N.M.J. Vermeulen

ISOLATION OF A NEUROTOXIN FROM THE SALIVARY GLANDS OF FEMALE RHIPICEPHALUS EVERTSI EVERTSI

A quantitative study of the changes in the protein pattern of the salivary glands of female Rhipicephalus evertsi evertsi during the entire repletion process was undertaken. These results, in conjunction with the previously determined toxic phase, indicated the presence of a toxic protein. The development of a sensitive in vitro assay using a Xenopus nerve-muscle preparation, made it possible to identify toxic phases during feeding and to assay fractions of salivary gland extracts during toxin isolation. Sufficient amounts of electrophoretically and chromatographically homogeneous toxin could be obtained through the use of chromatofocusing, enabling its characterization with respect to molecular weight (68 kDa; determined by gel permeation chromatography), pI (6.00), and amino acid composition. The toxin was inactivated by pronase digestion as well as by antiserum.

Kinetic properties of toxic protease inhibitors isolated from tick eggs

1. Egg-toxins from Rhipicephalus evertsi evertsi, Boophilus microplus, Boophilus decoloratus and Hyalomma truncatum were found to be inhibitors of trypsin and in two cases also of chymotrypsin. 2. Fast tight-binding and slow-binding inhibition were observed. 3. Immunological identity of the toxins were assessed with Ouchterlony immunodiffusion and ELISA. 4. The protease content of B. decoloratus and Amblyomma hebraeum tick eggs were determined by a linked enzyme assay. 5. The predictive value of the kinetic constants in inferring a possible physiological role was discussed.

Purification and properties of tick egg toxic proteins

Abstract A purification procedure involving chromatofocusing by means of which toxic components may be obtained in a pure form from crude egg extracts of the tick species Rhipicephalus evertsi evertsi , Hyalomma truncatum, Boophilus microplus and B. decoloratus is described. The molecular weight of the toxin from R. evertsi evertsi was shown to be approx. 5500 according to sedimentation equilibrium centrifugation, SDS gel electrophoresis and calculations from the amino acid composition. The mol. wt of the toxins from the other tick species were found to be between approx. 27,000 and 40,000. Large differences in the amino acid composition of the toxins exist. The pIs range from 6 in the case of R. evertsi evertsi to 9.2 for B. decoloratus . The MLD of the toxins are between 0.4 and 1.7 mg/kg as determined by subcutaneous injection into guinea-pigs. The histopathological symptoms caused by the crude egg extracts of purified toxins of the different tick species appear to be the same in guinea-pigs. Lesions include focal areas of necrosis in the liver, mineralization and oedema of the urinary bladder and vacuolation of the lining epithelium.

A toxic amino acid, 2(S)3(R)-2-amino-3-hydroxypent-4-ynoic acid from the fungus Sclerotium rolfsii

Abstract An amino acid, lethal to New Hampshire chickens (LD 50 , 150 mg/kg) was isolated from dried sclerotia of the fungus Sclerotium rolfsii (Sacc.). Purification of the rather unstable compound was effected on a cation exchange column by means of displacement chromatography and the amino acid was crystallised from 80% methanol. A structure was assigned to the compound on the basis of available chemical and physical data, namely 2( S ),3( R )-2- amino-3-hydroxypent-4-ynoic acid. Confirmation of this structure was gained by direct and indirect synthetic procedures.

Anti-protease from Amblyomma hebraeum

A purification procedure involving affinity chromatography by means of which a protease inhibitor may be obtained in pure form from crude egg extracts of Amblyomma hebraeum, is described. The molecular mass of the inhibitor is 8400 according to sedimentation equilibrium centrifugation. Anti-protease activity was studied with trypsin. Chymotrypsin was not inhibited. Stoichiometric inhibition of trypsin was obtained at a EOKi ratio in the order of 15. The protein was shown to be a competitive inhibitor of trypsin with a dissociation constant of about 10−7 M.