Novel endogenous inhibitor of sulfur mustard-stimulated protease in cultured human epidermal keratinocytes: possible application in vesicant intervention.
Protease stimulation at the dermal-epidermal junction may be responsible for the skin blistering (vesication) action of sulfur mustard (HD). We have purified a protease to homogeneity from cultured normal human epidermal keratinocytes (NHEK) exposed to 300 microM HD. In this report, we describe the results of our studies on purification and characterization of an endogenous inhibitor of HD-stimulated protease in NHEK. Purification to homogeneity was accomplished by chromatographic separation of the dialyzed Triton X-100-solubilized inhibitor using ion-exchange DEAE-cellulose. Analysis of the purified inhibitor by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed one polypeptide with an apparent molecular mass of 116 kDa. Activity of the inhibitor was screened by incubating different column elute fractions with protease purified from the same cells. Preliminary results showed that the purified inhibitor effectively inhibited the protease isolated from NHEK, whereas other naturally occurring inhibitors, e.g. soybean trypsin-chymotrypsin inhibitors, elafin and aprotinin, were ineffective. Although complete characterization and regulation of this inhibitor remain to be resolved, this purification may be a major step towards developing a specific protective measure against HD-induced toxicity.
Chakrabarti
A K
AK
Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA.
Ray
P
P
D001768:Blister / Q000139:chemically induced* / Q000503:physiopathology; D018929:Cell Culture Techniques; D003879:Dermatologic Agents / Q000633:toxicity*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes; D009151:Mustard Gas / Q000633:toxicity*; D010455:Peptides; D011041:Poisoning / Q000517:prevention & control; D020011:Protective Agents / Q000302:isolation & purification* / Q000378:metabolism*
D003879:Dermatologic Agents; D010455:Peptides; D020011:Protective Agents; D010450:Endopeptidases; D009151:Mustard Gas