Synthesis of pyridine nucleotides by mitochondrial fractions of yeast

BIOCHEMICAL

Vol. 76, No. 2, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SYNTHESIS OF PVRIDINE BY MITOCHONDRIAL

Richard

A. Lange

NUCLEOTIDES

FRACTIONS OF YEAST

and Myron

K. Jacobson

Departments of Chemistry and Basic Health Sciences North Texas State University Denton, Texas 76203

Received

April

11,1977 SUMMARY

Washed mitochondrial nicotinate mononucleotide tinate or nicotinamide. ruption of the fractions phosphoribosyl-1-pyrophosphate,

fractions from yeast catalyze the synthesis of and nicotinate adenine dinucleotide from nicoThe synthesis is stimulated 10 fold by sonic disand shows an absolute requirement for ATP, 5and MgC12. INTRODUCTION

The impermeability

of the yeast

nucleotides

(1)

of pyridine

nucleotide.

the

raises

intramitochondrial

tact

preparations

dine

nucleotide

here

the

in sonically

Recently

synthesis

liver in

of pyridine

disrupted

mitochondrial

of the origin Grunicke

synthesis of rat

synthesis

the question

inner

mitochondria sonically

mitochondrial MATERIALS

fractions

(2)

presented

nicotinamide

but were

disrupted

nucleotides

to pyridine

of the mitochondrial

et al.

of NAD+ from

membrane

unable

preparations.

from nicotinate

pools

evidence

for

in isolated, to detect

inpyri-

We describe and nicotinamide

of yeast.

AND METHODS

Saccharomyces cerevisiae was grown, converted to spheroplasts, and a mitochondrial fraction was isolated as described previously (3) with the following modifications: (i) Following Glusulase treatment, the spheroplasts were washed with 20 mM KH2P04-K2HP04, pH 6.8, 1.5 M sorbitol, 1 mM EDTA. (ii) Rather than utilizing a Waring blendor to aid lysis, the spheroplasts were lysed by gentle stirring for 15 to 30 min at 0-4oC in 20 HIM KH2PO K2HP04 pH 6.8, 250 I#! sucrose, 1 mM EDTA. We have found that even gen 0 le

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

424 ISSN

0006-291

X

Vol.76,

No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Waring blending converts a sizeable fraction of the total DNA to a form that does not sediment at either 1OOOg or lO,OOOg, indicating nuclear fragmentation. With the present methods of lysis, over 95% of the total DNA sediments at 10009. (iii) The isolated mitochondrial fraction was washed 5 times to minimize cross contamination with soluble cellular components. For analysis of pyridine nucleotide synthesis, mitochondrial fractions were incubated for 20 min at 300C in a medium (complete incubation mixture) containing 111 mM Tris (H3P04) pH 8.4, 4.4 mM ATP, 22 mM MgC12, 1.5 mM PRPP*, an ATP regenerating system containing 22 mM phos hoenol pyruvate, 22 mM KCl, and 100 pg/ml pyruvat kinase, and either 8 pM [ s H] nicotinate (2.2 x 108 cpm/pmole) or 15 uM [ 'i 4C] nicotinamide (1.3 x 108 cpm/pmole). Portions of the incubation mixtures were fractionated by chromatography on paper or thinlayers of microcrystalline cellulose. Authentic carrier compounds were cochromatographed with samples containing radioactivity. Development in nbutanol-acetic acid-water (4/l/2) was used to separate pyridine bases (nicotinate, nicotinamide) from pyridine mononucleotides (NMN, NaMN) and pyridine dinucleotides (NAD+, NaAD+). The two-dimensional system of Kahn and Blum (4) was used to separate nicotinate, nicotinamide, NMN, NaMN, NAD+, and NaAD+. Radioactivity migrating with authentic carrier compounds was quantified by liquid scintillation counting. RESULTS AND DISCUSSION Washed mitochondrial or after ['HI

sonic

disruption

nicotinate

version

tinate

pyridine

disruption

both

incorporation

into

both

to one or more of the indeed

adhering

associated cytoplasmic

the

was located

radioactivity

(NaMN and NaAD'). fractions

represented

the

formation

*Nonstandard abbreviations NMN, nicotinamide mononucleotide; nicotinate adenine dinucleotide,

(Table

result

suggests

Analysis

that

I).

observed

of 2.6 nmol/mg

of nico-

However, 10 fold

the activity

pyridine

of

activity barriers

observed represent

mononucleotides revealed

that

derivatives

sonically

protein

sonic

increase

The lower

nicotinate

in the

con-

Incubation

conversion

chromatography

in the

8 pM

for

and does not

of the

thin-layer

intact

of permeability

fractions

exclusively

The incorporation

chromatography

in detectable

the

the particulate

by two-dimensional

containing

and dinucleotides.

which

and dinucleotides

mixture

in an approximately

is probably

contamination.

either

and dinucleotides.

mononucleotides

with

by paper

resulted

substrates

incubated

or dinucleotides

resulted

fraction

were

incubation

examined

mononucleotides

fractions

in the non-disrupted

is

were

fractions

pyridine

of the

complete

mononucleotides

mitochondrial into

from yeast

in the

and portions

into

of intact

fractions

disrupted

of NaMN and 0.5

used: PRPP, 5-phosphoribosyl-l-pyrophosphate; NaMN, nicotinate mononucleotide, NaAD+, oxidized form.

425

Vol. 76, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Pyridine

Nucleotide

I

Synthesis

in Mitochondrial

Conversion Incubation Conditions

[3H]

nicotinate

Pyridine Mononucleotides

20 min,

30°C

minus

sonic sonic

disruption disruption

into

Pyridine Dinucleotides

CPM

CPM

104

73

912

253

No incubation

plus

of

Fractions

11,399

2,102

The incubation mixtures (100 ~1, 1.0 mg protein/ml), containing mitochondrial fractions, were incubated in the complete incubation mixture and 20 ul portions containing approximately 35,000 total cpm were subjected to chromatographic analysis.

nmol/mg

protein

pyridine

of NaAD+.

nucleotides

NaAD+ from

presence

are shown

nicotinate

1-pyrophosphate,

The requirements

showed

of radioactivity exchange

reactions.

and II were

observed

when

was observed

that

requirement

and the

synthesis

system.

These

Results

[3H] the

the --in vitro

extracts

for

strongly

NaMN and

by the suggest

nucleotide

to those

converted

of

5-phosphoribosyl-

pyridine

was replaced rapidly

of both

was stimulated data

net similar

nicotinate

synthesis

The formation

represented

than

and it

an absolute

of the ATP regenerating

rather

II.

in Table

ATP, and MgC12,

the conversion

for

shown

that synthesis

in Table

by [14C]

I

nicotinamide,

nicotinamide

into

nicotinate. These

observations

represent

nucleotide

biosynthesis

from

Previously

Griffiths

chondria

contain

pyridine

and Bernofsky a unique

the

isozyme

(5)

first

reported

ring

precursors

have demonstrated

of NAD kinase

426

that

evidence in yeast that catalyzes

for

pyridine

mitochondria. yeast

mito-

the synthesis

Vol. 76, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL

Table Requirements

for

RESEARCH COMMUNICATIONS

II

Pyridine

Nucleotide

Synthesis

Conversion Incubation Conditions

Incubation Mixture

No incubation

Complete

20 min,

Complete

30°C

of

r3H] nicotinate

Pyridine Mononucleotides

into

Pyridine Dinucleotides

CPM

CPM

227

79

10,564

1,992

Complete

minus

ATP

208

63

Complete

minus

PRPP

177

95

Complete

minus

MgC12

78

61

Complete minus regenerating

ATP system

6,255

679

The incubation mixtures (100 ul, 1.0 mg protein/ml) contained sonicallydisrupted mitochondrial fractions and 20 ul portions containing approximately 35,000 total cpm were subjected to chromatographic analysis.

of NADPH from fractions

differ

tides

in rat

not

in sonically

preparations

utilized

of nicotinate

NADf was not

distinct

may be the tional observed

either

here

for

with

liver

NADf formation,

fractions.

nucleotides the

mitochondrial of pyridine

nucleo-

preparations

utilized

and activity

was not

In contrast, or nicotinate

was observed.

in

yeast

synthesis

The rat

nicotinamide

was included

the yeast and the

synthesis

In two experiments

incubation

mixture,

in

the formation

of

detected.

those

case for

studies

here

reported

(2).

disrupted

mitochondria from

previously

nicotinate

pyridine

glutamine

Yeast

the

obtained

mitochondria

but

detected

The results

from

liver

nicotinamide

which

NADH.

will

are known of the the

cytoplasm,

synthesis

be needed

in mitochondrial

to contain

many metabolic

and these

of pyridine

to assess

studies

nucleotide

427

suggest

nucleotides.

the significance

pyridine

capabilities that

However,

this addi-

of the activities

metabolism.

Vol. 76, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS This work was supported in part by grants from the American Cancer Society (BC-184), the Robert A. Welch Foundation (B-633), and the North Texas State University Faculty Research Fund.

REFERENCES 1.

von Jagow,

2.

Grunicke, H., Keller, Eur. J. Biochem. 53,

3.

Jacobson, 277-291.

M. K.,

4.

Kahn,

and Blum,

5.

Griffiths, 1478.

V.,

G.,

and Klingenberg,

M. M.,

H. J., 41-45.

M. (1970) Puschendorf,

and Bernofsky,

J. J. (1968) and Bernofsky,

C. (1974)

J. Biol. C. (1972)

428

Eur. B.,

J. Biochem. and Benaguid,

Biochem.

Biophys.

12,

583-592.

A. (1975) Acta

Chem. 243,

1441-1447.

J. Biol.

Chem. 274,

350,

1473-