Determination of NAD pyrophosphorylase activity in biological samples

Edit Paulik, Hiremagalur N. Jayaram, George Weber

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

A sensitive and simple method was developed for the accurate measurement of NAD pyrophosphorylase (NMN adenylyltransferase; EC 2.7.7.1) activity in biological samples. The reaction product of [4-3H]NAD was separated from the substrates [4-3H]NMN and ATP by HPLC. Under the standardized conditions of the assay, the enzyme activity in human chronic myelogenous leukemia K562 cells was found mainly in the nucleus (97%) with a sp act of 183.5 ± 3.5 nmol/h/mg protein. The Km's for substrates NMN and ATP were 0.11 ± 0.01 mm and 0.55 ± 0.04 mm, respectively. This technique is highly reproducible with a 5% variation (SD) in five separate determinations. The lowest number of cells used for this enzyme assay was 41,000 with a protein content of 4 μg. The range of NAD produced during the assay was 2 to 200 μm. NAD pyrophosphorylase activities in the mononuclear cells of leukemic patients, human ovarian carcinoma cells, and rat liver were assayed.

Original languageEnglish
Pages (from-to)143-148
Number of pages6
JournalAnalytical Biochemistry
Volume197
Issue number1
DOIs
StatePublished - Aug 15 1991

Fingerprint

Nicotinamide-Nucleotide Adenylyltransferase
Assays
Enzyme Assays
NAD
Adenosine Triphosphate
Cells
K562 Cells
Enzyme activity
Substrates
Leukemia, Myelogenous, Chronic, BCR-ABL Positive
Reaction products
Human Activities
Liver
Rats
Hepatocellular Carcinoma
Proteins
Cell Count
High Pressure Liquid Chromatography
Enzymes

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Determination of NAD pyrophosphorylase activity in biological samples. / Paulik, Edit; Jayaram, Hiremagalur N.; Weber, George.

In: Analytical Biochemistry, Vol. 197, No. 1, 15.08.1991, p. 143-148.

Research output: Contribution to journalArticle

Paulik, Edit ; Jayaram, Hiremagalur N. ; Weber, George. / Determination of NAD pyrophosphorylase activity in biological samples. In: Analytical Biochemistry. 1991 ; Vol. 197, No. 1. pp. 143-148.
@article{e21cf0d44eef448ea315f7eafd1f75c6,
title = "Determination of NAD pyrophosphorylase activity in biological samples",
abstract = "A sensitive and simple method was developed for the accurate measurement of NAD pyrophosphorylase (NMN adenylyltransferase; EC 2.7.7.1) activity in biological samples. The reaction product of [4-3H]NAD was separated from the substrates [4-3H]NMN and ATP by HPLC. Under the standardized conditions of the assay, the enzyme activity in human chronic myelogenous leukemia K562 cells was found mainly in the nucleus (97{\%}) with a sp act of 183.5 ± 3.5 nmol/h/mg protein. The Km's for substrates NMN and ATP were 0.11 ± 0.01 mm and 0.55 ± 0.04 mm, respectively. This technique is highly reproducible with a 5{\%} variation (SD) in five separate determinations. The lowest number of cells used for this enzyme assay was 41,000 with a protein content of 4 μg. The range of NAD produced during the assay was 2 to 200 μm. NAD pyrophosphorylase activities in the mononuclear cells of leukemic patients, human ovarian carcinoma cells, and rat liver were assayed.",
author = "Edit Paulik and Jayaram, {Hiremagalur N.} and George Weber",
year = "1991",
month = "8",
day = "15",
doi = "10.1016/0003-2697(91)90370-9",
language = "English",
volume = "197",
pages = "143--148",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Determination of NAD pyrophosphorylase activity in biological samples

AU - Paulik, Edit

AU - Jayaram, Hiremagalur N.

AU - Weber, George

PY - 1991/8/15

Y1 - 1991/8/15

N2 - A sensitive and simple method was developed for the accurate measurement of NAD pyrophosphorylase (NMN adenylyltransferase; EC 2.7.7.1) activity in biological samples. The reaction product of [4-3H]NAD was separated from the substrates [4-3H]NMN and ATP by HPLC. Under the standardized conditions of the assay, the enzyme activity in human chronic myelogenous leukemia K562 cells was found mainly in the nucleus (97%) with a sp act of 183.5 ± 3.5 nmol/h/mg protein. The Km's for substrates NMN and ATP were 0.11 ± 0.01 mm and 0.55 ± 0.04 mm, respectively. This technique is highly reproducible with a 5% variation (SD) in five separate determinations. The lowest number of cells used for this enzyme assay was 41,000 with a protein content of 4 μg. The range of NAD produced during the assay was 2 to 200 μm. NAD pyrophosphorylase activities in the mononuclear cells of leukemic patients, human ovarian carcinoma cells, and rat liver were assayed.

AB - A sensitive and simple method was developed for the accurate measurement of NAD pyrophosphorylase (NMN adenylyltransferase; EC 2.7.7.1) activity in biological samples. The reaction product of [4-3H]NAD was separated from the substrates [4-3H]NMN and ATP by HPLC. Under the standardized conditions of the assay, the enzyme activity in human chronic myelogenous leukemia K562 cells was found mainly in the nucleus (97%) with a sp act of 183.5 ± 3.5 nmol/h/mg protein. The Km's for substrates NMN and ATP were 0.11 ± 0.01 mm and 0.55 ± 0.04 mm, respectively. This technique is highly reproducible with a 5% variation (SD) in five separate determinations. The lowest number of cells used for this enzyme assay was 41,000 with a protein content of 4 μg. The range of NAD produced during the assay was 2 to 200 μm. NAD pyrophosphorylase activities in the mononuclear cells of leukemic patients, human ovarian carcinoma cells, and rat liver were assayed.

UR - http://www.scopus.com/inward/record.url?scp=0025914729&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025914729&partnerID=8YFLogxK

U2 - 10.1016/0003-2697(91)90370-9

DO - 10.1016/0003-2697(91)90370-9

M3 - Article

C2 - 1952057

AN - SCOPUS:0025914729

VL - 197

SP - 143

EP - 148

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 1

ER -