A mechanism of resistance to methotrexate NADPH but not NADH stimulation of methotrexate binding to dihydrofolate reductase

Barton A. Kamen; Wendy Whyte-Bauer; Joseph R. Bertino

doi:10.1016/0006-2952(83)90047-3

A mechanism of resistance to methotrexate NADPH but not NADH stimulation of methotrexate binding to dihydrofolate reductase

Barton A. Kamen, Wendy Whyte-Bauer, Joseph R. Bertino

Research output: Contribution to journal › Article › peer-review

18 Scopus citations

Abstract

Characteristics of methotrexate (MTX) inhibition of dihydrofolic acid reductase (DHFR) enzyme activity and the effects of NADPH and NADH on enzyme-drug interaction were studied. Two highly sensitive assay procedures were used. The first utilized tritium-labeled MTX to measure direct binding properties of the enzyme and the second utilized tritium-labeled dihydrofolate (H₂PteGlu) and folate (PteGlu) to analyze kinetics of reduction of these substrates. NADPH was found to enhance DHFR binding of MTX (K_d = 2.6 × 1O^-11 M), whereas NADH was found to have no effect (K_d = 3.7 × 10^-9 M). However, NADH proved to be a good substrate for folate reduction compared to NADPH, especially in low salt buffer. The observation that NADH supports the reduction of folate and dihydrofolate but not MTX binding suggests that natural resistance to MTX could exist if NADH replaces NADPH as the main cofactor for DHFR.

Original language	English (US)
Pages (from-to)	1837-1841
Number of pages	5
Journal	Biochemical Pharmacology
Volume	32
Issue number	12
DOIs	https://doi.org/10.1016/0006-2952(83)90047-3
State	Published - Jun 15 1983
Externally published	Yes

All Science Journal Classification (ASJC) codes

Biochemistry
Pharmacology

Access to Document

10.1016/0006-2952(83)90047-3

Cite this

@article{a7797031fe1a46b4ad6a973e6629c64d,

title = "A mechanism of resistance to methotrexate NADPH but not NADH stimulation of methotrexate binding to dihydrofolate reductase",

abstract = "Characteristics of methotrexate (MTX) inhibition of dihydrofolic acid reductase (DHFR) enzyme activity and the effects of NADPH and NADH on enzyme-drug interaction were studied. Two highly sensitive assay procedures were used. The first utilized tritium-labeled MTX to measure direct binding properties of the enzyme and the second utilized tritium-labeled dihydrofolate (H2PteGlu) and folate (PteGlu) to analyze kinetics of reduction of these substrates. NADPH was found to enhance DHFR binding of MTX (Kd = 2.6 × 1O-11 M), whereas NADH was found to have no effect (Kd = 3.7 × 10-9 M). However, NADH proved to be a good substrate for folate reduction compared to NADPH, especially in low salt buffer. The observation that NADH supports the reduction of folate and dihydrofolate but not MTX binding suggests that natural resistance to MTX could exist if NADH replaces NADPH as the main cofactor for DHFR.",

author = "Kamen, {Barton A.} and Wendy Whyte-Bauer and Bertino, {Joseph R.}",

year = "1983",

month = jun,

day = "15",

doi = "10.1016/0006-2952(83)90047-3",

language = "English (US)",

volume = "32",

pages = "1837--1841",

journal = "Biochemical Pharmacology",

issn = "0006-2952",

publisher = "Elsevier Inc.",

number = "12",

}

TY - JOUR

T1 - A mechanism of resistance to methotrexate NADPH but not NADH stimulation of methotrexate binding to dihydrofolate reductase

AU - Kamen, Barton A.

AU - Whyte-Bauer, Wendy

AU - Bertino, Joseph R.

PY - 1983/6/15

Y1 - 1983/6/15

N2 - Characteristics of methotrexate (MTX) inhibition of dihydrofolic acid reductase (DHFR) enzyme activity and the effects of NADPH and NADH on enzyme-drug interaction were studied. Two highly sensitive assay procedures were used. The first utilized tritium-labeled MTX to measure direct binding properties of the enzyme and the second utilized tritium-labeled dihydrofolate (H2PteGlu) and folate (PteGlu) to analyze kinetics of reduction of these substrates. NADPH was found to enhance DHFR binding of MTX (Kd = 2.6 × 1O-11 M), whereas NADH was found to have no effect (Kd = 3.7 × 10-9 M). However, NADH proved to be a good substrate for folate reduction compared to NADPH, especially in low salt buffer. The observation that NADH supports the reduction of folate and dihydrofolate but not MTX binding suggests that natural resistance to MTX could exist if NADH replaces NADPH as the main cofactor for DHFR.

AB - Characteristics of methotrexate (MTX) inhibition of dihydrofolic acid reductase (DHFR) enzyme activity and the effects of NADPH and NADH on enzyme-drug interaction were studied. Two highly sensitive assay procedures were used. The first utilized tritium-labeled MTX to measure direct binding properties of the enzyme and the second utilized tritium-labeled dihydrofolate (H2PteGlu) and folate (PteGlu) to analyze kinetics of reduction of these substrates. NADPH was found to enhance DHFR binding of MTX (Kd = 2.6 × 1O-11 M), whereas NADH was found to have no effect (Kd = 3.7 × 10-9 M). However, NADH proved to be a good substrate for folate reduction compared to NADPH, especially in low salt buffer. The observation that NADH supports the reduction of folate and dihydrofolate but not MTX binding suggests that natural resistance to MTX could exist if NADH replaces NADPH as the main cofactor for DHFR.

UR - http://www.scopus.com/inward/record.url?scp=0020594031&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0020594031&partnerID=8YFLogxK

U2 - 10.1016/0006-2952(83)90047-3

DO - 10.1016/0006-2952(83)90047-3

M3 - Article

C2 - 6882460

AN - SCOPUS:0020594031

SN - 0006-2952

VL - 32

SP - 1837

EP - 1841

JO - Biochemical Pharmacology

JF - Biochemical Pharmacology

IS - 12

ER -

A mechanism of resistance to methotrexate NADPH but not NADH stimulation of methotrexate binding to dihydrofolate reductase

Abstract

All Science Journal Classification (ASJC) codes

Access to Document

Other files and links

Fingerprint

Cite this