Juvenile hormone-induced biosynthesis of vitellogenin in Leucophaea maderae

Isolated abdomens of virgin female Leucophaea maderae, a South American cockroach, could be induced by a single injection of juvenile hormone I to synthesize vitellogenin. Synthetic capacity was assayed by removal of the fat body at various times after induction and incubation in organ culture with radioactive leucine for 5 h. Under these conditions, active tissues secrete radioactive vitellogenin into the medium after a lag of 2 h and continue secretion at a fixed rate for up to 8 h. Vitellogenin in the tissue reaches a steady-state level after about 3 h. By 5 h, approximately 80% of the newly synthesized vitellogenin can be found in the medium and constitutes 75% of the secreted protein.Vitellogenin does not appear in the medium until 18 h after juvenile hormone induction, rises to a maximum between 72 and 96 h, and has declined to half by 120 h postinduction. At the maximum, about 75% of the total protein secreted is vitellogenin, a more than 50-fold stimulation over injected controls. Since virtually the same quantitative effect of juvenile hormone was found in abdomens with and without ovaries, secondary stimulation by ovarian hormones does not appear to be involved in vitellogenin synthesis in this insect.Oocyte vitellogenin exists as a 560,000 molecular weight monomer complex and a 1,590,000 molecular weight trimer. The smaller complex is only found in young oocytes. By sodium dodecyl sulfate-gel electrophoresis, we find that the 560,000 molecular weight 14S monomer consists of four discrete peptides in the ratio A₁B₁C₂D₂ with molecular weights 118,000, 87,000, 57,500, and 96,000, respectively. The 28S trimer contains only the three peptides A, B, and C in the ratio A₁B₃C₂. These stoichiometries satisfactorily account for the molecular weights of both complexes when corrected for the lipid content. Maturation of 14S to 28S probably involves specific proteolytic conversion of D to B^D, a different peptide of the same size as B, suggesting that the true composition of 28S is A₁B₁B₂^DC₂.The 14S vitellogenin synthesized and secreted by fat body in tissue culture consists mostly of larger polypeptides. There is a major fraction of 179,000 molecular weight, a minor fraction of >260,000 molecular weight, and other smaller polypeptides. Although the exact relationship between these polypeptides and the mature subunits of vitellogenin are not yet clear, it is evident that the multiple protein subunits of this complex protein are synthesized as one or more precursor proteins, followed by proteolytic processing to the mature size.     

Inhibitors of microsomal oxidations in insect homogenates

1. Homogenates of insect tissues were assayed for enzymes capable of oxidizing p-nitrotoluene to p-nitrobenzoic acid. 2. Locust fat-body homogenate 10000g supernatant was an effective enzyme and required no added cofactors. 3. Homogenates of other insects or locust organs and 10000g sediment from locust fat-body were not active and inhibited microsomal oxidations carried out by locust fat-body or rabbit liver enzyme. 4. Inhibitory power was high in homogenates of whole flies and of fly heads or thoraces. 5. Inhibition appeared to involve both irreversible inactivation of enzyme and the removal of essential cofactors.     

Vitellogenin synthesis and characterisation of the liver estrogen receptor in the neotenous salamander Ambystoma mexicanum

The neotenous salamander Ambystoma mexicanum reaches sexual maturity without completing metamorphosis. Females must, therefore, synthesize vitellogenin, the precursor of the egg-yolk proteins. We show that livers of female axolotls synthesize and secrete a phosphoprotein which migrates with Xenopus vitellogenin on SDS-gels and is precipitated by antibody prepared against Xenopus vitellogenin. The livers of male axolotls do not normally synthesize this protein but can be induced to do so by treatment in vivo with estradiol. A receptor with a high affinity for estradiol (K_d = 0.3 × 10^?9M) was found in the nuclei prepared from livers of male and female axolotls. It sediments at 3.7 S at 0°C in sucrose gradients containing 0.5 M KCl. Each nucleus contains about 1300 binding sites for estradiol, 13 times the number found in normal male Xenopus nuclei, but as axolotl nuclei are about 12 times larger, the concentrations of binding sites are similar. In contrast to Xenopus, there is no detectable increase in the number of nuclear binding sites following estrogen treatment. We conclude that the controls affecting both the appearance of vitellogenin inducibility and the induction of vitellogenin synthesis differ between the two species A. mexicanum and Xenopus laevis.     

Protection of tryptic-sensitive sites in the large subunit of ribulosebisphosphate carboxylase/oxygenase by catalysis

Limited tryptic proteolysis of spinach (Spinacia oleracea) ribulose bisphosphate carboxylase/oxygenase (ribulose-P₂ carboxylase) resulted in the ordered release of two adjacent N-terminal peptides from the large subunit, and an irreversible, partial inactivation of catalysis. The two peptides were identified as the N-terminal tryptic peptide (acetylated Pro-3 to Lys-8) and the penultimate tryptic peptide (Ala-9 to Lys-14). Kinetic comparison of hydrolysis at Lys-8 and Lys-14, enzyme inactivation, and changes in the molecular weight of the large subunit, indicated that proteolysis at Lys-14 correlated with inactivation, while proteolysis at Lys-8 occurred much more rapidly. Thus, enzyme inactivation is primarily the result of proteolysis at Lys-14. Proteolysis of ribulose-P₂ carboxylase under catalytic conditions (in the presence of CO₂, Mg²⁺, and ribulose-P₂) also resulted in ordered release of these tryptic peptides; however, the rate of proteolysis at lysyl residues 8 and 14 was reduced to approximately one-third of the rate of proteolysis of these lysyl residues under noncatalytic conditions (in the presence of CO₂ and Mg²⁺ only). The protection of these lysyl residues from proteolysis under catalytic conditions could reflect conformational changes in the N-terminal domain of the large subunit which occur during the catalytic cycle.     

Novel cathepsin L-like protease from dermestid beetle Dermestes frischii maggot

A novel cathepsin L-like protease from dermestid beetle Dermestes frischii maggot guts was obtained and investigated. The protease was isolated through affinity chromatography at arginine-diasorb followed by FPLC gel-filtration at Superdex 75. Protease is active against chromogenic peptide substrates, containing Arg or Leu in P1 position and a hydrophobic residue in P2 position. PH optimum is about 4,5 and temperature optimum at 40 °C. Enzyme is inhibited completely by HgCl₂ and leupeptin that prove it’s belonging to cysteine proteases of papain family.cDNA analysis of cathepsin L-like protease showed that protein sequence consists of 339 amino acid residues. Mature cysteine protease contains 219 amino acid residues corresponding to molecular mass 24027.20 Da. Residues of the active site were identified: Gln¹⁴⁰, Cys¹⁴⁶, His²⁸⁵, Asn³⁰⁶ and Trp³⁰⁸. Calculated pI is 4,73. The amino acid sequence of the cystein protease from dermestid beetle displays high structural homology with cathepsin L of other insects.     

The vitellogenin of Leucophaea maderae: Synthesis as a large phosphorylated precursor

Phosphorylation of vitellogenin (yolk protein precursor) and vitellin (major yolk protein) polypeptides of Leucophaea maderae was studied by [³²P]ortho phosphate labeling and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography. The vitellogenin molecule was isolated from the hemolymph and fat body by antibody precipitation and high-performance liquid chromatography (HPLC), and shown to consist of at least five polypeptides (“subunits”) which had apparent molecular masses of 155, 112, 95, 92 and 54 kD. Labeling studies with ³²P showed that the covalently attached phosphorus was distributed in an uneven fashion among the five polypeptides. The two heavily-phosphorylated polypeptides, 112 and 54 kD, corresponded to the large and small, mature vitellin subunits. Quantitative SDS-PAGE analysis of long-term ³²P-labeled vitellin showed that these large and small “subunits” contained 55 and 30%, respectively, of the total radioactivity.When fat body was pulse-labeled with ³²P we found a heavily-phosphorylated intracellular 215 kD polypeptide which was precipitable with anti-vitellogenin. The synthesis of this intracellular precursorform of vitellogenin (pre-Vg) was under absolute juvenile hormone control. In vitro³²P pulse-chase experiments showed that pre-Vg was proteolytically processed within the fat body into some (or possibly all) of the mature vitellogenin subnits. Furthermore, peptide mapping confirmed that all of the phosphorylated vitellogenin subunits were derived from pre-Vg. Since previous studies have shown that phosphoserine residues account for essentially all of the covalently-attached phosphorus of the native vitellogenin molecule, we speculate that the asymmetric pattern of vitellogenin and vitellin subunit-phosphorylation is due to an uneven distribution of phosphoserine residues along the initial pre-Vg polypeptide chain. Finally, we conclude that phosphorylation of vitellogenin occurred post-translationally in the fat body endoplasmic reticulum because we could identify ³²P-labeled pre-Vg in purified microsomal vesicles but not in nascent vitellogenin polypeptide chains attached to vitellogenin polyribosomes.     

Endocrine control of vitellogenin synthesis and vitellogenesis in Triatoma protracta.

The corpus allatum (CA) is required for vitellogenesis in the blood-sucking reduviid, Triatoma protracta, as seen by the total lack of yolk deposition in allatectomized females. Normally the CA becomes active within a day after emergence. After a period of activity during which unfed virgins may mature a few eggs, the CA is inhibited via its neural connectives from the brain. The CA is activated by mating, while a blood meal provides an additional stimulus for vitellogenesis. If the ventral nerve cord (VNC) is severed within 48 hr after copulation, the mating stimulus does not get through. However, the pathway of the feeding stimulus does not involve the VNC. The brain does not have any allatotropic or gonadotropic function in this species.A female-specific protein (vitellogenin) was identified by immunoelectrophoresis in the haemolymph of egg-maturing females. This protein is taken up by the oöcytes immunologically unaltered and forms the bulk of the yolk. Allatectomy at emergence prevents the appearance of the vitellogenin, and the topical application of JH_III to allatectomized females led to its synthesis de novo, as shown by the incorporation of labelled precursors into vitellogenin. From its mobility in polyacrylamide gels of different concentrations, the molecular weight of the yolk protein is estimated to be 4.37 × 10⁵ daltons.     

Studies on the structure of the calcium-dependent adenosine triphosphatase from rabbit skeletal muscle sarcoplasmic reticulum

The linear arrangement of the three fragments of Ca²⁺-ATPase from rabbit skeletal muscle sarcoplasmic reticulum with molecular weights of 20,000, 30,000, and 45,000 obtained by limited tryptic hydrolysis was determined by locating the NH₂-terminal acetylated methionyl residue of the original peptide in the M_r = 20,000 fragment. Since both the M_r = 20,000 and 30,000 polypeptides originate from a M_r = 55,000 fragment which is distinct from the M_r = 45,000 polypeptide, the sequence of these three fragments was determined to be 20,000, 30,000, and 45,000. The M_r = 20,000 fragment was further cleaved by cyanogen bromide to yield a M_r = 7,000 COOH-terminal fragment which is relatively hydrophilic. The NH₂-terminal portion is rich in glutamyl residues. The COOH-terminus of the M_r = 30,000 fragment was determined by both digestion with carboxypeptidases and cyanogen bromide cleavage. Using the partial amino acid sequence of the Ca²⁺-ATPase, it was deduced that the active site phosphoaspartyl residue is 154 amino acids from the COOH-terminus of the M_r = 30,000 fragment and hence approximately 35,000 M_r from the NH₂-terminus of the original Ca²⁺-ATPase molecule. Furthermore, it was shown that the two tryptic cleavages of the Ca²⁺-ATPase generating these three large fragments were both single hydrolyses of arginylalanine peptide bonds.     

Vitellogenesis and other physiological responses induced by 17-β-estradiol in males of freshwater fish Rhamdia quelen

This study investigated the effects of different doses of 17-β-estradiol (E₂) in Rhamdia quelen. Groups of males exposed to different doses of E₂ (0.1 mg kg^− 1, 1 mg kg^− 1 and 10 mg kg^− 1) were compared with non-exposed male and female fish groups. Among the considered biomarkers, no significant differences were observed for micronuclei test, reduced glutathione concentration and lipid peroxidation. All E₂-treated individuals had decreased glutathione S-transferase activity. Increased catalase and superoxide dismutase activities, increased vitellogenin expression and decreased metallothionein concentration were observed in males treated with the highest dose. Liver of all test groups showed necrotic areas, but cytoplasm vacuolization was again found only in the individuals exposed to highest dose. E₂ causes deleterious hepatic effects to R. quelen, and vitellogenin expression, catalase and superoxide dismutase activity and metallothionein concentration represent appropriate biomarkers for studying E₂ effects. Additionally, the response of some biomarkers was similar in males exposed to E₂ and unexposed females, and therefore exposure to endocrine disruptors may cause consequences for fish populations.     

Purification and properties of vitellogenin and vitellin from a tick,Ornithodoros moubata

Summary The vitellogenin (Vg) and vitellin (Vn) of a soft tick,Ornithodoros moubata, were purified from reproductive female haemolymph and from eggs, respectively. The Vg preparation displayed two components (Vg-1 and Vg-2) on polyacrylamide gel electrophoresis (PAGE), both of which reacted with anti-Vn serum. The Vn and Vg-2 preparations were homogeneous as judged by PAGE, electron microscopy, and immunodiffusion, whereas the Vg-1 always contained a small amount of Vg-2. The electron micrographs of Vn and Vg-2 showed a rugby-ball shape with a cleft at the middle of the molecules, while Vg-1 appeared as half of Vn or Vg-2 in shape and size. This together with the data on molecular weights (600, 000 for Vn and Vg-2, 300, 000 for Vg-1) suggests that the Vn and Vg-2 are dimers of Vg-1. Six polypeptides (P1–P6, mol wt 100, 000–215, 000) for Vgs and six polypeptides (P3–P8, mol wt 50, 000–160, 000) for Vn were demonstrated by SDS PAGE; P3–P6 were common to Vgs and Vn. These observations suggest proteolytic processing from larger to smaller polypeptides. The Vn contained 7.6% lipids with triacylglycerol as the major neutral lipid and 12.4% carbohydrates with mannose as the major sugar. The Vn and Vgs showed a reddish brown coloration due to the presence of haem compound(s). The amino acid composition of Vn was high in glutamic acid, proline, valine and leucine but low in methionine and isoleucine. The isoelectric point of Vn and Vgs was pH 6.9. Unlike Vg and Vn of insects, the Vgs and Vn of tick were soluble in distilled water.Abbreviations Vg vitellogenin - Vn vitellin - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

Short-term exposure to waterborne free silver has acute effects on membrane current of Xenopus oocytes

Waterborne free silver can cause osmo- and ionoregulatory disturbances in freshwater organisms. The effects of a short-term exposure to extracellular Ag⁺ ions on membrane currents were investigated in voltage-clamped defolliculated Xenopus oocytes. At a holding potential of − 60 mV, ionic silver (1 μM Ag⁺) increased inward currents (=I_Ag) from − 8 ± 2 nA to − 665 ± 41 nA (n = 74; N = 27). I_Ag activated within 2 min of silver exposure and then rose impetuously. This current was largely reversible by washout and repeatable. I_Ag reversed around − 30 mV and rectified slightly at more positive potentials. Na⁺-free bath conditions reduced the silver-induced current to a smaller but sustained current. The response to silver was abolished by the Cl⁻ channel blockers DIDS and SITS, whereas niflumic acid strongly potentiated I_Ag. Intraoocyte injection of AgNO₃ to about 1 mM [Ag]_i strongly potentiated I_Ag. Extracellular application of either dithiothreitol (DTT), a compound known to reduce disulfide bridges, or l-cysteine abolished Ag⁺-activated increase of membrane current. In contrast, n-ethylmaleimide (NEM) which oxidizes SH-groups potentiated I_Ag. Hypoosmotic bath solution significantly increased I_Ag whereas hyperosmolar conditions attenuated I_Ag. The activation of I_Ag was largely preserved after chelation of cytosolic Ca²⁺ ions with BAPTA/AM. Taken together, these data suggest that Xenopus oocytes are sensitive to short-term exposure to waterborne Ag⁺ ions and that the elicited membrane currents result from extra- and intracellular action of Ag⁺ ions on peptide moieties at the oocyte membrane but may also affect conductances after internalization.     

A Novel Glucagon-Related Peptide (GCRP) and Its Receptor GCRPR Account for Coevolution of Their Family Members in Vertebrates

The glucagon (GCG) peptide family consists of GCG, glucagon-like peptide 1 (GLP1), and GLP2, which are derived from a common GCG precursor, and the glucose-dependent insulinotropic polypeptide (GIP). These peptides interact with cognate receptors, GCGR, GLP1R, GLP2R, and GIPR, which belong to the secretin-like G protein-coupled receptor (GPCR) family. We used bioinformatics to identify genes encoding a novel GCG-related peptide (GCRP) and its cognate receptor, GCRPR. The GCRP and GCRPR genes were found in representative tetrapod taxa such as anole lizard, chicken, and Xenopus, and in teleosts including medaka, fugu, tetraodon, and stickleback. However, they were not present in mammals and zebrafish. Phylogenetic and genome synteny analyses showed that GCRP emerged through two rounds of whole genome duplication (2R) during early vertebrate evolution. GCRPR appears to have arisen by local tandem gene duplications from a common ancestor of GCRPR, GCGR, and GLP2R after 2R. Biochemical ligand-receptor interaction analyses revealed that GCRP had the highest affinity for GCRPR in comparison to other GCGR family members. Stimulation of chicken, Xenopus, and medaka GCRPRs activated Gα_s-mediated signaling. In contrast to chicken and Xenopus GCRPRs, medaka GCRPR also induced Gα_q/11-mediated signaling. Chimeric peptides and receptors showed that the K¹⁶M¹⁷K¹⁸ and G¹⁶Q¹⁷A¹⁸ motifs in GCRP and GLP1, respectively, may at least in part contribute to specific recognition of their cognate receptors through interaction with the receptor core domain. In conclusion, we present novel data demonstrating that GCRP and GCRPR evolved through gene/genome duplications followed by specific modifications that conferred selective recognition to this ligand-receptor pair.     

Chloride-dependent transmural potential in the rectal wall of Schistocerca gregaria

Rectum transmural potential (PD) and short-circuit current (I_sc) of the desert locust, Schistocerca gregaria, have been studied in vitro, with everted rectal wall preparations in solutions of different ionic composition. Initially, a PD of about 35 mV (lumen positive) and a I_sc of about 300 μA cm^?2 were recorded. Omission of sodium or potassium (Tris as substitute), from the luminal side or from both sides led to an increase of 4 to 6 mV in PD (lumen more positive) together with an increase in I_sc. In the absence of chloride alone (sulphate as substitute) the PD quickly dropped to nearly zero. In each case the control values were recovered on replacing the corresponding ions. Neither the PD nor the I_sc changed when substitutions affected only the haemocoelic solution. These findings corroborate the assumption that active transport of chloride ions from lumen to haemolymph is the major factor for transmural PD and account for the short-circuit current in the rectal wall of desert locust. A working scheme is given to explain the influence of sodium, potassium, and chloride ions on the PD.     

SDS-PAGE comparative studies on the polyhedral and viral polypeptides of the nuclear polyhedrosis viruses of Mamestra brassicae, Autographa californica, and Lymantria dispar

After solubilization of polyhedra of Autographa californica, Lymantria dispar, and Mamestra brassicae nuclear polyhedrosis viruses, PAGE showed at least eight distinct polyhedral polypeptide bands. Whereas the molecular weights of the major polypeptide were similar for the three NPVs (28.0–30.0 kdalton), characteristic differences between the species were found for the minor polypeptides having molecular weights in the range from 12.4 to 62.0 kdalton. It is assumed that these polypeptides are not generated by polyhedral alkaline protease since they are detected after protease inactivation. The data demonstrate that different baculoviruses can be distinguished from each other by SDS-PAGE of their polyhedral polypeptides.     

Studies on the structure of rabbit muscle aldolase: Amino acid sequence of cysteine-containing peptides

Five cysteine-containing peptides have been isolated in nearly stoichemometric yields from the tryptic digests of the NH₂^? and COOH-terminal BrCN peptides of rabhit muscle aldolase and their sequence determined. Peptides NS1, NS2, and NS3, from the NH₂-terminal part of the enzyme have the following sequences: NS1, Val-Asp-Pro-Cys-Ile-Gly-Gly-Val-Ile-Leu-Phe-His-Glu-Thr-Leu-Tyr-Gln-Lys; NS2, Cys-Val-Leu-Lys; NS3, Cys-Ala-Glu-Tyr-Lys. The two peptides isolated from the COOH-terminal region are: CS1, Ala-Leu-Ala-Asn-Ser-Leu-Ala-Cys-Gln-Gly-Lys and CS2, Cys-Pro-Leu-Leu-Trp-Pro-Lys-Ala-Leu-Thr-Phe-Ser-Tyr-Gly-Arg. The Lys-Ala bond in peptide CS2 was found to be resistant to tryptic hydrolysis. The results provide the basis for assigning the positions of cysteine residues in the polypeptide chain. Cys-72 in peptide NS1 and Cys-336 in peptide CS1 are the residues that form a disulfide bridge when the enzyme is inactivated by oxidation with an o-phenanthroline-Cu²⁺ complex; Cys-287 in peptide CS2 in one of the two exposed residues, while Cys-134 and Cys-149 in peptides NS2 and NS3, respectively, are buried in the native enzyme. All of eight cysteine-containing peptides of rabbit muscle aldolase have now been sequenced, and structural homology of the α and β subunits extended to these regions.     

Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity

The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X₃X₂X₁-aminomethylcoumarin) and by determining specificity constants (k_cat/K_m), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X₁ position, basic residues or Trp in the X₂ position, and Pro ≫ Ala > Trp in the X₃ position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.