scholarly journals Synthesis and secretion of alpha2-macroglobulin by cultured human fibroblasts.

1976 ◽  
Vol 143 (2) ◽  
pp. 462-467 ◽  
Author(s):  
D F Mosher ◽  
D A Wing
Keyword(s):  
Gel Electrophoresis ◽  
Human Fibroblasts ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Serum Free Medium ◽  
Free Medium ◽  
Major Band ◽  
Serum Free ◽  
Cultured Human Fibroblasts

The following observations indicate that cultured human WI-38 fibroblasts synthesize and secrete alpha2-macroglobulin into serum-free medium: (a) after incubation of cultures with [35S]L-methionine, a labeled protein appeared in the medium which was precipitated by antiserum directed against alpha2-macroglobulin; (b) after incubation of cultures with [35S]L-methionine, a major band of radioactivity detected by polyacrylamide gel electrophoresis of the proteins in medium co-migrated with alpha2-macroglobulin; and (c) the amount of alpha2-macroblobulin in the medium, estimated both functionally and immunologically, increased with time in normal but not not puromycin-treated cultures.

Isoenzymes of chloroperoxidase from Caldariomyces fumago

1982 ◽  
Vol 28 (12) ◽  
pp. 1382-1388 ◽  
Author(s):  
Michael A. Pickard ◽  
Atsumi Hashimoto
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Media Analysis ◽  
Polyacrylamide Gel ◽  
Prolonged Storage ◽  
Malt Extract ◽  
Major Band ◽  
Dodecyl Sulfate

Chloroperoxidase (EC 1.11.1.10) was purified from Caldaromyces fumago strains ATCC 16373 and CM1 89362 grown in defined and complex media. Analysis of the enzyme by polyacrylamide gel electrophoresis at pH 3, sodium dodecyl sulfate – polyacrylamide gel electrophoresis, and isoelectric focussing in polyacrylamide gels revealed two forms of the enzyme, the major form representing greater than 80% of the total protein. When the fungi were grown in synthetic media, more of the enzyme was found in the major band than when the fungi were grown in the presence of malt extract. Enzyme activity was demonstrated both by staining gels in situ with 3,3′,5,5′-tetramethylbenzidine and by spectrophometric assay of material eluted from gel slices. There were no obvious differences between the enzyme from C. fumago ATCC 16373 and from C. fumago CMI 89362. Commercial samples, included for comparative purposes, contained three or four enzymically active species which separated on polyacrylamide gel electrophoresis at pH 3 and electrofocussing but migrated as a close double band on sodium dodecyl sulfate – polyacrylamide gel electrophoresis. It is believed that prolonged storage in the unfrozen state is responsible for the conversion to multiple forms.

Primitive erythropoiesis of mouse teratocarcinoma stem cells PCC3/A/1 in serum-free medium

Development ◽  
1991 ◽  
Vol 111 (2) ◽  
pp. 543-549 ◽  
Author(s):  
Y. Miwa ◽  
T. Atsumi ◽  
N. Imai ◽  
Y. Ikawa
Keyword(s):  
Stem Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Red Cells ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Interleukin 3 ◽  
Globin Chains ◽  
Primitive Erythropoiesis ◽  
Mouse Teratocarcinoma

Mouse teratocarcinoma stem cells PCC3/A/1 differentiated into various types of cells, such as red cells, when they were grown in serum-free medium containing transferrin and bovine serum albumin on a KCF cell feeder layer. These red cells were stained well with 2,7-diaminofluorene (DAF), and therefore were erythroid cells. They were nucleated and contained embryonic globin chains, immunologically identified with antiembryonic hemoglobin antisera after acid urea Triton X-100 polyacrylamide gel electrophoresis (UT-PAGE). The addition of erythropoietin to the culture medium enhanced the production of both embryonic and adult globin chains. The addition of interleukin-3 also enhanced the production of embryonic globin chains, but not the production of adult globin chains. These results indicated that primitive erythropoiesis of PCC3/A/1 teratocarcinoma cells did not require exogenous addition of any hematopoietic factor such as erythropoietin or interleukin-3. This culture system will be a new model system for investigating the factors regulating the primitive erythropoiesis in yolk sac blood islands.

scholarly journals Proteins of the kidney microvillus membrane. Identification of subunits after sodium dodecylsullphate/polyacrylamide-gel electrophoresis

1976 ◽  
Vol 159 (2) ◽  
pp. 395-407 ◽  
Author(s):  
A G Booth ◽  
A J Kenny
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Neutral Endopeptidase ◽  
Polyacrylamide Gel ◽  
British Library ◽  
Major Band ◽  
Microvillus Membrane

The proteins of microvilli prepared from pig kidney were analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The typical pattern stained for protein revealed five major bands, four of which also stained for carbohydrate, and about 15 minor bands. For descriptive purposes the bands were designated numerically by their apparent molecular weights (X10(-3). Well-characterized proteins were identified with four of the five major bands. Dipeptidyl peptidase IV, a serine proteinase that may be specifically labelled with di-isopropyl [32P]phosphorofluoridate, was assigned to band 130. Aminopeptidase M was assigned to band 160, though when released from the membrane by a proteinase, this protein comprises three polypeptides each of lower apparent molecular weight than the native enzyme. Neutral endopeptidase can be assigned to band 95 and actin to band 42. The fifth major band (180) is an extrinsic glycoprotein that has not been identified with any microvillus enzyme activity. These four proteins contribute 21% of the microvillus-membrane protein. Kidney microvillus actin was characterized by a variety of properties and was similar to muscle actin. A computer analysis of the gel pattern indicates that it comprises 9.0% of the microvillus protein. Myosin is not present in the microvillus, but another protein associated with band 95, with properties that distinguish it from neutral endopeptidase, was tentatively identified as α-actinin. Alkaline phosphatase was identified as a monomeric polypeptide with an apparent molecular weight of 80000; it is a minor protein of the microvillus and is not discernible as a discrete band in the gel pattern. These and other results permit a model of the organization of the microvillus protein to be suggested. The computer program used has been deposited as Supplementary Publication SUP 50070 (12 pages) at the British Library Lending Division, Boston Spa. Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1976) 153,5.

Changes in isoenzyme patterns during imbibition and germination of lodgepole pine (Pinuscontorta var. latifolia)

1984 ◽  
Vol 14 (5) ◽  
pp. 743-746 ◽  
Author(s):  
J. A. Pitel ◽  
W. M. Cheliak ◽  
B. S. P. Wang
Keyword(s):  
Gel Electrophoresis ◽  
Leucine Aminopeptidase ◽  
Lodgepole Pine ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Glutamate Oxaloacetate Transaminase ◽  
Major Band ◽  
Tissue Specific ◽  
Isoenzyme Patterns

Isoenzymes of esterase (EST), glutamate-oxaloacetate transaminase (GOT), leucine aminopeptidase (LAP), and peroxidase (PER) were examined following various periods of imbibition and germination of lodgepole pine (Pinuscontorta var. latifolia Engelm.) seeds. The acidic and basic isoenzymes of embryos, and roots and shoots of germinating seedlings were analyzed by polyacrylamide gel electrophoresis. One major band of EST disappeared with imbibition, while some minor bands appeared and disappeared with imbibition and germination. Comparison of the roots and shoots after 7 and 14 days of germination showed several tissue-specific differences. The number of bands of GOT increased with imbibition and germination. The levels of activity of three isoenzymes differed between the root and shoot tissues. One band of LAP disappeared with imbibition. The levels of activity of two bands varied between the root and shoot tissues. The number of bands of PER increased dramatically following imbibition and germination. Also, many tissue-specific differences were observed between root and shoot tissues.

scholarly journals Isolation and renaturation of α2-macroglobulin receptor from diploid human fibroblasts

1983 ◽  
Vol 214 (2) ◽  
pp. 629-631 ◽  
Author(s):  
J Frey ◽  
E G Afting
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Binding ◽  
Human Fibroblasts ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Step ◽  
Polyacrylamide Gel ◽  
Human Diploid Fibroblasts ◽  
Α2 Macroglobulin

alpha 2-Macroglobulin receptor was extracted from human diploid fibroblasts and purified by affinity chromatography in a single step. The receptor had mol.wt. 125 000 after sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. The isolated receptor was separated by SDS/polyacrylamide-gel electrophoresis, transferred on to nitrocellulose sheets and subsequently renatured, as shown by a specific binding test, by incubation with Nonidet P40.

Ultrastructural localization of specific nucleoprotein fractions

1978 ◽  
Vol 36 (2) ◽  
pp. 204-205
Author(s):  
G. L. Brown
Keyword(s):  
Gel Electrophoresis ◽  
Mouse Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ultrastructural Localization ◽  
Polyacrylamide Gel ◽  
Acid Extraction ◽  
Acid Solutions ◽  
Low Salt ◽  
Liver Nuclei ◽  
Phosphate Groups

Bismuth (Bi) stains nucleoproteins (NPs) by interacting with available amino and primary phosphate groups. These two staining mechanisms are distinguishable by glutaraldehyde crosslinking (Fig. 1,2).Isolated mouse liver nuclei, extracted with salt and acid solutions, fixed in either formaldehyde (form.) or gl utaraldehyde (glut.) and stained with Bi, were viewed to determine the effect of the extractions on Bi stainina. Solubilized NPs were analyzed by SDS-polyacrylamide gel electrophoresis.Extraction with 0.14 M salt does not change the Bi staining characteristics (Fig. 3). 0.34 M salt reduces nucleolar (Nu) staining but has no effect on interchromatinic (IC) staining (Fig. 4). Proteins responsible for Nu and glut.- insensitive IC staining are removed when nuclei are extracted with 0.6 M salt (Fig. 5, 6). Low salt and acid extraction prevents Bi-Nu staining but has no effect on IC staining (Fig. 7). When nuclei are extracted with 0.6 M salt followed by low salt and acid, all Bi-staining components are removed (Fig. 8).

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