Isolation and characterization of a cytostatic histone H2B-like protein from fetal lungs of non-diabetic and diabetic rats

1993 ◽  
Vol 139 (1) ◽  
pp. 97-105
Author(s):  
P. R. Conliffe ◽  
H. P. J. Bennett ◽  
S. Mulay
Keyword(s):  
Amino Acid ◽  
Gel Filtration ◽  
Fetal Lung ◽  
Reversed Phase ◽  
Diabetic Rats ◽  
Histone H2b ◽  
Amino Terminal ◽  
Isolation And Characterization ◽  
Fetal Lungs ◽  
Cytostatic Factor

ABSTRACT It was observed in the course of other studies that rat fetal lung extracts inhibited proliferation of fetal lung cells in culture. The purpose of the present study was to isolate and characterize this cytostatic factor. It was found that fetal lungs contained a 16 kDa cytostatic factor and its concentration was twofold greater in fetal lungs of diabetic rats compared with control rats. This fetal lung cytostatic protein (FLCP) was purified by reversed-phase, heparin-affinity and gel filtration high-performance liquid chromatography and SDS-PAGE. The purified protein was electroblotted onto polyvinylidene difluoride membrane and subjected to sequence analysis. The amino-terminal sequence of this fetal lung cytostatic protein was PEPAKSAPAPXKGIGKQXXKAX XKA... and showed significant homology with histone H2B; however, the amino acid composition of FLCP suggested that it may be structurally distinct from histone H2B. Ion-spray mass spectrometry suggested that FLCP was made up of at least two species of the protein with molecular weights of 13 776·1 and 14 007·3 and was different from the molecular weight of rat histone H2B predicted by its cDNA sequence. The concentration of FLCP, based on amino acid compositions, was 0·32 nmol/g and 0·83 nmol/g wet fetal lung from non-diabetic and diabetic rats respectively. These findings suggest that the fetal rat lung produces a regulatory factor bearing considerable homology with but possibly different from histone H2B and that fetal lung immaturity during diabetic pregnancy might be contributed to by an increase in this factor. Journal of Endocrinology (1993) 139, 97–105

Isolation and Structural Characterization of Antioxidant Peptides from Degreased Apricot Seed Kernels

2018 ◽  
Vol 101 (5) ◽  
pp. 1661-1663 ◽  
Author(s):  
Haisheng Zhang ◽  
Jing Xue ◽  
Huanxia Zhao ◽  
Xinshuai Zhao ◽  
Huanhuan Xue ◽  
...  
Keyword(s):  
Amino Acid ◽  
Antioxidant Activities ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Seed Kernel ◽  
Antioxidant Peptides ◽  
Food Preservatives ◽  
Isolation And Purification ◽  
Rp Hplc

Abstract Background: The composition and sequence of amino acids have a prominent influence on the antioxidant activities of peptides. Objective: A series of isolation and purification experiments was conducted to explore the amino acid sequence of antioxidant peptides, which led to its antioxidation causes. Methods: The degreased apricot seed kernels were hydrolyzed by compound proteases of alkaline protease and flavor protease (3:2, u/u) to prepare apricot seed kernel hydrolysates (ASKH). ASKH were separated into ASKH-A and ASKH-B by dialysis bag. ASKH-B (MW < 3.5 kDa) was further separated into fractions by Sephadex G-25 and G-15 gel-filtration chromatography. Reversed-phase HPLC (RP-HPLC) was performed to separate fraction B4b into two antioxidant peptides (peptide B4b-4 and B4b-6). Results: The amino acid sequences were Val-Leu-Tyr-Ile-Trp and Ser-Val-Pro-Tyr-Glu, respectively. Conclusions: The results suggested that ASKH antioxidant peptides may have potential utility as healthy ingredients and as food preservatives due to their antioxidant activity. Highlights: Materials with regional characteristics were selected to explore, and hydrolysates were identified by RP-HPLC and matrix-assisted laser desorption ionization-time-of-flight-MS to obtain amino acid sequences.

scholarly journals α-Crystallin. The isolation and characterization of distinct macromolecular fractions

1971 ◽  
Vol 124 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Abraham Spector ◽  
Lu-Ku Li ◽  
Robert C. Augusteyn ◽  
Arthur Schneider ◽  
Thomas Freund
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Chemical Difference ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Different Populations ◽  
Molecular Weight Fractions

α-Crystallin was isolated from calf lens periphery by chromatography on DEAE-cellulose and gel filtration. Three distinct populations of macromolecules have been isolated with molecular weights in the ranges approx. 6×105−9×105, 0.9×106−4×106and greater than 10×106. The concentration of macromolecules at the molecular-weight limits of a population are very low. The members of the different populations do not appear to be in equilibrium with each other. Further, in those molecular-weight fractions investigated, no equilibrium between members of the same population was observed. The population of lowest molecular weight comprises 65–75% of the total material. The amino acid and subunit composition of the different-sized fractions appear very similar, if not identical. The only chemical difference observed between the fractions is the presence of significant amounts of sugar in the higher-molecular-weight fractions. Subunit molecular weights of approx. 19.5×103and 22.5×103were observed for all α-crystallin fractions.

scholarly journals Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

2001 ◽  
Vol 183 (15) ◽  
pp. 4468-4476 ◽  
Author(s):  
Franz Kaufmann ◽  
Derek R. Lovley
Keyword(s):  
Amino Acid ◽  
Electron Donor ◽  
Formate Dehydrogenase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Geobacter Sulfurreducens ◽  
Oxidoreductase Activity ◽  
Isolation And Characterization ◽  
Apparent Homogeneity

ABSTRACT NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 μmol · min−1 · mg−1. The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP+ oxidoreductase activity and catalyzed the reduction of NADP+ with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content.

scholarly journals The catalytic chain of human complement subcomponent C1. Purification and N-terminal amino acid sequences of the major cyanogen bromide-cleavage fragments

1982 ◽  
Vol 201 (1) ◽  
pp. 49-59 ◽  
Author(s):  
G J Arlaud ◽  
J Gagnon ◽  
R R Porter
Keyword(s):  
High Pressure ◽  
Amino Acid ◽  
Gel Filtration ◽  
Gel Permeation Chromatography ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Serine Proteinases ◽  
Human Complement ◽  
Terminal Sequence ◽  
Cnbr Cleavage

1. The a- and b-chains of reduced and alkylated human complement subcomponent C1r were separated by high-pressure gel-permeation chromatography and isolated in good yield and in pure form. 2. CNBr cleavage of C1r b-chain yielded eight major peptides, which were purified by gel filtration and high-pressure reversed-phase chromatography. As determined from the sum of their amino acid compositions, these peptides accounted for a minimum molecular weight of 28 000, close to the value 29 100 calculated from the whole b-chain. 3. N-Terminal sequence determinations of C1r b-chain and its CNBr-cleavage peptides allowed the identification of about two-thirds of the amino acids of C1r b-chain. From our results, and on the basis of homology with other serine proteinases, an alignment of the eight CNBr-cleavage peptides from C1r b-chain is proposed. 4. The residues forming the ‘charge-relay’ system of the active site of serine proteinases (His-57, Asp-102 and Ser-195 in the chymotrypsinogen numbering) are found in the corresponding regions of C1r b-chain, and the amino acid sequence around these residues has been determined. 5. The N-terminal sequence of C1r b-chain has been extended to residue 60 and reveals that C1r b-chain lacks the ‘histidine loop’, a disulphide bond that is present in all other known serine proteinases.

Primary Structure and Anticandidal Activity of the Major Histatin from Parotid Secretion of the Subhuman Primate, Macaca fascicularis

1990 ◽  
Vol 69 (11) ◽  
pp. 1717-1723 ◽  
Author(s):  
T. Xu ◽  
E. Telser ◽  
R.F. Troxler ◽  
F.G. Oppenheim
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Macaca Fascicularis ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Alpha Helix ◽  
Reversed Phase ◽  
Amino Acid Residues ◽  
Beta Turns ◽  
Anticandidal Activity

A major macaque histatin (M-histatin 1) from the parotid secretion of the subhuman primate, Macaca fascicularis, was isolated by gel filtration on Bio-Gel P-2 and purified to homogeneity by reversed-phase high-performance liquid chromatography on a TSK-ODS C18 column. The complete amino acid sequence of M-histatin 1, determined by automated Edman degradation, is: 1 10 20 Asp-Pse-His-Glu-Glu-Arg-His-His-Gly-Arg-His-Gly-His-His-Lys-Tyr-Gly-Arg-Lys-Phe 21 30 38 His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr-Arg-Ser-Asn-Tyr-Leu-Tyr-Asp-Asn M-histatin 1 contains 38 amino acid residues, a phosphoserine at residue 2, has a molecular weight of 4881.8, a calculated pI of 8.5, and histidine forms 26.3% of the mass. The hydropathicity plot of M-histatin 1 predicts that the molecule is entirely hydrophilic, and Chou-Fasman secondary prediction indicates that the polypeptide is devoid of alpha-helix and beta-sheet conformation in aqueous solutions but contains a series of beta turns. M-histatin 1 includes a six-amino-acid insert (residue 10-15) not present in human histatins and, with the introduction of gaps to maximize homology, it displays 89% and 91% sequence similarity with human histatins 1 and 3, respectively. M-histatin 1 exhibited fungicidal and fungistatic effects against the dimorphic pathogen, Candida albicans, in three separate bioassays. Its anticandidal effects were comparable with or greater than those of human histatins 1, 3, and 5. M-histatins 2, 3, and 4 were not sequenced directly because insufficient materials were available, but the amino acid composition of M-histatin 3 was nearly identical to that of the N-terminal 20 amino acid residues of M-histatin 1. There appears to be only one major histatin in macaque parotid secretion in contrast to the family of histatins in human parotid and submandibular secretions, and the significance of this in the context of evolution and mechanism of action in anticandidal assays is discussed.

scholarly journals Haemoglobins of the Shark, Heterodontus Portusjacksoni II. Amino Acid Sequence of the a-Chain

1976 ◽  
Vol 29 (2) ◽  
pp. 73 ◽  
Author(s):  
AR Nash ◽  
WK Fisher ◽  
EOP Thompson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Paper Chromatography ◽  
Cyanogen Bromide ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Tryptic Digestion ◽  
Amino Terminal ◽  
Core Peptide ◽  
A Chain

The amino acid sequence of the a-chain of the principal haemoglobin from the shark, H. portusjacksoni has been determined. The chain has 148 residues and is acetylated at the amino terminal. The soluble peptides obtained by tryptic and chymotryptic digestion of the protein or its cyanogen bromide fragments were isolated by gel filtration, paper ionophoresis and paper chromatography. The amino acid sequences were determined by the dansyl-Edman procedure. The insoluble 'core' peptide from the tryptic digestion contained 34 residues and required cleavage by several proteases before the sequence was established. Compared with human a-chain there are 88 amino acid differences including the additional seven residues which appear on the amino terminal of the shark chain. There is also one deletion and one insertion. The chain contains no tryptophan but has four cysteinyl residues which is the highest number of such residues recorded for a vertebrate globin.

A serum protease cleaves proANF into a 14-kilodalton peptide and ANF

1987 ◽  
Vol 252 (1) ◽  
pp. E147-E151
Author(s):  
K. D. Bloch ◽  
J. B. Zisfein ◽  
M. N. Margolies ◽  
C. J. Homcy ◽  
J. G. Seidman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Culture Medium ◽  
Rat Plasma ◽  
Biologically Active ◽  
Amino Terminal ◽  
Isolation And Characterization ◽  
Amino Acid Precursor ◽  
Acid Precursor

Proatrial natriuretic factor (proANF), the 126-amino acid precursor of ANF, is the major storage form in mammalian atria. In contrast, two ANF peptides containing the 28- and 24-carboxyterminal residues of proANF have been isolated from rat plasma. Whether the cleavage of proANF in vivo to these ANF peptides occurs during or after its release into the circulation has not been determined. The latter possibility was suggested by our previous study where, by using a cultured rat cardiocyte preparation, we demonstrated that proANF is secreted intact into the culture medium. We now report that serum, but not plasma, contains a protease that specifically cleaves the 17-kdalton proANF to a 14-kdalton amino-terminal peptide and the carboxyterminal 3-kdalton circulating forms of ANF. The role of this proANF-cleaving enzyme in the generation of the biologically active ANF peptides remains to be defined. Its isolation and characterization should provide insights into its site of production and whether in vivo it is involved in the processing of circulating proANF.

Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen

1987 ◽  
Vol 7 (9) ◽  
pp. 3221-3230
Author(s):  
N Beauchemin ◽  
S Benchimol ◽  
D Cournoyer ◽  
A Fuks ◽  
C P Stanners
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Carcinoembryonic Antigen ◽  
Full Length ◽  
Cdna Clones ◽  
Base Pairs ◽  
Amino Terminal ◽  
Isolation And Characterization

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.

Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7

1992 ◽  
Vol 38 (9) ◽  
pp. 891-897 ◽  
Author(s):  
Hiroshi Tsujibo ◽  
Yukio Yoshida ◽  
Katsushiro Miyamoto ◽  
Chiaki Imada ◽  
Yoshiro Okami ◽  
...  
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Amino Terminal

Chitinase (EC 3.2.1.14) was isolated from the culture supernatant of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (Chi-A) was purified by anion-exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephadex G-100). The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of Chi-A were 70 kDa and 3.9, respectively. The optimum pH and temperature of Chi-A were 8.0 and 50 °C, respectively. Chi-A was stable in the range of pH 5–10 up to 40 °C. Among the main cations, such as Na+, K+, Mg2+, and Ca2+, contained in seawater, Mg2+ stimulated Chi-A activity. N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide inhibited Chi-A activity. The amino-terminal 27 amino acid residues of Chi-A were sequenced. This enzyme showed sequence homology with chitinases from terrestrial bacteria such as Serratia marcescens QMB1466 and Bacillus circulons WL-12. Key words: marine bacterium, Alteromonas sp., chitinase.

scholarly journals Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen.

1987 ◽  
Vol 7 (9) ◽  
pp. 3221-3230 ◽  
Author(s):  
N Beauchemin ◽  
S Benchimol ◽  
D Cournoyer ◽  
A Fuks ◽  
C P Stanners
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Carcinoembryonic Antigen ◽  
Full Length ◽  
Cdna Clones ◽  
Base Pairs ◽  
Amino Terminal ◽  
Isolation And Characterization

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.

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