DISSERTATION. H. Costa submitted this dissertation for the degree of Master of Science in Cell and Molecular Biology publicly examined at the Institute of Biosciences of Rio Claro, São Paulo State University, Rio Claro, São Paulo, Brazil in 1996.
Advisor: Professor Mário Sérgio Palma
ABSTRACT. Studies of social Hymenoptera venoms are of great medical importance, since a great number of people are sensitive to the venom. In the present work, the composition and the physico-chemical characterization of the most important component parts of the social wasp Agelaia pallipes pallipes venom was studied.
The venom was obtained through the dissection of the venom reservoirs. The venom contained protein with molecular weights (MW) ranging from 13,000 to 80,000 Daltons, as well as a high concentration of low MW proteins. Phospholipase A2 and hyaluronidase, the high MW proteins were investigated as they are two important allergens present in this venom.
Phospholipase A2 was purified by reverse phase High-Performance Liquid Chromatography (RP-HPLC). The molecular weights were estimated by gel permeation in columns of Sephadex G-100, at pH 6.8, and by High-Performance Molecular Exclusion Chromatography (HPLC). Elution in HPLC produced three peaks (I, II and III) which were rechromatographed individually in the same conditions mentioned above, resulting in the fractionation of three different forms of PLA2: A, B and C. In peak I only form A (MW 42 KDa) was found, in peak II form A in equilibrium with form B (MW 74 KDa) was found and in peak III form A in equilibrium with form C (MW 14 KDa) was found. Therefore, form C is a monomer, form B is a pentamer and form A is a trimer, which seem to be in equilibrium. Phospholipase A2 represents 3.7% of the total content of proteins in the venom. All the forms of this enzyme presented a high sugar content (around 20% w/w).
In order to characterize PLA2, some kinetic parameters were investigated for the three different forms of this enzyme. It was verified that all forms presented Michaelian kinetics with Km = 2.10
M and Hill's coefficient = 1.0. However, the Vmax values for forms A, B and C were 500, 125 and 154 moles/ml/h, respectively. Thus, the dissociation of trimer (A) in monomer (C) and the polymerization in pentamer (B) causes an inhibition similar to the effect of a competitive inhibition. The optimum pH values for forms A, B and C were 7.5, 8.5 and 9.0, respectively. Comparisons of these results suggest that the equilibrium of association/dissociation among the three forms of PLA2 may be part of a regulatory mechanism of this enzyme.
Hyaluronidase was purified by using a cation exchange chromatography in CM-cellulose, at pH 4.5, and the elution produced only one peak. The molecular weight was estimated by molecular exclusion chromatography in Sephadex G-100, pH 6.8, and by SDS-PAGE. Elution in Sephadex G-100 produced one peak of MW 62 KDa, and SDS-PAGE analysis showed only one band of MW 64 KDa. Hyaluronidase represents 1.3% of the total content of proteins in the venom, and its total content of sugar was very high (58%w/w).
The steady-state kinetic study of condroitine sulfate A hydrolysis, pH 5.2, presented a Vmax value = 34 moles/ml/h, a S0.5 = 1,25.10 M, and Hill's coefficient = 1.5, showing that this enzyme presented a positive cooperative behavior, and in spite of being a monomer, it may have at least two substrate binding sites. However, another hypothesis is the existence of two different conformation states of this enzyme in equilibrium and with different substrate affinity.
Peptide fractions were purified through RP-HPLC and then incubated with Wistar rat platelets in order to identify the serotonin-releasing fractions. The peptides named APP-2, APP-3. APP-4, APP-5, APP-6 and APP-7 induced serotonin release from the platelets and they were submitted to hemolytic activity test, but only APP-5 presented this activity. The peptides APP-3, APP-4, APP-5, APP-6 and APP-7 were submitted to amino acid sequence analysis.
The sequence of APP-3 was similar to those of waspkinins; APP-4 was similar to sylverin (and they may form a new class of releasing peptides); APP-5 and APP-7 were similar to mastoporans and APP-6 was similar to chemotactic peptides. A simulation of peptide conformation tendencies showed that APP-3, APP-4, APP-6 and APP-7 did not present a definite secondary structure, and thus, they did not present a tendency of cell membrane insertion, which indicated that they probably needed a receptor to act properly.
APP-5 showed a rich 
-helical conformation presenting a great potential to direct cell membrane penetration. This result was experimentally confirmed, since it was verified that this peptide presents both the hemolytic and the secretagogue activities. The secretagogue activity seems to be due to mechanisms that cause cell membrane disturbance and lysis with consequent serotonin release.
CORRESPONDENCE TO:
H. COSTA - Departamento de Biologia, Instituto de Biociências, UNESP, Campus de Rio Claro, Caixa Postal 199, CEP 13506-900, Rio Claro, SP, Brasil.
Publication Dates
-
Publication in this collection
08 Jan 1999 -
Date of issue
1997
