scholarly journals A new h.p.l.c. isolation procedure for chicken and goose erythrocyte histones

1988 ◽  
Vol 255 (1) ◽  
pp. 23-27 ◽  
Author(s):  
W Helliger ◽  
H Lindner ◽  
S Hauptlorenz ◽  
B Puschendorf
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Histone Variants ◽  
Reversed Phase ◽  
Gradient System ◽  
Chicken Erythrocyte ◽  
Sequence Analyses ◽  
Electrophoretic Mobilities ◽  
Goose Erythrocyte ◽  
Short Time ◽  
Chicken Blood

Total chicken erythrocyte histones were separated by reversed-phase h.p.l.c. using a multi-step acetonitrile gradient in a very short time (35 min). The proteins were eluted in the following order: H1, H5, H2B, H2A.2, H4, H2A.1 and H3.2. Applying a special gradient system adapted for the separation of very-lysine-rich histones, chicken erythrocyte H5 was resolved into two subfractions. Their electrophoretic mobilities were identical in both SDS and acetic acid/urea/Triton polyacrylamide-gel electrophoresis, but different in free-flow electrophoresis. Amino-acid-sequence analyses revealed that the two components only differ with respect to position 15, one having glutamine in that position and the other arginine. A separation of histones prepared from goose erythrocytes disclosed no H5 subfractionation. Furthermore, histones obtained from anaemic-chicken blood were analysed by the above-mentioned h.p.l.c. conditions. An alteration in the relation of H1 to H5 was detected, but no further differences in the number and quantity of the histones and histone variants were observed as compared with the corresponding proteins processed from normal-chicken blood.

CLEAVAGE PATHWAY,AND SPECIFICITY OF LEUCOCYTE ELASTASE AS COMPARED TO PLASMIN DURING FIBRINOGEN DEGRADATION

1987 ◽  
Author(s):  
L Goretzki ◽  
E Miller ◽  
A Henschen
Keyword(s):  
Amino Acid ◽  
Time Course ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Reversed Phase ◽  
Cleavage Sites ◽  
Human Fibrinogen ◽  
Leucocyte Elastase ◽  
N Terminus

Plasmin and leucocyte elastase are regarded as the two medically most important fibrin(ogen)-degrading proteolytic enzymes. There is, however, a considerable difference in information available about the cleavage specificities and fragmentation pathways of these two enzymes. Degradation by plasmin has been studied already for a long time in great detail so that now the time course of the degradation, the cleavage sites and the functional properties of many fragments are well known. In contrast, relatively little is known about the degradation by leucocyte elastase, except that the overall cleavage pattern resembles that obtained with plasminIn this investigation the leucocyte elastase-mediated degradation of fibrinogen has been examined by means of proteinchemi-cal methods. Human fibrinogen was incubated with human enzyme material for various periods of time and at some different enzyme concentrations. The split products formed at the various stages were isolated in pure form by gel filtration followed by reversed-phase high-performance liquid chromatography. The fragments were identified by N-terminal amino acid sequence and amino acid composition. The course of the degradation was also monitored by sodium dodecylsulfate-polyacrylamide gel electrophoresis. All cleavage patterns were compared with the corresponding patterns from plasmic degradation. It could be confirmed that X-, D- and E-like fragments are formed also with elastase. However, several early elastolytic Aα-chain fragments are characteristically different from plasmic fragments. The previously identified N-terminal cleavage site in the Aα-chain, i.e. after position 21, was found to be the most important site in this region of fibrinogen. The very early degradation of the Aα-chain N-terminus by elastase is in strong contrast to the stability against plasmin. Several cleavage sites in N-terminal region of the Bβ-chain were observed, though the low amino acid specificity of elastase partly hampered the identification. The γ-chain N-terminus was found to be as highly stable towards elastase as towards plasmin. The results are expected to contribute to the understanding of the role of leucocyte elastase in pathophysiologic fibrino(geno)lysis

Isoperoxidases of (IAA oxidase) oxidase in oat coleoptiles

1973 ◽  
Vol 51 (11) ◽  
pp. 2047-2052 ◽  
Author(s):  
William R. Gordon ◽  
James H. M. Henderson
Keyword(s):  
Acetic Acid ◽  
Avena Sativa ◽  
Gel Electrophoresis ◽  
Enzyme Assay ◽  
Polyacrylamide Gel Electrophoresis ◽  
Acid Oxidase ◽  
Iaa Oxidase ◽  
Substrate Specificities ◽  
Electrophoretic Mobilities ◽  
Indole 3 Acetic Acid

Eight constitutive isoperoxidases were separated by the disc method of polyacrylamide gel electrophoresis from a lyophilized extract of 8-day-old oat (Avena sativa L., cv. Victory) coleoptiles. Both anodic and cathodic isoperoxidases were studied and differences in electrophoretic mobilities and hydrogen donor substrate specificities were revealed. In addition, by enzyme assay, cathodic and anodic isoenzymes were shown to possess differences in peroxidase and IAA (indole-3-acetic acid) oxidase activities.Treatment of coleoptiles with 0.07 mM IAA for 24 h resulted in the repression of two slow-migrating anodic isoperoxidases; however, the same treatment also resulted in the induction of two slow-migrating cathodic isoenzymes which were shown to exhibit peroxidase and IAA oxidase activities.

scholarly journals Characterization of cytoplasmic and chloroplast 5S ribosomal ribonucleic acid from broad-bean leaves

1971 ◽  
Vol 124 (1) ◽  
pp. 83-89 ◽  
Author(s):  
P. I. Payne ◽  
T. A. Dyer
Keyword(s):  
Mammalian Cells ◽  
Broad Bean ◽  
Polyacrylamide Gel Electrophoresis ◽  
Minor Component ◽  
5S Rna ◽  
E Coli ◽  
Electrophoretic Mobilities ◽  
Thermal Melting ◽  
Cytoplasmic Ribosomes

Green leaves of the broad bean (Vicia faba) contain two 5S RNA components that can be separated from each other by polyacrylamide-gel electrophoresis. The major component is located in the larger subunit of cytoplasmic ribosomes, whereas the minor component occurs in the larger subunit of chloroplast ribosomes. Their electrophoretic mobilities relative to those of Escherichia coli 5S RNA (120 nucleotides) and plant 4S RNA (78 nucleotides) suggest that they consist of 118 and 122 nucleotide residues respectively. Thermal ‘melting‘ profiles of plant cytoplasmic and chloroplast 5S RNA species at 260nm indicate the similarity of their secondary structures, not only to each other, but also to those of E. coli and mammalian 5S RNA species. The base compositions of the two plant 5S RNA species have more in common with each other than with the corresponding molecules from either E. coli or mammalian cells.

scholarly journals HPLC Fingerprint Analysis with the Antioxidant and Cytotoxic Activities of Selected Lichens Combined with the Chemometric Calculations

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4301
Author(s):  
Anna Hawrył ◽  
Mirosław Hawrył ◽  
Agnieszka Hajnos-Stolarz ◽  
Jagoda Abramek ◽  
Anna Bogucka-Kocka ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Tumor Cell Line ◽  
Reversed Phase ◽  
Total Phenolic ◽  
Cytotoxic Activities ◽  
Multivariate Techniques ◽  
Gradient System ◽  
Evernia Prunastri ◽  
Antioxidant Power ◽  
Hplc Fingerprint

The aim of this study was to evaluate the ability of multivariate techniques to predict antioxidant and cytotoxic activity of the selected lichens from the chromatographic data. A simple and reproducible HPLC-DAD technique has been used to obtain the chromatographic fingerprint profiles. Reversed phase high performance liquid chromatography (RP-HPLC) linear gradient system with methanol, water and phosphoric acid (V) (pH 2.3) as the mobile phase was used (50 min). Principal Component Analysis (PCA) has been applied to the evaluation of the phytochemical similarity between studied samples, especially between the same species collected in various places of Poland (Cetraria islandica (L.) Ach., CI, Cladina mitis Sandst., CM, Hypogymnia physodes (L.) Nyl., HP). The ability to scavenge free radicals was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods and the total phenolic content was determined by Folin-Ciocalteu (F-C) test. In the case of DPPH % of inhibition was higher for selected species (Pseudevernia furfuracea (L.) Zopf, H. physodes in comparison to the literature data. The FRAP test showed that the H. physodes extract had higher ability to scavenge free radical in comparison to Cladonia furcata (Huds.) Schrader and Evernia prunastri (L.) Ach., whereas P. furfuracea extract showed higher ability than C. islandica. The high content of phenolics in P. furfuracea and H. physodes confirms their high antioxidant activity. The cytotoxic activity of studied extracts was tested by cell culture method using the human HL-60 / MX2 acute CKL-22 (CRL-2257) promyelocytic leukemia tumor cell line. The lowest values of IC50 [µg∙mL−1] were obtained for: H. physodes (HP1)—99.4; C. digitate—122.6; H. physodes (HP)—136.5, C. subulata—142.6; C. mitis—180.2.

Liquid-chromatographic procedure for simultaneous analysis for eight benzodiazepines in serum.

1982 ◽  
Vol 28 (11) ◽  
pp. 2274-2278 ◽  
Author(s):  
G L Lensmeyer ◽  
C Rajani ◽  
M A Evenson
Keyword(s):  
High Performance ◽  
Reversed Phase ◽  
Gradient System ◽  
Active Metabolites ◽  
Reduced Pressure ◽  
Working Standard ◽  
Standard Curve ◽  
Chromatographic Procedure ◽  
Pharmacologically Active ◽  
Liquid Chromatographic

Abstract We describe an efficient extraction and liquid-chromatographic method for separating commonly encountered benzodiazepine drugs and their pharmacologically active metabolites. After a single extraction of the drugs from serum, chlordiazepoxide, demoxepam, N-desmethyl-chloriazepoxide, diazepam, N-desmethyldiazepam, N-desalkylflurazepam, oxazepam, and prazepam can be resolved and quantified by using a C18 reversed-phase "high-performance" column and a ternary-solvent gradient system. Three separate solutions [60 mmol/L ammonium acetate (pH 7.69), 60 mmol/L acetic acid (pH 2.8), and acetonitrile] were incorporated into a gradient mobile phase such that changes in pH and solvent composition occur. Complete chromatographic resolution of the benzodiazepines resulted, permitting quantification of all within 15 min. The standard curve is linear to at least 8 mg/L for each drug, and the detection limit for each was 0.05-0.10 mg/L. The day-to-day precision for both high and low concentrations yielded CVs of 5 to 9%. Extraction of each drug from serum was 95 to 100% complete. Exogenous and endogenous interferences are minimal. Finally, we circumvented the instability problem of benzodiazepine standards in solution by using a simple reduced-pressure drying process that produces a working standard that is stable for at least nine months.

scholarly journals Semisynthetic human [[3H2]Phe1]proinsulin

1987 ◽  
Vol 247 (3) ◽  
pp. 785-788 ◽  
Author(s):  
R M L Jones ◽  
K Rose ◽  
R E Offord
Keyword(s):  
Time Course ◽  
Recombinant Dna ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Specific Radioactivity ◽  
Reversed Phase ◽  
British Library ◽  
Recombinant Dna Techniques ◽  
Phenylalanine Residue

Biosynthetic human proinsulin (obtained by recombinant DNA techniques) was used as the starting material for the preparation, by semisynthetic methods, of [3H]proinsulin with the label at the N-terminal phenylalanine residue. The labelled proinsulin was characterized by its retention time on reversed-phase h.p.l.c., by polyacrylamide-gel electrophoresis, by the time course of its enzymic conversion into insulin and by chromatographic analysis after extensive proteolytic degradation. The specific radioactivity of the product was 5 Ci/mmol. Experimental details of the preparation of human [[3H]Phe1]proinsulin, the isolation of this product by isocratic h.p.l.c. and gel filtration, and further characterization of protein intermediates have been deposited as supplement SUP 50138 (12 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on prepayment [see Biochem. J. (1987) 241, 5].

scholarly journals GENETIC CONTROL OF MITOCHONDRIAL THYMIDINE KINASE IN HUMAN-MOUSE AND MONKEY-MOUSE SOMATIC CELL HYBRIDS

1974 ◽  
Vol 61 (1) ◽  
pp. 35-44 ◽  
Author(s):  
Saul Kit ◽  
Wai-Choi Leung
Keyword(s):  
Somatic Cell ◽  
Thymidine Kinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Molecular Forms ◽  
Kidney Cells ◽  
Somatic Cell Hybrids ◽  
Human Embryonic Lung ◽  
Cell Hybrids ◽  
Electrophoretic Mobilities ◽  
Mouse Somatic Cell

Distinctive thymidine (dT) kinase molecular forms are present in mouse, human, and monkey mitochondria. Disk polyacrylamide gel electrophoresis (disk PAGE) analyses have shown that the mitochondrial-specific dT kinases differ from cytosol dT kinases in relative electrophoretic mobilities (Rm). Furthermore, the mouse mitochondrial dT kinase differs in Rm value from primate mitochondrial dT kinases. The mouse and primate cytosol dT kinases can also be distinguished. Disk PAGE analyses have been carried out on the cytosol and mitochondrial dT kinases of human-mouse (WIL-8) and monkey-mouse (mK·CVIII) somatic cell hybrids in order to learn whether the mitochondria of the hybrid cells contained murine mitochondrial-specific, primate mitochondrial-specific, or both dT kinases. WIL-8 cells were derived from cytosol dT kinase-negative, mitochondrial dT kinase-positive mouse fibro blasts and from cytosol dT kinase-positive, mitochondrial dT kinase-positive human embryonic lung cells; they contained mostly mouse chromosomes and a few human chromosomes, including the determinant for human cytosol dT kinase. The mK·CVIII cells were derived from cytosol dT kinase-negative, mitochondrial dT kinase-positive mouse kidney cells and from cytosol dT kinase-positive, mitochondrial dT kinase-positive monkey kidney cells; they contained mostly mouse chromosomes and a few monkey chromosomes, including the determinant for monkey cytosol dT kinase. Disk PAGE analyses demonstrated that the mitochondria of human-mouse and monkey-mouse somatic cell hybrids contained the mouse-specific mitochondrial dT kinase but not the human- or monkey-specific mitochondrial dT kinase. These findings suggest that primate cytosol and mitochondrial thymidine kinase genes are coded on different chromosomes.

A Validated Method for the Quality Control of Andrographis paniculata Preparations

Planta Medica ◽  
2017 ◽  
Vol 83 (14/15) ◽  
pp. 1207-1213 ◽  
Author(s):  
Anastasia Karioti ◽  
Patricia Timoteo ◽  
Maria Bergonzi ◽  
Anna Bilia
Keyword(s):  
Quality Control ◽  
Time Course ◽  
Symptomatic Treatment ◽  
Reversed Phase ◽  
Andrographis Paniculata ◽  
Gradient System ◽  
Solvent Mixtures ◽  
Relative Standard ◽  
Precision And Accuracy

Abstract Andrographis paniculata is a herbal drug of Asian traditional medicine largely employed for the treatment of several diseases. Recently, it has been introduced in Europe for the prophylactic and symptomatic treatment of common cold and as an ingredient of dietary supplements. The active principles are diterpenes with andrographolide as the main representative. In the present study, an analytical protocol was developed for the determination of the main constituents in the herb and preparations of A. paniculata. Three different extraction protocols (methanol extraction using a modified Soxhlet procedure, maceration under ultrasonication, and decoction) were tested. Ultrasonication achieved the highest content of analytes. HPLC conditions were optimized in terms of solvent mixtures, time course, and temperature. A reversed phase C18 column eluted with a gradient system consisting of acetonitrile and acidified water and including an isocratic step at 30 °C was used. The HPLC method was validated for linearity, limits of quantitation and detection, repeatability, precision, and accuracy. The overall method was validated for precision and accuracy over at least three different concentration levels. Relative standard deviation was less than 1.13%, whereas recovery was between 95.50% and 97.19%. The method also proved to be suitable for the determination of a large number of commercial samples and was proposed to the European Pharmacopoeia for the quality control of Andrographidis herba.

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