A comparative analysis of strategies for isolation of matrix vesicles.

Matrix vesicles (MVs) are extracellular organelles involved in the initial steps of mineralization. MVs are isolated by two methods. The first isolation method of MVs starts with collagenase digestion of osseous tissues, followed by two differential centrifugations. The second isolation method does not use proteases but rather starts with differential centrifugation, followed by a fractionation on a sucrose gradient. The first method results in a homogeneous population of MVs with higher cholesterol/lipid content, alkaline phosphatase activity, and mineral formation rate as compared with MVs isolated by the second method. The second method leads to higher protein diversity as compared with MVs isolated according to the first method. Due to their distinct protein composition, lipid-to-protein and cholesterol-to-phospholipid ratios, and differences in rates of mineral formation, both types of isolated MVs are crucial for proteomic analysis and for understanding the regulation of mineralization process at the molecular level.

Activity increase after extraction of alkaline phosphatase from human osteoblastic membranes by nonionic detergents: influence of age and sex.

The solubilization of alkaline phosphatase (AP) from osteoblastic cell membranes obtained from human primary bone cell cultures was studied according to the age and sex of the donors (17 females, 11 males; age range: 2-77 years). Cell membranes were treated by non-ionic (n-octyl beta-D-glucopyranoside, OG), ionic or zwitterionic detergents, then centrifuged. When OG was used almost all the AP was solubilized. AP activity in supernatant of solubilization was compared to the activity of the suspension before centrifugation. The activity ratio (AR) increased in function of age for subjects between 65 and 74. Neither total nor specific AP activities were influenced by age or sex. Electrophoresis studies showed that the AP released was a GPI (glycosyl phosphatidylinositol)-anchored protein, amphipathic form, with 140 kDa as apparent molecular mass. The activity change of AP in the presence of OG may result from age-related modifications either in the AP structure or in the constituents of the plasma membranes (proteins or phospholipids).

Differential solubilization of osteoblastic alkaline phosphatase from human primary bone cell cultures.

Mineralization of cartilage and bone requires alkaline phosphatase activity. In order to study the enzymatic properties of bone alkaline phosphatase in bone disease and more particularly in patients with osteoporosis and osteoarthritis, we investigated the solubilization of alkaline phosphatase from primary bone cell cultures derived from human bone explants. To study the release of alkaline phosphatase from membranes, several detergents at a concentration above the critical micellar concentration and cholesterol were used. Solubilized alkaline phosphatase was characterized by enzymatic activity and electrophoresis analysis. Almost all the alkaline phosphatase was solubilized using non-ionic detergent as n-octylglucoside and hecameg. In comparison with initial membranous activity, the solubilized activity was increased by a factor, i.e. 2 +/- 0.05 (SEM, n = 3) (with n-octylglucoside), i.e. 2.1 +/- 0.05 (SEM, n = 3) (with Hecameg). With an ionic detergent (sodium dodecylsulfate), zwitterionic detergent ((cholamido propyl) dimethylammonio 1 propane sulfonate) and cholesterol, a fraction of alkaline phosphatase was resistant to solubilization. Electrophoresis studies showed that released alkaline phosphatase was a glycosylphosphatidylinositol protein (amphipatic form) with 140 kDa as apparent molecular weight. A hydrophilic form was obtained by treatment with a specific lipase. This study showed differential solubilization of osteoblastic alkaline phosphatase from human primary bone cell cultures. Better extractibility and higher activation of this membrane anchored enzyme were obtained with non-ionic detergents.

Evidence for incorporation of N-acetylglucosamine in endogenous nuclear acceptors during the nuclear matrix preparation.

1. The presence of glycoproteins within the nucleus of cell is now well established and the question arises on the nature of the nuclear glycosylation and the site of their glycosylation. 2. In order to study endogenous nuclear proteins acceptors, we have isolated a subnuclear fraction: nuclear matrix characterized by DNA, RNA, phospholipids and proteins content. Nuclear matrix acceptors were obtained from nuclei incubated with UDP-N-acetyl [14C]glucosamine. 3. In this report we describe the presence of three major glycoproteins labeled with N-acetyl [14C]glucosamine in the nuclear matrix fraction. We obtained gP32, gP67 and gP70 with pI values around 6.2, 6.5 and 8.2.

Transfer of N-acetylglucosamine to nuclear endogenous acceptors of rat hepatocytes.

1. Nuclei were prepared from rat hepatocytes. A biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. 2. The transfer of [14C]N-acetylglucosamine to endogenous acceptors were studied comparatively in the nuclei and in the other subcellular fractions of rat hepatocytes. 3. In this report we describe the presence of the transfer of N-acetylglucosamine within the nucleus of rat hepatocytes. We found 21% of this transfer in the nucleus fraction with an enrichment of 26 in comparison to homogenate.

Studies on the mechanism of different collagen glucosyltransferase reactions (Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum) in chick embryo liver.

1. The galactosylhydroxylysylglucosyltransferase (GGT) specific to collagen is located in the RER (rough endoplasmic reticulum), SER (smooth endoplasmic reticulum) and Golgi apparatus for the chick embryo liver. 2. The UDP-glucose collagen glucosyltransferase activities in chick embryo liver were solubilized by Nonidet P-40. 3. The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparation of Golgi apparatus CF2, smooth endoplasmic reticulum CF4 and rough endoplasmic reticulum CF8. 4. For the three fractions, data obtained in experiments were consistent with a sequential ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, collagen and UDP-glucose substrate, with different values for Km and Vmax.

Solubilization of UDP-glucose collagen glucosyltransferase activities from chick embryo liver: Golgi apparatus, smooth and rough endoplasmic reticulum.

1. The choice of a suitable detergent for solubilization of UDP-glucose collagen glucosyltransferase (GGT) activities from chick embryo liver has been investigated. Several detergents were used (zwitterionic detergent as Chaps, and non-ionic detergents as Triton X-100, Nonidet P 40, Brij 35). 2. All the detergents with GGT activities were tested in Golgi apparatus, smooth and rough endoplasmic reticulum (SER, RER). 3. 80-100% GGT Golgi apparatus activity was easily solubilized at low concentrations in surfactant (0.5 mg/ml). 25-78% of SER and RER GGT activities were extracted at this concentration. 4. A higher level of detergent (5 mg/ml) was necessary to release all GGT activities of SER and RER. Protein extraction was identical to GGT activities.

Alkaline phosphatase from Echinococcus multilocularis: purification and characterization.

1. Kinetic and physical parameters of purified alkaline phosphatase from Echinococcus multilocularis metacestodes, livers of infected gerbils and control animals were determined. 2. Km value for p-nitrophenyl phosphate was about 0.05 +/- 0.02 mM for the three enzymes. 3. Vmax values were 357 +/- 67 nmol/min/mg proteins for metacestode enzyme, and 6.7 +/- 1.1 and 6.7 +/- 0.8 nmol/min/mg proteins for liver enzyme of infected and control animals, respectively. 4. Mr and pI were different for the parasite and hepatic enzyme. 5. The parasite enzyme was less sensitive to the elevation of temperature than hepatic enzyme. 6. The isatin inhibition was a competitive inhibition type for parasite and uncompetitive type for host liver enzyme.

Characterization and purification of culture-derived soluble glycoproteins of Babesia canis.

The addition of [14C]-glucosamine to media of Babesia canis cultures causes the appearance of labeled glycoproteins in the culture supernatants. These radioactive soluble glycoproteins were separated according to their molecular weight by gel filtration and according to their (acidic) pI by preparative electrofocusing. The labeled fractions were then analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The results showed three series of glycoproteic antigens. The molecular weights for the three antigens determined by gel filtration and by SDS-PAGE were approximately 100, 40, and 12.5 kDa, and the preparative gel electrofocusing suggested that the antigens focus in the pH range of 3-5.

Effect of vitamin A deficiency on the in vitro transfer of mannose and N-acetylglucosamine in rat liver nuclei.

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.

Evidence of partial localization in Golgi apparatus of UDP-glucose collagen glucosyltransferase from chick embryo liver.

1. Collagens are the most important components of the connective tissue. 2. Collagen synthesis involves greater than 12 different enzymes whereas three enzymatic systems are involved in the ordered degradation. 3. Some enzymes are found in the rough endoplasmic reticulum (RER). The subcellular localization of disulfur isomerase, alpha D-glucosidase, proteases, galactosyltransferases and glucosyltransferases specific to collagen is unknown. 4. After having determined the best subcellular fractionation conditions for the chick embryo liver, we demonstrate that the galactosylhydroxylysyl glucosyltransferase specific to collagen is located in the RER and in the Golgi apparatus.

Echinococcus multilocularis: comparative study of glycogen synthase in metacestodes and in the livers of infected and control Meriones unguiculatus.

A comparative study of the kinetic parameters of glycogen synthase was performed on Echinococcus multilocularis metacestodes and on the livers of infected and control host (Meriones unguiculatus). The enzyme of the parasite was found to be different from the enzyme of infected host liver. The apparent Km for UDP-glucose is 100 microM for the parasite and 400 microM for the host liver. The apparent Km for glucose 6-phosphate is 4 mM for the parasite and 2 mM for the host liver. The apparent Km for glycogen is 16 mg/ml for the parasite and 125 mg/ml for the host liver. The influence of glucose 6-phosphate and exogenous glycogen on the activity of glycogen synthase differs between the metacestode and the host liver. The enzyme of the metacestodes apparently does not need exogenous glycogen to work, contrary to the case for the liver host enzyme. The glycogen synthase of the parasite seems to be present in forms I and D, whereas the enzyme of the host liver appears in form I and that of the control liver in form D.

A comparative study of UTP-D-glucose-1-phosphate uridylyl transferase in the cysts of Echinococcus multilocularis and the livers of infected and control Meriones unguiculatus.

Kinetic and physical parameters of UDP-glucose pyrophosphorylase were determined in Meriones unguiculatus infected with Echinococcus multilocularis metacestodes (cestoda). Studies were carried out on parasite cysts, and on livers from control and infected animals after purification of the enzyme by affinity chromatography on UTP-agarose. The enzyme from infected and control livers had km values for UTP of 0.01 mM and 0.5 mM, respectively; for glucose-1-phosphate values were 0.46 mM and 0.07 mM, respectively. On the other hand the enzyme from cysts was found to have a higher Km for UTP (1 mM) and for glucose-1-phosphate (1.5 mM) than from infected or non-infected livers. Physical characteristics (pI = 6 and Mr = 160,000) of UDP-glucopyrophosphorylases were the same in controls and infected host livers but were different from the cyst enzyme (pI = 7 and Mr = 251,000). These results provide evidence for the existence of significant differences between parasitic and host enzymes, which could possibly be exploited in chemotherapy.

Temperature and external electric field influence in membrane permeability of Babesia infected erythrocytes.

1. Modification of erythrocyte membrane properties infected by Babesia canis was studied using the effect of electric pulses of short duration. 2. This process induces the formation of pores in the membrane and the releasing of hemoglobin and other cytoplasmic proteins into the external medium. 3. The rate of molecular permeation across the electrically perforated membranes depends on several factors: electric-field strength, pulse number, pulse duration, temperature and cellular concentration. 4. Even for low parasitemia, differences in the effect of these parameters were observed between infected and non-infected erythrocytes. 5. Here we describe an influence of electric field intensity and temperatures on the opening pores.

Permeability of membrane of Babesia canis infected erythrocytes--influence of an external electric field.

The erythrocytes infection by a parasite (Babesia canis) induced a modification of the biological membrane which was studied using the effect of electric pulses of short duration. This process induces the formation of pores and during the opening hemoglobin and other cytoplasmic proteins diffuse out of the cells and are recovered in the external medium. The rate of molecular permeation across the electrically perforated membranes depends on several factors: electric-field strength, pulses number, pulse duration, temperature and cellular concentration. Even for low parasitemia, differences in the effect of these parameters were observed between infected and non-infected erythrocytes.

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.

Purification of a UTP:D-glucose-1-phosphate uridylyltransferase from Golgi apparatus of cat liver by affinity chromatography on UTP-agarose.

UDP-glucose pyrophosphorylase from Golgi apparatus solubilized by detergent has been purified 100-fold from microsomes by affinity chromatography on UTP-agarose. The purified enzyme has apparent Mr 270,000 and isoelectric pH 3.9 against 360,000 and 4.2 for soluble enzyme. According to these characteristics, UDP-glucose pyrophosphorylase from Golgi apparatus is different from cytosolic enzyme.

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.

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.