scholarly journals Separation of rat tissue histone H1 subtypes by reverse-phase h.p.l.c. Identification and assignment to a standard H1 nomenclature

1990 ◽  
Vol 269 (2) ◽  
pp. 359-363 ◽  
Author(s):  
H Lindner ◽  
W Helliger ◽  
B Puschendorf
Keyword(s):  
Acetic Acid ◽  
Amino Acid ◽  
Rat Liver ◽  
Reverse Phase ◽  
Rat Testis ◽  
Sds Page ◽  
Rat Tissue ◽  
First Time ◽  
Acetonitrile Gradient ◽  
Made In

H1 histones from rat liver and rat testis were separated by reverse-phase h.p.l.c. Within 40 min six subfractions (H1(0), H1b, H1a, H1d, H1e + H1c and H1c) and seven subfractions (H1(0), H1b, H1a, H1d, H1e + H1c, H1c and H1t) respectively were isolated by using a linear acetonitrile gradient. Each individual H1 subtype was identified either by comparing the H1 variants (contained in both tissues but in different quantities) or by SDS/PAGE and acetic acid/urea/PAGE. Moreover, all H1 variants were characterized by amino acid analyses. The amino acid compositions of rat histone subfractions H1(0), H1b and H1e were determined for the first time. It was possible to classify unambiguously the H1 subfractions obtained by h.p.l.c. by following the standardized H1 nomenclature for electrophoretic systems recommended by Lennox, Oshima & Cohen [(1982) J. Biol. Chem. 257, 5183-5189]. Incorrect assignments that have been made in various publications are discussed.

Comparison of plasma membrane FABP and mitochondrial isoform of aspartate aminotransferase from rat liver

1993 ◽  
Vol 265 (5) ◽  
pp. G894-G902 ◽  
Author(s):  
D. D. Stump ◽  
S. L. Zhou ◽  
P. D. Berk
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Amino Acid ◽  
Rat Liver ◽  
Aspartate Aminotransferase ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fatty Acid Binding ◽  
Sds Page

A relationship between plasma membrane fatty acid binding protein (FABPpm), a putative membrane transporter for long-chain fatty acids, and the mitochondrial isoform of aspartate aminotransferase (m-AspAT) has been reported. Accordingly, we have compared the chemical and immunological properties of rat liver m-AspAT with those of rat liver FABPpm isolated by two procedures: 1) detergent solubilization of the membranes followed by purification via fatty acid affinity chromatography (FABP-1) or 2) salt extraction of the membranes and subsequent purification by high-performance liquid chromatography (HPLC; FABP-2). Comparison of the three protein preparations revealed no differences with respect to NH2-terminal amino acid sequence, amino acid composition, peptides from tryptic digests, AspAT enzymatic activity, isoelectric point, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), retention on five different HPLC columns, and immunoprecipitation and immunoblotting of SDS-PAGE separated proteins with polyclonal antisera. Examination of the proteins by nondenaturing PAGE showed a consistent second band in FABP-1 and FABP-2 not always present in m-AspAT. However, whenever present, this band was immunoreactive with antibodies to both m-AspAT and FABP-1. Hence, FABP-1 and FABP-2 are indistinguishable from one another. They are also at least closely related, if not identical, to m-AspAT.

scholarly journals Purification of human erythropoietin to homogeneity by a rapid five- step procedure

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 71-79 ◽  
Author(s):  
G Krystal ◽  
HR Pankratz ◽  
NM Farber ◽  
JE Smart
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Assay ◽  
Tween 20 ◽  
Reverse Phase ◽  
Vitro Assay ◽  
Step Procedure ◽  
Sds Page

Abstract Human urinary erythropoietin (Ep) has been purified using a simple five- step procedure to yield preparations with potencies of 80,000 U/mg in 25% yield. The five steps involve: (1) affinity chromatography on CM Affi-Gel Blue, (2) chromatofocusing, (3) wheat germ lectin (or hydroxylapatite) chromatography, (4) reverse-phase high-performance liquid chromatography (HPLC) using a phenyl column, and (5) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Ep activity was determined at each stage using a highly sensitive and specific in vitro assay that measures [3H]-thymidine incorporation into erythroid cells from spleens of phenylhydrazine-treated mice. The step 5 material was also tested with the in vivo polycythemic mouse assay procedure and was found to have a similar potency to that obtained in the [3H]-thymidine in vitro assay. SDS-PAGE analysis of the step 5 material revealed a single 38.5-kd band that comigrated with Ep bioactivity. Homogeneity was confirmed by amino acid sequence analysis. Starting with urine containing approximately 13 U/mg of protein, the cumulative degrees of purification achieved with each step were: step 1,25-fold; step 2, 75-fold; step 3, 300-fold; step 4, 1,500-fold; and step 5, 5,000-fold. Corresponding overall recoveries after each step were: greater than 100%, 70%, 45%, 30%, and 25%. These recoveries could be obtained when as little as 5,000 U of starting urinary Ep were processed because of the introduction of Tween 20 and SDS into buffers used at various stages of the purification procedure. In addition, a rapid method for determining Ep purity which involves reverse-phase HPLC of trypsinized 125I-labeled Ep is presented. This allows the establishment of purity with far less material than is required for amino acid sequencing.

scholarly journals Purification of human erythropoietin to homogeneity by a rapid five- step procedure

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 71-79 ◽  
Author(s):  
G Krystal ◽  
HR Pankratz ◽  
NM Farber ◽  
JE Smart
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Assay ◽  
Tween 20 ◽  
Reverse Phase ◽  
Vitro Assay ◽  
Step Procedure ◽  
Sds Page

Human urinary erythropoietin (Ep) has been purified using a simple five- step procedure to yield preparations with potencies of 80,000 U/mg in 25% yield. The five steps involve: (1) affinity chromatography on CM Affi-Gel Blue, (2) chromatofocusing, (3) wheat germ lectin (or hydroxylapatite) chromatography, (4) reverse-phase high-performance liquid chromatography (HPLC) using a phenyl column, and (5) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Ep activity was determined at each stage using a highly sensitive and specific in vitro assay that measures [3H]-thymidine incorporation into erythroid cells from spleens of phenylhydrazine-treated mice. The step 5 material was also tested with the in vivo polycythemic mouse assay procedure and was found to have a similar potency to that obtained in the [3H]-thymidine in vitro assay. SDS-PAGE analysis of the step 5 material revealed a single 38.5-kd band that comigrated with Ep bioactivity. Homogeneity was confirmed by amino acid sequence analysis. Starting with urine containing approximately 13 U/mg of protein, the cumulative degrees of purification achieved with each step were: step 1,25-fold; step 2, 75-fold; step 3, 300-fold; step 4, 1,500-fold; and step 5, 5,000-fold. Corresponding overall recoveries after each step were: greater than 100%, 70%, 45%, 30%, and 25%. These recoveries could be obtained when as little as 5,000 U of starting urinary Ep were processed because of the introduction of Tween 20 and SDS into buffers used at various stages of the purification procedure. In addition, a rapid method for determining Ep purity which involves reverse-phase HPLC of trypsinized 125I-labeled Ep is presented. This allows the establishment of purity with far less material than is required for amino acid sequencing.

TWO ABNORMAL FIBRINOGENS DESIGNATED AS OSAKA II AND MORIOKA WITH A HITHERTO UNIDENTIFIED AMINO ACID SUBSTITUTION; γARG-275 BY CYS

1987 ◽  
Author(s):  
S Terukina ◽  
M Matsuda ◽  
N Yoshida ◽  
K Yamazumi ◽  
Y Takeda ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Sequence Data ◽  
Total Amino Acid ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Functional Studies ◽  
Sds Page ◽  
Two Populations ◽  
Fibrin Monomers

A hitherto unidentified amino acid substitution of γ Arg-275 by Cys has been found in two abnormal fibrinogens, Osaka II and Morioka. The propositi are both asymptomatic heterozygotes for the abnormality characterized by altered polymerization of fibrin monomers. Reducing SDS-PAGE revealed that fibrinogens derived from thé propositi both consist of two populations; one with a normal and the other with an abnormal longer γ-chain by 0.5 Kd.The γ-γ cross-linking took place nearly normally, however. Analyzing plasmic digests of fibrinogen by SDS-PAGE, we located the abnormality residing in the γ-chain remnant of fragment D. Chromatofocusing of D1 obtained by plasmic digestion in 5 mM Ca++ of purified fibrinogen separated the variant D1 (vD1) from the normal one (nD1) distinctly, as confirmed by SDS-PAGE and functional studies. As anticipated, vD1 failed to interfere with normal fibrin polymerization and thrombin clotting of normal fibrinogen, whereas nD1 inhibited these reactions significantly. After reduction and pyridylethylation, vD1 and nD1 were individually digested with lysylendopeptidase (lysEP). Analyzing the digests by reverse phase HPLC, we noted a single peak present in the digests of vD1 but missing in those of nD1, and vice versa. Analysis of N-terminal five cycles of these peptides suggested that both of them corresponded to the peptide with residues 274302 based on the known sequence data. Primary sequence and total amino acid analyses revealed that γ Arg-275 has been substituted by Cys in both of these abnormal fibrinogens. Analysis of the lysEP-digests of the isolated γ-chain also gave the same result. Since no free SH has been identified at the γ Cys-275 substitute, the variant γ-chain may be endowed with some additive by an S-S linkage. Even if so, elucidation of an apparent elongation by SDS-PAGE of the γ-chain variant must await further investigation. In any case, however, the substitution of γ Arg-275 by Cys may have induced critical alterations in the γ-chain-dependent polymerization site in the D domain in these two abnormal fibrinogens.

THE ACTIVATION OF GLUTAMIC ACID AND GLUTAMINE IN MAMMALIAN TISSUES

1963 ◽  
Vol 41 (5) ◽  
pp. 1135-1145 ◽  
Author(s):  
M. A. Alford ◽  
M. Brotman ◽  
M. A. Chudy ◽  
M. J. Fraser
Keyword(s):  
Amino Acid ◽  
Glutamine Synthetase ◽  
Glutamic Acid ◽  
Rat Liver ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Specific Enzyme ◽  
Ehrlich Ascites ◽  
Mammalian Tissues ◽  
Made In

Measurements of α-glutamyl-RNA synthetase and glutaminyl-RNA synthetase activities have been made in fractions derived from 105,000 × g supernatants of homogenates of rat liver and of mouse Ehrlich ascites carcinoma cells. Evidence is presented which indicates that each amino acid is activated by a specific enzyme. Both enzymes catalyze amino acid dependent ATP-32PP exchange. The α-glutamyl-RNA synthetase purified 42-fold from rat liver 105,000 × g supernatant is free of glutamine synthetase.

THE ACTIVATION OF GLUTAMIC ACID AND GLUTAMINE IN MAMMALIAN TISSUES

1963 ◽  
Vol 41 (1) ◽  
pp. 1135-1145 ◽  
Author(s):  
M. A. Alford ◽  
M. Brotman ◽  
M. A. Chudy ◽  
M. J. Fraser
Keyword(s):  
Amino Acid ◽  
Glutamine Synthetase ◽  
Glutamic Acid ◽  
Rat Liver ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Specific Enzyme ◽  
Ehrlich Ascites ◽  
Mammalian Tissues ◽  
Made In

Measurements of α-glutamyl-RNA synthetase and glutaminyl-RNA synthetase activities have been made in fractions derived from 105,000 × g supernatants of homogenates of rat liver and of mouse Ehrlich ascites carcinoma cells. Evidence is presented which indicates that each amino acid is activated by a specific enzyme. Both enzymes catalyze amino acid dependent ATP-32PP exchange. The α-glutamyl-RNA synthetase purified 42-fold from rat liver 105,000 × g supernatant is free of glutamine synthetase.

Increased expression of an amino acid transporter LAT1 in healing of acetic acid-induced chronic gastric ulcer in rats

2001 ◽  
Vol 120 (5) ◽  
pp. A153-A153
Author(s):  
S MIYAMOTO ◽  
K KATO ◽  
Y ISHII ◽  
S ASAI ◽  
T NAGAISHI ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Gastric Ulcer ◽  
Amino Acid ◽  
Amino Acid Transporter ◽  
Chronic Gastric Ulcer ◽  
Acid Transporter

Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

1989 ◽  
Vol 61 (03) ◽  
pp. 437-441 ◽  
Author(s):  
Cindra Condra ◽  
Elka Nutt ◽  
Christopher J Petroski ◽  
Ellen Simpson ◽  
P A Friedman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Salivary Gland ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Potent Inhibitor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Sds Page ◽  
Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

Purification and Characterization of Lupus Anticoagulant Like Protein from Agkistrodon Halys Brevicaudus Venom

1996 ◽  
Vol 76 (06) ◽  
pp. 0993-0997
Author(s):  
Zhao-Yan Li ◽  
Xiao-Wei Wu ◽  
Tie-Fu Yu ◽  
Eric C-Y Lian
Keyword(s):  
Amino Acid ◽  
Lupus Anticoagulant ◽  
Inhibitory Effect ◽  
Clotting Factor ◽  
Acid Oxidase ◽  
Crude Venom ◽  
Amino Acid Oxidase ◽  
Sds Page ◽  
The Individual ◽  
Reducing Condition

SummaryBy means of CM-Sephadex C-25, DEAE-Sephadex A-50, Sephadex G-200, and Sephadex G-75 chromatographies, a lupus anticoagulant like protein (LALP) from Agkistrodon halys brevicaudus was purified. On SDS-PAGE, the purified LALP had a molecular weight of 25,500 daltons under non-reducing condition and 15,000 daltons under reducing condition. The isoelectric point was pH 5.6. Its N terminal amino acid sequencing revealed a mixture of 2 sequences: DCP(P/S)(D/G)WSSYEGH(C/R)Q(Q/K). It was devoid of phospho-lipaseA, fibrino(geno)lytic, 5′-nucleotidase, L-amino acid oxidase, phosphomonoesterase, phosphodiesterase and thrombin-like activities, which were found in crude venom. In the presence of LALP, PT, aPTT, and dRVVT of human plasma were markedly prolonged and its effects were concentration-dependent but time-independent. The inhibitory effect of LALP on the plasma clotting time was enhanced by decreasing phospholipid concentration in TTI test. The individual clotting factor activity was not affected by LALP when higher dilutions of LALP-plasma mixture were used for assay. Russell’s viper venom time was shortened when high phospholipid confirmatory reagent was used. Therefore, the protein has lupus anticoagulant property.

Single Amino Acid Based Self-Assemblies of Cysteine and Methionine

2018 ◽  
Author(s):  
Nidhi Gour ◽  
Bharti Koshti ◽  
Chandra Kanth P. ◽  
Dhruvi Shah ◽  
Vivek Shinh Kshatriya ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Self Assembly ◽  
Aromatic Amino Acids ◽  
Neutral Condition ◽  
Single Amino Acid ◽  
Binding Assays ◽  
Human Neuroblastoma ◽  
Cytotoxicity Assays ◽  
First Time

We report for the very first time self-assembly of Cysteine and Methionine to discrenible strucutres under neutral condition. To get insights into the structure formation, thioflavin T and Congo red binding assays were done which revealed that aggregates may not have amyloid like characteristics. The nature of interactions which lead to such self-assemblies was purported by coincubating assemblies in urea and mercaptoethanol. Further interaction of aggregates with short amyloidogenic dipeptide diphenylalanine (FF) was assessed. While cysteine aggregates completely disrupted FF fibres, methionine albeit triggered fibrillation. The cytotoxicity assays of cysteine and methionine structures were performed on Human Neuroblastoma IMR-32 cells which suggested that aggregates are not cytotoxic in nature and thus, may not have amyloid like etiology. The results presented in the manuscript are striking, since to the best of our knowledge,this is the first report which demonstrates that even non-aromatic amino acids (cysteine and methionine) can undergo spontaneous self-assembly to form ordered aggregates.

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