engleski

Aminopeptidases present in various granules of human polymorphonuclear granulocytes

Aminopeptidases, proteolytic enzymes which might regulate physiological processes by changing peptide hormones activity, are found in human polymorphonuclear granulocytes, but their localization within subpopulations of granules is unknown, as well as a class of these enzymes. Polymorphonuclear granulocytes were isolated from fresh human blood buffy coat. Different subpopulations of their granules were separated from postnuclear supernatant by centrifugation in Percoll gradient and identified by marker enzymes and vitamin B_12-binding protein activity. Aminopeptidase activity was measured with 2-naphthylamides of various amino acids as substrates. Different aminopeptidases were separated by isoelectric focusing and gel filtration. The presence of aminopeptidases degrading Met-, Ala-, Leu-, Phe- and Arg-2-naphthylamide was established in primary, secondary, and alkaline phosphatase positive granules (secretory vesicles). The enzyme from primary granules is a basic protein (pI 8.7, M_r 185000) unstable in alkaline media, optimally active at pH 5.9-6.0. It is strictly dependent on activation by reducing thiols, resistant to EDTA and sensitive to p-hydroxymercuribenzoate and E-64. Consequently, it belongs to broad specificity cysteine aminopeptidases. Secondary granules aminopeptidase is an acidic protein (pI approx. 3.7, M_r 308000) most stable and active at neutral pH. According to substrates and sensitivity to EDTA, bestatin and puromycin, it is a broad specificity metallo-aminopeptidase. Aminopeptidases have been added to the list of constituents of the secondary, and light and heavy primary granules of human polymorphonuclear leukocytes, what broadens a current status of enzymatic capabilities and physiological relevance of these organelles. The presence of two different enzymes was revealed and offered as possible granule markers.

aminopeptidase; human granulocyte; primary granule; secondary granule; vesicle; separation; localization

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