A dendritic cell-like cell line, HIN-Ret-1(RET-1), was found to produce the highest level of tissue factor (TF) among several established cell lines from human lymphoma or leukemia. The TF level expressed by this cell line exceeded 5.6 times that expressed by a monocyte-like cell line, U-937. Unlike other TF producing cell line, i.e. RET-2, HL-60, ML-3, and U-937, the TF expression by RET-1 was spontaneous and unaffected with TP A, PHA, LPS, or MAF. The TF activity of RET-1 was markedly inhibited by Con A as well as that produced by LPS-stimulated monkey monocytes, whereas the TF activity of monkey brain and lung was hardly inhibited by the lectin. Hence, the RET-1 cell lysate solubilized in Triton X-100 was subjected to affinity chromatography on a Con A-Sepharose column, and TF-apoprotein (TF-Apo) was completely bound to the column and eluted with TBS containing 0.15 M α(-methylglucoside and 0.1 % Triton X-100. Further purification of this material was performed with combination of FPLC on a DEAE-5PW column and affinity chromatography using a factor VII-Sepharose column. By these methods, TF-Apo preparation with purification-fold of 9,400 and over-all yield of 7 % was obtained. Its apparent molecular weight was estimated to be 120 kDa by gel filtration in TBS containing 0.1 % Triton X-100. SDS-PAGE gave the value of 47 kDa, which was almost compatible with that of TF-Apo from brain or placenta. TF-Apo frcm the monocytes also bound to the lectin column, suggesting that the apoprotein of these macrophage-related cells has an oligosaccharide chain interacting with the lectin. RET-1 TF-Apo was unstable under acidic condition (<pH 4.0) or in organic solvents such as isopropanol (>18 %) and acetonitrile (>8 %).
The hyaluronate synthase from a eukaryotic cell line
