Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A rapid and efficient 2-step procedure is described for covalently attaching proteins to cell surfaces by using a heterobifunctional cross-linking agent, succinimidyl-4-(N-maleimidomethylcyclohexane)-1-carboxylate (SMCC). In the first step, protein is derivatized for about 30 min with a 1:1 (mole:mole) stoichiometric ratio of SMCC which creates 4-(N-maleimidomethylcyclohexane)-1-amidyl-protein (MCA-protein) covalent linkages with the primary amino groups of proteins. In the second step, cell-MCA-protein conjugates are rapidly prepared (in less than 30 min) by reacting MCA-protein with 'reduced' cells which have been pre-incubated (for about 1 h) with dithiothreitol (DTT). Stable protein-cell conjugates result from the covalent thioether linkages formed between the maleimido groups of the derivatized protein and the cell surface thiol groups created by DTT reduction. Protein-cell conjugates have been made in this way with several different proteins using several different types of cells. Such conjugates are characterized in this study by complement plus antibody-mediated cytotoxicity (CAMC) and by antibody-dependent cellular cytotoxicity (ADCC) with human effector lymphocytes. Because these protein-cell conjugates are shown to remain viable and to retain significant levels of coupled protein for at least 24 h in culture, they are potentially useful for probing various membrane-related phenomena such as the recognition of cell surface antigens by immune effector cells.
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PMID:Rapid covalent coupling of proteins to cell surfaces: immunological characterization of viable protein-cell conjugates. 609 68

Targeting studies using the anti-cancer agent neocarzinostatin (NCS), conjugated to anti-bodies have shown relatively poor specificity. From the literature, it is unclear whether NCS mediates its effects either in conjugated or unconjugated form. In the present work we have used a conjugate of NCS with transferrin, a biological ligand with a well defined endocytic route, to probe these mechanisms. NCS was covalently coupled to transferrin using the heterobifunctional reagent sulfo-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and 2-iminothiolane to give a stable thioether-linked conjugate with a ratio of 1.6 mol of NCS per mole of transferrin. The binding activity of transferrin was completely retained. Conjugation of NCS to transferrin resulted in an apparent enhancement of cytotoxicity. However, incubation with excess transferrin had no influence on the observed enhanced toxicity, indicating that endocytosis is not responsible. Further experiments demonstrated that the apparent enhancement was dependent on incubation conditions and not an effect due to endocytosis of ligand. Studies where apo-NCS competed with holo-NCS and transferrin strongly indicated that the cytotoxicity of both NCS and conjugate is mediated by direct entry of the dissociated chromophore into the cell.
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PMID:Mechanism of free and conjugated neocarzinostatin activity: studies on chromophore and protein uptake using a transferrin-neocarzinostatin conjugate. 916 76