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B J Bormann

Publications and source records attributed to B J Bormann.

24 records · Page 2Linked to original sources
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

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

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↗