Fractionation and quantification of calcium-dependent proteinase activity from small tissue samples

Almost all mammalian tissues contain a cytoplasmic Ca²⁺-dependent proteolytic system consisting of two different proteinases (CDP I and CDP II) and an endogenous inhibitor specific for these proteinases. It was difficult to determine the relative activities of CDP I and CDP II directly and accurately without extensive purification, requiring relatively large amounts of tissue. We developed a simple technique based on Reactive Red-agarose affinity chromatography for quantitatively measuring CDP II activities in small (< 100 mg) tissue samples. This technique is rapid, sensitive and highly reproducible. CDP II activities can be quantified in numerous tissue samples in a single day. Using this method, we analysed the components of the CDP system in various rat tissues and demonstrated quantitative differences in CDP II activities as well as relative differences in the amounts of CDP-inhibitor activity among the various tissues. This technique should prove useful in the efforts to define the currently unknown physiological function(s) of the Ca²⁺-dependent proteolytic system by allowing comparison of CDP activities in tissues under diverse conditions of protein metabolism.