scholarly journals Binding of ATP and ATP analogues to the uncoating ATPase Hsc70 (70 kDa heat-shock cognate protein)

1996 ◽  
Vol 318 (3) ◽  
pp. 923-929 ◽  
Author(s):  
Engelbert BUXBAUM ◽  
Philip G WOODMAN
Keyword(s):  
Heat Shock ◽  
Temperature Dependence ◽  
Mung Bean ◽  
Dissociation Constants ◽  
High Affinity ◽  
Pig Brain ◽  
High Affinity Site ◽  
Heat Shock Cognate Protein ◽  
Atp Analogues ◽  
Cognate Protein

Nucleotide binding to the 70 kDa heat-shock cognate protein (Hsc70) from mung bean seeds and pig brain was investigated, as well as the clathrin uncoating activity of Hsc70 in the presence of these nucleotides. The two enzymes were found to behave identically. ATP bound to two different forms of Hsc70, with dissociation constants of 1.1±0.1 µM and 1.4±0.7 mM respectively at 25 °C. This corresponds to ΔG0´ = -34 and -16 kJ/mol respectively. From the temperature-dependence of the dissociation constant of the high-affinity site, ΔH0´ was calculated to -36±2 kJ/mol. This gives ΔS0´ = 6.7 J/mol per K. Adenosine 5´-[γ-thio]triphosphate, ADP, adenosine 5´-[β,γ-imino]triphosphate and adenosine 5´-[β,γ-methylene]triphosphate showed dissociation constants of 2.3, 11, 31 and 284 µM respectively. The order of affinities corresponded to the order of effectiveness in uncoating of pig brain coated vesicles. The implications of these findings for the mechanism of Hsc70 action are discussed.

scholarly journals Identification of high-affinity (Kd 0.35 mumol/L) and low-affinity (Kd 7.9 mumol/L) platelet binding sites for ADP and competition by ADP analogues

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 110-116 ◽  
Author(s):  
JR Jefferson ◽  
JT Harmon ◽  
GA Jamieson
Keyword(s):  
Platelet Activation ◽  
Dissociation Constants ◽  
Partial Agonist ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Ionophore A23187 ◽  
High Affinity ◽  
Binding Isotherms ◽  
High Affinity Site ◽  
High Concentrations

Steady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2–3 H]ADP bound to two sites with a small amount (approximately 5% of total) of nonspecific binding: 410,000 +/- 40,000 sites of low affinity (Kd 7.9 +/- 2.0 mumol/L) and 160,000 +/- 20,000 sites of high affinity (Kd 0.35 +/- 0.04 mumol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1–0.5 mumol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3- aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 mumol/L and 200 mumol/L, respectively. The partial agonist 2′,3′-dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 mumol/L and 49 mumol/L, respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 mumol/L) approximately equal to ATP (Kd 0.45 mumol/L) much greater than AMP (Kd 360 mumol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuribenzene sulfonate (Ki 250 mumol/L) but only very weakly by 5′-p- fluorosulfonylbenzoyladenosine (Ki 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 mumol/L), 2-MeS ADP (200 mumol/L) and 2′,3′-dialdehyde ADP (270 mumol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 mumol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50 approximately 100 mumol/L) but not by collagen, thrombin, or ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS).

scholarly journals Exogenous heat shock cognate protein Hsc70 prevents axotomy-induced death of spinal sensory neurons

1996 ◽  
Vol 1 (3) ◽  
pp. 161 ◽  
Author(s):  
Lucien J. Houenou ◽  
Linxi Li ◽  
Ming Lei ◽  
Carol R. Kent ◽  
Michael Tytell
Keyword(s):  
Heat Shock ◽  
Sensory Neurons ◽  
Heat Shock Cognate Protein ◽  
Cognate Protein

Structural, thermodynamic and functional studies of human 71 kDa heat shock cognate protein (HSPA8/hHsc70)

2021 ◽  
Vol 1869 (12) ◽  
pp. 140719
Author(s):  
Noeli Soares Melo Silva ◽  
Luiz Fernando de Camargo Rodrigues ◽  
Paulo Roberto Dores-Silva ◽  
Carlos Alberto Montanari ◽  
Carlos Henrique Inácio Ramos ◽  
...  
Keyword(s):  
Heat Shock ◽  
Functional Studies ◽  
Heat Shock Cognate Protein ◽  
Cognate Protein

scholarly journals Exogenous Heat Shock Cognate Protein 70 Suppresses LPS-Induced Inflammation by Down-Regulating NF-κB through MAPK and MMP-2/-9 Pathways in Macrophages

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2124 ◽  
Author(s):  
Erna Sulistyowati ◽  
Mei-Yueh Lee ◽  
Lin-Chi Wu ◽  
Jong-Hau Hsu ◽  
Zen-Kong Dai ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Heat Shock ◽  
Critical Role ◽  
Matrix Metalloproteinase 2 ◽  
No Production ◽  
Raw 264.7 ◽  
Tnf Α ◽  
Heat Shock Cognate Protein ◽  
Anti Inflammatory ◽  
Cognate Protein

Heat shock cognate protein 70 (HSC70), a molecular chaperone, is constitutively expressed by mammalian cells to regulate various cellular functions. It is associated with many diseases and is a potential therapeutic target. Although HSC70 also possesses an anti-inflammatory action, the mechanism of this action remains unclear. This current study aimed to assess the anti-inflammatory effects of HSC70 in murine macrophages RAW 264.7 exposed to lipopolysaccharides (LPS) and to explain its pathways. Mouse macrophages (RAW 264.7) in 0.1 µg/mL LPS incubation were pretreated with recombinant HSC70 (rHSC70) and different assays (Griess assay, enzyme-linked immune assay/ELISA, electrophoretic mobility shift assay/EMSA, gelatin zymography, and Western blotting) were performed to determine whether rHSC70 blocks pro-inflammatory mediators. The findings showed that rHSC70 attenuated the nitric oxide (NO) generation, tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) expressions in LPS-stimulated RAW264.7 cells. In addition, rHSC70 preconditioning suppressed the activities and expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9. Finally, rHSC70 diminished the nuclear translocation of nuclear factor-κB (NF-κB) and reduced the phosphorylation of extracellular-signal regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPK), and phosphatidylinositol-3-kinase (PI3K/Akt). We demonstrate that rHSC70 preconditioning exerts its anti-inflammatory effects through NO production constriction; TNF-α, and IL-6 suppression following down-regulation of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and MMP-2/MMP-9. Accordingly, it ameliorated the signal transduction of MAPKs, Akt/IκBα, and NF-κB pathways. Therefore, extracellular HSC70 plays a critical role in the innate immunity modulation and mechanisms of endogenous protective stimulation.

Psychophysiological stress induces heat shock cognate protein (HSC)70 mRNA in the cerebral cortex and stomach of rats

1995 ◽  
Vol 675 (1-2) ◽  
pp. 98-102 ◽  
Author(s):  
Shin Fukudo ◽  
Koji Abe ◽  
Michio Hongo ◽  
Atsushi Utsumi ◽  
Yasuto Itoyama
Keyword(s):  
Cerebral Cortex ◽  
Heat Shock ◽  
Psychophysiological Stress ◽  
Hsc 70 ◽  
Heat Shock Cognate Protein ◽  
Cognate Protein
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