Subunit Mass Research Articles

L-Theanine Synthesis Using γ-Glutamyl Transpeptidase from Bacillus licheniformis ER-15

Recombinant γ-glutamyl transpeptidase (rBLGGT) from Bacillus licheniformis ER-15 was purified to homogeneity by ion-exchange chromatography. Molecular masses of large and small subunits were 42 and 22 kDa, respectively. The enzyme was optimally active at pH 9.0 and 60 °C and was alkali stable. K(m) and V(max) for γ-glutamyl-p-nitroanilide hydrochloride were 45 μM and 0.34 mM/min, respectively. L-Theanine synthesis was standardized using a one variable at a time approach followed by response surface methodology, which resulted in approximately 85-87% conversion of L-glutamine to L-theanine within 4 h. The standardized reaction contained 80 mM L-glutamine, 600 mM ethylamine, and 1.0 U/mL rBLGGTin 50 mM Tris-Cl (pH 9.0) at 37 °C. Similar conversions were also obtained with the enzyme immobilized in calcium alginate. Using immobilized enzyme, 35.2 g of L-theanine was obtained in three cycles of 1 L each. The product was purified by Dowex 50W X 8 hydrogen form resin and was confirmed by HPLC and proton NMR spectroscopy.

Collision-induced release, ion mobility separation, and amino acid sequence analysis of subunits from mass-selected noncovalent protein complexes.

In recent years, mass spectrometry has become a valuable tool for detecting and characterizing protein-protein interactions and for measuring the masses and subunit stoichiometries of noncovalent protein complexes. The gas-phase dissociation of noncovalent protein assemblies via tandem mass spectrometry can be useful in confirming subunit masses and stoichiometries; however, dissociation experiments that are able to yield subunit sequence information must usually be conducted separately. Here, we furnish proof of concept for a method that allows subunit sequence information to be directly obtained from a protein aggregate in a single gas-phase analysis. The experiments were carried out using a quadrupole time-of-flight mass spectrometer equipped with a traveling-wave ion mobility separator. This instrument configuration allows for a noncovalent protein assembly to be quadrupole selected, then subjected to two successive rounds of collision-induced dissociation with an intervening stage of ion mobility separation. This approach was applied to four model proteins as their corresponding homodimers: glucagon, ubiquitin, cytochrome c, and β-lactoglobulin. In each case, b- and y-type fragment ions were obtained upon further collisional activation of the collisionally-released subunits, resulting in up to 50% sequence coverage. Owing to the incorporation of an ion mobility separation, these results also suggest the intriguing possibility of measuring complex mass, complex collisional cross section, subunit masses, subunit collisional cross sections, and sequence information for the subunits in a single gas-phase experiment. Overall, these findings represent a significant contribution towards the realization of protein interactomic analyses, which begin with native complexes and directly yield subunit identities.

Top‐down protein identification of proteasome proteins with nanoLC‐FT‐ICR‐MS employing data‐independent fragmentation methods

A comparison of different data-independent fragmentation methods combined with LC coupled to high-resolution FT-ICR-MS/MS is presented for top-down MS of protein mixtures. Proteins composing the 20S and 19S proteasome complexes and their PTMs were identified using a 15 T FT-ICR mass spectrometer. The data-independent fragmentation modes with LC timescales allowed for higher duty-cycle measurements that better suit online LC-FT-ICR-MS. Protein top-down dissociation was effected by funnel-skimmer collisionally activated dissociation (FS-CAD) and CASI (continuous accumulation of selected ions)-CAD. The N-termini for 9 of the 14 20S proteasome proteins were found to be modified, and the α3 protein was found to be phosphorylated; these results are consistent with previous reports. Mass-measurement accuracy with the LC-FT-ICR system for the 20- to 30-kDa 20S proteasome proteins was 1 ppm. The intact mass of the 100-kDa Rpn1 subunit from the 19S proteasome complex regulatory particle was measured with a deviation of 17 ppm. The CASI-CAD technique is a complementary tool for intact-protein fragmentation and is an effective addition to the growing inventory of dissociation methods that are compatible with online protein separation coupled to FT-ICR-MS.

Interaction between 25S rRNA A Loop and Eukaryotic Translation Initiation Factor 5B Promotes Subunit Joining and Ensures Stringent AUG Selection

In yeast, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During the last step of translation initiation, eukaryotic initiation factor 5B (eIF5B) promotes the 60S subunit joining with the 40S initiation complex (IC). Malfunctional 60S subunits produced by misfolding or mutation may disrupt the 40S IC stalling on the start codon, thereby altering the stringency of initiation. Using several point mutations isolated by random mutagenesis, here we studied the role of 25S rRNA in start codon selection. Three mutations changing bases near the ribosome surface had strong effects, allowing the initiating ribosomes to skip both AUG and non-AUG codons: C2879U and U2408C, altering the A loop and P loop, respectively, of the peptidyl transferase center, and G1735A, mapping near a Eukarya-specific bridge to the 40S subunit. Overexpression of eIF5B specifically suppressed the phenotype caused by C2879U, suggesting functional interaction between eIF5B and the A loop. In vitro reconstitution assays showed that C2879U decreased eIF5B-catalyzed 60S subunit joining with a 40S IC. Thus, eIF5B interaction with the peptidyl transferase center A loop increases the accuracy of initiation by stabilizing the overall conformation of the 80S initiation complex. This study provides an insight into the effect of ribosomal mutations on translation profiles in eukaryotes.

Characterization of a periplasmic quinoprotein from Sphingomonas wittichii that functions as aldehyde dehydrogenase

The α-proteobacterium Sphingomonas wittichii RW1 is known for its ability to degrade dioxins and related toxic substances. Bioinformatic analysis of the genome indicated that this organism may contain the largest number of pyrroloquinoline quinone-dependent dehydrogenases of any bacteria sequenced so far. Sequence analysis also showed that one of these genes (swit_4395) encodes an enzyme that belongs to the class of periplasmic glucose dehydrogenases. This gene was fused to a pelB signal sequence and a strep-tag coding region at the 5' and 3' ends, respectively. The fusion product was cloned into the broad-host range expression vector pBBR1p264-Streplong and the corresponding protein was heterologously produced in Escherichia coli, purified via Strep-Tactin affinity chromatography, and characterized. The protein Swit_4395 had a subunit mass of 39.3kDa and formed active homooctamers and homododecamers. The enzyme showed the highest activities with short- and medium-chain aldehydes (chain length C1-C6) and ketoaldehydes, such as methylglyoxal and phenylglyoxal. Butyraldehyde was the best substrate, with V max and apparent K M values of 3,970U/mg protein and 12.3mM, respectively. Pyrroloquinoline quinone was detected using UV-Vis spectroscopy and was found to be a prosthetic group of the purified enzyme. Therefore, Swit_4395 was identified as a pyrroloquinoline quinone-dependent aldehyde dehydrogenase. The enzyme could be purified from the native host when the expression vector was introduced into S. wittichii RW1, indicating homologous protein production. Overproduction of Swit_4395 in S. wittichii RW1 dramatically increased the tolerance of the bacterium toward butyraldehyde and thus might contribute to the detoxification of toxic aldehydes.

Random mutagenesis of yeast 25S rRNA identify bases critical for 60S subunit structural integrity and function.

In yeast Saccharomyces cerevisiae, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During translation initiation, the 60S subunit joins the 40S initiation complex, producing the 80S initiation complex. During elongation, the 60S subunit binds the CCA-ends of aminoacyl- and peptidyl-tRNAs at the A-loop and P-loop, respectively, transferring the peptide onto the α-amino group of the aminoacyl-tRNA. To study the role of 25S rRNA in translation in vivo, we randomly mutated 25S rRNA and isolated and characterized seven point mutations that affected yeast cell growth and polysome profiles. Four of these mutations, G651A, A1435U, A1446G and A1587G, change a base involved in base triples crucial for structural integrity. Three other mutations change bases near the ribosomal surface: C2879U and U2408C alter the A-loop and P-loop, respectively, and G1735A maps near a Eukarya-specific bridge to the 40S subunit. By polysome profiling in mmslΔ mutants defective in nonfunctional 25S rRNA decay, we show that some of these mutations are defective in both the initiation and elongation phases of translation. Of the mutants characterized, C2879U displays the strongest defect in translation initiation. The ribosome transit-time assay directly shows that this mutation is also defective in peptide elongation/termination. Thus, our genetic analysis not only identifies bases critical for structural integrity of the 60S subunit, but also suggests a role for bases near the peptidyl transferase center in translation initiation.

Serum GlcNAc-binding IgM of fugu (Takifugu rubripes) suppresses the growth of fish pathogenic bacteria: A novel function of teleost antibody

N-acetyl-d-glucosamine (GlcNAc) is one of the components of peptidoglycan, a biopolymer in the bacterial cell wall. We purified a novel GlcNAc-binding protein, designated as fGBP-78, from sera of fugu (Takifugu rubripes). The fGBP-78 is a heteromer of 78- and 25-kDa subunits. Moreover, fGBP-78 exerted remarkable inhibitory effects on the growth of both Gram-positive and Gram-negative bacteria, including ones virulent for marine fish species as well as non-pathogenic Escherichia coli. These results suggest that fGBP-78 contributes to bacterial clearance in fugu. Furthermore, the nanoLC–MS/MS and Western blotting analyses reveal that the 78-kDa subunit is the fugu IgM heavy chain. In addition, the molecular mass of the other subunit (25kDa) was equal to that of the Ig light chain. Overall, results indicate that fGBP-78 is an IgM molecule presumably acts as a natural antibody. This paper reports a novel function of teleost IgM as a significant suppresser against bacterial growth.

Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity

Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0–400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay.

S-Mercuration of rat sorbitol dehydrogenase by methylmercury causes its aggregation and the release of the zinc ion from the active site

We previously developed a screening method to identify proteins that undergo aggregation through S-mercuration by methylmercury (MeHg) and found that rat arginase I is a target protein for MeHg (Kanda et al. in Arch Toxicol 82:803-808, 2008). In the present study, we characterized another S-mercurated protein from a rat hepatic preparation that has a subunit mass of 42kDa, thereby facilitating its aggregation. Two-dimensional SDS-polyacrylamide gel electrophoresis and subsequent peptide mass fingerprinting using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry revealed that the 42kDa protein was NAD-dependent sorbitol dehydrogenase (SDH). With recombinant rat SDH, we found that MeHg is covalently bound to SDH through Cys44, Cys119, Cys129 and Cys164, resulting in the inhibition of its catalytic activity, release of zinc ions and facilitates protein aggregation. Mutation analysis indicated that Cys44, which ligates the active site zinc atom, and Cys129 play a crucial role in the MeHg-mediated aggregation of SDH. Pretreatment with the cofactor NAD, but not NADP or FAD, markedly prevented aggregation of SDH. Such a protective effect of NAD on the aggregation of SDH caused by MeHg is discussed.

Characterization of dihydrolipoamide dehydrogenase from the mitochondria of Helicoverpa armigera, a pest resistant to insecticides

AbstractDihydrolipoamide dehydrogenase (DHLDH) was isolated from the mitochondria of Helicoverpa armigera, a destructive pest which has developed resistance to commonly used insecticides. The flavoenzyme was purified 17.98‐fold to homogeneity with an overall yield of 10.53% by employing ammonium sulfate precipitation, hydroxylapatite chromatography and CM‐Sephadex chromatography. The purified enzyme exhibited the specific activity of 18.7 U/mg and was characterized as a dimer with a subunit mass of 66 kDa. The enzyme showed specificity for nicotinamide adenine dinucleotide – hydrogen (NADH) and lipoamide, as substrates, with Michaelis‐Menten constants (Km) of 0.083 mmol/L and 0.4 mmol/L, respectively. The reduction reaction of lipoamide by the enzyme could be explained by ping‐pong mechanism. The spectra of DHLDH showed the maximum absorbance at 420 nm, 455 nm and 475 nm. The enzyme activity was strongly inhibited by mercurial and arsenical compounds. The N‐terminal sequence of Ha‐DHLDH showed homology with those of mammalian and arthropod DHLDH. Since H. armigera has developed high levels of resistance to commonly used insecticides, biochemical properties of the metabolic enzymes such as DHLDH, could be helpful to develop insecticidal molecules for the control of H. armigera, with a different mode of action.

Gentisate 1,2-dioxygenase, in the third naphthalene catabolic gene cluster of Polaromonas naphthalenivorans CJ2, has a role in naphthalene degradation

Polaromonas naphthalenivorans strain CJ2 metabolizes naphthalene via the gentisate pathway and has recently been shown to carry a third copy of gentisate 1,2-dioxygenase (GDO), encoded by nagI3, within a previously uncharacterized naphthalene catabolic gene cluster. The role of this cluster (especially nagI3) in naphthalene metabolism of strain CJ2 was investigated by documenting patterns in regulation, transcription and enzyme activity. Transcriptional analysis of wild-type cells showed the third cluster to be polycistronic and that nagI3 was expressed at a relatively high level. Individual knockout mutants of all three nagI genes were constructed and their influence on both GDO activity and cell growth was evaluated. Of the three knockout strains, CJ2ΔnagI3 showed severely diminished GDO activity and grew slowest on aromatic substrates. These observations are consistent with the hypothesis that nagI3 may prevent toxic intracellular levels of gentisate from accumulating in CJ2 cells. All three nagI genes from strain CJ2 were cloned into Escherichia coli: the nagI2 and nagI3 genes were successfully overexpressed. The subunit mass of the GDOs were ~36-39 kDa, and their structures were deduced to be dimeric. The K(m) values of NagI2 and NagI3 were 31 and 10 µM, respectively, indicating that the higher affinity of NagI3 for gentisate may protect the wild-type cells from gentisate toxicity. These results provide clues for explaining why the third gene cluster, particularly the nagI3 gene, is important in strain CJ2. The organization of genes in the third gene cluster matched that of clusters in Polaromonas sp. JS666 and Leptothrix cholodnii SP-6. While horizontal gene transfer (HGT) is one hypothesis for explaining this genetic motif, gene duplication within the ancestral lineage is equally valid. The HGT hypothesis was discounted by noting that the nagI3 allele of strain CJ2 did not share high sequence identity with its homologues in Polaromonas sp. JS666 and L. cholodnii SP-6.

DrosophilaSet1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription

Histone H3 lysine 4 trimethylation (H3K4me3) is a major hallmark of promoter-proximal histones at transcribed genes. Here, we report that a previously uncharacterized Drosophila H3K4 methyltransferase, dSet1, and not the other putative histone H3K4 methyltransferases (Trithorax; Trithorax-related protein), is predominantly responsible for histone H3K4 trimethylation. Functional and proteomics studies reveal that dSet1 is a component of a conserved H3K4 trimethyltransferase complex and polytene staining and live cell imaging assays show widespread association of dSet1 with transcriptionally active genes. dSet1 is present at the promoter region of all tested genes, including activated Hsp70 and Hsp26 heat shock genes and is required for optimal mRNA accumulation from the tested genes. In the case of Hsp70, the mRNA production defect in dSet1 RNAi-treated cells is accompanied by retention of Pol II at promoters. Our data suggest that dSet1-dependent H3K4me3 is responsible for the generation of a chromatin structure at active promoters that ensures optimal Pol II release into productive elongation.

An Extracellular Tetrathionate Hydrolase from the Thermoacidophilic Archaeon Acidianus Ambivalens with an Activity Optimum at pH 1.

Background: The thermoacidophilic and chemolithotrophic archaeon Acidianus ambivalens is routinely grown with sulfur and CO2-enriched air. We had described a membrane-bound, tetrathionate (TT) forming thiosulfate:quinone oxidoreductase. Here we describe the first TT hydrolase (TTH) from Archaea. Results: A. ambivalens cells grown aerobically with TT as sole sulfur source showed doubling times of 9 h and final cell densities of up to 8 × 108/ml. TTH activity (≈0.28 U/mg protein) was found in cell-free extracts of TT-grown but not of sulfur-grown cells. Differential fractionation of freshly harvested cells involving a pH shock showed that about 92% of the TTH activity was located in the pseudo-periplasmic fraction associated with the surface layer, while 7.3% and 0.3% were present in the soluble and membrane fractions, respectively. The enzyme was enriched 54-fold from the cytoplasmic fraction and 2.1-fold from the pseudo-periplasmic fraction. The molecular mass of the single subunit was 54 kDa. The optimal activity was at or above 95°C at pH 1. Neither PQQ nor divalent cations had a significant effect on activity. The gene (tth1) was identified following N-terminal sequencing of the protein. Northern hybridization showed that tth1 was transcribed in TT-grown cells in contrast to a second paralogous tth2 gene. The deduced amino acid sequences showed similarity to the TTH from Acidithiobacillus and other proteins from the PQQ dehydrogenase superfamily. It displayed a β-propeller structure when being modeled, however, important residues from the PQQ-binding site were absent. Conclusion: The soluble, extracellular, and acidophilic TTH identified in TT-grown A. ambivalens cells is essential for TT metabolism during growth but not for the downstream processing of the TQO reaction products in S°-grown cells. The liberation of TTH by pH shock from otherwise intact cells strongly supports the pseudo-periplasm hypothesis of the S-layer of Archaea.

Molecular masses and sedimentation coefficients of extracellular hemoglobin of Glossoscolex paulistus: Alkaline oligomeric dissociation

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) has a molecular mass ( M) of 3600 ± 100 kDa and a standard sedimentation coefficient ( s 20 , w 0 ) of 58 S, estimated by analytical ultracentrifugation (AUC). In the present work, further AUC studies were developed for HbGp, at pH 10.0, which favors oligomeric dissociation into lower M species. The HbGp oligomer is formed by globin chains a , b , c and d plus the linker chains. The pure monomeric fraction, subunit d , and HbGp at pH 10.0, in the presence of β-mercaptoethanol, were also studied. Our results indicate that for samples of pure subunit d , besides the monomeric species with s 20 , w 0 of 2.0 S, formation of dimer of subunit d is observed with s 20 , w 0 of around 2.9 S. For the whole HbGp at pH 10.0 contributions from monomers, trimers and linkers are observed. No contribution from 58 S species was observed for the sample of oxy-HbGp at pH 10.0, showing its complete dissociation. For cyanomet-HbGp form a contribution of 17% is observed for the un-dissociated oligomer, consistent with data from other techniques that show the cyanomet-form is more stable as compared to oxy-HbGp. Masses of HbGp subunits, especially trimer abc and monomeric chains a , b , c and d , were also estimated from sedimentation equilibrium data, and are in agreement with the results from MALDI-TOF-MS.

Characterization of Deinococcus radiophilus thioredoxin reductase active with both NADH and NADPH

Thioredoxin reductase (TrxR, EC 1.6.4.5) of Deinococcus radiophilus was purified by steps of sonication, ammonium sulfate fractionation, 2'5' ADP Sepharose 4B affinity chromatography, and Sephadex G-100 gel filtration. The purified TrxR, which was active with both NADPH and NADH, gave a 368 U/mg protein of specific activity with 478-fold purification and 18% recovery from the cell-free extract. An isoelectric point of the purified enzymes was ca. 4.5. The molecular weights of the purified TrxR estimated by PAGE and gel filtration were about 63.1 and 72.2 kDa, respectively. The molecular mass of a TrxR subunit is 37 kDa. This suggests that TrxR definitely belongs to low molecular weight TrxR (L-TrxR). The Km and Vmax of TrxR for NADPH are 12.5 μM and 25 μM/min, whereas those for NADH are 30.2 μM and 192 μM/min. The Km and Vmax for 5, 5'-dithio-bis-2-nitrobenzoic acid (DTNB, a substituted substrate for thioredoxin) are 463 μM and 756 μM/min, respectively. The presence of FAD in TrxR was confirmed with the absorbance peaks at 385 and 460 nm. The purified TrxR was quite stable from pH 3 to 9, and was thermo-stable up to 70°C. TrxR activity was drastically reduced (ca. 70%) by Cu(2+), Zn(2+), Hg(2+), and Cd(2+), but moderately reduced (ca. 50%) by Ag(+). A significant inhibition of TrxR by N-ethylmaleimide suggests an occurrence of cysteine at its active sites. Amino acid sequences at the N-terminus of purified TrxR are H(2)N-Ser-Glu-Gln-Ala-Gln-Met-Tyr-Asp-Val-Ile-Ile-Val-Gly-Gly-Gly-Pro-Ala-Gly-Leu-Thr-Ala-COOH. These sequences show high similarity with TrxRs reported in Archaea, such as Methanosarcina mazei, Archaeoglobus fulgidus etc.

The Subunit Composition of Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I) From Pichia pastoris

Respiratory complex I (NADH:quinone oxidoreductase) is an entry point to the electron transport chain in the mitochondria of many eukaryotes. It is a large, multisubunit enzyme with a hydrophilic domain in the matrix and a hydrophobic domain in the mitochondrial inner membrane. Here we present a comprehensive analysis of the protein composition and post-translational modifications of complex I from Pichia pastoris, using a combination of proteomic and bioinformatic approaches. Forty-one subunits were identified in P. pastoris complex I, comprising the 14 core (conserved) subunits and 27 supernumerary subunits; seven of the core subunits are mitochondrial encoded. Three of the supernumerary subunits (named NUSM, NUTM, and NUUM) have not been observed previously in any species of complex I. However, homologues to all three of them are present in either Yarrowia lipolytica or Pichia angusta complex I. P. pastoris complex I has 39 subunits in common with Y. lipolytica complex I, 37 in common with N. crassa complex I, and 35 in common with the bovine enzyme. The mitochondrial encoded subunits (translated by the mold mitochondrial genetic code) retain their N-α-formyl methionine residues. At least eight subunits are N-α-acetylated, but the N-terminal modifications of the nuclear encoded subunits are not well-conserved. A combination of two methods of protein separation (SDS-PAGE and HPLC) and three different mass spectrometry techniques (peptide mass fingerprinting, tandem MS and molecular mass measurements) were required to define the protein complement of P. pastoris complex I. This requirement highlights the need for inclusive and comprehensive strategies for the characterization of challenging membrane-bound protein complexes containing both hydrophilic and hydrophobic components.

Glutathionyl-biliverdin IXα, a new heme catabolite in a marine annelid: Sex and cell specific accumulation

We have isolated and characterized the green pigment accumulating in coelomic cells (eleocytes) of the common clam worm Nereis virens by means of RP-HPLC and ESI-tandem mass spectrometry. This pigment is a novel biliverdin-glutathione conjugate in which the glutathione is linked to the biliverdin-backbone via a thioether bond. The yolk precursor vitellogenin, a female-specific high-density lipoprotein ( ρ = 1179 kg/m 3) with a native molecular mass of ∼500 kDa and a subunit mass of ∼150 kDa, is capable of transporting this pigment as well as heme. The sex-independent large discoidal lipoprotein present in the coelomic fluid, which is sequestered by the eleocytes, could also be shown to transport heme. This renders both lipoproteins as heme-lipoproteins. As the vitellogenin is secreted by the eleocytes, this suggests the eleocytes as a metabolic hub linking the uptake of the potent pro-oxidant heme thought to arise from aged hemoglobin via the large discoidal lipoprotein and its conversion to a bile pigment-conjugate. The conjugate is exported from the eleocytes to the oocytes via vitellogenin leading to the accumulation of green yolk protein. In contrast, no such export route exists in male eleocytes resulting in an accumulation of the biliverdin-conjugate in these cells.

A new acylamidase from Rhodococcus erythropolis TA37 can hydrolyze N-substituted amides

A new acylamidase was isolated from Rhodococcus erythropolis TA37 and characterized. N-Substituted acrylamides (isopropyl acrylamide, N,N-dimethyl-aminopropyl acrylamide, and methylene-bis-acrylamide), acid para-nitroanilides (4'-nitroacetanilide, Gly-pNA, Ala-pNA, Leu-pNA), and N-acetyl derivatives of glycine, alanine, and leucine are good substrates for this enzyme. Aliphatic amides (acetamide, acrylamide, isobutyramide, n-butyramide, and valeramide) are also used as substrates but with less efficiency. The enzyme subunit mass by SDS-PAGE is 55 kDa. Maximal activity is exhibited at pH 7-8 and 55°C. The enzyme is stable for 15 h at 22°C and for 0.5 h at 45°C. The Michaelis constant (K(m)) is 0.25 mM with Gly-pNA and 0.55 mM with Ala-pNA. The acylamidase activity is suppressed by inhibitors of serine proteases (phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate) but is not suppressed by inhibitors of aliphatic amidases (acetaldehyde and nitrophenyl disulfides). The N-terminal amino acid sequence of the acylamidase is highly homologous to those of two putative amidases detected from sequenced R. erythropolis genomes. It is suggested that the acylamidase together with the detected homologs forms a new class within the amidase signature family.

The metabolic costs of building ant colonies from variably sized subunits

Ant colonies are superorganisms with emergent traits that, for some species, reflect the combined activity of physically distinct worker castes. Although larger castes have high production costs, they are thought to save their colonies energy by efficiently performing specialized tasks. However, because workers are generally idle until sensing specific stimuli, their maintenance costs may be an important component of colony-level investment. I used metabolic scaling to examine the maintenance costs of dimorphic major and minor Pheidole castes across levels of colony organization (e.g., individual, group, and colony). Majors from three species had lower mass-specific meta- bolic rates than minors because of allometries at both individual and group levels and subsequently lived longer when starved. Thus, large major castes may offset their production costs in both their idle and active states. The slope scaling metabolic rate from incipient to reproductive colonies of Pheidole dentata (∼colony mass 0.89 )f ell between the slopes for minor groups (∼group mass 1.04 ) and major groups (∼group mass 0.79 ) and appears to reflect developmental shifts in subunit mass and number and their offsetting effects on per capita energy demands. These results highlight how metabolic scaling may help visualize the energetic correlates of emergent behavior and unravel the mechanisms governing colony organization.

Cytoglobin conformations and disulfide bond formation

The oligomeric state and kinetics of ligand binding were measured for wild‐type cytoglobin. Cytoglobin has the classical globin fold, with an extension at each extremity of about 20 residues. The extended length of cytoglobin leads to an ambiguous interpretation of its oligomeric state. Although the hydrodynamic diameter corresponds to that of a dimer, it displays a mass of a single subunit, indicating a monomeric form. Thus, rather than displaying a compact globular form, cytoglobin behaves hydrodynamically like a tightly packed globin with a greater flexibility of the N‐ and C‐terminal regions. Cytoglobin displays biphasic kinetics after the photolysis of CO, as a result of competition with an internal protein ligand, the E7 distal histidine. An internal disulfide bond may form which modifies the rate of dissociation of the distal histidine and apparently leads to different cytoglobin conformations, which may affect the observed oxygen affinity by an order of magnitude.