The distribution and characterization of esterases in skin.

1 - Two techniques of homogenising skin tissue have been examined for their efficiency to produce homogenates of rat skin containing subcellular organelles with the minimun amount of damage. 2 - The subcellular distribution of three hydrolase enzymes with catalytic activity towards, two esters of thiocholine and p-nitrophenylace tate, have been determined in the subcellular fractions produced from rat skin by homogenisation using grinding in liquid nitrogen. All three hydrolases were found to be solubilised by this technique and remained in the 100,000xg supernatant. 3 - The 100,000xg supernatant from rat skin was subject to molecular exclusion chromatography and the three solubilised hydrolases were followed through this procedure. Each of the hydrolase activities was found to be associated with a number of proteins that were different by molecular size. The two thiocholine ester hydrolases were associated with the same proteins, but the p-nitrophenylacetate hydrolase was predominantly associated with other proteins. 4 - The 100,000xg supernatant was also subjected to analysis of by polyacrylamide gel electrophoresis and staining with naphthylacetate. This procedure showed the presence of at least eight different hydrolases, and some of these electrophoretic bands could be associated with specific proteins of specific molecular size as determined by molecular exclusion chromatography. 5 - The hydrolases present in the 100,000xg supernatant from rat skin were characterised using four chromogenic substrates and four selective inhibitors. A cholinesterase with a substrate selectivity similar to that of acetylcholinesterase but an inhibitor selectivity similar to butyrylcholinesterase was found. Different esterases were responsible for the hydrolysis of p-nitrophenylacetate and indoxylacetate. 6 - The 100,000xg supernant from human skin was found to contain hydrolases that were different to those in rat skin. An enzyme hydrolysing p-nitrophenyl acetate that was insensitive to diisopropylfluorophosphate and did not hydrolyse this inhibitor was detected. The cholinesterase present behaved in the same way as butyrylcholinesterase. Only one electrophoretic band of naphthylacetate hydrolysing activity could be detected by polyacrylamide gel electrophoresis. 7 - Naphthylacetate and 5-bromoindoxylacetate hydrolases were histochemically located in skin from rat and man