Molecular pathologies of the hepatic endoplasmic reticulum.
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Animals↗
Biomedical subjects
A Burchell
Publications and source records attributed to A Burchell.
A direct method for the diagnosis of human hepatic type 1b and type 1c glycogen-storage disease.
1. Type 1b and type 1c glycogen-storage disease are caused respectively by deficiencies of the glucose-6-phosphate translocase and the phosphate/pyrophosphate translocase of the human hepatic microsomal glucose-6-phosphatase system. 2. Current methods of unequivocally diagnosing type 1b and type 1c glycogen storage disease are indirect and complex. 3. We have therefore developed a simple, rapid and direct microfiltration assay for the glucose-6-phosphate translocase and the phosphate/pyrophosphate translocase. 4. We have demonstrated that the microfiltration assay can be used to directly diagnose type 1b and 1c glycogen-storage disease in microsomes isolated from hepatic needle-biopsy samples.
Antiporters↗
Identification of the human hepatic microsomal glucose-6-phosphatase enzyme.
The glucose-6-phosphatase enzyme protein of the human hepatic microsomal glucose-6-phosphatase system was identified as a 36.5 kDa polypeptide. The 36.5 kDa glucose-6-phosphatase enzyme protein was shown to be absent in the microsomes isolated from a patient previously diagnosed as having a type 1a glycogen storage disease.
Animals↗
The microsomal glucose-6-phosphatase enzyme of pancreatic islets.
Microsomal fractions isolated from pancreatic islet cells were shown to contain high specific glucose-6-phosphatase activity. The islet-cell glucose-6-phosphatase enzyme has the same Mr (36,500), similar immunological properties and kinetic characteristics to the hepatic microsomal glucose-6-phosphatase enzyme.
Animals↗
A new microtechnique for the analysis of the human hepatic microsomal glucose-6-phosphatase system.
A microtechnique has been developed which enables a complete kinetic analysis of the human hepatic microsomal glucose-6-phosphatase system to be carried out in microsomes isolated from very small liver samples. Complete or partial deficiencies of any of the proteins of the glucose-6-phosphatase system resulting in Type 1a, 1b, 1c or 1d glycogen storage disease can be therefore be diagnosed using hepatic needle biopsy samples, whereas previous methods of diagnosis needed large wedge biopsy samples requiring laparotomy.
Female↗
The identification of T2; the phosphate/pyrophosphate transport protein of the hepatic microsomal glucose-6-phosphatase system.
The phosphate/pyrophosphate translocase protein (T2) of the hepatic microsomal glucose-6-phosphatase system was identified and then purified using antibodies raised against the rat mitochondrial phosphate/hydroxyl ion antiport protein. The T2 protein was shown to be absent in the microsomes isolated from a patient previously diagnosed as having type lc glycogen storage disease.
Animals↗
The phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system is a 36.5-kilodalton polypeptide.
The phosphohydrolase component of the microsomal glucose-6-phosphatase system has been identified as a 36.5-kDa polypeptide by 32P-labeling of the phosphoryl-enzyme intermediate formed during steady-state hydrolysis. A 36.5-kDa polypeptide was labeled when disrupted rat hepatic microsomes were incubated with three different 32P-labeled substrates for the enzyme (glucose-6-P, mannose-6-P, and PPi) and the reaction terminated with trichloroacetic acid. Labeling of the phosphoryl-enzyme intermediate with [32P]glucose-6-P was blocked by several well-characterized competitive inhibitors of glucose-6-phosphatase activity (e.g. Al(F)-4 and Pi) and by thermal inactivation, and labeling was not seen following incubations with 32Pi and [U-14C]glucose-6-P. In agreement with steady-state dictates, the amount of [32P]phosphoryl intermediate was directly and quantitatively proportional to the steady-state glucose-6-phosphatase activity measured under a variety of conditions in both intact and disrupted hepatic microsomes. The labeled 36.5-kDa polypeptide was specifically immunostained by antiserum raised in sheep against the partially purified rat hepatic enzyme, and the antiserum quantitatively immunoprecipitated glucose-6-phosphatase activity from cholate-solubilized rat hepatic microsomes. [32P]Glucose-6-P also labeled a similar-sized polypeptide in hepatic microsomes from sheep, rabbit, guinea pig, and mouse and rat renal microsomes. The glucose-6-phosphatase enzyme appears to be a minor protein of the hepatic endoplasmic reticulum, comprising about 0.1% of the total microsomal membrane proteins. The centrifugation of sodium dodecyl sulfate-solubilized membrane proteins was found to be a crucial step in the resolution of radiolabeled microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Animals↗
The mechanism of histone activation of the hepatic microsomal glucose-6-phosphatase system: a novel method to assay glucose-6-phosphatase activity.
The mechanism of activation of hepatic microsomal glucose-6-phosphatase (EC 3.1.3.9) by histone 2A has been investigated in both intact and disrupted microsomes. Histone 2A increased the Vmax and decreased the Km of glucose-6-phosphatase in intact microsomes but had no effect on glucose-6-phosphatase activity in disrupted microsomes. Histone 2A was shown to activate glucose-6-phosphatase in intact microsomes by disrupting the membrane vesicles and thereby allowing the direct measurement of the activity of the latent glucose-6-phosphatase enzyme. The study demonstrated that disrupting microsomes with histone 2A is an excellent method for directly assaying glucose-6-phosphatase activity as it poses none of the problems encountered with all of the previously used methods.
Animals↗
Development of the rat hepatic microsomal glucose-6-phosphatase system and its glucocorticoid inducibility.
Rat liver glucose-6-phosphatase activity was determined in both fully activated and intact microsomal preparations during perinatal development. Activities increased rapidly in both preparations following birth, then gradually fell to adult values. The latency of the glucose-6-phosphatase system also increased neonatally whereas hepatic glycogen concentrations decreased postnatally. The effect of dexamethasone pretreatment on the hepatic glucose-6-phosphatase system was also investigated. Dexamethasone pretreatment failed to decrease the latency of glucose-6-phosphatase in microsomes from neonatal rats but decreased the latency of the enzyme in adult female rat liver from 44 to 12% of total activity.
Aging↗
Diagnosis of type 1a and type 1c glycogen storage diseases in adults.
The hepatic glucose-6-phosphatase system was studied with a novel microanalytical technique in adult patients undergoing liver biopsy. 4 patients were diagnosed as having type 1 glycogen storage disease (GSD). 3 of these patients, who had hypoglycaemic symptoms, had variations of type 1a GSD, which is caused by a defect in the hepatic microsomal glucose-6-phosphatase enzyme. The fourth, with hepatomegaly and no hypoglycaemic symptoms, had a normal glucose-6-phosphatase enzyme but a defect in the hepatic microsomal phosphate/pyrophosphate translocase T2; this is the first report of an adult with type 1c GSD. Adult type 1 GSD should be considered in patients with unresolved hypoglycaemic symptoms and/or unresolved hepatomegaly.
Adult↗
Aluminum ions are required for stabilization and inhibition of hepatic microsomal glucose-6-phosphatase by sodium fluoride.
Stabilization and inhibition of hepatic microsomal glucose-6-P phosphohydrolase (EC 3.1.3.9) by F- requires the presence of Al3+ ions. At millimolar concentrations, reagent grade NaF inhibited glucose-6-P hydrolysis and protected the enzyme against inactivation induced by heat in the presence of 0.025% (w/v) Triton X-100 or by reaction of the catalytic site with the histidine-specific reagent, diethyl pyrocarbonate. The presence of millimolar EDTA in all test systems abolished the effectiveness of NaF, yet EDTA by itself was without significant influence on the kinetics of phosphohydrolase reaction, the thermal stability of the enzyme or its reactivity with diethyl pyrocarbonate. Although ultrapure NaF was ineffectual in all test systems, its potency as a competitive inhibitor or protective agent was markedly increased by micromolar AlCl3 or when assays were carried out in flint glass test tubes. The latter response is explained by the well documented ability of fluoride solutions to extract Al3+ from glass at neutral pH. Our analysis indicates that the effectiveness of fluoride in all test systems derives from the formation of a specific complex with Al3+, most likely Al(F)4-. The apparent dissociation constant for interaction of the enzyme and Al(F)4- is 0.1 microM. The combination of NaF and AlCl3 holds promise as an unusually effective and versatile means to stabilize this notoriously labile enzyme during efforts to purify it.
Aluminum↗
Stabilization of glucose-6-phosphatase activity by a 21 000-dalton hepatic microsomal protein.
Hepatic microsomal glucose-6-phosphatase activity was rendered extremely unstable by a variety of techniques: (a) incubation at pH 5.0; (b) extraction of the microsomal fraction in the presence of 1% Lubrol; (c) various purification procedures. These techniques all result in the removal of a 21 kDa polypeptide from the fraction containing glucose-6-phosphatase activity. The 21 kDa protein was purified to apparent homogeneity by solubilization in the detergent Lubrol 12A-9 and chromatography on Fractogel TSK DEAE-650(S) and centrifugation at 105 000 g. The 21 kDa protein stabilizes glucose-6-phosphatase activity, whereas other purified hepatic microsomal proteins do not. The 21 kDa protein appears to be a potential regulator of glucose-6-phosphatase activity.
Animals↗
A simple method for purification of rat hepatic microsomal cytochrome b5.
Rat hepatic microsomal cytochrome b5 was purified to homogeneity by solubilization with the detergent Lubrol 12-A9 and chromatography on Fractogel TSK DEAE-650(S). The protein was obtained in high yield (52-87%) in 8 h, and only one polypeptide band, of Mr 16 600, was visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.
Animals↗
Rat hepatic microsomal glucose-6-phosphatase protein levels are increased in streptozotocin-induced diabetes.
Hepatic microsomal glucose-6-phosphatase activity levels and the hepatic output of glucose are increased in diabetes. We have used protein chemistry and immunological techniques to determine the mechanism by which the activity levels of the glucose-6-phosphatase system are increased in streptozotocin-induced diabetic rats. In the streptozotocin-induced diabetic rats, the activity of the glucose-6-phosphatase enzyme increased four-fold without appreciably altering the transport capacity of the glucose-6-phosphatase system. The solubilized diabetic rat liver glucose-6-phosphatase enzyme appeared to be very similar to the solubilized enzyme from control rat liver microsomes. They exhibit the same Km, are labile at 30 degrees C, are stabilized by sodium fluoride and they migrate to the same position during density gradient centrifugation. Immunological studies demonstrated that a greater amount of hepatic microsomal glucose-6-phosphatase enzyme protein is present in diabetic rats than in control rats. Thus, we have determined for the first time that increased levels of the glucose-6-phosphatase protein are present in streptozotocin-induced diabetes. The significance of this finding in relation to the regulation of the hepatic microsomal glucose-6-phosphatase system is discussed.
Animals↗
Specific inactivation of the phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system by diethyl pyrocarbonate.
We have examined the interactions of the histidine-specific reagent diethyl pyrocarbonate (DEPC) with the components of the rat hepatic glucose-6-phosphatase system (EC 3.1.3.9). DEPC is the first known reagent that satisfies the criteria of an active-site-specific label for the phosphohydrolase component. (a) It inactivates through formation of a stable covalent bond. (b) It is effective at reasonably low concentrations (2-4 mM) under relatively mild conditions (e.g. 30 degrees C at neutral pH). (c) Inactivation is substantially blocked by glucose 6-phosphate, Pi and NaF, compounds which are known to interact quite specifically with the phosphohydrolase. (d) Under conditions where glucose 6-phosphate and NaF protect the enzyme, no protection is provided against DEPC-mediated inactivation of two other functional components of the membrane, the glucose 6-phosphate translocase and UDP-glucuronyltransferase. DEPC also shows potential for use at 0 degree C as a label for UDP-glucuronyltransferase.
Animals↗
Cost-effectiveness of community psychiatric nurse or out-patient psychiatrist care of neurotic patients.
In a prospective controlled investigation chronic patients, the majority with neurotic conditions, were randomly allocated either to community psychiatric nurses (CPN) as the main therapist or to routine out-patient psychiatrist follow-up. There was no statistically significant difference in the mean public expenditure for the two modes of care. The direct costs of psychiatric care comprised a small proportion of total public expenditure and were initially greater in the nursing group. Subsequently, and over the whole 18-month study period, nursing was the cheaper option in terms of these direct costs. Clinical and social outcomes were comparable in both follow-up groups. Consumer satisfaction was significantly greater among CPN patients. On balance, these results confirm the benefit of community psychiatric nursing for this patient group.
Adolescent↗
Identification and purification of a liver microsomal glucose 6-phosphatase.
1. Hepatic glucose 6-phosphatase activity was purified 65-fold in good yield over that in cholate-solubilized microsomal fractions. 2. This preparation still contained five major polypeptides and numerous minor contaminants. 3. The smallest of the five major polypeptides (Mr approx. 18 500) could be purified from heat-treated microsomal fractions. 4. Antisera raised against the heat-stable protein doublet was used to immunoprecipitate specifically glucose 6-phosphatase activity from cholate-solubilized microsomal fractions. 5. This work indicates that hepatic microsomal glucose 6-phosphatase appears to be one or both of the low-molecular-weight heat-stable polypeptides.
Animals↗
A critical evaluation of the possible modulation of hepatic microsomal glucose-6-phosphatase activity by protein phosphorylation.
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Animals↗