sepharose and Leukemia--Lymphoid

The glycosylation of beta-hexosaminidase was investigated in the transformed cell-line, CCRF/CEM, derived from a human acute lymphoblastic leukaemia, and comparisons were made with enzyme from normal human skin fibroblasts. A series of studies including neuraminidase sensitivity, lectin chromatography, Biogel P4 chromatography of [3H]-mannose-labelled glycopeptides and endoglycosidase susceptibility, provided clear evidence that in CCRF/CEM cells, beta-hexosaminidase was abnormally glycosylated. The results indicate that leukemia-associated changes in beta-hexosaminidase expression are probably due to increased sialylation of highly-branched complex oligosaccharides.

Topics: Acetylglucosaminidase; beta-N-Acetylhexosaminidases; Cell Line, Transformed; Chromatography, Gel; Glycopeptides; Glycosylation; Humans; Leukemia, Lymphoid; Lysosomes; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Protein Processing, Post-Translational; Sepharose

Detergent-solubilized plasma-membrane proteins from lymphocytes of patients with chronic lymphocytic leukaemia were applied to phenylboronic acid-agarose. Some of the polypeptides were bound specifically and subsequently eluted with sorbitol. Immunoprecipitation analyses showed that no surface immunoglobulin M was bound, but that most of the histocompatibility antigens HLA-A, HLA-B, HLA-C and HLA-DR were.

Topics: alpha-Fetoproteins; Boronic Acids; Chromatography, Agarose; Chromatography, Gel; Deoxycholic Acid; HLA Antigens; Humans; Leukemia, Lymphoid; Lymphocytes; Membrane Proteins; Octoxynol; Polyethylene Glycols; Receptors, Antigen, B-Cell; Sepharose

A method is described by which an immunoaffinity matrix was constructed by binding antibody directly or indirectly to protein A-Sepharose 4B followed by cross-linking of the complex with dimethyl pimelimidate. This allows optimal spatial orientation of antibodies and, thus, maximum antigen binding efficiency. The affinity matrices were stable to high and low pH buffers without any significant antibody loss. The optimal conditions of antibody saturation, cross-linker concentration, and elution system were established and affinity columns made with the monoclonal antibodies J5, W6/32, and OKT9 for one-step isolation of the common acute lymphoblastic leukemia-associated antigen, HLA-AB antigens, and transferrin receptor, respectively, from cell lysates. The same methodology was also applied to immobilize transferrin with polyvalent anti-transferrin antibodies. This was then used to isolate the transferrin receptor from cell lysates.

Topics: Antibodies, Monoclonal; Antigen-Antibody Complex; Cell Line; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Humans; Leukemia, Lymphoid; Membrane Proteins; Molecular Weight; Sepharose; Staphylococcal Protein A; Transferrin

Human and mouse lymphocytes were surface-labeled by lactoperoxidase-catalyzed iodination, or by galactose oxidase oxidation followed by reduction with tritiated sodium borohydride. The labeled cells were lysed with Nonidet P-40. Proteins binding to Helix pomatia A hemagglutinin (HP) were isolated by affinity chromatography on HP-Sepharose and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. A major cell surface glycoprotein (apparent mol. wt. 150 000, using reducing conditions) on human lymphocytes was responsible for almost all binding of HP. This protein was present on normal and malignant thymus-derived lymphocytes, e.g. thymocytes, blood T cells and T leukemia cell lines. It was also found on chronic lymphocytic leukemia cells, one null cell leukemia line, one unidentified leukemia line, one lymphoblastoid cell line of B origin and on one stem cell lymphoma line. In contrast, this protein was not found on various B cells at different steps of differentiation, e.g. four B lymphoma lines or one myeloma line. It was also absent from a histiocytic leukemia line. However, two of the four B lymphoma lines and the myeloma line had another HP-binding surface glycoprotein (mol. wt. 200 000) instead of the 150 000 protein. Studies of mouse lymphocytes similarly showed that thymus-derived lymphocytes (normal and malignant) but not normal adult B cells expressed a major HP-binding surface glycoprotein of apparent mol. wt. 130 000 (reducing conditions).

Topics: Agglutinins; Animals; B-Lymphocytes; Binding Sites; Cell Line; Cell Membrane; Glycoproteins; Helix, Snails; Hemagglutinins; Hematopoietic Stem Cells; Humans; Lectins; Leukemia, Lymphoid; Mice; Sepharose; T-Lymphocytes

Neocarzinostatin (NCS) is an acidic protein (molecular weight, 10,700) isolated from Streptomyces carzinostaticus that has antitumor activity both in model rodent systems and in humans. In vitro it inhibits the growth of a human lymphoblastic leukemic cell line (CCRF-CEM) at a very low concentration (the amount of drug that causes a 50% inhibition of growth compared to control cultures as extrapolated from a dose-response curve (ID50), 2.4 X 10(-9) M). We covalently coupled NCS to the N-hydroxysuccinimide ester of agarose and obtained a product that, by a variety of biochemical and immunological criteria, has been demonstrated to be devoid of any free or loosely bound NCS. Agarose-bound NCS, which is unable to enter cells because of its size, retains a significant amount of inhibitory activity (ID50, 6 to 15 X 10(-9) M) and is also capable of inhibiting tritiated deoxythymidine incorporation into CCRF-CEM cells. Since agarose-bound NCS cannot enter mammalian cells, the above findings indicate that NCS is able to exert its toxic effects by binding to or reacting with receptors on the cell membrane.

Topics: Animals; Antibiotics, Antineoplastic; Cell Division; Cell Line; Cell Membrane; Humans; Leukemia, Experimental; Leukemia, Lymphoid; Sepharose; Zinostatin