scholarly journals Preparation and properties of a complex from rat liver of polyribosomes with components of the cytoskeleton

1983 ◽  
Vol 216 (1) ◽  
pp. 215-226 ◽  
Author(s):  
A Adams ◽  
E G Fey ◽  
S F Pike ◽  
C J Taylorson ◽  
H A White ◽  
...  
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Subcellular Fractionation ◽  
Cytoskeletal Proteins ◽  
Deoxyribonuclease I ◽  
Microsomal Membranes ◽  
Cytoskeletal Elements ◽  
Triton X

Gel filtration with 1% agarose (Bio-Gel A-150m) separates polyribosomes bound to microsomal membranes from ‘free’ polyribosomes when these fractions are prepared by standard centrifugal techniques. However, when polyribosomes contained in an unfractionated postmitochondrial supernatant are run on an identical column, over 90% of the total polyribosomes are present as aggregates, designated ‘membrane-cytomatrix’, which are eluted in the column void volume. Polyribosomes are not released from these aggregates on removal of microsomal phospholipids by treatment of postmitochondrial supernatant with 1% Triton X-100, a neutral detergent. The aggregates are disrupted by the usual ultracentrifugation techniques used in subcellular fractionation. After treatment of membrane-cytomatrix with Triton X-100 to remove phospholipids and membrane proteins, 58% of the polyribosomes still remain associated with protein-containing complexes in the form of a cytomatrix and are not ‘free’. Preparations of both membrane-cytomatrix and cytomatrix are capable of sustained protein synthesis. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that the cytoskeletal proteins actin and myosin are present in the cytomatrix. Incubation of cytomatrix preparations with the actin-depolymerizing agent deoxyribonuclease I caused release of the polyribosomes. Polyribosome release by deoxyribonuclease I was prevented by prior incubation with phalloidin, which is known to stabilize F-actin. Thus polyribosomes are associated with cytoskeletal elements in rat liver, and this association is dependent on polymeric forms of actin.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

Cross-linking of neuropeptide Y to its receptor on rat brain membranes

1989 ◽  
Vol 256 (3) ◽  
pp. G637-G643 ◽  
Author(s):  
P. J. Mannon ◽  
I. L. Taylor ◽  
L. M. Kaiser ◽  
T. D. Nguyen
Keyword(s):  
Neuropeptide Y ◽  
Rat Brain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Brain Membranes ◽  
Npy Receptor ◽  
Rat Brain Membranes ◽  
Immature Form ◽  
Triton X

The receptor for neuropeptide Y (NPY) was identified on rat brain membranes after covalent labeling with 125I-NPY using the homobifunctional cross-linkers disuccinimido suberate and disuccinimido dithiobis(propionate) and the heterobifunctional photoactive cross-linker succinimido 4-azidobenzoate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed the presence of two bands at Mr 62,000 and 39,000. Both species showed the same high affinity for 125I-NPY. Exposure to reducing agents did not change the migration of these bands. When the NPY receptor complex was solubilized from the membranes with 1% Triton X-100 and analyzed by gel filtration chromatography, it eluted from a Fractogel TSK 55F column as a peak at approximately 65 kDa. This peak was asymmetric with a shoulder of radioactivity that probably reflects the smaller receptor species. These data indicate that the NPY receptor on rat brain membranes is a monomeric 58-kDa unit (62 kDa minus the mass of the cross-linked NPY) without covalently or noncovalently linked subunits. The smaller 39-kDa species may be an immature form of the 62-kDa species, a second distinct receptor, or a degradation product of the 62-kDa band.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

scholarly journals Purification and properties of a new glutathione-dependent thiol:disulphide oxidoreductase from rat liver

1982 ◽  
Vol 207 (1) ◽  
pp. 133-138 ◽  
Author(s):  
M G Battelli ◽  
E Lorenzoni
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Xanthine Dehydrogenase ◽  
Enzymic Activity ◽  
Oxidized Glutathione ◽  
Adult Rats ◽  
Purification And Properties ◽  
Rat Liver Cytosol

A new GSSG-dependent thiol:disulphide oxidoreductase was extensively purified from rat liver cytosol. The enzymic protein shows molecular weight 40 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and 43 000 as determined by thin-layer gel filtration on Bio-Gel P-100. The pI is 8.1. This enzyme converts rat liver xanthine dehydrogenase into an oxidase, in the presence of oxidized glutathione. Other disulphide compounds are either inactive or far less active than oxidized glutathione in the enzymic oxidation of rat liver xanthine dehydrogenase. The enzyme also catalyses the reduction of the disulphide bond of ricin and acts as a thioltransferase and as a GSH:insulin transhydrogenase. The enzymic activity was measured in various organs of newborn and adult rats.

scholarly journals MPB70 and MPB83 as Indicators of Protein Localization in Mycobacterial Cells

1998 ◽  
Vol 66 (1) ◽  
pp. 289-296 ◽  
Author(s):  
Morten Harboe ◽  
Harald G. Wiker ◽  
Gunni Ulvund ◽  
Bent Lund-Pedersen ◽  
Åse Bengård Andersen ◽  
...  
Keyword(s):  
Protein Localization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Culture Fluid ◽  
Secreted Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sds Page ◽  
Shock Proteins ◽  
Triton X

ABSTRACT Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates ofMycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

Rat lingual lipase: partial purification, hydrolytic properties, and comparison with pancreatic lipase

1984 ◽  
Vol 247 (4) ◽  
pp. G385-G393 ◽  
Author(s):  
I. M. Roberts ◽  
R. K. Montgomery ◽  
M. C. Carey
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Pancreatic Lipase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Dodecyl Sulfate ◽  
Triton X

We have partially purified lingual lipase from the serous glands of rat tongue. With a combination of Triton X-100 extraction or Triton X-114 phase-separation techniques, Bio-Bead SM-2 treatment, dialysis, and gel filtration on Sephadex G-200 or Sephacryl S-300, we obtained a sparingly soluble lipid-free protein demonstrating hydrolytic activity against triglycerides and negligible phospholipase or cholesteryl esterase activities. Compared with homogenate, specific activities of the enzyme were enriched 3- to 5-fold prior to gel filtration and 10-fold after gel filtration. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions (6 M guanidine X HCl or 0.1% sodium dodecyl sulfate) revealed one major glycoprotein band with Mr approximately 50,000. Gel filtration of the active enzyme in 0.1% Triton X-100 gave an Mr approximately 270,000-300,000, suggesting extensive self-aggregation. With both tributyrin and triolein, the pH optimum of the purified enzyme was 4.0 and activity extended from pH 2.0 to 8.0. In contrast to purified human pancreatic lipase, lingual lipase hydrolyzed triglyceride emulsions and mixed micelles stabilized with both short-chain (dihexanoyl) and long-chain (egg) lecithin and were inhibited only slightly (18-25%) by micellar concentrations of two common bile salts, taurodeoxycholate and taurocholate. Our results suggest that the hydrolysis of dietary fat by lingual lipase may extend from the pharynx through the esophagus and stomach and into the upper small intestine.

scholarly journals Glycoproteins and glycosaminoglycans synthesized by human keratinocytes in culture. Their role in cell-substratum adhesion

1983 ◽  
Vol 212 (2) ◽  
pp. 355-363 ◽  
Author(s):  
G P Roberts ◽  
L Jenner
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Keratinocytes ◽  
Cytoskeletal Proteins ◽  
Galactose Oxidase ◽  
Surface Labelling ◽  
Triton X ◽  
Sulphated Glycosaminoglycan

Glycoproteins and proteoglycans synthesized by human keratinocytes in medium containing D-[1-14C]glucosamine were extracted and analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Extraction of the labelled keratinocytes with 0.5% Triton X-100 removed most of the glycoconjugates and left the cytoskeleton and nuclear residue adherent to the substratum. In addition to the cytoskeletal proteins, there was a relatively simple profile of glycoproteins and glycosaminoglycans associated with this adherent cytoskeleton. These consisted of eight glycoproteins in the mol.wt. range 99000-232000, five proteins in the keratin region (mol.wt. 42000-61000), hyaluronic acid and a sulphated glycosaminoglycan. Surface labelling of the keratinocytes with galactose oxidase (with or without neuraminidase)/KB3H4 revealed that many of the glycoproteins were exposed on the cell surface. The importance of the glycoproteins and proteoglycans in attaching the keratinocytes to the substratum was examined by studying their expression after incubation in medium containing tunicamycin and their degradation after digestion with trypsin and hyaluronidase. These studies, together with an examination of the glycoconjugates released by sequential extraction with 0.5% Triton X-100 followed by 0.2% sodium dodecyl sulphate, revealed that the glycoprotein of mol.wt. 232000 has an important role in mediating the attachment of keratinocytes to the substratum.

scholarly journals Lactogenic and somatogenic binding sites in intact and detergent-solubilized membrane preparations of female rat liver

1988 ◽  
Vol 252 (2) ◽  
pp. 509-514 ◽  
Author(s):  
L A Haldosén ◽  
J A Gustafsson
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Growth ◽  
Microsomal Membrane ◽  
Gel Chromatography ◽  
Female Rat ◽  
Microsomal Membranes ◽  
Triton X

The presence of lactogenic and somatogenic binding sites in intact microsomal membranes and in detergent-solubilized microsomal membrane preparations of female rat liver has been studied by affinity cross-linking-SDS/polyacrylamide-gel electrophoresis. In microsomal membrane preparations an Mr 40,000 lactogenic binder is present which is not disulphide-linked to another protein. Triton X-100 solubilization of membranes results in the appearance of three lactogenic 125I-human growth hormone (125I-hGH) binders with Mr values of 87,000, 40,000 and 35,000, and one somatogenic 125I-hGH binder with Mr 32,000. Treatment of rats with oestrogen increased the amount of lactogenic and somatogenic binding species in liver. The lactogenic binding sites are present as one entity in Triton X-100-solubilized preparations, clearly separated from the somatogenic binder as analysed by gel chromatography. Furthermore, 125I-hGH interacts with an Mr 95,000 somatogenic binder in membrane preparations to which the hormone can be cross-linked only following Triton X-100 solubilization.

Isolation and characterization of cancer-associated galactosyltransferase isoenzyme.

1984 ◽  
Vol 30 (10) ◽  
pp. 1656-1663 ◽  
Author(s):  
B P Ram ◽  
D D Munjal
Keyword(s):  
Cancer Patient ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Isolation And Characterization ◽  
Absolute Requirement ◽  
Triton X ◽  
Alpha Lactalbumin

Abstract We isolated galactosyltransferase (EC 2.4.1.22) from pleural effusions of a lung cancer patient and a patient with cirrhosis by precipitation with ammonium sulfate, followed by gel filtration on Sepharose 6B, and affinity chromatography on columns of alpha-lactalbumin-agarose and protein A-Sepharose. By this procedure the enzyme from both sources was purified 40 000-fold with approximate yields of 37% and 60%, respectively, and did not contain immunoglobulin. Electrophoresis on polyacrylamide gel of the enzyme from the cancer patient (slower moving) and from the non-cancer patient (faster moving) gave one sharp band for each. Their respective relative molecular masses, 74 131 and 107 151, were estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and gel filtration, respectively. The isoenzymes were active between pH 5 and 8, most active at 7, and showed no activity below pH 4 or above pH 9. Activity was greatest at temperatures between 37 and 40 degrees C. At 30 degrees C or 50 degrees C the activity was more than halved, and was lost completely above 60 degrees C. The isoenzymes had an absolute requirement for Mn2+. Omitting the surfactant Triton X-100 from the buffer resulted in considerable loss in activity of both isoenzymes. Glucose can be used as an acceptor for these isoenzymes if alpha-lactalbumin is present in the assay mixture. These isoenzymes had different Km values for UDP-galactose, N-acetylglucosamine, and Mn2+.

Identification of high-affinity calmodulin-binding proteins in rat liver

1987 ◽  
Vol 252 (3) ◽  
pp. C277-C284 ◽  
Author(s):  
R. M. Hanley ◽  
J. R. Dedman ◽  
S. Shenolikar
Keyword(s):  
Rat Liver ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Apparent Molecular Weight ◽  
Convincing Evidence ◽  
Calmodulin Binding ◽  
High Affinity

The Ca2+-dependent binding of [125I]calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or “acceptor” proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised greater than 80% of the Ca2+-dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding “subunit” of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca2+ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver.

Sign in / Sign up

Export Citation Format

Share Document