Characterization of hemoglobin-hydrolyzing acidic proteinases in human and rat neutrophils.
The nature and levels of hemoglobin (Hb)-hydrolyzing acidic proteinases including cathepsin D and cathepsin E, which were most active at pH 3.5-4.0, were enzymatically and immunochemically compared between human and rat neutrophils. By subcellular fractionation and immunoprecipitation with discriminative antibodies specific for each enzyme, cathepsin D was shown to be present in the granular content fraction of both human and rat neutrophils and to account for about 35% of the total Hb-hydrolyzing activity. Cathepsin E was observed mainly in the cytoplasmic fraction of rat neutrophils from peripheral blood and peritoneal exudates and accounted for about 65% of the total activity, but it was not detected in human blood neutrophils. Immunoelectron microscopy on rat neutrophils revealed that cathepsin D was exclusively confined to lysosomes, whereas cathepsin E was localized mainly in the cytoplasmic matrix and often in the perinuclear spaces and the rough endoplasmic reticulum. The non-cathepsin D activity in human neutrophils, which represented about 65% of the total activity, appeared to be due to a serine proteinase, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride and was not inhibited by agents specific for aspartic-, cysteine-, or metallo proteinases. The enzyme(s) responsible for this activity was largely associated with the granular membranes, and a half of it could be described as an integral membrane protein on the basis of phase separation with Triton X-114 at 35 degrees C. The levels of these Hb-hydrolases in gingival crevicular fluid from human chronic inflammatory periodontitis patients were examined in order to clarify their participation in the periodontal tissue breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)
Ichimaru
E
E
Department of Periodontology, Nagasaki University School of Dentistry.
Sakai
H
H
Saku
T
T
Kunimatsu
K
K
Kato
Y
Y
Kato
I
I
Yamamoto
K
K
D000818:Animals; D002402:Cathepsin D / Q000276:immunology / Q000378:metabolism*; D020059:Cathepsin E; D002403:Cathepsins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007531:Isoflurophate / Q000494:pharmacology; D016253:Microscopy, Immunoelectron; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011233:Precipitin Tests; D051381:Rats
D007531:Isoflurophate; D010664:Phenylmethylsulfonyl Fluoride; D002403:Cathepsins; D020059:Cathepsin E; D002402:Cathepsin D