Characterization of aminopeptidase in midgut of body louse pediculus humanus humanus

Abstract

Body louse Pediculus humanus humanus, belong to the group of blood-sucking insects that act as vectors of several deadly diseases to either Mankind or his Livestock, resulting in massive economic losses, especially in third world countries where these diseases are prevalent. Body louse are host specific and feed exclusively on blood. Because of their feeding habits they are therefore prone to blood-borne control agents. Proteins are by far the most abundant nutrients of a bloodmeal. Hence the characterization of enzymes involved in bloodmeal digestion is crucial in understanding their physiological role within the body louse. In this study, Aminopeptidase was characterized from Midgut of human body louse Pediculus humanus humanus which had been dissected 24 hrs after bloodmeal. In crude midgut homogenate, the aminopeptidase showed optimum activity at pH 8.0 (Alkaline) using Leucine para nitoanilide as the sub,strate. Body louse midgut aminopeptidase is fairly thermally stable, incubation at 50°C for 150 min resulted in 80% remaining activity. Supernatants from midgut homogenized in 1% Tween 20 showed 2.5 fold increase in enzyme activity; while midguts homogenized in 1% Triton X-IOO showed 8.0 fold increase in enzyme activity. Aminopeptidase inthe midgut of body louse is stimulated by bloodmeal and increases gradually with time after feeding. Maximal release is realized 48 hrs after bloodmeal. The activity persisted even 96 hrs after bloodrneal. The pattern of feeding in both female adults and newly hatched nymphs remained fairly similar. Kinetic studies showed that the enzyme exists in two forms of soluble and membrane bound. However it exhibited different catalytic activity with regard to Vmax but had the same Km value. The enzyme activity was inhibited by and l' 1a Phenanthroline and Mn2+. The enzyme activity in homogenate supernatant was restricted to one peak after partial purification using Gel Filtration Chromatography on Superose 6. Only one peak of 67 KDa - 69 KDa was observed after separation of homogenates extracted with or without Triton Xvl Ou. Fractions purified from Superose 6 revealed one band of 69 KDa on Native PAGE with homogenates extracted in saline buffer and two bands of 67 KDa and 69 KDa with homogenates extracted in 1% Triton X-lOa. In-gel staining of the enzyme activity revealed one band of Mr=70,OOO. These results indicate that Aminopeptidase is one of the major digestive enzyme in the body louse