Sources of larval salivary gland secretion in the dipteran Chironomus tentans.

The soluble proteins in the hemolymph, the salivary gland, and the salivary secretion of fourth instar Chironomus tentans were examined by disc electrophoresis in acrylamide gels. Of the 11 protein fractions detected in buffered saline extracts of the gland, 10 are present also in the hemolymph. Amino acid isotope incorporation experiments indicate that the protein fractions shared by the salivary gland and the hemolymph are not synthesized in the gland but are synthesized in other larval tissues. Immunochemical studies show that most of these proteins eventually are secreted from the gland. The salivary gland in vivo and in vitro is active in de novo protein synthesis. The protein synthesized tends to form large molecular weight aggregates. As demonstrated by radioautography, at least 80% of this protein is secreted from the 30 large cells forming most of the gland. The proteins synthesized in the salivary gland cannot be detected in the hemolymph. The results of this investigation are consistent with a mechanism of secretion formation involving both de novo synthesis of some secretion proteins and the selective uptake, transport, and secretion of hemal proteins by the salivary gland.

Female sterile (1) yolkless: a recessive female sterile mutation in Drosophila melanogaster with depressed numbers of coated pits and coated vesicles within the developing oocytes.

Ultrastructural analysis of developing oocytes produced by the recessive female sterile mutant, yolkless (yl), in Drosophila melanogaster shows that yl+ gene activity is necessary for coated pit and coated vesicle formation within these oocytes. 29 alleles of the mutation are known to exist, and they fall either within a strongly affected class or a weakly affected class. Analysis of oocytes produced by females homozygous for the strongly affected class of alleles shows a greater than 90% reduction in the numbers of coated pits and coated vesicles. These oocytes have very little proteinaceous yolk, and the females accumulate vitellogenin (the yolk protein precursor) within their hemolymph. Moreover, females homozygous or hemizygous for a given strong allele produce mature oocytes that are flaccid. Alternatively, females homozygous or hemizygous for weak alleles produce yolk-filled oocytes, but the number of coated pits and coated vesicles within these oocytes is 50% of that found in the oocytes of wild-type females. Despite the presence of yolk within these oocytes, females homozygous for weak yl- alleles remain sterile, and their mature oviposited eggs collapse with time.

Isolation and characterization of insect vitellogenin. Its identity with hemolymph lipoprotein II.

Several years ago, we isolated two major lipoproteins from the pupal hemolymph of the Cynthia silkworm, both of which contained diacylglycerol as a major lipid, and named them lipoproteins I and II. In this paper, we present definite evidence that the vitellogenin (female protein) of this insect is indeed identical with lipoprotein II.

Subunit heterogeneity of Cancer magister hemocyanin.

Hemocyanin from the Dungeness crab, Cancer magister, has been described as a 25-S two-hexamer assembly of two different 5-S subunits. We have found that at least six different 5-S polypeptide chains constitute this hemocyanin. They can be separated from one another by sodium dodecyl sulfate slab gel electrophoresis as well as by regular gel electrophoresis. The six 5-S polypeptides appear very different from one another when each SDS-treated subunit is partially digested with Staphylococcus aureus V8 protease. This pattern of six subunits is present both in hemolymph which has been examined immediately upon removal from the animal as well as in hemocyanin which has remained at room temperature for two weeks. Thus, it is unlikely that the heterogeneity is a result of proteolysis during preparation of the sample. Possible implications of the high degree of subunit heterogeneity on the protein's quaternary structure are discussed.

Chiral 1,2-diacylglycerols in the haemolymph of the locust, Locusta migratoria.

A 1H-NMR method using chiral shift reagents was applied in the stereochemical analysis of the haemolymph 1,2-diacylglycerols of Locusta migratoria. Conversion of the 1,2-diacylglycerols into 1,2-diacetyl-3-tritylglycerols allowed the accurate determination of the enantiomeric purity, whereas direct trimethylsilylation of the unmodified or hydrogenated haemolymph 1,2-diacylglycerols proved to be less suitable because of signal broadening. In the haemolymph of Locusta, sn-1,2-diacylglycerols with a remarkably high optical purity were found to be present. In the resting locust, at least 96% of the haemolymph 1,2-diacylglycerols have the sn-1,2-configuration, in locusts in which the haemolymph diacylglycerol concentration was elevated by fat body triacylglycerol mobilization induced by flight activity or injection of adipokinetic hormone, over 97% of the 1,2-diacylglycerols is the sn-1,2-enantiomer. The few percent sn-2,3-enantiomer may not have been present initially. Positional distribution of the fatty acids in the fat body triacylglycerols and in the haemolymph sn-1,2-diacylglycerols obtained from locusts after a 2 h flight revealed nearly identical occupation of the sn-2-positions in both acylglycerols. The distribution patterns in the sn-1-position of the 1,2-diacylglycerols and the combined sn-1 and sn-3 positions of the triacylglycerols are compatible with the possible existence of a stereospecific sn-3-triacylglycerol lipase.

Mössbauer spectroscopic studies of the cores of human, limpet and bacterial ferritins.

Ferritin cores from human spleen, limpet (Patella vulgata) haemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated using 57Fe Mössbauer spectroscopy. The Mössbauer spectra were recorded over a range of temperatures from 1.3 to 78 K, all the spectra are quadrupole-split doublets with similar quadrupole splittings and isomer shifts, characteristic of iron(III), while at sufficiently low temperatures the spectra of all the samples show well-resolved magnetic splitting. At intermediate temperatures, the spectra from the human ferritin exhibit typical superparamagnetic behaviour, while those from the bacterial ferritin show behaviour corresponding to a transition from a magnetically ordered to a paramagnetic state. The spectra from the limpet ferritin show a complex combination of the two effects. The results are discussed in terms of the magnetic behaviour of small particles. The data are consistent with magnetic ordering temperatures of about 3 and 30 K for the bacterial and limpet ferritin cores, respectively, while the data indicate that the magnetic ordering temperature for the human ferritin cores must be above 50 K. These differences are interpreted as being related to different densities of iron in the cores and to variations in the composition of the cores. The human ferritin cores are observed to have a mean superparamagnetic blocking temperature of about 40 K, while that of the limpet ferritin cores is about 25 K. This difference is interpreted as being due not only to different mean numbers of iron atoms in the two types of core but also to the higher degree of crystallinity in the cores of the human ferritin.

Galactose-specific lectin in the hemolymph of solitary ascidian, Halocynthia roretzi. Molecular, binding and functional properties.

Galactose-specific lectin isolated from the hemolymph of solitary ascidian, Halocynthia roretzi, has been further characterized. The hemagglutinating activity of the lectin is Ca2+-dependent. The lectin has a large molecular form as revealed by gel-permeation chromatography, sedimentation equilibrium and velocity measurement, and electron microscopic observation. The lectin is adsorbed to columns of blue-Sepharose and phenyl-Sepharose, and eluted with ethylene glycol, not with lactose or high concentration of NaCl. The lectin shows a stimulatory effect on the superoxide anion production by guinea-pig polymorphonuclear leukocytes, and the effect is inhibited, among various sugars, most strongly by melibiose.

Physiochemical study of rock crab lipoproteins.

Physicochemical studies have been carried out on the hemolymph and egg lipoproteins of the rock crab (Cancer antennarius). Analytical ultracentrifugal analyses of vitellogenic female HDL3 revealed the presence of two types of lipoproteins. The first with a sedimentation rate of 5.35 S was comparable to lipoproteins in male and non-vitellogenic female hemolymph. The second with a sedimentation rate of 10.74 S was comparable to the major lipoprotein of egg yolk. A similar comparison could be made following electrophoretic analyses in native polyacrylamide gels. Electrophoresis in SDS-polyacrylamide gels revealed three major apolipoproteins common to egg and vitellogenic HDL3. A fourth apolipoprotein was found in both male and female HDL3. In contrast to mammalian HDL, none of these crustacean apolipoproteins had a molecular weight less than 82 000. One of these apolipoproteins appears to be comparable physicochemically to the enteric form of apolipoprotein B in mammals.

Insect lipid transfer particle catalyzes diacylglycerol exchange between high-density and very-high-density lipoproteins.

Facilitated diacylglycerol exchange between Manduca sexta [3H]diacylglycerol-labeled high-density lipophorin and Heliothis zea very-high-density chromolipoprotein was studied. M. sexta lipid transfer particle was employed in assays which measured exchange of [3H]diacylglycerol. Following incubations with lipid transfer particle, donor and acceptor lipoproteins were reisolated by density-gradient ultracentrifugation to determine facilitated exchange. The reaction was limited to diacylglycerol exchange, while donor or acceptor particle apoprotein exchange did not occur. Lipid analysis of donor and acceptor lipoproteins after the lipid-exchange reaction revealed that the labeled diacylglycerol remained unchanged. Lipid transfer particle-catalyzed diacylglycerol exchange was linear up to 0.3 micrograms lipid transfer particle protein in a standard assay and exchange occurred at a rate of 2.5 micrograms diacylglycerol min-1.micrograms-1 lipid transfer particle protein. The assay method was used to show that the hemolymph concentration of lipid transfer particle increased during development.

gamma-Carboxyglutamic acid distribution.

The distribution of the vitamin K-dependent amino acid, gamma-carboxyglutamic acid was examined in proteins from a variety of sources. Proteins examined include purified rat and bovine coagulation proteins, barium citrate-adsorbing proteins from trout plasma, lamprey plasma, earthworm hemolymph, army worm hemolymph, lobster hemolymph, E. coli B/5, soybean leaf, the protein lysate from the hemolymph cell of the horseshoe crab and parathyroid extract. Other purified proteins examined included human alpha-1-antitrypsin, pepsinogen, S-100, fetuin, tropomyosin-troponin and complement protein C-3. Of these, only the blood-cotting proteins and the vertebrate plasma samples were shown to contain gamma-carboxyglutamic acid.

Agglutinin from Limulus polyphemus. Purification with formalinized horse erythrocytes as the affinity adsorbent.

We have purified an agglutinin from the hemolymph of Limulus polyphemus about 1500-3000-fold by adsorption to formalinized horse erythrocytes, elution with N-acetylneuraminic acid and subsequent fractionation on Sephadex G-200. Recovery was in the range of 50 percent. On ultracentrifugation the agglutinin behaves as an homogenous protein with a molecular weight of about 460 000. On polyacrylamide gel electrophoresis of the dissociated protein in sodium dodecylsulfate we found a single prominent diffuse band with an apparent molecular weight of 22 000 plus or minus 2000. This band contained carbohydrate as determined by periodic acid-Schiff staining. The intensity of staining compared with standards suggested a carbohydrate content of less than 4 percent. The protein contains a preponderance of acidic amino acids and has an isoelectric point of 4.83.5 residues per 1000 of glucosamine were detected on amino acid analysis. Agglutination of formalinized horse erythrocytes by the purified protein is inhibited not only by N-acetylneuraminic acid but also by D-glucuronic acid; but not by a number of other monosaccharides. D-Glucuronic acid may be used in place of N-acetylneuraminic acid as the eluting sugar in the purification procedure.

Structure and composition of ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells.

Ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated by high resolution transmission electron microscopy, electron diffraction and chemical analysis. Hemosiderin particles isolated from thalassemic spleens also have been studied. The results show that there is a marked difference in structure and composition of the biomineral phases. Human ferritin and hemosiderin particles are single domain crystals of hydrated iron (III) oxide (ferrihydrite). Lattice fringes were low in contrast and often discontinuous within the central regions of the core. Heat treatment of human ferritins results in a 5 A shrinkage in particle size and an increase in the single crystalline nature of the core. In contrast, lattice images and electron diffraction of limpet and bacterial cores show no evidence of long-range crystallographic order. Chemical analysis indicates a high inorganic phosphate (Pi) (Fe/Pi = 1.71) content in bacterial ferritin compared with human ferritin (thalassemic) (Fe/Pi = 21.0). The high Pi content of bacterial ferritin suggests a hydrated amorphous iron (III) phosphate mineral core. Structural disorder within the limpet and bacterial cores may be associated with increased Pi content and increased oxidation in Fe(II), resulting in rapid mineral deposition. Growth of the iron (III) oxide cores in human ferritin is discussed on the basis of high resolution electron microscopy results.

Genic variation in abundant soluble proteins of Drosophila melanogaster and Drosophila pseudoobscura.

Genic variation was surveyed for 20 proteins of Drosophila melanogaster and 18 proteins of D. pseudoobscura. Analysis was by extraction and one-dimensional polyacrylamide gel electrophoresis under nondenaturing conditions, followed by staining with Coomassie Brilliant Blue to detect soluble proteins present in relatively large amounts ("abundant soluble proteins"). D. melanogaster was polymorphic for 65% of its protein loci and an individual was heterozygous for 10% of its loci. The respective figures for D pseudoobscura were 61% and 11%. These estimates of genic variation fall between previously published estimates obtained for these species by one-dimensional electrophoresis of soluble enzymes and those obtained by two-dimensional electrophoresis of solubilized abundant proteins. However, variation for both species could be strongly partitioned between loci, on the basis of tissue and stage expression of the proteins. The results are discussed with respect to their bearing on the possibility that abundant proteins constitute a distinct class of proteins less polymorphic than soluble enzymes.

Interactions between sex-transformation mutants of Drosophila melanogaster. I. Hemolymph vitellogenins and gonad morphology.

In Drosophila, vitellogenins (yolk protein precursors) are synthesized by the female fat body, secreted into the hemolymph and subsequently taken up by the developing oocytes. The male fat body, on the other hand, does not do this even when immature ovaries are transplanted into the body cavity and grow. Thus, the hemolymph vitellogenins serve as an easily detectable sexually dimorphic biochemical marker.--We have examined hemolymph vitellogenins by SDS polyacrylamide gel electrophoresis in flies carrying various sex-transformation mutants (dsx, tra, tra-2 and tra-2OTF) singly and in all possible combinations. Chromosomal females homozygous for tra or tra-2 have no detectable hemolymph vitellogenins, while those homozygous for tra-2OTF exhibit appreciable levels of these proteins. Flies homozygous for dsx, both X/X and X/Y, have hemolymph vitellogenins, although the amount is consistently smaller in the latter. Indeed, X/Y; dsx/dsx is the only genotype in which hemolymph vitellogenins are detected in the X/Y flies. A clear hierarchy of epistasis exists among these sex-transformation mutants when they are examined in various combinations: dsx greater than tra, tra-2 greater than tra-2OTF. Moreover, an interaction between tra-2OTF and tra was seen in these experiments: X/X; tra-2OTF/tra-2OTF flies show the presence of only a trace of hemolymph vitellogenins when they are made heterozygous for tra. These results, combined with observations on gonad morphology, are discussed with respect to the Baker and Ridge (1980) hypothesis of sex determination.

Detection and microsequencing of juvenile hormone-binding proteins of an insect by the use of an iodinated juvenile hormone analog.

An [125I]iodinated juvenile hormone (JH) analog can be used as a sensitive and highly selective probe for the visualization of high-affinity, (JH)-specific binding proteins from insect hemolymph samples. The proteins can be detected in their native form using a two-dimensional (isoelectric focusing then native gradipore gel) separation of the crude protein mixture containing the 125I-labeled iodinated JH analog. The proteins can be transferred to activated glass fiber paper by electroblotting, and the location of the bound gamma-emitter can be found by exposure of the dried gel or the electroblot to X-ray film. The radiolabeled protein spot can be excised from the Coomassie-stained glass fiber paper and subjected directly to gas-phase N-terminal amino acid sequencing. This non-destructive, non-denaturing technique may have wide applicability in identifying and sequencing ligand-specific binding proteins in complex mixtures.

Biophysical studies on the lipid transfer particle from the hemolymph of the tobacco hornworm, Manduca sexta.

Hydrodynamic studies conducted in the analytical ultracentrifuge provided evidence for two populations of lipid transfer particle (LTP) when centrifuged in a buffer solution containing 10 mM Tris, pH 8.0/100 mM KCl. The apparent sedimentation coefficients of the two species was 23.3 S and 15.3 S. Upon changing the buffer pH to 7.0 or 5.7, two species of LTP were still present but the ratio of their relative abundance was altered. When the KCl concentration in the buffer was lowered to 50 mM the sample sedimented as a single species with an apparent S20,w of 22.9 S. In higher ionic strength buffers (10 mM succinate, pH 5.7/500 mM KCl) LTP sedimented with an apparent S20,w of 14.8 S. Further experiments revealed that these two forms are interconvertable as a function of buffer ionic strength. Given previous estimates of the molecular size of LTP we concluded that the slower sedimenting peak observed at high ionic strength represents monomeric LTP while the faster sedimenting material observed at low ionic strength is likely to be an aggregated state of LTP. This interpretation is supported by molecular weight determinations made by sedimentation equilibrium experiments conducted in 10 mM succinate, pH 5.7/500 mM KCl which yielded a particle Mr = 887,000. Circular dichroism spectra of monomeric LTP sample revealed 6% alpha-helix, 49% beta-sheet, 7% beta-turn and 35% random coil while aggregated LTP contained 13% alpha-helix, 66% beta-sheet and 21% random coil. The transfer activity of the two LTP forms was assayed and found to be the same indicating that either the state of LTP aggregation did not affect transfer activity or that upon exposure to a large excess of lipoprotein substrate disaggregation, without loss of activity, occurs.

Amino acid sequence around the thiolester of alpha 2-macroglobulin from plasma of the crayfish, Pacifastacus leniusculus.

Alpha 2-Macroglobulin (alpha 2 M) was isolated from plasma of the freshwater crayfish, Pacifastacus leniusculus, using ultracentrifugation, ion-exchange chromatography and gel filtration techniques. The Pacifastacus alpha 2 M molecule (P alpha 2 M) was radio-actively labeled in the thiol ester structure with iodo [14C]acetic acid in the presence of methylamine. After reduction and carboxymethylation of the protein, it was digested with trypsin. A 14C-labeled tryptic peptide was sequenced and contained an amino acid sequence very similar to other known thiol ester sequences from human alpha 2 M and related proteins. The N-terminal sequence of P alpha 2 M was related to that recently determined for lobster alpha 2 M [(1987) J. Biol. Chem. 262, 14606-14611].

Protease inhibitor controls prophenoloxidase activation in Manduca sexta.

Prophenoloxidase from the hemolymph of tobacco hornworm Manduca sexta can be activated by a specific activating enzyme found in the cuticle. Inhibition studies with benzamidine, diisopropyl phosphofluoridate and p-nitrophenyl-p'-guanidinobenzoate indicate that the activating enzyme is a trypsin-like serine protease. An endogenous protease inhibitor, isolated from the hemolymph of Manduca larvae, inhibits the prophenoloxidase activation mediated by this enzyme. These results indicate that the probable physiological role of endogenous protease inhibitor is to control the undesired activation of prophenoloxidase in the hemolymph.

Purification and partial characterization of an erythroagglutinin from the hemolymph of scorpion, Heterometrus bengalensis.

An erythroagglutinin from the hemolymph of the scorpion, Heterometrus bengalensis, has been purified by gel filtration and ion-exchange chromatography. Its homogeneity has been demonstrated by polyacrylamide gel electrophoresis. The purified agglutinin appears to be a monomeric protein having a possible molecular weight between 146,000 and 148,000. It has no divalent cation requirement for erythroagglutination. The erythroagglutination is not inhibited by saccharides, glycoproteins and mucin. Identical erythroagglutination pattern is obtained with normal as well as neuraminidase treated erythrocytes.

The interaction of lactose-specific lectins with acid-treated and lactose-substituted sepharose.

The binding of two lectins, one a galactosyl/lactosyl and the other a lactosyl binding protein, to various Sepharose 4B derivatives has been investigated. The adsorbents, lactose-substituted Sepharose, acid-treated Sepharose and acid-treated, lactose-substituted Sepharose, were each tested with regard to their overall binding capacity and for the ability to separate the lectins by differential elution with solutions of galactose and lactose. The binding capacity for both lectins decreased in the order Lac-acid-Sepharose greater than Acid-Sepharose greater than Lac-Sepharose much greater than Untreated Sepharose. The ability of the gels to bind both lectins with a sufficient affinity to allow the proteins to be purified by differential elution decreased in a similar order. Acid-treated, lactose-substituted Sepharose proved the most useful gel and was utilised to isolate each lectin in a pure form.