scholarly journals Isolation of human platelet plasma membranes with polylysine beads.

1979 ◽  
Vol 82 (3) ◽  
pp. 688-696 ◽  
Author(s):  
T Kinoshita ◽  
R L Nachman ◽  
R Minick
Keyword(s):  
Gel Electrophoresis ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Human Platelet ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Marker Enzymes ◽  
Nitrophenyl Phosphate ◽  
Full Complement

Human platelet plasma membranes were isolated with polylysine beads according to the technique developed by Jacobson and Branton (1977, Science [Wash. D. C.] 195:302--304). Lactoperoxidase-catalyzed surface iodination revealed that ninefold greater 125I specific activity was associated with the membranes isolated on beads than with whole platelets. Enrichment in the bead membrane preparation of the activities of membrane marker enzymes, bis(p-nitrophenyl)phosphate phosphodiesterase and Na,K-ATPase, was 8.0 and 4.4, respectively. Contamination with enzymes of other organelles, cytochrome oxidase and beta-glucuronidase, was relatively low as compared with membranes isolated by sucrose gradient centrifugation. Analysis by SDS polyacrylamide gel electrophoresis showed that a full complement of surface glycoproteins was present on the membranes isolated with polylysine beads. The polylysine bead technique is a rapid, reproducible and efficient method for the preparation of relatively pure platelet plasma membranes.

Purification and Characterization of a 3,17 β-Hydroxysteroid Dehydrogenase from Streptomyces hydrogenans

1979 ◽  
Vol 34 (7-8) ◽  
pp. 533-540 ◽  
Author(s):  
Helmut Duchmann ◽  
Lothar Träger
Keyword(s):  
Gel Electrophoresis ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Sedimentation Coefficient ◽  
Hydroxysteroid Dehydrogenase ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization

3,17 β-Hydroxysteroid dehydrogenase has been enriched and purified from cytosol of Streptomyces hydrogenans. After ammonium sulfate precipitation and filtration on Sephadex G-100 the enzyme was finally purified by preparative gel electrophoresis and DEAE-Sephadex A-50 chro­matography. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate gave a single band of mobility corresponding to molecular weight of 70 200 ± 2 500. 3 β-. 17 β- as well as 20 β-hydroxy steroids were dehydrogenated by the enzyme in the presence of NAD+. The dehydrogenation proceeded faster than the reduction of the corresponding ketosteroids in the presence of NADH. The enzyme does not accent NADP+ or NADPH as co-substrates. The apparent Km values were calculated to be 11 μᴍ for 5 α-dihydrotestosterone, 20 μᴍ for testosterone ana 68 μᴍ for epiandrosterone in the NAD+-driven reaction, 1.8 x 10-4 m for NADH+ and 1.9 x 10-4 ᴍ for NADH. The catalytic activity was influenced by the ratio of NAD+/ATP. The inhibition by ATP appears to be of a competitive type with respect to NAD+ (Ki 1.15 x 10-3 ᴍ).After sucrose gradient centrifugation in a preparative ultracentrifuge the enzyme sediments with 4.1 ± 0.1 S as estimated in comparison to other proteins of known sedimentation coefficient. The isoelectric point was determined to be 3.9 with the LKB preparative isoelectric focusing col­umn (pH 2-11) and 4.1 with the analytical flat bed polyacrylamide isofocusing (pH 3 - 5). The number of SH groups was determined to be 2 mol/mol enzyme. In the presence of 6 M urea the fig­ure inceases to 3 mol SH/mol enzyme. In the presence of an excess of p-chloromercuribenzoate the enzyme activity decreases only partially.

scholarly journals Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue

1984 ◽  
Vol 219 (1) ◽  
pp. 301-308 ◽  
Author(s):  
A A Davies ◽  
N M Wigglesworth ◽  
D Allan ◽  
R J Owens ◽  
M J Crumpton
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Insoluble Fraction ◽  
Insoluble Residue ◽  
Protein Ratio

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5′-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.

scholarly journals Purification and some properties of tartrate-sensitive acid phosphatase from rabbit kidney cortex

1978 ◽  
Vol 175 (1) ◽  
pp. 321-329 ◽  
Author(s):  
J J Helwig ◽  
A A Farooqui ◽  
C Bollack ◽  
P Mandel
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Hydrolysis Rate ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Basic Form ◽  
Nitrophenyl Phosphate ◽  
Acidic Form

Two forms of tartrate-sensitive acid phosphatases (EC 3.1.3.2) were purified from rabbit kidney cortex by a multiple-column-chromatography method. The basic form constituted 90% of the enzyme and migrated as a single band of protein on polyacrylamide-gel electrophoresis. The proteins contaminating the acidic form did not exceed 5% of the total protein. The specific activity towards p-nitrophenyl phosphate was 12 mumol/min per mg for the basic form and 0.7 mumol/min per mg for the acidic form. The basic form of the enzyme differs from the acidic form in its heat-stability, Km values, inhibition rates by tartrate and fluoride and substrate specificities. Relative to p-nitrophenyl phosphate hydrolysis rate, the acidic form hydrolysed a variety of physiological monophosphate esters, whereas the basic form hydrolysed only CMP and phosphoenolpyruvate. Bacterial neuraminidases had no effect on the activity and mobility of the acidic form on polyacrylamide-gel electrophoresis. Both forms have the same molecular weight (101000 +/- 4000) and are probably composed of two identical subunits. The question whether the two forms of the enzyme are different proteins or whether one is a modified form of the other is discussed.

Kinetic and electrophoretic studies on acid and alkaline phosphatases of the metacercariae of Clinostomum complanatum (Trematoda: Digenea)

1982 ◽  
Vol 56 (1) ◽  
pp. 17-22 ◽  
Author(s):  
Afzal A. Siddiqui ◽  
Wajih A. Nizami
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Arsenate ◽  
Alkaline Phosphatases ◽  
Acid And Alkaline Phosphatases ◽  
Nitrophenyl Phosphate ◽  
Two Peaks ◽  
Hydrolysis Of ◽  
Clinostomum Complanatum

ABSTRACTHomogenates of the metacercariae of Clinostomum complanatum showed two peaks for the hydrolysis of p-nitrophenyl phosphate at pH 4·5–5·0 and 11·0; specific activity at pH 5·0 was 0·017 µmol p-nitrophenol/mg protein/min and 0·0049 µmol p-nitrophenol/mg protein/min at pH 11·0 at 37°C. Maximal activities of acid and alkaline phosphatases were observed at 50° and 40°C respectively. Both enzymes reached maximal rates after 50 min of incubation. Linearity in enzyme activity was noticed with increasing homogenate concentrations for both enzymes. The Km for p-nitrophenyl phosphate was 1·1 mM with a Vmax of 0·018 units/mg protein for acid phosphatase and 1·6 mM with a Vmax of 0·0052 units/mg protein for alkaline phosphatase. Sodium arsenate and sodium fluoride inhibit and Mg++ and Co++ stimulate both enzymes. Polyacrylamide gel electrophoresis showed one cathodic band each for both the enzymes.

scholarly journals Human liver alkaline phosphatase purified by affinity chromatography, ultracentrifugation and polyacrylamide-gel electrophoresis

1976 ◽  
Vol 159 (3) ◽  
pp. 697-705 ◽  
Author(s):  
A L Latner ◽  
A W Hodson
Keyword(s):  
Alkaline Phosphatase ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Human Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Nitrophenyl Phosphate ◽  
Hydrolysis Of

A method is presented for the preparation of human liver alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1). The method gives a purification factor of 12.5 × 10(3) over the initial aq. butan-1-ol extract, a recovery of 6.0% and a specific activity for the preparation of 1450-1550 units/mg of protein, 1 unit being defined as the amount of enzyme catalysing the hydrolysis of 1mumol of p-nitrophenyl phosphate/min at 35 degrees C in 0.1 M-2-amino-2-methylpropan-1-ol/HCl buffer, pH 10.5, containing 10mM-p-nitrophenyl phosphate. Homogeneity was studied by ultracentrifugation, by immunoelectrophoresis and by polyacrylamide-gel electrophoresis. A single contaminating protein was present which was less than 5% of the total. Ultracentrifugation and equilibrium-gradient-pore electrophoresis techniques indicated a mol.wt. of 156000 and 160000 respectively. Equilibrium-gradient-pore electrophoresis indicated that the alkaline phosphatase molecule is possibly a dimer, comprising two subunits of about 80000 mol.wt. Amino acid analysis proved remarkably similar to that for alkaline phosphatase from other sources, regardless of species.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

scholarly journals LYSOSOMAL PHOSPHOLIPASES A1 AND A2 OF NORMAL AND BACILLUS CALMETTE GUERIN-INDUCED ALVEOLAR MACROPHAGES

1973 ◽  
Vol 56 (3) ◽  
pp. 621-627 ◽  
Author(s):  
Richard C. Franson ◽  
Moseley Waite
Keyword(s):  
Acid Phosphatase ◽  
Alveolar Macrophages ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Marker Enzymes ◽  
Sucrose Gradient Centrifugation ◽  
Control Distribution ◽  
Specific Activities ◽  
Selective Increase ◽  
Phospholipases A

A single intravenous injection of 0.1 mg of heat-killed Bacillus Calmette Guérin (BCG) in 0.1 ml of Bayol F produced an accumulation of activated alveolar macrophages (BCG induced). Cells were collected 3.5–4.0 wk after injection. Phospholipases A and three lysosomal marker enzymes (acid phosphatase, ß-glucuronidase, and lysozyme) were measured in homogenates, and the distribution of the phospholipases A and lysosomal, mitochondrial, and microsomal marker enzymes were examined after sucrose gradient centrifugation of a postnuclear (1,000 g) supernatant. Homogenates of normal and BCG-induced macrophages contained phospholipases A1 and A2 which had optimal activity at pH 4.0 in the presence of 2.0 mM ethylenediaminetetraacetate (EDTA). These activities were inhibited 50–70% by 2.0 mM CaCl2. Homogenates of BCG-induced macrophages had specific activities of ß-glucuronidase, acid phosphatase, and lysozyme, which were increased 1.5- to 3.0-fold over the controls, whether expressed as activity per mg protein or activity per 107 cells. The specific activities of the phospholipases A, on the other hand, were consistently lower than those of the control. Distribution of the phospholipases A and the lysosomal marker enzymes after sucrose gradient centrifugation suggested that the phospholipases A active at pH 4.0 in the presence of EDTA are of lysosomal origin since: (a) BCG treatment caused a selective increase in the density of particles which contained both the phospholipases A and three lysosomal marker enzymes; and (b) since the density of mitochondria and microsomes were not affected by BCG treatment. The increase in the density of lysosomes seen here may be related to previously described morphologic changes of BCG-induced alveolar macrophages.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

scholarly journals Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells

1986 ◽  
Vol 236 (3) ◽  
pp. 665-670 ◽  
Author(s):  
W P Gati ◽  
J A Belt ◽  
E S Jakobs ◽  
J D Young ◽  
S M Jarvis ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hepatoma Cells ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
Animal Cells ◽  
Novikoff Hepatoma

Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.

Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase

1973 ◽  
Vol 51 (11) ◽  
pp. 1551-1555 ◽  
Author(s):  
Tony C. M. Seah ◽  
A. R. Bhatti ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Protein ◽  
Wild Type ◽  
Major Band ◽  
Subunit Molecular Weight ◽  
Purification And Properties

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

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