Inactivation of dihydrofolate reductase from Lactobacillus casei by diethyl pyrocarbonate

Daron, Harlow H.; Aull, John L.

doi:10.1021/bi00533a024

Cited by 24 publications

(5 citation statements)

References 26 publications

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“…The assay mixture after incubation was diluted to 0.9 mL by 20 mM potassium phosphate buffer, pH 8, 3 mM Na2S03 was added to develop the color at 50 or 37 °C for 15 min, and the mixture was measured at different wavelengths ranging from 320 to 460 nm. -Acetylation and carbethoxylation of the enzymes by /V-acetylimidazole (Riordan et & Aull, 1982), respectively, were determined by UV absorption at 278 (for tyrosine) and 242 nm (for histidine).…”

Section: Methodsmentioning

confidence: 99%

Chemical modification of acetylcholinesterase from eel and basal ganglia: effect on the acetylcholinesterase and aryl acylamidase activities

Majumdar¹,

Balasubramanian²

1984

Biochemistry

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The effect of chemical modification on the acetylcholinesterase and the aryl acylamidase activities of purified acetylcholinesterase from electric eel and basal ganglia was investigated in the presence and absence of acetylcholine, the substrate of acetylcholinesterase, and 1,5-bis[4-(allyldimethylammonium)phenyl]pentan-3-one dibromide (BW284C51), a reversible competitive inhibitor of acetylcholinesterase. Trinitrobenzenesulfonic acid, pyridoxal phosphate, acetic anhydride, diethyl pyrocarbonate, and 2-hydroxy-5-nitrobenzyl bromide under specified conditions inactivated both acetylcholinesterase and aryl acylamidase in the absence of acetylcholine and BW284C51. Chemical modifications in the presence of acetylcholine and BW284C51 by all the above except diethyl pyrocarbonate selectively prevented the loss of acetylcholinesterase but not aryl acylamidase activity; modification by diethyl pyrocarbonate in the presence of acetylcholine and BW284C51 prevented the loss of both acetylcholinesterase and aryl acylamidase activities. Treatment with N-acetylimidazole resulted in the inactivation of acetylcholinesterase and the activation of aryl acylamidase. These changes in both the activities could be prevented by acetylcholine and BW284C51. Modification by phenylglyoxal, 2,4-pentanedione, or N-ethylmaleimide did not affect the enzyme activities. Indophenylacetate hydrolase activity followed a pattern similar to that of acetylcholinesterase in all the above modification studies. The results suggested essential lysine, tyrosine, tryptophan, and histidine residues for the active center of acetylcholinesterase and essential lysine, histidine, and tryptophan residues for the active center of aryl acylamidase.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodsmentioning

confidence: 99%

Chemical modification of acetylcholinesterase from eel and basal ganglia: effect on the acetylcholinesterase and aryl acylamidase activities

Majumdar¹,

Balasubramanian²

1984

Biochemistry

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“…Diethylpyrocarbonate (25 mM) was incubated with the enzyme in 20 mM potassium phosphate buffer, pH 7.0, at 22 mC for 15 min [16,19]. The enzyme was then dialysed against 20 mM phosphate buffer, pH 7.4, for 16 h. A 100 % loss of cholinesterase activity was observed after diethylpyrocarbonate treatment under these conditions [13].…”

Section: Diethylpyrocarbonate Treatment For Histidine Modificationmentioning

confidence: 99%

Evidence for a Zn2+-binding site in human serum butyrylcholinesterase

Bhanumathy

Balasubramanian

1996

Biochemical Journal

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Purified human serum butyrylcholinesterase after treatment with either of the metal chelators EDTA or NaCN was able to bind to a Zn(2+)-chelate-Sepharose affinity column and was eluted from the column by EDTA or imidazole. Prior EDTA treatment of the enzyme was essential for binding to this affinity column. The enzyme could be labelled with (65)Zn(2+) after EDTA treatment of the enzyme. Diethylpyrocarbonate modification of histidine residues in the EDTA-treated enzyme resulted in the abolition of both binding to the Zn(2+)-chelate-Sepharose column and labelling by (65)Zn(2+). Stoicheiometry of (65)Zn(2+) binding indicated approximately 0.85 mol of Zn(2+)/mol of subunit of the EDTA-treated enzyme. EDTA or NaCN treatment resulted in the loss of thermal stability of the enzyme at 37 degrees C which could not be reversed by Zn(2+). Whereas the cholinesterase activity of butyrlcholinesterase was not affected by EDTA, there was significant loss of its carboxypeptidase activity in the presence of EDTA, and the loss could be reversed by added ZnCl2. These results suggest the presence of a Zn(2+)-binding site on human serum butyrylcholinesterase and the involvement of histidine residues in the metal binding. The presence in human serum butyrylcholinesterase of a sequence HXXE...H found in many known Zn(2+)-containing enzymes supports these findings.

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“…This reagent has been used for the modification of histidine Carbethoxylation of histidine by diethylpyrocarbonate [15] and 0-acetylation by acetic anhydride or N-acetylimidazole of tyrosine residues [16] were determined by ultraviolet absorption at 242 nm (for histidine) and 278 nm (for tyrosine). The extent of trinitrophenylation following the treatment with (N02)3PhS03 was determined by the method of Goldfarb [17] with slight modification.…”

Section: Spectral Studiesmentioning

confidence: 99%

“…Diethylpyrocarbonate modification of histidine has been shown to be reversible by NH20H [15,231. When the enzyme inactivated using 10 mM diethylpyrocarbonate was treated with 0.5 M NHzOH at pH 7.5 as given under Methods the activity was restored to 80% of the original.…”

Section: Sds Polyacrylamide Gel Electrophoresismentioning

confidence: 99%

Chemical modification of the bifunctional human serum pseudocholinesterase

Boopathy

Balasubramanian

1985

European Journal of Biochemistry

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The effect of chemical modification on the pseudocholinesterase and aryl acylamidase activities of purified human serum pseudocholinesterase was examined in the absence and presence of butyrylcholine iodide, the substrate of pseudocholinesterase. Modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, diethylpyrocarbonate and trinitrobenzenesulfonic acid caused a parallel inactivation of both pseudocholinesterase and aryl acylamidase activities that could be prevented by butyrylcholine iodide. With phenylglyoxal and 2,4-pentanedione as modifiers there was a selective activation of pseudocholinesterase alone with no effect on aryl acylamidase. This activation could be prevented by butyrylcholine iodide. N-Ethylmaleimide and p-hydroxy-mercuribenzoate when used for modification did not have any effect on the enzyme activities. The results suggested essential tryptophan, lysine and histidine residues at a common catalytic site for pseudocholinesterase and aryl acylamidase and an arginine residue (or residues) exclusively for pseudocholinesterase. The use of N-acetylimidazole, tetranitromethane and acetic anhydride as modifiers indicated a biphasic change in both pseudocholinesterase and aryl acylamidase activities. At low concentrations of the modifiers a stimulation in activities and at high concentrations an inactivation was observed. Butyrylcholine iodide or propionylcholine chloride selectively protected the inactivation phase without affecting the activation phase. Protection by the substrates at the inactivation phase resulted in not only a reversal of the enzyme inactivation but also an activation. Spectral studies and hydroxylamine treatment showed that tyrosine residues were modified during the activation phase. The results suggested that the modified tyrosine residues responsible for the activation were not involved in the active site of pseudocholinesterase or aryl acylamidase and that they were more amenable for modification in comparison to the residues responsible for inactivation. Two reversible inhibitors of pseudocholinesterase, namely ethopropazine and imipramine, were used as protectors during modification. Unlike the substrate butyrylcholine iodide, these inhibitors could not protect against the inactivation resulting from modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide and trinitrobenzenesulfonic acid. But they could protect against the activation of pseudocholinesterase and aryl acylamidase by low concentrations of N-acetylimidazole and acetic anhydride thereby suggesting that the binding site of these inhibitors involves the non-active-site tyrosine residues.

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Inactivation of dihydrofolate reductase from Lactobacillus casei by diethyl pyrocarbonate

Cited by 24 publications

References 26 publications

Chemical modification of acetylcholinesterase from eel and basal ganglia: effect on the acetylcholinesterase and aryl acylamidase activities

Chemical modification of acetylcholinesterase from eel and basal ganglia: effect on the acetylcholinesterase and aryl acylamidase activities

Evidence for a Zn2+-binding site in human serum butyrylcholinesterase

Chemical modification of the bifunctional human serum pseudocholinesterase

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