scholarly journals Co2+-mediated time- and temperature-dependent activation of neutral α-d-mannosidase from monkey brain

1988 ◽  
Vol 253 (3) ◽  
pp. 677-685 ◽  
Author(s):  
R Mathur ◽  
K Panneerselvam ◽  
A S Balasubramanian
Keyword(s):  
Amino Acids ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Time Dependent ◽  
Aminopeptidase Activity ◽  
Temperature Dependent ◽  
Monkey Brain ◽  
Stimulation Of

Neutral alpha-D-mannosidase from monkey brain was purified by Co2+-chelate affinity chromatography and immunoadsorbent affinity chromatography. The purified enzyme, with subunit Mr 45,000, was essentially homogeneous with only traces of two contaminant proteins as revealed by SDS/polyacrylamide-gel electrophoresis and AgNO3 staining. The purified enzyme, on preincubation with Co2+ at 37 degrees C or 60 degrees C followed by assay, showed a time-dependent enhancement in activity. The enhanced activity of the enzyme persisted even after removal of the Co2+. Bacitracin could partially prevent the activation. An aminopeptidase activity that was stimulated by Co2+ both at 37 degrees C and at 60 degrees C was present in the purified enzyme. After preincubation of the enzyme with Co2+ there was evidence for the release of amino acids, as revealed by t.l.c., but the Mr determined by SDS/polyacrylamide-gel electrophoresis was not appreciably altered. It is suggested that a Co2+-stimulated thermostable aminopeptidase, inseparable from the neutral mannosidase, may be involved in the stimulation of neutral mannosidase activity during its preincubation with Co2+.

Polyacrylamide Gel Electrophoresis without a Stacking Gel: Use of Amino Acids as Electrolytes

2001 ◽  
Vol 291 (2) ◽  
pp. 300-303 ◽  
Author(s):  
Taeho Ahn ◽  
Sung-Kun Yim ◽  
Ho-Il Choi ◽  
Chul-Ho Yun
Keyword(s):  
Amino Acids ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel

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.

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 Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro

1981 ◽  
Vol 198 (2) ◽  
pp. 331-338 ◽  
Author(s):  
Ian A. King ◽  
Anne Tabiowo
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Cell Adhesion ◽  
Epidermal Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Membrane Glycoproteins ◽  
Glycosaminoglycan Synthesis ◽  
Core Proteins

1. When pig ear skin slices were cultured for 18h in the presence of 1mug of tunicamycin/ml the incorporation of d-[3H]glucosamine into the epidermis, solubilized with 8m-urea/5% (w/v) sodium dodecyl sulphate, was inhibited by 45–55%. This degree of inhibition was not increased by using up to 5mug of tunicamycin/ml or by treating the skin slices with tunicamycin for up to 8 days. The incorporation of (U-14C)-labelled l-amino acids under these conditions was not affected by tunicamycin. Polyacrylamide-gel electrophoresis indicated that the labelling of the major glycosaminoglycan peak with d-[3H]glucosamine was unaffected, whereas that of the faster migrating glycoprotein components was considerably decreased in the presence of tunicamycin. 2. Subcellular fractionation indicated that tunicamycin specifically inhibited the incorporation of d-[3H]glucosamine but not of (U-14C)-labelled l-amino acids into particulate (mainly plasma-membrane) glycoproteins by about 70%. The labelling of soluble glycoproteins was hardly affected. Polyacrylamide-gel electrophoresis of the plasma-membrane fraction showed decreased d-[3H]glucosamine incorporation into all glycoprotein components, indicating that the plasma-membrane glycoproteins contained mainly N-asparagine-linked oligosaccharides. 3. Cellulose acetate electrophoresis of both cellular and extracellular glycosaminoglycans showed that tunicamycin had no significant effect on the synthesis of the major component, hyaluronic acid. However, the incorporation of both d-[3H]glucosamine and 35SO42− into sulphated glycosaminoglycans was inhibited by about 50%. This inhibition was partially overcome, at least in the cellular fraction, by 2mm-p-nitrophenyl β-d-xyloside indicating that tunicamycin-treated epidermis retained the ability to synthesize sulphated glycosaminoglycan chains. Tunicamycin may affect the synthesis and/or degradation of proteoglycan core proteins or the xylosyltransferase. 4. Electron-microscopic examination of epidermis treated with tunicamycin for up to 4 days revealed no significant changes in cell-surface morphology or in epidermal-cell adhesion. Either N-asparagine-linked carbohydrates play little role in epidermal-cell adhesion or more probably there is little turnover of these components in epidermal adhesive structures such as desmosomes and hemidesmosomes during organ culture.

Improved polyacrylamide gel electrophoresis with different amino acids as the trailing constituent

1981 ◽  
Vol 117 (1) ◽  
pp. 6-11 ◽  
Author(s):  
Anna M. Parkinson ◽  
Allan R. Dorn ◽  
Phillip B. Maples ◽  
Robert H. Broyles
Keyword(s):  
Amino Acids ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel

scholarly journals Vitellogenesis in the stick insect Carausius morosus I. Specific protein synthesis during ovarian development

1980 ◽  
Vol 46 (1) ◽  
pp. 1-16
Author(s):  
F. Giorgi ◽  
F. Macchi
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Fat Body ◽  
Polyacrylamide Gel Electrophoresis ◽  
Stick Insect ◽  
Polyacrylamide Gel ◽  
Carausius Morosus

Vitellogenesis in the stick insect Carausius morosus (Br.) has been studied with the goal of identifying vitellogenin in various tissues. Following exposure to in vivo to radioactive amino acids, oocytes in the medium size range are labelled with a minimum delay of 6 h after the time of injection. Incorporation of radioactivity under these conditions is shown to depend upon accumulation of proteins rather than on a differential rate of protein synthesis in succeeding stages of oogenesis. By immunochemical analyses, it is shown that at least two antigens are common to both haemolymph and ovary and that one of these is also present in the fat body. Both antigens are labelled during exposure to radioactive amino acids. When analysed by the SDS polyacrylamide gel electrophoresis, extracts from both haemolymph and ovary appear to share a number of protein fractions which range in molecular weight from 40 000 to 200 000 Daltons. The labelling pattern exhibited by these fractions is clearly indicative of a protein transfer from the fat body to the oocyte. Fat body cultured in vivo for up to 4 h releases a major macromolecular complex in the external medium. The latter has been identified as vitellogenin by both immuno-precipitation assay and SDS polyacrylamide gel electrophoresis. The protein which is synthesized and secreted under these conditions results from the processing of a protein complex of higher molecular weight.

scholarly journals Purification of fibronectin from human plasma by affinity chromatography under non-denaturing conditions

1979 ◽  
Vol 183 (2) ◽  
pp. 331-337 ◽  
Author(s):  
M Vuento ◽  
A Vaheri
Keyword(s):  
Affinity Chromatography ◽  
Human Plasma ◽  
Gel Electrophoresis ◽  
Analytical Ultracentrifugation ◽  
Biological Activities ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
The Novel ◽  
Two Dimensional ◽  
Native State

Fibronectin was purified from human plasma by affinity chromatography under nondenaturing conditions. The method was based on the previously known binding of fibronectin to gelatin. The novel features of our method are the use of arginine in the elution of fibronectin from immobilized gelatin [Vuento & Vaheri (1978) Biochem. J. 175, 333-336] and the use of arginine-agarose as second affinity step. The purified protein was homogeneous as judged by polyacrylamide-gel electrophoresis, analytical ultracentrifugation and two-dimensional immunoelectrophoresis. The yield was 60%. We propose that the method would be useful in preparation of fibronectin for studies on its biological activities, where it is important that the protein is obtained in a native state.

Purification of the mitochondrial triiodothyronine (T3) receptor from rat liver

1984 ◽  
Vol 105 (3) ◽  
pp. 391-397 ◽  
Author(s):  
Kenneth Sterling ◽  
Gordon A. Campbell ◽  
Milton A. Brenner
Keyword(s):  
Amino Acid ◽  
Affinity Chromatography ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Partial Specific Volume ◽  
Rat Liver Mitochondria

Abstract. The thyroid hormone receptor of the inner membrane of rat liver mitochondria was purified by osmotic and freeze-thaw lyses followed by partial purification on Sephadex G-200, and then by affinity chromatography with T3-Sepharose 4B. A single predominant protein band demonstrable on sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis was present in the first 4 mm NaOH elution peak of affinity chromatography. This was collected from affinity peaks from about 30 rat livers followed by preparative polyacrylamide gel electrophoresis. A single absorbance peak was observed by high pressure liquid chromatography (HPLC). The purified protein was analyzed for binding constants, amino acid composition, and characterized by analytical ultracentrifugation. The association constant (KA) exceeded 1011 m−1. The sedimentation coefficient (S20,W) was 2.2S, partial specific volume (v) 0.72, frictional coefficient (f/fo)s m 1.68 and the molecular weight was estimated at 28000. The amino acid composition was obtained.

Sign in / Sign up

Export Citation Format

Share Document