Characterization of [3H]di-isopropyl phosphorofluoridate-binding proteins in hen brain. Rates of phosphorylation and sensitivity to neurotoxic and non-neurotoxic organophosphorus compounds

The experiments described in this paper were designed to isolate [3H]di-isopropyl phosphorofluoridate-binding proteins by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis for the purpose of characterizing and identifying potential initiation sites for organophosphorus-compound-induced delayed neurotoxicity. The major Paraoxon-insensitive Mipafox-sensitive binding protein (Mr 160 000) was found to be identical with one previously identified as neurotoxic esterase, an enzyme that has been proposed to be the target site for organophosphorus-compound-induced delayed neurotoxicity. However, two other binding proteins with suitable binding characteristics were also found in smaller amounts, one of which has not been detected previously. Di-isopropyl phosphorofluoridate was found to phosphorylate all three of these proteins at rates similar to the rate at which neurotoxic esterase is inhibited by di-isopropyl phosphorofluoridate. Varying the concentration of di-isopropyl phosphorofluoridate or the time of incubation produced similar increases in binding to each of the labelled proteins. This suggests that the reaction rates of di-isopropyl phosphorofluoridate with proteins may be described by first-order kinetics, and the concentration of the Michael is complex formed during binding is minimal for all the phosphorylated proteins. The recovery of the binding activity in the 160 000-Mr band was found to be similar to the recovery of neurotoxic esterase activity, lending further support to the contention that this band is identical with neurotoxic esterase.

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Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.164-171.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 164-171

Author(s):

M J Matunis ◽

W M Michael ◽

G Dreyfuss

Keyword(s):

Hela Cells ◽

Mammalian Cells ◽

Binding Proteins ◽

Rna Binding ◽

Consensus Sequence ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Predicted Amino Acid Sequence ◽

Cdna Clones ◽

Hnrnp Proteins

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.

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Cloning and characterization of E2F-2, a novel protein with the biochemical properties of transcription factor E2F

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7802-7812.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7802-7812

Author(s):

M Ivey-Hoyle ◽

R Conroy ◽

H E Huber ◽

P J Goodhart ◽

A Oliff ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Hela Cell ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Amino Acid Identity ◽

Biochemical Properties ◽

Binding Activity ◽

Dna Binding Activity ◽

Novel Protein

E2F is a mammalian transcription factor that appears to play an important role in cell cycle regulation. While at least two proteins (E2F-1 and DP-1) with E2F-like activity have been cloned, studies from several laboratories suggest that additional homologs may exist. A novel protein with E2F-like properties, designated E2F-2, was cloned by screening a HeLa cDNA library with a DNA probe derived from the DNA binding domain of E2F-1 (K. Helin, J. A. Lees, M. Vidal, N. Dyson, E. Harlow, and A. Fattaey, Cell 70:337-350, 1992). E2F-2 exhibits overall 46% amino acid identity to E2F-1. Both the sequence and the function of the DNA and retinoblastoma gene product binding domains of E2F-1 are conserved in E2F-2. The DNA binding activity of E2F-2 is dramatically enhanced by complementation with particular sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified components of HeLa cell E2F, and anti-E2F-2 antibodies cross-react with components of purified HeLa cell E2F. These observations are consistent with a model in which E2F binds DNA as a heterodimer of two distinct proteins, and E2F-2 is functionally and immunologically related to one of these proteins.

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Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein.

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.164 ◽

1992 ◽

Vol 12 (1) ◽

pp. 164-171 ◽

Cited By ~ 172

Author(s):

M J Matunis ◽

W M Michael ◽

G Dreyfuss

Keyword(s):

Hela Cells ◽

Mammalian Cells ◽

Binding Proteins ◽

Rna Binding ◽

Consensus Sequence ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Predicted Amino Acid Sequence ◽

Cdna Clones ◽

Hnrnp Proteins

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Cadmium-binding proteins of rat testes. Characterization of a low-molecular-mass protein that lacks identity with metallothionein

Biochemical Journal ◽

10.1042/bj2200811 ◽

1984 ◽

Vol 220 (3) ◽

pp. 811-818 ◽

Cited By ~ 50

Author(s):

M P Waalkes ◽

S B Chernoff ◽

C D Klaassen

Keyword(s):

Metal Binding ◽

Gel Electrophoresis ◽

Binding Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Polyacrylamide Gel ◽

Or Groups

Cadmium-binding proteins in the cytosol of testes from untreated rats were separated by Sephadex G-75 gel filtration. Three major testicular metal-binding proteins (TMBP), or groups of proteins, with relative elution volumes of approx. 1.0 (TMBP-1), 1.7 (TMBP-2) and 2.4 (TMBP-3) were separated. Elution of Zn-binding proteins exhibited a similar pattern. TMBP-3 has previously been thought to be metallothionein (MT), and hence this protein was further characterized and compared with hepatic MT isolated from Cd-treated rats. Estimation of Mr by gel filtration indicated a slight difference between MT (Mr 10000) and TMBP-3 (Mr 8000). Two major forms of MT (MT-I and MT-II) and TMBP-3 (TMBP-3 form I and TMBP-3 form II) were obtained after DEAE-Sephadex A-25 anion-exchange chromatography, with the corresponding subfractions being eluted at similar conductances. Non-denaturing polyacrylamide-gel electrophoresis on 7% acrylamide gels indicated that the subfractions of TMBP-3 had similar mobilities to those of the corresponding subfractions of MT. However, SDS (sodium dodecyl sulphate)/12% (w/v)-polyacrylamide-gel electrophoresis resulted in marked differences in migration of the two corresponding forms of MT and TMBP-3. Co-electrophoresis of MT-II and TMBP-3 form II by SDS/polyacrylamide-gel electrophoresis revealed two distinct proteins. Amino acid analysis indicated much lower content of cysteine in the testicular than in the hepatic proteins. TMBP-3 also contained significant amounts of tyrosine, phenylalanine and histidine, whereas MT did not. U.v.-spectral analysis of TMBP-3 showed a much lower A250/A280 ratio than for MT. Thus this major metal-binding protein in testes, which has been assumed to be MT is, in fact, a quite different protein.

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Heparin Binding Proteins of Black Bengal Buck Semen and Their Correlation with Sperm Characters and Freezability

Indian Journal of Animal Research ◽

10.18805/ijar.b-3849 ◽

2019 ◽

Author(s):

M Karunakaran ◽

Vivek C Gajare ◽

Ajoy Mandal ◽

Mohan Mondal ◽

S K Das ◽

...

Keyword(s):

Seminal Plasma ◽

Binding Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Heparin Binding ◽

Sds Page ◽

Significant Difference ◽

Sperm Characters

This experiment was conducted to study the electrophoretic characters of heparin binding proteins (HBP) of Black Bengal buck semen and their correlation with sperm characters and cryo-survivability. Semen ejaculates (n=20/buck) were collected from nine bucks and in vitro sperm characters were evaluated at collection, after equilibration and after freeze - thawing. HBP were isolated through heparin column and discontinuous Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed to assess molecular weight. Significant difference (plessthan0.01) were observed among the bucks in sperm characters and freezability. Eight protein bands of 17 to 180 kDa in seminal plasma and 7 bands in sperm were found. 180 -136 kDa HBP of seminal plasma and 134-101 kDa HBP of sperm had showed high correlation with in vitro sperm characters. Further studies on identification of these proteins and their correlation with in vivo pregnancy are needed to find their role as marker for buck selection.

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Studies of the heterogeneity of human pituitary LH by fast protein liquid chromatography

Journal of Endocrinology ◽

10.1677/joe.0.1050405 ◽

1985 ◽

Vol 105 (3) ◽

pp. 405-413 ◽

Cited By ~ 16

Author(s):

A. Stockell Hartree ◽

J. B. Lester ◽

R. C. Shownkeen

Keyword(s):

Liquid Chromatography ◽

Sialic Acid ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Binding Activity ◽

Fast Protein Liquid Chromatography ◽

Human Pituitary ◽

Sialic Acid Content ◽

Receptor Binding Activity

ABSTRACT The Pharmacia fast protein liquid chromatography system was employed to fractionate a purified preparation of human LH (hLH) on the anion exchanger Mono Q at pH 7·8 into 14 sub-fractions. Each of the sub-fractions was characterized by its behaviour on polyacrylamide gel electrophoresis, sodium dodecyl sulphate (SDS) gel electrophoresis, LH receptor binding activity and sialic acid content. All sub-fractions contained sialic acid, were active in binding to LH receptors, and exhibited components typical of hLH subunits on SDS gel electrophoresis. None of the sub-fractions was homogeneous with respect to charge. There is evidence that part of the heterogeneity results from the presence in some molecules of an internal proteolytic cleavage within the β-subunit, and fractions enriched in species containing such cleavages were prepared by this method. J. Endocr. (1985) 105,405–413

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Analysis of Nickel-Binding Proteins from Various Animal Sera

Folia Veterinaria ◽

10.2478/fv-2018-0017 ◽

2018 ◽

Vol 62 (2) ◽

pp. 59-66

Author(s):

J. Šimková ◽

M. Milkovičová ◽

M. Valko-Rokytovská ◽

Z. Kostecká ◽

E. Bencúrová ◽

...

Keyword(s):

Cervus Elaphus ◽

Sus Scrofa ◽

Binding Proteins ◽

Ursus Arctos ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Ovis Aries ◽

Nickel Binding ◽

Lower Abundance ◽

Abundance Proteins

Abstract Nickel-binding proteins play an important role in the biological processes and can also be utilized in several fields of biotechnology. This study was focused on analysing the nickel-binding proteins from the blood sera of humans (Homo sapiens), cattle (Bos taurus), sheep (Ovis aries), red deer (Cervus elaphus), mouflon (Ovis orientalis), fallow deer (Dama dama), horses (Equus ferus caballus), pigs (Sus scrofa domesticus), wildboars (Sus scrofa), brown bears (Ursus arctos) and pheasants (Phasianus colchicus). The presence of higher abundance proteins in the blood serum, such as albumins, may mask the detection of lower abundance proteins. The samples were depleted from these higher abundance proteins to facilitate the detection of those with lower abundance. For the characterization of these proteins, nickel cations bound to tetradentate ligand nitrilotriacetic acid(Ni-NTA)immobilized on agarose beads were incubated with animal sera to capture nickel-binding proteins and subsequently the proteins were eluted and fractionated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed a set of nickel-binding proteins with various molecular weights within different animal species. A unique ~42 kDa nickel-binding protein in the brown bear serum, which was not present in any of the other species, was further characterized and identified by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS). This protein was identified as ahaptoglobin-like protein. This result may provide some valuable clue for the physiological difference in the metal binding proteins in the serum of Ursus arctos and other animals.

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Discriminating between Cell Surface and Intracellular Plasminogen-binding Proteins: Heterogeneity in Profibrinolytic Plasminogen-binding Proteins on Monocytoid Cells

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614132 ◽

2000 ◽

Vol 84 (11) ◽

pp. 882-890 ◽

Cited By ~ 15

Author(s):

Michael Green ◽

Lindsey Miles ◽

Stephen Hawley

Keyword(s):

Cell Surface ◽

Peripheral Blood ◽

Gel Electrophoresis ◽

Binding Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Annexin Ii ◽

Two Dimensional ◽

Intact Cells ◽

Carboxyl Terminal

SummaryWhen plasminogen binds to cell surfaces, its activation is markedly enhanced compared to soluble plasminogen. Although several distinct molecules may contribute to plasminogen binding to a given cell type, the subset of plasminogen receptors responsible for enhancing plasminogen activation expose a carboxyl-terminal lysine on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). To distinguish this subset of plasminogen receptors from plasminogen-binding proteins that are not profibrinolytic, we treated intact U937 monocytoid cells and peripheral blood monocytes with CpB to remove exposed carboxyl-terminal lysines, and subjected the membrane proteins to two-dimensional gel electrophoresis followed by ligand blotting with 125I-plasminogen. Western blotting was performed with antibodies against previously characterized candidate plasminogen receptors to identify plasminogen-binding proteins on the two-dimensional ligand blots. Densitometry of autoradiograms of the 125I-plasminogen ligand blots of U937 cell membranes revealed that membraneassociated α-enolase, actin and annexin II showed minimal changes in 125I-plasminogen binding following CpB treatment of intact cells, suggesting that these proteins are not accessible to CpB on the U937 cell surface and most likely do not serve as profibrinolytic plasminogen receptors on U937 cells. In contrast, densitometry of autoradiograms of 125I-plasminogen ligand blots of monocyte membranes revealed that 125I-plasminogen binding to α-enolase was reduced 71% by treatment of intact cells with CpB, while binding to annexin II was reduced 14%. Thus, a portion of membrane-associated α-enolase and annexin II expose carboxyl terminal lysines that are accessible to CpB on the peripheral blood monocyte surface, suggesting that these molecules may serve as profibrinolytic plasminogen receptors on monocytes. Our data suggest that U937 cells and peripheral blood monocytes have distinct sets of molecules that constitute the population of cell surface profibrinolytic plasminogen-binding proteins. Furthermore, our data suggest that while several plasminogen-binding proteins with carboxyl terminal lysines are associated with cell membranes, only a small subset of these proteins expose a carboxyl terminal lysine that is accessible to CpB on the cell surface. The abbreviations used are: 2D, two-dimensional; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; BSA, bovine serum albumin; CpB, carboxypeptidase B; EACA, є-aminocaproic acid; HBSS, Hanks’ Balanced Salt Solution supplemented with 20 mM HEPES; HBSS-BSA, HBSS with 0.1% bovine serum albumin; HRP, horseradish peroxidase; IEF, isoelectric focusing; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBST, Tris buffered saline with 0.1% Tween 20; uPAR, urokinase-type plasminogen activator receptor.

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The γ-aminobutyrate/benzodiazepine receptor from pig brain. Purification and characterization of the receptor complex from cerebral cortex and cerebellum

Biochemical Journal ◽

10.1042/bj2330265 ◽

1986 ◽

Vol 233 (1) ◽

pp. 265-270 ◽

Cited By ~ 21

Author(s):

E F Kirkness ◽

A J Turner

Keyword(s):

Cerebral Cortex ◽

Dissociation Constants ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Benzodiazepine Receptor ◽

Binding Activity ◽

Exchange Chromatography ◽

Synaptic Membranes ◽

Receptor Complex ◽

A Minor

The gamma-aminobutyrate/benzodiazepine-receptor complex has been purified from a Triton X-100 extract of crude synaptic membranes from pig cerebral cortex and cerebellum by a combination of affinity and ion-exchange chromatography. [3H]Flunitrazepam binding activity was purified 2200-fold from cortex with an overall yield of 2%. The dissociation constants for the binding of [3H]muscimol and [3H]flunitrazepam to the receptor complex were 14 +/- 3 nM and 14 +/- 2 nM respectively. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was in the range 2.2-2.8. There appeared to be no selective inactivation of either binding site during the purification procedure. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed two major polypeptides of Mr 49 000 and 55 000 from both cortex and cerebellum. When the receptor from cortex was photoaffinity labelled with [3H]flunitrazepam, radioactivity was incorporated predominantly into the Mr-49 000 polypeptide, although some radioactivity was detectable in the Mr-55 000 band. The cerebellar receptor was photoaffinity labelled on the 49 000-Mr polypeptide but not on the polypeptide of Mr 55 000. In addition, some radioactivity was detected in a minor polypeptide of Mr 43 000. When purified in the presence of 3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate the same major polypeptide components (Mr 49 000 and 55 000) were isolated, but the receptor now retained its ability to be modulated by secobarbital and by the anaesthetic propanidid.

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Purification of PRL receptors from toad kidney: comparisons with rabbit mammary PRL receptors

AJP Cell Physiology ◽

10.1152/ajpcell.1988.254.3.c372 ◽

1988 ◽

Vol 254 (3) ◽

pp. C372-C382 ◽

Cited By ~ 3

Author(s):

M. Dunand ◽

J. P. Kraehenbuhl ◽

B. C. Rossier ◽

M. L. Aubert

Keyword(s):

Mammalian Species ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Structural Features ◽

Polyclonal Antiserum ◽

Partial Purification ◽

Molecular Characteristics ◽

Binding Characteristics ◽

Sds Page

The binding characteristics of the prolactin (PRL) receptors present in toad (Bufo marinus) kidneys were investigated and compared to those of PRL receptors present in rabbit mammary glands. The molecular characteristics of the Triton X-100 solubilized renal and mammary PRL receptors were assessed by gel filtration and by migration analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after affinity labeling of the binding sites with 125I-human growth hormone. Similar results were obtained for both receptors. Partial purification of the toad PRL receptor could be achieved by affinity chromatography. The molecular weight of this purified receptor could be determined by analysis on SDS-PAGE. With the use of a polyclonal antiserum raised against a purified preparation of rabbit mammary PRL receptor, one or several antigenic epitope(s) could be identified on the core of the toad renal PRL receptor. In conclusion, although the structure and the biological role(s) of PRL have substantially changed during evolution, the receptor for this hormone has retained many of its structural features as could be assessed between an amphibian and a mammalian species on functionally different target tissues.

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