Membranes Of Specific Granules Research Articles

The neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules.

The glycoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove absorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils.

Flavonoid impairment of neutrophil response

Flavonoids are a class of phenolic plant pigments which impair the oxidative burst of neutrophils to an extent dependent on their hydrophobicity. The distribution of quercetin and of morin in nitrogen-cavitated neutrophils paralleled their respective hydrophobic characteristics and respiratory burst inhibition. While both flavonoids were localized primarily in the specific granule membrane of neutrophils, the amount of quercetin was considerably greater than that of morin. We here demonstrate inhibition of the initial stimulation response, depolarization of the membrane potential as monitored by fluorescence of the membrane probe diS-C 3-(5), and of the respiratory burst, monitored by following the destruction of diS-C 3-(5), a reaction mediated by the H 2O 2 produced in the burst. The flavonoids kaempferol, morin, quercetin, or fisetin were preincubated with human neutrophils at a concentration of 100 μM per 2 × 10 6 cells/ml for 2–3 min and subsequently stimulated with 1 μg/ml of the tumor promoter phorbol myristate acetate (PMA) or with 60 μg/ml of immune complex. The effect of each compound differed, i.e. depolarization was enhanced by some and inhibited by others, while H 2O 2 generation was inhibited by each, supporting our previous findings that membrane potential depolarization and the respiratory burst are dissociable events. Concentration-response experiments, performed at flavonoid concentrations between 12.5 and 500 μM to determine the ic 50 values of these compounds for depolarization and burst activation, indicated that none of the flavonoids affected the resting potential, while all perturbed the stimulus-coupled response, the direction and extent of the perturbation depending upon the stimulus, and the function assessed. These data show that the effects of flavonoids on human neutrophils are complex and suggest several sites of action depending upon the flavonoid's subcellular distribution and pathway of stimulation.

Anomalous neutrophil granule distribution in a patient with lactoferrin deficiency: pertinence to the respiratory burst.

Neutrophils from a patient with lactoferrin deficiency were examined and the quantity and subcellular localization of protein markers were determined on Percoll density gradients. Distribution of azurophilic and specific granule markers was abnormal in that azurophilic granules were lighter than normal and appeared in the fraction of the gradient where normally the specific granules sediment. The specific granule membrane markers, cytochrome b-235 and its associated flavoprotein, were abnormally distributed in the gamma fraction, the site of the plasma membrane marker alkaline phosphatase. Thus, the b-cytochrome-flavoprotein complex had either been incorporated into the plasma membrane or was still present in the membranes of granules that were abnormally light and cosedimented with the plasma membranes. This is of particular interest in regard to the patient's respiratory burst oxidase function, since the b-cytochrome/flavoprotein complex normally translocates from the specific granules to the plasma membrane to constitute the active respiratory burst oxidase. The functional consequences of this abnormal distribution are discussed, as is the importance of characterizing both intragranular enzymatic markers and granule membrane proteins to define granular disorders.

Characterization of the chromobindins. Soluble proteins that bind to the chromaffin granule membrane in the presence of Ca2+.

A group of proteins that bind to the chromaffin granule membrane in the presence of Ca2+ has been isolated by affinity chromatography of bovine adrenal medullary cytosol on granule membranes coupled to Sepharose 4B. Twenty-two of these proteins were resolved into classes depending upon the Ca2+ concentration at which they were eluted from the affinity column (40 or 0.1 microM), upon their affinities for native granule membranes or for liposomes prepared from extracted granule lipids, and upon the requirement of seven of the proteins for ATP in the cytosol fraction and column buffers to promote binding. The molecular weights and isoelectric points of these proteins were determined by two-dimensional electrophoresis. Two of the granule-binding proteins were identified: synexin and calmodulin. Calmodulin was found to bind to seven specific granule membrane proteins after diffusion of 125I-labeled calmodulin into an acrylamide gel of membrane proteins separated by electrophoresis in the presence of sodium dodecyl sulfate. A phospholipid-activated protein kinase activity, possibly due to protein kinase C, was present in the granule-binding fraction. Two major granule-binding proteins were found to present a pattern in two-dimensional electrophoresis that was very similar to but shifted slightly toward the basic end of the gel from the pattern generated by light chains associated with clathrin in adrenal medullary coated vesicles. In the chromaffin cell, these proteins, by associating with the granule membrane in the presence of an increased cytosolic Ca2+ concentration, might play a variety of roles in the process of exocytosis.

The release of granule components from human polymorphonuclear leukocytes in response to both phagocytic and chemical stimuli

The differential effects of phagocytic and chemical stimuli on neutrophil enzyme and specific protein release were compared. Phorbol myristate acetate (PMA) stimulated release of the specific granule matrix marker, vitamin B-12-binding protein in a dose-dependent manner. Subcellular fractionation by sucrose density gradient centrifugation indicated that the residual vitamin B-12-binding protein is associated with the specific granule fraction. In contrast, neutral alpha-glucosidase and adenosine diphosphatase, associated with specific granule membranes, were not released by PMA. Subcellular fractionation studies suggest that fusion of the specific granule membrane and plasma membrane occurs, thus translocating the adenosine diphosphatase to the cell surface. The relevance of this finding to the possible role of nucleoside phosphatases in limiting platelet aggregation is discussed. Serum-treated zymosan particles also caused a selective release of vitamin B-12-binding protein from the specific granule without release of alpha-glucosidase and adenosine diphosphatase. Neither PMA nor opsonized zymosan caused significant release of azurophil, tertiary granule or cytosol marker enzymes.

Degranulation, membrane addition, and shape change during chemotactic factor-induced aggregation of human neutrophils.

Neutrophils stimulated by the chemotactic factor formyl-methionyl-leucyl-phenyl-alanine (FMLP) undergo a transient change in surface properties that permits the cells to adhere more readily to surfaces and to each other. This transient change can be monitored by light scattering as stimulated neutrophils form aggregates while stirred in a platelet aggregometer. Maximum change in light scattering occurs within 1 min and correlates with an increase in the percentage of cells that are in aggregates of four or more cells and a decrease in the percentage of single cells. With time (3-5 min), small aggregates disappear and single cells reappear. The transient change in adhesiveness is accompanied by a persistent change in cell shape; the cells become polarized and protrude ruffles from one sector of the cell surface. During aggregation the cells adhere to one another with smooth sides together and ruffles pointed outward. During disaggregation the cells dissociate laterally with the simultaneous internalization of membrane in the region opposite the ruffles. Particle bound to the surface by charge (thorotrast, cationized ferritin) are concentrated and internalized in this region. The change in cell shape from round to ruffled occurs within seconds, suggesting that membrane is added to the cell surface from an intracellular store. We therefore quantified surface membrane by electron microscopy morphometry and measured a 25% increase within 10 s of adding FMLP. The source of new membrane appeared to be the specific granule membrane since the kinetics of granule discharge (between 30% and 50% of all release occurs in the first 10 s) correlate with the appearance of new membrane. Furthermore, the amount of membrane that appears at the cell surface at 10 s correlates with that lost from intracellular granules in that time. Chemotaxin-induced aggregation thus begins with granule discharge and membrane addition followed by protrusion of ruffles. Adherence is maximal at 60 s and the gradual loss of adhesiveness that follows is associated with uropod formation and enhanced endocytic activity.

Studies on human polymorphonuclear leukocyte enzymes. III. Differential activation of primary and specific granules by phospholipase C and deoxycholate

1. 1. Human polymorphonuclear leukocyte cytoplasmic extracts were preincubated at pH 7.2 in 0.34 M sucrose with exogenous phospholipase C or with deoxycholate, and immediately free and soluble activities were measured for several primary and specific granule enzymes. 2. 2. Both treatments caused almost complete activation of latent lysosomal enzymes. 3. 3. Specific granule membrane was strongly resistant to phospholipase C and deoxycholate treatments, because free and soluble alkaline phosphatase activity, used as marker enzyme for these granules, did not increase significantly. 4. 4. The above results are evidence for a biochemical heterogeneity between primary and specific granule membranes.