Temperature and rates of protein degradation in the fish Gillichthys mirabilis

Comp. Biochem. PhysioL, 1973, Vol. 46B, pp. 463 to 474. Pergamon Press. Printed in Great Britain

TEMPERATURE AND RATES OF PROTEIN DEGRADAT I O N I N T H E F I S H GILLICHTHYS M I R A B I L I S GEORGE N. SOMERO and DEBORAH DOYLE Scripps Institution of Oceanography, Box 1529, La Jolla, California 92037, U.S.A.

(Received 30 ~anuary 1973) Abstract--1. Gill proteins were degraded more rapidly in 26°C-acclimated

Gillichthys mirabilis than in 8°C-acclimated specimens. Muscle proteins were degraded more rapidly in 8°C-acelimated fish. No effects of thermal acclimation were observed for liver and brain proteins. 2. Except for gill proteins, short-term changes in temperature did not appear to significantly alter rates of protein degradation, i.e. in most tissues the rates of protein degradation did not exhibit "typical" ~)10 responses. 3. These findings are consistent with the model for protein degradation control which proposes that rates of degradation are determined primarily by the rates at which proteins are rendered susceptible to the action of proteolytic enzymes. INTRODUCTION

P~LATIVEto the state of our knowledge about the mechanisms of protein synthesis, our understanding of the process(es) of protein degradation is very rudimentary. And, as is almost invariably the case in newly developing fields of biochemistry, most of the available data on protein degradation phenomena have come from studies of mammalian systems, especially rat liver (Schimke, 1969). We know very little about protein degradation phenomena in lower vertebrates and, in particular, we have essentially no information concerning the influences which various environmental factors might have on these processes. Because protein degradation is almost certainly effected by the activities of proteolytic enzymes (Schimke, 1969), one might predict, a priori, that the rates of protein degradation in vivo would be characterized by temperature coefficients (Qlo values) which are quantitatively similar to those described for a wide variety of other enzymic processes, including the reactions of protein synthesis (Das, 1967; Das & Prosser, 1967; Haschemeyer, 1968, 1969). To investigate this possibility we initiated studies of short- and long-term (acclimatory) changes in temperature on rates of protein degradation in the eurythermal fish, Gillichthys mirabilis. MATERIALS AND METHODS A. Experimental organisms and homing conda'tiom Gillichthys mirabilis (Gobiidae), commonly known as the "longjaw mudsucker", is found in sloughs and estuaries of mid- to southern California and parts of Baja California. Gillichthys is an extremely hardy and eurythermal fish, tolerating temperatures over the approximate range, 4-39°C (Somero, 1973). 463

464

G~OROE N. Sormmo Ar~, D~noa~a DOYLE

T h e fish used in these studies were purchased from local bait shops. Specimen weights ranged from 8 to 24 g, and most fish weighed approximately 20 g. Attempts were made to use fish of uniform weight in each experiment. Mortality was very limited, and when it occurred it usually was during the first day after purchase of the fish. T h e fish showed no signs of injury from the isotope injection procedures described below. Two acclimation regimes were used. "Warm-acclimated" fish were maintained in a 26°C constant temperature room under a 15-hr photoperiod. T h e fish were held in large plastic garbage cans, and the sea water was aerated and filtered continuously. Feeding on chopped squid was ad lib. Acclimation times were 3-4 weeks. "Cold-acclimated" fish were maintained in an 8°C constant temperature room, under an 11-hr photoperiod. Other holding conditions were identical to those used for 26°C acclimated fish.

B. Measurement of protein degradation rates 1. Theories and general experimental approaches. Since the initial discovery that proteins "turnover" (Schoenheimer, 1942; for a discussion of the terminology of this field, see Schimke, 1969), several experimental approaches have been developed for measuring the rates at which proteins are degraded in situ. However, in spite of considerable theoretical and experimental analysis of this phenomenon, the accurate determination of protein degradation rates remains a difficult goal to achieve. T h e major experimental difficulties entailed in studies of protein degradation stem from the fact that a fraction of the radioactively labeled amino acids released during protein degradation is rapidly reutilized by the translational machinery. A significant amount of isotope reutilization therefore biases the observed protein half-lives in the direction of abnormally long values. Isotope reutilization has thus made it extremely difficult to obtain absolute values for protein half-lives unless some means can be found for either quantifying the effects of reutilization or for eliminating reutilization altogether. Lucid treatments of the reutilization problem are found in papers by Sehimke (1969), Poole (1971) and Glass & Doyle (1972). Another inherent difficulty in measuring rates of protein degradation, particularly absolute rates, is that large numbers of individuals must be sacrificed to obtain points along a time-dependent incorporation-decay curve. Use of large numbers of experimental organisms entails great amounts of expense and effort and can create major problems in experimental variability. T o circumvent these two problems we employed experimental approaches which, while originally developed for studies of mammalian systems, seemed applicable for the study of a lower vertebrate. To obtain an estimate of the effects of isotope reutilization, we conducted "pulse-chase" experiments (described below) in an effort to decrease, if not block, the reincorporation of radioactive amino acids released from proteins undergoing degradation. T o reduce the number of specimens needed and, thereby, experimental efforts and variability, we used the "double label" procedure developed by Arias et al. (1969), which allows two points on a decay curve to be obtained from a single individual. 2. Incorporation kinetics. T o lay the groundwork for the studies of temperature effects on protein degradation, it was necessary to determine the kinetics of isotope incorporation into protein, and the decay therefrom. In particular, since these studies focused on decay phenomena, it was vital to first establish the times, at different temperatures, required for an injected isotope to be maximally incorporated into the proteins of different tissues. We therefore followed the changes in tissue protein and pool radioactivity as a fimction of time for tissues of fish acclimated to, and maintained at, temperatures of 8 and 26°C. In these experiments an initial injection ("pulse") of radioactive leucine (2 pCi of 4,5-3H leueine, injected in 0"05 ml of a buffered saline solution containing 1% NaCI in 0.05 M

TEMPERATURE AND RATES OF PROTEIN DEGRADATION IN G. M I R A B I L ] S

465

phosphate buffer, p H 7"5) was administered intraperitoneally, just anterior and lateral to the anus. Prior to the injection, the specimens had been starved for 16-18 hr. T h e fish were also starved during the experimental period. Specimens were sacrificed at different times over a 4--6 day period, and the specific radioactivity in protein and the soluble amino acid pools was determined as described below. 3. "Pulse-chase" studies. One procedure which, in theory, can reduce or eliminate isotope reutilization is the "pulse-chase" technique, wherein an initial "pulse" of radioactively labeled amino acid is followed by a "chase" with large quantifies of unlabeled ("cold") amino acid. This was the procedure utilized with Gillichthys to estimate the effects of isotope reincorporation into protein. It should be mentioned, parenthetically, that what appears to be the optimal procedure for controlling isotope reutilization, namely the use of guanido-labeled arginine (Schimke, 1969), which is rapidly degraded by the urea cycle, is a technique of very limited usefulness in the case of teleost fishes and, for that matter, most tissues other than the livers of ureotelic species. Our "pulse-chase" experiments were conducted with fish which had been acclimated to 19°C. Individuals were given an initial injection of radioactive leucine, as described above, and allowed to reach peak incorporation of label into protein. We adopted a pulsechase-decay regime in which the initial chase was given 2 days after the initial pulse of isotope. This was ample time for all tissues to have reached peak labeling at 19°C. Peak-labeled organisms were divided into two groups. A control group was given twice daily injections of buffered saline (described above). A n experimental group received twice daily injections of 30 F M of unlabeled leucine. Each chase injection represented a 10,000fold excess of cold leucine, relative to the amount of the amino acid given in the initial pulse. These two chase procedures were repeated for 4 days, and during this time the organisms were fed ad lib. T h e radioactivity in tissue proteins and soluble pools was determined as described below. 4. "Pulse-decay" studies. We were interested in two classes of temperature effects in these studies: (1) the immediate effect of a change in temperature on the velocities of protein degradation, and (2) the longer term effects of thermal acclimation on rates of protein degradation. T o approach the former question, i.e. to determine the Q10 characteristic of protein degradation for various tissues of Gillichthys, we conducted a series of studies in which peak-labeled specimens (see above) were transferred to waters of different temperatures, and protein radioactivity was subsequently monitored as a function of time. Organisms held at 18-19°C were injected as described, allowed to reach peak labeling over a 2-3 day period and then transferred to either 12 or 26°C sea water. Proteins were allowed to decay for an additional 4 days, during which time the animals were fed ad lib. Following sacrifice, tissue and pool radioactivities were determined as described below. 5. "Double-l~el" studies ~ t h acclimated fish. In the "double-label" procedure (Arias et aL, 1969; Glass & Doyle, 1972), a single individual is given sequential injections of two isotopic forms of a single amino acid. I n a typical double-label experiment, a l~C-labeled amino acid is injected and allowed to reach peak incorporation into protein. T h e n a second isotopic form of the amino acid, e.g. a SH-labeled form, is injected and allowed to reach peak incorporation. T h e animal is sacrificed shortly after decay begins. The 8FI radioactivity thus represents an initial point on a decay curve, whereas the 14C radioactivity represents a later point on this same curve. T h e ratio of 8I-I to 14C radioactivities is an estimate of the relative rate of degradation of the protein(s), since proteins which are being rapidly degraded and replaced will have large amounts of the SH-laheled amino acid, relative to the 14C form. Clearly, unless one can calibrate his 3I-I/14C ratios to known protein half-lives (Glass & Doyle, 1972), this method of measuring protein degradation rates can yield only relative values (in the form of different 8H/14C ratios). Nonetheless, in cases such as the present one where relative rates are of primary interest, this procedure is very useful and has the advantages of reducing effort, expense and a certain level of experimental variability.

466

GEORGE N , SOMEROAND DEBORAH DOYLE

T o be applicable, this technique can only be used in organisms which are in a steady state concerning the net protein metabolism, and the times selected for isotope injection must coincide with the times of protein decay. Good accounts of this procedure and its applications and limitations can be found in papers by Poole (1971) and Glass & Doyle (1972). In our experiments the following injection regime was used. Fish which had previously been starved for 24 hr were given an initial injection of 2/zCi of x4C leucine in buffered saline. Seventy-two hr after this initial injection, 8/~Ci of SH-labeled leucine were administered. Following both injections, feeding was resumed 24 hr later. After the second injection the fish were held at temperature for an additional 3 days and then sacrificed. T h e 3-day time interval between injections and between the second isotope injection and sacrifice was found to be sufficient to satisfy the criteria of experimental design (Schimke, 1969; Poole, 1971) for this procedure of measuring relative degradation rates. 6. Preparation of tissue samples. Fish were sacrificed by spinal cord transection. Liver, gill, brain and muscle tissues were immediately dissected out, rinsed well in cold 1% NaC1 and either frozen at once or placed on ice before weighing. It is worth noting that serum contamination was likely not a problem, as varying degrees of rinsing of the tissues did not alter radioactivity measurements. Liver, muscle and brain were homogenized in 10, 5 and 10 vol., respectively, of 0-05 M T r i s - H C l buffer, p H 7"4, using a motor-driven glass-glass DuaU homogenizer (Kontes Glassware, Inc.). Gills from a single individual were homogenized in 0.5 ml of this buffer. Aliquots of the homogenate were either frozen immediately or used at once for protein determinations (Lowry et al., 1951) and measurement of radioactivity. 7. Isolation of fibrous proteins of muscle. Muscle samples were homogenized in 6 vol. of a 0"02 M potassium phosphate buffer, p H 7"5, containing 0.45 M KCI. T h e samples were shaken overnight at 10°C and then centrifuged for 30 min at 3000 g. Nine vol. of distilled water were then added to the supernatant, and the fibrous proteins were allowed to precipitate for 4 hr at 4°C. T h e fibrous proteins were collected by centrifugation (3000 g for 20 rain), washed first with 10% T C A and then with absolute ethanol. T h e proteins were then digested overnight in N C S (Amersham-Searle) in the toluene cocktail described below. 8. Measurement of radioaetivities in tissue protein and soluble pools. T h e treatment given the homogenate fractions in isotope counting varied according to tissue and number of isotopes used. Because of the low radioactivity in muscle relative to the other tissues studied, 0"2 ml of the muscle homogenates was used, instead of 0.1 ml as in the case of the other three tissues. In experiments employing a single injection of SH leucine, homogenate aliquots were brought to a volume of 1"0 ml with cold 5% trichloroacetic acid (TCA). T h e acidified fraction was centrifuged at 12,000 g in a refrigerated Sorval RC2-B centrifuge, for 15 rain. T h e supernatant containing the radioactive free amino acids was decanted, and a 0'5-ml fraction was counted in 9"5 ml of Aquasol (New England Nuclear). T h e pellet was rinsed with cold 5 % T C A and then rinsed twice with absolute ethanol to remove all traces of the acid. Samples were centrifuged at 3000 g for 5 rain between each rinse. After the final ethanol rinse, the pellets were thoroughly dried and then dissolved in 0"5 ml of NCS, containing 0"1 m_l distilled water, b y overnight incubation at 40°C. This treatment rendered all samples completely miscible in 14"4 ml of scintillation fluid, containing 5 g/1. PPO (2,3-diphenyloxazole; New England Nuclear) and 100rag/1. P O P O P (p-bis(2-(5-phenyloxazolyl)benzene; New England Nuclear) in toluene. In experiments employing the double-label technique, samples were precipitated with cold absolute ethanol and collected on Whatman G F / C filters. W e found that cold ethanol precipitation resulted in finely divided precipitates which did not adhere to the sides or bottoms of the precipitation tubes, and which dispersed evenly over the surface of the glass fiber filters. T C A precipitation had neither of these advantages. Also, virtually all of the protein in the samples was insoluble in cold absolute ethanol.

TEMPERATURE AND RATIOS OF PROTEIN DI~GRADATION IN G. MIRABILIS

467

Following precipitation of the protein on filters, the samples were washed three times with 5 ml of cold 10% TCA and then given a final wash with cold absolute ethanol to remove the TCA. The filters were dried and then incubated overnight in 5 ml of NCS solution containing 0"1 ml distilled water. Fourteen point four millilltres of the toluene counting solution described above was then added to the samples. Two further points should be mentioned concerning preparation of the protein samples for counting. First, the effects of charged transfer RNA molecules appeared to be negligible. When the original protein precipitates were extracted with 1"0 N NaOH for 1 hr at 35°C, followed by a re-precipitation of the proteins by acidification with 10% TCA, less than 1 per cent of the radioactivity was lost. This finding suggests that only insignificant contamination by radioactive leueine-RNA complex was present; therefore, this extraction step was not routinely made. Lastly, to determine whether significant incorporation of radioactive label into lipids occurred, we extracted the tissue homogenates with chloroform-methanol (1 : 3). Only in liver tissue could counts be extracted by this procedure, and in liver at most a 10 per cent loss of radioactivity occurred, which did not correlate with the length of time following injection. The 8H/1~C ratios were not affected by lipid extraction. This procedure was therefore not routinely performed. 9. Radioisotopes and counting procedures. Uniformly labeled 1~C leucine (291 mCi/mM) and 4,5S-H leucine (31-9 Ci/mM) were purchased from New England Nuclear. The 3H labeled leucine was diluted directly in buffered saline for injection. The ~4C labeled leucine was supplied in an 0.01 M HCI solution and was lyophilized before being diluted to concentration. Counting efficiencies and differential quenching were monitored using both the external and internal methods of calibration. Counting was carried out to at least the 5 per cent level of confidence. A Beckman Liquid Scintillation System (LS-230) was used. The counting efficiency for tritium in the acid soluble pools in Aquasol medium was 36 per cent. Tritium counting efficiencies for the solubilized protein samples in the NCStoluene cocktail were approximately 48 per cent. When sH and t4C disintegrations were counted simultaneously in the double-label experiments, all 8H counts were removed from the 14C channel, and spill-over of 1 ~ counts into the tritium channel was only 19-20 per cent. At these settings, counting efficiencies for 3H and 14C were approximately 39 and 69 per cent, respectively. RESULTS

A. "Pulse-ch~e" experiments As discussed in Materials and Methods, reutilization of labeled amino acids can create major difficulties in studies of protein degradation. I n some studies more than 80 per cent of the labeled amino acids released during protein degradation were found to be reincorporated into protein (Righetti et al., 1971). U n d e r these conditions the estimated half-lives of proteins will be grossly longer than the true half-lives. I n these experiments we attempted to chase labeled leucine f r o m the amino acid pools drawn f r o m in protein synthesis b y administering massive amounts of unlabeled leucine after the tissue proteins had begun to decay. T h i s procedure for reducing the effects of isotope reutilization has been employed in whole organism (Goldberg, 1969) and tissue culture studies (Kleveez, 1971). I t is recognized, however, that this is a m u c h less effective means of controlling reutilization than, for example, the 14C-guanidoarginine m e t h o d (see Materials and Methods).

468

GEOROEN. S o l o

ANDD~mo~a-~DO~'LE

The data of Table 1 give no indication of a significant chase effect. The salineinjected and leucine chased proteins do not differ significantly in radioactivity. These results are open to two interpretations. Firstly, in spite of the fact that each twice-daily chase injection contained 10,000 times more leucine than was given in the radioactive pulse, the chase procedure may not have significantly diluted the amino acid pools actually being drawn from in protein synthesis. Alternatively, isotope reutilization may not have been a significant determinant of protein radioactivity in this study. TABLE 1 - - P a o a m l N SPECIFIC RADIOACTIVITY ( d l s / m i n p e r m g protein) IN TISSUES OF C,illichthys WHICH HAD RECEIVED TWICE DAILY ~CCHASES~ OF EITHER UNLABELED LEUCINE OR SALINE

4 days of saline chase (5)* 4 days of leucine chase (5)

Muscle

Liver

Gill

163 + 143t 99 + 64

351 + 147 337 + 116

575 + 160 624 + 209

* Number of individuals. t Mean + 1 S.D. Lacking data on the specific radioactivity of leucine in the amino acid pools being utilized in protein synthesis in the leucine- and saline-chased fish we cannot conclude that one or the other of these alternatives is correct. Regardless, because the chase procedure did not have a significant effect on protein radioactivity, we did not utilize a cold leucine chase in the subsequent studies of decay of radioactivity from pulse-labeled proteins.

B. "Pulse-decay" experiments To determine whether short-term changes in temperature altered rates of protein degradation, we performed a series of experiments in which fish were first peak-labeled at 19°C and then transferred to 12 and 26°C holding tanks for a period of 3 days. Our experimental rationale was that, under these conditions, we could measure the Qlo characteristics of protein degradative reactions, independent of any changes which might occur due to thermal acclimation. The results presented in Table 2 reveal that only in the ease of gill proteins was there a significant temperature effect on protein specific radioactivity. Thus, within the limitations of this experimental procedure, we find no evidence for a "typical" Q10 response of protein degradative processes. As we will discuss below, this finding appears consistent with the most tenable model of protein degradative mechanisms. The data in Table 2 also show that the radioactivity of the amino acid pools decreased much more rapidly at the higher temperature. This pattern is characteristic of all tissues. These data are interesting relative to concern with the effects of isotope reutilization. If the effects of isotope reutilization are greatest when the amount of radioactive amino acid in the tissues is highest, then one would predict

TEMPERATURE AND RATES OF PROTEIN DEGRADATION I N G. M I R A B I L 1 S

469

that the higher radioactivity in the 12°C amino acid pools would tend to give the 12°C tissue proteins longer apparent half-lives than in the 26°C fishes. These temperature-dependent pool effects could conceivably lead to significantly shorter apparent half-lives for the proteins of the 26°C fish in the absence of any real T A B L E 2 - - P R O T E I N SPECIFIC RADIOACTIVITY AND AMINO ACID POOL RADIOACTIVITY I N W H I C H WERE PULSED W I T H RADIOACTIVE LEUCINE AT 19°C, ALLOWEV TO ATTAIN

Gillichthys

PEAK LABELING OF TISSUE PROTEINS AT THIS TEMPERATURE, AND THEN WERE IMMEDIATELY TRANSFERRED TO EITHER 1 2 OR 26°C WATER AND HELD FOR AN ADDITIONAL 3 d a y s AT THESE TEMPERATURES

Protein* Liver

12°C (8)$ 26°C (7) Muscle 12°C 26°C Gill 12°C 26°C

P 1007_+ 529 - 644 + 177 127+ 5 0 - 142_+ 69 469_+ 187 0-05 318_+ 84

Poolst 73 + 9

38 _+20 40+7.6 25 + 5.8 63 _+10 33_+11

* Dis/rain per mg protein. t Dis/rain per mg tissue protein. $ Number of individuals. temperature effect on the actual rate of protein degradation. However, our data exhibit no consistent correlation between pool radioactivity and protein radioactivity, e.g. in muscle tissue the protein radioactivity is higher, albeit not significantly so, in 26°C tissue. Nonetheless, the observed effects of temperature on pool radioactivity should serve as a warning of the types of artifacts which can arise in experiments of this type.

C. Incorporation kinetics A series of experiments was run to determine the kinetics of isotope incorporation and decay in fish acclimated to 8 and 26°C. These data were necessary for determining the correct isotope administration regime for the double-label experiments (see Materials and Methods). A typical pattern of isotope incorporation-decay kinetics is illustrated in Fig. 1, for muscle of 8°C-acclimated Cn'llichthys. In common with all tissues at both temperatures, the radioactivity of the TCA-soluble amino acid pool increased rapidly, relative to TCA-preeipitable protein radioactivity. The only temperature effects observed in these studies were (i) a more rapid increase in both pool and protein radioactivity and (ii) a more rapid decay in pool radioactivity in the 26°C fishes. Peak radioactivity was reached in less than 2 days for all but the muscle of the 8°C-acclimated fish.

470

GEORGE N . SOMERO A ~

DFmORhn DOX~LE

Kinetic data comparable to those of Fig. 1 have been obtained by Dean & Berlin (1969).

"1000 0 0 r"

~30o

2..e r

4

=E o. .500 _z

bJ Q. xF; ~,

,'4

'

4~

'

-~

, '

~

, i

'100 0

TIME (hours)

FIG. 1. Incorporation-decay kinetics of pulse-labeled muscle of 8°C-acclimated Crillichthys. Open circles indicate protein specific radioactivity; closed circles indicate pool radioactivity, expressed as dis/re_in per mg protein. Vertical bars indicate standard errors; sample sizes are given at each point.

D. "Double-label" experiments The "double labd" technique developed by Arias et al. (1969) and recently refined by Glass & Doyle (1972) provides a means for measuring relative rates of protein degradation. This procedure was used to determine whether proteins of various tissues of Gillichthys were degraded at different rates in 8 and 26°C acclimated fish. The data of Table 3 indicate that gill proteins were degraded more rapidly at 26°C, whereas muscle proteins were degraded more rapidly at the lower temperature. No temperature effects on liver and brain protein degradation were observed. Tissue-specific degradation rates are also apparent in some cases. In the 8°C acclimated fish, liver proteins are degraded more rapidly than muscle proteins. In the 26°C-acclimated fish, gill, liver and muscle proteins all differ significantly in their rates of degradation. The finding that muscle proteins are degraded more rapidly at 8°C than at 26°C prompted us to examine the effects of thermal acclimation on the degradation rates of fibrous proteins (actin and myosin), since these proteins form a major

TEMPERATURE AND RATI~ OF PROTEIN DEGRADATION IN G. M1RABILI~q

471

component of the muscle protein mass. Our data suggest that, at least in the 8°C specimens, the fibrous proteins are being degraded at least as rapidly as the other mnscle proteins. TABLE 3--RELATIVE RATE8 OF PROTEIN DEGRADATION IN DI~'~'liitliNT T I ~ U E 8 0 E ACCLIMATED TO 8 AND 2 6 ° C

Cn'llichthys

SH/uC ratio 8°C-acclimated Muscle Totalprotein Fibrous protein Liver Gill Brain

f'3"29+0"89 ~ * ~ 3.62 _+0"82 [.4.51 _+1"27 3"71 _+0"77 ~ 4.06 + 1"16

26°C-acclimated ** **

- 1.40_+0.74"~ ") 1"27 + 1"14J *~ 4.48_+0"62 ~. f * ** ~ 8"18_+3"87J * ) 5"48 + 3"88

The SH/14Cratios represent the relative amounts of SH and 14C labeled leucine present in the tissues at the time of sacrifice. A high ratio indicates a high rate of protein degradation (see Materials and Methods). Statistically significant differences between pairs Of values are indicated as follows: ~ *~ (significant at the 95 per cent level); ** ~ (significant at the 99 per cent level). Values are means + 1 S.D. DISCUSSION With the exception of gill tissue, there appears to be no significant thermal acceleration of rates of protein degradation in G. mirabilis, at least over the temperature range employed in these studies. Bearing in mind the limitations of "pulse-chase-decay" techniques, one can tentatively conclude that the rate of protein degradation in this species is not determined primarily by the velocities at which the proteolytic enzymes are capable of functioning. Were the activities of proteolytic enzymes rate-limiting, i.e. if they were the primary determinant of protein degradation rates, then one would expect to observe measurable and uniform Q10 values for protein degradation in different tissues. Our data suggest, instead, that temperature affects rates of protein degradation in different ways in different tissues; Qt0 values may be less than, approximately equal to, or greater than unity, depending on the tissue studied. Whereas these conclusions may seem inconsistent with the fact that protein degradation is, ultimately, a process catalyzed by proteolytic enzymes, we feel that our interpretations are in agreement with the most reasonable current theory concerning control of protein degradation (Schimke, 1969). This theory proposes that the rate at which a given protein is degraded in situ is established primarily by the rate at which the protein is rendered susceptible to attack by proteolytic enzymes. The activities of the proteolytic enzymes are not rate-limiting, i.e. the enzymes operate well below their maximal velocities, in common with virtually all other enzymes.

472

GEORGE N. SOMEROAND DEBORAHDOYLE

The strength of this theory is due in large part to its ability to account for the widely different half-lives observed among different proteins and among populations of the same protein under different physiological conditions. Each protein may be rendered resistant or susceptible to proteolysis by unique sets of parameters, e.g. the presence or absence of substrates and cofactors, the pH and ionic composition of the protein's local environment, the extent to which the protein is being "used", and so forth. Certain changes in the local environment of a protein alter its quaternary and/or tertiary structure(s), and thereby render the protein a good substrate for proteolytic enzymes. Seen within the framework of this theory, differential effects of temperature on rates of protein degradation can be explained. Temperature is known to alter the aggregation states, the conformations and the abilities to bind substrates of numerous proteins (Hochachka & Somero, 1973). All of these temperaturedependent changes in protein structure and function could alter the resistance of a protein to degradation. And, since all of these temperature effects vary greatly among different proteins, e.g. some proteins lose their quaternary structures at low temperatures, some at high temperatures, one would predict that the resistance to proteolytic degradation may be greater at high temperatures for some proteins and greater for other proteins at low temperatures. For example, when the conformation or aggregation state of a protein is stabilized primarily by hydrophobic interactions, the protein may be more susceptible to proteolytic attack at temperatures near 0°C than at higher temperatures (see Brandts, 1967). Data bearing on the foregoing conclusion are found in Table 3. The rates of degradation of fibrous muscle proteins are higher in the 8°C-acclimated fish than in the 26°C-acclimated fish. Since the polymerization of G actin subunits to form F actin is stabilized by hydrophobic interactions, which are less effective in stabilizing protein structure at low, than at high, temperatures in the biological temperature range, one might predict that the G actin elements would be more apt to exist in a non-polymerized form at low temperatures and, perhaps as a result, be more susceptible to the activities of proteolytic enzymes. Whereas the foregoing speculations would seem to predict that a protein's half-life will be directly related to its in vitro thermal stability, such a correlation is not, in general, observed (Kuehl & Sumsion, 1970) and, in fact, such a correlation is not to be expected. In vitro studies, utilizing enzymes which are largely separated from the cellular constituents and structures which contribute to their stabilities/ instabilities, probably will be of limited usefulness in studying the effects of temperature on in situ protein degradation. One must also consider the determinants of protein degradation rates in the context of the organism's physiological state. For example, Goldberg (1969a, b) found that disuse of muscle in rats led to an increase in the rate of muscle protein degradation. Conversely, work hypertrophy of muscle was in part due to a reduced rate of muscle protein degradation. These findings suggest an alternative explanation for the observed higher rates of protein degradation in muscle of 8°C-acclim ated Gillichthys. Since the 8°C-acclimated fish were noticeably less active than the

TEMPERATURE AND RATES OF PROTEIN DEGRADATION I N G. M1RABILIS

473

26°C-acclimated specimens, even following 2-3 weeks of acclimation, the possibility exists that the apparent t e m p e r a t u r e - d e p e n d e n c y of muscle protein turnover is, in fact, due to activity differences between the two groups of fishes. T h e s e results and interpretations lead us to conclude that the effects of t e m perature on rates of protein degradation in situ are highly complex, and involve both direct physical effects of kinetic energy changes on protein structure and indirect effects which m a y stem f r o m temperature-related changes in the behavioral and physiological states of the organism.

`dcknowledgements---We thank Linnae Dayton for her help in the statistical analyses of our data. The study was supported by the National Science Foundation Grant No. GB31106. REFERENCES ARIAS I. M., DOYLE D. & SCHIMI~ R. T. (1969) Studies on the synthesis and degradation of proteins of the endoplasmic reticulum of rat liver..7, bioL Chem. 244, 3303-3315. BRANDTSJ. F. (1967) Heat effects on proteins and enzymes. In Thermobioloffy (Edited by Rose A. H.), pp. 25-72. Academic Press, New York. DAS A. B. (1967) Biochemical changes in tissues of goldfish acclimated to high and low temperatures--II. Synthesis of protein and RNA of subcellular fractions and tissue composition. Comp. Biochem. Physiol. 21, 469-485. D~s A. B. & PROSSERC. L. (1967) Biochemical changes in tissues of goldfish acclimated to high and low temperatures--I. Protein synthesis. Comp. Bioehem. Physiol. 21, 449-467. DEAN J. M. & B ~ I N J. D. (1969) Alterations in hepatocyte function of thermally acclimated rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 29, 307-312. GLASS R. D. & DOCLE D. (1972) On the measurement of protein turnover in animal cells. 3. biol. Chem. 247, 5234-5242. GOLDBERGA. L. (1969a) Protein turnover in skeletal muscle--I. Protein catabolism during work-induced hypertrophy and growth induced with growth hormone. ~t. biol. Chem. 244, 3217-3222. GOLDBERGA. L. (1969b) Protein turnover in skeletal muscle---II. Effects of denervation and cortisone on protein catabolism in skeletal muscle. ~t. biol. Chem. 244, 3223-3229. HASCrImm~R A. E. V. (1968) Compensation of liver protein synthesis in temperatureacclimated toadfish, Opsanus tau. Biol. Bull., Woods Hole 135, 130--140. HASCHEM~R A. E. V. (1969) Rates of polypeptide chain assembly in liver in vivo: relation to the mechanism of temperature acclimation in Opsanus tau. Proc. hath..dead. Sci. U.S..d. 62, 128-135. H o c r m c H ~ P. W. & SoMsnO G. N. (1973) Strategies of Biochemical .ddaptation. W . B . Saunders, Philadelphia. 358 pp. KI~wcz R. R. (1971) Rapid protein catabolism in mammalian ceils is obscured by reutilization of amino acids. Biochem. biophys. Res. Commun. 43, 76-81. KUEHL L. & SUMSION E. N. (1970) Turnover of several glycolytic enzymes in rat liver. jT. biol. Chem. 245, 6616-6623. LOWRY O., ROSERnOUGHN. J., FAnS A. L. & RA_~ALL R. J. (1951) Protein measurement with the Folin phenol reagent. ~. biol. Chem. 193, 265-275. POOLR B. (1971) The kinetics of disappearance of labeled leucine from the free leucine pool of rat liver and its effect on the apparent turnover of catalase and other hepatic proteins. .7. biol. Chem. 246, 6587--6591. RiGmrrTx P., LITTLEE. P. & WoLv G. (1971) Reutilization of amino acids in protein synthesis in HeLa cells. ~. biol. Chem. 246, 5724-5732.

474

GEOaOE N. S o l o

~

Dm~oRa~ DOYL~

SCHIMKE R. T. (1969) On the roles of synthesis and degradation in regulation of enzyme levels in mammalian tissues. In Current Topics in Cellular Regulation (Edited by H o a s e K ~ B. L. & STADT~L',NE. R.), Vol. I, pp. 77-124. Academic Press, New York. ScHomq~n~i~ R. (1942) Dynamic State of Body Constituents. Harvard University Press, Cambridge, Mass. So,taRO G. N. (1973) Thermal modulation of pyruvate metabolism in the fish Gillichthys mirabilis: the role of lactate dehydrogenases. Corap. Biochem. Physiol. 44B, 205-209.

Key Word Index--Temperature; temperature acclimation; protein degradation; protein turnover; Gillichthys mirabilis; acclimation; control of protein metabolism; adaptation.