RESUMEN
A chemotactic factor was identified in the supernatants of human large granular lymphocytes (LGL) activated by a glutaraldehyde-fixed NK-sensitive tumor, K562. The factor stimulated migration of human LGL, rat alveolar macrophage (RAM), and human monocytes and neutrophils (PMN). The locomotor response was chemotactic and chemokinetic on the basis of unidirectional migration in concentration gradients. The cell producing the factor was detected exclusively in LGL-rich Percoll fraction coincident with the peak of NK lytic activity and HNK-1+ cells. The monoclonal phenotype of the cell was HNK-1+, partially OKT-11+, OKM-1-, OKT-3-, OKT-4-, and OKT-8-. The factor was released by LGL within 20 min of incubation with Sr++, a cation that is able to induce LGL degranulation. A powerful chemoattractant was also detected in the granules of the rat LGL leukemia, RNK. Chemotactic activity coincided with granule enzyme beta-glucuronidase and cytolysin after RNK nitrogen cavitation and Percoll fractionation of subcellular constituents. The RNK granule chemoattractant induced unidirectional migration of human LGL and was also active against rat alveolar macrophages and human PMN. Anti-RNK granule antibody conjugated to Sepharose 4B was able to deplete the chemotactic activity from both K562-induced LGL supernatants and solubilized RNK granules. These observations indicate that a leukocyte chemotactic factor (NK-LCF) is present in NK cell granules and is probably released after tumor-induced granule exocytosis.
Asunto(s)
Factores Quimiotácticos/fisiología , Quimiotaxis de Leucocito , Células Asesinas Naturales/fisiología , Gránulos Citoplasmáticos/fisiología , Exocitosis/efectos de los fármacos , Humanos , Inmunidad Celular , Macrófagos/fisiología , Neutrófilos/fisiología , Estroncio/farmacologíaRESUMEN
The hypothesis that large granular lymphocytes (LGL) are capable of directed locomotion (chemotaxis) was tested. A population of LGL isolated from discontinuous Percoll gradients migrated along concentration gradients of N-formyl-methionyl-leucyl-phenylalanine (f-MLP), casein, and C5a, well known chemoattractants for polymorphonuclear leukocytes and monocytes, as well as interferon-beta and colony-stimulating factor. Interleukin 2, tuftsin, platelet-derived growth factor, and fibronectin were inactive. Migratory responses were greater in Percoll fractions with the highest lytic activity and HNK-1+ cells. The chemotactic response to f-MLP, casein, and C5a was always greater when the chemoattractant was present in greater concentration in the lower compartment of the Boyden chamber. Optimum chemotaxis was observed after a 1 hr incubation that made use of 12 micron nitrocellulose filters. LGL exhibited a high degree of nondirected locomotion when allowed to migrate for longer periods (greater than 2 hr), and when cultured in vitro for 24 to 72 hr in the presence or absence of IL 2 containing phytohemagluttinin-conditioned medium. The chemotactic LGL was HNK-1+, OKT11+ or HNK-1+, OKT11- on the basis of monoclonal antibody and complement depletion. They did not bear either T cell or monocyte cell surface markers, exhibiting an OKT3-, OKT4-, OKT8-, OKM1-, and MO2- phenotype, and did not form E rosettes at 29 degrees C, which is characteristic of lytic NK cells in contrast to T cells. Furthermore, a rat LGL leukemia (RNK) exhibited a chemotactic response to both f-MLP and casein. LGL chemotaxis to f-MLP could be inhibited in a dose-dependent manner by the inactive structural analog CBZ-phe-met, and the RNK tumor line specifically bound f-ML[3H]P, suggesting that LGL bear receptors for the chemotactic peptide.
Asunto(s)
Quimiotaxis de Leucocito , Células Asesinas Naturales/fisiología , Animales , Caseínas/farmacología , Línea Celular , Humanos , Células Asesinas Naturales/clasificación , Células Asesinas Naturales/metabolismo , Cinética , Leucemia Eritroblástica Aguda/inmunología , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Experimental/inmunología , Leucemia Experimental/metabolismo , Masculino , N-Formilmetionina Leucil-Fenilalanina/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Fenotipo , Ratas , Ratas Endogámicas F344 , Receptores de Formil Péptido , Receptores Inmunológicos/análisisRESUMEN
Uptake and metabolism of 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin) by L5178Y lymphoblasts in vitro was investigated, using both glucose- and chloroethyl-labeled chlorozotocin. A time course of uptake of total radioactivity revealed a greater cell/medium distribution ratio of activity in cells treated with chloroethyl-labeled chlorozotocin compared to cells treated with the glucose-labeled compound. Thin-layer chromatographic analysis showed that uptake of intact chlorozotocin was identical in cells treated with either glucose- or chloroethyl-labeled drug and that the cell/medium distribution ratio never exceeded unity. Accumulation of 14C-chlorozotocin was not inhibited by an excess of unlabeled chlorozotocin, the structural analogs glucose and glucosamine, or several metabolic inhibitors or by sodium ion depletion. These observations, together with the relatively low temperature quotient for the uptake process, suggested that chlorozotocin uptake occurs by passive diffusion. In cells treated with glucose-labeled chlorozotocin, a bicyclic urethan derivative and polar metabolites soluble in trichloroacetic acid were formed. In cells exposed to chloroethyl-labeled drug, nonpolar as well as polar metabolites were noted. Formation of metabolites from the glucose moiety was impeded by the presence of an excess of unlabeled chlorozotocin, the structural analogs glucose and glucosamine, the glucose transport inhibitors phlorizin and phloretin, the metabolic inhibitor m-chlorophenyl carbonyl cyanide hydrazone and by sodium depletion. Appearance of metabolites arising from the chloroethyl moiety was also blocked by the presence of m-chlorophenyl carbonyl cyanide hydrazone and by sodium ion depletion. These results suggested that metabolism of chlorozotocin in L51789Y lymphoblasts appears to be enzyme mediated.
Asunto(s)
Leucemia L5178/metabolismo , Leucemia Experimental/metabolismo , Estreptozocina/análogos & derivados , Animales , Transporte Biológico Activo/efectos de los fármacos , Células Cultivadas , Cinética , Linfocitos/metabolismo , Ratones , Sodio/farmacología , Estreptozocina/metabolismo , TemperaturaRESUMEN
Citrate synthase of Escherichia coli reacts rapidly with 1 equivalent of Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), per subunit, losing completely its sensitivity to the allosteric inhibitor, NADH. When the enzyme is treated instead with 4,4'-dithiodipyridine (4,4'-PDS), all activity is lost. Certain evidence in this paper is consistent with the belief that the sulfhydryl group modified by DTNB, and that whose modification by 4,4'-PDS inactivates the enzyme, are the same. (i) Both reagents abolish NADH fluorescence enhancement by the enzyme. (ii) Saturating levels of NADH and some other adenylic acid derivatives inhibit the reactions with both reagents. (iii) When the enzyme is modified with one equivalent of DTNB or 4,4'-PDS, subsequent reactivity toward the other reagent is greatly decreased. (iv) Following modifications, the DTNB and 4,4'-PDS derivatives spontaneously lose thionitrobenzoate (TNB) or pyridine-4-thione (PT), respectively, in reactions which are thought to involve displacement of TNB or PT by a second enzyme sulfhydryl group, so that an enzyme disulfide is introduced. The introduction of the disulfide bond, if this is what occurs, does not lead to cross-linking of citrate synthase polypeptide chains, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions. Certain evidence has also been found, however, that the sites of modification by DTNB and 4,4'-PDS are not the same. (i) DTNB modification desensitizes to NADH but does not inactivate, while 4,4'-PDS inactivates at least 99.9%. (ii) The presumed disulfide from elimination of TNB is also active, while that from PT modification is no more active than the original 4,4'-PDS modified product. (iii) Prior modification of the enzyme with DTNB affords no protection against later inactivation by 4,4'-PDS. The studies therefore indicate a close relationship between the DTNB desensitization and 4,4'-PDS inactivation, but they are unable to identify it exactly. Other properties of the DTNB reaction are also described, and a hypothesis is offered to explain quantitatively the finding that desensitization lags behind modification during the modification of citrate synthase by DTNB.
Asunto(s)
Citrato (si)-Sintasa/metabolismo , Ácido Ditionitrobenzoico/farmacología , Escherichia coli/enzimología , Nitrobenzoatos/farmacología , Oxo-Ácido-Liasas/metabolismo , Piridinas/farmacología , Reactivos de Sulfhidrilo/farmacología , Sitios de Unión , Disulfuros/farmacología , NADP/farmacologíaRESUMEN
Evidence is presented that a number of derivatives of adenylic acid may bind to the allosteric NADH binding site of Escherichia coli citrate synthase. This evidence includes the facts that all the adenylates inhibit NADH binding in a competitive manner and that those which have been tested protect an enzyme sulfhydryl group from reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) in the same way that NADH does. However, whereas NADH is a potent inhibitor of citrate synthase, most of the adenylates are activators. The best activator, ADP-ribose, increases the affinity of the enzyme for the substrate, acetyl-CoA, and saturates the enzyme in a sigmoid manner. A fluorescence technique, involving the displacement of 8-anilino-1-naphthalenesulfonate from its complex with citrate synthase, is used to obtain saturation curves for several nucleotides under nonassay conditions. It is found that acetyl-coenzyme A, coenzyme A, and ADP-ribose all bind to the enzyme cooperatively, and that the binding of each becomes tighter in the presence of KCl, the activator, and oxaloacetic acid (OAA), the second substrate. Another inhibitor, alpha-ketoglutarate, can complete with OAA in the absence of KCl but not in its presence. The nature of the allosteric site of citrate synthase, and the modes of action of several activators and inhibitors, are discussed in the light of this evidence.