RESUMO
Rat liver microsomal 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase was activated by 50% at a concentration of 0.4 mM 2,3-diphosphoglyceric acid (DPG) and by 11-fold at 10 mM DPG. DPG also prevented the inactivation of HMG-CoA reductase by ATP and Mg++. Rat liver microsomal HMG-CoA reductase prepared in the presence of 1 mM DPG was significantly more active than when prepared in the absence of DPG. Activation of the enzyme by DPG and protection of the enzyme against inhibition by ATP and Mg++ by DPG were also observed with solubilized HMG-CoA reductase.
Assuntos
Ácidos Difosfoglicéricos/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Microssomos Hepáticos/enzimologia , 2,3-Difosfoglicerato , Trifosfato de Adenosina/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases , Magnésio/farmacologia , Masculino , Ratos , Ratos EndogâmicosAssuntos
Fígado/metabolismo , Monoterpenos , Esteróis/biossíntese , Terpenos/farmacologia , Monoterpenos Acíclicos , Animais , Citosol/metabolismo , Dolicóis/farmacologia , Técnicas In Vitro , Ácido Mevalônico/metabolismo , Microssomos Hepáticos/metabolismo , Fitol/farmacologia , Ratos , Esqualeno/metabolismo , Ubiquinona/farmacologia , Vitamina E/farmacologiaRESUMO
The rate of formation of sterol from squalene in livers from suckling rats was less than one-third that of adults. This difference was due to a lesser activity of microsomal enzymes in the suckling rat livers, and not to any difference in cytosolic sterol carrier protein 1. The microsomal enzymes and sterol carrier protein 2 of the cytosol required for the conversion 7-dehydrocholesterol to cholesterol were both lower in suckling rats compared to adults. Both those activities paralleled the differences in HMG-CoA reductase activities between suckling and adult rats. Feeding of cholestryamine to adult rats increased the activities of the microsomal enzymes, sterol carrier protein 1 and sterol carrier protein 2 involved in the conversion of squalene to cholesterol.
Assuntos
Envelhecimento , Proteínas de Transporte/metabolismo , Resina de Colestiramina/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Proteínas de Plantas , Administração Oral , Animais , Resina de Colestiramina/administração & dosagem , Masculino , Microssomos Hepáticos/metabolismo , Ratos , Ratos Endogâmicos , Esqualeno/metabolismoRESUMO
Rat liver microsomes prepared in Tris buffer exhibited 3 to 10 times higher 3-hydroxy-3-methyl glutaryl CoA reductase specific activity than microsomes prepared with potassium phosphate buffer. This higher activity was observed in rats killed during mid-light cycle, but microsomes from rats killed during mid-dark cycle showed no significant difference in enzyme activity between buffers. When microsomes prepared in the 2 different buffers were preincubated with ATP and MG++, enzyme activity was inhibited to the same extent. The cytosol fraction in each of the 2 different buffer preparations possessed similar phosphatase activity. The higher 3-hydroxy-3-methyl reductase activity in Tris buffer, therefore, does not appear to be due to differences in phosphorylation or dephosphorylation activity.
Assuntos
Hidroximetilglutaril-CoA Redutases/metabolismo , Microssomos Hepáticos/enzimologia , Fosfatos/farmacologia , Compostos de Potássio , Potássio/farmacologia , Trometamina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Citosol/enzimologia , Técnicas In Vitro , Luz , Magnésio/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/metabolismo , RatosRESUMO
When rat liver cytosol, possessing sterol carrier protein (SCP) activity was incubated with [3H]-squalene-phospholipid liposomes, cofactors, and rat liver microsomes, squalene from the liposomes was converted into sterols. When cytosol was omitted from the incubation mixture, only insignificant amounts of sterols were produced. Liposomes of squalene with either phosphatidylserine or phosphatidylcholine were equally effective as substrates. The liposomes were stable at 4 degrees C for 3 weeks. The ratio of squalene to phospholipid in the liposomes could be varied over a range of 0.004 to 0.23. Multilamellar liposomes with squalene were not effective as a substrate for the conversion of squalene to sterols. The mechanism for transfer of squalene from the liposomes to the enzymes appears to be initial binding of liposomes to microsomes, with subsequent transfer of the substrate to the enzyme site by the SCP in the cytosol. Microsome-liposome complexes prepared in the absence or presence of cytosol are effective in converting squalene to sterols only if cytosol is added again, indicating that cytosol is not required for the binding of liposomes to microsomes.
Assuntos
Colesterol/metabolismo , Citosol/metabolismo , Microssomos Hepáticos/metabolismo , Proteínas de Plantas , Esqualeno/metabolismo , Animais , Proteínas de Transporte/farmacologia , Lipossomos/metabolismo , Masculino , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Ratos , Esteróis/farmacologiaRESUMO
The conversion of 3H-squalene to sterols by rat liver microsomes and cytosol was inhibited by individual rat and human plasma lipoproteins at various concentrations. This inhibition was also observed with added human high density apolipoprotein, but triglycerides, cholesterol, or cholesteryl esters had no inhibitory effects. Lipoproteins and apo high density lipoprotein (HDL) were demonstrated to bind 3H-squalene in vitro. The binding of 3H-squalene by apo HDL could be reversed by increasing concentration of liver cytosol containing sterol carrier protein.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Lipoproteínas/farmacologia , Fígado/metabolismo , Proteínas de Plantas , Esqualeno/metabolismo , Animais , Citosol/metabolismo , Lipoproteínas HDL/farmacologia , Lipoproteínas LDL/farmacologia , Lipoproteínas VLDL/farmacologia , Fígado/efeitos dos fármacos , RatosRESUMO
Experimental uremia was induced in rats by means of bilateral nephrectomy or bilateral ureteral ligation. Incorporation of acetate-1-14C into expired 14CO2 and into plasma and tissue lipids was studied immediately after surgery and at 48 hr, when the rats were uremic. In rats studied immediately after surgery, bilateral nephrectomy, but not bilateral ureteral ligation, significantly decreased the conversion of acetate-1-14C into expired 14CO2. In uremic rats at 48 hr, acetate-1-14C metabolism to 14CO2 was not significantly altered in either group. Plasma triglyceride concentrations and 14C-acetate incorporation into triglycerides were increased in the 48-hr uremic groups, but plasma and liver triglyceride specific radioactivities were not significantly altered. Plasma free fatty acid concentrations and incorporation of acetate into free fatty acids were lower in the 48-hr uremic groups than in controls. Plasma cholesterol concentrations and specific radioactivities were increased in these uremic groups, as were liver free cholesterol specific activities. These results suggest that increased triglyceride and cholesterol synthesis from acetate may contribute to the hypertriglyceridemia and hypercholesterolemia observed in uremic rats.
Assuntos
Acetatos/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Uremia/metabolismo , Animais , Dióxido de Carbono/metabolismo , Colesterol/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Lipídeos/sangue , Masculino , Nefrectomia , Ratos , Triglicerídeos/metabolismo , Ureter/fisiologiaAssuntos
Colágeno , Adesividade Plaquetária , Doenças de von Willebrand/sangue , Animais , Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/ultraestrutura , Diabetes Mellitus/sangue , Dipiridamol/farmacologia , Fator VIII , Humanos , Nefropatias/sangue , Adesividade Plaquetária/efeitos dos fármacos , Contagem de Plaquetas , Ratos , Sulfimpirazona/farmacologia , TemperaturaRESUMO
A group of 18 stable hemodialysis patients were dialyzed alternately for 4 week periods against acetate or bicarbonate solutions. Total plasma cholesterol and triglyceride concentrations and the content of these lipids in the VLDL, LDL, and HDL lipoprotein fractions were not significantly different when these dialysis periods were compared. When patients were subgrouped according to whether they were normo or hypertriglyceridemic or whether they had an adequate or deficient lipolytic response to heparin infusion, there were also no differences apparent between the acetate vs. bicarbonate dialysis periods. The data indicates, therefore, that acetate is probably not a contributory factor to the hypertriglyceridemias observed in some chronic hemodialysis patients.
Assuntos
Acetatos/farmacologia , Bicarbonatos/farmacologia , Lipídeos/sangue , Lipoproteínas/sangue , Diálise Renal/métodos , Uremia/sangue , Adulto , Colesterol/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Triglicerídeos/sangueAssuntos
Adenina/análogos & derivados , Plaquetas/efeitos dos fármacos , Colesterol/sangue , Hidroximetilglutaril-CoA Redutases/sangue , Adenina/farmacologia , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/enzimologia , Colágeno/farmacologia , Epinefrina/farmacologia , Feminino , Fígado/efeitos dos fármacos , Fígado/enzimologia , Agregação Plaquetária/efeitos dos fármacos , Coelhos , Fatores de TempoAssuntos
Oxirredutases do Álcool/isolamento & purificação , Resina de Colestiramina/farmacologia , Hidroximetilglutaril-CoA Redutases/isolamento & purificação , Microssomos Hepáticos/enzimologia , Animais , Cromatografia de Afinidade , Dextranos , Estabilidade de Medicamentos , Temperatura Alta , Hidroximetilglutaril-CoA Redutases/metabolismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Peso Molecular , Ratos , SefaroseRESUMO
The temperature sensitivity of crude solubilized 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, prepared from rat liver microsomes by different procedures, varies widely. When enzyme was solubilized by several techniques, HMG-CoA reductase activity in these extracts was rapidly and irreversibly inactivated at 4 degrees (Brown, M.S. Dana, S.E. Dietschy, J.M. and Siperstein, M.D. (1973) J. Biol. Chem 248, 4731-4738). In contrast, HMG-CoA reductase, solubilized from microsomes by a slow freeze-thaw method, was reversibility inactivated at 4 degrees over an interval of 2 h (Heller, R.A. and Gould, R.G. (1974) J. Biol. Chem 249, 5254-5260). In the present article irreversible inactivation at 4 degrees of crude solubilized HMG-CoA reductase, prepared from liver microsomes by a slow freeze-thaw technique, was investigated. In the absence of preincubation at 37 degrees, HMG-CoA reductase activity in the crude soluble extract had a half-life at 4 degrees of 4 h. Enzyme activity was more rapidly inactivated (t1/2=4 h) in extracts from younger (150 to 250 g) rats than in extracts from older (500 g) rats (t1/2=37 h). In contrast, partially purified HMG-CoA reductase was far more stable at 4 degrees (t1/2=312 h). However, when partially purified enzyme was treated with crude soluble extract, the enzyme was inactivated much more rapidly (t1/2=26 h). It is concluded that rapid irreversible inactivation of HMG-CoA reductase at 4 degrees is not an intrinsic property of this enzyme, but instead, this inactivation is caused by a factor or factors present in the crude soluble extract. While HMG-CoA reductase activity was rapidly inactivated in crude extracts from animals killed at the midpoint of the dark cycle, enzyme activity was inactivated much more slowly in extracts obtained from animals killed at the midpoint of the light cycle. These findings suggest that the concentration of the inactivating factor may vary, depending upon the physiological state of the animal. The nature of the inactivating factor is not known at the present time; however, it may be a protein since it is nondialyzable and destroyed by heat.
Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Inibidores de Hidroximetilglutaril-CoA Redutases , Microssomos Hepáticos/fisiologia , Proteínas/fisiologia , Animais , Ativação Enzimática , Cinética , Masculino , Microssomos Hepáticos/enzimologia , Ratos , TemperaturaRESUMO
Previous studies have demonstrated that both the 105,000 X g soluble supernatant (S105) and microsomal membranes from rat liver are required for the enzymatic conversion of squalene to cholesterol (Scallen, T.J., Dean, W.J., and Schuster, M.W. (1968) J. Biol. Chem. 243, 5202). It was postulated that S105 contained a noncatalytic carrier protein which was required for this enzymatic process (Scallen, T. J., Schuster, M.W., and Dhar, A.K. (1971) J. Biol. Chem. 246, 224). Later evidence demonstrated that S105 contained at least two proteins which were required for the microsomal conversion of squalene to cholesterol (Scallen, T.J., Srikantaiah, M.V., Seetharam, B., Hansbury, E., and Gavey, K.L. (1974) Fed. Proc. 33, 1733). This article describes the purification and properties of the first of these soluble proteins, sterol carrier protein1 (SCP1), which has been purified 575-fold from rat liver S105. While SCP1 specifically activated the enzymatic conversion of squalene to lanosterol by liver microsomal membranes, SCP1 possessed no capacity to activate the microsomal conversion of [3H-A14,4-dimethyl-delta8-cholestenol to C27 sterols or of [3H]7-dehydrocholesterol to cholesterol. Lanosterol was identified by silicic acid chromatography and mass spectrometry. The formation of lanosterol was a hyperbolic function of the concentration of SCP1 present in the incubation mixture. The Km observed for SCP1 was similar to the Km observed for squalene. The formation of lanosterol from squalene required FAD. The addition of phosphatidylserine increased enzymatic activity; however, phosphatidylserine was not required for this conversion. SCP1 was catalytically inactive when it was incubated with [3H] squalene and cofactors in the absence of microsomes. Substantial evidence supports the hypothesis that SCP1 operates as a noncatalytic carrier protein for the water-insoluble substrate squalene in the enzymatic conversion of squalene to lanosterol by liver microsomal membranes.