Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 7: assay and characterization of 7,8-diacetoxy-4-methylcoumarin:protein transacetylase from rat liver microsomes based on the irreversible inhibition of cytosolic glutathione S-Transferase

Raj, Hanumantharao G.; Parmar, Virinder S.; Jain, Subhash C.; Kohli, Ekta; Ahmad, Nizamuddin; Goel, Sanjay; Tyagi, Yogesh Kumar; Sharma, Sunil K.; Wengel, Jesper; Olsen, Carl Erik

doi:10.1016/s0968-0896(00)00104-8

Cited by 37 publications

(33 citation statements)

References 10 publications

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“…The ER was shown to contain a unique enzyme TAase, as described in our earlier reports [11,12], catalyzing the transfer of acetyl group to certain functional proteins. TAase was purified to homogeneity from human placental microsomes and exhibited M. wt.…”

Section: Discussionmentioning

confidence: 75%

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Autoacetylation of Purified Calreticulin Transacetylase Utilizing Acetoxycoumarin as the Acetyl Group Donor

Bansal

Ponnan

Raj

et al. 2008

Appl Biochem Biotechnol

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Our earlier reports documented that calreticulin, a multifunctional Ca2+-binding protein in endoplasmic reticulum lumen, possessed protein acetyltransferase function termed Calreticulin Transacetylase (CRTAase). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of receptor proteins by a model acetoxycoumarin, 7,8-Diacetoxy-4-methylcoumarin, was observed. Here, we have examined the autoacetylation property of CRTAase by immunoblotting and mass spectrometry. Ca2+ was found to inhibit CRTAase activity. The inhibition of both autoacetylation of CRTAase as well as acetylation of the receptor protein was apparent when Ca2+) was included in the reaction mixture as visualized by interaction with anti-acetyl lysine antibody. The acetylation of lysines residues: -48, -62, -64, -153, and -159 in N-domain and -206, -207, -209, and -238 in P-domain of CRTAase were located by high-performance liquid chromatography-electronspray ionization tandem mass spectrometry. Further, computer assisted protein structure modeling studies were undertaken to probe the effect of autoacetylation of CRTAase. Accordingly, the predicted CRTAase 3D model showed that all the loop regions of both N- and P-domain bear the acetylated lysines. Energy minimization of the acetylated residues revealed charge neutralization of lysines due to the N-epsilon-acetylation which may facilitate the interaction of CRTAase with the protein substrate and the subsequent transacetylase action.

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Section: Discussionmentioning

confidence: 75%

“…CRTAase in placental microsomes was assayed routinely using DAMC as the first substrate (unless otherwise mentioned) and cytosolic GST as the second substrate, using the method of Raj et al [12]. The percentage inhibition of cytosolic GST under the conditions of the assay was considered proportional to CRTAase activity.…”

Section: Assay Of Crtaasementioning

confidence: 99%

Autoacetylation of Purified Calreticulin Transacetylase Utilizing Acetoxycoumarin as the Acetyl Group Donor

Bansal

Ponnan

Raj

et al. 2008

Appl Biochem Biotechnol

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“…Several studies have documented the alterations in pattern of protein acetylation in aging and in several pathological conditions [17,23]. Protein acetylation is catalyzed by a wide range of acetyl transferases that transfer acetyl groups from acetyl CoA to largely the ɛ-amino group of lysine residues located at different position.…”

Section: Discussionmentioning

confidence: 99%

“…Protein acetylation is catalyzed by a wide range of acetyl transferases that transfer acetyl groups from acetyl CoA to largely the ɛ-amino group of lysine residues located at different position. The enzymatic protein acetylation independent of acetyl CoA was established for the first time by our identification of a unique enzyme termed TAase in the ER of animal tissues [17] utilizing PA as the acetyl group donors [5,18]. Our earlier work provided mass spectrometric evidence for the acetylation of GST 3-3 by DAMC, a model PA mediated by TAase purified from buffalo liver.…”

Section: Discussionmentioning

confidence: 99%

Calreticulin Transacetylase Mediates the Acetylation of Nitric Oxide Synthase by Polyphenolic Acetate

Bansal

Gaspari

Raj

et al. 2007

Appl Biochem Biotechnol

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Our earlier investigations identified acetoxy drug: protein transacetylase (TAase), a unique enzyme in the endoplasmic reticulum (ER) catalyzing the transfer of acetyl groups from polyphenolic acetates (PA) to certain functional proteins. Recently we have established the identity of TAase with ER protein calreticulin (CR) and subsequently transacetylase function of CR was termed calreticulin transacetylase (CRTAase). CRTAase was purified and characterized from human placenta. CRTAase catalyzed the acetylation of a receptor protein nNOS, by a model PA 7, 8-diacetoxy-4-methylcoumarin (DAMC), which was visually confirmed by using antiacetyl lysine. The aim of this report was to provide tacit proof by providing mass spectrometry evidence for CRTAase catalyzed acetylation of purified nNOS by DAMC. For this purpose, purified nNOS was incubated with DAMC and CRTAase, the modified nNOS was analyzed by nanoscale LC-MS/MS, which recorded 11 distinct peptides with a significant score as acetylated on lysine residues. The distribution was in order: lysines-24, -33, -38, -131, and -229 of the PDZ domain, Lys-245 of the oxygenase domain, Lys-754 and -856 of FMN binding domain, Lys-989 of connecting domain and Lys-1300, -1321, and -1371 of the NADPH-binding domain were acetylated. The results documented in this paper highlighted for the first time modification of nNOS by way of acetylation. Our earlier work recorded the profound activation of platelet NADPH cytochrome P-450 reductase and the acetylation of the reductase protein by DAMC, which also remarkably enhanced intracellular levels of nitric oxide. The results reported here coupled with the aforementioned previous observations strongly implicate the possible role of the acetylation of the reductase domain of nitric oxide synthase (NOS) in the NOS activation. In addition, the acetylation of nNOS can be expected to potentiate the interaction with CR, eventually leading to the augmented catalytic activity of NOS and expression of the related biological effects.

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“…Acetyl CoA-independent protein acetylation is also known, but is restricted to the action of aspirin-like drugs that would readily acetylate cyclooxygenase resulting in the inhibition of prostaglandin synthesis and, thus, induce anti-inflammatory effects [14]. Through extensive studies, we identified a microsomal enzyme, protein transacetylase (TAase) in mammalian cells and tissues, catalysing the transfer of acetyl groups from 7,8-diacetoxy-4-methylcoumarin (DAMC) to certain receptor proteins, viz., cytosolic glutathione S-transferase (GST), cytochrome P-450-linked mixed function oxidases (MFO), Nicotinamide Adenine Dinucleotide Phosphate (NADPH) cytochrome c-reductase, protein kinase C (PKC), nitric oxide synthase (NOS), and recombinant glutamine synthetase (rGlnA1) of Mycobacterium tuberculosis, resulting in the modulation of their catalytic activities and associated physiological effects [15][16][17][18][19][20]. The rat liver and human placental microsomal TAase was later identified as calreticulin, a calcium-binding protein in the lumen of the endoplasmic reticulum and, consequently, the acetyltransferase function of calreticulin was appropriately termed calreticulin transacetylase (CRTAase) [21][22][23][24].…”

Section: Introductionmentioning

confidence: 99%

Biocatalytic Synthesis of Novel Partial Esters of a Bioactive Dihydroxy 4-Methylcoumarin by Rhizopus oryzae Lipase (ROL)

et al. 2016

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Abstract:Highly regioselective acylation has been observed in 7,8-dihydroxy-4-methylcoumarin (DHMC) by the lipase from Rhizopus oryzae suspended in tetrahydrofuran (THF) at 45 • C using six different acid anhydrides as acylating agents. The acylation occurred regioselectively at one of the two hydroxy groups of the coumarin moiety resulting in the formation of 8-acyloxy-7-hydroxy-4-methylcoumarins, which are important bioactive molecules for studying biotansformations in animals, and are otherwise very difficult to obtain by only chemical steps. Six monoacylated, monohydroxy 4-methylcoumarins have been biocatalytically synthesised and identified on the basis of their spectral data and X-ray crystal analysis.

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Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 7: assay and characterization of 7,8-diacetoxy-4-methylcoumarin:protein transacetylase from rat liver microsomes based on the irreversible inhibition of cytosolic glutathione S-Transferase

Cited by 37 publications

References 10 publications

Autoacetylation of Purified Calreticulin Transacetylase Utilizing Acetoxycoumarin as the Acetyl Group Donor

Autoacetylation of Purified Calreticulin Transacetylase Utilizing Acetoxycoumarin as the Acetyl Group Donor

Calreticulin Transacetylase Mediates the Acetylation of Nitric Oxide Synthase by Polyphenolic Acetate

Biocatalytic Synthesis of Novel Partial Esters of a Bioactive Dihydroxy 4-Methylcoumarin by Rhizopus oryzae Lipase (ROL)

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