Biochemical characterization of GSK1070916, a potent and selective inhibitor of Aurora B and Aurora C kinases with an extremely long residence time1

2009 ◽  
Vol 420 (2) ◽  
pp. 259-265 ◽  
Author(s):  
Kelly Anderson ◽  
Zhihong Lai ◽  
Octerloney B. Mcdonald ◽  
J. Darren Stuart ◽  
Eldridge N. Nartey ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Enzyme Inhibitor ◽  
Competitive Inhibitor ◽  
Tumour Regression ◽  
Aurora B ◽  
Aurora Kinases ◽  
Chromosome Alignment ◽  
Xenograft Models ◽  
Protein Enzyme

The Aurora kinases AurA, B and C are serine/threonine protein kinases that play essential roles in mitosis and cytokinesis. Among them, AurB is required for maintaining proper chromosome alignment, separation and segregation during mitosis, and regulating a number of critical processes involved in cytokinesis. AurB overexpression has been observed in a variety of cancer cell lines, and inhibition of AurB has been shown to induce tumour regression in mouse xenograft models. In the present study we report the enzymatic characterization of a potent and selective AurB/AurC inhibitor. GSK1070916 is a reversible and ATP-competitive inhibitor of the AurB–INCENP (inner centromere protein) enzyme. It selectively inhibits AurB–INCENP (Ki*=0.38±0.29 nM) and AurC–INCENP (Ki*=1.5±0.4 nM) over AurA–TPX2 (target protein for Xenopus kinesin-like protein 2) (Ki=490±60 nM). Inhibition of AurB–INCENP and AurC–INCENP is time-dependent, with an enzyme-inhibitor dissociation half-life of >480 min and 270±28 min respectively. The extremely slow rate of dissociation from the AurB and AurC enzymes distinguishes GSK1070916 from two other Aurora inhibitors in the clinic, AZD1152 and VX-680 (also known as MK-0457).

scholarly journals Biochemical Characterization of Recombinant UDPG-Dependent IAA Glucosyltransferase from Maize (Zea mays)

2021 ◽  
Vol 22 (7) ◽  
pp. 3355
Author(s):  
Anna Ciarkowska ◽  
Maciej Ostrowski ◽  
Anna Kozakiewicz
Keyword(s):  
Biochemical Characterization ◽  
Competitive Inhibitor ◽  
Inhibitory Effect ◽  
Relative Activity ◽  
Dichlorophenoxyacetic Acid ◽  
Low Concentrations ◽  
Unique Effect ◽  
Potential Binding ◽  
2,4 Dichlorophenoxyacetic Acid

Here, we report a biochemical characterization of recombinant maize indole-3-acetyl-β-d-glucose (IAGlc) synthase which glucosylates indole-3-acetic acid (IAA) and thus abolishes its auxinic activity affecting plant hormonal homeostasis. Substrate specificity analysis revealed that IAA is a preferred substrate of IAGlc synthase; however, the enzyme can also glucosylate indole-3-butyric acid and indole-3-propionic acid with the relative activity of 66% and 49.7%, respectively. KM values determined for IAA and UDP glucose are 0.8 and 0.7 mM, respectively. 2,4-Dichlorophenoxyacetic acid is a competitive inhibitor of the synthase and causes a 1.5-fold decrease in the enzyme affinity towards IAA, with the Ki value determined as 117 μM, while IAA–Asp acts as an activator of the synthase. Two sugar-phosphate compounds, ATP and glucose-1-phosphate, have a unique effect on the enzyme by acting as activators at low concentrations and showing inhibitory effect at higher concentrations (above 0.6 and 4 mM for ATP and glucose-1-phosphate, respectively). Results of molecular docking revealed that both compounds can bind to the PSPG (plant secondary product glycosyltransferase) motif of IAGlc synthase; however, there are also different potential binding sites present in the enzyme. We postulate that IAGlc synthase may contain more than one binding site for ATP and glucose-1-phosphate as reflected in its activity modulation.

scholarly journals Identification and characterization of a neutral endopeptidase activity in Aplysia californica

1993 ◽  
Vol 296 (2) ◽  
pp. 459-465 ◽  
Author(s):  
W Bawab ◽  
R S Aloyz ◽  
P Crine ◽  
B P Roques ◽  
L DesGroseillers
Keyword(s):  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Enzyme Inhibitor ◽  
Molecular Form ◽  
Aplysia Californica ◽  
Neutral Endopeptidase ◽  
Bivalent Cation ◽  
Endopeptidase 24.11 ◽  
Endopeptidase Activity

Kidney plasma membranes of Aplysia californica were shown to contain an endopeptidase activity which cleaved [Leu]enkephalin (Tyr-Gly-Gly-Phe-Leu) and [Leu]enkephalinamide (Tyr-Gly-Gly-Phe-Leu-NH2) at the Gly3-Phe4 bond, as determined by reverse-phase h.p.l.c. analysis of metabolites. The optimal pH was shown to be 6.5. The bivalent cation chelating agent, 1,10-phenanthroline protected [Leu]enkephalin from degradation, suggesting that this enzyme is a metallopeptidase. The degradation of [Leu]enkephalin was also abolished by the neutral endopeptidase-24.11 inhibitors RB104 (2-[(3-iodo-4-hydroxyl)-phenylmethyl]-4-N-[3-(hydroxyamino-3-oxo-1- phenylmethyl)-propyl]amino-4-oxobutanoic acid), HABCO-Gly [(3-hydroxy-aminocarbonyl-2-benzyl-1-oxypropyl)glycine], phosphoramidon and thiorphan, with IC50 values of 1 nM, 1 microM, 20 microM and 30 microM respectively. By contrast, the angiotensin-converting enzyme inhibitor captopril and the serine proteinase inhibitor phenylmethanesulphonyl fluoride were without effect. Phase separation experiments using Triton X-114 showed that about 64% of the neutral endopeptidase activity in the Aplysia kidney membrane corresponds to an integral membrane protein. A specific radioiodinated inhibitor ([125I]RB104) was shown to bind the Aplysia endopeptidase with high affinity; the KD and Bmax. values were 21 +/- 5 pM and 20.3 +/- 5 fmol/mg of proteins respectively. This inhibitor was used to determine the molecular form of the enzyme, after separation of solubilized membrane proteins on SDS/PAGE and transfer on to nitrocellulose membranes. A single protein band with an apparent molecular mass of 140 kDa was observed. The labelling was abolished by specific neutral endopeptidase inhibitors. This study provides the first biochemical characterization of an endopeptidase with catalytic properties similar to those of neutral endopeptidase-24.11 in the mollusc Aplysia californica.

Plant Pathology Diagnosed by X-Ray Microanalysis

1987 ◽  
Vol 45 ◽  
pp. 974-975
Author(s):  
J. H. Resau ◽  
N. Howell ◽  
S. H. Chang
Keyword(s):  
Electron Microscopy ◽  
Plant Pathology ◽  
Biochemical Characterization ◽  
Nutrient Deficiency ◽  
Green Leaf ◽  
Parasitic Infestation ◽  
X Ray

Spinach grown in Texas developed “yellow spotting” on the peripheral portions of the leaves. The exact cause of the discoloration could not be determined as there was no evidence of viral or parasitic infestation of the plants and biochemical characterization of the plants did not indicate any significant differences between the yellow and green leaf portions of the spinach. The present study was undertaken using electron microscopy (EM) to determine if a micro-nutrient deficiency was the cause for the discoloration.Green leaf spinach was collected from the field and sent by express mail to the EM laboratory. The yellow and equivalent green portions of the leaves were isolated and dried in a Denton evaporator at 10-5 Torr for 24 hrs. The leaf specimens were then examined using a JEOL 100 CX analytical microscope. TEM specimens were prepared according to the methods of Trump et al.

Interaction effect of rootstocks on gas exchange parameters, biochemical changes and nutrientstatus in Sauvignon Blanc winegrapes

2014 ◽  
Vol 3 (3) ◽  
pp. 218-225
Author(s):  
R. G. Somkuwar ◽  
M. A. Bhange ◽  
A. K. Upadhyay ◽  
S. D. Ramteke
Keyword(s):  
Biochemical Characterization ◽  
Reducing Sugar ◽  
Wine Grape ◽  
Gas Exchange Parameters ◽  
Food Material ◽  
Total Carbohydrates ◽  
Salt Creek ◽  
Grape Varieties ◽  
Photosynthetic Activities

SauvignonBlanc wine grape was characterized for their various morphological, physiological and biochemical parameters grafted on different rootstocks. Significant differences were recorded for all the parameters studied. The studies on vegetative parameters revealed that the rootstock influences the vegetative growth thereby increasing the photosynthetic activities of a vine. The highest photosynthesis rate was recorded in 140-Ru grafted vine followed by Fercal whereas the lowest in Salt Creek rootstock grafted vines.The rootstock influenced the changes in biochemical constituents in the grafted vine thereby helping the plant to store enough food material. Significant differences were recorded for total carbohydrates, proteins, total phenols and reducing sugar. The vines grafted on1103-Pshowed highest carbohydrates and starch followed by 140-Ru,while the least amount of carbohydrates were recorded in 110-R and Salt Creek grafted vines respectively.Among the different rootstock graft combinations, Fercal showed highest amount of reducing sugar, proteins and phenols, followed by 1103-P and SO4, however, the lowest amount of reducing sugar, proteins and phenols were recorded with 110-R grafted vines.The vines grafted on different rootstocks showed changes in nutrient uptake. Considering this, the physico-biochemical characterization of grafted vine may help to identify particularrootstocks combination that could influence a desired trait in commercial wine grape varieties after grafting.

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