Accelerated conversion of human plasminogen activator inhibitor-1 to its latent form by antibody binding.

The serpin plasminogen activator inhibitor-1 (PAI-1) slowly converts to an inactive latent form by inserting a major part of its reactive center loop (RCL) into its beta-sheet A. A murine monoclonal antibody (MA-33B8), raised against the human plasminogen activator (tPA).PAI-1 complex, rapidly inactivates PAI-1. Results presented here indicate that MA-33B8 induces acceleration of the active-to-latent conversion. The antibody-induced inactivation of PAI-1 labeled with the fluorescent probe N, N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) at P9 in the RCL caused a fluorescence enhancement and shift identical to those accompanying the spontaneous conversion of the P9.NBD PAI-1 to the latent form. Like latent PAI-1, antibody-inactivated PAI-1 was protected from cleavage by elastase. The rate constants for MA-33B8 binding, measured by NBD fluorescence or inactivation, were similar (1.3-1.8 x 10(4) M-1 s-1), resulting in a 4000-fold faster inactivation at 4.2 microM antibody binding sites. The apparent antibody binding rate constant, at least 1000 times slower than one limited by diffusion, indicates that exposure of its epitope depends on an unfavorable equilibrium of PAI-1. Our observations are consistent with this idea and suggest that the equilibrium involves partial insertion of the RCL into sheet A: latent, RCL-cleaved, and tPA-complexed PAI-1, which are inactive loop-inserted forms, bound much faster than active PAI-1 to MA-33B8, whereas two loop-extracted forms of PAI-1, modified to prevent loop insertion, did not bind or bound much more weakly to the antibody.     

Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis

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Preformed frozen sucrose gradients--a new laboratory aid

A useful method for preparing large numbers of virtually identical gradients of sucrose has been described. This technique involves storage of preformed gradients at ?60°C for future use after a 90 min thaw at room temperature. The method is applicable to salt-containing sucrose gradients with certain limitations, noted above.