A METHOD FOR ISOLATION BY GEL ELECTROFOCUSING OF ISOHORMONES B AND C OF HUMAN PROLACTIN FROM AMNIOTIC FLUID

1980 ◽  
Vol 84 (1) ◽  
pp. 125-133 ◽  
Author(s):  
MENASHE BEN-DAVID ◽  
ANDREAS CHRAMBACH
Keyword(s):  
Amniotic Fluid ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Constant Ph ◽  
Human Prolactin ◽  
Isoelectric Points ◽  
Carrier Ampholytes

Human prolactin from amniotic fluid, consisting of isohormones B and C (major), was radio-iodinated after storage of the hormone for 3 years at −70 °C, and yielded a Ferguson plot in polyacrylamide gel electrophoresis that was indistinguishable from the original except that the zones of isohormone B and C were fused. However, isohormones B and C of 125I-labelled human prolactin were separated on isoelectric focusing in polyacrylamide gel, using Ampholine carrier ampholytes (pI range 5–8), taurine (pI 5·1) as anolyte and β-alanine (pI 6·9) as catholyte. After 20 h of electrofocusing at 0–4 °C, 1000 V, both isohormones reached constant pH (isoelectric) positions on the gel. The apparent isoelectric points of human prolactin B and C were 5·96 and 5·62 Micro-preparative gel electrofocusing followed by excision and re-electrofocusing of the gel slices containing human prolactin B and C, yielded zones of homogeneous isohormones B and C.

scholarly journals Isolation of pure anhydrotetracycline oxygenase from Streptomyces aureofaciens

1988 ◽  
Vol 253 (1) ◽  
pp. 263-267 ◽  
Author(s):  
I Vancurová ◽  
J Volc ◽  
M Flieger ◽  
J Neuzil ◽  
J Novotná ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Size Exclusion ◽  
Streptomyces Aureofaciens ◽  
Hydrophobic Chromatography ◽  
Step Procedure

Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.

scholarly journals Purification and properties of succinyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney

1978 ◽  
Vol 173 (3) ◽  
pp. 759-765 ◽  
Author(s):  
J A Sharp ◽  
M R Edwards
Keyword(s):  
Isoelectric Focusing ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Coenzyme A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Polyacrylamide Gel ◽  
Transferase Activity ◽  
Coa Transferase

CoA-transferase (succinyl-CoA-3-oxo acid CoA-transferase, EC 2.8.3.5) isolated from sheep kidney was purified to homogeneity. The purified enzyme has a specific activity of approx. 200 units/mg. A mol.wt. of 110000 was obtained by gel filtration on Sephadex G-200, and a lower mol.wt. of 102000 was determined by analytical ultracentrifugation. A sedimentation coefficient of 5.6S was also determined. A subunit mol.wt. of 56000 was obtained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing of sheep kidney extracts indicated the presence of a single band of CoA-transferase activity with pI9.0. However, isoelectric focusing of purified CoA-transferase showed the presence of two peaks of CoA-transferase activity with pI values of 8.7 and 8.4, suggesting the presence of proteolytic activity during purification. Evidence for sheep kidney CoA-transferase being a dimer of two identical subunits has been obtained from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the amino acid composition, peptide ‘mapping’ and N-terminal analysis.

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