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1.
Arch Biochem Biophys ; 393(1): 117-22, 2001 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-11516168

RESUMO

The effect of Ehrlich ascites tumor cells, in vivo, on the hepatic glucose-6-phosphatase (G6Pase) system was examined. The V(max) for glucose 6-phosphate hydrolysis by G6Pase was reduced by 40% and a greater than 15-fold decrease in mRNA encoding the catalytic unit of the G6Pase system was observed 8 days after injection with tumor cells. Blood glucose concentration was decreased from 169 +/- 17 to 105 +/- 9 mg/dl in tumor-bearing mice. There was no change in the G6P transporter (G6PT) mRNA level. However, there was a significant decrease in G6P accumulation into hepatic microsomal vesicles derived from tumor-bearing mice. Decreased G6P accumulation was also associated with a decrease in G6Pase hydrolytic activity in the presence of vanadate, a potent catalytic-unit inhibitor. In addition, G6P accumulation was nearly abolished in microsomes treated with N-bromoacetylethanolamine phosphate, an irreversible inhibitor of the G6PT. These results demonstrate that the catalytic unit and G6PT components of the G6Pase system can be discriminantly regulated, and that microsomal glucose 6-phosphate uptake is dependent on catalytic unit activity as well as G6PT action.


Assuntos
Carcinoma de Ehrlich/enzimologia , Glucose-6-Fosfatase/química , Glucose-6-Fosfatase/metabolismo , Fígado/enzimologia , Animais , Glicemia/metabolismo , Carcinoma de Ehrlich/genética , Carcinoma de Ehrlich/metabolismo , Domínio Catalítico , Etanolaminas/farmacologia , Glucose-6-Fosfatase/antagonistas & inibidores , Glucose-6-Fosfatase/genética , Glucose-6-Fosfato/metabolismo , Cinética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microssomos Hepáticos/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo
2.
Arch Biochem Biophys ; 377(1): 115-21, 2000 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-10775449

RESUMO

Hepatic microsomal glucose-6-phosphatase is a multicomponent system composed of substrate/product translocases and a catalytic subunit. Previously we (Foster et al. (1996) Biochim. Biophys. Acta 12, 244-254) demonstrated that N-bromoacetylethanolamine phosphate (BAEP) is a time-dependent, irreversible inhibitor of glucose-6-phosphate hydrolysis in intact but not disrupted microsomes. We proposed that BAEP manifests its inhibitory effect by binding with a glucose-6-phosphate translocase protein of the glucose-6-phosphatase system. Here we provide additional evidence that BAEP inhibits glucose-6-phosphate transport in microsomal vesicles and utilize [(32)P]BAEP as an affinity label in the identification of a glucose-6-phosphate transport protein. In this study, we identify 51-kDa rat and mouse liver microsomal proteins involved in glucose-6-phosphate transport into and out of microsomal vesicles by utilizing (1) an Ehrlich ascites tumor-bearing mouse model, which displays a decreased sensitivity to the time-dependent inhibitory effect of BAEP, and (2) another glucose-6-phosphate translocase inhibitor, tosyl-lysine chloromethyl ketone, in conjunction with [(32)P]BAEP as an affinity label.


Assuntos
Marcadores de Afinidade/metabolismo , Antiporters/metabolismo , Carcinoma de Ehrlich/metabolismo , Etanolaminas/metabolismo , Microssomos Hepáticos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Marcadores de Afinidade/farmacologia , Animais , Antiporters/antagonistas & inibidores , Transporte Biológico/efeitos dos fármacos , Difosfatos/metabolismo , Etanolaminas/farmacologia , Glucose-6-Fosfatase/antagonistas & inibidores , Glucose-6-Fosfatase/metabolismo , Glucose-6-Fosfato/metabolismo , Hidrólise/efeitos dos fármacos , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Peso Molecular , Proteínas de Transporte de Monossacarídeos/antagonistas & inibidores , Peptídeos/química , Peptídeos/metabolismo , Permeabilidade/efeitos dos fármacos , Ratos , Fatores de Tempo , Tosilina Clorometil Cetona/farmacologia
3.
Hum Genet ; 105(5): 515-7, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10598822

RESUMO

Glycogen storage disease type 1 (GSD-1) is a group of autosomal recessive disorders caused by deficiencies in glucose-6-phosphatase (G6Pase) and the associated substrate/product transporters. Molecular genetic studies have demonstrated that GSD-1a and GSD-1b are caused by mutations in the G6Pase enzyme and a glucose-6-phosphate transporter (G6PT), respectively. While kinetic studies of G6Pase catalysis predict that the index GSD-1c patient is deficient in a pyrophosphate/phosphate transporter, the existence of a separate locus for GSD-1c remains unclear. We have previously shown that the G6Pase gene of the index GSD-1c patient is intact; we now show that the G6PT gene of this patient is normal, strongly suggesting the existence of a distinct GSD-1c locus.


Assuntos
Antiporters/genética , Doença de Depósito de Glicogênio Tipo I/genética , Doença de Depósito de Glicogênio Tipo I/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Análise Mutacional de DNA , Glucose-6-Fosfatase/genética , Humanos , Cinética , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Annu Rev Nutr ; 19: 379-406, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10448530

RESUMO

Glucose is an essential nutrient for the human body. It is the major energy source for many cells, which depend on the bloodstream for a steady supply. Blood glucose levels, therefore, are carefully maintained. The liver plays a central role in this process by balancing the uptake and storage of glucose via glycogenesis and the release of glucose via glycogenolysis and gluconeogenesis. The several substrate cycles in the major metabolic pathways of the liver play key roles in the regulation of glucose production. In this review, we focus on the short- and long-term regulation glucose-6-phosphatase and its substrate cycle counter-part, glucokinase. The substrate cycle enzyme glucose-6-phosphatase catalyzes the terminal step in both the gluconeogenic and glycogenolytic pathways and is opposed by the glycolytic enzyme glucokinase. In addition, we include the regulation of GLUT 2, which facilitates the final step in the transport of glucose out of the liver and into the bloodstream.


Assuntos
Glucose/biossíntese , Homeostase , Fígado/metabolismo , Animais , Glicemia/metabolismo , Regulação da Expressão Gênica , Glucoquinase/genética , Glucoquinase/metabolismo , Gluconeogênese , Transportador de Glucose Tipo 2 , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Glicogênio/metabolismo , Humanos , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo
5.
Arch Biochem Biophys ; 357(1): 173-7, 1998 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-9721197

RESUMO

Many agents have been used to release the latent portion of the activities catalyzed by the glucose-6-phosphatase (Glc-6-Pase) system. Detergents, which disrupt the microsomal membrane concomitantly with Glc-6-Pase activation, have been the most widely used of these agents. The treatment of microsomes with alamethicin or histone II-A has also been reported to activate the Glc-6-Pase system to the same extent as detergent treatment. While alamethicin reportedly permeabilizes the microsomal membrane (R. Fulceri et al., 1995, Biochem. J. 307, 391-397), conflicting ideas as to histone II-A's mechanism of activation have been described (J. St.-Denis et al., 1995, Biochem. J. 310, 221-224 and J. Blair and A. Burchell, 1988, Biochim. Biophys. Acta 964, 161-167). We further investigated whether activation of the Glc-6-Pase system by histone II-A is due to permeabilization of the microsomal membrane. We treated rat liver microsomes with Triton X-100, alamethicin, or histone II-A and found them to be equally effective in maximally activating the Glc-6-Pase system. We also examined the modifying effects of alamethicin and histone II-A on the sensitivity of Glc-6-Pase activities to inhibition by N-bromoacetylethanolamine phosphate (BAEP) and 3-mercaptopicolinate (3-MP), both thiol-directed reagents. Alamethicin, but not histone II-A, abolished the inhibitory effects of BAEP and 3-MP on activities of the Glc-6-Pase system. Our studies support previous reports of Glc-6-Pase activation by alamethicin via permeabilization of microsomal membranes and histone II-A activation without microsomal membrane permeabilization.


Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Glucose-6-Fosfatase/metabolismo , Histonas/farmacologia , Membranas Intracelulares/enzimologia , Microssomos Hepáticos/enzimologia , Alameticina/farmacologia , Animais , Catálise , Ativação Enzimática/efeitos dos fármacos , Etanolaminas/farmacologia , Glucose-6-Fosfatase/antagonistas & inibidores , Glucose-6-Fosfatase/efeitos dos fármacos , Pirofosfatase Inorgânica , Membranas Intracelulares/efeitos dos fármacos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Octoxinol/farmacologia , Ácidos Picolínicos/farmacologia , Pirofosfatases/metabolismo , Ratos , Ratos Endogâmicos
6.
Biochem Cell Biol ; 76(1): 115-24, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9666313

RESUMO

The low-Km activity of mannose-6-phosphatase (Man-6-Pase) has been used for many years to measure the structural integrity of microsomes. Recently histone II-A has been shown to activate glucose-6-phosphatase (Glc-6-Pase) and Man-6-Pase activities. However, in contrast to detergents, this compound appears to activate without disrupting microsomal vesicles (J.-F. St-Denis, B. Annabi, H. Khoury, and G. van de Werve. 1995. Biochem. J. 310: 221-224). This suggests that Man-6-Pase latency can be abolished without disrupting microsomal integrity and that even normally microsomes may manifest some low-Km Man-6-Pase activity without being "leaky." We have studied the relationship of Man-6-Pase with microsomal integrity further by measuring the latency of several enzymes reported to reside within the lumen of endoplasmic reticulum. We have also correlated this latency with the microsomal permeability of substrates for these enzymes. We found that (i) lumenal enzymes have different degrees of latency when compared with each other, (ii) permeability, as determined via osmotically induced changes in light scattering, is not always consistent with enzymatic latency, (iii) increases in the hydrolysis of Glc-6-P and Man-6-P were not parallel when microsomes were treated with low but increasing concentrations of detergent, and (iv) kinetic studies suggest that mannose-6-phosphate is hydrolyzed by untreated microsomes by more than a single mechanism. We propose that Man-6-Pase is not a reliable index of the integrity of microsomes.


Assuntos
Membranas Intracelulares/enzimologia , Microssomos Hepáticos/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Aciltransferases/metabolismo , Animais , Biomarcadores , Ácido Edético/farmacocinética , Glucose 1-Desidrogenase , Glucose Desidrogenase/metabolismo , Hidrólise , Membranas Intracelulares/ultraestrutura , Cinética , Masculino , Microssomos Hepáticos/ultraestrutura , Permeabilidade , Ratos
7.
Arch Biochem Biophys ; 353(1): 141-51, 1998 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-9578609

RESUMO

Certain amino acids stimulate glycogenesis from glucose. The regulatory volume decrease mechanism explaining these effects was defined by Meijer et al. (1992, J. Biol. Chem. 267, 5823-5828). It involves amino acid-induced swelling of hepatocytes resulting in loss of chloride ions which leads to deinhibition of glycogen synthase phosphatase. This results in enhanced conversion of the inactive to active form of glycogen synthase and thus enhanced glycogen synthesis. We have studied the effects of amino acids and chloride ion on the glucose-6-phosphatase system (Glc-6-Pase) with rat liver microsomal preparations, and correlated our results with those reported by others with glycogen synthase. Glc-6-Pase activities are increased by elevated ionic strength varied by increasing the concentration of various buffers or charged amino acids but are not affected by changes in osmolarity, varied with disaccharides or uncharged amino acids. With undisrupted microsomes, chloride ion competitively inhibits carbamyl phosphate: glucose phosphotransferase (KCP,t,UMi,Cl- = 19 mM) more extensively than Glc-6-P phosphohydrolase (KG6P,h,UMi,Cl- = 117 mM). Inhibition by chloride ion and activation due to ionic strength may be important considerations when assessing in vitro Glc-6-Pase activities where an attempt is made to replicate physiologic conditions. Further we propose that amino acids may play a role in increasing biosynthetic activity of Glc-6-Pase, as well as previously characterized glycogen synthase (Meijer et al., op. cit.), via the regulatory volume decrease mechanism through diminished chloride ion inhibition. Reduced concentration of chloride ion will (1) deinhibit the biosynthetic activity of Glc-6-Pase, while still inhibiting Glc-6-P hydrolysis, leading to an increased cellular concentration of Glc-6-P (an important glycogenic intermediate as well as allosteric activator of glycogen synthase) and (2) increase the active form of glycogen synthase by deinhibiting glycogen synthase phosphatase both through the previously defined mechanism (see above) and via Glc-6-P-enhanced conversion of glycogen synthase from its inactive to active form. We propose that the biosynthetic activity of Glc-6-Pase may act in concert with glycogen synthase during amino acid-induced glycogenesis from glucose.


Assuntos
Aminoácidos/farmacologia , Cloretos/farmacologia , Glucose-6-Fosfatase/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Ácido Aspártico/farmacologia , Soluções Tampão , Ativação Enzimática , Glucose-6-Fosfatase/antagonistas & inibidores , Glicogênio Sintase/metabolismo , Homeostase , Cinética , Lisina/farmacologia , Modelos Químicos , Concentração Osmolar , Fosfotransferases/metabolismo , Prolina/farmacologia , Ratos
8.
Arch Biochem Biophys ; 354(1): 125-32, 1998 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-9633606

RESUMO

The insulin-like action of tungstate in diabetic rats (A. Barberà et al., 1994, J. Biol. Chem. 269, 20047-20053) prompted us to examine the effects of tungstate on the glucose-6-phosphatase system. Our results indicate that tungstate is a potent inhibitor of glucose-6-phosphatase, with a Ki in the 10-25 microM range determined with native microsomes and in the 1-7 microM range determined with detergent-treated microsomes. With both preparations, simple linear competitive inhibition was observed versus glucose 6-phosphate (glucose-6-P) as substrate with the glucose-6-P phosphohydrolase activity of the enzyme. Tungstate was a simple linear competitive inhibitor versus carbamyl phosphate (carbamyl-P) and a linear noncompetitive inhibitor versus glucose with the carbamyl-P:glucose phosphotransferase activity of the glucose-6-phosphatase system. These findings, in addition to the observation that tungstate protected the enzyme against thermal inactivation, indicate that tungstate binds with high affinity and competes at the active site of the enzyme where the substrates glucose-6-P and carbamyl-P bind prior to catalysis. Our results suggest that potent inhibition of glucose-6-P hydrolysis by tungstate is likely responsible, at least in part, for the normalization of glycemia and the rebound in hepatic glucose-6-P levels observed in earlier studies in which tungstate exhibited insulin-like action in diabetic rats.


Assuntos
Glucose-6-Fosfatase/antagonistas & inibidores , Compostos de Tungstênio/farmacologia , Animais , Catálise , Permeabilidade da Membrana Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Glucose/metabolismo , Glucose-6-Fosfatase/metabolismo , Glucose-6-Fosfato/metabolismo , Hidrólise/efeitos dos fármacos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Fosfotransferases/metabolismo , Ratos , Temperatura , Fatores de Tempo
9.
Proc Soc Exp Biol Med ; 215(4): 314-32, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9270716

RESUMO

Work on the glucose-6-phosphatase system has intensified and diversified extensively in the past 3 years. The gene for the catalytic unit of the liver enzyme has been cloned from three species, and regulation at the level of gene expression is being studied in several laboratories worldwide. More than 20 sites of mutation in the catalytic unit protein have been demonstrated to underlie glycogenesis type 1a. inhibition of glucose-6-P hydrolysis by several newly identified competitive and time-dependent, irreversible inhibitors has been demonstrated and in several instances the predicted effects on liver glycogen formation and/or breakdown and on blood glucose production have been shown. Refinements in and additions to the presently dominant "substrate transport-catalytic unit" topological model for the glucose-6-phosphatase system have been made. A new model alternative to this, based on the "combined conformational flexibility-substrate transport" concept, has emerged. Experimental evidence for the phosphorylation of glucose in liver by high-K(m),glucose enzyme(s) in addition to glucokinase has continued to emerge, and new in vitro evidence supportive of biosynthetic functions of the glucose-6-phosphatase system in this role has appeared. High levels of multifunctional glucose-6-phosphatase have been shown present in pancreatic islet beta cells. Glucose-6-P has been established as the likely insulin secretagog in beta cells. Interesting differences in the temporal responses of glucose-6-phosphatase in kidney and liver have been demonstrated. An initial attempt is made here to meld the hepatic and pancreatic islet beta-cell glucose-6-phosphatase systems, and to a lesser extent the kidney tubular and small intestinal mucosal glucose-6-phosphatase systems into an integrated, coordinated mechanism involved in whole-body glucose homeostasis in health and disease.


Assuntos
Glucose-6-Fosfatase , Animais , Regulação Enzimológica da Expressão Gênica , Glucose-6-Fosfatase/química , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/fisiologia , Humanos , Intestino Delgado/enzimologia , Ilhotas Pancreáticas/enzimologia , Rim/enzimologia , Microssomos Hepáticos/enzimologia , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
10.
Biochim Biophys Acta ; 1297(2): 244-54, 1996 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-8917628

RESUMO

N-Bromoacetylethanolamine phosphate (BAEP) has been used previously as an affinity label to study the hexose phosphate binding sites of fructose-6-P, 2-kinase:fructose-2, 6-bisphosphatase (Sakakibara et al. (1984) J. Biol. Chem. 259, 14023-14028). We have employed this compound to probe components of the glucose-6-phosphatase system using a combination of time-dependent and immediate inhibition kinetic techniques. Inhibition of D-glucose-6-phosphate (G6P) phosphohydrolase activity of native microsomes was irreversible and time- and inhibitor-concentration-dependent. Only a partial time-dependent, irreversible inhibition of the PPi phosphohydrolase activity of native microsomes was observed. BAEP inhibited PPi:glucose phosphotransferase activity of native microsomes in a concentration-dependent, irreversible manner which was more extensive than that seen with PPi phosphohydrolase, but less extensive than was observed with G6P phosphohydrolase. Disruption of microsomal integrity by detergent-treatment either prior to incubation with BAEP or subsequent to preliminary incubation with BAEP but prior to assay for activity abolished the time-dependent inhibition. These irreversible, time- and concentration-dependent inhibitory actions of BAEP thus are manifest at a site or sites where the intact membrane-bound enzyme first makes contact with substrates G6P and PPi. An additional site of inhibition by BAEP, through relatively weak, reversible competitive inhibition at the active catalytic site, is indicated by classical steady-state kinetic analysis. The irreversible, time- and concentration-dependent inhibitions by BAEP seen with G6P and PPi as substrates strongly suggest the potential utility of radio-labeled BAEP as an affinity label for the identification and ultimate isolation and study of uncharacterized auxiliary components of the glucose-6-phosphatase system.


Assuntos
Etanolaminas/farmacologia , Glucose-6-Fosfatase/metabolismo , Microssomos Hepáticos/enzimologia , Marcadores de Afinidade/metabolismo , Animais , Detergentes/farmacologia , Difosfatos/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Etanolaminas/metabolismo , Glucose/metabolismo , Glucose-6-Fosfato/metabolismo , Cinética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Octoxinol/farmacologia , Pirofosfatases/metabolismo , Ratos , Relação Estrutura-Atividade
11.
Biochem J ; 311 ( Pt 2): 537-40, 1995 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-7487892

RESUMO

The inhibitory interactions of orthophosphate (P1) with the glucose-6-phosphatase system of intact microsomes derived from the livers of normal and Ehrlich-ascites-tumour-bearing mice reveal the appearance of a novel form of the T2 beta translocase component of the glucose-6-phosphatase system in tumour-stressed mice. Kinetic studies, with and without 20 mM P1, show a strictly classical competitive inhibition, with a K1,P1 of 4.2 mM, with disrupted microsomes from both control and tumour-bearing mouse liver. Inhibition was also observed with intact microsomes from livers of control mice, and contributions by both competitive and non-competitive components of inhibition were quantified by calculation of Kis,P1 and Kii,P1 values respectively. However, little inhibition was noted with intact microsomes from the livers of tumour-bearing mice. It is concluded that this novel form of T2 beta is less able to transport Pi, from the cytosol to the endoplasmic reticulum lumen, perhaps because of the tumour-related increased Km for Pi transport in this direction.


Assuntos
Carcinoma de Ehrlich/enzimologia , Retículo Endoplasmático/enzimologia , Glucose-6-Fosfatase/metabolismo , Microssomos Hepáticos/enzimologia , Fosfotransferases/metabolismo , Animais , Antiporters , Membrana Celular , Hidrólise , Camundongos , Proteínas de Transporte de Monossacarídeos
12.
J Clin Invest ; 95(1): 234-40, 1995 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7814621

RESUMO

Glycogen storage disease (GSD) type 1, which is caused by the deficiency of glucose-6-phosphatase (G6Pase), is an autosomal recessive disease with heterogenous symptoms. Two models of G6Pase catalysis have been proposed to explain the observed heterogeneities. The translocase-catalytic unit model proposes that five GSD type 1 subgroups exist which correspond to defects in the G6Pase catalytic unit (1a), a stabilizing protein (1aSP), the glucose-6-P (1b), phosphate/pyrophosphate (1c), and glucose (1d) translocases. Conversely, the conformation-substrate-transport model suggests that G6Pase is a single multifunctional membrane channel protein possessing both catalytic and substrate (or product) transport activities. We have recently demonstrated that mutations in the G6Pase catalytic unit cause GSD type 1a. To elucidate whether mutations in the G6Pase gene are responsible for other GSD type 1 subgroups, we characterized the G6Pase gene of GSD type 1b, 1c, and 1aSP patients. Our results show that the G6Pase gene of GSD type 1b and 1c patients is normal, consistent with the translocase-catalytic unit model of G6Pase catalysis. However, a mutation in exon 2 that converts an Arg at codon 83 to a Cys (R83C) was identified in both G6Pase alleles of the type 1aSP patient. The R83C mutation was also demonstrated in one homozygous and five heterogenous GSD type 1a patients, indicating that type 1aSP is a misclassification of GSD type 1a. We have also analyzed the G6Pase gene of seven additional type 1a patients and uncovered two new mutations that cause GSD type 1a.


Assuntos
Glucose-6-Fosfatase/genética , Doença de Depósito de Glicogênio Tipo I/classificação , Doença de Depósito de Glicogênio Tipo I/genética , Mutação/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Genoma Humano , Doença de Depósito de Glicogênio Tipo I/enzimologia , Heterozigoto , Homozigoto , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Transfecção
13.
Biochim Biophys Acta ; 1208(2): 222-8, 1994 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-7947952

RESUMO

3-Mercaptopicolinate (3-MP) inhibits D-glucose-6-phosphate (G6P) phosphohydrolase activity of the glucose-6-phosphatase system (Bode et al. (1993) Biochem. Cell Biol. 71, 113-121). We therefore attempted to maximize the inhibition by varying the physical state of microsomes, the concentration of 3-MP, and the time of preliminary incubation of 3-MP with the enzyme. The inhibition was irreversible and time- and inhibitor-concentration-dependent, with G6P phosphohydrolase activity of intact rat liver microsomes, but there was no inhibition with detergent-treated microsomes. The effectiveness of 3-MP as a time-dependent inhibitor of glucose 6-phosphatase was demonstrated in situ by measuring glycogenolysis in isolated, perfused livers from fed rats. We first exposed the livers to 2 mM 3-MP for 40 min, and then assessed the inhibitory effects on glycogenolysis. It was lowered by 50%. These observations establish that 3-MP at the mM level may be useful as an experimental probe in the study of the role(s) of G6P in the regulation of glycogenolysis as well as glycogenesis. Further, they validate the use of much lower (microM) concentrations of 3-MP to block gluconeogenesis (at the phosphoenolpyruvate carboxykinase step) without interfering with glucose 6-phosphatase. We also explored the mechanism of 3-MP inhibition. The time-dependent inhibition of carbamoyl-phosphate:glucose phosphotransferase activity with microsomes incubated with 1 mM 3-MP for 60 or 90 min and then assayed with 1 mM carbamoyl phosphate and 180 mM glucose was modest compared with inhibition of G6P phosphohydrolase. When G6P production by carbamoyl-phosphate:glucose phosphotransferase was reduced by decreasing glucose concentration to 60 mM, no inhibition by 3-MP was discernible. There was no inhibition of inorganic pyrophosphatase activity. These studies support the model of time-dependent, irreversible reaction of 3-MP with the G6P translocase component of the glucose-6-phosphatase system.


Assuntos
Glucose-6-Fosfatase/antagonistas & inibidores , Ácidos Picolínicos/farmacologia , Animais , Relação Dose-Resposta a Droga , Glicogênio/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos , Fatores de Tempo
14.
J Biol Chem ; 269(11): 7879-86, 1994 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-8132505

RESUMO

The probable involvement of hepatic carbamyl-P in the reciprocal relationship between hepatic ureagenesis and glycogenesis from glucose was explored. Isolated perfused liver preparations from 48-h fasted rats were employed. Moderate (9.2 mM) and relatively high levels of glucose (34 mM) were perfused. Hepatic glycogenesis, glucose-6-P, carbamyl-P, and citrulline levels, hepatic urea formation, and ureagenesis based upon perfusate urea levels were measured. Experimental probes selected to modify hepatic ureagenesis and carbamyl-P production and utilization included: (a) NH4Cl, maintained at 5 mM by continuous infusion (NH4+ is a substrate for carbamyl-P synthase I and glutamate dehydrogenase); (b) norvaline, an inhibitor of ornithine transcarbamylase which catalyzes the first committed step in the urea cycle; and (c) ethoxyzolamide, an inhibitor of carbonic anhydrase which produces HCO3-, an essential substrate for carbamyl-P synthase I. NH4+ increased ureagenesis and decreased glycogenesis. The inclusion of norvaline with NH4+ decreased ureagenesis and increased glycogenesis. Ethoxyzolamide with or without NH4+ inhibited both ureagenesis and glycogenesis, and decreased the hepatic glucose-6-P level. Glycogenesis was greater at 34 mM than 9.2 mM glucose, increased in norvaline-containing preparations correlative with increased availability of carbamyl-P, and decreased when carbamyl-P formation was inhibited by ethoxyzolamide. Kinetic analysis indicated a Km, Glc of 31 mM for glucose phosphorylation preliminary to glycogenesis. Glycogen formation via the "indirect pathway" (i.e. involving extrahepatic glycolysis, transport of lactate to the liver, and glyconeogenesis therefrom) was quantitatively insufficient to account for the observed glycogenesis. Glucokinase is contraindicated by the inverse relationship between hepatic glycogenesis and ATP availability in the ethoxyzolamide-treated preparations. In contrast, carbamyl-P:glucose phosphotransferase activity of the glucose-6-phosphatase system has the characteristics to bridge hepatic ureagenesis and glycogenesis.


Assuntos
Cloreto de Amônio/farmacologia , Etoxzolamida/farmacologia , Glucose/metabolismo , Glicogênio Hepático/biossíntese , Fígado/metabolismo , Ureia/metabolismo , Valina/análogos & derivados , Animais , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Glucoquinase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Hexoquinase/metabolismo , Técnicas In Vitro , Cinética , Fígado/efeitos dos fármacos , Modelos Biológicos , Tamanho do Órgão , Ornitina Carbamoiltransferase/antagonistas & inibidores , Perfusão , Ratos , Valina/farmacologia
15.
J Biol Chem ; 268(22): 16298-301, 1993 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-8344917

RESUMO

L-Proline and L-glutamine were used to probe the inverse relationship between glycogenesis and ureagenesis in isolated, perfused livers from 48-h fasted rats. Both amino acids may provide nitrogen in the form of NH+4 for carbamyl-P synthesis. However, one molecule of glutamine may provide additionally for the synthesis of one molecule of the urea cycle substrate L-aspartate, but proline can provide for the synthesis of a molecule of NH+4 or one molecule of aspartate on an either/or basis only. In all perfusates, glucose was initially 30 mM (to favor phosphotransferase activity of glucose-6-phosphatase) and 0.5 mM 3-mercaptopicolinate was present (to inhibit glyconeogenesis from endogenous substrates, from the added amino acids, and via the indirect pathway). Glycogenesis from glucose, perfusate and hepatic urea formation, and levels of hepatic glucose-6-P, citrulline, PPi, and carbamyl-P were measured. The addition of glutamine to the perfusate markedly stimulated the urea cycle, but not glycogenesis. Hepatic urea level, perfusate urea concentration, and hepatic citrulline and PPi increased while carbamyl-P content decreased. In contrast, proline stimulated glycogenesis from glucose, but not ureagenesis. In the proline-supplemented compared with glutamine group, hepatic glycogenesis and carbamyl-P content increased; hepatic glucose-6-P levels showed a tendency toward increase; and hepatic urea formation, hepatic citrulline, and PPi levels were decreased. These observations are interpreted to support an hepatic mechanism whereby the relative availability of carbamyl-P to the urea cycle and as a substrate for glucose phosphorylation via phosphotransferase activity of the glucose-6-phosphatase system preliminary to glycogenesis from glucose is a major metabolic determinant.


Assuntos
Glucose/metabolismo , Glutamina/metabolismo , Glicogênio/biossíntese , Fígado/metabolismo , Prolina/metabolismo , Ureia/metabolismo , Animais , Carbamoil-Fosfato/metabolismo , Ratos
17.
Biochim Biophys Acta ; 1163(3): 297-302, 1993 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-8389592

RESUMO

Although Canfield and Arion (J. Biol. Chem. 263, 7458-7460 (1990)) have described the kinetics as hyperbolic, Traxinger and Nordlie (J. Biol. Chem. 262, 10015-10019 (1987)) reported sigmoid kinetics in the glucose-6-phosphatase system of intact microsomes at near-physiologic glucose-6-P concentrations. We show here that apparent sigmoidal kinetics, most clearly seen as sharp upward inflections in Hanes plots as substrate concentration approaches zero, are a consequence of the hysteretic lag in product formation during the first minutes of incubation of the enzyme with low concentrations of substrate. The appearance of sigmoidicity, observed when reaction velocities are calculated from changes in Pi concentration between 0 and 6 min of incubation, is not present when velocity is determined from slopes of [product]-time plots after linearity is achieved. The Km,glucose-6-P value, 0.86 mM, based on these hysteresis-corrected velocity values determined with intact microsomes from normal, control rats at low substrate concentrations, approached the upper limit of physiologic hepatic glucose-6-P concentrations. This suggests that glucose-6-phosphatase activity may be regulated by factors other than substrate concentrations alone. We propose that the hysteretic behavior, not sigmoid kinetics of the glucose-6-phosphatase enzyme system, may be a prime regulatory feature.


Assuntos
Diabetes Mellitus Experimental/enzimologia , Glucose-6-Fosfatase/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Glucose-6-Fosfato , Glucofosfatos/metabolismo , Cinética , Masculino , Fosfatos/análise , Ratos , Reprodutibilidade dos Testes , Estreptozocina , Fatores de Tempo
18.
Biochem Cell Biol ; 71(3-4): 113-21, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8398068

RESUMO

3-Mercaptopicolinae (3-MP) blocks gluconeogenesis from lactate, pyruvate, alanine, and other substrates through its inhibition of phosphoenolpyruvate carboxykinase. Nevertheless, we observed increased glycogenesis, net glucose uptake, and glucose-6-P levels in livers perfused with glucose in the presence of 3-MP. In perfusions with 20 mM dihydroxyacetone, increased glycogenesis and decreased glucose production were observed with 3-MP. These metabolic effects suggested additional site(s) of action of 3-MP. Further studies showed that 3-MP inhibits glucose-6-P phosphohydrolase activity of intact liver microsomes. Several compounds with structural similarities to 3-MP (2-mercaptonicotinic acid, picolinic acid, cysteine, reduced glutathione, nicotinic acid, quinolinic acid, tryptophan, and pyridine) were tested for their effect on glucose-6-P phosphohydrolase activity. Two of these compounds, 2-mercaptonicotinic acid and picolinic acid, were found to inhibit. In perfusions including 7.5 mM fructose, the addition of 3-MP, 2-mercaptonicotinic acid, or picolinic acid increased glycogenesis, decreased glucose production, and increased hepatic glucose-6-P concentrations. These observations indicate that the inhibition of glucose-6-P phosphohydrolase may play a role in enhanced glycogenesis from glucose, dihydroxyacetone, and fructose in isolated livers from 48-h fasted rats perfused with 3-MP or certain sulfhydryl-containing and sulfhydryl-devoid analogs.


Assuntos
Glucose-6-Fosfatase/efeitos dos fármacos , Fígado/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/antagonistas & inibidores , Ácidos Picolínicos/farmacologia , Animais , Di-Hidroxiacetona/farmacologia , Glucoquinase/efeitos dos fármacos , Glucose/metabolismo , Glicogênio/biossíntese , Glicogênio Sintase/efeitos dos fármacos , Técnicas In Vitro , Fígado/enzimologia , Masculino , Modelos Biológicos , Ácidos Nicotínicos/farmacologia , Perfusão , Fosforilases/efeitos dos fármacos , Ratos , Compostos de Sulfidrila/farmacologia
19.
Biochem J ; 290 ( Pt 3): 907-11, 1993 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-8384451

RESUMO

To examine the effects of the presence of Ehrlich ascites tumours on both the catalytic unit and the substrate/product translocase components of the glucose-6-phosphatase system in vivo, we isolated microsomes from the livers of control and tumour-bearing mice. Samples were analysed immunochemically for the quantity of catalytic unit, stabilizing protein and translocases T2 and T3 proteins. In comparison experiments, a variety of kinetic studies were performed. The most striking findings in tumour-bearing mice were: a 2.5-fold increase in the quantity of translocase T2 protein; increases in the Km and Vmax. for glucose 6-phosphate phosphohydrolase; and a decrease in the Km value for carbamoyl phosphate (carbamoyl-P) of carbamoyl-P:glucose phosphotransferase, all with intact microsomes. The percentage latency at Vmax. decreased for PPi phosphohydrolase and for glucose 6-phosphate phosphohydrolase, but was unaffected for carbamoyl-P:glucose phosphotransferase. These observations support a tumour-related increase in translocase T2 capacity in vivo, as it transports Pi from the microsomal lumen to the medium and carbamoyl-P or PPi from the medium to the microsomal lumen.


Assuntos
Carcinoma de Ehrlich/enzimologia , Glucose-6-Fosfatase/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Antiporters , Imuno-Histoquímica , Cinética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas de Transporte de Monossacarídeos , Fosfotransferases/metabolismo
20.
Eur J Pediatr ; 152 Suppl 1: S2-6, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8391443

RESUMO

The discovery of glucose-6-phosphatase (EC 3.1.3.9) and of its physiological function in releasing glucose from the liver are discussed briefly. The identification by the Coris of glucose-6-phosphatase deficiency as the underlying defect in certain cases of glycogenosis (type I glycogenosis; von Gierke disease) is described. Characteristics of the catalyst, with a focus on its multiplicity of functions and multicomponent character, are considered with an emphasis on the human liver enzyme. Pioneering studies from the author's laboratory leading to the characterization of two variants of type I glycogenosis, types Ib and Ic, are described.


Assuntos
Glucose-6-Fosfatase/metabolismo , Doença de Depósito de Glicogênio Tipo I/enzimologia , Microssomos Hepáticos/enzimologia , Doença de Depósito de Glicogênio Tipo I/classificação , Doença de Depósito de Glicogênio Tipo I/fisiopatologia , Humanos
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