PubMed Health⌕ Search

Biomedical subjects

D Ladant

Publications and source records attributed to D Ladant.

43 records · Page 3Linked to original sources
PubMed1988

Immunological relatedness between Bordetella pertussis and rat brain adenylyl cyclases.

A prokaryotic adenylyl cyclase, secreted by Bordetella pertussis, shares a common functional property with eukaryotic adenylyl cyclases, i.e., regulation by the eukaryotic protein calmodulin. Making use of polyclonal antibodies raised against the bacterial adenylyl cyclase and the rat brain adenylyl cyclase catalytic component, respectively, we showed an immunological cross-reactivity between the two enzymes. Furthermore, B. pertussis adenylyl cyclase was inhibited and immunoprecipitated by the homologous and one of the heterologous immune sera. These results suggest an evolutionary relationship between the B. pertussis enzyme and its eukaryotic counterpart.

Adenylyl Cyclases↗
PubMed1988

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coli.

The adenylate cyclase toxin of the prokaryote Bordetella pertussis is stimulated by the eukaryotic regulatory protein, calmodulin. A general strategy, using the adenylate-cyclase-calmodulin interaction as a tool, has permitted cloning and expression of the toxin in Escherichia coli in the absence of any B. pertussis trans-activating factor. We show that the protein is synthesized in a large precursor form composed of 1706 amino acids. The calmodulin-stimulated catalytic activity resides in the amino-terminal 450 amino acids of the adenylate cyclase. The enzyme expressed in E. coli is recognized in Western blots by antibodies directed against purified B. pertussis adenylate cyclase, and its activity is inhibited by these antibodies.

Adenylate Cyclase Toxin↗
PubMed1988

Bordetella pertussis adenylate cyclase: the gene and the protein.

Using the adenylate cyclase-calmodulin interaction as a tool, the B. pertussis cya gene was cloned in a cya defective E. coli strain harbouring a plasmid which expressed high levels of calmodulin. The determination of the nucleotide sequence of the gene showed that adenylate cyclase is synthesized as a large precursor of 1706 amino acids. The calmodulin-stimulated catalytic activity resides in the amino-terminal 400 amino acids whereas the 1300 amino acid carboxy-terminal part of the precursor is endowed with haemolytic activity. The catalytically active 43 kDa form of adenylate cyclase is organized in two domains: the N-terminal domain of 25 kDa harbors the catalytic site, and the 18 kDa C-terminal domain carries the main calmodulin-binding site. Immunological relatedness established between B. pertussis, B. anthracis and rat brain adenylate cyclases suggests a common evolutionary origin of a central domain of these calmodulin-stimulated enzymes. The secretion of the adenylate cyclase-haemolysin bifunctional protein (cyclolysin) requires the expression of three additional genes, contiguous to the cya gene. These four genes appear to form a single operon. The mechanism of secretion of the bifunctional protein should be similar to that described for E. coli alpha-haemolysin.

Adenylate Cyclase Toxin↗
PubMed1987

Gene isolation by direct in situ cAMP binding.

The regulatory subunit of the cAMP-dependent protein kinase expressed in clones isolated by immunoscreening of a lambda gt11 cDNA library from Dictyostelium discoideum exhibits high affinity for cAMP [Mutzel et al., Proc. Natl. Acad. Sci. USA 84 (1987) 6-10]. Based on this property, we have developed a screening procedure to detect in situ cAMP-binding activity directly on phage plaques transferred to nitrocellulose filters. Highly radioactive cAMP was synthesized using [alpha-32P]ATP at 3000 Ci/mmol as the substrate of purified adenylate cyclase from Bordetella pertussis. Filter replicas of the library plated at 3 X 10(4) pfu/dish, were incubated in the presence of 2 nM [32P]cAMP and then washed thoroughly. Three clones out of 1.2 X 10(5) were detected, all of which coded for the regulatory subunit, as judged by hybridization with a specific DNA probe. The cAMP binding to the purified clones was characterized in situ by displacement with specific analogues. The ability to displace labelled cAMP was in accord with the affinities of the analogues previously reported for the regulatory subunit of the Dictyostelium cAMP-dependent protein kinase. We are able to detect fmol levels of regulatory subunit contained in phage plaques and therefore the method could be used to screen libraries from other organisms for proteins exhibiting high affinities for cyclic nucleotides.

Bacteriophages↗
PubMed1986

Bordetella pertussis adenylate cyclase. Purification, characterization, and radioimmunoassay.

The extracellular adenylate cyclase of Bordetella pertussis was purified either as a free enzyme or as a complex with calmodulin. The purified enzyme has a specific activity of 1600 mumol of cAMP min-1 X mg-1 and exists under two molecular forms of 45 and 43 kDa which are apparently structurally related. Calmodulin increased considerably the resistance of adenylate cyclase to inactivation by trypsin. Although trypsin cleaved the adenylate cyclase-calmodulin complex, the digested fragments remained associated by noncovalent interactions in an active conformation. Specific mouse anti-adenylate cyclase antibodies inhibit adenylate cyclase activity and were used to develop a specific radioimmunoassay that allows detection of as little as 5 ng of adenylate cyclase in culture supernatants.

Adenylyl Cyclases↗
PubMed1986

Characterization of a beta-galactosidase hybrid protein carrying the catalytic domain of Escherichia coli adenylate cyclase.

A hybrid protein of Escherichia coli, exhibiting both adenylate cyclase and beta-galactosidase activities, was purified and characterized. This protein, obtained by genetic engineering, contained the first 556 amino acids of adenylate cyclase connected to the eighth-residue of beta-galactosidase through a pentapeptide Val-Gly-Asp-Pro-Val. The fusion protein was less stable than the native beta-galatosidase. Trypsin cleaved preferentially the adenylate cyclase moiety of the hybrid protein at a ratio of 1/50 (w/w). The kinetic properties of the hybrid protein were comparable, with a few exceptions, to those of native adenylate cyclase and beta-galactosidase. 'Truncated' adenylate cyclase was no longer sensitive to inhibition by excess ATP, which seems to indicate a second nucleotide binding site of wild-type adenylate cyclase. Photoirradiation of the hybrid protein with 8-azidoadenosine 5'-triphosphate inactivated the adenylate cyclase activity, leaving intact the beta-galactosidase activity. A radiolabeled ATP analog was incorporated after photoirradiation into the adenylate cyclase moiety of the fusion protein as shown by limited digestion with trypsin.

Adenosine Triphosphate↗