scholarly journals Identification of specific polypeptides of the nuclear envelope by iodination of mouse liver nuclei

1989 ◽  
Vol 261 (3) ◽  
pp. 733-738 ◽  
Author(s):  
S Pandey ◽  
V K Parnaik
Keyword(s):  
Nuclear Envelope ◽  
Gel Electrophoresis ◽  
Mouse Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rapid Identification ◽  
Polyacrylamide Gel ◽  
Sensitive Technique ◽  
Molecular Masses ◽  
Liver Nuclei ◽  
Triton X

A sensitive technique is described for the rapid identification of nuclear-envelope proteins. Mouse liver nuclei (purified on sucrose gradients) were iodinated with Na125I by the immobilized water-insoluble reagent Iodogen. Iodinated nuclei were digested with RNAase A and DNAase I and then salt-extracted to obtain labelled nuclear envelopes. Nuclear envelopes were characterized by morphological and biochemical criteria and by SDS/polyacrylamide-gel electrophoresis. In all, 13 polypeptides of molecular masses 145, 115, 98, 85, 75, 70, 65, 54, 50, 45, 40, 38 and 36 kDa were identified in the labelled nuclear envelopes. The labelled polypeptides were localized to the nuclear envelope by extraction of the envelope with Triton X-100 and different concentrations of salt. Iodination of intact nuclei was shown to be specific for the nuclear envelope by the absence of labelling of histones and cytoplasmic contaminants.

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).

scholarly journals Surface glycoproteins of human spermatozoa

1986 ◽  
Vol 82 (1) ◽  
pp. 11-22
Author(s):  
M. Kallajoki ◽  
I. Virtanen ◽  
J. Suominen
Keyword(s):  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Spermatozoa ◽  
Galactose Oxidase ◽  
Sperm Cells ◽  
Neuraminidase Treatment ◽  
Triton X

The surface membrane glycoprotein composition of human spermatozoa has been studied by introducing radioactive label into galactosyl (Gal) and N-acetylgalactosaminyl (GalNAc) residues by using the galactose oxidase/NaB3H4 method. Triton X-100 extracts and Triton X-100-resistant cytoskeletal residues were subjected to analysis by polyacrylamide gel electrophoresis. The distribution of the radiolabel in sperm cells was studied by light-microscopic auto-radiography. The grains were evenly distributed on the cells by the labelling methods used. The Triton X-100 treatment did not affect sperm morphology at the light-microscopic level, but in transmission electron microscopy the plasma membrane covering the acrosome was removed totally, together with most of the acrosomal membranes and acrosomal contents. Plasma membrane residues were, however, always found in the postacrosomal region. Borohydride alone without oxidative pretreatment labelled two polypeptides of molecular weights (Mr) 48,000 and 43,000 in the Triton X-100-soluble fraction. When the Gal/GalNAc residues were labelled by galactose oxidase pretreatment 120,000, 105,000, 78,000 and 68,000 Mr glycoproteins were revealed. When additional neuraminidase treatment was used to remove terminal sialic acid residues, the total labelling intensity was increased two- to fivefold and additional 36,000 and 20,000 Mr glycoproteins were revealed. The Triton X-100-resistant cytoskeletal residue contained 53–75% of the total radioactivity bound in sperm cells. When these components were analysed by polyacrylamide gel electrophoresis, all the major bands found in the Triton X-100-soluble fraction were detected and also some radioactivity was incorporated into the major bands visualized by protein staining. In the present study we describe several human sperm glycoproteins, which seem to be distributed evenly on the sperm cells. Detergent extraction, producing cytoskeletal models, appeared to leave most of the glycoproteins detectable in the extraction residues also with the apparent enrichment of a single 68,000 Mr glycoprotein.

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.

scholarly journals Lupus autoantibodies target ribosomal P proteins.

1985 ◽  
Vol 162 (2) ◽  
pp. 459-471 ◽  
Author(s):  
K B Elkon ◽  
A P Parnassa ◽  
C L Foster
Keyword(s):  
Western Blotting ◽  
Lupus Erythematosus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ribosomal Subunit ◽  
Polyacrylamide Gel ◽  
Antibody Activity ◽  
Composite Gel ◽  
Molecular Masses

All nine SLE (systemic lupus erythematosus) sera with antiribosomal antibody activity targeted the same three ribosomal protein antigens, of molecular masses 38 and 17/19 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. One serum reacted with an additional protein of approximately kD. Ribosomal subunit fractionation by composite gel electrophoresis and sucrose density ultracentrifugation showed that these proteins were part of the large subunit. Isoelectric focusing in agarose, and two-dimensional polyacrylamide gel electrophoresis revealed that the antigens had pI between 4.5 and 6.5, but that the 17/19 kD antigens were more acidic than the 38 kD antigen. Similarities in the molecular masses, charges, as well as the presence of highly conserved crossreactive epitopes, failure to bind to carboxymethylcellulose at pH 4.2, and extractability of the 17/19 kD proteins by 400 mM NH4Cl-ethanol at 0 degrees C indicated that these antigens were analogous to the proteins P0 (38 kD) and P1/P2 (17/19 kD) described previously (25, 36). Co-identity was confirmed using reference antibodies and antigen. Although antibodies to these proteins were only found in 5-10% of more than 50 sera screened by radioimmunoassay or Western blotting, the selective production of antibodies to epitopes on three (out of a total of more than 80) ribosomal proteins may provide further clues to autoantibody induction of SLE.

scholarly journals Purification and characterization of rat liver glycosylasparaginase

1989 ◽  
Vol 260 (1) ◽  
pp. 101-108 ◽  
Author(s):  
O K Tollersrud ◽  
N N Aronson
Keyword(s):  
Rat Liver ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Oligosaccharide Chain ◽  
Molecular Masses ◽  
High Mannose

1. Rat liver glycosylasparaginase [N4-(beta-N-acetylglucosaminyl)-L-asparaginase, EC 3.5.1.26] was purified to homogeneity by using salt fractionation, CM-cellulose and DEAE-cellulose chromatography, gel filtration on Ultrogel AcA-54, concanavalin A-Sepharose affinity chromatography, heat treatment at 70 degrees C and preparative SDS/polyacrylamide-gel electrophoresis. The purified enzyme had a specific activity of 3.8 mumol of N-acetylglucosamine/min per mg with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. 2. The native enzyme had a molecular mass of 49 kDa and was composed of two non-identical subunits joined by strong non-covalent forces and having molecular masses of 24 and 20 kDa as determined by SDS/polyacrylamide-gel electrophoresis. 3. The 20 kDa subunit contained one high-mannose-type oligosaccharide chain, and the 24 kDa subunit had one high-mannose-type and one complex-type oligosaccharide chain. 4. N-Terminal sequence analysis of each subunit revealed a frayed N-terminus of the 24 kDa subunit and an apparent N-glycosylation of Asn-15 in the same subunit. 5. The enzyme exhibited a broad pH maximum above 7. Two major isoelectric forms were found at pH 6.4 and 6.6. 6. Glycosylasparaginase was stable at 75 degrees C and in 5% (w/v) SDS at pH 7.0.

Isolation and Analysis of Non-Histone Chromatin Proteins from Rat-Liver Nuclei by Three Different Methods

1974 ◽  
Vol 52 (12) ◽  
pp. 1143-1153 ◽  
Author(s):  
D. Suria ◽  
C. C. Liew
Keyword(s):  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Soluble Proteins ◽  
Polyacrylamide Gel ◽  
Pulse Labelling ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Chromatin Proteins

Non-histone chromatin proteins were isolated from rat-liver nuclei by three different methods, and defined as (I) phenol-soluble proteins, (II) SDS-soluble proteins and (III) proteins not adsorbed by cation-exchange chromatography. About 62–70% of chromatin proteins were recovered from the total nuclear proteins. The yield of non-histone chromatin proteins varied from 17 to 26% of chromatin proteins, depending on the method used. The amino-acid composition of these proteins showed that they are acidic in nature. Their phosphorus content was found to be 0.9, 1.1, and 1.4%, respectively, according to method I, II, or III. In-vivo pulse-labelling experiments indicated that chromatin proteins were highly labelled with 3H-acetate and 32P-phosphoric acid. In particular, the specific activities of 32P incorporation were higher in all non-histone chromatin proteins isolated as compared with histones. One-dimensional SDS–polyacrylamide gel electrophoresis showed that at least 26 similar fractions can be detected in the samples prepared by these three methods.The similarity of some of the proteins obtained from methods I and III was further confirmed by fractionation of the non-histone chromatin proteins in an isoelectro-focusing system followed by a second-dimensional SDS–polyacrylamide gel electrophoresis. It was found that more than 100 components could be identified. However, some minor variations of the non-histone chromatin proteins were detected by this system. The differences in proteins isolated by these methods are mainly quantitative rather than qualitative. The methods examined are not specific for the fractionation of a certain class of non-histone chromatin proteins.

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