Abstract
Calcium-dependent inhibitory factor (CIF) is a major calcium binding protein in the plasmodia of a lower eukaryote, Physarum polycephalum, and exerts an inhibitory effect on protein kinases of Physarum such as the myosin light chain kinase. A cDNA encoding CIF was isolated, expressed in E. coli, and characterized. The CIF contained four EF-hand motifs and an Arg-Gly-Asp sequence. The molecular mass of CIF was calculated to be 40, 508 deduced from the nucleotide sequence. This CIF assembles in calcium-dependent manner. Polyclonal antibodies were raised against recombinant CIF. Using this antibody, I found that CIF was located in not only cytoplasm but also in nucleolus of Physarum plasmodia.
In an attempt to extend these observations in vertebrate cells, an immunofluorescence study of cultured vertebrate cells, CHO-K1 and 10 T 1/2 was performed. This antibody stained nucleoli of the both cell Types. Western blot analysis of the nuclear fraction of these cells indicated that the antibody recognized a protein band of 40 kDa, that is similar in mass to that of CIF in Physarum. The antibody also cross-reacted with polymorphonuclear leukocytes. The subcellular localization of this protein in these cells was found to be predominantly in the cytoplasm. The role of this protein in nucleolus has been discussed with special reference to cell cycle and motility.
