The role of proteolytic enzymes derived from crude bacterial collagenase in the liberation of hepatocytes from rat liver

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Abstract
Crude bacterial [Clostridium histolyticum] collagenase was chromatographed on DEAE-cellulose to yield 3 peaks with proteolytic activity: an arginine esterase (DEAE-1), gelatinase (DEAE-2) and a caseinolytic activity (DEAE-3). The arginine esterase and gelatinase activity fractions were slightly contaminated with each other but neither possessed caseinolytic activity; the caseinolytic fraction was devoid of arginine esterase and gelatinase activities. In addition, crude collagenase was fractionated by ZnII-affinity chromatography to produce a gelatinase peak (ZnII peak 1), which was free from arginine esterase and caseinolytic activities. The 4 fractions were compared to crude collagenase in their ability to liberate rat hepatocytes by using either liver slices or a standard perfusion technique. Compared to crude collagenase (0.05-0.1% wt/vol), which produced 70-80% liver digestion with .apprx. 80% cell viability, digestion with equivalent quantities of the isolated enzymic activities was relatively poor. Gelatinase activity (ZnII peak 1) was wholly ineffective and DEAE-1 and DEAE-2 each possessed only slight digestive properties. Hepatocyte liberation by the caseinolytic activity, DEAE-3, was partially successful (30-40% digestion, 25-30% viability) but only a portion of liver tissue was digested regardless of the quantity of DEAE-3 used. However, by mixing certain fractions before perfusion 2 gelatinase-dependent, cell-releasing mechanisms were identified: DEAE-3 with ZnII peak 1 and DEAE-1 mixed with either DEAE-2 or ZnII peak 1. Each system compared creditably with the digestive properties of an equivalent activity of crude collagenase. Differences between hepatocytes produced by the 2 enzymic mechanisms are being investigated.