Translocation of a small cytosolic calcium-binding protein (MRP-8) to plasma membrane correlates with human neutrophil activation.
To further understand the mechanisms involved in phagocyte activation in general and in NADPH oxidase activation in particular, a polyclonal antibody was raised in rabbit against a partially purified oxidase preparation. The enzyme was solubilized from zymosan-activated human neutrophils and resting cells and separated by preparative isoelectric focusing electrophoresis. A polyclonal antibody was raised in rabbit against the pI 5.0 fraction, which had the maximum superoxide-producing capacity. Analysis of the polyclonal antibody revealed marked differences between activated and resting neutrophils. The antibody recognized in particular an 8-kDa protein (p8) in resting human neutrophil cytosol and in the membrane of zymosan-activated cells. A polyclonal antibody (anti-p8) was raised against the pure cytosolic p8 protein. This anti-p8 reacted not only with p8, but also with cytosolic proteins of 14 kDa and 6 kDa. N-terminal amino acid sequence analysis of p8 revealed homology with the calcium-binding myeloid related protein (MRP-8). Upon neutrophil activation, translocation of the 8- and 14-kDa proteins to the membrane was observed with stimuli known to depend on extracellular calcium. In calcium-depleted medium, the absence of translocation correlated with a depression of superoxide production, supporting a role for the calcium-binding protein in cellular activation.
Lemarchand
P
P
Central Laboratory of Clinical Chemistry, CHUV, Lausanne, Switzerland.
Vaglio
M
M
Mauël
J
J
Markert
M
M
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D040501:Calgranulin A; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism* / Q000648:ultrastructure; D016897:Respiratory Burst; D013481:Superoxides / Q000378:metabolism
D002135:Calcium-Binding Proteins; D040501:Calgranulin A; D013481:Superoxides; D002118:Calcium