Formation of the Ca2+-activated photoprotein obelin from apo-obelin and mRNA inside human neutrophils.
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.
Campbell
A K
AK
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, U.K.
Patel
A K
AK
Razavi
Z S
ZS
McCapra
F
F
D001059:Apoproteins / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008081:Liposomes / Q000378:metabolism; D008164:Luminescent Proteins / Q000097:blood* / Q000235:genetics; D009504:Neutrophils / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*
D001059:Apoproteins; D008081:Liposomes; D008164:Luminescent Proteins; D012333:RNA, Messenger; C010497:obelin; D002118:Calcium