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The non-nuclear membranes and the nuclei prepared from L1210 cells catalyze the in vitro transfer of N-acetyl(14C)glucosamine from UDP-N-acetyl(14C)glucosamine to endogenous glycoprotein acceptors. Adequate analysis of these acceptors have demonstrated that the nucleus has its own N-acetylglucosaminyltransferase system that leads to the formation of N-N'-diacetylchitobiosylated proteins.

Aspergillus niger postmitochondrial fraction, which contains high GTPase activity and high GTP binding capacity, has been subjected to subcellular fractionation on a sucrose gradient. A cytosolic and four membranous populations have been separated according to their relative density. The main difficulty has been the characterization of the plasma membrane of the fungus. This fraction, which does not contain any typical enzyme, has been identified after iodination of the outer proteins of protoplasts from A. niger. The immunological detection has shown the occurrence of cytosolic G proteins and membranous small G proteins located not only in the plasma membrane but also in the membranes of the endoplasmic reticulum.

In this report, we describe the main characteristics of the transfer of N-acetylglucosamine within the nucleus of rat hepatocytes. The glycosylation pathway includes the presence of lipids which mediate the nuclear proteins glycosylation. The level of dolichylphosphate seems low and thus could be a regulation factor in the nuclear glycosylations. The discussion deals with the membranous character of the acceptors and the N-acetylglucosaminyltransferase, the N-linkage of the sugar moiety to nuclear proteins and the function of such glycosylation.

Liver nuclei, prepared from normal and vitamin A-deficient rats, were incubated in the presence of GDP-(14C)mannose or UDP-N-acetyl(14C)glucosamine and the labelled glycoproteins analysed by SDS PAGE. Fluorographic analysis has shown that (14C) mannose labelling is enhanced by vitamin A deficiency whereas N-acetyl(14C)glucosamine transfer remains approximately at the same level regardless of the vitamin A status; we did not notice any modification when the proteins were monitored by Coomassie blue or by silver nitrate.

Resume 1. 1. On decrit un essai de localiser trois glycosyl transferases dans les hepatocytes de la roussette ( Scyliorhinus caniculus ), en utilisant le fractionnement cellulaire et la determination par marquage des enzymes. 2. 2. Nous n'avons trouve aucune galactosyltransferase, meme en presence d'un detergent comme le Triton X-100. 3. 3. La N-acetyle glucoseaminyl transferase est entierement localisee dans la fraction microsomique. La methode de Dallner montre que cette enzyme est egalement contenue dans les microsomes lisses et ceux irreguliers. 4. 4. Une activite mannosyl transferase a ete trouvee dans les trois fractions isolees: 18,000 g et 105,000 g sediments et 105,000 g surnageant. 1. 1. An attempt to localize three glucosyl transferases in dogfish ( Scyliorhinus caniculus ) hepatocytes is described, using cellular fractionation and marker enzyme determination. 2. 2. We did not find any galactosyltransferases, even in the presence of a detergent such as Triton X-100. 3. 3. N-Acetyl glucosaminyl transferase is entirely localized in the microsomal fraction. Dallner's method shows that this enzyme is equally contained in rough and smooth microsomes. 4. 4. A mannosyl transferase activity was found in the three fractions isolated: 18,000 g and 105,000 g sediments and 105,000 g supernatant.

Growth of Aspergillus niger Van Tieghem in presence of 32p1 was followed by cell fractionation; the subcellular fractions were identified using marker enzymes and their phospholipid content was analyzed. Microsomes are characterized by high PC and PS values, whereas the largest amount of cardiolipin is recovered in the mitochondria. Polyprenyl phosphate was found mainly in mitochondria (46%) (microsomal content: 38%) and, more precisely, the highest ratio was in the outer membrane. This situation raises the problem of polyprenyl phosphate transfer from mitochondria to microsomes where the enzyme responsible for polyprenyl phosphate mannosylation is located.

Glycoprotein biosynthesis in culture cells in vitro 1. 1. Protein macromolecules in which the d -[ i - 14 C]glucosamine is incorporated exist in non-specialized cancerous ( kb strain) or normal (chick embryo fibroblasts) cells cultivated in vitro . 2. 2. The preferential sites of incorporation of this precursor are located at the endoplasmic membranes and especially at rough membranes. 3. 3. The ratio of specific radioactivity of the cell sap to that of the endoplasmic membranes is quantitatively different in normal and cancerous cells. 4. 4. Only a few parts of the d -glucosamine are incorporated in the cell sap and in the ribosome fraction. No “precursor-derivative” relation is observed among the three cytoplasmic fractions studied.

Summary Microsomal glycoprotein-mannosyltransferase, -galactosyltransferase and -N-acetyl-glucosaminyltransferase utilizing endogenous acceptors were characterized in the epithelial cells of rat small intestine. Galactosyltransferase gave a Km value of 14,6 × 10 −6 M with respect to UDP-galactose. A pH optimum of 6,7 and a temperature optimum of 22°C were observed. The enzyme shows an absolute requirement for Mn ++ , for which the optimal concentration is 0,01 M. On the other hand, mannosyltransferase and N-acetyl-glucosaminyltransferase did not require divalent cations and were not inhibited by EDTA 10 −3 M. They have a pH optimum of 6,2 and a temperature optimum respectively of 37°C and 28°C. The Km value for the glycosyl donors, GDP-mannose and UDP-N-acetylglucosamine, were 6 × 10 −5 and 4,35 × 10 −4 M. Each of these enzymes is strongly bound to the membranous structure; Triton X-100 causes a 2 to 3 fold enhancement of incorporation of mannose, galactose and N-acetylglucosamine when a suitable detergent: protein ratio is applied. ATP, UTP and GTP had no significant effect on incorporation. A discontinuity in heat denaturation curves reveals at least two enzymatic activities for each glycosyltransferase.

Resume L'incorporation in vivo de d -[1-14C]glucosamine ou de d -[1-14C]galactose ou de d -[1-14C]mannose permet de marquer un constituant glycoproteique de la muqueuse intestinale de rat. Extrait par trypsination moderee, ce constituant est purifie par filtration en gel de Sephadex G-200, suivie de chromatographie sur hydroxylapatite et d'electrophorese preparative sur bloc d'acetate de cellulose. Cette glycoproteine, qui migre comme une α2-globuline, a une constante de sedimentation de 14 S; son poids moleculaire apparent, estime par filtration sur Sepharose 6 B, est voisin de 560 000. Sa composition en acides amines et sa teneur en oses neutres (galactose plus mannose:7.8%), en 6-desoxyhexose (fucose:2.6%), en osamines (glucosamine plus des traces de galactosamine:7.7%), en acide sialique (3.3%) sont determinees. Elle ne contient ni sulfate, ni acides uroniques. Elle ne presente aucune reaction d'identite immunologique avec les proteines seriques. Cette etude confirme l'existence d'une biosynthese glycoproteique active et donne une methode d'obtention d'un accepteur exogene specifique pour les glycosyltransferases mises en evidence dans un precedent travail (J. Frot-Coutaz and R. Got, Biochimie, 53 (1971) 595) au niveau de la muqueuse intestinale de rat.

The present studies are concerned with the subcellular localisation of an active multiglycosyltransferase system involved in the transfer of galactose, N-acetylglucosamine and mannose to various glycoprotein acceptors. Smooth microsomes, obtained according to the method of Dallner, were the main loci of the three glycosyltransferases. After placing smooth microsomes in the bottom of a sucrose gradient, we recovered galactosyltransferase and mannosyltransferase in a fraction (density 1.12) containing Golgi apparatus and endoplasmic reticulum and N-acetylglucosaminyltransferase in a non-identified fraction with a density from 1.14 to 1.16; these results are obtained when performing all enzyme assays on endogenous acceptors. When exogenous acceptors was used, galactosyltransferase N-acetylglucosaminlytransferase and mannosyltransferase appear to be present in fractions having density of 1.14–1.16, 1.18 and 1.12, respectively.

By means of polyacrylamide gel electrophoresis, four fractions with amylase activity have been shown in human colostrum, pancreatic juice and saliva. Colostrum was fractionated by gel fitration on dextran gel (Sephadex G-100); amylolytic activity was eluted as two peaks well after the elution of the other proteins. Six different amylase-active components could be found in the second peak.

Cow's milk has been shown to contain a protein complex which is able to bind vitamin K1 in a reversible manner. This binding property has been investigated by the celite method which consists in creating a dynamic equilibrium between the adsorbent, the celite and the protein complex for the ligand (vitamin K1). Based on competition experiment, the binding is specific and the vitamin K1 binding protein complex has a molecular weight equal to or higher than 7.5 X 10(2) KD.

Rat liver microsomal membranes have been shown to contain an UDP-glucose binding protein. Its mol. wt was estimated to be 120 000 by gel filtration and by ultracentrifugation in a sucrose gradient. The receptor activity was purified by gel filtration on Sephadex G200 and analysed by gel-electrofocusing.

Rat liver microsomal membranes have been shown to contain a biosynthetic pathway of UDP-glucose. In addition, they are able to bind UDP-glucose in a reversible manner, As UDP-glucose is also metabolized in these membranes, the study of the binding has been performed with a microsomal Triton X 100 extract. This reversible binding depends on pH (maximum at pH 8.1) and manganous ions, and disappears at pH 6.5. It exhibits a high affinity (K-diss equals 3 mu M), and a narrow specifity for UDP-glucose. Proteolytic digestion inhibits the binding up to 90%, showing that the UDP-glucose receptor has a proteic nature. These binding characteristics have been also found in the membranes themselves, indicating that the detergent solubilization does not destroy the protein binding capacity.

Nuclei and non-nuclear membranes were tested for their ability to transfer in vitro (14C)mannose from GDP-(14C)mannose to endogenous glycoprotein acceptors in the presence and in the absence of exogenous retinyl-phosphate. Electrophoretic analysis shows that retinylphosphate is responsible for the labeling of a few endogenous acceptors only in the non-nuclear membranes; in the nuclei the mannosylation reaction is not retinylphosphate dependent and the electrophoretic profile of the labeled protein acceptors is different from that of the non-nuclear membranes.

The transfer of N-acetyl(14C)glucosamine from UDP-N-acetyl(14C)glucosamine to endogenous glycoproteins acceptors were studied comparatively in the nuclei and in the non-nuclear membranes of rat hepatocytes. Electrophoretic and autoradiographic analysis show that most of the glycoprotein acceptors of the nuclei differ from those of the non-nuclear membranes in terms of molecular weight. In addition, it may interesting to mention that in the nuclear fraction a 30% inhibition by tunicamycin is obtained for concentrations as low as 0.03 microM, whereas at this concentration no effect is detected in the non-nuclear membranes. In the presence of 0.2 microM tunicamycin, the inhibition does not go beyond 25% in the latter fraction but goes up to 80% in the former. The previous results demonstrate clearly that a particular glycosylation reaction occurs in the nucleus.

The present report is concerned with an N-acetylglucosaminyltransferase which catalyzes the transfer of N-acetylglucosamine from UDP-N-acetyl-glucosamine to nascent protein chains. This enzyme is localized in a rough microsomes fraction prepared according to the method ofDallner3.

Protein macromolecules in which the 1-14C-l-fucose is incorporated exist in non-specialized cancerous (KB strain) or normal (chick embryo fibroblasts) cells cultivated in vitro. The preferential site of incorporation of this precursor is located at the endoplasmic membranes for cancerous cells and in the cell sap for normal cells.