A polypeptide that causes pore formation in target-cell membranes is implicated in the potent cytolytic activity of pathogenic Entamoeba histolytica. Pore-forming material was purified to apparent homogeneity by a multistep procedure, and its analysis by NaDodSO4/PAGE revealed one peptide of 4-5 kDa under nonreducing or under reducing conditions. Pore-forming activity was measured by depolarization of liposome membrane potential and was found to be optimally expressed at low pH. Active material preferentially inserted into negatively charged lipid vesicles. Treatment of purified amoeba peptide in solution or bound to liposomes with glutaraldehyde revealed oligomers upon NaDodSO4/PAGE, suggesting functionally relevant peptide-peptide interactions. The NH2-terminal amino acid sequence of the amoeba peptide was determined by protein sequencing and revealed a structural similarity to melittin, the membranolytic peptide of bee venom.
Limited proteolysis of Cl-inhibitor was observed with human skin chymase, human cathepsin G, and bovine chymotrypsin. In each case., the inhibitor was degraded to one major product migrating slightly faster than the native inhibitor in an SDS-polyacrylamide gel. The inhibitory activity of Cl-inhibitor against human plasma kallikrein was not altered by the modification with chymase. Edman degradation of the proteolyzed inhibitor revealed two sequences in a 1:l ratio: NPNATSSSQ, the N-terminus of native Cl-inhibitor, and VEPILEVSSL. This second sequence showed that the Phe'"-Val% bond was hydrolyzed. Our results provide another example of the susceptibility of the N-terminal region of Cl-inhibitor to proteolytic cleavage.
Summary:The catalytic concentration of N-acetylalanine aminopeptidase was determined in erythrocytes, polymorphonuclear leukocytes, lymphocytes, alveolar macrophages, human lung fibroblasts, human lung cancer cells, human umbilical vein endothelial cells, mice Leydig cells, rat tumour cells, and endothelial cells from hog and bovine lung. The catalytic concentration ranged from % 0.5 ±0.1 nU/cell (erythrocytes) to 35 nU/cell (rat tumour cell line B Sp 73 ASML). Almost all tumour cells showed higher activity levels. No activity was found in human plasma.
N-Acetylalanm-aminopeptidase-Aktivität in normalen Zellen und in TumorzellenZusammenfassung: Die katalytische Konzentration von N-Acetylalanin-aminopeptidase bestimmten wir in Erythrocyten, polymorphkemigen Leukocyten, Lymphocyten, Alveolar-Makrophagen, menschlichen Lungenfibroblasten, menschlichen Lungentumorzellen, menschlichen Nabelschnurendothelzellen, Leydig-Zdlen von Mäusen, Ratten-Tumorzellen und Endothelzellen aus Schweine-und Rinderlunge. Die katalytische Konzentration reichte von 0,5 ± 0,1 nU/Zelle (Erythrocyten) bis zu 35 nU/Zelle (Ratten Tumorzell-Linie B Sp 73 ASML). In fast allen Tumorzellen wurde eine erhöhte Aktivität gefunden. Keine Aktivität konnte im menschlichen Plasma nachgewiesen werden.
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