38 research outputs found
Antiphospholipid Antibodies Bind ATP: A putative Mechanism for the Pathogenesis of Neuronal Dysfunction
Antiphospholipid antibodies (aPL) generated in experimental animals
cross-react with ATP. We therefore examined the possibility that aPL IgG from
human subjects bind to ATP by affinity column and an enzyme linked
immunosorbent assay (ELISA). Sera with high levels of aPL IgG were collected
from 12 patients with the antiphospholipid syndrome (APS). IgG fractions from
10 of 12 APS patients contained aPL that could be affinity-bound to an ATP
column and completely eluted with NaCl 0.5 M. A significant (>50%) inhibition
of aPL IgG binding by ATP 5 mM was found in the majority. Similar inhibition
was obtained with ADP but not with AMP or cAMP. All the affinity purified
anti-ATP antibodies also bound β2-glycoprotein-I (β2-GPI, also known as
apolipoprotein H) suggesting that, similar to most pathogenic aPL, their binding
depends on this serum cofactor. We further investigated this possibility and found
that the binding of β2-GPI to the ATP column was similar to that of aPL IgG in
that most was reversed by NaCl 0.5 M. Furthermore, addition of β2-GPI to aPL
IgG significantly increased the amount of aPL binding to an ATP column. We
conclude that aPL IgG bind ATP, probably through β2-GPI. This binding could
interfere
with the normal extracellular function of ATP and similar neurotransmitters
Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions
Nature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe a very simple and fast one-pot synthesis to phosphorylate an alcohol with phosphoric acid using trichloroacetonitrile as activating agent. Using this procedure, we efficiently attached the radioactive phosphorus isotope 32P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme—an RNA sequence that catalyzes the eponymous reaction. We used the 32P-substrate for the measurement of RNA-catalyzed reaction kinetics of several dye-labeled ribozyme variants for which precise optical activity determination (UV/vis, fluorescence) failed due to interference of the attached dyes. The reaction kinetics were analyzed by thin-layer chromatographic separation of the 32P-labeled reaction components and densitometric analysis of the substrate and product radioactivities, thereby allowing iterative optimization of the dye positions for future single-molecule studies. The phosphorylation strategy with trichloroacetonitrile may be applicable for labeling numerous other compounds that contain alcoholic hydroxyl groups