Regulation of mitochondrial respiration in liver.
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Animals↗
Biomedical subjects
J M Tager
Publications and source records attributed to J M Tager.
Peroxisomal disorders: a newly recognised group of genetic diseases.
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Bile Acids and Salts↗
A prenatal test for the cerebro-hepato-renal (Zellweger) syndrome by demonstration of the absence of catalase-containing particles (peroxisomes) in cultured amniotic fluid cells.
In this paper we show that whereas acyl-CoA: dihydroxyacetone phosphate acyltransferase, a membrane-bound peroxisomal enzyme, is deficient in homogenates of cultured amniotic fluid cells of fetuses with Zellweger syndrome, catalase a soluble peroxisomal matrix enzyme is present in normal amounts. Digitonin titration experiments revealed a striking difference in the percentage of particle-bound catalase in control and Zellweger amniocytes: in Zellweger amniocytes all catalase activity was found to be present in the soluble cytoplasm, (less than 5% particle-bound), whereas in control amniocytes catalase was found to be predominantly particle-bound (62% +/- 8%, n = 5). Measurement of the percentage of particle-bound catalase by means of digitonin titrations thus provides a simple prenatal test for Zellweger syndrome via the direct demonstration of the presence or absence of catalase-containing particles (peroxisomes).
Acyltransferases↗
Infantile Refsum disease: deficiency of catalase-containing particles (peroxisomes), alkyldihydroxyacetone phosphate synthase and peroxisomal beta-oxidation enzyme proteins.
In recent years a number of biochemical abnormalities have been described in patients with the infantile form of Refsum disease, including the accumulation of very long chain fatty acids, trihydroxycoprostanoic acid and pipecolic acid. In this paper we show that catalase-containing particles (peroxisomes), alkyl dihydroxyacetone phosphate synthase and acyl-CoA oxidase protein are deficient in patients with infantile Refsum disease. These findings suggest that in the infantile form of Refsum disease, as in the cerebro-hepato-renal (Zellweger) syndrome the multiplicity of biochemical abnormalities is due to a deficiency of peroxisomes and hence to a generalized loss of peroxisomal functions. As a consequence the infantile form of Refsum disease can be diagnosed biochemically by methods already available for the prenatal and postnatal diagnosis of the cerebro-hepato-renal (Zellweger) syndrome.
Acatalasia↗
Age-related differences in plasmalogen content of erythrocytes from patients with the cerebro-hepato-renal (Zellweger) syndrome: implications for postnatal detection of the disease.
Phosphatidylethanolamine plasmalogen levels were determined in erythrocytes from controls and 13 patients with the cerebro-hepato-renal (Zellweger) syndrome. It was found that in Zellweger patients 20 weeks of age or younger, erythrocyte phosphatidylethanolamine plasmalogen levels were lowered whereas in older patients (except in one) normal levels were found. The results obtained suggest a close relationship between the age of the patients at sampling and the phosphatidylethanolamine plasmalogen levels in their erythrocytes. A possible explanation for these findings and the implications for the postnatal detection of Zellweger syndrome are discussed.
Brain↗
Biosynthesis and maturation of peroxisomal beta-oxidation enzymes in fibroblasts in relation to the Zellweger syndrome and infantile Refsum disease.
The biosynthesis of the peroxisomal enzymes acyl-CoA oxidase, 3-oxoacyl-CoA thiolase (acetyl-CoA acyl-transferase, EC 2.3.1.16), and catalase (EC 1.11.1.6) was studied in cultured skin fibroblasts from a control subject and from patients with Zellweger syndrome and the infantile form of Refsum disease, inherited disorders in which peroxisomes are deficient and certain peroxisomal functions are impaired. The results of continuous labeling and pulse-chase experiments indicate that in control fibroblasts, as in rat liver, acyl-CoA oxidase is synthesized as a 72-kDa percursor that is converted to two polypeptides of 52 and 20 kDa and 3-oxoacyl-CoA thiolase is synthesized as a 44-kDa precursor that is converted to the 41-kDa mature protein. In fibroblasts from the patients the precursors of the two enzymes are formed but their maturation is impaired, and they are rapidly degraded. In contrast, the biosynthesis of catalase is not impaired. We conclude that functional peroxisomes are required for the maturation and stability of acyl-CoA oxidase and 3-oxoacyl-CoA thiolase but not for catalase.
Abnormalities, Multiple↗
A specific acid alpha-glucosidase in lamellar bodies of the human lung.
In the present investigation, we have demonstrated that three lysosomal-type hydrolases, alpha-glucosidase, alpha-mannosidase and a phosphatase, are present in lamellar bodies isolated from adult human lung. The hydrolase activities that were studied, all showed an acidic pH optimum, which is characteristic for lysosomal enzymes. The properties of acid alpha-glucosidase in the lamellar body fraction and that in the lysosome-enriched fraction were compared. Using specific antibodies against lysosomal alpha-glucosidase from human placenta, two alpha-glucosidases could be distinguished in the lamellar body fraction: one with a high affinity to the antibodies as found in the lysosome-enriched fraction and another with a much lower affinity. Both forms showed an acidic pH optimum. The same heterogeneity of alpha-glucosidase in the lamellar body fraction could be observed using immobilized concanavalin A. The lectin was able to precipitate nearly all alpha-glucosidase activity of the lysosome-enriched fraction. In contrast, 30% of the alpha-glucosidase activity in the lamellar body fraction was not precipitable. Furthermore, the lamellar body alpha-glucosidase with the low antibody affinity could not be bound to concanavalin A. The results suggest that lamellar bodies contain at least two acid alpha-glucosidases: one similar to the lung lysosomal alpha-glucosidase, and another lamellar body-specific isoenzyme with a different immunoreactivity and lectin affinity. The lamellar body-specific alpha-glucosidase should prove useful as a lamellar body-specific marker enzyme.
Antibodies↗
Mechanism of the stimulation of respiration by fatty acids in rat liver.
The mechanism of stimulation of hepatic respiration by fatty acids was studied in isolated rat hepatocytes. Stimulation of respiration by fatty acids varied from about 35% to about 105% depending on chain length. The stimulatory effect of octanoate (1 mM) or oleate (0.5 mM) was prevented by oligomycin (2 micrograms/ml). With carboxyatractyloside (100 microM) and ouabain (2 mM) the stimulation of respiration was partially inhibited (by 50-70 and 50-60%, respectively). From these results it can be concluded that the increased rate of respiration after addition of fatty acids is coupled to ATP synthesis. A large part (50-60%) of this ATP is utilized by the (Na+ + K+)-ATPase.
2,4-Dinitrophenol↗
Biosynthesis and intracellular transport of alpha-glucosidase and cathepsin D in normal and mutant human fibroblasts.
In order to study the intracellular localization of the proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D in cultured human skin fibroblasts we have used incubation with glycyl-L-phenylalanine-beta-naphthylamide (Gly-Phe-NH-Nap) as described by Jadot et al. [Jadot, M., Colmant, C., Wattiaux-de Coninck, S. & Wattiaux, R. (1984) Biochem. J. 219,965-970] for the specific lysis of lysosomes. When a homogenate of fibroblasts was incubated for 20 min with 0.5 mM Gly-Phe-NH-Nap, a substrate for the lysosomal enzyme cathepsin C, the latency of the lysosomal enzymes alpha-glucosidase and beta-hexosaminidase decreased from 75% to 10% and their sedimentability from 75% to 20-30%. In contrast, treatment with Gly-Phe-NH-Nap had no significant effect on the latency of galactosyltransferase, a marker for the Golgi apparatus, and on the sedimentability of glutamate dehydrogenase and catalase, markers for mitochondria and peroxisomes, respectively. The maturation of alpha-glucosidase and cathepsin D in fibroblasts was studied by pulse-labelling with [35S]methionine, immunoprecipitation, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and fluorography. When homogenates of labelled fibroblasts were incubated with Gly-Phe-NH-Nap prior to immunoprecipitation, 70-80% of all proteolytically processed forms of metabolically labelled alpha-glucosidase and cathepsin D was recovered in the supernatant. The earliest proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D appeared to be coupled to their transport to the lysosomes. Although both enzymes are transported via the mannose-6-phosphate-specific transport system, the velocity with which they arrived in the lysosomes was consistently different. Whereas newly synthesized cathepsin D was found in the lysosomes 1 h after synthesis, alpha-glucosidase was detected only after 2-4 h. When a pulse-chase experiment was carried out in the presence of 10 mM NH4Cl there was a complete inhibition of the transport of cathepsin D and a partial inhibition of that of alpha-glucosidase to the lysosomes. Leupeptin, an inhibitor of lysosomal thiol proteinases, had no effect on the transport of labelled alpha-glucosidase to the lysosomes. However, the early processing steps in which the 110-kDa precursor is converted to the 95-kDa intermediate form of the enzyme were delayed, a transient 105-kDa form was observed and the conversion of the 95-kDa intermediate form to the 76-kDa mature form of the enzyme was completely inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)
Ammonium Chloride↗
Role of plasma membrane transport in hepatic glutamine metabolism.
In livers of fed rats and in perfused livers supplied with a physiological portal glutamine concentration of 0.6 mM, the mitochondrial and cytosolic glutamine concentrations are 20 mM and 7 mM, respectively, thus, the mitochondrial/cytosolic glutamine concentration gradient is 2-3. Uptake and release of glutamine by periportal and perivenous hepatocytes occurs predominantly by an Na+-dependent transport system (so-called system 'N'). Histidine in near-physiological concentrations inhibits both glutamine uptake by periportal hepatocytes and its release by perivenous hepatocytes. This is not due to an inhibition of glutamine-metabolizing enzymes by histidine or its metabolites. With physiological portal glutamine concentrations (0.6 mM), stimulation of glutaminase flux or of glutamine transaminase flux is followed by a decrease of hepatic glutamine levels to about 80% or 30%, respectively, glutamine levels are further decreased to 50% or 20% in the presence of histidine. When glutamine is synthesized endogenously (no glutamine added), the histidine-induced inhibition of glutamine release is paralleled by a 210% increase of the hepatic tissue level of glutamine. In experiments with and without methionine sulfoximine and in the absence of added glutamine, the glutamine content in the small perivenous hepatocyte population containing glutamine synthetase is estimated to be about 3.5 mumol/g wet weight and that in the periportal hepatocytes as low as 0.1 mumol/g wet weight. In contrast to the prevailing view, it is concluded that glutamine transport across the plasma membrane of hepatocytes is a potential regulatory site in glutamine degradation and synthesis, especially under the influence of effectors like histidine.
Amino Acids↗
Deficiency of acyl-CoA:dihydroxyacetone phosphate acyltransferase in thrombocytes of Zellweger patients: a simple postnatal diagnostic test.
Activity of acyl-CoA: dihydroxyacetone phosphate acyltransferase, a membrane-bound peroxisomal enzyme in mammalian liver cells catalyzing the first step in the biosynthesis of etherphospholipids, is detectable in thrombocytes isolated from blood of human controls. However, in thrombocytes from patients affected by the cerebro-hepato-renal (Zellweger) syndrome, the activity of this enzyme is severely reduced, permitting rapid postnatal biochemical detection of this severe inborn disease, by measuring the enzyme activity in patients' thrombocytes.
Acyltransferases↗
The occurrence of two immunologically distinguishable beta-glucocerebrosidases in human spleen.
The beta-glucosidase activity in spleen from control subjects and patients with different clinical phenotypes of Gaucher's disease was characterized. The occurrence of a soluble non-specific beta-glucosidase with a neutral pH optimum and two membrane-associated beta-glucocerebrosidases with an acid pH optimum was demonstrated. The two beta-glucocerebrosidases can be distinguished on the basis of their ability to react with anti-(placental beta-glucocerebrosidase) antibodies bound to protein-A--Sepharose 4B beads. One of the splenic beta-glucocerebrosidases (form I) is precipitated by the immobilized antibodies and the other (form II) is not. The two forms also differ in binding affinity to concanavalin A, degree of stimulation of enzymic activity by taurocholate and isoelectric point. In contrast, the Km values of the two beta-glucocerebrosidases for natural and artificial substrates are similar and both are inhibited by conduritol B-epoxide. In spleen from three patients with type 1, one patient with type 2 and one patient with type 3 Gaucher's disease form I beta-glucocerebrosidase was found to be clearly deficient, whereas the activity of form II was 25-50% of that in control spleen. The non-specific, neutral beta-glucosidase was not deficient in these Gaucher spleens. The distinct biochemical and immunological properties of non-specific beta-glucosidase and the fact that normal levels of the enzyme are present in patients with Gaucher's disease indicate, in confirmation of previous reports, that non-specific beta-glucosidase is not related to beta-glucocerebrosidase.
Chromatography↗
The effect of detergents on immunoprecipitability of lysosomal sphingomyelinase.
Antibodies raised against the soluble form of acid sphingomyelinase from human urine and placenta are able to precipitate about 70% of the sphingomyelinase activity present in preparations of urinary sphingomyelinase. In contrast, no precipitation of sphingomyelinase activity occurs in detergent-containing preparations from placenta or splenic membranes. The formation of immune complexes between the antibodies and urinary sphingomyelinase is inhibited if detergents are added. With the non-ionic detergent Triton X-100 significant inhibition occurs only above the critical micellar concentration of the detergent. With the anionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (Chaps) substantial inhibition is already observed below the critical micellar concentration of the detergent.
Animals↗
Defects in synthesis, phosphorylation, and maturation of acid alpha-glucosidase in glycogenosis type II.
Glycogenosis type II is an inherited lysosomal storage disease with acid alpha-glucosidase deficiency as the primary defect. Using cultured skin fibroblasts, we have studied the biosynthesis of acid alpha-glucosidase in clinically different forms of this disease. Three unrelated patients were identified (one with an infantile, one with a juvenile, and one with an adult form of the disease) producing normal quantities of the 110-kDa precursor form of acid alpha-glucosidase. However, post-translational modification to mature 76-kDa enzyme protein was either completely deficient or extremely inefficient. No abnormalities were observed in glycosylation of the mutant precursors, as measured by the incorporation of [3H]mannose, but phosphorylation was only detectable for the precursor synthesized by fibroblasts from the juvenile patient. In three other patients (one with a juvenile and two with adult forms of glycogenosis type II) apparently reduced synthesis of precursor protein was observed, but the processing to mature enzyme seemed to be undisturbed. Finally, neither precursor nor mature forms of acid alpha-glucosidase were detectable in one particular case of infantile glycogenosis type II. The studies reveal an unexpected degree of genetic heterogeneity in this disease and identify various mutants which could be of importance to further elucidate the biosynthetic events during lysosomal enzyme formation.
Adolescent↗
Peroxisomal beta-oxidation enzyme proteins in the Zellweger syndrome.
The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities, including an accumulation of very long-chain fatty acids. We show by immunoblotting that there is a marked deficiency in livers from patients with the Zellweger syndrome of the peroxisomal beta-oxidation enzyme proteins acyl-CoA oxidase, the bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase. Using anti-(acyl-CoA oxidase), increased amounts of cross-reactive material of low Mr were seen in the patients. With anti-(oxoacyl-CoA thiolase), high Mr cross-reactive material, presumably representing precursor forms of 3-oxoacyl-CoA thiolase, was detected in the patients. Catalase protein was not deficient, in accordance with the finding that catalase activity is not diminished in the patients. Thus in contrast to the situation with catalase functional peroxisomes are required for the stability and normal activity of peroxisomal beta-oxidation enzymes.
3-Hydroxyacyl CoA Dehydrogenases↗
Effects of thyroid hormone on mitochondrial oxidative phosphorylation.
In order to locate sites of action of thyroid hormone on mitochondrial oxidative phosphorylation we have used an experimental application of control analysis as previously described [Groen, Wanders, Westerhoff, Van der Meer & Tager (1982) J. Biol. Chem. 257, 2754-2757]. Rat-liver mitochondria were isolated from hypothyroid rats or from hypothyroid rats 24 h after treatment with a single dose of 3,3',5-triiodothyronine (T3). The amount of control exerted by four different steps on State-3 respiration with succinate as respiratory substrate was quantified by using specific inhibitors. The hormone treatment resulted in an increase in the flux control coefficient of the adenine nucleotide translocator, the dicarboxylate carrier and cytochrome c oxidase and a decrease in the flux control coefficient of the bc1-complex. The results of this analysis indicate that thyroid hormone treatment results in an activation of the bc1-complex and of at least one other enzyme, possibly succinate dehydrogenase. Measurement of the extramitochondrial ATP/ADP ratio at different rates of respiration (induced by addition of different amounts of hexokinase in the presence of glucose and ATP) showed that the adenine nucleotide translocator operates at a higher (ATP/ADP)out after T3 treatment, which supports previous reports on stimulation of this step by thyroid hormone.
Adenosine Diphosphate↗
Kinetic analysis of short-term effects of alpha-agonists on gluconeogenesis in isolated rat hepatocytes.
Isolated hepatocytes from fasted rats were perifused with glycerol as gluconeogenic substrate. Stimulation of gluconeogenesis with phenylephrine (10(-5) M) as alpha-adrenergic agonist consisted of two distinct phases. The first phase was a transient stimulation of gluconeogenesis and was accompanied by transient changes in cytosolic and mitochondrial redox state; this phase was abolished by the transaminase inhibitor aminooxyacetate. The second phase was a stable stimulation of less magnitude, without change in redox state and insensitive to addition of aminooxyacetate. It is concluded that the first phase is due to a transient enhancement of flux through the malate/aspartate shuttle and that the stable phase is probably due to a stimulation of mitochondrial glycerol-3-phosphate dehydrogenase and glycerol kinase.
Adrenergic alpha-Agonists↗
Soluble sphingomyelinase from human urine as antigen for obtaining anti-sphingomyelinase antibodies.
A soluble form of lysosomal sphingomyelinase was partially purified from human urine using concanavalin A-Sepharose 4B, Sephadex G-100 and octyl-Sepharose 4B chromatography. The octyl-Sepharose 4B eluate was used to immunise a rabbit. The antiserum obtained was able to precipitate about 70% of the sphingomyelinase activity present in urine from control subjects. Both the immunoprecipitable and non-precipitable activities were found to be deficient in urine from patients with Niemann-Pick disease Type A and Type B. In contrast, both activities were present in urine from patients with Niemann-Pick disease Type C. The antiserum was able to precipitate about 80% of the sphingomyelinase activity present in an aqueous extract of placenta.
Animals↗