scholarly journals Acyl-CoA-binding protein (ACBP) can mediate intermembrane acyl-CoA transport and donate acyl-CoA for β-oxidation and glycerolipid synthesis

1994 ◽  
Vol 299 (1) ◽  
pp. 165-170 ◽  
Author(s):  
J T Rasmussen ◽  
N J FÆrgeman ◽  
K Kristiansen ◽  
J Knudsen
Keyword(s):  
Dissociation Constants ◽  
Binding Protein ◽  
Nitrocellulose Membrane ◽  
Beta Oxidation ◽  
Titration Microcalorimetry ◽  
Kd Values ◽  
Glycerolipid Synthesis

The dissociation constants for octanoyl-CoA, dodecanoyl-CoA and hexadecanoyl-CoA binding to acyl-CoA-binding protein (ACBP) were determined by using titration microcalorimetry. The KD values obtained, (0.24 +/- 0.02) x 10(-6) M, (0.65 +/- 0.2) x 10(-8) M and (0.45 +/- 0.2) x 10(-13) M respectively, were much lower than expected. ACBP was able to extract hexadecanoyl-CoA from phosphatidylcholine membranes immobilized on a nitrocellulose membrane. The acyl-CoA/ACBP complex formed was able to transport acyl-CoA to mitochondria or microsomes in suspension, or to microsomes immobilized on a nitrocellulose membrane, and to donate them to beta-oxidation or glycerolipid synthesis in mitochondria or microsomes, respectively.

scholarly journals Quin 2: the dissociation constants of its Ca2+ and Mg2+ complexes and its use in a fluorimetric method for determining the dissociation of Ca2+-protein complexes

1985 ◽  
Vol 226 (2) ◽  
pp. 613-616 ◽  
Author(s):  
D T W Bryant
Keyword(s):  
Vitamin D ◽  
Good Accuracy ◽  
Dissociation Constants ◽  
Protein Complexes ◽  
Binding Protein ◽  
Fluorimetric Method ◽  
Kd Values ◽  
Spectrophotometric Titrations

Fluorimetric or spectrophotometric titrations with the appropriate cations gave Kd values of 2.9 +/- 0.2 nM and 89 +/- 5 microM respectively for the Ca2+ and Mg2+ complexes of quin 2 at pH 7.5. Mixtures of quin 2 and vitamin D-dependent Ca2+-binding protein from pig duodenum were titrated fluorimetrically with Ca2+ in the absence or presence of Mg2+. These measurements were used with the Kd values of the Ca2+ and Mg2+ complexes of quin 2 to obtain Kd or apparent Kd values for Ca2+-protein complexes ranging from 5 nM to 5 microM with good accuracy.

scholarly journals Studies on the Interaction between Poly-Phosphane Gold(I) Complexes and Dihydrofolate Reductase: An Interplay with Nicotinamide Adenine Dinucleotide Cofactor

2019 ◽  
Vol 20 (7) ◽  
pp. 1802
Author(s):  
Stefania Pucciarelli ◽  
Silvia Vincenzetti ◽  
Massimo Ricciutelli ◽  
Oumarou Camille Simon ◽  
Anna Teresa Ramadori ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Protein Interactions ◽  
Dissociation Constants ◽  
Gold Compound ◽  
Inhibitory Effects ◽  
Binding Properties ◽  
Enzyme Active Site ◽  
E Coli ◽  
Kd Values ◽  
Gold Compounds

A class of gold(I) phosphane complexes have been identified as inhibitors of dihydrofolate reductase (DHFR) from E. coli, an enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), using NADPH as a coenzyme. In this work, to comprehend the nature of the interaction at the basis of these inhibitory effects, the binding properties of bis- and tris-phosphane gold(I) chloride compounds in regards to DHFR have been studied by emission spectroscopy and spectrophotometric assays. The lack of cysteine and seleno-cysteine residues in the enzyme active site, the most favorable sites of attack of Au(I) moieties, makes this work noteworthy. The interaction with the gold compounds results into the quenching of the DHFR tryptophan’s emissions and in an enhancement of their intrinsic emission intensities. Moreover, a modulating action of NADPH is highlighted by means of an increase of the gold compound affinity toward the enzyme; in fact, the dissociation constants calculated for the interactions between DHFR and each gold compound in the presence of saturating NADPH were lower than the ones observed for the apo-enzyme. The fluorimetric data afforded to Kd values ranged from 2.22 ± 0.25 µM for (PPh3)2AuCl in the presence of NADPH to 21.4 ± 3.85 µM for 4L3AuTf in the absence of NADPH. By elucidating the energetic aspects of the binding events, we have attempted to dissect the role played by the gold phosphane/protein interactions in the inhibitory activity, resulting in an exothermic enthalpy change and a positive entropic contribution (ΔH° = −5.04 ± 0.08 kcal/mol and ΔS° = 7.34 ± 0.005 cal/mol·K).

scholarly journals Binding of ADP to rat liver cytosolic proteins and its influence on the ratio of free ATP/free ADP

1989 ◽  
Vol 259 (1) ◽  
pp. 117-124 ◽  
Author(s):  
S Mörikofer-Zwez ◽  
P Walter
Keyword(s):  
Ethylene Glycol ◽  
Rat Liver ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Cellular Protein ◽  
Poly Ethylene Glycol ◽  
Cytosolic Proteins ◽  
Kd Values ◽  
Cytosolic Extract ◽  
Poly Ethylene

In a cytosolic extract from rat liver, the number and the concentration of ADP-binding sites as well as their dissociation constants were determined by using the rate-of-dialysis technique. Interfering cytosolic adenylate kinase was extracted from the cytosol by affinity chromatography on Ap5A-agarose, and remaining traces of enzyme activity were inhibited with (+)-catechin. Binding of ADP to cytosolic proteins was increased by poly(ethylene glycol) and decreased by EDTA. The effect of 0.1 mM-EDTA could be reversed by addition of equimolar concentrations of Mn2+ or Mg2+. In presence of 5% poly(ethylene glycol), added to increase local protein concentration, two binding sites for ADP were observed, with KD values of 1.9 microM (site I) and 10.8 microM (site II). The concentration of these binding sites, when extrapolated to cellular protein concentrations, were 30 microM (site I) and 114 microM (site II). It is concluded that a minimum of about 50% of total cytosolic ADP is bound to proteins, and that the ratio of free ATP/free ADP is at least twice that of total ATP/total ADP.

scholarly journals Acyl-Coenzyme A–Binding Protein Regulates Beta-Oxidation Required for Growth and Survival of Non–Small Cell Lung Cancer

2014 ◽  
Vol 7 (7) ◽  
pp. 748-757 ◽  
Author(s):  
Fredrick T. Harris ◽  
S.M. Jamshedur Rahman ◽  
Mohamed Hassanein ◽  
Jun Qian ◽  
Megan D. Hoeksema ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Coenzyme A ◽  
Binding Protein ◽  
Small Cell ◽  
Small Cell Lung ◽  
Beta Oxidation ◽  
Growth And Survival ◽  
Acyl Coenzyme A

scholarly journals Microfluidic Affinity Profiling reveals a Broad Range of Target Affinities for Anti-SARS-CoV-2 Antibodies in Plasma of Covid Survivors

2020 ◽  
Author(s):  
Matthias M. Schneider ◽  
Marc Emmenegger ◽  
Catherine K. Xu ◽  
Itzel Condado Morales ◽  
Priscilla Turelli ◽  
...  
Keyword(s):  
Immune Response ◽  
Dissociation Constants ◽  
Total Plasma ◽  
Antibody Affinity ◽  
Unrelated Control ◽  
Receptor Complexes ◽  
Kd Values ◽  
Antibody Titres ◽  
Over Time

The clinical outcome of SARS-CoV-2 infections can range from asymptomatic to lethal, and is thought to be crucially shaped by the quality of the immune response which includes antibody titres and affinity for their targets. Using Microfluidic Antibody Affinity Profiling (MAAP), we determined the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 23 of whom were confirmed to be SARS-CoV-2-positive by PCR testing. We found that dissociation constants (Kd) of anti-RBD antibodies spanned more than two orders of magnitude from 80 pM to 25 nM, despite having similar antibody concentrations. Individual patients showed progressively higher antibody concentrations but constant Kd values, suggesting that affinities did not mature over time. 33 sera showed affinities higher than that of the CoV2 spike for its ACE2 receptor. Accordingly, addition of seropositive plasma to pre-formed spike-ACE2 receptor complexes led to their dissociation. Finally, we observed that the RBD of HKU1, OC43, and SARS-CoV coronaviruses, but not unrelated control proteins, were able to compete substantially with the RBD of SARS-CoV-2 in solution. Therefore, the affinity of total plasma immunoglobulins to SARS-CoV-2 is an indicator of the quality of the immune response to SARS-CoV-2, and may help select the most efficacious samples for therapeutic plasmapheresis.

scholarly journals Structural Investigations of a New Familial Dysalbuminemic Hyperthyroxinemia Genotype

1999 ◽  
Vol 45 (8) ◽  
pp. 1248-1254 ◽  
Author(s):  
Charles E Petersen ◽  
Chung-Eun Ha ◽  
Krishna Harohalli ◽  
David S Park ◽  
Jimmy B Feix ◽  
...  
Keyword(s):  
Amino Acid ◽  
Human Serum Albumin ◽  
Dissociation Constants ◽  
High Serum ◽  
Wild Type ◽  
Japanese Family ◽  
Structural Investigations ◽  
Kd Values ◽  
Total Thyroxine ◽  
Caucasian Patients

Abstract Background: In a previous study, we found that the amino acid substitution R218H in human serum albumin (HSA) was the cause of familial dysalbuminemic hyperthyroxinemia (FDH) in several Caucasian patients. Subsequently the substitution R218P was shown to be the cause of FDH in several members of a Japanese family. This study attempts to resolve discrepancies in the only other study of R218P HSA and identifies two new Japanese R218P FDH patients unrelated to those described previously. Methods and Results: Recombinant R218H, R218P, and wild-type HSA were synthesized in yeast, and the affinities of these HSA species for l- and d-thyroxine were determined using fluorescence spectroscopy. The dissociation constants for the binding of wild-type, R218P, and R218H HSA to l-thyroxine were 1.44 × 10−6, 2.64 × 10−7, and 2.49 × 10−7 mol/L, respectively. The circular dichroism spectra of thyroxine bound to R218H and R218P HSA were markedly different, indicating that the structure of the thyroxine/HSA complex is different for either protein. Conclusions: The Kd values for l-thyroxine bound to R218P and R218H HSA determined in this study were similar. The extremely high serum total-thyroxine concentrations reported previously for R218P FDH patients (10-fold higher than those reported for R218H FDH patients) are not consistent with the Kd values determined in this study. Possible explanations for these discrepancies are discussed.

scholarly journals The binding of natural and fluorescent lysophospholipids to wild-type and mutant rat liver fatty acid-binding protein and albumin

1995 ◽  
Vol 307 (1) ◽  
pp. 305-311 ◽  
Author(s):  
A E A Thumser ◽  
D C Wilton
Keyword(s):  
Fatty Acid ◽  
Rat Liver ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Liver Fatty Acid ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
Kd Values ◽  
Fluorescence Characteristics ◽  
Wide Range

Rat liver fatty acid-binding protein (FABP) is able to bind a wide range of non-polar anionic ligands, including lysophospholipids. In order to understand the nature of lysophospholipid interactions with liver FABP, the binding of natural lysophospholipids and two fluorescent analogues, N-(5-dimethylaminonaphthalenesulphonyl)-1-palmitoyl-sn-glycero-3- phosphoethanolamine (dansyl lysoPE) and 1-(O-[11-(5-dimethylaminonaphthalene-sulphonyl)amino]undecyl)-sn-glycero -3- phosphocholine (dansyl-C11-lysoPAF), has been investigated. The results confirmed the ability of liver FABP to bind lysophospholipids with KD values in the range of 1-2 microM, and a 1:1 binding stoichiometry was indicated. Binding of fluorescent lysophospholipids was enhanced with the FABP mutant, R122Q, possibly due to increased flexibility of the binding cavity as a result of reduced hydrogen-bonding constraints. The fluorescent lysophospholipids also bound to albumin, with KD values in the range 0.1-1.0 microM, and could be displaced by oleic acid. The fluorescence characteristics of the dansyl lysophospholipid analogue dansyl-C11-lyso-PAF suggested that this probe binds to the same site(s) on albumin as the fluorescent fatty acid probe 11-(5-dimethylaminonaphthalene-sulphonylamino)-undecanoic acid (DAUDA).

Cyclosporin G and metabolite binding to cyclophilin and a 50-kDa binding protein related to in vitro immunosuppression

1993 ◽  
Vol 39 (1) ◽  
pp. 122-125 ◽  
Author(s):  
J G Donnelly ◽  
Y Chen ◽  
R W Yatscoff ◽  
K R Copeland ◽  
E W Palaszynski ◽  
...  
Keyword(s):  
Cyclosporin A ◽  
Dissociation Constants ◽  
Binding Protein ◽  
Potency Ratio ◽  
Immunosuppressive Potency ◽  
Metabolite Binding ◽  
Suppression Assay

Abstract Seven purified metabolites of cyclosporin G (CsG) were studied for binding to cyclophilin and a 50-kDa binding protein (50-kDa BP). The ratios of the metabolite dissociation constants with respect to CsG were compared with in vitro immunosuppression by using the primary mixed lymphocyte suppression assay. The immunosuppressive potency ratio of the parent compounds, both cyclosporin A (CsA) and CsG, compared favorably with the drug dissociation constants for cyclophilin and the 50-kDa BP. Three of the seven metabolites had comparable binding and potency ratios for the 50-kDa BP. In contrast, none of the seven metabolites appreciably bound to cyclophilin in the concentration range tested.

scholarly journals A tobacco (Nicotiana tabaccum) calmodulin-binding protein kinase, NtCBK2, is regulated differentially by calmodulin isoforms

2003 ◽  
Vol 376 (1) ◽  
pp. 291-302 ◽  
Author(s):  
Wei HUA ◽  
Shuping LIANG ◽  
Ying-Tang LU
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Dissociation Constants ◽  
Binding Protein ◽  
Enzymic Activity ◽  
Protein Motif ◽  
Independent Manner ◽  
Nicotiana Tabaccum ◽  
Calmodulin Binding ◽  
Dependent Protein

A calcium (Ca2+)/calmodulin (CaM)-binding protein kinase (CBK) from tobacco (Nicotiana tabaccum), NtCBK2, has been characterized molecularly and biochemically. NtCBK2 has all 11 conserved subdomains of the kinase-catalytic domain and a CaM-binding site as shown by other kinases, including Ca2+-dependent protein kinase and chimaeric Ca2+/CaM-dependent protein kinases. However, this kinase does not contain an EF-hand motif for Ca2+ binding, and its activity was not regulated by Ca2+. Whereas NtCBK2 phosphorylated both itself and other substrates, such as histone IIIS and syntide-2, in a Ca2+/CaM-independent manner, as also shown by OsCBK, a CaM-binding protein kinase from rice (Oryza sativa), the kinase activity of NtCBK2 was greatly stimulated by Ca2+/CaM, whereas that of OsCBK was not. By molecular dissection analyses, the CaM-binding domain of NtCBK2 has been localized in a stretch of 30 amino acid residues at residue positions 431–460 as a 1-5-10 protein motif. Three tobacco CaM isoforms (NtCaM1, NtCaM3 and NtCaM13) used in the present study have been shown to bind to NtCBK2, but with different dissociation constants (Kds), as follows: NtCaM1, 55.7 nM; NtCaM3, 25.4 nM; and NtCaM13, 19.8 nM, indicating that NtCBK2 has a higher affinity for NtCaM3 and NtCaM13 than for NtCaM1. The enzymic activity of NtCBK2 was also modulated differently by various CaM isoforms. Whereas the phosphorylation activity of NtCBK2 was shown by assay to be enhanced only ≈2–3-fold by the presence of NtCaM1, the activity could be amplified up to 8–9-fold by NtCaM3 or 10–11-fold by NtCaM13, suggesting that NtCaM3 and NtCaM13 are better activators than NtCaM1 for NtCBK2.

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