Anthony J. Lawrence

High resolution of honey bee (Apis mellifera) venom peptides by propionic acid/urea polyacrylamide gel electrophoresis after ethanol precipitation

A new and simple gel electrophoretic method is described which enables the protein and polypeptide components of bee venom to be resolved on a single gel. The electrophoretic method allows octapeptides to be resolved and species as small as decapeptides can be detected at high sensitivity using the Coomassie blue staining method without prior fixation. This has been achieved by replacing acetic acid by propionic acid in acid/urea polyacrylamide gels and by controlling the amount of TEMED catalyst for the polymerisation of high concentration gels in order to obtain a low effective pore size. We demonstrated the value of ethanol precipitation as a rapid and efficient desalting the fractionation technique and propose that it could be used in combination with gel filtration to purify many of the peptides to homogeneity.

Conductimetric assay of phospholipids and phospholipase A

Direct measurement of chemical change during enzyme-catalysed hydrolysis is, in principle, an attractive method for qualitative and quantitative analysis of phosphatidyl phospholipids. Phospholipase type AZ (EC 3.1.1.4) of high activity and broad specificity is available from bee venom, the source used in this work, and from mammalian pancreas. We have developed a rapid conductimetric assay which has a detection level below 10 pg of phospholipid (l-2 pg Pi) and below 10 ng of purified enzyme. The reaction: lecithin -+lysolecithin + fatty acid anion -+ H+ has been followed by titration [l] or by turbidity change in protein-rich solution [2], but neither of these methods has been adapted for routine estimation of phospholipids. The conductimetric method [3,4] requires minimum sample preparation and the apparatus is simple and readily adapted for routine largescale determinations. Bee venom enzyme has highest activity with substrates in true solution [l] , and for naturally occurring phospholipids this is obtained in dilute aqueous solutions of organic solvents; however, the intrinsic activity of the enzyme is decreased by organic solvents and optimum rates are given in 12-2% n-propanol : water mixtures. In addition activity is increased 5-lO-fold by 0.1 mM Ca2+ but abolished by excess EDTA [ l] .

Activation of bee venom phospholipase A2 by fatty acids, aliphatic anhydrides and glutaraldehyde.

The phospholipase from Bee venom, a small cationic protein (mol. wt 14 629, p1 10.5 ? 1.0) of known primary structure [ 1,2] hydrolyses long-chain phosphatidyl phospholipids most rapidly in dilute aqueous solutions of organic solvents (e.g. n-propanol) where it shows’product activation [3] which becomes more pronounced as the solvent concentration is decreased. Activation of lipases is a common phenomenon of possible importance in many regulatory processes [4,5] and the explanations proposed concentrate on possible modification of the substrate by the activator to increase attraction to, or penetration of the surface by the enzyme [6,7]. The Bee venom enzyme is activated by fatty acid anions (e.g. palmitate, oleate) with a minor contribution from lysolecithin, fig. la, and both products are inhibitory although this is masked at low concentrations. The kinetics of the system are deceptively simple, an individual progress curve having a near linear slow phase followed after a brief transition by a fast, activated phase. Whilst the rate in the activated phase is highly dependent on substrate concentration, that in the slow phase is not, showing that activation, which increases V,,, approx. 25fold appears to decrease affinity for the substrate. This result is more readily explained (given in detail in a later paper) by postulating that activators modify the enzyme not the substrate, a conclusion strongly reinforced by the experiments presented here.

Signal response transduction in rabbit neutrophil leucocytes: The effects of exogenous phospholipase A2 suggest two pathways exist

Rabbit neutrophils stimulated by chemotactic peptide (fMLP) or phorbol ester (PMA) respond with a metabolic burst which can be assayed by following luminol-enhanced chemiluminescence. Depending upon the agonist used, exogenous bee-venom phospholipase A2 (PLA2) will enhance or inhibit the response. Neutrophil activation by fMLP is enhanced by PLA2 or by the addition of arachidonic acid, but unaffected by lysophosphatide. The cellular response to PMA is markedly inhibited by PLA2 or by lysophosphatide, though not completely abrogated, but is enhanced by arachidonic acid. The lysophosphatide inhibition overrides the arachidonic acid potentiation of the PMA-induced response. Neither PLA2 nor arachidonic acid alone will activate the cells; it seems that agonist is essential. We interpret these results to mean that at least two signal-response transduction systems are involved in agonist-induced metabolic activation of rabbit neutrophil leucocytes.

The preparation of activated bee venom phospholipase A2.

Many phospholipase enzymes are activated by long-chain fatty acids, but the sensitivity to activation depends on the nature of the substrate. It therefore has been concluded that fatty acids intercalate into the lipid phase and modify the primary interaction of the enzyme with its substrate [l] . Bee venom phospholipase A* is activated by fatty acids [2] , but the kinetics of activation do not support this substratemediated mechanism (unpublished data); however the high affinity of both substrate and enzyme for free long-chain fatty acids makes investigation by kinetic means both difficult and unreliable. This enzyme can be activated irreversibly by treatment with acid anhydrides presumed to add longchain acyl residues to nucleophilic groups in the protein [2]. If all of the effects of fatty acid activation were produced by covalent addition of a single acyl residue, fatty acid activation could be attributed to allosteric properties of the enzyme. It would then be feasible to design reagents for selectively activating or blocking the activation of this and related enzymes in vivo. Lauroyl ethyl carbonate (the mixed anhydride of lauric acid and ethyl carbonic acid) was chosen as activator for this study because it gave rapid activation with little non-specific inactivation (tested against a non-activating substrate) and free lauric acid is a relatively weak activator at corresponding concentrations. Dimethyl maleic anhydride, a reversible blocking agent for amino-groups [3] was used initially to protect the protein against non-specific acylation, but its direct action on fatty acid activation became of greater interest here.

Activation of bee venom phospholipase A2 by oleoyl imidazoline produces a thiol- and proteinase-resistant conformation

Assay methods for bee venom phospholipase A2 are presented which respond to different aspects of enzymic behaviour and which allow basal activity, fatty acid activation and acyl-group activation to be distinguished. The stability of the enzyme to thiols and proteinases is dramatically increased by activation with the selective acylating agent, oleoyl imidazolide. These results support the model of activation by conformation change. Limited-fixation studies indicate that enzyme conformation is determined by interaction with the substrate. The oleoyl-enzyme is partially inactivated by trypsin, but its electrophoretic mobility is unchanged. This protective effect is highly selective and only one other component of the venom is protected against trypsin by oleoyl imidazolide. Combination of trypsin and thiol treatment produces a large fragment of the activated enzyme which could be used for structural studies of the activation site.

Lysolecithin inhibits an action of bee venom phospholipase A2 in erythrocyte membrane

Bee venom phospholipase Aa does not alter the permeability of erythrocyte membranes unless exogenous long-chain fatty acids are also present [l] . The acids that are effective are also direct activators of the enzyme, but this may not explain their action here since enzyme activated by treatment with decanoic anhydride [2] does not enhance membrane leakiness. An alternative possibility is that the exogenous fatty acid brings the membrane close to a threshold level for high permeability that may be exceeded when further lytic products are formed. The kinetics of the leakage process indicate a complex underlying mechanism. Briefly, leakiness increases very rapidly after addition of either fatty acid or enzyme to cells that have been incubated with the other component, but then falls and, depending on the concentration of reagents may rise again and is then accompanied by lysis. The initial phase of rapid leakage is subject to a number of possible experimental artefacts (of mixing or of cell heterogeneity) which make interpretation difficult, but it is very strongly inhibited by lysolecithin and as this effect is unlikely to be influenced by the same artefacts it is of more immediate interest. The enzyme can be activated (20-50-fold) by both fatty acid and lysolecithin [2,3] reaction products and, after making an appropriate correction for activation, an underlying competitive and weak product inhibition is observed. No conditions have been found where either product is as strong an inhibitor as the action of lysolecithin outlined above would suggest.

Albumin mediates lysis of erythrocytes by bee venom phospholipase A2 activated with oleoyl imidazolide.

In a study of the activation of bee venom phospholipase AZ by long-chain fatty acids we showed that the enzyme was irreversibly activated by treatment with reactive derivatives of these acids [ 1,2]. This indicated that fatty acids were allosteric activators of the enzyme and that the binding site contains a nucleophilic residue which can form a covalent linkage to an acyl group. Activity measurements were made using a defined chemical assay, the hydrolysis of dioleoyl phosphatidy1 choline, but under non-physiological conditions in the presence of a dilute organic solvent [3]. Chemically modified enzyme was tested in a sensitive variant of the erythrocyte lysis assay for phospholipase AZ involving conductimetric determination of electrolyte release from the cells [4,5], but initial results failed to show any increase in enzymic activity. Subsequent work showed that acyl imidazolides were more potent activators than other fatty-acid derivatives and they produced less extensive, but presumably more specific modification of the protein than other reagents [6]. When enzyme, activated by oleoyl imidazolide, was tested in the erythrocyte leakage assay the results proved positive and formed the basis for a new and sensitive assay of covalent activation of venom phospholipase A enzymes.

A factor released by monocytes in the presence of dexamethasone stimulates neutrophil locomotion.

1 Steroid‐treated monocyte supernatants cause a dramatic increase in the speed of locomotion of human neutrophils and a significant decrease in their adhesion to protein‐coated glass. In contrast, control monocyte supernatants have a smaller effect on the speed of locomotion, but cause a large increase in their adhesiveness. 2 This supernatant activity was produced equally well in the presence or absence of serum after 24 h culture at 37°C with 10−6 m dexamethasone. 3 The effect of the steroid‐treated monocyte supernatants on the speed of locomotion of human peripheral blood neutrophils was not altered by rabbit polyclonal antisera against lipocortins 1–6. 4 Rabbit anti‐interleukin‐8 antibody which blocked the effect of IL‐8 on the speed of locomotion of neutrophils did not antagonize the locomotion stimulating action of steroid‐treated monocyte supernatants. 5 The exocellular release of this factor(s) by human mononuclear leucocytes suggests that it may be an in vivo mediator of the anti‐inflammatory effect of glucocorticoids.

Purification and activation of phospholipase A2 isoforms from Naja mossambica mossambica (spitting cobra) venom

Isoforms of phospholipase A2 (PLA2) from the venom of Naja mossambica mossambica (the spitting cobra) were purified by a combination of gel filtration on Bio-gel P-30 and ion exchange chromatography on DE-52 Cellulose and the purification followed by three types of polyacrylamide gel electrophoresis. SDS PAGE failed to resolve the active band into separate isoforms. Acid/urea PAGE, resolved the peptides and major protein components of the venom and was able to separate two PLA2 bands in the whole venom. Alkali/urea PAGE resolved four PLA2 bands in whole venom, but could resolve six distinct purified PLA2 species. Of the known isoforms, the acidic form (CM-1) was purified to homogeneity. The basic non-toxic isoform (CM-II) was shown to migrate as a close doublet of PLA2 isoforms. A novel minor purified isoform was identified with mobility intermediate between CM-I and the basic non-toxic isoform CM-II. CM-III was shown to contain a minor PLA2 contaminant. The analysis was facilitated by the fact that all of the isoforms could be eluted from the gels with > 60% recovery of activity. The venom therefore contains at least six isoforms of PLA2 which differ largely by their content of acidic acids. Oleoyl imidazolide treatment increased the haemolytic activity of all but the toxic PLA2 isoform in Naja mossambica mossambica, but partially inhibited the catalytic activity of acidic, toxic and newly purified isoforms whilst partially activating the non-toxic isoform.