scholarly journals Characterization of a rat liver Golgi sulphotransferase responsible for the 6-O-sulphation of N-acetylglucosamine residues in β-linkage to mannose: role in assembly of sialyl-galactosyl-N-acetylglucosamine 6-sulphate sequence of N-linked oligosaccharides

1996 ◽  
Vol 319 (1) ◽  
pp. 209-216 ◽  
Author(s):  
Robert G SPIRO ◽  
Yuichiro YASUMOTO ◽  
Vishnu BHOYROO
Keyword(s):  
Liver Enzyme ◽  
Rat Liver ◽  
Nitrous Acid ◽  
Bovine Milk ◽  
Ph Optimum ◽  
Golgi Membranes ◽  
Triton X

Rat liver Golgi membranes were found to contain an enzyme that can transfer sulphate from 3´-phosphoadenosine 5´-phosphosulphate (PAPS) to C-6 of the terminal GlcNAc in β-linkage to mannose and has properties indicating that it is involved in the synthesis of the NeuAcα2-3(6)Galβ1-4GlcNAc(6-SO4) sequences observed in the N-linked carbohydrate units of various glycoproteins. Assays performed with [35S]PAPS (Km 0.67 µM) and GlcNAcβ1-6Manα1-O-Me (GnMaMe) acceptor (Km 0.71 mM) indicated that the sulphotransferase had a pH optimum of approx. 7.0 and is markedly stimulated by Mn2+ ions (maximum approx. 15 mM) and Triton X-100 (0.05-0.1%). Hydrazine/nitrous acid/NaBH4 treatment of the 35S-labelled product yielded radiolabelled 2,5-anhydromannitol(6-SO4). The sulphated GnMaMe product of the GlcNAc-6-O-sulphotransferase could be galactosylated by a rat liver Golgi enzyme that was shown to have the same properties as the UDP-Gal:GlcNAc β-1,4-galactosyltransferase from bovine milk. Competition studies performed with GlcNAc and GlcNAc-6-SO4 furthermore indicated that the same liver enzyme acted on both acceptors to produce Galβ1-4GlcNAc and Galβ1-4GlcNAc(6-SO4) with Km values of 1.04 and 1.68 mM respectively. Because the sulphated N-acetyl-lactosamine could in turn serve as an acceptor for rat liver sialyltransferase, it seems that this enzyme, together with the Golgi galactosyltransferase and the GlcNAc-6-O-sulphotransferase, could act in concert in assembling the NeuAcα2-3(6)Galβ1-4GlcNAc(6-SO4) branches of complex N-linked oligosaccharides.

scholarly journals Characterization of vitamin K-dependent carboxylase from the livers from the adult ox and dicoumarol-treated calf.

1982 ◽  
Vol 201 (2) ◽  
pp. 249-258 ◽  
Author(s):  
L Uotila ◽  
J W Suttie
Keyword(s):  
Rat Liver ◽  
Vitamin K ◽  
Pyridoxal Phosphate ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Ph Optimum ◽  
Carboxylase Activity ◽  
Triton X ◽  
Assay Conditions

The properties of the microsomal vitamin K-dependent carboxylase from the livers of the adult ox and dicoumarol-treated calf were investigated. The enzymes from both sources utilized glutamic residues of synthetic peptides as substrates and could be solubilized with Triton X-100 similarly to the enzyme from vitamin K-deficient rat liver. Under the optimal assay conditions, the microsomes from calf liver had peptide carboxylase activity comparable with that of the rat liver microsomes and 6.5-fold that of adult ox liver microsomes. The apparent Km for reduced vitamin K and the ionic strength optima of the calf and adult ox enzyme clearly differ from those of the rat enzyme. Pyridoxal phosphate activated the adult ox carboxylase only slightly, whereas the calf enzyme was activated by pyridoxal phosphate as effectively as was the enzyme from the vitamin K-deficient rat. Mn2+ activated the adult ox enzyme 9-fold and calf enzyme 22-fold under optimal conditions (no KCl). Three other divalent metal cations (Ca2+, Ba2+, and Mg2+) activated the adult ox and calf enzymes to about half the extent caused by Mn2+, KCl inhibited this activation. The vitamin K-dependent carboxylase from the dicoumarol-treated calf is apparently more tightly bound to the microsomal membrane than is the adult ox enzyme. In many other respects (pH optimum), temperature optimum, Km values for peptide substrate, substrate specificity, inhibitor effects), the properties of the adult ox and calf enzymes resemble closely those of the rat enzyme.

scholarly journals Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles

1987 ◽  
Vol 244 (1) ◽  
pp. 219-224 ◽  
Author(s):  
J M Jacobs ◽  
N J Jacobs
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chlorophyll Synthesis ◽  
Yeast Mitochondria ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Haem Biosynthesis ◽  
Triton X ◽  
Polypeptide Band

The protoporphyrinogen-oxidizing enzyme from Triton X-100 extracts of the mitochondrial and etioplast fractions of etiolated barley was purified by using ion-exchange and hydroxyapatite chromatography. The purified enzyme from both organelle fractions exhibited a Km of 5 microM and was labile to mild heat and acidification. The pH optimum (5-6) and the substrate-specificity (mesoporphyrinogen was oxidized as rapidly as protoporphyrinogen) revealed properties very different from the protoporphyrinogen-oxidizing enzyme of rat liver or yeast mitochondria, which is specific for protoporphyrinogen as substrate. The purest fractions showed a polypeptide band corresponding to an Mr of approx. 36,000 on SDS/polyacrylamide-gel electrophoresis. This is the first purification and characterization of the enzyme from a plant, and indicates no readily detectable differences between the enzyme isolated from mitochondrial or etioplast fractions, although only the latter organelle has the capacity for both haem and chlorophyll synthesis.

scholarly journals Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver

1995 ◽  
Vol 308 (3) ◽  
pp. 983-989 ◽  
Author(s):  
I N Fleming ◽  
S J Yeaman
Keyword(s):  
Phosphatidic Acid ◽  
Rat Liver ◽  
Lysophosphatidic Acid ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Sds Page ◽  
Chromatography Columns ◽  
Triton X ◽  
Second Peak

N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.

scholarly journals Characterization of the interaction between p100, a novel G-protein-related protein, and rat liver endosomes

1991 ◽  
Vol 280 (1) ◽  
pp. 171-178 ◽  
Author(s):  
L M Traub ◽  
E Shai ◽  
R Sagi-Eisenberg
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Trypsin Treatment ◽  
Putative Receptor ◽  
Microsomal Membranes ◽  
Direct Binding ◽  
Triton X ◽  
Binding Sequence

p100 is a recently identified 100 kDa protein which shares a putative receptor-binding sequence with the signal transducing G-proteins Gt and Gi. In liver, p100 immunoreactivity is distributed between the cytosolic and the microsomal fractions [Traub, Evans & Sagi-Eisenberg (1990) Biochem. J. 272, 453-458; Udrisar & Rodbell (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6321-6325]. More specifically, we have localized the membrane-associated form of p100 to an endosomal subfraction of rat liver microsomes. In this study we have investigated the nature of the interaction between p100 and microsomal membranes. p100 was located on the cytoplasmic surface of the microsomal vesicles, and could be released by treatment with 0.5 M-NaCl or 0.5 M-Tris/HCl, pH 7.0. However, p100 was not released by non-ionic detergents, such as Triton X-100. Binding of p100 to the membrane was reversible, as both membrane-released and cytosolic p100 could re-bind stripped (Tris-washed) microsomes. Soluble p100 could not, however, bind to untreated microsomes. Binding to stripped microsomes approached saturation and was inhibited by up to 60% by either heat treatment or mild trypsin treatment of the vesicles. This implies that the interaction between p100 and the microsomal vesicles involves the direct binding of p100 to vesicular proteins. This binding was regulated by both adenine and guanine nucleotides. As p100 contains a region similar to the C-terminal decapeptide of alpha i, (the alpha-subunit of Gi) and has a localization that is restricted to an endosomal subfraction, we propose that cytosolic p100 may bind to cytoplasmically exposed domains of internalized receptors. Thus, like the adaptins, p100 may be involved in the process of sorting and receptor trafficking through the endosomal compartment of the cells.

scholarly journals Solubilization and characterization of pellet-associated human brain α-L-fucosidase activity

1985 ◽  
Vol 229 (3) ◽  
pp. 679-685 ◽  
Author(s):  
R L Hopfer ◽  
J A Alhadeff
Keyword(s):  
Human Brain ◽  
Isoelectric Focusing ◽  
Human Liver ◽  
Gel Filtration ◽  
Compelling Evidence ◽  
Supernatant Fluid ◽  
Ph Optimum ◽  
Triton X ◽  
Soluble Supernatant

The pellet-associated portion of human brain alpha-L-fucosidase (which represents approx. 20% of the homogenate activity) was solubilized with 0.5% (w/v) Triton X-100, characterized with regard to several properties and compared with the corresponding properties of the soluble supernatant-fluid enzyme in an attempt to find a second alpha-L-fucosidase in human brain. The solubilized and soluble alpha-L-fucosidase activities exhibited complete stability after storage at 2-4 degrees C for up to 29 days, comparable thermostability after preincubation at 50 degrees C, comparable apparent Km values (0.07-0.08 mM) for 4-methylumbelliferyl alpha-L-fucopyranoside, comparable hydrophobicity, comparable isoelectric-focusing profiles (six major forms, with pI values between 4.5 and 5.8) and comparable immunoprecipitation curves (with the IgG fraction of antisera prepared against human liver alpha-L-fucosidase). Differences in three properties were found between solubilized and soluble alpha-L-fucosidase activities: the solubilized activity was less stable to storage at −20 degrees C, had a 0.5-pH-unit neutral shift in its pH optimum (6.0) and had smaller Mr forms after gel filtration on Sephadex G-200. The overall results indicate that the pellet-associated and soluble portions of human brain alpha-L-fucosidase are quite similar in most of their properties. Thus there is still no compelling evidence for the existence of a second mammalian alpha-L-fucosidase.

Purification and characterization of rat liver enzyme catalyzing stereoselective reduction of acetylpyridines

Chirality ◽  
2005 ◽  
Vol 17 (8) ◽  
pp. 494-500 ◽  
Author(s):  
Koji Uwai ◽  
Noboru Konno ◽  
Shigeyuki Kitamura ◽  
Shigeru Ohta ◽  
Mitsuhiro Takeshita
Keyword(s):  
Liver Enzyme ◽  
Rat Liver ◽  
Purification And Characterization ◽  
Stereoselective Reduction

scholarly journals Subcellular localization of the sulphation reaction of heparan sulphate synthesis and transport of the proteoglycan to the cell surface in rat liver

1988 ◽  
Vol 252 (2) ◽  
pp. 437-445 ◽  
Author(s):  
J M Graham ◽  
D J Winterbourne
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Heparan Sulphate ◽  
Membrane Vesicles ◽  
Membrane System ◽  
Glycoprotein Synthesis ◽  
Golgi Membranes ◽  
Two Populations

We report on the incorporation of radiolabelled sulphate into proteoglycan in the ‘in situ’-perfused rat liver. After 5 min virtually all of the [35S]sulphate was incorporated into heparan sulphate; no partially sulphated precursors were detected. Pulse-chase experiments, followed by centrifugation in gradients of sucrose and metrizamide, showed that, at 5 min, the heparan sulphate was associated predominantly with the Golgi membranes. Over the next 20 min, intact proteoglycan appeared at the plasma membrane. At intermediate times the heparan sulphate was detected simultaneously in two distinct populations of membrane vesicles. Whether the heparan sulphate in these two populations has two different destinies (e.g. plasma membrane or secretion) is not yet clear. Subfractionation of the Golgi membranes showed that the N-sulphotransferase co-purified with the heparan [35S]sulphate and was separable from the galactosyltransferase of glycoprotein synthesis, confirming that the Golgi membrane system is functionally segregated. Subfractionation also permitted an almost 100-fold purification of the N-sulphotransferase over the homogenate: this will provide an excellent starting material for isolation and further characterization of the enzyme.

scholarly journals Purification and Characterization of UDP-glucose:Ceramide Glucosyltransferase from Rat Liver Golgi Membranes

1996 ◽  
Vol 271 (4) ◽  
pp. 2287-2293 ◽  
Author(s):  
Pascal Paul ◽  
Yasushi Kamisaka ◽  
David L. Marks ◽  
Richard E. Pagano
Keyword(s):  
Rat Liver ◽  
Purification And Characterization ◽  
Golgi Membranes

scholarly journals The topographical location and unique nature of a glucokinase associated with the Golgi apparatus of rat liver

1976 ◽  
Vol 154 (1) ◽  
pp. 193-201 ◽  
Author(s):  
G Berthillier ◽  
R Coleman ◽  
D G Walker
Keyword(s):  
Golgi Apparatus ◽  
Rat Liver ◽  
Freezing And Thawing ◽  
Golgi Membranes ◽  
Unique Nature ◽  
Triton X

A particulate glucokinase was recovered in the Golgi-rich fraction of rat liver prepared by the method of Morré [Methods Enzymol. (1971) 22, 130-148], thus extending the demonstration by Berthillier et al. [Biochim. Biophys. Acta (1973), 293, 370-378] of particulate glucokinase activity in a microsomal subfraction that showed enrichment in Golgi characteristics. The purity of this fraction was examined and it was then subjected to several treatments, the action of Triton X-100, freezing and thawing, and sonication to establish the topographical location of the glucokinase activity thus solubilized. The evidence suggests that the glucokinase activity is either soluble in the lumen of the Golgi apparatus or loosely associated with the inside of the Golgi membranes.

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