Two l-amino acid oxidase enzyme isoforms, Cc-LAAOI and Cc-LAAOII were purified to apparent homogeneity from Cerastes cerastes venom in a sequential two-step chromatographic protocol including; gel filtration and anion exchange chromatography. The native molecular weights of the isoforms were 115 kDa as determined by gel filtration on calibrated Sephacryl S-200 column, while the monomeric molecular weights of the enzymes were, 60, 56 kDa and 60, 53 kDa for LAAOI and LAAOII, respectively. The tryptic peptides of the two isoforms share high sequence homology with other snake venom l-amino acid oxidases. The optimal pH and temperature values of Cc-LAAOI and Cc-LAAOII were 7.8, 50 °C and 7, 60 °C, respectively. The two isoenzymes were thermally stable up to 70 °C. The Km and Vmax values were 0.67 mM, 0.135 μmol/min for LAAOI and 0.82 mM, 0.087 μmol/min for LAAOII. Both isoenzymes displayed high catalytic preference to long-chain, hydrophobic and aromatic amino acids. The Mn2+ ion markedly increased the LAAO activity for both purified isoforms, while Na+, K+, Ca2+, Mg2+ and Ba2+ ions showed a non-significant increase in the enzymatic activity of both isoforms. Furthermore, Zn2+, Ni2+, Co2+, Cu2+ and AL3+ ions markedly inhibited the LAAOI and LAAOII activities. l-Cysteine and reduced glutathione completely inhibited the LAAO activity of both isoenzymes, whereas, β-mercaptoethanol, O-phenanthroline and PMSF completely inhibited the enzymatic activity of LAAOII. Furthermore, iodoacitic acid inhibited the enzymatic activity of LAAOII by 46% and had no effect on the LAAOI activity.
In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 µg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species.
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