Dissociation of enzymatic and toxic activities by the use of antibodies. Disociación de las actividades enzimáticas y tóxicas mediante la utilización de anticuerpos

Tosimn Vol. 28, No. 11, pp. 1245- 1246. 1990. Printed in Great Britain.

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LETTERS TO THE EDITOR DISSOCIATION OF ENZYMATIC AND TOXIC ACTIVITIES BY THE USE OF ANTIBODIES (Acceptedfor publication 19 July 1990)

IN A RECENT article KASTURi and GOWDA,(1990) described the dissociation of enzymatic and toxic activities of phospholipases A, (PLA) from Vipera russelli venom by neutralization experiments using rabbit polyclonal antibodies. The authors conclude that their results "have shown the presence of distinct sites on the same PLA2 molecule responsible for neurotoxicity, catalytic activity, anticoagulant activity and myotoxicity" . Also they state that "there are no reports explaining the relationship between the catalytic activity and pharmacological properties making use of these (polyclonal) antibodies", and "For the first time we are reporting the presence of multiple pharmacological sites on the PLA2 molecule apart from the catalytic site using polyclonal antibodies". In the study of a myotoxic PLA2 from Bothrops asper, we described the dissociation of catalytic and myotoxic activities by antibody neutralization experiments (LomoNTE et al., 1987). For instance, when antiserum was mixed with toxin at a ratio of 2 ml antiserum/mg toxin, PLA2 activity was completely neutralized, while myotoxic activity remained unaffected . However, antibody-neutralization experiments of different activities of a single molecule must be interpreted with caution. Dissociations observed, although suggestive, do not always imply the existence of distinct or multiple active sites. Since each effect (or its neutralization) is measured on a particular test system, many extrinsic factors may contribute to the results obtained. In this regard the evidence provided by COLMAN et al. (1987) is very illustrative . These authors studied the ability of monoclonal antibodies (their Fab fragments) to neutralize the enzymatic activity of influenza virus neuraminidase . They observed that a single monoclonal antibody (NC 10) could neutralize completely the enzymatic activity of neuraminidase if a large substrate (fetuin) was utilized, but could not inhibit this activity if a small substrate (neuraminyl-lactose) was employed as the test system . Thus, if we assume, for instance, that these results imply distinct molecular sites, we would have to conclude that influenza virus neuraminidase has two different active sites for this enzymatic activity, which would be a wrong interpretation . The presence of multiple pharmacological sites of venom PLA2s in addition to the catalytic site is being studied by many groups, and undoubtedly, polyclonal and monoclonal antibodies are useful tools in this area of research . However, the complexity of neutralization mechanisms at the molecular level, and the influence of extrinsic factors in each particular test system, urge caution in the interpretation of results. REFERENCES COLMAN, P . M ., AIR, and LAVER, W . G .

G. M .,

WEBSTER,

R . G .,

VARGHESE,

J . N .,

BAKER,

A . T.,

LENTZ,

M . R .,

TULLOCH,

(1987) How antibodies recognize virus proteins . Immunology Today 8, 323-326. 1245

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Letters to the Editor

S. and GOWDA, T . V . (1990) Detection, using antibodies, of pharmacologically active sites, apart from the catalytic site, on venom phospholipase A_ . Toxicon 28, 91-99 . LOMONTE, B ., GuTibRREz, J . M ., MORENO, E . and CERDÁs, L . (1987) Antibody neutralization of a myotozin from the venom of Botbrops asper (terciopelo). Toxicon 25, 443-449 . KAmiu,

BRUNO LOMONTE and JOSÉ MARIA GUnÉRREz Instituto Clodomiro Picado Facultad de Microbiología Universidad de Costa Rica San José Costa Rica

Taxico Vol. 28, No . 11, pp. 1246-1247,1990 . Printed in Great Britain.

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COMMENT ON THE LETTER TO THE EDITOR BY LOMONTE AND GUTIÉRREZ (Accepted for publication 19 July 1990)

happy to respond to the Letter to the Editor by LOMONTE and GUTIÉRREZ about our recent paper which appeared in Toxicon (KASTURI and GOWDA, 1990). We have reviewed the paper of LomoNTE et al. (1987) where they have shown the neutralization of myotoxic, PLA2 and lethal activities of a myotoxin by polyclonal antibodies. They have particularly emphasized the myotoxic and PLA2 activity and have suggested that myotoxicity does not depend on the enzyme activity of the toxin. Their contribution in understanding the structure-function relationship among venom PLA2 is important but they have not mentioned the sites that are responsible for myotoxic and PLA 2 activity . We had earlier suggested the possible presence of a separate myotoxic site apart from the neurotoxic and catalytic sites (JAYANTm et al., 1989). In our paper we repeatedly mentioned that there have been many attempts relating PLA 2 activity to pharmacological properties . There were no reports explaining the relatonship between catalytic activity and pharmacological properties making use of polyclonal antibodies. For the first time we reported the presence of multiple pharmacologically active sites on the PLA, molecule apart from the catalytic site using polyclonal antibodies. We considered all the pharmacological properties (neurotoxicity, myotoxicity, edema formation, anticoagulant activity, hemoglobinurea) of a PLA2 and tried to find their relationship with each other and with the PLA 2 activity . During this approach we have suggested the possible presence of distinct functional sites. LomoNTE and GUTdRREZ mentioned a paper of COLMAN et al. (1987) which showed the selective neutralization by monoclonal antibodies of influenza virus neuraminidase activity when fetuin was used as a substrate. They have also suggested wrong interpretations that can be made using as an example the results of COLMAN et al. (1987) . Our interpretation is, however, not comparable to the assumption suggested by LomoNTE and GUTIÉRREZ, and is thought to be clear and correct. WE ARE