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E L Becker

Publications and source records attributed to E L Becker.

At least 73 records · Page 4Linked to original sources
PubMed1984

Conformationally constrained chemotactic peptide analogs of high biological activity.

The stereochemically constrained chemotactic peptide analogs, formylmethionyl-alpha-aminoisobutyryl-phenylalanine (formyl-Met-Aib-Phe-OH) and formylmethionylcycloleucinylphenylalanine (formyl-Met-Cyl-Phe-OH) are highly effective in inducing lysosomal enzyme release from rabbit neutrophils. NMR studies of the Aib2 analog in (CD3)2SO favor a folded conformation in which the Phe NH group is inaccessible to solvent. Intramolecularly hydrogen-bonded conformations involving a Met-Aib-beta-turn or a gamma-turn centered at Aib2 are considered. The results suggest that folded conformations may allow highly active interactions with the neutrophil formylpeptide receptor.

Animals↗
PubMed1984

Characterization of rabbit neutrophil membrane proteins. A 140K major membrane protein is the predominant Con A-binding protein.

The major plasma membrane proteins of rabbit neutrophils were characterized by SDS-PAGE, surface iodination, 125I-concanavalin A binding, and detergent extraction. Neutrophil membranes were prepared which lacked significant intracellular contamination with good retention of protease-sensitive proteins. The major protein and predominant Con A-binding protein was as surface exposed, 140,000 D (gp 140) protein which was solubilized by nonionic detergents but not low ionic strength. Actin and myosin but not other cytosol proteins were prominently associated with the isolated membrane particularly in a Triton-insoluble form. Membranes were also prepared from surface-iodinated neutrophils previously stimulated with a chemotactic peptide or degranulated. The granule membrane enzyme alkaline phosphatase was incorporated into the plasma membrane fraction of degranulated neutrophils. However, the membrane proteins in the different membrane preparations were identical on SDS-PAGE and autoradiography. Therefore, using these techniques, no major alterations in protein composition of the plasma membrane could be detected following stimulation or degranulation of rabbit neutrophils.

Animals↗
PubMed1984

Receptor-mediated activation of a phospholipase A2 in rabbit neutrophil plasma membrane.

Using the exogenous substrate [1-14C]oleate-labeled autoclaved Escherichia coli, we have demonstrated that the chemotactic factors fMet-Leu-Phe, complement component C5a, and leukotriene B4 [(5S,12R)-dihydroxy-6-cis,8-trans,11-trans,14-cis-icosatetraenoic acid] stimulate a phospholipase A2 of isolated plasma membranes of rabbit peritoneal neutrophils. Each of the chemotactic factors shows a biphasic concentration dependence with the optimal concentrations occurring at 1, 10, and 0.1 nM, respectively. The specific antagonists of fMet-Leu-Phe binding, carbobenzoxy-Phe-Met and t-butoxycarbonyl-Phe-Leu-Phe, effectively block the stimulation by fMet-Leu-Phe, indicating that the activation is receptor mediated. delta 6-trans-leukotriene [(5S-12R)-dihydroxy-all-trans-6,8,10,14-icosatetraenoic acid], a biologically inactive stereoisomer of leukotriene B4, does not stimulate phospholipase activity, suggesting that the enhancement by leukotriene B4 is also receptor mediated. The unstimulated and activated phospholipase exhibit a broad range of maximal activity between pH 7.0 and pH 8.5, both with an optimal pH of 8.5. The activation of the phospholipase by fMet-Leu-Phe is completely calcium dependent; no increase in activity is demonstrable if fMet-Leu-Phe is added in the absence of exogenous calcium or in the presence of EGTA. In contrast, the unstimulated plasma membrane activity of the phospholipase, as well as the activity arising after stimulation, is relatively insensitive to the concentration of calcium, being inhibited by less than 50% in the presence of 10 mM EGTA. The phospholipase hydrolyzes 1-[1-14C]palmitoyl-2-acyl-sn-glycerophosphoethanolamine to form only radioactive lysophosphatidylethanolamine as the product, indicating that the enzyme has an A2 specificity.

Animals↗
PubMed1983

Endogenous substrates for cyclic AMP-dependent and calcium-dependent protein phosphorylation in rabbit peritoneal neutrophils.

As in other cells, cAMP-dependent (protein kinase A) and calcium-dependent protein kinases are present in the rabbit peritoneal neutrophil. The major substrates for protein kinase A in the cytosol of rabbit peritoneal neutrophil is a 43 kDa protein which appears to be actin (pI 5.7). The other substrates for protein kinase A in the cytosol are very acidic proteins with molecular weights of 135000 (pI 4.6) and 130 000 (pI 4.8). Two classes of calcium-dependent protein kinases are present in the rabbit peritoneal neutrophil: one is calcium, calmodulin-dependent, the other is calcium, phosphatidylserine-dependent. Phosphatidylserine appears to be much more effective than calmodulin in stimulating calcium-dependent protein kinase activity. The phospholipid-sensitive, calcium-dependent protein kinase (protein kinase C), present only in the cytosol fraction, exhibits much higher activity than the cAMP-dependent protein kinase from the same source. At least four substrates (Mr 130 000 (pI 4.6) 43 000 (pI 4.8), 41 000 (pI 6.3) and 34 000) of the protein kinase C in the cytosol were identified. Trifluoperazine, a compound which inhibits the degranulation, aggregation and stimulated oxygen consumption of rabbit peritoneal neutrophils. (Alobaidi, T., Naccache, P.H. and Sha'afi, R.I. (1981) Biochim. Biophys. Acta 675, 316-321), also inhibits the activity of protein kinase C. The possible role of cAMP-dependent and calcium-dependent phosphorylation system in neutrophil function is discussed.

Animals↗
PubMed1983

The photoaffinity probe 8-N3[alpha-32P]ATP labels the ATP-binding sites of rabbit neutrophil and skeletal muscle actin.

8-Azido-[alpha-32P]ATP (8-N3-ATP) was used as a photoaffinity label for ATP binding sites in the subcellular fractions of rabbit peritoneal neutrophils. The radioactive 8-N3-ATP was specifically incorporated into one major protein of 43 kDa. The isoelectric point, molecular mass and subcellular distribution of this labeled protein closely resemble those of the actin. 8-N3-[alpha-32P]ATP was further tested as a photoaffinity label for the ATP binding site in the purified rabbit skeletal muscle G-actin. The radioactive 8-N3-ATP was specifically incorporated into the actin band in SDS-polyacrylamide gel. The results indicate that 8-N3-ATP can be used as a photoaffinity label for actin.

Actins↗
PubMed1983

Inhibition of chemotactic factor-induced neutrophil responsiveness by arachidonic acid.

Arachidonic acid when added simultaneously with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) inhibits the ability of the latter to initiate several but not all of its effects on rabbit peritoneal neutrophils. Stimulated neutrophil aggregation, calcium uptake, and increases in the steady state level of exchangeable calcium are all inhibited by 1-10 microM arachidonic acid. The binding of f-Met-Leu-Phe and the parameters of intracellular calcium redistribution (calcium efflux and changes in the steady state level of exchangeable calcium in the absence of extracellular calcium) and of stimulated sodium uptake are, on the other hand, unaffected by the same concentrations of arachidonic acid. Arachidonic acid, the saturated analog of arachidonic acid, was found not to inhibit f-Met-Leu-Phe-stimulated aggregation and calcium uptake. Arachidonic acid, therefore, in addition to its well-described agonist properties, also possesses antagonist activities toward rabbit neutrophils. These results add a new level of complexity to the study of the role of arachidonic acid in cell activation.

Animals↗
PubMed1983

A simple and rapid assay for measuring radiolabeled ligand binding to purified plasma membranes.

A simple, rapid assay for measuring radiolabeled ligand binding to purified plasma membranes was developed. In this assay, membrane proteins and ligand are mixed atop a nonmiscible silicone oil (density = 1.029 g/cm3) and incubated to establish equilibrium. The membrane proteins and bound ligand are then rapidly separated (30-60 s) from unbound ligand by centrifugation at 100,000g in a Beckman airfuge. A small amount of unbound ligand is contained in the pellet as extramembranous fluid so that the bound and free ligand remain essentially in equilibrium. Thus, this binding assay is suitable for the characterization of low-affinity (Kd greater than 10(-8) M) binding sites with rapid dissociation rate constants. In addition, measurements and comparisons of the binding of the synthetic chemotactic peptide formylnorleucyl-leucyl-[3H]phenylanine to purified rabbit neutrophil membranes have been made using the silicone oil centrifugation assay and a filtration binding assay. The results of these experiments illustrate the problems and potential errors associated with nonequilibrium binding assays and emphasize the advantage of using the silicone oil centrifugation binding assay.

Animals↗
PubMed1983

The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils: change of receptor affinity and number by L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK).

Pretreatment of rabbit peritoneal neutrophils at 37 degrees with 10-35 microM L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) decreases by 20-50% the detectable number of f Met-Leu-[3H]Phe binding sites. Greater TPCK concentrations, between 50 and 100 microM, cause less of a decrease or actually increase peptide binding activity to a level greater than that of untreated cells. Furthermore, Scatchard analysis indicates that the sites detected on neutrophils after TPCK treatment have 1.2-3.2 fold lower apparent Kd (higher affinity) than those detected on untreated, control cells (1.1 +/- 1.7 X 10(-8) M vs 1.7 +/- 1.5 X 10(-8) M, P less than 0.02). Thus, TPCK treatment of rabbit peritoneal neutrophils causes both a decrease in f Met-Leu-[3H]Phe receptors and increases the affinity of the remaining sites. In addition, peritoneal neutrophils incubated at 37 degrees without TPCK were found to rapidly express additional f Met-Leu-[3H]Phe receptors. These additional sites, however, were not evident on neutrophils incubated at 37 degrees with TPCK. Concomitantly with the expression of additional sites, neutrophils placed at 37 degrees were found to spontaneously release small amounts of lysozyme. However, since equivalent amounts of lysozyme were released by cells incubated with or without TPCK, we are unable to state whether expression of the additional sites is due to neutrophil degranulation. Finally, although rabbit peripheral blood neutrophils also show an increase in binding sites at 37 degrees, treatment of these cells with TPCK does not cause a decrease in their f Met-Leu-[3H]Phe binding activity.

Amino Acid Chloromethyl Ketones↗
PubMed1983

Release of phospholipase A2 activity from rabbit peritoneal neutrophils by f-Met-Leu-Phe.

Rabbit peritoneal neutrophils secrete phospholipase A2 activity in response to f-Met-Leu-Phe (FMLP). The secretion of phospholipase A2, like that of the known granule enzymes, requires cytochalasin B and is enhanced by extracellular Ca2+, and the time course of the release is rapid, being completed in less than a minute. The concentration dependence of the secretion of the phospholipase is the same as that of the known granule enzymes up to 10(-9) M FMLP. At this concentration, the release of the known granule enzymes reaches a maximum, but that of phospholipase A2 does not until 10(-8) M FMLP. The amount of enzyme activity released plus the amount of enzyme activity remaining in the pellet is distinctly greater than the total enzyme activity extractable from the cell before release. The phospholipase secreted is active in the neutral to alkaline range, requires Ca2+, is inhibited by low levels of detergent, and is of A2 specificity.

Animals↗
PubMed1983

Cyclic AMP receptor protein and cyclic AMP-dependent protein kinase activity in rabbit peritoneal neutrophils.

The cAMP receptor protein and cAMP-dependent protein kinase activity in rabbit peritoneal neutrophils have been identified. The cAMP receptor protein in either the plasma membrane or cytosol fractions, identified by photoaffinity labeling with 8-N3-[32P]cAMP, has an apparent molecular weight of 54,000. The cytosol and membrane receptor proteins have apparent dissociation constants for 8-N3-[32P]cAMP of 0.20 microM and 0.06 microM, respectively. The molecular weight and dissociation constant for 8-N3-[32P]cAMP of this cAMP receptor protein are similar to what has been known for RII, the regulatory subunit of the type II cAMP-dependent protein kinase. Unlike the human neutrophils, no evidence of RI activity was detected. cAMP-dependent protein kinase activity was identified by using histone as a substrate. Subcellular fractionation studies showed that the cAMP receptor protein and the cAMP-dependent protein kinase activity are most enriched in the cytosol fraction.

Animals↗
PubMed1982

Specificity of the effect of lipoxygenase metabolites of arachidonic acid on calcium homeostasis in neutrophils. Correlation with functional activity.

The ability of the major neutrophil-derived lipoxygenase metabolites of arachidonic acid to increase the rate of 45Ca influx in rabbit neutrophils was examined. The results obtained demonstrate that (5S),(12R)-dihydroxy-6,8,11,14-(cis,trans,trans,cis)-eicosatetraenoic acid (leukotriene B4) is the most active of the arachidonic acid metabolites. The activity of leukotriene B4 is highly stereospecific in that its three nonenzymatically derived isomers are essentially inactive. The omega-hydroxylation of leukotriene B4 results in a compound that is nearly as active as leukotriene B4 as far as its ability to stimulate calcium influx and neutrophil aggregation while being a much weaker secretagogue. The further conversion of leukotriene B4 into a dicarboxylic acid removes all detectable biological activity. 5,6-Oxido-7,9,11,14-eicosatetraenoic acid (leukotriene A4) methyl ester was also found to increase the rate of calcium influx, while the degradation products of native leukotriene A4 were essentially inactive. These results demonstrate that a close correlation exists between the ability of the various lipoxygenase products to alter calcium homeostasis in rabbit neutrophils and their biological activities.

Animals↗
PubMed1982

Characterization of the secretory activity of leukotriene B4 toward rabbit neutrophils.

We have described in det ail the secretory activity of leukotriene B4 toward rabbit neutrophils. Leukotriene B4 rapidly and vigorously degranulates rabbit neutrophils. This activity is stereospecific, cytochalasin B-dependent, and is enhanced by extracellular calcium. Pretreatment with leukotriene B4 deactivates rabbit neutrophils, i.e., cells so treated do not respond to stimulation by an additional bolus of leukotriene B4. In addition, the secretory activity of leukotriene B4 is sharply dependent on the simultaneous presence of cytochalasin B. Rabbit neutrophils therefore exhibit the previously described desensitization to the effect of cytochalasin B. In these and other discussed respects the characteristics of the leukotriene B4-induced degranulation of rabbit neutrophils are strikingly similar to those of the chemotactic factors. These results support the hypothesis that leukotriene B4 mediates, at least in part, the secretory, and possibly other, activities of chemotactic factors.

Animals↗
PubMed1982

The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils. I. Evidence for two binding sites with different affinities.

F Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe binding to rabbit peritoneal neutrophils and purified membranes were measured at 4 degrees C silicone oil centrifugation assays, and the results were analyzed by the LIGAND computer program, which permits analysis of ligand binding to multiple classes of binding sites. LIGAND analysis of peptide binding to intact neutrophil indicated that both f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe detected two population of binding sites. The apparent Kd values for f Met-Leu-[3H]Phe binding were 1.6 +/- 1.0 X 10(-9) M and 2.2 +/- 0.9 X 10(-8) M, respectively, and 3.1 +/- 0.2 X 10(-9) M and 1.2 +/- 0.6 X 10(-7) M for f Nle-Leu-[3H]Phe. Furthermore, the higher affinity sites detected on whole cells comprised approximately 15 to 30% of the total sites. Two populations of binding sites were also detected on purified neutrophil plasma membranes by both radiolabeled chemotactic peptides. LIGAND analysis of peptide binding to purified membranes yielded apparent Kd values of 5.0 +/- 2.5 X 10(-10) M and 4.8 +/- 0.6 X 10(-8) M for f Met-Leu-[3H]Phe binding, and 4.7 +/- 4.2 X 10(-10) M and 3.0 +/- 1.3 X 10(-8) M for f Nle-Leu-[3H]Phe. The percentage of higher affinity sites detected by f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe on purified membranes were 1 to 5% of the total sites detected. These data are consistent either with the existence of two independent binding sites for formylpeptides on rabbit neutrophils or receptor negative cooperativity.

Animals↗
PubMed1982

Membrane activity and topography of F-Met-Leu-Phe-Treated polymorphonuclear leukocytes. Acute and sustained responses to chemotactic peptide.

The chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe) causes a dramatic stimulation of membrane ruffling and a fluid pinocytosis in polymorphonuclear leukocytes (PMNs). These responses are maximal by 1 minute and subside within 5-10 minutes. The same immediate responses characterize cells exposed to several peptide hormones and may thus represent an essential component of target cell activation by peptides. The stimulation of the whole membrane following f-Met-Leu-Phe binding is succeeded by the development of a polarized cell shape with a posterior uropod and a broad anterior lamellipodium, both subtended by microfilaments. Membrane components and functions segregate into distinct domains on polarized PMNs. Thus, succinyl concanavalin A-receptor complexes are capped and internalized by receptor-mediated endocytosis at the uropod; the uptake by fluid pinocytosis of fluorescein-dextran is restricted to the uropod; and coated pits and coated vesicles are concentrated at the uropod. The lamellipodium excludes coated pits and lacks pinocytic activity but shows preferential binding of immunoglobulin aggregates, presumably to Fc receptors. The origin and physiologic implications of these asymmetries of membrane molecular and functional topography on polarized cells are discussed.

Adsorption↗
PubMed1982

Anti-f Met-Leu-Phe: similarities in fine specificity with the formyl peptide chemotaxis receptor of the neutrophil.

We have prepared antisera in both rabbits and rats against f Met-Leu-Phe conjugated to a variety of carrier proteins. Over 40 peptides with widely varying reactivity for the neutrophil formylpeptide receptor have been tested for their ability to bind to rabbit antibody raised against fMLP10-BSA. Structure-activity studies of peptides structurally related to f Met-Leu-Phe demonstrate that the N-formyl group is mandatory for maximum antibody binding activity. Methionine in position 1 and phenylalanine in position 3 are found to confer maximum binding activity. Stereoselectivity of the antibody-combining site also has been demonstrated. Comparison of the ability of the peptides to bind to the antibody receptor with their reactivity for the neutrophil has demonstrated a strong correlation in the rank order of reactivity of the numerous synthetic peptides: for the alpha NH2-acyl group, r = 0.94; for position 1, r = 0.90; for position 2, r = 0.97; and for position 3, r = 0.78. This strong correlation is seen across species lines with both rabbit and rat antibodies. Significant differences, however, in the specificity of the antibody and neutrophil receptors are seen at the carboxyl terminus of phenylalanine, and beyond the phenylalanine ring, r = 0.29. In addition, bacterial chemotactic factor-enriched butanol extracts from Escherichia coli culture filtrates can also bind to anti-f Met-Leu-Phe, affording additional evidence for the similarity in the structure of the bacterial chemotactic factor to the synthetic chemotactic peptides.

Animals↗