Purification of gelatin-specific neutral protease from human skin by conventional and high-performance liquid chromatography.
Human skin, maintained in serum-free organ culture, secretes a neutral metalloendopeptidase which is remarkably specific for gelatin. Because the product peptides from the action of collagenase on collagen become denatured into random coil polypeptides of 25000 and 75000 daltons at physiological temperature, it is thought that this "gelatinase" is the second, and possibly the only other enzyme in the pathway of extracellular collagen degradation. New types of high-performance liquid chromatography (HPLC) columns have enabled us to improve the yields of active gelatinase from skin culture medium. Raw medium, which has been dialyzed and lyophilized, is fractionated with ammonium sulfate, and applied to Pharmacia Blue Sepharose in a batch step. The 0.4 M sodium chloride eluate is then subjected to gel filtration on Sephacryl S-200, followed by gradient elution from Amicon Green Sepharose. The fractions with gelatinolytic activity are applied to a Bio-Rad TSK-Phenyl-5PW HPLC column for mild hydrophobic chromatography with a gradient of decreasing ammonium sulfate concentration. In the final step, the enzyme is applied to a Pharmacia Mono-Q FPLC column and eluted with a gradient of sodium chloride. At this point, the enzyme appears as two bands, corresponding to enzymatic activity zymograms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Seltzer
J L
JL
Eschbach
M L
ML
Eisen
A Z
AZ
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D018093:Gelatinases; D006801:Humans; D009924:Organ Culture Techniques; D010434:Pepsin A / Q000302:isolation & purification*; D012709:Serum Albumin / Q000032:analysis; D012867:Skin / Q000201:enzymology*
D012709:Serum Albumin; D010434:Pepsin A; D018093:Gelatinases