Surface biochemical changes accompanying primary infection with Rous sarcoma virus. II. Proteolytic and glycosidase activity and sublethal autolysis

Changes in the cellular adenine nucleotide contents of larval salivary glands of Drosophila hydei as a result of treatments affecting the respiratory metabolism were established and correlated with changes in the activity of four genome loci. The results demonstrate that the activation of the genome loci is not a necessary consequence of a reduction in the ATP level or changes in ADP or AMP levels. Other regulatory mechanisms for the activation of these genome loci are discussed.     

Electrokinetic properties of asparaginase sensitive or resistant mouse leukemic cells treated with L-asparaginase

The electrophoretic mobility of L5178Y cells in 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO₃, pH 7.2, at 25°C was — 1.78 μ·s^?1·V^?1·cm^?1 while that of an L-asparaginase resistant subline, L5178Y/ASN, was — 1.11 μm·s^?1·V^?1·cm^?1. Both cell lines were characterized by terminal sialic acid residues on their surfaces. Treatment of L5178Y cells for 90 min with 10 units of L-asparaginase per ml in saline decreased the electrophoretic mobility of the cells to — 1.65 μm·s^?1·V^?1·cm^?1 while treatment in Fischer's medium decreased the mobility to — 1.25 μm·s^?1·V^?1·cm^?1; neither treatment had a significant effect on the L5178Y/ASN electrophoretic mobility. The results suggest that L-asparaginase has an immediate and specific effect on synthesis of cell surface asparaginyl glycoproteins.     

SYNTHESIS OF GLYCOPROTEINS IN BRAIN: IDENTIFICATION, PURIFICATION AND PROPERTIES OF GLYCOSYL TRANSFERASES FROM PURIFIED SYNAPTOSOMES OF GUINEA PIG CEREBRAL CORTEX

Abstract— Four glycoprotein:glycosyl transferases (a fetuin:N-acetylglucosaminyl transferase; a bovine submaxillary mucin: N-acetylgalactosaminyl transferase; a collagen: glucosyl transferase and an orosomucoid: galactosyl transferase) were purified 34-, 45-, 37- and 47-fold, respectively, from synaptosomes prepared from guinea pig cerebral cortex. Purifications were achieved by centrifugation and by column chromatography on Sephadex G-100 and G-150 of 0 , 1% (w/v) Triton X-100 extractsof the purified cerebral cortical synaptosomes. The enzymes were separated from endogenous acceptors and were highly specific for specific macromolecular acceptors; small molecules were ineffective as acceptors. The fetuin: N-acetylglucosaminyl transferase functioned only with fetuin minus N-acetylneuraminic acid, galactose and N-acetylglucosamine; the bovine submaxillary mucin: N- acetylgalactosaminyl transferase with bovine submaxillary much minus N-acetylneuraminic acid and N-acetylgalactosamine; the collagen: glucosyl transferase with collagen minus glucose; and the orosomucoid: galactosyl transferase with either orosomucoid minus N-acetylneuraminic acid and galactose or fetuin minus N-acetylneuraminic acid and galactose. Each transferase required a specific (XDP)-monosaccharide for transfer. The transferases were entirely dependent on either Mn²⁺ or Mg²⁺ for activation and Fe²⁺ and Hg²⁺ inhibited each of the four enzymes. The optimum pH's for the enzymes were: for fetuin: N-acetylglucosaminyl transferase, 7 , 4–8.0; for bovine submaxillary mucin: N-acetylgalactosaminyl transferase, 7 , 7; for collagen: glucosyl transferase, 7 , 7 and for orosomucoid: galactosyl transferase, 6 , 6. The enzymes were distributed subsynaptosomally primarily in the synaptosomal plasma membrane and in the mitochondria of the synaptosome. The respective values for K_m (μM) and V_mex (pmoles/h/mg of protein) for the transferases were: fetuin: N-acetylglucosaminyl transferase, 12 and 143; for bovine submaxillary mucin: N-acetylgalactosaminyl transferase, 25 and 166; for collagen: glucosyl transferase, 4 and 10 and for orosomucoid:galactosyl transferase, 8 and 111.     

Membrane glycoprotein biosynthesis: Changes in levels of glycosyl transferases in fibroblasts transformed by oncogenic viruses

Fibroblasts transformed by oncogenic viruses were found to have greater enzymic activities of four membrane glycoprotein:glycosyl transferases on a cell or protein basis then two non-transformed fibroblastic lines. These enzymes are responsible for the synthesis of membrane glycoproteins; each of the four transferases studied, the polypeptide:N-acetylgalactosaminyl, glycoprotein:galactosyl, fetuin:fucosyl and PSM:fucosyl transferases, was more than twice as active in the transformed cell lines using both endogenous and added receptor. The most pronounced differences occurred with the doubly (SV-PY-3T3) transformed fibroblasts in all cases; with the N-acetylgalactosaminyl and galactosyl transferases the increase was 8–16 fold over the non-transformed cells. It was demonstrated that these results do not arise from a changed level of glycosidase activities.     

Electrokinetic properties of isolated cerebral-cortex synaptic vesicles

Synaptic vesicles isolated from guinea-pig cerebral cortex had an electrophoretic mobility of -3.55mum.s(-1).V(-1).cm in saline-sorbitol, pH7.2, at 25 degrees C (ionic strength 0.015g-ions/1). The mobility was pH-dependent, varied with ionic strength and indicated that the vesicular surface contained weak acidic functions with a pK(a) in the range 3.0-3.8. Although the vesicular surface was determined to be highly negatively charged, treatment with neuraminidase had no effect on mobility and indicated that the relatively strong carboxyl groups of sialic acid do not contribute significantly to vesicular electrokinetic properties. Treatment of synaptic vesicles with trypsin or trypsinized concanavalin A resulted in increases in mobility, but treatment with ribonuclease, deoxyribonuclease, chrondroitinase ABC or hyaluronidase had no significant effect on mobility. Mn(2+) or Ca(2+) was more effective in decreasing vesicle mobility than was Mg(2+), Sr(2+) or Ba(2+). The electrokinetic properties of the synaptic vesicle surface are discussed and contrasted with the properties of the synaptosomal membrane.     

MOLECULES AT THE EXTERNAL NUCLEAR SURFACE : Sialic Acid of Nuclear Membranes and Electrophoretic Mobility of Isolated Nuclei and Nucleoli

The molecules occurring as terminal residues on the external surfaces of nuclei prepared from rat liver by either sucrose-CaCl₂ or citric acid methods and nucleoli derived from the sucrose-CaCl₂ nuclei were studied chemically and electrokinetically. In 0.0145 M NaCl, 4.5% sorbitol, and 0.6 mM NaHCO₃ with pH 7.2 ± 0.1 at 25°C, the sucrose-CaCl₂ nuclei had an electrophoretic mobility of -1.92 µm/s/V/cm, the citric acid nuclei, -1.63 µm/s/V/cm, and the nucleoli, -2.53 µm/s/V/cm. The citric acid nuclei and the nucleoli contained no measurable sialic acid. The sucrose-CaCl₂ nuclei contained 0.7 nmol of sialic acid/mg nuclear protein; this was essentially located in the nuclear envelope. Treatment of these nuclei with 50 µg neuraminidase/mg protein resulted in release of 0.63 nmol of sialic acid/mg nuclear protein; treatment with 1 % trypsin caused release of 0.39 nmol of the sialic acid/mg nuclear protein. The pH-mobility curves for the particles indicated the sucrose-CaCl₂ nuclei surface had an acid-dissociable group of pK. ~2.7 while the pK for the nucleoli was considerably lower. Nucleoli treated with 50 µg neuraminidase/mg particle protein had a mobility of -2.53 µm/s/V/cm while sucrose-CaCl₂ nuclei similarly treated had a mobility of -1.41 µm/s/V/cm. Hyaluronidase at 50 µg/mg protein had no effect on nucleoli mobility but decreased the sucrose-CaCl₂ nuclei mobility to -1.79 µm/s/V/cm. Trypsin at 1 % elevated the electrophoretic mobility of the sucrose-CaCl₂ nuclei slightly but decreased the mobility of the nucleoli to -2.09 µm/s/V/cm. DNase at 50 µg/mg protein had no effect on the mobility of the isolated sucrose-CaCl₂ nuclei but decreased the electrophoretic mobility of the nucleoli to -1.21 µm/s/V/cm. RNase at 50 µg/mg protein also had no effect on the electrophoretic mobility of the sucrose-CaCl₂ nuclei but decreased the nucleoli mobility to -2.10 µm/s/V/cm. Concanavalin A at 50 µg/mg protein did not alter the nucleoli electrophoretic mobility but decreased the sucrose-CaCl₂ nuclei electrophoretic mobility to -1.64 µm/s/V/cm. The results are interpreted to mean that the sucrose-CaCl₂ nuclear external surface contains terminal sialic acid residues in trypsin-sensitive glycoproteins, contains small amounts of hyaluronic acid, is completely devoid of nucleic acids, and binds concanavalin A. The nucleolus surface is interpreted to contain a complex made up of protein, RNA, and primarily DNA, to be devoid of sialic acid and hyaluronic acid, and not to bind concanavalin A.     

Cell plasma membrane external surface glycosyltransferases: Activity in the cell mitotic cycle

Activity levels of three cell surface glycoprotein:glycosyltransferases were found to be S-peak enzymes in the cell mitotic cycle of L5178Y cells. These enzymes, which may be important in cell—cell adhesion, had virtually no activity in the M-period of the cell mitotic cycle.