Peter W. Riddles

Jack Bean Urease VI. Determination of thiol and disulfide content: Reversible inactivation of the enzyme by the blocking of the unique cysteine residue

The total thiol content of highly pure jack bean urease (urea amidohydrolase, EC 3.5.1.5) has been determined by titration with 5,5′-dithiobis(2-nitrobenzoic acid) in the presence of 6 M guanidinium chloride. Urease contains 15 thioi groups per 96.6-kDa subunit. Coupled with amino acid analysis data, this result establishes that urease contains one cystine disulfide bond per 96.6-kDa subunit. Slow loss of enzymatic activity in the presence of β-mercaptoethanol and oxygen is due to the formation of a mixed disulfide which involves the unique active-site cysteine residue. Enzymatic activity can be fully restored by treatment of inactive urease with 0.1 M dithiothreitol at pH 7.3. Urease is quite stable when stored in 0.05 M sulfite/0.02 M phosphate buffer (pH 7.2, 1 mM in EDTA).

A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.

Separation and characterization of the pyrethroid-hydrolyzing esterases of the cattle tick, Boophilus microplus

Abstract The principal esterases present in homogenates of cattle tick larvae have been separated by gel filtration and preparative isoelectric focusing. Substrate specificities have been determined using trans -permethrin, trans -cypermethrin, p -nitrophenyl butyrate, and the pyrethroid analog, p -nitrophenyl-(1 R,S )-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate ( t -NPDC). One of the esterases, with p I = 4.6, and molecular weight ∼67,000, hydrolyzed the α-cyano-substituted pyrethroid, trans -cypermethrin, but not permethrin. The major esterase activity was found in the p I 5.6–5.8 region, and corresponded to a molecular weight of ∼89,000. Small differences in substrate specificity and differences in the banding pattern after isoelectric focusing were detected between esterases of ticks of a pyrethroid-resistant strain (Malchi) and a pyrethroid-susceptible strain (Yeerongpilly). Rate constants were determined for the inhibition of the different esterases by the organophosphate coroxon and by naphthyl N -propylcarbamate, using p -nitrophenyl butyrate and t -NPDC as substrates.

An estimate of the number of serine protease genes expressed in sheep blowfly larvae (Lucilia cuprina)

A large and diverse family of serine protease genes was identified in first‐instar larval cDNA of the sheep blowfly (Lucilia cuprina). This complex repertoire of genes was identified via a PCR approach using highly degenerate primers based on structurally conserved regions which surround the active site His and Ser residues found in all serine proteases. PCR products from entire first‐instar larval cDNA, or from third‐instar larval salivary glands or cardia, generated using a microscale RT‐PCR method, were cloned into a plas‐mid vector. Comparison of the restriction fragment patterns of PCR products generated from the three different sources suggests a highly diverse tissue‐specific pattern of serine protease expression in this organism. Detailed analysis of the restriction fragment patterns of sixty‐nine randomly selected clones from entire first‐instar larvae revealed forty‐nine different classes of PCR product. Maximum likelihood analysis of these data indicate that between 125 and 220 different serine protease genes are expressed in first‐instar larvae of L. cuprina. DNA sequence analysis of ten randomly‐selected clones, derived from the three tissue sources, indicated that all ten encoded serine protease gene fragments. A frequently occurring PCR product, generated from both first‐instar total cDNA and third‐instar cardia cDNA, showed 73% amino acid identity to a digestive protease expressed in Droso‐phila melanogaster larval gut cells.

Cloning and analysis of a cDNA encoding a human liver carboxylesterase

A human liver carboxylesterase (CE)-encoding cDNA has been cloned using synthetic oligodeoxyribonucleotides (oligos) based on the known amino acid (aa) sequences of rabbit and rat liver CEs. The oligos hybridize specifically to DNA encoding liver CEs. The longest cDNA obtained from screening several cDNA libraries encodes about 80% of the protein and translates into an aa sequence which has a high degree of similarity with the sequences of liver CEs from other species. On hybridization to mRNA isolated from human liver, the cDNA gave a single band of about 2.0 kb consistent with its encoding a protein of less than 68 kDa. DNA obtained from a number of human livers and probed with the CE cDNA gave identical hybridization patterns. These patterns were moderately complex by comparison with published data.

Carboxylesterases from Boophilus microplus hydrolyze trans-permethrin

Abstract An esterase which hydrolyzes the ester bond of trans -permethrin has been partially purified from homogenates of larvae of the cattle tick. The final (gel filtration) step showed two distinct peaks of p -nitrophenylbutyrate-hydrolyzing activity. The trans -permethrin-hydrolyzing activity of the lower-molecular-weight enzyme cochromatographed with the p -nitrophenylbutyrate-hydrolyzing activity of that enzyme. Little trans -permethrin hydrolysis was observed in the high-molecular-weight peak. The yield of the low-molecular-weight enzyme increased on extraction of the homogenates with Triton X-100. Inhibition studies using the low-molecular-weight enzyme and trans -permethrin as substrate indicated that hydrolysis was due largely to a carboxylesterase (EC 3.1.1.1).

Application of trans and cis isomers of p-nitrophenyl-(1R, S)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate to the assay of pyrethroid-hydrolyzing esterases.

A continuous-rate assay for the detection of esterases which hydrolyze synthetic pyrethroids is described. The assay is based on the release of p-nitrophenolate ion upon hydrolysis of the pyrethroid-like compound, trans- or cis-p-nitrophenyl-(1R,S)-3-(2,2-dichlorovinyl)-2, 2-dimethylcyclopropanecarboxylate, at pH 7.4 where spontaneous hydrolysis is not detected. The reagent is solubilized by 0.02% Triton X-100 in the presence of 1.0% ethanol. A simple procedure for the synthesis and separation of the isomers is described. The application of the reagent to the assay of esterases which detoxify synthetic pyrethroids in the cattle tick Boophilus microplus is reported.

Prospects for the management of arthropod resistance to pesticides

Abstract Extensive use has been made of mathematical modelling to examine many of the factors which are involved in the development of pesticide resistance in arthropods. These models have demonstrated that the emergence of resistance can be delayed if more attention is given to planned use of pesticides at the time of their introduction. However the practical application of such delaying strategies at the national or even regional level may be difficult. It is unfortunate that the suggestions made have not been subjected to more extensive testing in the field situation. It is suggested that the contribution of molecular biology to the management of pests and pesticide resistance in arthropod livestock pests will be significant and will be seen in a variety of ways. Very definitely, a greater understanding of the basic molecular processes involved in the development of resistance will be seen. Such work, always in conjunction with the other biological disciplines, will provide new techniques for the control, prediction, detection and prevention of pesticide resistance. A few entirely conjectural examples of the practical application of molecular biology to pesticide resistance and its management have been presented.

Babesia bovis: Biosynthesis and localisation of the 12D3 antigen in bovine erythrocytes

The 12D3 antigen of Babesia bovis was found to be synthesised rapidly in cultured parasites, and localised to both the apical complex of the merozoite and the cytoplasm of the parasitised erythrocyte. Amino-terminal sequencing suggested that the nascent protein had been processed and differences between the predicted and measured molecular weights suggested post-translational modification. The major proportion of 12D3 appeared in the soluble compartment of the parasitised erythrocytes with a molecular weight consistent with no further processing. A significant proportion of the protein required extraction by sodium carbonate, suggesting association with membranous components. The timing of release of soluble 12D3 was coincident with haemoglobin release and this probably reflects a non-specific lysis of the erythrocyte. Synthesis of recombinant BV12D3 was achieved in baculovirus-infected SF9 insect epithelial cells. The product was of the same molecular weight as the native 12D3 and polyclonal antibodies raised against the recombinant protein reacted with both the recombinant and native forms of the antigen.

Choline acetyltransferase in organophosphorus-resistant and -susceptible strains of the cattle tick, Boophilus microplus

Abstract Choline acetyltransferase was demonstrated in homogenate extracts of larvae and adult brains of the cattle tick. The enzyme activity was approximately 15 and 100 μmol of acetylcholine synthesized/g of tissue/hr for larvae and adult brains, respectively. Comparisons of five strains showed that, despite organophosphate selection for acetylcholinesterases with wide differences in activity and inhibition kinetics, the choline acetyltransferase activity was statistically uniform between strains. it is concluded that the two enzymic components of the cholinergic system are controlled independently.