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.

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.

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.

[Quantitative analysis of a tetraribonucleotide mixture by ultraviolet spectrophotometry]. / Analyse quantitative d'un mélange tétraribonucléotidique par spectrophotométrie dans l'ultraviolet.

Using the least squares method we have calculated the proportions of each nucleotide of a mixture of AMP, CMP, GMP and UMP, after measuring the absorbance of the mixture every ten nanometers from 230 to 290 nm at pH 12.7 and 2. The method is very simple and rapid (the calculations are made in less than one minute), does not require a highly sensitive spectrophotometer to obtain reasonably precise results and, in contrast to the other methods of the literature, it can be applied to quantities as little as 50 micrograms of nucleotides.

[Simple method for preparation of rat liver DNA]. / Technique simple de préparation des acides désoxyribonucléiques de foie de rat.

The frozen tissue was sliced and then homogenized at 20 degree C in LiCl, 2 M; lauryl-trimethyl ammonium chloride (K & K No. 4484), 5%; pronase, B grade (calbiochem), 1 mg/ml and Tris-HCl 10 mM pH 7.5. The homogenate was left to stand 15 min at 20 degree C with occasional shaking. After centrifugation at 35 000 X g for 30 min at 0 degree C, the supernatant containing the crude DNA was purified by filtration on Ultrogel A 2 (LKB, Sweden). The Ultrogel A 2 column (2.5 X 45 cm) was equilibrated with a solution containing NaCl 2 M, EDTA 2.5 mM, Tris-HCl 10 mM pH 7.5. The flow rate was 3 ml cm-2 h-1. Five ml of the supernatant were placed on the column. The first peak contained highly polymerized (as demonstrated by ultracentrifugation) pure DNA (A260/A230 = 3.19; A260/A280 = 1.82). The yield was 2.26 mg of DNA/g of fresh liver.

[Isolation and characterization of beef thyroid giant RNA]. / Isolement et caractérisation des RNA géants de la glande thyroide de boeuf.

Total HMW-RNAs were prepared by three different methods (method with phenol, method with NaClO4, method without phenol using Ultrogel AcA 22 filtration). Giant RNAs were obtained in the void volume by filtration on Sepharose 2B. The giant RNAs/total HMW-RNA ratio is higher (6.77%) with the gel filtration method than with phenol or NaClO4 methods (1.41% and 1.00% respectively). The nucleotide composition of these RNAs is DNA-like and the sedimentation constants are approximately 70-100 S.

[Rapid technic for preparation of thyroid ribonucleic acids without phenol]. / Technique rapide de preparation des acides ribonucleiques thyroidiens sans phenol.

Thyroid tissue was homogenized in 2 M LiCl. The homogenate was alloued to stand 1 h 30 min at 2 degrees C and then centrifuged. The pellet was suspended in 5% triisopropylnaphthalene sulfonic acid (the sodium salt), 0.05 M Tris--HCl, and 0.1 M NaCl (pH 8). After stirring and centrifuging, the supernatant containing the crude RNA was purified by filtration on Ultrogel AcA 22 (LKB, Sweden). Before adding the sample of crude RNA to the column, pronase was placed on the column. When pronase had entered the gel, we added the sample. The first peak contained pure RNA plus some DNA. The former was precipitated with 2 M LiCl. The RNA species obtained by this technique were undegraded and the yield was 30% better than that of the phenol technique.

Ribosomal and transfer RNA contents of the normal and pathologic human thyroid gland.

One hundred and seventy seven pieces of normal or pathologic thyroid tissue from 17 patients were assayed for rRNA, tRNA and DNA content. The tRNA/DNA and rRNA/DNA ratios in pathologic tissue were statistically compared with the same ratios in normal tissue. In toxic adenoma (5 cases) and anoplastic cancer (2 cases), both ratios were increased. In cold nodules (9 cases), there was in increase of the tRNA/DNA ratio only in 1 case, of the rRNA/DNA ratio only in 4 cases, and of both ratios in 3 cases. In one case of a cold nodule in a Basedow's disease gland, there was no modification of these ratios. In Basedow's disease (3 cases), there was an increase of rRNA/DNA ratio only in one case.