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Properties of muscle phosphofructokinase of cold- and warm-acclimated Carassius auratus
Como. Biochem. Physiol., 1971, VoL 39B,pp. 747 to 764. Pergamon Press. Printed in Great Britain
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF COLD- AND WARM-ACCLIMATED C A R A S S I U S A U R A T U S JAMES M. F R E E D * Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois 61801 (Received 21 December 1970)
Abstract--1. Phosphofructokinase (PFK) activity of homogenate super-
natants of 5°C-acclimated goldfish is slightly more than that of 25°C-acclimated goldfish when measured at 5° and 15°C, but not at 25°C. 2. Muscles of warm-acclimated goldfish have a large increase relative to 5°C fish in several glycolytic intermediates and adenine nudeotides; the increases in fructose-6-phosphate (F-6-P) and glucose-6-phosphate (G-6-P) appear within 3 hours after the fish are exposed to the selected temperature. 3. Magnesium inhibits PFK at higher temperatures (25°C); citrate inhibits less at higher than at lower assay temperatures. 4. Goldfish muscle PFK activity changes markedly within a small pH range, and it is suggested that if goldfish muscle intraceUular pH decreases in warm acclimation, this pH decrease, by its action on PFK, would cause the observed effects of decreased glyeolytic rate and increased levels of F-6-P and G-6-P. INTRODUCTION WHEN a poikilotherm is transferred from one temperature to another, its physiology and biochemistry may undergo a series of adaptations or compensations so that the organism may live successfully at the new temperature. Part of these compensations include changes in enzyme activity and changes in pathways. Increased activity of an enzyme in cold acclimation may be due to increased amounts of the enzyme or of specific isozymes or to changes in various co-factors or modulators. Relative changes in rates of carbon flow through alternate pathways are indicated by a fivefold increase in glycolysis in muscle of cold-acclimated over warm-acclimated trout (Hochachka & Hayes, 1962). Since the enzyme phosphofruetokinase (ATP: D-fructose-6-phosphate-l-phosphotransferase, E.C. 2.7.1.11) is considered to be the first rate-controlfing enzyme unique to glyeolysis (Frenkel, 1965 ; Wilson et al., 1967), it appeared to be the enzyme most likely to be affected in temperature adaptation. P F K is a cytoplasmic enzyme and not known to be present in isozymes (Kopperschl/iger et al., 1968; LindeU & Stellwagen, 1968), but to be regulated by its substrates and various modulators.t * Present address: Department of Zoology, Ohio Wesleyan University, Delaware, Ohio 43015. t Abbreviations used: EDTA = ethylenediaminetetraacetic acid; FDP = fructose-l,6diphosphate; F-6-P = fructose-6-phosphate; G-6-P = glucose-6-phosphate; PFK = phosphofructokinase; Tris-C1 = tris(hydroxymethyl)arninomethane-chloride. 747
748
JAMESM. FREED MATERIALS AND METHODS
Animals Goldfish (Carassius auratus, L), obtained from Auburndale Goldfish Co. (Chicago) and averaging 33 g in weight and 15 cm in length, were kept in aerated well water at 15:C (12-hr photoperiod) for at least 1 week after arrival before being transferred to either 25 or 5°C. The fish were kept at these temperatures at least 2 weeks during which they were fed Conditioner Goldfish Food (Wardley) twice each day at 25°C; once each day at 15°C; and once every other day at 5°C. For the time course studies of changes in ATP, G-6-P and F-6-P, with temperature of acclimation, fish were transferred in 15°C water to 5° and 25°C rooms. By gradual addition of colder or warmer water, the temperature of the aquaria in which they were placed reached the selected temperature of 5 or 25°C within 30 rain. For measurements at 3, 6 and 12 hr at the new temperatures, the fish were not fed after transfer to the new temperature. Fish at the new temperatures for 24 hr were fed once during this time ; fish at 96 hr were fed seven times at 25°C and twice at 5°C. No fish was fed during a period of 12 hr prior to its sacrifice.
Measurement of nucleotides and glycolytic intermediates Nucleotides and glycolytic intermediates were measured in muscles from cold- and warm-acclimated goldfish. Preparation of the solutions to be tested followed methods adapted from Adam (1963b). Fish were obtained from the temperatures of acclimation, decapitated as quickly as possible and a slice of dorsal trunk musculature was dropped into liquid nitrogen. A second slice was weighed and placed in a 110°C oven, and the dry weight was determined after a m i n i m u m of 12 hr. About 1"5 g of the frozen tissue was pulverized with a mortar and pestle cooled with liquid nitrogen, and frozen 0'9 N percholoric acid equivalent in volume to one-and-a-half times the weight of the tissue was added to the pulverized muscle. After being allowed to thaw at 5°C, the mixture was homogenized, centrifuged at 3000g for 10rain, the precipitate resuspended in 0"9 N perchloric acid, recentrifuged and the combined supernatants neutralized to pH 6"5 with 3"75 M potassium carbonate. After 1 hr at 5°C, the preparation was centrifuged and the volume of the supernatant was measured and subsequently used in the assays. Several experiments with small fish (4-5 cm in length) dropped directly into liquid nitrogen indicated that no significant change in ratios of the adenine nucleotides occurred during the few seconds required to decapitate the fish and remove the muscle before freezing. Enzymatic methods for measurement of nucleotides and glycolytic intermediates were based on the conversion of either N A D H to NAD, or of N A D P to N A D P H as measured at 340 m/~. Assays were performed in a Beckman DB spectrophotometer with a Sargent Recorder model SRL. Adenine nucleotides were measured with kits prepared by Boehringer. Procedures followed that of Adam (1963b) for A T P and that of Adam (1963a) for ADP and AMP. Procedures for the measurements of glucose, G-6-P and F-6-P were adapted from the method of Hohorst (1963). NADP, ATP, G-6-P dehydrogenase and hexokinase were obtained from Sigma; phosphoglucose isomerase was obtained from both Sigma and Calbiochem. Wriethanolamine was obtained from Matheson, Coleman and Bell. The sum of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate (triose phosphates) and the concentration of fructose-l,6-diphosphate were determined by the methods of Biicher & Hohorst (1963). Aldolase and a mixture of glycerophosphate dehydrogenase and triosephosphate isomerase were obtained from Sigma. Phospho-enol-pyruvate was measured by procedures adapted from Czok & Eckert (1963). Pyruvate kinase and lactate dehydrogenase were purchased from Boehringer. The concentration of pyruvate was measured with the same kit from Boehringer used to measure nucleoside diphosphates and monophosphates using the procedure of Adam (1963a), or by the procedure of BiJcher et al.
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF C A R A S S I U S A U P ~ g T U S
749
(1963). Citric acid was measured using the procedure of Stem (1957). Magnesium was measured on a nitric acid digest of muscle by the use of an atomic absorption flame photometer (Evans Electroselenium Ltd., England). Results are expressed in terms of micromoles of the specified substance/gram tissue either dry weight or wet weight. Preparation and assay of enzyme Homogenates of goldfish muscle, 15 per cent by weight, were prepared in 0"1 M K2HPO4 and centrifuged at 25,000 g for 30 rain. Ammonium sulfate was added to the supernatant and the resulting precipitate from a 25% saturated ammonium sulfate solution was resuspended in 0"01 M Tris-C1, pH 7"6, 1 mM in EDTA, and dialyzed against 300 ml 0-01 M Tris-C1, pH 7"6, for 3-5 hr with three changes of the buffer. This preparation produced a tenfold increase in specific activity of the enzyme above the original supematant and with 90-100 per cent recovery. Protein was analyzed by the Lowry method (Layne, 1957) using bovine serum albumin (Sigma) as the standard; measurements were at 750 m/z with a Beckman DB spectrophotometer. Assays for the goldfish muscle PFK followed the method of Ling et al. (1966), in which the FDP produced by the PFK from F-6-P was converted by aldolase, triose phosphate isomerase and glycerophosphate dehydrogenase, to glycerophosphate with the concomitant oxidation of NADH. The change in absorbance at 340 m/z, as NADH was oxidized, was measured by a Beckman DB spectrophotometer and recorded on a Sargent SRL recorder. The standard concentrations for the assay included 50 mM Tris-C1 buffer, pH 7"6; variable F-6-P, ATP, MgC12; 1-0mM dithiothreitol; 0-15 mM NADH; and auxiliary enzymes (aldolase, triose phosphate isomerase and glycerophosphate dehydrogenase) added in excess and PFK to a total of 1-5 ml. Magnesium chloride was obtained from Mallinckrodt; dithiothreitol (Cleland's reagent) from Calbiochem; all others from Sigma. The F-6-P was obtained as the sodium salt and its concentration in solution standardized using the method of Hohorst (1963). Results were expressed in change in absorbance/min (AO.D./min) or/zmoles F-6-P converted to FDP/min per mg protein. The concentration of substrate at which 50 per cent maximal activity was observed was designated as (S)0.5 (Koshland et al., 1966). The cuvette temperature was controlled by a circulating methanol-water mixture kept at a constant temperature by a Lauda K-2/R circulator (Brinkman). The temperature of the cuvette contents was monitored intermittently by a YSI telethermometer. Studies on pH effects on goldfish PFK were performed substituting imidazole CI (Sigma) for the Tris-Cl buffer. The pH of the cuvette contents was measured immediately after the conclusion of the assay by a Beckman Zeromatic pH meter. RESULTS Nucleotides and intermediates Measurements of the concentration of goldfish muscle glycolytic intermediates, nucleotides, citrate and magnesium are given in Table 1. T h e concentrations of glycolytic intermediates (with the exception of glucose and F D P ) and of citrate are higher in the warm-acclimated goldfish than in the cold-acclimated goldfish when expressed on a wet weight basis. For all measurements except citrate and magnesium a paired student's t-test (Steel & Torrie, 1960) was used in the analysis of the data. T h e citrate and magnesium measurements were performed with a different series of unpaired fish and a non-paired Student's t-test (Steel & Torrie, 1960) was used to test the significance. Energy charge was calculated from the formula ( A T P + ½ A D P ) / ( A M P + A D P + A T P ) (Atkinson & Walton, 1967). T h e
750
JAMES M . FREED
TABLE 1 - - C O N C E N T R A T I O N
OF GLYCOLYTIC INTERMEDIATES, CITRATE, MAGNESIUM ADENINE NUCLEOTIDES IN COLD- AND WARM-ACCLIMATED GOLDFISH
AND
No. of fish
Acclimation temperature (°C)
M e a n + S.E.
P-value
/~moles/g w e t wt.
6 6
5 25
0"505 _+0"133 0"294 + 0'060
N.S.
/zmoles/g d r y wt.
6 6
5 25
2"88 + 0"84 1"39 + 0'29
<0"1
/zmoles/g w e t wt.
6 6
5 25
0"202 + 0"036 0'837 + 0-127
<0"01
/zmoles/g d r y wt.
6 6
5 25
1'18 + 0-21 4'11 +0"56
<0"01
/zmoles/g w e t wt.
6 6
5 25
0"0337 + 0"0039 0"160 + 0-024
<0'01
/zmoles/g d r y wt.
6 6
5 25
0-205 + 0"0346 0"749 + 0"105
<0"01
/zmoles/g w e t wt.
7 7
5 25
0"451 + 0"092 0"405 + 0-063
/~moles/g d r y wt.
7 7
5 25
2"51 + 0-57 1"98 + 0"36
N.S.
/zmoles/g w e t wt.
7 7
5 25
0"0716 + 0-0092 0"137 + 0-019
< 0"05
/~moles/g d r y wt.
7 7
5 25
0-393 + 0"060 0'673 + 0"110
N.S.
/zmoles/g w e t wt.
7 7
5 25
0"0457 + 0-0041 0"298 + 0'061
< 0"01
/zmoles/g d r y wt.
7 7
5 25
0-267 + 0"030 1 "52 _+0'30
< 0-01
Citrate
/zmoles/g w e t wt.
8 8
5 25
0"223 + 0"025 0"334 + 0'18
< 0-05
M g ~+
/zmoles/g w e t wt.
6 5
5 25
AMP
/zmoles/g w e t wt.
7 7
5 25
0" 123 + 0"021 0"195 + 0-041
<0-05
/zmoles/g d r y wt.
7 7
5 25
0"664 + 0-112 0"923 _+0"179
< 0-05
/zmoles/g w e t wt.
7 7
5 25
0"682 + 0"061 0"799 _+0"045
< 0'05
/zmoles/g d r y wt.
7 7
5 25
3"68 + 0"31 3"81 + 0'17
N.S.
Compound Glucose
G-6-P
F-6-P
FDP
Triose-PO**
Pyruvate
ADP
Measurement
17'0 _+ 1 "0 19"1 + 1"9
N.S.t
N.S.
751
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINA~E OF CA.RASSIUS AURATUS T.ad3LE 1 (Cont.)
Compound ATP
D r y wt.
No. of fish
Acclimation temperature (°C)
/~moles/g wet wt. /~moles/g dry wt.
7 7 7 7
5 25 5 25
3"67 + 0"33 4"86 + 0-23 19-8 + 1-61 23"2 + 0"77
Per cent dry wt.
7 7
5 25
18"6 + 0.61 21.0 + 0"48
Measurement
Mean + S.E.
P-value < 0-05 < 0"1
<0"05
* Triose-PO4 is equivalent to the sum of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. t N.S. = not significant.
energy charge showed no significant difference; for muscle of 5°C fish it was 0.894 and for 25°C fish, 0.898. I n order to ascertain whether or not these changes in concentration are dependent on a 1-2 week acclimation period or are a direct effect of t e m p e r a t u r e on the fish metabolism, the time course of change in three substances, A T P , G - 6 - P and F - 6 - P was measured. Results are shown in Figs. 1-3. Although Fig. 1 indicates
1.6
0
I 0
I I0
I 20
I 30
I 40
i 50
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Hours
FIG. 1. Changes in goldfish muscle ATP concentration as a function of time after transfer of the fish from 15 to 5°C and 25°C; V], 15°C; O, 25°C; O, 5°C. Mean _+S.E. for three fish except 15°C at which temperature five fish were used. an early drop in A T P in the fish transferred to 25°C, the only point that is significantly different between the fish at 5 ° and 15°C is at 6 hr. T h e 25°C fish was significantly higher than the 5°C fish in both G - 6 - P concentration (Fig. 2) and F - 6 - P concentration (Fig. 3) at all time periods except at 1 2 h r (0.1>P>0.05).
752
JAMES M. FREED
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96
FIG. 2. Changes in G-6-P concentration in goldfish muscle as a function of time after transfer of fish from 15 to 5°C and to 25°C; D, 15°C; O, 25°C; O, 5°C. Mean + S.E. at each point for three fish.
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FIG. 3. Changes in F-6-P concentration of goldfish muscle as a function of time after transfer of the fish from 15 to 5°C and to 25°C; [3, 15°C; O, 25°C; O, 5°C. Mean + S.E. at each point for three fish.
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF C A R A S S I U 8 A U R A T U S
753
Even at 3 hr after the temperature change, the levels of G-6-P and F-6-P were significantly higher in the 25°C fish muscle.
Phosphofructokinase, total activity To compare the total activity of muscle PFK in cold- and warm-acclimated goldfish, assays were conducted with the supernatant of a 25,000 g centrifugation of the initial 15% homogenate. To avoid errors produced by endogenous oxidation of NADH, the activity of the PFK without added F-6-P was subtracted from the activity with F-6-P present to give the results shown in Fig. 4. The trend shows the .08 .07 .06
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Acclimotion Temperature
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I 25
FIG. 4. Phosphofructokinase from muscle of goldfish acclimated to 5, 15 and 25°C and assayed at 5, 15 and 25°C; 25,000g supematant of a 15~ homogenate. (1.0 mM F-6-P, 0-1 mM ATP, 1.0 mM MgCI=.) 5°C-acclimated fish to be higher at all temperatures of assay, but the only significantly different points (P< 0.05) on this graph are between the fish acclimated to 5 and 15°C; and 5 and 25°C, both assayed at 5°C; and between 5 and 25°C, assayed at 15°C.
Phosphofructokinase, kinetic studies Fig. 5 gives results typical of several experiments showing the responses at different temperatures of measurement of goldfish muscle to changes in F-6-P
754
JAMES M. FREED
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FiG. 5. Goldfish P F K activity at various concentrations of F-6-P and at various temperatures. A, 5°C-acclimated goldfish; B, 25°C-acclimated goldfish. (0"5 m M ATP, 1"0 m M MgC12.)
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FIG. 6. Goldfish P F K activity at various concentrations of F-6-P and ATP; © and O, 5°C-acclimated fish assayed at 5°C; [] and i , 25 °C-acclimated goldfish assayed at 25°C. Unshaded symbols, 0"5 m M ATP ; shaded symbols, 0"1 m M ATP. (1"0 m M MgCla.)
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF C A R A S S I U S .4URATUS
755
concentration in the presence of 0.5 m M ATP (a concentration which does not inhibit PFK, Fig. 7). There was no significant difference in responses to the changes in concentration of F-6-P for fish from the two temperatures of acclimation. In both cases, however, an increase in (8)o. 5 is noted with increasing temperature of assay. A I00
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FzG. 7. Goldfish muscle PFK activity at different concentrations of ATP measured at 25°C (A) and 5°C (B). O and O, 5°C-acclimated goldfish; [] and II, 25°C-acdimated goldfish. (1"0 mM F-6-P, 1"0 mM MgClv) Fig. 6, typical of several studies, shows the effect of ATP on the changes in F-6-P saturation curve. In both the 5- and 25°C-acclimated fish, measured at their respective acclimation temperature, an increase in (S)o. 5 is noted with increase in ATP concentration. The effects of increasing ATP concentration on P F K are shown in Fig. 7, typical of several studies. No difference in action of ATP in activating the enzyme is apparent between the 5 and 25°C-acclimated goldfish; however, the (S)0. 5 values for A T P in both 5 and 25°C-acclimated fish are higher when assayed at 25°C than at 5°C. It appears that in the 25°C fish P F K is more inhibited by high ATP than in the 5°C-acclimated fish; but this difference was not consistently observed.
JAMES M. FREED
756
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mM F - 6 - P
.4
o.5
i.o ATP
; .5 2.o
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3.0
,/'
s.o
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FIG. 8. Muscle P F K activity from 5°C-acclimated goldfish at varying concentrations of ATP and at 0"2 mM and 1"0 mM F-6-P, and assayed at 15°C. (1"0 mM MgC12.) T h e effect of F - 6 - P concentration on A T P saturation curves for a 5°C-accli mated goldfish is shown in Fig. 8 ; to obtain the same percentage inhibition a m u c h higher concentration of A T P is necessary at 1"0 m M F - 6 - P than at 0.2 m M F-6-P. A similar result was observed for the 25°C-acclimated goldfish. Changes in m a g n e s i u m ion concentration seem not to affect the 5- and 25°C acclimated fish differently (Fig. 9). I t is apparent, however, that at the higher .30
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FIO. 9. Muscle PFK activity from 5°C-(O, I ) and 25°C-(D, , ) acclimated goldfish with varying concentrations of MgCI,. Shaded symbols assayed at 25°; unshaded symbols at 5°C. (1"0 mM F-6-P, 0"5 mM ATP.)
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF C A R A S S I U S A U R A T U S
757
assay temperature the percentage inhibition by high magnesium was greater than at the lower assay temperature. In contrast to the results with magnesium, citrate inhibited more at the 5°C assay temperature than at either 15 or 25°C assay temperature (Fig. 10). No apparent difference in 5 and 25°C-acclimated goldfish in their response to citrate was noted. ~00!~" ['~
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5 ° Acclimoted Golcrfish
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60
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FIc. 10. Muscle PFK activity from 5 and 25°C-acclimated goldfish with varying concentrations of citrate and assayed at various temperatures; 0 and O, at 5°(2; A and A, at 15°C; [] and n, at 25°C. (1"0 mM F-6-P, 0-5 mM ATP.)
Phosphofructokinase, pH studies Fig. 11 shows the effects of pH on muscle P F K from 5°C-acclimated goldfish. A sharp decline in activity was produced with small decreases in pH. Under physiological conditions of substrate, activity changed from 80 to 20 per cent of maximum activity within 0.1 pH unit. With decreasing concentration of F-6-P (Fig. llA) and magnesium (Fig. 11C), the pH curves shift to the right. With decreasing A T P concentration, the curve shifts to the left (Fig. 11B). Experiments done on the effect of pH upon activity of P F K from 25°C-acclimated fish gave similar results to those with the 5°C fish. 26
758
JAMES M.
FREED
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Fro. 11. Muscle PFK activity from 5°C-acclimated goldfish assayed at 15°C and at various pH's. A: 4"0mM ATP; 5"0mM MgC12; ~, l'0mM F-6-P; ©, 0"1 mM F-6-P; ~ , 0"05mM F-6-P. B: 0"1 mM F-6-P; 5.0mM MgCI~; V, 0"1 mM ATP; e, 1-0mM ATP; ©, 4-0raM ATP. C: 0"l mM F-6-P; 4"0raM ATP; [2, 20raM MgC12; V, 10raM MgC12; ©, 5 mM MgC12; l , l'0mM MgCI~.
DISCUSSION In vivo levels of metabolites The levels of cellular compounds given in Table 1 [comparing favorably with levels in rat heart (Williamson, 1956)], indicate that the concentration of most glycolytic intermediates is extremely low, when compared with concentrations necessary for maximum activity of the enzymes associated with them. Consequently, one must be cautious in interpreting results of enzyme studies performed at substrate levels producing maximum velocity. Furthermore, comparison of the measured concentration of intermediates with the activity curves show that phosphofructokinase would be markedly inhibited if the concentrations of intermediates in the microenvironment of the enzyme were the same as given by tissue analysis. CompartmentaSzation of the intermediates within the cell is, therefore, very probable and such compartmentalization makes difficult the extrapolation of data obtained in vitro to the in vivo situation.
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF C A R A S S I U S A U R A T U S
759
Properties of goldfish muscle P F K Phosphofructokinase catalyzes a rate-controlling step in glycolysis, and is markedly affected by the concentrations of a number of modulators. ATP has the most significant effect because it is a necessary and second substrate for the reaction (F-6-P to FDP) and also is an inhibitor at higher concentrations (sheep heart PFK, Ahlfors & Mansour, 1969; sheep brain PFK, Lowry & Passonneau, 1966; rabbit skeletal muscle PFK, Ui, 1968 and Hofer & Pette, 1968; calf lens PFK, Lou & Kinoshita, 1967; erythrocyte PFK, Kiihn et al., 1968; cockroach muscle PFK, Grasso & Natalizi, 1968; desert locust fat body and flight muscle PFK, Walker & Bailey, 1969; sheep liver fluke PFK, Stone & Mansour, 1967; and PFK from ascites carcinoma cells, Freyer et al., 1967; among others). ATP also inhibits king crab (Freed, 1971) and goldfish muscle PFK as shown in Figs. 6-8. The levels of ATP which produce inhibition are well below the levels which may be present in vivo (Table 1). A TCA cycle intermediate, citrate, is an inhibitor of PFK and has been studied extensively (in ascites carcinoma cells, Freyer et al., 1967; rabbit muscle, Garfinkel, 1965; adipose tissue, Denton & Randle, 1966; rat heart, Pogson & Randle, 1966; rat kidney cortex, Underwood & Newsholme, 1967; rat liver, Underwood & Newsholme 1965; among others). Citrate markedly affects the activity of both king crab (Freed, 1971) and goldfish PFK as seen in Fig. 10. The levels of citrate in goldfish (Table 1) may be high enough to have an effect on PFK control-particularly at higher ATP levels. Activity of PFK increases markedly with a rise in pH and decreases with a fall in pH over a very narrow range (Fig. 11). The pH at which activity increases changes with the concentration of substrates and modulators of the reaction. An increase in magnesium concentration shifts the pH of increased activity in goldfish PFK (Fig. 11C) to lower values. Similarly, Lowry & Passonneau (1966) observed in sheep brain that at pH 8.0, a molar ratio of magnesium : ATP of 2 : 1 was optimal whereas at pH 7.0, much higher ratios were necessary for optimal activity. Increased concentration of F-6-P shifts the pH curves of PFK toward lower values (Fig. l lA). Although in yeast, the pH optimum shifted to higher pH values at increased concentrations of F-6-P (Kopperschl~iger et al., 1969), animal systems show a marked lowering of the pH at which PFK activity is first noticed as a function of increasing F-6-P (rabbit skeletal muscle, Hofer & Pette, 1968; frog muscle, Trivedi & Danforth, 1966). That the lowering of pH optimum with increased F-6-P results from a release from ATP inhibition is shown by the further shift toward lower pH values on addition of 5' AMP (Trivedi & Danforth, 1966). ATP markedly shifts the pH optimum for goldfish PFK (Fig. llB), but this has not been consistently observed in other systems. No influence of pH on ATP inhibition occurs in monkey sperm PFK (Hoskins & Stephens, 1969). A decreased pH optimum as ATP concentration increases was found in yeast by Kopperschl~iger et al. (1968) and Lindell & Stellwagen (1968), but an increase in pH optimum as ATP concentration increases was found for frog skeletal muscle (Danforth, 1965); brussels sprouts (Dennis & Coultate, 1967); red blood cells (Kiihn et al., 1968);
760
JAMES 1~'I. FREED
and rabbit muscle (Hofer & Pette, 1968). Undoubtedly the affinity of ATP for inhibitory sites on P F K decreases markedly as the pH increases (Ui, 1968).
Temperature adaptation and control of metabolism Because P F K is the first rate-controlling enzyme unique to glycolysis and glycolytic rates change during temperature acclimation, it is expected that some changes in this enzyme may occur in the process of temperature acclimation. One possible way in which temperature may affect P F K is directly on the enzyme affinity for substrates and modulators. However, the compensatory responses in glycolysis (Hochachka & Hayes, 1962) during temperature acclimation of goldfish is not completely explained by the direct effect of temperature on the enzyme's affinity for F-6-P (Fig. 5). Higher temperatures do, however, decrease the affinity of P F K for ATP in the concentration range of A T P that activates the enzyme (Fig. 7); they increase the inhibition at higher concentrations of magnesium (Fig. 9); and decrease the amount of citrate inhibition (Fig. 10). These examples indicate a direct effect of temperature on enzyme-modulator affinity that may explain a change in glycolytic rate in temperature acclimation. That temperature does affect the affinity of modulators for other enzymes has been observed in the effect of fructose diphosphate and manganese on lungfish fructose diphosphatase (Behrisch & Hochachka, 1969a), and magnesium and AMP on trout fructose diphosphatase (Behrisch & Hochachka, 1969b). This temperature dependence of modulator action, however, is not shown by fructose diphosphate and ATP on pyruvate kinase of Trematomus bernacchii (Somero & Hochachka, 1968) ; AMP and magnesium on lungfish fructose diphosphatase (Behrisch & Hochachka, 1969a); fructose diphosphate and manganese on trout fructose diphosphatase (Behrisch & Hochachka, 1969b); and AMP in salmon fructose diphosphatase (Behrisch, 1969). A second possible explanation of changes in P F K activity in temperature acclimation is that ions may change which affect the P F K enzyme. Magnesium does not appear to change in goldfish (Table 1), but potassium does increase in cold acclimation in carp (Houston et al., 1970). That this increase in potassium may increase P F K activity has been shown in rabbit muscle (Paetkau & Lardy, 1967) and sheep liver and brain (Passonneau & Lowry, 1964). Another possible explanation of glycolytic compensation with temperature acclimation is the effect of metabolite changes on PFK. Table 1 indicates that 25°C-acclimated fish have increased concentrations of glycolytic intermediates and that the most significant differences are in the levels of G-6-P, F-6-P and pyruvate. At 25°C, F-6-P levels are about four times greater than at 5°C. Does this indicate increased PFK activity in the 5°C-acclimated fish--or is some other factor responsible for this difference ? Perhaps goldfish PFK has a lower Qlo than succeeding enzymatic steps in glycolysis. The concentration of citrate and of ATP are both slightly higher in the 25°C-acclimated fish than in the 5°C-acclimated fish; this could cause more inhibition of P F K in the 25°C fish than the 5°C fish; however, the slightly higher values of AMP and ADP in the 25°C-acclimated fish
PROPERTIES OF MUSCLE PHOSPHOFltUCTOKINASE OF C A R A S S I U S . A U R A T U S
761
may counteract the effect of the increased ATP. The similarity of changes for the two temperatures indicates that changes in amount of ATP are balanced by AMP and ADP; this suggests some conservation of energy levels in nucleotides. In all likelihood, then, the change in metabolites with temperature acclimation cannot completely explain increased muscle glycolysis in cold acclimation (Hochachka & Hayes, 1962). An additional possible explanation of how PFK activity may control glycolytic changes in temperature acclimation may be related to a change in pH with temperature. That temperature affects the pH of buffers (pH varying inversely with temperature) has been known for some time--particularly for amine buffers (Bates, 1961). That temperature changes may and do affect the plasma pH of a poikilotherm is becoming increasingly apparent (Howell, 1970). In frogs adapted to selected temperature for a minimum of 3 days the intracellular pH of skeletal muscle (but not in cardiac muscle) decreased 0.0154 units/degC temperature increase [pH 7.2 and 6.9 at 5 and 25°C, respectively (Reeves & Wilson, 1969; Reeves, personal communication)]. The effect of pH changes on glycolysis has been known since Ronzoni & Kerly (1933) demonstrated that high carbon dioxide concentrations inhibit the conversion of hexosemonophosphate to lactate. Danforth (1965) ascribed the pH effects on glycolysis in frog skeletal muscle to PFK. When concentrations of glycolytic intermediates were measured as a function of pH in supernatant fractions and in intact polymorphonuclear leukocytes of guinea pig (Halperin et al., 1969) and in erythrocytes (Kloppick et al., 1967), it was possible to pinpoint the site of pH effects on glycolysis at the PFK step. A similar conclusion was reached by Scheuer & Berry (1967) who noted decreased G-6-P and F-6-P and increased FDP and dihydroxyacetone phosphate with higher pH's in the isolated rat heart. An hypothesis to explain some of the effects of temperature acclimation on glycolysis in goldfish muscle may be presented as follows: goldfish PFK is markedly affected by pH (Fig. 11); as the temperature of the fish decreases, intracellular pH may increase and thus PFK is activated and a rise in the glycolytic rate results. Conversely, as the fish temperature rises, the pH decreases, causing an inhibition of PFK, and the glycolytic rate decreases. These effects would be direct and rapid, not requiring days or weeks for "acclimation". This hypothesis can also explain the time course and changes in amounts in F-6-P and G-6-P. As muscle temperature of fish increases over a period of a few hours, PFK is gradually inhibited and intermediates prior to PFK begin to increase in concentration. Lowered pH in glycolysis also inhibits glyceraldehyde phosphate dehydrogenase and/or diphosphoglycerate kinase in leukocytes (Halperin et al., 1969). Inhibition of either of these two enzymes in goldfish muscles at 25°C may explain the increased levels of triose phosphates. Changes in the rate of glycolysis in temperature acclimation may be dependent on types of isozymes, amount of each enzyme present, the concentration of modulators, the direct effect of temperature on the binding of enzymes and modulators
762
JAMES M. FREED
and ions. T o this list must now be added the possibility of p H change with temperature acclimation, particularly with respect to a p H change on the ratecontrolling enzyme, phosphofructokinase. For phosphofructokinase the direct effects of temperature on the many modulators may overbalance possible longterm acclimation changes.
Acknowledgements--Especial appreciation is expressed to Dr. C. Ladd Prosser of the University of Illinois for research guidance; also to a traineeship provided by National Institutes of Health (USPH 2G-619) for support and to research Grant GB 4005 from the National Science Foundation to Dr. C. Ladd Prosser. REFERENCES ADAM H. (1963a) Adenosine-5'-diphosphate and adenosine-5'monophosphate. In Methods of Enzymatic Analysis (Edited by BEaCMEYERH. U.), pp. 573-577. Academic Press, New York. ADAM H. (1963b) Adenosine-5'-triphosphate; determination with phosphoglycerate kinase. In Methods of Enzymatic Analysis (Edited by BERGMEYERH. U.), pp. 539-543. Academic Press, New York. AIaLFORSC. E. & MANSOURT. E. (1969) Studies on heart phosphofructokinase. Desensitization of the enzyme to adenosine triphosphate inhibition. J. biol. Chem. 244, 12471251. ATKINSOND. E. & WALTONG. M. (1967) Adenosine triphosphate conservation in metabolic regulation. J. biol. Chem. 242, 3239-3241. BATESR. G. (1961) Amine buffers for pH control. Ann. N. Y. Acad. Sci. 92, 341-356. BEHmSCH H. W. (1969) Temperature and the regulation of enzyme activity in poikilotherms. Fructose diphosphatase from migrating salmon. Biochem. J. 115, 687-696. BEHRISCHH. W. & HOCHACHKAP. W. (1969a) Temperature and the regulation of enzyme activity in poikilotherms. Properties of lungfish fructose diphosphatase. Biochem. ft. 112, 601-607. BEHRISCHH. W. & HOCHACHKAP. W. (1969b) Temperature and the regulation of enzyme activity in poikilotherms. Properties of rainbow-trout fructose diphosphatase. Biochem. ft. I I I , 287-295. BI]CHERT., CZOK R., LAMPRECHTW. & LATZKOF. (1963) Pyruvate. In Methods of Enzymatic Analysis (Edited by BERGM~ER H. U.), pp. 253-259. Academic Press, New York. B0CHER T. & HOHORSTH. J. (1963) Dihydroxyacetone phosphate, fructose-l,6-diphosphate and D-glyceraldehyde-3-phosphate; Determination with glycerol-l-phosphate dehydrogenase, aldolase and triosephosphate isomerase. In Methods of Enzymatic Analysis (Edited by BEaCMEYERH. U.), pp. 246-252. Academic Press, New York. CzoK R. & ECK~T L. (1963) D-3-phosphoglycerate, D-2-phosphoglycerate, phosphoenolpyruvate. In Methods of Enzymatic Analysis (Edited by BERGME'~R H. U.), pp. 224233. Academic Press, New York. DANFORTHW. H. (1965) Activation of glycolytic pathway in muscle. In Control of Energy Metabolism (Edited by CHANCEB., ESTAImOOKR. W. & WILLIAMSONJ. R.), pp. 287297. Academic Press, New York. DENNIS D. T. & COULTATET. P. (1967) The regulatory properties of a plant phosphofructokinase during leaf development. Biochim. biophys. Acta 146, 129-137. DENTON R. M. & RANDLEP. J. (1966) Citrate and the regulation of adipose-tissue phosphofructokinase. Biochem. ft. 100, 420-423. FREED J. M. (1971) Temperature effects on muscle phosphofructokinase of the Alaskan king crab, Paralithodes camtschatica. Comp. Biochem. Physiol. (In press.)
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF C A R A S S I U S
AURATUS
763
R. (1965) Enzyme profile of beef heart supernatant fraction. In Control of Energy Metabolism (Edited by CHANCEB., ESTAnROOKR. W. & WILLIAMSONJ. R.), pp. 123-
F~L
125. Academic Press, New York. F ~ R., HOFMANN E. & FRANK K. (1967) Substrat- und Effektorwirkungen auf die Phosphofructokinase yon Aszitesturnorzellen. Acta biol. reed. germ. 19, 737-749. GARFINKELD. (1965) A computer simulation study of the metabolic control behavior of phosphofructokinase. In Control of Energy Metabolism (Edited by CHANCE B., ESTABROOKR. W. & WILLIAMSONJ. R.), pp. 101-106. Academic Press, New York. GRASSO A. & NATALIZIG. M. (1968) Studies on insect (Periplaneta americana L.) phosphofructokinase. Comp. Biochem. Physiol. 26, 979-984. HALPZRINM. L., CONNORSH. P., R~LMANA. S. & KARNOVSKYM. L. (1969) Factors that control the effect of pH on glycolysis in leukocytes..7, biol. Chem. 244, 384--390. HAZEL J. & PROSSER C. L. (1970) Interpretation of inverse acclimation to temperature. Z. vergl. Physiol. 67, 217-228. HOCHACHKAP. W. & HAYESF. R. (1962) The effect of temperature acclimation on pathways of glucose metabolism in the trout. Can. J. Zool. 40, 261-270. HOFER H. W. & PETTE D. (1968) Wirkungen and Wechselwirkungen yon Substraten and Effektoren an der Phosphofructokinase des Kaninchen-Skeletmuskels. Hoppe-Seyler's Z. physiol. Chem. 349, 1378-1392. HOHOP~STH. J. (1963) D-glucose-6-phosphate and D-fructose-6-phosphate. In Methods of Enzymatic Analysis (Edited by B]mG~YER H. U.), pp. 134-138. Academic Press, New York. HOSKINSD. D. & STEPH~S D. T. (1969) Regulation of primate sperm phosphofructokinase. Fedn Proc. Fedn Am. Socs exp. Biol. 28, 705. HOUSTONA. H., MADDENJ. A. & DEWILDEM. A. (1970) Environmental temperature and the body fluid system of the fresh-water teleostJIV. Water-electrolyte regulation in thermally acclimated carp, Cyprinus carpio. Comp. Biochem. Physiol. 34, 805-818. HOWELL B. J., BAUMGARDNERF. W., BONDI K. & RAHN H. (1970) Acid-base balance in cold-blooded vertebrates as a function of body temperature. Am. ft. Physiol. 218, 600-606. KLOPPICK E., JACOBASCHG. & RAPOPORTS. (1967) Steigerung der Glykolyse dutch den Einfluss yon Ammonium ionen auf die Phosphofruktokinaseaktivit~t. Acta biol. med. germ. 18, 37-42. KOPPlmSCHLXGERG., FREY~RR., DIF.Z]~LW. & HOFMANNE. (1968) Some kinetic and molecular properties of yeast phosphofructokinase. FEBS Letters 1, 137-141. KOSHLAND D. E., JR., NEMETHY G. & FILMER D. (1966) Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry, N.Y. 5, 365-385. KOHN B., JACOBASCHC. & RAPOPORT S. (1968) Kinetische Eigenschaften der Phosphofruktokinase toter Blutzellen. Folia haemat., Lpz. 89, 436 A.A,1. LAY~ ENNXS(1957) Spectxophotometric and turbidimetric methods for measuring proteins. In Mcthods in Enzymology (Edited by COLOWlCKS. P. & KAPLANN. O.), Vol. 3, pp. 447-454. Academic Press, New York. LINDELLT. J. & STELLWAGENE. (1968) Purification and properties of phosphofructokinase from yeast..7, biol. Chem. 243, 907-912. LING K. H., PA~TKAUV., MARCUS F. & LARDY H. A. (1966) Phosphofructokinase---I. Skeletal muscle. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPL~U~N. O.), Vol. 9, pp. 4-25-429. Academic Press, New York. Lou 1VL F. & KINOSHITAJ. H. (1967) Control of lens glycolysis. Biochim. biophys. Acta 141, 547-559. LowRY O. H. & PASSONNEAUJ. V. (1966) Kinetic evidence for multiple binding sites on phosphofructokinase. ~. biol. Chem. 241, 2268-2279.
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JAMES M. FREED
PAETKAU V. & LARDY H. A. (1967) Phosphofructokinase--correlation of physical and enzymatic properties, ft. biol. Chem. 242, 2035-2042. PASSONNEAU J. V. & LOWRY O. H. (1964) The role of phosphofructokinase in metabolic regulation. In Advances in Enzyme Regulation (Edited by WEBER G.), Vol. 2, pp. 265274. POGSON C. I. & RANDLE P. J. (1966) The control of rat-heart phosphofructokinase by citrate and other regulators. Biochem. ft. 100, 683-693. REEVES R. B. & WILSON T. L. (1969) Intracellular p H in bullfrog striated and cardiac muscle as a function of body temperature. Fedn Proc. Fedn Am. Socs exp. Biol. 28, 782. RONZONI E. & KERLY M. (1933) The disappearance of hexosephosphate from intact frog muscle, ft. biol. Chem. 103, 175-181. SCHEUER J. & BERRY M. (1967) Effect of alkalosis on glycolysis in the isolated rat heart. Am. ft. Physiol. 213, 1143-1148. SOMERO G. N. & HOCHACrlKA P. W. (1969) Isoenzymes and short-term temperature compensation in poikilotherms: activation of lactate dehydrogenase isoenzymes by temperature decreases. Nature, Lond. 223, 194-195. STEEL R. G. D. & TORRIE J. H. (1960) Principles and Procedures of Statistics. McGraw-Hill, New York. STERN J. R. (1957) Assay of tricarboxylic acids. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. 3, 425-431. Academic Press, New York. STONE D. B. & MANSOUR T. E. (1967) Phosphofructokinase from the liver fluke Fasciola hepatica--II. Kinetic properties of the enzyme. Mol. Pharmacol. 3, 177-187. TRIWDI B. & DANEORTH W. H. (1966) Effect of p H on the kinetics of frog muscle phosphofructokinase, ft. biol. Chem. 241, 4110-4112. UI MICHIO (1968) Multiple inhibitor sites for A T P on muscle phosphofructokinase as influenced by a change of p H : a computer analysis of nonlinear kinetic data. Biochim. biophys. Acta 159, 50-63. UNDERWOOD A. H. & NEWSHOLME E. A. (1965) Properties of phosphofructokinase from rat liver and their relation to the control of glycolysis and gluconeogenesis. Biochem. J. 95, 868-875. UNDERWOOD A. H, & NEWSHOLME E. A. (1967) Some properties of phosphofructokinase from kidney cortex and their relation to glucose metabolism. Biochem. J. 104, 296-299. WALLER P. R. & BAILEY E. (1969) A comparison of the properties of the phosphofructokinase of the fat body and flight muscle of the adult male desert locust. Biochem. J. 111,365-369. WILLIAMSON J. R. (1966) Glycolytic control m e c h a n i s m s - - I I . Kinetics of intermediate changes during the aerobic-anoxic transition in perfused rat heart. J. biol. Chem. 241, 5026-5036. WILSON J. E., SACKTORB. & TIEKERT C. G. (1967) In situ regulation of glycolysis in tetanized cat skeletal muscle. Arehs Biochem. Biophys. 120, 542-546.
Key Word Index'--Acclimation; Carassius auratus ; control mechanisms ; fish; glycolysis; goldfish; p H ; phosphofructokinase; temperature effects.
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF COLD- AND WARM-ACCLIMATED C A R A S S I U S A U R A T U S JAMES M. F R E E D * Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois 61801 (Received 21 December 1970)
Abstract--1. Phosphofructokinase (PFK) activity of homogenate super-
natants of 5°C-acclimated goldfish is slightly more than that of 25°C-acclimated goldfish when measured at 5° and 15°C, but not at 25°C. 2. Muscles of warm-acclimated goldfish have a large increase relative to 5°C fish in several glycolytic intermediates and adenine nudeotides; the increases in fructose-6-phosphate (F-6-P) and glucose-6-phosphate (G-6-P) appear within 3 hours after the fish are exposed to the selected temperature. 3. Magnesium inhibits PFK at higher temperatures (25°C); citrate inhibits less at higher than at lower assay temperatures. 4. Goldfish muscle PFK activity changes markedly within a small pH range, and it is suggested that if goldfish muscle intraceUular pH decreases in warm acclimation, this pH decrease, by its action on PFK, would cause the observed effects of decreased glyeolytic rate and increased levels of F-6-P and G-6-P. INTRODUCTION WHEN a poikilotherm is transferred from one temperature to another, its physiology and biochemistry may undergo a series of adaptations or compensations so that the organism may live successfully at the new temperature. Part of these compensations include changes in enzyme activity and changes in pathways. Increased activity of an enzyme in cold acclimation may be due to increased amounts of the enzyme or of specific isozymes or to changes in various co-factors or modulators. Relative changes in rates of carbon flow through alternate pathways are indicated by a fivefold increase in glycolysis in muscle of cold-acclimated over warm-acclimated trout (Hochachka & Hayes, 1962). Since the enzyme phosphofruetokinase (ATP: D-fructose-6-phosphate-l-phosphotransferase, E.C. 2.7.1.11) is considered to be the first rate-controlfing enzyme unique to glyeolysis (Frenkel, 1965 ; Wilson et al., 1967), it appeared to be the enzyme most likely to be affected in temperature adaptation. P F K is a cytoplasmic enzyme and not known to be present in isozymes (Kopperschl/iger et al., 1968; LindeU & Stellwagen, 1968), but to be regulated by its substrates and various modulators.t * Present address: Department of Zoology, Ohio Wesleyan University, Delaware, Ohio 43015. t Abbreviations used: EDTA = ethylenediaminetetraacetic acid; FDP = fructose-l,6diphosphate; F-6-P = fructose-6-phosphate; G-6-P = glucose-6-phosphate; PFK = phosphofructokinase; Tris-C1 = tris(hydroxymethyl)arninomethane-chloride. 747
748
JAMESM. FREED MATERIALS AND METHODS
Animals Goldfish (Carassius auratus, L), obtained from Auburndale Goldfish Co. (Chicago) and averaging 33 g in weight and 15 cm in length, were kept in aerated well water at 15:C (12-hr photoperiod) for at least 1 week after arrival before being transferred to either 25 or 5°C. The fish were kept at these temperatures at least 2 weeks during which they were fed Conditioner Goldfish Food (Wardley) twice each day at 25°C; once each day at 15°C; and once every other day at 5°C. For the time course studies of changes in ATP, G-6-P and F-6-P, with temperature of acclimation, fish were transferred in 15°C water to 5° and 25°C rooms. By gradual addition of colder or warmer water, the temperature of the aquaria in which they were placed reached the selected temperature of 5 or 25°C within 30 rain. For measurements at 3, 6 and 12 hr at the new temperatures, the fish were not fed after transfer to the new temperature. Fish at the new temperatures for 24 hr were fed once during this time ; fish at 96 hr were fed seven times at 25°C and twice at 5°C. No fish was fed during a period of 12 hr prior to its sacrifice.
Measurement of nucleotides and glycolytic intermediates Nucleotides and glycolytic intermediates were measured in muscles from cold- and warm-acclimated goldfish. Preparation of the solutions to be tested followed methods adapted from Adam (1963b). Fish were obtained from the temperatures of acclimation, decapitated as quickly as possible and a slice of dorsal trunk musculature was dropped into liquid nitrogen. A second slice was weighed and placed in a 110°C oven, and the dry weight was determined after a m i n i m u m of 12 hr. About 1"5 g of the frozen tissue was pulverized with a mortar and pestle cooled with liquid nitrogen, and frozen 0'9 N percholoric acid equivalent in volume to one-and-a-half times the weight of the tissue was added to the pulverized muscle. After being allowed to thaw at 5°C, the mixture was homogenized, centrifuged at 3000g for 10rain, the precipitate resuspended in 0"9 N perchloric acid, recentrifuged and the combined supernatants neutralized to pH 6"5 with 3"75 M potassium carbonate. After 1 hr at 5°C, the preparation was centrifuged and the volume of the supernatant was measured and subsequently used in the assays. Several experiments with small fish (4-5 cm in length) dropped directly into liquid nitrogen indicated that no significant change in ratios of the adenine nucleotides occurred during the few seconds required to decapitate the fish and remove the muscle before freezing. Enzymatic methods for measurement of nucleotides and glycolytic intermediates were based on the conversion of either N A D H to NAD, or of N A D P to N A D P H as measured at 340 m/~. Assays were performed in a Beckman DB spectrophotometer with a Sargent Recorder model SRL. Adenine nucleotides were measured with kits prepared by Boehringer. Procedures followed that of Adam (1963b) for A T P and that of Adam (1963a) for ADP and AMP. Procedures for the measurements of glucose, G-6-P and F-6-P were adapted from the method of Hohorst (1963). NADP, ATP, G-6-P dehydrogenase and hexokinase were obtained from Sigma; phosphoglucose isomerase was obtained from both Sigma and Calbiochem. Wriethanolamine was obtained from Matheson, Coleman and Bell. The sum of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate (triose phosphates) and the concentration of fructose-l,6-diphosphate were determined by the methods of Biicher & Hohorst (1963). Aldolase and a mixture of glycerophosphate dehydrogenase and triosephosphate isomerase were obtained from Sigma. Phospho-enol-pyruvate was measured by procedures adapted from Czok & Eckert (1963). Pyruvate kinase and lactate dehydrogenase were purchased from Boehringer. The concentration of pyruvate was measured with the same kit from Boehringer used to measure nucleoside diphosphates and monophosphates using the procedure of Adam (1963a), or by the procedure of BiJcher et al.
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF C A R A S S I U S A U P ~ g T U S
749
(1963). Citric acid was measured using the procedure of Stem (1957). Magnesium was measured on a nitric acid digest of muscle by the use of an atomic absorption flame photometer (Evans Electroselenium Ltd., England). Results are expressed in terms of micromoles of the specified substance/gram tissue either dry weight or wet weight. Preparation and assay of enzyme Homogenates of goldfish muscle, 15 per cent by weight, were prepared in 0"1 M K2HPO4 and centrifuged at 25,000 g for 30 rain. Ammonium sulfate was added to the supernatant and the resulting precipitate from a 25% saturated ammonium sulfate solution was resuspended in 0"01 M Tris-C1, pH 7"6, 1 mM in EDTA, and dialyzed against 300 ml 0-01 M Tris-C1, pH 7"6, for 3-5 hr with three changes of the buffer. This preparation produced a tenfold increase in specific activity of the enzyme above the original supematant and with 90-100 per cent recovery. Protein was analyzed by the Lowry method (Layne, 1957) using bovine serum albumin (Sigma) as the standard; measurements were at 750 m/z with a Beckman DB spectrophotometer. Assays for the goldfish muscle PFK followed the method of Ling et al. (1966), in which the FDP produced by the PFK from F-6-P was converted by aldolase, triose phosphate isomerase and glycerophosphate dehydrogenase, to glycerophosphate with the concomitant oxidation of NADH. The change in absorbance at 340 m/z, as NADH was oxidized, was measured by a Beckman DB spectrophotometer and recorded on a Sargent SRL recorder. The standard concentrations for the assay included 50 mM Tris-C1 buffer, pH 7"6; variable F-6-P, ATP, MgC12; 1-0mM dithiothreitol; 0-15 mM NADH; and auxiliary enzymes (aldolase, triose phosphate isomerase and glycerophosphate dehydrogenase) added in excess and PFK to a total of 1-5 ml. Magnesium chloride was obtained from Mallinckrodt; dithiothreitol (Cleland's reagent) from Calbiochem; all others from Sigma. The F-6-P was obtained as the sodium salt and its concentration in solution standardized using the method of Hohorst (1963). Results were expressed in change in absorbance/min (AO.D./min) or/zmoles F-6-P converted to FDP/min per mg protein. The concentration of substrate at which 50 per cent maximal activity was observed was designated as (S)0.5 (Koshland et al., 1966). The cuvette temperature was controlled by a circulating methanol-water mixture kept at a constant temperature by a Lauda K-2/R circulator (Brinkman). The temperature of the cuvette contents was monitored intermittently by a YSI telethermometer. Studies on pH effects on goldfish PFK were performed substituting imidazole CI (Sigma) for the Tris-Cl buffer. The pH of the cuvette contents was measured immediately after the conclusion of the assay by a Beckman Zeromatic pH meter. RESULTS Nucleotides and intermediates Measurements of the concentration of goldfish muscle glycolytic intermediates, nucleotides, citrate and magnesium are given in Table 1. T h e concentrations of glycolytic intermediates (with the exception of glucose and F D P ) and of citrate are higher in the warm-acclimated goldfish than in the cold-acclimated goldfish when expressed on a wet weight basis. For all measurements except citrate and magnesium a paired student's t-test (Steel & Torrie, 1960) was used in the analysis of the data. T h e citrate and magnesium measurements were performed with a different series of unpaired fish and a non-paired Student's t-test (Steel & Torrie, 1960) was used to test the significance. Energy charge was calculated from the formula ( A T P + ½ A D P ) / ( A M P + A D P + A T P ) (Atkinson & Walton, 1967). T h e
750
JAMES M . FREED
TABLE 1 - - C O N C E N T R A T I O N
OF GLYCOLYTIC INTERMEDIATES, CITRATE, MAGNESIUM ADENINE NUCLEOTIDES IN COLD- AND WARM-ACCLIMATED GOLDFISH
AND
No. of fish
Acclimation temperature (°C)
M e a n + S.E.
P-value
/~moles/g w e t wt.
6 6
5 25
0"505 _+0"133 0"294 + 0'060
N.S.
/zmoles/g d r y wt.
6 6
5 25
2"88 + 0"84 1"39 + 0'29
<0"1
/zmoles/g w e t wt.
6 6
5 25
0"202 + 0"036 0'837 + 0-127
<0"01
/zmoles/g d r y wt.
6 6
5 25
1'18 + 0-21 4'11 +0"56
<0"01
/zmoles/g w e t wt.
6 6
5 25
0"0337 + 0"0039 0"160 + 0-024
<0'01
/zmoles/g d r y wt.
6 6
5 25
0-205 + 0"0346 0"749 + 0"105
<0"01
/zmoles/g w e t wt.
7 7
5 25
0"451 + 0"092 0"405 + 0-063
/~moles/g d r y wt.
7 7
5 25
2"51 + 0-57 1"98 + 0"36
N.S.
/zmoles/g w e t wt.
7 7
5 25
0"0716 + 0-0092 0"137 + 0-019
< 0"05
/~moles/g d r y wt.
7 7
5 25
0-393 + 0"060 0'673 + 0"110
N.S.
/zmoles/g w e t wt.
7 7
5 25
0"0457 + 0-0041 0"298 + 0'061
< 0"01
/zmoles/g d r y wt.
7 7
5 25
0-267 + 0"030 1 "52 _+0'30
< 0-01
Citrate
/zmoles/g w e t wt.
8 8
5 25
0"223 + 0"025 0"334 + 0'18
< 0-05
M g ~+
/zmoles/g w e t wt.
6 5
5 25
AMP
/zmoles/g w e t wt.
7 7
5 25
0" 123 + 0"021 0"195 + 0-041
<0-05
/zmoles/g d r y wt.
7 7
5 25
0"664 + 0-112 0"923 _+0"179
< 0-05
/zmoles/g w e t wt.
7 7
5 25
0"682 + 0"061 0"799 _+0"045
< 0'05
/zmoles/g d r y wt.
7 7
5 25
3"68 + 0"31 3"81 + 0'17
N.S.
Compound Glucose
G-6-P
F-6-P
FDP
Triose-PO**
Pyruvate
ADP
Measurement
17'0 _+ 1 "0 19"1 + 1"9
N.S.t
N.S.
751
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINA~E OF CA.RASSIUS AURATUS T.ad3LE 1 (Cont.)
Compound ATP
D r y wt.
No. of fish
Acclimation temperature (°C)
/~moles/g wet wt. /~moles/g dry wt.
7 7 7 7
5 25 5 25
3"67 + 0"33 4"86 + 0-23 19-8 + 1-61 23"2 + 0"77
Per cent dry wt.
7 7
5 25
18"6 + 0.61 21.0 + 0"48
Measurement
Mean + S.E.
P-value < 0-05 < 0"1
<0"05
* Triose-PO4 is equivalent to the sum of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. t N.S. = not significant.
energy charge showed no significant difference; for muscle of 5°C fish it was 0.894 and for 25°C fish, 0.898. I n order to ascertain whether or not these changes in concentration are dependent on a 1-2 week acclimation period or are a direct effect of t e m p e r a t u r e on the fish metabolism, the time course of change in three substances, A T P , G - 6 - P and F - 6 - P was measured. Results are shown in Figs. 1-3. Although Fig. 1 indicates
1.6
0
I 0
I I0
I 20
I 30
I 40
i 50
p. i *" 9 6
Hours
FIG. 1. Changes in goldfish muscle ATP concentration as a function of time after transfer of the fish from 15 to 5°C and 25°C; V], 15°C; O, 25°C; O, 5°C. Mean _+S.E. for three fish except 15°C at which temperature five fish were used. an early drop in A T P in the fish transferred to 25°C, the only point that is significantly different between the fish at 5 ° and 15°C is at 6 hr. T h e 25°C fish was significantly higher than the 5°C fish in both G - 6 - P concentration (Fig. 2) and F - 6 - P concentration (Fig. 3) at all time periods except at 1 2 h r (0.1>P>0.05).
752
JAMES M. FREED
i
.30
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T
I .15 (b .io
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I
20
I
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I
st
50 H
I
96
FIG. 2. Changes in G-6-P concentration in goldfish muscle as a function of time after transfer of fish from 15 to 5°C and to 25°C; D, 15°C; O, 25°C; O, 5°C. Mean + S.E. at each point for three fish.
.04
~ .03 \
I
T
I
!
i ,02
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r 20
i ~30
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I 96
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FIG. 3. Changes in F-6-P concentration of goldfish muscle as a function of time after transfer of the fish from 15 to 5°C and to 25°C; [3, 15°C; O, 25°C; O, 5°C. Mean + S.E. at each point for three fish.
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF C A R A S S I U 8 A U R A T U S
753
Even at 3 hr after the temperature change, the levels of G-6-P and F-6-P were significantly higher in the 25°C fish muscle.
Phosphofructokinase, total activity To compare the total activity of muscle PFK in cold- and warm-acclimated goldfish, assays were conducted with the supernatant of a 25,000 g centrifugation of the initial 15% homogenate. To avoid errors produced by endogenous oxidation of NADH, the activity of the PFK without added F-6-P was subtracted from the activity with F-6-P present to give the results shown in Fig. 4. The trend shows the .08 .07 .06
.05
.04
Acclimotion Temperature
~
t'C]
~.03
=~.oz
15 ;'5
~.,
I 15 Assay T e m p e r a t u r e ('C)
I 25
FIG. 4. Phosphofructokinase from muscle of goldfish acclimated to 5, 15 and 25°C and assayed at 5, 15 and 25°C; 25,000g supematant of a 15~ homogenate. (1.0 mM F-6-P, 0-1 mM ATP, 1.0 mM MgCI=.) 5°C-acclimated fish to be higher at all temperatures of assay, but the only significantly different points (P< 0.05) on this graph are between the fish acclimated to 5 and 15°C; and 5 and 25°C, both assayed at 5°C; and between 5 and 25°C, assayed at 15°C.
Phosphofructokinase, kinetic studies Fig. 5 gives results typical of several experiments showing the responses at different temperatures of measurement of goldfish muscle to changes in F-6-P
754
JAMES M. FREED
80 A5Acea
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i
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FiG. 5. Goldfish P F K activity at various concentrations of F-6-P and at various temperatures. A, 5°C-acclimated goldfish; B, 25°C-acclimated goldfish. (0"5 m M ATP, 1"0 m M MgC12.)
.8
-S
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FIG. 6. Goldfish P F K activity at various concentrations of F-6-P and ATP; © and O, 5°C-acclimated fish assayed at 5°C; [] and i , 25 °C-acclimated goldfish assayed at 25°C. Unshaded symbols, 0"5 m M ATP ; shaded symbols, 0"1 m M ATP. (1"0 m M MgCla.)
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF C A R A S S I U S .4URATUS
755
concentration in the presence of 0.5 m M ATP (a concentration which does not inhibit PFK, Fig. 7). There was no significant difference in responses to the changes in concentration of F-6-P for fish from the two temperatures of acclimation. In both cases, however, an increase in (8)o. 5 is noted with increasing temperature of assay. A I00
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FzG. 7. Goldfish muscle PFK activity at different concentrations of ATP measured at 25°C (A) and 5°C (B). O and O, 5°C-acclimated goldfish; [] and II, 25°C-acdimated goldfish. (1"0 mM F-6-P, 1"0 mM MgClv) Fig. 6, typical of several studies, shows the effect of ATP on the changes in F-6-P saturation curve. In both the 5- and 25°C-acclimated fish, measured at their respective acclimation temperature, an increase in (S)o. 5 is noted with increase in ATP concentration. The effects of increasing ATP concentration on P F K are shown in Fig. 7, typical of several studies. No difference in action of ATP in activating the enzyme is apparent between the 5 and 25°C-acclimated goldfish; however, the (S)0. 5 values for A T P in both 5 and 25°C-acclimated fish are higher when assayed at 25°C than at 5°C. It appears that in the 25°C fish P F K is more inhibited by high ATP than in the 5°C-acclimated fish; but this difference was not consistently observed.
JAMES M. FREED
756
1.2 ~~'/"-~l.0
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mM F - 6 - P
.4
o.5
i.o ATP
; .5 2.o
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FIG. 8. Muscle P F K activity from 5°C-acclimated goldfish at varying concentrations of ATP and at 0"2 mM and 1"0 mM F-6-P, and assayed at 15°C. (1"0 mM MgC12.) T h e effect of F - 6 - P concentration on A T P saturation curves for a 5°C-accli mated goldfish is shown in Fig. 8 ; to obtain the same percentage inhibition a m u c h higher concentration of A T P is necessary at 1"0 m M F - 6 - P than at 0.2 m M F-6-P. A similar result was observed for the 25°C-acclimated goldfish. Changes in m a g n e s i u m ion concentration seem not to affect the 5- and 25°C acclimated fish differently (Fig. 9). I t is apparent, however, that at the higher .30
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FIO. 9. Muscle PFK activity from 5°C-(O, I ) and 25°C-(D, , ) acclimated goldfish with varying concentrations of MgCI,. Shaded symbols assayed at 25°; unshaded symbols at 5°C. (1"0 mM F-6-P, 0"5 mM ATP.)
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF C A R A S S I U S A U R A T U S
757
assay temperature the percentage inhibition by high magnesium was greater than at the lower assay temperature. In contrast to the results with magnesium, citrate inhibited more at the 5°C assay temperature than at either 15 or 25°C assay temperature (Fig. 10). No apparent difference in 5 and 25°C-acclimated goldfish in their response to citrate was noted. ~00!~" ['~
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5 ° Acclimoted Golcrfish
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FIc. 10. Muscle PFK activity from 5 and 25°C-acclimated goldfish with varying concentrations of citrate and assayed at various temperatures; 0 and O, at 5°(2; A and A, at 15°C; [] and n, at 25°C. (1"0 mM F-6-P, 0-5 mM ATP.)
Phosphofructokinase, pH studies Fig. 11 shows the effects of pH on muscle P F K from 5°C-acclimated goldfish. A sharp decline in activity was produced with small decreases in pH. Under physiological conditions of substrate, activity changed from 80 to 20 per cent of maximum activity within 0.1 pH unit. With decreasing concentration of F-6-P (Fig. llA) and magnesium (Fig. 11C), the pH curves shift to the right. With decreasing A T P concentration, the curve shifts to the left (Fig. 11B). Experiments done on the effect of pH upon activity of P F K from 25°C-acclimated fish gave similar results to those with the 5°C fish. 26
758
JAMES M.
FREED
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o
.I
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.I I
d 0
I 7.0 7.1 7.2 7.3 7.4 7 5
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Fro. 11. Muscle PFK activity from 5°C-acclimated goldfish assayed at 15°C and at various pH's. A: 4"0mM ATP; 5"0mM MgC12; ~, l'0mM F-6-P; ©, 0"1 mM F-6-P; ~ , 0"05mM F-6-P. B: 0"1 mM F-6-P; 5.0mM MgCI~; V, 0"1 mM ATP; e, 1-0mM ATP; ©, 4-0raM ATP. C: 0"l mM F-6-P; 4"0raM ATP; [2, 20raM MgC12; V, 10raM MgC12; ©, 5 mM MgC12; l , l'0mM MgCI~.
DISCUSSION In vivo levels of metabolites The levels of cellular compounds given in Table 1 [comparing favorably with levels in rat heart (Williamson, 1956)], indicate that the concentration of most glycolytic intermediates is extremely low, when compared with concentrations necessary for maximum activity of the enzymes associated with them. Consequently, one must be cautious in interpreting results of enzyme studies performed at substrate levels producing maximum velocity. Furthermore, comparison of the measured concentration of intermediates with the activity curves show that phosphofructokinase would be markedly inhibited if the concentrations of intermediates in the microenvironment of the enzyme were the same as given by tissue analysis. CompartmentaSzation of the intermediates within the cell is, therefore, very probable and such compartmentalization makes difficult the extrapolation of data obtained in vitro to the in vivo situation.
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF C A R A S S I U S A U R A T U S
759
Properties of goldfish muscle P F K Phosphofructokinase catalyzes a rate-controlling step in glycolysis, and is markedly affected by the concentrations of a number of modulators. ATP has the most significant effect because it is a necessary and second substrate for the reaction (F-6-P to FDP) and also is an inhibitor at higher concentrations (sheep heart PFK, Ahlfors & Mansour, 1969; sheep brain PFK, Lowry & Passonneau, 1966; rabbit skeletal muscle PFK, Ui, 1968 and Hofer & Pette, 1968; calf lens PFK, Lou & Kinoshita, 1967; erythrocyte PFK, Kiihn et al., 1968; cockroach muscle PFK, Grasso & Natalizi, 1968; desert locust fat body and flight muscle PFK, Walker & Bailey, 1969; sheep liver fluke PFK, Stone & Mansour, 1967; and PFK from ascites carcinoma cells, Freyer et al., 1967; among others). ATP also inhibits king crab (Freed, 1971) and goldfish muscle PFK as shown in Figs. 6-8. The levels of ATP which produce inhibition are well below the levels which may be present in vivo (Table 1). A TCA cycle intermediate, citrate, is an inhibitor of PFK and has been studied extensively (in ascites carcinoma cells, Freyer et al., 1967; rabbit muscle, Garfinkel, 1965; adipose tissue, Denton & Randle, 1966; rat heart, Pogson & Randle, 1966; rat kidney cortex, Underwood & Newsholme, 1967; rat liver, Underwood & Newsholme 1965; among others). Citrate markedly affects the activity of both king crab (Freed, 1971) and goldfish PFK as seen in Fig. 10. The levels of citrate in goldfish (Table 1) may be high enough to have an effect on PFK control-particularly at higher ATP levels. Activity of PFK increases markedly with a rise in pH and decreases with a fall in pH over a very narrow range (Fig. 11). The pH at which activity increases changes with the concentration of substrates and modulators of the reaction. An increase in magnesium concentration shifts the pH of increased activity in goldfish PFK (Fig. 11C) to lower values. Similarly, Lowry & Passonneau (1966) observed in sheep brain that at pH 8.0, a molar ratio of magnesium : ATP of 2 : 1 was optimal whereas at pH 7.0, much higher ratios were necessary for optimal activity. Increased concentration of F-6-P shifts the pH curves of PFK toward lower values (Fig. l lA). Although in yeast, the pH optimum shifted to higher pH values at increased concentrations of F-6-P (Kopperschl~iger et al., 1969), animal systems show a marked lowering of the pH at which PFK activity is first noticed as a function of increasing F-6-P (rabbit skeletal muscle, Hofer & Pette, 1968; frog muscle, Trivedi & Danforth, 1966). That the lowering of pH optimum with increased F-6-P results from a release from ATP inhibition is shown by the further shift toward lower pH values on addition of 5' AMP (Trivedi & Danforth, 1966). ATP markedly shifts the pH optimum for goldfish PFK (Fig. llB), but this has not been consistently observed in other systems. No influence of pH on ATP inhibition occurs in monkey sperm PFK (Hoskins & Stephens, 1969). A decreased pH optimum as ATP concentration increases was found in yeast by Kopperschl~iger et al. (1968) and Lindell & Stellwagen (1968), but an increase in pH optimum as ATP concentration increases was found for frog skeletal muscle (Danforth, 1965); brussels sprouts (Dennis & Coultate, 1967); red blood cells (Kiihn et al., 1968);
760
JAMES 1~'I. FREED
and rabbit muscle (Hofer & Pette, 1968). Undoubtedly the affinity of ATP for inhibitory sites on P F K decreases markedly as the pH increases (Ui, 1968).
Temperature adaptation and control of metabolism Because P F K is the first rate-controlling enzyme unique to glycolysis and glycolytic rates change during temperature acclimation, it is expected that some changes in this enzyme may occur in the process of temperature acclimation. One possible way in which temperature may affect P F K is directly on the enzyme affinity for substrates and modulators. However, the compensatory responses in glycolysis (Hochachka & Hayes, 1962) during temperature acclimation of goldfish is not completely explained by the direct effect of temperature on the enzyme's affinity for F-6-P (Fig. 5). Higher temperatures do, however, decrease the affinity of P F K for ATP in the concentration range of A T P that activates the enzyme (Fig. 7); they increase the inhibition at higher concentrations of magnesium (Fig. 9); and decrease the amount of citrate inhibition (Fig. 10). These examples indicate a direct effect of temperature on enzyme-modulator affinity that may explain a change in glycolytic rate in temperature acclimation. That temperature does affect the affinity of modulators for other enzymes has been observed in the effect of fructose diphosphate and manganese on lungfish fructose diphosphatase (Behrisch & Hochachka, 1969a), and magnesium and AMP on trout fructose diphosphatase (Behrisch & Hochachka, 1969b). This temperature dependence of modulator action, however, is not shown by fructose diphosphate and ATP on pyruvate kinase of Trematomus bernacchii (Somero & Hochachka, 1968) ; AMP and magnesium on lungfish fructose diphosphatase (Behrisch & Hochachka, 1969a); fructose diphosphate and manganese on trout fructose diphosphatase (Behrisch & Hochachka, 1969b); and AMP in salmon fructose diphosphatase (Behrisch, 1969). A second possible explanation of changes in P F K activity in temperature acclimation is that ions may change which affect the P F K enzyme. Magnesium does not appear to change in goldfish (Table 1), but potassium does increase in cold acclimation in carp (Houston et al., 1970). That this increase in potassium may increase P F K activity has been shown in rabbit muscle (Paetkau & Lardy, 1967) and sheep liver and brain (Passonneau & Lowry, 1964). Another possible explanation of glycolytic compensation with temperature acclimation is the effect of metabolite changes on PFK. Table 1 indicates that 25°C-acclimated fish have increased concentrations of glycolytic intermediates and that the most significant differences are in the levels of G-6-P, F-6-P and pyruvate. At 25°C, F-6-P levels are about four times greater than at 5°C. Does this indicate increased PFK activity in the 5°C-acclimated fish--or is some other factor responsible for this difference ? Perhaps goldfish PFK has a lower Qlo than succeeding enzymatic steps in glycolysis. The concentration of citrate and of ATP are both slightly higher in the 25°C-acclimated fish than in the 5°C-acclimated fish; this could cause more inhibition of P F K in the 25°C fish than the 5°C fish; however, the slightly higher values of AMP and ADP in the 25°C-acclimated fish
PROPERTIES OF MUSCLE PHOSPHOFltUCTOKINASE OF C A R A S S I U S . A U R A T U S
761
may counteract the effect of the increased ATP. The similarity of changes for the two temperatures indicates that changes in amount of ATP are balanced by AMP and ADP; this suggests some conservation of energy levels in nucleotides. In all likelihood, then, the change in metabolites with temperature acclimation cannot completely explain increased muscle glycolysis in cold acclimation (Hochachka & Hayes, 1962). An additional possible explanation of how PFK activity may control glycolytic changes in temperature acclimation may be related to a change in pH with temperature. That temperature affects the pH of buffers (pH varying inversely with temperature) has been known for some time--particularly for amine buffers (Bates, 1961). That temperature changes may and do affect the plasma pH of a poikilotherm is becoming increasingly apparent (Howell, 1970). In frogs adapted to selected temperature for a minimum of 3 days the intracellular pH of skeletal muscle (but not in cardiac muscle) decreased 0.0154 units/degC temperature increase [pH 7.2 and 6.9 at 5 and 25°C, respectively (Reeves & Wilson, 1969; Reeves, personal communication)]. The effect of pH changes on glycolysis has been known since Ronzoni & Kerly (1933) demonstrated that high carbon dioxide concentrations inhibit the conversion of hexosemonophosphate to lactate. Danforth (1965) ascribed the pH effects on glycolysis in frog skeletal muscle to PFK. When concentrations of glycolytic intermediates were measured as a function of pH in supernatant fractions and in intact polymorphonuclear leukocytes of guinea pig (Halperin et al., 1969) and in erythrocytes (Kloppick et al., 1967), it was possible to pinpoint the site of pH effects on glycolysis at the PFK step. A similar conclusion was reached by Scheuer & Berry (1967) who noted decreased G-6-P and F-6-P and increased FDP and dihydroxyacetone phosphate with higher pH's in the isolated rat heart. An hypothesis to explain some of the effects of temperature acclimation on glycolysis in goldfish muscle may be presented as follows: goldfish PFK is markedly affected by pH (Fig. 11); as the temperature of the fish decreases, intracellular pH may increase and thus PFK is activated and a rise in the glycolytic rate results. Conversely, as the fish temperature rises, the pH decreases, causing an inhibition of PFK, and the glycolytic rate decreases. These effects would be direct and rapid, not requiring days or weeks for "acclimation". This hypothesis can also explain the time course and changes in amounts in F-6-P and G-6-P. As muscle temperature of fish increases over a period of a few hours, PFK is gradually inhibited and intermediates prior to PFK begin to increase in concentration. Lowered pH in glycolysis also inhibits glyceraldehyde phosphate dehydrogenase and/or diphosphoglycerate kinase in leukocytes (Halperin et al., 1969). Inhibition of either of these two enzymes in goldfish muscles at 25°C may explain the increased levels of triose phosphates. Changes in the rate of glycolysis in temperature acclimation may be dependent on types of isozymes, amount of each enzyme present, the concentration of modulators, the direct effect of temperature on the binding of enzymes and modulators
762
JAMES M. FREED
and ions. T o this list must now be added the possibility of p H change with temperature acclimation, particularly with respect to a p H change on the ratecontrolling enzyme, phosphofructokinase. For phosphofructokinase the direct effects of temperature on the many modulators may overbalance possible longterm acclimation changes.
Acknowledgements--Especial appreciation is expressed to Dr. C. Ladd Prosser of the University of Illinois for research guidance; also to a traineeship provided by National Institutes of Health (USPH 2G-619) for support and to research Grant GB 4005 from the National Science Foundation to Dr. C. Ladd Prosser. REFERENCES ADAM H. (1963a) Adenosine-5'-diphosphate and adenosine-5'monophosphate. In Methods of Enzymatic Analysis (Edited by BEaCMEYERH. U.), pp. 573-577. Academic Press, New York. ADAM H. (1963b) Adenosine-5'-triphosphate; determination with phosphoglycerate kinase. In Methods of Enzymatic Analysis (Edited by BERGMEYERH. U.), pp. 539-543. Academic Press, New York. AIaLFORSC. E. & MANSOURT. E. (1969) Studies on heart phosphofructokinase. Desensitization of the enzyme to adenosine triphosphate inhibition. J. biol. Chem. 244, 12471251. ATKINSOND. E. & WALTONG. M. (1967) Adenosine triphosphate conservation in metabolic regulation. J. biol. Chem. 242, 3239-3241. BATESR. G. (1961) Amine buffers for pH control. Ann. N. Y. Acad. Sci. 92, 341-356. BEHmSCH H. W. (1969) Temperature and the regulation of enzyme activity in poikilotherms. Fructose diphosphatase from migrating salmon. Biochem. J. 115, 687-696. BEHRISCHH. W. & HOCHACHKAP. W. (1969a) Temperature and the regulation of enzyme activity in poikilotherms. Properties of lungfish fructose diphosphatase. Biochem. ft. 112, 601-607. BEHRISCHH. W. & HOCHACHKAP. W. (1969b) Temperature and the regulation of enzyme activity in poikilotherms. Properties of rainbow-trout fructose diphosphatase. Biochem. ft. I I I , 287-295. BI]CHERT., CZOK R., LAMPRECHTW. & LATZKOF. (1963) Pyruvate. In Methods of Enzymatic Analysis (Edited by BERGM~ER H. U.), pp. 253-259. Academic Press, New York. B0CHER T. & HOHORSTH. J. (1963) Dihydroxyacetone phosphate, fructose-l,6-diphosphate and D-glyceraldehyde-3-phosphate; Determination with glycerol-l-phosphate dehydrogenase, aldolase and triosephosphate isomerase. In Methods of Enzymatic Analysis (Edited by BEaCMEYERH. U.), pp. 246-252. Academic Press, New York. CzoK R. & ECK~T L. (1963) D-3-phosphoglycerate, D-2-phosphoglycerate, phosphoenolpyruvate. In Methods of Enzymatic Analysis (Edited by BERGME'~R H. U.), pp. 224233. Academic Press, New York. DANFORTHW. H. (1965) Activation of glycolytic pathway in muscle. In Control of Energy Metabolism (Edited by CHANCEB., ESTAImOOKR. W. & WILLIAMSONJ. R.), pp. 287297. Academic Press, New York. DENNIS D. T. & COULTATET. P. (1967) The regulatory properties of a plant phosphofructokinase during leaf development. Biochim. biophys. Acta 146, 129-137. DENTON R. M. & RANDLEP. J. (1966) Citrate and the regulation of adipose-tissue phosphofructokinase. Biochem. ft. 100, 420-423. FREED J. M. (1971) Temperature effects on muscle phosphofructokinase of the Alaskan king crab, Paralithodes camtschatica. Comp. Biochem. Physiol. (In press.)
PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE OF C A R A S S I U S
AURATUS
763
R. (1965) Enzyme profile of beef heart supernatant fraction. In Control of Energy Metabolism (Edited by CHANCEB., ESTAnROOKR. W. & WILLIAMSONJ. R.), pp. 123-
F~L
125. Academic Press, New York. F ~ R., HOFMANN E. & FRANK K. (1967) Substrat- und Effektorwirkungen auf die Phosphofructokinase yon Aszitesturnorzellen. Acta biol. reed. germ. 19, 737-749. GARFINKELD. (1965) A computer simulation study of the metabolic control behavior of phosphofructokinase. In Control of Energy Metabolism (Edited by CHANCE B., ESTABROOKR. W. & WILLIAMSONJ. R.), pp. 101-106. Academic Press, New York. GRASSO A. & NATALIZIG. M. (1968) Studies on insect (Periplaneta americana L.) phosphofructokinase. Comp. Biochem. Physiol. 26, 979-984. HALPZRINM. L., CONNORSH. P., R~LMANA. S. & KARNOVSKYM. L. (1969) Factors that control the effect of pH on glycolysis in leukocytes..7, biol. Chem. 244, 384--390. HAZEL J. & PROSSER C. L. (1970) Interpretation of inverse acclimation to temperature. Z. vergl. Physiol. 67, 217-228. HOCHACHKAP. W. & HAYESF. R. (1962) The effect of temperature acclimation on pathways of glucose metabolism in the trout. Can. J. Zool. 40, 261-270. HOFER H. W. & PETTE D. (1968) Wirkungen and Wechselwirkungen yon Substraten and Effektoren an der Phosphofructokinase des Kaninchen-Skeletmuskels. Hoppe-Seyler's Z. physiol. Chem. 349, 1378-1392. HOHOP~STH. J. (1963) D-glucose-6-phosphate and D-fructose-6-phosphate. In Methods of Enzymatic Analysis (Edited by B]mG~YER H. U.), pp. 134-138. Academic Press, New York. HOSKINSD. D. & STEPH~S D. T. (1969) Regulation of primate sperm phosphofructokinase. Fedn Proc. Fedn Am. Socs exp. Biol. 28, 705. HOUSTONA. H., MADDENJ. A. & DEWILDEM. A. (1970) Environmental temperature and the body fluid system of the fresh-water teleostJIV. Water-electrolyte regulation in thermally acclimated carp, Cyprinus carpio. Comp. Biochem. Physiol. 34, 805-818. HOWELL B. J., BAUMGARDNERF. W., BONDI K. & RAHN H. (1970) Acid-base balance in cold-blooded vertebrates as a function of body temperature. Am. ft. Physiol. 218, 600-606. KLOPPICK E., JACOBASCHG. & RAPOPORTS. (1967) Steigerung der Glykolyse dutch den Einfluss yon Ammonium ionen auf die Phosphofruktokinaseaktivit~t. Acta biol. med. germ. 18, 37-42. KOPPlmSCHLXGERG., FREY~RR., DIF.Z]~LW. & HOFMANNE. (1968) Some kinetic and molecular properties of yeast phosphofructokinase. FEBS Letters 1, 137-141. KOSHLAND D. E., JR., NEMETHY G. & FILMER D. (1966) Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry, N.Y. 5, 365-385. KOHN B., JACOBASCHC. & RAPOPORT S. (1968) Kinetische Eigenschaften der Phosphofruktokinase toter Blutzellen. Folia haemat., Lpz. 89, 436 A.A,1. LAY~ ENNXS(1957) Spectxophotometric and turbidimetric methods for measuring proteins. In Mcthods in Enzymology (Edited by COLOWlCKS. P. & KAPLANN. O.), Vol. 3, pp. 447-454. Academic Press, New York. LINDELLT. J. & STELLWAGENE. (1968) Purification and properties of phosphofructokinase from yeast..7, biol. Chem. 243, 907-912. LING K. H., PA~TKAUV., MARCUS F. & LARDY H. A. (1966) Phosphofructokinase---I. Skeletal muscle. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPL~U~N. O.), Vol. 9, pp. 4-25-429. Academic Press, New York. Lou 1VL F. & KINOSHITAJ. H. (1967) Control of lens glycolysis. Biochim. biophys. Acta 141, 547-559. LowRY O. H. & PASSONNEAUJ. V. (1966) Kinetic evidence for multiple binding sites on phosphofructokinase. ~. biol. Chem. 241, 2268-2279.
764
JAMES M. FREED
PAETKAU V. & LARDY H. A. (1967) Phosphofructokinase--correlation of physical and enzymatic properties, ft. biol. Chem. 242, 2035-2042. PASSONNEAU J. V. & LOWRY O. H. (1964) The role of phosphofructokinase in metabolic regulation. In Advances in Enzyme Regulation (Edited by WEBER G.), Vol. 2, pp. 265274. POGSON C. I. & RANDLE P. J. (1966) The control of rat-heart phosphofructokinase by citrate and other regulators. Biochem. ft. 100, 683-693. REEVES R. B. & WILSON T. L. (1969) Intracellular p H in bullfrog striated and cardiac muscle as a function of body temperature. Fedn Proc. Fedn Am. Socs exp. Biol. 28, 782. RONZONI E. & KERLY M. (1933) The disappearance of hexosephosphate from intact frog muscle, ft. biol. Chem. 103, 175-181. SCHEUER J. & BERRY M. (1967) Effect of alkalosis on glycolysis in the isolated rat heart. Am. ft. Physiol. 213, 1143-1148. SOMERO G. N. & HOCHACrlKA P. W. (1969) Isoenzymes and short-term temperature compensation in poikilotherms: activation of lactate dehydrogenase isoenzymes by temperature decreases. Nature, Lond. 223, 194-195. STEEL R. G. D. & TORRIE J. H. (1960) Principles and Procedures of Statistics. McGraw-Hill, New York. STERN J. R. (1957) Assay of tricarboxylic acids. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. 3, 425-431. Academic Press, New York. STONE D. B. & MANSOUR T. E. (1967) Phosphofructokinase from the liver fluke Fasciola hepatica--II. Kinetic properties of the enzyme. Mol. Pharmacol. 3, 177-187. TRIWDI B. & DANEORTH W. H. (1966) Effect of p H on the kinetics of frog muscle phosphofructokinase, ft. biol. Chem. 241, 4110-4112. UI MICHIO (1968) Multiple inhibitor sites for A T P on muscle phosphofructokinase as influenced by a change of p H : a computer analysis of nonlinear kinetic data. Biochim. biophys. Acta 159, 50-63. UNDERWOOD A. H. & NEWSHOLME E. A. (1965) Properties of phosphofructokinase from rat liver and their relation to the control of glycolysis and gluconeogenesis. Biochem. J. 95, 868-875. UNDERWOOD A. H, & NEWSHOLME E. A. (1967) Some properties of phosphofructokinase from kidney cortex and their relation to glucose metabolism. Biochem. J. 104, 296-299. WALLER P. R. & BAILEY E. (1969) A comparison of the properties of the phosphofructokinase of the fat body and flight muscle of the adult male desert locust. Biochem. J. 111,365-369. WILLIAMSON J. R. (1966) Glycolytic control m e c h a n i s m s - - I I . Kinetics of intermediate changes during the aerobic-anoxic transition in perfused rat heart. J. biol. Chem. 241, 5026-5036. WILSON J. E., SACKTORB. & TIEKERT C. G. (1967) In situ regulation of glycolysis in tetanized cat skeletal muscle. Arehs Biochem. Biophys. 120, 542-546.
Key Word Index'--Acclimation; Carassius auratus ; control mechanisms ; fish; glycolysis; goldfish; p H ; phosphofructokinase; temperature effects.