scholarly journals Conformational states of the (Na+ + K+)-transporting ATPase. Formation of 240000-Mr and 116000-Mr polypeptides in the presence of a bifunctional thiol probe

1984 ◽  
Vol 218 (2) ◽  
pp. 331-339 ◽  
Author(s):  
W E Harris ◽  
W L Stahl
Keyword(s):  
Atpase Activity ◽  
Adenine Nucleotide ◽  
Conformational State ◽  
Cross Linking ◽  
Beta Subunits ◽  
Specific Antibodies ◽  
Alpha Subunits ◽  
Phosphorylated Form ◽  
Kidney Enzyme ◽  
Thiol Reagent

Interpeptide cross-linking of alpha-subunits with concomitant loss of Na+ + K+-transporting ATPase (Na+, K+-ATPase) activity was found when the purified lamb kidney enzyme was treated with the bifunctional thiol reagent 4,4′-difluoro-3,3′-dinitrodiphenyl sulphone (F2DNS). Several forms of the enzyme could be clearly distinguished: one binding ATP (non-phosphorylated enzyme, E1 X ATP), a phosphorylated form (E2-P) and a phosphoenzyme-ouabain complex (E2P X ouabain). A polypeptide of approx. Mr 240 000 and probable alpha 2 composition comprised up to 5-20% of the total polypeptides after reaction of the lamb kidney Na+, K+-ATPase with F2DNS. The amount of this polypeptide formed was related to the conformational state of the enzyme. The presence of adenine nucleotide greatly diminished the amount of 240 000-Mr polypeptide formed and provides evidence for an enzyme-adenine-nucleotide complex under conditions where the enzyme is not phosphorylated. F2DNS reacted with the enzyme in the presence of Mg2+, Pi and ouabain to form a new polypeptide with an approx. Mr of 116 000, and comprised 23% of the total, whereas the 240 000-Mr polypeptide comprised 9% of the total. This suggests that the 116 000-Mr polypeptide is a characteristic marker of the E2P X ouabain complex. By using specific antibodies it was established that both the 240 000- and 116 000-Mr polypeptides contained alpha-, but not beta-, subunits of the Na+, K+-ATPase.

scholarly journals Isolation of α-subunits of factor F1 from submitochondrial particles and the reconstitution of active ATPase from isolated α-subunits and β-subunits bound to the mitochondrial membrane

1980 ◽  
Vol 192 (2) ◽  
pp. 483-488 ◽  
Author(s):  
I A Kozlov ◽  
Y M Milgrom ◽  
I S Tsybovski
Keyword(s):  
Atpase Activity ◽  
Active Site ◽  
Mitochondrial Membrane ◽  
Iodoacetic Acid ◽  
Reduced Form ◽  
Beta Subunits ◽  
Submitochondrial Particles ◽  
F1 Atpase ◽  
Alpha Subunits ◽  
Α Subunits

The alpha-subunits of factor-F1 ATPase are removed by extraction of submitochondrial particles with 1.75 M-LiCl, with the consequent loss of ATPase activity. ATPase activity is reconstituted by incubation of LiCl-extracted particles with purified alpha-subunits, and the reconstituted ATPase activity is oligomycin-sensitive. Reconstitution is enhanced by maintenance of the alpha-subunits in reduced form by dithiothreitol or NaBH4 and by modification of the alpha-subunits by p-chloromercuribenzoate, iodoacetic acid or N-ethylmaleimide. Experiments with the mixed anhydride of ATP and mesitylene-carboxylic acid, which was previously shown to interact with the F1 active site, localized on the beta-subunits, indicate that the active site of ATPase is shielded by the alpha-subunits.

scholarly journals Impact of Immunoglobulin Isotype and Epitope on the Functional Properties of Vibrio cholerae O-Specific Polysaccharide-Specific Monoclonal Antibodies

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Robert C. Kauffman ◽  
Oluwaseyi Adekunle ◽  
Hanyi Yu ◽  
Alice Cho ◽  
Lindsay E. Nyhoff ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Functional Properties ◽  
Diarrheal Disease ◽  
Cross Linking ◽  
Specific Antibodies ◽  
Immunoglobulin Isotype ◽  
High Affinity ◽  
O Antigen ◽  
The Impact ◽  
Motility Inhibition

ABSTRACT Vibrio cholerae causes the severe diarrheal disease cholera. Clinical disease and current oral cholera vaccines generate antibody responses associated with protection. Immunity is thought to be largely mediated by lipopolysaccharide (LPS)-specific antibodies, primarily targeting the O-antigen. However, the properties and protective mechanism of functionally relevant antibodies have not been well defined. We previously reported on the early B cell response to cholera in a cohort of Bangladeshi patients, from which we characterized a panel of human monoclonal antibodies (MAbs) isolated from acutely induced plasmablasts. All antibodies in that previous study were expressed in an IgG1 backbone irrespective of their original isotype. To clearly determine the impact of affinity, immunoglobulin isotype and subclass on the functional properties of these MAbs, we re-engineered a subset of low- and high-affinity antibodies in different isotype and subclass immunoglobulin backbones and characterized the impact of these changes on binding, vibriocidal, agglutination, and motility inhibition activity. While the high-affinity antibodies bound similarly to O-antigen, irrespective of isotype, the low-affinity antibodies displayed significant avidity differences. Interestingly, despite exhibiting lower binding properties, variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies, suggesting that how the MAb binds to the O-antigen may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition. IMPORTANCE Immunity to the severe diarrheal disease cholera is largely mediated by lipopolysaccharide (LPS)-specific antibodies. However, the properties and protective mechanisms of functionally relevant antibodies have not been well defined. Here, we have engineered low and high-affinity LPS-specific antibodies in different immunoglobulin backbones in order to assess the impact of affinity, immunoglobulin isotype, and subclass on binding, vibriocidal, agglutination, and motility inhibition functional properties. Importantly, we found that affinity did not directly dictate functional potency since variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies. This suggests that how the antibody binds sterically may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition.

scholarly journals The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

1991 ◽  
Vol 278 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T L Kirley
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Rabbit Skeletal Muscle ◽  
Cross Linking ◽  
Low Concentrations ◽  
Sds Page ◽  
Molecular Masses ◽  
Relationship Of ◽  
Peptide Core

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.

Thyroid hormone induction of Na(+)-K(+)-ATPase and its mRNAs in a rat liver cell line

1990 ◽  
Vol 258 (3) ◽  
pp. C544-C551 ◽  
Author(s):  
G. G. Gick ◽  
F. Ismail-Beigi
Keyword(s):  
Atpase Activity ◽  
Cell Line ◽  
Rat Liver ◽  
Liver Cell ◽  
Northern Blot Analysis ◽  
Fold Increase ◽  
Beta Subunits ◽  
Inhibit Protein Synthesis ◽  
Liver Cell Line ◽  
Stimulation Of

The expression of mRNAs encoding the alpha- and beta-subunits of Na(+)-K(+)-ATPase (Na(+)-K+ pump) was examined in a rat liver cell line, Clone 9, in various thyroidal states. Northern blot analysis of total RNA isolated from cells incubated in hypothyroid serum-containing medium revealed the expression of mRNAs encoding Na(+)-K(+)-ATPase alpha 1-(mRNA alpha 1) and beta- (mRNA beta) subunits; mRNAs encoding the alpha 2- and alpha 3-subunits were undetectable. There was a discrepancy in the abundance of mRNA alpha 1 relative to mRNA beta such that mRNA alpha 1 exceeded the sum of the multiple mRNA beta bands by approximately 35-fold. 3,3',5-Triiodothyronine (T3) produced a coordinate augmentation of mRNA alpha 1 and mRNA beta contents that was demonstrable within 2 h and preceded the stimulation of Na(+)-K(+)-ATPase activity. After incubation of cells with T3 for 48 h, Na(+)-K(+)-ATPase activity was stimulated by 1.32-fold, whereas mRNA alpha 1 and mRNA beta abundances were increased 1.46- and 2.87-fold, respectively. Treatment of cells for 6 h with 10 micrograms/ml cycloheximide, a concentration sufficient to inhibit protein synthesis by 95%, elicited a 3.5- and 5.1-fold increase in mRNA alpha 1 and mRNA beta content, respectively. Cycloheximide abrogated the stimulatory effect of T3 on mRNA beta abundance, whereas the T3-induced increase in mRNA alpha 1 content was not prevented.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase.

1984 ◽  
Vol 99 (2) ◽  
pp. 734-741 ◽  
Author(s):  
W A Braell ◽  
D M Schlossman ◽  
S L Schmid ◽  
J E Rothman
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Low Ph ◽  
Coated Vesicles ◽  
Magnesium Concentration ◽  
Cross Linking ◽  
Dependent Atpase ◽  
Event Triggering ◽  
Hydrolysis Of

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles. The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity. Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages. The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly. The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein. Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released. This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release.

scholarly journals Beta Subunits Bridge Two Alpha Subunits Within the BK Channel Tetramer

2010 ◽  
Vol 98 (3) ◽  
pp. 128a
Author(s):  
Guoxia Liu ◽  
Roland Wu ◽  
Xin Jin ◽  
Xiaowei Niu ◽  
Neelesh Chudasama ◽  
...  
Keyword(s):  
Bk Channel ◽  
Beta Subunits ◽  
Alpha Subunits

Characterization of three erythropoietin (Epo)-binding proteins in various human Epo-responsive cell lines and in cells transfected with human Epo-receptor cDNA [see comments]

Blood ◽  
1995 ◽  
Vol 85 (1) ◽  
pp. 106-114 ◽  
Author(s):  
T Takahashi ◽  
S Chiba ◽  
N Hirano ◽  
Y Yazaki ◽  
H Hirai
Keyword(s):  
Molecular Cloning ◽  
Cell Lines ◽  
Cross Linking ◽  
Specific Antibodies ◽  
Epo Receptor ◽  
Receptor Complexes ◽  
Receptor Cdna ◽  
Cloned Cdna ◽  
Responsive Cell

Molecular cloning of a cDNA for a mouse erythropoietin (Epo) receptor (EpoR) has facilitated the understanding of the structure of this receptor. However, there is, as yet, no explanation for the discrepancy between the protein recognized by specific antibodies against mouse EpoR and the unexpectedly larger species that can be cross-linked to labeled Epo. It is unclear whether the product of an unidentified gene is included in the EpoR complex. In the present study, we directly compared the cross-linking patterns for human EpoR that were endogenously expressed in three types of Epo-responsive cell, and that was artificially expressed in nonhematopoietic cells after transfection with cDNA for human EpoR. We observed that 85-kD and 105-kD proteins formed ligand-receptor complexes in all the human Epo-responsive cells and, furthermore, that the formation of a complex derived from the 70- kD protein was dependent on the level of expression of the cloned EpoR mRNA in these cells. By contrast, a prominent cross-linked band derived from the 70-kD protein and a weaker band derived from the 80- to 85-kD protein, but no band derived from the 105-kD protein, could be shown in the case of nonhematopoietic cells transfected with the human EpoR cDNA. These observations suggest that the cloned cDNA for human EpoR alone does not allow generation of the complete EpoR in nonhematopoietic cells and that the 105-kD Epo-binding protein may represent the product of an as yet unidentified gene that is expressed in hematopoietic cells.

scholarly journals Homology between legumin-like polypeptides from cereals and pea

1985 ◽  
Vol 226 (3) ◽  
pp. 847-852 ◽  
Author(s):  
L S Robert ◽  
C Nozzolillo ◽  
I Altosaar
Keyword(s):  
Triticum Aestivum ◽  
Zea Mays ◽  
Pisum Sativum ◽  
Western Blot ◽  
Secale Cereale ◽  
Avena Sativa ◽  
Blot Analysis ◽  
Beta Subunits ◽  
Two Dimensional ◽  
Alpha Subunits

The presence of legumin-like constituents within the globulin fractions of wheat (Triticum aestivum), rye (Secale cereale) and corn (maize, Zea mays) was demonstrated. Two-dimensional analysis of wheat globulins in the presence and absence of a reducing agent revealed the existence of reducible approximately 60 kDa polypeptides. Western-blot analysis with 125I-labelled antibodies raised against the oat (Avena sativa) 12S globulin holoprotein or its alpha-subunits demonstrated, firstly, the immunological homology between the alpha- and beta-subunits of pea (Pisum sativum) legumin and oat 12S globulin, and secondly, the similar occurrence in wheat of antigenically homologous approximately 20kDa and approximately 40 kDa polypeptides that associate via disulphide linkage to form approximately 60 kDa dimers. Western blotting also showed the presence of disulphide-linked approximately 20 kDa and approximately 40 kDa legumin-like subunits within the globulin fractions of rye and corn.

The phosphorylated form of the enhancer-binding protein NTRC has an ATPase activity that is essential for activation of transcription

Cell ◽  
1991 ◽  
Vol 67 (1) ◽  
pp. 155-167 ◽  
Author(s):  
David S. Weiss ◽  
Jacques Batut ◽  
Karl E. Klose ◽  
John Keener ◽  
Sydney Kustu
Keyword(s):  
Atpase Activity ◽  
Binding Protein ◽  
Phosphorylated Form ◽  
Activation Of Transcription ◽  
Enhancer Binding Protein
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