scholarly journals Localization of the forskolin photolabelling site within the monosaccharide transporter of human erythrocytes

1990 ◽  
Vol 272 (1) ◽  
pp. 151-158 ◽  
Author(s):  
B E Wadzinski ◽  
M F Shanahan ◽  
K B Seamon ◽  
A E Ruoho
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Glucose Transporter ◽  
Transmembrane Helix ◽  
Transport Protein ◽  
Tryptophan Residue ◽  
Amino Acid Residues ◽  
Monosaccharide Transporter ◽  
Tryptic Fragment ◽  
Transporter Protein

Chemical and proteolytic digestion of intact erythrocyte glucose transporter as well as purified transporter protein has been used to localize the derivatization site for the photoaffinity agent 3-[125I]iodo-4-azido-phenethylamino-7-O-succinyldeacetylforskol in [(125I]IAPS-forskolin). Comparison of the partial amino acid sequence of the labelled 18 kDa tryptic fragment with the known amino acid sequence for the HepG2 glucose transporter confirmed that the binding site for IAPS-forskolin is between the amino acid residues Glu254 and Tyr456. Digestion of intact glucose transporter with Pronase suggests that this site is within the membrane bilayer. Digestion of labelled transporter with CNBr generated a major radiolabelled fragment of Mr approximately 5800 putatively identified as residues 365-420. Isoelectric focusing of Staphylococcus aureus V8 proteinase-treated purified labelled tryptic fragment identified two peptides which likely correspond to amino acid residues 360-380 and 381-393. The common region for these radiolabelled peptides is the tenth putative transmembrane helix of the erythrocyte glucose transporter, comprising amino acid residues 369-389. Additional support for this conclusion comes from studies in which [125I]APS-forskolin was photoincorporated into the L-arabinose/H(+)-transport protein of Escherichia coli. Labelling of this transport protein was protected by both cytochalasin B and D-glucose. The region of the erythrocyte glucose transporter thought to be derivatized with IAPS-forskolin contains a tryptophan residue (Trp388) that is conserved in the sequence of the E. coli arabinose-transport protein.

Amino Acid Sequence Studies of Horseradish Peroxidase. II. Thermolytic Peptides

1972 ◽  
Vol 50 (1) ◽  
pp. 63-90 ◽  
Author(s):  
K. G. Welinder ◽  
L. B. Smillie
Keyword(s):  
Amino Acid ◽  
Horseradish Peroxidase ◽  
Amino Acid Sequence ◽  
Cation Exchanger ◽  
Paper Electrophoresis ◽  
Tryptophan Residue ◽  
Amino Acid Residues ◽  
Disulfide Bridges ◽  
Serine Residue ◽  
C Terminus

Horseradish peroxidase (HRP) was digested with thermolysin. On fractionation on Sephadex G-25, Fine Chromobeads type P (Dowex 50 type resin) and by high-voltage paper electrophoresis, we isolated about 120 thermolytic peptides. Some experimentation on the composition of the pyridine acetate gradient, used for elution of the cation exchanger, is reported. All peptides were characterized with respect to amino acid composition, N-terminal residue, and pH 6.5 mobility. Unknown peptides or peptides not corresponding unambiguously to previously established tryptic sequences were subjected to dansyl-Edman analysis. Thermolytic peptides accounting for all tryptic sequences except a dipeptide and a tripeptide, and unique thermolytic sequences accounting for about 100 amino acid residues, were obtained. Nine convincing and several indicative overlaps were established for known tryptic sequences. The sequences around all four disulfide bridges, the three histidine residues, and the only tryptophan residue have been elucidated. Eight sites of carbohydrate attachment have been identified. For seven of these sites we have evidence for attachment to asparagine, and for six of the sites the carbohydrate-bound asparagine was found in the well-known sequences Asn–X–Ser/Thr. The remaining two sequences, though incomplete, are compatible with this pattern. Tentatively we suggest a pyrrolidone carboxyl N-terminal for HRP. The specificity of trypsin implicates a sequence found in two varieties, differing only by a C-terminal serine residue at the C-terminus of HRP. A discussion of the possible complications of the acidic heme extraction on the results obtained is included.

Amino acid sequence of cyanogen bromide fragment CB5 of human hemopexin

1989 ◽  
Vol 54 (3) ◽  
pp. 803-810 ◽  
Author(s):  
Ivan Kluh ◽  
Ladislav Morávek ◽  
Manfred Pavlík
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Acid Hydrolysis ◽  
Cyanogen Bromide ◽  
Polypeptide Chain ◽  
Amino Acid Residues ◽  
Mild Acid Hydrolysis ◽  
Methionine Residue ◽  
Cyanogen Bromide Fragment ◽  
Mild Acid

Cyanogen bromide fragment CB5 represents the region of the polypeptide chain of hemopexin between the fourth and fifth methionine residue (residues 232-352). It contains 120 amino acid residues in the following sequence: Arg-Cys-Ser-Pro-His-Leu-Val-Leu-Ser-Ala-Leu-Thr-Ser-Asp-Asn-His-Gly-Ala-Thr-Tyr-Ala-Phe-Ser-Gly-Thr-His-Tyr-Trp-Arg-Leu-Asp-Thr-Ser-Arg-Asp-Gly-Trp-His-Ser-Trp-Pro-Ile-Ala-His-Gln-Trp-Pro-Gln-Gly-Pro-Ser-Ala-Val-Asp-Ala-Ala-Phe-Ser-Trp-Glu-Glu-Lys-Leu-Tyr-Leu-Val-Gln-Gly-Thr-Gln-Val-Tyr-Val-Phe-Leu-Thr-Lys-Gly-Gly-Tyr-Thr-Leu-Val-Ser-Gly-Tyr-Pro-Lys-Arg-Leu-Glu-Lys-Glu-Val-Gly-Thr-Pro-His-Gly-Ile-Ile-Leu-Asp-Ser-Val-Asp-Ala-Ala-Phe-Ile-Cys-Pro-Gly-Ser-Ser-Arg-Leu-His-Ile-Met. The sequence was derived from the data on peptides prepared by cleavage of fragment CB5 by mild acid hydrolysis, by trypsin and chymotrypsin.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

scholarly journals Diarrhea-Inducing Activity of Avian Rotavirus NSP4 Glycoproteins, Which Differ Greatly from Mammalian Rotavirus NSP4 Glycoproteins in Deduced Amino Acid Sequence, in Suckling Mice

2002 ◽  
Vol 76 (11) ◽  
pp. 5829-5834 ◽  
Author(s):  
Yoshio Mori ◽  
Mohammed Ali Borgan ◽  
Naoto Ito ◽  
Makoto Sugiyama ◽  
Nobuyuki Minamoto
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Suckling Mice

ABSTRACT Avian rotavirus NSP4 glycoproteins expressed in Escherichia coli acted as enterotoxins in suckling mice, as did mammalian rotavirus NSP4 glycoproteins, despite great differences in the amino acid sequences. The enterotoxin domain of PO-13 NSP4 exists in amino acid residues 109 to 135, a region similar to that reported in SA11 NSP4.

Plasmin-platelet interaction involves cleavage of functional thrombin receptor

1996 ◽  
Vol 271 (1) ◽  
pp. C54-C60 ◽  
Author(s):  
M. Kimura ◽  
T. T. Andersen ◽  
J. W. Fenton ◽  
W. F. Bahou ◽  
A. Aviv
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Platelet Activation ◽  
Synthetic Peptide ◽  
Time Dependent ◽  
Thrombin Receptor ◽  
Amino Acid Residues ◽  
Enzymatic Action ◽  
High Concentrations ◽  
Inhibition Of Thrombin

We tested the hypothesis that the inhibition of thrombin-induced platelet activation by plasmin is mediated via the enzymatic action of plasmin on the functional thrombin receptor. We monitored the binding of the anti-thrombin receptor antibody [anti-TR-(34-46)] to platelets; this binding is sensitive to the cleavage of the thrombin receptor at amino acid residues Arg-41 to Ser-42. Plasmin inhibited anti-TR-(34-46) binding in dose- and time-dependent manners. The inactive synthetic peptide with the amino acid sequence 40-55 of the thrombin receptor (D-FPRSFLLRNPNDKYEPF) was similarly cleaved by thrombin and plasmin to an active peptide (SFLLRNPNDKYEPF) that produced robust cytosolic Ca2+ responses. At high concentrations, plasmin itself can activate platelets. We explored this effect with the use of anti-TR-(1-160). This antibody abolished the cytosolic Ca2+ responses to thrombin and to the thrombin receptor-activating peptide SFLLRN but did not attenuate the plasmin-induced cytosolic Ca2+ response. Thus plasmin inhibits thrombin-evoked platelet activation by cleaving the thrombin receptor, but the plasmin-induced cytosolic Ca2+ response is not due to the generation of the tethered peptide of the thrombin receptor.

scholarly journals Hyaluronan-binding region of aggrecan from pig laryngeal cartilage. Amino acid sequence, analysis of N-linked oligosaccharides and location of the keratan sulphate

1992 ◽  
Vol 286 (3) ◽  
pp. 761-769 ◽  
Author(s):  
F P Barry ◽  
J U Gaw ◽  
C N Young ◽  
P J Neame
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Signal Peptidase ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Binding Region ◽  
Keratan Sulphate ◽  
Laryngeal Cartilage ◽  
Hyaluronan Binding

The hyaluronan-binding region (HABR) was prepared from pig laryngeal cartilage aggrecan and the amino acid sequence was determined. The HABR had two N-termini: one N-terminal sequence was Val-Glu-Val-Ser-Glu-Pro (367 amino acids in total), and a second N-terminal sequence (Ala-Ile-Ser-Val-Glu-Val; 370 amino acids in total) was found to arise due to alternate cleavage by the signal peptidase. The N-linked oligosaccharides were analysed by examining their reactivity with a series of lectins. It was found that the N-linked oligosaccharide on loop A was of the mannose type, while that on loop B was of the complex type. No reactivity was detected between the N-linked oligosaccharide on loop B' and any of the lectins. The location of keratan sulphate (KS) in the HABR was determined by Edman degradation of the immobilized KS-containing peptide. The released amino acid derivatives were collected and tested for the presence of epitope to antibody 5-D-4. On the basis of 5-D-4 reactivity and sequencing yields, the KS chains are attached to threonine residues 352 and 357. There is no KS at threonine-355. This site is not in fact in G1, but about 16 amino acid residues into the interglobular domain. Comparison of the structure of the KS chain from the HABR and from the KS domain of pig laryngeal cartilage aggrecan was made by separation on polyacrylamide gels of the oligosaccharides arising from digestion with keratanase. Comparison of the oligosaccharide maps suggests that the KS chains from both parts of the aggrecan molecule have the same structure.

The Heparin Binding Site Of Antithrombin: Identification Of A Critical Tryptophan Residue Within The Amino Acid Sequence

1981 ◽  
Author(s):  
M Blackburn
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Tryptophan Fluorescence ◽  
Tryptophan Residue ◽  
Affinity Column ◽  
Heparin Binding ◽  
High Affinity ◽  
Heparin Binding Site

Chemical modification of antithrombin III with the tryptophan reagent, dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide, results in the incorporation of one hydroxynitrobenzyl (HNB) moiety per molecule of antithrombin III. Heparin protects against tryptophan modification, particularly at low reagent concentrations. Unlike native antithrombin, which has high affinity for heparin, HNB-anti- thrombin does not bind to a heparin-agarose affinity column. Furthermore, the heparin-induced increase in tryptophan fluorescence, obtained with native antithrombin, is not observed with the singly modified inhibitor. HNB-anti- thrombin does not exhibit heparin-promoted rate enhancement in the inactivation of thrombin and Factor Xa. However, in the absence of heparin, HNB-antithrombin and native antithrombin possess progressive antithrombin activity, inactivating these proteases at identical rates. These results indicate that the integrity of a specific tryptophan residue is required for the binding of heparin to antithrombin III. Chemical and enzymatic cleavage techniques have been used to isolate peptides containing this tryptophan from both HNB-labeled and native antithrombin and to identify this critical tryptophan residue within the amino acid sequence of the antithrombin molecule.

scholarly journals The human erythrocyte anion-transport protein. Partial amino acid sequence, conformation and a possible molecular mechanism for anion exchange

1983 ◽  
Vol 213 (3) ◽  
pp. 577-586 ◽  
Author(s):  
C J Brock ◽  
M J A Tanner ◽  
C Kempf
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mechanism ◽  
Anion Exchange ◽  
Human Erythrocyte ◽  
Exchange Process ◽  
Anion Transport ◽  
Transport Protein ◽  
British Library ◽  
Alpha Helices

The N-terminal 72 residues of an integral membrane fragment, P5, of the human erythrocyte anion-transport protein, which is known to be directly involved in the anion-exchange process, was shown to have the following amino acid sequence: Met-Val-Pro-Lys-Pro-Gln-Gly-Pro-Leu-Pro-Asn-Thr-Ala-Leu-Leu-Ser-Leu-Val-Leu-Met -Ala-Gly-Thr-Phe-Phe-Phe-Ala-Met-Met-Leu-Arg-Lys-Phe-Lys-Asn-Ser-Ser-Tyr-Phe-Pro-Gly-Lys-Leu-Arg-Arg-Val-Ile-Gly-Asp-Phe-Gly-Val-Pro-Ile-Ser-Ile-Leu-Ile-Met-Val-Leu-Val-Asp-Phe-Phe-Ile-Gln-Asp-Thr-Tyr-Thr-Gln- The structure of this fragment was analysed, with account being taken of the constraints that apply to the folding of integral membrane proteins and the topographical locations of various sites in the sequence. It was concluded that this sequence forms two transmembrane alpha-helices. These are probably part of a cluster of amphipathic transmembrane alpha-helices, which could comprise that part of the protein responsible for transport activity. The presently available evidence relating to the anion-exchange process was considered with the structural features noted in this study and a possible molecular mechanism is proposed. In this model the rearrangement of a network of intramembranous charged pairs mediates the translocation of an anion between anion-binding regions at each surface of the membrane, which are composed of clusters of positively charged amino acids. This model imposes a sequential exchange mechanism on the system. Supplementary material, including Tables and Figures describing the compositions of peptides determined by amino acid analysis and sequence studies, quantitative and qualitative data that provide a residue-by-residue justification for the sequence assignment and a description of modifications to and use of the solid-phase sequencer has been deposited as Supplementary Publication SUP 50123 (12 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained as indicated in Biochem. J. (1983) 209, 5.

scholarly journals The amino acid sequence of plastocyanin from Cucurbita pepo L. (vegetable marrow)

1974 ◽  
Vol 143 (2) ◽  
pp. 257-264 ◽  
Author(s):  
Michael D. Scawen ◽  
Donald Boulter
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Phenyl Isothiocyanate ◽  
Amino Acid Residues ◽  
Higher Plant ◽  
Single Polypeptide Chain ◽  
Vegetable Marrow ◽  
Single Polypeptide ◽  
Phylogenetic Affinities ◽  
Good Agreement

The amino acid sequence of plastocyanin from marrow was determined. It consists of a single polypeptide chain of mol.wt. 10284 containing 99 amino acid residues. The sequence was determined by using a Beckman 890C automatic sequencer and by dansyl–phenyl isothiocyanate analysis of peptides obtained by the enzymic digestion of purified CNBr fragments. The sequence is in good agreement with the amino acid composition, except that fewer residues of glutamic acid were found in the sequence than were suggested by the composition. Evidence for histidine-37 was weaker than for the rest of the sequence. A ‘tree’ of phylogenetic affinities was constructed by using several higher-plant plastocyanin sequences.

scholarly journals Peptidase E, a Peptidase Specific for N-Terminal Aspartic Dipeptides, Is a Serine Hydrolase

2000 ◽  
Vol 182 (9) ◽  
pp. 2536-2543 ◽  
Author(s):  
Rachel A. L. Lassy ◽  
Charles G. Miller
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Serine Hydrolase ◽  
Genome Sequences ◽  
New Family ◽  
The Family ◽  
Serovar Typhimurium ◽  
Structural Classes

ABSTRACT Salmonella enterica serovar Typhimurium peptidase E (PepE) is an N-terminal Asp-specific dipeptidase. PepE is not inhibited by any of the classical peptidase inhibitors, and its amino acid sequence does not place it in any of the known peptidase structural classes. A comparison of the amino acid sequence of PepE with a number of related sequences has allowed us to define the amino acid residues that are strongly conserved in this family. To ensure the validity of this comparison, we have expressed one of the most distantly related relatives (Xenopus) in Escherichia coli and have shown that it is indeed an Asp-specific dipeptidase with properties very similar to those of serovar Typhimurium PepE. The sequence comparison suggests that PepE is a serine hydrolase. We have used site-directed mutagenesis to change all of the conserved Ser, His, and Asp residues and have found that Ser120, His157, and Asp135 are all required for activity. Conversion of Ser120 to Cys leads to severely reduced (104-fold) but still detectable activity, and this activity but not that of the parent is inhibited by thiol reagents; these results confirm that this residue is likely to be the catalytic nucleophile. These results suggest that PepE is the prototype of a new family of serine peptidases. The phylogenetic distribution of the family is unusual, since representatives are found in eubacteria, an insect (Drosophila), and a vertebrate (Xenopus) but not in the Archaea or in any of the other eukaryotes for which genome sequences are available.

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