Abstract
Previous studies have established that mature neutrophils from the peritoneal cavity, blood, and bone marrow of beige (Chédiak-Higashi syndrome) mice essentially lack activities of two lysosomal proteinases: elastase and cathepsin G. There are, however, significant levels of each enzyme in early neutrophil precursors in bone marrow. In the present experiments, it was found that the addition of extracts from mature beige neutrophils to extracts of normal neutrophils or to purified human neutrophil elastase and cathepsin G resulted in a significant inhibition of elastase and cathepsin G G activities. 125I-Labeled human neutrophil elastase formed high molecular mass complexes at 64 and 52 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis when added to beige neutrophil extracts. The molecular masses of the inhibitor-125I-elastase complexes suggested that the molecular masses of the inhibitors are approximately 36 and 24 kDa, respectively. These results were confirmed by gel filtration on Superose 12 under nondenaturing conditions. Cathepsin G was inhibited only by the 36-kDa component. The inhibitors formed a covalent complex with the active sites of elastase and cathepsin G. No inhibitory activity was present in mature neutrophil extracts of genetically normal mice or in extracts of bone marrow of beige mice. These results thus represent an unusual example of an enzyme deficiency state caused by the presence of excess inhibitors. Inactivation of neutrophil elastase and cathepsin G in mature circulating and tissue neutrophils may contribute to the increased susceptibility of Chédiak-Higashi patients to infection.
Highlights
Caused by the presence of excess inhibitors
Evidence for Inhibitors oEf lastase and CathepsinG in Beige (Chidiak-Higashi)NeutrophilExtracts-Positiveindication of inhibitorsofhumanneutrophil elastase (Fig. l.4) and humancathepsin G (Fig. 1B) in beigeneutrophilswas obtained in mixingexperiments.Humanneutrophil elastase activity was 50% inhibited when 1.2 pg of beige extract was preincubated with0.02 pg of purified human neutrophilelastase (Fig. U),and 90%inhibition occurred at about 20 pg of beige extract
(Fig. 2 B ) of normal mouse neutrophils were likewise diminished when excess extract from beige neutrophils was added to extract from normalneutrophils.Significantinhibitionwas observed when equal amounts of normal and beige extracts werepreincubated.Only 10%of elastase activity remained when 100 pg of beige protein was preincubated with18 pg of normal extractprotein (Fig. 2 A ), and 40% ofcathepsin G
Summary
Na'25Z-Purified humanneutrophilelastase (100 pg in 100 pl of iodination buffer (see below)) was radiolabeled by incubating with. Human neutrophil elastase was incubated with cell extracts containing the inhibitor in 50 pl of 0.15 M NaCl, 20 mM Tris-HC1, pH 7.5, 0.2%Triton X-100, 5 mM EDTA, and 1 mg/ml BSA a t 4 "C for 10 min. The cathepsin G and extracts containing the inhibitor were preincubated 10 min a t 4 "C in 0.15 M NaCl, 20 mM Tris-HC1, pH 7.5, 0.2% Triton X-100,5 mM EDTA in 50 pl. The complete reaction mixture (500 p l ) [2, 15] was incubated 20 min a t 50 "C to assay cathepsin G activity, and the reaction was stopped by the addition of 50 pgof soybean trypsin inhibitor. A unit of inhibitor is defined as the amount which inhibits 50% human cathepsin G activity under the described conditions
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