[Effects of basic proteins of low molecular weight on the phosphohydrolase and phosphotransferase activities of microsomal glucose-6-phosphatase in adult monkey hepatocytes (author's transl)]. / Effet de petites protéines basiques sur les activitiés phosphohydrolase et phosphotransférase de la glucose-6-phosphatase microsomique des hépatocytes de singe.

The effects of histone 2A and some polycations on microsomal carbamylphosphate:D-glucose phosphotransferase and glucose-6-phosphate phosphohydrolase activities (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9), have been investigated. 1. Histone 2A and polycations activate the two enzymic activities. At a constant cation concentration, this activation increases with the number of cationic groups per molecule. 2. Activation by histone 2A is related to its fixation on microsomal membranes. This fixation varies with quantities of histones and pH. 3. The nature of the interactions between histones and microsomal membranes is shown to be electrostatic, probably between the cationic groups of histones and the anionic group of membranous lipids. 4. Kinetic analysis reveal that histone 2A increases the maximal reaction velocity but does not affect the apparent Michaelis constant values for the substrates. 5. The role played by the cationic groups of histone 2A on the microsomal glucose 6-phosphatase, is discussed.

[Galactose transfer in the membranes of human milk fat globules (author's transl)]. / Transfert du galactose dans les membranes des globules lipidiques du lait humain

Galactosyltransferase which catalyzes the transfer from UDP-galactose to either endogeneous glycoproteins, free N-acetylglucosamine or N-acetylglucosaminyl residues in the carbohydrate portion of glycoproteins, or to glucose when alpha-lactalbumin is added, occurs in human milk fat globule membranes. Various treatments (washing of membranes, freezing and thawing) did not affect this activity. In the presence of Triton X-100, the enzyme shows appreciable latency, This detergent was then used to solubilize the enzyme and to study its main characteristics. A competition and a heat stability experiment show that only one enzyme acts on two substrates (free N-acetylglucosamine or desialyzed and degalactosylated fetuin). UDP-galactose hydrolase activities were very low compared to those of the bovine milk fat globule membranes. Other characteristic enzymes of Golgi vesicles were found in human milk fat globules membranes. It is of interest to find out whether this is the result of contamination with cytoplasmic particles or whether it reflects the participation of Golgi vesicles in human milk fat globule secretion.

Topology of glucosylceramide synthesis in Golgi membranes from porcine submaxillary glands.

The topology of ceramide glucosyltransferase and de novo synthesized glucosylceramide was studied in sealed and 'right-side-out' vesicles of porcine submaxillary glands derived from Golgi apparatus. Pronase treatment which did not cause any breakdown of the luminal glycoprotein galactosyltransferase activity, inhibited the ceramide glucosyltransferase to more than 50% at a ratio proteinase to Golgi protein 1:100. Trypsin at the same concentration, while producing no inactivation of luminal galactosyltransferase, caused a complete loss of ceramide glucosyltransferase activity. The membrane-impermeable compound, DIDS, which did not cause any inhibition of the galactosyltransferase, inhibited the ceramide glucosyltransferase (70% reduction at 80 microM DIDS). Thus, the enzyme ceramide glucosyltransferase is accessible from the cytoplasmic side of the Golgi vesicles. The orientation of the newly synthesized glucosylceramide is studied by the ability of the enzyme glucosylceramidase to hydrolyse this compound both on intact and on disrupted vesicles. The same percentage (respectively, 36 and 30%) of hydrolysis was obtained during an incubation of 3 h, showing that glucosylceramide is not at all protected from external hydrolysis. Pronase-treated vesicles revealed an increase in glucosylceramidase hydrolysis (up to 45%), which indicates that glucosylceramide that glucosylceramide may be cryptic. All these results indicate that the ceramide glucosyltransferase, as well as related glucosylceramide, are cytoplasmically oriented in Golgi vesicles from porcine submaxillary glands.

[Comparative study of microsomal enzymic activities in adult and foetal monkey hepatocytes (author's transl)]. / Etude comparative d'activités enzymatiques microsomiques dans les hepatocytes de singe adulte et foetal.

Differential centrifugation was applied to adult and foetal liver of monkey. Obtained fractions were: F1 (800 X g); F2 (12 500 X g); F3 (200 000 X g); and cell sap. Analysis of chemical compounds of these fractions shows that: (1) adult and foetal nucleic acids levels are similar; (2) there are more proteins in adult than in foetal hepatocytes; (3) most of the glycogen is located in F3; the foetal level is twenty times higher than the adult level. Plasma membrane enzymes (5'-nucleotidase, adenylate cyclase) show a nucleomicrosomic distribution. The distribution of alkaline phosphatase is not significant. Mitochondrial enzymes (monoamine oxydase, succinate cytochrome c reductase, cytochrome oxydase) are enriched in F2 without any sedimentation in F3. There is more malate dehydrogenase liberated in cell sap during foetal liver fractionation. This indicates the foetal mitochondria are more sensitive to the homogenisation method. Lysosomal enzymes (acid phosphatase, N-acetylglucosaminidase) are enriched in F2. The same observation for N-acetylglucosaminidase as for malate dehydrogenase leads to the same conclusion for foetal lysosomes. Endoplasmic reticulum and Golgi enzymes (glucose-6-phosphatase and related phosphotransferase activity, NADPH-cytochrome c reductase and sialytransferase) are much enriched in F3. Thus this fraction F3 is pure enough to allow the observation of the modification produced on endoplasmic reticulum and Golgi apparatus during foetal and neonatal development.

[The presence of glycosyltransferases on chromatin acceptors in monkey liver nuclear membranes (author's transl)]. / Présence de glycosyltransferases à accepteurs chromatiniens dans les membranes nucléaires d'hépatocytes de singe.

Nuclei were prepared from monkey hepatocytes by centrifugation of the homogenate on a cushion of 2.3 M sucrose, during 45 min at 100000 X g. The yield was 2.2 x 10(7) nuclei per g of liver, and 70% of te homogenate DNA was recovered in these nuclei. An electron microscopic study as well as a biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. A mannosyltransferase and an N-acetylglucosaminyltransferase, working on endogenous glycoproteic acceptors, are present in the nuclei for 1.4 and 6.5% of the homogenate activities, respectively. The nuclei are hydrolysed by DNAse I. The suspension, adjusted in 1.9 M sucrose, was centrifuged for 2 h at 100000 X g, under buffer layer. Purified nuclear membranes were collected at the interface. These membranes did not contain any more endoplasmic reticulum enzyme activities, but the mannosyl and N-acetylglucosaminyltransferase activities were still present. They essentially work on an exogenous chromatin acceptor, prepared by lysis of the nuclei. The eventual role of these glycosyltransferases in the glycosylation of non-histone proteins is discussed.

Solubilization of UDPglucose-ceramide glucosyltransferase from the Golgi apparatus.

The choice of a suitable detergent for solubilization of UDPglucose-ceramide glucosyltransferase from Golgi membranes has been investigated. Among the various classes of detergent, CHAPS, a zwitterionic detergent, was used as it produced a substantial activation of the enzyme activity. 30% of the enzyme activity and 50% of proteins were solubilized in the first attempts. Further experiments were conducted with addition of a second detergent, Zwittergent 3-14 which increased enzyme recovery to 45%. Lastly, reducing the concentrations of buffer and divalent cations Mn2+, Mg2+ and introducing glycerol (20%, v/v) allowed 80% of proteins to be solubilized together with 68% of the ceramide glucosyltransferase activity.

Properties of uridine diphosphate glucose pyrophosphorylase from Golgi apparatus of liver.

Golgi apparatus isolated from cat liver contained UDPglucose pyrophosphorylase (UTP:alpha-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.9) activity. The results of washing suggested that pyrophosphorylase was bound firmly to Golgi membranes. Moreover, the enzyme was activated by Triton X-100 in the same extent as galactosyltransferase, a typical Golgi apparatus enzyme. Two-substrate kinetic studies were performed with the enzymes from cytosol and Golgi fractions. The soluble enzyme showed an apparent 2.5-fold greater activity for the glucose 1-phosphate than for UTP, while pyrophosphorylase of Golgi apparatus had the same affinity for the two substrates. A random mechanism was observed with a direct dependence of apparent Michaelis constant values on the concentration of second substrate for soluble enzyme. In contrast, with Golgi enzyme one ligand had no effect on the binding of the other.

Evidence for coupling between transport of UDP-glucose and its synthesis by membrane-bound pyrophosphorylase in Golgi apparatus of cat liver.

Incubation of sealed vesicles of cat-liver Golgi apparatus with UDP[14C]glucose showed that the vesicles accumulated radioactivity. After Triton X-100 treatment or sonication of washed vesicles, soluble radiolabeled species were released and identified by paper chromatography as UDP[14C]glucose, [14C]glucose 1-phosphate and free glucose. In the incubation medium, UDPglucose was effectively protected by addition of dimercaptopropanol and UTP. Presence of glucose 1-phosphate and glucose within the vesicles most probably arose from luminal pyrophosphatase and phosphatase. A portion of the [14C]glucose moiety became covalently linked to endogenous acceptors. Uptake of UDPglucose was saturable and dependent on time and on the concentration of sugar nucleotide. Together, these results were consistent with a transport system for UDPglucose in Golgi vesicles. Furthermore, penetration rate was considerably higher with UDPglucose synthetized in situ from glucose 1-phosphate by membrane-bound pyrophosphorylase than from added UDPglucose: Vmax values were respectively 10 and 2 pmol/15 min per mg protein. This result allows the conclusion that a coupling between translocase and synthetase is involved in UDPglucose transport through Golgi apparatus membranes. The mechanism of this 'kinetic advantage' is discussed.

UDPglucose-ceramide glucosyltransferase from porcine submaxillary glands is associated with the Golgi apparatus.

Subcellular distribution of pig submaxillary gland UDPglucose-ceramide glucosyltransferase (EC 2.4.1.80), the enzyme which catalyses the first step during the sequential addition of carbohydrate moieties for ganglioside biosynthesis, was studied. The results presented strongly suggest that in pig submaxillary gland, the transfer of glucose on endogenous or exogenous ceramides takes place in the Golgi apparatus: the specific activity of UDPglucose-ceramide glucosyltransferase increased in parallel with the activity of a known marker of the Golgi apparatus, UDPgalactose-ovomucoid galactosyltransferase. The specific activity of the glucosyltransferase was 18-times higher in the purified Golgi membranes than in the postnuclear supernatant and the yield was over 30%. An apparent Km of 22 microM for UDPglucose and 54 microM for ceramides was determined. Maximal glucosylation of endogenous ceramides was achieved at pH 6.5 in the presence of NADH (1 mM) as inhibitor of pyrophosphatases and with Mn2+ (5 mM). It was found that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) is an efficient activator for the glucosylation of exogenous ceramides.

Protein-mediated fusion of liposomes with microsomal membranes of Aspergillus niger: evidence for a complex mechanism dealing with membranous and cytosolic fusogenic proteins.

Membrane fusion is a fundamental and wide-spread phenomenon in the functioning of cells. Many studies were carried out concerning fusion of plasma membranes as for example cell-cell fusions or uptake by cells of lipid-enveloped viruses. The present study deals with the interaction of intracellular membranes of Aspergillus niger with artificial membranes (liposomes). Association is monitored by the uptake of radioactive liposomes by fungal microsomal membranes. The discrimination between aggregation and pure fusion is done by layering the liposomes-microsomes mixture on a continuous sucrose gradient. The accurate quantitation of the fusion phenomenon is monitored with a fluorescent assay based on resonance energy transfer (Struck, D.K. et al. (1981) Biochemistry 20, 4093-4099). Both methods show that, at physiological pH, there is a spontaneous fusion of microsomes with cholesterol-free liposomes. This phenomenon is protein dependent as trypsinized microsomal membranes are no longer able to fuse with liposomes. Biological significance of the fusion process has been demonstrated using microsomal intrinsic protein mannosylation assay; the enhancement of the lipid to protein ratio due to the fusion of liposomes with microsomes of A. niger results in an increase in the rate of endogenous proteins mannosylation. Moreover, cytosolic proteins of A. niger promote the fusion of any kind of liposomes with microsomes.

Spatial aspects of mannosyl phosphoryl retinol formation.

Rat liver microsomes catalyze the transfer of mannose from GDPmannose to both retinyl phosphate and dolichyl phosphate to form mannosylphosphorylretinol, mannosylphosphoryldolichol and GDP. The two reactions differ in term of reversibility. In fact, a 200-fold isotopic dilution of GDP[14C]mannose by unlabeled GDPmannose causes mannosylphosphoryldolichol labeling to disappear almost completely, while mannosylphosphorylretinol labeling remains at the same level. The same observation can be made if the mannose donor is removed by centrifugation and replaced by excess GDP; again mannosylphosphorylretinol is stable, but mannosylphosphoryldolichol drops down to one-third of its initial level, as expected for, respectively, a non-reversible and a reversible reaction. Placed in an aqueous medium, mannosylphosphorylretinol releases mannose 1-phosphate (beta configuration) whereas it is quite stable when kept in a membranous environment. These results strongly suggest that mannosylphosphorylretinol as soon as it is formed is segregated in such a way that it is no longer available to the back-reaction; the functional consequence of this segregation would be the possibility for mannosylphosphorylretinol to mannosylate some non-polar regions of certain protein chains.

[Effect of low molecular weight basic proteins on mannose transport in Aspergillus niger microsomes]. / Effet de protéines basiques de faible poids moléculaire sur le transfert de mannose dans les microsomes d'Aspergillus niger.

Basic proteins of low molecular weight activate the transfer of mannose to endogenous glycoprotein acceptors in microsomal membranes of Aspergillus niger. The enhancement of mannosyltransferase activity is linked to the activation of the transport of mannose across the membrane. The role of these polycationic proteins on the membrane permeability is discussed.

[Isolation by affinity chromatography of specialized membrane fractions from cat liver microsomes]. / Isolement par chromatographie d'affinité de fractions membranaires specialisées, à partir de microsomes d'hepatocytes de chat.

Microsomal glucokinase is solubilized by incubation in the presence of several metabolites. After solubilization of the enzymes, the membranes present free sites for specific binding of glucokinase, therefore, they can be purified by affinity chromatography on Sepharose--ATP-glucokinase. This method yields membranous vesicles which contain, in addition to glucokinase, uridylyl-transferase, phosphoglucomutase, sialyl-transferase and adenylate cyclase. Galactosyl-transferase, glucose-6-phosphatase and NADPH cytochrome c reductase are absent. It appears that functionally related enzyme from UDP-glucose biosynthesis are aggregated onto specific patches of the membrane, most likely from Golgi apparatus.

[A receptor for UDP-glucose in the microsomal membranes of rat hepatocytes]. / Mise en évidence d'un récepteur d'UDP-glucose dans les membranes microsomiques des hepatocytes de rat

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.

[Molecular weight of a membrane receptor of UDP-glucose]. / Poids moléculaire d'un récepteur membranaire d'udp-glucose

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.

[Presence of a pathway for biosynthesis of UDP-glucose in the Golgi apparatus of rat hepatocytes]. / Présence d'une voie de biosynthèse de l'UDP-glucose dans l'appareil de Golgi des hépatocytes de rat

Glucokinase, phosphoglucomutase and glucose-1-phosphate uridylyltransferase are the three enzymes involved in a microsomic pathway for the synthesis of UDP glucose. Evidence is given, in this paper, for the localization of these three enzymes in a Golgi-rich fraction of rat liver. This fraction is prepared, from smooth microsomes, by the means of a discontinuous four-step sucrose gradient. Three of the lighter fractions (d = 1.08-1.13) are enriched in the Golgi markers (galactosyltransferase, sialytransferase and thiamin pyrophosphatase), especially the one with density 1.13. The three enzymes we are interested in are enriched in the two upper hands (d 1.08-1.11), which display an activity for the biosynthesis of UDP-glucose from glucose equivalent to the one obtained in a crude microsomic preparation, and which are not contaminated by other subcellular components.

[Liberation and reassociation of the microsomal membrane glucokinase in cat liver]. / Libération et réassociation à la membrane de la glucokinase microsomique du foie de chat.

The particulate glucokinase of cat liver is shown to be microsomal. The activity is readily solubilized by glucose-6-phosphate, ATP, pyrophosphate, high salt concentrations and, to a lesser extent, ribonucleoside triphosphates. The solubilization by glucose-6-phosphate is inhibited by Pi. Solubilizations by ATP and glucose-6-phosphate differ in their sensitivity to temperature changes; they are relatively specific for glucokinase as compared to solubilization by detergent (Triton X 100). The enzyme can be bound again to previously eluted microsomal membranes. Treatment of membrane with trypsin, at 0 degrees C, destroys the ability to rebind the enzyme to the membrane. It is suggested that electrostatic forces are of considerable importance for the binding of glucokinase to a possible protein binding site in the membrane.

[Presence of enzymes catalyzing UDP-glucose biosynthesis in a low density Golgi fractions of cat hepatocytes]. / Présence des enzymes catalysant la biosynthèse d'UDP glucose dans une fraction légère de l'appareil de Golgi des hépatocytes de chat.

A Golgi-rich fraction is prepared from cat hepatocytes by the means of a four-step sucrose density gradient. The material applied to this gradient is composed either of smooth microsomes prepared from healthy animals, or of total microsomes prepared from cat treated by 50 per cent ethanol (0.6 g/100 g body weight, administered by stomach tube). A light fraction (d : 1.10) is obtained by the two procedures. It does not show any glucose-6-phosphatase activity, but is enriched in sialyltransferase, known as a marker enzyme for Golgi apparatus. It also contains the three enzymes implicated in the biosynthetic pathway for UDP-glucose (glucokinase, phosphoglucomutase and UTP : glucose-1-phosphate uridylyltransferase). UDP-glucose being the ultimate substrate in membranous glucosylation reactions, these results could support the hypothesis that sugar-nucleotides necessary for the glycoprotein biosynthesis are produced in the Golgi vesicles directly.

[Monkey liver microsomal glucose-6-phosphatase]. / Glucose-6-phosphatase microsomique d'hépatocytes de singe.

Kinetic studies indicate that glucose-6-phosphatase is a multifunctional enzyme. a) Phosphohydrolase activities. The mannose-6-phosphatase activity is low (Km = 8 mM, VM = 90 nmoles. min-1mg-1). The enzyme shows a strong affinity for glucose-6-phosphate (Km = 2.5 mM, VM = 220 nmoles.min-1mg-1). beta-glycerophosphate (K1 = 30 mM), D-glucose (Ki = 120 mM) are mixed type inhibitors; pyrophosphate (Ki = 2 mM) is a non competitive one. b) Phosphotransferase activities. Di and triphosphate adenylic nucleosides or phosphoenol pyruvate are not substrates. Carbamylphosphate serves as a phosphoryl donor with D-glucose as acceptor. The phosphate transfer is consisstent with a random mechanism in which the binding of one substrate increases the enzymes affinity for the second substrate. Apparent Km values for carbamyl-phosphate range from 5.2 mM (D-glucose concentration leads to infinity) to 8 mM (D-glucose concentration leads to 0). The corresponding apparent Km values for D-glucose are 59 mM (carbamyl-phosphate concentration leads to infinity) to 119 mM (carbamyl-phosphate concentration leads to 0). Maximal reaction velocity with infinite levels of both substrates is 270 nmoles.min-1.mg-1. Pyrophosphate is a poor phosphoryl donnor (Km = 55 mM with D-glucose concentration 250 mM). In addition we do not find any latency; detergents, namely sodium deoxycholate, Triton X 100 do not affect or inhibit glucose-6-phosphatase activity.

[Perparation and comparative analytical study of cat liver microsomes]. / Préparation et étude analytique comparée des microsomes de foie de chat

Cat liver homogenates have been fractionated by differential centrifugation. Four particulate fractions (1 000 X g, 10 000 X g, and 145 000 X g) and a supernatant have been obtained. The biochemical composition of these fractions has been established from the assay and distribution pattern of 22 enzymatic and chemical constituents including marker enzymes for mitochondria, lysosomes, peroxisomes, plasma membranes, endoplasmic reticulum, Golgi apparatus and cell sap. The microsomal fraction was characterized by a moderate contamination with large cytoplasmic granules and by a low yield in protein and cholesterol. It contained 50 per cent of Golgi complex and about 40 per cent of plasma membranes. Morphological analysis of subcellular fractions was performed and confirmed biochemical results.