In vitro inhibition of carrot chalcone synthase by 3′-nucleotidase: The role of the 3′-phosphate group of malonyl-coenzyme A in flavonoid biosynthesis

1986 ◽  
Vol 246 (1) ◽  
pp. 217-224 ◽  
Author(s):  
Walter Hinderer ◽  
Hanns Ulrich Seitz
Keyword(s):  
Chalcone Synthase ◽  
Coenzyme A ◽  
Flavonoid Biosynthesis ◽  
Phosphate Group ◽  
In Vitro Inhibition

Purification of Chalcone Synthase from Tulip Anthers and Comparison with the Synthase from Cosmos Petals

1981 ◽  
Vol 36 (1-2) ◽  
pp. 30-34 ◽  
Author(s):  
Rainer Sütfeld ◽  
Rolf Wiermann
Keyword(s):  
Chalcone Synthase ◽  
In Vitro Assay ◽  
Flavonoid Biosynthesis ◽  
Chalcone Isomerase ◽  
Gel Chromatography ◽  
Purification Procedure ◽  
Reaction Products ◽  
Complete Elimination ◽  
Vitro Assay

Abstract Chalcone synthase was isolated from both anthers of Tulipa cv. “Apeldoorn” and petals of Cosmos sulphureus Cav. After certain prepurification steps, the enzymes were further purified using gel chromatography on Sephadex G-200 followed by repeated hydroxylapatite absorption chromatography. Both the enzymes showed the same chromatographic properties. After gel chromatography as well as after the first hydroxylapatite fractionation, the reaction products appeared as flavanones. However, after the second hydroxylapatite step, production of chalcones was observed. Like the enzyme from tulip anthers, the synthase from Cosmos petals produced the correspondingly substituted chalcones when p-coumaroyl-CoA, caffeoyl-CoA and feruloyl-CoA, respectively, were used as substractes. In both the cases, the ratios of the different chalcones produced were found to be about the same. The appearance of chalcone synthesis in this in vitro assay is caused by the complete elimination of chalcone isomerase in the purification procedure. The importance of the isomerase for flavonoid biosynthesis, particularly in plant systems which are accumulating chalcones, is discussed.

scholarly journals Biochemical and Mutational Analyses of AcuA, the Acetyltransferase Enzyme That Controls the Activity of the Acetyl Coenzyme A Synthetase (AcsA) in Bacillus subtilis

2008 ◽  
Vol 190 (14) ◽  
pp. 5132-5136 ◽  
Author(s):  
Jeffrey G. Gardner ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Bacillus Subtilis ◽  
Coenzyme A ◽  
Effective Means ◽  
Wild Type ◽  
Modification System ◽  
Vitro Assay ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme

ABSTRACT The acuABC genes of Bacillus subtilis comprise a putative posttranslational modification system. The AcuA protein is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, the AcuC protein is a class I histone deacetylase, and the role of the AcuB protein is not known. AcuA controls the activity of acetyl coenzyme A synthetase (AcsA; EC 6.2.1.1) in this bacterium by acetylating residue Lys549. Here we report the kinetic analysis of wild-type and variant AcuA proteins. We contrived a genetic scheme for the identification of AcuA residues critical for activity. Changes at residues H177 and G187 completely inactivated AcuA and led to its rapid turnover. Changes at residues R42 and T169 were less severe. In vitro assay conditions were optimized, and an effective means of inactivating the enzyme was found. The basic kinetic parameters of wild-type and variant AcuA proteins were obtained and compared to those of eukaryotic GNATs. Insights into how the isolated mutations may exert their deleterious effect were investigated by using the crystal structure of an AcuA homolog.

scholarly journals Organoselenium mild electrophiles in the inhibition of Mpro and SARSCoV-2 replication

2020 ◽  
Author(s):  
Luca Sancineto ◽  
Francesca Mangiavacchi ◽  
Agnieszka Dąbrowska ◽  
Agata Pacuła ◽  
Magdalena Obieziurska-Fabisiak ◽  
...  
Keyword(s):  
Cytopathic Effect ◽  
Selenium Atom ◽  
Inhibition Activity ◽  
Exchange Processes ◽  
Main Protease ◽  
In Vitro Inhibition ◽  
Complex Scenario ◽  
Detoxification Process

New Ebselen-like derivatives resulted to be very strong in vitro inhibitors of SARS-CoV-2 main protease. We demonstrated that this activity mainly depends on the electrophilicity of the selenium atom that is considerably higher in the N-substituted 1,2- benzoselenazol-3(2H)-ones respect to the corresponding diselenides allowing it to be rapidly attached by free thiols affording sulfur-selenium intermediates that are further subjected to thiol exchange processes. This data paints a very complex scenario that requires us to consider Ebselen and Ebselen-like derivatives as potential electrophilic substrates for the several other free thiols present in the cell beside the target free cysteine. The sulfur selenium intermediates are milder electrophiles that could be theoretically implicated in both the detoxification process as well as in the final enzymatic inhibition. We here demonstrated that the in vitro inhibition activity is not fully reproduced in the prevention of viral replication in the cell-based assay. This indicates that the structure of the substituents introduced in the Ebselen scaffold is a crucial factor to control the reactivity of the selenated molecule in the network of thiol exchanges, as well as for molecular recognition of the targeted enzymatic cysteine. For this reason, an in-depth investigation is strongly desirable to better understand how to increase the activity and the selectivity of Ebselen derivatives overcoming the issues of the apparent PAINS-like role of Ebselen. Furthermore, besides the antiviral activity, thee selected compounds also showed a different ability to reduce the virus-induced cytopathic effect, indicating that other mechanisms could be implicated. One may consider here the well-known cytoprotective antioxidant activity of Ebselen and its derivatives.<p></p>

scholarly journals The effect of isoferritins on granulopoiesis

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 436-443 ◽  
Author(s):  
G Sala ◽  
M Worwood ◽  
A Jacobs
Keyword(s):  
Monoclonal Antibody ◽  
Colony Formation ◽  
Colony Growth ◽  
Conditioned Media ◽  
Regulatory Role ◽  
Consistent Effect ◽  
Cell Extracts ◽  
In Vitro Inhibition

Abstract The evidence for a regulatory role of acidic isoferritins on hemopoiesis is not entirely consistent with our knowledge of ferritin biochemistry, and no clear picture of this phenomeonon has emerged. In the present study, we have been unable to confirm a consistent effect of purified heart (acidic), spleen (basic), or serum (glycosylated) isoferritins on CFU-GM colony formation in vitro. Inhibition of colony formation by cell extracts or conditioned media does not relate to the presence of acidic isoferritins, nor is this effect neutralized by a monoclonal antibody to acidic isoferritins. The composition of ferritin preparations previously described as inhibitory to CFU-GM colony growth could not be confirmed, and they were not found to be predominantly acidic in nature. Our data do not support a role for acidic isoferritins as inhibitors of granulopoiesis.

Organoselenium mild electrophiles in the inhibition of Mpro and SARSCoV-2 replication

2020 ◽  
Author(s):  
Luca Sancineto ◽  
Francesca Mangiavacchi ◽  
Agnieszka Dąbrowska ◽  
Agata Pacuła ◽  
Magdalena Obieziurska-Fabisiak ◽  
...  
Keyword(s):  
Cytopathic Effect ◽  
Selenium Atom ◽  
Inhibition Activity ◽  
Exchange Processes ◽  
Main Protease ◽  
In Vitro Inhibition ◽  
Complex Scenario ◽  
Detoxification Process

New Ebselen-like derivatives resulted to be very strong in vitro inhibitors of SARS-CoV-2 main protease. We demonstrated that this activity mainly depends on the electrophilicity of the selenium atom that is considerably higher in the N-substituted 1,2- benzoselenazol-3(2H)-ones respect to the corresponding diselenides allowing it to be rapidly attached by free thiols affording sulfur-selenium intermediates that are further subjected to thiol exchange processes. This data paints a very complex scenario that requires us to consider Ebselen and Ebselen-like derivatives as potential electrophilic substrates for the several other free thiols present in the cell beside the target free cysteine. The sulfur selenium intermediates are milder electrophiles that could be theoretically implicated in both the detoxification process as well as in the final enzymatic inhibition. We here demonstrated that the in vitro inhibition activity is not fully reproduced in the prevention of viral replication in the cell-based assay. This indicates that the structure of the substituents introduced in the Ebselen scaffold is a crucial factor to control the reactivity of the selenated molecule in the network of thiol exchanges, as well as for molecular recognition of the targeted enzymatic cysteine. For this reason, an in-depth investigation is strongly desirable to better understand how to increase the activity and the selectivity of Ebselen derivatives overcoming the issues of the apparent PAINS-like role of Ebselen. Furthermore, besides the antiviral activity, thee selected compounds also showed a different ability to reduce the virus-induced cytopathic effect, indicating that other mechanisms could be implicated. One may consider here the well-known cytoprotective antioxidant activity of Ebselen and its derivatives.<p></p>

scholarly journals The effect of isoferritins on granulopoiesis

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 436-443
Author(s):  
G Sala ◽  
M Worwood ◽  
A Jacobs
Keyword(s):  
Monoclonal Antibody ◽  
Colony Formation ◽  
Colony Growth ◽  
Conditioned Media ◽  
Regulatory Role ◽  
Consistent Effect ◽  
Cell Extracts ◽  
In Vitro Inhibition

The evidence for a regulatory role of acidic isoferritins on hemopoiesis is not entirely consistent with our knowledge of ferritin biochemistry, and no clear picture of this phenomeonon has emerged. In the present study, we have been unable to confirm a consistent effect of purified heart (acidic), spleen (basic), or serum (glycosylated) isoferritins on CFU-GM colony formation in vitro. Inhibition of colony formation by cell extracts or conditioned media does not relate to the presence of acidic isoferritins, nor is this effect neutralized by a monoclonal antibody to acidic isoferritins. The composition of ferritin preparations previously described as inhibitory to CFU-GM colony growth could not be confirmed, and they were not found to be predominantly acidic in nature. Our data do not support a role for acidic isoferritins as inhibitors of granulopoiesis.

scholarly journals The role of N-linked glycosylation in the regulation of activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and proliferation of SV40-transformed 3T3 cells

1989 ◽  
Vol 260 (2) ◽  
pp. 597-600 ◽  
Author(s):  
O Larsson ◽  
W Engström
Keyword(s):  
Cell Proliferation ◽  
Dna Synthesis ◽  
Coenzyme A ◽  
Inhibitory Effects ◽  
3T3 Cells ◽  
Hmg Coa Reductase ◽  
Regulation Of Activity ◽  
Coa Reductase

The effects of glycosylation inhibitors on the proliferation of SV40-transformed 3T3 cells (SV-3T3) were examined in vitro. Whereas swainsonine and castanospermine, which inhibit distal steps in the glycosylational processing, exerted marginal or no effects on cell proliferation, a proximal inhibitor, tunicamycin, efficiently decreased the rate of DNA synthesis and also inhibited the activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. The inhibitory effects of tunicamycin on cell proliferation could be partially reversed by addition of dolichol, a metabolite in the pathway regulated by HMG-CoA reductase. This finding suggests that tunicamycin exerts at least one of its effects on cell proliferation by modulating the activity of HMG-CoA reductase.

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