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Changes in activity of cytochrome oxidase during adaptation of goldfish to different temperatures
Comp. Biochem. Physiol., 1965, Vol. 14, pp. 651 to 659. Pergamon Press Ltd. Printed in Great Britain
C H A N G E S I N A C T I V I T Y OF C Y T O C H R O M E O X I D A S E D U R I N G A D A P T A T I O N OF G O L D F I S H T O D I F F E R E N T TEMPERATURES J A M E S FREED* Department of Physiology and Biophysics, University of Illinois, Urbana (Received 4 November 1964)
A b s t r a c t - - 1 . Cytochrome oxidase from muscle of goldfish showed an increased activity with cold acclimation and decreased activity with warm acclimation when measured at an intermediate temperature. Activity of the enzyme from 5°C fish was three to five times greater than from 30 ° fish when measured at 15 °. 2. Measurements at various temperatures showed a translation toward the left of the rate-temperature curves with cold acclimation. The Q1o over the linear range was 2-1 for enzyme preparations from fish acclimated to 5, 15, 25 or 30 °. 3. Enzyme activity measured at the temperatures of acclimation was maximal at 25 °. 4. After 2 hr of heating at 45 ° the cytochrome oxidase was 80 per cent inactivated, irrespective of acclimation temperature. 5. After 5-10 min of heat treatment, the activity of the enzyme from 5° fish was higher than its activity without heating, decline in activity of enzyme from 15 ° fish was delayed, but activity from fish acclimated to 25-30 ° declined directly. 6. It is concluded that cold acclimation causes an increase and warm acclimation a decrease in amount of cytochrome oxidase in goldfish muscle, also that in cold acclimation there is either an increase in a heat-sensitive inhibitor or a change which causes prolongation of thermal activation.
INTRODUCTION
TEMPE~,TUREacclimation of metabolism in poikilotherms has been studied in two respects: (1) resistance adaptation, tolerance or denaturation at temperature limits and (2) capacity adaptation or rate functions measured at either (a) temperatures of acclimation or (b) intermediate temperatures for various acclimation temperatures. Many examples of compensatory changes in metabolism of intact animals, of tissues a n d of e n z y m e s isolated f r o m acclimated a n i m a l s are k n o w n ( s u m m a r i e s i n P r e c h t et al., 1955; Prosser, 1962; H o c h a c h k a & Hayes, 1963). Classifications of * Present address: Department of Biology, Manchester College, North Manchester, Indiana. Supported in part by grant G-8795 from National Science Foundation and U S P H Training Grant 5TI-GM-619. The author is indebted to Professor C. Ladd Prosser for research guidance. 651
652
JAMESFaEEI}
capacity adaptation have been presented (Precht, 1958; Prosser & Brown, 1961). Information is poor or lacking concerning (1) the relation between resistance and capacity adaptation, (2) why some enzymes and tissues show acclimation while others do not or may even show reverse changes, (3) the mechanisms by which environmental (and body) temperatures bring about long-term metabolic changes and (4) whether there may be qualitative changes in enzymatic proteins or whether adaptation results only from quantitative changes in specific proteins or in co-factors. It is hoped that a study of both capacity and resistance changes in one key enzyme, cytochrome oxidase, may be of help in answering some of the preceding questions. Adaptive changes in cytochrome oxidase in differently acclimated fish have not been reported, although Ekberg (1958) found greater sensitivity to cyanide of gills and liver from cold- than from warm-acclimated goldfish. MATERIALS AND METHODS Goldfish (Carassius auratus) obtained from Auburndale Goldfish Co. of Chicago, mostly 14-18 cm long and 30-45 g in weight, were acclimated in aquaria kept in rooms of the following constant temperatures: 5 °, 15 °, 25 ° and 30°C, all with 12 hr photoperiod. The goldfish were fed fish-food pellets (Glencoe Mills, Inc.). The fish at 30 ° were fed twice each day, those at 15 ° and 25 ° once each day and those at 5 ° on alternate days; this schedule corresponded nearly to the maximum the fish would eat. Cytochrome oxidase activity was measured on muscle extracts. In a few preliminary experiments liver was used, but due to the small amount of tissue per fish, the turbidity when the whole homogenate was used, and interfering color when the supernatant was used, liver was replaced by muscle. Muscle is more plentiful and gives a clearer supernatant than liver. To prepare the muscle extract, the fish were decapitated and trunk muscles rapidly removed in a 5° room. The muscle tissue was first squeezed through a stainless steel tissue-press and then homogenized in an amount of water four times the wet weight to make a 20% homogenate. Homogenization was performed with a teflon pestle for fifteen up-and-clown strokes. The homogenate either was used directly for assay by the manometric method or was centrifuged at 3000 g for 10 min and the supernatant fluid was used for spectrophotometric assays. The manometric procedure for cytochrome oxidase was that of Potter (1957) in which cytochrome-c is the substrate and sodium ascorbate is the reductant. The following substances were present in the assay medium: Phosphate buffer (pH 7-4) Cytochrome-c (horse heart from Sigma) Aluminum chloride Sodium ascorbate
3-3 x 10-2 M 8-0 x 10 -~ M 4"0 x 10 -4 M 1'1 x 10 -2 M
The spectrophotometric method was that of Smith (1955a, 1955b) in which the decrease in absorbance at 550 m/~ is measured spectrophotometrically as cytochrome oxidase oxidizes reduced cytochrome-c. Reduced cytochrome-c was
CHANGBSIN ACTIVITYOF CYTOCHROMBOXIDASE
653
prepared by bubbling nitrogen through cytochrome-c solution for 5 rain, hydrogen for 1 hr and then nitrogen for 5 more rain. T h e results were expressed as velocity constant per mg protein. T h e velocity constant (K) was calculated from the formula K = l°g(ODt'-
OD®)-l°g(ODt'- OD®) ×
2.303,
t~ - t I where ODt, is the initial optical density at T i m e 1 and ODt, the optical density at T i m e 2. A Zeiss spectrophotometer with a variable temperature cell was used. T h e decrease in absorbance was recorded on a Varian recorder. T h e velocity constants at various intervals of time during the assay were calculated and averaged to give a mean velocity constant. T h e reaction mixture consisted of phosphate buffer, p H 7.0, and cytochrome-c 2.1 × 10 -5 M, plus enzyme solution for a total of 3 ml. Assays were made at 15 ° except for one series run at different temperatures. Protein was measured by the method of Ennis (1957). Statistical validation of all results was by the M a n n - W h i t n e y U-test for small samples (Auble, 1953). RESULTS
Cytochrome oxidase activity T h e activity of cytochrome oxidase was measured in some experiments at an intermediate temperature (15 °) and in others over a range of temperatures (5-44°). T h e results of spectrophotometric assays of cytochrome oxidase of supernatants of muscle extracts from fish acclimated to different temperatures and measured at 15 ° are given in Table 1. T h e activity of the enzyme/mg protein in the extract TABLE 1--ACTIVITY OF CYTOCHROMEOXIDASEOF SUPERNATANTSFROMMUSCLEHOMOGENATES FROM FISH ACCLIMATEDTO DIFFERENTTEMPERATURES. MEASUREMENTSBY SPECTROPHOTOMETRIC METHODAT 15°C
Acclimation temperature
No. of fish
Activity constant/mg protein x 10-s (mean + S.E.)
5° 15°
20 19
13"0+ 1-12 8'6 +0'83
25 ° 30 °
18 19
4"6 +0-40 2"4+0"32
p values for given temperature pairs 5-30 ° 5-250 5-15 ° 15-300 15-25 ° 25-30 °
<0-002 <0.002 <0"01 <0"002 <0.002 <0.002
decreased significantly throughout the series from cold- to warm-acclimated goldfish. T h e activity for muscle from 5°-acclimated fish was five times greater than for muscle from 30°-acclimated fish when both were measured at 15 °. T o check whether or not there was loss of some active component during the centrifugation of the extract and whether the results could be due to a greater loss 42
654
JAMES Fm~D
from extracts of warm-acclimated fish, manometric assays were performed with both the homogenates and supernatants from the same fish in a second series of experiments. Results are given in Tables 2A and 2B. These tables indicate that, although the difference between activity of supernatants of the 5 ° and 30 ° fish as measured manometrically was not as great as the difference measured spectrophotometrically, the difference between supernatants of cold- and warm-acclimated T A B L E 2 - - C Y T O C H R O M E OXlDASE ACTIVITY OF 20 PER CENT MUSCLE HOMOGENATES AND SUPERNATANTS OF MUSCLES FROM ACCLIMATED GOLDFISH. MEASUREMENTS BY MANOMETRIC METHOD AT 1 5 ° C
A Acclimation temperature
No. of fish
Qo, of homogenate in/xl O~/mg wet wt./hr + S.E.
5° 15 °
8 6
4.0 + 0.35 3.5 +0.37
25 ° 30°
6 8
2.1 -+0-18 1.6 _+0.14
p values for given temperature pairs 5-30 ° 5-25 ° 5-15 ° 15-30 ° 15-25 ° 25-30 °
<0.002 <0.010 <0.20 <0.002 <0.020 <0-10
5-30 ° 5-25 ° 5-15 ° 15-30 ° 15-25 ° 25-30 °
<0.01 <0.01 >0.20 <0.01 >0.20 < 0.04
B
Qo, of supernatant in/~10z/mg protein/hr _+S.E. 5° 15° 25 °
6 6 6
21 _+2.04 18_+3.26 11 +1.63
30°
7
5_+ 1.14
fish were significant. T h e differences obtained with homogenates were similar to those obtained with supernatants (Table 2). It is concluded that the differences found in the first series (Table 1) were not due to differential losses during centrifugation. Assays of the supernatant fluid were also conducted over a range of temperature from 5 ° to 44 ° . At 44o the activity constants were calculated only for the first 2 min of assay because of inactivation after longer times. Results are given in Fig. 1. T h e Q10 from 5 ° to 25 ° is 2.1 for fish from all acclimation temperatures. The effect of cold acclimation is to shift the rate-temperature curve to the left without rotation. Fig. 1 also shows that values of enzyme activity extrapolated to the temperatures of acclimation pass through a maximum at 25 °. Heat inactivationofcytochrome oxidase. Resistance of cytochrome oxidase to heat was measured by placing tubes containing the muscle supernatant in a bath at 45 ° for various lengths of time; the samples were then transferred directly to an ice-bath
30 °
25 °
15 °
5°
14"0+1"37 (9) 8"8_+1"3 (9) 2.9_+0-49 (6) 1.5 + 0-3 (9)
10 rain 11"00+1"3 (5) 7"2 _+1"18 (6) 3-1 +0-49 (6) 0.92 + 0.26 (5)
30 m i n 7"7 ___1-02 (6) 5"8 -+1-21 (6) 3.0 _+0.28 (6) 0.74 + 0-09 (5)
40 m i n
T i m e of heating
10 m i n < 0.002 <0.002 <0-01 < 0-002 <0.04 <0-10 N.S.
5-30 ° 5-25 ° 5-15 ° 15-30 ° 15-25 ° 25-30 °
< 0.002 <0.002 <0-10 N.S. < 0-08 N . S . >0.20 N.S. >0.10 N.S.
30 m i n
< 0-002 >0-20 N.S. >0-20 N.S. < 0-002 >0.20 N.S. <0.002
40 m i n
< 0-002 >0-20 N.S. N.S. N.S. N.S. N.S.
1 hr
6"3 _+0-62 (5) 4-0 _+0-69 (6) 2-0 + 0 . 4 0 (6) 0.61 + 0-035 (4)
1 hr
2 hr 2"8 _+0-50 (4) 2"0 _+0-40 (4) 0-87_+0.17 (4) 0.20 + 0-08 (4)
N.S. N.S. N.S. N.S. N.S. N.S.
2 hr
PROTEIN X 10 -3 + S.E. NUMBER OF FISH IN PARENTHESES
(B) P V~LVES ~OR PERCENTAGE REMAINING ACTIVITY AFTER HEATING
12"0+1"15 (12) 8.2_+0"92 (12) 4-1_+0.315 (12) 2.2 + 0.43 (12)
0 rain
Temperature comparison
Acclimation temperature
(A) ACTIVITY IN VELOCITY CONSTANT PER m g
TAm.~ 3--CYTOCHROME OXIDASE ACTIVITY BEFORE AND AFTER TREATMENT WITH HEAT (45°C) FOR STATED TIMES
656
JAMES FREED 80 60 40
20
~
I0
o_ 8 x
6 _
tlA
'~
~
Iz Q.
~
~
~.
T FIG.
1_.
I
5
I
I
I
I
15 25 35 45 TEMPERATURE OF ASSAY, =C
Cytochrome oxidase activity of supernatant of muscle homogenates
f r o m fish acclimated to 5 °, 15 °, 25 ° or 30°C, m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y at various t e m p e r a t u r e s . Points are averages of five to seven fish. Broken line gives extrapolated rates for acclimation t e m p e r a t u r e s .
1 2 0 ~
° Acclimotion Te,'np
,oo \ ~ ~o
~0
0
,
0
,
I
50 60 90 LENGTH OF HEAT TREATMENT,Min.
120
FIG. 2. P e r c e n t a g e of initial activity of c y t o c h r o m e oxidase r e m a i n i n g after h e a t i n g to 45°C for various t i m e s for fish acclimated to 5 °, 15 °, 25 ° or 30°C.
CHANGES IN ACTIVITY OF CYTOCHROME OXIDASE
657
where they remained for at least 5 min before assay at 15 °. Results are given in Table 3 in terms of the measured activity after heat treatment. The percentage of activity remaining after different durations of heating is plotted in Fig. 2. Table 3A shows that, as in the experiments of Tables 1 and 2, the zero-time (unheated) activity shows compensatory changes with temperature acclimation of the fish. Inactivation was approx. 80 per cent complete in all samples after 2 hr at 45 ° and there was little difference remaining between the samples at I hr (Table 3A and Fig. 2). However, the rate of inactivation of the supernatant from warmacclimated fish was faster than from cold-acclimated ones. At 10 and 30 min the muscle extracts from 5 ° fish showed more activity than before heat treatment and the extract from 15 ° fish did not start to decline in activity until after 10 min at which time the extract from the 30 ° fish was 25 per cent inactivated (Fig. 2 and Table 3). DISCUSSION Previous studies have shown marked compensatory changes in activity of the hexose monophosphate shunt in fish under different conditions of temperature acclimation (Kkberg, 1952; Hochachka& Hayes, 1953), less marked compensations in glycolytic enzymes (Ekberg, 1952). Oxidative enzymes have shown less effect although succinic dehydrogenase activity in liver from eels acclimated to 11 ° was 80 per cent greater than that from eels acclimated to 20 ° (Precht, 1951 ; Kruger, 1902). The data of Tables I and 2 show activity of cytochrome oxidase three to five times greater for 5°-acclimated fish than for 30°-acclimated fish when measured at 15 °. The increase in cytochrome oxidase activity in goldfish muscle as given in rate of activity/mg protein might be due to a decrease in all other proteins. However, this is most unlikely since cytochrome oxidase constitutes a very small fraction of the total cellular protein and no quantitative change in total protein concentration/ ml of supernatant or homogenate was observed. The increase in cytochrome-c oxidase activity after acclimation to cold and the decrease with acclimation to heat appears to be due to quantitative rather than to qualitative changes in the enzyme. This conclusion is based on the translation of the rate-temperature curves without change in 010 which indicates change~ in amount rather than in kind of enzyme (Prosser, 1958, 1951). Similar shift of the rate-temperature curve was observed for cocarboxylase from eel liver (Carlsen, 1953) and for succinic dehydrogenase from muscle of Rhodeu~ (Kruger, 1952). The maximum activity at 25 ° when measurements were made at the temperatures of acclimation is similar to observations of Fry & Hart (1948) on metabolism of intact goldfish. However, the activity of cytochrome oxidase from each group of fish does not decline when measured at high temperatures as much as does the total metabolism (Kanungo & Prosser, 1959). The similarity between cold- and warm-acclimated fish in the percentage of cytochrome oxidase activity remaining after I and 2 hr of heating to 4.5° indicates that there is no marked change in thermostabillty of the enzyme with acclimation. This is in agreement with evidence for constancy of thermostahility of proteins and
658
JAMES I:R~-~{I,
with evidence that "resistance adaptation" of proteins is less affected by temperature acclimation than is "capacity adaptation" (Ushakov, 1964). T h e significant increase in activity of cytochrome oxidase after heat treatment for 10 min in the 5 ° fish and the delay in inactivation of the enzyme from 15 ~ fish are in marked contrast to the abrupt inactivation of the enzyme from the warmacclimated fish. A similar increase in activity of hexokinase after heat treatment occurs in yeast cultured at low temperatures but not in yeast from higher temperatures {Christophersen, 1963). T h e increase in activity after heat treatment may be due to removal of an inhibitor that is present in cold-acclimated fish or it may be due to longer persistence of thermal activation of the enzyme from cold-acclimated fish. One aspect of thermal activation may be removal of an intrinsic inhibitor. Similar increase in activity of an enzyme due to loss of inhibition after heat treatment was found by Gerhart & Pardee (1962); heating a purified preparation of aspartate transcarbamytase from Escherichia coli removed the inhibiting effect of cytidine triphosphate and increased the m a x i m u m velocity of the enzyme reaction twofold. Other enzymes in which "heat activation" is due to the destruction of an inhibitor that is more t e m p e r a t u r e labile than the enzyme being measured have been reported by Swartz et al. (1957). It is concluded that there is not only an increase in amount of cytochrome oxidase in muscle in cold-acclimated goldfish but also either an increase in a heat-sensitive inhibitor or a change in the enzyme complex such that a brief heating increases its activity. Tests on purified enzymes m a y reveal the nature of these differences between cold- and warm-acclimated fish. REFERENCES AUBLE D. (1953) Extended tables for the Mann-Whitney statistic, Bull. Inst. Educ. Res. (Indiana University) 1, No. 2. CAaLSEN H. (1953) Die Cocarboxylasegehalt des Aales (Anquilla vulgaris). Z. vergl. Physiol. 35, 199-208. CHRISTO~'HERSnNJ. (1963) Untersuchungen fiber den Einfluss der Adaptationstemperatur auf die Hitzeresistenz und Aktivit~it der Hexokinase yon Hefezellen. Arch. Mikrobiol. 45, 58-64. EKBImCD. R. (1958) Respiration in tissues of goldfish adapted to high and low temperatures. Biol. Bull, Woods Hole. 114, 308-316. EKB~aG D. R. (1962) Anaerobic and aerobic metabolism in gills of Crucian carp adapted to low and high temperature. Comp. Biochem. Physiol. 5, 123-128. ENNIS L. (1957) Spectrophotometric and turbidometric methods for measuring proteins. In Methods in En~ymology (Edited by COLOWmR S. B. & KAPt.^N N. O.), 3, 447-454. Academic Press, New York. Fay F. E. J. & HAsT J. S. (1948) Relation of temperature to oxygen consumption in goldfish. Biol. Bull, Woods Hole. 94, 66-77. GERn^m" J. C. & PARD~mA. B. (1962) The enzymology of control of feedback inhibition. J. Biol. Chem. 237, 891-896. HocrIACn~,A P. W. & H^,t-~s F. R. (1963) Effect of temperature acclimation on pathways of glucose metabolism in trout. Canad. J. Zool. 40, 261-270. KANUNGO M. S. & PROSSm~ C. L. (1959) Physiological and biochemical adaptation of goldfish to cold and warm temperatures. I. Standard and active oxygen consumption of cold- and warm-acclimated goldfish at various temperatures, ft. Cell. Comp. Physiol. 54, 259-264.
CHANGES IN ACTIVITY OF CYTOCHROME OXIDASE
659
KRUGER G. (1962) l~ber die Temperaturadaptation des Bitterlings (Rhodeus amarus). Z. Wiss. Zool. 167, 87-104. POTTER V. P. (1957) The homogenate technique. In Manometric Techniques (Edited by UMERE~T W. W., BuRros R. H. & STAUFFERJ. F.), 170-187. Burgess Publishing Co., Minneapolis. PRECHT H. (1951) Der Einfluss der Temperatur auf die Atmung und auf einige Fermente bein Aal (Anquilla vulgaris). Biol. Zbl. 70, 71-85. PRECHT H. (1958) Concepts of the temperature adaptation of. unchanging reaction systems of cold-blooded animals. In Physiological Adaptation (Edited by PROSSERC. L.), 50-78. Amer. Physiol. Soc., Washington. PRECHT H . , CHRISTOPHERSENJ. & HENSEL H. (1955) Temperatur und Leben. Springer, Berlin. PROSSER C. L. (1958) The nature of physiological adaptation. In Physiological Adaptation (Edited by PROSSEI~C. L.), 167-180. Amer. Physiol. Soc., Washington. PROSSER C. L. (1962) Acclimation of poikilothermic vertebrates to low temperatures. In Symposium on the Comparative Physiology o.f Temperature Regulation, pp. 1-43. Arctic Aeromedical Laboratory, Wainwright, Alaska. PROSSER C. L. & BROWN F. A. (1961) Comparative Animal Physiology. W. B. Saunders, Philadelphia. SMITH L. (1955a) Cytochromes-a, -a, -as and -aa. In Methods in Enzymology (Edited by COLOW~CKS. P. & KA~'LANN. O.), 2, 732-740. Academic Press, New York. SMITH L. (1955b) Spectrophotometric assay of cytochrome-c oxidase. In Methods of Biochemical Analysis (Edited by GLICK D.), 2, 427--448. Interscience, New York. SWARTZ M. N., KAPLANN. O. & FRENCHM. E. (1957) Mechanism of heat activation of enzymes. In Influence of Temperature on Biological Systems (Edited by JOHNSONF. H.), pp. 61-70. Amer. Physiol. Soc., Washington. USHAKOVB. P. (1964) Thermostability of cells and proteins of poikilotherrns and its significance in speciation. Physiol. Rev. 44, 518-560.
C H A N G E S I N A C T I V I T Y OF C Y T O C H R O M E O X I D A S E D U R I N G A D A P T A T I O N OF G O L D F I S H T O D I F F E R E N T TEMPERATURES J A M E S FREED* Department of Physiology and Biophysics, University of Illinois, Urbana (Received 4 November 1964)
A b s t r a c t - - 1 . Cytochrome oxidase from muscle of goldfish showed an increased activity with cold acclimation and decreased activity with warm acclimation when measured at an intermediate temperature. Activity of the enzyme from 5°C fish was three to five times greater than from 30 ° fish when measured at 15 °. 2. Measurements at various temperatures showed a translation toward the left of the rate-temperature curves with cold acclimation. The Q1o over the linear range was 2-1 for enzyme preparations from fish acclimated to 5, 15, 25 or 30 °. 3. Enzyme activity measured at the temperatures of acclimation was maximal at 25 °. 4. After 2 hr of heating at 45 ° the cytochrome oxidase was 80 per cent inactivated, irrespective of acclimation temperature. 5. After 5-10 min of heat treatment, the activity of the enzyme from 5° fish was higher than its activity without heating, decline in activity of enzyme from 15 ° fish was delayed, but activity from fish acclimated to 25-30 ° declined directly. 6. It is concluded that cold acclimation causes an increase and warm acclimation a decrease in amount of cytochrome oxidase in goldfish muscle, also that in cold acclimation there is either an increase in a heat-sensitive inhibitor or a change which causes prolongation of thermal activation.
INTRODUCTION
TEMPE~,TUREacclimation of metabolism in poikilotherms has been studied in two respects: (1) resistance adaptation, tolerance or denaturation at temperature limits and (2) capacity adaptation or rate functions measured at either (a) temperatures of acclimation or (b) intermediate temperatures for various acclimation temperatures. Many examples of compensatory changes in metabolism of intact animals, of tissues a n d of e n z y m e s isolated f r o m acclimated a n i m a l s are k n o w n ( s u m m a r i e s i n P r e c h t et al., 1955; Prosser, 1962; H o c h a c h k a & Hayes, 1963). Classifications of * Present address: Department of Biology, Manchester College, North Manchester, Indiana. Supported in part by grant G-8795 from National Science Foundation and U S P H Training Grant 5TI-GM-619. The author is indebted to Professor C. Ladd Prosser for research guidance. 651
652
JAMESFaEEI}
capacity adaptation have been presented (Precht, 1958; Prosser & Brown, 1961). Information is poor or lacking concerning (1) the relation between resistance and capacity adaptation, (2) why some enzymes and tissues show acclimation while others do not or may even show reverse changes, (3) the mechanisms by which environmental (and body) temperatures bring about long-term metabolic changes and (4) whether there may be qualitative changes in enzymatic proteins or whether adaptation results only from quantitative changes in specific proteins or in co-factors. It is hoped that a study of both capacity and resistance changes in one key enzyme, cytochrome oxidase, may be of help in answering some of the preceding questions. Adaptive changes in cytochrome oxidase in differently acclimated fish have not been reported, although Ekberg (1958) found greater sensitivity to cyanide of gills and liver from cold- than from warm-acclimated goldfish. MATERIALS AND METHODS Goldfish (Carassius auratus) obtained from Auburndale Goldfish Co. of Chicago, mostly 14-18 cm long and 30-45 g in weight, were acclimated in aquaria kept in rooms of the following constant temperatures: 5 °, 15 °, 25 ° and 30°C, all with 12 hr photoperiod. The goldfish were fed fish-food pellets (Glencoe Mills, Inc.). The fish at 30 ° were fed twice each day, those at 15 ° and 25 ° once each day and those at 5 ° on alternate days; this schedule corresponded nearly to the maximum the fish would eat. Cytochrome oxidase activity was measured on muscle extracts. In a few preliminary experiments liver was used, but due to the small amount of tissue per fish, the turbidity when the whole homogenate was used, and interfering color when the supernatant was used, liver was replaced by muscle. Muscle is more plentiful and gives a clearer supernatant than liver. To prepare the muscle extract, the fish were decapitated and trunk muscles rapidly removed in a 5° room. The muscle tissue was first squeezed through a stainless steel tissue-press and then homogenized in an amount of water four times the wet weight to make a 20% homogenate. Homogenization was performed with a teflon pestle for fifteen up-and-clown strokes. The homogenate either was used directly for assay by the manometric method or was centrifuged at 3000 g for 10 min and the supernatant fluid was used for spectrophotometric assays. The manometric procedure for cytochrome oxidase was that of Potter (1957) in which cytochrome-c is the substrate and sodium ascorbate is the reductant. The following substances were present in the assay medium: Phosphate buffer (pH 7-4) Cytochrome-c (horse heart from Sigma) Aluminum chloride Sodium ascorbate
3-3 x 10-2 M 8-0 x 10 -~ M 4"0 x 10 -4 M 1'1 x 10 -2 M
The spectrophotometric method was that of Smith (1955a, 1955b) in which the decrease in absorbance at 550 m/~ is measured spectrophotometrically as cytochrome oxidase oxidizes reduced cytochrome-c. Reduced cytochrome-c was
CHANGBSIN ACTIVITYOF CYTOCHROMBOXIDASE
653
prepared by bubbling nitrogen through cytochrome-c solution for 5 rain, hydrogen for 1 hr and then nitrogen for 5 more rain. T h e results were expressed as velocity constant per mg protein. T h e velocity constant (K) was calculated from the formula K = l°g(ODt'-
OD®)-l°g(ODt'- OD®) ×
2.303,
t~ - t I where ODt, is the initial optical density at T i m e 1 and ODt, the optical density at T i m e 2. A Zeiss spectrophotometer with a variable temperature cell was used. T h e decrease in absorbance was recorded on a Varian recorder. T h e velocity constants at various intervals of time during the assay were calculated and averaged to give a mean velocity constant. T h e reaction mixture consisted of phosphate buffer, p H 7.0, and cytochrome-c 2.1 × 10 -5 M, plus enzyme solution for a total of 3 ml. Assays were made at 15 ° except for one series run at different temperatures. Protein was measured by the method of Ennis (1957). Statistical validation of all results was by the M a n n - W h i t n e y U-test for small samples (Auble, 1953). RESULTS
Cytochrome oxidase activity T h e activity of cytochrome oxidase was measured in some experiments at an intermediate temperature (15 °) and in others over a range of temperatures (5-44°). T h e results of spectrophotometric assays of cytochrome oxidase of supernatants of muscle extracts from fish acclimated to different temperatures and measured at 15 ° are given in Table 1. T h e activity of the enzyme/mg protein in the extract TABLE 1--ACTIVITY OF CYTOCHROMEOXIDASEOF SUPERNATANTSFROMMUSCLEHOMOGENATES FROM FISH ACCLIMATEDTO DIFFERENTTEMPERATURES. MEASUREMENTSBY SPECTROPHOTOMETRIC METHODAT 15°C
Acclimation temperature
No. of fish
Activity constant/mg protein x 10-s (mean + S.E.)
5° 15°
20 19
13"0+ 1-12 8'6 +0'83
25 ° 30 °
18 19
4"6 +0-40 2"4+0"32
p values for given temperature pairs 5-30 ° 5-250 5-15 ° 15-300 15-25 ° 25-30 °
<0-002 <0.002 <0"01 <0"002 <0.002 <0.002
decreased significantly throughout the series from cold- to warm-acclimated goldfish. T h e activity for muscle from 5°-acclimated fish was five times greater than for muscle from 30°-acclimated fish when both were measured at 15 °. T o check whether or not there was loss of some active component during the centrifugation of the extract and whether the results could be due to a greater loss 42
654
JAMES Fm~D
from extracts of warm-acclimated fish, manometric assays were performed with both the homogenates and supernatants from the same fish in a second series of experiments. Results are given in Tables 2A and 2B. These tables indicate that, although the difference between activity of supernatants of the 5 ° and 30 ° fish as measured manometrically was not as great as the difference measured spectrophotometrically, the difference between supernatants of cold- and warm-acclimated T A B L E 2 - - C Y T O C H R O M E OXlDASE ACTIVITY OF 20 PER CENT MUSCLE HOMOGENATES AND SUPERNATANTS OF MUSCLES FROM ACCLIMATED GOLDFISH. MEASUREMENTS BY MANOMETRIC METHOD AT 1 5 ° C
A Acclimation temperature
No. of fish
Qo, of homogenate in/xl O~/mg wet wt./hr + S.E.
5° 15 °
8 6
4.0 + 0.35 3.5 +0.37
25 ° 30°
6 8
2.1 -+0-18 1.6 _+0.14
p values for given temperature pairs 5-30 ° 5-25 ° 5-15 ° 15-30 ° 15-25 ° 25-30 °
<0.002 <0.010 <0.20 <0.002 <0.020 <0-10
5-30 ° 5-25 ° 5-15 ° 15-30 ° 15-25 ° 25-30 °
<0.01 <0.01 >0.20 <0.01 >0.20 < 0.04
B
Qo, of supernatant in/~10z/mg protein/hr _+S.E. 5° 15° 25 °
6 6 6
21 _+2.04 18_+3.26 11 +1.63
30°
7
5_+ 1.14
fish were significant. T h e differences obtained with homogenates were similar to those obtained with supernatants (Table 2). It is concluded that the differences found in the first series (Table 1) were not due to differential losses during centrifugation. Assays of the supernatant fluid were also conducted over a range of temperature from 5 ° to 44 ° . At 44o the activity constants were calculated only for the first 2 min of assay because of inactivation after longer times. Results are given in Fig. 1. T h e Q10 from 5 ° to 25 ° is 2.1 for fish from all acclimation temperatures. The effect of cold acclimation is to shift the rate-temperature curve to the left without rotation. Fig. 1 also shows that values of enzyme activity extrapolated to the temperatures of acclimation pass through a maximum at 25 °. Heat inactivationofcytochrome oxidase. Resistance of cytochrome oxidase to heat was measured by placing tubes containing the muscle supernatant in a bath at 45 ° for various lengths of time; the samples were then transferred directly to an ice-bath
30 °
25 °
15 °
5°
14"0+1"37 (9) 8"8_+1"3 (9) 2.9_+0-49 (6) 1.5 + 0-3 (9)
10 rain 11"00+1"3 (5) 7"2 _+1"18 (6) 3-1 +0-49 (6) 0.92 + 0.26 (5)
30 m i n 7"7 ___1-02 (6) 5"8 -+1-21 (6) 3.0 _+0.28 (6) 0.74 + 0-09 (5)
40 m i n
T i m e of heating
10 m i n < 0.002 <0.002 <0-01 < 0-002 <0.04 <0-10 N.S.
5-30 ° 5-25 ° 5-15 ° 15-30 ° 15-25 ° 25-30 °
< 0.002 <0.002 <0-10 N.S. < 0-08 N . S . >0.20 N.S. >0.10 N.S.
30 m i n
< 0-002 >0-20 N.S. >0-20 N.S. < 0-002 >0.20 N.S. <0.002
40 m i n
< 0-002 >0-20 N.S. N.S. N.S. N.S. N.S.
1 hr
6"3 _+0-62 (5) 4-0 _+0-69 (6) 2-0 + 0 . 4 0 (6) 0.61 + 0-035 (4)
1 hr
2 hr 2"8 _+0-50 (4) 2"0 _+0-40 (4) 0-87_+0.17 (4) 0.20 + 0-08 (4)
N.S. N.S. N.S. N.S. N.S. N.S.
2 hr
PROTEIN X 10 -3 + S.E. NUMBER OF FISH IN PARENTHESES
(B) P V~LVES ~OR PERCENTAGE REMAINING ACTIVITY AFTER HEATING
12"0+1"15 (12) 8.2_+0"92 (12) 4-1_+0.315 (12) 2.2 + 0.43 (12)
0 rain
Temperature comparison
Acclimation temperature
(A) ACTIVITY IN VELOCITY CONSTANT PER m g
TAm.~ 3--CYTOCHROME OXIDASE ACTIVITY BEFORE AND AFTER TREATMENT WITH HEAT (45°C) FOR STATED TIMES
656
JAMES FREED 80 60 40
20
~
I0
o_ 8 x
6 _
tlA
'~
~
Iz Q.
~
~
~.
T FIG.
1_.
I
5
I
I
I
I
15 25 35 45 TEMPERATURE OF ASSAY, =C
Cytochrome oxidase activity of supernatant of muscle homogenates
f r o m fish acclimated to 5 °, 15 °, 25 ° or 30°C, m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y at various t e m p e r a t u r e s . Points are averages of five to seven fish. Broken line gives extrapolated rates for acclimation t e m p e r a t u r e s .
1 2 0 ~
° Acclimotion Te,'np
,oo \ ~ ~o
~0
0
,
0
,
I
50 60 90 LENGTH OF HEAT TREATMENT,Min.
120
FIG. 2. P e r c e n t a g e of initial activity of c y t o c h r o m e oxidase r e m a i n i n g after h e a t i n g to 45°C for various t i m e s for fish acclimated to 5 °, 15 °, 25 ° or 30°C.
CHANGES IN ACTIVITY OF CYTOCHROME OXIDASE
657
where they remained for at least 5 min before assay at 15 °. Results are given in Table 3 in terms of the measured activity after heat treatment. The percentage of activity remaining after different durations of heating is plotted in Fig. 2. Table 3A shows that, as in the experiments of Tables 1 and 2, the zero-time (unheated) activity shows compensatory changes with temperature acclimation of the fish. Inactivation was approx. 80 per cent complete in all samples after 2 hr at 45 ° and there was little difference remaining between the samples at I hr (Table 3A and Fig. 2). However, the rate of inactivation of the supernatant from warmacclimated fish was faster than from cold-acclimated ones. At 10 and 30 min the muscle extracts from 5 ° fish showed more activity than before heat treatment and the extract from 15 ° fish did not start to decline in activity until after 10 min at which time the extract from the 30 ° fish was 25 per cent inactivated (Fig. 2 and Table 3). DISCUSSION Previous studies have shown marked compensatory changes in activity of the hexose monophosphate shunt in fish under different conditions of temperature acclimation (Kkberg, 1952; Hochachka& Hayes, 1953), less marked compensations in glycolytic enzymes (Ekberg, 1952). Oxidative enzymes have shown less effect although succinic dehydrogenase activity in liver from eels acclimated to 11 ° was 80 per cent greater than that from eels acclimated to 20 ° (Precht, 1951 ; Kruger, 1902). The data of Tables I and 2 show activity of cytochrome oxidase three to five times greater for 5°-acclimated fish than for 30°-acclimated fish when measured at 15 °. The increase in cytochrome oxidase activity in goldfish muscle as given in rate of activity/mg protein might be due to a decrease in all other proteins. However, this is most unlikely since cytochrome oxidase constitutes a very small fraction of the total cellular protein and no quantitative change in total protein concentration/ ml of supernatant or homogenate was observed. The increase in cytochrome-c oxidase activity after acclimation to cold and the decrease with acclimation to heat appears to be due to quantitative rather than to qualitative changes in the enzyme. This conclusion is based on the translation of the rate-temperature curves without change in 010 which indicates change~ in amount rather than in kind of enzyme (Prosser, 1958, 1951). Similar shift of the rate-temperature curve was observed for cocarboxylase from eel liver (Carlsen, 1953) and for succinic dehydrogenase from muscle of Rhodeu~ (Kruger, 1952). The maximum activity at 25 ° when measurements were made at the temperatures of acclimation is similar to observations of Fry & Hart (1948) on metabolism of intact goldfish. However, the activity of cytochrome oxidase from each group of fish does not decline when measured at high temperatures as much as does the total metabolism (Kanungo & Prosser, 1959). The similarity between cold- and warm-acclimated fish in the percentage of cytochrome oxidase activity remaining after I and 2 hr of heating to 4.5° indicates that there is no marked change in thermostabillty of the enzyme with acclimation. This is in agreement with evidence for constancy of thermostahility of proteins and
658
JAMES I:R~-~{I,
with evidence that "resistance adaptation" of proteins is less affected by temperature acclimation than is "capacity adaptation" (Ushakov, 1964). T h e significant increase in activity of cytochrome oxidase after heat treatment for 10 min in the 5 ° fish and the delay in inactivation of the enzyme from 15 ~ fish are in marked contrast to the abrupt inactivation of the enzyme from the warmacclimated fish. A similar increase in activity of hexokinase after heat treatment occurs in yeast cultured at low temperatures but not in yeast from higher temperatures {Christophersen, 1963). T h e increase in activity after heat treatment may be due to removal of an inhibitor that is present in cold-acclimated fish or it may be due to longer persistence of thermal activation of the enzyme from cold-acclimated fish. One aspect of thermal activation may be removal of an intrinsic inhibitor. Similar increase in activity of an enzyme due to loss of inhibition after heat treatment was found by Gerhart & Pardee (1962); heating a purified preparation of aspartate transcarbamytase from Escherichia coli removed the inhibiting effect of cytidine triphosphate and increased the m a x i m u m velocity of the enzyme reaction twofold. Other enzymes in which "heat activation" is due to the destruction of an inhibitor that is more t e m p e r a t u r e labile than the enzyme being measured have been reported by Swartz et al. (1957). It is concluded that there is not only an increase in amount of cytochrome oxidase in muscle in cold-acclimated goldfish but also either an increase in a heat-sensitive inhibitor or a change in the enzyme complex such that a brief heating increases its activity. Tests on purified enzymes m a y reveal the nature of these differences between cold- and warm-acclimated fish. REFERENCES AUBLE D. (1953) Extended tables for the Mann-Whitney statistic, Bull. Inst. Educ. Res. (Indiana University) 1, No. 2. CAaLSEN H. (1953) Die Cocarboxylasegehalt des Aales (Anquilla vulgaris). Z. vergl. Physiol. 35, 199-208. CHRISTO~'HERSnNJ. (1963) Untersuchungen fiber den Einfluss der Adaptationstemperatur auf die Hitzeresistenz und Aktivit~it der Hexokinase yon Hefezellen. Arch. Mikrobiol. 45, 58-64. EKBImCD. R. (1958) Respiration in tissues of goldfish adapted to high and low temperatures. Biol. Bull, Woods Hole. 114, 308-316. EKB~aG D. R. (1962) Anaerobic and aerobic metabolism in gills of Crucian carp adapted to low and high temperature. Comp. Biochem. Physiol. 5, 123-128. ENNIS L. (1957) Spectrophotometric and turbidometric methods for measuring proteins. In Methods in En~ymology (Edited by COLOWmR S. B. & KAPt.^N N. O.), 3, 447-454. Academic Press, New York. Fay F. E. J. & HAsT J. S. (1948) Relation of temperature to oxygen consumption in goldfish. Biol. Bull, Woods Hole. 94, 66-77. GERn^m" J. C. & PARD~mA. B. (1962) The enzymology of control of feedback inhibition. J. Biol. Chem. 237, 891-896. HocrIACn~,A P. W. & H^,t-~s F. R. (1963) Effect of temperature acclimation on pathways of glucose metabolism in trout. Canad. J. Zool. 40, 261-270. KANUNGO M. S. & PROSSm~ C. L. (1959) Physiological and biochemical adaptation of goldfish to cold and warm temperatures. I. Standard and active oxygen consumption of cold- and warm-acclimated goldfish at various temperatures, ft. Cell. Comp. Physiol. 54, 259-264.
CHANGES IN ACTIVITY OF CYTOCHROME OXIDASE
659
KRUGER G. (1962) l~ber die Temperaturadaptation des Bitterlings (Rhodeus amarus). Z. Wiss. Zool. 167, 87-104. POTTER V. P. (1957) The homogenate technique. In Manometric Techniques (Edited by UMERE~T W. W., BuRros R. H. & STAUFFERJ. F.), 170-187. Burgess Publishing Co., Minneapolis. PRECHT H. (1951) Der Einfluss der Temperatur auf die Atmung und auf einige Fermente bein Aal (Anquilla vulgaris). Biol. Zbl. 70, 71-85. PRECHT H. (1958) Concepts of the temperature adaptation of. unchanging reaction systems of cold-blooded animals. In Physiological Adaptation (Edited by PROSSERC. L.), 50-78. Amer. Physiol. Soc., Washington. PRECHT H . , CHRISTOPHERSENJ. & HENSEL H. (1955) Temperatur und Leben. Springer, Berlin. PROSSER C. L. (1958) The nature of physiological adaptation. In Physiological Adaptation (Edited by PROSSEI~C. L.), 167-180. Amer. Physiol. Soc., Washington. PROSSER C. L. (1962) Acclimation of poikilothermic vertebrates to low temperatures. In Symposium on the Comparative Physiology o.f Temperature Regulation, pp. 1-43. Arctic Aeromedical Laboratory, Wainwright, Alaska. PROSSER C. L. & BROWN F. A. (1961) Comparative Animal Physiology. W. B. Saunders, Philadelphia. SMITH L. (1955a) Cytochromes-a, -a, -as and -aa. In Methods in Enzymology (Edited by COLOW~CKS. P. & KA~'LANN. O.), 2, 732-740. Academic Press, New York. SMITH L. (1955b) Spectrophotometric assay of cytochrome-c oxidase. In Methods of Biochemical Analysis (Edited by GLICK D.), 2, 427--448. Interscience, New York. SWARTZ M. N., KAPLANN. O. & FRENCHM. E. (1957) Mechanism of heat activation of enzymes. In Influence of Temperature on Biological Systems (Edited by JOHNSONF. H.), pp. 61-70. Amer. Physiol. Soc., Washington. USHAKOVB. P. (1964) Thermostability of cells and proteins of poikilotherrns and its significance in speciation. Physiol. Rev. 44, 518-560.