Amino acid sequence of locust neuroparsins

Neuroparsins A and B were isolated from the nervous part of the corpus cardiaca of Locusta migratoria via a two-step purification procedure. Both consist of two polypeptide chains linked by disulfide bridges. The N-terminal sequence of both native neuroparsins was determined: the N-terminal end of neuroparsin B was unique while that of neuroparsin A showed three different sequences. These sequences were that of neuroparsin B and two others having five and two extra N-terminal residues. Neuroparsin B was found as a homodimer and the complete sequence of the monomer, determined from peptide fragments generated by treatment with cyanogen bromide and endoprotease Glu-C, comprises 78 residues.     

Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I: CONSTRAINTS AND FINE-TUNING IN THE EVOLUTION OF A PROTEIN FAMILY*

Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6—A11, A7–B7, and A20—B19) maintained by IGF-binding proteins; IGF-swap has alternative pairing (A7–A11, A6—B7, and A20—B19) and impaired activity. Studies of mini-domain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, His^B5 → Thr markedly destabilizes the hormone (ΔΔG_u 2.0 ± 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution Thr^B5 → His (residue 4) specifies a unique structure with native ¹H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, His^B5-IGF-I retains canonical pairing. Chemical denaturation studies indicate that His^B5 does not significantly enhance thermodynamic stability (ΔΔG_u 0.2 ± 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the activity of Thr^B5-insulin is decreased 5-fold, His^B5-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation of Thr^B5 in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast, the conservation of His^B5 in insulin highlights its critical role in insulin biosynthesis.     

cDNA sequence analysis and mutagenesis studies on the a chain of Β-bungarotoxin from Taiwan banded krait

The cDNA encoding the A chain ofΒ-bungarotoxin (Β-Bgt) was constructed from the cellular RNA isolated from the venom glands ofBungarus multicinctus (Taiwan banded krait). The deduced amino acid sequence encoding the A chain revealed that the determined chain was different from the known A chains (A1, A2, A3, A4, and A5). Nevertheless, the amino acid sequence and the cDNA sequence of the novel A chain were highly homologous with those of other A chains. The gene encoding the A chain ofΒ-Bgt was subjected to mutagenesis, and the Tyr-11, Cys-15, and Leu-72 of the A chain were substituted by Cys-11, Ser-15, and Cys-72, respectively. Instead of the six disulfide bonds observed with the A chain, the resulting mutant contained seven disulfide linkages in its molecular structure which simulated those of presynaptic PLA₂ neurotoxins and PLA₂ enzymes. However, the mutant did not exhibit a higher phospholipase activity than that noted with the recombinant A chain. These results seem to suggest that, in the absence of the B chain, the six pairs of disulfide bonds in the recombinant A-chain molecule are enough to maintain its active conformation for exerting the phospholipase activity.     

Anti-juvenile effect of neuroparsin A,a neuroprotein isolated from locust corpora cardiaca

Neuroparsin A, a sulfur-containing protein synthesized by the medial part of the brain of Locusta and transported to the corpora cardiaca (CC) via the nervi corporis cardiaci I (Girardie et al., 1987), was satisfactorily isolated using electro-elution. A specific immune serum against electro-eluted neuroparsin A was generated. On serial histological sections of the brain treated with the immune serum, only the median neurosecretory cells [stained in blue following the double staining Victoria blue-paraldehyde fuchsin (Al type)] were revealed using peroxidase-antiperoxidase technique. Inactivation of neuroparsin A by antigen-antibody complex formation following injections of immune serum induced green pigmentation, intermediary forms and precocious sexual maturation. These symptoms also follow juvenile hormone (JH) injections. Injections of immune serum antineuroparsin A or the electro-eluted neuroparsin A produced opposite effects on oocyte growth but had no effect on the rate of JH biosynthesis evaluated by radiochemical assay. The neurohormone neuroparsin A could be the median humoral inhibiting factor of the JH system which was previously demonstrated (Girardie, 1966, 1967) in the central area of the pars intercerebralis.     

Genetic control of protein synthesis in white lupine (Lupinus albus L.) seeds

Using polyacrylamide gel electrophoresis in the glycine-acetic acid system (pH 3.2), variants of proteins of white-lupine seeds were revealed. The study of conglutin polymorphism in the culture of the autogamous population F_→∞ (var. Dega) revealed two loci, Con A and Con B, which control protein synthesis. The loci were situated in the same linkage group within a distance of 11.48 ± 3.4% of recombination. Natural selection in favor of genotypes that contain Con A1 and Con B2 alleles is proposed. It is established that conglutins A and B (CON A and CON B) contain cysteine residues, which form intermolecular disulfide bonds between peptides.     

Structural Analysis and Molecular Model of a Self-Incompatibility RNase from Wild Tomato

Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated with rejection of self-pollen in solanaceous plants. The nucleotide sequences of S-RNase alleles from several genera have been determined, but the structure of the gene products has only been described for those from Nicotiana alata. We report on the N-glycan structures and the disulfide bonding of the S₃-RNase from wild tomato (Lycopersicon peruvianum) and use this and other information to construct a model of this molecule. The S₃-RNase has a single N-glycosylation site (Asn-28) to which one of three N-glycans is attached. S₃-RNase has seven Cys residues; six are involved in disulfide linkages (Cys-16-Cys-21, Cys-46-Cys-91, and Cys-166-Cys-177), and one has a free thiol group (Cys-150). The disulfide-bonding pattern is consistent with that observed in RNase Rh, a related RNase for which radiographic-crystallographic information is available. A molecular model of the S₃-RNase shows that four of the most variable regions of the S-RNases are clustered on one surface of the molecule. This is discussed in the context of recent experiments that set out to determine the regions of the S-RNase important for recognition during the self-incompatibility response.     

Is the heme pocket region modulated by disulfide-bridge formation in fish and amphibian neuroglobins as in humans?

Neuroglobin, a globin characterized by a bis-histidine ligation of the heme iron, has been identified in mammalian and non-mammalian vertebrates, including fish, amphibians and reptiles. In human neuroglobin, the presence of an internal disulfide bond in the CD loop (CD7–D5) is found to modulate the ligand binding through a change in the heme pocket structure. Although the neuroglobin sequences mostly display conserved Cys at positions CD7, D5 and G18/19, a number of exceptions are known. In this study, neuroglobins from amphibian (Xenopus tropicalis) and fish (Chaenocephalus aceratus, Dissostichus mawsoni and Danio rerio) are investigated using electron paramagnetic resonance and optical absorption spectroscopy. All these neuroglobins differ from human neuroglobin in their Cys-positions. It is demonstrated that if disulfide bonds are formed in fish and amphibian neuroglobins, the reduction of these bonds does not result in alteration of the heme pocket in these globins. Furthermore, it is shown that mutagenesis of the Cys residues of X. tropicalis neuroglobin influences the protein structure. The amphibian neuroglobin is also found to be more resistant to H₂O₂-induced denaturation than the other neuroglobins under study, although all show an overall large stability in high concentrations of this oxidant. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Transnitrosylating Nitric Oxide Species Directly Activate Type I Protein Kinase A, Providing a Novel Adenylate Cyclase-independent Cross-talk to ��-Adrenergic-like Signaling

The transnitrosylating nitric oxide (NO) donor nitrocysteine (CysNO) induced a disulfide bond between the two regulatory RI subunits of protein kinase A (PKA). The conventional NO donor S-nitroso-N-acetylpenicillamine failed to do this, consistent with our observation that it also did not promote protein S-nitrosylation. This disulfide oxidation event activated PKA and induced vasorelaxation independently of the classical β-adrenergic or NO signaling pathway. Activation of PKA had also been anticipated to exert a positive inotropic effect on the myocardium but did not. The lack of positive inotropy was explained by CysNO concomitantly activating protein kinase G (PKG) Iα. PKG was found to exert a partial negative inotropic influence regardless of whether PKA was activated by classical β-receptor stimulation or by disulfide bond formation. This work demonstrates that NO molecules that can induce S-nitrosylation directly activate type I PKA, providing a novel cross-talk to β-adrenergic-like signaling without receptor or adenylate cyclase stimulation. However, the expected positive inotropic consequences of PKA activation by this novel mechanism are countermanded by the simultaneous dual activation of PKGIα, which is also activated by CysNO.Nitric oxide (NO) initiates cell signaling by binding and activating soluble guanylate cyclase (sGC)² to produce the second messenger cGMP. cGMP primarily allosterically activates protein kinase G (PKG) but can also regulate other proteins. Although this NO-sGC-cGMP-PKG pathway is well defined (1), a second major mechanism of NO-dependent regulation has subsequently emerged. This involves NO covalently adducting to protein thiols, a process known as S-nitrosylation or S-nitrosation (2).Significant evidence continues to accumulate supporting protein S-nitrosylation as a fundamental regulator of protein and thus cell function (3). NO is produced in a regulated way (4), with a defined structural basis for selectivity in the proteins it covalently modifies (5, 6). Additional regulatory control can be achieved by the localization of NO synthase enzymes proximal to target proteins (6) and by reverse denitrosylation being enzymatically controlled (7). Indeed, many proteins appear to be basally S-nitrosylated, offering the potential for attenuation (8) as well as potentiation of signaling.Although stable regulatory S-nitrosylation occurs in some proteins, in others, it serves as an intermediate prior to transition to other redox states, especially disulfides (9). Previously, we searched for proteins that form interprotein disulfides in response to hydrogen peroxide (H₂O₂), identifying the regulatory RI subunit of protein kinase A (PKA) as such a protein (10, 11). This appears to activate the kinase (11), although the mechanism is not yet precisely defined. There is a rational structural basis for interprotein disulfide formation in PKA RI in response to H₂O₂. The RI dimer is held together by an N-terminal amphipathic leucine zipper in which the monomers are aligned antiparallel to each other with both Cys¹⁷ residues directly facing the corresponding Cys³⁸ residues on the opposite chains (12). H₂O₂-mediated RI disulfide formation is likely via protein sulfenic acid formation by one thiol in the Cys¹⁷ and Cys³⁸ disulfide-forming pair, prior to reduction by the other cysteine to yield the covalently conjugated dimer. Intriguingly, this pair of thiol-disulfide switches in RI is located directly on either side of the protein kinase A anchor protein-binding domain (13). This provides a rational structural basis for the PKA RI-protein kinase A anchor protein interaction being redox-modulated, as the interaction is strongly anticipated to change depending on the oxidation state of the cysteine switches, which flank the interaction locus (11).We hypothesized that NO may also be able to drive RI disulfide formation via an S-nitrosylated catalytic redox intermediate in a mechanism analogous to transient sulfenation formation during H₂O₂-induced covalent conjugation. This conceptual link between NO and PKA was investigated by comparing the biochemical and functional responses of cardiovascular tissue to the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and nitrocysteine (CysNO). The authentic NO donor SNAP did not promote RI disulfide formation, whereas CysNO did so efficiently, consistent with its established thiol-oxidizing transnitrosylating ability. We show that disulfide-mediated activation of PKA significantly contributes to vasorelaxation induced by CysNO. However, disulfide activation of PKA failed to exert a positive inotropic influence in isolated hearts exposed to CysNO, which was difficult to reconcile with the kinase being truly activated by oxidation. Further investigations showed that this lack of positive inotropy following CysNO-induced oxidation is explained by the co-activation of PKGIα, which we demonstrated previously can be disulfide-activated (15). PKGIα serves as a master regulator of cardiac inotropy, dominating the system to prevent increases in cardiac contractility. Thus, thiol-oxidizing derivatives of NO can activate PKA and so exert β-adrenergic-like signaling, although dual activation of PKG prevents the anticipated positive inotropy.     

Bakers' yeast protease a purification and enzymatic and molecular properties

It has been previously demonstrated in our laboratory that uridine nucleosidase (EC 3.2.2.3) is inactivated by yeast protease A (EC 3.4.23.8). A complete purification procedure for protease A from bakers' yeast, which lacks the acidic activation step used by other workers, and the major properties of the enzyme are shown. The enzyme is homogeneous as judged by disc gel electrophoresis. Its molecular weight, calculated from both sodium dodecyl sulfate-disc gel electrophoresis and gel filtration experiments, is around 45,000. The protein does not possess quaternary structure. The isoelectric point is 4.1. Carbohydrate content is around 8%. Amino acids analysis and sulfur analysis reveal the presence of 1-SH group and two disulfide bridges. The free-SH group does not seem to be involved in catalysis. Amino terminal analysis shows that isoleucine is at the amino terminal position. The pH optima are 2.4 for the hydrolysis of azocasein and casein, and 3.3 for the hydrolysis of hemoglobin. The K_m value for hemoglobin is 1.7 × 10^?5m. The inhibition exerted by pepstatin on the proteolytic activity of protease A is pH dependent. Among various yest enzyme substrates only uridine nucleosidase is inactivated by protease A.     

Cloning, Sequencing and Characterization of FAE1 Genes of Brassica juncea cv Pusa Bold and the Mutant fae1 of B. rapa cv Tobin

Level of erucic acid (22:1), the major storage fatty acid of oil seed Brassica, is controlled by the activity of the Fatty Acid Elongation1 (FAE1) gene. Southern hybridization revealed the presence of two FAE1 genes in B. juncea. The two FAE1 genes of B. juncea and the mutant fae1 of B. rapa cv Tobin were isolated from genomic libraries of the respective species and sequenced. The two BjFAE1 gene sequences shared more than 98% homology and contained ORF capable of coding for 509/510 amino acid polypeptides. One of the FAE1 genes of B. juncea was found to be nearly identical (99.6%) to the mutant formof B. rapa suggesting its origin from the later species. Comparison of the sequences generated with one another and with other FAE1 sequences in the database revealed that substitution of C₂₃₃ (cysteine) with G (glycine) might be responsible for the loss of enzyme activity in B. rapa cv Tobin.     

Purification and Characterization of a Low-Molecular-Weight Phospholipase A2 from Developing Seeds of Elm

Phospholipase A₂ (PLA₂) was purified about 180,000 times compared with the starting soluble-protein extract from developing elm (Ulmus glabra) seeds. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified fraction showed a single protein band with a mobility that corresponded to 15 kD, from which activity could be recovered. When analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry, the enzyme had a deduced mass of 13,900 D. A 53-amino acid-long N-terminal sequence was determined and aligned with other sequences, giving 62% identity to the deduced amino acid sequence of some rice (Oryza sativa) expressed sequence tag clones. The purified enzyme had an alkaline pH optimum and required Ca²⁺ for activity. It was unusually stable with regard to heat, acidity, and organic solvents but was sensitive to disulfide bond-reducing agents. The enzyme is a true PLA₂, neither hydrolyzing the sn-1 position of phosphatidylcholine nor having any activity toward lysophosphatidylcholine or diacylglycerol. The biochemical data and amino acid sequence alignments indicate that the enzyme is related to the well-characterized family of animal secretory PLA₂s and, to our knowledge, is the first plant enzyme of this type to be described.     

Studies on synthesis and activation mechanism of mitomycin dimers connected by 1,2-dithiolane and diol linkers

We report the synthetic and mechanistic studies on a new cyclic disulfide mitomycin dimer, 7-N,7′-N′-(1″,2″-dithiolanyl-3″,5″-dimethylenyl)bismitomycin C (8), and a diol mitomycin dimer, 7-N,7′-N′-(2″,4″-dihydroxy-1″,5″-pentanediyl)bismitomycin C (9). Mitomycin 8 is a dimer connected by a 1,2-dithiolane (a five-membered cyclic disulfide) linker, and was specifically designed to undergo nucleophilic activation and double DNA alkylations leading to efficient production of DNA interstrand cross-link (DNA ISC) adducts. Disulfide cleavage in 8 would generate two thiol groups that could serve as probes to activate two mitomycin rings. At first, the target mitomycin 8 was synthesized using mitomycin A (1) and the key intermediate, cyclic disulfide (10), which was prepared through a seven-step synthetic sequence. Diol mitomycin 9 was also synthesized from 1 and diamine salt 13. Next, kinetic studies using solvolysis reaction revealed that the activation rates of 8 were much higher than those of 9 and mitomycin C (2) under nucleophilic conditions provided by Et₃P presumably due to the presence of a cyclic disulfide unit in 8. These findings led us to propose a nucleophilic activation pathway for 8. Then, DNA ISC experiments further revealed that the levels of DNA ISC caused by 8 in the presence of Et₃P were much higher (97%) than those by 9 (5%) and 2 (4%). More importantly, mitomycin 8 underwent much faster activation and produced slightly higher levels of DNA ISC than the previously reported mitomycins 5–7. Overall, we concluded that 8 was highly efficient for both nucleophilic activation and corresponding DNA ISC formation, and that this differentiation came from the crucial function of the cyclic disulfide unit in 8.     

Cloning and sequencing of a copper-containing,topa quinone-containing monoamine oxidase from human placenta

A 4040-bp cDNA was cloned from a human placenta library by screening with a polymerase chain reaction-amplified fragment. The fragment was generated from the library using primers corresponding to conserved sequences encompassing the topa quinone (TPQ) cofactor sites of the copper-containing proteins, bovine serum amine oxidase (BSAO) and human kidney diamine oxidase (DAO). The cloned cDNA contains a coding sequence from positions 161 to 2449. Between bases 2901 and 2974, in a very long 1591-bp 3′-untranslated region, there is a G/A-rich region in the minus strand, which contains a (AGG)₅ tandem repeat. The human placenta cDNA sequence and its translated amino acid sequence are 84% and 81% identical to the corresponding BSAO sequences, while the identities for the placenta sequences and those for human kidney DAO are 60% and 41%, respectively. The TPQ consensus nucleotide and protein sequences are identical for the placenta enzyme and BSAO, but the corresponding sequences for human kidney DAO are nonidentical. Three His residues that have been identified as Cu(II) ligands in other amine oxidases are conserved in the human placenta amine oxidase protein sequence. It was concluded that the placenta cDNA open-reading frame codes for a copper-containing, TPQ-containing monoamine oxidase. A putative 19-amino acid signal peptide was identified for human placenta amine oxidase. The resulting mature protein would be composed of 744 amino acids, and would have a M_r of 82 525. Comparison of the human placenta amine oxidase with DNA sequences found in GenBank suggests that the gene for this enzyme is located in the q21 region of human chromosome 17, near the BRCA1 gene.     

Going through the Barrier: COUPLED DISULFIDE EXCHANGE REACTIONS PROMOTE EFFICIENT CATALYSIS IN QUIESCIN SULFHYDRYL OXIDASE*

The quiescin sulfhydryl oxidase (QSOX) family of enzymes generates disulfide bonds in peptides and proteins with the reduction of oxygen to hydrogen peroxide. Determination of the potentials of the redox centers in Trypanosoma brucei QSOX provides a context for understanding catalysis by this facile oxidant of protein thiols. The CXXC motif of the thioredoxin domain is comparatively oxidizing (E′₀ of −144 mV), consistent with an ability to transfer disulfide bonds to a broad range of thiol substrates. In contrast, the proximal CXXC disulfide in the ERV (essential for respiration and vegetative growth) domain of TbQSOX is strongly reducing (E′₀ of −273 mV), representing a major apparent thermodynamic barrier to overall catalysis. Reduction of the oxidizing FAD cofactor (E′₀ of −153 mV) is followed by the strongly favorable reduction of molecular oxygen. The role of a mixed disulfide intermediate between thioredoxin and ERV domains was highlighted by rapid reaction studies in which the wild-type CGAC motif in the thioredoxin domain of TbQSOX was replaced by the more oxidizing CPHC or more reducing CGPC sequence. Mixed disulfide bond formation is accompanied by the generation of a charge transfer complex with the flavin cofactor. This provides thermodynamic coupling among the three redox centers of QSOX and avoids the strongly uphill mismatch between the formal potentials of the thioredoxin and ERV disulfides. This work identifies intriguing mechanistic parallels between the eukaryotic QSOX enzymes and the DsbA/B system catalyzing disulfide bond generation in the bacterial periplasm and suggests that the strategy of linked disulfide exchanges may be exploited in other catalysts of oxidative protein folding.     

Engineering a Disulfide Bond in the Lid Hinge Region of Rhizopus chinensis Lipase: Increased Thermostability and Altered Acyl Chain Length Specificity

The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for “interfacial activation” is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of R. chinensis lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from R. chinensis in the hinge region of the lid according to the prediction of the “Disulfide by Design” algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the t _1/2 value at 60°C and a 7°C increase of T _m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (k _cat) and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from R. chinensis by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.     

The position of the disulfide bonds in human plasma α2HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma α₂HS-glycoprotein were determined. α₂HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)-Cys-14 (A-chain), Cys-71-Cys-82, Cys-96-Cys-114, Cys-128-Cys-131, Cys-190-Cys-201 and Cys-212-Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of α₂HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the SS bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two SS bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second SS loops and the end of domains A and B, and the positions of the ordered structures.     

Uncovering the Ancestry of B Chromosomes in Moenkhausia sanctaefilomenae (Teleostei,Characidae)

B chromosomes constitute a heterogeneous mixture of genomic parasites that are sometimes derived intraspecifically from the standard genome of the host species, but result from interspecific hybridization in other cases. The mode of origin determines the DNA content, with the B chromosomes showing high similarity with the A genome in the first case, but presenting higher similarity with a different species in the second. The characid fish Moenkhausia sanctaefilomenae harbours highly invasive B chromosomes, which are present in all populations analyzed to date in the Parana and Tietê rivers. To investigate the origin of these B chromosomes, we analyzed two natural populations: one carrying B chromosomes and the other lacking them, using a combination of molecular cytogenetic techniques, nucleotide sequence analysis and high-throughput sequencing (Illumina HiSeq2000). Our results showed that i) B chromosomes have not yet reached the Paranapanema River basin; ii) B chromosomes are mitotically unstable; iii) there are two types of B chromosomes, the most frequent of which is lightly C-banded (similar to euchromatin in A chromosomes) (B₁), while the other is darkly C-banded (heterochromatin-like) (B₂); iv) the two B types contain the same tandem repeat DNA sequences (18S ribosomal DNA, H3 histone genes, MS3 and MS7 satellite DNA), with a higher content of 18S rDNA in the heterochromatic variant; v) all of these repetitive DNAs are present together only in the paracentromeric region of autosome pair no. 6, suggesting that the B chromosomes are derived from this A chromosome; vi) the two B chromosome variants show MS3 sequences that are highly divergent from each other and from the 0B genome, although the B₂-derived sequences exhibit higher similarity with the 0B genome (this suggests an independent origin of the two B variants, with the less frequent, B₂ type presumably being younger); and vii) the dN/dS ratio for the H3.2 histone gene is almost 4–6 times higher for B chromosomes than for A chromosome sequences, suggesting that purifying selection is relaxed for the DNA sequences located on the B chromosomes, presumably because they are mostly inactive.     

Structural studies of Bacillus subtilis glutamine synthetase. Further purification, sulfhydryl groups, and the NH2-terminal amino acid sequence.

A new procedure for the isolation of Bacillus subtilis glutamine synthetase in a high state of purity is described. Automated Edman degradation of the reduced and carboxy-methylated protein revealed a single NH₂-terminal amino acid sequence: H₂N-Ala-Lys- Tyr-Thr-Arg⁵-Glu-Asp-Ile-Gln-Lys¹⁰-Leu-Val-Ser-Glu-Ser¹⁵-CM-Cys-Val-Thr- Tyr-Ile²⁰-Ser-Leu-Gly-Phe-Ser²⁵-Asn-Ser-Leu-Gly- -. The recovery of phenylthiohydantoin(PTH)-amino acids and the single sequence obtained are consistent with the view that the dodecameric enzyme of molecular weight 600,000 is composed of identical subunits. Earlier observations of multiple sequences (80% PTH-Ala and 20% PTH-Gly as NH₂ terminal residues) appear to have been due to impurities removed by the final purification step described herein, which involves column chromatography on hydroxyapatite. Evidence for the existence of one disulfide bond and two free cysteine residues per subunit of dodecameric glutamine synthetase was obtained by alkylation of the denatured enzyme in the presence and absence of reducing agents. This distribution of the four cysteine residues in the enzyme monomer was confirmed by titration of the enzyme denatured in sodium dodecyl sulfate with 5,5′-dithiobis(2-nitrobenzoic acid).