Human neutrophil N-acetyl-beta-D-glucosaminidase: granule localization. Further evidence for two azurophil granules.
The heterogeneity of human neutrophil granules, particularly of azurophil granules, defined as peroxidase containing, was examined by measuring N-acetyl-beta-D-glucosaminidase in fractions of isopycnic sucrose gradients of such granules. Neutrophil granules were prepared from normal human blood, monodispersed in heparinized sucrose, and centrifuged by established methods. N-Acetyl-beta-D-glucosaminidase was previously shown to be exquisitely sensitive to small amounts of heparin; paradoxically, larger amounts restore activity. The inhibition is reversed with protamine. N-Acetyl-beta-D-glucosaminidase activity had its peak at density 1.20 gm/ml (band B) and a lesser peak at density 1.22 gm/ml (band A). Mean specific enzyme activity was 899 +/- 217 nmol p-nitrophenol released per minute per milligram protein in gradient fraction 12 (band B), and 507 +/- 149 nmol p-nitrophenol released per minute per milligram protein in fraction 8 (band A) (p less than 0.01), which correlated in part to the significantly higher protein content in band A. These specific activities are 31-fold and 17-fold greater, respectively, then mean neutrophil lysate specific activity of 28.8 +/- 7.0 nmol p-nitrophenol released per minute per milligram protein, indicating a considerable concentration of granule enzyme activity in these gradient bands. The gradient distribution of N-acetyl-beta-D-glucosaminidase is nearly identical to that of myeloperoxidase and beta-glucuronidase, thus providing another enzyme marking the existence of two populations of azurophil granules, separable by density. Of total gradient enzyme activity, the mean percentage distribution of N-acetyl-beta-D-glucosaminidase in the pellet was 0.3%. Because the mean percentage of total gradient activity in the pellet was on an order of magnitude higher for myeloperoxidase (4.7%), beta-glucuronidase (3.7%), lysozyme (3.9%), and protein (2.8%), our data suggest that N-acetyl-beta-D-glucosaminidase has a possibly unique site within or an affinity to one or both of the two populations of azurophilic granules that is distinct from that for myeloperoxidase and beta-glucuronidase.
D000118:Acetylglucosaminidase / Q000097:blood*; D000328:Adult; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D003594:Cytoplasmic Granules / Q000201:enzymology*; D005260:Female; D005966:Glucuronidase / Q000097:blood; D006023:Glycoproteins / Q000097:blood; D006596:Hexosaminidases / Q000097:blood*; D006801:Humans; D008297:Male; D009113:Muramidase / Q000097:blood; D009504:Neutrophils / Q000201:enzymology* / Q000648:ultrastructure; D009195:Peroxidase / Q000097:blood; D011486:Protein C
D006023:Glycoproteins; D011486:Protein C; D009195:Peroxidase; D006596:Hexosaminidases; D009113:Muramidase; D005966:Glucuronidase; D000118:Acetylglucosaminidase