Professional Documents
Culture Documents
net/publication/10150760
CITATIONS READS
108 3,083
2 authors, including:
Jack Preiss
Michigan State University
373 PUBLICATIONS 17,160 CITATIONS
SEE PROFILE
Some of the authors of this publication are also working on these related projects:
All content following this page was uploaded by Jack Preiss on 03 June 2014.
EXPERIMENTAL
Materials-ATP, GMP, and DPN were obtained from the Pabst Labo-
ratories, adenine and erotic acid from the California Foundation for
Biochemical Research, guanosine, inosine, ribose, and hypoxanthine from
the Nutritional Biochemicals Corporation, and guanine hydrochloride
hydrate from the Distillation Products Industries. IMP and R5P were
obtained as barium salts from the Schwarz Laboratories, Inc., and con-
verted to their sodium salts by treatment with Na2S04 in dilute acid solu-
tion. NMN was prepared from DPN by the procedure of Plaut and Plaut
(4) by using potato pyrophosphatase (5). PRPP2 was prepared by the
method of Kornberg et al. (6) and purified by the procedure of Remy,
Remy, and Buchanan (7). Nicotinamide riboside was prepared by hy-
drolyzing NMN with snake venom (8).
MethodsHemolysates were prepared by alternately freezing and
thawing freshly collected erythrocytes which had been washed four times
in 0.15 M NaCl. 1 volume of 0.5 M buffer (phosphate or Tris), pH 7.4,
* These studies were supported in part by Contract AT-(40.l)-289 between Duke
University and the United States Atomic Energy Commission and by Grant RG-91
from the National Institutes of Health.
The data herein are taken from a dissertation by Jack Preiss presented to the
Graduate School of Duke University in partial fulfilment of the requirements for the
degree of Doctor of Philosophy.
t Predoctoral Fellow of the National Institutes of Health.
1 The following abbreviations are employed: nicotinamide mononucleotide, NMN;
nicotinamide, NAm; nicotinamide riboside, NR; 5-phosphoribosyl 1-pyrophosphate,
PRPP; inorganic orthophosphate, Pi; inorganic pyrophosphate, P-P;; ribose 5-phos-
phate, R5P; adenosine triphosphate, ATP; diphosphopyridine nucleotide, DPN;
adenosine 5’-phosphate, AMP; guanosine 5’-phosphate, GMP; inosine 5’-phosphate,
IMP; tris(hydroxymethyl)aminomethane, Tris.
2 An initial sample of PRPP was generously provided by Dr. Arthur Hornberg.
759
760 NICOTINAMIDE MONONUCLEOTIDE SYNTHESIS
per 5 volumes of red cells was added and the stroma were removed at 5’
by centrifugation at 3000 r.p.m.
Acetone powder was prepared by homogenizing washed erythrocytes in
5 volumes of acetone at -12”. The insoluble material was collected on
a Biichner funnel, twice rehomogenized in acetone, and dried in air. Such
powders retained activity for at least 6 months when stored at -10”.
Suitable extracts were prepared by stirring 2 gm. of powder with 10 ml.
of 0.1 M buffer (phosphate or Tris), pH 7.4, for 10 minutes at 3”. Insoluble
material was removed by centrifugation at 8000 r.p.m.
DPN was assayed with alcohol dehydrogenase, essentially by Racker’s
procedure (9). NMN was determined by conversion to DPN with DPN
pyrophosphorylase (10) and calculated as the increment in DPN due to
when 0.4 pmole of NMN had accumulated per 0.5 ml. of extract. Table
II shows that NMN synthesis by such extracts is dependent upon Pi,
Mg++, and R5P and is stimulated by, but not absolutely dependent upon,
ATP. Maximal activity was obtained at 0.005 M Pi.
TABLE I
NMN Synthesis by Hemolysates
The reaction mixtures contained 0.5 ml. of hemolysate, 164 pmoles of NAm, 10
pmoles of Mg++, 4 rmoles of ATP, and the ribose source shown in a total volume of
1.0 ml. of the indicated 0.05 M buffer. The reaction vessels were maintained at 35”
for 16 hours with occasional shaking.
NMN synthesized
TABLE II
NMN Synthesis by Extract of Acetone-Powdered Erythrocytes
The complete system, containing NAm 164 pmoles, R5P 2 pmoles, ATP 4 pmoles,
Pi 50 pmoles, Mg ++ 5 pmoles, and extract of acetone-powdered human erythrocytes
0.5 ml. in a total volume of 1.0 ml., was maintained at 35” for 5 hours.
System I NMN
I
system
I
NMN
* At zero time the system contained 0.021 pmole of NMN and in the absence of
Mg++ 0.008 pmole of NMN disappeared.
t The effect of ATP varied in individual experiments; occasionally in its absence
NMN synthesis was limited to about half that of the complete system.
After incubation, 2.5 pmoles of NMN and 5 pmoles of P-Pi were added as
carriers and the deproteinized mixture was placed on a Dowex 2 column.
NMN, ATP, and P-Pi were separated by the procedure of Deutsch and
Nielsen (18) ; NMN and NAm were eluted from the column with water.
Each of these was determined in the usual manner and aliquots were con-
centrated, evaporated to dryness, and counted. As shown in Table III,
the acid-labile phosphates of ATP completely equilibrated with the Pi
during the incubation, as did P-Pi, whereas the specific activity of NMN
was less than 7 per cent of that of the Pi.
Thus, despite the absolute dependence upon Pi, the phosphate moiety
of NMN does not arise from the Pi of the medium in the system here em-
this reaction sequence could explain the presence of P32in the R5P moiety
of NMN, the data do not permit a decision as to whether reaction (2) is
operative or whether NMN is formed by the reaction
NAm + PRPP -+ NMN + P-Pi (31
in analogy with the systems described for synthesis of pyrimidine and
purine nucleotides (22-26). However, the low specific activity of the
phosphate of NMN rendered it relatively unlikely that the reactions lead-
ing to ribose 1,5-diphosphate formation were proceeding rapidly enough to
account for NMN production.
TABLE IV
* The PRPP content of the enzyme sources at zero time varied from 0.120 to 0.160
pmole. In all instances in which NMN synthesis was ineffective there was a net loss
of PRPP during incubation.
pWdt?
Hemolysate Pi 0.063*
Tris 0.070
Acetone powder Pi 0.092*
Tris 0.114t
volume of buffer and the precipitation was repeated twice. The final
solution was dialyzed overnight against phosphate buffer and then placed
in a water bath at 73” and allowed to come to 70”. After 5 minutes at this
temperature, the solution was cooled and the insoluble protein was removed
by centrifugation and discarded. The supernatant solution, designated
“partially purified enzyme” (PPE), was usedin the studies described below.
PPE was 36.7 times as active as the initial extract and 53 per cent of the
initial activity remained at this stage. No activity was lost during the
heat treatment which effected a 5-fold purification.
In Table VI the ability of various fractions to synthesize NMN either
from R5P + ATP + NAm or NAm + PRPP and also the synthesis of
PRPP from R5P + ATP is compared. It will be seenthat the heat treat-
ment, which effected 5-fold purification of the NMN-synthesizing enzyme,
completely inactivated the PRPP-synthesizing system. Since NMN
synthesis was then possible only with PRPP and NAm as the substrate
J. PREISS AND P. HANDLER 765
* The protein present in each incubation was equivalent to the total protein of
0.5 ml. of extract of acetone powder.
TABLE VII
Purine Inhibition of NMN Synthesis
The reaction mixture contained extract of acetone-powdered erythrocytes 0.5
ml., NAm 164 pmoles, Mg+f 5 pmoles and either PRPP 0.3 rmole or R5P 2 pmoles $
ATP 4 pmoles in a total volume of 1.0 ml. of 0.05 M Pi, pH 7.4. The incubation time
was 5 hours at 35’.
NMN synthesized
Addition
RSP + ATP PRPP
-
pm&s /m3lc pmole
None ............................ 0 0.138 0.130
Hypoxanthine. ................... 0.4 0.005 0.000
TABLE VIII
Stoichiometry of Nucleotide Syntheses by PPE
0.5 ml. of PPE was incubated with PRPP in the amount shown plus 5 rmoles of
Mg++ and 0.05 M phosphate, pH 7.4. Data are not given for NAm which was present
in great excess.
- -
Incubation time Initial A
_-
hrs. /O?df2 pmole /mole
12 NMN 0.01 0.17 +0.16
PRPP 0.33 0.12 -0.21
0.25 Guanine 0.18 0.00 -0.18
PRPP 0.20 0.00 -0.20
GMP 0.00 0.16 $0.16
0.25 Hypoxanthine 0.56 0.24 -0.32
PRPP 0.33 0.00 -0.33
IMP 0.00 0.29 +0.29
3 Adenine 0.70
PRPP 0.33 0.26 -0.07
AMP 0.00 0.00 0.00
-
J. PREISS AND P. HANDLER 767
TABLE IX
Separation of NMN and GMP Pyrophosphorylases
GMP pyrophosphorylase activity was assayed by incubating guanine 0.18 pmole,
MgC12 10 pmoles, PRPP 0.19 pmole, 0.5 ml. of dialyzed enzyme fraction, and Tris 50
rmoles, pH 7.4, in a total volume of 2.9 ml. at 35” for 15 minutes. NMN pyrophos-
phorylase activity was measured by incubating NAm 164 rmoles, PRPP 0.3 pmole,
MgClz 5 @moles, 0.5 ml. of dialyzed enzyme fraction, and 50 pmoles of Tris in a total
volume of 1.2 ml. at 35” for 5 hours.
Poole p9d*
PPE 0.056 0.18
TABLE X
Separation of NMN and IMP Pyrophosphorylases
Assay incubations contained PRPP 0.3 pmole, MgClz 5 pmoles, Tris 50 rmoles,
pH 7.4, 0.5 ml. of dialyzed enzyme fraction, and either hypoxanthine 0.17 pmole or
NAm 164 bmoles. The incubation times were 5 hours for NMN and 15 minutes for
IMP.
- -
Gel Fraction NMN synthesis IMP synthesis
.- .-
pmoie pmolc
PPE 0.067 0.17
Alumina CT Supernatant fluid 0.000 0.00
0.01 M PO4 eluate 0.017 0.00
0.05 “ “ “ 0.039 0.17
0.10 “ I‘ ‘I 0.055 0.17
0.20 ‘I “ ‘< 0.055 0.055
0.30 “ “ “ 0.047 0.014
Ca8(PO41 2 Supernatant fluid 0.007 0.00
0.05 M PO, eluate 0.043 0.17
0.10 ‘I ‘I “ 0.037 0.14
0.20 “ “ “ 0.033 0.00
0.30 I‘ “ <‘ 0.037 0.00
- - -
768 NICOTINAMIDE MONONUCLEOTIDE SYNTHESIS
The relatively slow synthesis of NMN and high K, for NAm, as com-
pared with the rapid synthesis of IMP and GMP at low substrate concen-
trations, suggested the possibility that NMN synthesis was accomplished
by an enzyme which “normally” operates with a different substrate, per-
haps guanine or hypoxanthine. However, as shown in Tables IX and X,
NMN-synthesizing activity was clearly separated from both GMP- and
IMP-synthesizing activity by adsorption of the protein of PPE on calcium
phosphate gel or alumina Cy and elution with phosphate buffers of in-
TABLE XI
Pyrophosphorolysis of NMN
The incubation mixtures contained PPE 0.5 ml., MgClz 5 pmoles, Tris 50 pmoles,
DISCUSSION
In keeping with the convention employed for those enzymes which syn-
thesize pyrimidine nucleotides, it is suggested that this enzyme be desig-
nated NMN pyrophosphorylase, even though the demonstration of reversi-
bility was not satisfactory and the equilibrium position for this system
could not be ascertained. The relatively slow and difficult pyrophos-
phorolysis of the purine nucleotides has been discussed by Kornberg et al.
SUMMARY
BIBLIOGRAPHY
1. Leder, I. G., and Handler, P., J. Biol. Chem., 189, 889 (1951).
2. Leder, I. G., and Handler, P., in McElroy, W. D., and Glass, B., Phosphorus