scholarly journals Molecular cloning and immunological characterization of the gamma polypeptide, a small protein associated with the Na,K-ATPase.

1993 ◽  
Vol 121 (3) ◽  
pp. 579-586 ◽  
Author(s):  
R W Mercer ◽  
D Biemesderfer ◽  
D P Bliss ◽  
J H Collins ◽  
B Forbush
Keyword(s):  
Northern Blot Analysis ◽  
Beta Subunits ◽  
Gamma Subunit ◽  
Hydrophobic Domain ◽  
Subunit Mrna ◽  
Nephron Segments ◽  
Gamma Subunits ◽  
Specific Association ◽  
Hydropathy Analysis

The gamma subunit of the Na,K-ATPase is a small membrane protein that copurifies with the alpha and beta subunits of the enzyme. Strong evidence that the gamma subunit is a component of the Na,K-ATPase comes from studies indicating that the subunit is involved in forming the site for cardiac glycoside binding. We have isolated and characterized the cDNAs coding the gamma subunit from several species. The gamma subunit is a highly conserved protein consisting of 58 amino acids with a molecular weight of 6500. Hydropathy analysis reveals the presence of a single hydrophobic domain that is sufficient to cross the membrane. There are no sites for N-linked glycosylation. Northern blot analysis revealed that the gamma subunit mRNA is expressed in a tissue-specific fashion and is present in all tissues characterized. gamma-specific antibodies have been used to verify that the sequenced protein is the same protein labeled by [3H]nitroazidobenzoyl-ouabain (NAB-ouabain), and that this protein, the gamma subunit of the Na,K-ATPase, has a distribution pattern along nephron segments that is identical with the alpha subunit. In addition, coimmunoprecipitation of the alpha, beta and gamma subunits demonstrate specific association of the subunits. These results are consistent with the notion that the gamma subunit is specifically associated with and may be an important component of the Na,K-ATPase.

scholarly journals Characterization of the cDNA and genomic sequence of a G protein gamma subunit (gamma 5).

1992 ◽  
Vol 12 (4) ◽  
pp. 1585-1591 ◽  
Author(s):  
K J Fisher ◽  
N N Aronson
Keyword(s):  
G Protein ◽  
Genomic Sequence ◽  
Cdna Clones ◽  
Gamma Subunit ◽  
Bovine Gene ◽  
Gamma Subunits ◽  
Sequence Encoding ◽  
Rat Livers ◽  
Exon Structure

A cDNA from human placenta and liver tissues that contained both sequence for the lysosomal glycosidase di-N-acetylchitobiase and sequence homologous to the gamma subunit of GTP-binding proteins was previously isolated. Here we have shown that the gamma-subunit-homologous portion of this unusual cDNA is derived from a member of the gamma-subunit multigene family. The partial human gamma-subunit sequence was used to isolate the corresponding full-length cDNA clones from bovine and rat livers. The two cDNAs encode identical 68-amino-acid proteins (7.3 kDa) homologous to previously cloned G protein gamma subunits. The bovine gene sequence encoding this new gamma-subunit isoform (gamma 5) was determined and found to have an intron-exon structure consistent with the original human chitobiase-gamma 5-subunit hybrid mRNA being a product of alternative splicing. Genomic cloning also resulted in the isolation of a human gamma 5 pseudogene.

Characterization of the cDNA and genomic sequence of a G protein gamma subunit (gamma 5)

1992 ◽  
Vol 12 (4) ◽  
pp. 1585-1591
Author(s):  
K J Fisher ◽  
N N Aronson
Keyword(s):  
G Protein ◽  
Genomic Sequence ◽  
Cdna Clones ◽  
Gamma Subunit ◽  
Bovine Gene ◽  
Gamma Subunits ◽  
Sequence Encoding ◽  
Rat Livers ◽  
Exon Structure

A cDNA from human placenta and liver tissues that contained both sequence for the lysosomal glycosidase di-N-acetylchitobiase and sequence homologous to the gamma subunit of GTP-binding proteins was previously isolated. Here we have shown that the gamma-subunit-homologous portion of this unusual cDNA is derived from a member of the gamma-subunit multigene family. The partial human gamma-subunit sequence was used to isolate the corresponding full-length cDNA clones from bovine and rat livers. The two cDNAs encode identical 68-amino-acid proteins (7.3 kDa) homologous to previously cloned G protein gamma subunits. The bovine gene sequence encoding this new gamma-subunit isoform (gamma 5) was determined and found to have an intron-exon structure consistent with the original human chitobiase-gamma 5-subunit hybrid mRNA being a product of alternative splicing. Genomic cloning also resulted in the isolation of a human gamma 5 pseudogene.

scholarly journals Influence of the α-, β- and γ-subunits of the energy-transducing adenosine triphosphates from Micrococcus lysodeikticus in the immunochemical properties of the protein and in their reconstitution studied by a radioimmunoassay method

1981 ◽  
Vol 193 (3) ◽  
pp. 729-735 ◽  
Author(s):  
V Larraga ◽  
F Mollinedo ◽  
N Rubio ◽  
E Muñoz
Keyword(s):  
Antigenic Determinants ◽  
Beta Subunits ◽  
Gamma Subunit ◽  
F1 Atpase ◽  
Antigenic Sites ◽  
Specific Radioimmunoassay ◽  
Gamma Subunits ◽  
Micrococcus Lysodeikticus ◽  
Alpha Beta ◽  
Radioimmunoassay Method

We developed a sensitive and specific radioimmunoassay of the energy-transducing adenosine triphosphatase (F1-ATPase, EC 3.6.1.3) of Micrococcus lysodeikticus and extended the assay to the alpha-, beta- and gamma-subunits of the enzyme. We isolated these subunits and studied cross-reactions. We found the immunochemical properties of alpha- and beta-subunits to differ, and gamma-subunits showed an intermediate behaviour between that of alpha- and beta-subunits. Our findings indicate that each subunit of M. lysodeikticus F1-ATPase has its own identity and that conformational antigenic determinants and/or co-operative antigenic sites-arise from subunit assembly. Equimolecular amounts of alpha- and beta-subunits (up to three copies of each) reconstituted partially the immunochemical properties of the ATPase molecule, and addition of 2 mol of gamma-subunit per mol of alpha 3 beta 3 complex improved reconstitution. Our findings describe the first reconstitution of biological activity of this ATPase by assembly of the isolated subunits, and provide support for earlier proposals on the stoicheiometry of the alpha 3 beta 3 gamma 2 type for M. lysodeikticus F1-ATPase. The radioimmunoassay method affords opportunities to study the homologies between different energy-transducing ATPases and their constituent polypeptides before the primary structure of these complex proteins has been determined.

Expression and molecular regulation of Na(+)-K(+)-ATPase after renal ischemia

1994 ◽  
Vol 267 (1) ◽  
pp. F75-F85 ◽  
Author(s):  
S. K. Van Why ◽  
A. S. Mann ◽  
T. Ardito ◽  
N. J. Siegel ◽  
M. Kashgarian
Keyword(s):  
Enzyme Production ◽  
Time Course ◽  
Apical Membrane ◽  
Proximal Tubules ◽  
Renal Ischemia ◽  
Beta Subunit ◽  
Molecular Regulation ◽  
Beta Subunits ◽  
Renal Epithelia ◽  
Subunit Mrna

Renal ischemia causes redistribution of Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) to the apical membrane of proximal tubules. We determined the time course of regeneration of Na(+)-K(+)-ATPase polarity and sought evidence of increased enzyme production during recovery as a means to restore polarity. Anesthetized rats underwent 45 min renal ischemia and reflow of 15 min, 2 h, 6 h, and 24 h. Immunofluorescent and electron microscopy showed loss of strict basolateral localization of Na(+)-K(+)-ATPase at 15 min reflow with repolarization by 24 h in sublethally injured cells. Both alpha 1- and beta-subunits were only in microsomal fractions at all reflow intervals. Immunodetectable levels of both subunits declined to 60-70% of control by 24 h reflow. Levels of mRNA for each subunit declined in parallel through 24 h to 55% of control. Overall transcription was profoundly depressed through 6 h but had recovered to near control by 24 h. Specific transcription of alpha 1- and beta-subunit mRNA was markedly decreased after ischemia and only partially recovered by 24 h. These results suggest that recycling of misplaced units rather than new Na(+)-K(+)-ATPase production is the means by which renal epithelia initially repolarize after ischemic injury.

scholarly journals Characterization of Mycoplasma gallisepticum pyruvate dehydrogenase alpha and beta subunits and their roles in cytoadherence

PLoS ONE ◽  
2018 ◽  
Vol 13 (12) ◽  
pp. e0208745 ◽  
Author(s):  
Jingjing Qi ◽  
Fanqing Zhang ◽  
Yu Wang ◽  
Ting Liu ◽  
Lei Tan ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Mycoplasma Gallisepticum ◽  
Beta Subunits

Open channel block of human heart hKv1.5 by the beta-subunit hKv beta 1.2

1997 ◽  
Vol 272 (6) ◽  
pp. H2932-H2941 ◽  
Author(s):  
M. De Biasi ◽  
Z. Wang ◽  
E. Accili ◽  
B. Wible ◽  
D. Fedida
Keyword(s):  
Human Heart ◽  
Open Channel ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Channel Block ◽  
Open Channel Block ◽  
Pharmacological Characterization ◽  
Kinetics Of ◽  
K Currents

Voltage-gated K+ currents in human heart are likely to derive from multisubunit complexes of pore-forming alpha-subunits with one or more auxiliary beta-subunits. We recently cloned a novel beta-subunit from human atrium, hKv beta 1.2 (K. Majumder, M. De Biasi, Z. Wang, and B. A. Wible. FEBS Lett. 361: 13-16, 1995), and showed that it interacts with channels in the Kv1 family. Here we characterize the interaction of hKv beta 1.2 with hKv1.5 in terms of a two-closed-state and one-open-state open channel block model. After coexpression in Xenopus oocytes, hKv1.5 currents were reduced in the presence of hKv beta 1.2, and at positive potentials an inactivation process was introduced. Deactivation kinetics of hKv1.5 were slowed, and there was an increased steepness with a -14-mV hyperpolarizing shift in the midpoint of steady-state activation. The model was able to predict all the above features of the interaction of hKv1.5 and hKv beta 1.2 as a result of rapid open channel block of activated channels. Understanding the mechanism of hKv beta 1.2 action on heart K+ channels will further aid the development of the functional and pharmacological characterization of native cardiac K+ currents.

Cloning and characterization of a type III Na-dependent phosphate cotransporter from mouse intestine

2000 ◽  
Vol 279 (4) ◽  
pp. C1135-C1143 ◽  
Author(s):  
Liqun Bai ◽  
James F. Collins ◽  
Fayez K. Ghishan
Keyword(s):  
Small Intestine ◽  
Northern Blot Analysis ◽  
Kinetic Studies ◽  
Michaelis Constant ◽  
Phosphate Homeostasis ◽  
Type Iii ◽  
Mouse Small Intestine ◽  
Mouse Intestine

Intestinal and renal absorption of inorganic phosphate (Pi) is critical for phosphate homeostasis in mammals. We have isolated a cDNA that encodes a type III Na-dependent phosphate cotransporter from mouse small intestine (mPit-2). The nucleotide sequence of mPit-2 predicts a protein of 653 amino acids with at least 10 putative transmembrane domains. Kinetic studies, carried out in Xenopus oocytes, showed that mPit-2 cRNA induces significant Na-dependent Piuptake with an apparent Michaelis constant ( Km) for phosphate of 38 μM. The transport of phosphate by mPit-2 is inhibited at high pH. Northern blot analysis demonstrated the presence of mPit-2 mRNA in various tissues, including intestine, kidney, heart, liver, brain, testis, and skin. The highest expression of mPit-2 in the intestine was found in the jejunum. In situ hybridization revealed that mPit-2 mRNA is expressed throughout the vertical crypt-villus axis of the intestinal epithelium. The presence of mPit-2 in the mouse intestine and its unique transport characteristics suggest that multiple Na-dependent cotransporters may contribute to phosphate absorption in the mammalian small intestine.

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)

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