Irreversible inhibition of folate transport in Lactobacillus casei by covalent modification of the binding protein with carbodiimide-activated folate

1982 ◽  
Vol 216 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Gary B. Henderson ◽  
Suzana Potuznik
Keyword(s):  
Lactobacillus Casei ◽  
Binding Protein ◽  
Covalent Modification ◽  
Folate Transport ◽  
Irreversible Inhibition

scholarly journals The plasma membrane Ca2+ pump mutant lysine591 → arginine retains some activity, but is still inactivated by fluorescein isothiocyanate

1996 ◽  
Vol 317 (1) ◽  
pp. 41-44 ◽  
Author(s):  
Hugo P. ADAMO ◽  
Adelaida G. FILOTEO ◽  
John T. PENNISTON
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Human Plasma ◽  
Fluorescein Isothiocyanate ◽  
Covalent Modification ◽  
Transport Activity ◽  
Wild Type ◽  
Pump Function ◽  
Irreversible Inhibition ◽  
Lysine Residues

Inactivation of the wild-type human plasma membrane Ca2+ pump (isoform 4b) by fluorescein isothiocyanate is accompanied by covalent modification of Lys591. The mutation of Lys591 to arginine reduced the Ca2+ transport activity to 35% of the wild-type, and diminished the amount of acylphosphate formed from ATP by a corresponding amount. When this mutant was treated with fluorescein isothiocyanate, the enzyme was still irreversibly inactivated, even though no reactive residue was available at position 591. The results show that, although Ca2+ pump function is sensitive to the residue at position 591, Lys591 is not essential for enzyme activity. They also demonstrate that irreversible inhibition of the plasma membrane Ca2+ pump by fluorescein isothiocyanate does not require the covalent modification of Lys591. This indicates that fluorescein isothiocyanate reacts with lysine residues at other positions in addition to Lys591.

scholarly journals Covalent Modification of Epithelial Fatty Acid-binding Protein by 4-Hydroxynonenalin Vitroandin Vivo

2002 ◽  
Vol 277 (52) ◽  
pp. 50693-50702 ◽  
Author(s):  
Assumpta Bennaars-Eiden ◽  
LeeAnn Higgins ◽  
Ann V. Hertzel ◽  
Rebecca J. Kapphahn ◽  
Deborah A. Ferrington ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Covalent Modification ◽  
Fatty Acid Binding ◽  
Acid Binding Protein

Trimethoprim interferes with serum methotrexate assay by the competitive protein binding technique.

1980 ◽  
Vol 26 (11) ◽  
pp. 1617-1619 ◽  
Author(s):  
K Hande ◽  
J Gober ◽  
R Fletcher
Keyword(s):  
Protein Binding ◽  
Dihydrofolate Reductase ◽  
Lactobacillus Casei ◽  
Binding Assay ◽  
Binding Protein ◽  
Concomitant Administration ◽  
Protein Binding Assay ◽  
Competitive Protein Binding Assay ◽  
Competitive Protein Binding ◽  
Similar Combination

Abstract Administration of Bactrim (a combination of trimethoprim and sulfamethoxazole) to a patient who also was receiving methotrexate caused a significant increase in apparent plasma methotrexate concentrations as determined by competitive protein binding assay with use of dihydrofolate reductase (EC 1.5.1.3) from Lactobacillus casei as the binding protein. This spurious increase was caused by trimethoprim in the patient's plasma. A plasma trimethoprim concentration of 0.1 mg/L inhibited binding of radiolabeled methotrexate to dihydrofolate reductase by 50%. In contrast, radioimmunoassay for methotrexate was not affected by concomitant administration of trimethoprim. The competitive protein binding assay for methotrexate should not be used in patients being treated with Bactrim or Septra (a similar combination). However, the L. casei competitive protein binding assay technique can be used to assay plasma trimethoprim concentrations with sensitivity to 0.02 mg of trimethoprim per liter.

scholarly journals TFIIF, a basal eukaryotic transcription factor, is a substrate for poly(ADP-ribosyl)ation

1997 ◽  
Vol 324 (1) ◽  
pp. 249-253 ◽  
Author(s):  
Jean M. RAWLING ◽  
Rafael ALVAREZ-GONZALEZ
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Polymerase Activity ◽  
Tata Binding Protein ◽  
Covalent Modification ◽  
Eukaryotic Transcription ◽  
Calf Thymus ◽  
Associated Proteins ◽  
Protein Transcription

We have examined the susceptibility of some of the basal eukaryotic transcription factors as covalent targets for poly(ADP-ribosyl)ation. Human recombinant TATA-binding protein, transcription factor (TF)IIB and TFIIF (made up of the 30 and 74 kDa RNA polymerase II-associated proteins RAP30 and RAP74) were incubated with calf thymus poly(ADP-ribose) polymerase and [32P]NAD+ at 37 °C. On lithium dodecyl sulphate/PAGE and autoradiography, two bands of radioactivity, coincident with RAP30 and RAP74, were observed. No radioactivity co-migrated with TATA-binding protein or TFIIB. The phenomenon was dependent on the presence of nicked DNA, which is essential for poly(ADP-ribose) polymerase activity. Covalent modification of TFIIF increased with time of incubation, with increasing TFIIF concentration and with increasing NAD+ concentration. High-resolution PAGE confirmed that the radioactive species associated with RAP30 and RAP74 were ADP-ribose polymers. From these observations, we conclude that both TFIIF subunits are highly specific substrates for covalent poly(ADP-ribosyl)ation.

scholarly journals Modeling covalent-modifier drugs

2017 ◽  
Author(s):  
Ernest Awoonor-Williams ◽  
Andrew G Walsh ◽  
Christopher N Rowley
Keyword(s):  
Computer Modeling ◽  
Covalent Binding ◽  
Covalent Bond ◽  
Bioinformatic Analysis ◽  
Covalent Modification ◽  
Binding Affinities ◽  
Chemical Bonds ◽  
Irreversible Inhibition ◽  
Large Sets ◽  
Covalent Inhibitor

In this review, we present a summary of how computer modeling has been used in the development of covalent modifier drugs. Covalent modifier drugs bind by forming a chemical bond with their target. This covalent binding can improve the selectivity of the drug for a target with complementary reactivity and result in increased binding affinities due to the strength of the covalent bond formed. In some cases, this results in irreversible inhibition of the target, but some targeted covalent inhibitor (TCI) drugs bind covalently but reversibly. Computer modeling is widely used in drug discovery, but different computational methods must be used to model covalent modifiers because of the chemical bonds formed. Structural and bioinformatic analysis has identified sites of modification that could yield selectivity for a chosen target. Docking methods, which are used to rank binding poses of large sets of inhibitors, have been augmented to support the formation of protein--ligand bonds and are now capable of predicting the binding pose of covalent modifiers accurately. The pKa's of amino acids can be calculated in order to assess their reactivity towards electrophiles. QM/MM methods have been used to model the reaction mechanisms of covalent modification. The continued development of these tools will allow computation to aid in the development of new covalent modifier drugs.

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