Joshua O. Ajele

Carbamylation of N-terminal proline.

Protein carbamylation is of great concern both in vivo and in vitro. Here, we report the first structural characterization of a protein carbamylated at the N-terminal proline. The unexpected carbamylation of the α-amino group of the least reactive codified amino acid has been detected in high-resolution electron density maps of a new crystal form of the HIV-1 protease/saquinavir complex. The carbamyl group is found coplanar to the proline ring with a trans conformation. The reaction of N-terminal with cyanate ion derived from the chaotropic agent urea was confirmed by mass spectra analysis on protease single crystals. Implications of carbamylation process in vitro and in vivo are discussed.

Some physicochemical properties of two major soluble hepatic glutathione transferases of tilapia (Tilapia zilli)

Two distinct glutathione transferases from the liver of adult Tilapia zilli were identified and purified to apparent homogeneity by ion-exchange chromatography on DEAE-cellulose and by gel filtration on Sephadex G-150. These major GST forms labeled tzGST1 and tzGST2 accounted for approximately 42% of the activity detectable with 1-chloro-2,4-dinitrobenzene (CDNB) as a typical electrophilic substrate. Apparent subunit and molecular mass values, substrate specificities and sensitivity to inhibitors as well as kinetic studies were used to differentiate the GST forms. SDS/PAGE indicated subunit molecular masses of 22.0 kDa (tzGST1) and 26.1 kDa (tzGST2) while native molecular weight by gel-filtration on sephadex G-100 indicated native molecular masses of 46.8 kDa and 48.0 kDa for tzGST1 and tzGST2 respectively. They appeared to be homodimers. Inhibition studies showed that tzGST1 was more sensitive to ethacrynic acid (EA), hematin, tributyltinacetate (TBTA), triethyltinbromide (TETB), and triphenyltinchloride (TPTC) than tzGST2 with TPTC being the most potent inhibitor. T. zilli GSTs could conjugate CDNB, DCNB, ρ-NBC, and EA with GSH but displayed no observable conjugating activity with NBDCl. The K(m) and V(max) for tzGST1 and tzGST2 with CDNB were 0.56 ± 0.05 mM; 0.24 ± 0.03 μmol/min/ml and 0.91 ± 0.07 mM; 0.14 ± 0.05 μmol/min/ml respectively while K(m) and V(max) with GSH were 0.46 ± 0.02 mM; 0.19 ± 0.20 μmol/min/ml and 1.32 ± 0.15 mM; 0.21 ± 0.07 μmol/min/ml respectively. Denaturation and renaturation studies with guanidine hydrochloride (Gdn-HCl) revealed that concentration of 4.0 M Gdn-HCl completely denatured tzGST1 and the possible isoenzyme was able to renature to 92% of the original activity. The renaturation process was dependent on temperature. The outcome of this study indicated that tzGSTs are possible GST isoenzymes actively present and involve in the detoxification process in the liver of tilapia when the subject is exposed to chemical toxins. The wide range of chemical toxins encountered in the polluted environment may have directed the selection of multiple tilapia GST isoforms with broad substrate specificity via gene duplication. Consequently, tzGST1 has a better chemical toxin bio-transforming capacity than tzGST2 due to its higher affinity for its substrates--a form of adaption to the polluted environment.

Activity of the Antioxidant Defense System in a Typical Bioinsecticide- and Synthetic Insecticide-treated Cowpea Storage Beetle Callosobrochus maculatus F. (Coleoptera: Chrysomelidae)

The non-enzymatic and enzymatic antioxidant defense systems play a major role in detoxification of pro-oxidant endobiotics and xenobiotics. The possible involvement of beetle non-enzymatic [α-tocopherol, glutathione (GSH), and ascorbic acid] and enzymatic [catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), and polyphenol oxidase (PPO)] antioxidant defense system on the insecticidal activity of synthetic insecticides (cypermethrin, 2,2-dicholorovinyl dimethyl phosphate, and λ-cyhalothrin) and ethanolic plant extracts of Tithonia diversifolia, Cyperus rotundus, Hyptis suaveolens leaves, and Jatropha Curcas seeds was investigated. 2,2-Dicholorovinyl dimethyl phosphate (DDVP; 200 ppm, LC50 = 13.24 ppm) and T. diversifolia (20,000 ppm) resulted in 100% beetle mortality at 96-hour post-treatment. The post-treatments significantly increased the beetle α-tocopherol and GSH contents. Activities of CAT, SOD, POX, and PPO were modulated by the synthetic insecticides and bioinsecticides to diminish the adverse effect of the chemical stresses. Quantitative and qualitative allelochemical compositions of bioinsecticides and chemical structure of synthetic insecticides possibly account and for modulation of their respective enzyme activities. Altogether, oxidative stress was enormous enough to cause maladaptation in insects. This study established that oxidative imbalance created could be the molecular basis of the efficacy of both insecticides and bio-insecticides. Two, there was development of functional but inadequate antioxidant defense mechanism in the beetle.