BBA - General Subjects (v.1523, #2-3)
Inactivation of a novel three-cistronic operon tcaR-tcaA-tcaB increases teicoplanin resistance in Staphylococcus aureus by Marcel Brandenberger; Martin Tschierske; Philipp Giachino; Akihito Wada; Brigitte Berger-Bächi (135-139).
A novel teicoplanin-associated operon termed tcaR-tcaA-tcaB was identified by Tn917-mediated insertional mutagenesis. Resistance to teicoplanin rose 4-fold by insertional inactivation of tcaA or by deletion of the entire operon. tcaA encodes a hypothetical transmembrane protein with a metal-binding motif, possibly a sensor–transducer. tcaB codes for a membrane-associated protein, which has sequence homologies to a bicyclomycin resistance protein. The two genes are preceded by tcaR encoding a putative regulator with sequence homologies to the transcriptional regulator MarR. The fact that tcaA inactivation as well as deletion of tcaRAB produced the same increase in teicoplanin resistance confirmed the association of tcaRAB with teicoplanin susceptibility. Cotransductional crosses showed that the level of teicoplanin resistance produced by these insertions was strain-dependent and that in the methicillin-resistant strain COL, it was paired with a remarkable decrease in methicillin resistance. This allowed to postulate that tcaRAB may be involved in some way in cell wall biosynthesis, and that teicoplanin may interact with TcaA and/or TcaB either directly or indirectly.
Keywords: Teicoplanin; Methicillin; Resistance; tcaRAB operon; Inactivation; Staphylococcus aureus;
Melatonin reduces rat hepatic macromolecular damage due to oxidative stress caused by δ-aminolevulinic acid by Małgorzata Karbownik; Russel J. Reiter; Joaquin J. Garcia; Dun Xian Tan; Wenbo Qi; Lucien C. Manchester (140-146).
δ-Aminolevulinic acid, precursor of heme, accumulates in a number of organs, especially in the liver, of patients with acute intermittent porphyria. The potential protective effect of melatonin against oxidative damage to nuclear DNA and microsomal and mitochondrial membranes in rat liver, caused by δ-aminolevulinic acid, was examined. Changes in 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels, an index of DNA damage, and alterations in membrane fluidity (the inverse of membrane rigidity) and lipid peroxidation in microsomal and mitochondrial membranes, as indices of damage to lipid and protein molecules in membranes, were estimated. Measurements were made in rat liver after a 2 week treatment with δ-aminolevulinic acid (40 mg/kg b.w., every other day). To test the potential protective effects of melatonin, the indole was injected (i.p. 10 mg/kg b.w.) 3 times daily for 2 weeks. 8-OHdG levels and lipid peroxidation in microsomal membranes increased significantly whereas microsomal and mitochondrial membrane fluidity decreased as a consequence of δ-aminolevulinic acid treatment. Melatonin completely counteracted the effects of δ-aminolevulinic acid. Melatonin was highly effective in protecting against oxidative damage to DNA as well as to microsomal and mitochondrial membranes in rat liver and it may be useful as a cotreatment in patients with acute intermittent porphyria.
Keywords: Melatonin; Porphyria; Free radical; DNA; Membrane;
On the modulation of photoinduced fluorescence enhancement and conformational stability of albumin-bound bilirubin: by Mohd.Mushahid Khan; Saad Tayyab (147-153).
Photoinduced fluorescence enhancement of bilirubin bound to primary binding site on human serum albumin (HSA) was completely ceased when ϵ-NH2 groups of its internal lysine residues were covalently blocked by acetylation or succinylation though the pigment bound to these derivatives in a folded conformation akin to that bound to HSA. These photoinduced fluorescence modulations cannot be ascribed to the binding of bilirubin to secondary low affinity sites as the CD spectrum of bilirubin bound to these derivatives showed complete inversion upon addition of chloroform which binds to subdomain IIA in HSA where high affinity bilirubin binding site is located. Presence of chloroform reconciled the photoinduced alterations in the CD spectrum observed in its absence, suggesting that chloroform stabilized the bound ligand against light but the fluorescence properties of bilirubin complexed with acetylated or succinylated derivatives remained unchanged. Guanidination of internal ϵ-NH2 groups in HSA by O-methylisourea did not alter the spectral properties of the bound ligand. These results suggest that salt linkage(s) existing between ϵ-NH2 groups of lysine residues in HSA and carboxyl groups of bilirubin, act(s) as a potential barrier during conformational rotation of the bound ligand assisted by photoactivation and their abolishment can alter its dynamics and stereoselectivity, a hitherto unnoticed implication of salt linkage(s) in BR–HSA complex.
Keywords: Albumin-bound bilirubin; Chemical modification; Chloroform-induced stabilization; Conformational dynamics; Fluorescence modulation; Photoirradiation; Salt linkage;
The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and its analogues prevent damage to 2-deoxyribose mediated by ferric iron plus ascorbate by Marcelo Hermes-Lima; Prem Ponka; Herbert M. Schulman (154-160).
Iron chelating agents are essential for treating iron overload in diseases such as β-thalassemia and are potentially useful for therapy in non-iron overload conditions, including free radical mediated tissue injury. Deferoxamine (DFO), the only drug available for iron chelation therapy, has a number of disadvantages (e.g., lack of intestinal absorption and high cost). The tridentate chelator pyridoxal isonicotinoyl hydrazone (PIH) has high iron chelation efficacy in vitro and in vivo with high selectivity and affinity for iron. It is relatively non-toxic, economical to synthesize and orally effective. We previously demonstrated that submillimolar levels of PIH and some of its analogues inhibit lipid peroxidation, ascorbate oxidation, 2-deoxyribose degradation, plasmid DNA strand breaks and 5,5-dimethylpyrroline-N-oxide (DMPO) hydroxylation mediated by either Fe(II) plus H2O2 or Fe(III)–EDTA plus ascorbate. To further characterize the mechanism of PIH action, we studied the effects of PIH and some of its analogues on the degradation of 2-deoxyribose induced by Fe(III)–EDTA plus ascorbate. Compared with hydroxyl radical scavengers (DMSO, salicylate and mannitol), PIH was about two orders of magnitude more active in protecting 2-deoxyribose from degradation, which was comparable with some of its analogues and DFO. Competition experiments using two different concentrations of 2-deoxyribose (15 vs. 1.5 mM) revealed that hydroxyl radical scavengers (at 20 or 60 mM) were significantly less effective in preventing degradation of 2-deoxyribose at 15 mM than 2-deoxyribose at 1.5 mM. In contrast, 400 μM PIH was equally effective in preventing degradation of both 15 mM and 1.5 mM 2-deoxyribose. At a fixed Fe(III) concentration, increasing the concentration of ligands (either EDTA or NTA) caused a significant reduction in the protective effect of PIH towards 2-deoxyribose degradation. We also observed that PIH and DFO prevent 2-deoxyribose degradation induced by hypoxanthine, xanthine oxidase and Fe(III)–EDTA. The efficacy of PIH or DFO was inversely related to the EDTA concentration. Taken together, these results indicate that PIH (and its analogues) works by a mechanism different than the hydroxyl radical scavengers. It is likely that PIH removes Fe(III) from the chelates (either Fe(III)–EDTA or Fe(III)–NTA) and forms a Fe(III)–PIH2 complex that does not catalyze oxyradical formation.
Keywords: Chelator; Iron; Iron overload; Free radical; Antioxidant; Deferoxamine; Pyridoxal isonicotinoyl hydrazone;
Introduction of the human growth hormone gene into the guinea pig mammary gland by in vivo transfection promotes sustained expression of human growth hormone in the milk throughout lactation by Julie R. Hens; Michael D. Amstutz; Floyd L. Schanbacher; Ian H. Mather (161-171).
We tested the feasibility of transfecting mammary tissue in vivo with an expression plasmid encoding the human growth hormone (hGH) gene, under the control of the cytomegalovirus promoter. Guinea pig mammary glands were transfected with plasmid DNA infused through the nipple canal and expression was monitored in control and transfected glands by radioimmunoassay of milk samples for hGH. Sustained expression of hGH throughout lactation was attained with a polyion transfection complex shown to be optimal for the transfection of bovine mammary cells, in vitro. However, contrary to expectations, hGH expression was consistently 5- to 10-fold higher when DEAE–dextran was used alone for transfection. Thus polyion complexes which are optimal for the transfection of cells in vitro may not be optimal in vivo. The highest concentrations of hGH in milk were obtained when glands were transfected within 3 days before parturition. This method may have application for studying the biological role or physical properties of recombinant proteins expressed in low quantities, or for investigating the regulation of gene promoters without the need to construct viral vectors or produce transgenic animals.
Keywords: In vivo transfection; Mammary gland; Expression of recombinant protein; Lactation; Charge-shifting polyions; DEAE–dextran;
The biochemical mode of inhibition of DNA polymerase β by α-rubromycin by Yoshiyuki Mizushina; Takamasa Ueno; Masako Oda; Toyofumi Yamaguchi; Mineo Saneyoshi; Kengo Sakaguchi (172-181).
Quinone antibiotics, α- and β-rubromycin, were originally found as inhibitors of retroviral reverse transcriptase. We investigated the effects of these agents on DNA metabolic enzymes including DNA and RNA polymerases as retroviral reverse transcriptase is a kind of the polymerase. As expected, we found that α- and β-rubromycin strongly inhibited not only the retroviral reverse transcriptase activity, but the activities of the mammalian DNA polymerases, telomerase and terminal deoxynucleotidyl transferase in vitro. These agents should therefore be classified as DNA polymerase inhibitors. The Ki values of α-rubromycin against nucleotide substrate were 0.66 and 0.17 μM for DNA polymerase α and β (pol. α and β), respectively, and those of β-rubromycin was 2.40 and 10.5 μM, respectively. α-Rubromycin strongly inhibited the pol. β activity, and showed the strongest pol. β inhibitory effect reported to date. At least on pol. β, α-rubromycin was suggested to bind to the active region competing with the nucleotide substrate, and subsequently inhibit the catalytic activity. α-Rubromycin directly competed with the nucleotide substrate, and indirectly but simultaneously and non-competitively disturbed the template-DNA interaction with pol. β.
Keywords: α-Rubromycin; β-Rubromycin; DNA polymerase β; Enzyme inhibitor; Trypsin digestion;
Differed preferential iron-binding lobe in human transferrin depending on the presence of bicarbonate detected by HPLC/high-resolution inductively coupled plasma mass spectrometry by Megumi Hamano Nagaoka; Tamio Maitani (182-188).
The binding of iron (Fe) to human serum transferrin (Tf) was analyzed with an HPLC system equipped with an anion exchange column and directly connected with a high-resolution inductively coupled plasma mass spectrometer for metal detection. The 56Fe level in the eluate was monitored at resolution m/Δm=3000. Two monoferric Tfs were assigned based on the results of urea–PAGE and desferrioxamine experiments. When Fe was added as Fe-citrate stepwise to an apo-Tf solution in the presence of bicarbonate, the N-lobe site was the preferential Fe-binding site, while the C-lobe site was preferred in the absence of bicarbonate. In both cases, the Fe-peak areas of the preferential site and Fe2-Tf increased up to an Fe/Tf molar ratio of 1, and then the peak area of the monoferric Tf decreased while the peak area of Fe2-Tf increased. When the Fe/Tf molar ratio was below 1, the amount of Fe bound to the lobe with a weaker affinity was higher in Fe2-Tf than in the monoferric Tf in each case. Namely, Fe2-Tf was the preferential binding state of Fe to human serum Tf. The preference is reasonable for transferring Fe ions effectively to Tf-receptors.
Keywords: Transferrin; Iron; High-resolution inductively coupled plasma mass spectrometry; High-performance liquid chromatography; Binding site;
Dopamine-melanin protects against tyrosine nitration, tryptophan oxidation and Ca2+-ATPase inactivation induced by peroxynitrite by Krystyna Stępień; Alicja Zajdel; Adam Wilczok; Tadeusz Wilczok; Agnieszka Grzelak; Agnieszka Mateja; Mirosław Soszyński; Grzegorz Bartosz (189-195).
The effects of dopamine-melanin (DA-melanin), a synthetic model of neuromelanin, on peroxynitrite-mediated 3-nitrotyrosine formation, oxidation of tryptophan in bovine serum albumin and inactivation of erythrocyte membrane Ca2+-ATPase activity were investigated in the absence and in the presence of bicarbonate. DA-melanin inhibited nitration of free tyrosine, loss of tryptophan residues and Ca2+-ATPase inactivation by peroxynitrite in a dose dependent manner. In the presence of bicarbonate, this inhibitory effect was lower for nitration and insignificant for oxidative protein modifications. These results suggest that neuromelanin can protect against nitrating and oxidizing action of peroxynitrite but is a worse protector against the peroxynitrite–CO2 adduct. As peroxynitrite may be a mediator of neurotoxic processes, the obtained results suggest that neuromelanin may be important as a physiological protector against peroxynitrite.
Keywords: Neuromelanin; Dopamine-melanin; Peroxynitrite; 3-Nitrotyrosine; Tryptophan oxidation; Ca2+-ATPase;
Ovotransferrin antimicrobial peptide (OTAP-92) kills bacteria through a membrane damage mechanism by Hisham R Ibrahim; Yasushi Sugimoto; Takayoshi Aoki (196-205).
Ovotransferrin antimicrobial peptide (OTAP-92) is a cationic fragment of hen ovotransferrin (OTf). OTAP-92 consists of 92 amino acid residues located within the 109–200 sequence of the N-lobe of OTf. This study was aimed to delineate the antimicrobial mechanism of OTAP-92 and to identify its interaction with bacterial membranes. OTAP-92 caused permeation of Escherichia coli outer membrane (OM) to 1-N-phenylnaphthylamine fluorescent probe in a dose-dependent manner. These results suggested that OTAP-92 crossed the bacterial OM by a self-promoted uptake. Cytoplasmic membrane of E. coli was found to be the target for OTAP-92 bactericidal activity, as assayed by the unmasking of cytoplasmic β-galactosidase due to membrane permeabilization in a kinetic manner. Pretreatment of bacteria with uncoupler, carbonyl cyanide m-chlorophenylhydrazone, markedly enhanced permeation of cytoplasmic membrane, suggesting that the membrane permeation due to OTAP-92 is independent of the transmembrane potential. In an E. coli phospholipid liposome model, it was demonstrated that OTAP-92 has the ability to dissipate the transmembrane electrochemical potential. Intrinsic fluorescence spectra of the two tryptophan residues in OTAP-92, using liposomal membrane, have identified the lipid-binding region as a helix-sheet motif, and suggested an adjacent Ca2+-sensitive site within OTAP-92. These data indicated that OTAP-92 possesses a unique structural motif similar to the insect defensins. Further, this cationic antimicrobial peptide is capable of killing Gram-negative bacteria by crossing the OM by a self-promoted uptake and cause damage to the biological function of cytoplasmic membrane.
Keywords: Ovotransferrin; Antibacterial activity; Bactericidal peptide; Conformation; Liposome; Membrane permeation; Fluorescence; Defensin;
Determination of H2S solubility via the reaction with ferric hemoglobin I from the bivalve mollusc Lucina pectinata by Alberto Boffi; Menico Rizzi; Fabrizio Monacelli; Paolo Ascenzi (206-208).
A new, simple and fast spectrophotometric method for the determination of the H2S concentration is reported. This method, based on the 1:1 reaction between H2S and the ferric derivative of hemoglobin I (HbI) from the bivalve mollusc Lucina pectinata, allows the quantitative determination of H2S dissolved in a given solution even at concentrations as low as 1×10−6 M. Note that L. pectinata HbI is considered the physiological receptor of H2S.
Keywords: H2S solubility; Lucina pectinata hemoglobin I; H2S binding to Lucina pectinata hemoglobin I;
Time course and quantitative analysis of the adaptive responses to 85% oxygen in the rat lung and heart by Pablo Evelson; Beatriz González-Flecha (209-216).
The phenomenon of oxygen tolerance (resistance to 100% O2 in rats previously exposed to 85% O2) constitutes one of the few models of adaptive responses to oxidative stress in mammals. In vitro studies suggest that reactive oxygen species mediate this response. To test this hypothesis in vivo, we followed the time course of oxidative stress, enzyme induction, and edema in the lung, heart and liver of rats exposed to 85% O2 for 1 to 5 days. Interestingly, not only the lung, but also the heart of rats exposed to 85% O2 showed increases in the production of O⋅− 2 (aconitase inactivation) early during the exposure. Increases in O⋅− 2 were associated to oxidative stress (increased in situ chemiluminescence) and transient edema in both tissues. Both the lung and heart displayed induction of superoxide dismutase and reversion of the oxidative stress and damage. The adaptive response in the heart was faster and more efficient, suggesting that this tissue is at a more critical risk when exposed to elevated O2 concentrations.
Keywords: Reactive oxygen species; Oxidative stress; Hyperoxia; Antioxidant;
Modulation of luteinizing hormone subunit gene expression by intracerebroventricular microinjection of gonadotropin-releasing hormone or β-endorphin in female rats by Alina Gajewska; Kazimierz Kochman; Yannick Lerrant; Helena Kochman; Raymond Counis (217-224).
The effects of gonadotropin-releasing hormone (GnRH), β-endorphin and its antagonist naloxone on the expression of luteinizing hormone (LH) subunit genes and LH secretion were examined in ovariectomized and/or cycling female rats through their direct microinjection into the third cerebral ventricle, in the proximity of the hypothalamus-pituitary complex. GnRH (1 nM) induced a significant augmentation of the pituitary content of α mRNA when administered 15, 30 or 60 min intervals over 5 h to ovariectomized rats whereas only the 30 and 60 min intervals were effective in increasing LHβ mRNA, and the 60 min intervals for LH release. This was in agreement with the established concept of a pulse-dependent regulation of gonadotropin synthesis and release. Hourly pulses of GnRH also increased α and LHβ mRNA levels when microinjected in female cycling rats during proestrus or diestrus II. Using this model we observed a marked negative influence of hourly intracerebral microinjections of β-endorphin on LH mRNA content and LH release in ovariectomized rats while naloxone had no effect. This suggests that endogenous β-endorphin was unable to exert its negative action on β-endorphin receptors that were present and responded to the ligand. The present approach would be valuable for the exploration of the mechanisms of action of β-endorphin or other substances on the functions of the gonadotrophs.
Keywords: Gonadotropin-releasing hormone; β-Endorphin; Intracerebroventricular microinjection; Luteinizing hormone subunit mRNA; Luteinizing hormone release;
Hemolysis of human red blood cells by riboflavin-Cu(II) system by Iyad Ali; Mansour K.M Gatasheh; Imrana Naseem (225-229).
The photodynamic action of riboflavin is generally considered to involve the generation of reactive oxygen species, whose production is enhanced when Cu(II) is present in the reaction. In the present study we report that photoactivated riboflavin causes K+ loss from fresh human red blood cells (RBC) in a time dependent manner. Addition of Cu(II) further enhances the K+ loss and also leads to significant hemolysis. Riboflavin in a 2:1 stoichiometry with Cu(II) leads to maximum K+ loss and up to 45% hemolysis. Bathocuproine, a specific Cu(I)-sequestering agent, when present in the reaction, inhibits the hemolysis completely. Free radical scavengers like superoxide dismutase, potassium iodide and mannitol inhibited the hemolysis up to 55% or more. However, thiourea was the most effective scavenger showing 90% inhibition. These results suggest that K+ leakage and hemolysis of human RBC are basically free radical mediated reactions.
Keywords: Riboflavin; Copper; Oxygen radical; Red blood cell; Hemolysis;
Specific ganglioside changes in extraneural tissues of adult rats with hypothyroidism by Megumi Saito; Kiyoshi Sugiyama (230-235).
Adults rats with hypothyroidism were prepared by administration of 6-propyl-2-thiouracil (PTU) or methimazole, and the tissues were examined for their gangliosides through methods including glycolipid-overlay techniques. Normal thyroid tissue contained GM3, GD3, and GD1a as the major gangliosides, with GM1, GD1b, GT1b, and GQ1b in lesser amounts. The goitrous tissue of PTU-induced hypothyroid rats had higher concentrations of GM1 and GD1a with a concomitant decrease of GM3. The amount of GT3 in thyroid tissue was increased in hypothyroid animals. While normal liver tissue had a complex ganglioside pattern with a- and b-series gangliosides, the PTU-induced hypothyroid tissue showed a simpler ganglioside profile that consisted mainly of a-series gangliosides with almost undetectable amounts of b-series gangliosides. The expression of c-series gangliosides was suppressed in the hypothyroid liver tissue. Heart tissue had higher contents of GM3 and GT3 than control. No apparent change was observed in the compositions of major and c-series gangliosides in other extraneural tissues (i.e., kidney, lung, spleen, thymus, pancreas, testis, skeletal muscle, and eye lenses), and neural tissues (i.e., cerebrum and cerebellum) from PTU-induced hypothyroid rats. The ganglioside changes of thyroid, liver, and heart tissues were reproduced in corresponding tissues of methimazole-induced hypothyroid rats. These results suggest that hypothyroid conditions affect the biosynthesis and expression of gangliosides in specific tissue and cell types.
Keywords: Ganglioside; Hypothyroidism; Propylthiouracil; Methimazole; Thyroid; A2B5;
Structural analysis of N-glycans from yellow lupin (Lupinus luteus) seed diphosphonucleotide phosphatase/phosphodiesterase by Mariusz Olczak; Wies l ̵ aw W a ̨ torek (236-245).
N-Linked oligosaccharide chains released by hydrazinolysis from yellow lupin seed diphosphonucleotide phosphatase/phosphodiesterase were fluorescence labeled and separated by high performance liquid chromatography (GlycoSep N and GlycoSep H columns). Exoglycosidase sequencing elucidated the structures of 24 separated N-glycans. Thirty percent of isolated glycans were found to be of high-mannose type (three to eight mannosyl residues), 42% were complex type and 26% belonged to paucimannosidic type. Among complex type glycans, structures with Lewisa epitope were identified. It is very unusual to find all types of plant N-glycans in one protein. Possible reasons for such a broad spectrum of N-glycan structures are discussed.
Keywords: N-Glycan; Exoglycosidase sequencing; Diphosphonucleotide phosphatase/phosphodiesterase; Yellow lupin;
Chorion peroxidase-mediated NADH/O2 oxidoreduction cooperated by chorion malate dehydrogenase-catalyzed NADH production: a feasible pathway leading to H2O2 formation during chorion hardening in Aedes aegypti mosquitoes by Qian Han; Guoyu Li; Jianyong Li (246-253).
A specific chorion peroxidase is present in Aedes aegypti and this enzyme is responsible for catalyzing chorion protein cross-linking through dityrosine formation during chorion hardening. Peroxidase-mediated dityrosine cross-linking requires H2O2, and this study discusses the possible involvement of the chorion peroxidase in H2O2 formation by mediating NADH/O2 oxidoreduction during chorion hardening in A. aegypti eggs. Our data show that mosquito chorion peroxidase is able to catalyze pH-dependent NADH oxidation, which is enhanced in the presence of Mn2+. Molecular oxygen is the electron acceptor during peroxidase-catalyzed NADH oxidation, and reduction of O2 leads to the production of H2O2, demonstrated by the formation of dityrosine in a NADH/peroxidase reaction mixture following addition of tyrosine. An oxidoreductase capable of catalyzing malate/NAD+ oxidoreduction is also present in the egg chorion of A. aegypti. The cooperative roles of chorion malate/NAD+oxidoreductase and chorion peroxidase on generating H2O2 with NAD+ and malate as initial substrates were demonstrated by the production of dityrosine after addition of tyrosine to a reaction mixture containing NAD+ and malate in the presence of both malate dehydrogenase fractions and purified chorion peroxidase. Data suggest that chorion peroxidase-mediated NADH/O2 oxidoreduction may contribute to the formation of the H2O2 required for chorion protein cross-linking mediated by the same peroxidase, and that the chorion associated malate dehydrogenase may be responsible for the supply of NADH for the H2O2 production.
Keywords: Chorion peroxidase; Malate dehydrogenase; H2O2; NAD+; NADH; Nicotinamide adenine dinucleotide; Dityrosine;
Amino acid sequence and some properties of phytolacain G, a cysteine protease from growing fruit of pokeweed, Phytolacca americana by Tetsuya Uchikoba; Kazunari Arima; Hiroo Yonezawa; Masayuki Shimada; Makoto Kaneda (254-260).
A protease, phytolacain G, has been found to appear on CM-Sepharose ion-exchange chromatography of greenish small-size fruits of pokeweed, Phytolacca americana L, from ca. 2 weeks after flowering, and increases during fruit enlargement. Reddish ripe fruit of the pokeweed contained both phytolacain G and R. The molecular mass of phytolacain G was estimated to be 25.5 kDa by SDS–PAGE. Its amino acid sequence was reconstructed by automated sequence analysis of the peptides obtained after cleavage with Achromobacter protease I, chymotrypsin, and cyanogen bromide. The enzyme is composed of 216 amino acid residues, of which it shares 152 identical amino acid residues (70%) with phytolacain R, 126 (58%) with melain G, 108 (50%) with papain, 106 (49%) with actinidain, and 96 (44%) with stem bromelain. The amino acid residues forming the substrate binding S2 pocket of papain, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Trp, Met, His, Ala, and Ser in phytolacain G, respectively. As a consequence of these substitutions, the S2 pocket is expected to be less hydrophobic in phytolacain G than in papain.
Keywords: Cysteine protease; Plant endopeptidase; Substrate specificity; Amino acid sequence; Pokeweed fruit; Phytolacca americana;
Comparative characterization of two serine endopeptidases from Nocardiopsis sp. NCIM 5124 by V.S Dixit; A Pant (261-268).
A protease-producing, crude oil degrading marine isolate was identified as Nocardiopsis sp. on the basis of the morphology, cell wall composition, mycolic acid analysis and DNA base composition. The Nocardiopsis produces two extracellular proteases, both of which are alkaline serine endopeptidases. Protease I was purified to homogeneity by chromatography on CM-Sephadex at pH 5.0 and pH 9.0. Protease II was purified using DEAE-cellulose, Sephadex G-50, phenyl–Sepharose and hydroxyapatite chromatography. Protease I and II had almost similar M r of 21 kDa (Protease I) and 23 kDa (Protease II), pI of 8.3 and 7.0 respectively with pH and temperature optima for activity between 10.0 and 11.0 and about 60°C. Specific activities were 152 and 14 U/mg respectively on casein. However, Protease I was antigenically unrelated to Protease II. Both proteases were endopeptidases and required extended substrate binding for catalysis. Both proteases had collagenolytic and fibrinolytic activity but only Protease I had elastinolytic activity. The proteases were chymotrypsin-like with respect to their amino acid compositions and N-terminal sequences.
Keywords: Protease; Endopeptidase; Actinomycete; Substrate specificity; Nocardiopsis;
Functional expression and genomic structure of human N-acetylglucosamine-6-O-sulfotransferase that transfers sulfate to β-N-acetylglucosamine at the nonreducing end of an N-acetyllactosamine sequence 1 1 The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL/DDBJ data bank and are available under the accession numbers AB021124 and AB021125. by Hiromi Sakaguchi; Hiroshi Kitagawa; Kazuyuki Sugahara (269-276).
The cDNA and gene encoding human N-acetylglucosamine-6-O-sulfotransferase (Gn6ST) have been cloned. Comparative analysis of this cDNA with the mouse Gn6ST sequence indicates 96% amino acid identity between the two sequences. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active sulfotransferase, which transferred sulfate to the terminal GlcNAc in GlcNAcβ1-O-CH3, GlcNAcβ1-3Galβ1-O-CH3 and GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc but not in GlcNAcα1-4GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser. In addition, neither Galβ1-4GlcNAcβ1-O-naphthalenemethanol nor GalNAcβ1-4GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser were utilized as acceptors. These findings indicate that a terminal β-linked GlcNAc residue is necessary for acceptor substrates of Gn6ST. The human Gn6ST gene spans about 7 kb, consists of two exons and exhibits an intron-less coding region.
Keywords: Gene structure; Glycosaminoglycan; L-selectin ligand; Recombinant enzyme; Sulfotransferase;