The clotting of the lysed white cells of Limulus induced by endotoxin—I: Preparation and characterization of clot-forming proteins1

Publisher Website
Google Scholar
Abstract
Attempts were made to isolate and further characterize some of the active agents in the Limulus white blood cell lysate, with the ultimate aim of preparing a better endotoxin assay material. Chloral hydrate at pH3 and 6 mM was used to inhibit the clot reaction and to provide reversible denaturing conditions during separation procedures; the reversibility did not occur, but .alpha.-clot protein [CP] was isolated. The untreated lysate showed at least 23 distinct bands on SDS [sodium dodecyl sulfate] electrophoresis; the most abundant material had a MW of 21,000 (.alpha.-CP) and was totally absent in the clot. A 14,000 MW (.beta.-CP) minor component in the untreated lysate was the single most abundant material in the clot (34%); a small amount was present in the supernate. .alpha.-CP was active after isolation. The turbidity of a solution containing endotoxin and whole Limulus lysate (which contains the endotoxin-sensitive enzyme) was much greater than that of the endotoxin and lysate alone. .alpha.-CP did not form a clot in the presence of endotoxin alone and the presence of bovine serum albumin instead of .alpha.-CP led to a decrease in turbidity of the clot. Pure .beta.-CP was isolated from the clot by Sephadex G-200 chromatography with SDS. On the basis of differences in amino acid composition between .alpha.-CP and .beta.-CP, > 80% of the fragment liberated in the clot reaction seemed to be composed of only glutamic and aspartic acids, proline and tyrosine. The fragment was probably a single unit of 7000 MW as only a single type of .beta.-CP of 14,000 MW was produced. There were apparently no intermediates. There was no evidence for the involvement in the clotting reaction of any protein in the lysate other than the specific enzyme, .alpha.-CP and .beta.-CP. UV spectra of .alpha.-CP and .beta.-CP which showed prominent phenylalanine absorption maxima were rare; the amino acid analyses of .alpha.-CP and .beta.-CP were significantly different from other proteins which bound Ca2+ and Mg2+ such as troponin C, a light subunit of myosin, fish myogen and vitamin D-dependent Ca-binding protein; no homology with them could be inferred.

This publication has 0 references indexed in Scilit: