USE OF ENZYME OVERLAY MEMBRANES TO SURVEY PROTEINASE ACTIVITY IN FROZEN-SECTIONS - CATHEPSIN-LIKE AND PLASMIN-LIKE ACTIVITY IN REGENERATINGNEWT LIMBS

We present a method that permits extremely simple and rapid screening of proteolytic enzyme activity in sectioned tissues. Enzyme overlay me mbranes (EOMs) are custom-made membranes designed to fluoresce at site s of specific proteolytic enzyme activity after separation of proteins by gel electrophoresis. EOMs, selected to detect either plasmin-like or cathepsin B-like activity, have been used in a novel way to documen t the distribution of enzyme activity in frozen sectioned tissues. Whe n moistened membranes were placed in contact with sectioned regenerati ng newt limbs, a fluorescent pattern of enzyme activity was generated. In limbs at 3 hr post amputation, cathepsin B-like activity was promi nent across the amputation site but plasmin-like activity was distribu ted in dermal and deeper proximal tissues, suggesting different roles for these two classes of enzymes. EOM enzymology in situ (EEl) on froz en sectioned tissues may be a widely useful technique to display distr ibution and level of activity of proteolytic enzymes in various system s.