Comparison of sialyl- and alpha-1,3-galactosyltransferase activity in NIH3T3 cells transformed with ras oncogene: increased beta-galactoside alpha-2,6-sialyltransferase.

Previous studies have indicated that transfection of NIH3T3 cells with the ras oncogene induced modifications of the terminal glycosylation of N-linked glycans which appeared in the early stage after transfection. These changes affected especially the terminal part of N-linked glycans which is substituted with alpha-1,3-Gal residues in NIH3T3 and with Neu5Ac residues in the ras-transformed counterpart. We have transformed NIH3T3 cells with the human c-Ha-ras oncogene, evaluated tumorigenicity and metastatic capacity in vivo and compared alpha-1,3-galactosyltransferase, alpha-2,3- and alpha-2,6-sialyltransferases activities. By using different specific acceptors, we detected the enhancement of sialic acid transfer in transformed cells while the activity of alpha-1,3-galactosyltransferase remained unchanged. We showed that the higher sialyltransferase activity was due to the increase of beta-galactoside alpha-2,6-sialyltransferase in ras-transfectant although alpha-2,3-sialyltransferase was weakly expressed in these cells. On the basis of binding of different lectins, we correlated these observations with changes of protein glycosylation. We concluded that altered glycosylation of ras-transformed NIH3T3 is the result of a competitive effect of the enzymes acting for terminal glycosylation of N-linked glycans and the reflection of the higher expression of alpha-2,6-sialyltransferase.     

Structure of neutral glycolipids in bovine thyroid tissue

Four asialo glycolipid fractions have been isolated from bovine thyroid glands. The structures were elucidated by partial hydrolysis, periodate oxidation, permethylation analysis and sequential enzymatic degradation studies. The following structures were identified: GL-1a glucosyl-beta-(1 leads to 1)ceramide; GL-1b galactosyl-beta-(1 leads to 1)ceramide: GL-2 galactosyl-beta-(1 leads to 4)glucosyl-beta-(1 leads to 1)ceramide: GL-3 galactosyl-alpha-(1 leads to 4)galactosyl-beta-(1 leads to 4)glucosyl-beta-(1 leads to 1)-ceramide; GL-4 N-acetylgalactosaminyl-beta-(1 leads to 3)galactosyl-alpha-(1 leads to 4)galactosyl-beta-(1 leads to 4)glucosyl-geta-(1 leads to 1)ceramide.     

Dolichyl-pyrophosphoryloligosaccharide protein oligosaccharide transferase in neuronal ceroid-lipofuscinosis.

The kinetics of the oligosaccharide transfer from oligosaccharyl pyrophosphoryldolichol to endogenous protein acceptors in human fibroblasts were studied. No alterations in the transferase activity and enzyme characteristics could be observed in fibroblasts from neuronal ceroid-lipofuscinosis (NCL) patients. Analysis of urinary dolichol of two NCL patients also did not reveal substantial differences with respect to controls.     

Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.