scholarly journals The structure of postsynaptic densities isolated from dog cerebral cortex: II. characterization and arrangement of some of the major proteins within the structure

1977 ◽  
Vol 74 (1) ◽  
pp. 204-225 ◽  
Author(s):  
F Blomberg ◽  
RS Cohen ◽  
P Siekevitz
Keyword(s):  
Cerebral Cortex ◽  
Gel Electrophoresis ◽  
Disulfide Bonds ◽  
Structural Changes ◽  
Sodium Dodecyl ◽  
Nerve Impulse ◽  
Major Protein ◽  
Neurofilament Protein ◽  
10 Nm Filaments ◽  
Opening Up

An attempt was made to identify some of the proteins of the postsynaptic density (PSD) fraction isolated from dog cerebral cortex. The major protein has been tentatively labeled "neurofilament" protein, on the basis of its 51,000 mol wt correspondence to a protein found in neurofilament preparations. Other proteins are akin to some dog myofibrillar proteins, on the basis if immunological crossreaction and equal sodium dodecyl sulfate (SDS)-gel electrophoretic mobilities. While a protein similar to dog muscle myosin is not present in the PSD fraction, a major protein present is actin, as evident from reactivity with antiactin serum, from SDS-gel mobility, and from amino acid composition. Only very little tubulin may be present in the PSD fraction, as determined by gel electrophoresis. Various treatments of the PSD fraction were attempted in order to extract some proteins, as revealed by gel electrophoresis, and to observe the structural changes of the PSD fraction residue after extraction of these proteins. The PSD is remarkably resistant to various extraction conditions, with only 4 M guanidine being found to extract most of the proteins, except the 51,000 mol wt protein. Disulfide reducing agents such as dithiothreitol (DTT), blocking agents such as p-chloromercuribenzoate (PCMB) (both in the presence of deoxycholate [DOC]), a Ca++ extractor, ethylene glycol-bis (beta- aminoethyl ether) N,N,N',N'-tetraacetate (EGTA), and guanidine caused an opening up of the native dense PSD structure, revealing approximately 10-nm filaments, presumably consisting of "neurofilament" protein. Both DTT-DOC and PCMB-DOC removed chiefly actin but also some other proteins. EGTA, in greatly opening up the structure, as observed in the electron microscope, revealed both 10-nm and 3- to 5-nm filaments; the later could be composed of actin, since actin was still in the residue after the treatment. EGTA removed a major 18,000 mol wt component and two minor proteins of 68,000 and 73,000 mol wt. Based on the morphological and biochemical evidence, a picture is presented of the PSD as a structure partly made up of 10-nm and 3- to 5-nm filaments, held together through Ca++ interaction and by bonds amendable to breakage by sulfhydrylblocking and disulfide-reducing reagents; either removal of Ca++ and/or rupture of these disulfide bonds opens up the structure. On the basis of the existence of filamentous proteins and the appearance of the PSD after certain treatments as a closed or open structure, a theory is presented with envisages the PSD to function as a modulator in the conduction of the nerve impulse, by movements of its protein relative.

scholarly journals Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration

1977 ◽  
Vol 163 (2) ◽  
pp. 369-378 ◽  
Author(s):  
P R Dunkley ◽  
H Holmes ◽  
R Rodnight
Keyword(s):  
Cerebral Cortex ◽  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Synaptic Membrane ◽  
Hydroxyl Groups ◽  
Phosphorylated Proteins

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories.

Urease. X. A study by gel electrophoresis of its dissociation by sodium dodecyl sulfate

1969 ◽  
Vol 47 (10) ◽  
pp. 989-991 ◽  
Author(s):  
D. P. Blattler ◽  
George Gorin
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Disulfide Bonds ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Dodecyl Sulfate ◽  
Polypeptide Chains

Urease, m.w. 480 000, treated with an excess of sodium dodecyl sulfate is converted to a product of greatly increased mobility in polyacrylamide gel electrophoresis. We estimate its weight to be about 80 000. Treatment with excess thiol and detergent yielded the same product as detergent alone, indicating that the subunit or subunits do not contain polypeptide chains linked by disulfide bonds.

Rapid detection and initial characterization of genetic variants of human serum albumin

1991 ◽  
Vol 37 (7) ◽  
pp. 1221-1224 ◽  
Author(s):  
J Merle Sheat ◽  
Robert J Peach ◽  
Peter M George
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Genetic Variants ◽  
Gel Electrophoresis ◽  
Structural Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl

Abstract We have studied the detection and classification of genetic variants of human serum albumin by electrophoresis. Samples from 10 patients who were heterozygous for eight different albumin variants were studied by two methods. In agarose gel electrophoresis, each of these variants has an abnormal mobility and can be classified on the basis that structural changes at the N-terminus abolish 63Ni binding. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole serum, glycosylated variants are easily detected because of their greater apparent molecular mass.

scholarly journals Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus

1979 ◽  
Vol 177 (1) ◽  
pp. 49-62 ◽  
Author(s):  
C M Clarke ◽  
B S Hartley
Keyword(s):  
Enzyme Activity ◽  
Restriction Endonuclease ◽  
Dna Cleavage ◽  
Gel Electrophoresis ◽  
Bacillus Stearothermophilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Major Protein ◽  
Activity Restriction

The restriction endonuclease BstI was purified from 70kg of Bacillus stearothermophilus. The final product is at least 97% pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; this major protein species co-migrates with the enzyme activity on native polyacrylamide-gel electrophoresis and isoelectric focusing. Pure restriction endonuclease BstI has a subunit mol.wt. of 26,000 and is probably a loosely associated dimer. The enzyme shows maximum activity at pH values between 7 and 9.5, and in the presence of 0.5-2mM-Mg2+. NaCl inhibits the restriction enzyme activity. Restriction endonuclease BstI cleaves DNA in a position identical with that cleaved by endonuclease BamHI (for Bacillus amyloliquefaciens), i.e.: (formula: see text). In the presence of high concentrations of enzyme, DNA cleavage occurs at secondary sites. This side-specificity is enhanced by the addition of glycerol. Preliminary studies indicate that these sites are of the type: (formula: see text).

Identification of circulating growth hormone-binding proteins in domestic poultry: an initial characterization

1991 ◽  
Vol 130 (1) ◽  
pp. 115-NP ◽  
Author(s):  
R. Vasilatos-Younken ◽  
B. J. Andersen ◽  
R. W. Rosebrough ◽  
J. P. McMurtry ◽  
W. L. Bacon
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Sodium Dodecyl ◽  
Mouse Serum ◽  
Nucleotide Sequence Analysis ◽  
Major Protein ◽  
Western Blots ◽  
Major Band

ABSTRACT Multiple growth hormone (GH)-binding proteins (GHBPs) were identified in serum and plasma samples from domestic chickens and turkeys. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis on 10% acrylamide, 2·7% bis discontinuous gels under reducing conditions and electrotransferred to nitrocellulose paper. Western blots were incubated with 125I-labelled recombinant chicken GH (cGH) or bovine GH and GHBPs visualized by means of autoradiography. In fresh samples (< 2 h from collection to gel electrophoresis), multiple minor high Mr bands were evident between approximately 72 000 and 175 000. Two major bands were observed at approximately 69 500 and 27 500. The latter is consistent with previous reports for the rat and mouse serum GHBPs based on nucleotide sequence analysis. The minor bands were essentially undetectable after storage at −25 °C for several months, and an additional major band at Mr approximately 52 500 appeared. The Mr-69 500 major protein contained N-linked carbohydrate, as determined by a reduction in molecular size by treatment with peptide N-glycosidase F. Binding of 125I-labelled GH was partially inhibited by co-incubation with 50 μg unlabelled pituitary-derived cGH/ml and excess unlabelled porcine GH as well as ovine prolactin, but not by bovine insulin. Non-specific binding of 125I-labelled GH by serum albumin was also observed. A comparison was made between these GHBPs and the hepatic GH receptor (e.g. molecular weight estimates, affinity for homologous versus heterologous GHs, cross-reactivity with prolactin, presence of N-linked carbohydrate). The origin and relationship among the various molecular weight species of GHBPs identified, and their potential role in regulation of the biological activity of GH in birds, remain to be determined. Journal of Endocrinology (1991) 130, 115–122

scholarly journals Contribution of Chemical Modifications and Conformational Epitopes to IgE Binding by Ara h 3

Foods ◽  
2018 ◽  
Vol 7 (11) ◽  
pp. 189 ◽  
Author(s):  
Scott Dyer ◽  
Jacqueline Nesbit ◽  
Beatriz Cabanillas ◽  
Hsiaopo Cheng ◽  
Barry Hurlburt ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Secondary Structure ◽  
Disulfide Bonds ◽  
Structural Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cd Spectroscopy ◽  
Chemical Modifications ◽  
Ige Binding ◽  
Ara H 3

Roasting is known to change the allergenic properties of peanuts. To study these observations at a molecular level, the relationship of IgE binding to the structure of Ara h 3 from raw and roasted peanuts was assessed. Ara h 3 (A3) was purified from raw (R), light roast (LR) and dark roast (DR) peanuts, the purity was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the secondary structures were compared with circular dichroism (CD) spectroscopy. In order to understand the contribution of structure to IgE binding, the R A3 was partially denatured (PD) by heat treatment (65 °C for 2 h), subjected to CD spectroscopy and IgE spot blot analysis with sera from peanut- allergic individuals. While we observed that the secondary structure of purified A3 from R and LR peanut in solution was affected by the reduction of disulfide bonds and heat treatment when purified from the peanut following the roasting process, only small alterations were seen in the secondary structure. The purified LR A3 bound higher levels of IgE than the RA3. CD spectroscopy of PD A3 revealed a reduction in the percentage of alpha helices, and serum IgE binding. Therefore, while A3 purified from roasted peanuts did not show significant changes in secondary structure, it showed higher IgE binding than R A3. Therefore, the higher IgE binding to LR A3 was more likely to be due to chemical modifications than structural changes. However, a decrease in the IgE binding was seen if R A3 was deliberately unfolded, indicating that the structure played an important role in IgE binding to A3.

The effect of ageing cooled milk on the composition of the fat globule membrane

1972 ◽  
Vol 39 (1) ◽  
pp. 95-105 ◽  
Author(s):  
M. Anderson ◽  
G. C. Cheeseman ◽  
Dorothy J. Knight ◽  
W. F. Shipe
Keyword(s):  
Gel Electrophoresis ◽  
Milk Fat ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Protein Band ◽  
Major Protein ◽  
Fresh Milk ◽  
Major Protein Band ◽  
Fat Globule

SummaryThe effect of ageing fresh milk for 24 h at 4°C on the composition of 4 fractions of milk fat globule membrane (FGM) – microsomal pellet membrane (MPM), deoxycholate soluble membrane (DOCM), high density membrane (HDM) and low density membrane (LDM) – prepared from washed cream treated with sodium deoxycholate (DOC), was studied for milk of individual cows. Total FGM and its fractions were solubilized by treatment with sodium dodecyl sulphate (SDS), EDTA and β-mercaptoethanol, and the dissociated FGM proteins were separated by polyacrylamide gel electrophoresis in the presence of SDS.Ageing resulted in a greater loss of phospholipid during cream preparation than was found with fresh milk, and also in a reduction in the total amount of FGM isolated. Loss of FGM material was confined to MPM, DOCM and LDM and was accompanied by compositional changes in DOCM and LDM, quantitatively the most significant fractions. Ageing also produced changes in the gel electrophoresis patterns of DOCM, where the band of greatest mobility (mol. wt about 16000) was partially lost, and of LDM, where there was an increase observed in the major protein band.The implication of the results on the proposed models of FGM structure is discussed.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis of serum after protein denaturation in the presence or absence of 2-mercaptoethanol.

1984 ◽  
Vol 30 (3) ◽  
pp. 475-479 ◽  
Author(s):  
T Marshall
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Serum Proteins ◽  
Protein Denaturation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Major Protein ◽  
Dodecyl Sulfate ◽  
Electrophoretic Techniques

Abstract Human serum proteins were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis after protein denaturation in the presence or absence of 2-mercaptoethanol. Both electrophoretic techniques give characteristic and reproducible banding patterns and achieve a high degree of resolution within the limits of a one-dimensional separation. The major protein bands have been identified, and the merits of the two techniques are compared. Protein denaturation in the absence of 2-mercaptoethanol gives more discrete bands for the purpose of protein identification by maintaining the disulfide-bridge-dependent polypeptide associations characteristic of many serum proteins. However, simultaneous use of both electrophoretic techniques enhances identification by exploiting the response of an individual protein to mercaptoethanol treatment. The clinical potential of this approach for detecting protein disorders is discussed.

scholarly journals Electrophoretic and immunological charactorization of rat neurophysin

1971 ◽  
Vol 122 (5) ◽  
pp. 671-676 ◽  
Author(s):  
A. Norström ◽  
J. Sjöstrand ◽  
B. G. Livett ◽  
L. O. Uttenthal ◽  
D. B. Hope
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Polyacrylamide Gel ◽  
Male Rats ◽  
Major Protein ◽  
Starch Gel Electrophoresis ◽  
Neural Lobe ◽  
Starch Gel

1. The electrophoretic properties of rat posterior pituitary proteins have been compared on starch gel with those of bovine and porcine neurophysins. 2. [35S]-Cysteine was injected into the supraoptic nucleus of male rats and 16–24h later the distribution of labelled neural-lobe protein in starch and polyacrylamide gels was determined. In both systems a single major protein component was found to contain more than 80% of the total recovered radioactivity. Between 5 and 10% of the radioactivity was found in a minor component in polyacrylamide gel. 3. In agar, microimmuno-diffusion and -electrophoresis of the rat neural-lobe proteins gave a single arc with neurophysin antiserum, and after starch-gel electrophoresis this arc was shown to be due to the major labelled component. 4. The molecular weights of the rat neural-lobe proteins were estimated by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the major labelled component was found to be 12000. 5. It is concluded that the rat neurophysin consists of one major and possibly one minor component.

Degradation of neurofilament protein in cerebral ischemia

1989 ◽  
Vol 70 (1) ◽  
pp. 103-107 ◽  
Author(s):  
Nobuyoshi Ogata ◽  
Yasuhiro Yonekawa ◽  
Waro Taki ◽  
Reiji Kannagi ◽  
Takashi Murachi ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cytoskeletal Proteins ◽  
Transient Ischemia ◽  
Neurofilament Protein ◽  
Vessel Occlusion ◽  
Content Type ◽  
Selective Degradation ◽  
Fine Print

✓ Degradation of neurofilament (NF) triplet proteins: NF200 (molecular weight (MW) 200,000), NF150 (MW 150,000), and NF68 (MW 68,000) as well as of other cytoskeletal proteins in the rat brain during ischemia was investigated. Sodium dodecyl sulfate-gel electrophoresis and immunoblot methods with anti-NF200 antibody were used for the study. Selective degradation of NF200 and NF150 was observed during the initial 10 to 15 minutes of ischemia. The degradation was demonstrated both in permanent ischemia caused by decapitation and in transient ischemia induced by four-vessel occlusion followed by reperfusion after 30 minutes of occlusion (modified Pulsinelli method). The degradation suggests that the activation of a protease occurs in the first 15 minutes of cerebral ischemia, which is the earliest irreversible neuronal change ever to be reported.

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