New aspects of the mechanism and regulation of pyridine nucleotide metabolism

NEW ASPECTS OF AND REGULATION NUCLEOTIDE

THE MECHANISM OF PYRIDINE METABOLISM

HANS GRUNICKE, HEINZ J. KELLER, MICHAELLIERSCHAND ABDELMOGHITBENAGUID BiochcmischcsInstitut dcr Univcrsit~t Freiburg im Brcisgau, Germany INTRODUCTION SEVERALaspects of the biochemistry of the pyridine nucleotides have remained subject to question. Among these are the route of biosynthesis of NAD from nicotinamide, the mechanism and the biological meaning of the so called pyridine nucleotide cycle, the quantitative contribution of the quinolinic acid dependent pathway in rat river, and the origin of the mitochondrial NAD. This paper summarizes results of our studies on the above mentioned subjects. Due to its ability to synthesize quinorini¢ acid from tryptophan, liver plays a central role in the pyridine nucleotide metabolism of the organism. Our studies have, therefore, been confined to liver tissue. In order to avoid an interference of non_hepatic tissues, all studies were performed with isolated rat river perfused with an erythrocyte free medium or with isolated rat liver mitochondda.

MATERIAL

AND

METHODS

7- t , C Nicotinic acid (specific activity 59.1 mC/mmole), 7- t 4C nicotinamide (specific activity 59.6 mC/mmole) and adenosine-2-3H-triphosphate (specific activity 10 C/mmole) were obtained from Amersham, Buckinghamshire, England. The nicotinamide was purified by ion exchange chromatography on a Dowex 1--× 2 column, formate form, eluted with 0.01 N HCOOH. Nicotinamide mononucleotide and carboxyatractyloside were purchased from Boehringer, Mannheim, Germany. Azaserine (diazoacetylserine) was a ~ft from Dr. R. R. Engle, Cancer Chemotherapy National Service Center, National Institutes of Health, Bethesda, Md., USA. Nicotinic acid adenine dinucleotide was prepared according to Lamborg et aL (1). Nicotinic acid mononucleotide was prepared from the dinucleotide using a purified snake venom diesterase as described previously (2). Rat rivers were obtained from female Wistar rats weighing 150-170 g. Liver perfusion, isolation and identi397

398

HANS GRUNICKE e t aL

fieation of the pyridine nucleotides, of nicotinic acid and of nicotinamide and its metabolites were performed as described previously (3). Mitchondria were prepared as described by Johnson and Lardy (4).

RESULTS AND DISCUSSION

1. Biosynthesis of NAD from Nicotinamide Rat liver utilizes quinolinic acid, nicotinic acid and nieotinamide for the synthesis of N A D (Fig. 1). The biosynthetic routes leading from quinolinie .

.

.

.

.

.

T,Y-~%--h--~V--- :~.,. (~ CO2 Vl~ "ATP NaAD f " R-NH2sATP ~) J ~.-R-OH, AHP~PP

NAD ~ , ~ . 2 o . ~

NHN

Nan~

Fro. 1. Biosynthesis of NAD in rat liver. Q,: quinolinic acid; N,: nicotimc acid; N,,: nicotinamide;N,MN: nicotinicacid mononucleotide;NMN: nicotinamidemononucleotide; N,AD: nicotinic acid adenine dinucleotide; NAD: nicotinamide adenine dinucleotide; PRPP: 5-phosphoribosyl-l-pyrophosphate;ARPPR: adenosine diphosphate ribose. acid or nicotinic acid to NAD are well established (for a review see Chayldn (5)). However, the utilization of nicotinamide has remained open to question. According to a widespread belief, nieotinamide has to be deamidated before entering the biosynthetic route. This assumption is mainly based on the appearance of labeled nicotinic acid derivatives after application of radioactive nieotinamide to mice (6-8), rats (9) and cats (10) and on the presence of a nicotinamide deamidase (11, 12). However, the formation of nicotinic acid derivatives from injected nicotinamide has been demonstrated only after administration of hyperphysiologieal concentrations of the vitamin greater than 1 mmole/kg. Furthermore, the nicotinamide deamidase exhibits a K,, value :for nicotinamide of about O.1 M (12) which is four orders of magnitude

PYRIDINE NUCLEOTIDE METABOLISM

399

above the normal biological concentration of the vitamin. It seemed interesting, therefore, to investigate the biosynthetic route of NAD from nicotinamide at normal nicotinamide concentrations. Figure 2 demonstrates the incorporation of 14C-nicotinamide and 14Cnicotinic acid into the NAD ofisolated perfused rat fiver as a function of time. In these experiments both precursors were used at biological concentrations. As can be seen, there is a considerable incorporation of nicotinamide, although at the concentration used, nicotinic acid is more rapidly converted to NAD than nicotinamide.

6'S4

O

O

10

20min ~

x

3 21. 5

FK]. 2. Incorporationof 14C-nicotinicacid ( 0 - 0 - 0 ) and 14C-nicotinamideinto NAD of isolated peffusedrat liver. The initial concentrationsof nicotinic acid and nicotinArrfide were 5 / ~ or 10/a~, respectively.Both precursors had the samespecificradioactivity.Each point represents the average of three experiments. In order to investigate if the nicotinamide is deamidated before its incorporation into NAD, the following experiments were performed: (a) The incorporation of nicotinamide was studied in the presence of an excess of unlabeled nicotinic acid. If the nicotinamide is deamidated prior to the incorporation into NAD, the addition of an excess of unlabeled nicotinic acid should decrease the radioactivity appearing in NAD. (b) The effect of azaserine on the incorporation of nicotinamide was investigated. Azaserine is known to inhibit the nicotinic acid dependent pathway of NAD biosynthesis by blocking the amidation of nicotinic acid adenine dinucleotide (13). If nicotinamide is incorporated into NAD via nicotinic acid, the incorporation of nicotinamide should be as sensitive to the inhibition as the incorporation of nicotinic acid. (c) The tissue was analyzed for the presence of radioactive intermediates of the nicotinic acid pathway after perfusion with 14C.nicotinamide"

400

HANS GRUNICKEet al.

The effect of an excess of non labeled nicotinic acid on the incorporation of 14C-nicotinamide and 1'~C-nicotinic acid is demonstrated in Table 1. As should be expected, addition of a 200-fold excess of unlabeled nicotinic acid results in a corresponding decrease in the radioactivity from 14C-nicotinic acid into N A D to 0.5 % of the control. Contrary to the effect on the incorporation of labeled nicotinic acid, the uptake of radioactivity from t acnicotinamide is lowered to only 50 % by the addition of unlabeled nicotinic acid. As high concentrations of nicotinic acid are known to inhibit the synthesis o f N A D from nicotinamide (7) even the observed decrease in the uptake o f radioactivity from nicotinamide does not necessarily reflect a deamidation of the nicotinamide. T~LE 1. EFFECT OF AN EXCESS OF UNLABELED NICOTINIC ACID ON THE INCORPORATION OF t4C-NICOTINIC ACID AND 14C-NICOTINAMIDE INTO N A g ) OF ISOLATED PERFUSED RAT LIVER

Addition None Nicotinic acid (I0- 3 M)

None Nicotinic acid (10- ~'M)

Labeled precursor "C-nicotinic acid (5×10-6M) *C-nicotinicacid (5x10-6 M) 1,C.nicotinamide (10- s M) 1,C.nicotinamide (10- s M)

NAD cpm#anole × 10-6 3.85 + 0.96 (6)

0.019 0.017 1.78+0.16 (9) 1.01 0.76

%

loo 0.5 0.4

100 57 43

Values obtained from experiments where no unlabeled nicotinic acid was added are given as means+ s.d. The number of experiments are shown in parenthesis. Experimental details are described in ref. (3).

That indeed the amount of nicotinic acid formed from nicotinamide is either very small or zero is demonstrated by the lack of any effect of azaserine on the incorporation of nicotinamide into N A D (Table 2). The conclusion that nicotinamide is incorporated into N A D without deamidation to nicotinic acid is further supported by the fact that no radioactive nicotinic acid or nicotinic acid nucleotide is detectable after peffusion with 14C-nicotinamide (Fig. 3). Figure 3A shows the labeling pattern after peffnsion with 14C_nicotini c acid. As can be seen, this pattern is characterized by a prominent peak of radioactive nicotinic acid adenine dinucleotide which is the most sensitive parameter for the function of the nicotinic acid pathway. Neither nicotinic acid adenine dinucleotide nor any other nicotinic acid compound could be observed after peffusion with nicotinamide.

PYRIDINE NUCLEOTIDEMETABOLISM

401

The data presented so far demonstrate that nicotinic acid is not an intermediate in the incorporation of nicotinamide into NAD. An incorporation o f nicotinamide into N A D without deamidation may proceed by two alternative path~vays: (a) by the biosynthetic route via nicotinamide mononucleotide (Fig. 1) or (b) by a nicotinamide exchange catalyzed by N A D glycohydrolase (E.C.3.2.2.5). A decision between these two alternatives is possible if conditions can be prepared which inhibit the biosynthetic route without affecting the N A D glycohydrolase. An inhibition of the biosynthetic pathway should occur after lowering the ATP level of the cell. Anoxia is known to cause a rapid decrease in the ATP content of isolated perfused liver. The metabolic

TASLE2. E~,ecr oF ~z~gn~E ONTHEINCORPORATIONOF 4C-~,~C0TtNICACXDAND~4C=NICOT~A~VEroT0 NAD oF PERFUSEDRATLIVER

Addition

Labeled precursor

Azaserine (2x10 -3 M)

14C.nicotinic acid (5XI0-eM) 14C.nicotinic acid (5xl0-eu)

None

x4C-nicotinamide

None

"C-NAD (counts/ min//zmole)x 10-e

(10- 5 M)

Azaserine (2x10-3 M)

14C-nicotinamide (10- 5 M)

6.71 5.90 0.68 0.45

3.15 2.99 3.63 2.60

Experimental details are described in ref. (3).

alterations produced by anaerobic conditions are reversible, provided the anoxia does not exceed 15 vain (14). Anoxia was produced by replacing the oxygenated medium by a perfusate gassed with N2/COz. To reduce glycolytic A T P production, livers from starved animals were employed and glucose was omitted from the medium. Control experiments indicated that these conditions do not impair the incorporation of nicotinic acid or nicotinamide into NAD. The effect of anoxia on the incorporation o f nicotinamide into N A D and on the intracellular ATP levels is pictured in Table 3. Within I rain after the switch to anaerobic conditions, the ATP level has dropped to about 20 % o f the initial value. No incorporation of nicotinamide is observed during this anaerobic time interval As soon as oxygen is made available again, the initial incorporation rates are resumed and the ATP level increases. The same effects are obtained if the incorporation o f 14C.nicotini c acid is observed under anaerobic conditions (3). o

HANS GRUNICRE et al.

40

20

FRACTION

.

.

,

Na

20

40

FRACTION

100

80

60

NUMBER

NaAD

60

80

100

NUMBER

FIG. 3. Separation of the intermediates of NAD biosynthesis in rat liver by ion exchange chromatography after perfusion with 14C-nicotinic acid (A), 14C-nicotinamide (B). Chromatography was performed on a Dowex 1 x 2 formate column with unlabeled carrier compounds. Ten ml fractions were collected and the radioactivity of each fraction determined in 0.5 ml aliquots in a liquid scintillation spectrometer. The fractions from each peak were combined and the material further identified with the aid of authentic compounds by paper chromatography and electrophoresis as described previously (3). w radioactivity. C-C-0, A-A-A optical density at 256 nm. The initial concentrations were 5 PM for nicotinic acid and 10 p~for nicotinamide. Both precursors had the same specific radioactivity. Perfusion was terminated 5 min after addition of the labeled substrate. The abbreviations are the same as in Figure 1.

PYPADINE~-t3CL~OT~- M~rABOLISM

403

It can be concluded from the data shown in Figure 4 that anoxia does not inhibit the N A D glycohydrolase activity of the liver. The figure shows the production of labeled nicotinamide during perfusion with 14C.nicotini c acid. After administration of 14C-nicotinic acid, labeled nicotinamide can only occur as a product of a N A D glycohydrolase reaction. Under these conditions the rate o f formation of labeled nicotinamide should therefore be proportional to the intracellular N A D glycohydrolase activity. The data demonstrate that anoxia causes an increase rather than a decrease o f the formation o f labeled

TAme 3. EFFECTOF ANOXtAON ~ ISCOXPOltATIONOF X4C. ~COTnqAMIDE n~ro NAD AND O~q THE ATP co~lg~rr oF I$0LAT-1~DPER.lq.~DLIVER

Peffusion

x4C-Nicotinamide incorporation cpm#anol × 10- •

ATP content /anole/g

10 min O~/CO2

2.5

3.03

20 rain O2lCO2

4.6

10 min O2/CO~ then 1 rain N2/CO2 10 rain N~/CO2

2.6

0.66 0.17

10 mln O2/CO2 then 10 mln 1%/O3z then 10 mln 02/C0~,

4.2

2.27

For experimental conditions see ref. (3).

nicotinamide during perfusion with 14C.nicotini c acid. These results strongly argue against an inhibition of N A D glycohydrolase (E.C.3.2.2.5) during anaerobic conditions. Anoxia does not prevent the permeation o f nicotinic acid into the cell. It the liver is perfused for 10 rain with the oxygenated medium containing 5 #M 14C-nicotinic acid, no intracellular nicotinic acid can be detected by the procedure used. Switching to anaerobic conditions leads to an intracellulal accumulation of labeled nicotinic acid which is probably due to lack o f utilization of the incoming precursor (Fig. 4). This observation argues against an inhibition of the nicotinic acid transport during anoxia. Principally the same observation is made after perfusion with 14C.nicotinamide" Here again anoxia causes an accumulation o f the labeled precursor in the cell.

404

HANS GRUNICKE e t

al.

The results demonstrate that isolated perfused rat liver incorporates nicotinamide into NAD exclusively via the ATP consuming biosynthetic pathway and not by an NAD glycohydrolase catalyzed exchange reaction. If this conclusion is valid, labeled nicotinamide mononucleotide should appear after perfusion with 14C_nicotinamide" As shown in Figure 3B, this is indeed the case. The same results had been obtained previously from Ehdich ascites tumor cells (2). In part our results confirm previous findings by Hagino et aL (15). These authors reported that little or no deamidation of nicotinamide occurs in isolated perfused rat liver. However, as the authors stated, their results did not permit a decision whether the limited uptake into N A D observed in their

~ 2

500

~o x

/.O0-

"- 300E 200-

N/jo

100l_

10

20

30

rain

FIG. 4. Effect of anoxia on the intracellular levels of l#C-nicotinamideand 14C-nicotinic acid in isolated rat liver perfused with a medium containing 5 #M ~'C-nicotinic acid. x - x - x nicotinamide, O-O-© nicotinic acid. The arrows marked with N2 or 02 indicate a change to a medium gassed with N2/CO~ or O2/CO~, respectively.For experimental details see ref. (3). studies occurred after a slow and undetected deamidation, whether the uptake proceeded by an exchange reaction catalyzed by NAD glycohydrolase or whether the incorporation requires the prior formation of nicotinamide mononucleotide. Hagino et al. (15) considered the last alternative unlikely as they could not detect any labeled nicotinamide mononucleotide after perfusion with 14C-nicotinamide. However, in our studies nicotinamide mononucleotide could clearly be demonstrated after perfusion with labeled nicotinamide (Fig. 3). These results are in contrast to findings of other authors who demonstrated the appearance of labeled intermediates of the nicotinic acid pathway after administration of radioactive nicotinamide (6, 7, 9, 10). They are also in

PYRIDINE NUC~,EOTIDEMETABOLISM

40~

conflict to the findings of Narrod et al. (16) who demonstrated that doubly labeled nicotinamide is incorporated into the NAD of mice liver with considerable dilution of the I SN=labeled amide nitrogen but little dilution of the 14C.labeled carboxyl carbon, thus indicating an intermediate deamidation of the nicotinamide. However, all these studies were performed with hyperphysiological concentrations of the vitamin greater than 1 mmole/kg. The nicotinamide deamidase of rat liver exhibits a Km value for nicotinamide of about 0.1 M (12). It is conceivable, therefore, that at high nicotinamide concentrations, the enzyme is saturated enough to produce measurable amounts of nicotinic acid whereas at the normal tissue content of nicotinamide of about 10-100 pM (17, 18) almost no deamidation occurs. Another explanation for the appearance of labeled nicotinic acid metabolites after administration of radioactive nicotinamide is offered by the results obtained by Ijichi et al. (8). These authors injected 82-164 #moles of nicotinamide within 20 sec into the portal vein of mice and found most of the material to be excreted into the intestinal tract where it was reabsorbed and recovered in the liver as nicotinic acid. Ijichi and coworkers reported that the radioactive material which is excreted into the intestine is found in the content of the tract rather than the intestinal walls. One has to assume, therefore, that the material was excreted into the bile. As hyperphysiological levels of nicotinamide are reached under the conditions used by Ijichi et al., it was investigated how the liver handles nicotinamide which is present at normal concentrations. Our studies demonstrated that at normal nicotinamide concentrations the excretion of nicotinamide into the bile is negligible. Only 1.8 ~o of the total radioactivity is excreted into the bile during 150 min of perfusion with 0.1 ram 14C-nicotinamide. The bile production of the perfused liver was 1 ml per 2.5 hr which is close to the values reported for conditions in vivo (19). Only 43 ~o of the excreted material represents nicotinamide. The bulk of the remaining material consists of non-reutilizable products mainly Nl-methyl nicotinamide. Smaller fractions behaved chromatographically like nicotinamide-N-oxide, N-methyl-4-pyridone-3-carboxamide and N-methyl-2pyridone-5-carboxamide if the system described by Chaykin (20) is used to separate these compounds. One has to conclude, therefore, that the excretion reported by Ijichi et al. (8) is a response to hyperphysiological concentrations of nicotinamide and not a mechanism which functions under normal conditions. The studies presented so far can be summarized by stating that isolated perfused rat liver utilizes nlcotinamide for the biosynthesis of NAD without deamidation and that the exchange reaction catalyzed by NAD glycohydrolase (E.C.3.2.2.5) is not involved in the incorporation of nicotinamide into NAD. Considering these results, the regulatory function ascribed to the liver nicotinamide deamidase (11, 12) is questionable. Results similar to our

406

HANSGRU~¢I~ et al.

findings have recently been obtained from isolated perfused kidney by Lin and Henderson (21).

2. The Pyridine Nucleotide Cycle The term "pyridine nucieotide cycle" was introduced by Gholson (22) describing a series of reactions indicated by the numbers 2, 3, 4, 5 and 8 in Figure 1. As our studies demonstrate that nicotinamide is not deamidated, the pyridine nucleotide cycle of rat liver should consist of reactions 5, 6 and 7 in Figure 1. Table 4 demonstrates that such a cycle indeed exists. The table shows the incorporation of radioactivity from 14C_nieotinic acid into various pyridine nucieotides during a period of 150 min. The initial concentration of 14C.nicotinie acid was 1 #M. As can be seen, there is a rapid decline in the radioactivity of the intermediates of the nicotinic acid pathway. But in the same time an increase in labeled nieotinamide mononueleotide can be observed. After 150 rain, nicotinamide mononueleotide is the only detectable pyridine nucleotide intermediate and the total radioactivity of the nicotinamide mononucleotide approaches the values obtained after perfusion with 14C_nicotinamide" From these data it can be concluded that nicotinamide produced during the degradation of NAD is reutilized via nicotinamide mononucleotide. A nicotinamide mononueleotide deamidase has recently been described to occur in bacteria (23). This enzyme would generate a cycle involving nicotinic acid mononucleotide besides nicotinamide mononucleotide. However, no evidence for the existence of this enzyme in rat liver has been obtained in our studies. Approximative data on the rate of the nicotinamide dependent NAD synthesis can be computed from the incorporation rate of the labeled precursor. Table 4 demonstrates that the immediate precursor of NAD--namely nicotinamide mononucleotide has reached a constant level of radioactivity after 5 min of perfusion with 14C-nicotinamide. It can, therefore, be assumed that the specific radioactivity of nicotinamide mononucleotide and nicotinamide are identical after this time interval. It is furthermore assumed that due to the rapid equilibrium between NAD and NADH, the specific radioactivities of these two compounds are also the same. Under these conditions the rate of NAD synthesis from nicotinamide is calculated as 0.16 #moles/ hr x g by using the data given in Table 5 and a NAD + NADH content of 0.9 #mole/g. NADP and NADPH were not considered for this approximative calculation as the rate of conversion of NAD to NADP is rather small compared to the rate of nicotinamide incorporation into NAD. The rate of NAD synthesis from nicotinic acid is difficult to determine as steady state conditions were not achieved during perfusion with 14C-nicotinic acid (Table 4). But if a dilution of the specific radioactivity of the labeled

5 10 20

1aC-nicotinamide x4C-nicotinamide

N,MN

NMN

N.AD

NAD

x

< 1.0 <1.0 < 1.0

23.6 5.5 2.9 < 1.0

I0-"

g liver

6.5 5.1 5.3

< 1.0 2.1 4.7 4.1

g liver xlO-4

< 1.0 < 1.0 < 1.0

245.0 106.1 48.8 < 1.0

g liver xlO-4

68.5 131.0 265.0

215.0 380.0 690.0 541.0

g liver xlO-4

(counts/rain) (counts/rain) (counts/rain) (cotmts/min)

Experimental details are described in ref. (3). F o r abbreviations see legend to Figure 1.

14C-nicotinamide

5 10 20 150

Time mill

1"C-nicotinic acid 4C-nicotinic acid 14C.]]~cotinic acid 14C.nicotini c acid

Pl'eCOl~or

OF ISOLATED PERFUmeD RAT LIVER

0.626 0.684 0.649

0.883 0.829 0.824

NAD pmoles g liver

TABLE 4. INCORPORATION OF x 4C.NICO,rlNIC ACID AND 14C.NIODTINAMIDE INTO PYRIDINE NUCLEOTIDE$

HANS GRUNICKE e t al.

408

nicotinic acid by intermediate pools is neglected and if otherwise the same assumptions are made as in the calculation of the rate of NAD synthesis from nicotinamide, the rate of NAD synthesis from nicotinic acid is computed as 0.28 #moles/hr x g liver. These rough calculations are questionable. However, despite all limitations they do indicate that NAD exhibits a considerable turnover. What is the biological function of this turnover and of the pyridine nucleotide cycle ? Considering the central role of the pyridine nucleotides one should expect that the organism is equipped with a regulatory mechanism which keeps these coenzymes at a constant level. This requires a constant availability of the pyridinium precursors. As quinolinic acid can only be utilized by liver and 40-

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30"

-3.0

u

:eu 10

"1.0 "~

.-~

30

60

80

120 rain

FZO. 5. Appearance of radioactive nicotinamide in the perfusate after perfusion with

t4C-nicotinic acid. x - x - x 14C-nicotinic acid. © - O - © 14C-nicotinamide. The initial concentration of z4C-nicotinic acid in the medium was 5/zM.

kidney, the remaining tissues depend on the availability of nicotinic acid or nicotinamide. If there is no exogenous supply with these vitamins, the organism is capable of generating nicotinamide with the aid of the NAD glycohydrolase (E.C.3.2.2.5). As there does not seem to be a nicotinamide deamidase outside the intestinal tract which is sufficiently active at normal substrate levels (24), no free nicotinic acid is formed under these conditions. Thus, mechanisms regulating the pyridine nucleotide metabolism of the whole organism should operate by providing a constant supply with nicotinamide. It is in accordance with this hypothesis that the amide is the predominant form of the vitamin in the blood (25, 26). As nicotinamide can only be produced at the expense of NAD, this can only work as part of a regulatory machinery if there is another source of NAD. This source is provided by the tryptophan-quinolinic acid pathway (27). As this pathway operates only in

PYRID~ ~CLEOTIDE METABOLISM liver and kidney it is postulated that these organs excrete nicotinamide into the blood for the supply of non-hepatic or nonrenal tissues. This mechanism would render the organism at least partially independent of alterations in the food supply with nicotinamide or nicotinic acid. The nicotinamide which is not used to restore normal NAD levels is reutilized in the pyridine nucieotide cycle. Thus, the NAD turnover and the pyridine nucleotide cycle act as a pump to provide the organism with a constant level of nicotinamide. From this point of view one of the main functions of the nicotinic acid pathway described by Preiss and Handler (28, 13) is to convert nicotinic acid to nicotinamide. This should be especially the case in liver as the nicotinic acid which is absorbed in the intestinal tract has to pass this organ. Our data shown in Figure 4 demonstrate that isolated perfused rat liver rapidly converts nicotinic acid to nicotinamide. A considerable part of this nicotinamide is excreted into the perfusate (Fig. 5). In order to accept this model one has to postulate that all tissues have the capacity to utilize nicotinamide at normal concentrations for the biosynthesis of NAD. Dietrich et al. (29) have demonstrated that all tissues examined so far have the enzymic capacity to convert nicotinamide to NAD via nicotinamide mononucleotide. The same results have been obtained from mouse tissues by Streffer and Bene~ (30). Collins and Chaykin (31) have reported that in mice all tissues except liver and intestine prefer nicotinamide to nicotinic acid for the synthesis of NAD. If the nicotinamide dependent pathway of HAD synthesis is of such an importance, one should expect this pathway to be under a stringent control. Dietrich et al. (32) have demonstrated that NAD, NADP and NADPH at their normal intracellular concentration exert a strong feedback inhibition on the nicotinamide phosphoribosyltransferase (E.C.2.4.2.12), whereas the nicotinic acid pathway is not known to be under an effective feedback control by any pyridine nucleotide under normal conditions. The model would also require an increase in NAD turnover in response to low nicotinamide levels. Further studies will show if this mechanism exists. Figure 6 gives a schematic presentation of the role of the liver in the control of pyridine nucleotide metabolism. A similar scheme has been presented by Dietrich (24). However, in the latter scheme, excretion of nicotinamide into the intestine and reabsorption as nicotinic acid was pictured as a central part of the so-called systemic pyridine nucleotide cycle. According to our studies this excretion does not occur under normal conditions. Similar conclusions concerning the biological significance of the synthesis of NAD via nicotinamide mononudeotide and about the function of the nicotinic acid dependent pathway have been reached by Lin and Henderson (21) who studied the synthesis of NAD by isolated peffused rat kidney and by Collins and Chaykin (31) who investigated the in vivo metabolism of labeled nicotinic acid and nicotinamide in mice.

HANS GRUNICKEet al.

410

3. The Tryptophan-Quinolinic Acid Pathway As outlined above the tryptophan--quinolinic acid pathway m a y be an important part o f the mechanism regulating the pyridine nucleotide metabolism. The following experiments were designed in order to permit an approximative calculation of the rate of N A D synthesis starting with quinolinic acid in the perfused liver system. Intestine

Brood Gen. Circut.

Liver

Non -hepatic Tissues

N~,m Na

.

.

.

T~y

.

.

.

.

..), N B

I I

._<. Na

.

.N a . . . . . . NMN PNC

~'~,.~.JJ

Nam~

~

N~

-e

~'

~N-methyl.Na m m..~

FIG. 6. Model of nicotinamide and nicotinic acid metabofism. The model shows nicotinamide as the predominant pyridinium precursor for the biosynthesis of NAD in non-hepatic tissues. Nicotinamide is produced by NAD breakdown and excreted into the general circulation. The tryptophan--quinolinie acid pathway acts as an anaplerotic sequence for the replenishment of nicotinarnide. Free nicotinamide is reutilized by the pyridine nueleotide cycle (PNC) which thus serves as a pump for the generation of nicotlnamide. The methylation of nicotinamide may act as another control mechanism for the regulation of the nicotinarnide level (37). Most of the dietary nicotinamide is probably deamidated in the intestinal tract by microbial deamidases. The resulting nicotinic acid and the nicotinic acid taken up with the food are absorbed and transported to the liver. Liver converts the nicotinic acid to nicotinarnide with the aid of the nicotinic acid dependent pathway of NAD synthesis and the NAD glycohydrolase. The scheme does not consider the special role of the kidney. The N A D of perfused liver was pulse labeled with 14C-nicotinic acid and subsequently perfused with a nicotinic acid and nicotinamide free medium until radioactive intermediates o f the nicotinic acid pathway were no longer detectable. After this time point, the specific radioactivity o f the N A D should at first decrease with a velocity which is proportional to the sum o f the rates of N A D formation by the nicotinamide and the quinolinic acid pathways. (Nicotinic acid was not present as indicated by measurements with 14Cnicotinic acid whereas the liver cell contains nicotinamide and presumably

PYRIDINE NUCLEOTIDEMITrABOLISM

411

qulnolinic acid.) Due to the pyridine nucleotide cycle, the specific radioactivities of nicotinamide and the pyridine nucleotides should equilibrate after some time. This effect will slow down the rate of decrease of the specific activity of the NAD. As soon as the equilibrium has been achieved (i.e., the specific activities of the pyridine nucleotides and of the nicotinamide are the same) the specific radioactivity of the N A D should either remain at a constant level or decrease at a new constant rate. A decrease would indicate that some other unlabeled pyridinium precursor is fueled into the system. As there is no nicotinic acid present and as the nicotinamide should have the same specific

0

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80 •

J . ~ a

T

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T

.

.~ s0

T 0

60

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|

90 120 150 180 210 240 min

FIG. 7. Specificradioactivity of NAD in perfused rat liver as a function of time after pulse labeling with 1*C-nicot/nicacid. Isolated rat liver was peffused with a medium containing an initial concentration of 5/~M 14C-nicotinicacid. After 10 rain the perfusion was continued with a nicotinic acid and nicotinamide free medium. Fifteen rain later the first sample was take~. This time point has been designatedas zero time in the graph. Each point represents the mean of 10 experiments + s.d. The slope of the linear part of the curve was computed by the method of the least squares. activity as the NAD, this unlabeled precursor should be quinolinic acid. The experimental results are pictured in Figure 7. In agreement with our expectation there is a rapid initial decrease in the specific radioactivity of the HAD. However, the curve does not level off but continues to decfine. After 60 min the decline proceeds at a constant rate following first order kinetics as indicated by the linear slope in the semilogarithmic plot. As outlined above, this continued decrease in the specific activity of the NAD points to the synthesis of an unlabeled pyridine base. From the slope of the linear part of the curve shown in Figure 7 which yields a half-life of 12.3 hr, and the total pyridine nucleotide content per g liver (1.27/=moles) the rate of this synthesis is computed as 73 m/~moles per hr per g. Of course, there are other ways to interpret the curve shown in Figure 7. However, the data presented in Table 5 support the concept outlined above. Table 5 demonstrates that after pulse labeling of the N A D with 14C.nicotinic acid, the liver excretes radioactivity into the medium. About one half of

412

HANS GRUNICKE

et

al.

material corresponds to nicotinamide, the other half represents nonreutilizable metabolites of nicotinamide of which Nl-methyl-nicotinamide is the predominant form. Using the paper chromatographic system described by Chaykin et al. (20) smaller fractions exhibit R I values like nicotinamideN-oxide, N-methyl-4-pyridone-3-earboxamide and N-metbyl-2-pyridone-5carboxamide. All these radioactive compounds must have been produced by NAD degradation. As the NAD levels remain constant, NAD is being synthesized. Since neither nicotinic acid nor nicotinamide are offered, the liver has to use the tryptophan-quinolinic acid pathway to compensate for the loss of pyridine bases into the medium. The rate of this replenishing synthesis TABLE 5. RECOVERY OF RADIOACTIVITY IN THE PERFUSATE DURING PERFUSION OF ISOLATED RAT LIVER PULSE LABELED WITH t 4C.NICOTINIC ACID

Radioactivityin the perfusate

Time rain 0 60 120 180

NAD dpm//tmole x10-e

NAD

nicotinamide and nicotinamide metabolites

/anoles/g

dpm x 10- ~

8.3

0.596

6.1 5.6

0.62 4.88

7.23 0.600

11.68

nicotinic acid dpm × 10- 6 < 0.01 < 0.01 < 0.01 < 0.01

Experimentalconditionsare the sameas describedin the legendof Figure7. Nicotinamide metabolltes include: N-methyl-nicotinamide,nicotinamide-N-oxide,N-methyl-4-pyridon~ 3-carboxamide and N-methyl-2-pyridone-5-carboxamide. should be equal to the rate at which pyridine bases are lost. As these compounds are breakdown products of NAD they should have the same specific radioactivity as NAD. This permits a quantitative calculation yielding 0.56 #mole per hr per total liver which corresponds to 7 % of the total pyridine nucleotide content of the liver. This value is close to the figure obtained from the data presented in Figure 7. From these data a value of 73 m#moles/hr x g liver was obtained corresponding to 6 % of the pyridine nucleotide content. It should be emphasized again that these figures represent first approximations and that more data are required to prove the validity of these results.

4. Biosynthesis of N A D in Mitochondria Many authors have reported that the inner mitochondrial membrane is almost impermeable to intact pyridine nucleotides (review by Ernster and Kuylenstierna (33)). The origin of the intramitochondrial NAD is obscure.

PYRIDM

413

HUCLI~YIIDE METABOLISM

One possibility would be the existence o f an intramitochondrial N A D synthesizing system. Studies have therefore been initiated to investigate the capacity of isolated rat fiver mitochondria to incorporate labeled precursors into their NAD. Our first experiments indicate that isolated mitochondria incorporate nicotinamide into N A D (Fig. 8). However, to our surprise no incorporation o f radioactive nicotinic acid could be observed (Table 6).

S ~

x

t:3 4,< 3"

~2-

/

/

g 1-

K

.

=

4'

/

x |

t

2

i

,

I

|

I

4

6

8

10

!

I

FIG. 8. Incorporation of 14C-nicotinamideinto NAD of isolated mitochondrla from rat fiver. Mitochondria (15 mg of protein) were incubated in 1.1 ml of the medium described by Wheeldon and Lehni%,er (38). The medium contained: KCI 100 mM; sucrose 100 ms; Tris, pH 7.5, 33 raM; MgCI2 10 raM; KH2PO4 10 raM; succinate 10 raM; ATP 2 mM; phosphoenolpyruvate 5 mM; and 0.1 mg of pyruvate kln~e. Temperature was 37°C. Incubation was stopped by the addition of 0.2 ml 3 N HCI04. After centrifugafion, 5 vol of acetone ( - 10°) were added to the supernatant. After 12 hr at - 10°, the precipitate was collected by ¢entrifugation, washed once with diethylether, dried in a desiccator and soinbWwedin 0.25 ml H20. NAD was assayed enzymaticallywith alcohol defiydrogenaso. The NAD was isolated by pape~ chromatography and electrophoresis as described previously (3). The mitochondrial preparations used in our studies are contaminated with microsomes. One has to exclude, therefore, that the nicotinamide incorporation is not due to an exchange reaction catalyzed by the microsonml N A D glycohydrolase (E.C.3.2.2.5). However, Figure 9 demonstrates that labeled nicotinamide mononucleotide can also be observed after incubation with 14C-nicotinamide. Furthermore, conditions which increase the production o f A T P especially the generation of intramitochondrial ATP, enhance the incorporation of nicotinamide into the NAD (Table 6). It is unlikely that the incorporation of nicotinamide into nicotinamide mononucleotide and N A D is due to a contamination with cytoplasmic or nuclear material. This follows from the fact that no incorporation o f nicotinic

HANS GRUNICKE et al.

414

TABLE6. INCORPORATION OF 14C-NICOTINAMIDE AND 14CNICOTINIC ACID INTO THE NA.I) OF ISOLATED MITOCHONDRIA

Incubation

Incorporation of 14C.nicotinamide into NAD cpm/~nole × 10~ s

- Succinate -ATP - ATP reg. system

0.6

+ Succinate -ATP - ATP reg. system

1.6

- Succinate +ATP + ATP reg. system

+ Succinate +ATP + A T P reg. system

Incorporation of acid into NAD cpm/pmole × 10- 5 l,C.nicotinic

3.8

6.7

< 0.001

Experimental conditions are the same as described in the legend of Figure 8. The concentrations of nicotinamide and nicotinic acid were 10 pM or 5 / ~ respectively. Incubation was 10 rain at 37°C.

S

7 x

o

5

3 2 1

2 /. 6 8 10 12 1/, 16 18 20 22 24 26 28 30 32 34 3 6 3 8 1.0 cm from the start I D, I • . NADP NAD NMN Nam

FIO. 9. Paper chromatography of a perchloric acid extract from mitochondria after incubation in he pze.umce of 14C-nicotinamide. For experimental details see legend to Figure 8.

P Y R I D I N E NUCLEOTIDE METABOLISM

415

acid into any pyridine nucleotide could be observed. In rat liver the activity o f the nicotinic acid dependent pathway which is localized in the cytoplasm and in the nucleus is not lower than the capacity o f these cell constituents to synthesize nicotiuamide mononucleotide and N A D from nicotinamide. Thus, the lack o f any incorporation of nicotinic acid argues against a contamination with cytoplasmic or nuclear material. Table 7 supports the assumption that the incorporation o f nicotinamide into N A D observed in these mitochondrial preparations is due to a mitoTAeLe 7. ~ OF CARBOXYATRACTYLOSIDEON THE INCORPORATION OF RADIOACTIVITY FROM x 4C..NICOTINAMID E SH-ATP INTONAD oF ~OLATED~rOCHONVRIA

Labeled precursor

Atractyloside

14C.nicotjn2mide

x4C.nico.tmamide SH-ATP SH-ATP

+ +

Incorporation of radioactivity into NAD % 100 87 100 17

Experimental conditions were the same as described in the legend of Figure 8 with the exception that 40 mg of mitochondrial protein per assay were used in a total vol. of 2.7 ml. Incubation was 10 rain at 37°C. Where indicated, SH-ATP was added to a final concentration of 40 mC/mmole. All assays contained 10/~ nicotinamide. Where indicated, x4C-nlcotinamideWas added at a specific activity of 59.6 mC/mmole. The concentration of carboxyatractylc~ide was 95 nmoles/mg of protein. After incubation with SH-ATP the SHlabeled NAD was separated from the adenine nucleotides by paper electrophoresis (2). The spots were eluted with water, lyophylized, solubilized in a small vol. of H20, mixed with scintillator and the radioactivity determined in a scintillation spectrophotometer. chondrial N A D synthesis. The table shows that the incorporation o f radioactivity from labeled extramitochondrial A T P into the N A D is inhibited by carboxyatractyloside. This agent is known as a highly specific inhibitor of the transiocation of adenine nucleotides through the inner mitochondrial membrane (34). The observation that this compound inhibits the incorporation of labeled A T P into N A D demonstrates that the adenine nucleotide had to pass the inner membrane of the mitochondrium before it is incorporated into N A D . The biological significance of this intramitochondrial synthesis o f N A D is difficult to evaluate on the basis of the results present so far. According to Purvis and Lowenstein (35) and K u n et al. (36) there is some exchange between intra- and extramitochondrial pyridine nucleotides. The biological

416

HANS GRUNICKEet al.

significance of the intramitochondrial synthesis of N A D depends on the ratio of the transfer rates to the rates of synthesis under various metabolic conditions. These determinations have not yet been completed.

SUMMARY Isolated perfused rat liver incorporates nicotinamide at biological concentrations into N A D . This incorporation proceeds exclusively via nicotinamide mononucleotide. Evidence is presented for the function of a pyridine nucleotide cycle by which nicotinamide produced during N A D breakdown is reutifized via nicotinamide mononucleotide. Isolated perfused rat liver rapidly converts nicotinic acid to nicotinamide. Part of this nicotinamide is excreted into the medium. The production of quinolinic acid from tryptophan serves as an anaplerotic sequence for the replenishment of nicotinamide. Approximate calculations of the rate of the quinolinic acid dependent N A D synthesis yields 73 m/~moles pyridine nucleotide synthesized per hr per g fiver in the perfused fiver system. Based on these results a concept of the regulation o f pyridine nucleotide metabolism is suggested. Evidence for the existence o f an intramitochondrial synthesis of N A D is presented.

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417

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