Characterization of in vitro proteolytic processing of β-endorphin by reversed-phase HPLC

Peptides ◽  
1984 ◽  
Vol 5 (6) ◽  
pp. 1037-1042 ◽  
Author(s):  
Thomas P. Davis ◽  
Hans Schoemaker ◽  
Alison J. Culling-Berglund
Keyword(s):  
Reversed Phase ◽  
Proteolytic Processing ◽  
Reversed Phase Hplc

Characterization of Highly Polar Dna Adducts Derived from Dibenz[A,H]Anthracene (DBA), 3,4-Dihydroxy-3,4-Dihydro-DBA, and 3,4,10,11-Tetrahydroxy-3,4,10,11-Tetrahydro-DBA

1993 ◽  
Vol 9 (3) ◽  
pp. 503-509 ◽  
Author(s):  
Juergen Fuchs ◽  
Jiri Mlcoch ◽  
Franz Oesch ◽  
Karl Ludwig Platt
Keyword(s):  
Dna Adducts ◽  
Liver Microsomes ◽  
Metabolic Activation ◽  
Reversed Phase ◽  
Reversed Phase Hplc ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Calf Thymus

Two highly polar DNA adducts were found after metabolic activation of 3,4,10,11-tetrahydroxy-3,4,10,11-tetrahydrodibenz[ a,h]anthracene(DBA-3,4;10, 11-bisdiol) by liver microsomes isolated from male Sprague-Dawley rats pretreated with Aroclor 1254 in presence of calf thymus DNA. These DNA adducts could be assigned to the metabolites of dibenz[ a,h]anthracene (DBA), of 3R,4R,10R,11R-tetrahydroxy-3,4,10,11-tetrahydro-DBA and of 3R,4R,10S,US-tetrahydroxy-3,4,10,11-tetrahydro-DBA. DNA adducts derived from metabolites of 3S,4S,10S,11S-tetrahydroxy-3,4,10,11-tetrahydro-DBA were not found. These highly polar adducts also could be detected by reversed phase HPLC after incubation of dibenz[ a,h]anthracene, 3R,4R-dihydroxy-3,4-dihydro-DBA ((-)-DBA-3,4-diol) and 3S,4S- dihydroxy-3,4-dihydro-DBA ((+)-DBA-3,4-diol) with DNA in presence of the activating system. After incubation of 14C labelled DBA DNA adducts derived from DBA-3,4;10,11-bisdiol were found in a fraction of 38% and bay region 3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-DBA-DNA adducts at a level of 25%. DBA-3,4; 10,11-bisdiol exhibited a higher DNA binding yield (38 × 12 pmollmg DNA) than (-)-DBA-3,4-diol (23 × 6 pmol/mg DNA), the most mutagenic 3,4-diol enantiomer. For (+)-DBA-3,4-diol the highly polar DNA adducts derived from DBA-3,4;10,11-bisdiol were by far the most predotmnant adducts in vitro.

Comparative Profiling of Four Lignans (Phyllanthin, Hypophyllanthin, Nirtetralin, and Niranthin) in Nine Phyllanthus Species from India Using a Validated Reversed Phase HPLC-PDA Detection Method

2020 ◽  
Author(s):  
Jinal Patel ◽  
Padamnabhi Shanker Nagar ◽  
Kalpana Pal ◽  
Raghuraj Singh ◽  
Tushar Dhanani ◽  
...  
Keyword(s):  
Reversed Phase ◽  
Reversed Phase Hplc ◽  
Extraction Solvent ◽  
Plant Parts ◽  
Commercial Cultivation ◽  
Wide Range ◽  
Development And Validation ◽  
Herbal Formulations

Abstract Background Phyllanthus species exhibit a wide range of in vitro and in vivo pharmacological activities; however, little is known about the compounds present in the extracts that are responsible for such actions. Objective Development and validation of a simple reversed phase HPLC-PDA method for profiling of phyllanthin, hypophyllanthin, nirtetralin, and niranthin in extracts of Phyllanthus species was carried out. Methods Separation was achieved using an XBridge column® (150 × 4.6 mm, 5.0 µm id) in an isocratic elution mode with mobile phase comprising of a mixture of acetonitrile and water with TFA (0.05%, v/v, pH = 2.15) at ambient temperature with a flow rate of 1 mL/min. Results Phyllanthin, hypophyllanthin, nirtetralin, and niranthin were eluted at mean retention times of 10.47, 11.10, 13.67, and 14.53 min, respectively. LOD and LOQ for all four analytes were 0.75 and 3.00 μg/mL, respectively. RSDr values for intraday and interday precision for phyllanthin, hypophyllanthin, nirtetralin, and niranthin were 0.38–1.32 and 0.45–1.77%; 0.22–3.69 and 0.24–3.04%, 0.73–2.37 and 0.09–0.31%, and 1.56–2.77 and 0.12–0.68%, respectively. Conclusions The developed and validated HPLC-PDA method was applied for identification and quantification of phyllanthin, hypophyllanthin, nirtetralin, and niranthin in extracts of different plant parts of selected Phyllanthus species. The outcome of the present investigation could be useful for selection of best species to promote its commercial cultivation and suitable extraction solvent for preparation of lignan-enriched fractions. This HPLC-PDA method could be useful for quality control of herbal formulations containing plants from Phyllanthus species.

Biological characterization of charge isomers of human growth hormone

1988 ◽  
Vol 118 (1) ◽  
pp. 14-21 ◽  
Author(s):  
A. Skottner ◽  
A. Forsman ◽  
B. Skoog ◽  
J. L. Kostyo ◽  
C. M. Cameron ◽  
...  
Keyword(s):  
Biological Activity ◽  
Biological Activities ◽  
Human Growth ◽  
Proteolytic Processing ◽  
Biological Characterization ◽  
Growth Promoting ◽  
Rat Adipose Tissue ◽  
Insulin Like Activity

Abstract. Since deamidation of the human GH molecule may alter the manner and extent to which the hormone is cleaved by proteases, and since it has been repeatedly suggested that proteolytic processing is required for the expression of certain of the activities of GH, the present study was conducted to determine whether the biological activity profiles of more acidic forms of human GH are altered. Three charge isomers, GH-b, GH-c and GH-d, representing primarily deamidated forms, were isolated from a native human GH preparation (Crescormon®) in amounts adequate for characterization of their biological activities. All three were essentially equipotent in a radioimmunoassay for human GH. When assessed for growth-promoting activity in the hypophysectomized rat, the isomers were again equipotent with each other and with the GH preparation from which they were derived. The charge isomers also had significant in vitro insulin-like activity on isolated rat adipose tissue and diabetogenic activity in the ob/ob mouse. Thus, the biological activity profiles of these charge isomers of human GH do not differ greatly from one another.

scholarly journals Purification and characterization of an L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)-specific suppressor factor from genetic responder mice.

1983 ◽  
Vol 158 (4) ◽  
pp. 1034-1047 ◽  
Author(s):  
C M Sorensen ◽  
C W Pierce ◽  
D R Webb
Keyword(s):  
Specific Activity ◽  
Reversed Phase ◽  
Biologically Active ◽  
Reversed Phase Hplc ◽  
Purification And Characterization ◽  
Suppressor Factor ◽  
Chemical Homogeneity ◽  
T Cell Factor ◽  
Molecular Weights

A hybridoma-derived, GAT-specific suppressor T cell factor (GAT-TsFR) from responder C57BL/10 mice has been purified to apparent chemical homogeneity using reversed phase HPLC techniques. 40 l of starting material yielded approximately 880 micrograms protein with a specific activity of 28.4 X 10(3) S50 U/ng protein representing a purification factor of 4.2 X 10(6). Purified GAT-TsFR is a hydrophobic protein with a minimum molecular weight of 18,000 that is capable of forming biologically active aggregates with molecular weights of 28,000, 64,000 and approximately 84,000 and has a pI of 6.4. GAT-TsFR is a glycoprotein that binds GAT and GT, but not GA, and bears determinants encoded by the I-J subregion of the H-2 complex. This GAT-TsFR derived from an H-2b responder haplotype to GAT is compared with GAT-TsF derived from the nonresponder H-2q haplotype on the basis of biochemical and some serological properties.

Intracellular proteolytic processing of proopiomelanocortin in heterologous COS-1 cells by the yeast KEX2 endoprotease

1990 ◽  
Vol 68 (3) ◽  
pp. 635-640 ◽  
Author(s):  
L. Zollinger ◽  
C. Racine ◽  
P. Crine ◽  
G. Boileau ◽  
D. Germain ◽  
...  
Keyword(s):  
High Pressure ◽  
Liquid Chromatography ◽  
High Pressure Liquid Chromatography ◽  
Reversed Phase ◽  
Proteolytic Processing ◽  
Amino Terminal ◽  
Cell Extracts ◽  
Gel Permeation

We have transiently expressed the yeast KEX2 gene together with the proopiomelanocortin (POMC) cDNA in COS-1 cells. Characterization of the POMC-related immunoreactive peptides by gel permeation and reversed-phase high pressure liquid chromatography showed that the KEX2 enzyme was active and capable of carrying out cleavage of POMC to release the authentic maturation product β-endorphin(1–31). Peptides resembling β-lipotropin, the amino terminal glycopeptide, and ACTH(1–39) were also detected as major products in the cell extracts. Our results indicate that the KEX2 enzyme can proteolytically release from POMC a set of peptides similar to that normally found in interior pituitary.Key words: prohormone proteolytic processing, KEX2 expression, COS-1 cells.

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