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Levels of intermediates and cofactors of the glycolytic pathway and the citric acid cycle in rat platelets
SHORT COMMUNICATIONS ~BA
IbI
23481
Levels of intermediates and cofactors of the 91ycolytic pathway and the citric acid cycle in rat platelets Understanding of metabolism and its regulation in intact cells and tissues has been advanced b y analyses of levels of metabolic intermediates under various steadystate conditions. This type of analysis could be valuable in investigations of metabolism of blood platelets, which have a wide range of energy requirements and metabolic activity. This paper reports the results of measurements of intermediates and cofactors of the glycolytic and citric acid cycle pathways in rat platelets. Equipment used for collection of blood and isolation of platelets was siliconecoated. Temperature was maintained at o-4 °. Blood was taken from the abdominal aorta of ether anesthetized rats into cold syringes containing 2.o ml of 1% E D T A o.7% NaC1. Red ceils and white cells were sedimented b y centrifugation at I8o × g for 4o min and platelets were obtained from the resulting platelet-rich plasma b y centrifugation at IOOO × g for 3o rain. The platelets were washed once with 2.o ml of o.9% NaC1 and were finally suspended in o.9% NaC1 to give a concentration of about 7" I°9 platelets per ml. Glycogen was extracted from o.oi ml of platelet suspension with 3o% K O H essentially b y the method of PFLEmERER6. Reduced pyridine nucleotides were extracted from o.2 ml platelet suspension with o.2 ml of I M K O H in ethanol as described b y KLINC,ENBERG7 for mitochondria. For extraction of all other metabolites measured, o.2 vol. of 3 M perchloric acid was rapidly added to the platelet suspension with mixing and the suspension was allowed to stand at room temperature, with occasional mixing, for 2o rain. The precipitate was removed b y centrifugation and washed with o.5 ml of o.5 M perchIoric acid. The combined supernatant solutions were neutralized with o.o5 voh of 5 M K~COa. After ~5 min in ice, the precipitated potassium perchlorate was removed b y centrifugation. All extracts were stored at - - I 5 ° until used. Metabolites were measured b y enzymatic analysis s using the fluorimetric method of MAITRA AND ESTABROOK9 with a commercial (Brinkman Instrument Co., Westbury, N.Y.) adaptation of the instrument described b y t h e m ~.° and modifications of published procedures~, s-~°. Results from a series of platelet suspensions, each prepared from blood pooled from IO-I2 rats, are shown in Table I. Each Platelet suspension was divided into halves and extraction and complete analyses were made of each half. Thus, variation between halves (standard deviation of the difference) is due only to analytical error, whereas variation between a series of suspensions (standard deviation of the mean) represents differences in the various platelet preparations in addition to analytical error. To establish that the compounds measured were actually inside the platelets and not in the suspending medium due to contamination from plasma or to leakage from platelets, levels of metabolites in a platelet suspension were compared with levels in the supernatant solution following centrifugation of the suspension. Only glucose, pyruvate, and lactate were present in the supernatant solution at levels that would represent an appreciable error. D a t a for these-compounds are not given in Table I. Results of measurement of metabolic intermediates m a y be analyzed b y comparing mass action ratios with apparent equilibrium constants for individual reactions ~iochSm. Biophys. Acts, 177 (19-69) i 6 i - i 6 3
SHORT COMMUNICATIONS
102
TABLE
I
L E V E L S OF G L Y C O L Y T I C AND
C I T R I C AC][D C Y C L E I N T E R M I S D t A T E S A N D C O F A C T O R S I X I U A S H E D 7Fl 'f
PLATGLETS
~,3-Diphosphoglycera~e, glyceratdehyde-3-phosphate, and oxatoacetate were present at ievels too low to measure. Snccinate was not measured because the enzymes required for the assay are not commercially available.
.MetaboliZe
Numbe~, of duplicate ae~alyses
Glycogen Glucose 1-phosphate Glucose 0-phosphate Fructose 6-phosphate Fructose 1,6-diphosphate Dihydroxyacetone phosphate ~-Glycerophosphate 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Citrate Isocitrate ~-Keloglutarate Fumarate Malate NAD + NADH NADP+ NADPH ATP ADP AMP
6 5 6 5
P/IeaT¢ (nmoles/zo ~° platdets)
Standard deviation ~f mea~.z
Standard devia~io~¢ of differ>once
~ / E z~ N--z
~
6 6 7 4 7 7 6 3 6 6 6
232o 0.2 2.5 o.0 3.4 1.2 ~. 6 2.6 o.4 1,4 89 ~.2 i~2 2.1 14 26
6
5 .0
['4
i .O
6 6
Lo 6. 3 454 ~9 8.3
o.2 2.2 75 57 5
o.6 0.6 22 2.2 ~-3
6
0
6 6
I43o 0.09 1.3 o 3 ~.o 0. 3 o. 7 0.9 o. ~ 0.5 26 o. ~ o.~ ~. ~ 6 ~I
~':-r
24 ° 0,4 0.05 o.I o, ~ o. i o 3 0-3 4 o !
o.o~ o. i o.2 2.8
* Duplicate values for this metabolite were not obtained with several of the platelet prepar a t i o n s so t h e r e is i n s u f f i c i e n t d a t a t o c a l c u l a t e s t a n d a r d d e v i a t i o n o f t h e d i f f e r e n c e . TABLE
I1
COMPARISON
OF
5lASS
ACTION
RATIOS
WITH
Reaction
PhosphogIucomutase Phosphoglucoisomerase Phosphofructokinase Phosphoglyceromutase EnoIase Pyruvate kinase Aconitase Fumarase
APPARENT
EQUILIBRIUM
Apparent equilibrium constanX ~ (EC (EC (EC (EC (EC (EC (EC (EC
2.7.5.~ ) 5.3,~-9) 2.7.zo11) 2.7.5.3 ) 4.2.Lll) 2 . 7 . ~ . 4 o) 4.2~.3) 4.2. ~.2)
i8 0.28 1.2" ~o a o. ~ - o . 17 4 .6 6.3 2" z o a - 1 5 " I o a ~5 4
CONSTANTS
3/Iass action raazo ~~ 12.5 o.24 ~.5 o. ~ 5 3.5 <35 ~ 68 6 -
* V a l u e s u s e d b y o t h e r s ~,5 f o r s i m i l a r c o m p a r i s o n s . ** C a l c u l a t e d f r o m T a b l e I a s p r o d u c t / s u b s t r a t e with the reaction in the direction glycogen t o CO~. *** C a l c u l a t e d o n t h e b a s i s t o t o t a l p y r u v a t e i n t h e s u s p e n s l o n .
Biochim. Biophys. Act& 177 ( I 9 6 9 ) 1 6 1 - I 6 3
I~3
SHO~R.T COMMUNICATIONS
in a metabolic pathway. Mass action ratios could not be calculated for those reactions where either the substrate or product was not measured or where the reaction includes pyridine nucleotides, which are compartmented in the cell so that total amounts are not proportional to levels at a particular enzyme. Mass action tatios that could be calculated are compared with equilibrium constants in Table II. Mass action ratios are far displaced from equilibrium constants only for the reactions of phosphofructokinase and pyruvate kinase. This is typical of other tissues and is consistent with the ~cole of these enzymes as major control points of glycolysis. The reactions of aconitase and fumarase are also displaced from equilibrium but not to an extent that would suggest a control point. Levels of metabolic intermediates v a r y with the metabolic state of the tissue. Although data here were obtained from platelets with a reduced rate of metabolism due to low temperatures, they probably approximate the levels in metabolically active pla}elets. This research was supported b y grant H E 10099 from the National Heart Institute., U.S. Public Health Service. I am grateful for the technical assistance of Mr. Michael De Jesus, Jr.
Department of Biochemistry, State University of New York, Downstate Medical Center, Brooklyn, N.Y., 112o3 IUoS.A.)
THOMAS C. DETWILEI~
I T. ~BI~cHER AND W . RI~SSMANN, Angew. Chem. Intern. Ed. Engl., 3 (1964) 426. 2 B. H E s s AND IK. BRAND, Clin. Chem., i i (1965) 223. 3 0 . ~ . LowRY, J. V. PASSONN/~AU, F. X. HASS/~LBERGER AND D. W. SCHULZ, ./. Biol. Chem., 239 (1964) 18. 4 N. D. C~OLDBERG,J. ~'. PASSONNEAIJ AND O. I-I. LOWRY, J. Biol. Chem., 241 (1966) 39975 J. 1~. WILLIAMSON, ./. Biol. Chem., 240 (1965) 2308. 6 C'r. PFLI!:IDERER, in K . U. BERGMEYER, Methods of Enzymatic Analysis, A c a d e m i c Press, N e w York, 1965, p. 597 ~ . KLINGENB•RG, in H. U. BERGMEYER, Methods of Enzymatic Analysis, A c a d e m i c Press, N e w York, 1965, p. 531. 8 H. U. BERGMEYI~R, Methods of Enzymatic Analysis, A c a d e m i c Press, N e w York, 1965. 9 P. K. MAITRA AND R. W . ESTABROOK, Anal. Biochem., 7 (1964) 472. IO ~ . W. ]~STABROOK AND P. K. MAITRA, Anal. Biochem., 3 (1962) 369.
Reoeived September 26th, 1968 Biochim. Biophys. Acta, 177 (1969) 161-163
BBA 23483
The: significance of ribokinase for ribose utilization by Escherichia coli It has been establishedZ, ~ that Escherichia colt take up a variety of sugars from their media via a phosphoenolpyruvate-dependent phosphotransferase system ~ through which the sugars are converted to sugar phosphates. This implies that ATPdependent hexokinases are not necessarily involved in sugar utilization, as also shown b y the observation that mutants lacking glucokinase grow normally on glucose ~, Biochim. Biophys. Acta, 177 (1969) 163-165
IbI
23481
Levels of intermediates and cofactors of the 91ycolytic pathway and the citric acid cycle in rat platelets Understanding of metabolism and its regulation in intact cells and tissues has been advanced b y analyses of levels of metabolic intermediates under various steadystate conditions. This type of analysis could be valuable in investigations of metabolism of blood platelets, which have a wide range of energy requirements and metabolic activity. This paper reports the results of measurements of intermediates and cofactors of the glycolytic and citric acid cycle pathways in rat platelets. Equipment used for collection of blood and isolation of platelets was siliconecoated. Temperature was maintained at o-4 °. Blood was taken from the abdominal aorta of ether anesthetized rats into cold syringes containing 2.o ml of 1% E D T A o.7% NaC1. Red ceils and white cells were sedimented b y centrifugation at I8o × g for 4o min and platelets were obtained from the resulting platelet-rich plasma b y centrifugation at IOOO × g for 3o rain. The platelets were washed once with 2.o ml of o.9% NaC1 and were finally suspended in o.9% NaC1 to give a concentration of about 7" I°9 platelets per ml. Glycogen was extracted from o.oi ml of platelet suspension with 3o% K O H essentially b y the method of PFLEmERER6. Reduced pyridine nucleotides were extracted from o.2 ml platelet suspension with o.2 ml of I M K O H in ethanol as described b y KLINC,ENBERG7 for mitochondria. For extraction of all other metabolites measured, o.2 vol. of 3 M perchloric acid was rapidly added to the platelet suspension with mixing and the suspension was allowed to stand at room temperature, with occasional mixing, for 2o rain. The precipitate was removed b y centrifugation and washed with o.5 ml of o.5 M perchIoric acid. The combined supernatant solutions were neutralized with o.o5 voh of 5 M K~COa. After ~5 min in ice, the precipitated potassium perchlorate was removed b y centrifugation. All extracts were stored at - - I 5 ° until used. Metabolites were measured b y enzymatic analysis s using the fluorimetric method of MAITRA AND ESTABROOK9 with a commercial (Brinkman Instrument Co., Westbury, N.Y.) adaptation of the instrument described b y t h e m ~.° and modifications of published procedures~, s-~°. Results from a series of platelet suspensions, each prepared from blood pooled from IO-I2 rats, are shown in Table I. Each Platelet suspension was divided into halves and extraction and complete analyses were made of each half. Thus, variation between halves (standard deviation of the difference) is due only to analytical error, whereas variation between a series of suspensions (standard deviation of the mean) represents differences in the various platelet preparations in addition to analytical error. To establish that the compounds measured were actually inside the platelets and not in the suspending medium due to contamination from plasma or to leakage from platelets, levels of metabolites in a platelet suspension were compared with levels in the supernatant solution following centrifugation of the suspension. Only glucose, pyruvate, and lactate were present in the supernatant solution at levels that would represent an appreciable error. D a t a for these-compounds are not given in Table I. Results of measurement of metabolic intermediates m a y be analyzed b y comparing mass action ratios with apparent equilibrium constants for individual reactions ~iochSm. Biophys. Acts, 177 (19-69) i 6 i - i 6 3
SHORT COMMUNICATIONS
102
TABLE
I
L E V E L S OF G L Y C O L Y T I C AND
C I T R I C AC][D C Y C L E I N T E R M I S D t A T E S A N D C O F A C T O R S I X I U A S H E D 7Fl 'f
PLATGLETS
~,3-Diphosphoglycera~e, glyceratdehyde-3-phosphate, and oxatoacetate were present at ievels too low to measure. Snccinate was not measured because the enzymes required for the assay are not commercially available.
.MetaboliZe
Numbe~, of duplicate ae~alyses
Glycogen Glucose 1-phosphate Glucose 0-phosphate Fructose 6-phosphate Fructose 1,6-diphosphate Dihydroxyacetone phosphate ~-Glycerophosphate 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Citrate Isocitrate ~-Keloglutarate Fumarate Malate NAD + NADH NADP+ NADPH ATP ADP AMP
6 5 6 5
P/IeaT¢ (nmoles/zo ~° platdets)
Standard deviation ~f mea~.z
Standard devia~io~¢ of differ>once
~ / E z~ N--z
~
6 6 7 4 7 7 6 3 6 6 6
232o 0.2 2.5 o.0 3.4 1.2 ~. 6 2.6 o.4 1,4 89 ~.2 i~2 2.1 14 26
6
5 .0
['4
i .O
6 6
Lo 6. 3 454 ~9 8.3
o.2 2.2 75 57 5
o.6 0.6 22 2.2 ~-3
6
0
6 6
I43o 0.09 1.3 o 3 ~.o 0. 3 o. 7 0.9 o. ~ 0.5 26 o. ~ o.~ ~. ~ 6 ~I
~':-r
24 ° 0,4 0.05 o.I o, ~ o. i o 3 0-3 4 o !
o.o~ o. i o.2 2.8
* Duplicate values for this metabolite were not obtained with several of the platelet prepar a t i o n s so t h e r e is i n s u f f i c i e n t d a t a t o c a l c u l a t e s t a n d a r d d e v i a t i o n o f t h e d i f f e r e n c e . TABLE
I1
COMPARISON
OF
5lASS
ACTION
RATIOS
WITH
Reaction
PhosphogIucomutase Phosphoglucoisomerase Phosphofructokinase Phosphoglyceromutase EnoIase Pyruvate kinase Aconitase Fumarase
APPARENT
EQUILIBRIUM
Apparent equilibrium constanX ~ (EC (EC (EC (EC (EC (EC (EC (EC
2.7.5.~ ) 5.3,~-9) 2.7.zo11) 2.7.5.3 ) 4.2.Lll) 2 . 7 . ~ . 4 o) 4.2~.3) 4.2. ~.2)
i8 0.28 1.2" ~o a o. ~ - o . 17 4 .6 6.3 2" z o a - 1 5 " I o a ~5 4
CONSTANTS
3/Iass action raazo ~~ 12.5 o.24 ~.5 o. ~ 5 3.5 <35 ~ 68 6 -
* V a l u e s u s e d b y o t h e r s ~,5 f o r s i m i l a r c o m p a r i s o n s . ** C a l c u l a t e d f r o m T a b l e I a s p r o d u c t / s u b s t r a t e with the reaction in the direction glycogen t o CO~. *** C a l c u l a t e d o n t h e b a s i s t o t o t a l p y r u v a t e i n t h e s u s p e n s l o n .
Biochim. Biophys. Act& 177 ( I 9 6 9 ) 1 6 1 - I 6 3
I~3
SHO~R.T COMMUNICATIONS
in a metabolic pathway. Mass action ratios could not be calculated for those reactions where either the substrate or product was not measured or where the reaction includes pyridine nucleotides, which are compartmented in the cell so that total amounts are not proportional to levels at a particular enzyme. Mass action tatios that could be calculated are compared with equilibrium constants in Table II. Mass action ratios are far displaced from equilibrium constants only for the reactions of phosphofructokinase and pyruvate kinase. This is typical of other tissues and is consistent with the ~cole of these enzymes as major control points of glycolysis. The reactions of aconitase and fumarase are also displaced from equilibrium but not to an extent that would suggest a control point. Levels of metabolic intermediates v a r y with the metabolic state of the tissue. Although data here were obtained from platelets with a reduced rate of metabolism due to low temperatures, they probably approximate the levels in metabolically active pla}elets. This research was supported b y grant H E 10099 from the National Heart Institute., U.S. Public Health Service. I am grateful for the technical assistance of Mr. Michael De Jesus, Jr.
Department of Biochemistry, State University of New York, Downstate Medical Center, Brooklyn, N.Y., 112o3 IUoS.A.)
THOMAS C. DETWILEI~
I T. ~BI~cHER AND W . RI~SSMANN, Angew. Chem. Intern. Ed. Engl., 3 (1964) 426. 2 B. H E s s AND IK. BRAND, Clin. Chem., i i (1965) 223. 3 0 . ~ . LowRY, J. V. PASSONN/~AU, F. X. HASS/~LBERGER AND D. W. SCHULZ, ./. Biol. Chem., 239 (1964) 18. 4 N. D. C~OLDBERG,J. ~'. PASSONNEAIJ AND O. I-I. LOWRY, J. Biol. Chem., 241 (1966) 39975 J. 1~. WILLIAMSON, ./. Biol. Chem., 240 (1965) 2308. 6 C'r. PFLI!:IDERER, in K . U. BERGMEYER, Methods of Enzymatic Analysis, A c a d e m i c Press, N e w York, 1965, p. 597 ~ . KLINGENB•RG, in H. U. BERGMEYER, Methods of Enzymatic Analysis, A c a d e m i c Press, N e w York, 1965, p. 531. 8 H. U. BERGMEYI~R, Methods of Enzymatic Analysis, A c a d e m i c Press, N e w York, 1965. 9 P. K. MAITRA AND R. W . ESTABROOK, Anal. Biochem., 7 (1964) 472. IO ~ . W. ]~STABROOK AND P. K. MAITRA, Anal. Biochem., 3 (1962) 369.
Reoeived September 26th, 1968 Biochim. Biophys. Acta, 177 (1969) 161-163
BBA 23483
The: significance of ribokinase for ribose utilization by Escherichia coli It has been establishedZ, ~ that Escherichia colt take up a variety of sugars from their media via a phosphoenolpyruvate-dependent phosphotransferase system ~ through which the sugars are converted to sugar phosphates. This implies that ATPdependent hexokinases are not necessarily involved in sugar utilization, as also shown b y the observation that mutants lacking glucokinase grow normally on glucose ~, Biochim. Biophys. Acta, 177 (1969) 163-165