scholarly journals Biochemical and Mutational Characterization of the Heme Chaperone CcmE Reveals a Heme Binding Site

2003 ◽  
Vol 185 (1) ◽  
pp. 175-183 ◽  
Author(s):  
Elisabeth Enggist ◽  
Michael J. Schneider ◽  
Henk Schulz ◽  
Linda Thöny-Meyer
Keyword(s):  
Amino Acids ◽  
Covalent Bond ◽  
Resonance Structure ◽  
Visible Range ◽  
Nuclear Magnetic Resonance Structure ◽  
Heme Binding ◽  
Alanine Scanning Mutagenesis ◽  
Isolation And Characterization ◽  
Vinyl Group

ABSTRACT CcmE is a heme chaperone that binds heme transiently in the periplasm of Escherichia coli and delivers it to newly synthesized and exported c-type cytochromes. The chemical nature of the covalent bond between heme and H130 is not known. We have purified soluble histidine-tagged CcmE and present its spectroscopic characteristics in the visible range. Alanine scanning mutagenesis of conserved amino acids revealed that H130 is the only residue found to be strictly required for heme binding and delivery. Mutation of the hydrophobic amino acids F37, F103, L127, and Y134 to alanine affected CcmE more than mutation of charged and polar residues. Our data are in agreement with the recently solved nuclear magnetic resonance structure of apo-CcmE (PDB code 1LIZ) and suggest that heme is bound to a hydrophobic platform at the surface of the protein and then attached to H130 by a covalent bond. Replacement of H130 with cysteine led to the formation of a covalent bond between heme and C130 at a low level. However, the H130C mutant CcmE was not active in cytochrome c maturation. Isolation and characterization of the heme-binding peptides obtained after a tryptic digest of wild-type and H130C CcmE support the hypothesis that heme is bound covalently at a vinyl group.

scholarly journals Isolation and Characterization of Mutations in theEscherichia coli Regulatory Protein XapR

1999 ◽  
Vol 181 (14) ◽  
pp. 4397-4403 ◽  
Author(s):  
Casper Jørgensen ◽  
Gert Dandanell
Keyword(s):  
Amino Acids ◽  
Purine Nucleoside Phosphorylase ◽  
Mutational Analysis ◽  
Regulatory Protein ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Wild Type ◽  
Isolation And Characterization ◽  
In The Wild

ABSTRACT In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase inEscherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

ISOLATION AND CHARACTERIZATION OF RIBONUCLEIC ACID FROM RAT LENS

1962 ◽  
Vol 40 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Anima Devi
Keyword(s):  
Amino Acids ◽  
The Other ◽  
Ocular Lens ◽  
E Coli ◽  
Isolation And Characterization ◽  
Coiled Structure ◽  
Calf Thymus ◽  
Original Procedure ◽  
Polynucleotide Chain

RNA from rat ocular lens has been isolated by a method based on Kirby's original procedure (7), but greatly modified so as to avoid any degradation of RNA by RNase during the process of its extraction from lenses. The absorption at 260 mμ of a 1.0% solution of this purified material in a 1-cm cell is 1.95. Its N/P ratio is 1.58. It has 20 to 25% activity of that of yeast-soluble RNA in accepting activated amino acids. When this RNA (like all other RNA's) is heated and cooled the polynucleotide chain can again form loops, thus suggesting a randomly coiled structure for this RNA. On the other hand, DNA preparations from calf thymus, rat liver, and E. coli showed irreversible changes when heated and cooled.

Amino acid sequence of penicillopepsin. I. Isolation and characterization of the chymotryptic peptides

1976 ◽  
Vol 54 (10) ◽  
pp. 872-884 ◽  
Author(s):  
Alexander Kurosky ◽  
Theo Hofmann
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Acid Protease ◽  
Terminal Region ◽  
Isolation And Characterization

The amino acid sequences of 48 peptides obtained from a chymotryptic digest of the mould acid protease, penicillopepsin (EC 3.4.23.7), have been determined. These peptides established the sequences of 26 unique fragments of up to 28 residues in length. The 28-residue fragment was identified as the N-terminal region. The C-terminal region is represented by a 13-residue fragment. The amino acids contained in these fragments account for some 85% of the residues of the enzyme.

scholarly journals Alternative primary structures in the transmembrane domain of the chicken erythroid anion transporter

1988 ◽  
Vol 8 (3) ◽  
pp. 1327-1335
Author(s):  
J V Cox ◽  
E Lazarides
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Cytoplasmic Domain ◽  
Band 3 ◽  
Transporter Protein ◽  
Isolation And Characterization ◽  
Anion Transporter ◽  
Primary Structures ◽  
Membrane Spanning

Isolation and characterization of the chicken erythroid anion transporter (band 3) cDNA clone, pCHB3-1, revealed that the chicken erythroid band 3 polypeptide is 844 amino acids in length with a predicted mass of 109,000 daltons. This polypeptide is composed of a hydrophilic N-terminal cytoplasmic domain and a hydrophobic C-terminal transmembrane domain. The approximately 90 N-terminal amino acids of the human and murine erythroid band 3 polypeptides are absent in the predicted sequence of the chicken erythroid band 3 polypeptide. The absence of this very acidic N-terminal region is consistent with the lack of binding of glyceraldehyde-3-phosphate dehydrogenase to chicken erythroid band 3, as well as the relatively basic isoelectric point observed for this molecule. The remainder of the cytoplasmic domain shows little similarity to the cytoplasmic domain of the murine and human erythroid band 3, with the exception of the putative ankyrin-binding site, which is highly conserved. In contrast, the transmembrane domain of the chicken band 3 polypeptide is very similar to that of the murine erythroid and human nonerythroid band 3 polypeptides. The transmembrane domain contains 10 hydrophobic regions that could potentially traverse the membrane 12 to 14 times. In addition, a variant of chicken erythroid band 3, pCHB3-2, was cloned in which one of the hydrophobic regions of pCHB3-1 is lacking. The transcript complementary to pCHB3-2 accumulated in chicken erythroid cells in a similar manner as the transcript complementary to pCHB3-1 during embryonic development. This is the first example of a transporter protein or ion channel with alternative primary structures in its membrane-spanning segments.

scholarly journals Produksi, isolasi dan karakterisasi superoksida dismutase dari Spirulina platensis yang dibiakkan dalam serum lateks Production, isolation, and characterization of superoxyde dismutase from Spirulina platensis cultured on latex serum

2016 ◽  
Vol 77 (1) ◽  
Author(s):  
. TRI-PANJI ◽  
. SUHARYANTO ◽  
Marini WIJAYANTI
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Spray Drying ◽  
Spirulina Platensis ◽  
Active Agent ◽  
Pilot Scale ◽  
Isolation And Characterization ◽  
The Stability ◽  
Major Amino Acid

AbstractSpirulina platensis is a blue-green microalgawhich is frequently used for food and feedsupplements and cosmetic active agent. Thismicroalga also produces a strong antioxidantnamely superoxide dismutase (SOD) used ascosmetic active agent for anti aging and anti freeradicals. SOD was isolated from S. platensis cellbiomass from local isolate grown on latex serumon semipilot (3.5 m 3 ) and pilot scale (40 m 3 )then dried with spray drying or sun drying andcharacterized. SOD was purified with sequentialtwo-stage sedimentation using ammoniumsulphate and fractionated in chromatographiccolumn containing Sephadex G 200. Thefractions were analysed to determine the activity,cofactor metal and amino acid composition of theantioxidant. The results showed thatsedimentation of SOD extract with 80%ammonium sulphate produced SOD with higheractivity compared to that of SOD fromcommercial S. platensis biomass. This SOD wassuccessfully isolated and purified. MetaloenzymeSOD was composed of subunits with molecularweight of 77.78; 71.74; and 19.2 kDa, whichcontained nine types of amino acids with tyrosineand lysine as the major amino acid components.Zn was the most predominant metal on SOD, thenfollowed by Fe and Mn. The main subunitcofactors consisted of Zn 72%, Fe 25%, Mn 2%,and Cu 1%, which were different from thesmall subunit that contained of Zn 55%, Mn 31%,Fe 14%, and Cu 4%. The stability of SOD wasachieved on pH 7.5 and temperature below 25 o C.AbstrakSpirulina platensis adalah mikroalga hijaubiru yang banyak digunakan sebagai suplemenpangan, pakan, dan bahan aktif kosmetika.Mikroalga ini juga menghasilkan antioksidankuat yaitu superoksida dismutase (SOD), yangmerupakan bahan aktif kosmetika anti penuaandini dan pencegah efek radikal bebas. SODdiisolasi dari biomassa sel S. platensis isolat lokalyang dibiakkan dalam serum lateks skalasemipilot (3,5 m 3 ) dan pilot (40 m 3 ) sertadikeringkan dengan cara pengeringan kabut(spray drying) atau penjemuran untuk kemudiandikarakterisasi. SOD dimurnikan dengan peng-endapan bertingkat menggunakan ammoniumsulfat dan dipisahkan dengan kolom kromatografiberisi Sephadex G 200. Hasil pemisahankemudian dianalisis untuk menentukan aktivitas,logam kofaktor serta komposisi asam amino antioksidan tersebut. Hasil penelitian menunjukkanbahwa pengendapan ekstrak SOD denganSOD lebih tinggi dari SOD asal biomassaS. platensis komersial. SOD tersebut telahberhasil diisolasi dan dimurnikan. MetaloenzimSOD tersusun atas subunit dengan BM 77,78;71,74; dan 19,2 kDa, yang mengandungsembilan jenis asam amino dengan tirosin danlisin sebagai komponen asam amino utama.Logam yang dominan pada SOD adalah Zn,disusul kemudian Fe dan Mn. Kofaktor sub unitbesar terdiri dari Zn 72%, Fe 25%, Mn 2%, danCu 1%, berbeda dengan sub unit kecil yangmengandung Zn 55%, Mn 31%, Fe 14%, dan Cu4%. Stabilitas SOD S. platensis dicapai pada pH7,5 dan suhu di bawah 25 o Cammonium sulfat 80% menghasilkan aktivitas

scholarly journals ISOLATION AND CHARACTERIZATION OF AMBER SUPPRESSORS IN YEAST

Genetics ◽  
1976 ◽  
Vol 82 (2) ◽  
pp. 251-272
Author(s):  
Susan W Liebman ◽  
Fred Sherman ◽  
John W Stewart
Keyword(s):  
Amino Acids ◽  
Cytochrome C ◽  
Yeast Strain ◽  
Normal Amount ◽  
Amber Mutation ◽  
Genetic Analyses ◽  
Isolation And Characterization ◽  
And Function ◽  
Nonsense Suppressors

ABSTRACT Nonsense suppressors were obtained in a haploid yeast strain containing eight nutritional mutations, that are assumed to be amber or ochre, and the cyc1-179 amber mutation that has a UAG codon corresponding to position 9 in iso-1-cytochrome c. Previous studies established that the biosynthesis and function of iso-1-cytochrome c is compatible with replacements at position 9 of amino acids having widely different structures (Stewart and Sherman 1972). UV-induced revertants, selected on media requiring the reversion of one or two of the amber nutritional markers, were presumed to contain a suppressor if there was the unselected reversion of at least one other marker. The 1088 suppressors that were isolated could be divided into 78 phenotypic classes. Only 43 suppressors of three classes caused the production of more than 50% of the normal amount of iso-1-cytochrome c in the cyc1-179 strain. Genetic analyses indicated that all of these highly efficient amber suppressors are allelic to one or another of the eight suppressors which cause the insertion of tyrosine at ochre (UAA) codons (Gilmore, Stewart and Sherman 1971). Furthermore, only tyrosine has been identified at position 9 in iso-1-cytochrome cin cyc1-179 strains suppressed with these efficient amber suppressors.

scholarly journals Isolation and characterization of six human hepatic isometallothioneins

1985 ◽  
Vol 231 (2) ◽  
pp. 375-382 ◽  
Author(s):  
P E Hunziker ◽  
J H R Kägi
Keyword(s):  
Amino Acids ◽  
Aromatic Amino Acids ◽  
Reversed Phase ◽  
Low Ph ◽  
Autopsy Material ◽  
Isolation And Characterization ◽  
Cysteine Content ◽  
Hepatic Metallothionein ◽  
Mammalian Metallothioneins

Human hepatic metallothionein (MT) was separated into six isoforms by using reversed-phase h.p.l.c. at the analytical and preparative levels. By comparison with the h.p.l.c. elution profiles of the charge-separable species MT-1 and MT-2 isolated by the procedure of Bühler & Kägi [(1974) FEBS Lett. 39, 229-234], five of these isoproteins are identified as hitherto unresolved subforms of MT-1, and one is identical with MT-2. The six isoforms have distinct and reproducible retention times at neutral pH, where the metal remains bound to the protein, and at low pH, where the metal is removed. Their amino acid compositions display the high cysteine content and the lack of aromatic amino acids and of histidine typical of mammalian metallothioneins, but they differ significantly with respect to all other amino acids. A survey of autopsy material indicates that in adult human liver all six isoforms are usually expressed, albeit in somewhat variable relative proportions.

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