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In the present study, attempts were made to clarify the role of an elastase-like enzyme in Arthus reaction by using rabbit-peritoneal polymorphonuclear leukocytes (PMNs). It was first confirmed that the injection of large amounts of PMNs with antibody can increase tissue damage in reversed Arthus reaction. The five various fractions, which were intact PMNs, whole homogenate, granule fraction, nuclei and cell debris, and membrane extract, were prepared from rabbit-peritoneal exudate. Each fraction was intracutaneously injected into the abdominal wall of rabbit. It was of particular interest that the tissue damage induced by membrane extract occurred earlier and was more severe than that caused by the others. Furthermore, membrane extract possessed remarkably higher caseinolytic and elastinolytic activities than the others. The membrane extract was partially purified by Sephadex G-100 column chromatography. The protein concentration of the elastase-like enzyme solution was 3.64 mg/ml, and its specific activity was 195 caseinolytic units and 0.45 elastinolytic units per milligram of protein. The cutaneous reaction by membrane extract was dependent on the concentration of this elastase-like enzyme and could be strongly inhibited by Elastatinal. From these results, the authors suggest that an elastase-like enzyme which originates in PMN granules is the key enzyme that promotes the severity of tissue damage in Arthus reaction.

GTP and GTP-gamma-S enhanced several-fold the NADPH-dependent superoxide production induced by sodium dodecyl sulfate in a cell-free system of pig neutrophils consisting of the membrane fraction and two cytosolic fractions separated by gel filtration. The enhanced activity was decreased by the addition of GDP in a dose-dependent manner, but 70% of the activity in the absence of GTP remained even at 1 mM GDP. Only one cytosol fraction besides the membrane fraction was required for the activation in the presence of GTP. The cytosol fraction was analyzed by chromatography on 2',5'-ADP agarose and two components responsible for the GTP-dependent and independent activation were separated. These results suggest that at least two pathways are available for the activation of superoxide production in the cell-free system of pig neutrophils.

We studied the effect of the 2',3'-dialdehyde derivative of NADPH on the activation of superoxide-producing oxidase in a cell-free system of pig neutrophils. The system consisted of a membrane fraction, two cytosolic fractions prepared by gel filtration, and arachidonic acid. Preincubation of one of the cytosolic fractions with the derivatives of NADPH and NADP+ caused the loss of its ability to activate the enzyme. The inactivation was effectively prevented by the addition of NADPH and NADP+. Neither the membrane fraction nor the other cytosolic fraction was affected by the derivatives. The results indicate that the NADPH binding component of the oxidase is present in the cytosolic fraction and may be translocated to the membrane fraction during the activation process in the cell-free system.