Neutrophil leukocytes are the body's major defence against bacteria, which they phagocytose and kill. It has been found that phagocytosis and killing are accompanied by a dramatic rise in non-mitochondrial respiration; and that the efficiency of killing is impaired in the absence of oxygen. It is also impaired in neutrophils from patients with chronic granulomatous disease (CGD), where the respiratory burst is absent. This has been difficult to reconcile with their normal content of granule proteins that kill bacteria in vitro. Indeed, CGD cells are essentially normal both morphologically and constitutionally except that they lack a functional very low potential cytochrome b (b-245), which is a component of the oxidase system responsible for the respiratory burst of normal cells. Activation of the oxidase is associated with the generation of various reduced oxygen species which have been widely thought to be responsible for the killing of phagocytosed microorganisms either directly, or by acting as substrates for myeloperoxidase-mediated halogenation. We report here, however, that a major consequence of the defective function of this oxidase in neutrophils and monocytes from CGD patients is an absence of the normal initial rise, and an unusually rapid and extensive fall in pH which is itself associated with the impairment of the killing and digestion of intracellular staphylococci.
Stimulation of bovine adrenal chromaffin cells results in a rise in the concentration of cytosolic calcium which triggers the release of catecholamines by exocytosis. Several cytosolic proteins that bind to secretory granule membranes in a calcium-dependent manner have been implicated in exocytosis and some belong to a family of calcium-binding proteins, the annexins. One of these, calpactin, is a tetramer consisting of two heavy and two light chains (relative molecular masses 36,000 and 10,000 respectively) and can aggregate and fuse membranes in vitro in the presence of arachidonic acid. Calpactin is found at the cell periphery and is phosphorylated when chromaffin cells are stimulated. We show here that both calpactin and calpactin heavy chain (p36) reconstitute secretion in permeabilized chromaffin cells in which secretion has been reduced as a result of leakage of cellular components. This effect is inhibited by an affinity-purified antibody against p36. Secretion from permeabilized cells is inhibited by a synthetic annexin-consensus peptide, but not by a nonspecific hydrophobic peptide; this inhibition is reversed by p36. Our results indicate that either calpactin or p36 is essential for exocytosis.
A group of calcium-binding proteins which bind to biomembranes has recently been identified in widely different cells and tissues (refs 1-7, reviewed in ref. 8). Three of these proteins (p70, p36 and p32.5) cross-react with antiserum to calelectrin, a Ca2+-binding protein (relative molecular mass 34,000 (34K] from the ray Torpedo marmorata, giving rise to their designation as calelectrin-related proteins. We now report that calelectrin, p36 and p32.5 contain a 17-amino-acid consensus sequence which is conserved and present in multiple copies. We suggest that this sequence may be common to other members of this new group of Ca2+-binding proteins and may underlie their unusual mode of combination with biomembranes.
Intravacuolar pH was measured within the lysosomes and newly formed phagosomes in cultured mouse peritoneal macrophages. The kinetics of pH change in both vacuolar systems was quantitatively determined within a large cell population by fluorescence spectroscopy . Additionally, pH changes within individual phagosomes were followed semiquantitatively using indicator dyes .Two novel findings were made . Firstly, the pH in new phagosomes was transiently driven alkaline (higher than physiological) even when the external medium was buffered at pH 6.5.Secondly, perturbations of phagosome-lysosome fusion had little effect upon phagosomal pH changes, even though the compounds used markedly altered the pH of the lysosomes in resting and phagocytosing cells .A pH-sensitive fluorescent probe has been employed to measure pH and "microviscosity" within living macrophage lysosomes and phagosomes during the fusion of these organelles (P-1,F). Fluorescein was specifically introduced to phagosomes or lysosomes by conjugation with the surface of yeasts or dextran, respectively. The resting pH of lysosomes in mouse peritoneal macrophages and baby hamster kidney (BHK21) cells in culture was 4.8 ± 0.1, in close agreement with the value reported by Ohkuma and Poole (1) for macrophage lysosomes . Phagocytosis of yeasts provoked a rapid rise in lysosomal pH, and the subsequent P-LF was characterized by a chronically raised pH (5.4 ± 0.2) recorded by the fluorescent probe . The initially high fluorescence polarization of the probe (0.33) gradually decreased during P-LF, reflecting its transfer within the cells to a less restricted environment (i.e., the phagosomes) . Conversely, fluorescein conjugated to yeasts experienced a rapid fall in pH after entering the cells, finally monitoring the same average pH as the lysosomal probe (5 .4 t 0.4). When phagocytosis of fluorescent yeasts was synchronized by prebinding the yeasts at 4°C, there was a transient rise in fluorescent emission that was maximal 2 min after the initiation of phagocytosis . The maximum recorded pH was 7.75, higher than that in the extracellular medium . Thereafter the pH of the phagocytic vacuoles decreased as before. The kinetics of this pH change are reminiscent of those ofthe respiratory burst of neutrophilic leukocytes (2) .
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