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Formation of the Ca2+-activated photoprotein obelin from apo-obelin and mRNA inside human neutrophils.


ABSTRACT: 1. A method has been developed to incorporate the apoprotein of the Ca2+-activated photoprotein obelin, and mRNA purified from the hydroid Obelia, into the cytoplasm of intact human neutrophils. This was based on internal release from pH-sensitive immunoliposomes taken up initially by phagocytosis. 2. Addition of the prosthetic group of obelin, coelenterazine, to these cells containing apo-obelin or Obelia mRNA resulted in formation of active Ca2+-activated obelin. 3. The obelin formed within the neutrophils responded to the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (1 microM) and to the membrane attack complex of complement (C5B6789n). 4. The formation of the apo-obelin from mRNA within neutrophils was inhibited by over 80% in the absence of added amino acids, and by over 90% by the protein-synthesis inhibitor puromycin (100 micrograms/ml). 5. The translation of Obelia mRNA inside cells provides a method for circumventing consumption of Ca2+-activated photoproteins during cell activation or injury, and for monitoring protein synthesis in living cells.

SUBMITTER: Campbell AK 

PROVIDER: S-EPMC1149117 | BioStudies | 1988-01-01

SECONDARY ACCESSION(S): 10.1042/bj2520143

REPOSITORIES: biostudies

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