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Pyridine nucleotides
8
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25 772 P Y R I D I N E NUCLEOTIDES III. RESPONSE OF DOG T H Y R O I D P Y R I D I N E NUCLEOTIDES TO HORMONAL CONTROL F R E D R. B U T C H E R * AND G E O R G E S. S E R I F
The Department of Biochemistry, The Ohio State University, Columbus, Ohio (U,S.A.) (Received November 22nd, 1966)
SUMMARY
TPN + levels in dog thyroid slices appear to be elevated above control values when these tissues are exposed to thyroid-stimulating hormone (TSH) and cholinergic agents. It has been uncertain as to whether these hormone effects are due to an increased synthesis de novo of TPN + or an inhibition of TPN + degradation. This question has been evaluated through a study of the rate of incorporation of E32plorthophosphate and [14Clnicotinic acid into DPN + and TPN-- in the presence and absence of TSH and cholinergic agents. The specific activity data show an enhanced synthesis de novo of TPN+ with no apparent concomitant enhancement of DPN + formation. Although these data suggest a specific stimulation of the DPN + kinase step, the possibility of pool dilution effects does not permit such an absolute assignment.
INTRODUCTION
The study of iodinated derivatives has been a fruitful chapter in the investigation of thyroid metabolism. More recently, however, considerable attention has been focused on non-iodinated thyroid metabolites 1-6 with the concept that an understanding of their control by hormone action may give some insight into control mechanisms for thyroid hormone biogenesis. One of the more intriguing relationships developed by this approach is concerned with the observation that glucose metabolism in thyroid tissue responds to several hormones. Thus FIELD and PASTAN with their colleagues have shown increased [14CJglucose oxidation to 14C02 by dog thyroid slices in the presence of thyroid-stimulating hormone (TSH) and acetylcholineS,7,s. This group of investigators has observed an increase in thyroid TPN+ levels as a response to these hormones as well9. They suggest that increased glucose utilization is a secondary response to an increase in TPN + levels which promotes [i-14Clglucose oxidation to 14CO2 by means of the hexose monophosphate pathway. Since DPN + concentrations decrease concomitantly with TPN + increase FIELD and co-workers 10 suggest that the higher titers of TPN + may be brought about through specific stimulaAbbreviation: TSH, thyroid-stimulating hormone. * These studies are to be presented to Ohio State University by F.R.B. in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Biochim. Biophys..4eta, 141 (1967) 8-18
PYRIDINE NUCLEOTIDES
9
tion of a D P N + kinase responsible for the conversion of DPN + to T P N +. Although this hypothesis is appealing, it has been observed that a fairly large group of nonspecific reagents m a y induce TPN+ increases in dog thyroid slices. Thus in addition to TSH and acetylcholine the PASTAN--FIELD group has observed elevated T P N + levels with epinephrine, serotonin, and menadione TM. I t is possible, also, that elevated T P N + levels m a y simply reflect a disruption in the steady-state levels of T P N + due to decreased degradation of the nucleotide. Indeed FIELD has not excluded this possibility as a potential explanation of his data TM. Alternatively, the hormones might act b y elevating the 'extractable' T P N + from a relatively constant T P N + pool. This latter effect might be accomplished b y actions of the hormones to 'release' TPN+ from protein association, making it more easily solubilized, or, by actions of the hormones to convert an unstable and poorly extracted form of T P N ÷ to a stable, extractable form. With respect to this latter possibility it is of interest that LOWRY and his coworkers TMhave recently suggested the existence of 'acid-labile' and 'acid-stable' forms of T P N ÷ in a number of mammalian tissues, although this acid-labile form is controversial 24. Attempts b y the Field group to validate the hypothesis that a hormoneinduced stimulation of T P N + synthesis de novo is occurring have been indirect and inconclusive10, n, 19. I t would appear that this hypothesis might be critically evaluated through the use of tracer techniques. In particular a study of the incorporation of [32plorthophosphate or [14C~nicotinic acid into DPN+ and T P N + should permit an evaluation of potential stimulation of label incorporation as a response to hormone action. This paper reports the results of such a study. METHODS
Thyroid slices were prepared as previously described TM,17 using tissue obtained from mongrel male dogs. Tissue samples, in the weight range 2oo-3o0 mg, each consisting of several thyroid slice sections, were added to 25-ml erlenmeyer incubation flasks. These flasks contained 2.o ml of Krebs-Ringer bicarbonate buffer with o.6 ~mole of [32p~orthophosphate possessing a specific activity of 263 ~C/~mole, or 0. 3 vmole of [carboxy-14Clnicotinic acid with specific activities of 17, or 27. 9 ~C/~mole. Glucose is normally omitted from the medium. However, in appropriate experiments, [14Clglucose was added to the incubation medium at a concentration of o.oi M. Incubations were conducted for the appropriate time intervals with shaking at 37 ° using 95 % 02, 5 %CO2 as the gas phase. Immediately after termination of incubation pyridine nucleotides were extracted with either 0.2 M perchloric acid or o.I M HC1 depending upon subsequent use of the extracts. Since perchlorate ion interferes with the TPN + assay, tissues to be assayed directly for pyridine nucleotides were extracted with hot o.I M HC1 according to the procedure of GLOCK AND McLEAN 25. Following this treatment, the extracts were rapidly cooled to o °, diluted with IO ml of 0.2 M imidazole-HC1 buffer (pH 6.5), and adjusted to p H 6.5 with o.I M NaOH. After appropriate dilutions with distilled water these extracts were assayed directly for T P N +. Aliquots of the original HC1 extracts were prepared as tissue blanks b y adjusting the pH to I I with o.I M NaOH and heating these samples in a boiling-water bath for IO min. These blanks were then adjusted to p H 6.5 using imidazole-HC1 buffer and o.I M HC1. Since mild HC1 extraction does not destroy all TPN+-degrading enzymes, aqueous perchloric acid extraction was used where neutralized extracts were Biochim. Biophys. Acta, 141 (1967) 8-18
I0
F . R . BUTCHER, G. S. SERIF
to be handled for more prolonged periods. This situation obtained with tissue extract from which DPN + and T P N + were to be isolated b y column chromatography, paper chromatography, and paper electrophoresis for specific activity studies. In this extraction procedure the flask contents were adjusted to 0.2 M perchloric acid in the cold and homogenized in a Potter-Elvehjem homogenizer. Cell debris was removed at o ° b y centrifugation and the supernatant adjusted in the cold to a phenol red endpoint using 2 M KOH. In some experiments it was necessary to determine initial T P N + titers and isolate DPN + and TPN+ for specific activity studies as well. In these cases separate flasks were incubated with tissue slices from the same thyroid gland. One of these was then extracted for T P N + titers (HCI extraction) and the other extracted for specific activity studies (perchloric acid extraction). Experiments established that the method of extraction was without influence upon the specific activity of the isolated pyridine nucleotides. In DPN ÷ and T P N + isolation a separation of labeled DPN+ and TPN÷ from each other and miscellaneous phosphorus-containing compounds present in the perchloric acid extract was accomplished in the following fashion. The cold neutralized extracts were added to a Dowex I-X8 formate column (IO cm × I cm) and subjected to a convex formic acid gradient (water ~ 4 M formic acid) using a I25-ml mixing flask according to the procedure of HURLBERT et al. 14. Having previously established the elution pattern with unlabeled DPN ÷ and T P N +, the appropriate tubes were collected and lyophilized. The residues were taken up in o.ooi M phosphate buffer (pH 7.0) and spotted to W h a t m a n No. I chromatography paper. The chromatograms were developed by the descending technique with Solvent A containing 95 % ethanol and i M ammonium acetate buffer (pH 5.0) (7:3, v/v). The chromatograms were radioautographed and the DPN + and TPN + components developed in a second dimension with an ascending Solvent B composed of 0.i M sodium phosphate buffer containing 600 g (NH4)2SO4 per 1 and 2 % propanol, or, alternatively, paper electrophoresis was performed (Savant Model HV-3ooo) using o.I M ammonium acetate buffer (pH 5.0) and a potential drop of 20 V/cm. Since S042- interferes with the T P N + assay the second procedure was used most frequently when preparing samples for this assay whereas the sulfate system was employed primarily to establish purity of DPN+ and TPN + components. After second dimension development, the chromatograms were radioautographed, the DPN + and T P N + components identified and eluted from the paper with o.ooi M phosphate buffer (pH 7.0). Aliquots of the eluants were counted for 3,p or 14C content b y liquid scintillation techniques and then assayed for DPN + or TPN÷ content. Specific activities were expressed as counts/min per t ~ m o l e of pyridine nucleotide. Pyridine nucleotide assays are radiometric procedures developed b y the authors and are sensitive for DPN + and T P N + sample amounts of IO-1~ and lO-13 moles, respectively ~°,21. In the DPN + assay, limiting concentrations of DPN + are used to control the rate of pyruvate formation from lactate in the presence of lactate dehydrogenase. The resultant D P N H is utilized to convert EIJ4C]-a-ketoglutarate to [I-14C~-L-glutamate through the action of L-glutamate dehydrogenase. Regenerated DPN + recycles. 14CO~ is evolved from EIJ4C~-L-glutamate by L-glutamate decarboxylase and counted as a measure of DPN ÷ concentration. TPN÷ is measured in a similar system with glucose 6-phosphate and glucose-6-phosphate dehydrogenase replacing lactate and lactate dehydrogenase. Biochim. Biophys. Acta, 141 (1967) 8-18
PYRIDINE NUCLEOTIDES
II
TSH was obtained from Sigma and Armour (Thytropar). These preparations possessed activities of the order I unit per mg and appeared essentially comparable in their effects. Acetylcholine was used at a level of 2.8. lO-5 M in the presence of eserine sulfate (3.6.1o-4). In order to eliminate the potential effects of eserine sulfate, carbamylcholine, a cholinesterase-resistant analogue of acetylcholine, was frequently substituted for the acetylcholine--eserine pair at a level of 2.8. lO-5 M. RESULTS AND DISCUSSION
[32p~Ortho~hosfihate studies Orthophosphate incorporation reflects synthesis of pyridine nucleotides de novo ~3. The use of [3~p]orthophosphate, therefore, permits a convenient approach to the study of a specific stimulation of the conversion of DPN + to TPN + by a DPN + kinase. It is to be expected that [3zP]orthophosphate should rapidly enter cells of thyroid slices exposed to it with consequent formation of 32P-labeled ATP. If the DPN+ to TPN + step is stimulated by hormone action it would be expected that increased quantities of 32p-labeled ATP would be incorporated into TPN + in stimulated tissues. As a consequence of this action the specific activities of the resultant labeled TPN + should be higher in stimulated tissues as compared to control thyroid slices. In a study of this type the use of a non-specific precursor necessitates that considerable care be exercised to establish the radiochemical purity of isolated 3ZP-labeled pyridine nucleotides. Although the column separation of HURLBERT AND POTTER effectively separates DPN + from TPN +, each of these separate fractions contains additional 32p-labeled nucleotides. Fig. I contains reproductions of radioautographs of a DPN+ column fraction after additional chromatography with Solvent A and subsequent development by electrophoresis. Fig. 2 shows a similar separation for TPN +. After electrophoresis both DPN + and TPN + travel as single components in Solvent B. Further verification of the purity of the electrophoretic samples was obtained by splitting each spot into a front and back portion and determining specific activities for both halves separately. Table I contains the results of a study of this type. Within experimental error the two halves of each component have the same specific activities. In general, specific activities determined in this way are within the experimental range of + 7 %. These data suggest that the purified 32p-labeled DPN + and 3~p-labeled TPN + samples are homogeneous single components. Table II depicts typical data demonstrating the effects of TSH, acetylcholine and carbamylcholine upon TPN + titers and the effects of TSH upon glucose metabolism in dog thyroid slices. The influence of cholinergic agents on glucose metabolism has previously been determined 8,17. It is apparent from these studies that the TSH preparations used are effective in elevating 'extractable' TPN+ levels as are the cholinergic agents. In addition the TSH preparations stimulate [14C3glucose conversion to 14CO2. Duplication of the results of the FIELD--PASTAN groups suggests that the experimental materials and conditions employed in this study are suitable and permit further evaluation of these elevated TPN + titers by specific activity measurements. Fig. 3 contains the results of a study designed to determine the influence of time upon 3zp incorporation into pyridine nucleotides. With the exception of an initial lag phase for DPN+ it is seen that the specific activities of both DPN + and Biochim. Biophys. Acta, 141 (1967) 8-18
12
F. R, BUTCHER, G. S. SERIF
T P N + i n c r e a s e i n a n e a r l y l i n e a r f a s h i o n f o r p e r i o d s of a t l e a s t o n e h o u r . S i n c e PASTAN et al. 1~ r e p o r t a s t e a d y i n c r e a s e i n T P N + t i t e r s for t h y r o i d slices e x p o s e d t o T S H f o r i - h p e r i o d s , t h i s t i m e w a s a d o p t e d as a n o p t i m u m i n c u b a t i o n p e r i o d t o p e r m i t e f f e c t i v e i n c o r p o r a t i o n of 3zp i n t o p y r i d i n e n u c l e o t i d e s . I t is of i n t e r e s t t h a t t h e r a t i o of specific a c t i v i t i e s of D P N + t o T P N + is of t h e o r d e r of I : I0. A t first g l a n c e , s u c h a
Fig. i. Composite radioautographs of the further separation of asp-labeled I)PN + from 3zp_ containing contaminants after preliminary separation from 32P-labeled TPN + by column chromatography on Dowex I-X8 as described under METHODS. Vertical separation represents the initial z*P p a t t e r n developed by paper chromatography in Solvent A. Horizontal separation represents final separation of the z2P-labeled DPN + segment of the paper chromatogram by paper electrophoresis as described under METHODS. Rp refers to distance traveled by DPN + as a ratio to the distance traveled by a picric acid standard. Fig. 2. Composite radioautographs of the further separation of z~P-labeled T P N + from 3ZP-labeled TPN + from zzP-containing contaminants after preliminary separation from 3zp-labeled DPN + by column chromatography on Dowex I-X8. Procedures used are similar to those described in the legend to Fig. i and under METHODS. TABLE I U N I F O R M I T Y OF P U R I F I E D 3 2 p - L A B E L E D
DPN+
AND 32p-LABELED
TPN +
Terminal radioactive spots for 3ZP-labeled DPN + and TPN + isolated from dog thyroid by a sequence of column chromatography, paper chromatography and paper electrophoresis were split into front and back components and each assayed for specific activity in order to establish t h a t the isolation procedure produces nucleotide spots which are homogeneous with respect to 3zp and pyridine nucleotide content.
Nucleotide
Front Back (counts/min per i~l~mole) (counts/min per i~l~mole)
32P-labeled DPN + s'P-labeled TPN +
1.6 9.6
Biochim. Biophys. Acta, 141 (1967) 8-18
1.5 9.8
PYRIDINE
13
NUCLEOTIDES
wide disparity in specific activities might appear unusual. However, at least two explanations of this phenomenon are readily apparent. Thus, for example, DPN + and TPN+ might be labeled from ATP pools with different specific activities. Alternatively DPN + and TPN + might be labeled from a common ATP pool but the pool size of DPN + might be considerably greater than that of TPN +. In such circumstances the 3,p incorporated into the two phosphate groups of DPN + might be considerably diluted whereas the 3~p incorporated into the third phosphate group of TPN + would be less so. As a result the specific activities of TPN + would be greater than those of DPN +. Since the DPN + pool size in dog thyroid slices9 has been established to be approximately one order of magnitude greater than that of TPN+, the latter explanation would adequately explain the data.
o7
S
t--
•~
°
*~o~O~7,
°~2-~°-"~° 0
20
40
• 60
MINUTES
F i g . 3. A t i m e s t u d y o f t h e i n c o r p o r a t i o n (0--0) of dog thyroid slices. Specific pyridine nucleotide.
TABLE
of [3~PJorthophosphate activities are expressed
into DPN + (©--O) as counts/min per
and TPN + #/,mole of
II
I N F L U E N C E OF T S H A N D C H O L I N E R G I C AGENTS ON T P N + DOG T H Y R O I D SLICES
T I T E R S AND [14C]GLUCOSE O X I D A T I O N I N
F~xpt. No.
Hormone
[l*C]glucose (o.ox M)
TPN + (mpmoles/g)
14C02 (counts/rain)
I
Control TSH Acetylcholine
----
3.2 12. 5 I3.6
----
2
Control TSH Carbamylcholine
----
8.0 19.o 20.8
----
3*
Control TSH
+ +
---
14 o 5 8 41 0 8 0
4" *
Control TSH
+ +
---
29 260 47 034
* Krebs-Ringer ** K r e b s - R i n g e r
phosphate buffer. bicarbonate buffer.
Biochim. Biophys. Acta,
141 ( 1 9 6 7 ) 8 - 1 8
14
F. R. B U T C H E R ,
G. S. S E R I F
Since the existence of a DPN kinase is compatible with the above results it remains to establish whether T P N formation is stimulated by hormones, and if possible to determine whether the stimulatory effect is at a DPN kinase step. Table I I I contains data from experiments attempting to resolve these points. Thus specific activities were determined for DPN+ and T P N + in control tissues and tissues treated with TSH, acetylcholine, or carbamylcholine. I t is apparent that the specific activities of DPN+h (hormone treated) are for the most part slightly elevated as compared to DPNc + (control). Surprisingly, however, the specific activities for TPNh + are seen to TABLE
III
INFLUENCE OF T S H AND CHOLINERGIC AGENTS ON I32p]ORTHOPHOSPHATI~ INCORPORATION INTO D P N + AND T P N + OF DOG THYROID
Expt. No.
Treatment
Stimulation of D P N + T P N + levels (counts/min (%) . per iq~mole)
TPN + (counts]min per t~l~mole)
TPNh+/DPNn +* TPNc+/DPNc+ i probable error**
I
Control TSH Carbamylcholine
-208
1.26 1.35 1.35
12.5 II.I 9.6
-0.86 i 0.06 o.71 ± 0 . 0 7
2
Control TSH
-N***
1.57 1.64
11.6 lO.2
-0.84 ± 0.07
3
Control Carbamylcholine
-44
1.14 § 1-14 §
13. 5 12.9
-0.96 ~ 0.o9
4
Control Carbamylcholine
-N***
1.25 1.59
18.8 13.6
-0.57 -- 0.08
158
* S u b s c r i p t s c a n d h r e f e r t o c o n t r o l a n d h o r m o n e - t r e a t e d slices, r e s p e c t i v e l y . ** C o m p o s i t e p r o b a b l e e r r o r d e r i v e d f r o m t h e s u m m a t i o n of t h e i n d i v i d u a l p r o b a b l e e r r o r s o f each specific activity involved in a given ratio. *** N o t m e a s u r e d d u e t o i n a d e q u a t e q u a n t i t i e s o f t i s s u e . § S p e c i f i c a c t i v i t i e s b a s e d o n a s i n g l e d e t e r m i n a t i o n . T h e m a x i m u m p r o b a b l e e r r o r is a s s u m e d . A l l o t h e r s p e c i f i c a c t i v i t y f i g u r e s a r e a v e r a g e s of t w o o r m o r e c l o s e l y a g r e e i n g d e t e r m i n a t i o n s .
be generally lower than the specific activities for TPNc +. Since hormone action does appear to have some small effect on the overall synthesis of pyridine nucleotides de novo as represented b y the specific activities of DPN +, it is not adequate to compare simple changes in specific activities of T P N + and conclude that such changes represent an effect on the DPN + kinase step. Rather it is necessary to eliminate the influence of hormones on synthesis of pyridine nucleotides de novo prior to the DPN + to T P N + step by comparing the ratios of T P N + to DPN + in hormone-treated and control thyroid slices. Thus, if for the expression TPN+h/DPNh + TPN+c/DPNe +
an experimental value greater than one were obtained a specific stimulation of the DPN + kinase step would be indicated. Alternatively, if experimental values of one or less were obtained, no effect or inhibiting effects of hormone, respectively, would be deduced. As a first approximation these ratios will hold if hormone induced Bioehim. Biophys. Acta, 141 (1967) 8 - 1 8
P Y R I D I N E NUCLEOTIDES
15
changes in specific activities of DPN + are small. Since such changes are seen to be small the ratios given in Table I I I m a y be used to weigh the influence of the various hormones on the D P N + kinase step. I t is apparent that the greater number of these ratios are one or less than one. Thus, under the experimental conditions, these data might be interpreted to signify the absence of a stimulatory hormonal effect on T P N + formation 22.
[3~PJOrthophosphate studies with preimubation It is conceivable that the response to hormonal agents m a y be of short duration. In such an event stimulatory effects on TPN + formation m a y be induced before the precursor pool of DPN + is appreciably labeled with 32p. Thus increased incorporation of radioactivity might be masked despite an actual stimulation of T P N + formation. In order to test this possibility, dog thyroid slices were incubated with [a~P]orthophosphate for 3o-min periods followed by a 3o-min exposure to the appropriate hormone. D a t a from these studies are presented in Table IV. In each instance, the ratio values are now seen to be considerably in excess of one. Clearly, under these conditions, stimulation of asP-labeled T P N + formation is occurring as a consequence of hormone action. If the original postulate for the preincubation experiment is correct, these data would imply that the stimulatory influence of these hormones is maximally effected, or apparent, over a limited time interval.
[14C~Nicotinic acid studies with j~reincubation It is possible to explain the 3zp data as a consequence of a hormonally induced heightened turnover of 3~p in ATP without an actual increase in T P N + formation. Thus it was considered advisable to repeat crucial experiments with a more immeT A B L E IV INFLUENCE OF T S H AND CARBAMYLCHOLINEON [32P]ORTHOPHOSPHATE INCORPORATION INTO D P N + AND T P N + IN DOG THYROID WITH PREINCUBATION T h e e x p e r i m e n t a l p r o t o c o l (see METHODS) w a s followed w i t h e x c e p t i o n t h a t a 3o-min p r e i n c u b a t i o n of t h e slices w i t h t h e m e d i u m c o n t a i n i n g [3~P]orthophosphate w a s used. A t t h e e n d of t h i s p e r i o d h o r m o n e w a s i n j e c t e d t h r o u g h t h e i n c u b a t i o n flask s t o p p e r a n d i n c u b a t i o n c o n t i n u e d for a t e r m i n a l 3o-min period.
Expt. Treatment No.
3
Stimulation of D P N + T P N + levels (counts]rain (%) per iH~mole)
TPN + (counts]rain per ttttmole)
TPNh+/DPNh + TPNe+/DPNe+ d- probable error*
Control TSH Carbamylcholine
-119 144
0.87 0.84 0.94
I 1.3 I7.8 15.7
-1.6 2_ 0.2 1. 3 4- o.I
Co ntro l TSH Carbamylcholine
-28 I16
0.93 0.87 0.53
5-4 7.5 7.6
-1. 5 _a 0.2 2.5 + 0.3
Control Carbamylcholine
-76
0.85 0.64
4.4 7.4
-2.2 4- 0.2
* C o m p o s i t e p r o b a b l e error d e r i v e d from t h e s u m m a t i o n of t h e i n d i v i d u a l p r o b a b l e e rrors of each specific a c t i v i t y i n v o l v e d in a g i v e n ratio. E a c h specific a c t i v i t y figure is a n a v e r a g e of t w o or m o r e closely a g r e e i n g d e t e r m i n a t i o n s .
Biochim. Biophys. Acta, 141 (1967) 8-18
16
F . R . BUTCHEI~, G. S. SERIF
diate and specific precursor. Additional experiments were carried out with [carboxyl14C]nicotinic acid using the identical isolation and assay procedure employed on the 3~p studies. Nicotinic acid was chosen instead of nicotinamide since the incorporation of the former derivative into DPN + and TPN + appears to reflect synthesis of these nucleotides de novo ~3. The influence of TSH and carbamylcholine upon [14Clnicotinic acid incorporation into pyridine nucleotides is shown in Table V. Consideration of the ratio values indicates that these data are consistent with those obtained from 32p studies and clearly demonstrate a stimulation of TPN + formation in the presence of TSH and carbamylcholine. Since an examination of the specific activities of DPN + in Table V reveals little hormone influence upon DPN + formation, these data might be interpreted to suggest that the stimulation of TPN + formation occurs at the DPN + TABLE V INFLUENCE AND TPN
OF
TSH
A N D C A R B A M Y L C H O L I N E ON [ 1 4 C ] N I C O T I N I C ACID I N C O R P O R A T I O N
+ IN DOG THYROID
INTO
DPN +
WITH PREINCUBATION
The experimental protocol of the METHODS section was followed with the exception t h a t a 3o-min p r e i n c u b a t i o n of the slices with the m e d i u m containing [14C]nitotinic acid was used. At the end of this period h o r m o n e was injected t h r o u g h the incubation flask s t o p p e r and incubation continued for a terminal 3o-min period.
Expt. Treatment No.
Stimulation of D P N + T P N + levels (counts/min (%) per ##mole)
TPN + (counts/min per tv2mole)
TPNh+/DPNh + TPNc+/DPNc+ ± probable error*
Control TSH Carbamylcholine
-lO 9 69
6.4 6.1 6.4
3.4 5.6 5.8
-1.8 ± o.2 1. 7 ~ o.2
Control TSH Carbamylcholine
-12o lO 3
6.8 6.6 7.2
6.6 9.7 9.8
-1.5 ± o.i 1. 4 ± o.i
* Composite probable error derived from the s u m m a t i o n of the individual probable errors of each specific activity involved in a given ratio. E a c h specific activity figure is an average of two or more closely agreeing determinations.
/
//
// /0
'~
i
0
o/
/
12
n
20
o/
o --
i
i
i
40
60
80
MINUTES
Fig. 4- A time s t u d y of the incorporation of [14Clnicotinic acid into D P N + ( [] ) and T P N + ( O ) in the presence (closed points) and absence (open points) of TSH. Specific activities are expressed as c o u n t s / m i n per # # m o l e of pyridine nucleotide. Arrows indicate T S H addition.
Biochim. Biophys. Acta, 141 (1967) 8 - I 8
17
PYRIDINE NUCLEOTIDES
kinase step. I t is thought provoking, however, to examine the specific activities of T P N + in Expt. 2 of Table V as compared to the corresponding DPN + values. In the presence of T S H and carbamylcholine both T P N + specific activities exceed those of DPN +. This phenomenon was examined further as a function of time. Figs. 4 and 5 depict time studies showing the influence of T S H and carbamylcholine upon DPN + and TPN+ specific activities. Again, a hormone-induced elevation of tile rate of incorporation of radioactivity into T P N + is obvious, with little apparent hormonal influence upon DPN+ formation. In addition, with extended time, the increase in T P N + specific activities above and beyond those of DPN + become consistent and pronounced. This departure from precursor-product relationships m a y be explained through the thesis, (a) that DPN+ is not a direct precursor of TPN + or (b) that a pool dilution effect has occurred. With the latter explanation it is necessary to propose that precursor DPN + and TPN+ are synthesized at a common restricted site removed from the major nucleotide pools, in order to prevent DPN+ dilution prior to its conversion to T P N +. Thus, as precursor DPN + of high specific activity is formed, it is converted to a somewhat lower, but nonetheless high specific activity of T P N +.
/
/ 24
e//
20
O/
//
~.
/
~ 0
i
= 20
i
i 40
i
i 60
i
i 80
MINUTES
Fig. 5. A time study of the incorporation of E14C~nicotinic acid into D P N + [D) and T P N + (O) in the presence (closed points) and absence (open points) of carbamylcholine. Specific activities are expressed as counts/min per /~#mole of pyridine nucleotide. Arrows indicate carbamylcholine addition.
Release of these nucleotides to the main pool, by normal cell metabolism or extraction techniques, then permits more extensive dilution of DPN + than T P N + and hence higher TPN+ specific activity values. Because of this potential interpretation, care must be exercised in the assessment that stimulation of DPN+ formation is not occurring. Expressed as specific activity, the average stimulatory response observed with T P N + is of the order of 60 To. Since the total DPN + pool is approx. Io-fold that of the T P N + pool in dog thyroid, a similar response with respect to an increased DPN + formation might be reflected, through dilution, as an increase in DPN + specific activity of approx. 6%. Under conditions of preincubation the average observed influence of hormones on DPN + synthesis is reflected as a decrease in specific activity of 8 %. The range of values encountered encompasses a possible 6 % increase. Thus no statistical significance can be applied to the difference between these two numbers. Support for a lack of stimulatory effect on DPN + formation, however, is available Biochim. Biophys. Acta, 141 (1967) 8-18
I8
F . R . BUTCHER, G. S. SERIF
from the observation that DPN + and D P N H levels decrease in thyroid slices as a response to TSH and cholinergic agents 9. It is improbable that a stimulation of DPN + formation would be propagated through DPN + to T P N + under conditions of net DPN + decrease. In summation, the data of these studies clearly demonstrate a stimulation of labeled T P N + formation when dog thyroid slices are preincubated with [32P~orthophosphate or [14Clnicotinic acid and exposed to TSH or cholinergic agents. Thus increased titers of TPN+, as a response to these reagents, m a y be accounted for through enhanced synthesis of TPN+ de novo. The apparent lack of a concomitant increase in labeled DPN+ formation, taken in conjunction with the fact that DPN + levels are depressed by T S H and cholinergic agents 9 is suggestive of a hormoneinduced activation of a parameter associated with DPN + kinase. However, no absolute assignment of such an activation is permissible because of the possibility of interfering pool dilution effects. ACKNOWLEDGEMENTS
This investigation was supported in part b y research grant AM-II43O from the Division of Arthritis and Metabolic Diseases of the U.S. Public Health Service, and GB-42I 7 from the National Science Foundation.
REFERENCES
I 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21 22 23 24 25
N. FREINKEL, J. Endocrinol., 14 (i956) X X . N. FREINKEL, Endocrinology, 61 (1957) 448. J. E. DUMONT, Biochim. Biophys. Acta, 46 (1961) 195. J. E. DUMONT, Biochim. Biophys. Acta, 5 ° (1961) 506. J- B. FIELD, B. HERRING AND P. JOHNSON,Biochim. Biophys. Acta, 5 ° (1961) 513 . F. MALOOF AND M. SOODAK,Pharmacol. Rev., 15 (1963) 43. J. ]3. FIELD, I. PASTAN, P. JOHNSON AND B. HERRING, J. Biol. Chem., 235 (196o) 1863. I. PASTAN, B. HERRING, P. JOHNSON AND J. ]3. FIELD, J. Biol. Chem., 236 (1961) 34 o. I. PASTAN, B. HERRING AND J. B. FIELD, J. Biol. Chem., 236 (1961) P.C. 25. R. J. JARR~TT, S. M. EPSTEIN, A. K. REMER AND J. B. FIELD, Endocrinology, 77 (1965) 290. J. B. FIELD, A. I<. REMER AND R. j. JARRETT, J. Biol. Chem., 240 (1965) 883. G. S. SERIF, Proc. Soc. Exptl. Biol. Med., IiO (1962) 356. B. V~. O'MALLEY AND J. ]3. FIELD, Biochim. Biophys. Acta, 90 (1964) 349R. B. HURLBERT, H. SCHMITZ, A. F. BRUMM AND V. R. POTTER, J . Biol. Chem., 2o9 (1954) 23. O. H. LowRY AND J. V. PASSONNEAU, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 6, A c a d e m i c Press, N e w York, 1963, p. 792. I. PASTAN, P. JOHNSON, E. KENDIG AND J. }3. FI]SLD, J, Biol. Chem., 238 (1963) 3366. G. S. SERIF, Biochim. Biophys. Acta, 119 (1961) 229. H. B. BURCH, O. H. LOWRY AND P. VON DIPPE, J. Biol. Chem., 238 (1963) 2838. J. ]3. FIELD, S. M. EPSTEIN, A. K. REMER AND C. BOYLE, Biochim. Biophys. Acta, 121 (1966) 241. G. S. SERIF AND F. R. BUTCHER, Anal. Biochem., 15 (1966) 278. G. S. SERIF, L. SCHMOTZER AND F. R. BUTCHER, Anal. Biochem., 17 (1966) 125. G. S. SERIF AND F. R. BUTCHER, Federation Proe., 25 (1966) 763 . L. S. DIETRICH AND L. FULLER, J. Biol. Chem., 239 (1964) 1653. H. W. HELDT, N. GREIF AND M. KLINGENBERG, J . Biol. Chem., 240 (1965) 4659. G. E. CLOCK AND P. MCLEAN, Biochem. J., 61 (1955) 381.
Biochim. Biophys. Acta, 141 (1967) 8-18
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25 772 P Y R I D I N E NUCLEOTIDES III. RESPONSE OF DOG T H Y R O I D P Y R I D I N E NUCLEOTIDES TO HORMONAL CONTROL F R E D R. B U T C H E R * AND G E O R G E S. S E R I F
The Department of Biochemistry, The Ohio State University, Columbus, Ohio (U,S.A.) (Received November 22nd, 1966)
SUMMARY
TPN + levels in dog thyroid slices appear to be elevated above control values when these tissues are exposed to thyroid-stimulating hormone (TSH) and cholinergic agents. It has been uncertain as to whether these hormone effects are due to an increased synthesis de novo of TPN + or an inhibition of TPN + degradation. This question has been evaluated through a study of the rate of incorporation of E32plorthophosphate and [14Clnicotinic acid into DPN + and TPN-- in the presence and absence of TSH and cholinergic agents. The specific activity data show an enhanced synthesis de novo of TPN+ with no apparent concomitant enhancement of DPN + formation. Although these data suggest a specific stimulation of the DPN + kinase step, the possibility of pool dilution effects does not permit such an absolute assignment.
INTRODUCTION
The study of iodinated derivatives has been a fruitful chapter in the investigation of thyroid metabolism. More recently, however, considerable attention has been focused on non-iodinated thyroid metabolites 1-6 with the concept that an understanding of their control by hormone action may give some insight into control mechanisms for thyroid hormone biogenesis. One of the more intriguing relationships developed by this approach is concerned with the observation that glucose metabolism in thyroid tissue responds to several hormones. Thus FIELD and PASTAN with their colleagues have shown increased [14CJglucose oxidation to 14C02 by dog thyroid slices in the presence of thyroid-stimulating hormone (TSH) and acetylcholineS,7,s. This group of investigators has observed an increase in thyroid TPN+ levels as a response to these hormones as well9. They suggest that increased glucose utilization is a secondary response to an increase in TPN + levels which promotes [i-14Clglucose oxidation to 14CO2 by means of the hexose monophosphate pathway. Since DPN + concentrations decrease concomitantly with TPN + increase FIELD and co-workers 10 suggest that the higher titers of TPN + may be brought about through specific stimulaAbbreviation: TSH, thyroid-stimulating hormone. * These studies are to be presented to Ohio State University by F.R.B. in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Biochim. Biophys..4eta, 141 (1967) 8-18
PYRIDINE NUCLEOTIDES
9
tion of a D P N + kinase responsible for the conversion of DPN + to T P N +. Although this hypothesis is appealing, it has been observed that a fairly large group of nonspecific reagents m a y induce TPN+ increases in dog thyroid slices. Thus in addition to TSH and acetylcholine the PASTAN--FIELD group has observed elevated T P N + levels with epinephrine, serotonin, and menadione TM. I t is possible, also, that elevated T P N + levels m a y simply reflect a disruption in the steady-state levels of T P N + due to decreased degradation of the nucleotide. Indeed FIELD has not excluded this possibility as a potential explanation of his data TM. Alternatively, the hormones might act b y elevating the 'extractable' T P N + from a relatively constant T P N + pool. This latter effect might be accomplished b y actions of the hormones to 'release' TPN+ from protein association, making it more easily solubilized, or, by actions of the hormones to convert an unstable and poorly extracted form of T P N ÷ to a stable, extractable form. With respect to this latter possibility it is of interest that LOWRY and his coworkers TMhave recently suggested the existence of 'acid-labile' and 'acid-stable' forms of T P N ÷ in a number of mammalian tissues, although this acid-labile form is controversial 24. Attempts b y the Field group to validate the hypothesis that a hormoneinduced stimulation of T P N + synthesis de novo is occurring have been indirect and inconclusive10, n, 19. I t would appear that this hypothesis might be critically evaluated through the use of tracer techniques. In particular a study of the incorporation of [32plorthophosphate or [14C~nicotinic acid into DPN+ and T P N + should permit an evaluation of potential stimulation of label incorporation as a response to hormone action. This paper reports the results of such a study. METHODS
Thyroid slices were prepared as previously described TM,17 using tissue obtained from mongrel male dogs. Tissue samples, in the weight range 2oo-3o0 mg, each consisting of several thyroid slice sections, were added to 25-ml erlenmeyer incubation flasks. These flasks contained 2.o ml of Krebs-Ringer bicarbonate buffer with o.6 ~mole of [32p~orthophosphate possessing a specific activity of 263 ~C/~mole, or 0. 3 vmole of [carboxy-14Clnicotinic acid with specific activities of 17, or 27. 9 ~C/~mole. Glucose is normally omitted from the medium. However, in appropriate experiments, [14Clglucose was added to the incubation medium at a concentration of o.oi M. Incubations were conducted for the appropriate time intervals with shaking at 37 ° using 95 % 02, 5 %CO2 as the gas phase. Immediately after termination of incubation pyridine nucleotides were extracted with either 0.2 M perchloric acid or o.I M HC1 depending upon subsequent use of the extracts. Since perchlorate ion interferes with the TPN + assay, tissues to be assayed directly for pyridine nucleotides were extracted with hot o.I M HC1 according to the procedure of GLOCK AND McLEAN 25. Following this treatment, the extracts were rapidly cooled to o °, diluted with IO ml of 0.2 M imidazole-HC1 buffer (pH 6.5), and adjusted to p H 6.5 with o.I M NaOH. After appropriate dilutions with distilled water these extracts were assayed directly for T P N +. Aliquots of the original HC1 extracts were prepared as tissue blanks b y adjusting the pH to I I with o.I M NaOH and heating these samples in a boiling-water bath for IO min. These blanks were then adjusted to p H 6.5 using imidazole-HC1 buffer and o.I M HC1. Since mild HC1 extraction does not destroy all TPN+-degrading enzymes, aqueous perchloric acid extraction was used where neutralized extracts were Biochim. Biophys. Acta, 141 (1967) 8-18
I0
F . R . BUTCHER, G. S. SERIF
to be handled for more prolonged periods. This situation obtained with tissue extract from which DPN + and T P N + were to be isolated b y column chromatography, paper chromatography, and paper electrophoresis for specific activity studies. In this extraction procedure the flask contents were adjusted to 0.2 M perchloric acid in the cold and homogenized in a Potter-Elvehjem homogenizer. Cell debris was removed at o ° b y centrifugation and the supernatant adjusted in the cold to a phenol red endpoint using 2 M KOH. In some experiments it was necessary to determine initial T P N + titers and isolate DPN + and TPN+ for specific activity studies as well. In these cases separate flasks were incubated with tissue slices from the same thyroid gland. One of these was then extracted for T P N + titers (HCI extraction) and the other extracted for specific activity studies (perchloric acid extraction). Experiments established that the method of extraction was without influence upon the specific activity of the isolated pyridine nucleotides. In DPN ÷ and T P N + isolation a separation of labeled DPN+ and TPN÷ from each other and miscellaneous phosphorus-containing compounds present in the perchloric acid extract was accomplished in the following fashion. The cold neutralized extracts were added to a Dowex I-X8 formate column (IO cm × I cm) and subjected to a convex formic acid gradient (water ~ 4 M formic acid) using a I25-ml mixing flask according to the procedure of HURLBERT et al. 14. Having previously established the elution pattern with unlabeled DPN ÷ and T P N +, the appropriate tubes were collected and lyophilized. The residues were taken up in o.ooi M phosphate buffer (pH 7.0) and spotted to W h a t m a n No. I chromatography paper. The chromatograms were developed by the descending technique with Solvent A containing 95 % ethanol and i M ammonium acetate buffer (pH 5.0) (7:3, v/v). The chromatograms were radioautographed and the DPN + and TPN + components developed in a second dimension with an ascending Solvent B composed of 0.i M sodium phosphate buffer containing 600 g (NH4)2SO4 per 1 and 2 % propanol, or, alternatively, paper electrophoresis was performed (Savant Model HV-3ooo) using o.I M ammonium acetate buffer (pH 5.0) and a potential drop of 20 V/cm. Since S042- interferes with the T P N + assay the second procedure was used most frequently when preparing samples for this assay whereas the sulfate system was employed primarily to establish purity of DPN+ and TPN + components. After second dimension development, the chromatograms were radioautographed, the DPN + and T P N + components identified and eluted from the paper with o.ooi M phosphate buffer (pH 7.0). Aliquots of the eluants were counted for 3,p or 14C content b y liquid scintillation techniques and then assayed for DPN + or TPN÷ content. Specific activities were expressed as counts/min per t ~ m o l e of pyridine nucleotide. Pyridine nucleotide assays are radiometric procedures developed b y the authors and are sensitive for DPN + and T P N + sample amounts of IO-1~ and lO-13 moles, respectively ~°,21. In the DPN + assay, limiting concentrations of DPN + are used to control the rate of pyruvate formation from lactate in the presence of lactate dehydrogenase. The resultant D P N H is utilized to convert EIJ4C]-a-ketoglutarate to [I-14C~-L-glutamate through the action of L-glutamate dehydrogenase. Regenerated DPN + recycles. 14CO~ is evolved from EIJ4C~-L-glutamate by L-glutamate decarboxylase and counted as a measure of DPN ÷ concentration. TPN÷ is measured in a similar system with glucose 6-phosphate and glucose-6-phosphate dehydrogenase replacing lactate and lactate dehydrogenase. Biochim. Biophys. Acta, 141 (1967) 8-18
PYRIDINE NUCLEOTIDES
II
TSH was obtained from Sigma and Armour (Thytropar). These preparations possessed activities of the order I unit per mg and appeared essentially comparable in their effects. Acetylcholine was used at a level of 2.8. lO-5 M in the presence of eserine sulfate (3.6.1o-4). In order to eliminate the potential effects of eserine sulfate, carbamylcholine, a cholinesterase-resistant analogue of acetylcholine, was frequently substituted for the acetylcholine--eserine pair at a level of 2.8. lO-5 M. RESULTS AND DISCUSSION
[32p~Ortho~hosfihate studies Orthophosphate incorporation reflects synthesis of pyridine nucleotides de novo ~3. The use of [3~p]orthophosphate, therefore, permits a convenient approach to the study of a specific stimulation of the conversion of DPN + to TPN + by a DPN + kinase. It is to be expected that [3zP]orthophosphate should rapidly enter cells of thyroid slices exposed to it with consequent formation of 32P-labeled ATP. If the DPN+ to TPN + step is stimulated by hormone action it would be expected that increased quantities of 32p-labeled ATP would be incorporated into TPN + in stimulated tissues. As a consequence of this action the specific activities of the resultant labeled TPN + should be higher in stimulated tissues as compared to control thyroid slices. In a study of this type the use of a non-specific precursor necessitates that considerable care be exercised to establish the radiochemical purity of isolated 3ZP-labeled pyridine nucleotides. Although the column separation of HURLBERT AND POTTER effectively separates DPN + from TPN +, each of these separate fractions contains additional 32p-labeled nucleotides. Fig. I contains reproductions of radioautographs of a DPN+ column fraction after additional chromatography with Solvent A and subsequent development by electrophoresis. Fig. 2 shows a similar separation for TPN +. After electrophoresis both DPN + and TPN + travel as single components in Solvent B. Further verification of the purity of the electrophoretic samples was obtained by splitting each spot into a front and back portion and determining specific activities for both halves separately. Table I contains the results of a study of this type. Within experimental error the two halves of each component have the same specific activities. In general, specific activities determined in this way are within the experimental range of + 7 %. These data suggest that the purified 32p-labeled DPN + and 3~p-labeled TPN + samples are homogeneous single components. Table II depicts typical data demonstrating the effects of TSH, acetylcholine and carbamylcholine upon TPN + titers and the effects of TSH upon glucose metabolism in dog thyroid slices. The influence of cholinergic agents on glucose metabolism has previously been determined 8,17. It is apparent from these studies that the TSH preparations used are effective in elevating 'extractable' TPN+ levels as are the cholinergic agents. In addition the TSH preparations stimulate [14C3glucose conversion to 14CO2. Duplication of the results of the FIELD--PASTAN groups suggests that the experimental materials and conditions employed in this study are suitable and permit further evaluation of these elevated TPN + titers by specific activity measurements. Fig. 3 contains the results of a study designed to determine the influence of time upon 3zp incorporation into pyridine nucleotides. With the exception of an initial lag phase for DPN+ it is seen that the specific activities of both DPN + and Biochim. Biophys. Acta, 141 (1967) 8-18
12
F. R, BUTCHER, G. S. SERIF
T P N + i n c r e a s e i n a n e a r l y l i n e a r f a s h i o n f o r p e r i o d s of a t l e a s t o n e h o u r . S i n c e PASTAN et al. 1~ r e p o r t a s t e a d y i n c r e a s e i n T P N + t i t e r s for t h y r o i d slices e x p o s e d t o T S H f o r i - h p e r i o d s , t h i s t i m e w a s a d o p t e d as a n o p t i m u m i n c u b a t i o n p e r i o d t o p e r m i t e f f e c t i v e i n c o r p o r a t i o n of 3zp i n t o p y r i d i n e n u c l e o t i d e s . I t is of i n t e r e s t t h a t t h e r a t i o of specific a c t i v i t i e s of D P N + t o T P N + is of t h e o r d e r of I : I0. A t first g l a n c e , s u c h a
Fig. i. Composite radioautographs of the further separation of asp-labeled I)PN + from 3zp_ containing contaminants after preliminary separation from 32P-labeled TPN + by column chromatography on Dowex I-X8 as described under METHODS. Vertical separation represents the initial z*P p a t t e r n developed by paper chromatography in Solvent A. Horizontal separation represents final separation of the z2P-labeled DPN + segment of the paper chromatogram by paper electrophoresis as described under METHODS. Rp refers to distance traveled by DPN + as a ratio to the distance traveled by a picric acid standard. Fig. 2. Composite radioautographs of the further separation of z~P-labeled T P N + from 3ZP-labeled TPN + from zzP-containing contaminants after preliminary separation from 3zp-labeled DPN + by column chromatography on Dowex I-X8. Procedures used are similar to those described in the legend to Fig. i and under METHODS. TABLE I U N I F O R M I T Y OF P U R I F I E D 3 2 p - L A B E L E D
DPN+
AND 32p-LABELED
TPN +
Terminal radioactive spots for 3ZP-labeled DPN + and TPN + isolated from dog thyroid by a sequence of column chromatography, paper chromatography and paper electrophoresis were split into front and back components and each assayed for specific activity in order to establish t h a t the isolation procedure produces nucleotide spots which are homogeneous with respect to 3zp and pyridine nucleotide content.
Nucleotide
Front Back (counts/min per i~l~mole) (counts/min per i~l~mole)
32P-labeled DPN + s'P-labeled TPN +
1.6 9.6
Biochim. Biophys. Acta, 141 (1967) 8-18
1.5 9.8
PYRIDINE
13
NUCLEOTIDES
wide disparity in specific activities might appear unusual. However, at least two explanations of this phenomenon are readily apparent. Thus, for example, DPN + and TPN+ might be labeled from ATP pools with different specific activities. Alternatively DPN + and TPN + might be labeled from a common ATP pool but the pool size of DPN + might be considerably greater than that of TPN +. In such circumstances the 3,p incorporated into the two phosphate groups of DPN + might be considerably diluted whereas the 3~p incorporated into the third phosphate group of TPN + would be less so. As a result the specific activities of TPN + would be greater than those of DPN +. Since the DPN + pool size in dog thyroid slices9 has been established to be approximately one order of magnitude greater than that of TPN+, the latter explanation would adequately explain the data.
o7
S
t--
•~
°
*~o~O~7,
°~2-~°-"~° 0
20
40
• 60
MINUTES
F i g . 3. A t i m e s t u d y o f t h e i n c o r p o r a t i o n (0--0) of dog thyroid slices. Specific pyridine nucleotide.
TABLE
of [3~PJorthophosphate activities are expressed
into DPN + (©--O) as counts/min per
and TPN + #/,mole of
II
I N F L U E N C E OF T S H A N D C H O L I N E R G I C AGENTS ON T P N + DOG T H Y R O I D SLICES
T I T E R S AND [14C]GLUCOSE O X I D A T I O N I N
F~xpt. No.
Hormone
[l*C]glucose (o.ox M)
TPN + (mpmoles/g)
14C02 (counts/rain)
I
Control TSH Acetylcholine
----
3.2 12. 5 I3.6
----
2
Control TSH Carbamylcholine
----
8.0 19.o 20.8
----
3*
Control TSH
+ +
---
14 o 5 8 41 0 8 0
4" *
Control TSH
+ +
---
29 260 47 034
* Krebs-Ringer ** K r e b s - R i n g e r
phosphate buffer. bicarbonate buffer.
Biochim. Biophys. Acta,
141 ( 1 9 6 7 ) 8 - 1 8
14
F. R. B U T C H E R ,
G. S. S E R I F
Since the existence of a DPN kinase is compatible with the above results it remains to establish whether T P N formation is stimulated by hormones, and if possible to determine whether the stimulatory effect is at a DPN kinase step. Table I I I contains data from experiments attempting to resolve these points. Thus specific activities were determined for DPN+ and T P N + in control tissues and tissues treated with TSH, acetylcholine, or carbamylcholine. I t is apparent that the specific activities of DPN+h (hormone treated) are for the most part slightly elevated as compared to DPNc + (control). Surprisingly, however, the specific activities for TPNh + are seen to TABLE
III
INFLUENCE OF T S H AND CHOLINERGIC AGENTS ON I32p]ORTHOPHOSPHATI~ INCORPORATION INTO D P N + AND T P N + OF DOG THYROID
Expt. No.
Treatment
Stimulation of D P N + T P N + levels (counts/min (%) . per iq~mole)
TPN + (counts]min per t~l~mole)
TPNh+/DPNn +* TPNc+/DPNc+ i probable error**
I
Control TSH Carbamylcholine
-208
1.26 1.35 1.35
12.5 II.I 9.6
-0.86 i 0.06 o.71 ± 0 . 0 7
2
Control TSH
-N***
1.57 1.64
11.6 lO.2
-0.84 ± 0.07
3
Control Carbamylcholine
-44
1.14 § 1-14 §
13. 5 12.9
-0.96 ~ 0.o9
4
Control Carbamylcholine
-N***
1.25 1.59
18.8 13.6
-0.57 -- 0.08
158
* S u b s c r i p t s c a n d h r e f e r t o c o n t r o l a n d h o r m o n e - t r e a t e d slices, r e s p e c t i v e l y . ** C o m p o s i t e p r o b a b l e e r r o r d e r i v e d f r o m t h e s u m m a t i o n of t h e i n d i v i d u a l p r o b a b l e e r r o r s o f each specific activity involved in a given ratio. *** N o t m e a s u r e d d u e t o i n a d e q u a t e q u a n t i t i e s o f t i s s u e . § S p e c i f i c a c t i v i t i e s b a s e d o n a s i n g l e d e t e r m i n a t i o n . T h e m a x i m u m p r o b a b l e e r r o r is a s s u m e d . A l l o t h e r s p e c i f i c a c t i v i t y f i g u r e s a r e a v e r a g e s of t w o o r m o r e c l o s e l y a g r e e i n g d e t e r m i n a t i o n s .
be generally lower than the specific activities for TPNc +. Since hormone action does appear to have some small effect on the overall synthesis of pyridine nucleotides de novo as represented b y the specific activities of DPN +, it is not adequate to compare simple changes in specific activities of T P N + and conclude that such changes represent an effect on the DPN + kinase step. Rather it is necessary to eliminate the influence of hormones on synthesis of pyridine nucleotides de novo prior to the DPN + to T P N + step by comparing the ratios of T P N + to DPN + in hormone-treated and control thyroid slices. Thus, if for the expression TPN+h/DPNh + TPN+c/DPNe +
an experimental value greater than one were obtained a specific stimulation of the DPN + kinase step would be indicated. Alternatively, if experimental values of one or less were obtained, no effect or inhibiting effects of hormone, respectively, would be deduced. As a first approximation these ratios will hold if hormone induced Bioehim. Biophys. Acta, 141 (1967) 8 - 1 8
P Y R I D I N E NUCLEOTIDES
15
changes in specific activities of DPN + are small. Since such changes are seen to be small the ratios given in Table I I I m a y be used to weigh the influence of the various hormones on the D P N + kinase step. I t is apparent that the greater number of these ratios are one or less than one. Thus, under the experimental conditions, these data might be interpreted to signify the absence of a stimulatory hormonal effect on T P N + formation 22.
[3~PJOrthophosphate studies with preimubation It is conceivable that the response to hormonal agents m a y be of short duration. In such an event stimulatory effects on TPN + formation m a y be induced before the precursor pool of DPN + is appreciably labeled with 32p. Thus increased incorporation of radioactivity might be masked despite an actual stimulation of T P N + formation. In order to test this possibility, dog thyroid slices were incubated with [a~P]orthophosphate for 3o-min periods followed by a 3o-min exposure to the appropriate hormone. D a t a from these studies are presented in Table IV. In each instance, the ratio values are now seen to be considerably in excess of one. Clearly, under these conditions, stimulation of asP-labeled T P N + formation is occurring as a consequence of hormone action. If the original postulate for the preincubation experiment is correct, these data would imply that the stimulatory influence of these hormones is maximally effected, or apparent, over a limited time interval.
[14C~Nicotinic acid studies with j~reincubation It is possible to explain the 3zp data as a consequence of a hormonally induced heightened turnover of 3~p in ATP without an actual increase in T P N + formation. Thus it was considered advisable to repeat crucial experiments with a more immeT A B L E IV INFLUENCE OF T S H AND CARBAMYLCHOLINEON [32P]ORTHOPHOSPHATE INCORPORATION INTO D P N + AND T P N + IN DOG THYROID WITH PREINCUBATION T h e e x p e r i m e n t a l p r o t o c o l (see METHODS) w a s followed w i t h e x c e p t i o n t h a t a 3o-min p r e i n c u b a t i o n of t h e slices w i t h t h e m e d i u m c o n t a i n i n g [3~P]orthophosphate w a s used. A t t h e e n d of t h i s p e r i o d h o r m o n e w a s i n j e c t e d t h r o u g h t h e i n c u b a t i o n flask s t o p p e r a n d i n c u b a t i o n c o n t i n u e d for a t e r m i n a l 3o-min period.
Expt. Treatment No.
3
Stimulation of D P N + T P N + levels (counts]rain (%) per iH~mole)
TPN + (counts]rain per ttttmole)
TPNh+/DPNh + TPNe+/DPNe+ d- probable error*
Control TSH Carbamylcholine
-119 144
0.87 0.84 0.94
I 1.3 I7.8 15.7
-1.6 2_ 0.2 1. 3 4- o.I
Co ntro l TSH Carbamylcholine
-28 I16
0.93 0.87 0.53
5-4 7.5 7.6
-1. 5 _a 0.2 2.5 + 0.3
Control Carbamylcholine
-76
0.85 0.64
4.4 7.4
-2.2 4- 0.2
* C o m p o s i t e p r o b a b l e error d e r i v e d from t h e s u m m a t i o n of t h e i n d i v i d u a l p r o b a b l e e rrors of each specific a c t i v i t y i n v o l v e d in a g i v e n ratio. E a c h specific a c t i v i t y figure is a n a v e r a g e of t w o or m o r e closely a g r e e i n g d e t e r m i n a t i o n s .
Biochim. Biophys. Acta, 141 (1967) 8-18
16
F . R . BUTCHEI~, G. S. SERIF
diate and specific precursor. Additional experiments were carried out with [carboxyl14C]nicotinic acid using the identical isolation and assay procedure employed on the 3~p studies. Nicotinic acid was chosen instead of nicotinamide since the incorporation of the former derivative into DPN + and TPN + appears to reflect synthesis of these nucleotides de novo ~3. The influence of TSH and carbamylcholine upon [14Clnicotinic acid incorporation into pyridine nucleotides is shown in Table V. Consideration of the ratio values indicates that these data are consistent with those obtained from 32p studies and clearly demonstrate a stimulation of TPN + formation in the presence of TSH and carbamylcholine. Since an examination of the specific activities of DPN + in Table V reveals little hormone influence upon DPN + formation, these data might be interpreted to suggest that the stimulation of TPN + formation occurs at the DPN + TABLE V INFLUENCE AND TPN
OF
TSH
A N D C A R B A M Y L C H O L I N E ON [ 1 4 C ] N I C O T I N I C ACID I N C O R P O R A T I O N
+ IN DOG THYROID
INTO
DPN +
WITH PREINCUBATION
The experimental protocol of the METHODS section was followed with the exception t h a t a 3o-min p r e i n c u b a t i o n of the slices with the m e d i u m containing [14C]nitotinic acid was used. At the end of this period h o r m o n e was injected t h r o u g h the incubation flask s t o p p e r and incubation continued for a terminal 3o-min period.
Expt. Treatment No.
Stimulation of D P N + T P N + levels (counts/min (%) per ##mole)
TPN + (counts/min per tv2mole)
TPNh+/DPNh + TPNc+/DPNc+ ± probable error*
Control TSH Carbamylcholine
-lO 9 69
6.4 6.1 6.4
3.4 5.6 5.8
-1.8 ± o.2 1. 7 ~ o.2
Control TSH Carbamylcholine
-12o lO 3
6.8 6.6 7.2
6.6 9.7 9.8
-1.5 ± o.i 1. 4 ± o.i
* Composite probable error derived from the s u m m a t i o n of the individual probable errors of each specific activity involved in a given ratio. E a c h specific activity figure is an average of two or more closely agreeing determinations.
/
//
// /0
'~
i
0
o/
/
12
n
20
o/
o --
i
i
i
40
60
80
MINUTES
Fig. 4- A time s t u d y of the incorporation of [14Clnicotinic acid into D P N + ( [] ) and T P N + ( O ) in the presence (closed points) and absence (open points) of TSH. Specific activities are expressed as c o u n t s / m i n per # # m o l e of pyridine nucleotide. Arrows indicate T S H addition.
Biochim. Biophys. Acta, 141 (1967) 8 - I 8
17
PYRIDINE NUCLEOTIDES
kinase step. I t is thought provoking, however, to examine the specific activities of T P N + in Expt. 2 of Table V as compared to the corresponding DPN + values. In the presence of T S H and carbamylcholine both T P N + specific activities exceed those of DPN +. This phenomenon was examined further as a function of time. Figs. 4 and 5 depict time studies showing the influence of T S H and carbamylcholine upon DPN + and TPN+ specific activities. Again, a hormone-induced elevation of tile rate of incorporation of radioactivity into T P N + is obvious, with little apparent hormonal influence upon DPN+ formation. In addition, with extended time, the increase in T P N + specific activities above and beyond those of DPN + become consistent and pronounced. This departure from precursor-product relationships m a y be explained through the thesis, (a) that DPN+ is not a direct precursor of TPN + or (b) that a pool dilution effect has occurred. With the latter explanation it is necessary to propose that precursor DPN + and TPN+ are synthesized at a common restricted site removed from the major nucleotide pools, in order to prevent DPN+ dilution prior to its conversion to T P N +. Thus, as precursor DPN + of high specific activity is formed, it is converted to a somewhat lower, but nonetheless high specific activity of T P N +.
/
/ 24
e//
20
O/
//
~.
/
~ 0
i
= 20
i
i 40
i
i 60
i
i 80
MINUTES
Fig. 5. A time study of the incorporation of E14C~nicotinic acid into D P N + [D) and T P N + (O) in the presence (closed points) and absence (open points) of carbamylcholine. Specific activities are expressed as counts/min per /~#mole of pyridine nucleotide. Arrows indicate carbamylcholine addition.
Release of these nucleotides to the main pool, by normal cell metabolism or extraction techniques, then permits more extensive dilution of DPN + than T P N + and hence higher TPN+ specific activity values. Because of this potential interpretation, care must be exercised in the assessment that stimulation of DPN+ formation is not occurring. Expressed as specific activity, the average stimulatory response observed with T P N + is of the order of 60 To. Since the total DPN + pool is approx. Io-fold that of the T P N + pool in dog thyroid, a similar response with respect to an increased DPN + formation might be reflected, through dilution, as an increase in DPN + specific activity of approx. 6%. Under conditions of preincubation the average observed influence of hormones on DPN + synthesis is reflected as a decrease in specific activity of 8 %. The range of values encountered encompasses a possible 6 % increase. Thus no statistical significance can be applied to the difference between these two numbers. Support for a lack of stimulatory effect on DPN + formation, however, is available Biochim. Biophys. Acta, 141 (1967) 8-18
I8
F . R . BUTCHER, G. S. SERIF
from the observation that DPN + and D P N H levels decrease in thyroid slices as a response to TSH and cholinergic agents 9. It is improbable that a stimulation of DPN + formation would be propagated through DPN + to T P N + under conditions of net DPN + decrease. In summation, the data of these studies clearly demonstrate a stimulation of labeled T P N + formation when dog thyroid slices are preincubated with [32P~orthophosphate or [14Clnicotinic acid and exposed to TSH or cholinergic agents. Thus increased titers of TPN+, as a response to these reagents, m a y be accounted for through enhanced synthesis of TPN+ de novo. The apparent lack of a concomitant increase in labeled DPN+ formation, taken in conjunction with the fact that DPN + levels are depressed by T S H and cholinergic agents 9 is suggestive of a hormoneinduced activation of a parameter associated with DPN + kinase. However, no absolute assignment of such an activation is permissible because of the possibility of interfering pool dilution effects. ACKNOWLEDGEMENTS
This investigation was supported in part b y research grant AM-II43O from the Division of Arthritis and Metabolic Diseases of the U.S. Public Health Service, and GB-42I 7 from the National Science Foundation.
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Biochim. Biophys. Acta, 141 (1967) 8-18