scholarly journals Purification and subunit structure of phosphoglycerate dehydrogenase from rabbit liver

1986 ◽  
Vol 238 (3) ◽  
pp. 919-922 ◽  
Author(s):  
K Lund ◽  
D K Merrill ◽  
R W Guynn
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Molecular Sieve ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Rabbit Liver ◽  
Subunit Structure ◽  
Phosphoglycerate Dehydrogenase

D-3-Phosphoglycerate dehydrogenase (EC 1.1.1.95) was purified from rabbit liver by (NH4)2SO4 fractionation, DEAE-Sephacel chromatography, affinity chromatography on AMP-agarose and molecular-sieve h.p.l.c. The purified enzyme was homogeneous as judged by SDS/polyacrylamide-slab-gel electrophoresis. On the basis of molecular-sieve h.p.l.c. and SDS/polyacrylamide-gel electrophoresis, the enzyme is a tetramer composed of subunits of Mr 60,000.

scholarly journals Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with α2-macroglobulin

1980 ◽  
Vol 191 (3) ◽  
pp. 799-809 ◽  
Author(s):  
R G Sutcliffe ◽  
B M Kukulska-Langlands ◽  
J R Coggins ◽  
J B Hunter ◽  
C H Gore
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Plasma Protein ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Carbohydrate Content ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Tryptic Fragment

Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000–820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A.

scholarly journals The subunit structure of the arom multienzyme complex of Neurospora crassa. Evidence from peptide ‘maps’ for the identity of the subunits

1978 ◽  
Vol 169 (2) ◽  
pp. 441-444 ◽  
Author(s):  
J Lumsden ◽  
J R Coggins
Keyword(s):  
Neurospora Crassa ◽  
Gel Electrophoresis ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Subunit Structure ◽  
Multienzyme Complex ◽  
Peptide Maps

Evidence was obtained, from polyacrylamide-gel electrophoresis in the presence of urea and from peptide ‘mapping’ of specifically labelled cysteine-and methionine-containing peptides, that the two subunits of the arom multienzyme complex of Neurospora crassa are chemically very similar and possibly identical.

Polyacrylamide Gel-electrophoresis across a Molecular Sieve Gradient

Nature ◽  
1967 ◽  
Vol 214 (5095) ◽  
pp. 1334-1336 ◽  
Author(s):  
J. MARGOLIS ◽  
K. G. KENRICK
Keyword(s):  
Gel Electrophoresis ◽  
Molecular Sieve ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel

scholarly journals Purification and subunit structure of legumin of Vicia faba L. (broad bean)

1974 ◽  
Vol 141 (2) ◽  
pp. 413-418 ◽  
Author(s):  
David J. Wright ◽  
Donald Boulter
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Broad Bean ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Subunit Structure ◽  
Isoelectric Precipitation ◽  
Molecular Weights

Zonal isoelectric precipitation was shown to be an effective method for the preparation of legumin which was homogeneous as judged by ultracentrifugation and polyacrylamide-gel electrophoresis. The subunit structure of legumin was investigated by preparative sodium dodecyl sulphate–polyacrylamide-gel electrophoresis and ion-exchange chromatography in urea. Five distinct subunits, of which two were acidic (α) and had a molecular weight of 37000, and three were basic (β) with molecular weights of 20100, 20900 and 23800, were identified. The α and β subunits were present in equimolar amounts in the legumin molecule and, in view of this and molecular-weight considerations, an α6β6 subunit model was proposed for legumin.

Isolation of carboxypeptidase N by affinity chromatography on column of CNBr-activated Sepharose with immobilized antibody

1979 ◽  
Vol 44 (2) ◽  
pp. 626-630 ◽  
Author(s):  
Eva Simonianová ◽  
Marie Petáková
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rabbit Antiserum ◽  
Serum Analysis ◽  
Polyacrylamide Gel ◽  
Rat Serum ◽  
Carboxypeptidase N

The isolation of rat serum carboxypeptidase N (EC 3.4.2.2) by affinity chromatography on a column of CNBr-activated Sepharose with immobilized antibody is described. The ligands used were either rabbit antiserum to rat carboxypeptidase N or the IgG fraction prepared from this serum. The coupling of the isolated antibodies to CNBr-activated Sepharose increased the capacity of the column approximately three times. The specific activity of the enzyme prepared by this method was 109-times higher than the activity of the serum. Analysis of the final product by polyacrylamide gel electrophoresis showed carboxypeptidase N and traces of albumin.

scholarly journals The exocellular dd-carboxypeptidase-endopeptidase from Streptomyces albus G. Purification and chemical properties

1978 ◽  
Vol 175 (3) ◽  
pp. 793-800 ◽  
Author(s):  
C Duez ◽  
J M Frère ◽  
F Geurts ◽  
J M Ghuysen ◽  
L Dierickx ◽  
...  
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Molecular Sieve ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chemical Properties ◽  
Polyacrylamide Gel ◽  
Streptomyces Albus ◽  
Enzyme I ◽  
And Chemical Properties

The exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G was purified to protein homogeneity and compared with the exocellular DD-carboxypeptidases-transpeptidases of Streptomyces R61 and Actinomadura R39. The S. albus G enzyme, as it is isolated, occurs in two forms. Enzyme I (30% of the total amount) and enzyme II (70% of the total amount) are identical in all respects, except that, by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, enzyme I has an apparent mol. wt. (9000) that is half of that found by molecular-sieve filtration under non-denaturing conditions. Irrespective of the technique used, enzyme II has an apparent mol. wt. of about 18500.

γ’-Dimer Antibody and its Specificity Against Fibrinogen, FR-a and Carboxymethylated chains from Fibrinogen and Cross-linked Fibrin

1975 ◽  
Author(s):  
N. T. Clausen ◽  
J. Gormsen
Keyword(s):  
Ionic Strength ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Acetate Buffer ◽  
Subunit Structure ◽  
Weak Reaction ◽  
Subcutaneous Injections ◽  
L 96 ◽  
Heterologous Antibody

γ’-dimers (M: 80.000) were obtained from fully cross-linked fibrin (Kabi, grade L, 96% clottable) by digestion with porcine plasmin (0.5 CTA units/mg protein) for 18 h., carboxymethylation and subsequent isolation on Whatman CM-52 with a combined pH (4.8 → 5.5) and ionic strength (0.05 → 0.25 M) gradient in Na-acetate buffer with 8 M urea*).The γ’-dimers were recycled once and exhibited purity on SDS polyacrylamide gel electrophoresis. These γ’-dimers showed weak reaction in immunoprecipitation assays with anti-fibrinogen and anti-D but not with anti-E rabbit immunserum.Heterologous antibody was obtained from rabbit after subcutaneous injections of γ’-dimer antigen and bleeding after 4 months, 14 days after last injection.This crude antibody reacted in immunodiffusion and monorocket immunoelectrophoresis with γ’-dimers and weakly with fibrinogen and fragment D but not with fragment E (DEAE purified). After absorption with fibrinogen and fractional desalting with (NH4)2SO4 (25% and 50%) there was no reaction with fibrinogen, D or E fragments, but still with γ’-dimers.Testing against isolated chains (SDS-pure) from fibrinogen revealed Aα cross-reaction but only reaction with γ and γ’-dimer chains remained after proper absorption. Possible differentiation between these two subunit structure is reported.* Murano, G. et al. FEBS-letters 14, 37–41, 1971.

scholarly journals Purification of multiple forms of the soluble 17α-hydroxy steroid dehydrogenase or rabbit liver.

1975 ◽  
Vol 147 (3) ◽  
pp. 457-461 ◽  
Author(s):  
S Hasnain ◽  
D G Williamson
Keyword(s):  
Substrate Specificity ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Rabbit Liver ◽  
Multiple Forms ◽  
Hydroxy Steroid ◽  
Steroid Dehydrogenase

Eight distinct forms of the soluble 17alpha-hydroxy steroid dehydrogenase of rabbit liver were resolved by DEAE-cellulose chromatography and isoelectric focusing. Five of these enzymes were homogeneous as judged by polyacrylamide-gel electrophoresis. Substrate-specificity studies carried out with oestradiol-17alpha and oestradiol-17alpha 3-glucuronide revealed a variation in activity toward these substrates among the different purified enzyme forms. Three forms of the 17alpha-hydroxy steroid dehydrogenase exhibited equal activity toward both oestrogen substrates, whereas three forms of the enzyme displayed a greater activity toward the glucuronide derivative of oestradiol-17alpha. One enzyme in particular is essentially specific for oestradiol-17alpha 3-glucuronide, its activity toward oestradiol-17alpha being only one-thirtieth that observed with the 3-glucuronide derivative.

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