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.

scholarly journals Differential effects of interleukin-15 and interleukin-2 on differentiation of bipotential T/natural killer progenitor cells.

1996 ◽  
Vol 184 (2) ◽  
pp. 325-336 ◽  
Author(s):  
G Leclercq ◽  
V Debacker ◽  
M de Smedt ◽  
J Plum
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Cell Number ◽  
Gamma Delta ◽  
Fetal Thymus ◽  
Low Concentrations ◽  
Alpha Beta ◽  
High Concentrations ◽  
Delta Cells ◽  
Gamma Delta Cells

Bipotential T/natural killer (NK) progenitor cells are destined to differentiate mainly into T cell receptor (TCR) alpha beta and TCR gamma delta cells in a thymic microenvironment, whereas extrathymically they selectively develop into NK cells. The exact environmental conditions that are required for differentiation into these three leukocyte populations are largely unknown. In this report, we have investigated and compared the effect of interleukin (IL)-15 and IL-2 in this process. The IL-15 receptor is composed of the gamma and beta chains of the IL-2 receptor (IL-2R gamma and IL-2R beta) and of a specific alpha chain (IL-15R alpha). Here, it is shown that IL-15 mRNA is mainly expressed in thymic epithelial stromal cells, whereas IL-2 mRNA is exclusively expressed in thymocytes. IL-2R beta-expressing cells were present in the fetal thymus with a CD25-CD44+Fc gamma R+HSA-/low TCR- phenotype, which is characteristic of progenitor cells. These cells also expressed IL-15R alpha messenger RNA. Sorted IL-2R beta + TCR- cells differentiated into TCR alpha beta and TCR gamma delta cells after transfer to alymphoid thymic lobes, whereas culture of the same sorted cells in cell suspension in the presence of IL-15 resulted in the generation of functional NK cells. This shows that IL-2R beta +TCR- cells of the fetal thymus contain bipotential T/NK progenitors. Addition of low concentrations of IL-15 to fetal thymic organ culture (FTOC) resulted in an increase of all T cell subpopulations. The largest expansion occurred in the TCR gamma delta compartment. In contrast, low concentrations of IL-2 did not result in a higher total cell number and did not induce outgrowth of TCR gamma delta cells. High concentrations of IL-15 blocked TCR alpha beta development and shifted differentiation towards NK cells. Differentiation towards TCR gamma delta cells still proceeded. High concentrations of IL-2 similarly induced development into NK cells, but the cell number was fourfold lower than in IL-15 cultures. Importantly, blocking of IL-2R alpha in IL-2-treated FTOC resulted in a drastic increase in cell number, indicating that IL-2R alpha negatively regulates cell expansion. Collectively, these experiments provide direct evidence that IL-15 and IL-2 differentially affect the differentiation of bipotential T/NK progenitors.

scholarly journals A novel architecture of 600 kDa bacterial citrate lyase complex

2014 ◽  
Vol 70 (a1) ◽  
pp. C1284-C1284
Author(s):  
Farhad Forouhar ◽  
Scott Lew ◽  
Jayaraman Seetharaman ◽  
Rong Xiao ◽  
Thomas Acton ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Gel Filtration ◽  
Single Step ◽  
Bacterial Genomes ◽  
Citrate Lyase ◽  
Gamma Subunits ◽  
Genes Encoding ◽  
Lyase Activity ◽  
Entire Complex ◽  
Alpha Beta

Citrate lyase activity exists in eukaryotes, bacteria, and archaea and plays a central role in cellular energy metabolism. There are two types of citrate lyases, ATP-dependent and ATP-independent. The latter, which exists only in bacteria, converts citrate to acetate and oxaloacetate using a two-step reaction catalyzed by a stable heterotrimeric protein complex comprising alpha-beta-gamma subunits. We selected three bacterial genomes in which the alpha-beta-gamma subunits of citrate lyase are all encoded on the chromosome in a continuous, co-cistronic geometry. The genes encoding the entire complex were subsequently amplified together using a single PCR reaction on genomic DNA. This approach enabled the entire citrate lyase complex from three organisms to be purified to homogeneity using a single step of Ni-NTA chromatography followed by gel-filtration chromatography. One of the three citrate lyase complexes purified yielded crystals diffracting to 4 Å. Six-fold non-crystallographic symmetry averaging enabled a high quality structure to be determined for the 18-subunit alpha-beta-gamma citrate lyase complex. This structure provides insight into the multistep catalytic mechanism employed by this enzyme.

scholarly journals Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells

1989 ◽  
Vol 263 (1) ◽  
pp. 223-229 ◽  
Author(s):  
K C Cawley ◽  
C G Akita ◽  
D A Walsh
Keyword(s):  
Kinase Activity ◽  
Phosphorylase Kinase ◽  
3T3 Cells ◽  
Gamma Delta ◽  
Gamma Subunit ◽  
Mammalian Expression ◽  
Mammalian Expression Vector ◽  
Muscle Phosphorylase ◽  
Nih 3T3 ◽  
Delta Subunit

Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.

scholarly journals Trigonometric approximation of functions $f(x,y)$ of generalized Lipschitz class by double Hausdorff matrix summability method

2020 ◽  
Vol 2020 (1) ◽  
Author(s):  
Abhishek Mishra ◽  
Vishnu Narayan Mishra ◽  
M. Mursaleen
Keyword(s):  
Double Fourier Series ◽  
Degree Of Approximation ◽  
Trigonometric Approximation ◽  
Lipschitz Class ◽  
Gamma Delta ◽  
Approximation Of Functions ◽  
Matrix Summability ◽  
Alpha Beta ◽  
Generalized Lipschitz Class ◽  
Hausdorff Matrix

AbstractIn this paper, we establish a new estimate for the degree of approximation of functions $f(x,y)$ f ( x , y ) belonging to the generalized Lipschitz class $Lip ((\xi _{1}, \xi _{2} );r )$ L i p ( ( ξ 1 , ξ 2 ) ; r ) , $r \geq 1$ r ≥ 1 , by double Hausdorff matrix summability means of double Fourier series. We also deduce the degree of approximation of functions from $Lip ((\alpha ,\beta );r )$ L i p ( ( α , β ) ; r ) and $Lip(\alpha ,\beta )$ L i p ( α , β ) in the form of corollary. We establish some auxiliary results on trigonometric approximation for almost Euler means and $(C, \gamma , \delta )$ ( C , γ , δ ) means.

scholarly journals Analysis of serum lipoproteins by high performance gel filtration chromatography.

1996 ◽  
Vol 45 (1) ◽  
pp. 103-106 ◽  
Author(s):  
Takashi KITAMURA ◽  
Seiji ITO ◽  
Yoshio KATO ◽  
Keiko SASAMOTO ◽  
Mitsuyo OKAZAKI
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Serum Lipoproteins ◽  
Gel Filtration Chromatography
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