Radiometric enzyme-inhibition technique for measuring acivicin in plasma

H. N. Jayaram, T. W. Kensler, B. Ardalan

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

A sensitive radiometric enzyme-inhibition assay is described for the determination of acivicin in plasma; it is based on the potent inhibition of carbamyl phosphate synthetase II (CPS) by the drug. Plasma is heated at 95°C for 5 minutes to quantitatively detach bound acivicin. After centrifugation, free drug is quantitated by exposing purified CPS from Escherichia coli to representative aliquots or subdilutions of the resultant supernatants in the presence of L-glutamine, L-aspartic acid, ATP-MgCl2, NaH[14C]O3, and purified L-aspartate transcarbamylase (ATC) from E. coli. Carbamyl phosphate is first synthesized from L-glutamine, ATP-MgCl2, and NaH[14C]O3 by the action of CPS. The unstable carbamyl phosphate thus generated is quickly and quantitatively converted to [14C]carbamyl-L-aspartic acid by the action of ATC utilizing [14C]carbamyl phosphate and L-aspartic acid as substrates. After a 15-minute incubation at 37°C, unreacted NaH[14C]O3 is dissipated at acidic pH and the newly formed [14C]carbamyl-L-aspartic acid is quantitated by scintillation spectrometry. The percent inhibition of the formation of carbamyl-L-aspartic acid through the conjoint actions of CPS and ATC responds in a linear way to the logarithm of the concentration of acivicin between 20 and 200 μM. The unknown concentration of acivicin is determined indirectly by matching the percent inhibition produced by the unknown to the percent inhibition produced by a series of acividin standards extending over the linear range. This assay is sensitive, adequately reproducible, and easy. It can be used to measure acivicin in the plasma of subjects treated with this new oncolytic agent.

Original languageEnglish (US)
Pages (from-to)491-494
Number of pages4
JournalCancer Treatment Reports
Volume65
Issue number5-6
StatePublished - 1981
Externally publishedYes

Fingerprint

acivicin
Carbamyl Phosphate
Aspartic Acid
Aspartate Carbamoyltransferase
Ligases
Enzymes
Glutamine
Adenosine Triphosphate
Escherichia coli
Enzyme Assays
Centrifugation
Pharmaceutical Preparations
Spectrum Analysis

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Jayaram, H. N., Kensler, T. W., & Ardalan, B. (1981). Radiometric enzyme-inhibition technique for measuring acivicin in plasma. Cancer Treatment Reports, 65(5-6), 491-494.

Radiometric enzyme-inhibition technique for measuring acivicin in plasma. / Jayaram, H. N.; Kensler, T. W.; Ardalan, B.

In: Cancer Treatment Reports, Vol. 65, No. 5-6, 1981, p. 491-494.

Research output: Contribution to journalArticle

Jayaram, HN, Kensler, TW & Ardalan, B 1981, 'Radiometric enzyme-inhibition technique for measuring acivicin in plasma', Cancer Treatment Reports, vol. 65, no. 5-6, pp. 491-494.
Jayaram, H. N. ; Kensler, T. W. ; Ardalan, B. / Radiometric enzyme-inhibition technique for measuring acivicin in plasma. In: Cancer Treatment Reports. 1981 ; Vol. 65, No. 5-6. pp. 491-494.
@article{80b3a53ef83c4ff7ac2e35c3fdf6de5d,
title = "Radiometric enzyme-inhibition technique for measuring acivicin in plasma",
abstract = "A sensitive radiometric enzyme-inhibition assay is described for the determination of acivicin in plasma; it is based on the potent inhibition of carbamyl phosphate synthetase II (CPS) by the drug. Plasma is heated at 95°C for 5 minutes to quantitatively detach bound acivicin. After centrifugation, free drug is quantitated by exposing purified CPS from Escherichia coli to representative aliquots or subdilutions of the resultant supernatants in the presence of L-glutamine, L-aspartic acid, ATP-MgCl2, NaH[14C]O3, and purified L-aspartate transcarbamylase (ATC) from E. coli. Carbamyl phosphate is first synthesized from L-glutamine, ATP-MgCl2, and NaH[14C]O3 by the action of CPS. The unstable carbamyl phosphate thus generated is quickly and quantitatively converted to [14C]carbamyl-L-aspartic acid by the action of ATC utilizing [14C]carbamyl phosphate and L-aspartic acid as substrates. After a 15-minute incubation at 37°C, unreacted NaH[14C]O3 is dissipated at acidic pH and the newly formed [14C]carbamyl-L-aspartic acid is quantitated by scintillation spectrometry. The percent inhibition of the formation of carbamyl-L-aspartic acid through the conjoint actions of CPS and ATC responds in a linear way to the logarithm of the concentration of acivicin between 20 and 200 μM. The unknown concentration of acivicin is determined indirectly by matching the percent inhibition produced by the unknown to the percent inhibition produced by a series of acividin standards extending over the linear range. This assay is sensitive, adequately reproducible, and easy. It can be used to measure acivicin in the plasma of subjects treated with this new oncolytic agent.",
author = "Jayaram, {H. N.} and Kensler, {T. W.} and B. Ardalan",
year = "1981",
language = "English (US)",
volume = "65",
pages = "491--494",
journal = "Journal of the National Cancer Institute",
issn = "0027-8874",
publisher = "Oxford University Press",
number = "5-6",

}

TY - JOUR

T1 - Radiometric enzyme-inhibition technique for measuring acivicin in plasma

AU - Jayaram, H. N.

AU - Kensler, T. W.

AU - Ardalan, B.

PY - 1981

Y1 - 1981

N2 - A sensitive radiometric enzyme-inhibition assay is described for the determination of acivicin in plasma; it is based on the potent inhibition of carbamyl phosphate synthetase II (CPS) by the drug. Plasma is heated at 95°C for 5 minutes to quantitatively detach bound acivicin. After centrifugation, free drug is quantitated by exposing purified CPS from Escherichia coli to representative aliquots or subdilutions of the resultant supernatants in the presence of L-glutamine, L-aspartic acid, ATP-MgCl2, NaH[14C]O3, and purified L-aspartate transcarbamylase (ATC) from E. coli. Carbamyl phosphate is first synthesized from L-glutamine, ATP-MgCl2, and NaH[14C]O3 by the action of CPS. The unstable carbamyl phosphate thus generated is quickly and quantitatively converted to [14C]carbamyl-L-aspartic acid by the action of ATC utilizing [14C]carbamyl phosphate and L-aspartic acid as substrates. After a 15-minute incubation at 37°C, unreacted NaH[14C]O3 is dissipated at acidic pH and the newly formed [14C]carbamyl-L-aspartic acid is quantitated by scintillation spectrometry. The percent inhibition of the formation of carbamyl-L-aspartic acid through the conjoint actions of CPS and ATC responds in a linear way to the logarithm of the concentration of acivicin between 20 and 200 μM. The unknown concentration of acivicin is determined indirectly by matching the percent inhibition produced by the unknown to the percent inhibition produced by a series of acividin standards extending over the linear range. This assay is sensitive, adequately reproducible, and easy. It can be used to measure acivicin in the plasma of subjects treated with this new oncolytic agent.

AB - A sensitive radiometric enzyme-inhibition assay is described for the determination of acivicin in plasma; it is based on the potent inhibition of carbamyl phosphate synthetase II (CPS) by the drug. Plasma is heated at 95°C for 5 minutes to quantitatively detach bound acivicin. After centrifugation, free drug is quantitated by exposing purified CPS from Escherichia coli to representative aliquots or subdilutions of the resultant supernatants in the presence of L-glutamine, L-aspartic acid, ATP-MgCl2, NaH[14C]O3, and purified L-aspartate transcarbamylase (ATC) from E. coli. Carbamyl phosphate is first synthesized from L-glutamine, ATP-MgCl2, and NaH[14C]O3 by the action of CPS. The unstable carbamyl phosphate thus generated is quickly and quantitatively converted to [14C]carbamyl-L-aspartic acid by the action of ATC utilizing [14C]carbamyl phosphate and L-aspartic acid as substrates. After a 15-minute incubation at 37°C, unreacted NaH[14C]O3 is dissipated at acidic pH and the newly formed [14C]carbamyl-L-aspartic acid is quantitated by scintillation spectrometry. The percent inhibition of the formation of carbamyl-L-aspartic acid through the conjoint actions of CPS and ATC responds in a linear way to the logarithm of the concentration of acivicin between 20 and 200 μM. The unknown concentration of acivicin is determined indirectly by matching the percent inhibition produced by the unknown to the percent inhibition produced by a series of acividin standards extending over the linear range. This assay is sensitive, adequately reproducible, and easy. It can be used to measure acivicin in the plasma of subjects treated with this new oncolytic agent.

UR - http://www.scopus.com/inward/record.url?scp=0019418137&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0019418137&partnerID=8YFLogxK

M3 - Article

C2 - 7237469

AN - SCOPUS:0019418137

VL - 65

SP - 491

EP - 494

JO - Journal of the National Cancer Institute

JF - Journal of the National Cancer Institute

SN - 0027-8874

IS - 5-6

ER -