Characterization, purification, and subcellular localization of bovine thyroid sialidases

Sialidase activities have been studied in bovine thyroid using sialoglycolipids, sialoglycoproteins, sialo-oligosaccharides and fluorogenic 4-methylumbelliferyl-alpha-D-N-acetylneuraminate as substrates. No sialidase activity could be detected towards native glycoprotein substrates. From enzyme kinetics, effector data and more convincingly from subcellular studies it became clear that in bovine thyroid at least two sialidase activities were present, a sialyllactitol sialidase confined to the lysosomal membrane and a glycolipid sialidase residing in the plasma membrane and displaying the features of a true ectoenzyme. The lipid requirement for full enzyme activity supported the membrane bound character of both sialidase activities. A soluble sialidase activity could not be demonstrated. After solubilization by CHAPS treatment, partial purification of the sialyllactitol sialidase could be achieved by affinity chromatography (Sepharose diamino dipropylamino-N-acetylneuraminic acid). The purified enzyme was extremely labile. Titration of the sialidase preparation with amino acid modifying agents revealed that sulfhydryl- and tryptophanyl groups were essential for the sialidase action.     

Regulation of glucose 6-phosphatase in hepatic microsomes by thyroid and corticosteroid hormones

The regulation of glucose 6-phosphatase in hepatic microsomes by thyroid and corticosteroid hormones has been studied following the administration of 3,3',5-triiodo-L-thyronine and/or triamcinolone to hypophysectomized rats. The apparent Km for glucose-6-P in isolated ("intact") microsomes increased following administration of either hormone; there was little or no difference in the apparent Km when microsomes were treated with sodium deoxycholate ("disrupted"). In intact microsomes, triiodothyronine caused a 2.3-fold increase in the Vmax of glucose 6-phosphatase; triamcinolone, a 4-fold increase; and both hormones together, a 4.4-fold increase. Corresponding values for disrupted microsomes were: triiodothyronine, 3.7-fold; triamcinolone, 1.8-fold; both hormones, 3.3-fold. After triiodothyronine treatment, disruption of microsomes caused an over 5-fold increase in Vmax; after triamcinolone treatment, the increase was only 1.5-fold. This difference could not be explained by a change in the energy of activation of glucose 6-phosphatase in either intact or disrupted microsomes following hormone treatment. Glucose 6-phosphatase was localized by a cytochemical procedure; the reaction product was associated with 90% of the profiles in all microsomal preparations, except for those from triiodothyronine-treated rats, where less than 50% contained lead precipitate. Vesicles free of lead phosphate were isolated from sucrose gradients and accounted for less than 10% of the protein and glucose 6-phosphatase in all preparations, again except for those from triiodothyronine-treated rats, where they represented 40% of both the protein and glucose 6-phosphatase. The results are consistent with a model for glucose 6-phosphatase in which the substrate is transported across the microsomal membrane by a specific carrier before hydrolysis within the cisternae by a phosphohydrolase. It is suggested that the effect of triiodothyronine is mainly on the activity of the phosphohydrolase, and triamcinolone, on that of the carrier.     

Subcellular localization of glucose-6-phosphatase in animal tissues.

1. Glucose-6-phosphatase (EC 3.1.3.9 D-glucose-6-phosphate phosphohydrolase) was found to be localized mainly in the endoplasmic reticulum (microsomal fraction) of all species of vertebrate liver tissue examined. 2. Hepatopancreas tissue from gastropod molluscs was found to be unique in showing the localization of glucose-6-phosphatase in the cytosol (soluble fraction).     

Sensitivity of glucose 6-phosphatase activity to glutaraldehyde.

The effect of glutaraldehyde fixation on glucose 6-phosphatase activity in mouse liver was investigated. After transparenchymal perfusion with 2% glutaraldehyde for 1.5 minutes, the activity of the recovered enzyme was higher than those reported for acid phosphatase and aryl sulfatase activities after fixation under similar condition, and an abundant deposition of reaction product was observed in hepatocytes. Subsequent immersion in the same fixative solution for 30 minutes after 4 degrees C resulted in only a slight decrease in the activity. However, the activity was almost completely destroyed after 3 hours of immersion fixation at 4 degrees C following the perfusion. Therefore, the enzyme can be said to be aldehyde-sensitive when a long fixation time is used, but not aldehyde-sensitive during a short fixation time.     

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.     

Subcellular structure of bovine thyroid gland. The localization of the peroxidase activity in bovine thyroid.

1. After differential pelleting of bovine thyroid tissue the highest relative specific activities for plasma membrane markers are found in the L fraction whereas those for peroxidase activities (p-phenylenediamine, guaiacol and 3,3'-diaminobenizidine tetrachloride peroxidases) are found in the M fraction. 2. When M + L fractions were subjected to buoyant-density equilibration in a HS zonal rotor all peroxidases show different profiles. The guaiacol peroxidase activity always follows the distribution of glucose 6-phosphatase. 3. When a Sb fraction is subjected to Sepharose 2B chromatography three major peaks are obtained. The first, eluted at the void volume, consists of membranous material and contains most of the guaiacol peroxidase activity. Most of the protein (probably thyroglobulin) is eluted with the second peak. Solubilized enzymes are recovered in the third peak. 4. p-Phenylenediamine peroxidase activity penetrates into the gel on polyacrylamidegel electrophoresis, whereas guaiacol peroxidase activity remains at the sample zone. 5. DEAE-Sephadex A-50 chromatography resolves the peroxidase activities into two peaks, displaying different relative amounts of the different enzymic activities in each peak. 6. The peroxidase activities may be due to the presence of different proteins. A localization of guaiacol peroxidase in rough-endoplasmic-reticulum membranes (or in membranes related to them) seems very likely.     

Immunocytochemical localization of glucose 6-phosphatase and cytosolic phosphoenolpyruvate carboxykinase in gluconeogenic tissues reveals unsuspected metabolic zonation

Immunohistochemical analysis was used to define the precise cell-specific localization of Glucose-6-phosphatase (Glc6Pase) and cytosolic form of the phosphoenolpyruvate carboxykinase (PEPCK-C) in the digestive system (liver, small intestine and pancreas) and the kidney. Co-expression of Glc6Pase and PEPCK-C was shown to take place in hepatocytes, in proximal tubules of the cortex kidney and at the top of the villi of the small intestine suggesting that these tissues are all able to perform complete gluconeogenesis. On the other hand, intrahepatic bile ducts, collecting tubes of the nephron and the urinary epithelium in the calices of the kidney, as well as the crypts of the small intestine, express Glc6Pase without significant levels of PEPCK-C. In such cases, the function of Glc6Pase could be related to the transepithelial transport of glucose characteristic of these tissues, rather than to the neoformation of glucose. Lastly, PEPCK-C expression in the absence of Glc6Pase was noted in both the exocrine pancreas and the endocrine islets of Langerhans. Possible roles of PEPCK-C in exocrine pancreas might be the provision of gluconeogenic intermediates for further conversion into glucose in the liver, whereas PEPCK-C would be instrumental in pyruvate cycling, which has been suggested to play a regulatory role in insulin secretion by the β-cells of the islets. An erratum to this article can be found at     

Ultrastructural demonstration of glucose 6-phosphatase in cerebral cortex

Summary A two-stage fixation technique has been developed to obtain morphological preservation and retention of glucose 6-phosphatase (G6-Pase) activity for its demonstration in rat cerebral cortex. The technique was then employed to localize the enzyme in the cortex where it produced a dense reaction over the well developed granular endoplasmic reticulum cisternae in nerve cells and oligodendrocytes which contrasted with a thin reaction in astrocytes. Other membranous organelles showed no reaction.     

Ultrastructural demonstration of glucose 6-phosphatase in cerebral cortex

A two-stage fixation technique has been developed to obtain morphological preservation and retention of glucose 6-phosphatase (G6-Pase) activity for its demonstration in rat cerebral cortex. The technique was then employed to localize the enzyme in the cortex where it produced a dense reaction over the well developed granular endoplasmic reticulum cisternae in nerve cells and oligodendrocytes which contrasted with a thin reaction in astrocytes. Other membranous organelles showed no reaction.     

New lessons in the regulation of glucose metabolism taught by the glucose 6-phosphatase system.

The operation of glucose 6-phosphatase (EC 3.1.3.9) (Glc6Pase) stems from the interaction of at least two highly hydrophobic proteins embedded in the ER membrane, a heavily glycosylated catalytic subunit of m 36 kDa (P36) and a 46-kDa putative glucose 6-phosphate (Glc6P) translocase (P46). Topology studies of P36 and P46 predict, respectively, nine and ten transmembrane domains with the N-terminal end of P36 oriented towards the lumen of the ER and both termini of P46 oriented towards the cytoplasm. P36 gene expression is increased by glucose, fructose 2,6-bisphosphate (Fru-2,6-P2) and free fatty acids, as well as by glucocorticoids and cyclic AMP; the latter are counteracted by insulin. P46 gene expression is affected by glucose, insulin and cyclic AMP in a manner similar to P36. Accordingly, several response elements for glucocorticoids, cyclic AMP and insulin regulated by hepatocyte nuclear factors were found in the Glc6Pase promoter. Mutations in P36 and P46 lead to glycogen storage disease (GSD) type-1a and type-1 non a (formerly 1b and 1c), respectively. Adenovirus-mediated overexpression of P36 in hepatocytes and in vivo impairs glycogen metabolism and glycolysis and increases glucose production; P36 overexpression in INS-1 cells results in decreased glycolysis and glucose-induced insulin secretion. The nature of the interaction between P36 and P46 in controling Glc6Pase activity remains to be defined. The latter might also have functions other than Glc6P transport that are related to Glc6P metabolism.     

Inhibition of microsomal glucose 6-phosphatase by unsaturated aliphatic aldehydes and ketones.

Aldehydes and ketones with one double bond conjugated to the carbonyl group inhibited the enzyme glucose 6-phosphatase, which is embedded in the microsomal membrane. The Michaelis constant, Km and the maximal rate of reaction, V, were affected in a way dependent on the inhibitor's chain-length: trans-2-pentenal and 1-penten-3-one increased Km linearly with concentration and had almost no effect on V, whereas trans-2-nonenal caused a large increase in V but only a small and non-linear change in Km. The effect of the short-chain aldehydes on the kinetic parameters increased with chain-length, but pentenone increased Km more than did trans-2-heptenal and conjugated dienals did not act as inhibitors. Therefore, sterical effects apparently are of importance. Washing the microsomes after incubation with hexenal or heptenal did not substantially decrease the inhibition, but with nonenal the inhibition was reduced by washing. Inhibition by the SH-group blocking reagent p-hydroxymercuribenzoate was competitive to inhibition by the alkenals. It is concluded that the alpha-beta unsaturated oxo-compounds inhibit glucose 6-phosphatase by binding covalently to an important mercapto group and that perturbation of the enzyme's membrane environment also plays a part in the inhibition.     

The stereochemical course of phosphoryl transfer catalysed by glucose 6-phosphatase.

Rat liver microsomal glucose 6-phosphatase catalyses phosphoryl transfer between D-glucose 6-[(R)-16O,17O,18O]phosphate and D-glucose with retention of configuration at the phosphorus atom. Since individual phosphoryl-transfer steps appear in general to occur with inversion of configuration, this observation is most simply interpreted in terms of a double-displacement mechanism with a phosphoryl-enzyme intermediate. Such an intermediate has been proposed previously from kinetic and 32P-labelling experiments.