The structure of a juvenile hormone-binding lipophorin from the hemolymph of Diploptera punctata

The high molecular weight, high affinity juvenile hormone binding protein from the hemolymph of Diploptera punctata was identified as a lipophorin by gradient KBr ultracentrifugation and SDS gradient PAGE. This juvenile hormone binding lipophorin (JHBL) was composed of two subunits, apolipoprotein I (230 kDa mol. wt) and apolipoprotein II (80 kDa mol. wt). The density of the native protein was 1.15 g/ml. Photoaffinity labeling using the JH analog [³H]EFDA demonstrated that the JH binding site resides on apolipoprotein I. The amino acid composition of both native lipophorin and its two subunits was determined and the N-terminal sequence of the 80 kDa apolipoprotein described for 19 of the first 21 amino acids. This sequence did not have similarity to any known protein. The N-terminus of the 230 kDa apolipoprotein was blocked. The specificity of a monoclonal antibody to purified native JHBL was also demonstrated. We show that the monoclonal antibody was specific to the 230 kDa subunit and did not recognize the 80 kDa apolipoprotein.     

Purification and properties of the very high density lipoprotein from the hemolymph of adult Triatoma infestans

The very high density lipoprotein (VHDL) of Triatoma infestans hemolymph from adult males has been isolated and purified by two-step density gradient ultracentrifugation. It appears to be homogeneous as judged by native polyacrylamide gel electrophoresis. The content of VHDL in hemolymph was estimated to be 8 mg protein/ml. The purified protein has a molecular weight (Mr) of 450,000, is composed of six subunits of Mr approximately equal to 77,000, and possesses a high content of aromatic amino acids. This protein is glycosylated and contains 3% of lipids by weight with a remarkable amount of free fatty acids (25% of total lipids). The T. infestans VHDL has a different lipid and amino acid composition from lipophorin. The lipid composition and the spectroscopic studies using cis-parinaric acid indicated a high fatty acid binding affinity. It has nine binding sites per mol of VHDL. Competence studies revealed that VHDL has its highest affinity for the binding of palmitic acid followed by stearic and arachidonic acids.     

越冬松针瘿蚊幼虫整体携脂蛋白 的分离和纯化

采用KBr密度梯度超速离心并结合常规Sepharose CL-4B凝胶柱层析,从越冬松针瘿蚊Thecodiplosis japonensis(Uchida et Inouye) 幼虫整体中,分离并纯化了一种携脂蛋白。这是第二例从昆虫整体分离并纯化出携脂蛋白的报道。采用凝胶柱层析确定该携脂蛋白的相对分子质量为638 kD,它是由分别为240 kD和52 kD的两个亚基组成 。整体分子中含有52.8%的蛋白和47.2%的脂类 。苏丹黑B和希夫氏试剂染色显示阳性,说明它是一种糖脂复合蛋白。采用超速离心确定它的密度为1.11 g/mL,表明它是一种高密度的脂蛋白。     

Purification and characterization of vitellogenin from the American cockroach,Periplaneta americana

• 1.1. Vitellogenin (VG) was isolated and purified from the hemolymph of female American cockroaches.
• 2.2. The purification method used in this study comprises two steps: the first step is based on the method originally developed for purifying lipophorin from hemolymph, and the second step is the separation of VG from lipophorin by a KBr density gradient ultracentrifugation.
• 3.3. The purified VG was characterized according to molecular weight, substructure, shape and size, and lipid composition.
• 4.4. The VG molecule is almost globular in shape with the diameter of about 15.5 nm and is indistinguishable from lipophorin in shape and size.
• 5.5. The native molecular weight determined by light scattering method was 560 kDa.
• 6.6. The VG consists of four subunits with molecular weights of approximately 102, 81, 49 and 40 kDa, respectively.
• 7.7. VG is a lipoprotein and comprises 92% protein and 8% lipid.
• 8.8. Major lipid components were found to be diacylglycerol (25%) and phospholipids (71%).

     

Isolation and fluorescence studies on a lipophorin from the weevil diaprepes abbreviatus

Lipophorin was isolated from larvae of a root weevil, Diaprepes abbreviatus (Coleoptera: Curculionidae), using density gradient ultracentrifugation. D. abbreviatus lipophorin contained two apoproteins, apolipophorin-I (M_r = 226,000) and apolipophorin-II (M_r = 72,100) and had a density of 1.08. Relative to other larval lipophorins, D. abbreviatus lipophorin contained little cysteine (determined as cysteic acid) and methionine. Fluorescence spectroscopy of intrinsic tyrosine and tryptophan residues excited at 290 nm revealed a single broad emission peak at 330 nm. Upon denaturing and delipidating lipophorin in guanidine HCl, this peak resolved into two peaks with maxima at 305 and 350 nm. Excitation spectra suggested that the two peaks were due to tyrosine and tryptophan, respectively. Fluorescence quenching agents, iodide and acrylamide, were used to determine accessibility of tyrosine and tryptophan residues to the aqueous environment. Iodide, a polar quenching agent, did not quench fluorescent emission from native lipophorin; quenching by iodide increased to moderate levels when lipophorin was denatured in guanidine HCl. Acrylamide quenched the fluorescence of native lipophorin moderately and very efficiently quenched fluorescence of denatured lipophorin. No difference was observed between fluorescence quenching of denatured vs. denatured and delipidated lipophorin by either iodide or acrylamide.     

Regulatory Properties of Sucrose Synthetase in Sweet Potato Roots

The molecular weight of the yeast tannase [E.C. 3.1.1.20, tannin acyl-hydrolase] of Candida sp. was determined to be 250,000 by gel filtration on Sephadex G–200. The enzyme was dissociable into two identical subunits with molecular weight of 120,000 on SDS-polyacrylamide gel electrophoresis. The amino acid analysis revealed that the enzyme consisted of 786 amino acid residues per protein molecule. The polypeptide moiety of the enzyme was 38 % by the Lowry-Folin reaction and 35% by the amino acid analysis. The enzyme contained 62% neutral sugars, which were identified as mannose and galactose on cellulose thin-layer chromatogram and 2.2 % hexosamines.     

Lipophorin of female Blattella germanica (L.): characterization and relation to hemolymph titers of juvenile hormone and hydrocarbons

High density lipophorin (HDLp) from the hemolymph of the German cockroach, Blattella germanica (L.) (Family Blattellidae), has an apparent molecular weight of 670kDa, with an isoelectric point of 7.0 and a density of 1.109g/ml. It is composed of two subunits, apolipoprotein-I (212kDa) and apolipoprotein-II (80kDa), and consists of 51.4% lipid, 46.2% protein and 2.4% carbohydrate. Hydrocarbons constitute 42.2% of the total lipids which also contain diacylglycerol, cholesterol and phospholipid. Lipophorin is rich in the amino acids glutamic acid, aspartic acid, lysine, valine, and leucine. Specificity of a polyclonal antibody was demonstrated by Western blotting and Ouchterlony immunodiffusion: the antiserum recognized native HDLp and apolipoprotein-I, but not apolipoprotein-II, purified vitellin, or other hemolymph proteins. It also recognized a protein in the hemolymph of Supella longipalpa (Blattellidae) but did not cross-react with hemolymph proteins from Periplaneta americana (Blattidae) or Diploptera punctata (Blaberidae). An enzyme-linked immunosorbent assay was developed to measure the HDLp titer in the hemolymph of adult females. The titer of HDLp, a juvenile hormone binding protein, exhibited no clear relationship to the changing titer of juvenile hormone in hemolymph. The hemolymph titer of hydrocarbon, which is also carried by HDLp, showed some functional relation to the concentration of HDLp in the hemolymph. Because it concurrently serves multiple functions in insect development and reproduction, lipophorin titer might covary with the titers of lipid ligands that occur at high concentrations and require extensive shuttling through the hemolymph.     

Histidinoalanine, a naturally occurring cross-link derived from phosphoserine and histidine residues in mineral-binding phosphoproteins

Native mineral-containing phosphoprotein particles were isolated from the Heterodont bivalve Macrocallista nimbosa. The native particles are discrete structures about 40 nm in diameter which migrate as a single band during electrophoresis in agarose gels. Removal of the mineral component with ethylenediaminetetraacetic acid dissociates the native protein into nonidentical subunits. The lower molecular weight subunits, representing 8% of the total protein, were obtained by differential centrifugation. The native protein is characterized by a high content of aspartic acid, phosphoserine, phosphothreonine, histidine, and the bifunctional cross-linking residue histidinoalanine. The low molecular weight subunits have the same amino acid composition except for a reduction in histidinoalanine and a corresponding increase in phosphoserine and histidine residues, demonstrating that the alanine portion of the cross-link is derived from phosphoserine residues. Ion-exchange chromatography and molecular sieve chromatography show that the low molecular weight subunits have a similar charge density but differ in molecular weight, and the relative mobilities of the subunits on agarose gels indicate that they are polymers of a single phosphoprotein molecule. The minimum molecular weight of the monomer is about 140 000 on the basis of the amino acid composition. The high molecular weight subunits are rich in histidinoalanine and too large to be resolved by either molecular sieve chromatography or gel electrophoresis. On the basis of the ultrastructural, electrophoretic, chromatographic, and compositional evidence, native phosphoprotein particles are composed of subunits ionically cross-linked via divalent cations. These subunits are variable molecular weight aggregates of a single phosphoprotein molecule covalently cross-linked via histidinoalanine residues. Evidence for a nonenzymatic cross-linking mechanism is discussed.     

Polymeric structure of pig small-intestinal mucus glycoprotein. Dissociation by proteolysis or by reduction of disulphide bridges.

Pig small-intestinal mucus glycoprotein, of molecular weight 1.72 X 10(6), is cleaved by Pronase digestion into glycoprotein subunits of molecular weight 4.5 X 10(5). Of the protein component of the native glycoprotein 29% by weight was lost on Pronase digestion, with no loss of carbohydrate. The non-glycosylated region of the protein that was lost with proteolytic digestion had a broad spectrum of amino acid residues, in contrast with the glycosylated region of the protein, which was resistant to proteolysis and was rich in serine, threonine and proline residues. Reduction with 0.2M-mercaptoethanol dissociated the Pronase-digested glycoprotein subunits into smaller glycoprotein subunits of molecular weight 2.7 X 10(5). On reduction, the native glycoprotein was dissociated into subunits of molecular weight 2.4 X 10(5), a similar size to those obtained from reduction of the Pronase-digested glycoprotein. On reductive dissociation of the native glycoprotein, in addition to glycoprotein subunits, protein was also released principally as a component of 90000 molecular weight. This protein was separated quantitatively from the reduced glycoprotein in amounts compatible with one 90000-mol.wt. protein molecule per 1.72 X 10(6)-mol.wt. native glycoprotein molecule. No 90000-mol.wt. protein was released on reduction of the isolated Pronase-digested glycoprotein. Pig small-intestinal mucus glycoprotein is therefore a covalent polymer of glycoprotein subunits joined by disulphide bridges. This polymeric structure differs in important respects from that previously shown for gastric mucus, in particular with respect to the size and number of component subunits per native molecule.     

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.     

Ribulose Diphosphate Carboxylase from Autotrophic Euglena gracilis

Ribulose 1,5-diphosphate carboxylase (RUDPcase) from autotrophically grown Euglena gracilis was purified to homogeneity as measured by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and immunoprecipitation reactions. The enzyme represented about 9% of total protein and 24% of soluble protein in the autotrophic cell. Light-grown, heterotrophic cells seemed to contain considerably less RUDPcase. Native carboxylase from autotrophic Euglena showed an s_{20, w} at low protein concentrations of 17 to 17.5, suggesting a molecular weight of >500,000 daltons. Upon denaturation, the enzyme dissociated into two subunits having different amino acid compositions and molecular weights of 59,000 and 12,000 daltons. Based upon the amino acid mass ratios, a quaternary organization of 7 to 8 large and 8 to 10 small subunits per native enzyme molecule was indicated.     

Isolation and characterization of the major androgen-dependent glycoprotein of canine prostatic fluid

Canine prostatic fluid, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, is characterized by the presence of a single diffuse band (Mr approximately 30,000) which accounts for over 90% of the total protein. The biosynthesis of this protein is under androgen control. Castration results in the disappearance of this protein, whereas its presence in the prostate can be maintained in the castrated animal with exogenous androgen. Analysis of the native protein by isoelectric focusing revealed the presence of 10-13 charged variants with pI values in the range of 6.5 to 8.4. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed that each isoform is constructed of two dissimilar polypeptide subunits covalently linked through disulfide bonds. One subunit has a molecular weight of 15,000 (H chain); the second subunit (L chain) has a variable molecular weight in the 12,000-14,000 range. The H and L subunits have been purified by preparative isoelectric focusing and chemically characterized. Based on tryptic peptide mapping, NH2-terminal analysis, amino acid and carbohydrate composition, the H and L subunits are structurally unrelated and consequently appear to be unique gene products. Furthermore, the L subunit is glycosylated which potentially accounts for its size heterogeneity. Quantitative NH2-terminal analysis indicated that the H and L subunits are present in the native molecule in a ratio of 2:1, suggesting that the native molecule is a trimer with an apparent molecular weight of 43,000. Based on electrophoretic data, the glycoprotein also constitutes the major fraction of the soluble protein in canine prostatic tissue; its presence is organ specific. This glycoprotein should prove useful as a marker for prostatic function under varying hormonal and environmental conditions.     

Purification and properties of alcohol oxidase from <Emphasis Type="Italic">Pichia putida</Emphasis>

Alcohol oxidase (AO) was extracted from the methylotrophic yeast Pichia putida and purified using various methods. AO purified by crystallization was homogeneous based on analytical centrifugation with subsequent gel filtration and SDS-PAGE. The molecular weight of the enzyme was around 600 kDa. SDS-PAGE revealed a single protein band (74 ± 4 kDa), and 8–9 bands of native protein with similar specific AO activities and substrate specificities were identified by PAGE without SDS. Electron microscopy of a single molecule revealed eight subunits located on the top of a regular tetragon with dotted symmetry of 422D4 providing evidence that AO consists of eight subunits. Apparently, each molecule of AO has two types of subunits with very similar molecular weights and differing from each other by the number of acidic and basic amino acid residues. Each subunit includes one molecule of FAD and 2–3 cysteine residues. The pH optimum was within 8.5–9.0. Specific activity of the enzyme varied from 10 to 50 μmol methanol/min per mg protein from batch to batch depending on separation methods and had linear relationship with protein concentration. The AO was quickly inactivated at 20°C and seemed to be stable in phosphate-citrate buffer with 30–50% (w/v) of sucrose. Different forms of 0.1–1 mm crystals of the enzyme were obtained. However the crystals did not yield X-ray reflections, apparently as a result of their molecular microheterogeneity.     

Major hemolymph proteins from larvae of the black swallowtail butterfly,Papilio polyxenes

Three major hemolymph proteins of Papilio polyxenes larvae were isolated and characterized. Density gradient ultracentrifugation of hemolymph resulted in flotation of the major lipoprotein, lipophorin. P. polyxenes larval lipophorin is composed of two apoproteins, apolipophorin-I and apolipophorin-II, plus a mixture of lipids, to give a density of 1.13 g/ml. Immunoblotting experiments using antisera directed against Manduca sexta apolipophorin-I and apolipophorin-II, respectively, revealed cross-reactivity of apoLp-I with Manduca sexta apoLp-I, and apoLp-II with M. sexta apoLp-II. Gel permeation chromatography of the subnatant obtained following density gradient ultracentrifugation revealed the presence of a major protein peak which was shown to contain three major serum proteins, two of which were isolated and characterized. One of these proteins was purified by lectin affinity chromatography. Both proteins have native molecular weights in the range of 450,000 and appear to be hexamers of a single subunit type. Major serum protein-1 is nonglycosylated and has a subunit molecular weight of 75,000. Major serum protein-2 is glycosylated and has a subunit molecular weight of 74,000. Amino acid analysis of this protein revealed a tyrosine plus phenylalanine content of 20 mole percent, characteristic of the arylphorin class of insect storage proteins. Using antibodies against M. sexta larval hemolymph proteins, both the P. polyxenes major serum proteins were shown to be immunologically related to serum proteins of other lepidopteran species.     

Enzymes in atlatoxin B1 biosynthesis: Strategies for identifying pertinent genes

Recent work on the aflatoxin biosynthetic pathway is reviewed, with special emphasis on the enzymes of the late stages of the pathway involving conversion of sterigmatocystin (ST) to aflatoxin B₁ (AFB₁) through an O-methylsterigmatocystin intermediate. Two enzyme activities were discovered in subcellular fractions of cell-free extracts of a mutant strain ofAspergillus parasiticus (SRRC 163): 1)A post-microsomal methyltransferase (MT) catalyzed conversion of ST to OMST, and 2) a microsomal-associated activity (oxido-reductase) converted OMST to AFB₁. The 168 KDa, anionic MT was purified to homogeneity and characterized (two subunits, 110 KDa and 58 KDa). Preliminary evidence indicated the presence of a cationic isozyme of the MT in mycelial extracts. The oxido-reductase has been partially purified and characterized. Polyclonal antibodies were prepared to the anionic MT and the enzyme's amino acid composition determined. A cDNA library has been constructed from mRNA isolated fromAspergillus parasiticus mycelia during the onset of AFB₁ biosynthesis for the purpose of identifying the genes responsible for aflatoxin biosynthesis.     

Molecular characterization of the bifunctional VHDL-CP from the hemolymph of white shrimp Penaeus vannamei

A very high-density lipoprotein (VHDL) purified from the hemolymph of the white shrimp Penaeus vannamei is shown to be identical to the clotting protein (CP) previously reported from the same organism based on size, subunits and N-terminal amino acid sequence. The approximately 440-kDa protein, a homodimer of approximately 200-kDa subunits, was present in KBr gradient fractions ranging in density from 1.155 to 1.212 g/ml. Samples of VHDL after purification by strong cation exchange chromatography were subjected to electrophoresis on native polyacrylamide gels. Lipids associated with the VHDL were detected by Sudan Black and Oil Red O staining and comprise 9-15% of the purified protein. Circular dichroism of VHDL-CP indicates that the alpha-helix content of the VHDL-CP is 32%, while beta-sheets correspond to 33%, closely resembling the secondary structure of CP from the shrimp Penaeus monodon and, remarkably, the secondary structure of very high-density lipophorin E (VHDLpE) from the tobacco hornworm, Manduca sexta.     

Lipid transfer particle in locust hemolymph: purification and characterization

A lipid transfer particle (LTP) from the hemolymph of adult male locusts, Locusta migratoria, was isolated and purified. The locust LTP exhibited its capacity to catalyze the exchange of diacylglycerol between low density lipophorin (LDLp) and high density lipophorin (HDLp). Contrary to the LTP reported for the tobacco hornworm, M. sexta, the locust LTP appeared to lack the capacity to promote net transfer of diacylglycerol to form an intermediate density lipophorin, although it seems premature to conclude the complete lack of such a capacity in locust LTP. The original concentration of LTP in hemolymph is assumed to be extremely low compared to that of lipophorin; only a catalytic amount of LTP may be present in the hemolymph (e.g., only 160 micrograms of LTP was obtained from the original hemolymph containing 400 mg protein). The molecular weight of intact LTP was estimated to be about 600,000 and the LTP was comprised of three glycosylated apoproteins, apoLTP-I (mol wt 310K), apoLTP-II (mol wt 89K), and apoLTP-III (mol wt 68K). The locust LTP contained significant amounts of lipids; the total lipid content amounted to 14.4% and the lipids were comprised of 17% hydrocarbons, 44% diacylglycerol, 8% cholesterol, 13% free fatty acid, and 18% phospholipids. The above molecular properties of locust LTP are essentially similar to those reported for M. sexta LTP.     

Physical and surface properties of insect apolipophorin III

Apolipophorin III (apoLp-III) from Manduca sexta has a molecular weight of 18,100. Based on its hydrodynamic properties (sedimentation and diffusion coefficients, frictional ratio, intrinsic viscosity) and its behavior during gel permeation chromatography, we concluded that apoLp-III is a prolate ellipsoid with an axial ratio of about 3. The circular dichroic spectrum of apoLp-III suggests that the protein contains approximately 50% alpha-helix. At the air-water interface, apoLp-III forms a monolayer which is gaseous at surface pressures less than or equal to 1 dyne/cm. The isotherm of this phase yields an excluded molecular area of 3800 A2/molecule (23 A2/amino acid). At a surface pressure of 22.1 dynes/cm, the monolayer undergoes a phase transition reminiscent of a first-order phase transition of pure lipids. The monolayer can be compressed in this surface pressure range to an area per molecule of 480 A2 (2.9 A2/amino acid). Since a globular protein of molecular weight 18,100 could occupy an area of only about 2000 A2 when bound to a surface, it is suggested that in the expanded state, apoLp-III must unfold on the surface, whereas in the compressed state, the molecule is oriented with its minor axis parallel to the water surface. ApoLp-III binds with high affinity (Kd = 1.9 X 10(-7)M) to both phosphatidylcholine- and diacylglycerol-coated polystyrene beads. All of these results are consistent with the proposal that apoLp-III plays a key role in increasing the capacity of the insect lipoprotein, lipophorin, to transport diacylglycerol by stabilizing the increment of lipid-water interface that results from diacylglycerol uptake.     

Subunit structure of biodegradative threonine deaminase.

The molecule weight of the biodegradative threonine deaminase from Escherichia coli was determined to be approximately 147,000 by sedimentation equilibrium ultracentrifugation. Similar experiments using 5 M guanidinium chloride gave a value of 39,000 for the molecular weight of the enzyme subunit. On sodium dodecyl sulfate-gel electrophoresis the enzyme also dissociated into a single subunit with an estimated molecular weight of 38,000. The NH2 terminus of the enzyme was determined to be methionine by the dinitrophenylation procedure. Quantitative analysis revealed that 3.6 mol of methionine were detected per 147,000 g of enzyme. The selective tritium labeling method established alanine as the COOH-terminal residue. The sequence of residues at the NH2 terminus, determined using an automated sequence analyzer, was: (formula: see text). The fact that a single amino acid was released at each degradation step in the above experiment strongly suggests that the subunits in the enzyme contain the same amino acid sequence. Therefore, the native enzyme with a molecular weight of 147,000 appears to be composed of four identical polypeptide subunits.     

Lipophorin from Rhodnius prolixus: Purification and partial characterization

The lipophorin of adult females of Rhodnius prolixus was radioactively labelled with ³²P exclusively in the phospholipid moiety and purified on a KBr ultracentrifugation gradient. The density of purified [³²P]phospholipid labelled lipophorin on the fifth day after a blood meal was 1.1211 ± 0.0017 g/ml. By weight it contained 51.7% protein, 0.7% sugar and 47.6% lipid. The protein moiety was composed of three apoproteins of 226 ± 11, 86 ± 2 and 16 ± 1 kDa. Mannose and N-acetylglucosamine were the only sugars detected. Among the lipids, 66.3% were neutral lipids and 33.8% were phospholipids. Analysis by thin-layer chromatography showed that in the total phospholipids fraction ³²P was distributed as follows: phosphatidylethanolamine (54.4%), phosphatidylcholine (44.7%), cardiolipin (2.1%), phosphatidylserine (0.7%), phosphatidylinositol (0.4%), sphingomyelin (0.3%) and phosphatidic acid (0.2%). The total phosphate content was 0.53 ± 0.03 nmol/μg of protein.