Turnover of acyl-CoA-binding protein in four different cell lines measured by using two-dimensional polyacrylamide-gel electrophoresis

Acyl-CoA-binding protein (ACBP), also named diazepam-binding inhibitor or endozepine, is a 10 kDa protein for which a surprisingly large number of biological activities has been suggested. Some of these would seem to require a rapid intracellular turnover of the protein. In this paper we report on the turnover of ACBP in cell lines derived from mouse, rat and man. ACBP was identified in two-dimensional gels by using specific antibodies. Cells were labelled with [35S]methionine and chased for various periods of time. Total protein was extracted, subjected to two-dimensional PAGE, and radioactivity in the spot containing ACBP was determined by liquid-scintillation counting. ACBP half-life was determined, and varied from 25 to 53 h depending on the cell line and the growth conditions. In all cases, radioactivity in ACBP was lost slightly faster than radioactivity in total protein. These results are discussed in relation to the possible function suggested for ACBP.

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Quantificação de [32P] fosfoproteínas em gel de pollacrilamida pela Radiação Cerenkov: vantagens sobre outros procedimentos

Ciência e Natura ◽

10.5902/2179460x26714 ◽

1997 ◽

Vol 19 (19) ◽

pp. 21

Author(s):

Carlos A. Gonçalves ◽

Guido Lenz ◽

Christianne Salbego ◽

Carmem Gottfried

Keyword(s):

Liquid Scintillation Counting ◽

Gel Electrophoresis ◽

Liquid Scintillation ◽

Polyacrylamide Gel Electrophoresis ◽

Scintillation Counting ◽

Polyacrylamide Gel ◽

Cerenkov Counting ◽

Phosphorylation Of Proteins ◽

Phosphate Incorporation

The phosphorylation of proteins has been recognized as an important mechanism for controlling cellular activities. There are many ways to measure 32P-labellingof phosphoproteins resolved by polyacrylamide gel electrophoresis, including densitometry of autoradiographs, liquid scintillation counting and Cerenkov counting. This report compares such different procedures and indicates the advantages of Cerenkov counting to determine radioactive phosphate incorporation into proteins.

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Protein Profile of Human Perilymph: In Search of Markers for the Diagnosis of Perilymph Fistula and other Inner Ear Disease

Otolaryngology ◽

10.1177/01945998941113p117 ◽

1994 ◽

Vol 111 (3P1) ◽

pp. 273-280 ◽

Cited By ~ 16

Author(s):

Isolde Thalmann ◽

Robert I. Kohut ◽

Jay Ryu ◽

Thomas H. Comegys ◽

Masamitsu Senarita ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Inner Ear ◽

Total Protein ◽

Gel Electrophoresis ◽

Protein Profile ◽

Polyacrylamide Gel Electrophoresis ◽

Density Lipoprotein ◽

Polyacrylamide Gel ◽

Computer Assisted ◽

Two Dimensional

Recent developments in high-resolution two-dimensional polyacrylamide gel electrophoresis, combined with amino acid sequencing and computer-assisted image analysis, have allowed separation of approximately 100 proteins and identification and quantitation of some 30 proteins in human perilymph. The majority of proteins were found to be present in perilymph at levels in basic agreement with the total protein gradient between perilymph and plasma (1:35). However, several striking differences were observed: (1) β2-transferrin, known to be absent from normal plasma but present in cerebrospinal fluid, was detected in perilymph at a concentration roughly equal to that in cerebrospinal fluid; and (2) two high-density lipoprotein-associated apolipoproteins—apo D (formerly PLS:33) and apo J or NA1 and NA2 (formerly PSL:29/30), the latter showing identity with 5P40/40, or cytolysis inhibitor—were found to be present at concentrations 1 to 2 orders of magnitude higher when examined in terms of total protein and to be comparable with or higher than plasma levels when examined in terms of absolute concentrations. The functional significance of the extremely high levels of the two apolipoproteins is not known at this time. An attempt was made to use β2-transferrin, as well as apo D and apo J (NA1/NA2), as markers for the diagnosis of perilymph fistula, one of the most controversial and challenging problems for the otologist today. It was determined that the technique is indeed applicable when relatively pure fistula samples are analyzed. Limitations and potential improvements of the technique are discussed. In addition, the potential usefulness of two-dimensional polyacrylamide gel electrophoresis in other pathologic conditions of the inner ear is discussed briefly.

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Purification of fibronectin from human plasma by affinity chromatography under non-denaturing conditions

Biochemical Journal ◽

10.1042/bj1830331 ◽

1979 ◽

Vol 183 (2) ◽

pp. 331-337 ◽

Cited By ~ 378

Author(s):

M Vuento ◽

A Vaheri

Keyword(s):

Affinity Chromatography ◽

Human Plasma ◽

Gel Electrophoresis ◽

Analytical Ultracentrifugation ◽

Biological Activities ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

The Novel ◽

Two Dimensional ◽

Native State

Fibronectin was purified from human plasma by affinity chromatography under nondenaturing conditions. The method was based on the previously known binding of fibronectin to gelatin. The novel features of our method are the use of arginine in the elution of fibronectin from immobilized gelatin [Vuento & Vaheri (1978) Biochem. J. 175, 333-336] and the use of arginine-agarose as second affinity step. The purified protein was homogeneous as judged by polyacrylamide-gel electrophoresis, analytical ultracentrifugation and two-dimensional immunoelectrophoresis. The yield was 60%. We propose that the method would be useful in preparation of fibronectin for studies on its biological activities, where it is important that the protein is obtained in a native state.

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A Sensitive Peroxidase Staining Immunoblotting Method for Measuring Total Protein S in Human Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651607 ◽

1993 ◽

Vol 69 (04) ◽

pp. 331-334 ◽

Cited By ~ 3

Author(s):

Xiangan Li ◽

Kaoru Hatanaka ◽

Ling Guo ◽

Motoo Tsushima ◽

Yukihiko Kitamura ◽

...

Keyword(s):

Human Plasma ◽

Total Protein ◽

Gel Electrophoresis ◽

Binding Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Apparent Molecular Weight ◽

Protein S ◽

Polyacrylamide Gel ◽

Free Form

SummaryIn plasma, protein S is found in its free form and as a complex with C4b-binding protein. After 125I-protein S was added tonormal human plasma and applied to SDS-8% polyacrylamide gel electrophoresis, the autoradiogram of the gel showed only one single band at free protein S position. Applying this evidence, we have developed a peroxidase staining Western Blotting method to quantitate total protein S in human plasma which consists of sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting to nitrocellulose membrane and a sensitive avidin-biotinylated peroxidase staining method (ABC technique). The measurement of protein S by the immunoblotting was reproducible and the coefficient of variation was 7%. As little as 1 ng of protein S could be detected. C4b-binding protein did not affect the measurement of protein S. Compared to other immunoassays, this peroxidase staining immunoblotting method has the advantage of directly estimating the apparent molecular weight of protein of interest, eliminating nonspecific stain and having high sensitivity without using radioisotope.

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Non-Specific Binding of Thiocholine Ester of Cinnamic Acid to Fibrin

10.1055/s-0039-1680582 ◽

1977 ◽

Author(s):

T. Seelich ◽

B. A. Perret ◽

M. Furlan ◽

E. A. Beck

Keyword(s):

Cinnamic Acid ◽

Gel Electrophoresis ◽

Liquid Scintillation ◽

Specific Binding ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Scintillation Counting ◽

Human Fibrinogen ◽

Α Chain ◽

Β Chain

Thiocholine esters inhibit the enzymatic crosslinking of fibrin (1). Thiocholine ester of 14C-labelled cinnamic acid (2-diethylbenzylaminoethyl thioltranscinnamate bromide) was prepared and incubated with human fibrinogen in the presence of factor XIII and thrombin. Polyacrylamide gel electrophoresis with sodium dodecyl sulphate, followed by liquid scintillation counting of the separated protein bands, showed that all three chains of fibrinogen had bound the crosslinking inhibitor(2 moles/mol α chain, 1.5 moles/mol β chain, 2.2 moles/mol γ chain). Cyanogen bromide cleavage of the isolated α chain resulted in 6 major fragments all of whichwere radioactive. These results indicate that the binding of the thiocholine ester of cinnamic acid was not restricted to the specific donor crosslinking sites of fibrin(ogen). This thiocholine ester differs in this respect from dansyl-cadaverine which binds exclusively to the acceptor sites involved in fibrin(ogen) crosslinking.

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Synthesis of stress-induced protein in isolated and perfused rat hearts

Biochemistry and Cell Biology ◽

10.1139/o86-059 ◽

1986 ◽

Vol 64 (5) ◽

pp. 418-426 ◽

Cited By ~ 7

Author(s):

R. William Currie

Keyword(s):

Protein Synthesis ◽

Heat Shock ◽

Total Synthesis ◽

Gel Electrophoresis ◽

Liquid Scintillation ◽

Scintillation Counting ◽

Two Dimensional ◽

Two Dimensional Gel Electrophoresis ◽

Rat Hearts ◽

Perfused Rat Hearts

Isolated and perfused rat hearts were examined by two-dimensional gel electrophoresis and liquid scintillation counting for alterations in protein synthesis following incubation with L-[3H]leucine at 0.5–2.5, 2.5–4.5, or 4.5–6.5 h of perfusion. When 35-mL volumes of three different buffers were recycled for a 2-h period from 0.5 to 2.5 h, by fluorography little effect was seen on the normal patterns of protein synthesis and there was a moderate synthesis of a stress-induced protein (heat-shock protein) with a molecular mass of 71 × 103 daltons (SP71). However, hearts perfused with Krebs-improved Ringer 1 bicarbonate had the highest incorporation of L-[3H]leucine. When buffers were recycled for 30-min periods from 0.5 to 2.5 h, SP71 was synthesized in hearts perfused with Krebs–Henseleit original Ringer bicarbonate. Hearts perfused in a similar fashion with Krebs-improved Ringer 1 bicarbonate had the lowest incorporation of label into SP71 and in fact SP71 was undetectable on fluorograms. Overall protein synthesis was decreased and the ratio of SP71 to the total synthesis was increased at 4.5–6.5 h of perfusion when 35-mL volumes of Krebs-improved Ringer 1 bicarbonate was recycled for 2-h periods. A similar result was observed at 2.5–4.5 h of perfusion when this buffer was recycled for either the duration of the experiment or 30-min periods.

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Demonstration of structural polymorphism among HLA-DR light chains by two-dimensional gel electrophoresis.

Journal of Experimental Medicine ◽

10.1084/jem.151.1.144 ◽

1980 ◽

Vol 151 (1) ◽

pp. 144-165 ◽

Cited By ~ 106

Author(s):

D A Shackelford ◽

J L Strominger

Keyword(s):

Cell Lines ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Light Chains ◽

Two Dimensional ◽

Structural Polymorphism ◽

Two Dimensional Gel Electrophoresis ◽

Hla Dr ◽

Sds Page

Human HLA-DR antigens were immunoprecipitated from Nonidet P-40 extracts of [35S]methionine-labeled B lymphoblastoid cell lines and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Two-dimensional (2-D) gel analyses, combining SDS-PAGE in the first dimension and IEF in the second dimension, revealed that the heavy (alpha) and light (beta) chains of each DRw specificity displays microheterogeneity of charge. However, the pattern of the heavy chain did not vary among different DRw specificities. In contrast, the light chains of different DRw types varied both in apparent size and charge distribution. Removal of sialic acids with neuraminidase or inhibition of glycosylation with tunicamycin reduced the microheterogeneity of both DR subunits. However, the heavy and light chains each still focused as two major bands, suggesting that other post-translational modifications contribute to the microheterogeneity or that there are two nonallelic DR-like molecules. After treatment with either neuraminidase or tunicamycin, the DR light chains, but not the heavy chains, were still structurally polymorphic. The DR light chains of serologically cross-reactive specificities displayed similar 2-D gel patterns suggesting that the structural polymorphism of the DR light chains is the basis for the serologically detected polymorphism of the HLA-DR antigens. Two additional polypeptides were observed in immunoprecipitates of DR antigens. These proteins, designated M1 and M2, both had a basic isoelectric point and were invariant among different cell lines. The protein M1 may be intracellular because it can not be immunoprecipitated from the cell surface.

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