An inhibitor of blood coagulation, a new protein with an apparent molecular weight of 34,000 and an isoelectric point of 4.9, was purified from human placental tissue by EDTA extraction. Five cDNA clones were isolated from the human placental lambda gt11 cDNA library using the mouse monoclonal antibody raised against the coagulation inhibitor as the probe. The longest insert consists of 1,566 nucleotides, and contains 960 nucleotides entirely encoding the 320 amino acids of the inhibitor, and a poly A tail. The deduced amino acid sequence was corroborated by chemical analyses of the protein. The entire amino acid sequence shows homology to those of lipocortin I, lipocortin II, and endonexin-related proteins. The cDNA for the inhibitor was expressed in Escherichia coli under the regulation of the trc promotor of the plasmid pKK233-2. The resulting recombinant protein manifested inhibitory activities against both blood coagulation and phospholipase A2 activity, as did the coagulation inhibitor isolated from human placenta.
A direct measurement method for the enzymatic determination of cholesteryl esters (CEs) without measuring total cholesterol (TC) and free cholesterol (FC) is described. In the first step, hydrogen peroxide generated by cholesterol oxidase from FC was decomposed by catalase. In the second step, CE was measured by enzymatic determination using a colorimetric method or a fluorometric method. The measurement sensitivity of the fluorometric method was more than 20 times that of the colorimetric method. Optimal conditions of the assay were determined, and examples of measured CE in human plasma, rat liver, and cultured cells are indicated. The method of directly measuring CE was simple and has exceptional reproducibility compared with the technique of subtracting FC from TC using each measured TC and FC. Free cholesterol (FC) has an important role as a component of cell membranes and a starting material for bile acid synthesis. However, cholesteryl ester (CE) is inactive when it is stored. In the progression of arteriosclerosis, CE accumulates in macrophages and smooth muscle cells and leads to the formation of foam cells. Determination of CE in cells or various tissues is of great importance in the fundamental research into atherosclerosis and the development of anti-atherosclerotic drugs.Several methods of enzymatic determination for FC and total cholesterol (TC) have been published (1-5). Measurement of TC has measured FC resulting from the decomposition of CE by cholesterol esterase, and FC was contained in the native sample. To measure CE, individual TC and FC are measured, and FC is then subtracted from TC (indirect assay). Measuring CE by indirect assay is difficult in a sample solution with a low ratio of CE to FC.This report describes a direct assay of CE by enzymatic determination without measuring TC and FC. In the first step, FC is oxidized by cholesterol oxidase to yield the corresponding cholest-4-en-3-one and hydrogen peroxide. Hydrogen peroxide is decomposed into water and oxygen by catalase. At the second step, CE is measured by enzymatic determination using a colorimetric method or a fluorometric method. CE is hydrolyzed to FC by cholesterol esterase. FC is oxidized by cholesterol oxidase to yield the corresponding cholest-4-en-3-one and hydrogen peroxide. Thus, the enzymatic method for assaying FC is based on the measurement of hydrogen peroxide by way of peroxidase-coupled oxidation of hydrogen-sensitive probes. The hydrogen peroxide reacts with 4-aminoantipyrine or Amplex Red in the presence of peroxide to form a pigment or fluorescent products. The enzymatic reactions involved in the assay are as follows:CE content in human plasma, rat liver, and cultured cells was measured using this method, and its usefulness was evaluated.
Two major molecular forms of thrombomodulin fragments present in urine were isolated from human urine by four sequential steps of column chromatography. The apparent molecular weights of these thrombomodulins estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis were 98,000 (type I) and 90,000 (type II) with dithiothreitol, and 60,000 (type I) and 55,000 (type II) without dithiothreitol. The isoelectric points of the type I and type II molecules were 2.5 and 3.8, respectively. From sequence analyses, both forms were revealed to have identical 468 amino acid sequences, Ala1-Asp468, lacking 29 amino acids of the carboxyl-terminal sequence of intact cellular thrombomodulin. Major structural differences between type I and type II were observed in the carbohydrate composition: type II had less galactosamine content than type I. Both types were active in thrombin inhibition and protein C activation, although the activities were significantly less than those of intact cellular thrombomodulin. Type I had twice as much inhibitory activity on thrombin clotting activity as type II, whereas type II was more effective as a cofactor for thrombin-catalyzed protein C activation than type I.
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