Calcium binding to porcine pancreatic prophospholipase A<sub>2</sub> studied by <sup>43</sup>Ca NMR

Andersson, Thomas; Drakenberg, Torbjörn; Forsén, Sture; Wieloch, Tadeusz; Lindström, Mats

doi:10.1016/0014-5793(81)80032-4

Cited by 35 publications

(13 citation statements)

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“…The changes in the apparent 43Ca relaxation rate below pH 6.5 are most likely due to the titration of groups responsible for the binding of CaZf. [19,20]. The increase in the 43Ca relaxation rate observed between pH 7 and 10 can be attributed to an increased number of CaZ + binding sites, in agreement with results obtained with a CaZ + selective electrode [I 71, while the steep drop in the relaxation rate at pH 10.5 is likely to be due to dissociation into subunits.…”

Section: Discussionsupporting

confidence: 77%

Characterization of Cation‐Binding Sites on Panulirus interruptus Hemocyanin by ⁴³Ca and ²³Na NMR

Andersson¹,

Chiancone

Forsén³

1982

European Journal of Biochemistry

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The binding of N a + and Ca2+ ions to Panulirus interruptus hemocyanin has been studied by nuclear magnetic resonance. The combined use of 43Ca and 23Na N M R has allowed two different classes of sites to be distinguished. The sites are characterized by a relatively fast chemical exchange, local mobility and binding constants of the order of lo3 -lo4 M-' for Ca2+ and Mg2+ and z 10' M-' for Na+. These features and the observed pH dependence of the Ca2+ binding are consistent with the protein ligands being carboxylate groups, some of which may occur in clusters.Hemocyanins are the copper-containing respiratory proteins of arthropods and molluscs. In the proteins from both phyla the oxygen binding site comprises two copper atoms but the sizes of the polypeptide chains differ. Molluscan hemocyanins are made up of large polypeptide chains ( M , ~2 -3 x lo5) each carrying seven or eight oxygen-binding sites while arthropod hemocyanins are made up of smaller subunits ( M , = 7 -8 x lo4) each carrying one oxygen binding site [I].The effects of calcium, magnesium, sodium and hydrogen ions on the structure and biological function of hemocyanins have been known for many years [I]. Only recently, however, has attention been directed to the analysis of the specific and reciprocal effects arising from the interplay of all these allosteric ligands in the relatively small hemocyanin from the spiny lobster Panulirus interruptus (a hexamer of M , = 4.5 x lo5). This task demands a complete characterization of the different sets of binding sites for each ligand. The present paper focuses on the binding of Ca2+ to Panulirus hemocyanin. This was measured directly by means of 43Ca N M R and indirectly by studying the effect of Ca2+ on the 23Na line width. The combined use of 43Ca and 23Na N M R allowed two different classes of Ca2+ binding sites, differing approximately 10-fold in affinity, to be distinguished. Information on the dynamics of the interaction at the protein binding sites has been obtained. MATERIALS A N D METHODSHemocyanin was isolated and fractionated as described by Kuiper et al. [2] and van der Berg 131. For this study, reassociated fraction 11, which showed the same physicochemical and functional properties as the unfractionated material was used. The lyophilized protein was dissolved in a 0.02 M Tris buffer at pH 7.2 containing 0.08 M NaCl and 5 mM EDTA and dialysed against one change of the buffer without EDTA. The protein was then dialysed against several changes of NaCl solutions at the desired concentration. EDTA. is known to bind strongly to proteins [4] but no residual EDTA was detected after this procedure using 'I3Cd NMR. (Cd2+ is known to bind very strongly to EDTA and produces a signal with a characteristic chemical shift.) The concentration of the hemocyanin solutions was determined spectrophotometrically and is expressed in terms of mol mol subunit-' I-' (subunit M , = 7 . 5~ lo4) 121. The pH of the hemocyanin solution was changed by adding small amounts of 1 M NaOH or 1 M HCI. All experiments were per...

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Section: Discussionsupporting

confidence: 77%

Characterization of Cation‐Binding Sites on Panulirus interruptus Hemocyanin by ⁴³Ca and ²³Na NMR

Andersson¹,

Chiancone

Forsén³

1982

European Journal of Biochemistry

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“…If this could fully describe the functional involvement of Ca 2+ in the catalytic mechanism of PLA2, one might expect that a number of cations could substitute for Ca 2+ in this catalytic action. Andersson et al (1981) noted that Ba 2+, Eu 3+, Tb 3+, and Gd 3+ could bind at the Ca2+-binding site of porcine pancreatic PLA2, but only Gd 3+ supported catalytic activity. Moreover, UV spectra of Crotalus adamanteus PLA2 were perturbed by the binding of Ca 2+ and Zn 2+, but Zn 2+ itself was an inhibitor ion of PLA2 (Well, 1973).…”

Section: Introductionmentioning

confidence: 99%

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2

1996

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In order to address the mechanism whereby Ca2+ wad crucial for the manifestation of the enzymatic activity of phospholipase A2 (PLA2), four divalent cations were used to assess their influences on the catalytic activity and the fine structures of Naja naja atra PLA2. It was found that substitution of Mg2+ or Sr2+ for Ca2+ in the substrate solution caused a decrease in the PLA2 activity to 77.5% or 54.5%, respectively, of that in the presence of Ca2+. However, no PLA2 activity was observed with the addition of Ba2+. With the exception of Mg2+, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA2 markedly increased with the binding of cations to PLA2. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate and p-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca2+, Sr2+, and Ba2+, but not by Mg2+. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba2+ > Sr2+ > Ca2+ > Mg2+, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA2 and that Tyr-3, Lys-6, and Tyr-63 of PLA2 are involved in the binding with the substrate, suggest that the binding of Ca2+ to PLA2 induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg2+ or the bigger atomic radii with Sr2+ and Ba2+ might render the conformation improperly rearranged after their binding to PLA2 molecule.

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“…The finding that modification of bovine pancreas PLA 2 and Agkistrodon halys PLA 2 by p-bromophenacyl bromide led to conformational changes outside the catalytic site [8,9] supports this proposition. Noticeably, Anderson et al [10] noted that Ba 2+ , Eu 3+ , Tb 3+ , and Gd 3+ could bind at the Ca 2+ -binding site of porcine pancreatic PLA 2 , but only Gd 3+ supported catalytic activity. Likewise, fluorescence measurement showed that the structure of N. naja atra PLA 2 is perturbed by the binding of Ca 2+ , Sr 2+ and Ba 2+ , but Sr 2+ and Ba 2+ are inhibitor ions of PLA 2 [11].…”

Section: Introductionmentioning

confidence: 99%

Mutations on N‐terminal region of Taiwan cobra phospholipase A₂ result in structurally distorted effects

Chiou¹,

Lin²,

Chang³

2008

Journal of Peptide Science

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In the present study, three Taiwan cobra PLA(2) variants were prepared by adding an extra N-terminal Met, substituting Asn-1 by Met or deleting the N-terminal heptapeptide. Recombinant PLA(2) mutants were expressed in Escherichia coli (E. coli), and purified to homogeneity by reverse phase HPLC. Fluorescence measurement showed that the hydrophobic character of the catalytic site, the microenvironment of Trp residues and energy transfer from excited Trp to 8-anilinonaphthalene sulfonate (ANS) were affected by N-terminal mutations. An alteration in the structural flexibility of the active site was noted with the mutants lacking the N-terminal heptapeptide or with an extra N-terminal Met added as evidenced by the inability of the two variants to bind with Ba(2+). Moreover, modification of Lys residues and energy transfer within the protein-ANS complex revealed that the Ca(2+)-induced change in the global structure of PLA(2) was different from that in N-terminal variants. Together with the fact that an 'activation network' connects the N-terminus with the active site, our data suggest that mutagenesis on the N-terminal region affects directly the fine structure of the catalytic site, which subsequently transmits its influence in altering the structure outside the active site of PLA(2).

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Calcium binding to porcine pancreatic prophospholipase A₂ studied by ⁴³Ca NMR

Cited by 35 publications

References 11 publications

Characterization of Cation‐Binding Sites on Panulirus interruptus Hemocyanin by ⁴³Ca and ²³Na NMR

Characterization of Cation‐Binding Sites on Panulirus interruptus Hemocyanin by ⁴³Ca and ²³Na NMR

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2

Mutations on N‐terminal region of Taiwan cobra phospholipase A₂ result in structurally distorted effects

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Calcium binding to porcine pancreatic prophospholipase A2 studied by 43Ca NMR

Cited by 35 publications

References 11 publications

Characterization of Cation‐Binding Sites on Panulirus interruptus Hemocyanin by 43Ca and 23Na NMR

Characterization of Cation‐Binding Sites on Panulirus interruptus Hemocyanin by 43Ca and 23Na NMR

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2

Mutations on N‐terminal region of Taiwan cobra phospholipase A2 result in structurally distorted effects

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Calcium binding to porcine pancreatic prophospholipase A₂ studied by ⁴³Ca NMR

Characterization of Cation‐Binding Sites on Panulirus interruptus Hemocyanin by ⁴³Ca and ²³Na NMR

Characterization of Cation‐Binding Sites on Panulirus interruptus Hemocyanin by ⁴³Ca and ²³Na NMR

Mutations on N‐terminal region of Taiwan cobra phospholipase A₂ result in structurally distorted effects