Activation and inhibition of phosphorylase kinase by monospecific antibodies against preparatively isolated alpha, beta and gamma subunits

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

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.102 ◽

1995 ◽

Vol 15 (1) ◽

pp. 102-111 ◽

Cited By ~ 86

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.

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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

Biochemical Journal ◽

10.1042/bj2650655 ◽

1990 ◽

Vol 265 (3) ◽

pp. 655-658 ◽

Cited By ~ 22

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.

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Functional reconstitution of Chlamydomonas outer dynein arms from alpha-beta and gamma subunits: requirement of a third factor.

The Journal of Cell Biology ◽

10.1083/jcb.126.3.737 ◽

1994 ◽

Vol 126 (3) ◽

pp. 737-745 ◽

Cited By ~ 97

Author(s):

S Takada ◽

R Kamiya

Keyword(s):

Cross Section ◽

Cross Sections ◽

Attachment Site ◽

The Other ◽

Wild Type ◽

Heavy Chains ◽

Sds Page ◽

Gamma Subunits ◽

Dynein Arms ◽

Alpha Beta

The outer dynein arm of Chlamydomonas flagella, when isolated under Mg(2+)-free conditions, tends to dissociate into an 11 to 12S particle (12S dynein) containing the gamma heavy chain and a 21S particle (called 18S dynein) containing the alpha and beta heavy chains. We show here that functional outer arms can be reconstituted by the addition of 12S and 18S dyneins to the axonemes of the outer armless mutants oda1-oda6. A third factor that sediments at integral 7S is required for efficient reconstitution of the outer arms on the axonemes of oda1 and oda3. However, this factor is not necessary for reconstitution on the axonemes of oda2, oda4, oda5, and oda6. SDS-PAGE analysis indicates that the axonemes of the former two mutants lack a integral of 70-kD polypeptide that is present in those of the other mutants as well as in the 7S fraction from the wild-type extract. Furthermore, electron micrographs of axonemal cross sections revealed that the latter four mutants, but not oda1 or oda3, have small pointed structures on the outer doublets, at a position in cross section where outer arms normally occur. We suggest that the 7S factor constitutes the pointed structure on the outer doublets and facilitates attachment of the outer arm. The discovery of this structure raises a new question as to how the attachment site for the outer arm dynein is determined within the axoneme.

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Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells.

The Journal of Cell Biology ◽

10.1083/jcb.114.4.799 ◽

1991 ◽

Vol 114 (4) ◽

pp. 799-807 ◽

Cited By ~ 80

Author(s):

Y Gu ◽

J R Forsayeth ◽

S Verrall ◽

X M Yu ◽

Z W Hall

Keyword(s):

Acetylcholine Receptor ◽

Surface Binding ◽

Mouse Muscle ◽

Cos Cells ◽

Gamma Subunits ◽

Alpha Beta ◽

High Concentrations ◽

Alpha Bungarotoxin ◽

Transfection Mixture ◽

In Cells

We have investigated the mechanisms of assembly and transport to the cell surface of the mouse muscle nicotinic acetylcholine receptor (AChR) in transiently transfected COS cells. In cells transfected with all four subunit cDNAs, AChR was expressed on the surface with properties resembling those seen in mouse muscle cells (Gu, Y., A. F. Franco, Jr., P.D. Gardner, J. B. Lansman, J. R. Forsayeth, and Z. W. Hall. 1990. Neuron. 5:147-157). When incomplete combinations of AChR subunits were expressed, surface binding of 125I-alpha-bungarotoxin was not detected except in the case of alpha beta gamma which expressed less than 15% of that seen with all four subunits. Immunoprecipitation and sucrose gradient sedimentation experiments showed that in cells expressing pairs of subunits, alpha delta and alpha gamma heterodimers were formed, but alpha beta was not. When three subunits were expressed, alpha delta beta and alpha gamma beta complexes were formed. Variation of the ratios of the four subunit cDNAs used in the transfection mixture showed that surface AChR expression was decreased by high concentrations of delta or gamma cDNAs in a mutually competitive manner. High expression of delta or gamma subunits also each inhibited formation of a heterodimer with alpha and the other subunit. These results are consistent with a defined pathway for AChR assembly in which alpha delta and alpha gamma heterodimers are formed first, followed by association with the beta subunit and with each other to form the complete AChR.

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The Genes for Mouse Salivary Androgen-Binding Protein (ABP) Subunits Alpha and Gamma Are Located on Chromosome 7

Genetics ◽

10.1093/genetics/115.3.535 ◽

1987 ◽

Vol 115 (3) ◽

pp. 535-543

Author(s):

Stephen R Dlouhy ◽

Benjamin A Taylor ◽

Robert C Karn

Keyword(s):

Binding Protein ◽

Inbred Strains ◽

Beta Subunit ◽

Chromosome 7 ◽

Glucose Phosphate ◽

Gamma Subunits ◽

Androgen Binding Protein ◽

Free Translation ◽

Alpha Beta ◽

Androgen Binding

ABSTRACT We demonstrate that the previously described gene Androgen binding protein (Abp; Dlouhy and Karn, 1984) codes for the Alpha subunit of ABP and rename the locus Androgen binding protein alpha (Abpa). A study of recombinant inbred strains demonstrates that Abpa is located on chromosome 7 near Glucose phosphate isomerase-1 (Gpi-1). Biochemical and genetic evidence indicates the existence of another ABP subunit, Gamma, and its locus, Androgen binding protein gamma (Abpg), that is closely linked to Abpa. Although no polymorphism has yet been found for the previously described Beta subunit of ABP (Dlouhy and Karn, 1983; 1984), we suggest that it represents a third locus, Androgen binding protein beta (Abpb). ABP subunits appear to dimerize randomly and a model is presented demonstrating the origin of six ABP dimers in the salivas of AbpaaAbpga/AbpabAbpgb heterozygous mice. The results of cell-free translation studies in which the pre-ABP subunits are identified specifically by immunoprecipitation with anti-ABP antibody supports the idea that independent mRNAs code for the Alpha, Beta and Gamma subunits.

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Proteins of the membrane skeleton in rat Sertoli cells

Journal of Cell Science ◽

10.1242/jcs.86.1.145 ◽

1986 ◽

Vol 86 (1) ◽

pp. 145-154

Author(s):

E. Ziparo ◽

B.M. Zani ◽

A. Filippini ◽

M. Stefanini ◽

V.T. Marchesi

Keyword(s):

Plasma Membrane ◽

Sertoli Cell ◽

Sertoli Cells ◽

Primary Cultures ◽

Membrane Skeleton ◽

Permeabilized Cells ◽

Protein 4.1 ◽

Molecular Weights ◽

Gamma Subunits ◽

Alpha Beta

Analogues of the alpha, beta and gamma subunits of human spectrin and erythroid protein 4.1 have been detected in rat Sertoli cell primary cultures. Immunofluorescence of permeabilized cells showed that erythroid type spectrin, protein 4.1 and actin co-distribute within the cells as filamentous structures. Fodrin-like molecules were distributed in a diffuse manner, mostly associated with the plasma membrane. Immunoprecipitation and immunoblotting experiments indicated that the polypeptides present in rat Sertoli cells are immunologically related and display molecular weights similar to their analogues in the human erythroid and non-erythroid membrane skeleton.

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Bilin attachment sites in the alpha, beta, and gamma subunits of R-phycoerythrin. Structural studies on singly and doubly linked phycourobilins.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)89151-7 ◽

1985 ◽

Vol 260 (8) ◽

pp. 4864-4868 ◽

Cited By ~ 7

Author(s):

J O Nagy ◽

J E Bishop ◽

A V Klotz ◽

A N Glazer ◽

H Rapoport

Keyword(s):

Structural Studies ◽

Attachment Sites ◽

Gamma Subunits ◽

Alpha Beta

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The role of heterotrimeric G proteins in the control of symbiosis development in legume plants

BIO Web of Conferences ◽

10.1051/bioconf/20202303004 ◽

2020 ◽

Vol 23 ◽

pp. 03004

Author(s):

Andrey D. Bovin ◽

Irina V. Leppyanen ◽

Olga A. Pavlova ◽

Elena A. Dolgikh

Keyword(s):

Gene Expression ◽

G Proteins ◽

Heterotrimeric G Proteins ◽

Subunit Gene ◽

Gene Suppression ◽

Gamma Subunits ◽

Genes Encoding ◽

Specific Proteins ◽

Alpha Beta

Heterotrimeric G proteins are involved in the regulation of signaling pathways in eukaryotes. Previously, the data about possible participation of heterotrimeric G proteins in the regulation of nodulation in legumes were obtained, however, specific proteins, their composition and role in this process remain poorly understood. In this work searching of the genes encoding the alpha, beta, and gamma subunits of heterotrimeric G proteins based on an analysis of the Pisum sativum L. genome was performed, as well as the dynamics of the gene expression encoding the particular subunits of G proteins in the process of symbiosis was studied. In addition, a significant effect of beta 1-subunit gene suppression by RNA interference on the nodulation process was revealed.

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Baculovirus-directed expression of the γ-subunit of phosphorylase kinase: purification and calmodulin dependence

Biochemical Journal ◽

10.1042/bj2990183 ◽

1994 ◽

Vol 299 (1) ◽

pp. 183-189 ◽

Cited By ~ 10

Author(s):

R A Lanciotti ◽

P K Bender

Keyword(s):

Skeletal Muscle ◽

Insect Cells ◽

Protein A ◽

Recombinant Baculovirus ◽

Phosphorylase Kinase ◽

Purification Procedure ◽

Gamma Subunit ◽

Regulatory Subunits ◽

Γ Subunit ◽

Alpha Beta

A recombinant baculovirus containing a cDNA encoding the gamma-subunit of phosphorylase kinase from mouse skeletal muscle was constructed. Cultures of Sf-9 insect cells infected with the gamma-baculovirus produce an intact and soluble gamma-protein. A purification procedure is presented that yields a sample of gamma-protein which is devoid of interfering enzyme activity and which is not associated with calmodulin from the insect cells. The isolated gamma sample has a Km for phosphorylase b of 36 (+/- 6, S.E.M) microM at pH 8.2 and 140 (+/- 25) microM at pH 6.8. These values are similar to those reported for the activated phosphorylase kinase holoenzyme isolated from skeletal muscle tissue. However, the Vmax. of the baculovirus-expressed gamma is 65 and 80% of that of the activated holoenzyme at pH 6.8 and 8.2 respectively. These results indicate that one or more of the regulatory subunits alpha, beta, or calmodulin stimulate the activity of the catalytic subunit gamma in the activated holoenzyme. Addition of calmodulin to the baculovirus-expressed gamma stimulates its activity 1.5-2.0 fold at pH 6.8 in both the presence and absence of calcium. At pH 8.2, calmodulin has only minor stimulatory affects. The stimulation by calmodulin at pH 6.8 results from an increase in the Vmax of gamma with little effect on its Km. This result is unlike that for most calmodulin-stimulated kinases which bind calmodulin only in the presence of calcium and exhibit a decrease in their Km upon binding calmodulin. The change in Vmax. of gamma in the presence of calmodulin and in the absence of calcium presents a novel mechanism for the regulation of a calmodulin-stimulated kinase.

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