Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The extracellular adenylate cyclase of Bordetella pertussis was partially purified and found to contain high- and low-molecular-weight species. The high-molecular-weight form had a variable molecular weight with a peak at about 700,000. The smaller species had a molecular weight of 60 to 70,000 as determined by gel filtration. The low-molecular-weight form could be derived from the high-molecular-weight species. The high-molecular-weight complex purified from the cellular supernatant was highly stimulated by calmodulin, while the low-molecular-weight enzyme was much less stimulated. Active enzyme could be recovered from sodium dodecyl sulfate (SDS) gels at positions corresponding to molecular weights of about 50,000 and 65,000. Active low-molecular-weight enzyme recovered from SDS gels migrated with a molecular weight of about 50,000, which coincides with a coomassie blue-stained band. However, when both high- and low-molecular weight preparations were analyzed in 8 M urea isoelectrofocusing gels, the enzyme activity recovered did not comigrate with stained protein bands. The enzyme recovered from denaturing isoelectrofocusing or SDS gels was activated by calmodulin, indicating a direct interaction of calmodulin and enzyme. The high-molecular-weight form of the enzyme showed increasing activity with calmodulin concentrations ranging from 0.1 to 500 nM, while the low-molecular-weight form was fully activated by calmodulin at 20 nM. Adenylate cyclase on the surface of living cells was activated by calmodulin in a manner which resembled that found for the high-molecular-weight form.
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PMID:Secreted adenylate cyclase of Bordetella pertussis: calmodulin requirements and partial purification of two forms. 287 55

Calmodulin-activated, adenylate cyclase toxin, a virulence factor produced by the human respiratory pathogen Bordetella pertussis, elicits marked accumulation of cyclic AMP in cell lines from rat pituitary tumors. This effect is associated with and apparently responsible for an enhanced release of prolactin and/or growth hormone from GH3, GH4C1 and 235-1 cells. The utility of this novel toxin in probing cyclic AMP-mediated responses is supported by these observations and studies with pertussis and cholera toxins.
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PMID:Bacterial adenylate cyclase increases cyclic AMP and hormone release in pituitary tumor cells. 287 35

A simple method for the simultaneous assay of both substrate utilization and product formation by Bordetella pertussis adenylate cyclase has been developed. This method involves measurement of ATP remaining in the reaction mixture and cyclic 3',5'-AMP (cAMP) formation by 31p-NMR spectroscopy. No separation of the nucleotides is required. The measurement of the rate of cAMP formation compared very well with other methods that require separation of product from the substrate. With this method it has been possible to show calmodulin activation of B. pertussis adenylate cyclase and to demonstrate an inhibition of calmodulin activation by melittin. The inhibition of calmodulin-activated adenylate cyclase by melittin is not permanent and can be overcome by long-term incubation.
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PMID:A simple method for the assay of Bordetella pertussis adenylate cyclase employing 31P nuclear magnetic resonance spectroscopy. 287 63

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.
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PMID:Bordetella pertussis adenylate cyclase. Purification, characterization, and radioimmunoassay. 287 86

Purified preparations of adenylate cyclase were obtained from crude urea extracts of Bordetella pertussis by a one-step calmodulin affinity chromatography technique. Diluted extract was loaded onto the column and washed, and adenylate cyclase was eluted with 10mM EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. A 104-fold purification was accomplished in one step. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the affinity-purified adenylate cyclase was dissociated into one major protein band with an apparent molecular weight of 60,000 and a minor band at 200,000. The affinity-purified adenylate cyclase was observed to have adenylate cyclase enzymatic activity which was activated by calmodulin, to bind 125I-calmodulin, and to be free of pertussis toxin as determined by in vivo and in vitro assays.
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PMID:Bordetella pertussis adenylate cyclase: isolation and purification by calmodulin-sepharose 4B chromatography. 287 83

Highly purified tryptic fragments of calmodulin were tested for their ability to stimulate adenylate cyclase activity of Bordetella pertussis spheroplast membranes and were compared to their activities on brain Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase. The C-terminal fragment, consisting of residues 78-148, was a full agonist for the cyclase with 0.1-0.15 the potency of calmodulin but did not stimulate phosphodiesterase. Fragments 1-77, 1-90, and 107-148 stimulated adenylate cyclase (and not phosphodiesterase) at low potency; this was not due to calmodulin contamination, but contamination by fragment 78-148 could not be excluded with certainty. An adduct of norchlorpromazine isothiocyanate and calmodulin showed full agonist activity for adenylate cyclase at 0.01-0.02 the potency of calmodulin. Stimulation of adenylate cyclase by a number of the fragments occurred in the absence of Ca2+, but stimulator potency was enhanced 20-60-fold in its presence. The similarity of Ca2+ requirements of fragment 78-148 and calmodulin suggests that occupancy of the two C-terminal Ca2+ binding sites of calmodulin accounts for most of the Ca2+ enhancement of calmodulin stimulation of adenylate cyclase.
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PMID:Activation of Bordetella pertussis adenylate cyclase by the carboxyl-terminal tryptic fragment of calmodulin. 287 60

Bordetella pertussis, the pathogen responsible for whooping cough, releases a soluble calmodulin-sensitive adenylate cyclase into its culture medium which enters several different types of animal cells and elevates intracellular cAMP. In this study, the influence of B. pertussis adenylate cyclase on intracellular cAMP levels of cultured chick cardiac cells and the beating rates of chick cardiac cell aggregates was examined. Treatment with B. pertussis adenylate cyclase caused up to a 60-fold increase in intracellular cAMP which was significantly greater than that caused by forskolin or isoproterenol. Increases in intracellular cAMP caused by B. pertussis adenylate cyclase were observed within 2 min after treating cells with the enzyme, and binding of calmodulin to the enzyme inhibited these effects. In addition, high concentrations of the enzyme completely inhibited the beating of cardiac cells. However, lower concentrations of the adenylate cyclase accelerated beating rates 30-40% and cardiac cells continued to beat at an accelerated rate for at least 30 min. These data indicate that B. pertussis adenylate cyclase invades chick cardiac cells and catalyzes significant increases in intracellular cAMP. It is proposed that the effect of the enzyme on the beating rates of heart cell aggregates may be due to alteration of intracellular cAMP levels.
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PMID:Alteration of intracellular cAMP levels and beating rates of cultured chick cardiac cells by Bordetella pertussis adenylate cyclase. 288 70

Bordetella pertussis, the causative organism of whooping cough, produces a calmodulin-sensitive adenylate cyclase. Confer & Eaton [(1982) Science 217, 948-950] have shown that an extract from B. pertussis increases intracellular cyclic AMP levels in neutrophils and suggested that this increase is caused by the bacterial adenylate cyclase which penetrates these cells. We demonstrate in the present study that adenylate cyclase activity in lysates from lymphocytes exposed to a partially purified preparation of the bacterial enzyme has properties completely different from those of the intrinsic membrane-bound enzyme. Adenylate cyclase activity in lysates from lymphocytes exposed to the invasive enzyme is insensitive to N-ethylmaleimide, readily inactivated by acetic anhydride and relatively stable to SDS. Similar properties are exhibited by the bacterial enzyme itself. By contrast, the intrinsic membrane-bound enzyme activated by forskolin and guanosine 5'-gamma-thiotriphosphate is sensitive to N-ethylmaleimide and SDS and relatively stable to acetic anhydride. This strongly supports the notion that B. pertussis adenylate cyclase penetrates cells. Using the partially purified preparation of the invasive enzyme, we have studied the kinetics of its penetration. The intracellular catalytic activity reaches a steady state within 20 min, irrespective of enzyme or cell concentration. Steady-state levels are maintained for at least 2 h provided that the invasive enzyme is present in the incubation medium. Upon its removal, a rapid decrease (t1/2 approximately equal to 15 min) in the intracellular cyclase level is observed. This decrease reflects intracellular inactivation of the bacterial enzyme and is not caused by the release of the enzyme to the cell medium.
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PMID:The invasive adenylate cyclase of Bordetella pertussis. Properties and penetration kinetics. 288 19

The structural organization of Bordetella pertussis adenylate cyclase was examined by limited proteolysis with trypsin and/or cross-linking with azido-calmodulin a photoactivable derivative of its activator, calmodulin (CaM). Adenylate cyclase (which consists of three structurally related peptides of 50, 45, and 43 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) formed a 1:1 complex with CaM or azido-CaM. CaM-bound adenylate cyclase was cleaved by trypsin into two separate trypsin-resistant fragments of 25 and 18 kDa which both interacted with CaM as judged by their ability to be cross-linked with azido-CaM. These two fragments remained associated with CaM in a catalytically active conformation resembling that of the undigested complex. When proteolysis was carried out in the absence of CaM, the adenylate cyclase was completely inactivated in less than 3 min. Sodium dodecyl sulfate-polyacrylamide gel revealed a single 24-kDa trypsin-resistant fragment. Since this fragment cannot be cross-linked with azido-CaM we suggest that the CaM-binding site on the 25-kDa moiety of the adenylate cyclase is located on a short segment of 1 kDa.
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PMID:Interaction of Bordetella pertussis adenylate cyclase with calmodulin. Identification of two separated calmodulin-binding domains. 289 92

An engineered calmodulin differs from vertebrate calmodulin in its ability to activate Bordetella pertussis adenylate cyclase, and this difference has been utilized as the basis for a new purification protocol for the adenylate cyclase. VU-8 calmodulin, in which 3 glutamic acid residues (residues 82-84) have been substituted with 3 lysine residues, has a 1000-fold lower apparent affinity for the adenylate cyclase, compared to vertebrate calmodulin, and decreased maximal activity. Because of the relatively calcium-independent nature of the interaction between calmodulin and the cyclase, the use of calmodulin-Sepharose conjugates in the purification of the cyclase requires the use of chaotropic agents for elution. However, when immobilized VU-8 calmodulin was tested as a calcium-dependent, affinity-based, adsorption chromatography step in the purification of the cyclase from culture media or bacterial extracts, the enzyme bound to the column in a calcium-dependent manner, and a nearly homogeneous enzyme was obtained in high yield. These results demonstrate the feasibility of using engineered calmodulins that have selective differences in activity for the rational design of rapid purification protocols for calmodulin-binding proteins as well as indicate the importance of the conserved negative charge cluster at residues 82-84 of calmodulin for activation of this cyclase.
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PMID:Affinity-based chromatography utilizing genetically engineered proteins. Interaction of Bordetella pertussis adenylate cyclase with calmodulin. 289 77


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