High-affinity calcium and substrate-binding sites on protein kinase C .alpha. as determined by nuclear magnetic resonance spectroscopy

Biochemistry ◽  
1992 ◽  
Vol 31 (33) ◽  
pp. 7714-7721 ◽  
Author(s):  
Muriel C. Maurer ◽  
Julianne J. Sando ◽  
Charles M. Grisham
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Protein Kinase C ◽  
Magnetic Resonance ◽  
Protein Kinase ◽  
Magnetic Resonance Spectroscopy ◽  
Binding Sites ◽  
Resonance Spectroscopy ◽  
High Affinity ◽  
Kinase C ◽  
Substrate Binding Sites

scholarly journals Effects of phorbol esters and secretagogues on nitrobenzylthioinosine binding to nucleoside transporters and nucleoside uptake in cultured chromaffin cells

1991 ◽  
Vol 279 (3) ◽  
pp. 651-655 ◽  
Author(s):  
E G Delicado ◽  
R P Sen ◽  
M T Miras-Portugal
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Sites ◽  
Chromaffin Cells ◽  
Phorbol Esters ◽  
Binding Studies ◽  
High Affinity ◽  
Kinase C ◽  
Adenosine Uptake ◽  
Protein Kinase C Activators

Secretagogues inhibited adenosine uptake in chromaffin cells without causing apparent changes in the uptake affinity. The inhibition caused by carbachol, nicotine and acetylcholine reached 50%. This inhibition was reproduced by the action of protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA; 100 nM), phorbol 12,13-dibutyrate (PDBu; 100 nM), dicaproin (10 micrograms/ml) and tricaprylin (10 micrograms/ml), with inhibitions of Vmax. of 18, 20, 37 and 47% respectively. No changes in the affinity of uptake were observed with these effectors. Down-regulation of protein kinase C by phorbol esters decreased the inhibitory effects of carbachol on adenosine uptake. Binding studies with nitrobenzylthioinosine (NBTI) showed a similar decrease in the number of transporters when chromaffin cells were treated with the same effectors used for the uptake studies. The high-affinity dissociation constants showed minor changes with respect to the control. The ratio between maximal uptake capacity and the transporter number per cell was not significantly modified by the action of secretagogues or direct effectors of protein kinase C. The number of high-affinity binding sites for NBTI was decreased in cellular homogenates by the direct action of protein kinase C activators, with staurosporine able to reverse this action. Protein kinase C from bovine brain in the presence of ATP and effectors, decreased the number of high-affinity NBTI-binding sites in purified chromaffin cell plasma membranes. These data suggest the possibility of a molecular modification at the transporter level.

scholarly journals Sevoflurane Binds and Allosterically Blocks Integrin Lymphocyte Function-associated Antigen-1

2010 ◽  
Vol 113 (3) ◽  
pp. 600-609 ◽  
Author(s):  
Koichi Yuki ◽  
Nathan S. Astrof ◽  
Clay Bracken ◽  
Sulpicio G. Soriano ◽  
Motomu Shimaoka
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Magnetic Resonance Spectroscopy ◽  
Ligand Binding ◽  
Nuclear Magnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Lymphocyte Function ◽  
Wild Type ◽  
Allosteric Inhibition ◽  
High Affinity

Background Volatile anesthetics have been shown to modify immune cell functions via several mechanisms, some of which have been only partially elucidated. We demonstrated that isoflurane inhibits primary leukocyte integrin lymphocyte function-associated antigen-1 (LFA-1) by binding to the allosteric cavity critical for conformational activation to its high-affinity form. It remains to be determined whether the allosteric inhibition of LFA-1 by isoflurane can be generalized to other anesthetics such as sevoflurane. Methods The effects of sevoflurane on the ability of LFA-1 to bind to its counter-ligand, intercellular adhesion molecule-1, was studied in leukocytes by flow cytometry. To examine whether sevoflurane acts directly on LFA-1, we measured ligand-binding using beads coated with purified LFA-1 protein. To distinguish between competitive versus allosteric inhibition, we analyzed the effects of sevoflurane on both wild-type and mutant-locked high-affinity LFA-1. One-way analysis of variance was employed for statistical analysis of the data. Nuclear magnetic resonance spectroscopy was used to identify sevoflurane binding site(s). Results Sevoflurane at clinically relevant concentrations inhibited the ligand-binding function of LFA-1 in leukocytes as well as in cell-free assays (P<0.05). Sevoflurane blocked wild-type but not locked high-affinity LFA-1, thereby demonstrating an allosteric mode of inhibition. Nuclear magnetic resonance spectroscopy revealed that sevoflurane bound to the allosteric cavity, to which LFA-1 allosteric antagonists and isoflurane also bind. Conclusions This study suggests that sevoflurane also blocks the activation-dependent conformational changes of LFA-1 to the high-affinity form. The allosteric mode of action exemplified by sevoflurane and isoflurane via LFA-1 might represent one of the underlying mechanisms of anesthetic-mediated immunomodulation.

scholarly journals Neurotensin stimulates inositol trisphosphate-mediated calcium mobilization but not protein kinase C activation in HT29 cells. Involvement of a G-protein

1989 ◽  
Vol 264 (3) ◽  
pp. 871-878 ◽  
Author(s):  
J C Bozou ◽  
N Rochet ◽  
I Magnaldo ◽  
J P Vincent ◽  
P Kitabgi
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
G Proteins ◽  
Binding Sites ◽  
Inositol Phosphate ◽  
Calcium Mobilization ◽  
Scatchard Analysis ◽  
High Affinity ◽  
Ht29 Cells ◽  
Kinase C

It has previously been shown that neurotensin binds to high-affinity receptors in the adenocarcinoma HT29 cell line, and that receptor occupancy leads to inositol phosphate formation. The present study was designed to investigate further the effects of neurotensin on calcium mobilization and protein kinase C (PKC) activation in HT29 cells, and to assess the role of GTP-binding proteins (G-proteins) in the neurotensin response. Direct measurements of cytosolic Ca2+ variations using the fluorescent indicator quin 2 showed that neurotensin (0.1-1 microM) elicited Ca2+ transients in HT29 cells. These transients occurred after the neurotensin-stimulated formation of Ins(1,4,5)P3, as measured by means of a specific radioreceptor assay. In addition, the peptide induced a decrease in the 45Ca2+ content of cells previously equilibrated with this isotope. The peptide effect was rapid, long-lasting and concentration-dependent, with an EC50 of 2 nM. Phorbol 12-myristate 13-acetate (PMA) inhibited by 50% the neurotensin effects on both intracellular Ca2+ and inositol phosphate levels. The inhibition by PMA was abolished in PKC-depleted cells. Pertussis toxin had no effect on either the Ca2+ or inositol phosphate responses to neurotensin. Epidermal growth factor (EGF) receptors which are present in HT29 cells have been shown to be down-regulated through phosphorylation by PKC in a variety of systems. Here, PMA markedly (70-80%) inhibited EGF binding to HT29 cells. Scatchard analysis revealed that PMA abolished the high-affinity component of EGF binding, an effect that was totally reversed in PKC-depleted cells. In contrast, neurotensin slightly (10-20%) inhibited EGF binding to HT29 cells, and its effect was only partly reversed by PKC depletion. Neurotensin had no detectable effect on sn-1,2-diacylglycerol levels in HT29 cells, as measured by a specific and sensitive enzymic assay. In membranes prepared from HT29 cells, monoiodo[125I-Tyr3]neurotensin bound to a single population of receptors with a dissociation constant of 0.27 nM. Sodium and GTP inhibited neurotensin binding in a concentration-dependent manner. Maximal inhibition reached 80% with Na+ and 35% with GTP.IC50 values were 20 mM and 0.2 microM for Na+ and GTP respectively. Li+ and K+ were less effective than Na+ and the effects of GTP were shared by GDP and guanosine-5′-[beta gamma- imido]triphosphate but not by ATP. Scatchard analysis of binding data indicated that Na+ and GTP converted the high-affinity neurotensin-binding sites into lower affinity binding sites. The properties of the effects of Na+ and GTP on neurotensin-receptor interactions are characteristic of those receptors which interact with G-proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

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