Biochimica et Biophysica Acta, 542 (1978) 1--11 © Elsevier/North-Holland Biomedical Press
BBA 28571 IDENTIFICATION OF THE REGULATORY STEPS IN GLUCONEOGENESIS IN COTYLEDONS OF C U C U R B I T A PEPO
R.C. LEEGOOD and T. AP REES Botany School, University of Cambridge, Cambridge CB2 3EA (U.K.)
(Received January 23rd, 1978)
Summary 1. The aim of this work was to discover the steps at which the conversion of oxaloacetate to glucose 6-phosphate during gluconeogenesis is regulated in the cotyledons of 5-day-old seedlings of Cucurbita p e p o . 2. We estimated the m a x i m u m catalytic activities of all the enzymes in the above sequence and also the amounts of their substrates present in vivo. The results show that the reactions catalysed by fructose-l,6-bisphosphatase and phosphoenolpyruvate carboxykinase are the only ones in the sequence that are substantially displaced from equilibrium in vivo. 3. We also determined the effects of 3-mercaptopicolinic acid, an inhibitor of gluconeogenesis, on the amounts of the gluconeogenic intermediates present in vivo. The results show that the enzyme system, fructose-l,6-bisphosphatase: phosphofructokinase, and the system phosphoenolpyruvate carboxykinase: phosphoenolpyruvate carboxylase make major contributions to the regulation of gluconeogenesis in the cotyledons. 4. Possible mechanisms for the above regulation are discussed.
Introduction Gluconeogenesis during the germination of f a t t y seedlings involves the conversion of considerable quantities of oxaloacetate to glucose 6-phosphate. Available evidence indicates t h a t this occurs in the soluble phase of the cytoplasm in the presence of relatively small amounts of phosphofructokinase and appreciable amounts of pyruvate kinase [1--3]. The manner in which interaction between gluconeogenesis and glycolysis is controlled during germination is n o t known. Kobr and Beevers [4] identified phosphofructokinase and pyruvate kinase as major points of control of glycolysis during gluconeogenesis in castor bean endosperm, but we have n o t direct knowledge of the fine control of the
conversion of oxaloacetate to glucose 6-phosphate. The aim of the prese~t work has been to discover where the latter sequence is regulated. We chose the cotyledons of 5-day-old seedlings of marrow as our experimental material: such cotyledons are at their peak of gluconeogenesis [5]. The rationale of the experimental approach which we used has been discussed in detail by Newsholme and Start [6]. First, we determined which reactions in the sequence from oxaloacetate to glucose 6-phosphate are appreciably displaced from equilibrium in vivo. An indication of which reactions are far from equilibrium is given by comparison of the maximum catalytic activities of the enzymes in the sequence. More definitive evidence is provided by comparison of the apparent equilibrium constants of the enzymes with the relative amounts of their substrates and products present in vivo [6]. Thus we measured the enzyme activities and the amounts of the intermediates in the cotyledons. We then investigated whether any reaction, found to be far from equilibrium, was regulatory. Proof that a non-equilibrium reaction is regulatory can be provided by the demonstration that, in vivo, its substrate changes in the direction opposite to that of the flux when the latter is varied. We used 3-mercaptopicolinic acid to vary the rate of gluconeogenesis. We have shown previously that treatment of thc cotyledons of 5-day-old marrows with this c o m p o u n d severely reduces the conversion of acetate units to sugars, and that it does so by inhibiting phosphoenolpyruvate carboxykinase [3]. Thus we determined the effects of this inhibitor on the amounts of the intermediates of gluconeogenesis in the cotyledons. The measurement of phosphorylated intermediates in plant tissues calls for particular care, and for evidence that such measurements represent the amounts of the compounds present in vivo [7]. We obtained this evidence from the following type of recovery experiment. For each test of the technique we prepared duplicate samples of cotyledons, freeze-clamped them and killed them with cold HC104. Measured amounts of the intermediates were added to one of the samples immediately after freeze-clamping and just before the HC104 was added. The amounts of intermediates added were comparable to those present in the sample of cotyledons. Comparison of the amounts of the intermediates recovered from the two samples is taken as a measure of the reliability of the complete process of killing, extraction and measurement. Materials and Methods Materials 3-Mercaptopicolinic acid was a gift from Dr. H.L. Saunders, Smith Kline and French Laboratories, Philadelphia, Pa. Substrates, cofactors and enzymes were obtained from Boehringer Mannheim except that 3-phosphoglyceraldehyde and glutamic-oxaloacetic transaminase came from the Sigma Chemical Company. Marrows (Cucurbita pepo L. var. medullosa Alef.) were grown and harvested as described previously except that they were grown in continuous light [5]. E n z y m e assays Freshly harvested cotyledons, fresh weight 2 g , were homogenized as described previously [5] in 40 mM glycylglycine buffer (pH 7.4) that contained
10 mM EDTA and 10 mM dithiothreitol. The homogenate was centrifuged at 105 000 X g for 30 min and the supernatant was assayed for enzyme activity at once. Enzymes were measured at 25 ° C, as indicated in the following references, in reaction mixtures of 3.0 ml. These mixtures contained: glucosephosphate isomerase [8], 3 mM fructose 6-phosphate, 0.2 mM NADP ÷, 5 mM MgC12 and 3.5 units glucose-6-phosphate dehydrogenase in 50 mM Tris-HC1, pH 8.1; fructose-l,6-bisphosphate aldolase [9], 3.4 mM fructose 1,6-bisphosphate, 0.35 mM NADH, 20 pg of a mixture of a-glycerolphosphate dehydrogenase and triosephosphate isomerase in 40 mM glycylglycine, pH 7.7; triosephosphate isomerase [10], 0.48 mM 3-phosphoglyceraldehyde, 0.25 mM NADH and 1.2 units a-glycerolphosphate dehydrogenase in 40 mM glycylglycine buffer, pH 7.9; phosphoglycerate kinase [11], 15 mM 3-phosphoglycerate, 1 mM ATP, 0.25 mM NADH, 5 mM MgC12 and 4 units glyceraldehydephosphate dehydrogenase in 40 mM 2-(N-morpholino)ethanesulphonic acid, pH 6.9; lactate dehydrogenase [12], 0.33 mM pyruvate and 0.1 mM NADH in 40 mM glycylglycine, pH 7.4. NADP-specific malic enzyme was assayed as described by Johnson and Hatch [13] except that malate was 5 mM and the buffer was 40 mM glycylglycine (pH 7.5). For phosphate, pyruvate dikinase the extracts were prepared according to Hatch and Slack [14] and assayed as described by Hatch and Slack [15] except that 5 mM 2-mercaptoethanol replaced the dithiothreitol, and the phosphoenolpyruvate carboxylase (2 units per assay) came from Boehringer. For assay of phosphoenolpyruvate carboxylase the extract was desalted on a column (1 × 15 cm) of Sephade~ G-25 and assayed as described by Wong and Davies [16] in a reaction mixture that contained: 2 mM phosphoenolpyruvate, 7.5 mM MgC12, 2.5 mM KHCO3, 0.35 mM NADH, 2 pCi NaH14CO3 (spec. act. 58.5 mCi/mmol) in 40 mM glycylglycine, pH 7.9. Measu remen ts o f su bstra tes Measurements for whole cotyledons were made on samples of 1 g fresh weight which were prepared and killed within 5 min of harvest. For t r e a t m e n t with 3-mercaptopicolinic acid cotyledons were excised and their upper and lower surfaces were cut away with a razor blade. This left central portions of the cotyledons (each approx. 0.5 mm thick and approx. 25 mg fresh weight) t h a t were used to prepare replicate samples of 500--700 mg fresh weight. Each sample was placed on a filter paper (Whatman No. 1, 5 cm diameter) in a Petri dish. For treatment with the inhibitor the filter paper had been moistened with 0.5 ml 0.02 M KH2PO4 (pH 5.2) which contained 2 mM 3-mercaptopicolinic acid; the same solution was also used to moisten the uppermost cut surfaces of the cotyledons. The samples were then incubated in the light at 25°C (the conditions under which the seedlings had been grown) for 150 min before being taken for analysis. The control samples were treated similarly except that the 3-mercaptopicolinic acid was omitted from the 0.02 M KH2PO4. We adopted this procedure because the oxygen uptake of marrow cotyledons is so high in relation to the diffusion coefficient of oxygen in water t h a t immersion of the cotyledons in aqueous solution leads to anaerobiosis and some diminution of gluconeogenesis [ 3]. All samples of tissue were freeze-clamped as described previously [7]. The freeze-clamped material was dropped into liquid nitrogen at once and then
fragmented with a glass rod. Before the nitrogen had evaporated 3.0 ml 1.41 M HC104 at 4°C was added to the above mixture. The frozen mass which resulted was transferred to ....5°C and kept at this temperature for 18 h except for measurement of oxaloacetate when the time was reduced to 3 h. Next, the suspension that had formed during the time at --5°C was centrifuged at 27 000 × g for 2 min, and the sediment that formed was extracted thrice, each time with 1 ml 1.41 M HC104. All the extracts were combined, neutralized with 5 M K2CO3, and then centrifuged as above. The sediment was washed twice, each time with 1.0 ml 1.41 M HC104 which had been neutralized with 5 M K2CO3. During the above manipulations the temperature of the extract did not exceed 2°C. For measurement of oxaloacetate the HC104 was neutralized, as described by Lowry and Passonneau [17], with a mixture that was 2.0 M, 0.4 M and 0.4 M with respect to KOH, imidazole and KC1, respectively. In all instances the neutralized extracts were assayed at once by the spectrophotometric techniques described by Lowry and Passonneau [17]. The sum of the amounts of d i h y d r o x y a c e t o n e phosphate and 3-phosphoglyceraldehyde was determined by Method II from a single measurement made in the presence of triosephosphate isomerase and 3-phosphoglyceraldehyde dehydrogenase. 3-Phosphoglycerate was determined by Method I. For the measurement of aspartate the reaction mixture was altered so that it contained: 0.47 mM NADH, 0.26 mM a-ketoglutarate, 120 units malate dehydrogenase and 18.6 units glutamic oxaloacetic transaminase in 50 mM potassium phosphate buffer, pH 7.2. Gas exchange of the cotyledons was measured by Warburg's direct manometric method. Fisher's P values were calculated by Student's t-test. Results and Discussion
Enzyme activities We already know the m a x i m u m catalytic activities of some of the enzymes of gluconeogenesis in the cotyledons of 5-day-old marrows [1]. We assayed the outstanding enzymes and the complete list of activities is given in Table I. In the present work, except in the case of lactate dehydrogenase and phosphate, pyruvate dikinase, we optimized each assay by varying the concentration of each c o m p o n e n t of the assay mixture and by varying the pH. We investigated whether loss or activation of the gluconeogenic enzymes occurred during the preparation of the extracts. For each test we prepared duplicate samples of cotyledons; we extracted one sample in the usual way and the other in buffer which contained measured amounts of pure enzymes. The amounts of pure enzymes added were comparable to the activities found in the cotyledons. Comparison of the activities found in the extracts of the two samples showed the extent to which the pure enzymes had survived the processes of homogenization and extraction. Our estimates of the recovery of 3-phosphoglycerate kinase, fructose-l,6-bisphosphate aldolase, and glucosephosphate isomerase were 126%, 89% and 109%, respectively, of the amounts added. The previously published data in Table I are also supported by optimization and recovery experiments. Therefore we suggest that the values for the enzymes of gluconeogenesis shown in Table I reflect the m a x i m u m catalytic activities of the cotyledons.
TABLE I A C T I V I T I E S OF ENZYMES OF G L U C O N E O G E N E S I S IN C O T Y L E D O N S OF 5-DAY-OLD S E E D L I N G S OF MARROW V a l u e s m a r k e d * are f r o m ap R e e s e t al. [1] a n d t h a t f o r phosphoenolpyruvate c a r b o x y k i n a s e is f r o m L e e g o o d a n d ap R e e s [ 3 ] ; for t h e r e m a i n d e r , s a m p l e s o f c o t y l e d o n s w e r e e x t r a c t e d a n d a s s a y e d as d e s c r i b e d in t h e t e x t . V a l u e s are m e a n s +- S.E. T h e n u m b e r o f s a m p l e s a s s a y e d is s h o w n in p a r e n t h e s e s . R e l a t i v e a c t i v i t i e s are b a s e d o n a value o f 1 / ~ m o l / m i n p e r g for f r u c t o s e - l , 6 - b i s p h o s p h a t a s e ; t h e d a t a for liver are averages o f m e a s u r e m e n t s f r o m a r a n g e o f v e r t e b r a t e s a n d w e r e o b t a i n e d f r o m N e w s h o l m e a n d Start [ 6 ] . Enzyme
G l u c o s e p h o s p h a t e i s o m e r a s e (EC 2 . 7 . 1 . 1 ) F r u c t o s e - l , 6 - b i s p h o s p h a t a s e (EC 3 . 1 . 3 . 1 1 ) F r u c t o s e - l , 6 - b i s p h o s p h a t e aldolase (EC 4 . 1 . 2 . 7 ) T r i o s e p h o s p h a t e i s o m e r a s e (EC 5 . 3 . 1 . 1 ) NAD-specific glyceraldehydephosphate dehydrogenase (EC 1 . 2 . 1 . 1 2 ) P h o s p h o g l y c e r a t e k i n a s e (EC 2 . 7 . 2 . 3 ) P h o s p h o g l y c e r o m u t a s e (EC 2 . 7 . 5 . 3 ) E n o l a s e (EC 4 . 2 . 1 . 1 1 ) PhosphoenolPyruvate c a x b o x y k i n a s e (EC 4 . 1 . 1 . 4 9 ) Phosphoenotpyruvate c a x b o x y l a s e (EC 4 . 1 . 1 . 3 1 ) P y r u v a t e k i n a s e (EC 2 . 7 . 1 . 4 0 ) P h o s p h o f r u c t o k i n a s e (EC 2 . 7 . 1 . 1 1 ) N A D P - s p e c i f i c m a l i c e n z y m e (EC 1 . 1 . 1 . 3 9 ) P h o s p h a t e , p y t u v a t e d i k i n a s e (EC 2 . 7 . 9 . 1 ) L a c t a t e d e h y d r o g e n a s e (EC 1 . 1 . 1 . 2 7 )
A c t i v i t y in c o t y l e d o n s (#mol substrate consumed ]min per g fresh w t . )
Cotyledons
Liver
17.15 3.21 1.69 425.1
5.3 1 0.5 132
14" 1 0.3 --
4.6 14.4
7.5 6.5
± 1.11 -+ 0 . 2 4 + 0.05 +_ 27.1
(4) (6) * (4)
1 4 . 7 5 + 2.97 46.20 + 2.25 1 9 . 7 8 -+ 3 . 1 4 2 0 . 7 5 -+ 2.54 7.68 + 0.31 0 . 3 2 -+ 0 . 0 2 9 . 7 4 + 1.24 0 . 0 9 t 0.03 1 . 1 4 -+ 0 . 0 6 None detected None detected
(6) * (4)
(3)
Relative activity
(6) *
6.2
--
(6) * (6)
6.5 2.4
2.7 0.7
(4)
0.1
--
(6) *
3.0
1.9
(6) *
0.03
0.3
(3)
--
-
-
Examination of Table I shows that the gluconeogenic enzymes with the lowest activities are phosphoenolpyruvate carboxykinase, aldolase, and fructose-l,6-bisphosphatase. This suggests that possibly these three reactions are appreciably displaced from equilibrium in vivo. The activities of enolase, phosphoglyceromutase, glyceraldehydephosphate dehydrogenase and glucosephosphate isomerase are all comparable, appreciably higher than those of the first mentioned enzymes, and much lower than that of triosephosphate isomerase. The activities of the other enzymes in Table I are given as indicators of possible alternative routes of metabolism of gluconeogenic intermediates. Our inability to detect phosphate, pyruvate dikinase accords with the considerable evidence that there is no direct route from pyruvate to sugar in such tissues [181. Data comparable to those in Table I are not available for any other plant tissue. Kagawa et al. [19] report the activities of pyruvate kinase, phosphoglyceromutase, enolase, phosphoglycerate kinase, glyceraldehydephosphate dehydrogenase, triosephosphate isomerase and aldolase for cotyledons of 4-day-old water melon seedlings. Their values, when expressed per g fresh weight, are very similar to those in Table I. More extensive data are available for animal tissues [6]. This permits comparison of the relative activities of the enzymes in marrow cotyledons with those of liver (Table I). The comparison reveals a remarkable similarity between the two. The only appreciable differences are
that the relative activity of phosphoenolpyruvate carboxykinase is higher, and that o f p h o s p h o f r u c t o k i n a s e lower, in marrows.
Measurements of intermediates The recoveries o f the c o m p o u n d s added to the freeze-clamped cotyledons (Table II) show that there was significant loss of AMP, and some loss of triosephosphate during extraction and analysis. The recoveries of all the other compounds were satisfactory. We argue that freeze-clamping ruptures the tissue and stops significant biochemical and chemical change. Thus the c o m p o u n d s we added in our recovery experiments would have been subjected to the same e n v i ro n men t as the endogenous c o m p o u n d s during the killing and extraction of the cotyledons. The high recoveries and the consistency of our data provide sound evidence that, AMP excepted, our estimates represent the amounts of gluconeogenic intermediates which were present in vivo. When expressed per g fresh weight these a m o u n t s are appreciably higher than those report ed for plant cells in general [20] and significantly higher than those report ed for castor bean endosperm [4], the only ot her gluconeogenic plant tissue for which data are available. We attribute these high values to the high metabolic activity of the c o t y l e d o n s and to the dense packing of the cells and their contents [1]. Table III compares the apparent equilibrium constants of the enzymes of gluconeogenesis with the relative am ount s of some of their substrates and pr o d u c t s present in vivo. For some of the reactions the latter constitute the mass-action ratios. It is clear that the reactions catalysed by enolase, phosphoglyceromutase and glucosephosphate isomerase are very close to equilibrium in vivo. Thus we suggest that none of these enzymes plays a d o m i n a n t direct role in the regulation of gluconeogenesis in marrows. The aldolase reaction is n o t very far from equilibrium and our results provide no firm ground for believing th at this step is regulatory. Determination of the mass-action ratios for the remaining reactions of gluconeogenesis is made difficult by the fact that some of the reactants are almost certainly unequally distributed between the different c o m p a r t m e n t s of the cells of the cotyledons. For the reaction catalysed by fructose-l,6-bisphosphatase it is possible to use the apparent equilibrium constant, and our values for fructose 1,6-bisphosphate and fructose 6-phosphate, to calculate the a m o u n t of o r t h o p h o s p h a t e which would have to be present in 1 g of c o t y l e d o n s if the reaction were at equilibrium in vivo. Values of 20.1, 14.3 and 56.6 m m o l of phosphate per g c o t y l e d o n are obtained for intact, cut and treated cotyledons, respectively. The likelihood that such amounts of inorganic phosphate are present in the cytoplasm of marrow cotyledons is so r e m o t e [24] that we may conclude that the reaction catalysed by fructose-l,6-bisphosphatase is some considerable distance from equilibrium in these cotyledons. It is more difficult to assess the state of the reaction catalysed by phosphoenolpyruvate carboxykinase. In order to do so, we have assumed t hat the concentration of bicarbonate in the cytoplasm is 0.4 mM (roughly equivalent to the c o n c e n t r a t i o n o f COs in air) [23]. By using our estimates of oxaloacetate and phosphoenolpyruvate, we have calculated the ratio ATP : ADP that would exist in the cytoplasm of the c o t y l e d o n s if the phosphoenolpyruvate carboxykinase reaction was at equilibrium. The values obtained are 7 . 4 . 1 0 -3 , 5 . 4 . 1 0 -3 ,
OF
GLUCONEOGENESIS
IN COTYLEDONS
OF
5-DAY-OLD
MARROW
SEEDLINGS,
AND
EFFECTS
THEREON
OF
3-MERCAPTO-
phosphate
96 90 109 70 100 103 93 104 95 93
Glucose 6-phosphate Fructose 6-phosphate Fructose 1,6-bisphosphate 3-Phosphoglyceraldehyde + dihydroxyacetone 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Oxaloacetate Pyruvate Malate 106 + 10 100-+ 12 118 + 8 4 7 +_ 5
Aspartate ATP
ADP AMP
+ 10 + 10 ± 13 -+ 3 ± 6 + 10 ± 3 ± 17 ± 1 ± 6
Recovery (%)
Compound
n.s. ( n o t s i g n i f i c a n t ) .
(9)
(5) (9) (9)
36 2 (5)
± 45 + 21 ± 8 ± 6 + 56 + 7 + 8 1 3 + "11 + 344 + 88 ± 21
234 ± 32 ±
2113 467 54 46 235 37 111 10 91 4998 2281 447
1495 376 31 38 148 37 65 8 88 2796 1918 356 258 48
± 89 +- 1 8 + 5 _* 6 + 18 t 12 + 6 + 1 + 14 z 304 + 281 ± 28 *_ 4 1 ± 6
II Cut cotyledons
(nmol per g fresh wt.)
Whole cotyledons
I
Amount
(5) (5)
(9)
(5) (5)
712 169 55 49 91 8 28 15 89 8127 1780 488 281 62
± 102 -+ 1 4 + 8 ± 9 -~ 2 5 ~ 1 (5) -+ 5 (5) + 2 - 16 (8) ± 837 + 200 ± 56 * 4 0 (5) ± 15(5)
III Cut cotyledons in 3-mercaptopicolinic acid
n.s. 40.001 n.s.
n.s. n.s. n.s.
n.s.
BBA 28571 IDENTIFICATION OF THE REGULATORY STEPS IN GLUCONEOGENESIS IN COTYLEDONS OF C U C U R B I T A PEPO
R.C. LEEGOOD and T. AP REES Botany School, University of Cambridge, Cambridge CB2 3EA (U.K.)
(Received January 23rd, 1978)
Summary 1. The aim of this work was to discover the steps at which the conversion of oxaloacetate to glucose 6-phosphate during gluconeogenesis is regulated in the cotyledons of 5-day-old seedlings of Cucurbita p e p o . 2. We estimated the m a x i m u m catalytic activities of all the enzymes in the above sequence and also the amounts of their substrates present in vivo. The results show that the reactions catalysed by fructose-l,6-bisphosphatase and phosphoenolpyruvate carboxykinase are the only ones in the sequence that are substantially displaced from equilibrium in vivo. 3. We also determined the effects of 3-mercaptopicolinic acid, an inhibitor of gluconeogenesis, on the amounts of the gluconeogenic intermediates present in vivo. The results show that the enzyme system, fructose-l,6-bisphosphatase: phosphofructokinase, and the system phosphoenolpyruvate carboxykinase: phosphoenolpyruvate carboxylase make major contributions to the regulation of gluconeogenesis in the cotyledons. 4. Possible mechanisms for the above regulation are discussed.
Introduction Gluconeogenesis during the germination of f a t t y seedlings involves the conversion of considerable quantities of oxaloacetate to glucose 6-phosphate. Available evidence indicates t h a t this occurs in the soluble phase of the cytoplasm in the presence of relatively small amounts of phosphofructokinase and appreciable amounts of pyruvate kinase [1--3]. The manner in which interaction between gluconeogenesis and glycolysis is controlled during germination is n o t known. Kobr and Beevers [4] identified phosphofructokinase and pyruvate kinase as major points of control of glycolysis during gluconeogenesis in castor bean endosperm, but we have n o t direct knowledge of the fine control of the
conversion of oxaloacetate to glucose 6-phosphate. The aim of the prese~t work has been to discover where the latter sequence is regulated. We chose the cotyledons of 5-day-old seedlings of marrow as our experimental material: such cotyledons are at their peak of gluconeogenesis [5]. The rationale of the experimental approach which we used has been discussed in detail by Newsholme and Start [6]. First, we determined which reactions in the sequence from oxaloacetate to glucose 6-phosphate are appreciably displaced from equilibrium in vivo. An indication of which reactions are far from equilibrium is given by comparison of the maximum catalytic activities of the enzymes in the sequence. More definitive evidence is provided by comparison of the apparent equilibrium constants of the enzymes with the relative amounts of their substrates and products present in vivo [6]. Thus we measured the enzyme activities and the amounts of the intermediates in the cotyledons. We then investigated whether any reaction, found to be far from equilibrium, was regulatory. Proof that a non-equilibrium reaction is regulatory can be provided by the demonstration that, in vivo, its substrate changes in the direction opposite to that of the flux when the latter is varied. We used 3-mercaptopicolinic acid to vary the rate of gluconeogenesis. We have shown previously that treatment of thc cotyledons of 5-day-old marrows with this c o m p o u n d severely reduces the conversion of acetate units to sugars, and that it does so by inhibiting phosphoenolpyruvate carboxykinase [3]. Thus we determined the effects of this inhibitor on the amounts of the intermediates of gluconeogenesis in the cotyledons. The measurement of phosphorylated intermediates in plant tissues calls for particular care, and for evidence that such measurements represent the amounts of the compounds present in vivo [7]. We obtained this evidence from the following type of recovery experiment. For each test of the technique we prepared duplicate samples of cotyledons, freeze-clamped them and killed them with cold HC104. Measured amounts of the intermediates were added to one of the samples immediately after freeze-clamping and just before the HC104 was added. The amounts of intermediates added were comparable to those present in the sample of cotyledons. Comparison of the amounts of the intermediates recovered from the two samples is taken as a measure of the reliability of the complete process of killing, extraction and measurement. Materials and Methods Materials 3-Mercaptopicolinic acid was a gift from Dr. H.L. Saunders, Smith Kline and French Laboratories, Philadelphia, Pa. Substrates, cofactors and enzymes were obtained from Boehringer Mannheim except that 3-phosphoglyceraldehyde and glutamic-oxaloacetic transaminase came from the Sigma Chemical Company. Marrows (Cucurbita pepo L. var. medullosa Alef.) were grown and harvested as described previously except that they were grown in continuous light [5]. E n z y m e assays Freshly harvested cotyledons, fresh weight 2 g , were homogenized as described previously [5] in 40 mM glycylglycine buffer (pH 7.4) that contained
10 mM EDTA and 10 mM dithiothreitol. The homogenate was centrifuged at 105 000 X g for 30 min and the supernatant was assayed for enzyme activity at once. Enzymes were measured at 25 ° C, as indicated in the following references, in reaction mixtures of 3.0 ml. These mixtures contained: glucosephosphate isomerase [8], 3 mM fructose 6-phosphate, 0.2 mM NADP ÷, 5 mM MgC12 and 3.5 units glucose-6-phosphate dehydrogenase in 50 mM Tris-HC1, pH 8.1; fructose-l,6-bisphosphate aldolase [9], 3.4 mM fructose 1,6-bisphosphate, 0.35 mM NADH, 20 pg of a mixture of a-glycerolphosphate dehydrogenase and triosephosphate isomerase in 40 mM glycylglycine, pH 7.7; triosephosphate isomerase [10], 0.48 mM 3-phosphoglyceraldehyde, 0.25 mM NADH and 1.2 units a-glycerolphosphate dehydrogenase in 40 mM glycylglycine buffer, pH 7.9; phosphoglycerate kinase [11], 15 mM 3-phosphoglycerate, 1 mM ATP, 0.25 mM NADH, 5 mM MgC12 and 4 units glyceraldehydephosphate dehydrogenase in 40 mM 2-(N-morpholino)ethanesulphonic acid, pH 6.9; lactate dehydrogenase [12], 0.33 mM pyruvate and 0.1 mM NADH in 40 mM glycylglycine, pH 7.4. NADP-specific malic enzyme was assayed as described by Johnson and Hatch [13] except that malate was 5 mM and the buffer was 40 mM glycylglycine (pH 7.5). For phosphate, pyruvate dikinase the extracts were prepared according to Hatch and Slack [14] and assayed as described by Hatch and Slack [15] except that 5 mM 2-mercaptoethanol replaced the dithiothreitol, and the phosphoenolpyruvate carboxylase (2 units per assay) came from Boehringer. For assay of phosphoenolpyruvate carboxylase the extract was desalted on a column (1 × 15 cm) of Sephade~ G-25 and assayed as described by Wong and Davies [16] in a reaction mixture that contained: 2 mM phosphoenolpyruvate, 7.5 mM MgC12, 2.5 mM KHCO3, 0.35 mM NADH, 2 pCi NaH14CO3 (spec. act. 58.5 mCi/mmol) in 40 mM glycylglycine, pH 7.9. Measu remen ts o f su bstra tes Measurements for whole cotyledons were made on samples of 1 g fresh weight which were prepared and killed within 5 min of harvest. For t r e a t m e n t with 3-mercaptopicolinic acid cotyledons were excised and their upper and lower surfaces were cut away with a razor blade. This left central portions of the cotyledons (each approx. 0.5 mm thick and approx. 25 mg fresh weight) t h a t were used to prepare replicate samples of 500--700 mg fresh weight. Each sample was placed on a filter paper (Whatman No. 1, 5 cm diameter) in a Petri dish. For treatment with the inhibitor the filter paper had been moistened with 0.5 ml 0.02 M KH2PO4 (pH 5.2) which contained 2 mM 3-mercaptopicolinic acid; the same solution was also used to moisten the uppermost cut surfaces of the cotyledons. The samples were then incubated in the light at 25°C (the conditions under which the seedlings had been grown) for 150 min before being taken for analysis. The control samples were treated similarly except that the 3-mercaptopicolinic acid was omitted from the 0.02 M KH2PO4. We adopted this procedure because the oxygen uptake of marrow cotyledons is so high in relation to the diffusion coefficient of oxygen in water t h a t immersion of the cotyledons in aqueous solution leads to anaerobiosis and some diminution of gluconeogenesis [ 3]. All samples of tissue were freeze-clamped as described previously [7]. The freeze-clamped material was dropped into liquid nitrogen at once and then
fragmented with a glass rod. Before the nitrogen had evaporated 3.0 ml 1.41 M HC104 at 4°C was added to the above mixture. The frozen mass which resulted was transferred to ....5°C and kept at this temperature for 18 h except for measurement of oxaloacetate when the time was reduced to 3 h. Next, the suspension that had formed during the time at --5°C was centrifuged at 27 000 × g for 2 min, and the sediment that formed was extracted thrice, each time with 1 ml 1.41 M HC104. All the extracts were combined, neutralized with 5 M K2CO3, and then centrifuged as above. The sediment was washed twice, each time with 1.0 ml 1.41 M HC104 which had been neutralized with 5 M K2CO3. During the above manipulations the temperature of the extract did not exceed 2°C. For measurement of oxaloacetate the HC104 was neutralized, as described by Lowry and Passonneau [17], with a mixture that was 2.0 M, 0.4 M and 0.4 M with respect to KOH, imidazole and KC1, respectively. In all instances the neutralized extracts were assayed at once by the spectrophotometric techniques described by Lowry and Passonneau [17]. The sum of the amounts of d i h y d r o x y a c e t o n e phosphate and 3-phosphoglyceraldehyde was determined by Method II from a single measurement made in the presence of triosephosphate isomerase and 3-phosphoglyceraldehyde dehydrogenase. 3-Phosphoglycerate was determined by Method I. For the measurement of aspartate the reaction mixture was altered so that it contained: 0.47 mM NADH, 0.26 mM a-ketoglutarate, 120 units malate dehydrogenase and 18.6 units glutamic oxaloacetic transaminase in 50 mM potassium phosphate buffer, pH 7.2. Gas exchange of the cotyledons was measured by Warburg's direct manometric method. Fisher's P values were calculated by Student's t-test. Results and Discussion
Enzyme activities We already know the m a x i m u m catalytic activities of some of the enzymes of gluconeogenesis in the cotyledons of 5-day-old marrows [1]. We assayed the outstanding enzymes and the complete list of activities is given in Table I. In the present work, except in the case of lactate dehydrogenase and phosphate, pyruvate dikinase, we optimized each assay by varying the concentration of each c o m p o n e n t of the assay mixture and by varying the pH. We investigated whether loss or activation of the gluconeogenic enzymes occurred during the preparation of the extracts. For each test we prepared duplicate samples of cotyledons; we extracted one sample in the usual way and the other in buffer which contained measured amounts of pure enzymes. The amounts of pure enzymes added were comparable to the activities found in the cotyledons. Comparison of the activities found in the extracts of the two samples showed the extent to which the pure enzymes had survived the processes of homogenization and extraction. Our estimates of the recovery of 3-phosphoglycerate kinase, fructose-l,6-bisphosphate aldolase, and glucosephosphate isomerase were 126%, 89% and 109%, respectively, of the amounts added. The previously published data in Table I are also supported by optimization and recovery experiments. Therefore we suggest that the values for the enzymes of gluconeogenesis shown in Table I reflect the m a x i m u m catalytic activities of the cotyledons.
TABLE I A C T I V I T I E S OF ENZYMES OF G L U C O N E O G E N E S I S IN C O T Y L E D O N S OF 5-DAY-OLD S E E D L I N G S OF MARROW V a l u e s m a r k e d * are f r o m ap R e e s e t al. [1] a n d t h a t f o r phosphoenolpyruvate c a r b o x y k i n a s e is f r o m L e e g o o d a n d ap R e e s [ 3 ] ; for t h e r e m a i n d e r , s a m p l e s o f c o t y l e d o n s w e r e e x t r a c t e d a n d a s s a y e d as d e s c r i b e d in t h e t e x t . V a l u e s are m e a n s +- S.E. T h e n u m b e r o f s a m p l e s a s s a y e d is s h o w n in p a r e n t h e s e s . R e l a t i v e a c t i v i t i e s are b a s e d o n a value o f 1 / ~ m o l / m i n p e r g for f r u c t o s e - l , 6 - b i s p h o s p h a t a s e ; t h e d a t a for liver are averages o f m e a s u r e m e n t s f r o m a r a n g e o f v e r t e b r a t e s a n d w e r e o b t a i n e d f r o m N e w s h o l m e a n d Start [ 6 ] . Enzyme
G l u c o s e p h o s p h a t e i s o m e r a s e (EC 2 . 7 . 1 . 1 ) F r u c t o s e - l , 6 - b i s p h o s p h a t a s e (EC 3 . 1 . 3 . 1 1 ) F r u c t o s e - l , 6 - b i s p h o s p h a t e aldolase (EC 4 . 1 . 2 . 7 ) T r i o s e p h o s p h a t e i s o m e r a s e (EC 5 . 3 . 1 . 1 ) NAD-specific glyceraldehydephosphate dehydrogenase (EC 1 . 2 . 1 . 1 2 ) P h o s p h o g l y c e r a t e k i n a s e (EC 2 . 7 . 2 . 3 ) P h o s p h o g l y c e r o m u t a s e (EC 2 . 7 . 5 . 3 ) E n o l a s e (EC 4 . 2 . 1 . 1 1 ) PhosphoenolPyruvate c a x b o x y k i n a s e (EC 4 . 1 . 1 . 4 9 ) Phosphoenotpyruvate c a x b o x y l a s e (EC 4 . 1 . 1 . 3 1 ) P y r u v a t e k i n a s e (EC 2 . 7 . 1 . 4 0 ) P h o s p h o f r u c t o k i n a s e (EC 2 . 7 . 1 . 1 1 ) N A D P - s p e c i f i c m a l i c e n z y m e (EC 1 . 1 . 1 . 3 9 ) P h o s p h a t e , p y t u v a t e d i k i n a s e (EC 2 . 7 . 9 . 1 ) L a c t a t e d e h y d r o g e n a s e (EC 1 . 1 . 1 . 2 7 )
A c t i v i t y in c o t y l e d o n s (#mol substrate consumed ]min per g fresh w t . )
Cotyledons
Liver
17.15 3.21 1.69 425.1
5.3 1 0.5 132
14" 1 0.3 --
4.6 14.4
7.5 6.5
± 1.11 -+ 0 . 2 4 + 0.05 +_ 27.1
(4) (6) * (4)
1 4 . 7 5 + 2.97 46.20 + 2.25 1 9 . 7 8 -+ 3 . 1 4 2 0 . 7 5 -+ 2.54 7.68 + 0.31 0 . 3 2 -+ 0 . 0 2 9 . 7 4 + 1.24 0 . 0 9 t 0.03 1 . 1 4 -+ 0 . 0 6 None detected None detected
(6) * (4)
(3)
Relative activity
(6) *
6.2
--
(6) * (6)
6.5 2.4
2.7 0.7
(4)
0.1
--
(6) *
3.0
1.9
(6) *
0.03
0.3
(3)
--
-
-
Examination of Table I shows that the gluconeogenic enzymes with the lowest activities are phosphoenolpyruvate carboxykinase, aldolase, and fructose-l,6-bisphosphatase. This suggests that possibly these three reactions are appreciably displaced from equilibrium in vivo. The activities of enolase, phosphoglyceromutase, glyceraldehydephosphate dehydrogenase and glucosephosphate isomerase are all comparable, appreciably higher than those of the first mentioned enzymes, and much lower than that of triosephosphate isomerase. The activities of the other enzymes in Table I are given as indicators of possible alternative routes of metabolism of gluconeogenic intermediates. Our inability to detect phosphate, pyruvate dikinase accords with the considerable evidence that there is no direct route from pyruvate to sugar in such tissues [181. Data comparable to those in Table I are not available for any other plant tissue. Kagawa et al. [19] report the activities of pyruvate kinase, phosphoglyceromutase, enolase, phosphoglycerate kinase, glyceraldehydephosphate dehydrogenase, triosephosphate isomerase and aldolase for cotyledons of 4-day-old water melon seedlings. Their values, when expressed per g fresh weight, are very similar to those in Table I. More extensive data are available for animal tissues [6]. This permits comparison of the relative activities of the enzymes in marrow cotyledons with those of liver (Table I). The comparison reveals a remarkable similarity between the two. The only appreciable differences are
that the relative activity of phosphoenolpyruvate carboxykinase is higher, and that o f p h o s p h o f r u c t o k i n a s e lower, in marrows.
Measurements of intermediates The recoveries o f the c o m p o u n d s added to the freeze-clamped cotyledons (Table II) show that there was significant loss of AMP, and some loss of triosephosphate during extraction and analysis. The recoveries of all the other compounds were satisfactory. We argue that freeze-clamping ruptures the tissue and stops significant biochemical and chemical change. Thus the c o m p o u n d s we added in our recovery experiments would have been subjected to the same e n v i ro n men t as the endogenous c o m p o u n d s during the killing and extraction of the cotyledons. The high recoveries and the consistency of our data provide sound evidence that, AMP excepted, our estimates represent the amounts of gluconeogenic intermediates which were present in vivo. When expressed per g fresh weight these a m o u n t s are appreciably higher than those report ed for plant cells in general [20] and significantly higher than those report ed for castor bean endosperm [4], the only ot her gluconeogenic plant tissue for which data are available. We attribute these high values to the high metabolic activity of the c o t y l e d o n s and to the dense packing of the cells and their contents [1]. Table III compares the apparent equilibrium constants of the enzymes of gluconeogenesis with the relative am ount s of some of their substrates and pr o d u c t s present in vivo. For some of the reactions the latter constitute the mass-action ratios. It is clear that the reactions catalysed by enolase, phosphoglyceromutase and glucosephosphate isomerase are very close to equilibrium in vivo. Thus we suggest that none of these enzymes plays a d o m i n a n t direct role in the regulation of gluconeogenesis in marrows. The aldolase reaction is n o t very far from equilibrium and our results provide no firm ground for believing th at this step is regulatory. Determination of the mass-action ratios for the remaining reactions of gluconeogenesis is made difficult by the fact that some of the reactants are almost certainly unequally distributed between the different c o m p a r t m e n t s of the cells of the cotyledons. For the reaction catalysed by fructose-l,6-bisphosphatase it is possible to use the apparent equilibrium constant, and our values for fructose 1,6-bisphosphate and fructose 6-phosphate, to calculate the a m o u n t of o r t h o p h o s p h a t e which would have to be present in 1 g of c o t y l e d o n s if the reaction were at equilibrium in vivo. Values of 20.1, 14.3 and 56.6 m m o l of phosphate per g c o t y l e d o n are obtained for intact, cut and treated cotyledons, respectively. The likelihood that such amounts of inorganic phosphate are present in the cytoplasm of marrow cotyledons is so r e m o t e [24] that we may conclude that the reaction catalysed by fructose-l,6-bisphosphatase is some considerable distance from equilibrium in these cotyledons. It is more difficult to assess the state of the reaction catalysed by phosphoenolpyruvate carboxykinase. In order to do so, we have assumed t hat the concentration of bicarbonate in the cytoplasm is 0.4 mM (roughly equivalent to the c o n c e n t r a t i o n o f COs in air) [23]. By using our estimates of oxaloacetate and phosphoenolpyruvate, we have calculated the ratio ATP : ADP that would exist in the cytoplasm of the c o t y l e d o n s if the phosphoenolpyruvate carboxykinase reaction was at equilibrium. The values obtained are 7 . 4 . 1 0 -3 , 5 . 4 . 1 0 -3 ,
OF
GLUCONEOGENESIS
IN COTYLEDONS
OF
5-DAY-OLD
MARROW
SEEDLINGS,
AND
EFFECTS
THEREON
OF
3-MERCAPTO-
phosphate
96 90 109 70 100 103 93 104 95 93
Glucose 6-phosphate Fructose 6-phosphate Fructose 1,6-bisphosphate 3-Phosphoglyceraldehyde + dihydroxyacetone 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Oxaloacetate Pyruvate Malate 106 + 10 100-+ 12 118 + 8 4 7 +_ 5
Aspartate ATP
ADP AMP
+ 10 + 10 ± 13 -+ 3 ± 6 + 10 ± 3 ± 17 ± 1 ± 6
Recovery (%)
Compound
n.s. ( n o t s i g n i f i c a n t ) .
(9)
(5) (9) (9)
36 2 (5)
± 45 + 21 ± 8 ± 6 + 56 + 7 + 8 1 3 + "11 + 344 + 88 ± 21
234 ± 32 ±
2113 467 54 46 235 37 111 10 91 4998 2281 447
1495 376 31 38 148 37 65 8 88 2796 1918 356 258 48
± 89 +- 1 8 + 5 _* 6 + 18 t 12 + 6 + 1 + 14 z 304 + 281 ± 28 *_ 4 1 ± 6
II Cut cotyledons
(nmol per g fresh wt.)
Whole cotyledons
I
Amount
(5) (5)
(9)
(5) (5)
712 169 55 49 91 8 28 15 89 8127 1780 488 281 62
± 102 -+ 1 4 + 8 ± 9 -~ 2 5 ~ 1 (5) -+ 5 (5) + 2 - 16 (8) ± 837 + 200 ± 56 * 4 0 (5) ± 15(5)
III Cut cotyledons in 3-mercaptopicolinic acid
n.s. 40.001 n.s.
n.s. n.s. n.s.
n.s.
