Resolution of Granules From Rabbit Heterophil Leukocytes Into Distinct Populations by Zonal Sedimentation

Abstract

Postnuclear supernates from homogenates of essentially pure rabbit heterophil leukocytes were fractionated by means of zonal differential centrifugation through a discontinuous sucrose gradient at various speeds. Three distinct groups of granules were characterized biochemically and morphologically. They were, in order of decreasing sedimentation coefficient: (a) Large, relatively dense granules, identified morphologically as the azurophil or primary granules, and containing essentially all of the myeloperoxidase activity of the preparations, about one-third of their lysozyme activity, and between 50 and 80% of their content in five acid hydrolases typically associated with lysosomes in other cells; (b) smaller, less dense granules, with the morphological appearance of the specific or secondary granules, and carrying most of the alkaline phosphatase and the remainder of the lysozyme activity of the preparations; (c) a second group of lysosome-like particles, associated with a morphologically heterogeneous fraction, and containing the remainder of the acid hydrolases, but little or no myeloperoxidase. When p-nitrophenyl phosphate was used instead of j-glycerophosphate for the assay of acid phosphatase, only small proportions of the total activity accompanied the two main lysosomal bands, and considerable activity was found in a zone slightly retarded with respect to the slowly moving band of acid hydrolases.

Isolation and Properties of Phagocytic Vesicles from Polymorphonuclear Leukocytes

“…All of our findings are consistent with the view that particle uptake and granule interaction with the PV are 2 A similar distribution of enzyme activities was noted among fractions of granules separated from rabbit PMN (23,24). An acid phosphatase which was associated with slowly sedimenting particles from the rabbit cells, however, hydrolyzed p-nitrophenyl phosphate much more readily than it did 8-glycerophosphate, whereas the enzyme from guinea pig cells exhibited little preference (15).…”

Antibacterial activity of cationic proteins from human granulocytes.

“…Biochemical and morphological studies of granulocytes from several species have revealed two types of cytoplasmic granules, namely, peroxidase-positive (azurophil or primary granules) and peroxidase-negative (specific or secondary granules) (30)(31)(32)(33). The cationic proteins of rabbit polymorphonuclear leukocytes are associated with the peroxidase-containing granules (32).…”

Demonstration of ricin-binding sites on the outer face of azurophil and specific granules of rabbit polymorphonuclear leukocytes.

“…The cells were homogenized, and a postnuclear (4,000 g-rain) supernate was prepared as described previously (3). Granules were obtained by fractionating 2.5-ml aliquots of the postnuclear supernates through discontinuous sucrose gradients consisting of three layers (8.0, 5.5, and 4.0 ml) with densities 1.08, I.…”

“…The two types of granules arise from opposite faces of the Golgi apparatus (i) during successive stages of PMN maturation (1,2). The azurophils contain myeloperoxidase, lysozyme, and a wide variety of lysosomal hydrolases, while the specifics contain alkaline phosphatase, iysozyme, and lactoferrin, but are devoid of lysosomal hydrolases (3)(4)(5).…”