ABSTRACT
The kinetics of [125I]Endothelin-1 ([125I]ET-1) binding were studied using membranes from rat heart, rat lung, rat brain, and porcine vascular smooth muscle at 37 degrees C in 0.05M Tris-HCl buffer (pH = 7.4). The dissociation half-life (t1/2, diss.) for bound [125I]ET-1 was in excess of 30 hours for each tissue studied. Equilibrium-time requirements for proper Scatchard analysis of [125I]ET-1 were also far in excess of 30 hours for each tissue. These data suggest that determination of dissociation constants, Kd, and receptor concentrations, Bmax, by conventional Scatchard analysis is not feasible with [125I]ET-1. Kinetic analyses may provide a more accurate means for determining [125I-ET-1] binding characteristics including Kd and Bmax.
Subject(s)
Endothelins/metabolism , Receptors, Endothelin/metabolism , Animals , Cerebellum/metabolism , Iodine Radioisotopes , Kinetics , Ligands , Lung/metabolism , Male , Muscle, Smooth, Vascular/metabolism , Myocardium/metabolism , Rats , Rats, Sprague-Dawley , SwineSubject(s)
Corpus Striatum/enzymology , Dopamine/analogs & derivatives , Synaptosomes/enzymology , Tyrosine 3-Monooxygenase/metabolism , Animals , Bucladesine/pharmacology , Dopamine/pharmacology , In Vitro Techniques , Kinetics , Male , Rats , Synapses/physiology , Synaptosomes/drug effects , Tyrosine/metabolismABSTRACT
A method for affinity chromatography of plasma and platelet factor XIII has been developed, based on known structural characteristics of these molecules. Plasma factor XIII is composed of a and b subunits which are held together by noncovalent interactions; platelet factor XIII has only a subunits. a subunit contains free sulfhydryl groups, while in b subunit all the cystines form disulfide bonds. The affinity gel is an organomercurial agarose with p-chloromercuribenzoate as the reactive group. Both the zymogen and activated forms of a subunit reversibly bind to the ligand by forming covalent mercaptide bonds and are eluted by reducing agents. b subunit does not bind to the affinity gel and is held to it only through interaction with a subunit. Affinity chromatography can be used to purify plasma and platelet factor XIII and to study interactions of the subunits. Experiments on the affinity chromatography of purified plasma factor XIII in several stages of activation agree with earlier observations that activation is a two-step procedure in which b subunit is not quantitatively released from the complex until the final stage of activation by Ca2+.
