scholarly journals Immunochemical characterization of proteins from mouse liver plasma membranes

1973 ◽  
Vol 135 (4) ◽  
pp. 827-832 ◽  
Author(s):  
James W. Gurd ◽  
W. Howard Evans ◽  
Harold R. Perkins
Keyword(s):  
Mouse Liver ◽  
Plasma Membranes ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Extraction Procedure ◽  
Relative Distribution ◽  
Two Dimensional ◽  
Nucleotidase Activity ◽  
Triton X

1. Antiserum was prepared in rabbits against a purified mouse liver plasma-membrane fraction. 2. The antiserum was made to react with an125I-labelled alkaline-EDTA extract of the plasma membranes, and the immunoprecipitate analysed by polyacrylamide-gel electrophoresis. Seven proteins were immunoprecipitated and a single glycoprotein present in the alkaline-EDTA-soluble fraction was found to be a major component. 3. The alkaline-EDTA-soluble fraction was analysed by two-dimensional immunoelectrophoresis and this procedure indicated the presence of six antigenic components. 4. The plasma membranes were also extracted with 1% deoxycholate–1% Triton X-100; 50% of the protein, 80% of the alkaline phosphodiesterase activity and 30% of the 5′-nucleotidase activity were solubilized. 5. Two-dimensional immunoelectrophoresis of the deoxycholate–Triton X-100 extract indicated the presence of six antigens. 6. The relative distribution of the six antigens among the fractions obtained during the extraction procedure was examined immunoelectrophoretically to provide information on their disposition within the membrane.

scholarly journals Purification and characterization of a major glycoprotein in rat hepatoma plasma membranes. One of the membrane proteins released by phosphatidylinositol-specific phospholipase C

1987 ◽  
Vol 241 (1) ◽  
pp. 63-70 ◽  
Author(s):  
Y Ikehara ◽  
Y Hayashi ◽  
S Ogata ◽  
A Miki ◽  
T Kominami
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase C ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Soluble Form ◽  
Microsomal Membranes ◽  
Hydrophobic Nature ◽  
Rat Hepatoma

A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen.

scholarly journals Surface glycoproteins of human spermatozoa

1986 ◽  
Vol 82 (1) ◽  
pp. 11-22
Author(s):  
M. Kallajoki ◽  
I. Virtanen ◽  
J. Suominen
Keyword(s):  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Spermatozoa ◽  
Galactose Oxidase ◽  
Sperm Cells ◽  
Neuraminidase Treatment ◽  
Triton X

The surface membrane glycoprotein composition of human spermatozoa has been studied by introducing radioactive label into galactosyl (Gal) and N-acetylgalactosaminyl (GalNAc) residues by using the galactose oxidase/NaB3H4 method. Triton X-100 extracts and Triton X-100-resistant cytoskeletal residues were subjected to analysis by polyacrylamide gel electrophoresis. The distribution of the radiolabel in sperm cells was studied by light-microscopic auto-radiography. The grains were evenly distributed on the cells by the labelling methods used. The Triton X-100 treatment did not affect sperm morphology at the light-microscopic level, but in transmission electron microscopy the plasma membrane covering the acrosome was removed totally, together with most of the acrosomal membranes and acrosomal contents. Plasma membrane residues were, however, always found in the postacrosomal region. Borohydride alone without oxidative pretreatment labelled two polypeptides of molecular weights (Mr) 48,000 and 43,000 in the Triton X-100-soluble fraction. When the Gal/GalNAc residues were labelled by galactose oxidase pretreatment 120,000, 105,000, 78,000 and 68,000 Mr glycoproteins were revealed. When additional neuraminidase treatment was used to remove terminal sialic acid residues, the total labelling intensity was increased two- to fivefold and additional 36,000 and 20,000 Mr glycoproteins were revealed. The Triton X-100-resistant cytoskeletal residue contained 53–75% of the total radioactivity bound in sperm cells. When these components were analysed by polyacrylamide gel electrophoresis, all the major bands found in the Triton X-100-soluble fraction were detected and also some radioactivity was incorporated into the major bands visualized by protein staining. In the present study we describe several human sperm glycoproteins, which seem to be distributed evenly on the sperm cells. Detergent extraction, producing cytoskeletal models, appeared to leave most of the glycoproteins detectable in the extraction residues also with the apparent enrichment of a single 68,000 Mr glycoprotein.

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

scholarly journals Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles

1987 ◽  
Vol 244 (1) ◽  
pp. 219-224 ◽  
Author(s):  
J M Jacobs ◽  
N J Jacobs
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chlorophyll Synthesis ◽  
Yeast Mitochondria ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Haem Biosynthesis ◽  
Triton X ◽  
Polypeptide Band

The protoporphyrinogen-oxidizing enzyme from Triton X-100 extracts of the mitochondrial and etioplast fractions of etiolated barley was purified by using ion-exchange and hydroxyapatite chromatography. The purified enzyme from both organelle fractions exhibited a Km of 5 microM and was labile to mild heat and acidification. The pH optimum (5-6) and the substrate-specificity (mesoporphyrinogen was oxidized as rapidly as protoporphyrinogen) revealed properties very different from the protoporphyrinogen-oxidizing enzyme of rat liver or yeast mitochondria, which is specific for protoporphyrinogen as substrate. The purest fractions showed a polypeptide band corresponding to an Mr of approx. 36,000 on SDS/polyacrylamide-gel electrophoresis. This is the first purification and characterization of the enzyme from a plant, and indicates no readily detectable differences between the enzyme isolated from mitochondrial or etioplast fractions, although only the latter organelle has the capacity for both haem and chlorophyll synthesis.

scholarly journals Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits

1986 ◽  
Vol 239 (3) ◽  
pp. 679-683 ◽  
Author(s):  
A McElduff ◽  
A Watkinson ◽  
J A Hedo ◽  
P Gorden
Keyword(s):  
Insulin Receptor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Beta Subunits ◽  
Relative Distribution ◽  
Single Chain ◽  
Complex Type ◽  
Mannose Residue ◽  
Predominant Species ◽  
High Mannose

The insulin receptor is synthesized as a 190,000-Mr single-chain precursor that contains exclusively asparagine-N-linked high-mannose-type carbohydrate chains. In this study we have characterized the structure of the pro-receptor oligosaccharides. IM-9 lymphocytes were pulse-chase-labelled with [3H]mannose, and the insulin pro-receptor was isolated by immunoprecipitation and SDS/polyacrylamide-gel electrophoresis. The pro-receptor oligosaccharides were removed from the protein backbone with endoglycosidase H and analysed by h.p.l.c. Immediately after a [3H]mannose pulse the largest oligosaccharide found in the pro-receptor was Glc1Man9GlcNAc2; this structure represented only a small fraction (3%) of the total. The predominant oligosaccharides present in the pro-receptor were Man9GlcNAc2 (25%) and Man8GlcNAc2 (48%). Smaller oligosaccharides were also detected: Man7GlcNAc2 (18%), Man6GlcNAc2 (3%) and Man5GlcNAc2 (3%). The relative distribution of the different oligosaccharides did not change at 1, 2 or 3 h after the pulse with the exception of the rapid disappearance of the Glc1Man9GlcNAc2 component. The mature alpha- and beta-subunits of the insulin receptor are known to contain both high-mannose-type and complex-type oligosaccharides. We have also examined here the structure of the high-mannose chains of these subunits. The predominant species in the alpha-subunit was Man8GlcNAc2 whereas in the beta-subunit it was Man7GlcNAc2. These results demonstrate that most (approx. 75%) oligosaccharides of the insulin pro-receptor are chains of the type Man8GlcNAc2 or Man9GlcNAc2. Thus, assuming that a Glc3Man9GlcNAc2 species is transferred co-translationally, carbohydrate processing of the pro-receptor appears to be very rapid and limited to the removal of the three glucose residues and one mannose residue. Further mannose removal does not occur until the pro-receptor has been proteolytically cleaved. In addition, the degree of mannose trimming appears to be different in the alpha- and beta-subunits.

Timed photoaffinity labeling and characterization of bile acid binding and transport proteins in rat ileum

1993 ◽  
Vol 265 (1) ◽  
pp. G56-G62 ◽  
Author(s):  
M. C. Lin ◽  
E. Mullady ◽  
F. A. Wilson
Keyword(s):  
Bile Acid ◽  
Microsomal Fraction ◽  
Flash Photolysis ◽  
Basolateral Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Microsomal Protein ◽  
Bile Acid Binding ◽  
Triton X

Rat ileal enterocytes were radiolabeled by flash photolysis with a photolabile derivative of taurocholate (7,7-azo-[3H]TC) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal labeling of the bile acid binding proteins (BABPs) was achieved between 15 and 90 s. When enterocytes were pulsed with 7,7-azo-[3H]TC for 2 min, and then 0.5 mM TC was added to chase the radiolabel, the radioactivity in the BABPs was displaced by 50% after 2 min. The 99-kDa brush-border membrane (BBM) protein had the highest initial labeling rate, followed by 43-kDa actin, 35- and 14-kDa cytosolic proteins, 54-kDa basolateral membrane (BLM) protein, 59-kDa BLM-associated protein, and 20-kDa microsomal protein. When a mixed microsomal and cytosolic fraction was photolabeled with 7,7-azo-[3H]TC and then separated, the 20-kDa microsomal protein was labeled. However, if the microsomal fraction alone was photolabeled, the 20-kDa protein was not labeled, suggesting this protein required a cytosolic cofactor for labeling. Using Triton X-114 phase separation and EDTA extraction, the BABPs were separated into amphiphilic integral membrane proteins (99- and 54-kDa proteins) and hydrophilic proteins (14-, 35-, 43-, and 59-kDa proteins). From these data, a model is proposed for transcellular bile acid transport in rat ileal enterocytes.

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