The primary sequence of phospholipase‐A from bee venom

Shipolini, Rudolf A.; Callewaert, George L.; Cottrell, Richard C.; Vernon, C. A.

doi:10.1016/0014-5793(71)80558-6

Cited by 57 publications

(8 citation statements)

References 2 publications

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“…But, no reagent which could inactivate both proteins, Mel and PLA 2 was commercially available. The peptide melittin does not have a H residue (Shipolini et al, 1971), which is an important amino acid for the PLA 2 active site. Then, it was necessary to use more than one chemical modifier to inhibit both, the PLA 2 and Mel activities.…”

Section: Tablementioning

confidence: 99%

“…It is important to remember that within BV, all the membrane lytic proteins (PLA 2 and Lysophospholipase) and peptide (Mel) were active, which explains the rhodamine leak-out. The pbb reagent modifies the H present in PLA 2 (Andriao-Escarso et al, 2000;Dempsey, 1990), but absent in Mel (Shipolini et al, 1971); thus, it was to be expected that the A-BV derivative induced rhodamine leak-out ( Figure 7B), meaning that here, the Mel of BV was still active. The residue W is present within all the lytic proteins and peptides and, after NBS oxidation (which promotes Mel inhibition), they could not induce rhodamine leak-out ( Figure 7C).…”

Section: Tablementioning

confidence: 99%

“…Then, it was necessary to use more than one chemical modifier to inhibit both, the PLA 2 and Mel activities. Mel has one W residue at the position 19 (Shipolini et al, 1971). It is known that the extremely reactive protein reagent NBS modifies mainly W residues producing their oxidation (Scheme 1).…”

Section: Tablementioning

confidence: 99%

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Design of a Modern Liposome and Bee Venom Formulation for the Traditional VIT-Venom Immunotherapy

Silva

Moura²,

Ramos

et al. 2008

Journal of Liposome Research

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Traditional venom immunotherapy uses injections of whole bee venom in buffer or adsorbed in Al (OH) 3 in an expensive, time-consuming way. New strategies to improve the safety and efficacy of this treatment with a reduction of injections would, therefore, be of general interest. It would improve patient compliance and provide socio-economic benefits. Liposomes have a long tradition in drug delivery because they increase the therapeutic index and avoid drug degradation and secondary effects. However, bee venom melittin (Mel) and phospholipase (PLA 2 ) destroy the phospholipid membranes. Our central idea was to inhibit the PLA 2 and Mel activities through histidine alkylation and or tryptophan oxidation (with pbb, para-bromo-phenacyl bromide, and/or NBS-N-bromosuccinimide, respectively) to make their encapsulations possible within stabilized liposomes. We strongly believe that this formulation will be nontoxic but immunogenic. In this paper, we present the whole bee venom conformation characterization during and after chemical modification and after interaction with liposome by ultraviolet, circular dichroism, and fluorescence spectroscopies. The PLA 2 and Mel activities were measured indirectly by changes in turbidity at 400 nm , rhodamine leak-out, and hemolysis. The native whole bee venom (BV) presented 78.06% of a-helical content. The alkylation (A-BV) and succynilation (S-BV) of BV increased 0.44 and 0.20% of its a-helical content. The double-modified venom (S-A-BV) had a 0.74% increase of a-helical content. The BV chemical modification induced another change on protein conformations observed by Trp that became buried with respect to the native whole BV. It was demonstrated that the liposomal membranes must contain pbb (SPC:Cho:pbb, 26:7:1) as a component to protect them from aggregation and/or fusion. The membranes containing pbb maintained the same turbidity (100%) after incubation with modified venom, in contrast with pbb-free membranes that showed a 15% size decrease. This size decrease Journal of Liposome Research Downloaded from informahealthcare.com by University of Melbourne on 11/17/14 For personal use only.354 Silva et al. was interpreted as membrane degradation and was corroborated by a 50% rhodamine leak-out. Another fact that confirmed our interpretation was the observed 100% inhibition of the hemolytic activity after venom modification with pbb and NBS (S-A-BV). When S-A-BV interacted with liposomes, other protein conformational changes were observed and characterized by the increase of 1.93% on S-A-BV a-helical content andthe presence of tryptophan residues in a more hydrophobic environment. In other words, the S-A-BV interacted with liposomal membranes, but this interaction was not effective to cause aggregation, leak-out, or fusion. A stable formulation composed by S-A-BV encapsulated within liposomes composed by SPC:Cho:pbb, at a ratio of 26:7:1, was devised. Large unilamellar vesicles of 202.5 nm with a negative surface charge (-24.29 mV) encapsulated 95% of S-A-BV. This formulation ca...

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Section: Tablementioning

confidence: 99%

Section: Tablementioning

confidence: 99%

See 1 more Smart Citation

Design of a Modern Liposome and Bee Venom Formulation for the Traditional VIT-Venom Immunotherapy

Silva

Moura²,

Ramos

et al. 2008

Journal of Liposome Research

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“…It is a heat-stable widely distributed enzyme playing a central role in lipid metabolism and having an important application in several fields of investigation (Dawson, 1973). The isolation and complete primary-structure determination of phospholipases A2 have been accomplished by several laboratories from material extracted from pig (de Haas et al, 1970) and horse (Evenberg et al, 1977) pancreas, from bee venom (Shipolini et al, 1971(Shipolini et al, , 1974 and…”

Section: (Received 23 August 1979)mentioning

confidence: 99%

Venom from the snake Bothrops asper Garman. Purification and characterization of three phospholipases A2

Alagón

Molinar

Possani

et al. 1980

Biochemical Journal

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The water-soluble venom of Bothrops asper Garman (San Juan Evangelista, Veracruz, México) showed 15 polypeptide bands on polyacrylamide-gel electrophoresis. This material exhibited phospholipase, hyaluronidase, N-benzoyl-l-arginine ethyl hydrolase, N-benzoyl-l-tyrosine ethyl hydrolase and phosphodiesterase activity, but no alkaline phosphatase or acid phosphatase activity. Fractionation on Sephadex G-75 afforded seven protein fractions, which were apparently less toxic than the whole venom (LD(50)=4.3mug/g mouse wt.). Subsequent separation of the phospholipase-positive fraction (II) on DEAE-cellulose with potassium phosphate buffers (pH7.55) gave several fractions, two being phospholipase-positive (II.6 and II.8). These fractions were further purified on DEAE-cellulose columns with potassium phosphate buffers (pH8.6). Fraction II.8.4 was rechromatographed in the same DEAE-cellulose column, giving a pure protein designated phospholipase 1. The fraction II.6.3 was further separated by gel disc electrophoresis yielding two more pure proteins designated phospholipase 2 and phospholipase 3. Analysis of phospholipids hydrolysed by these enzymes have shown that all three phospholipases belong to type A(2). Amino acid analysis has shown that phospholipase A(2) (type 1) has 97 residues with a calculated mol.wt. of 10978+/-11. Phospholipase A(2) (type 2) has 96 residues with a mol.wt. of 10959+/-11. Phospholipase A(2) (type 3) has 266 residues with 16 half-cystine residues and a calculated mol.wt of 29042+/-31. Automated Edman degradation showed the N-terminal sequence to be: Asx-Leu-Trp-Glx-Phe-Gly-Glx-Met-Met-Ser-Asx-Val- Met-Arg-Lys-Asx-Val-Val-Phe-Lys-Tyr-Leu- for phospholipase A(2) (type 2).

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“…Best characterized BV allergens are PLA 2 [ 8, 9], hyaluronidase [ 10, 11] and acid phosphatase [ 12] with molecular weights (MW) of 16–20, 41 and 45 kDa, respectively. Currently, complete protein and cDNA sequence data for PLA 2 and hyaluronidase are available [ 9, 11, 13]. The N‐terminal sequence and the sequence of some proteolytic peptides derived from acid phosphatase have also been reported [ 14] and PLA 2 crystal structure has recently been resolved [ 15].…”

Section: Introductionmentioning

confidence: 99%