scholarly journals Characterization of calmodulin-dependent cyclic nucleotide phosphodiesterase isoenzymes

1994 ◽  
Vol 299 (1) ◽  
pp. 97-100 ◽  
Author(s):  
R K Sharma ◽  
J Kalra
Keyword(s):  
Cyclic Nucleotide ◽  
Catalytic Properties ◽  
Bovine Brain ◽  
Fine Tuning ◽  
Kinetic Properties ◽  
Complex Interactions ◽  
Second Messenger Systems ◽  
The Brain ◽  
Phosphodiesterase Isoenzymes

Calmodulin-dependent phosphodiesterase (CaMPDE) is one of the key enzymes involved in the complex interactions which occur between the cyclic-nucleotide and Ca2+ second-messenger systems. Calmodulin-dependent phosphodiesterase exists in different isoenzymic forms, which exhibit distinct molecular and/or catalytic properties. The kinetic properties suggest that the 63 kDa brain isoenzyme is distinct from the brain 60 kDa and heart and lung CaMPDE isoenzymes. The CaMPDE isoenzymes of 60 kDa from brain, heart and lung are regulated by calmodulin, but the affinities for calmodulin are different. At identical concentrations of calmodulin, the bovine heart CaMPDE isoenzyme is stimulated at a much lower Ca2+ concentration than the bovine brain or lung isoenzymes. The bovine lung CaMPDE isoenzyme contains calmodulin as a tightly bound subunit, so that a change in calmodulin concentration had no effect on the [Ca2+]-dependence of activation of this isoenzyme. These observations are consistent with the notion that differential regulation by calmodulin and Ca2+ is an important function of these isoenzymes, which provide fine-tuning mechanisms for calmodulin action.

scholarly journals Characterization of calcium-dependent membrane binding proteins of brain cortex

1985 ◽  
Vol 229 (3) ◽  
pp. 587-593 ◽  
Author(s):  
A R Rhoads ◽  
M Lulla ◽  
P B Moore ◽  
C E Jackson
Keyword(s):  
Brain Cortex ◽  
Peptide Mapping ◽  
Bovine Brain ◽  
Particulate Fraction ◽  
Structural Homology ◽  
One Dimensional ◽  
Calcium Dependent ◽  
Stokes Radius ◽  
The Brain

Proteins of Mr 68 000, 34 000 and 32 000 were selectively extracted by EGTA from brain cortex. The three proteins that were extracted along with calmodulin were acidic, monomeric, and did not exhibit structural homology, as demonstrated by one-dimensional peptide mapping. The Mr-68 000 protein was purified to homogeneity and had a Stokes radius of 3.54 nm and S20,W value of 5.1S. Purified calmodulin, Mr-68 000 protein and two proteins of Mr 34 000 and Mr 32 000, interacted with the brain particulate fraction, with half-maximal binding occurring at 3.5 microM, 8.3 microM and 150 microM-Ca2+ respectively. Proteins were bound independently of each other and calmodulin. Pretreatment of the particulate fraction with trypsin prevented the Ca2+-dependent binding of calmodulin; however, the binding of the Mr-68 000 protein or the Mr−32 000 and −34 000 proteins was unaffected. The Mr-68 000 protein of bovine brain did not cross-react immunologically with Mr-67 000 calcimedin from chicken gizzard.

scholarly journals Purification and characterization of phosphatidylinositol 4-phosphate 5-kinases

1992 ◽  
Vol 288 (2) ◽  
pp. 637-642 ◽  
Author(s):  
N Divecha ◽  
C E L Brooksbank ◽  
R F Irvine
Keyword(s):  
Bovine Brain ◽  
Kinetic Properties ◽  
Type Ii ◽  
Purification And Characterization ◽  
Kinase C ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Single Polypeptide ◽  
Phosphatidylinositol 4

We detail the purification and characterization of three distinct isoforms of PtdIns4P 5-kinase present in bovine brain. One of these, PtdIns4P 5-kinase C, was purified to apparent homogeneity, and SDS/PAGE analysis demonstrated a single polypeptide and molecular mass 53 KDa. These three isoforms were shown to differ in their kinetic properties, and immunological characterization with an antibody raised to PtdIns4P 5-kinase C demonstrated that this isoform was unrelated to the other two. Furthermore, PtdIns4P 5-kinase C was shown to be the bovine brain homologue of the Type II PtdIns4P 5-kinase previously purified from human erythrocytes [Bazenet, Ruano, Brockman & Anderson (1990) J. Biol. Chem. 265, 18012-18022].

scholarly journals Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]oregulation

1997 ◽  
Vol 273 (5) ◽  
pp. C1516-C1525 ◽  
Author(s):  
John A. Payne
Keyword(s):  
Cell Lines ◽  
Functional Characterization ◽  
Cell Swelling ◽  
Dual Function ◽  
Kinetic Properties ◽  
Hek 293 ◽  
Apparent Affinity ◽  
293 Cells ◽  
The Brain

The neuronal K-Cl cotransporter isoform (KCC2) was functionally expressed in human embryonic kidney (HEK-293) cell lines. Two stably transfected HEK-293 cell lines were prepared: one expressing an epitope-tagged KCC2 (KCC2–22T) and another expressing the unaltered KCC2 (KCC2–9). The KCC2–22T cells produced a glycoprotein of ∼150 kDa that was absent from HEK-293 control cells. The 86Rb influx in both cell lines was significantly greater than untransfected control HEK-293 cells. The KCC2–9 cells displayed a constitutively active86Rb influx that could be increased further by 1 mM N-ethylmaleimide (NEM) but not by cell swelling. Both furosemide [inhibition constant ( K i) ∼25 μM] and bumetanide (Ki∼55 μM) inhibited the NEM-stimulated86Rb influx in the KCC2–9 cells. This diuretic-sensitive86Rb influx in the KCC2–9 cells, operationally defined as KCC2 mediated, required external Cl−but not external Na+ and exhibited a high apparent affinity for external Rb+(K+) [Michaelis constant ( K m) = 5.2 ± 0.9 (SE) mM; n = 5] but a low apparent affinity for external Cl−( K m >50 mM). On the basis of thermodynamic considerations as well as the unique kinetic properties of the KCC2 isoform, it is hypothesized that KCC2 may serve a dual function in neurons: 1) the maintenance of low intracellular Cl− concentration so as to allow Cl− influx via ligand-gated Cl− channels and 2) the buffering of external K+ concentration ([K+]o) in the brain.

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