PubMed · 2286443
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V N Tibrewala, L M Ambani, S B Shah, B N Apte. 1990. Pyruvicacidemia.. https://pubmed.ncbi.nlm.nih.gov/2286443/
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The transmembrane topology of the serotonin transporter (SERT) has been examined by measuring the reactivity of selected lysine and cysteine residues with extracellular reagents. An impermeant biotinylating reagent, sulfosuccinimidyl 2-(biotinamido)ethyl-1, 3-dithiopropionate (NHS-SS-biotin), was shown to label SERT transiently expressed in cultured cells. Replacement of four lysine residues that were predicted to lie in external hydrophilic loops (eK-less) largely prevented the biotinylation reaction. Likewise, the cysteine-specific biotinylation reagent N-biotinylaminoethylmethanethiosulfonate (MTSEA-biotin) labeled wild type SERT but not a mutant in which Cys-109, predicted to lie in the first external loop, was replaced with alanine. These two mutant transporters reacted with the biotinylating reagents in digitonin-permeabilized cells, demonstrating that the abundant lysine and cysteine residues predicted to lie in intracellular hydrophilic domains were reactive but not accessible in intact cells. Mutants containing a single external lysine at positions 111, 194, 243, 319, 399, 490, and 571 reacted more readily with NHS-SS-biotin than did the eK-less mutant. Similarly, mutants with a single cysteine at positions 109, 310, 406, 489, and 564 reacted more readily with MTSEA-biotin than did the C109A mutant. All of these mutants were active and therefore likely to be folded correctly. These results support the original transmembrane topology and argue against an alternative topology proposed recently for the related glycine and gamma-aminobutyric acid transporters.
Clusterin is a highly conserved mammalian glycoprotein which has been predicted to contain heparin-binding sites. We tested this prediction by studying the interactions between heparin and clusterin using ELISA and heparin affinity chromatography methodologies. Two forms of biotinylated heparin were used in ELISA: heparin which had been directly biotinylated with a biotin-N-hydroxysuccinimide ester and heparin which had been activated using epichlorohydrin and 1,6-diaminohexane prior to biotinylation. Both gave dose-dependent increases in ELISA signal with increasing concentrations of biotinylated heparin, with the latter giving signals an order of magnitude greater than the former. There was a dose-dependent increase in the ELISA signal from bound biotinylated heparin with increasing concentrations of plate-bound clusterin. The apparent affinity constant for binding of biotinylated heparin to plate-bound clusterin at pH 6.0 was estimated as 0.06 +/- 0.02 microM. Unlabeled heparin blocked the binding of biotinylated heparin to clusterin over a concentration range similar to that of the binding of biotinylated heparin to plate-bound clusterin. The binding of biotinylated heparin to clusterin was independent of the presence or absence of Ca2+. The binding of biotinylated heparin to plate-bound clusterin increased with decreasing pH over the range 5.5-8.0 and was characterized by an apparent pKa of 6.9. Clusterin in human serum bound to heparin-Sepharose at pH 6.0 but not at pH 7.4. Dot-blot experiments showed that one of the polypeptide chains of clusterin which had been reduced and alkylated under denaturing conditions bound to heparin-Sepharose. This chain was identified as the alpha chain from its N-terminal amino acid sequence.
A cell separation system based on the calcium-dependent interaction of calmodulin (CM) with a calmodulin-binding peptide (CBP) has been developed. The prototype of this system utilizes an indirect method to label the target cell population. Cells are first labeled with a primary monoclonal antibody directed to a specific cell surface antigen, then with a secondary affinity reagent, consisting of a polyclonal goat anti-mouse IgG (GAM-IgG) that has been cross-linked to a CBP derived from the sequence of the rabbit skeletal muscle myosin light chain kinase. In the presence of Ca2+, the CBP on the cells labeled with GAM-IgG-CBP binds to biotinylated calmodulin (CM-Biotin) with high affinity. The target cells are then captured with a solid-phase streptavidin. The unbound non-target cells are washed away and the immobilized target cells are released by chelating Ca2+ with EGTA. The specificity of the GAM-IgG-CBP and CM-Biotin and the feasibility of using this system to separate cells was demonstrated using the KG-1 human acute myelogenous leukemia cell line. KG-1 cells were fractionated on the basis of cell surface expression of HLA-DR. The cell selection reagents and the cell separation process did not affect KG-1 cell viability while cells selected by this procedure were 90% pure with a yield of 75%. This cell separation system also was used for rare cell isolation from normal human peripheral blood mononuclear cells. T cells expressing the Vbeta5 T cell receptor, which represent < 5% of the unfractionated cells, were isolated with 89% viability, 72% purity, 80% yield, and retained the ability to respond to activation signals as measured by blast transformation. The results from this study show that a cell selection system based on the reversible interaction between CM and a CBP can be applied to gently and efficiently isolate cells from a heterogeneous starting population that are free of the solid matrix without exposure to the stresses of mechanical or enzymatic release.