scholarly journals Purified human liver acid β-d-galactosidases possessing activity towards G(M1)-ganglioside and lactosylceramide

1977 ◽  
Vol 165 (3) ◽  
pp. 591-594 ◽  
Author(s):  
A L Miller ◽  
R G Frost ◽  
J S O'Brien
Keyword(s):  
Affinity Chromatography ◽  
Enzyme Activities ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Single Band

Our studies with purified human liver acid beta-D-galactosidases (EC 3.2.1.23) indicate that 4-methylumbelliferyl beta-D-galactosidase and G(M1)-ganglioside beta-D-galactosidase activities are identical with lactosylceramidase II activity. Evidence for this includes co-purification of all enzyme activities by affinity chromatography to yield a single band on polyacrylamide-gel electrophoresis and coincident elution from Sepharose 6B of all three enzyme activities.

Purification Of Factors IX And X From Clinical Concentrate

1981 ◽  
Author(s):  
G C Russell ◽  
G Kemble ◽  
E G D Tuddenham
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
Factor Ix ◽  
Factor X ◽  
Clinical Factor

Human factors IX and X have been purified to homogeneity from clinical factor IX concentrate that had been rejected for therapeutic use due to particulate contamination. (It was necessary to start with this material since in the UK, plasma is not commercially available). The procedure involved barium citrate adsorption followed by ammonium sulphate elution, DEAE- cellulose chromatography, gel filtration on Sephacryl S-200 and affinity chromatography on heparin sepharose gel. The preparation of factor IX at this stage showed a single band on SDS-polyacrylamide gel electrophoresis, of molecular weight 58,000. No change in molecular weight was observed in the presence of 2-mercaptoethanol. A further affinity chromatography column - poly (homoarginine) Sepharose or dextran sulphate sepharose - was necessary to obtain homogeneous factor X. The preparation obtained showed a single band on SDS-polyacrylamide gel electrophoresis of molecular weight 67,000. In the presence of 2-mercaptoethanol, two bands were obtained of molecular weights 49000 and 17000 representing the heavy and light chains respectively of factor X. The purified coagulation proteins contained no activated species detectable by nonactivated partial thromboplastin time or by chromogenic substrate (S2222) assay. Prothrombin protein Sand protein C are by-products of this purification procedure.

scholarly journals Heparin-sepharose affinity chromatography for purification of bull seminal-plasma hyaluronidase

1979 ◽  
Vol 183 (3) ◽  
pp. 531-537 ◽  
Author(s):  
P N Srivastava ◽  
A A Farooqui
Keyword(s):  
Hyaluronic Acid ◽  
Affinity Chromatography ◽  
Seminal Plasma ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
National Formulary ◽  
Turnover Number

Bull seminal-plasma hyaluronidase was purified 180-fold by chromatography on concanvalin A-Sepharose, heparin Sepharose, Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 mumol/min per mg (104000 National Formulary units/mg of protein) and 214 min-1 (mol of product formed/mol of enzyme per min) respectively. Polyacrylamide-gel electrophoresis indicated that the purified enzyme migrated as a single band on 7.5 and 10% (w/v) gels at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenylhydrazine and semicarbazide. Purified hyaluronidase (1.25 munits; 1 unit = 1 mumol of N-acetylglucosamine liberated/min at 37 degrees C) dispersed the cumulus clot of rabbit ova in 1 h at 22 degrees C.

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.

A three step purification procedure for human liver ferritin

1980 ◽  
Vol 58 (6) ◽  
pp. 494-498 ◽  
Author(s):  
M. Pagé ◽  
J. Lagueux ◽  
C. Gauthier
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Purification Procedure ◽  
Human Liver Ferritin ◽  
Normal Human Liver ◽  
Normal Human ◽  
Liver Ferritin

We describe a method for the purification of normal human liver ferritin by ultrafiltration, gel filtration on Sephacryl S-300, and affinity chromatography on DEAE-Affi Gel Blue. The purity of the ferritin obtained was verified by immunoelectrophoresis, Ouchterlony immunodiffusion, polyacrylamide gel electrophoresis, and electrofocusing. This rapid method yields 32% of the original ferritin.

scholarly journals Antibody-affinity purification of novel α-l-fucosidase from mouse liver

1987 ◽  
Vol 245 (2) ◽  
pp. 589-593 ◽  
Author(s):  
L D Laury-Kleintop ◽  
I Damjanov ◽  
J A Alhadeff
Keyword(s):  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Mouse Liver ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Antibody Affinity ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Sepharose 4B

Previous studies have documented the presence of a novel alpha-L-fucosidase in mouse liver that contains unique basic isoelectric forms and that is antigenically similar to, but not identical with, human liver alpha-L-fucosidase [Laury-Kleintop, Damjanov & Alhadeff (1985) Biochem. J. 230, 75-82]. In the present investigation, mouse liver alpha-L-fucosidase was purified approx. 26,500-fold in 10% overall yield by antibody-affinity chromatography with the IgG fraction of goat anti-(human alpha-L-fucosidase) antibody coupled to Sepharose 4B. Native polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis indicated that the mouse fucosidase is highly purified if not homogeneous. Isoelectric focusing demonstrated that all enzymic forms found in crude mouse liver supernatant fluids were purified by the antibody-affinity procedure.

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.

Ontogeny of hepatocyte proliferation inhibitor activity during human liver development and its effect on cell proliferation in in vivo and in vitro studies

1993 ◽  
Vol 71 (5-6) ◽  
pp. 241-247 ◽  
Author(s):  
B. Geetha Devi ◽  
C. M. Habeebullah ◽  
P. D. Gupta
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Hepg2 Cells ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Hepatocyte Proliferation ◽  
Liver Development ◽  
Dodecyl Sulfate

Ontogeny of hepatocyte proliferation inhibitory (HPI) activity was studied during human liver development. HPI activity was first noticed in the cytosolic fraction of 20-week-old fetal liver and thereafter it began to increase with the liver maturation. An inverse correlation between the ontogeny of HPI activity and mitotic counting of the hepatocytes during human liver development was established. The crude HPI fraction from fetal and adult human liver inhibited the incorporation of [3H]thymidine into the DNA of 2-day-old rat liver and HepG2 cells and the inhibitory effect was directly proportional to the age of the HPI source. Prolonged exposure of HepG2 cells to the HPI fraction was also found to be cytotoxic. Preliminary characterization of the crude HPI fraction revealed an age-dependent increase in proteins of 18, 28, and 60 kilodaltons following silver chloride staining of sodium dodecyl sulfate - polyacrylamide gel electrophoresis and densitometric scanning.Key words: human liver, hepatocyte proliferation inhibitor, ontogeny, sodium dodecyl sulfate - polyacrylamide gel electrophoresis, HepG2 cells, cytotoxic.

scholarly journals Purification and characterization of mouse α-lactalbumin and preparation of its antibody

1980 ◽  
Vol 185 (1) ◽  
pp. 227-237 ◽  
Author(s):  
Y Nagamatsu ◽  
T Oka
Keyword(s):  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
Major Species ◽  
Alpha Lactalbumin

alpha-Lactalbumin was purified to apparent homogeneity from mouse milk by combined use of gel filtration, chromatography on DEAE-cellulose and hydroxyapatite, and concanavalin A-Sepharose affinity chromatography. Mouse alpha-lactalbumin exists in several species with different charges and in two molecular-size forms. The smaller form, which constituted over 90% of total alpha-lactalbumin, included two major and two minor species, each of which showed different electrophoretic mobility on polyacrylamide-gel electrophoresis, but gave the same single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in two different buffer systems and over the range 10-15% acrylamide concentrations. The molecular weight was estimated as 14 100. The two major species of the smaller form had the same amino acid composition and contained no significant amount of carbohydrate. The larger form of alpha-lactalbumin, consisting of two species with different charges, was present in a small amount (less than 10%) in the milk and was isolated by its ability to interact with concanavalin A-Sepharose. Each of the two species also gave the same single band of apparent mol.w.t 18 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, this value may be anomalous, since this larger form appears to be glycosylated, and glycoproteins can behave anomalously on sodium dodecyl sulphate/polyacrylamide gels by binding less sodium dodecyl sulphate. All species of mouse alpha-lactalbumin from milk were active in the lactose synthase reaction and showed identical immunological properties, as determined by the mono-specific antibody prepared against the small major species. The presence of both the larger and the smaller forms, each in a percentage concentration similar to that found in milk, was also demonstrated in alpha-lactalbumin induced by hormones in organ cultureof pregnant-mouse mammary gland.

Separation of horseradish peroxidase isoenzymes by preparative polyacrylamide gel electrophoresis

1977 ◽  
Vol 55 (18) ◽  
pp. 2474-2477
Author(s):  
Sheikh A. Saeed ◽  
Josephine Cuthbert
Keyword(s):  
Horseradish Peroxidase ◽  
Electrophoretic Mobility ◽  
Peroxidase Activity ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Step ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
Peroxidase Isoenzymes ◽  
Crude Material

Seven peroxidase isoenzymes were purified from crude horseradish peroxidase (EC 1.11.1.7) (HRP) in a single step by preparative polyacrylamide gel electrophoresis. The peroxidase activity in seven isoenzymes accounted for 90% of the activity in the crude material. Each isoenzyme (except HRP7) after separation migrated as a single band with characteristic electrophoretic mobility. All of the purified isoenzymes were found to retain complete peroxidase activity after storage at −20 °C or 4 °C for 3 months.

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