Silkworm midgut proteins interacting with a hemolymph protease inhibitor, CI-8

CI-8 is the chymotrypsin inhibitor in hemolymph from the silkworm, Bombyx mori. It occurs in the midgut at the spinning stage of larva, but little information on the mechanism of its uptake in the midgut is available. We found that two polypeptides interacting with CI-8 are in the midgut membrane, and we purified them using a biotinylated CI-8, viz., p29 and p60, having molecular sizes of 29 kDa and 60 kDa respectively. The structures of p29 and p60 were examined by N-terminal amino acid sequencing and peptide mass mapping, including tryptic digestion. p29 was highly similar to the matured 19G1-30K lipoprotein from hemolymph, but p60 was novel. Purified p29 was recognized by anti-19G1-30K antibody, and was confirmed to be similar to 19G1-30K. The antibody also neutralized the CI-8 binding ability of p29 in the midgut membrane. p29 and p60 are perhaps proteinaceous factors involved in the uptake of CI-8 into the midgut through the membrane.     

Selective transport of the mulberry leaf urease from the midgut into the larval hemolymph of the silkworm,Bombyx mori

Just before spinning, larvae of the silkworm, Bombyx mori, absorb intact urease of the host plant (mulberry leaf) from the midgut lumen into the hemolymph. In order to investigate whether the transport of the mulberry leaf urease is selective, crude proteins extracted from the mulberry leaves were labeled with biotin and orally administered to the fifth instar larvae. The biotinylated proteins transported into the hemolymph were detected by ligand blotting using streptavidin. When the biotinylated proteins were administered to 5-day-old fifth instar larvae, a strong signal of a biotinylated protein was detected in the hemolymph 2 days after the administration. In contrast, when the biotinylated mulberry leaf proteins were administered to 3-day-old fifth instar larvae, no signal derived from the biotinylated proteins was detected in the hemolymph. The signal weakened when the biotinylated proteins had been immunoprecipitated before administering to the larvae, indicating that the signal came from the mulberry leaf urease. These results show that the transport of the mulberry leaf urease from the midgut into the hemolymph is selective and larval-stage specific. Subsequently, binding assays were carried out to test the binding ability of the mulberry leaf urease to the brush border membrane in the epithelial cells of larval midgut. The urease was not bound to the brush border membrane vesicles (BBMV) from the midgut of 3-day-old fifth instar larvae, while more than 60% of the total amount of incubated urease was bound to the BBMV from the midgut of 6-day-old fifth instar larvae. The urease binding ability of BBMV correlated with the uptake of the mulberry leaf urease. This suggests that a urease binding molecule(s) exists in the BBM of the midgut epithelium, which is involved in the uptake of the mulberry leaf urease. In addition, the uptake of the mulberry leaf urease into the hemolymph was induced by 20-hydroxyecdysone.     

Host hemolymph proteins and protein digestion in larval Habrobracon hebetor (Hymenoptera: braconidae)

Host plasma proteins and protein digestion in larval parasitoids were studied during trophic interactions of the ectoparasitoid Habrobracon hebetor Say (Hymenoptera: Braconidae), with a host, larvae of the Indianmeal moth, Plodia interpunctella Hübner (Lepidoptera: Pyralidae). We could detect no apparent differences in host hemolymph protein patterns up to 72 h after paralysation and/or parasitization by H. hebetor. A 190 kDa putative apolipophorin I present in host hemolymph could not be detected in the midguts of feeding H. hebetor larvae indicating that it is rapidly digested. The major 60 kDa storage proteins (putative hexamerins) in host hemolymph were detected in the parasitoid midgut and were completely digested 24 h after cessation of feeding and the beginning of cocoon formation. Host hemolymph had a pH of about 6.4. The pH optima of the midgut proteinases in the larval parasitoid were in the alkaline region, but midgut fluid in feeding parasitoid larvae was about pH 6. 8. Based on enzyme activity against selected artificial proteinase substrates including azocasein, N-alpha-benzoyl-L-Arg p-nitroanilide (BApNA), succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAAPFpNA), succinyl-Ala-Ala-Pro-Leu p-nitroanilide (SAAPLpNA), and inhibition by selected proteinase inhibitors, serine proteinases appear to be the predominant class of enzymes involved in protein digestion in the midguts of H. hebetor. There is also an active aminopeptidase (LpNA) associated with the microsomal fraction of midgut preparations. There was no evidence for preoral digestion or ingestion of proteinases from host hemolymph by the parasitoid larva. There was a very active BApNAase in the soluble fraction of midgut extracts. This activity increased on a per midgut basis up to 24 h after the beginning of cocoon formation but decreased rapidly by 48 h. Two major (P1 and P3) and several minor proteinases were detected in midgut extracts of H. hebetor analysed with gelatin zymograms. The apparent molecular mass of P1 varied from 95 to 49 kDa depending on protein loading. P3 had an apparent molecular mass of 39 kDa that was independent of protein loading. In summary, electrophoretic evidence indicates that host hemolymph protein patterns do not change significantly for at least 72 h after paralysation by H. hebetor. The role, if any, of envenomization in preventing breakdown of hemolymph proteins during this time remains to be determined. Because the predominant host hemolymph proteins, a putative apolipophorin I and the putative hexamerins, are readily digested by the serine proteinases present in the midguts of this parasitoid larva, these or similar proteins would provide an easily digested source of dietary amino acids that could be used for development of artificial diets for this beneficial insect.     

Ornithodoros moubata: host immunoglobulin G in tick hemolymph

Hemolymph proteins of a soft tick, Ornithodoros moubata, were analyzed immunochemically and biochemically. The components of tick hemolymph proteins were shown to be totally different from the host (rabbit) serum proteins by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and Coomassie blue or silver stain. However, in the hemolymph of ticks engorged from rabbits immunoglobulin G was detected by immunoblotting analysis with goat anti-rabbit immunoglobulin G. The concentration of rabbit Immunoglobulin G in tick hemolymph changed with the physiological stages after a blood meal. Immunoglobulin G was isolated from tick hemolymph by affinity chromatography on a Protein A-Sepharose 4B column. Analysis of the isolated immunoglobulin G from tick hemolymph with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Ouchterlony double diffusion test showed it to be composed of the same subunits as heavy and light chains of host (rabbit) immunoglobulin G. Tracer experiments showed that 125I-labeled heavy and light chains of immunoglobulin G were detected in an intact form in hemolymph from ticks that sucked 125I-labeled rabbit immunoglobulin G through an artificial membrane. These facts suggested that the host rabbit immunoglobulin G ingested in the tick midgut passed through the gut wall without digestion. By solid-phase enzyme immunoassay, immunoglobulin in the hemolymph was shown to retain its antibody activity.     

Purification and Properties of Ferritin from the Last Instar Larval Hemolymph of Protaetia brevitarsis (Coleoptera)

Ferritin, an iron storage protein, has been purified from the last larval hemolymph of Protaetia brevitarsis (coleoptera) by KBr density gradient ultracentrifugation and resource Q (anion exchange chromatography) using fast performance liquid chromatography (FPLC) system. The iron content of ferritin was determined by atomic emission spectroscopy and FereneS stain. Ferritin of P. brevitarsis is shown to have two different subunits presented on a SDSPAGE in normal (N) and ironinjected (I) hemolymph. SDS PAGE revealed that the ferritin consists of two major polypeptides of 27 and 28 kDa in normal hemolymph. Interestingly, however, 30 kDa subunit was substituted for 28 kDa when iron was injected into the hemolymph. Apporximate isoelectric points of 27 kDa, 28 kDa, and 30 kDa ferritin subunits were 6.7, 6.75, and 6.8, respectively. Ferritin of P. brevitarsis was detected by FereneS stain and confirmed by Western blotting using its polyclonal antibody. Other characteristics such as amino acid composition and Nterminal amino acid sequence were investigated. Amino acid composition of ferritin (N and I) was rich in alanine, glycine, glutamine or glutamic acid and serine, but poor in histidine, arginine, methionine and phenylalanine.     

Vitellogenesis in spiders: first analysis of protein changes in different reproductive stages of Polybetes pythagoricus

Vitellogenesis represents one of the most vital processes of oviparous species during which various proteins, carbohydrates, and lipids are synthesized and stored inside the developing oocytes. Through analyzing protein changes in the midgut diverticula, hemolymph, and ovaries of females throughout the different vitellogenic stages of the spider Polybetes pythagoricus, we determined the origin of the different proteins involved in the formation of lipovitellins (LVs) along with the existence of a linkage between the hemocyanin and this vital process. An increase in the total protein content of the midgut diverticula, hemolymph, and ovary occurred throughout vitellogenesis followed by a decrease in those levels after laying. The presence of hemocyanin in egg and in LV2, as well as its accumulation in the ovary throughout the vitellogenesis process, was determined. Considering that all biologic processes depend on the correct structure and function of proteins, this study establishes, for the first time for the Order Araneae, the coexistence of three different origins of vitellogenesis-related proteins: one predominantly ovarian involving peptides of 120, 75, 46, and 30 kDa; another extraovarian one originated from the midgut diverticula and represented by a 170 kDa peptide, and a third hemolymphatic one, represented by the 67 kDa peptide.

     

Synthesis and resorption of a humoral chymotrypsin inhibitor, CI-8, by fat body of the silkworm, Bombyx mori

The Bombyx mori hemolymph contains up to 16 chymotrypsin inhibitors (CIs). The present in vitro culture of tissues in Grace's medium indicated that CI-8, which belongs to the largest molecular-size group of CIs with sugar moiety, is synthesized in the fat body and secreted from it during the feeding period. When the fat body from other strain which synthesizes an allelic component (CI-7) instead of CI-8 was incubated in vitro in hemolymph from the strain which has CI-8, the fat body was found to receive CI-8. Thus it was concluded that CI-8, once secreted into the hemolymph, was again sequestered into the fat body after the onset of spinning. Protein granules isolated from the pupal fat body were shown to contain CI-8, indicating that the sequestered CI-8 is present in the protein granules.     

Absorption of dietary triacylglycerol by lipolysis and lipid resynthesis in the mesenteron of larvalAeshna cyanea (insecta,odonata)

Summary Voluntary uptake of triolein, margarine, and lipid-rich natural food (Tubifex) by fasting dragonfly larvae (Aeshna cyanea) led to heavy accumulations of lipid absorption droplets in the enterocytes within 2 days, while subsequent lipid clearance of the midgut epithelium took several weeks depending on the ingested lipid load. No endocytotic lipid uptake was observed after application of a molecular-dispersed fat dye. The smallest lipid droplets first appeared in the subapical groundplasm of the enterocytes and showed a reversible increase in size on their way towards the base. Lipid droplets were also observed at appropriate intervals after oral administration of oleic acid, after feeding margarine in the cold, and after injection of triolein into the isolated midgut.Comparative biochemical analysis after triolein feeding evidenced release of lipase and hydrolytic liberation of FA from TG in the midgut lumen, as well as time-dependent accumulations of TG in the midgut epithelium and of DG in the hemolymph.Oral injection of [¹⁴C] oleic acid was followed by its rapid absorption into the midgut epithelium, where it was utilized for the synthesis of MG and esterification to DG and TG. Discharge of radioactive lipid into the hemolymph occurred in the form of FA and DG, while the rectal fat body showed approximately equal labeling of the FA, DG, and TG fractions.Abbreviations AG acylglycerol - DG diacylglycerol - ER endoplasmic reticulum - FA fatty acid - MG monoacylglycerol - TG triacylglycerol Dedicated to Prof. Dr. Dr. R.Lehmensick, Bonn, in honor of his 85^th birthday.     

Cadherin-like receptor binding facilitates proteolytic cleavage of helix alpha-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin

Cry toxins form lytic pores in the insect midgut cells. The role of receptor interaction in the process of protoxin activation was analyzed. Incubation of Cry1Ab protoxin with a single chain antibody that mimics the cadherin-like receptor and treatment with Manduca sexta midgut juice or trypsin, resulted in toxin preparations with high pore-forming activity in vitro. This activity correlates with the formation of a 250 kDa oligomer that lacks the helix alpha-1 of domain I. The oligomer, in contrast with the 60 kDa monomer, was capable of membrane insertion as judged by 8-anilino-1-naphthalenesulfonate binding. Cry1Ab protoxin was also activated to a 250 kDa oligomer by incubation with brush border membrane vesicles, presumably by the action of a membrane-associated protease. Finally, a model where receptor binding allows the efficient cleavage of alpha-1 and formation of a pre-pore oligomeric structure that is efficient in pore formation, is presented.     

Multicellular-vesicle-promoting polypeptide from Trichoplusia ni: Tissue distribution and N-terminal sequence

An N-terminal amino acid sequence of a 16.9 kDa hemolymph polypeptide, “Vesicle Promoting Factor” (VPF) from Trichoplusia ni, revealed a high sequence homology (70%) with Manduca sexta apolipophorin-III. A polyclonal antibody developed against VPF, however, was not immunoreactive with either purified M. sexta or T. ni apolipophorin-III. Immunoblots of tissue homogenates of T. ni indicated that VPF was present in imaginal wing discs, central nervous system (CNS), silk glands, midgut and hemocytes from fifth instar larvae, and also in the IAL-TND1 cell line which can grow as either fluid-filled multicellular vesicles or multicellular aggregates. VPF was also detected immunologically in the hemolymph of adults of T. ni, and in hemolymph of adults and larvae of Galleria mellonella and Heliothis virescens. Testes, midgut, hemocytes, and wing discs, but not Malpighian tubules, of T. ni released VPF into tissue culture medium during a 3 h incubation period. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Alterations in Biomphalaria glabrata plasma induced by infection with the digenetic trematode Echinostoma paraensei

Alterations in the noncellular hemolymph components of M line Biomphalaria glabrata snails infected with the trematode Echinostoma paraensei for 1, 2, 4, 8, 15, 30, or 60 days were monitored by direct microscopical examination, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with quantitative densitometry. A prominent particulate substance was first noted in the hemolymph of infected snails at 1 day postexposure (PE), persisted through day 15, and subsided by day 30. This substance, which was not observed in control snails, contained 2 major polypeptides of 190-200 and 80-120 kDa. Infection with E. paraensei also induced substantial changes in soluble hemolymph polypeptides. PAGE lanes loaded with plasma samples from M line snails infected for 4, 8, 15, and 30 days exhibited a generalized increase in staining intensity relative to controls. A diffuse band centered at approximately 100 kDa, but of variable width, was selectively enriched relative to control preparations in snails with 4-, 8-, 15-, and 30-day-old infections. Standard protein assays also indicated an increase in total protein content of plasma samples from snails infected for 2-60 days, with significant increases noted at 4, 8, and 30 days. Infected snails of the 10-R2 strain of B. glabrata also contained particulate material in their hemolymph. However, soluble hemolymph components of 10-R2 snails exhibited relatively little change, or declined, as a result of infection. For either strain, no new bands could be detected in plasma samples from infected snails, nor were any bands consistently deleted as a result of infection. Although both snail strains exhibit alterations in hemolymph components as a result of infection, their responses differ qualitatively and quantitatively.     

The characterization of ferritin in an insect

In mammals, the iron storage protein ferritin is predominantly synthesized on free polysomes and accumulates in the cytosol but some is secreted and circulates in the blood as serum ferritin. In insect tissues, on the other hand, iron-containing holoferritin accumulates in the vacuolar system and can be secreted through the Golgi complex. The midgut can secrete it to the gut lumen and other tissues to the hemolymph.Ferritin was isolated from the midgut and hemolymph of fifth instar larvae of Calpodes ethlius, Lepidoptera, Hesperiidae. This holoferritin is stable to heat (75°C) or in the presence of SDS, proteinase K, or urea, has an Mr above 600,000, contains iron and resembles mammalian ferritins in appearance by electron microscopy. Calpodes ferritin is a glycoprotein having N-linked high-mannose oligosaccharides. It is not antigenically related to horse ferritin but is related to that from Manduca sexta, Lepidoptera, Sphingidae. In its native form, Calpodes ferritin has only 3 isoforms with a pI 6.5–7 suggesting a more uniform subunit composition than that in vertebrates. It has two principle subunits, with relative Mrs of 24,000 (L) and 31,000 (G) and two minor subunits with Mrs of 26,000 and 28,000 all of which cross-react with antibody to Manduca ferritin. The 24 kDa subunit is the only one that is not glycosylated. Iron injections induce an increase in the proportion of the 24 kDa subunit. We conclude that Calpodes has ferritin and that it is glycosylated like mammalian serum ferritin.     

Lipophorin interaction with the midgut of Rhodnius prolixus: characterization and changes in binding capacity

Several classes of lipids are transported in insect hemolymph by lipophorin, a major hemolymphatic lipoprotein. The binding of lipophorin to the midgut of the hematophagous insect Rhodnius prolixus was characterized in a midgut membrane preparation, using purified lipophorin radiolabelled in protein moiety ((125)I-HDLp). Lipophorin specific binding to membranes achieved equilibrium after 30-40 min, was sensitive to pH, and was maximal at pH 7.0. In the presence of increasing concentrations of membrane protein, corresponding increases in lipophorin binding were observed. The specific binding of lipophorin to the membrane preparation was a saturable process, with K(d)=0.9+/-0.06 x 10(-7) M and a maximal binding capacity of 70+/-11 ng lipophorin/microg of membrane protein. Lipophorin binding did not depend on calcium, but it was affected by ionic strength and was inhibited in the presence of increasing salt concentrations. Suramin interfered with lipophorin binding to the midgut receptor, and it was abolished in the presence of 2 mM suramin, but at concentrations between 0.05 and 0.2 mM it was slightly increased. Condroitin 4-sulfate also affected lipophorin binding, which was reduced to 56% of control. Pre-incubation of the midgut membrane preparation with trypsin or at high temperature inhibited binding. Midgut capacity to bind lipophorin varied at different days after blood meal. It was highest at second day after feeding, and then gradually decreased.     

Enhanced production of recombinant protein inEscherichia coli using silkworm hemolymph

The effect of silkworm hemolymph on the expression of recombinant protein inEscherichia coli was investigated. The addition of silkworm hemolymph to the culture medium increased the production of recombinant β-galactosidase inE. coli. The production was dependent on the concentration of the added silkworm hemolymph, which increased 2-, 5-, and 8-fold in media supplemented with 1,3, and 5% silkworm hemolymph, respectively. To identify the effective component, the silkworm hemolymph was fractionated by gel filtration column chromatography. A fraction, with a molecular weight of about 30 K was identified as the effective component.     

Metabolic pathways for diacylglycerol biosynthesis and release in the midgut of larval Manduca sexta

The pathway for the synthesis of diacylglycerol in larval Manduca sexta midgut was studied. Fifth instar larvae were fed with [9,10–³H]–oleic acid–labeled triolein and the incorporation of the label into lipid intermediates was analyzed as a function of time. The results showed that the triacylglycerol was hydrolyzed to fatty acids and glycerol in the midgut lumen. In midgut tissue, the labeled fatty acids were rapidly incorporated into phosphatidic acid, diacylglycerol and triacylglycerol, but no significant labeling of monoacylglycerol was observed. Dual-labeling experiments were performed in order to characterize the kinetics of diacylglycerol biosynthesis in the midgut, its incorporation into hemolymph lipophorin and its clearance from hemolymph. The results were best described by a model in which the rate-limiting step in diacylglycerol biosynthesis was the uptake of fatty acid from the lumen of the midgut. Once in the cell the fatty acid was rapidly incorporated in phosphatidic acid and diacylglycerol. Diacylglycerol was converted to triacylglycerol or exported into hemolymph. The interconversion of diacylglycerol and triacylglycerol was fairly rapid, suggesting that triacylglycerol serves as a reservoir from which diacylglycerol can be produced. This mechanism permits the cell to maintain a low steady-state concentration of diacylglycerol and yet efficiently absorb fatty acids from the lumen of the midgut.     

Na-dependent uptake of phenylalanine in the midgut of a cockroach (Blabera gigantea)

Summary A net absorption of sodium ions and ofl-phenylalanine, in the absence of chemical gradients, occurs across the isolated midgut of the cockroachBlabera gigantea. Both sodium and amino acid net fluxes were abolished by the haemolymphatic addition of the Na–K ATPase inhibitor ouabain, at a concentration 1 mM. A purified fraction of brush border membranes, prepared from the midgut tissue by Ca-precipitation, was used to investigate the occurrence of a cotransport system in the luminal membrane of the cockroach enterocyte. An inwardly directed Na gradient (100 mM outside the vesicles, 0 mM inside) drives the uphill movement of phenylalanine into the vesicular space, whilst other monovalent cations fail to induce the concentrative uptake of the amino acid. Moreover, the amino acid uptake seems to be dependent on the transmembrane potential, since inwardly directed gradients of different sodium salts determine a decreasing rate of phenylalanine uptake in agreement with the presumptive permeabilities of Na counterions. These data suggest the presence of a Na-phenylalanine cotransport system located on the brush border membrane ofB. gigantea midgut.Abbreviations BBMV brush border membrane vesicles - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - PD transepithelial electrical potential difference - TMA tetramethylammonium - Tris tris-(hydroxymethyl) aminomethane     

Amblyomma americanum: specific uptake of immunoglobulins into tick hemolymph during feeding

The passage of immunoglobulins in the blood meal into hemolymph prompts development of vaccines against internal antigens of ticks, but little is known about kinetics and specificity of the immunoglobulin uptake. We used capillary feeding of adult Amblyomma americanum hard ticks to introduce compounds into the midgut and then examined the hemolymph after various times for their presence and concentration. Immunoglobulins of different sources, albumin, choramphenicol acetyltransferase, inulin, and mannitol were labeled with (125)I, (14)C, or biotin. With the exception of the carbohydrate inulin, all the compounds entered the hemolymph of tick during capillary feeding. The small molecule mannitol had the highest rate of entry at 9% after 6 h. Among proteins, the entry of immunoglobulin G (IgG) of different species into the hemolymph was greater at 6% after 6 h than for the smaller proteins albumin or choramphenicol acetyltransferase at 1 and 3%, respectively. The entry of denatured IgG was equal to that of nondenatured protein. There was no evidence of degradation of the IgG or of its binding to cells once it entered the hemolymph. A monoclonal IgG antibody labeled with biotin entered the hemolymph and retained its ability to bind to its specific antigen in an immunoassay. Although different proteins entered the hemolymph after capillary feeding, there was evidence of a specific mechanism for immunoglobulin uptake.     

The homeodomain protein PBX participates in JH-related suppressive regulation on the expression of major plasma protein genes in the silkworm, Bombyx mori

In the silkworm, Bombyx mori, major plasma proteins referred to as 30K proteins are the most abundant proteins in the hemolymph of final (fifth) instar larvae. Surgical extirpation of corpora allata, the source of a juvenile hormone (JH), causes rapid accumulation of 30K proteins in the hemolymph of fourth instar larvae. The 30K protein 6G1 (30K6G1) gene was repressed in primary cultured fat body cells treated with a JH analog (JHA), methoprene. To identify the JH response element present in the promoter region of the 30K6G1 gene, we performed transfection analyses of the 5'-deletion mutants of the 30K6G1 gene using primary cultured fat body cells, gel retardation assays and in vivo footprinting analysis. The results from those analyses revealed that a JH response element exists in the sequence between positions -147 and -140. When the promoter construct mutated at positions -143, -142, and -141 was transfected to fat body primary cultured cells, the suppression effect on the reporter gene expression caused by JHA was reduced. Gel retardation assay using specific antibody revealed that a PBX protein binds to the JH response element. Northern blot analysis revealed that the gene expression of Bombyx PBX is enhanced in the fat body cells by JHA treatment. These results indicate that PBX proteins are involved in the JH signaling pathway and play an important role in suppressing 30K protein gene expression in the fat body of B. mori.     

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrite uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.Abbreviations EDTA ethylenediaminetetraacetic acid - FAD flavine-adenine dinucleotide - IgG immunoglobulin G - NR nitrate reductase - PM plasma membrane - TX-100 Triton X-100     

Characterization of the carotenoid-binding protein of the Y-gene dominant mutants of Bombyx mori

Carotenoids play important and diverse roles in insects. Recently, we purified and partially characterized a carotenoid-binding protein (CBP) from the wild type of Bombyx mori. In this report, we utilized immunoblotting, ELISA and immunocytochemistry to further characterize and localize the expression of CBP in the larval midgut and silk gland obtained from the wild type and four naturally occurring mutants linked to carotenoids transport. CBP was expressed throughout the 5th stadium, with highest expressions on days 4-5 in the silk gland and days 3-5 in the midgut. Immunoblotting analyses demonstrated the presence of CBP along the middle part of the midgut. Microscopic immunocytochemistry demonstrated that the 33 kDa CBP was uniformly expressed along the brush border of columnar cells in the epithelium of the midgut typifying its function in aiding absorption of dietary carotenoids. Similarly, CBP was highly expressed along the distal membrane of the middle part of the silk gland demonstrating its function in uptake of carotenoids from lipophorin. When the middle silk glands and midguts of the four mutants were incubated with rabbit anti-CBP antibody, only proteins of the Y-gene dominant mutants cross reacted with the antibody further accentuating the hypothesis that the CBP is a Y-gene dependent protein.