Further studies on polyacrylamide gel electrophoresis of water-soluble proteolipid-protein from bovine brain white matter

1971 ◽  
Vol 27 (8) ◽  
pp. 881-882 ◽  
Author(s):  
Y. Komai ◽  
H. Sato ◽  
N. Hiraiwa ◽  
Y. Sawaishi
Keyword(s):  
White Matter ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Proteolipid Protein ◽  
Bovine Brain ◽  
Polyacrylamide Gel ◽  
Water Soluble ◽  
Brain White Matter

scholarly journals Spectrophotometric assay, solubilization and purification of brain 2′:3′-cyclic nucleotide 3′-phosphodiesterase

1980 ◽  
Vol 191 (1) ◽  
pp. 71-82 ◽  
Author(s):  
Y Nishizawa ◽  
T Kurihara ◽  
Y Takahashi
Keyword(s):  
White Matter ◽  
Cyclic Nucleotide ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Spectrophotometric Assay ◽  
Polyacrylamide Gel ◽  
Hexadecyltrimethylammonium Bromide ◽  
Gel Chromatography ◽  
Brain White Matter

1. A spectrophotometric assay of 2′:3′-cyclic nucleotide 3′-phosphodiesterase (EC 3.1.4.37) based on the use of an acid-base indicator and a buffer having identical pKa values is described. The assay is simple and rapid; it was particularly convenient for monitoring the enzyme activity at various stages of purification. 2. Several proteinases were examined for their ability to solubilize 2′:3′-cyclic nucleotide 3′-phosphodiesterase from delipidated brain white matter. Trypsin (EC 3.4.21.4) and elastase (EC 3.4.21.11) appeared to be more effective than the other proteinases examined. Trypsin, however, caused inactivation; elastase was therefore chosen to solubilize 2′:3′-cyclic nucleotide 3′-phosphodiesterase. When a partially purified preparation of 2′:3′-cyclic nucleotide 3′-phosphodiesterase was treated with elastase, 2′:3′-cyclic nucleotide 3′-phosphodiesterase was solubilized nearly quantitatively. Elastatinal, a specific inhibitor of elastase, specifically inhibited the solubilization with elastase. 3. 2′:3′-cyclic nucleotide 3′-phosphodiesterase was purified from bovine brain white matter by: (i) delipidation; (ii) solubilization with hexadecyltrimethylammonium bromide; (iii) gel chromatography on Sepharose; (iv) ethanol precipitation and resolubilization by digestion with elastase; (v) chromatography on DEAE-Sephadex; (vi) affinity chromatography on 8-(6-aminohexyl)amino-2′-AMP-Sepharose. 4. The purified enzyme migrated as a single protein band on polyacrylamide-gel electrophoresis at pH 4.3 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the estimated mol.wt. in the latter electrophoresis was 27000-31000. Gel filtration of the purified enzyme through Sephadex G-150 indicated a mol.wt. of 31000. Therefore the purified enzyme is a monomer protein with a mol.wt. of approx. 30000.

scholarly journals Two low-molecular-weight Ca2+-binding proteins isolated from squid optic lobe by phenothiazine-Sepharose affinity chromatography

1983 ◽  
Vol 209 (3) ◽  
pp. 797-802 ◽  
Author(s):  
J F Head ◽  
S Spielberg ◽  
B Kaminer
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Content ◽  
Bovine Brain ◽  
Polyacrylamide Gel ◽  
Dependent Manner

We have isolated two Ca2+-binding proteins from squid optic lobes, each of which is also able to bind phenothiazines in a Ca2+-dependent manner. These proteins have each been purified and partly characterized. One of the proteins corresponds to calmodulin, in that it has a similar amino acid content to bovine brain calmodulin, including a single residue of trimethyl-lysine, it co-migrates with bovine calmodulin both on alkaline-urea- and on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis, and will activate calmodulin-dependent phosphodiesterase. The second protein has the same subunit molecular weight as calmodulin, as determined by SDS/polyacrylamide-gel electrophoresis, Mr 17 000, but migrates more slowly than this protein on alkaline-urea-gel electrophoresis. It has an amino acid composition distinct from calmodulin, containing no trimethyl-lysine, its CNBr fragments migrate on alkaline gels in a pattern distinct from those of calmodulin and it shows little ability to activate phosphodiesterase. The u.v.-absorption spectra of the proteins indicate the absence of tryptophan and the presence of a high phenylalanine/tyrosine ratio in each. Both proteins also bind 3-4 calcium ions/mol at 0.1 mM-free Ca2+ and each binds chlorpromazine in a Ca2+-dependent manner.

scholarly journals Purification of bovine brain inositol 1,4,5-trisphosphate 3-kinase. Identification of the enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis

1989 ◽  
Vol 261 (2) ◽  
pp. 483-488 ◽  
Author(s):  
K Takazawa ◽  
H Passareiro ◽  
J E Dumont ◽  
C Erneux
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bovine Brain ◽  
Polyacrylamide Gel ◽  
Maximal Velocity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Maximal Activation

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). A method is presented for the rapid purification of InsP3 3-kinase from bovine brain by calmodulin (CaM)-Sepharose affinity chromatography. Maximal activation of the purified InsP3 3-kinase by Ca2+/CaM was 6-7-fold as compared with the activity measured in the presence of EGTA (1 mM) and 10 microM-InsP3. At 10 microM-InsP3 and 0.1 mM free Ca2+, half-maximal activation required about 2 nM-CaM. The mechanism of activation by CaM appeared to be an increase in the maximal velocity of the enzyme without a substantial change in the Km for InsP3. Further purification was achieved by phosphocellulose chromatography eluted with ATP. Specific activity of the purified enzyme at 37 degrees C and 10 microM-InsP3 was 10-20 mumol/min per mg. The apparent Mr of the enzyme, determined by f.p.l.c.-gel filtration, was estimated as about 44,000. The purified InsP3 3-kinase was subjected to SDS/10%-polyacrylamide-gel electrophoresis. InsP3 3-kinase activity was associated with three silver-stained bands, which migrated with apparent Mr values of approx. 52,000, 38,000 and 35,000.

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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