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

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.

Effect of testicular hormones on synthesis of soluble proteins by dog prostate slices

1983 ◽  
Vol 61 (7) ◽  
pp. 756-763 ◽  
Author(s):  
Jean Y. Dubé ◽  
Pierre Chapdelaine ◽  
Roland R. Tremblay
Keyword(s):  
Seminal Plasma ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Blue Staining ◽  
Secretory Proteins ◽  
Molecular Weights ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

We have submitted adult mongrel dogs to various endocrine manipulations. Prostate slices from these animals were then incubated in vitro in the presence of [3H]leucine or [35S]methionine. We have analyzed the cytosolic proteins by polyacrylamide gel electrophoresis. In intact adult uncastrated dogs, the radioactive amino acids were incorporated into three major bands having respective molecular weights (MW) of 32 000,16 000, and 15 000 in one-dimensional gels in presence of sodium dodecyl sulfate and mercaptoethanol. Two-dimensional electrophoresis revealed heterogeneity of each of these bands, both in isoelectric focussing (IEF) or nonequilibrium pH gel electrophoresis (NEpHGE) conditions. The 32 000 MW proteins showed five to six major radioactive spots and the 15 000 – 16 000 MW proteins showed six to seven spots by IEF. However, the highest incorporation of radioactivity occurred in a 16 000 MW protein seen only in NEpHGE. The lower MW proteins corresponded to some of the major proteins of dog seminal plasma as observed by immunoprecipitation of prostate proteins with antibodies against whole seminal plasma. By contrast, the 32 000 MW proteins were minor proteins of prostate cytosol and seminal plasma by Coomassie blue staining. Castration for 2 weeks completely abolished the synthesis of all these proteins. When castrated animals were treated with 5α-androstane-3α-17β-diol (10 mg/day for 2 weeks), the pattern of protein synthesis returned to the one observed in intact uncastrated animals. These observations show that testicular androgens control the synthesis of dog prostate major secretory proteins.

The reactivity to N-ethyl maleimide of the subunits of cytochrome oxidase

1977 ◽  
Vol 55 (9) ◽  
pp. 988-994 ◽  
Author(s):  
A. McGeer ◽  
B. Lavers ◽  
G. R. Williams
Keyword(s):  
Electron Transport ◽  
Cytochrome Oxidase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Blue Staining ◽  
Beef Heart ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Beef heart cytochrome oxidase (EC 1.9.3.1) prepared in this laboratory consistently presents 10 Coomassie blue staining zones on SDS–polyacrylamide gel electrophoresis. At pH 7.0 only two of these polypeptides (III and VIa) are labelled by radioactive N-ethyl maleimide (NEM). The labelling of VIa is variable and correlates with the activity of particular oxidase preparations. When cytochrome oxidase is isolated from alkylated membranes, either mitochondria or electron transport particles, polypeptide VIa is found not to be labelled; polypeptide III is more strongly labelled than when isolated oxidase is alkylated, and label now appears in polypeptide I which is not alkylated upon treatment of isolated oxidase with NEM.

scholarly journals Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1212-1219 ◽  
Author(s):  
N Kieffer ◽  
B Boizard ◽  
D Didry ◽  
JL Wautier ◽  
AT Nurden
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Blue Staining ◽  
Igg Antibody ◽  
Immune Disorder ◽  
Immunochemical Characterization ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Abstract We report the immunochemical characterization of a new platelet- specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.

Characterization of PIG Platelet Granule Proteins

1979 ◽  
Author(s):  
M.H. Fukami ◽  
J.L. Daniel ◽  
J.S. Bauer
Keyword(s):  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dense Granule ◽  
Release Mechanism ◽  
Molecular Weights ◽  
Dense Granules ◽  
Coomassie Blue ◽  
Pas Staining

Platelet granules contain glycoproteins similar to those found in platelet membranes (Hagen et al , BBA , 445, 21 4 , 1 976 ). Pig platelet granule fractions enriched in mitochondria, α-granules or dense granules were analyzed by SDS Polyacrylamide gel electrophoresis to determine if there are differences among the organelles. In a reduced system (5Ϊ OTT) the proteins of the ï-granules and dense granules showed staining patterns with Coomassie blue that were distinctly different from whole platelets, isolated membranes or mitochondria. In the granules about 10 to 12 bands with less mobility than actin were visualized. Staining with PAS was obtained in bands with apparent molecular weights of 250, 225, 185, 170, 150, 120, 55, 4B and 40 K. The 185 K band appeared to be the same as “thrombin sensitive protein”. The mobility of the 55 and 48 K hands were identical with the B (B) and γ-bands of bovine fibrinogen. The PAS staining of the granule components was more intense than that of whole platelets for the same amount of protein, indicating that granule membranes may be as rich in glycoproteins as external plasma membranes. With both PAS and Coomassie blue, the a-granule and dense granule staining patterns were almost identical. This observation may be relevant to recent studies which showed that both granule types exhibited similar release characteristics, suggesting that they share a common release mechanism. NIH-JSPHS Grant No. 14217

Oviductal fluid protein patterns in the rhesus monkey (Macaca mulatta) during the menstrual cycle

1992 ◽  
Vol 4 (2) ◽  
pp. 249 ◽  
Author(s):  
A Paliwal ◽  
B Malaviya ◽  
VP Kamboj
Keyword(s):  
Menstrual Cycle ◽  
Protein Profile ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Blue Staining ◽  
Protein Patterns ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Oviductal Fluid

Oviducts were obtained from monkeys on Days 8, 14, 19 and 25 of the menstrual cycle and changes in the pattern of luminal fluid proteins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis after periodic acid Schiff's reagent (PAS) and coomassie blue staining of the gels revealed 85 and 95 kDa proteins only up to Day 14 whereas a 130 kDa glycoprotein persisted up to Day 19 and reached a nadir at mid-menstrual cycle (Day 14). The absence of the 130 kDa glycoprotein in the serum and its presence in cytosolic preparations up to Day 19 suggest that it is of oviductal origin. The 130 kDa glycoprotein is of particular interest since it was present in the oviductal fluid during mid cycle, a period when the oviduct participates in gamete transport, fertilization and embryo development. The conclusion drawn from this study is that the protein profile of monkey oviductal fluid changes during the menstrual cycle.

Simplified sodium dodecyl sulfate-polyacrylamide gel electrophoresis of unconcentrated urine with enhanced resolution and detection sensitivity.

1987 ◽  
Vol 33 (10) ◽  
pp. 1886-1887 ◽  
Author(s):  
T Marshall ◽  
K M Williams
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Detection Sensitivity ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Dodecyl Sulfate

Abstract We applied a simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis method to urine. The method, developed for serum protein analysis (Clin Chem 1984;30:475-9), has a high sample throughput and gives excellent resolution with unconcentrated urine. It clearly distinguishes and characterizes proteinuric urine (7.5 microL) by Coomassie Blue staining and gives complex silver-stained patterns with nonproteinuric urine (2 microL). The former is recommended for routine clinical screening, the latter for research purposes.

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

1990 ◽  
Vol 271 (1) ◽  
pp. 171-178 ◽  
Author(s):  
C Enrich ◽  
P Tabona ◽  
W H Evans
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Plasma Membranes ◽  
Blue Staining ◽  
Membrane Domains ◽  
Two Dimensional ◽  
Liver Plasma Membrane ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Ph Range

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.

The Reduced Aggregation Response Of Bernard-Soulier Platelets To Thrombin May Be Related To An Abnormal Glycoprotein V

1981 ◽  
Author(s):  
A T Nurden ◽  
D Dupuis
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Surface Proteins ◽  
Galactose Oxidase ◽  
Blue Staining ◽  
Lag Phase ◽  
Coomassie Blue Staining ◽  
Aggregation Response ◽  
Coomassie Blue ◽  
Sds Page

Both platelet membrane GP Ib and GP V have been proposed as receptors for the activation of human platelets by thrombin. Bernard-Soulier (B-S) platelets exhibit a reduced aggregation response to thrombin with a lag phase that precedes aggregation. When B-S platelets, whose surface proteins had been labelled with (125I), were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by PAS-staining, Coomassie blue staining or autoradiography, the apparent absence of GP Ib and the normal presence of GP IIb, IIIa and IIIb was demonstrated. On the basis of such studies several authors have stated that “GP I” is the thrombin receptor. However, GP V is not located by the above procedure, requiring more sensitive analytical methods for its detection. To meet this requirement washed platelets isolated from 3 B-S patients have been treated sequentially with neuraminidase, galactose oxidase and sodium(3H,)-boro- hydride. The labelled platelets were analysed by SDS-PAGE using 7-12% gradient acrylamide gels and the (3H,)-labelled GP’s located by fluorography. In addition to the GP Ib defect the platelets of each B-S patient were lacking the band corresponding to GP V of normal platelets. In agreement with previous studies we observed that when (3H,)-labelled normal human platelets were incubated with thrombin GP V (Mr=82,000) was hydrolysed,and that this was accompanied by the appearance of a labelled glycopeptide (Mr=69,500) in the supernatant. When (3H)-labelled B-S platelets were treated with thrombin no labelled glycopeptide was located. GP V could therefore be either absetit from B-S platelets or have a modified carbohydrate composition rendering it insensitive to the analytical procedure used. Interpretations into the reduced aggregation response of B-S platelets to thrombin should be extended to include a possible GP V defect.

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

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.

Studies on the Biochemistry of Urokinase

1977 ◽  
Vol 38 (04) ◽  
pp. 0801-0808 ◽  
Author(s):  
Eng Bee Ong ◽  
Mercedes E. Soberano ◽  
Alan J. Johnson ◽  
Guenther Schoellmann
Keyword(s):  
Affinity Chromatography ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Blue Staining ◽  
Single Chain ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Sds Page ◽  
Excess Substrate

SummaryDirect evidence for an active center histidine residue in urokinase (UK) was obtained with use of newly synthesized peptide chloroketones Ac-Gly-Lys-CH2C1 and Nle-Gly-Lys-CH2C1. Stoichiometric inactivation by DFP provided further evidence that UK is a serine protease. Essential histidine and serine residues were both located in the heavy chain of the 47,0 M. W.UK. The high M.W. form can be converted (catalytically) to the low M. W. form.9 partially purified human urinary UK preparations (5 with predominantly high M. W. UK), varying in purity and proportion of high and low M. W. forms, were found to be heterogeneous by a number of acrylamide electrophoretic procedures. 7 preparations had strikingly similar molar activities at excess substrate, except for the lower values found in 2 predominantly high M. W. UK preparations from the same supplier. 2 high M. W. UK preparations from another supplier showed a definite increase in activity when assayed at low plasminogen concentration, but this effect was abolished after gel filtration (Sephadex G-25), by further purification with affinity chromatography, or when assayed with excess plasminogen.The high and low M. W. forms of UK (47,000 and 33,400 M. W.), isolated and purified by Sepharose-EACA-agmatine affinity chromatography were shown to be homogeneous by Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis (PAGE) and by 14C-DFP and 14C-NPGB incorporation before SDS-PAGE. Comparative properties of the high M. W. vs low M. W. forms were as follows: specific activity (104,000 IU/mg vs 226,000) ; 2 chains (33,100 and 18,600 M.W.) linked by disulfide(s) vs a single chain; pi 8.60 (major subform) and pi 8.90 (minor subform) vs pi 8.35, 8.60, 8.70 (major subform) and pi 8.05 (minor subform); and second order kinetics for DFP inactivation (400 vs 770 M−1 min−1). The molar activities were similar (9.6 × 109 and 10.2 × 109IUm/mole) for each form.

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