Kinetic properties and regulation of glycerol-3-phosphate dehydrogenase from the overwintering, freezing-tolerant gall fly larva, Eurosta solidaginis

CRYOBIOLOGY

19,

185-194

(1982)

Kinetic Properties and Regulation of Glycerol-3-phosphate Dehydrogenase from the Overwintering, Freezing-Tolerant Gall Fly Larva, Eurosta solidaginis JANET Institute

of

M. STOREY

Biochemistry,

Carleton

KENNETH

AND

University,

The third instar larvae of the gall fly, Eurosta soliduginis, overwinter inside stem galls on goldenrod plants surviving ambient temperatures as low as -40°C. The larvae, which are freezing tolerant, accumulate high levels of glycerol and sorbitol for cryoprotection, the two polyols being produced in a sequential, temperature-dependent manner during low temperature acclimation (5, 14). Glycogenolysis initially fuels glycerol biosynthesis, with levels of up to 0.6 M glycerol accumulating in hemolymph, but glycerol synthesis ceases, and sorbitol synthesis is initiated instead, when ambient temperature drops to about 5°C (5, 14). Sorbitol synthesis continues with subsequent low temperature acclimation but is stopped at about -lO”C, the point at which extracellular freezing takes place (5). An observed accumulation of glycerol-3-P, coincident with the cessation of glycerol biosynthesis in E. soliduginis, suggests that the production of glycerol results from the diversion of triose phosphates from the glycolytic pathway at the level of dihydroxyacetone-P (DHAP) with the actions of glycerol-39 dehydrogenase (G3PDH) and glycerol-3-phosphatase converting DHAP to glycerol (13, 14). The metabolic roles of G3PDH in E. soliduginis are at least threefold: (i) the enzyme is key in the production of cryoprotectant glycerol, (ii) the enzyme functions in the product ion of glycerol-3-Z’ for use in Received 1981.

September

2, 1981;

accepted

November

Ottawa,

B. STOREY Ontario

KlS

5B6, Canada

lipid synthesis, major triglyceride reserves being accumulated by the larvae to fuel the pupal and nonfeeding adult stages of the insect (14), and (iii) the enzyme participates as one-half of the glycerol-3-P cycle, the means used in insect tissues for the transfer of cytoplasmic reducing equivalents into the mitochondria (7). In the present study, we have examined the kinetic properties of cytoplasmic glycerol-3-P dehydrogenase (EC 1.1.1.8) from E. soliduginis with particular interest in studying temperature effects on enzyme kinetic and regulatory properties in an effort to elucidate some of the controls governing glycerol synthesis during low temperature acclimation in this species. MATERIALS

AND

METHODS

Chemicals and animals. All biochemicals were purchased from Sigma Chemical Company, except for rabbit muscle G3PDH which was from Boehringer-Mannheim Corporation. DHAP used was the Li+ salt; preliminary tests showed that Li+, at concentrations of up to 10 mM, had no effect on G3PDH activity. Round galls containing third instar larvae of E. soliduginis were collected from fields around Ottawa in September and October. Larvae were held at either 24, 15, or 0°C for 2 weeks followed by acclimation to lower temperatures using a 1°C decrease per day until -30°C was reached as previously described (5, 14). At various temperatures, larvae were sampled, quickly dissected out of their galls, frozen in liquid nitrogen, and

6,

18.5 001 l-2240/82/020185-10$02.00/O Copyright @ 1982 by Academic Ress, Inc. All rights of reproduction in any form reserved.

186

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AND

stored at -80°C until used. The larvae routinely used in this study were those which had been acclimated to 15°C for 2 weeks. Enzyme preparation. Larvae were homogenized (15 w/v, approximately 0.2 g per preparation) in ice-cold 20 r&l4 imidazole buffer, pH 7.0, containing 15 mM 2-mercaptoethanol and 1 n&f EDTA using a glass/glass homogenizer. The homogenate was centrifuged at 27,OOOg for 30 min at 4°C and the clear supernatant removed and dialyzed overnight at 4°C against 20 mZt4 imidazole buffer, pH 7.0, containing 15 mM 2-mercaptoethanol . The dialyzed supematant was used as the source of enzyme for kinetic studies. Rabbit muscle G3PDH was diluted for use in 20 r&f imidazole buffer, pH 7.0, containing 15 mM 2-mercaptoethanol and 1 mg/ml bovine serum albumin (to equal the protein concentration of the E. solidaginis supematant and to maintain enzyme stability) and dialyzed overnight as for the larval enzyme. Enzyme assay. Enzyme activity was monitored at 340 nm using a Unicam SP 8-100 recording spectrophotometer with water-jacketed cell holder for temperature control. Standard assay conditions for G3PDH were 20 n&f imidazole buffer, pH 7.0, with 0.5 m&f DHAP and 0.1 m&f NADH in the forward direction and 8 n&f m-glycerol-3-P and 2 n&f NAD+ in the reverse direction in a final volume of 1 ml. Assays were started by the addition of enzyme preparation. Control assays, omitting DHAP or glycerol-3-P, demonstrated the absence of nonspecific activities utilizing NAD(H). The pH of the imidazole buffer was set to 7.0 at 25°C and was allowed to fluctuate with changing temperature (for imidazole buffer this results in an approximately 0.02 pH unit increase in pH per 1°C decrease in temperature (10)). Michaelis constants were determined from direct linear, Lineweaver- Burk or Hanes plots while &‘s were determined from Dixon plots. I,,,‘s were estimated using the method of Job et al. (4) using subsaturat-

STOREY

ing substrate concentrations, 0.05 n&f DHAP and 0.005 m&f NADH. Kinetic constants are the means of determination on at least three separate preparations of enzyme and values are reproducible to within + 10%. Heat stability experiments. Enzyme was heated at 45 or 50°C in 20 n&f imidazole buffer, pH 7.0, containing 15 mM 2mercaptoethanol in the presence or absence of glycerol, sorbitol, or both polyols. At timed intervals, aliquots of G3PDH were removed and assayed immediately for enzyme activity at 25°C. Zsoelectrofocusiizg. Isoelectrofocusing, using an LKB 8108 column (110 ml) with pH 3.5 to 10.0 Ampholines in a sucrose density gradient, was carried out at 500 V for 16 hr at 4°C (17). RESULTS

lsozymes

and physical

properties.

Iso-

electrofocusing revealed the presence of three forms of G3PDH in the third instar larvae of E. solidaginis with pi’s of 5.68, 5.42, and 5.25 comprising 57.7, 23.6, and 18.8% of the total enzyme activity, respectively. No differences in the number of forms present, the pi’s or the relative activities of the three forms were found when larvae from two acclimation temperatures, 24 and -30°C were compared. Polyacrylamide gel electrophoresis and polyacrylamide gel isoelectrofocusing showed similar results when larvae from four acclimation temperatures were compared (15). The pH optimum of the enzyme was 7.0 in the forward direction in either Tris or imidazole buffers similar to that reported for other insect G3PDHs (2, 12). Imidazole buffer was used in all kinetic studies of the enzyme because the effect of temperature on the pH of imidazole buffer is virtually identical to the change in intracellular pH with temperature seen in poikilotherms (10). Arrhenius plots. Figure 1 shows an Arrhenius plot of E. solidaginis G3PDH. The relationship is linear over the temperature range 30 to 0°C with a slope of - 2.723 and a

CiLYCEROL-3-PHOSPHATE

DEHYDROGENASE

11°K x 1O-3 1. Arrhenius plot of E. solidaginis and rabbit muscle G3PDH. Enzyme activity was measured under standard assay conditions outlined under Materials and Methods. Symbols are: 0, E. sofidaginis G3PDH; 0, E. sofidaginis G3PDH plus 0.5 M glycerol and 0.25 M sorbitol; q , rabbit muscle G3PDH. FIG.

calculated activation energy of 12,630 ? 185 cal/mol. The Q10 for the enzyme was 2.16. The addition of polyols, glycerol (0.5 M), or sorbitol (0.25 M) lowered the maximal velocity of the enzyme by 5- 10% or by 15-20% when both polyols were added together at all assay temperatures. However, the addition of polyols did not alter the shape of the Arrhenius plot or significantly alter the calculated activation energy or the Q10 of the reaction. Figure 1 also shows an Arrhenius plot for rabbit muscle G3PDH; the plot is linear over the 30 to 0°C range with an activation energy of 12,805 cal/mol and a Q10 of 2..19. Kinetic con.stants. The Michaelis constants for/Z. soliduginis G3PDH were found to be independent of cosubstrate concentration. The effects of assay temperature

FROM

Emma

solidaginis

187

and polyol concentration on the K,a’s for DHAP and NADH are shown in Table 1 for the enzyme from larvae acclimated to 15°C. The K, for DHAP was not significantly altered by decreasing assay temperature over the range 30 to 10°C but was increased by 75% at 0°C. The K, for NADH, by contrast, showed an opposite behavior with respect to temperature; the K, was constant at the lower temperatures but increased at 30°C. The KmcDHAP) ofE. solidaginis G3PDH is considerably lower (5- to 7-fold lower) than the ~&DHAP~ reported for various insect flight muscle G3PDHs (2, 3, 12) although the KT,,‘s for NADH of all these enzymes are similar. Senkbeil and White (8) note that liver type G3PDHs show an approximately IO-fold lower K, for DHAP than do muscle forms of the enzyme, a property which they believe is related to the functions of the two enzyme forms. The low K, for DHAP displayed by the E. solidaginis larval enzyme may be related to the biosynthetic functions of the enzyme in glycerol or lipid synthesis as opposed to the major role of the enzyme in muscle in redox regulation during aerobic muscle work. The effects of temperature on the Km’s for DHAP and NADH were also tested on G3PDH isolated from larvae acclimated to 24 or 0°C. In these experiments, all homogenization, centrifugation, dialysis, and storage of the two enzymes were performed at room temperature (24°C) or on ice (0-3”(Z), respectively, a procedure which was designed to detect kinetically different conformational isomers of the enzyme (6, 11). The K,,‘s for the two enzymes, isolated in this fashion, were the same for the enzyme from the 24 or the 0°C acclimation groups when assayed at either 24 or 0°C and were not statistically different from those shown in Table 1. Hemolymph concentrations of glycerol and sorbitol in naturally overwintering populations of the gall fly larvae are reported to be up to 0.6 and 0.2 M, respectively (5). One function of polyols at low

188

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Effect of Temperature

AND

STOREY

TABLE 1 and Polyols on the Michaelis Constants for E. soliduginis Glycerol-3-phosphate Dehydrogenase Temperature 30°C

20°C

10°C

0°C

Control +Glycerol (0.5 M) +Sorbitol (0.25 M) +Glycerol and sorbitol (O.VO.25 M)

56 55 71

K, DHAP (pLM) 52 49 51 48 51 54

87 80 88

61

58

54

88

Control +Glycerol (0.5 M) +Sorbitol (0.25 M) +Glycerol and sorbitol (0.5/0.25 M)

5.2 6.9 -

3.1 2.8 2.7

2.5 2.8 3.3

2.4 2.6 -

6.5

3.2

3.1

2.4

K,

Note. Results are the means of determinations

NADH (PM)

on at least three preparations of enzyme with a variability of

temperature could be to regulate enzyme kinetic properties. The effects of added glycerol (0.5 M) or sorbitol(0.25 M) or both polyols on the Michaelis constants of E. sofidaginis were tested therefore and found to be minimal (Table 1). At 3o”C, addition of polyols produced a slight increase in the K,‘s for DHAP or NADH but at lower temperatures, those at which polyol accumulation occurs in vivo, no effects of polyols on K,‘s were seen. However, glycerol and sorbitol did, as mentioned above, lower the maximal velocity of the enzyme. The effects of temperature and polyols on purified rabbit muscle G3PDH were also tested. K,r,‘s for DHAP of the rabbit enzyme were 12, 7, 14, and 32 pM at 30, 20, 10, and O”C, respectively, significantly lower overall than the K,‘s for the E. solidaginis enzyme but exhibiting the same effect of temperature on K,,,, in particular the twofold increase in KmcDHAPjbetween 10 and 0°C. The addition of glycerol, sorbitol, or both polyols did not significantly alter the KmcDHAPjat any temperature. Kinetic constants for the reverse reaction of E. solidaginis G3PDH (determined in

imidazole buffer, pH 7.0) were 0.34 mM for glycerol-3-Z’ and 0.012 mM for NAD+. The K,‘s were unaffected by assay temperature (25 versus SC). Enzyme inhibitors. A variety of compounds were tested for possible inhibitory effects on the forward reaction of E. solidaginis G3PDH. Proline (50 n&Q, alanine (20 mM), and glucose (100 mA4), all of which increase in concentration during low temperature acclimation in E. soliduginis (14), had no effect on enzyme activity. Arginine phosphate (IS,, = 25 mM), fructose6-P (ISo = 50 mM), fructose-1,6-P, (I,, = 4 r&f), and AMP (I,,, = 5.3 mM) had slight inhibitory effects on enzyme activity but only at levels of these compounds which were well outside of the physiological range in vivo. Glycerol-3-P and NAD+ were product inhibitors of G3PDH with effects competitive with respect to DHAP and NADH, respectively. The Ki for glycerol-3-P was 2.4 mM while KLcNAD+)was 0.2 m&f. Neither Ki was altered by assay temperature (25 versus 5°C) or by the addition of polyols. ATP, Mg.ATP, ADP, citrate, Mgacitrate, and Pi were also inhibitors of the en-

GLYCEROL-3-PHOSPHATE

DEHYDROGENASE

zyme. ATP was a mixed competitive inhibitor with respect to both DHAP and NADH with &‘s of 9.5 and 0.25 mM, respectively. Addition of Mg2+ in a 1: 1 ratio with ATP altered these &‘s to 6.7 and 2 mA4. Pi inhibition had a Ki of 10.5 n&f with respect to DHAP. Assay temperature had no effect on the inhibition of larval G3PDH by ATP, Mg*ATP, Mgscitrate, or Pi but altered the inhibitions by ADP and citrate. Figure 2 shows the effects of temperature on ATP, ADP, and citrate inhibitions while the effects of temperature and polyols on the inhibitor constants for all six inhibitors are shown in Table 2. ADP inhibition was increased at 5 a:s compared to 25°C while the opposite effect was seen for citrate, the I,, for citrate increasing as temperature decreased. The addition of polyols had no significant effect on the I5,,‘s for any of the inhibitors at either 25 or 5°C assay temperatures. Heat stability experiments. The thermal stability of E. .rofidaginis and rabbit muscle G3PDHs were compared by monitoring the decay of enzyme activity with time at different temperatures. E. solidaginis G3PDH had a considerably higher thermal stability than did the rabbit muscle enzyme. At 45”C, the larval enzyme showed 97 and 84% of initial activity after 5 and 10 min, respectively, while the rabbit enzyme was much more rapidly inactivated to levels of 54 and 27% of initial activity after these same incubation times. Data for 50°C are shown in Fig. 3. The rabbit muscle enzyme is almost totally inactivated by 5 min at 50°C (a tllz of about 1 min) ,while the E. solidagini.s enzyme showed a tl,* of 5.5 min. The addition of 0.5 M glycerol had no significant effect on the thermal stability of larval G3PDH but the addition of 0.25 M sorbitol signiticantly increased the stability of the enzyme, increasing the tliz to 7.5 min. While glycerol had nlo effect on its own, its addition along with sorbitol further increased enzyme stability, raising the tl12 to 10.5 min.

FROM

Eurosra

soliduginis

189

Similar effects of the polyols were seen for rabbit muscle G3PDH when the enzyme was studied at 45°C: no effect of glycerol on its own but increased stability with the addition of sorbitol and a further increase in stability in the presence of both sorbitol and glycerol. Very high levels of glycerol and sorbitol(2 and 1 M, respectively) produced even greater thermal stability of the E. soliduginis enzyme (Fig. 3), an effect which could be important in protecting intracellular enzymes at temperatures below the freezing point of hemolymph when intracellular polyols become highly concentrated. DISCUSSION

Intermediary metabolism in E. solmust be controlled and coordinated over a temperature range which can vary from over 30°C in summer to winter lows approaching -40°C. Within this range, further controls must operate to induce first glycerol and then sorbitol synthesis during low temperature acclimation. Temperature change could have extremely disruptive effects on the sorts of fine controls needed to maintain this integrated metabolism and to acheive the accumulations of cryoprotectants. Somero and Hochachka (11) suggest four ways in which enzyme rates can be modified in response to temperature in order to maintain an integrated metabolic rate: (i) changing the amounts of enzymes present with changing temperature, (ii) changing the concentrations of substrates and effecters, (iii) changing the isozymic forms of enzymes present, and (iv) direct effects of temperature on enzyme kinetics. The first strategy, when analyzed in terms of changes in enzyme activities, is used by E. soliduginis during low temperature acclimation but applies mostly to low activity, regulatory enzymes such as hexokinase and phosphofructokinase (13). G3PDH showed no change in measured activity during low temperature acclimation from 15 to -30°C (13). Changes in the concentrations of substrates (glycolytic and iduginis

190

STOREY AND STOREY

%V 60

1.0

21)

[ATPI

a

mM

zo-

05

1.0

[ADPI

mM

b

12

3

[citrate]

4

ml4

5

Krebs cycle intermediates) or effecters (such as the adenylates) are not apparent during low temperature acclimation in the gall fly larvae (14) and it is unlikely, therefore, that the second strategy plays a role in modulating enzyme activity with temperature. The third strategy, that of altering isozymic forms of enzymes, also does not appear to occur with low temperature acclimation in E. soliduginis (15); for G3PDH, no change in the number of forms expressed (three), the pi’s or the relative activities of the forms were seen in larvae acclimated to high or low temperatures. Direct interactions of temperature with the kinetic and regulatory properties of enzymes would appear, therefore, to be a major control operating to maintain regulatory control with changing temperature in the gall fly larvae. A study of the kinetic properties of E. sofidaginis G3PDH, a key enzyme in glycerol biosynthesis, therefore, seemed appropriate. It has been suggested that certain enzymes exhibit multiple conformational states, the thermodynamically most stable conformer changing with ambient temperature (6, 9, 11). If the conformers differ in their kinetic properties, then a single polypeptide chain can function as what would appear to be multiple enzyme variants. Pyruvate kinase from the king crab shows this behavior, the two conformers having distinct and separate K, versus temperature curves (9). To test the possible existence of temperature-dependent conformers of G3PDH in E. solidaginis, we

FIG. 2. Effect of inhibitors on E. solidaginis G3PDH at 25 and 5°C. Enzyme activity was assayed at subsaturating substrate levels, 0.05 m&I DHAP and 0.005 mM NADH. Open symbols indicate assay at 2X, ftied symbols at 5°C. (a) ATP effects. (b) ADP effects. (c) Citrate effects.

GLYCEROL-3-PHOSPHATE

Inhibitor

DEHYDROGENASE

FROM

Eurosru

191

solidaginis

TABLE 2 Effects on E. solidaginis Glycerol-3-phosphate Dehydrogenase and Interactions of Inhibitors with Temperature and Polyols Inhibitor constant, I,, (mM)

Inhibitor

Temperature (“Cl

Control

+0.5 A4 glycerol

+0.25 M sorbitol

+Both polyols

ATP

25 5

0.55 0.47

0.48 0.51

0.45 0.60

0.52 0.50

Mg .ATP

25 5

2.1 2.3

2.0 2.5

1.4 2.2

1.5 -

ADP

25 5

1.6 0.71

1.3 0.91

1.2 0.83

1.2 -

Citrate

25 5

0.60 1.2

0.55 1.0

0.77 1.1

0.76 1.7

Mg. Citrate

25 5

0.48 0.60

0.47 0.50

0.42 0.72

0.50 0.54

pi

25 5

9.5 11.8

9.8 10.5

11.1 13.3

11.6 12.2

Note. I,,‘s, the inhibitor concentrations producing 50% inhibition compared to control activities, were determined a constant substrate concentrations, 0.05 mM DHAP and 0.005 mM NADH. Mg*+, when added, was present in a 1: 1 ratio with ATP or citrate. Results are means of duplicate determinations with variability + 10%.

prepared the enzyme from larvae acclimated to 24 versus o”C, performing the entire preparation and assay procedures at the two respective temperatures. However, our analysis of the kinetics of G3PDH showed no differences in the kinetic constants of these two enzymes when assayed at either 24 or 0°C suggesting that kinetically differing temperature-dependent conformers of the larval G3PDH do not occur. Many early studies of temperature effects on the k.inetics of enzymes from poikilothermic animals have demonstrated large effects of temperature on the Km’s of substrates (IO, 11). Minimal K,,,‘s generally occurred at the normal habitat temperature of the animal, a strategy which appears to be highly adaptive to optimal enzyme function. More recent studies have shown that many of these effects were exaggerated by assay conditions in which pH was held constant with changing temperature. It is now well known that the intracellular pH of poikilotherms varies with temperature follow-

ing the pK, of histidine, the major intracellular buffering agent (10) and recent studies allowing the pH of imidazole buffer to change with temperature have now shown that enzyme kinetic constants are much more strongly conserved with changing temperature. When buffer pH was allowed to change with temperature, thus maintaining a constant charge state on the essential histidyl groups of the active site of G3PDH (16), little or no change in the Michaelis constants or in the inhibitor constants was seen for E. soliduginis G3PDH over a wide temperature range. Regulation of gall fly larva G3PDH may rely largely on substrate availability, flux through this locus and into glycerol biosynthesis being determined by overall glycogenolytic flux. The Km’s for DHAP and NADH are both likely well within the physiological concentration range of these compounds in viva and major temperature effects on these K,‘s are lacking. The increase in KmcDHAP, at low (OC) temperature

192

STOREY

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STOREY

ulators of the enzyme were also unaffected by temperature suggesting that the enzyme is not subject to differential regulation by modulators with changing temperature. Two effecters, however, did show altered effects with varying temperature. ADP ef80 fects were relatively stronger at low temperatures although the physiological importance of ADP effects is questionable as the inhibitor constants for ADP are well 60 above the in viva levels of this compound. Citrate, however, could be an important effector of the enzyme; citrate levels in E. solidaginis are 4 to 5 pmollg wet wt compared to an I,, for citrate of 0.7 to 1.5 mZt4. Citrate effects, which are enhanced at high temperatures, could be of importance in regulating enzyme activity with respect to the role of G3PDH in lipid synthesis. Extramitochondrial levels of citrate would reflect the availability of acetyl-CoA for fatty acid synthesis and determine the need for glycerol-3-Z’ for glyceride synthesis. E. 2 10 20 solidaginis G3PDH is also under strong TIME (mid control by the redox and energy status of FIG. 3. Heat inactivation of G3PDH: effect of incuthe cell although the kinetic constants for bation time at 50°C on the measured activity of E. NAD+ and ATP are not affected by tempersolidaginis and rabbit muscle G3PDH. Enzyme activ- ature change. The low Ki for NAD’ (0.2 ity was measured at 24°C under standard assay condirnM) and for ATP (0.25 mM with respect to tions as described under Materials and Methods. SymNADH) would allow regulation of G3PDH bols are: for E. solidaginis G3PDH-0, control; 0, +0.5 M glycerol; Cl, +0.25 M sorbitol; n , +0.5 A4 activity in the enzyme’s role in the glycerol and 0.25 A4 sorbitol; a, +2 M glycerol and 1 glycerol-3-P cycle. Insect tissues utilize the M sorbitol; for rabbit muscle G3PDH-A, control: A, glycerol-3-P cycle as the means for trans+0.5 M glycerol and 0.25 M sorbitol. ferring cytoplasmic reducing equivalents, generated at the glyceraldehyde-3-P dehymay, however, be one factor limiting drogenase reaction of glycolysis, into the glycerol synthesis as acclimation temperamitochondria during aerobic carbohydrate ture declines in this species. The parallel catabolism (7). Effects of NAD+ and ATP increase in the KmcDHAP)of the rabbit en- coordinate G3PDH activity with cellular zyme at this temperature would indicate, redox levels and with the demand for celhowever, that this is a general property of lular energy production. Like the insect flight muscle or the squid mantle muscle, G3PDH enzymes and is not a specific both of which utilize the glycerol-3-P cycle adaptation of the E. solidaginis enzyme. The linear relationship of the Arrhenius plot for this purpose, but unlike various other over the range 30 to O’C, with a Q1,, of about tissues lacking this cycle (7, 12) E. sof2, shows that there are no “catastrophic” idaginis larvae contain a G3PDH which effects of temperature on enzyme maximal shows strong inhibitory effects by ATP. Among the presumed effects of cryoprovelocity. The actions of most metabolite mod-

GLYCEROL-3-PHOSPHATE

DEHYDROGENASE

tectant polyols in species capable of surviving subzero temperatures is an effect of polyols in stabilizing protein structure (1). Protein structure and enzyme-ligand interactions often rely upon various types of weak bonds for stability; hydrophobic bonds are weakened at low temperatures and stabilized at high temperatures, hydrophillic bonds behave oppositely. Accumulations of polyols could counteract some of the effects of low temperature on weak bonds. Glycerol and sorbitol accumulate to levels of about 500 and 200 FmoUg wet wt in E. solidaginis (14) with even higher intracellular levels of polyol occurring below - 10°C once extracellular freezing takes place. Polyols, however, had little effect on G3PDH kinetics, enzyme kinetic constants being little altered by temperature whether or not polyols were present. Polyols d.id have an effect on the V,,, activity of the enzyme and they had a prounounced effect on the structural stability of the enzyme protein as examined in the thermal inactivation studies. Sorbitol significantly increased the stability of E. solidaginis G3PDH while glycerol, which had no significant effect on its own, enhanced the protection of the enzyme when added along with sorbitol. These effects of polyols in protecting protein structure may be of considerable importance at subzero temperatures in stabilizing enzyme structures to preserve enzyme activity during over-wintering. The greater thermal stability of the E. soliduginis enzyme compared to the rabbit muscle enzyme suggests that the larval G3PDH is structurally adapted for function over a wide temperature range. E. solidaginis G3PDH, while showing no startling effects of temperature on enzyme properties, is in fact well suited to function in an animal which faces wide variation in ambient temperature on both a daily and a seasonal basis. The constancy of K,‘s and inhibitor effects over a wide temperature range allows efficient regulation of enzyme activity at all temperatures and keeps the activity of this enzyme-coordinated with

FROM

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solidaginis

193

others in the pathway of glycerol synthesis. While probably not a controlling reaction in glycerol synthesis, the enzyme is well adapted to responding to changes in flux through glycolysis at all temperatures and also shows a greater structural stability than does its homeothermic homolog which suits the enzyme for maintaining structural integrity over a wide temperature range. SUMMARY

The kinetic properties of cytoplasmic glycerol-3-Z’ dehydrogenase from the third instar larva of the gall fly, Eurosta solidaginis, were studied with emphasis on temperature effects on the enzyme and the regulation of enzyme activity during the synthesis of the cryoprotectant, glycerol. Isoelectrofocusing revealed one major and two minor forms of the enzyme with no alteration in the pi’s or relative activities of the forms in larvae acclimated to 24 versus -30°C. Kinetic properties of the enzyme were also the same in larvae acclimated to high and low temperatures. Arrhenius plots were linear over a 30 to 0°C range with an activation energy of 12,630 & 185 cal/mol and a Qr,, of 2.16. The K, for dihydroxyacetone-P was constant, at 50 pM, between 30 and 10°C but increased by 75% at 0°C; this increase may be a factor in the cessation of glycerol synthesis which occurs below 5°C in this species. The KmcNADHj, by contrast, was higher (5-6 pM) at 30°C but decreased (3 pM) at lower temperatures. In the reverse direction, Km’s were 340 pM for glycerol-3-P and 12 pM for NAD+. Effects of most inhibitors (of the forward reaction), glycerol-3-P (Ki = 2.4 mM), NAD+ (Ki = 0.2 mM), ATP, Mg.ATP, and Pi, were unaltered by assay temperature but ADP effects were potentiated by low temperature while citrate inhibition was greatest at high temperatures. Glycerol and sorbitol, which accumulate as cryoprotectants in E. solidaginis, had no significant effects on kinetic constants at any temperature but decreased the V,,, activity of the enzyme. Thermal inactivation studies showed an in-

194

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creased thermal stability of the larval enzyme compared to the homologous enzyme from rabbit muscle while added polyols stabilized enzyme activity, decreasing the rate of enzyme inactivation at 50°C. ACKNOWLEDGMENT Supported by an NSERC operating grant to K.B.S. REFERENCES 1. Baust, .I. G. Mechanisms of cryoprotection in freezing tolerant animal systems. Cryobiology 10, 197-206 (1973). 2. Brosemer, R. W., and Marquardt, R. R. Insect extramitochondrial glycerophosphate dehydrogenase. II. Enzymic properties and amino acid composition of the enzyme from honeybee (Apis mellifera) thoraces. Biochim. Biophys. Acta 128, 464-473 (1966). 3. Fernandez-Sousa, J. M., Gavelanes, .I. G., Municio, A. M., and Perez-Aranda, A. LGlycerol-3-phosphate dehydrogenase from the insect, Ceratitis capitata. Purification, physicochemical and enzymic properties. Biochim. Biophys. Acta 481, 6-24 (1977). 4. Job, D., Cachet, C., Dhien, A., and Chambaz, E. M. A rapid method for screening inhibitor effects: Determination of I,,, and its standard deviation. Anal. Biochem. 84, 68-77 (1978). 5. Morrissey, R. E., and Baust, J. G. The ontogeny of cold tolerance in the gall fly, Eurosta solidagensis. J. Insect Physiol. 22, 431-437 (1976). 6. Nickerson, K. W. Biological functions of multistable proteins. J. Theor. Biol. 40, 507-515 (1973). 7. Sacktor, B. Biochemical adaptations for flight in the insect. Biochem. Sot. Symp. 41, 111-131 (1976). 8. Senkbeil, E., and White, H. B. Parallel evolution

of pairs of dehydrogenase isoenzymes. J. Mol. 11, 57-66 (1978). 9. Somero, G. N. Pyruvate kinase variants of the Alaskan king crab-Evidence for a temperature-dependent interconversion between two forms having distinct and adaptive kinetic properties. Biochem. J. 114, 237-241 (1969). 10. Somero, G. N. Temperature adaptation of enzymes: Biological optimization through structure-function compromises. Annu. Rev. Ecol. Syst. 9, l-29 (1978). 11. Somero, G. N., and Hochachka, P. W. Biochemical adaptations to temperature. In “Adaptation to Environment” (R. C. Newell, Ed.), pp. 125-190. Butterworths, London, 1976. 12. Storey, K. B., and Hochachka, P. W. The kinetic requirements of cytoplasmic alpha-glycerophosphate (a-GP) dehydrogenase in muscles with active o-GP cycles. Comp. Biochem. Physiol. B 52, 175-178 (1975). 13. Storey, K. B., and Storey, J. M. Biochemical strategies of overwintering in the gall fly, Eurosta solidaginis: Effect of low temperature acclimation on the activities of enzymes of intermediary metabolism. J. Comp. Physiol. 144, 191-199 (1981). 14. Storey, K. B., Baust, J. G., and Storey, J. M. Intermediary metabolism during low temperature acclimation in the overwintering gall fly larva, Eurosta solidaginis. J. Comp. Physiol. 144, 183-190 (1981). 15. Storey, K. B., Park, I. R. A., and Storey, J. M. Isozyme composition and low temperature acclimation in the overwintering gall fly larva, Evol.

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(1981). 16. Suarez, Z., and Apitz-Castro, R. a-Glycerophosphate dehydrogenase: A regulatory enzyme. Biochim. Biophys. Acta 258, 339-342 (1972). 17. Vesterberg, 0. Isoelectrofocusing of proteins. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, Eds.), Vol. 22, pp. 389-412. Academic Press, New York, 1971.