A quantitative assay for the hydrolysis of structurally intact basement membranes

Abstract

A quantitative assay has been developed for the hydrolysis of native bovine anterior lens capsule basement membrane fragments. The rationale for using structurally intact membrane fragments as the substrate in enzyme assays is that the proteinase susceptibility of the various basement membrane components differs when examined individually compared to when they are present in their native state. The assay is based upon the solubilization of 3H-bound protein from a finely ground suspension of [ 3H]acetylated basement membranes. The acetylation reaction and the fragmentation procedure do not alter the morphology or proteinase susceptibility of the membranes. The initial rate of release of radiolabeled digestion fragments by six different proteinases is approximately linear over the first 15% of hydrolysis, and the initial rates obtained are proportional to the amount of enzyme over a wide range of enzyme concentrations. The labeling index of 3810 cpm/μg of basement membrane used in this study permits the solubilization of 50 ng of protein to be detected easily. Some information about the size of the protein fragments solubilized can be obtained by addition of trichloroacetic (5% w v )-tannic acid (0.25% w v ) reagent to the supernatants from the assays, since this reagent appears to selectively precipitate larger fragments. An additional feature of this assay is that, since the substrate is radiolabeled, one can selectively visualize and analyze the size distribution of the digestion products by carrying out sodium dodecyl sulfate-gel electrophoresis with fluorographic detection. Based on the observation that these intact membranes are extensively digested by a number of common proteinases which have widely different substrate specificities, it appears that the hypothesis that a highly specific proteinase or class of proteinases is necessary for basement membrane catabolism is specious.