scholarly journals Common epitopes of bovine lens multicatalytic-proteinase-complex subunits

1989 ◽  
Vol 257 (1) ◽  
pp. 265-269 ◽  
Author(s):  
B J Wagner ◽  
J W Margolis
Keyword(s):  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyclonal Antiserum ◽  
Antibody Affinity ◽  
Multicatalytic Proteinase ◽  
Bovine Lens ◽  
The Common ◽  
Multicatalytic Proteinase Complex ◽  
Monospecific Antibodies

Component polypeptides of both the bovine lens and pituitary multicatalytic proteinase complexes demonstrate different immunoreactivities with a polyclonal antiserum raised against the purified pituitary enzyme. Four (Mr 24000, 26000, 34000 and 38000) of eight bands that have been resolved by SDS/polyacrylamide-gel electrophoresis are stained in immunoblot experiments. Monospecific antibodies obtained from this antiserum by affinity purification from the 38000- and 34000-Mr bands of the lens enzyme bound equally well to either band, but showed little or no binding to the 26000- and 24000-Mr bands upon immunoblotting. Antibody affinity-purified from the 24000-Mr band showed comparable binding to the 24000-, 34000- or 38000-Mr band. One explanation of these results is that the 24000-Mr polypeptide is derived from the higher-Mr polypeptide(s) and has lost some of the common immunodeterminants.

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 Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

1990 ◽  
Vol 63 (03) ◽  
pp. 439-444 ◽  
Author(s):  
C Kuyas ◽  
A Haeberli ◽  
P Walder ◽  
P W Straub
Keyword(s):  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ease Of Use ◽  
Terminal Sequence ◽  
Purification Method ◽  
Human Fibrinogen ◽  
Isolation Methods ◽  
One Step ◽  
Complementary Binding

SummaryWith an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

scholarly journals Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

1981 ◽  
Vol 197 (3) ◽  
pp. 629-636 ◽  
Author(s):  
J L McKenzie ◽  
A K Allen ◽  
J W Fabre
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Antibody Affinity ◽  
Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

scholarly journals Affinity purification and subunit structures of β-N-acetylhexosaminidases A and B from boar epididymis

1984 ◽  
Vol 219 (3) ◽  
pp. 1009-1015 ◽  
Author(s):  
H C Parkes ◽  
J L Stirling ◽  
P Calvo
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B ◽  
Electrophoretic Transfer ◽  
Peptide Maps

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

scholarly journals Bovine lens aldehyde reductase (aldose reductase). Purification, kinetics and mechanism

1984 ◽  
Vol 219 (1) ◽  
pp. 33-39 ◽  
Author(s):  
A B Halder ◽  
M J C Crabbe
Keyword(s):  
Aldose Reductase ◽  
Gel Electrophoresis ◽  
Ternary Complex ◽  
Minimum Degree ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Aldehyde Reductase ◽  
Complex Mechanism ◽  
Bovine Lens ◽  
Initial Binding

Aldehyde reductase (aldose reductase) was purified to homogeneity (as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) from bovine lens by affinity chromatography on NADP+-Sepharose. The enzyme, a monomer of Mr about 40000, was active with a variety of alpha- hydroxyketones, including fructose. The minimum degree of the rate equation was 2:2 in the case of DL-glyceraldehyde, but linear kinetics were observed for glucose and NADPH over the concentration range studied. The enzyme largely followed a ternary-complex mechanism, with initial binding of NADPH before glucose and final release of NADP+.

Bovine Lens Multicatalytic Proteinase Complex

1993 ◽  
Vol 47 (4-6) ◽  
pp. 202-209 ◽  
Author(s):  
B.J. Wagner ◽  
Joyce W. Margolis ◽  
Inderpal Singh
Keyword(s):  
Multicatalytic Proteinase ◽  
Bovine Lens ◽  
Multicatalytic Proteinase Complex

Does the B820 Subcomplex of the B880 Complex Retain Carotenoids?

1996 ◽  
Vol 51 (5-6) ◽  
pp. 309-318 ◽  
Author(s):  
Andrey Moskalenko ◽  
Olga Toropygina ◽  
Nina Kuznetsova
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
High Performance ◽  
Rhodospirillum Rubrum ◽  
Complex Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
The Common

Abstract A study is reported on the modification of the B880-RC assembly of Chromatium minutissimum during octyl-β-D -glucopyranoside/dodecyl-β-D -maltoside/Deriphat polyacrylamide gel electrophoresis followed by electroelution with dodecyl-β-D-maltoside and high performance liquid chromatography with octyl-β-D-glucopyranoside according to the method developed by Kerfeld et al. (Biochim. Biophys. Acta 1185, 193-202 [1994a]) for isolation of the B820 subcomplexes of Chromatium purpuratum. The B880-RC assembly of Chromatium minutissimum isolated by electrophoresis was contaminated by the B 800-850 complex. It was further separated into four components, three of which were in agreement with the cited work: (i) colorless contaminations, (ii) the B880-RC assembly, (iii) the B 800-850 complex. In contrast with Kerfeld et al. (1994a), the fourth band was a band of free pigments (Bchl or Bchl-t-carotenoids) which had the same molecular mass as the B820 subcomplex of Chromatium purpuratum. For comparison, the B880-RC enriched fraction of Rhodospirillum rubrum modified by lyophilization in the presence of octyl-β-D-glucopyranoside with or w ithout carotenoids was separated by high performance liquid chromatography with octyl-β-D-glucopyranoside. The apparent molecular mass of the B820 subcomplex was 30 kD a for the sample without carotenoids and 245 kD a for that with carotenoids. The common principles of organization of the B880 complex, the interaction of the B 800- 850 complex with the B880-RC assembly, the participation of carotenoids in the stabilization of the B880 complex structure and the ability of different isolation steps to modify the structure of the B880 complex are discussed. It was concluded that there are other explanations for the presence of carotenoids in the B820 subcomplex. Hence, the question of whether the B820 subcomplex retains carotenoids remains open.

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