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.

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 Orientation of glycoprotein galactosyltransferase and sialyltransferase enzymes in vesicles derived from rat liver Golgi apparatus.

1981 ◽  
Vol 89 (2) ◽  
pp. 246-255 ◽  
Author(s):  
B Fleischer
Keyword(s):  
Golgi Apparatus ◽  
Rat Liver ◽  
Three Dimensional ◽  
Small Molecular Weight ◽  
Protein Substrates ◽  
Molar Ratios ◽  
Golgi Vesicles ◽  
Luminal Side ◽  
Triton X ◽  
Cis And Trans

UDP-galactose: N-acetylglucosamine galactosyltransferase (GT) and CMP-sialic:desialylated transferrin sialyltransferse (ST) activities of rat liver Golgi apparatus are membrane-bound enzymes that can be released by treatment with Triton X-100. When protein substrates are used to assay these enzymes in freshly prepared Golgi vesicles, both activities are enhanced about eightfold by the addition of Triton X-100. When small molecular weight substrates are used, however, both activities are only enhanced about twofold by the addition of detergent. The enzymes remain inaccessible to large protein substrates even after freezing and storage of the Golgi preparation for 2 mo in liquid nitrogen. Accessibility to small molecular and weight substrates increases significantly after such storage. GT and ST activities in Golgi vesicles are not destroyed by treatment with trypsin, but are destroyed by this treatment if the vesicles are first disrupted with Triton X-100. Treatment of Golgi vesicles with low levels of filipin, a polyene antibiotic known to complex with cholesterol in biological membranes, also results in enhanced trypsin susceptibility of both glycosyltransferases. Maximum destruction of the glycosyltransferase activities by trypsin is obtained at filipin to total cholesterol weight ratios of approximately 1.6 or molar ratios of approximately 1. This level of filipin does not solubilize the enzymes but causes both puckering of Golgi membranes visible by electron microscopy and disruption of the Golgi vesicles as measured by release of serum albumin. When isolated Golgi apparatus is fixed with glutaraldehyde to maintain the three-dimensional orientation of cisternae and secretory vesicles, and then treated with filipin, cisternal membranes on both cis and trans faces of the apparatus as well as secretory granule membranes appear to be affected about equally. These results indicate that liver Golgi vesicles as isolated are largely oriented with GT and ST on the luminal side of the membranes, which corresponds to the cisternal compartment of the Golgi apparatus in the hepatocyte. Cholesterol is an integral part of the membrane of the Golgi apparatus and its distribution throughout the apparatus is similar to that of both transferases.

scholarly journals Mannosyl carrier functions of retinyl phosphate and dolichyl phosphate in rat liver endoplasmic reticulum

1983 ◽  
Vol 210 (2) ◽  
pp. 541-547 ◽  
Author(s):  
K E Creek ◽  
D J Morré ◽  
C S Silverman-Jones ◽  
Y Shidoji ◽  
L M De Luca
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Subcellular Fractions ◽  
Transfer Activity ◽  
Dolichyl Phosphate ◽  
Endoplasmic Reticulum Protein ◽  
Triton X

Of the subcellular fractions of rat liver the endoplasmic reticulum was the most active in GDP-mannose: retinyl phosphate mannosyl-transfer activity. The synthesis of retinyl phosphate mannose reached a maximum at 20-30 min of incubation and declined at later times. Retinyl phosphate mannose and dolichyl phosphate mannose from endogenous retinyl phosphate and dolichyl phosphate could also be assayed in the endoplasmic reticulum. About 1.8 ng (5 pmol) of endogenous retinyl phosphate was mannosylated per mg of endoplasmic reticulum protein (15 min at 37 degrees C, in the presence of 5 mM-MnCl2), and about 0.15 ng (0.41 pmol) of endogenous retinyl phosphate was mannosylated with Golgi-apparatus membranes. About 20 ng (13.4 pmol) of endogenous dolichyl phosphate was mannosylated in endoplasmic reticulum and 4.5 ng (3 pmol) in Golgi apparatus under these conditions. Endoplasmic reticulum, but not Golgi-apparatus membranes, catalysed significant transfer of [14C]mannose to endogenous acceptor proteins in the presence of exogenous retinyl phosphate. Mannosylation of endogenous acceptors in the presence of exogenous dolichyl phosphate required the presence of Triton X-100 and could not be detected when dolichyl phosphate was solubilized in liposomes. Dolichyl phosphate mainly stimulated the incorporation of mannose into the lipid-oligosaccharide-containing fraction, whereas retinyl phosphate transferred mannose directly to protein.

scholarly journals Isolation of a matrix that binds medial Golgi enzymes

1994 ◽  
Vol 124 (4) ◽  
pp. 405-413 ◽  
Author(s):  
P Slusarewicz ◽  
T Nilsson ◽  
N Hui ◽  
R Watson ◽  
G Warren
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Dissociation Constant ◽  
Rat Liver ◽  
Proteinase K ◽  
Apparent Dissociation Constant ◽  
Low Salt ◽  
The Matrix ◽  
Matrix Removal ◽  
Triton X

Rat liver Golgi stacks were extracted with Triton X-100 at neutral pH. After centrifugation the low speed pellet contained two medial-Golgi enzymes, N-acetylglucosaminyltransferase I and mannosidase II, but no enzymes or markers from other parts of the Golgi apparatus. Both were present in the same structures which appeared, by electron microscopy, to be small remnants of cisternal membranes. The enzymes could be removed by treatment with low salt, leaving behind a salt pellet, which we term the matrix. Removal of salt caused specific re-binding of both enzymes to the matrix, with an apparent dissociation constant of 3 nM for mannosidase II. Re-binding was abolished by pretreatment of intact Golgi stacks with proteinase K, suggesting that the matrix was present between the cisternae.

scholarly journals Strain differences in rat liver (UDP-glucuronyltransferase activity towards androsterone

1979 ◽  
Vol 179 (3) ◽  
pp. 483-487 ◽  
Author(s):  
M Matsui ◽  
F Nagai ◽  
S Aoyagi
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Strain Differences ◽  
Transferase Activity ◽  
Sprague Dawley ◽  
Freezing And Thawing ◽  
Sprague Dawley Rats ◽  
Long Evans ◽  
Triton X

Male Donryu, Wistar King rats showed discontinuous variations in hepatic microsomal UDP-glucuronyltransferase activities towards androsterone, but not towards testosterone, bilirubin, phenolphthalein and 4-nitrophenol. Fresh microsomal fraction with a low transferase activity towards androsterone formed 0.049–0.080 nmole of glucuronide/min per mg of protein, whereas fresh microsomal fraction with a high transferase activity towards androsterone formed 0.335–0.557 nmol of glucuronide/min per mg of protein. The microsomal fraction with low enzyme activity towards androsterone was not stimulated by treatment with Triton X-100 or freezing and thawing. In contrast, male Long Evans and Sprague-Dawley rats did not exhibit such diversity.

scholarly journals Studies on the structure-bound sedimentability of some rat liver lysosome hydrolases

1971 ◽  
Vol 122 (3) ◽  
pp. 363-371 ◽  
Author(s):  
F. M. Baccino ◽  
G. A. Rita ◽  
Maria Franca Zuretti
Keyword(s):  
Acid Phosphatase ◽  
Rat Liver ◽  
High Speed ◽  
Particulate Material ◽  
The Other ◽  
Acid Proteinase ◽  
Freezing And Thawing ◽  
Clear Cut ◽  
Almost All ◽  
Triton X

1. Lysosome-rich fractions from rat liver were subjected to several disruptive procedures: osmotic lysis or freezing and thawing in different media, shearing forces in a high-speed blender, treatment with Triton X-100. 2. The soluble and particulate phases were then separated by high-speed centrifugation and assayed for their content of acid phosphatase, β-galactosidase, β-N-acetylglucosaminidase, acid proteinase, acid ribonuclease, acid deoxyribonuclease and protein. 3. The degree of elution of these hydrolases appeared to depend on both the enzyme species and the treatment. The resulting patterns of solubilization were rather complex, so that a clear-cut discrimination between soluble and structure-bound enzymes could not always be traced. 4. Although only β-galactosidase was readily solubilizable after all treatments, acid proteinase could also be extensively eluted from the sedimentable material in the presence of EDTA and acid phosphatase was fully extracted by Triton X-100. On the other hand, considerable proportions of the other activities could not be solubilized by any of the procedures used. 5. In other experiments, the adsorbability of hydrolases on subcellular structures was investigated by measuring the partition between sedimentable particles and soluble fraction of solubilized enzymes added to ‘intact’ liver homogenates. 6. Large proportions of acid proteinase, ribonuclease and deoxyribonuclease, and almost all of β-N-acetylglucosaminidase, were found to be adsorbed on the particulate material.

Fractionation of rat liver mitochondrial components after short treatments with triton x-100

1982 ◽  
Vol 14 (10) ◽  
pp. 933-940 ◽  
Author(s):  
M.C. Barbero ◽  
E. Rial ◽  
J.J. Otamendi ◽  
J.I.G. Gurtubay ◽  
F.M. Goni
Keyword(s):  
Rat Liver ◽  
Triton X
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