scholarly journals Further properties and possible mechanism of action of adenosine 5′-triphosphate–d-glucose 6-phosphotransferase from rat liver

1967 ◽  
Vol 105 (2) ◽  
pp. 473-482 ◽  
Author(s):  
M. J. Parry ◽  
D G Walker
Keyword(s):  
Catalytic Properties ◽  
Gel Filtration ◽  
Thiol Group ◽  
Acceptor Molecule ◽  
Magnesium Ions ◽  
Purification Procedure ◽  
Inhibitory Effects ◽  
Bivalent Ions ◽  
Low Glucose ◽  
Yeast Hexokinase

1. Magnesium ions are the most effective bivalent ions in the glucokinase reaction. 2. The molecular weight of rat hepatic glucokinase is 48000–49000 as assessed by gel filtration on Sephadex G-100. 3. Anomalous kinetic behaviour at low glucose concentrations appears to be due to the formation during the purification procedure of fragments possessing modified catalytic properties, but is unlikely to be of physiological significance. 4. Extension of previous studies (Parry & Walker, 1966) suggests that glucokinase catalyses a reaction of the random Bi Bi type similar to that of yeast hexokinase. 5. The inhibitory effects of various thiol reagents suggest that a thiol group may be involved at or near the binding site of the acceptor molecule.

scholarly journals Production and comparison of mature single-domain ‘trefoil’ peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58

1995 ◽  
Vol 308 (3) ◽  
pp. 1001-1007 ◽  
Author(s):  
M P Chadwick ◽  
F E B May ◽  
B R Westley
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Gel Filtration ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Exchange Chromatography ◽  
Single Domain ◽  
Trefoil Peptides ◽  
Trefoil Peptide

The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the ‘trefoil’ family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2.

Na+-K+-ATPase-G protein coupling in myocardial sarcolemma: separation and reconstitution

1991 ◽  
Vol 261 (4) ◽  
pp. L87-L91
Author(s):  
Mikhail P. Danilenko ◽  
Vera C. Turmukhambetova ◽  
Oleg V. Yesirev ◽  
Vsevolod A. Tkachuk ◽  
Mikhail P. Panchenko
Keyword(s):  
Atpase Activity ◽  
Pertussis Toxin ◽  
Inhibitory Concentration ◽  
Gel Filtration ◽  
Protein S ◽  
Half Maximum ◽  
Inhibitory Effects ◽  
Cardiac Sarcolemma ◽  
Dependent Inhibition ◽  
Zwitterionic Detergent

The cholinergic agonist carbachol produces a concentration-dependent (half-maximum inhibitory concentration = 0.9 μM) decrease in the Na+-K+-adenosine triphosphatase (ATPase) activity of rabbit cardiac sarcolemma that occurred only in the presence of guanosine 5'-[ggr-thio]triphosphate (0.1 μM GTPggrS) and reached 40% inhibition. The inhibition is blocked by the muscarinic receptor antagonist atropine (10 μM) and is abolished in sarcolemma treated with pertussis toxin (20 μg/ml) in the presence of 100 μM NAD. GTPggrS alone reduces Na+-K+-ATPase activity by 45% (half-maximum inhibitory = 1 μM). The apparent affinity of the enzyme for GTPgγS is increased ≈10-fold in the presence of 1 μM carbachol. In sarcolemma solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS, 10 mM), the GTPgγS-dependent inhibition of the Na+-K+-ATPase is also observed. Gel filtration of a CHAPS extract of sarcolemma on a Sepharose CL-6B column resulted in a separation of Na+-K+-ATPase and pertussis toxin-sensitive Gi activities. Na+-K+-ATPase activity that was separated on the column lost its sensitivity to the inhibitory action of guanine nucleotides. Inhibitory effects (20–30%) of guanosine 5'-triphosphate analogues [Gpp(NH)p, GTPggrS, or Gpp(CH2)p] at micromolar concentrations were restored when the Na+-K+-ATPase activity was recombined with fractions that contained the pertussis toxin-sensitive Gi protein(s). Similar concentrations of guanosine 5'-triphosphate, guanosine 5'-diphosphate, guanosine-5' -[beta-thio]diphosphate, or App(NH)p were unable to induce the Gi protein-mediated attenuation of Na+-K+-ATPase activity in the reconstitution system. These results suggest that a pertussis toxin-sensitive Gi protein may act as a transducer of the inhibitory hormonal signals on Na+-K+-ATPase in the sarcolemma. cardiac sarcolemma

scholarly journals Cathepsin B, the lysosomal thiol proteinase of calf liver

1969 ◽  
Vol 114 (4) ◽  
pp. 673-678 ◽  
Author(s):  
O. Snellman
Keyword(s):  
Cathepsin B ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Chelating Agent ◽  
Thiol Group ◽  
Maximum Activity ◽  
Active Centre ◽  
Peptide Bonds ◽  
Hydrolysis Of ◽  
Thiol Proteinase

Cathepsin B from calf liver was obtained by a method involving preparation of a lysosomal–mitochondrial pellet and treatment of this pellet with acetone. The material was extracted with an acid buffer, pH4·0, and then precipitated from the extract with acetone. The precipitate was dissolved in phosphate buffer, pH7·4, and subjected to gel filtration on Sephadex G-200 and G-100. The cathepsin B emerged in a range of molecular weight much lower than 50000 as a well-defined component. The purity of this material was checked by electrophoresis. To obtain maximum activity the enzyme had to be activated with a chelating agent and a reducing agent (i.e. EDTA and cysteine). A number of different substrates were used. The enzyme was active for the hydrolysis of both peptide bonds and ester bonds and had approximately equal reactivity in the two cases. The pH-dependence of the hydrolysis was the same with both substrates. The binding of the substrates was half-maximal at pH4·5 and at pH6·8. A thiol group occurred in the active centre but this group ought to have a much higher pK than that found in this enzyme.

A three step purification procedure for human liver ferritin

1980 ◽  
Vol 58 (6) ◽  
pp. 494-498 ◽  
Author(s):  
M. Pagé ◽  
J. Lagueux ◽  
C. Gauthier
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Purification Procedure ◽  
Human Liver Ferritin ◽  
Normal Human Liver ◽  
Normal Human ◽  
Liver Ferritin

We describe a method for the purification of normal human liver ferritin by ultrafiltration, gel filtration on Sephacryl S-300, and affinity chromatography on DEAE-Affi Gel Blue. The purity of the ferritin obtained was verified by immunoelectrophoresis, Ouchterlony immunodiffusion, polyacrylamide gel electrophoresis, and electrofocusing. This rapid method yields 32% of the original ferritin.

scholarly journals Neutral maltase/glucoamylase from rabbit renal cortex

1989 ◽  
Vol 261 (1) ◽  
pp. 43-47 ◽  
Author(s):  
B Pereira ◽  
S Sivakami
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Renal Cortex ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Rabbit Kidney ◽  
Purification Procedure ◽  
Glucoamylase Activity ◽  
Maltase Activity ◽  
Triton X

Maltase activity (EC 3.2.1.20) was solubilized from rabbit kidney brush-border membrane by using 1.0% Triton X-100 and purified 230-fold with an overall recovery of 30%. The purification procedure makes use of heat precipitation, chromatography on DE-52 DEAE-cellulose and gel filtration on Sephacryl S-300. Rabbit kidney brush border exhibited glucoamylase activity with a maltase/glucoamylase ratio of 1.5:1 to 2.0:1. During purification the maltase and glucoamylase activities behaved identically. The Mr of the complex is 590,000, and it appears to be composed of eight identical subunits linked by disulphide bridges.

scholarly journals Alkaline phosphatase from pig kidney. Method of purification and molecular properties

1974 ◽  
Vol 141 (1) ◽  
pp. 273-282 ◽  
Author(s):  
Ernst D. Wachsmuth ◽  
Kunio Hiwada
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Molecular Properties ◽  
Purification Procedure ◽  
Brush Border Membranes ◽  
Pig Kidney ◽  
Critical Ph

Alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes was solubilized from membrane precipitates by butan-1-ol at a critical pH of 7.0. The 12000-fold purification procedure included (NH4)2SO4 precipitation, DEAE-and TEAE-cellulose chromatography, Sephadex G-200 gel filtration and neuraminidase digestion followed by DEAE-cellulose chromatography. The purified protein contained 20% (w/w) carbohydrate and had mol.wt. 150000–156000 as estimated by Sephadex filtration and ultracentrifuge analysis. It was a tetrameric glycoprotein consisting of identical subunits, and it had a molecular activity at 25°C of 2600s-1 per tetramer. Its concentration in kidney was estimated to be 8.5–8.8mg/kg.

scholarly journals Oestrogen receptor of calf mammary gland. Purification by use of sodium bromide and heparin-sepharose

1979 ◽  
Vol 178 (3) ◽  
pp. 581-587 ◽  
Author(s):  
A Rotondi ◽  
F Auricchio
Keyword(s):  
Mammary Gland ◽  
Binding Sites ◽  
Oestrogen Receptor ◽  
Large Scale ◽  
Gel Filtration ◽  
Purification Procedure ◽  
Receptor Aggregation ◽  
Low Salt ◽  
Number Of Binding Sites ◽  
Stokes Radius

1. Calf mammary-gland cytosol apparently has a single oestrogen receptor capable of auto- and/or hetero-association of varying complexity. Computation of the dissociation constant for oestradiol-17beta gives Kd = 0.5 nM. The number of binding sites is 40 fmol/mg of cytosol protein. The oestrogen receptor in the presence of NaBr, a chaotropic salt that inhibits the interaction of receptor with other cytosol components, sediments through sucrose density gradients as a single sharp peak at 4S, and it has a Stokes radius of 3.4 nm measured by gel filtration. 2. A large-scale purification procedure of the calf mammary-gland oestrogen receptor based on the inhibition of receptor aggregation by NaBr and interaction with heparin-Sepharose is reported. The receptor is purified more than 1500-fold over that in the 27,000g supernatant of the homogenate, with a 30% yield. In ‘low-salt’ buffer the purified receptor sediments through sucrose gradients at 4S and the Stokes radius, measured by gel filtration in the presence of heparin, is 3.4 nm. The mol.wt computed from these values is about 60,000, and the frictional ratio is 1.3.

Enolase from Lobster (Homarus americanus)

1971 ◽  
Vol 28 (6) ◽  
pp. 879-882 ◽  
Author(s):  
M. John Chapman ◽  
Christopher Chin ◽  
Finn Wold
Keyword(s):  
Heat Treatment ◽  
Ion Exchange ◽  
Ammonium Sulfate ◽  
Catalytic Properties ◽  
Gel Filtration ◽  
Homarus Americanus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Acetone Fractionation

Enolase has been isolated from lobster muscle by acetone fractionation, heat treatment, ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Preliminary characterization of the pure enzyme shows that the catalytic properties are very similar to those of the enolases from rabbit and fish.

scholarly journals Purification and characterization of histidine decarboxylase from mouse kidney

1986 ◽  
Vol 234 (2) ◽  
pp. 349-354 ◽  
Author(s):  
S A M Martin ◽  
J O Bishop
Keyword(s):  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Histidine Decarboxylase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Mouse Kidney ◽  
Purification Procedure ◽  
Purification And Characterization ◽  
A Subunit

Histidine decarboxylase was purified 800-fold from the kidneys of thyroxine-treated mice. The purification procedure included precipitation of protein from a crude supernatant after heating it to 55 degrees C at pH 5.5, fractionation with (NH4)2SO4, phosphocellulose column chromatography, chromatofocusing, DEAE-Sepharose column chromatography, gel filtration on Sephacryl S-300 and preparative polyacrylamide-gel electrophoresis. The native enzyme had an estimated Mr of 113 000. The protein was analysed in SDS/10%-polyacrylamide gels and formed a single band corresponding to a subunit Mr of 55 000, indicating that it is a dimer. Three forms of the enzyme were resolved on isoelectrofocusing gels, with pI 5.3, 5.5 and 5.7.

Physiological control of alkylsulfatase synthesis in Pseudomonas aeruginosa: effects of glucose, glucose analogs, and sulfur

1977 ◽  
Vol 23 (10) ◽  
pp. 1456-1464 ◽  
Author(s):  
J. W. Fitzgerald ◽  
Lynda C. Kight
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dehydrogenase Activity ◽  
Glucose Transport ◽  
Parent Strain ◽  
Sodium Sulfide ◽  
Inhibitory Effects ◽  
Physiological Control ◽  
Marked Enhancement ◽  
Low Glucose ◽  
Glucose 6 Phosphate Dehydrogenase

Pseudomonas aeruginosa (isolated from soil) synthesizes an alkylsulfatase allowing this bacterium to utilize sodium hexan-1-yl sulfate as a source of carbon and sulfur for growth. The formation of the enzyme was induced by this and by other (C4–C16) primary alkylsulfate esters as well as by some (C-8 and C-9) primary alkylsulfonates. Secondary (2-yl) alkylsulfate esters did not act as inducers. The induction of alkylsulfatase was markedly inhibited by L-cysteine, L-methionine, sodium sulfide, and by high (>2 mM) concentrations of D-glucose and other related monosaccharides. Similar inhibitory effects by four glucose analogs which will not support growth suggest that prior metabolism was not a requirement for glucose-mediated inhibition. The inhibition by D-glucose of the same inducible system in P. aeruginosa (PAO-57) supported this conclusion since this glucose transport-positive mutant is deficient in the further metabolism of the monosaccharide. At low (0.1–1.0 mM) concentrations, D-glucose or D-glucose 6-O-phosphate (20 mM) caused a marked enhancement of alkylsulfatase induction in the isolate. This novel enhancement was reproduced using P. aeruginosa strain PAO. However, both monosaccharides acted as potent inhibitors of alkylsulfatase formation occurring in mutant PAO-57 which, unlike the parent strain PAO, exhibits low glucose-6-phosphate dehydrogenase activity. These results suggest that D-glucose (0.1–1.0 mM) must be metabolized to enhance the synthesis of the enzyme.

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