Use of enzyme overlay membranes to survey proteinase activity in frozen sections: cathepsin-like and plasmin-like activity in regenerating newt limbs.
We present a method that permits extremely simple and rapid screening of proteolytic enzyme activity in sectioned tissues. Enzyme overlay membranes (EOMs) are custom-made membranes designed to fluoresce at sites 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 document the distribution of enzyme activity in frozen sectioned tissues. When moistened membranes were placed in contact with sectioned regenerating newt limbs, a fluorescent pattern of enzyme activity was generated. In limbs at 3 hr post amputation, cathepsin B-like activity was prominent across the amputation site but plasmin-like activity was distributed in dermal and deeper proximal tissues, suggesting different roles for these two classes of enzymes. EOM enzymology in situ (EEI) on frozen sectioned tissues may be a widely useful technique to display distribution and level of activity of proteolytic enzymes in various systems.
Day
F A
FA
University of South Dakota School of Medicine, Department of Anatomy and Structural Biology, Vermillion 57069, USA.
Neufeld
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D002401:Cathepsin B / Q000032:analysis / Q000378:metabolism*; D005121:Extremities / Q000502:physiology*; D005341:Fibrinolysin / Q000032:analysis / Q000378:metabolism*; D005629:Frozen Sections; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence; D019185:Notophthalmus; D012038:Regeneration / Q000502:physiology*
D008567:Membranes, Artificial; D005341:Fibrinolysin; D002401:Cathepsin B