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67 k calcimedin (67 kDa) is distinct from p67 calelectrin and lymphocyte 68 kDa Ca2+-binding protein.

ABSTRACT: The 67 k calcimedin is a Ca2+-binding protein present in both muscle cells and peritoneal macrophages. Many tissues, including lymphoid tissues, liver and lymphocytes, have been shown to contain Ca2+-binding proteins of similar molecular size, such as the p67(67 kDa) calelectrin or the 68 kDa lymphocyte protein. We have tested affinity-purified antibodies raised to the smooth-muscle 67 k calcimedin in these several tissues and here report that the 67 k calcimedin is not detectable in liver, thymus, spleen or thymic lymphocytes. These findings support recent biochemical evidence, discussed here, suggesting that the 67 k calcimedin is a protein different from calelectrin and the 68 kDa lymphocyte protein. The more limited tissue distribution of the 67 k calcimedin, which includes muscle and macrophages, suggests that the 67 k calcimedin may function in Ca2+-mediated events special to these cell types. The affinity-purified antibodies to the 67 k calcimedin will be useful in obtaining information concerning the special roles of this Ca2+-binding protein in these cells.

SUBMITTER: Morse SS

PROVIDER: S-EPMC1148979 | biostudies-other | 1988 Apr

REPOSITORIES: biostudies-other

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Project description:BackgroundBacillus thuringiensis (Bt), an ubiquitous gram-positive spore-forming bacterium forms parasporal proteins during the stationary phase of its growth. Recent findings of selective human cancer cell-killing activity in non-insecticidal Bt isolates resulted in a new category of Bt parasporal protein called parasporin. However, little is known about the receptor molecules that bind parasporins and the mechanism of anti-cancer activity. A Malaysian Bt isolate, designated Bt18 produces parasporal protein that exhibit preferential cytotoxic activity for human leukaemic T cells (CEM-SS) but is non-cytotoxic to normal T cells or other cancer cell lines such as human cervical cancer (HeLa), human breast cancer (MCF-7) and colon cancer (HT-29) suggesting properties similar to parasporin. In this study we aim to identify the binding protein for Bt18 in human leukaemic T cells.MethodsBt18 parasporal protein was separated using Mono Q anion exchange column attached to a HPLC system and antibody was raised against the purified 68-kDa parasporal protein. Receptor binding assay was used to detect the binding protein for Bt18 parasporal protein in CEM-SS cells and the identified protein was sent for N-terminal sequencing. NCBI protein BLAST was used to analyse the protein sequence. Double immunofluorescence staining techniques was applied to localise Bt18 and binding protein on CEM-SS cell.ResultsAnion exchange separation of Bt18 parasporal protein yielded a 68-kDa parasporal protein with specific cytotoxic activity. Polyclonal IgG (anti-Bt18) for the 68-kDa parasporal protein was successfully raised and purified. Receptor binding assay showed that Bt18 parasporal protein bound to a 36-kDa protein from the CEM-SS cells lysate. N-terminal amino acid sequence of the 36-kDa protein was GKVKVGVNGFGRIGG. NCBI protein BLAST revealed that the binding protein was Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Double immunofluorescence staining showed co-localisation of Bt18 and GAPDH on the plasma membrane of the CEM-SS cells.ConclusionsGAPDH has been well known as a glycolytic enzyme, but recently GAPDH was discovered to have roles in apoptosis and carcinogenesis. Pre-incubation of anti-GAPDH antibody with CEM-SS cells decreases binding of Bt18 to the susceptible cells. Based on a qualitative analysis of the immunoblot and immunofluorescence results, GAPDH was identified as a binding protein on the plasma membrane of CEM-SS cells for Bt18 parasporal protein.

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67 k calcimedin (67 kDa) is distinct from p67 calelectrin and lymphocyte 68 kDa Ca2+-binding protein.

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