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D R Storm

Publications and source records attributed to D R Storm.

At least 127 records · Page 7Linked to original sources
PubMed1985

Calmodulin inhibits entry of Bordetella pertussis adenylate cyclase into animal cells.

Bordetella pertussis, the pathogen responsible for whooping cough, releases a soluble calmodulin-sensitive adenylate cyclase into its culture medium. Recently, Confer and Eaton [Confer, D., & Eaton, J. (1982) Science (Washington, D.C.) 217, 948-950], as well as Hanski and Farfel [Hanski, E., & Farfel, Z. (1985) J. Biol. Chem. 290, 5526-5536], have shown that crude extracts from B. pertussis containing adenylate cyclase activity cause elevations in intracellular cAMP when incubated with human neutrophils or lymphocytes. These investigators proposed that the bacterial enzyme enters animal cells and catalyzes the formation of cAMP from intracellular ATP. In this study, B. pertussis adenylate cyclase was purified to remove contaminating islet activating protein and examined for its effects on intracellular cAMP levels of human erythrocytes and N1E-115 mouse neuroblastoma cells. In both cases, the enzyme catalyzed the formation of intracellular cAMP. Addition of calmodulin to the adenylate cyclase preparations completely inhibited formation of intracellular cAMP catalyzed by the bacterial enzyme, indicating that cAMP was not synthesized extracellularly and then taken up by the cells. These experiments illustrate that the bacterial enzyme does enter animal cells and that the enzyme-calmodulin complex does not.

Adenylyl Cyclases↗
PubMed1985

Purification and characterization of a calmodulin-sensitive adenylate cyclase from Bordetella pertussis.

Bordetella pertussis, the bacterium responsible for whooping cough, releases a soluble, calmodulin-sensitive adenylate cyclase into its culture medium. B. pertussis mutants deficient in this enzyme are avirulent, indicating that the adenylate cyclase contributes to the pathogenesis of the disease. It has been proposed that B. pertussis adenylate cyclase may enter animal cells and increase intracellular adenosine cyclic 3',5'-phosphate (cAMP) levels. We have purified the enzyme extensively from culture medium using anion-exchange chromatography in the presence and absence of calmodulin and gel filtration chromatography. The enzyme was purified 1600-fold to a specific activity of 608 mumol of cAMP min-1 mg-1 and was free of islet activating protein. The molecular weight of the enzyme was 43 400 in the absence of calmodulin and 54 200 in the presence of calmodulin. The Km of the bacterial enzyme for adenosine 5'-triphosphate was 2.0 mM, whereas the Km of the calmodulin-sensitive adenylate cyclase from bovine brain was 0.07 mM. Although the enzyme was not purified to homogeneity, its turnover number of 27 000 min-1 is the highest documented for any adenylate cyclase preparation.

Adenylyl Cyclases↗
PubMed1985

P-57 is a neural specific calmodulin-binding protein.

P-57 is a novel calmodulin-binding protein which has recently been isolated from bovine cerebral cortex (Andreasen, T. J., Luetje, C. W., Heideman, W., and Storm, D. R. (1983) Biochemistry 22, 4615-4618). In contrast to all other calmodulin-binding proteins characterized thus far, P-57 has equivalent or higher affinity for calmodulin in the absence of free Ca2+ compared to the presence of Ca2+. In this study, the distribution of P-57 in other tissues and within brain was examined using a radioimmune assay and photoaffinity labeling with azido-125I-calmodulin. P-57 was not found in tissues other than brain, retina, and spinal cord. Within brain, P-57 levels varied from 0.1% of the total protein in white matter regions to about 0.5% in cell body-rich fractions. The protein was found in both membrane and soluble fractions. P-57 is the most abundant calmodulin-binding protein in brain and appears to be neural specific. The concentrations of P-57 in brain and its affinity for calmodulin in the absence of Ca2+ are sufficient to complex a significant fraction of the total calmodulin present.

Animals↗
PubMed1985

Purification of the calmodulin-sensitive adenylate cyclase from bovine cerebral cortex.

A calmodulin-sensitive adenylate cyclase was purified 3000-fold from bovine cerebral cortex using DEAE-Sephacel, calmodulin-Sepharose, and two heptanediamine-Sepharose column steps. The purified enzyme activity was stimulated by calmodulin, forskolin, 5'-guanylyl imidodiphosphate, and NaF. The molecular weight of the protein component was estimated as 328 000 with a smaller form of Mr 153 000 obtained in the presence of Mn2+. The most highly purified preparations contained major polypeptides of 150 000, 47 000, and 35 000 daltons on sodium dodecyl sulfate (SDS) gels. Photoaffinity labeling of the preparation with azido[125I]iodocalmodulin gave one product of 170 000 daltons on SDS gels. It is proposed that the catalytic subunit of the calmodulin-sensitive enzyme is 150 000 +/- 10 000 daltons and that the enzyme exists as a complex of one catalytic subunit and the stimulatory guanyl nucleotide regulatory complex. These data are consistent with the previous report that the catalytic subunit of this enzyme has a molecular weight of 150 000 +/- 10 000 [Andreasen, T.J., Heideman, W., Rosenberg, G.B., & Storm, D.R. (1983) Biochemistry 22,2757].

Adenylyl Cyclases↗
PubMed1985

Reconstitution of calmodulin-sensitive adenylate cyclase from bovine brain with phosphatidylcholine liposomes.

A partially purified calmodulin (CaM)-sensitive adenylate cyclase from bovine cerebral cortex was reconstituted with a series of phosphatidylcholine liposomes having variable fatty acid composition. The enzyme was successfully associated with dimyristoyl, dipalmitoyl, distearoyl, and dioleoylphosphatidylcholine liposomes. The specific activity of the enzyme in the various liposomes varied over a 4.6-fold range indicating some degree of specificity for fatty acid composition. The adenylate cyclase-liposome preparation retained sensitivity to both CaM and 5'-guanylylimidodiphosphate (GppNHp). Arrhenius plots of enzyme activity in the four different liposome preparations all exhibited a pronounced discontinuity at 30 degrees C +/- 2, even though the bulk-phase thermal transition points for the liposomes varied from -20 to 54 degrees C. Fluorescence anisotropy studies of reconstituted liposome systems illustrated that incorporation of protein did not alter the normal-phase transition point of these lipids. Since Arrhenius plots of the enzyme in Lubrol PX, prior to reconstitution with lipids, were strictly linear, it is concluded that the breaks at 30 degrees C may be the effect of a local enzyme-phospholipid environment. It appears that this adenylate cyclase is not particularly sensitive to phase transitions of the bulk lipid phase. The phospholipid reconstituted enzyme system appears suitable for examination of the influence of lipids on the CaM-sensitive adenylate cyclase.

Adenylyl Cyclases↗
PubMed1984

Quantitation of energy coupling between Ca2+, calmodulin, skeletal muscle myosin light chain kinase, and kinase substrates.

Interactions between Ca2+, calmodulin, rabbit skeletal muscle myosin light chain kinase, and kinase substrates were investigated by microequilibrium dialysis using 45CaCl2 and by fluorescence anisotropy using fluorescent labeled calmodulin. We have determined the free energy coupling for the interaction of Ca2+ and skeletal muscle myosin light chain kinase with calmodulin (delta GCM), and the free energy coupling for the interaction of calmodulin and enzyme substrates with myosin light chain kinase (delta GSCaM). The mean dissociation constants for Ca2+ interaction with calmodulin in the presence and absence of enzyme were 0.40 and 14 microM, respectively, yielding a minimal estimate for delta GCM of -2.11 kcal/mol of Ca2+ (-8.44 kcal/4 mol of Ca2+). Dissociation constants for the interaction of fluorescent 5-iodo-amino-ethyl-amino-naphthalene-1-sulfonic acid-labeled calmodulin and native calmodulin with skeletal muscle myosin light 100 and 15 nM, respectively. In the presence of substrates (phosphorylatable skeletal muscle myosin light chain and App(NH)p) these dissociation constants were 30 and 3 nM, respectively. The free energy coupling for binding of calmodulin and substrates to myosin light chain kinase was -0.95 kcal/mol of calmodulin. These data confirm that interaction of Ca2+ with calmodulin is highly cooperative in the presence of myosin light chain kinase with the degree of cooperativity dependent upon the concentrations of Ca2+, enzyme, and substrates.

Animals↗
PubMed1983

Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes.

A new calmodulin (CaM) binding protein, designated P-57, has been purified to apparent homogeneity from bovine cerebral cortex membranes. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin in the absence of bound Ca2+ than in its presence. The protein was purified by DEAE-Sephacel chromatography and two CaM-Sepharose affinity column steps. The first CaM-Sepharose column was run in the presence of Ca2+; the second was run in the presence of chelator in excess of Ca2+. P-57 was adsorbed by CaM-Sepharose only in the absence of bound Ca2+ and was eluted from the second column by buffers containing Ca2+. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the purified protein showed only one band at Mr 57 000. The major form of the protein on Bio-Gel A-1.5m and native polyacrylamide gradient gel electrophoresis ran with an apparent Stokes radius of 41 A. Photoaffinity labeling of P-57 with azido[125I]calmodulin yielded one cross-linked product on SDS gels with an Mr of 70 000. This interaction occurred only when excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was present and was inhibited by the presence of Ca2+ in excess of chelator. It appears that P-57 has novel binding properties for calmodulin distinct from all other calmodulin binding proteins described thus far.

Animals↗
PubMed1983

Prostaglandin E-induced heterologous desensitization of hepatic adenylate cyclase. Consequences on the guanyl nucleotide regulatory complex.

Prostaglandin E (PGE) receptor density in hepatic plasma membranes can be down-regulated by in vivo exposure to the 16,16-dimethyl analog of PGE2, and this is associated with desensitization of PGE-sensitive adenylate cyclase. These studies examined adenylate cyclase response to other agonists in membranes whose PGE receptor density was 51% decreased and whose maximal PGE-stimulated adenylate cyclase activity was 31% decreased. Down-regulated membranes had a 37% decrease in their maximal response to glucagon, indicating that treatment with the PGE analog had induced both homologous and heterologous desensitization. To determine whether adenylate cyclase had been affected, stimulation with NaF, guanyl 5'-yl imidodiphosphate (GppNHp), and forskolin was examined in both intact and solubilized membranes. Intact membranes had decreased adenylate cyclase responses to all three stimulators (NaF, -41%; GppNHp, -25%; forskolin, -41%) as did solubilized membranes (NaF, -51%; GppNHp, -50%; forskolin, -50%), suggesting alterations in adenylate cyclase rather than indirect membrane effects. Cholera toxin activation and labeling were examined to more directly assess whether the guanine nucleotide (G/F) regulatory component of adenylate cyclase had been affected. Cholera toxin activation was 42% less in down-regulated membranes, and these membranes incorporated less label when the incubation was performed in the presence of [32]NAD. Solubilized G/F subunit activity from down-regulated membranes was less effective in reconstitution of adenylate cyclase activity from cyc- cell membranes than G/F activity from control membranes. These data indicate that in vivo exposure to the PGE analog causes both homologous and heterologous desensitization of adenylate cyclase as well as an apparent quantitative decrease in G/F.

Adenylyl Cyclases↗
PubMed1983

Photoaffinity labeling of brain adenylate cyclase preparations with azido[125 I]iodocalmodulin.

A partially purified calmodulin-sensitive adenylate cyclase from bovine cerebral cortex was photoaffinity labeled with azido[125 I]iodocalmodulin. Sodium dodecyl sulfate gel electrophoresis followed by autoradiography revealed several cross-linked polypeptides ranging in molecular weight from 37000 to 200000. The calmodulin-sensitive enzyme was submitted to a number of purification steps to determine if any of the calmodulin binding polypeptides copurified with adenylate cyclase activity. Fractionation procedures used included Bio-Gel A5M and Ultragel AcA 34 gel chromatography, isoelectric focusing, and native gradient gel electrophoresis. One cross-linked peptide having a molecular weight of 170000 correlated with adenylate cyclase activity through all purification steps. Native gradient gel electrophoresis in the presence of 0.03% deoxycholate gave one peak of adenylate cyclase activity with a Stokes radius of 40 A, consistent with a molecular weight of 140000-150000. It is proposed that the molecular weight of the adenylate cyclase catalytic subunit is 150,000 and that each catalytic subunit interacts with one calmodulin.

Adenylyl Cyclases↗
PubMed1983

Interrelationships between PGE1 and PGI2 binding and stimulation of adenylate cyclase.

The effects of prostaglandin E1 (PGE1) and prostacyclin (PGI2) on hepatic adenylate cyclase were studied in plasma membranes isolated from Sprague-Dawley rat livers. Both PGE1 and PGI2 stimulated this enzyme complex to the same maximal levels and with approximately the same EC50 (10(-7) M). Maximally stimulating concentrations of PGE1 and PGI2 were examined alone and together; their effects were not additive, indicating that the same enzyme complex was shared. Although a receptor for PGE1 could be demonstrated with a dissociation constant of 1 X 10(-8) M, PGI2 was only 1/100 as effective in competing for PGE1 binding sites (KD, 1 X 10(-6) M), indicating that these two prostaglandins may act via separate membrane receptors. PGI2 is known to be unstable at neutral pH; however, we have determined its half-life during these assays by a sensitive bioassay and concluded that the degradation of PGI2 is not sufficient to account for its inability to dissociate [3H]PGE1 binding. Further evidence that PGI2 might act through a distinct receptor was found in animals whose PGE1 receptors were 40% downregulated with a corresponding 28% decrease in PGE1-sensitive adenylate cyclase activity. These membranes had no such decrease in PGI2-sensitive adenylate cyclase activity. We conclude that 1) hepatic adenylate cyclase is equally sensitive to PGE1 and PGI2; 2) the same adenylate cyclase complex responds to both prostaglandins; and 3) PGE1 and PGI2 interact with separate membrane receptors in rat liver.

Adenylyl Cyclases↗
PubMed1982

Interaction of a fluorescent N-dansylaziridine derivative of troponin I with calmodulin in the absence and presence of calcium.

Rabbit skeletal muscle troponin I was covalently labeled with N-dansylaziridine, resulting in a fluorescent labeled protein. This derivative (DANZTnI) and native troponin I (TnI) inhibited calmodulin (CaM) stimulation of bovine heart Ca2+-sensitive cyclic nucleodite phosphodiesterase with identical inhibition constants. Association of DANZTnI with calmodulin was monitored directly by changes in flourescence intensity in the presence of Ca2+ and by changes in fluorescence anisotropy in the absence of Ca2+. Quantitation of the affinity of calmodulin for calmodulin-binding proteins in both the presence and absence of Ca2+ is necessary for prediction of the extent of interaction of both Ca2+ and calmodulin-binding proteins with calmodulin in vivo. The dissociation constants for the DANZTnI-calmodulin-l4Ca2+ and DANZTnI-calmodulin complexes were 20 nM and 70 micrometers, respectively. These dissociation constants define a free energy coupling of-4.84 kcal/mol of troponin I for binding of Ca2+ and troponin I to calmodulin. The Ca2+ dependence for troponin I-calmodulin complex formation predicted from these experimentally determined parameters was closely approximated by the Ca2+ dependence for complex formation between troponin I and fluorescent 5-[[[(iodoacetyl)amino]ethyl]-amino]-1-napthalenesulfonic acid derivatized calmodulin as determined by fluorescence anisotropy. Complex formation occurred over a relatively narrow range of Ca2+ concentration, indicative of positive heterotropic cooperativity for Ca2+ and troponin I binding to calmodulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Calcium-independent stimulation of Bordetella pertussis adenylate cyclase by calmodulin.

Bordetella pertussis produces an extracellular adenylate cyclase activity that is present in the culture medium of exponentially growing cells. We have determined that calmodulin (CaM) stimulates this enzyme both in the presence and in the absence of free Ca2+. In the presence of 90 micron Ca2+ half-maximal stimulation of the enzyme occurred at 95 pM calmodulin. Comparable levels of calmodulin stimulation were observed when free Ca2+ levels were minimized by using EGTA-containing buffers. However, the concentration of CaM required for half-maximal stimulation of B. pertussis adenylate cyclase in the presence of 1 nM free Ca2+ was 24 nM. The apparent affinity of the enzyme for calmodulin was also significantly enhanced by Mn2+. In addition, troponin I inhibited calmodulin stimulation of the bacterial adenylate cyclase. Photoaffinity cross-linking experiments using azido[125I]calmodulin and B. pertussis adenylate cyclase revealed only one major cross-linked product having a molecular weight of 97000. It is proposed that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is 77000.

Adenylyl Cyclases↗
PubMed1982

Determination of the free-energy coupling for binding of calcium ions and troponin I to calmodulin.

Regulation of a wide variety of biological systems by Ca2+ is now known to be mediated through calmodulin, a Ca2+-binding protein. Calmodulin forms Ca2+-dependent complexes with several proteins, including troponin I. We have determined the free-energy coupling (delta GoCT) for binding of Ca2+ and troponin I to calmodulin by measuring Ca2+ binding to calmodulin and to the 1:1 calmodulin-troponin I complex by equilibrium dialysis. The dissociation constant for the Ca2+4.calmodulin and troponin I complex was also determined directly by monitoring fluorescence intensity changes accompanying complex formation between Ca2+4.CaM and N-dansylaziridine-troponin I, a fluorescent derivative of troponin I. Calmodulin displayed four Ca2+-binding sites of similar affinity with a geometric mean dissociation constant of 14 microM. In the presence of saturating troponin I, the geometric mean dissociation constant for the four Ca2+-binding sites was shifted to 1.7 microM. delta GoCT was therefore -1.25 kcal/mol of Ca2+. Saturation of calmodulin with Ca2+ would therefore be expected to increase its affinity for troponin I about 4500-fold. A dissociation constant of 20 nM was determined for the Ca2+4.calmodulin.N-dansylaziridine-troponin I complex. The dissociation constant of the calmodulin.N-dansylaziridine-troponin I complex in the absence of Ca2+ should therefore be about 90 microM. A prediction of this data is that binding of Ca2+ to calmodulin will show strong positive cooperativity when Ca2+ binding to a substoichiometric number of sites is sufficient to promote calmodulin.troponin I complex formation.

Calcium↗
PubMed1982

Bacitracin.

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Bacillus↗
PubMed1982

GTP is not required for calmodulin stimulation of bovine brain adenylate cyclase.

The importance of guanyl nucleotides for calmodulin stimulation of bovine cerebral cortex adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was examined by using a partially purified calmodulin-sensitive adenylate cyclase that was resolved from calmodulin-insensitive forms of the enzyme. By using 5'-adenylyl imidodiphosphate as a substrate, in the absence of an ATP-regenerating system, it was determined that GTP is not required for calmodulin stimulation of the enzyme. Maximal activation by 5'-guanylyl imidodiphosphate (p[NH]ppG) was 5.3-fold, whereas the combination of p[NH]ppG and calmodulin stimulated the enzyme 27-fold. Although GDP inhibited p[NH]ppG stimulation of the calmodulin-sensitive adenylate cyclase, it did not affect calmodulin stimulation. In addition, calmodulin did not alter the kinetics for activation of the enzyme by p[NH]ppG. It is concluded that GTP is not required for calmodulin stimulation of brain adenylate cyclase and that calmodulin regulation of this enzyme is probably not due to effects of calmodulin on the affinity of the guanyl nucleotide regulatory complex for guanyl nucleotides.

Adenylyl Cyclases↗
PubMed1981

Stereochemistry of the mammalian adenylate cyclase reaction.

Adenosine 5'-O-(1-thiotriphosphate), Sp-diastereomer, is cyclized by adenylate cyclase from bovine brain to adenosine 3',5'-cyclic phosphorothioate, Rp diastereomer, establishing inversion of configuration for this reaction. This result can most easily be explained by a direct nucleophilic attack of the 3'-OH group on alpha-phosphorus without involving a covalent enzyme intermediate.

Adenosine Triphosphate↗