| Calmodulin(CaM), widely distributed in almost all eukaryotic cells,is a major intracellular calcium receptor responsible for mediating the Ca2+ signal to a multitude of different enzyme systems and is thought to play a vital role in the regulation of cell proliferation and differentiation. Recently, many studies showed that CaM is also present in extracellular fluids such as cell culture media and normal body fluids and has been reported to stimulate proliferation in a range of normal and neoplastic cells,apparently acting as an extracellular signal molecular. So far, how extracellular CaM achieves its effects is yet unclear. It has been suggested that there may have some calmodulin-binding proteins(CaMBPs) on cell surface, acting as calmodulin receptors to mediate extracellular CaM signaling into cellsCaMBPs has been found to exsist on cell wall of some plant cells.In order to confirm the existence of CaMBPs on human lymphocyte as well as other tumor cell surface. Fresh human peripheral blood lymphocytes, mouse myeloma cells (Sp2/0),and human stomach cancer cells (MGC803) were incubated with recombinant human calmodulin (rhCaM) labeled with 10nm colloidal gold. After 24h of incubation, the cells were detected for colloidal gold particle distribution by transmission electron microscope. The results showed that the gold particles dispersed on cell surfaces of human lymphocyte, Sp2/0, and MGC803 after incubation with rhCaM-labeled colloidal-gold, while no gold particles were found in the cell surfaces of those cells incubated with BSA labeled colloidal-gold. This immuno-gold transmission electron observation directly verified the existence of calmodulin binding-site (CaMBS) on the cell surface.Recent researches have indicated that CaM exsist both in eucaryotic andprokaryotic cells. Our previous works found that extracellular CaM could promote the growth of Mycobacterium bovis. To confirm the existence of calmodulin binding-site on the cell wall of Mycobacterium bovis. Cultured fresh Mycobacterium bovis was incubated with rhCaM labeled colloidal-gold, and observed as above. Results showed that several gold particles were dispersed on the cell wall of Mycobacterium bovis after incubated with rhCaM-labeled colloidal-gold, indicating the existence of calmoduJin binding-site on the Mycobacterium bovis.The abnormalities both in CaM structure and CaM function,as well as changes of intracellular CaM levels were correlated with the development and progress of many human diseases. Determinations of CaM levels and its correlation with diseases are very useful in further investigation of CaM biologic activities and its pathogenetic significances. By using rhCaM and rabbit anti-CaM antibody, a quantitative ELISA for detecting CaM was established. The polystyrene microtiter plates were treated with ultraviolet radiation to improve the immobilazation of CaM antigen. The constitution of coating buffer and coating time were optimized as well. The standard curve of the method for CaM assay was satisfactory, with 40ng/ml of the lowest detection limit. The CaM levels of lymphocytes in the patient with lymphocytic leukemia(n=9), non-lymphocytic leukemia(n=7), and lymphoma(n=7) were 1349.3+206.38 fg/cell, 886.06+108.02 fg/cell, and 1746.05+547.84 fg/cell respectively, significantly higher than in normal control(107.16+56.07 fg/cell),P0.01.Production of high specific antibody may take an important role during the development of an ELISA technique. As low molecule weight and without species differences of CaM protein, it is quite difficult to obtain high liter and specific anti-CaM antibody by immunization of animals with CaM. The development of phage display technology provides us another feasible strategy to produce antibody against some antigens which are not easy to produce the specific antibodies by immunization. By using humanized phage display antibody library, after 3 rounds of panning, we obtained specific CaM-binding phage clones. After identifying the specificities of phage ScFv antibody by ELISA technique, Ca2+ sens...
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