desaminase/antagonistas & inibidores , Trifosfato de Adenosina/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Cinética , Lipossomos , SuínosRESUMO
Trout gill AMP deaminase is inhibited by liposomes made of synthetic phosphatidylcholines containing higher saturated fatty acids. A preincubation of 1 hr, at 4 degrees C, was necessary to obtain the maximal effect. At 4 or 25 degrees C, these phospholipids modified essentially the substrate affinity of the enzyme by increasing the Michaelis constant proportionally to the length of the fatty acid chain. At 13 degrees C, the liposomes decreased the Hill coefficient also, thus inducing a negative cooperativity. Natural phosphatidylcholine and phosphatidylserine were without significant effect on gill AMP deaminase while natural sphingomyelin exhibited a similar effect to that shown in the presence of synthetic phosphatidylcholines. These results are discussed in relation to a possible effect of sphingomyelins in vivo.
Assuntos
AMP Desaminase/metabolismo , Brânquias/enzimologia , Bicamadas Lipídicas , Nucleotídeo Desaminases/metabolismo , Fosfatidilcolinas/farmacologia , Animais , Cinética , Relação Estrutura-Atividade , Temperatura , Termodinâmica , TrutaRESUMO
AMP deaminase (EC 3.5.4.6) from pig kidney was purified about 1200-fold by chromatography on cellulose phosphate. The enzyme showed a sigmoid-shaped substrate saturation curve which was converted to hyperbolic by addition of ATP. The ATP-activated enzyme was sensitive to phosphatidylcholine-containing liposomes which caused a further increase of activity by lowering the S0.5 value and increasing V max. In the absence of ATP, the enzyme was not sensitive to phosphatidylcholine-containing liposomes. Phosphatidate-containing liposomes exerted an inhibitory effect both in the presence and absence of ATP. In the presence of ATP phosphatidate was a non-competitive inhibitor. Orthophosphate was found to be a competitive inhibitor of AMP deaminase from pig kidney. When the phosphatidylcholine/phosphatidic acid ratio in liposomes was 2.7, AMP deaminase was activated, whereas when this ratio dropped below 2.1, liposomes exerted a non-competitive inhibitory effect.
Assuntos
AMP Desaminase/metabolismo , Rim/enzimologia , Nucleotídeo Desaminases/metabolismo , AMP Desaminase/isolamento & purificação , Animais , Ativação Enzimática , Cinética , Lipossomos , Ácidos Fosfatídicos/farmacologia , SuínosRESUMO
Adenylate deaminase (AMP deaminase, EC 3.5.4.6) of a high substrate specificity was purified from pig heart by chromatography on cellulose phosphate. The enzyme shows a co-operative binding of AMP [h (Hill coefficient) 2.35, with SO.5 (half-saturating substrate concentration) 5mM]. ATP and ADP act as positive effectors, lowering h to 1.55 and SO.5 to 1 mM. The addition of liposomes (phospholipid bilayers) to ATP-activated or ADP-activated enzyme causes a further shift of the h value to 1.04 and SO.5 to 0.5 mM. For ATP-activated enzyme the addition of liposomes increases Vmax. by about 100%, and for ADP-activated enzyme by 50%. Liposomes have no effect on the kinetics of AMP deaminase in the absence of ATP and ADP, and neither do they influence the inhibitory effect of orthophosphate on heart muscle AMP deaminase. Metabolic implications of these findings are discussed.
