scholarly journals Multiple phosphorylation of pp30, a rat brain polyribosomal protein, sensitive to polyamines and corticotropin

1984 ◽  
Vol 224 (3) ◽  
pp. 747-753 ◽  
Author(s):  
L H Schrama ◽  
G Weeda ◽  
P M Edwards ◽  
A B Oestreicher ◽  
P Schotman
Keyword(s):  
Rat Brain ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Pulse Labelling ◽  
Ribosomal Protein S6 ◽  
Endogenous Protein ◽  
Gradient Gel Electrophoresis ◽  
Peptide Maps

A rat brain polyribosomal protein with an apparent Mr of 30 000, designated pp30, was further characterized. The protein was identified by its phosphorylation by an endogenous protein kinase sensitive to both corticotropin and spermine. Two-dimensional separation of a polyribosomal fraction was applied, combining non-equilibrium pH-gradient-gel electrophoresis in the first and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the second dimension. In this system, pp30 was separated into at least five defined phosphoprotein spots. Pulse-labelling with [gamma-32P]ATP followed by a chase for various time periods with excess unlabelled ATP resulted in a shift of the distribution of radioactivity and protein staining along the spots towards the anode. This suggests that the various spots of pp30 may represent multiple phosphorylation states. Limited proteolysis of the five spots with three different proteinases resulted in the same one-dimensional peptide maps with a given proteinase, indicating that all five spots represent different forms of a single phosphoprotein. Inhibition of the overall phosphorylation of pp30 by corticotropin or spermine was accompanied by a shift in the recovery of labelled phosphate towards spots nearer the cathode. Immunoblotting with monoclonal antibodies directed against ribosomal protein S6 stained only one band, a protein that had an apparent Mr of 34 000 and was clearly distinct from pp30.

scholarly journals Affinity purification and subunit structures of β-N-acetylhexosaminidases A and B from boar epididymis

1984 ◽  
Vol 219 (3) ◽  
pp. 1009-1015 ◽  
Author(s):  
H C Parkes ◽  
J L Stirling ◽  
P Calvo
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B ◽  
Electrophoretic Transfer ◽  
Peptide Maps

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

scholarly journals The acylation of megakaryocyte proteins: glycoprotein IX is primarily myristoylated while glycoprotein Ib is palmitoylated

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1377-1384 ◽  
Author(s):  
PK Schick ◽  
J Walker
Keyword(s):  
Fatty Acids ◽  
Myristic Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amide Bond ◽  
Endogenous Protein ◽  
Sds Page ◽  
Palmitic Acids ◽  
Protein Acylation

The acylation of megakaryocyte proteins was studied with special emphasis on the myristoylation and palmitoylation of the glycoprotein (GP) Ib complex. Guinea pig megakaryocytes were purified and separated into subpopulations at different phases of maturation. Cells were incubated with [3H]myristate, [3H]palmitate, or [3H]acetate to study endogenous protein acylation. Cycloheximide was used to distinguish between cotranslational and posttranslational acylation and hydroxylamine to distinguish between thioester and amide linkages. After incubations, delipidated proteins or GPIb complex subunits, immunoprecipitated with PG-1, AN-51 or FMC-25 monoclonal antibody, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assessed by fluorography. Radiolabeled fatty acids bound to GPIX and GPIb were also analyzed by high pressure liquid chromatography (HPLC) and scintillation spectrometry. With [3H]myristic acid and [3H]acetate, GPIX was found to be a major myristoylated protein in megakaryocytes and CHRF-288 cells. Myristic acid was linked to GPIX by an amide bond, and this process occurred cotranslationally. With [3H]acetate, GPIb was primarily palmitoylated, but with [3H]myristate, GPIb was acylated with about equal mounts of myristic acid and palmitic acids. Both fatty acids were linked to GPIb by thioester bonds, and acylation was posttranslational. The myristoylation of GPIX while the palmitoylation of GPIb occurred throughout megakaryocyte maturation. Myristoylation and palmitoylation may have different functions relevant to the assembly of the GPIb complex in megakaryocytes.

Transport and proteolytic processing of rabbit cardiac cathepsin D

1985 ◽  
Vol 248 (1) ◽  
pp. C135-C144
Author(s):  
A. M. Samarel ◽  
A. G. Ferguson ◽  
S. W. Worobec ◽  
M. Lesch
Keyword(s):  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Proteolytic Processing ◽  
Time Dependent ◽  
Low Density ◽  
Enzyme Maturation

Rabbit cardiac cathepsin D is synthesized as a 53,000-mol wt precursor that undergoes limited proteolysis at an unknown intracellular site to a 48,000-mol wt active form. To examine the site of proteolytic processing, isolated perfused rabbit hearts were fractionated by differential centrifugation 150 or 300 min after pulse labeling with [35S]methionine. Newly synthesized precursor and processed cathepsin D were quantitatively isolated from each fraction by extraction, immunoadsorption, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After 30-min pulse perfusions, all of the 35S-labeled cathepsin D was present as precursor, with the greatest amounts found in low-density subcellular fractions. Proteolytic processing of cathepsin D precursor occurred after chase perfusions that were coincident with the subcellular redistribution of newly synthesized enzyme from sites of synthesis to heavier subcellular structures. Pulse-chase perfusions with chloroquine (10 microM) inhibited precursor proteolytic processing and the time-dependent subcellular redistribution of newly synthesized cathepsin D. The data are consistent with a model for cardiac lysosomal enzyme maturation in which limited proteolytic processing occurs coincident with or soon after the transport of precursors to an acidic intracellular compartment. The results thus suggest that cathepsin D proteolytic processing occurs within cardiac lysosomes.

scholarly journals Dog and human acid β-d-galactosidases are structurally similar

1983 ◽  
Vol 213 (2) ◽  
pp. 473-478 ◽  
Author(s):  
J J Hubert ◽  
J S O′Brien
Keyword(s):  
Gel Electrophoresis ◽  
Human Liver ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Major Band ◽  
Chymotryptic Peptide ◽  
Human Acid ◽  
Dog Liver ◽  
Peptide Maps

The purification of dog liver acid β-galactosidase is described. The dog enzyme migrated as a single major band on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, with a molecular weight of 60000. Antiserum raised against purified human liver acid β-galactosidase cross-reacted with β-galactosidase from dog liver, but not with those from cat liver or Escherichia coli. Tryptic peptide maps of the dog and human acid β-galactosidases indicate that 21 of the 24 peptides observed were homologous; a similar result was obtained after chymotryptic peptide mapping. We conclude that dog and human acid β-galactosidases are structurally similar, and that canine GM1 gangliosidosis (acid β-galactosidase deficiency) is an excellent model for the same disease in man.

scholarly journals Purification and physical characterization of glutathione S-transferase K. Differential use of S-hexylglutathione and glutathione affinity matrices to isolate a novel glutathione S-transferase from rat liver

1986 ◽  
Vol 233 (3) ◽  
pp. 789-798 ◽  
Author(s):  
J D Hayes
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Structural Data ◽  
Glutathione S Transferase ◽  
Purification Scheme ◽  
Peptide Maps ◽  
Limited Sequence

A novel hepatic enzyme, glutathione S-transferase K, is described that, unlike previously characterized transferases, possesses little affinity for S-hexylglutathione-Sepharose 6B but can be isolated because it binds to a glutathione affinity matrix. A purification scheme for this new enzyme was devised, with the use of DEAE-cellulose, S-hexylglutathione-Sepharose 6B, glutathione-Sepharose 6B and hydroxyapatite chromatography. The final hydroxyapatite step results in the elution of three chromatographically interconvertible forms, K1, K2 and K3. The purified protein has an isoelectric point of 6.1 and comprises subunits that are designated Yk (Mr 25,000); during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it migrates marginally faster than the Ya subunit but slower than the pulmonary Yf monomer (Mr 24,500). Transferase K displays catalytic, immunochemical and physical properties that are distinct from those of other liver transferases. Tryptic peptide maps suggest that transferase K is a homodimer, or comprises closely homologous subunits. The tryptic fingerprints also demonstrate that, although transferase K is structurally separate from previously described hepatic forms, a limited sequence homology exists between the Yk, Ya and Yc polypeptides. These structural data are in accord with the immunochemical results presented in the accompanying paper [Hayes & Mantle (1986) Biochem. J. 233, 779-788].

scholarly journals Analysis of the heterogeneity of human collagens by two-dimensional polyacrylamide-gel electrophoresis

1981 ◽  
Vol 197 (2) ◽  
pp. 377-383 ◽  
Author(s):  
W G Cole ◽  
D Chan
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Relative Increase ◽  
Polyacrylamide Gel ◽  
Type I ◽  
Two Dimensional ◽  
Gradient Gel Electrophoresis ◽  
Cnbr Cleavage ◽  
Dimensional Electrophoresis

The heterogeneity of the CNBr-cleavage peptides of human types I, II, III and V collagens were studied by using two-dimensional electrophoresis combining non-equilibrium pH-gradient-gel electrophoresis and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Specific ‘maps’ were produced by the peptides obtained from the chains of each type of collagen, and most peptides had at least three charged forms of the same molecular weight. Specific ‘maps’ were also produced by the peptides of types I, III and V collagens from insoluble dermis and the peptides of types I and V collagens from decalcified bone. The alpha 1(I) CB7 and alpha 1(I) CB8 and the alpha 2 CB4 peptides obtained from the type I collagens of these tissues contained the same number of charged components, but there was a relative increase in the more basic components in bone. Some aspects of the involvement of the alpha 1(I) CB6 and the alpha 1(III) CB9 peptides in cross-linkages were also studied. The recovery of the alpha 1(I) CB6 peptide from bone and dermis was decreased and the alpha 1(III) CB9 peptide was not detected in dermis. Additional peptides, which were probably cross-linked peptides involving the alpha 1(I) CB6 peptide, were also observed.

scholarly journals The characterization of the non-histone chromosomal proteins of the main classes of nuclei from rat brain fractionated by zonal centrifugation

1979 ◽  
Vol 177 (1) ◽  
pp. 331-346 ◽  
Author(s):  
Sonia G. Tsitilou ◽  
David Cox ◽  
Anthony P. Mathias ◽  
David Ridge
Keyword(s):  
Rat Brain ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Brain Cells ◽  
Adult Rats ◽  
Chromosomal Proteins ◽  
Neuronal Nuclei ◽  
Infant Rats

1. Non-histone chromosomal proteins were isolated from the cell nuclei of whole rat brain and nuclei from different types of brain cells. 2. Brain nuclei were fractionated by zonal centrifugation into five zones deriving from five main categories of brain cells. These are the neuronals, astrocytes I, astrocytes II, oligodendrocytes I and oligodendrocytes II. 3. The non-histone chromosomal proteins were analysed by (a) sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, (b) electrofocusing electrophoresis and (c) two-dimensional electrophoresis. The results of this analysis showed a limited specific pattern of non-histone chromosomal proteins from the different classes of nuclei. Differences were found to exist between the proteins from neuronal and glial nuclei. In particular one polypeptide band with mol.wt. 10000 and pI8.5 was found to be present in the non-histone protein fractions of neuronal nuclei, and absent from the corresponding fractions of nearly all the other classes of nuclei. 4. Two other classes of nuclear proteins, buffered-saline-soluble and 0.35m-NaCl-soluble, were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis along with the non-histone chromosomal. The similarities and differences among these groups of proteins are discussed. 5. The patterns of non-histone chromosomal proteins during development were investigated by studying them in two age groups of animals: in infant rats (10 days old) and adult rats. The polypeptide that was found to be specific for the proteins of neuronal nuclei of adult rats is present in all the classes of nuclei of infant rats.

scholarly journals Isolation of Methylophaga spp. from Marine Dimethylsulfide-Degrading Enrichment Cultures and Identification of Polypeptides Induced during Growth on Dimethylsulfide

2007 ◽  
Vol 73 (8) ◽  
pp. 2580-2591 ◽  
Author(s):  
Hendrik Schäfer
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Large Subunit ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Butyl Ether ◽  
Bacterial Populations ◽  
Enrichment Cultures ◽  
Gradient Gel Electrophoresis ◽  
Insight Into

ABSTRACT Dimethylsulfide (DMS)-degrading enrichment cultures were established from samples of coastal seawater, nonaxenic Emiliania huxleyi cultures, and mixed marine methyl halide-degrading enrichment cultures. Bacterial populations from a broad phylogenetic range were identified in the mixed DMS-degrading enrichment cultures by denaturing gradient gel electrophoresis (DGGE). Sequences of dominant DGGE bands were similar to those of members of the genera Methylophaga and Alcanivorax. Several closely related Methylophaga strains were obtained that were able to grow on DMS as the carbon and energy source. Roseobacter-related populations were detected in some of the enrichment cultures; however, none of the Roseobacter group isolates that were tested were able to grow on DMS. Oxidation of DMS by Methylophaga sp. strain DMS010 was not affected by addition of the inhibitor chloroform or methyl tert-butyl ether, suggesting that DMS metabolism may occur by a route different from those described for Thiobacillus species and other unidentified marine isolates. Addition of DMS and methanethiol to whole-cell suspensions of strain DMS010 induced oxygen uptake when strain DMS010 was grown on DMS but not in cells grown on methanol. The apparent Km s of strain DMS010 for DMS and for methanethiol were 2.1 and 4.6 μM, respectively, when grown on DMS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the biomass of strain DMS010 and analysis of peptide bands by mass spectrometry techniques and N-terminal sequencing provided the first insight into the identity of polypeptides induced during growth on DMS. These included XoxF, a homolog of the large subunit of methanol dehydrogenase for which a biological role has not been identified previously.

scholarly journals Three immunoidentical cytochromes P-450 from liver microsomes of phenobarbital-treated rats

1983 ◽  
Vol 215 (1) ◽  
pp. 83-89 ◽  
Author(s):  
H Sakai ◽  
Y Hino ◽  
S Minakami
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Liver Microsomes ◽  
Polyacrylamide Gel ◽  
Tryptic Digestion ◽  
Post Translational Modification ◽  
Cytochrome P 450

Three forms of cytochrome P-450 were purified to homogeneity from liver microsomes of Wistar-strain rats treated with phenobarbital. They had minimum mol.wts. of 52 000, 53 000 and 54 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and are designated as P-450(L), P-450(M) and P-450(H) respectively. They were shown to be immunoidentical by Ouchterlony double-diffusion analysis. Several criteria, such as isoelectric points, substrate specificities and sensitivities to tryptic digestion, however, indicated that these cytochromes are distinct isoenzymes of cytochrome P-450. Whereas P-450(L) was highly active on various substrates, P-450(H) had generally low catalytic activities, except on aminopyrine. The cytochromes purified by immunoaffinity chromatography using anti-P-450(L) showed a marked variation in their distribution depending on the strain and colony of rat. Limited tryptic digestion of P-450(H) gave one tryptic peptide showing the same mobility as P-450(L) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their primary structures were very similar. The result suggests a possibility that such limited proteolysis is involved in the post-translational modification of the cytochrome or its destruction.

Sign in / Sign up

Export Citation Format

Share Document