Identification of elastase as a secretory protease from cultured rat microglia.
In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7-8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 microM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N-methoxysuccinyl-Ala-Ala-Pro-Val-methyl chloride, eluted at a position corresponding to a molecular mass of approximately 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase-like protease. This suggestion was confirmed by the finding that the 25-kDa protein band was stained with anti-elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopolysaccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.
Nakajima
K
K
Department of Neurochemistry, National Institute of Neuroscience, Tokyo, Japan.
Shimojo
M
M
Hamanoue
M
M
Ishiura
S
S
Sugita
H
H
Kohsaka
S
S
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003470:Culture Media; D010450:Endopeptidases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008070:Lipopolysaccharides; D008970:Molecular Weight; D009457:Neuroglia / Q000201:enzymology* / Q000378:metabolism; D010196:Pancreatic Elastase / Q000032:analysis* / Q000096:biosynthesis; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D013379:Substrate Specificity
D003470:Culture Media; D008070:Lipopolysaccharides; D011480:Protease Inhibitors; D010450:Endopeptidases; D010196:Pancreatic Elastase