Terukatsu Sasaki

A new assay system of phospholipid exchange activities using concanavalin a in the separation of donor and acceptor liposomes

A new assay system of phospholipid exchange activities is described. The exchange activities were quantitated by measuring the stimulation of phospholipid transfer between two separate populations of liposomes, which contained, as the major constituents, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, and cholesterol in molar ratios of 6 :2 : 1: 1: 5. One population of the liposomes was made reactive to concanavalin A by the incorporation of 1.8 mol% alpha-D-mannosyl-(1 leads to 3)-alpha-D-mannosyl-sn-1, 2-diglyceride from Micrococcus lysodeikticus. The concanavalin A-reactive liposomes, a phospholipid donor, were doubly labelled with [6-3H] galactosylglucosyl ceramide and that class of 32P-labelled phospholipids whose exchange was being measured. The 3H-labelled glycolipid served as a non-exchangeable reference marker. The other population of the liposomes, a phospholipid acceptor, was concanavalin A nonreactive. These two populations of liposomes were incubated with the cytosol protein of rat liver in a total volume of 0.2 ml. After the incubation, two different procedures were used to separate the two liposomal populations. In one procedure concanavalin A was added to agglutinate the reactive liposomes; the flocculated lectin . liposome complex was separated from the non-reactive liposomes by brief centrifugation. In the other procedure the reactive liposomes were trapped by binding to concanavalin A covalently coupled to Sepharose 2B; the complex was separated from the non-reactive liposomes by filtration through a filter paper under suction. In both assay procedures the amount of phospholipid transferred from the donor to the acceptor liposomes was calculated from the decrease of 32P/3H ratio of the concanavalin A-reactive liposomes during the incubation. By the assasy system it is possible to determine phosphatidylcholine and phosphatidylinositol exchange activities in 100 micrograms of rat liver cytosol protein.

Effects of anchorage-modulating doses of concanavalin A, microtubule-disrupting drugs and microfilament perturbants, cytochalasins, on the phosphatidylinositol response of rat lymph-node cells

In lymphocytes isolated from rat lymph nodes, concanavalin A stimulated the 32PO4 incorporation into phosphatidylinositol and phosphatidic acid in a dose-dependent manner up to 200 micrograms of the lectin per ml of the lymphocyte culture. [3H]Thymidine incorporation was found to be optimal at 2 micrograms concanavalin A per ml of the culture when the incorporation was examined at the same cell density as was used in the determination of the 32PO4 incorporation. As previously described (Wang, J.L. and Edelman, G.M. (1978) J. Biol. Chem. 253, 3000-3007), the [3H]thymidine incorporation was inhibited at doses higher than 5 micrograms/ml in a dose-dependent manner. These results indicate that concanavalin A produced the phosphatidylinositol PI response of rat lymph-node cells in the dose range in which the mobility and distribution of lymphocyte surface receptors were modulated by the lectin (Yahara, I. and Edelman, G.M. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 608--612). Colchicine and vinblastine at a concentration of 10(-4) M did not inhibit the concanavalin A-induced PI response of rat lymph-node cells. Cytochalasins B and D at a concentration of 10(-5) M enhanced the concanavalin A-induced PI response to some degree. All the results obtained suggest that submembranous assemblies of microtubules and microfilaments do not play an indispensable role in the sequence of events involved in the PI response of rat lymph-node cells.

Hydrolysis of phosphatidylinositol 4,5-bisphosphate and increase in cytosolic free Ca2+-concentration induced in a human T cell leukemia line, JURKAT, by monoclonal antibodies against the T3 complex.

The monoclonal antibodies against the T3 complex on human T lymphocytes, anti‐Leu‐4, OKT3, and T3, induced an accumulation of inositol phosphates in a human T cell leukemia line, JURKAT, in the presence of LiCl. The monoclonal antibodies also induced an increase in the cytosolic free Ca2+‐concentration ([Ca2+]i) in JURKAT. The accumulation of inositol phosphates and the increase in [Ca2+]i were specifically induced by the monoclonal antibodies against the T3 complex. Other monoclonal antibodies against differentiation antigens on human T lymphocytes were not active in inducing these responses in JURKAT. Stimulation of JURKAT by anti‐Leu‐4 induced a rapid and immediate decrease in phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] and an increase in the 32P‐labeling of phosphatidic acid, which occurred after a short lag period. An analysis of inositol phosphates formed in the anti‐Leu‐4‐stimulated JURKAT indicated the formation of inositol trisphosphate. These results strongly suggested that the T3 complex or T3/antigen receptor (Ti) complex functions as a receptor which transduces antigen signal, presented by either antigen‐presenting cells or target cells, into the hydrolysis of PtdIns(4,5)P2. Fetal bovine serum at a dose of 1–20 μl/ml induced a marked and transient [Ca2+]i increase in JURKAT immedately after addition. However, the level of formation of inositol phosphates was very small in cells stimulated by fetal bovine serum. Fetal bovine serum induced an immediate increase in the 32P‐labeling of phosphatidic acid in JURKAT. These and other results suggested that serum increased [Ca2+]i in JURKAT by a mechanism different from that for the anti‐Leu‐4‐induced [Ca2+]i response.

Extensive radioactive labeling of glycolipids in cultured hamster fibroblasts by incubation of whole cells with UDP-[14C]glucose and UDP-[14C]galactose.

NIL 2cl cells, a cloned hamster cell line, are known to have seven classes of neutral glycosphingolipids in addition to hematoside. In this paper, I examined the participation of cell surface glycosyltransferases in the glycolipid synthesis in NIL cells. The cells, harvested from culture bottles by the use of chelating agents of divalent cations, incorporated radioactive glucose and galactose from labeled UDPglucose and UDPgalactose into endogenous glycolipid acceptors to form all eight classes of glycosphingolipids and dolichyl phosphate glucose. The major part of the incorporation occurred by glycosyltransferase reactions from nucleotide sugars, as indicated by a slight inhibition of the incorporation observed in the presence of a large excess of sugars or sugar phosphates. Acid hydrolysis of the glycolipid products showed that 91.4% of the 14C label was in the galactose and glucose moieties of the glycolipids. Exogenously added lactosylceramide stimulated the synthesis of trihexosylceramide in the absence of detergents. Th results presented in this paper suggest that incubation of whole cells with an equilibrium mixture of radioactive UDPglucose and UDPgalactose is a useful radioactive labeling method to determine the glycolipid composition of a cultured cell.

Sequential glycosylations of endogenous glycosphingolipids in hamster fibroblasts incubated with nucleotide sugars

Hamster fibroblasts, NIL 2cl cells, which were harvested from culture bottles by the use of chelating agents of divalent cations, incorporated [14C]galactose and N-acetyl[14C]galactosamine (GalnAc) from corresponding nucleotide 14-C-labeled sugars into endogenous glycosphingolipid acceptors. Incorporation of [14C]-GalNAc from UDP-[14C]GalNAc into endogenous acceptors to form tetra- and pentahexosylcermide with terminal GalNAc was enhanced 3.8-gold by preincubation of the cells with UDPglucose and UDPgalactose. In the whole cell reactions, sequential glycosylations of endogenous glycosphingolipids were observed by addition of whole cell reactions, sequential glycosylations of endogenous glycosphingolipids were observed by addition of appropriate nucleotide sugars to the reaction mixture: in a two step incubation experiment, mono-, di= and trihexosylceramides newly glycosylated from UDP-[14C]glucose and UDP-[14C] galactose in the first step of the whole cell reaction were effectively converted to hematoside by addition of CMP-N-[3H]acetylneuraminic acid in the second step. In the sequential glycosylation of endogenous glycolipids observed in the whole cell reaction, quantitative differences were found between cells harvested from confluent cultures and cells harvested from sparse cultures. Furthermore, a result was obtained which indicates the presence of two distinct pools of each glycosphingolipid in NIL cells. Glycolipids in the one pools represent newly glycosylated glycolipids and act as substrates for further glycosylations. On the other hand, precursor-product relationships do not exist among the main pools of each glycolipid.