NMR solution structure and dynamics of an exchangeable apolipoprotein,Locusta migratoria apolipophorin III

We report here the NMR structure and backbone dynamics of an exchangeable apolipoprotein, apoLp-III, from the insect Locusta migratoria. The NMR structure adopts an up-and-down elongated five-helix bundle, which is similar to the x-ray crystal structure of this protein. A short helix, helix 4', is observed that is perpendicular to the bundle and fully solvent-exposed. NMR experimental parameters confirm the existence of this short helix, which is proposed to serve as a recognition helix for apoLp-III binding to lipoprotein surfaces. The L. migratoria apoLp-III helix bundle displays several characteristic structural features that regulate the reversible lipoprotein binding activity of apoLp-III. The buried hydrophilic residues and exposed hydrophobic residues readily adjust the marginal stability of apoLp-III, facilitating the helix bundle opening. Specifically, upon lipoprotein binding the locations and orientations of the buried hydrophilic residues modulate the apoLp-III helix bundle to adopt a possible opening at the hinge that is opposite the recognition short helix, helix 4'. The backbone dynamics provide additional support to the recognition role of helix 4' and this preferred conformational adaptation of apoLp-III upon lipid binding. In this case, the lipid-bound open conformation contains two lobes linked by hinge loops. One lobe contains helices 2 and 3, and the other lobe contains helices 1, 4, and 5. This preferred bundle opening is different from the original proposal on the basis of the x-ray crystal structure of this protein (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608), but it efficiently uses helix 4' as the recognition short helix. The buried interhelical H-bonds are found to be mainly located between the two lobes, potentially providing a specific driving force for the helix bundle recovery of apoLp-III from the lipid-bound open conformation. Finally, we compare the NMR structures of Manduca sexta apoLp-III and L. migratoria apoLp-III and present a united scheme for the structural basis of the reversible lipoprotein binding activity of apoLp-III.     

Different isoforms of an apoprotein (apolipophorin III) associate with lipoproteins in Locusta migratoria

Insects transport lipid for flight in the form of diacylglycerol-rich low-density lipoproteins (low-density lipophorin, LDLp), which in the hemolymph are produced from high-density lipophorin (HDLp) by reversible association with several molecules of an apolipoprotein, apolipophorin III (apoLp-III, Mr approximately 18,000-20,000) during lipid loading. Two isoforms of apoLp-III (a and b) were purified both from adult Locusta migratoria migratorioides hemolymph and LDLp, which have identical apparent Mr but differ in amino acid composition, NH2-terminal amino acid sequence, and isoelectric points (5.35 +/- 0.01 for apoLp-IIIa, 5.10 +/- 0.01 for apoLp-IIIb). The NH2-terminal sequence of apoLp-IIIb is identical to the primary structure of apoLp-III deduced from cloned cDNA [Kanost et al. (1988) J. Biol. Chem. 263, 10,568-10,573], whereas the NH2-terminal sequence of apoLp-IIIa is identical to that of apoLp-IIIb but preceded by Arg-Pro-, which is the C-terminal of the putative signal peptide coded by cDNA upstream from that coding for apoLp-IIIb. The ratio apoLp-IIIa apoLp-IIIb free in hemolymph is identical to that in LDLp (5:9); since 14 molecules of apoLp-III appear to be bound in one molecule of LDLp, an average of 5 molecules of apoLp-IIIa and 9 of apoLp-IIIb are involved in formation of each LDLp particle. In vivo studies using 35S-labeled apoLp-IIIa and b demonstrate that each of the isoforms can associate with HDLp to produce LDLp reversibly; in an in vitro system, production of LDLp containing exclusively apoLp-IIIa or apoLp-IIIb demonstrates independent participation of each isoform in LDLp formation.     

Conformational changes of an exchangeable apolipoprotein, apolipophorin III from Locusta migratoria, at low pH: correlation with lipid binding.

Locusta migratoria apolipophorin III (apoLp-III) is a helix bundle exchangeable apolipoprotein that reversibly binds to lipoprotein surfaces. Structural reorganization of its five amphipathic alpha-helices enables the transition from the lipid-free to lipid-bound state. ApoLp-III-induced transformation of dimyristoylphosphatidylcholine (DMPC) bilayer vesicles into smaller discoidal complexes is enhanced as a function of decreasing pH, with maximal transformation occurring at pH 3.5. Over the entire pH range studied, apoLp-III retains nearly all of its secondary structure content. Whereas no changes in fluorescence emission maximum of the two Trp residues in apoLp-III were observed in the pH range from 7.0 to 4.0, a further decrease in pH resulted in a strong red shift. Near-UV circular dichroism spectra of apoLp-III showed well-defined extrema (at 286 and 292 nm) between pH 7.0 and pH 4.0, which were attributed to Trp115. Below pH 4.0, these extrema collapsed, indicating a less rigid environment for Trp115. Similarly, the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate in the presence of apoLp-III increased 4-fold below pH 4.0, indicating exposure of hydrophobic sites in the protein in this pH range. Taken together, the data suggest two conformational states of the protein. In the first state between pH 7.0 and pH 4.0, apoLp-III retains a nativelike helix bundle structure. The second state, found between pH 3.0 and pH 4.0, is reminiscent of a molten globule, wherein tertiary structure contacts are disrupted without a significant loss of secondary structure content. In both states DMPC vesicle transformation is enhanced by lowering the solution pH, reaching an optimum in the second state. The correlation between tertiary structure and lipid binding activity suggests that helix bundle organization is a determinant of apoLp-III lipid binding activity.     

Isolation and identification of the AKH III precursor-related peptide from Locusta migratoria

We have isolated an 8770Da peptide from extracts of corpora cardiaca of adult male and female Locusta migratoria. The N-terminal amino-acid sequence as partially established by Edman degradation is Ala-Leu-Gly-Ala-Pro-Ala-Ala-Gly-Asp. These nine amino acids correspond to the first nine N-terminal amino acids of the adipokinetic hormone precursor-related peptide gamma-chain (APRP-gamma), a peptide that is predicted from the gene encoding the adipokinetic hormone III precursor. The APRP-gamma chain has a monoisotopic mass of 4387Da and contains two cysteine residues. It is known that both AKH I and AKH II precursors occur as dimers. After processing they give rise to the active hormones and three dimeric (two homodimers and one heterodimer) adipokinetic hormone precursor related peptides (APRPs). Based on the mass of 8770Da and the established N-terminal sequence tag, we conclude that the isolated peptide is a homodimer consisting of two APRP-gamma units, covalently linked to each other by two disulphide bounds. In analogy with the previous identified APRPs (APRP-1, APRP-2, and APRP-3), this APRP will be designated as APRP-4.     

东北地区亚洲飞蝗染色体核型分析

采用常规压片法对吉林省亚洲飞蝗Locusta migratoria migratoria(L.)的染色体核型进行分析.研究结果表明:亚洲飞蝗性别决定机制为X0型,染色体数目为2n♂=23,染色体组式4L+4M+3S+X,全部为端部着丝粒染色体,NF=23.染色体中最长(L1)与最短(S11)染色体之比大于4:1,臂比大...     

Inhibition of diacylglycerol uptake from the fat body by a monoclonal antibody against apolipophorin II in Locusta migratoria

Twenty monoclonal antibodies raised against locust native lipophorin were screened by testing their capacity to inhibit diacylglycerol (DG) uptake from fat body by lipophorin in vitro. One of the monoclonal antibodies clearly inhibits the loading of DG by lipophorin from the fat body. This antibody cross reacts only with apolipophorin-II(apoLp-II), one of the two apoproteins of lipophorin. By using proteolytic apoLp-II fragments, we have shown that the epitope for the antibody against apoLp-II contains lysine. Furthermore, both the apoproteins, apoLp-I and apoLp-II, were almost equally labeled with biotin when the native lipophorin was incubated with modified biotin-reagent. These observations strongly suggest that apoLp-II, at least in part, is localized on the outer surface of lipophorin and may contribute to the lipid loading process from fat body.     

Expression and kinetic analysis of carboxylesterase LmCesA1 from Locusta migratoria

Objective

To investigate the biochemical characterization of the carboxylesterase LmCesA1 from Locusta migratoria.

Results

We expressed recombinant LmCesA1 in Sf9 cells by using the Bac-to-bac baculovirus expression system. Enzyme kinetic assays showed that the K_m values of LmCesA1 for α-naphthyl acetate (α-NA) and β-naphthyl acetate (β-NA) were 0.08?±?0.01 mM and 0.22?±?0.03 mM, respectively, suggesting that LmCesA1 has a higher affinity for α-NA. LmCesA1 retained its enzymatic activity during incubations at pH 7–10 and at 10–30 °C. In an inhibition experiment, two organophosphate pesticides (malaoxon and malathion) and one pyrethroid pesticide (deltamethrin) showed different inhibition profiles against purified LmCesA1. Recombinant LmCesA1 activity was significantly inhibited by malaoxon in vitro. UPLC analysis showed that no metabolites were detected.

Conclusions

These results suggest that overexpression of LmCesA1 enhances malathion sequestration to confer malathion tolerance in L. migratoria.

     

Crystallization and preliminary analysis of crystals of cytochrome c2 from Rhodopseudomonas capsulata

Two crystal forms of the cytochrome c2 isolated from Rhodopseudomonas capsulata have been obtained. One crystal form (type I), grown from ammonium sulfate solutions at pH 7.5, belongs to the space group R32 with unit cell dimensions of a = b = 100.0 A, and c = 162.2 A in the hexagonal setting. These crystals most likely contain two molecules in the asymmetric unit. The other crystal form (type II) was obtained from polyethylene glycol 6000 solutions at pH 6.5. Type II crystals belong to the space group P3(1)21 or P3(2)21 with one molecule per asymmetric unit and unit cell dimensions of a = b = 52.4 A, and c = 87.9 A. Both crystal forms diffract to at least 1.8 A resolution and appear to be resistant to radiation damage.     

Crystallization and preliminary analysis of crystals of high potential iron-sulfur protein from Rhodospirillum tenue

Large single crystals of the high potential iron-sulfur protein isolated from Rhodospirillum tenue strain 3761 have been obtained. They belong to the space group P2(1) with unit cell dimensions of a = 36.7 A, b = 52.6 A, c = 27.6 A, and beta = 90.8 degrees. There are two molecules in the asymmetric unit. Based on oscillation photographs, the crystals diffract to at least 1.6 A resolution. They are stable in the x-ray beam and appear suitable for a high resolution x-ray structure analysis.     

东亚飞蝗谷胱甘肽S-转移酶分离纯化

通过硫酸铵沉淀技术和GSH-agarose亲和层析对东亚飞蝗Locusta migratoria manilensis(Meyen)5龄若虫谷胱甘肽S-转移酶(glutathione S-transferases,GSTs)进行了分离纯化。结果表明GSTs活性在硫酸铵各沉淀段均有分布,但在55%～100%沉淀段活性较高,在硫酸铵饱和度为85%时比活力最高,达到420.33μmol/min/mg protein,纯化倍数为18.86。根据硫酸铵粗沉淀谷胱甘肽S-转移酶结果,选择硫酸铵浓度为60%～90%沉淀段进行GSH-agarose亲和层析,纯化后比活力最高达到1365.29μmol/min/mg protein,纯化倍数达到61.25。经SDS-PAGE鉴定,得到的GST为1条带,亚基的分子量约为24kDa。     

Interaction of an exchangeable apolipoprotein with phospholipid vesicles and lipoprotein particles. Role of leucines 32, 34, and 95 in Locusta migratoria apolipophorin III.

Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein that binds reversibly to lipid surfaces. In the lipid-free state this 164-residue protein exists as a bundle of five elongated amphipathic alpha-helices. Upon lipid binding, apoLp-III undergoes a significant conformational change, resulting in exposure of its hydrophobic interior to the lipid environment. On the basis of x-ray crystallographic data (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608), it was proposed that hydrophobic residues, present in loops that connect helices 1 and 2 (Leu-32 and Leu-34) and helices 3 and 4 (Leu-95), may function in initiation of lipid binding. To examine this hypothesis, mutant apoLp-IIIs were designed wherein the three Leu residues were replaced by Arg, individually or together. Circular dichroism spectroscopy and temperature and guanidine hydrochloride denaturation studies showed that the mutations did not cause major changes in secondary structure content or stability. In lipid binding assays, addition of apoLp-III to phospholipid vesicles caused a rapid clearance of vesicle turbidity due to transformation to discoidal complexes. L34R and L32R/L34R/L95R apoLp-IIIs displayed a much stronger interaction with lipid vesicles than wild-type apoLp-III. Furthermore, it was demonstrated that the mutant apoLp-IIIs retained their ability to bind to lipoprotein particles. However, in lipoprotein competition binding assays, the mutants displayed an impaired ability to initiate a binding interaction when compared with wild-type apoLp-III. The data indicate that the loops connecting helices 1 and 2 and helices 3 and 4 are critical regions in the protein, contributing to recognition of hydrophobic defects on lipoprotein surfaces by apoLp-III.     

High-molecular-weight serum proteins from Locusta migratoria: identification of a protein specifically binding juvenile hormone III

ABSTRACT. Tritiated 10,11-epoxyfarnesyl diazoacetate (EFDA), a photoaffinity label, can be covalently attached to the binding site of a JH-III-specific binding protein in the haemolymph of Locusta migratoria migratorioides (R & F). The specificity of the binding of EFDA to the binding protein is verified by displacement with excess unlabelled JH-III, and EFDA can be used to identify the binding protein in native pore-limiting gradient poly(acrylamide) gel electrophoresis (PAGE) and sodium dodecyl sulphate-PAGE. The native binding protein has a molecular weight of 575,000 and is composed of seemingly identical subunits of molecular weight 81,000.
Three other high-molecular weight serum proteins are identified by native PAGE: a lipophorin, composed of two kinds of apolipophorins, a larval storage protein and a cyanoprotein. The molecular weights and subunit structures of these proteins are investigated, but none of these other high-molecular weight proteins bind JH-III to an appreciable extent.     

Characterization of apolipophorin III from Barytettix psolus and Melanoplus differentialis

Apolipophorin III has been isolated and characterized from two orthopteran species, Barytettix psolus and Melanoplus differentialis. Both apoLp-IIIs have M_r ∼ 20,000 and contain 5% covalently bound sugar. Compositional analysis of the carbohydrate component of these apoLp-IIIs, as well as Locusta migratoria apoLp-III, revealed the presence of manose, fucose and N-acetyl glucosamine. In all three proteins the ratios of these sugar residues was similar (∼2:2:1). Amino acid analysis of B. psolus and M. differentialis apoLp-IIIs was performed and the results compared with those from L. migratoria and M. sexta apoLp-IIIs. The three orthopteran apoLp-IIIs possessed very similar compositions that were distinct from that of M. sexta. Similarly, N-terminal sequence analysis revealed ∼50% sequence identity between the three orthopteran apoLp-IIIs but only 9–18% identity was observed when B. psolus or M. differentialis apoLp-IIIs were compared to M. sexta apoLp-III. The implications of these findings, with respect to flight-related physiology and adipokinetic hormone stimulated lipid mobilization in these different species, is discussed.     

Flight orientation of swarming Locusta migratoria

ABSTRACT. High-speed films of four swarms of Locusta migratoria in Australia and one swarm in New Guinea were analysed. Measurements were made of the locust's body orientation and flight track in the horizontal relative to wind direction, and of height and speed of flight. In all swarms mean course angle and mean track angle in relation to wind direction were significantly different from zero, although all indicated an upwind direction. No evidence was found for orientation to compass direction or sun. Considerable fluctuations in flight direction were measured in some individuals as they traversed the field of view. A modification of Kennedy's (1951) theory is adopted to explain the angled orientations to wind. It is suggested that this could be the result of an optical orientation mechanism.     

Identification of an arginine vasopressin-like diuretic hormone from Locusta migratoria

We have identified two neuropeptides (F1 and F2) from suboesophageal and thoracic ganglia of Locusta migratoria, which we isolated earlier based on their immunological similarity to arginine vasopressin. The more abundant and hydrophilic factor, F1, has sequence Cys-Leu-Ile-Thr-Asn-Cys-Pro-Arg-Gly-NH2, but its biological role is unknown. The less abundant factor, F2, is an antiparallel dimer of F1, and functions as a diuretic hormone of this species. It appears to act through the intermediacy of cyclic AMP. The properties of the native neuropeptides were identical with those of samples synthesized from appropriately protected L-amino acids.     

Purification and characterization of Locusta migratoria chymotrypsin

A chymotrypsin-like enzyme (CTLE) was isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides by ion-exchange chromatography on diethylaminoethyl (DEAE) cellulose followed by affinity chromatography on phenylbutylamine (PBA) Sepharose. The purity and homogeneity of CTLE have been shown by SDS-PAGE and on cellulose acetate strips. The enzyme has a molecular weight of 24,000, determined by SDS-PAGE and on a Sephadex G-75 calibrated column. It has an isoelectric point of 10.1 and contains 0-1 half cystine residues. Sequence analysis of the first 20 N-terminal amino acids has shown 25% homology with bovine chymotrypsin and 40% homology with Vespa crabo and Vespa orientalis chymotrypsins and with Hypoderma lineatum trypsin. The optimal pH for enzyme activity and stability was in the range of 8.5-9.0. The Km and kcat values, determined on substrates for proteolytic, esterolytic and amidolytic activity, similar to those for bovine chymotrypsin. CTLE was inactivated by PMSF and TPCK indicating the involvement of serine and histidine in its active site. The enzyme was fully inhibited by the proteinaceous, double-headed, chymotrypsin-trypsin inhibitors BBI from soybeans and CI from chickpeas, by chicken ovomucoid (COM) and turkey ovomucoid (TOM), as well as by the Kunitz soybean trypsin inhibitor (STI) which hardly inhibits bovine chymotrypsin. Inhibition studies of CTLE with amino acid and peptide-chloromethylketones point towards the existence of an extended binding site.     

Purification and characterization of prophenoloxidase from the haemolymph of Locusta migratoria

Prophenoloxidase (proPO) was purified from plasma of the locust, Locusta migratoria. This was achieved in three steps (gel filtration on 5300, anion exchange on QMA Memsep, and affinity chromatography on blue Trisacryl) without the use of anticoagulant buffer or inhibitors. The native protein had an apparent molecular mass of 250 kDa as determined by gel filtration and was likely composed of three non-covalently associated subunits of 81 kDa. Its amino acid composition was found to be very similar to that of Bombyx mori proPO. Purified locust proPO could be converted into phenoloxidase (PO) by α-chymotrypsin. Using L-dopa as substrate, K_in and V_max were determined to be 1.5 mM and 5 μM/s, respectively. © 1996 Wiley-Liss, Inc.