Cellular Distribution of P68, a New Calcium-binding Protein from Lymphocytes

A Ca2+-binding protein of mol. wt. 68 000 ( p68 ) is a major component of a Nonidet P-40 insoluble fraction of human and pig lymphocyte plasma membrane. An affinity-purified rabbit antibody has been produced against p68 and used to study its cellular distribution. The antibody stained fixed and permeabilised human B lymphoblastoid cells, peripheral blood lymphocytes and sections of human tonsil. Whole cells, however, were not stained, indicating that the protein was not represented at the cell surface. This assignment was consistent with the detection of p68 in immunoprecipitates from biosynthetically- but not surface-labelled cells. It is concluded that p68 is located on the cytoplasmic face of the plasma membrane. Subcellular fractionation experiments confirmed that p68 was largely membrane-bound in lymphocytes, although a small soluble fraction (approximately 10% of the total) was detected. Sub-fractionation of lymphocyte membranes revealed that p68 was associated not only with the plasma membrane but also with other endomembrane systems. As judged by immunoprecipitation, p68 was present in a variety of cultured cell lines of both lymphoid and non-lymphoid origin. p68 demonstrated a diffuse distribution in fixed and permeabilised fibroblasts which did not correspond to the distribution of either microfilaments or intermediate filaments. However, in detergent-extracted cells the protein was localised in a lamina-like network. A similar immunofluorescent staining pattern has recently been observed for spectrin-related proteins in the detergent-resistant cytoskeleton of fibroblasts. It is suggested that p68 is part of a sub-membranous cytoskeletal complex not only in lymphocytes but also in other cell types.