Norah K. Olembo

FAT BODY TRIACYLGLYCEROL LIPASE IN SOLITARY AND GREGARIOUS PHASES OF SCHISTOCERCA GREGARIA (FORSKAL) (ORTHOPTERA: ACRIDIDAE)

The activities of the fat body triacylglycerol lipase were determined using 14C-trioleoylglycerol. Resting activities were estimated at 1.98 ± 0.24 and 1.95 ± 0.53 nmol free fatty acid (FFA)/hr/mg protein for gregarious and solitary locusts, respectively. Administration of adipokinetic hormone (AKH) I led to the activation of lipase with peak activities occurring after 30 min. In the solitary locusts, activities of 2.28 ± 0.16 and 2.30 ± 0.43 nmol FFA/hr/mg were obtained following administration of 10 and 2 pmol AKH I, respectively. In the gregarious locusts, enzyme activities of 3.17 ± 0.66 and 2.47 ± 0.39 nmol FFA/hr/mg were obtained after administration of 10 and 2 pmol AKH I, respectively. The Km values were estimated at 46.67 and 18.75 μM for gregarious and solitary locusts, respectively. Similarly, the Vmax values were estimated at 10.29 and 2.52 nmol/hr/mg for gregarious and solitary locusts, respectively. These results confirm phase-dependent differences in lipase properties with the gregarious form having a higher catalytic ability, but a lower affinity for the substrate than the solitary type.

The effect of host blood in the in vitrotransformation of bloodstream trypanosomes by tsetse midgut homogenates

Abstract. Midgut homogenates prepared from Glossina morsitans morsitans, that had previously been fed on different host blood samples, were tested for their abilities to transform bloodstream Trypanosoma bruceiinto procyclic (midgut) forms in vitro.Compared to rat and goat blood samples, eland blood had the least capacity to support trypanosome transformation, whereas buffalo blood showed intermediate capacity. Fractionation of rat blood showed the importance of the cellular portion since both rat and eland red blood cells (RBCs) supported the process. Virtually no transformation was observed in rat and eland plasma or serum fractions. Suspending rat blood cells in eland plasma led to a reduction in parasite transformation rates. Further experiments showed that the RBC membranes were also capable of supporting the process. These results clearly show the important role played by blood, especially the red blood cells, in the transformation of bloodstream trypanosomes. In addition, the low transformation rates observed in eland blood is due to an inhibitory factor(s) present in the plasma fraction.

Changes in the contents of intermediates of proline and carbohydrate metabolism in flight muscle of the tsetse fly Glossina morsit ans and the fleshfly Sarcophaga tibialis

Abstract 1. 1. The contents of intermediates of proline metabolism, the tricarboxylate cycle and glycolysis were measured over the initial phases of flight in the thorax of the tsetse fly Glossina morsitans and the fleshfly Sarcophaga tibialis . 2. The initial rates of proline loss and alanine increase in G. morsitans were approx. twelve times those found in S. tibialis . 3. In G. morsitans there was a sharp initial decrease in the contents of glutamate, ammonia and citrate. Pyruvate content increased rapidly and those of malate and α-oxoglutarate more slowly. Similar initial changes in the contents of these intermediates were observed in S. tibialis . The total concentration of measured tricarboxylate cycle compounds did not change greatly in either insect when flight commenced. 4. Hexose monophosphate concentrations were much lower in G. morsitans than in S. tibialis , but in both insects an increase in the fructose-1,6-diphosphate/fructose-6-phosphate ratio occurred at the onset of flight. 5. The control of the tricarboxylate cycle and the proline-alanine pathway is considered. The utilisation of proline by S. tibialis during flight is discussed.

Catabolism of proline by procyclic culture forms of Trypanosoma congolense

The effect of various metabolic inhibitors on the rate of oxygen consumption by procyclic culture forms of Trypanosoma congolense utilizing proline as substrate was investigated. Cyanide inhibited the rate of oxygen consumption by 81.0 +/- 6.7%, malonate inhibited the rate by 51.6 +/- 1.6% and Antimycin A by 73.1 +/- 5.9%. A combination of cyanide and malonate inhibited the rate of oxygen consumption by 84.9 +/- 6.7% while a combination of antimycin A and malonate inhibited the rate by 81.6 +/- 7.6%. Rotenone had no effect on the rate of respiration except when the intact cells were first permeabilized by digitonin after which rotenone decreased the rate of respiration by 20-30%. Salicylhydroxamate (SHAM) did not have any effect on the rate of oxygen consumption. Enzymes involved in the catabolism of proline with high activities were: proline dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase, NADP-linked malic enzyme, alanine aminotransferase and malate dehydrogenase. Activities of 1-pyrroline-5 carboxylate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase and NAD-linked malic enzyme were detectable but lower. The end products of proline catabolism were alanine and glutamate. Unlike the case in Trypanosoma brucei brucei aspartate was not detected. Possible pathways of proline catabolism in procyclic culture forms of T. congolense and of electron transfer are proposed.

Lipophorin and apolipophorin-III in solitary and gregarious phases of Schistocerca gregaria

Abstract Lipophorin was isolated from the hemolymph by KBr density gradient ultracentrifugation. Apolipophorin-III (apoLp-III) was isolated from low-density lipophorin (LDLp). High-density lipophorin (HDLp) (Mr ∼-620,000) consisted of two apoproteins, apoLp-I (Mr ∼-224,000) and apoLp-II (Mr ∼-81,000). Isolated apoLp-III (Mr ∼-20,000) had two isoforms in non-denaturing gels. Injection of adipokinetic hormone-I resulted in the formation of LDLp in both locust phases. Lipid content of HDLp was estimated at 48.4% and 51.8% for solitary and gregarious locusts, respectively. In LDLp, lipids constituted 59.8% and 57.1% for gregarious and solitary locusts, respectively. The results suggest that the gregarious locusts were more efficient in the conversion of HDLp to LDLp.

Lipophorin from the tsetse fly, Glossina morsitans morsitans.

1. Lipophorin was isolated from the haemolymph of adult tsetse fly, Glossina morsitans morsitans, by ultracentrifugation in a potassium bromide density gradient. 2. The tsetse fly lipophorin (Mr congruent to 600,000) has a density of congruent to 1.11 g/ml and consists of two apoproteins, apolipophorin-I (apoLp-I, Mr congruent to 250,000) and apolipophorin-II (apoLp-II, Mr congruent to 80,000), both of which are glycosylated as shown by staining with periodate-Schiff reagent. The protein complex is composed of 49% protein and 51% lipids. 3. The finding of lipophorin in tsetse fly haemolymph suggests that, although these flies primarily utilize proline for their energy needs, there is an active transport mechanism for the supply of lipid requirements.

Isolation and Properties of 600‐kDa and 23‐kDa Haemolymph Proteins from the Tsetse Fly, Glossina morsitans: their Possible Role as Biological Insecticides

The haemolymph of the tsetse fly. Glossina morsitans morsilans. contains a high (lipophorin) and a low molecular weight protein of high densities. I. 11 and 1.29 g/ml. respectively. The purification of the proteins was achieved by a combination of density gradient ultracentrifugation and reported gel permeation chromatography. The lipophorin is of high molecular weight (Mr∼ 600.000) and consists of two apoproteins. apolipophorin I (M Mr∼250.250,000) and apolipo‐phorin II (M Mr∼ 80.000) both of which are glycosylated. Lipophorin also has a pi of 6.1. However, electrophoresis under non‐denaturing and denaturing conditions showed the low molecular weight protein to be a single polypeptide chain (Mr∼ 23,000). Amino acid analysis revealed a relatively high content of the acidic amino acids as well as serine and glycine. The protein contained lipids as shown by Sudan Black staining but was unglycosylated. Using rabbit antiserum against the isolated protein in immunodiffusion and immunoblotting experiments, no cross‐reactivity was detected with haemolymph samples from insects representing six orders. In conclusion, the finding of lipophorin suggests that, although flies primarily utilize proline for their energy needs, there is an active transport mechanism for the supply of lipid requirements. However, the results for the low molecular weight protein indicate that the protein is unique to Glossina, suggesting that it may have an important role in the physiology of this insect and is therefore a significant target for vector management.

Temporal synthesis of cuticle proteins during larval development in Glossina morsitans

1. Larval development in Glossina species occurs in utero with the mature third instar larva being deposited after a developmental period of 7 days. 2. In this study, the patterns of cuticular protein synthesis during larval development were analysed by two-dimensional gel electrophoresis. 3. From the results, four types of cuticle proteins were identified: those specific to larval, pupal and adult cuticles, and others common to all the stages. 4. Few cuticular proteins were synthesized between the first and second larval instars. By the third larval instar (two days before larviposition), a large number of proteins (Mr < or = 30 kDa) were induced. These proteins persisted up to the brown pupal stage and showed a rapid decline thereafter. Most of the proteins with molecular weights Mr < or = 30 kDa were undetectable at apolysis (5 days after larviposition). 5. By day 15 of the pupal stage, the number of cuticle proteins was very small. The protein profile during the pupal stages remained relatively constant. This was probably due to the fact that the pupal cuticle does not provide any protection since it is itself enclosed at all times within the protective puparium.

Enzyme activities in the fat body of the tsetse fly Glossina morsitans and the fleshfly Sarcophaga tibialis in relation to proline metabolism

Enzymes were assayed in the fat body of the tsetse fly Glossina morsitans and compared with those in the fat body of the fleshfly Sarcophaga tibialis. Alanine aminotransferase, 3-hydroxyacyl-CoA dehydrogenase, NADP-linked isocitrate dehydrogenase and proline synthetase were much more active in G. morsitans. NADP-linked malic enzyme, pyruvate carboxylase, citrate synthase and aconitase were also more active in G. morsitans, but α-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase and various glycolytic enzymes were more active in S. tibialis. Pyruvate carboxylase was mainly mitochondrial in the fat body of both insects, whereas NADP-linked malic enzyme was cytoplasmic. Alanine aminotransferase and NADP-linked isocitrate dehydrogenase were more predominantly mitochondrial in G. morsitans than in S. tibialis. In the fat body of G. morsitans the Km values of pyruvate carboxylase for pyruvate and bicarbonate were quite low, whereas those of NADP-linked malic enzyme were high. Alanine aminotransferase from G. morsitans fat body had lower apparent Km values for alanine and α-oxoglutarate than flight muscle enzyme from the same species. The proline synthesizing potential of G. morsitans fat body is discussed. It is suggested that the pathway involves the enzymes pyruvate carboxylase and NADP-linked isocitrate dehydrogenase.

Purification and characterization of two fibrinolysins from the midgut of adult female Glossina morsitans centralis

1. Adult female tsetse flies (Glossina morsitans centralis) have at least five midgut fibrinolytic proteases, the two most active of which we have purified using DE-52 cellulose. 2. The purified proteases appeared as single bands in sodium dodecylsulphate polyacrylamide gels and had mol. wts of 24,000 and 23,500 and pI values of 6.0 and 5.3, respectively. 3. Both proteases hydrolyse Tosyl-Gly-Pro-Arg-pNA optimally at pH 8.0 (with Km of 20 and 30 microM) and were inhibited by diisopropylfluorophosphate, alpha 1-protease inhibitor, aprotinin, soybean trypsin inhibitor, benzamidine and tosyllysine chloromethylketone. 4. Compared to bovine plasmin, these enzymes digest fibrinogen or fibrin at a slower rate but give similar products. 5. Thus these enzymes are serine proteases similar to the trypsin-like enzymes detected in G. m. morsitans.