Pyruvate carboxylase: isolation of the biotin-containing tryptic peptide and the determination of its primary sequency

The biotin-containing tryptic peptides of pyruvate carboxylase from sheep, chicken, and turkey liver mitochondria have been isolated and their primary structures determined. The amino acid sequences of the 19 residue peptides from chicken and turkey are identical and share a common sequence of 14 residues around biocytin with the 24-residue peptide isolated from sheep. The sequences obtained were: residue 1 → 11 Avian: Gly Ala Pro Leu Val Leu Ser Ala Met Biocytin Met Sheep: Gly Gln Pro Leu Val Leu Ser Ala Met Biocytin Met residues 12 → 19 or 24 Avian: Glu Thr Val Val Thr Ala Pro Arg Sheep: Glu Thr Val Val Thr Ser Pro Val Thr Glu Gly Val Arg A sensitive radiochemical assay for biotin was developed based on the tight binding of biotin by avidin. The ability of zinc sulfate to precipitate, without dissociating, the avidin-biotin complex provided a convenient procedure for separating free and bound biotin, and hence, for back-titrating a standard amount of avidin with [¹⁴C]biotin.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

RESTRICTED ROTATION IN CHIRAL PEPTIDE NUCLEIC ACID (PNA) MONOMERS - INFLUENCE OF SUBSTITUENTS STUDIED BY MEANS OF 1H NMR

The influence of amino acid side chains [derived from: Ala, Val, Leu, Ile, Phe, Tyr(Bzl), Ser(Bzl), Thr(Bzl), Pro, Trp], incorporated into “aminoalkyl” part of PNA monomers, on the temperature-dependent distributions of rotamers about the tertiary amide bond was studied by means of ¹H NMR at 0, 25 and 40°C in CDCl₃. The ΔG⁰ values of the energy differences between individual rotamers were calculated. The results may be helpful in the designing of monomers with desirable properties.     

Identification of coding polymorphisms in human circadian rhythm genes PER1, PER2, PER3, CLOCK, ARNTL, CRY1, CRY2 and TIMELESS in a multi-ethnic screening panel.

STUDY OBJECTIVE: In this study, the exonic regions of the circadian rhythm genes PER1, PER2, PER3, CLOCK, ARNTL, CRY1, CRY2 and TIMELESS were re-sequenced and coding changes identified in a panel of 95 individuals varying in ethnicity. STUDY PARTICIPANTS: DNA screening panel consisting of 95 DNA samples (17 American Caucasians, 17 African Americans, 8 Ashkenazi Jews, 8 Chinese, 8 Japanese, 5 Mexican Indians, 8 Mexicans, 8 Northern Europeans, 8 Puerto Ricans, and 8 South Americans) selected from the Coriell Institute Human Variation Panel. RESULTS: In addition to coding changes already identified in the database dbSNP, novel coding changes were identified, including PER1: Pro37Ser, Pro351Ser, Gln988Pro, Ala998Thr; PER2: Leu83Arg, Leu157Leu, Thre174Ile, Phe400Phe, Pro822Pro, Ala828Thr, Ala861Val, Phe876Leu, Val883Met, Val903Ile, Ala923Pro; PER3: Pro67Pro, Val90Ile, His638His, Ala820Ala, Leu929Leu; ARNTL: Arg166Gln, Ser459Phe; CLOCK: Ala34Ala, Ser208Cys, Phe233Phe, Ser632Thr, Ser816Ser; TIMELESS: Met870Val and CRY2: His35His. No coding polymorphisms were identified in CRY1. CONCLUSIONS: Considerable genetic variation occurs within the coding region of the genes regulating circadian rhythm. Many of the non-synonymous coding polymorphisms could affect protein structure/function with the potential to affect molecular regulation of the sleep/wake cycle. Many of the potential functional effects could be ethnic group specific.     

Stabilization of plant formate dehydrogenase by rational design

Recombinant formate dehydrogenase (FDH, EC 1.2.1.2) from soy Glycine max (SoyFDH) has the lowest values of Michaelis constants for formate and NAD⁺ among all studied formate dehydrogenases from different sources. Nevertheless, it also has the lower thermal stability compared to enzymes from bacteria and yeasts. The alignment of full sequences of FDHs from different sources as well as structure of apo- and holo-forms of SoyFDH has been analyzed. Ten mutant forms of SoyFDH were obtained by site-directed mutagenesis. All of them were purified to homogeneity and their thermal stability and substrate specificity were studied. Thermal stability was investigated by studying the inactivation kinetics at different temperatures and by differential scanning calorimetry (DSC). As a result, single-point (Ala267Met) and double mutants (Ala267Met/Ile272Val) were found to be more stable than the wild-type enzyme at high temperatures. The stabilization effect depends on temperature, and at 52°C it was 3.6- and 11-fold, respectively. These mutants also showed higher melting temperatures in DSC experiments — the differences in maxima of the melting curves (T _m) for the single and double mutants were 2.7 and 4.6°C, respectively. For mutations Leu24Asp and Val127Arg, the thermal stability at 52°C decreased 5- and 2.5-fold, respectively, and the T _m decreased by 3.5 and 1.7°C, respectively. There were no differences in thermal stability of six mutant forms of SoyFDH — Gly18Ala, Lys23Thr, Lys109Pro, Asn247Glu, Val281Ile, and Ser354Pro. Analysis of kinetic data showed that for the enzymes with mutations Val127Arg and Ala267Met the catalytic efficiency increased 1.7- and 2.3-fold, respectively.     

Stereodynamics of tetramezine

The antidepressant drug tetramezine [1,2‐bis‐(3,3‐dimethyldiaziridin‐1‐yl)ethane] consists of two bridged diaziridine moieties with four stereogenic nitrogen centers, which are stereolabile and, therefore, are prone to interconversion. The adjacent substituents at the nitrogen atoms of the diaziridines moieties exist only in an antiperiplanar conformation, which results in a coupled interconversion. Therefore, three stereoisomers exist (meso form and two enantiomeric forms), which epimerize when the diaziridine moieties are regarded as stereogenic units due to the coupled interconversion. Here, we have investigated the epimerization between the meso and enantiomeric forms by dynamic gas chromatography. Temperature‐dependent measurements were performed, and reaction rate constants were determined using the unified equation of chromatography implemented in the software DCXplorer. The activation barriers of the epimerization were found to be ΔG^≠ = 100.7 kJ mol^?1 at 25°C and ΔG^≠ = 104.5 kJ mol^?1 at 37°C, respectively. The activation enthalpy and entropy were determined to be ΔH^≠ = 70.3 ± 0.4 kJ mol^?1 and ΔS^≠ = ?102 ± 2 J mol^?1 K^?1. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Epitope analysis of the multiphosphorylated peptide αs1‐casein(59–79)

The multiphosphorylated tryptic peptide α_s1‐casein(59–79) has been shown to be antigenic with anti‐casein antibodies. In an approach to determine the amino acyl residues critical for antibody binding we undertook an epitope analysis of the peptide using overlapping synthetic peptides. With α_s1‐casein(59–79) as the adsorbed antigen in a competitive ELISA only two of five overlapping synthetic peptides at 1 mM significantly inhibited binding of the anti‐casein antibodies. Peptides Glu‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu and Ile‐Val‐Pro‐Asn‐Ser(P)‐Val‐Glu‐Glu inhibited antibody binding by 20.0±3.6% and 60.3±7.9%, respectively. The epitope of Glu⁶³‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu⁷⁰ was further localised to the phosphoseryl cluster as the peptide Ser(P)‐Ser(P)‐Ser(P) significantly inhibited binding of the anti‐casein antibodies to α_s1‐casein(59–79) by 29.5±7.4%. Substitution of Ser(P)⁷⁵ with Ser⁷⁵ in the second inhibitory peptide Ile‐Val‐Pro‐Asn‐Ser(P)⁷⁵‐Val‐Glu‐Glu also abolished inhibition of antibody binding to α_s1‐casein (59–79) demonstrating that Ser(P)⁷⁵ is also a critical residue for recognition by the antibodies. These data show that the phosphorylated residues in the cluster sequence ‐Ser(P)⁶⁶‐Ser(P)‐Ser(P)⁶⁸ and in the sequence ‐Pro⁷³‐Asn‐Ser(P)‐Val‐Glu⁷⁷‐ are critical for antibody binding to α_s1‐casein(59–79) and further demonstrate that a highly phosphorylated segment of a protein can be antigenic. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

CHARACTERISTICS OF AMINO ACID ACCUMULATION BY SYNAPTOSOMAL PARTICLES ISOLATED FROM RAT BRAIN

—The characteristics of the accumulation of 14 L-amino acids (Leu, Ileu, Val, His, Tyr, Phe, Gly, Ala, Ser, Thr, Asp, Pro, Arg and Lys) by synaptosomal fractions prepared from rat brains were studied. Distinct differences were observed in the ion requirements for the accumulation of these amino acids. The accumulation of Asp and Pro alone showed a total requirement for Na⁺; uptakes of the other amino acids were either maximal in Na⁺-free media or only partially dependent on the presence of external Na⁺. With brain maturation, two types of developmental alterations could be distinguished: (1) changes in rates of influx, and (2) changes in the effects of ions. Synaptosomal fractions prepared from brains of immature rats accumulated Leu, Arg and Lys to a greater extent and Val, Tyr, Pro and Asp to a lesser extent than did the fractions prepared from brains of mature animals. The accumulation of Ser and Thr by immature fractions was partially dependent on external Na⁺, whereas their accumulation by adult fractions was Na⁺-independent. These alterations in Na⁺ requirements coincided with developmental changes in mutual inhibitions of amino acid transport.     

Cyclopeptidic Constituents from Arenaria oreophila J. D. Hooker （Caryophyllaceae）

To further investigate the cyclopeptldes of the Caryophyllaceae family, two new cyclopeptldes, named Arenarlphilin E （compound 1） and Arenariphilin F （compound 2）, were obtained from Arenaria oreophUe J. D. Hooker using some Isolation methods, e. g. normal and reverse silica gel. By detailed spectroscopic analysis, such as FAB^＋-MS, 1 D NMR, 2D NMR, the structures of Arenariphilin E （compound 1） and Arenariphilln F （compound 2） were determined as cyclo（lle^1-Gly-Val^1-Ala-Leu-lle^3-lle^2-Val^2-Pro） and cyclo（Pro^2-Pro^1-Gly^2-lle-Val-Leu-Gly^1-AiaThr- Gly^3）, respectively.     

Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential in producing valuable chiral epoxides and β-substituted alcohols. The wild-type (WT) enzyme displays a high R-enantiopreference toward most aromatic substrates, whereas no S-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement in R-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from an E_R of 65 [WT] to an E_S of 101) and approximately 100-fold-enhanced catalytic efficiency toward (S)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (S)-2-CPE bound at a different location than that of its R-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference.     

The stereodynamics of 1,2‐dipropyldiaziridines

N‐alkylated trans‐diaziridines are an intriguing class of compounds with two stereogenic nitrogen atoms which easily interconvert. In the course of our investigations of the nature of the interconversion process via nitrogen inversion or electrocyclic ring opening ring closure, we synthesized and characterized the three constitutionally isomeric diaziridines 1,2‐di‐n‐propyldiaziridine 1 , 1‐isopropyl‐2‐n‐propyldiaziridine 2 , and 1,2‐diisopropyldiaziridine 3 to study the influence of the substituents on the interconversion barriers. Enantiomer separation was achieved by enantioselective gas chromatography on the chiral stationary phase Chirasil‐β‐Dex with high separation factors α (1‐isopropyl‐2‐n‐propyldiaziridine: 1.18; 1, 2‐diisopropyldiaziridine: 1.24; 100°C 50 kPa He) for the isopropyl substituted diaziridines. These compounds showed pronounced plateau formation between 100 and 150°C, and peak coalescence at elevated temperatures. The enantiomerization barriers ΔG? and activation parameters ΔH? and ΔS? were determined by enantioselective dynamic gas chromatography (DGC) and direct evaluation of the elution profiles using the unified equation implemented in the software DCXplorer. Interestingly, 1‐isopropyl‐2‐n‐propyldiaziridine and 1,2‐diisopropyldiaziridine exhibit similar high interconversion barriers ΔG? (100°C) of 128.3 ± 0.4 kJ mol^?1 and 129.8 ± 0.4 kJ mol^?1, respectively, which indicates that two sterically demanding substituents do not substantially increase the barrier as expected for a distinct nitrogen inversion process. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Co-permeability of 3H-labelled water and 14C-labelled organic acids across isolated Prunus laurocerasus cuticles: effect of temperature on cuticular paths of diffusion

Co‐permeability of ³H‐labelled water and ¹⁴C‐labelled benzoic acid or 2,4‐dichlorophenoxyacetic acid across isolated cuticular membranes of Prunus laurocerasus L. was measured at temperatures ranging from 15 to 50 °C. The water and benzoic acid permeances were highly correlated over the whole temperature range investigated, whereas water and 2,4‐dichlorophenoxyacetic acid permeances were only correlated between 15 and 30 °C. The activation energies of cuticular permeability calculated from Arrhenius plots were 40 kJ mol^?1 for water and benzoic acid and 115 kJ mol^?1 for 2,4‐dichlorophenoxyacetic acid. The slopes of the Arrhenius plots of 2,4‐dichlorophenoxyacetic acid were linear between 15 and 50 °C, whereas pronounced phase transitions around 30 °C were observed for water and benzoic acid permeability. However, with isolated polymer matrix membranes, where cuticular waxes forming the transport‐limiting barrier of cuticles have been extracted, phase transitions were not observed for water and benzoic acid. It is concluded that temperatures above 30 °C caused structural changes in the transport‐limiting barrier of the cuticles leading to additional paths of diffusion for water and benzoic acid but not for 2,4‐dichlorophenoxyacetic acid.     

Site-Directed Mutagenesis and Thermostability of Xylanase XYNB from <Emphasis Type="Italic">Aspergillus niger</Emphasis> 400264

Xylanase is one of the most important hemicellulases in industry. However, its low thermostability limits its applications. In this study, one thermostable xylanase-producing strain 400264 was obtained from screening 11 Aspergillus niger strains (producing thermotolerant xylanase), and the optimum temperature of crude xylanase extracted from it was 55°C. Original activity of the crude xylanase is 64% at 60°C and 55% at 85°C with an incubation time of 30 min, respectively. After the expression of recombinant xylanase gene (xynA/xynB), the XYNB (xylanase B) showed higher thermostability than XYNA (xylanase A). Recombinant enzyme XYNB retained 94% of its activity for 10 min at 85°C, while XYNA with no activity left. Site-directed mutagenesis was performed to replace Ala33 of XYNB by Ser33 resulting 19% decrease in enzyme activity after incubating at 85°C for 30 min. It suggested that the Ala33 residue may have a certain effect on the thermophilic adaptation of xylanase.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

The amino acid sequence of the C- terminal cyanogen bromide peptide of human J chain

The carboxyl-terminal peptide obtained from human J chain treated with cyanogen bromide (CNBr)¹ has been isolated and the amino acid sequence determined as Val - Glx - Thr - Ala - Leu - Thr - Pro - Asx - Ala - CMCys - Tyr - Pro - Asx. Comparisons with the primary structures of human lambda, kappa, alpha, or mu chains failed to disclose analogous regions with these immunoglobulins.     

Stability and identification of active-site residues of carboxymethylcellulases fromAspergillus niger andCellulomonas biazotea

Determination of the apparent pK _a's of purified carboxymethylcellulases fromAspergillus niger andCellulomonas biazotea at different temperatures and in the presence of dioxane indicated two side chain carboxyl groups which controlled the limiting rate in both organisms. The thermostability of both enzymes slightly decreased with increasing pH from 5 to 7.5 but was unaffected in the presence of 0.5 mmol/L Mn²⁺. The CMCase fromC. biazotea had an activation energy of 35 kJ/mol and a half-life of 89 min in the presence of 8 mol/L urea at 40°C. The half-life of CMCase fromA. niger in 8 mol/L urea and at 37°C was 125 min as determined by a 0–9 mol/L transverse urea gradient PAGE. The CMCases fromA. niger andC. biazotea had the same thermostabilities in the absence of CMC although the enzyme from the former was more thermostable in the presence of the substrate. The CMCase fromA. niger was also more efficient in hydrolyzing CMC than the enzyme fromC. biazotea.