A two-dimensional electrophoretic analysis of the proteins and glycoproteins of liver plasma membrane domains and endosomes. Implications for endocytosis and transcytosis

1. Polypeptides of liver plasma membrane fractions enriched in three surface domains of hepatocytes, blood-sinusoidal, lateral and bile canalicular, were analysed by isoelectric focusing (IEF) and non-equilibrium pH gel electrophoresis (NEPHGE) across a wide pH range, followed by SDS/PAGE. The overall Coomassie Blue-stained polypeptide patterns in the fractions were different. lateral plasma membrane fractions contained a characteristically higher number of polypeptides focusing at the basic pH range, whereas few basic polypeptides were present in sinusoidal plasma membrane fractions. The glycoproteins in these plasma membrane fractions stained by a lectin overlay technique with radio-iodinated concanavalin A, wheat-germ agglutinin and a slug lectin, were also different. 2. The polypeptides and glycoproteins of ‘early’ and ‘late’ endosome fractions were also compared by two-dimensional electrophoresis. Their composition was shown by Coomassie Blue staining, lectin overlay staining and in membranes metabolically labelled with [35S]methionine to be generally similar. The glycoproteins of sinusoidal plasma membranes and early and late endosomes were generally similar, but major differences in polypeptides of molecular mass 20-50 kDa, pI 7.5-8.5, in plasma membranes and endosomes were demonstrated, with a specific population of basic (pI 8-9) low-molecular-mass polypeptides being present at highest levels in ‘late’ endosomal fractions (shown by Coomassie Blue staining). 3. Analysis of the distribution of three specific membrane glycoproteins identified by using immunoblotting techniques showed that the asialoglycoprotein and the divalent-cation-sensitive mannose 6-phosphate receptors were present in sinusoidal plasma membrane and in early and late endocytic fractions: they were not detected in canalicular plasma membrane fractions. In contrast, 5′-nucleotidase was detected in all fractions examined. The role of the endocytic compartment in regulating trafficking pathways between the plasma membrane domains of the hepatocyte is discussed.

Download Full-text

A biochemical dissection of the cardiac intercalated disk: isolation of subcellular fractions containing fascia adherentes and gap junctions

Journal of Cell Science ◽

10.1242/jcs.52.1.313 ◽

1981 ◽

Vol 52 (1) ◽

pp. 313-325

Author(s):

C.A. Colaco ◽

W.H. Evans

Keyword(s):

Gap Junctions ◽

Plasma Membranes ◽

Polyacrylamide Gel Electrophoresis ◽

Intercellular Junctions ◽

Blue Staining ◽

Molecular Weights ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

Insoluble Material ◽

Rats And Mice

In view of our limited knowledge of the biochemical composition of intercellular junctions, a method was developed for the preparation from rats and mice of plasma membranes containing cardiac intercalated disks. When these membranes were extracted with detergents, e.g. N-lauryl sarcosinate or deoxycholate, the detergent-insoluble material contained structures derived mainly from fascia adherentes junctions, but a few gap junctions and maculae adherentes were also present. When the detergent extraction was carried out at an alkaline pH, the maculae adherentes junctions were dissolved. Fractionation of the detergent-insoluble extract on a sucrose gradient yielded a fraction containing fascia adherentes junction of density 1.20-1.26 g/cm3. Gap junctions banded at a lower density, 1.16-1.20 g/cm3. Polyacrylamide gel electrophoresis showed that the major polypeptide bands in the fascia adherentes-enriched fraction were of molecular weights 134000, 108000, 62–64000, 58000, 47000 and 43000. Although fractions with the gap junctions were contaminated by fascia adherentes junctions, the major polypeptides were calculated by subtraction to be of mol. wt 37000, 26000 and 19000. Two glycoproteins corresponding to minor polypeptides visualized by Coomassie Blue staining were present in the fascia adherentes fraction. Comparison of the fascia adherentes-enriched fraction with a Z-disc fraction prepared from rabbit hearts indicated a different morphology and polypeptide composition.

Download Full-text

Isolation and electrophoretic characterization of the plasma membrane of sea-urchin sperm

Journal of Cell Science ◽

10.1242/jcs.59.1.13 ◽

1983 ◽

Vol 59 (1) ◽

pp. 13-25

Author(s):

N.L. Cross

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Specific Activity ◽

Periodic Acid ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Blue Staining ◽

Coomassie Blue Staining ◽

Electrophoretic Patterns ◽

Sperm Plasma Membrane

A subcellular fraction containing plasma membranes was isolated from flagella of the sperm of Strongylocentrotus purpuratus by differential centrifugation, and analysed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Coomassie Blue staining revealed nine major bands and 14 minor species. Five bands of apparent molecular weights approximately 200 X 10(3), 149 X 10(3), 120 X 10(3), 75 X 10(3) and 59 X 10(3) also stained with periodic acid-Schiff's reagent and so are probably glycoproteins. These five components are externally exposed, as determined by lactoperoxidase-catalysed radio-iodination. Isolation of membranes from radio-iodinated sperm results in an enrichment of about tenfold in the specific activity of 125I. Comparison of the electrophoretic patterns of labelled sperm and of the membranes isolated from 125I-labelled sperm suggests that no major labelled proteins are lost during the isolation procedure, and so to this extent the membrane fraction is representative of the entire sperm plasma membrane.

Download Full-text

Microscale multisample two-dimensional electrophoresis of proteins in human serum, cerebrospinal fluid, and urine.

Clinical Chemistry ◽

10.1093/clinchem/28.4.824 ◽

1982 ◽

Vol 28 (4) ◽

pp. 824-827 ◽

Cited By ~ 49

Author(s):

T Manabe ◽

E Hayama ◽

T Okuyama

Keyword(s):

Cerebrospinal Fluid ◽

Human Serum ◽

Distribution Patterns ◽

Blue Staining ◽

Two Dimensional ◽

Protein Distribution ◽

Two Dimensional Electrophoresis ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

Dimensional Electrophoresis

Abstract In this technique, in which no denaturing agent is used, proteins in human serum, cerebrospinal fluid, and urine are separated by isoelectric focusing in cylindrical 40 g/L polyacrylamide gels of capillary size (1.3 x 35 mm) for 40 min, followed by electrophoresis in 40--170 g/L polyacrylamide linear gradient gel, with use of 38 x 35 x 1 mm slab gel, for 1 h. Only 2 microL of untreated human serum is required to obtain clear protein-distribution patterns, made visible by Coomassie Blue staining. By use of silver staining, proteins in unconcentrated cerebrospinal fluid can be made visible. An apparatus we devised for microscale two-dimensional electrophoresis enables us to analyze eight protein samples simultaneously.

Download Full-text

Computerized quantitative analysis of coomassie-blue-stained serum proteins separated by two-dimensional electrophoresis.

Clinical Chemistry ◽

10.1093/clinchem/35.12.2297 ◽

1989 ◽

Vol 35 (12) ◽

pp. 2297-2304 ◽

Cited By ~ 43

Author(s):

A Burgess-Cassler ◽

J J Johansen ◽

D A Santek ◽

J R Ide ◽

N C Kendrick

Keyword(s):

Serum Proteins ◽

Protein Isoforms ◽

Blue Staining ◽

Two Dimensional ◽

Two Dimensional Electrophoresis ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

Single Standard ◽

Microcomputer Software ◽

Dimensional Electrophoresis

Abstract Two-dimensional electrophoresis in combination with Coomassie Blue staining was refined for use as a quantitative method. Microcomputer software was developed for use with the IBM AT and compatible computers for analyzing the gels. To test the refined method to determine its usefulness in simultaneous measurements of 28 human serum proteins, we measured each protein relative to a single standard (bovine serum albumin) polymerized at different concentrations in a calibration scale, rather than using 28 individual standards. All samples were analyzed in triplicate. We evaluated calibration, linearity of response, recoveries, units, within-run CV, and between-run CV. The five isoforms of apolipoprotein A-I were analyzed in samples from 16 healthy donors and the isoform ratios determined. The method as presented here should prove useful for diagnosis of non-urgent disease states and for analysis for protein isoforms in relation to disease; it should also be applicable to assays of proteins in other fluids and tissues.

Download Full-text

Distribution of G-proteins in rat liver plasma-membrane domains and endocytic pathways

Biochemical Journal ◽

10.1042/bj2610905 ◽

1989 ◽

Vol 261 (3) ◽

pp. 905-912 ◽

Cited By ~ 28

Author(s):

N Ali ◽

G Milligan ◽

W H Evans

Keyword(s):

Plasma Membrane ◽

G Proteins ◽

Rat Liver ◽

Plasma Membranes ◽

Alpha Subunit ◽

Membrane Domains ◽

Liver Plasma Membrane ◽

Golgi Membranes ◽

Late Endosomes ◽

Plasma Membrane Domains

1. The distribution of the alpha- and beta-subunits of nucleotide-binding G-proteins among rat liver sinusoidal, lateral and canalicular plasma membranes, endosomes, Golgi membranes and lysosomes was investigated. 2. Pertussis-toxin-catalysed ADP-ribosylation identified a 41 kDa inhibitory alpha-subunit in all liver plasma-membrane functional domains as well as in endosomes. An antibody to a synthetic peptide corresponding to a C-terminal sequence of the inhibitory alpha-subunit also identified the 41 kDa polypeptide in all plasma-membrane domains, in ‘early’ and ‘late’ endosomes and in Golgi membranes; this polypeptide was not detected in lysosomes. The antibody-binding studies showed that bile-canalicular plasma membranes had the highest content of the inhibitory alpha-subunit. 3. Immunofluorescent microscopy confirmed the presence of the inhibitory alpha-subunit in all regions of the hepatocyte's cell surface. 4. An antibody recognizing the beta-subunit showed that a 36 kDa polypeptide was present in all plasma membranes and in ‘early’ and ‘late’ endosomes; it was not detected in lysosomes. The relative distribution among the fractions of this polypeptide was similar to the distribution of the inhibitory alpha-subunit. 5. The presence of high levels of the G-protein inhibitory alpha-subunit in bile-canalicular plasma membranes was confirmed by demonstration of its co-fractionation with marker enzymes in Nycodenz gradients and by free-flow electrophoresis. The significance of this location is discussed.

Download Full-text

Proteomic analysis ofEntamoeba histolytica

Parasitology ◽

10.1017/s0031182006001442 ◽

2006 ◽

Vol 134 (2) ◽

pp. 289-298 ◽

Cited By ~ 11

Author(s):

J. TOLSTRUP ◽

E. KRAUSE ◽

E. TANNICH ◽

I. BRUCHHAUS

Keyword(s):

Ubiquitin Proteasome System ◽

Blue Staining ◽

Two Dimensional Gel Electrophoresis ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

Peptide Mass ◽

Resultant Data ◽

Ubiquitin Proteasome ◽

Ph Range ◽

First Time

In this study, the proteome of axenically grownEntamoeba histolyticaparasites was explored by two-dimensional gel electrophoresis (2-DE), employing a practical and effective procedure for the solubilization ofE. histolyticaproteins. Approximately 900 protein species in the pH range between 4 and 7 were detected by Coomassie Blue staining. Ninety-five spots were excised, trypsinated and subjected to mass spectrometry. The resultant data from peptide mass fingerprints were compared with those available in theE. histolyticagenome and the (non-redundant) National Center for Biotechnology Information (NCBI) databases for the identification and categorization of proteins. Sixty-three of the proteins identified were predicted to relate to the cytoskeleton, surface, glycolysis, RNA/DNA metabolism, the ubiquitin-proteasome system, vesicular trafficking and signal transduction. The present study demonstrates, for the first time, that corresponding genes are indeed expressed inE. histolyticaand provides a foundation for further proteomic studies of this parasite.

Download Full-text

Purification and characterization of human liver dehydroepiandrosterone sulphotransferase

Biochemical Journal ◽

10.1042/bj2600641 ◽

1989 ◽

Vol 260 (3) ◽

pp. 641-646 ◽

Cited By ~ 128

Author(s):

C N Falany ◽

M E Vazquez ◽

J M Kalb

Keyword(s):

Affinity Chromatography ◽

Molecular Mass ◽

Human Liver ◽

Polyacrylamide Gel Electrophoresis ◽

Active Form ◽

Blue Staining ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

A Subunit

A form of sulphotransferase capable of sulphating dehydroepiandrosterone and other steroids was purified from cytosol prepared from human liver. Dehydroepiandrosterone sulphotransferase was purified 621-fold when compared with the activity in cytosol using DEAE-Sepharose CL-6B and adenosine 3′,5′-bisphosphate-agarose affinity chromatography. During affinity chromatography, dehydroepiandrosterone sulphation activity could be resolved from p-nitrophenol sulphation activity catalysed by phenol sulphotransferase by using a gradient of adenosine 3′-phosphate 5′-phosphosulphate. The purified enzyme was most active towards dehydroepiandrosterone but was capable of conjugating a number of other steroids, including pregnenolone, androsterone and beta-oestradiol. No activity towards p-nitrophenol or dopamine, substrates for the phenol sulphotransferase, was observed with the pure enzyme. A single band with a subunit molecular mass of 35 kDa was observed by Coomassie Blue staining following SDS/polyacrylamide-gel electrophoresis of the purified enzyme. A molecular mass of 68-70 kDa was calculated for the active form of the enzyme by chromatography on Sephacryl S-200, suggesting that the active form of the enzyme is a dimer.

Download Full-text

Membrane microdomains: lessons from microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140257 ◽

1995 ◽

Vol 53 ◽

pp. 776-777

Author(s):

J.M. Robinson ◽

J.M Oliver

Keyword(s):

Spatial Distribution ◽

Plasma Membrane ◽

Epithelial Cells ◽

Plasma Membranes ◽

Cell Types ◽

Imaging Modalities ◽

Membrane Microdomains ◽

Membrane Domains ◽

Lateral Heterogeneity ◽

Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

Download Full-text