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.

scholarly journals The effect of the γ-subunit of the cyclic GMP phosphodiesterase of bovine and frog (Rana catesbiana) retinal rod outer segments on the kinetic parameters of the enzyme

1990 ◽  
Vol 265 (3) ◽  
pp. 655-658 ◽  
Author(s):  
M M Whalen ◽  
M W Bitensky ◽  
D J Takemoto
Keyword(s):  
Catalytic Activity ◽  
Cyclic Gmp ◽  
Active Sites ◽  
Outer Segment ◽  
Maximal Activity ◽  
Inhibitory Effect ◽  
Gamma Subunit ◽  
Rod Outer Segment ◽  
Gamma Subunits ◽  
Alpha Beta

Rod-outer-segment cyclic GMP phosphodiesterase (PDE) (subunit composition alpha beta gamma 2) contains catalytic activity in alpha beta. The gamma-subunits are inhibitors. Removal of the gamma-subunits increases Vmax. without affecting the Km. The inhibitory effect of a single gamma-subunit (alpha beta gamma) on the Vmax. of alpha beta is much greater in bovine than in frog (Rana catesbiana) PDE. Bovine PDE in the alpha beta gamma 2 state has a Vmax. that is 2.6 +/- 0.4% of the Vmax. of alpha beta. The removal of one gamma-subunit to give alpha beta gamma results in a Vmax. 5.2 +/- 1% of that for maximal activity. Frog alpha beta gamma 2 has a Vmax. 10.8 +/- 2%, and alpha beta gamma has a Vmax. 50 +/- 18%, of the Vmax. of alpha beta. These data suggest that a single gamma-subunit can inhibit the catalytic activity of active sites on both alpha- and beta-subunits in bovine, but not in frog, rod-outer-segment PDE.

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 Role of the subunits of the energy-transducing adenosine triphosphatase from Micrococcus lysodeikticus membranes studied by proteolytic digestion and immunological approaches

1980 ◽  
Vol 186 (3) ◽  
pp. 713-723 ◽  
Author(s):  
F Mollinedo ◽  
V Larraga ◽  
F J Coll ◽  
E Muñoz
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Specific Activity ◽  
Relative Proportion ◽  
Inhibitory Effect ◽  
Beta Subunit ◽  
Gamma Subunit ◽  
Micrococcus Lysodeikticus ◽  
Alpha Beta

An energy-transducing adenosine triphosphatase (ATPase, EC 3.6.1.3) that contains an extra polypeptide (delta) as well as three intrinsic subunits (alpha, beta, gamma) was purified from Micrococcus lysodeikticus membranes. The apparent subunit stoichiometry of this soluble ATPase complex is alpha 3 beta 3 gamma delta. The functional role of the subunits was studied by correlating subunit sensitivity to trypsin and effect of antibodies raised against holo-ATPase and its alpha, beta and gamma subunits with changes in ATPase activity and ATPase rebinding to membranes. A form of the ATPase with the subunit proportions 1.67(alpha):3.00(beta:0.17(gamma) was isolated after trypsin treatment of purified ATPase. This form has more than twice the specific activity of native enzyme. Other forms with less relative proportion of alpha subunits and absence of gamma subunit are not active. Of the antisera to subunits, only anti-(beta-subunit) serum shows a slight inhibitory effect on ATPase activity, but its combination with either anti-(alpha-subunit) or anti-(gamma-subunit) serum increases the effect. The results suggest that beta subunit is required for full ATPase activity, although a minor proportion of alpha and perhaps gamma subunit(s) is also required, probably to impart an active conformation to the protein. The additional polypeptide not hitherto described in Micrococcus lysodeikticus ATPase had a molecular weight of 20 000 and was found to be involved in ATPase binding to membranes. This 20 000-dalton component can be equated with the delta subunit of other energy-transducing ATPases and its association with the (alpha, beta, gamma) M. lysodeikticus ATPase complex appears to be dependent on bivalent cations. The present results do not preclude the possibility that the gamma subunit also plays a role in ATPase binding, in which, however, the major subunits do not seem to play a role.

Common Antigenic Sites in Human, Bovine and Porcine Fibrinogens

1981 ◽  
Vol 45 (02) ◽  
pp. 169-172 ◽  
Author(s):  
Czeslaw S Cierniewski
Keyword(s):  
Antigenic Determinants ◽  
Antigenic Sites ◽  
Specific Antisera ◽  
Antigenic Homology ◽  
Polypeptide Chains ◽  
Fibrinogen Molecule

SummarySpecific antisera to the Aα, Bβ and γ chains of porcine fibrinogen were used to characterize an antigenic homology of human, bovine, and porcine fibrinogens. Antigenic determinants shared by these fibrinogens were mostly formed by the Aα chain. However, in the case of bovine and porcine fibrinogens they were also found in the Bβ and γ polypeptide chains. The results reported here show that the Aα chain determinants exposed on the intact fibrinogen molecule are conserved to a considerably larger extent than those of the Bβ and γ chains.

A spectrin-like protein from mouse brain membranes: phosphorylation of the 235,000-dalton subunit

1984 ◽  
Vol 247 (1) ◽  
pp. C61-C73 ◽  
Author(s):  
S. R. Goodman ◽  
I. S. Zagon ◽  
C. F. Whitfield ◽  
L. A. Casoria ◽  
S. B. Shohet ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Mouse Erythrocyte ◽  
Alpha Beta ◽  
Beta 2 ◽  
Independent Protein ◽  
Brain Spectrin

A mouse brain spectrin-like protein, which was an immunoreactive analogue of erythrocyte spectrin, has been isolated from demyelinated membranes. This spectrin analogue was a 10.5 S, 972,000 molecular weight (Mr) (alpha beta)2 tetramer containing subunits of 240,000 (alpha) and 235,000 (beta) Mr. We demonstrated that in vivo only the 235,000 Mr beta subunit of the mouse brain spectrin-like protein was phosphorylated, which was an analogous situation to mouse erythrocyte spectrin in which only the 220,000 Mr beta subunit was phosphorylated. Incubation of isolated membrane fractions with [gamma-32P]ATP +/- adenosine 3',5'-cyclic monophosphate (cAMP) indicated that mouse brain spectrin-like protein, mouse erythrocyte spectrin, and human erythrocyte spectrin's beta subunits were all phosphorylated in vitro by membrane-associated cAMP-independent protein kinases.

scholarly journals Four structurally distinct, non-DNA-binding subunits of human nuclear respiratory factor 2 share a conserved transcriptional activation domain.

1995 ◽  
Vol 15 (1) ◽  
pp. 102-111 ◽  
Author(s):  
S Gugneja ◽  
J V Virbasius ◽  
R C Scarpulla
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Deletion Mapping ◽  
Rnase Protection ◽  
Nuclear Respiratory Factor ◽  
Gamma Subunits ◽  
Nuclear Respiratory Factor 2 ◽  
Alpha Beta ◽  
Beta 2

Nuclear respiratory factor 2 (NRF-2) was previously purified to near homogeneity from HeLa cells on the basis of its ability to bind tandem recognition sites in the rat cytochrome oxidase subunit IV (RCO4) promoter. It consisted of five subunits, alpha, beta 1, beta 2, gamma 1, and gamma 2. Sequencing of tryptic peptides from alpha and from mixtures of the two beta or two gamma subunits revealed sequence identities with subunits of the mouse GA-binding protein (GABP), a ubiquitously expressed ETS domain activator composed of three subunits, alpha, beta 1, and beta 2. To understand the precise relationship between NRF-2 and GABP, cDNAs for all five NRF-2 subunits have now been cloned and their products have been overexpressed. The results establish that the two additional NRF-2 subunits are molecular variants that differ from GABP beta 1 and beta 2 by having a 12-amino-acid insertion containing two serine doublets. PCR and RNase protection assays show that mRNAs for these variants are expressed in the human but not the rodent cells and tissues examined. The insertion did not alter the ability of the beta and gamma subunits to associate with alpha, the DNA-binding subunit, nor did it affect the ability of NRF-2 beta 1 or beta 2 to direct high-affinity binding of alpha to tandem sites in the RCO4 promoter. In addition, the four NRF-2 beta and gamma subunits were equally proficient in activating transcription in transfected cells when fused to a GAL4 DNA-binding domain. The domain responsible for this transcriptional activation was localized by deletion mapping to a region of approximately 70 amino acids that is conserved in all four NRF-2 beta and gamma subunits. The repeated glutamine-containing hydrophobic clusters within this region bear a strong resemblance to those recently implicated in protein-protein interactions within the transcriptional apparatus.

scholarly journals The quaternary structure of phosphorylase kinase as influenced by low concentrations of urea. Evidence suggesting a structural role for calmodulin

1990 ◽  
Vol 268 (2) ◽  
pp. 393-399 ◽  
Author(s):  
H K Paudel ◽  
G M Carlson
Keyword(s):  
Quaternary Structure ◽  
Gel Filtration ◽  
Phosphorylase Kinase ◽  
Gamma Delta ◽  
Gel Filtration Chromatography ◽  
Structural Role ◽  
Low Concentrations ◽  
Gamma Subunits ◽  
Alpha Beta ◽  
Delta Subunit

Skeletal-muscle phosphorylase kinase is a hexadecameric oligomer composed of equivalent amounts of four different subunits, (alpha beta gamma delta)4. The delta-subunit, which is calmodulin, functions as an integral subunit of the oligomer, and the gamma-subunit is catalytic. To learn more about intersubunit contacts within the hexadecamer and about the roles of individual subunits, we induced partial dissociation of the holoenzyme with low concentrations of urea. In the absence of Ca2+ the quaternary structure of phosphorylase kinase is very sensitive to urea over a narrow concentration range. Gel-filtration chromatography in the presence of progressively increasing concentrations of urea indicates that between 1.15 M- and 1.35 M-urea the delta-subunit dissociates, allowing extensive formation of complexes larger than the native enzyme that contain equivalent amounts of alpha-, β- and gamma-subunits. As the urea concentration is increased to 2 M and 3 M, nearly all of the enzyme aggregates to the heavy species devoid of delta-subunit. Addition of Ca2+, which is known to block dissociation of the delta-subunit [Shenolikar, Cohen, Cohen, Nairn & Perry (1979) Eur. J. Biochem. 100, 329-337], also blocks aggregation of the enzyme induced by the low concentrations of urea. These results suggest that in native phosphorylase kinase the delta-subunit, in addition to activating the catalytic subunit and conferring upon it Ca2(+)-sensitivity, may also serve a structural role in preventing aggregation of the alpha-, β- and gamma-subunits, thus limiting to four the number of alpha beta gamma delta protomers that associate under standard conditions. In gel-filtration chromatography with urea a protein peak containing equivalent amounts of alpha- and gamma-subunits is also observed, as is a peak containing only β-subunits. Increasing concentrations of urea have a biphasic effect on the activity of the holoenzyme, being stimulatory up to 1 M and then inhibitory. The concentration-dependence of urea in the inhibitory phase parallels its ability to induce dissociation of the delta-subunit.

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