Insect Biochemistry最新文献

Radiolabeled photoaffinity analogs can be used to purify and characterize proteins involved in pheromone perception, juvenile hormone (JH) action, and neuropeptide reception. Several photoaffinity analogs and purification strategies are described for each of these physiological targets. First, a diazoacetate photoaffinity label is used to selectively modify the pheromone binding protein of the gypsy moth, Lymantria dispar. Reverse-phase HPLC is then employed to fractionate the male antennal proteins. Second, a tandem procedure involving preparative isoelectric focusing (IEF) and ion-exchange (IEX) HPLC is employed for the purification of the Manduca sexta hemolymph juvenile hormone binding protein (JHBP), which has now been cloned and sequenced. A separate application of this strategy for the purification of the 29 kDa JH I/methoprene receptor proteins from epidermal nuclei of M. sexta larvae is outlined. A new photolabel, farnesyl diazoketone, has been employed for the characterization of crustacean hemolymph methyl farnesoate binding proteins. Third, the development of neuropeptide photoaffinity labels is described for two systems: (1) the red pigment concentrating hormone (RPCH) of shrimp and (2) the allatostatins isolated from the brain of the cockroach Diploptera punctata.

Secretion from female reproductive accessory glands of the dipteran Ceratitis capitata was found to have antibacterial properties against E. coli. At least two basic polypeptides with mol. wt 15.5 and 4.7 kDa respectively, were identified as responsible for such activity. Furthermore, the 15.5 kDa protein is active against a number of Gram-positive and -negative bacterial strains. Lysozyme activity is also present in the secretion.

A method is described for the isolation of nerve endings from the central nervous system of the cricket (Acheta domesticus). The fraction is relatively free of contaminating particles and releases transmitter efficiently in response to depolarising stimuli. The procedure which employs Percoll in isoosmotic mannitol results in an average yield of 5.66 mg synaptosomes per gram wet weight and is highly reproducible. The separation technique is also reasonably rapid, taking <2h and has the advantage that a large proportion of the nerve membrane vesicles generated during homogenization and tissue fractionation effectively separate from the synaptosomal layer during centrifugation. Neuronal mitochondria can also be isolated in good purity using this procedure.

The imidazoline, 2,3-xylylaminomethyl-2′-imidazoline (XAMI) was evaluated for its effects on octopamine-sensitive adenylate cyclase (OSAC) in crude membrane preparations of neural and non-neural tissues of the American cockroach, Periplaneta americana and ventral-nerve cord homogenates of the tobacco hornworm, Manduca sexta. In the cockroach nerve cord, XAMI was found to be a partial agonist with a V_max 80% of p-octopamine (OA) and a K_a of 30 nM. The affinity is 185 times greater than that of OA. Additivity studies suggest that at maximally stimulating concentrations, XAMI interacts primarily with the OSAC. The antagonist profile for XAMI mimics that of OA with mianserin being the best antagonist, followed by the α-adrenergic antagonist phentolamine. These antagonists were shown to act competitively at the XAMI-binding site. β-adrenergic and dopaminergic antagonists were ineffective. These data indicate that XAMI has the highest reported affinity of any OA-receptor agonist and may be a suitable ligand for studies of the OA receptor.

α-Amylase was purified from adults of the lesser grain borer, Rhyzopertha dominica (F.), by ammonium sulfate precipitation, glycogen complex formation, and gel filtration chromatography. Specific activity increased from 16 AU/mg protein in the crude extract to 705 AU/mg protein in the final sample (1 AU = 1 mg maltose hydrate/min at 30°C). Two major protein bands, active in starch zymograms, were present at R_m 0.71 and 0.79 when the sample was examined by polyacrylamide gel electrophoresis (PAGE) on 7.5% gels. In addition, several minor proteins that had α-amylase activity were also present. Molecular masses of the two major allozymes were estimated to be 57 and 55 kDa under dissociating conditions. Isoelectric points of the allozymes were at pH 3.4 and 3.5. The amylases were most active at pH 7 and the presence of 20 mM NaCl resulted in a 10.7-fold increase in V_max. K_m for soluble starch was 0.127%.

Saline extracts of wheat (“Florida 302”) were 2- and 3-fold more inhibitory on a weight basis towards the amylases from R. dominica than were extracts prepared from two cultivars of triticale, “Morrison” and “CT-4161”, respectively. Interaction of purified α-amylase inhibitors from wheat, inhibitor-0.28 and a sample of the inhibitor-0.19 family of isoinhibitors, with the α-amylases from R. dominica was studied. Complex formation between the amylases and inhibitor-0.28 was demonstrated by PAGE, although the protein-protein complexes that formed were not completely stable during electrophoresis. K_i values were estimated to be 2.6 nM for inhibitor-0.28 and 2.9 nM for inhibitor-0.19. Binding of these inhibitors to α-amylases from R. dominica was not as tight compared with the interaction of these inhibitors with amylases from Sitophilus weevils and Tenebrio molitor.

In vivo and in vitro experiments were performed to examine the role of succinate and other potential precursors of the methylmalonyl-CoA used for methyl-branched hydrocarbon biosynthesis in the termite Zootermopsis nevadensis. The in vivo incorporation of [1,4-¹⁴C]succinate and [2,3-¹⁴C]succinate into hydrocarbon confirmed that succinate is a direct precursor to the methyl branch unit. The other likely precursors, the branched chain amino acids valine and isoleucine, were not efficiently incorporated into hydrocarbon. Carbon-13 NMR showed that one of the labeled carbons of [1,4-¹³C]succinate labeled position 6 of 5-methylalkanes and positions 6 and 18 of 5,17-dimethylalkanes, indicating that succinate, as a methylmalonyl-CoA unit, was incorporated as the third unit to form 5-methylheneicosane and as both the third and ninth units to form 5,17-dimethylheneicosane. Analysis of organic acids after the in vivo metabolism of [2,3-¹⁴C]succinate showed that succinate was converted to propionate and methylmalonate. Labeled succinate injected into the hemolymph was readily taken up by the gut tract. Isolated gut tissue efficiently converted succinate to acetate and propionate, both of which were released into the incubation media. Mitochondria from termite tissue (minus gut tract) converted succinate to methylmalonate and propionate only in the presence of malonic acid, an inhibitor of succinate dehydrogenase. The results of these studies show that while termite mitochondria are able to convert succinate to propionate and methylmalonate, most of the propionate used for methyl-branched hydrocarbon biosynthesis is produced by gut tract microorganisms. The propionate is then presumably transported through the hemolymph to epidermal cells for use in methyl-branched hydrocarbon biosynthesis.

The Ca²⁺/calmodulin and Ca²⁺/phospholipid-dependent protein kinases have been purified and characterized from neural tissue of the honeybee Apis mellifera. Ca²⁺/calmodulin-dependent protein kinase appeared as a multisubunit complex composed of three subunits that co-migrate with kinase activity during all purification steps. The three subunits had molecular weights of 52,000, 57,000 and 60,000, termed α, β′ and β, respectively. The α and β subunits are distinct peptides whereas β′ may have been generated from β by proteolysis. The Ca²⁺/calmodulin-dependent protein kinase required 0.1 μM calmodulin and about 1 μM Ca²⁺ for half-maximal activation. The Ca²⁺/phospholipid-dependent protein kinase (protein kinase C) was purified from honeybee neural tissue by using DEAE-Sephacel and phosphatidylserine-affinity chromatography. The molecular weight of the protein kinase C was about 80,000 as estimated by gelfiltration. Subjection to SDS-PAGE gave a single band with $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 80,000}$, indicating that the enzyme exists as a monomer. The enzyme was fully activated by diacylglycerol in the presence of phospholipid and Ca²⁺.

The Bombyx mori larval serum protein (BmLSP) is a major component of larval hemolymph proteins until early in the last instar. The cDNA for BmLSP was cloned from a library constructed from fat body RNA of penultimate instar larvae, and the complete nucleotide sequence of the 909 base pair cDNA insert was determined. The deduced 262 amino acid polypeptide included a 16 amino acid residue signal peptide and a 15 amino acid sequence prosegment. A homology search showed that BmLSP has significant similarity with microvitellogenin of Manduca sexta and the 30K proteins of B. mori. Tissue distribution and developmental profile of BmLSP mRNA were analyzed by northern hybridization. BmLSP mRNA was abundant in fat body but not detected in midgut and silk gland. BmLSP mRNA was present during the feeding periods of the fourth and fifth instar larvae, but absent during the larval molt and after the onset of cocoon spinning.

The synthesis and secretion of egg-specific protein (ESP) were investigated using the follicle cells isolated from the developing ovary of the silkworm, Bombyx mori. The follicle cells were isolated manually from a follicle into a cell layer by thoroughly extruding the oocyte contents through a small hole. Whole follicles and isolated follicle cells were incubated in vitro with [³⁵S]methionine, and ESP and its precursors were immunochemically isolated using antiserum raised to ESP. The isolated follicle cells incorporated label into ESP but the incorporation rate was about one-fifth of that found in whole follicles. About 20% of the total radioactivity of ESPs were recovered from the incubation medium of the isolated follicle cells while only trace activity (<2%) was found in the incubation medium of whole follicles. These results clearly showed that follicle cells synthesize and release ESP to be taken up by the developing oocyte.