Membrane-bound protein kinase activity in acetylcholine receptor-enriched membranes

Membrane protein phosphorylation may be a general regulatory mechanism mediating the response of cells to exogenous metabolic and physical signals. We have determined that the membrane-bound acetylcholine receptor is the major substrate phosphorylated in situ by a nearby membrane protein kinase. Moreover, these same membranes also contain phosphoprotein phosphatase activity which dephosphorylates the membrane-bound receptor. These findings suggest that reversible phosphorylation of the actylcholine receptor may be critical for receptor function at the synapse. Therefore, it is necessary to define the properties of the enzymes which mediate this phosphorylation-dephosphorylation mechanism. In this report we describe the properties of the first component of this system, the membrane-bound protein kinase in receptor-enriched membranes from the electric organ of Torpedo californica. Only ATP is effective as a phosphate donor for this cyclic AMP-independent membrane kinase; GTP does not support phosphorylation of the receptor. Both casein and histone can also be phosphorylated by the membrane protein kinase, but casein is a better substrate. Although phosphorylation of the receptor appears to be regulated by cholinergic ligands and K+, casein phosphorylation is not specifically affected by these agents. Moreover, while phosphorylation of the acetylcholine receptor is maximal in receptor=enriched membranes, casein phosphorylation is similar in all membrane fractions prepared from the electric organ. Taken together, these findings suggest that the membrane protein kinase activity in receptor-enriched membranes is similar to most other membrane kinases. Therefore, the unique characteristics of membrane-bound acetylcholine receptor phosphorylation appear to be determined by the receptor and its availability as a substrate for the membrane kinase.     

Glycolysis in quiescent cultures of 3T3 cells. Stimulation by serum, epidermal growth factor, and insulin in intact cells and persistence of the stimulation after cell homogenization.

Addition of serum to quiescent cultures of 3T3 cells rapidly increases lactic acid formation and subsequently stimulates cell division. The stimulation of lactic acid production is seen at high, saturating concentrations of extra-cellular glucose. It is dependent on the time of exposure and on the dose of serum and is not blocked by the addition of cycloheximide, puromycin, or actinomycin D. In contrast, serum only marginally affects glycolysis by rapidly growing 3T6 or SV40-3T3 cells. In addition to serum, epidermal growth factor (0.1 to 10 ng/ml) and insulin (10 to 500 ng/ml) cause a striking stimulation of glycolysis in quiescent 3T3 cells. Neither exogenous cyclic nucleotides nor ouabain effect the glycolytic response, but the presence of Ca2+ markedly influences the activation of glycolysis by epidermal growth factor and by insulin. A novel finding in this study is that homogenates prepared from quiescent cells treated with serum, epidermal growth factor, or insulin show increased glycolysis as compared with homogenates from nonstimulated cultures. This finding will allow further experimental analysis of the cause of increased glycolysis in rapidly proliferating cells.     

DNA-DNA hybridization assay for detection of Salmonella spp. in foods

We have developed a DNA-DNA hybridization test for the presence of Salmonella spp. in foods. This test requires an initial pre-enrichment of food samples in nutrient broth but does not require selective enrichment. Samples of food cultures are collected on membrane filters and assayed by molecular hybridization to labeled probes. The probes consist of DNA sequences which are unique to the genus Salmonella and are widely distributed in the genus. A diverse panel of foods was assayed successfully by this methodology.     

Leishmania promastigotes require opsonic complement to bind to the human leukocyte integrin Mac-1 (CD11b/CD18)

Previous reports have suggested that Leishmania spp. interact with macrophages by binding to Mac-1 (CD1 1b/CD18), a member of the leukocyte integrin family. To better define this interaction, we tested the ability of leishmania promastigotes to bind to purified leukocyte integrins and to cloned integrins expressed in COS cells. We show that leishmania promastigotes bind to cellular or purified Mac-1 but not lymphocyte function-associated antigen-1 in a specific, dose-dependent manner that requires the presence of serum. Binding is inhibited with specific monoclonal antibodies to Mac-1. In the absence of complement opsonization, three different species of leishmania tested fail to bind directly to any of the three leukocyte integrins. We show that binding to Mac-1 requires the third component of complement (C3). Organisms incubated in heat-inactivated serum or serum that has been immunologically depleted of C3 fail to bind to Mac-1. Because the addition of purified C3 to C3-depleted serum restores leishmania binding to Mac-1, we suggest that parasites gain entry into macrophages by fixing complement and subverting a well-characterized adhesive interaction in the immune system between Mac-1 and iC3b.     

High level of surface CD4 prevents stable human immunodeficiency virus infection of T-cell transfectants.

CD4 is the principal receptor for the human immunodeficiency virus (HIV). We have isolated and studied CD4-expressing tumor cell clones made by expressing CD4 in the T-cell tumor line HSB. Two clones, one designated HSBCD4, a clone expressing low levels of CD4, and the other, HSB10xCD4, a high-expresser CD4+ clone, were studied for their ability to bind and replicate HIV. In contrast to many other CD4+ cells that down-modulate CD4 following HIV infection, the HSB10xCD4 clones continued to express high levels of surface CD4 following infection with HIV. Unlike infection of HSBCD4 or many other human CD4+ cells, HIV infection of HSB10xCD4 clone was short lived: p24 antigen, provirus, or coculturable virus was present for less than 14 days following infection with several strains of HIV-1 or with HIV-2. When infection was initiated by transfection of proviral DNA, high and low CD4 expressers initially produced p24 antigen at approximately the same level. However, high CD4 expressers produced coculturable virus only during the first few days following transfection, whereas low CD4 expressers transfected with HIV continued to produce virus beyond 6 weeks. Monoclonal antibody-mediated down-modulation of CD4 surface expression on HSB10xCD4 clones permitted these formerly HIV-resistant cells to become persistently infected with HIV. Thus, high concentrations of CD4 on the surface of an HIV-infected cell prevent persistent HIV infection of CD4+ cells.     

Flow cytometric kinetic measurements of neutrophil phospholipase A activation.

The interrelationships between activation of phospholipases and neutrophil stimulus-induced Ca2+ responses remain unclear. We report here that immune complexes activate a phosphatidylcholine-specific phospholipase A in a neutrophil only after the cytoplasmic Ca2+ transient has been initiated in the same cell, while chemotactic peptide activation does not proceed via such a phospholipase A-mediated mechanism. Measurements of [Ca2+] changes and of phosphatidylcholine-specific phospholipase A activity were made by flow cytometry, using Indo-1 for Ca2+ indication, and a new fluorescent probe, bis-BODIPY-phosphatidylcholine, localized in the inner leaflet of the plasma membrane, to measure phospholipase A activation. Both 100 nM formyl-methionyl-leucyl-phenylalanine (with or without cytochalasin B) and 60 micrograms/ml insoluble immune complexes elicited cytoplasmic Ca2+ transients, but only insoluble immune complexes stimulated phospholipase A activation in a subpopulation of cells exhibiting an elevation of [Ca2+]in. Phospholipase A activation followed the Ca2+ transient, starting, in each cell, after [Ca2+]in had begun to decrease as Ca2+ redistributed in the activated cell. The products of this phospholipase activation were confirmed by thin layer chromatography. We conclude that neutrophils respond to immune complexes with an elevated cytoplasmic Ca(2+)-requiring phosphatidylcholine-specific phospholipase A activation and to chemotactic peptides by a different mechanism.