Resolution of the phosphoenolpyruvate: fructose phosphotransferase system of Escherichia coli into two components; enzyme IIfructose and fructose-induced HPr-like protein (FPr)

1980 ◽  
Vol 58 (10) ◽  
pp. 1144-1146 ◽  
Author(s):  
E. Bruce Waygood
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Membrane Protein ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Phosphotransferase System ◽  
Sugar Phosphorylation

A protein that substitutes for histidine-containing protein (HPr) in the phosphoenolpyruvate: fructose phosphotransferase system has been found in Escherichia coli grown on fructose. The impure preparation of the fructose-induced HPr-like protein (FPr) appears to be an extrinsic membrane protein which differs from HPr on the basis of its apparent molecular weight (45 000 vs. 9600, respectively), its affinity for DEAE-cellulose and its ability to promote sugar phosphorylation which is specific for fructose, rather than for glucose.

An integral membrane protein antigen associated with the membrane attachment sites of actin microfilaments is identified as an integrin beta-chain

1988 ◽  
Vol 8 (2) ◽  
pp. 564-570
Author(s):  
P A Maher ◽  
S J Singer
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Protein Complex ◽  
Apparent Molecular Weight ◽  
Integral Membrane Protein ◽  
Beta Chain ◽  
Attachment Sites ◽  
Wide Range ◽  
Membrane Attachment

A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101:785-801, 1985) which identified an integral membrane glycoprotein of chicken cells that was associated with a wide variety of sites of actin microfilament attachments to membranes. In this report, we present a further characterization of this integral protein. An immunochemical comparison was made of MAb 30B6 binding properties with those of two other MAbs, JG9 and JG22, which identify a component of a membrane protein complex that interacts with extracellular matrix proteins including fibronectin. We showed that the 110-kilodalton protein recognized by MAb 30B6 in extracts of chicken gizzard smooth muscle is identical, or closely related, to the protein that reacts with MAbs JG9 and JG22. These 110-kilodalton proteins are also structurally closely similar, if not identical, to one another as demonstrated by 125I-tryptic peptide maps. However, competition experiments showed that MAb 30B6 recognizes a different epitope from those recognized by MAbs JG9 and JG22. In addition, the 30B6 antigen is part of a complex that can be isolated on fibronectin columns. These results together establish that the 30B6 antigen is the same as, or closely similar to, the beta-chain of the protein complex named integrin, which is the complex on chicken fibroblast membranes that binds fibronectin. Although the 30B6 antigen is present in a wide range of tissues, its apparent molecular weight on gels varies in different tissues. These differences in apparent molecular weight are due, in large part, to differences in glycosylation.

The major glycoprotein in germinating bean seeds

1971 ◽  
Vol 49 (12) ◽  
pp. 2107-2111 ◽  
Author(s):  
David Racusen ◽  
Murray Foote
Keyword(s):  
Molecular Weight ◽  
Phaseolus Vulgaris ◽  
Total Protein ◽  
Soluble Protein ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Disc Electrophoresis ◽  
Total Soluble Protein ◽  
Combined Analysis

Bean seeds (Phaseolus vulgaris) yielded a soluble glycoprotein that accounted for about 35% of the total protein as determined by combined analysis with DEAE-cellulose and disc electrophoresis. Germination for up to 114 h had little effect on this glycoprotein or on the total soluble protein. The glycoprotein had an apparent molecular weight of 130 000 (6.1 S), contained 14.7% nitrogen, and yielded mannose, glucosamine, and some pentose upon hydrolysis.

scholarly journals Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes' strain)

1973 ◽  
Vol 131 (4) ◽  
pp. 833-841 ◽  
Author(s):  
Donald Stribling ◽  
Richard N. Perham
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Metal Ions ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Class Ii ◽  
Class I ◽  
E Coli ◽  
Km Value ◽  
Bivalent Metal Ions

Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca2+, Zn2+. It is a dimer with a molecular weight of approx. 70000, and the Km value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH4 in the presence of substrate. The Km value for fructose diphosphate is about 20μm (although the Lineweaver–Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.

Purification of Ristocetin Willebrand Factor (RWF)

1975 ◽  
Author(s):  
J. D. Olson ◽  
D. N. Fass ◽  
W. J. Brockway ◽  
E. J. W. Bowie ◽  
K. G. Mann
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Amino Sugar ◽  
Procoagulant Activity ◽  
Factor Viii Inhibitor ◽  
Induced Aggregation

A component required for the ristocetin-induced aggregation of platelets was isolated in yields of 20-30% from pooled human and porcine plasma by cryoethanol concentration of the RWF, after removal of the vitamin K dependent coagulation factors on barium citrate. The concentrate was further purified using gel filtration (4% agarose) and ion exchange (DEAE-cellulose) chromatography. Sodium dodecyl sulfate polyacrylamide gels of the isolated factor indicated an apparent molecular weight of greater than 500,000. After reduction of RWF with mercaptoethanol, a single band is resolved with an apparent molecular weight of 230,000. The purified component had no factor VIII procoagulant activity and did not compete for the activity of naturally occurring factor VIII inhibitor (human). Antisera raised in rabbits directed against the purified component inhibited the RWF activity but not the factor VIII procoagulant activity of plasma. Amino acid analysis indicated the presence of all normal amino acids and failed to detect any amino sugar. Analysis of lipid revealed a significant amount of lipid composed of mono, di and triglycerides, cholesterol, cholesterol esters and free fatty acid, with small portions of phospholipids.

scholarly journals Growth Inhibition by External Potassium of Escherichia coli Lacking PtsN (EIIANtr) Is Caused by Potassium Limitation Mediated by YcgO

2016 ◽  
Vol 198 (13) ◽  
pp. 1868-1882 ◽  
Author(s):  
Ravish Sharma ◽  
Tomohiro Shimada ◽  
Vinod K. Mishra ◽  
Suchitra Upreti ◽  
Abhijit A. Sardesai
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Phosphorylation Site ◽  
Phosphotransferase System ◽  
Inner Membrane ◽  
Content Type ◽  
E Coli ◽  
External Potassium ◽  
Inner Membrane Protein ◽  
External K

ABSTRACTThe absence of PtsN, the terminal phosphoacceptor of the phosphotransferase system comprising PtsP-PtsO-PtsN, inEscherichia coliconfers a potassium-sensitive (Ks) phenotype as the external K+concentration ([K+]e) is increased above 5 mM. A growth-inhibitory increase in intracellular K+content, resulting from hyperactivated Trk-mediated K+uptake, is thought to cause this Ks. We provide evidence that the Ksof the ΔptsNmutant is associated with K+limitation. Accordingly, the moderate Ksdisplayed by the ΔptsNmutant was exacerbated in the absence of the Trk and Kup K+uptake transporters and was associated with reduced cellular K+content. Conversely, overproduction of multiple K+uptake proteins suppressed the Ks. Expression of PtsN variants bearing the H73A, H73D, and H73E substitutions of the phosphorylation site histidine of PtsN complemented the Ks. Absence of the predicted inner membrane protein YcgO (also called CvrA) suppressed the Ks, which was correlated with elevated cellular K+content in the ΔptsNmutant, but the ΔptsNmutation did not alter YcgO levels. Heterologous overexpression ofycgOalso led to Ksthat was associated with reduced cellular K+content, exacerbated by the absence of Trk and Kup and alleviated by overproduction of Kup. Our findings are compatible with a model that postulates that Ksin the ΔptsNmutant occurs due to K+limitation resulting from activation of K+efflux mediated by YcgO, which may be additionally stimulated by [K+]e, implicating a role for PtsN (possibly its dephosphorylated form) as an inhibitor of YcgO activity.IMPORTANCEThis study examines the physiological link between the phosphotransferase system comprising PtsP-PtsO-PtsN and K+ion metabolism inE. coli. Studies on the physiological defect that renders anE. colimutant lacking PtsN to be growth inhibited by external K+indicate that growth impairment results from cellular K+limitation that is mediated by YcgO, a predicted inner membrane protein. Additional observations suggest that dephospho-PtsN may inhibit and external K+may stimulate K+limitation mediated by YcgO. It is speculated that YcgO-mediated K+limitation may be an output of a response to certain stresses, which by modulating the phosphotransfer capacity of the PtsP-PtsO-PtsN phosphorelay leads to growth cessation and stress tolerance.

Some Physical Parameters of Succinyl-Coenzyme A Synthetase of Escherichia coli

1974 ◽  
Vol 52 (7) ◽  
pp. 594-598 ◽  
Author(s):  
Anita Krebs ◽  
William A. Bridger
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Coenzyme A ◽  
Apparent Molecular Weight ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Physical Parameters ◽  
Equilibrium Studies ◽  
Α Helix ◽  
Succinyl Coenzyme A Synthetase

A physical study of succinyl-coenzyme A synthetase of Escherichia coli has been conducted. The extinction coefficient for the enzyme at 280 nm [Formula: see text] has been evaluated by two independent methods and found to be equal to 4.9 ± 0.2. Sedimentation equilibrium studies show that there is a marked dependence of the apparent molecular weight upon the concentration of the enzyme. At concentrations above 1 mg/ml, the enzyme exists predominantly as an α2β2 tetramer of overall molecular weight near 140 000; at lower concentrations, a significant fraction of the enzyme dissociates to an αβ dimer. The circular dichroism spectrum of the enzyme suggests a high proportion of random coil structure, with small contributions of α-helix and β-structure.

Comparison of the binding of 59Fe- and 239Pu-transferrin to rat liver cell membranes

1990 ◽  
Vol 9 (1) ◽  
pp. 17-24 ◽  
Author(s):  
F. Planas-Bohne ◽  
W. Rau
Keyword(s):  
Molecular Weight ◽  
Cell Membrane ◽  
Membrane Protein ◽  
Rat Liver ◽  
Liver Cell ◽  
Cell Membranes ◽  
Apparent Molecular Weight ◽  
Protein S ◽  
Membrane Protein Complex ◽  
Isolated Cell

The binding of the 59Fe and 239Pu complexes of transferrin and 125I labelled transferrin [Tf (125I) ] to isolated cell membranes of rat liver has been studied. Transferrin forms a complex with an integral protein of the membrane which has an apparent molecular weight of about 180 kDa and is stable only at pH 7.4. Iron-59 is eluted from Sephacryl S 300 columns together with Tf (125I) or the Tf-membrane protein complex while 239Pu seems to be bound to different membrane protein(s). After isolation of the Tf-binding protein from 35S-labelled membranes and incubation with one of the metal-Tf complexes 59Fe elutes from a Sephacryl S 300 column together with 35S at an apparent molecular weight of ca. 250 kDa while 239Pu is found in fractions of lower molecular weight. It is concluded from these results that there are Tf-receptors in the liver cell membrane to which iron transferrin may bind. Plutonium, however, seems to be dissociated from Tf and bound directly to other membrane proteins.

scholarly journals The separation and characterization of marmoset kidney β-d-galactosidase and β-d-glucosidase

1977 ◽  
Vol 167 (3) ◽  
pp. 765-773 ◽  
Author(s):  
R J Pierce ◽  
R G Price
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Starch Gel Electrophoresis ◽  
Lysosomal Fraction ◽  
Glucosidase Activity ◽  
Starch Gel

beta-D-Galactosidase and beta-D-glucosidase activities were determined in homogenates of marmoset kidney by using the appropriate 4-methylumbelliferyl glycoside, beta-D-Galactosidase activity was separated into two main components by ion-exchange chromatography on DEAE-cellulose, starch-gel electrophoresis, isoelectric focusing and gel filtration on Sephadex G-200. One form designated A had a pI of 5.1, was loosely bound to DEAE-cellulose at pH7.0, remained near the origin on starch-gel electrophoresis at pH 7.0 and had an apparent molecular weight of 160000. The second beta-D-galactosidase component, designated B, was associated with the total beta-D-glucosidase activity, had a pI of 4.3, was firmly bound to DEAE-cellulose, migrated rapidly towards the anode on starch-gel electrophoresis and had an apparent molecular weight of 50000. The optimum pH values of beta-D-galactosidase A and B were 4.5 and 6.0 respectively. beta-D-Galactosidase A was activated by 0.1 M-NaC1 but the activity of the B form was inhibited by 1 M-NaC1 at pH 4.5. beta-D-galactosidase had a bimodal distribution, the A form being recovered in the lysosomal fraction whereas the B form was present in the soluble fraction, as was the major portion of the beta-D-glucosidase activity. The lysosomal and soluble forms were further characterized by DEAE-cellulose chromatography.

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