Assay of nucleoside-diphosphate kinase activity by the coupled nucleotidyltransferase

ANAL

k-1 ICAL

166. 184-286

BIOCHEMIS-I’Rt

Assay

( 19x7)

of Nucleoside-Diphosphate by the Coupled

Kinase

Nucleotidyltransferase

NORIMITSUL. Facilities,fi~r

Cornparativr

School

Medinne

qf Afedicinr.

Activity

SATO

& Animal

Ichihancho.

Esperirnentution,

Asahimachi-dori.

Received

March

Niigata Niigata

951,

University Japan

4, 1987

A method coupled with Exhckhia co/i RNA polymerase is described for assaying nucleoside-diphosphate kinase activity. The principle ofthe procedure is indirect but allows processing ofa large number ofsamples by employing filter-paper disk techniques. 1~ 19x7 Academic PESS. IIIC.

(4,5) or is detected by thin-layer chromatography of the [y-3’P]NTP produced (6). The former involves three distinct enzymes in the reaction and the latter requires [y-3’P]NTP and autoradiography. In this communication I report a method coupled with a nucleotidyltransferase such as RNA polymerase to detect NDP-kinase. It is indirect but allows processing of a large number of samples.

Nucleoside-diphosphate kinase (NDPkinase.’ EC 2.7.4.6) catalyzes phosphate transfer between a wide variety of nucleoside 5’-di- and triphosphates (1,2) and plays an important role in the supply of nucleoside 5’-triphosphate during RNA and DNA synthesis in living cells (3). NDP-kinase activity is measured indirectly by the coupled pyruvate kinase-lactate dehydrogenase method

DP kinase

[3H+DP

yfI NTP

\

' LFH&TP -J--NDP

7

IlTP>

RNA +

DNA

0003.3697187

used: NDP. triphosphate;

$3.00

---+

DNA

6% (NTPs? CTPJ

A standard reaction mixture (125 ~1) contained 10 pmol Tris-HCl (pH 8.0): 8 Fmol KCl; 0.5 ymol MgCl,: 0.5 prnol 2-mercaptoethanol; 0.05 pmol GTP, UTP, and CTP; 1.0 &I of [3H]ADP (22 Ci/mmol); 320 pg

’ Abbreviations NTP. nucleoside acid.

polymerase

nuclcosidc diphosphate: TCA. trichloroacetic

284

DNA: 0.01 U E.cclwrichia co/i RNA polymerase; and 5-20-~1 NDP-kinase samples. All reaction mixtures were incubated for 20 min at 37°C. Reactions were terminated by pipetting loo-p1 portions onto Whatman filter-paper disks (3MM, 2.4 cm diameter) which were numbered with a lead pencil and mounted on straight pins. After I or 2 min, the disks were placed in a beaker of wash solution, ice-cold 10% TCA. After standing

ASSAY

OF

TABLE

1

NUCLEOSIDE-DIPHOSPHATE

KINASE

285

ACTIVITY

EFFECT OF VARUOUS CHEMICALS ON NDP-KINASE ACTIVITY BY I-HE COUPLED RNA POLYMERASE 40

Reaction

mixture”

Activityh

Complete -NDP-kinase -RNA polymerase -Mg?+

372.060 12,065 3,360 9,180 349.550 I.266 9, I30

-Mg”, +Mn2+ -Salmon sperm DNA - GTP. -UTP. -1CTP -NDP-kinase,

-[‘H]ADP,

-‘H[ADP], + ATP. -CTP, -NDP-kinase. -[‘H]ADP. --CTP, +[‘H]CTP

+[jH]ATP

153.9 IO

+[‘H]CDP +ATP,

367,585

in ice for 45 min, the wash solution was replaced three t?,mes with cold 5% TCA for 15 min each time. Finally, the disks were placed

0.4

20

10

370.028

IV~I/~. [“HIATP. 1.O gCi of 33 Ci/mmol: [‘H]CDP. I .O @Zi of 17.1 Ci/mmol; [‘H]CTP. I .O ~Lci of 28 Ci/mmol. ’ Complete, standard reaction mixture described under Reactions. NDP-kinase ( I .O pg included) was purified from Ehrlich ascites tumor cells by the method described in (6). h Activity (cpm I retained on filter paper disks.

NDP KINASE

30

0.8 ('dg/125

1.2 ~1)

FIG. I Dose dependency of [ ‘H JADP incorporation by the coupled NDP-hinase-RNA polymerasc reaction. Activity was assayed using the standard reaction mixture described under Reactions. The reaction took place at 37°C for 10 min.

19

20 TIME

30

(MINI

FIG. 2. Time course study of [‘H]ADP incorporation by the coupled NDP-kinase-RNA polymerase reaction. Activity was assayed using the standard reaction mixture described under Reactions (0) in the presence of 1 .O peg of NDP-kmase, or (0) in the absence of NDP-kinase.

in an ethanol/ether mixture (l/ 1, v/v) and in ether for 15 min each time. After the disks were dried by the blow of a hair dryer. the radioactivity retained on each disk was measured in toluene scintillation fluid as described previously (7). Results are summarized in Table 1. RNA polymerase can not use [jH]ADP as a substrate without NDP-kinase. In the presence of NDP-kinase, [“H JADP quickly converted to [jH]ATP by y-phosphate transfer reaction from the other cold NTPs that were included and was used as one of the donor substrates for RNA polymerization. The reaction required Mg’+ (4 mM) or Mn” (3 mM) ion. Optimal pH was 7.4-8.2. Other labeled nucleotides such as [‘H]CDP, [‘HIGDP, or [jH]UDP were available instead of [‘H]ADP. This reaction depended on the amount of NDP-kinase as is shown in Fig. 1 and the time course of [“H]ADP incorporation as is shown in Fig. 2. The principle can be applied also to systems using other types of nucleotidyltransferase such as DNA polymerase,

286

NORIMITSU

poly(A) polymerase, tRNA nucleotidyltransferase, etc. Although it was possible to detect the NDP-kinase activity of the crude cytoplasmic fraction by the present assay method, severe contamination with RNase and/or DNase must be avoided because of the characteristics of the reaction.

H. A..

Bi~ph~t~.

Acta

and

Hems, 12, 172.

2. Berg, P.. and Joclih.

W. K. (1953)

&‘arr,w

R. ( 1953)

Biochem.

IL~wtlonj

172,100X.

3. Reddy.

G. P. U., and Pardce.

h:atl.

4. Ratliff, S.

.kad.

Si.

L’S.1

R., Weaver,

I-. (1964)

5. Mourad.

J. Bid

77.

R. H.. Lardy. C’hmm

N.. and Parks.

C’hcw~. 241,

A. B. ( 1980)

239,

H. A., and Kuby. 301.

R. E.. Jr. (1966)

300.

J. Bio!.

3838.

7. Sate, N. L., and Yamada, 127,

Proc,.

33 I?.

6. Koyama, K.. Yokoyama. M., Koike, K., and Ishida. N. (I 984) J. Biochern.

REFERENCES I. Krebs,

L. SAT0

T. (1982)

T.. Ohtsuki.

Anai.

95,

925.

Biochtv~.