Use of Enzyme Overlay Membranes to Survey Proteinase Activity in Frozen Sections: Cathepsin-like and Plasmin-like Activity in Regenerating Newt Limbs

Abstract

SUMMARYWe 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. (J Histochem Cytochem 45:779-783, 1997)

A competitive hexapeptide inhibitor of annexin A2 prevents hypoxia-induced angiogenic events

“…7D). For the detection of plasmin activity in the frozen sections, we used enzyme overlay membranes (EOMs) incorporated with the fluorogenic plasmin substrate D-Val-Leu-Lys-7-amino-4-trifluoro-methylcoumarin (D-Val-Leu-Lys-AFC) (Day and Neufeld, 1997). When the EOMs were placed in contact with the frozen sections, a fluorescent pattern of enzymatic activity was detected, which is a representation of localization of plasmin activity in the tissue sections.…”

“…The assay was preformed as described previously (Berndt et al, 2000;Day and Neufeld, 1997). Frozen sections (8-10 m) of the Matrigel plugs were placed on coverslips, air dried for 1 hour, fixed in methanol for 10 minutes at -20°C and rinsed with PBS.…”

In Vivo Localization of Gelatinases (MMP-2 and -9) by in Situ Zymography with a Selective Gelatinase Inhibitor

“…However, both gelatinolytic and caseinolytic activity is usually accomplished by a wide variety of enzymes, and determination of enzyme-specific origin of the proteolytic activity has been difficult if not impossible. Recently, this problem has been overcome by the use of enzyme overlay membranes (19,20). We however, circumvented this problem by utilizing the recently described specific and selective inhibitor for MMP-2 and -9 (21,22), which belongs to a novel class of cyclic tissue-permeable peptides containing an HWGF motif.…”

A critical role for thrombin in vertebrate lens regeneration

“…To detect thrombin activity at a tissue level we have overlaid frozen sections of tissue with a membrane impregnated with a fluorogenic thrombin substrate (Day & Neufeld 1997). This molecule is a coumarin ester of the canonical thrombin substrate phe-pro-arg.…”