Phosphoglucose isomerase isozymes and allozymes of the brown trout, Salmo trutta L

Comp. Biochem. Physiol. Vol. 88B, No. 3, pp. 751-756, 1987

0305-0491/87 $3.00+ 0.00 © 1987 Pergamon Journals Ltd

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PHOSPHOGLUCOSE ISOMERASE ISOZYMES A N D ALLOZYMES OF THE BROWN TROUT, S A L M O T R U T T A L. T. HENRY and A. FERGUSON Department of Zoology, The Queen's University, Belfast BT7 1NN, UK (Tel.: 0232-661111) (Received 12 January 1987)

Abstraet--l. In brown trout the Pgi-1 and Pgi-2 loci are predominantly expressed in white skeletal muscle; Pgi-3 being mainly expressed in most other tissues. 2. Total PGI activity determinations revealed that the allele formerly designated Pgi-2(65) is a null allele, Pgi-2(n). 3. Enzyme kinetic studies on the partially purified PGI homodimeric isozymes revealed that from 5 to 25°C both IKH-I and PGI-2 had significantly higher mean Km(F6P) values compared to PGI-3. 4. Distinct metabolic roles for the "muscle" (PGI-I, PGI-2) and "liver" (PGI-3) isozymes are proposed. 5. Significant K~(F6P) differences were found among the PGI-2 allozymes and among the PGI-3 allozymes.

INTRODUCTION

Phosphoglucose isomerase (PGI; D-glucose-6-phosphate ketol isomerase; EC5.3.1.9) catalyses the interconversion of glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P). In fishes, P G I is coded for by one to five loci with 54% of species in a wide,ranging survey showing two loci (Coppes, 1986). Brown trout, Salmo trutta L., commonly exhibits a six-banded zymogram which can be explained on the basis of three loci coding for a dimeric enzyme resulting in three homodimeric and three heterodimeric isozymes (Taggart et al., 1981; Taggart and Ferguson, 1984a). Polymorphism has been described at two of these loci in brown trout populations involving five alleles at Pgi-2 (65, 100, 122, 130 and 135) and three alleles for Pgi-3(80, 100, 110). The allele described as Pgi-2(65) was found in a single population at a frequency of 0.4 (Ferguson and Fleming, 1983). The designation of this allele was based on the assumption that the PGI-2(65/65) allozyme had the same electrophoretic mobility as the PGI-1 isozyme but the possibility that a null allele is involved was not discounted. The Pgi-2(135) allele was found in 47% of British and Irish brown trout populations (Ferguson and Fleming, 1983) and was found to be fixed in one population in a small tributary river (Ferguson, unpublished). The Pgi3(110) allele was shown to have a very limited distribution in trout populations in Britain and Ireland with only two populations, in northern Scotland, having frequencies greater than 0.14 (Taggart and Ferguson, 1984a). The Pgi-2(122), Pgi-2(130) and Pgi-3(80) alleles are rare variants found in one or two populations (unpublished studies). Studies on the kinetic properties of fish isozymes have helped explain their observed tissue distributions (Lira et aL, 1975; Palumbi et al., 1980; Henry and Ferguson, 1986), Similarly, allozyme investigations have shed light on the possible selective mech-

anisms involved in transient and balanced enzyme polymorphisms (Kao and Farley, 1978; Place and Powers, 1979, 1984; Henry and Ferguson, 1985). This paper describes the partial purification of the major isozymes and allozymes of PGI from the brown trout and their kinetic characterisation at one pH and a range of temperatures. In all descriptions PGI refers to the enzyme product of the particular Pgi locus.

MATERIALSAND METHODS Brown trout were obtained from various natural Irish populations. Due to the low frequency of the Pgi-3(ll0) allele in natural populations, a Pgi-3(100/l10) xPgi3(100/110) cross was set up using four male and four female parents and the progeny used for this study. Purification of the PGI isozymes and aUozymes was carried out by starch gel electrophoresis using the buffer system of Ridgway et al. (1970). Vertical starch gel electrophoresis of white skeletal muscle extracts was used for isolation of PGI- 1 and PGI-2 and horizontal starch gel electrophoresis of liver was used for purification of PGI-3. After electrophoresis, a thin slice of the gel was stained for PGI and subsequently used as a template for the excision of specific isozymes and aUozymes from the unstained portion of the gel. All three intralocus allozymes were excised as a group in the case of heterozygotes. Purified isozymes and allozymes were removed from the gel by homogenlsation and centrifugation and were subsequently dialysed. All these procedures were carried out at 2-4°C in 50raM Tris-HC1 pH 8.0 buffer containing I mM EDTA and 23 mM MgC12. Final purity was checked by re-electrophoresis. Starch gels stained for PGI were scanned using an LKB 2202 Ultroscan laser densitometer. Care was taken that the mount of formazan deposition was linearly proportional to the enzyme units applied to the gel using a calibration curve as described by Lira and Bailey (1977). Linearity was found to exist up to 0.13 units of enzyme activity at 25°C. Kinetic measurements

PGI kinetic studies were carried out by measuring the change in optical density at 340nm in a Pye-Unicam SP6-550 spectrophotometer with thermostated cell holder. 751

752

T. HENRYand A. FEROUSON

The standard assay mixture was 3 ml of 50 mM Tris-HC1 pH 8.0 containing 1 mM EDTA, 23 mM MgCI 2, 1.3 mM F6P, 0.2 mM NADP and 3.3 U G6PDH. It was verified initially that the concentrations of NADP and G6PDH were in excess at all temperatures used. Reactions were initiated by the addition of PGI and absorbance changes were recorded at 15 sec intervals. Initial velocities (A~0/min) were calculated by linear regression of the rate from 2 to 5 rain since a lag phase was observed over the first 1.5 rain of the reaction. Only rates with correlation coefficients >0.99 were accepted. Sufficient purified PGI was added to give AOD of 0.1--0.2units/rain. Apparent Km for fructose-6-phosphate [K~(F6P)] was determined at 5, 10, 15, 20 and 25°C while varying the substrate concentration over the empirically determined range of (0.1--0.2) × Km to (2--4) × Kin. At least triplicate assays were conducted at 6--8 substrate concentrations. Estimates of K~ were obtained by the nonparametric direct linear plot method (Eisenthal and Cornish-Bowden, 1974) and by a parametric non-linear least squares regression analysis (Roberts, 1977).

mobility variant [Pgi-2(65)] as previously described by Taggart et al. (1981). Although in most cases the presumed heterozygote [Pgi-2(100/n)] gave a shift in staining intensity towards the two most cathodal PGI bands it was not always possible to confidently distinguish between Pgi-2(100/100) and Pgi-2(100/n) individuals and thus the enzyme activity results for these two groups were pooled. The mean total PGI activity of this combined group was significantly (P < 0.01) higher than that of the Pgi-2(n/n) individuals (Table 1). The mean wts of the fish in the two groups were compared since it has been reported that for some enzymes specific activity per unit wt of wet tissue changes with fish size (Somero and Childress, 1980). However, the mean wts were not found to be significantly different. For further analysis it was necessary to exclude the PGI-3 activity contribution for each of the two groups. Based on densitometry data for three individuals of each genotype, the PGI-3 activity contributions of the two groups PGI-2(100/100) + PGI2(100/n) and PGI-2(n/n) were 12.4 and 20.6% respectively (Table 2). Densitometry also revealed equal production and random association of PGI-1 and PGI-2 polypeptides in white skeletal muscle tissue of Pgi-2(100/100) individuals. Assuming Hardy-Weinberg proportions the expected number of individuals in each of the two genotypic classes, Pgi-2(100/100) and Pgi-2(100/n) was calculated. Also assuming that there was no compensation from Pgi-1 when the activity of Pgi-2 was reduced, the mean number of functional Pgi alleles per individual and the activity per allele were calculated. Under the null allele hypothesis the activity per allele is identical for both groups. However, if a functional mobility variant allele [Pgi-2(65)] is assumed, the total activity per allele for the two groups is markedly different (Table 3).

RESULTS

Tissue distribution of PGI isozymes The PGI-1 and PGI-2 isozymes were predominant in white skeletal and lateral line muscles, these isozymes being present in greatly reduced amounts or absent in most other tissues (Fig. 1). Both PGI-2 and PGI-3 isozymes were present in heart. The remainder of the tissues showed predominant expression of Pgi-3. In the tissues where all three loci were active a six-banded zymogram was found as a result of the three homodimeric and three heterodimeric isozymes.

PGI polymorphism Allozymic variation of PGI-2 and PGI-3 is shown in Fig. 2. In the case of the individuals producing a three-banded zymogram, for the purpose of presentation of results, it is assumed that these are homozygous for a null allele at Pgi-2 rather than a

PGI-8(IO0)

PGI-2(IO0) PGI-I(100) o

a

b

c

d

e

f

g

h

i

j

k

I

m

n

o

P

q

r

Fig. 1. Phosphogiucose isomerase isozymes of various tissues of brown trout separated by horizontal starch gel electrophoresis. The tissues arc: (a) white skeletal muscle; (b) lateral line (red) muscle; (c) heart; (d) liver; (e) kidney; (f) brain; (g) eye; (h) gonad; (i) pyloric caecae; (j) gill; (k) intestines; (1) oesophagus; (In) anal fin; (n) adipose fin; (o) pectoral fin; (p) pelvic fin; (q) caudal fin; (r) integument; (s) tongue. O indicates the sample origin and the anodal end of the gel is at the top.

Brown trout PGI

753

PGI--3(110) PGI-3(100)

PGI--2(135) PGI--2(IO0)

PGI- 1(100)

O

a

b

c

d

e

g

f

Fig. 2. Phosphogiucose isomerase zymograms of brown trout tissue extracts separated by horizontal starch gel eleetropboresis. Samples a - f are white skeletal muscle and sample g is heart muscle. Pgi genotypes are:

(~i) Pgi-2(100/100), Pgi-3(100/100); (b) Pgi-2(100/135); (e) Pgi-2(135/135); (d) Pgi-2(n/n); (e) Pgi-2(100/n); (f) and (g) Pgi-3(100/110). O indicates the sample origin and the anodal end of the gel is at the top. Km(F6P) estimates Km estimates obtained from separate extracts of a particular isozyme and calculations based on the parametric and nonparametric methods were generally in good agreement (Table 4). For all PGI isozymes and aUozymes there was a significant (P < 0.05) linear relationship of K~(F6P) with temperature from 5 to 25°C. The results of an analysis using combined two-way analysis of variance with regression revealed highly significant (P <0.001)

heterogeneity in the mean K~(F6P) values of the isozymes and allozymes. The PGI-3 allozymes had significantly (P < 0.05) lower mean K~(F6P) values compared to the PGI-1 and PGI-2 isozymes (Fig. 3). While there were no significant differences in mean Km(F6P) among PGI- I (100/100), PGI-2(100/135) and PGI-2(135/135), these all showed significantly (P < 0.05) larger mean values compared to the PGI2(100/100) homodimer. The PGI-3(100/100) allozyme exhibited a significantly (P <0.05) smaller mean Km(F6P) compared with the PGI-3(I10/ll0)

Table 1. PGI specific activities in white skeletal muscle of brown trout. Units of PGI activity are/~mol NADP converted to NADPH/min at 10°C. Weights refer to total fish wt

Genotype

N

Mean units PGl/g wet weight of tissue ± SD

PGI-2(100/100) + PGI-2(100/n) PGI-2(n/n)

58

0.979 + 0.429

22.021 ± 14.578

12

0.633 + 0.218

18.400 + 12.033

Mean weights (g) ± SD

One-way ANOVA: activities Fl,68= 7.35 (P < 0.01). One-way ANOVA: wts FI,~ = 0.65 (ns).

Table 2. PGI specific activities in white skeletal muscle of brown trout corrected for PGI-3 contribution. Units of PGI activity are/Jmol NADP converted to NADPH/min at 10°C

Genotype PGI-2(100/100) PGI-2(100/n) PGI-2(n/n)

Percentage PGI-3 contribution*

Mean percentage PGI-3 contribution for PGI-2(100/100) + PGI-2(100/n)

Total PGI activityt (corrected for PGI-3 contribution)

12.4

0.858

12.7 12.1 20.6

*From densitometry analysis of starch gels. tMean U PGI activity/g wet wt white muscle tissue.

0.503

754

T. HENRY a n d A. FEKOUSON

Table 3. Functional alleles per individual and activity per functional allele assuming (1) a null allele and (2) a functional allele at PGI-2 Genotype PGI-2(100/100) PGI-2(100/n) PGI-2(n/n) *Calculated assuming tAssuming null allele :l:Corrected for PGI-3 §Assuming functional

N 24* 34* 12

Functional allelesI per individualt ,

Total activity:l: per allelet

Functional alleles2 per individual§

Total activity:[: per allele~

0.251 0.251

4 4

0.214 0.126

3.414 2

Hardy-Weinberg equilibrium. at PGI-2. activity and expressed as mean U PGI activity/g wet wt white muscle tissue. allele at PGI-2.

form while that for PGI-3(100/110) was intermediate, but not significantly different compared to the two PGI-3 homozygote allozymes.

"muscle" locus. A three-banded zymogram for brown trout from West German hatcheries was interpreted on the basis of the expression of only two Pgi loci (Engel et al., 1975, 1977; Schmidtke et al., 1975).

DISCUSSION

Tissue distribution and occurrence o f P G I isozymes and allozymes

The initial duplication of Pgi loci is thought to have occurred early in the evolution of the teleost fishes (Fisher et al., 1980). On the basis of tissue distribution and relative electrophoretic mobilities it would appear that PGI-1 and PGI-2 are produced by loci which are duplicate partners, the result of the tetraploidisation event within the salmonid lineage (Ohno et al., 1968; Massaro and Markert, 1968). Avise and Kitto (1973) have suggested that two electrophoretically coincident loci may code for what Allendorf et al. (1977) and Taggart et al. (1981) have designated Pgi-3. Inheritance data (Taggart and Ferguson, 1984b) have been used to verify that the most anodal isozyme is the product of only a single Pgi locus. It would seem then that the duplicate partner of this locus has been silenced within the salmonid lineage at some stage following tetraploidisation. The PGI specific activity determinations of muscle extracts reported here strongly suggest that the originally designated Pgi-2(65) (Taggart et aL, 1981) is in fact a null allele Pgi-2(n). Fixation of this allele would thus lead to a silencing of this duplicate

0.31 0.32

1

0.30 0.29 0.28

-J 0.27 0.26 i

0.25 -

~

0.24 -

t

0.23-

E 0.22 i 0.21 0.20 0.19 0.18 0.17 -

t i

i

i

i

IIIO~=/,IIA.O~ME

Fig, 3. K~(F6P) v a l u e s (i.e. the m e a n Km over the s t a n d a r d t e m p e r a t u r e r a n g e ) ± ½ L . S . R , for the P G I isozymes a n d a l l o z y m e s ( P --- 0.05).

Table 4. Km(F6P)* (mM) estimates for PGI isozymes and allozymcs at varying temperatures

PGI-I(100/100)

PGI-2(100/100)

PGI-3(100/100)

PGI-2(100/135) PGI-2(135/135) PGI-3(100/110) PGI-3(110/110)

(1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2)

5

10

Temperature (°C) 15

20

25

0.230 0.219 ± 0.002 0.268 0.264 + 0.015 0.208 0.242 _+ 0.004

0.249 0.238 + 0.019 0.269 0.306 + 0.026 0.250 0.248 _+ 0.002 0.196 0.191 + 0.009 0.157 0.166 + 0.017 0.175 0.162+0.005 0.268 0.272 ± 0.014 0.266 0.238 + 0.010 0.164 0.187 _+ 0.010 0.190 0.204 _+0.011

0.307 0.29t + 0.009 0.329 0.314 + 0.016 0.248 0.256 ± 0.025 0.258 0.307 _+ 0.003 0.149 0.162 + 0.002 0.191 0.204_+0.00,1 0.293 0.311 _+ 0.005 0.308 0.261 + 0.011 0.195 0.198 _+ 0.005 0.212 0.205 _+ 0.008

0.312 0.370 + 0.015 0.356 0.351 5:0.015 0.334 0.323 + 0.018 0.312 0.342 + 0.003 0.222 0.222 + 0.008 0.211 0.207±0.008 0.315 0.322 ± 0.006 0.336 0.346 ± 0.009 0.215 0.219 5:0.010 0.223 0.237 _+ 0.004

0.345 0.376 _+ 0.027 0.389 0.395 "t" 0.007 0.329 0.325 _+0.041 0.394 0.399 + 0.014 0.265 0.208 + 0.009 0.223 0.224+0.015 0.353 0.351 ± 0.008 0.365 0.369 _+ 0.006 0.214 0.215 5:0.003 0.245 0.227 + 0.008

0.144 0.147 + 0.009 0.161 0.163+0.008 0.274 0.279 _+ 0.007 0.222 0.238 + 0.010 0.179 0.179 _+ 0.012 0.177 0.181 _+0.004

(1) Nonparametric estimates (Eisenthal and Cornish-Bowden, 1974). (2) Parametric estimates + SD (Roberts, 1977). Where there is more than one Km determination for any isozyme these refer to separate extracts. *The conditions of assay were 50 mM Tris-HCl buffer pH 8.0, 1 mM EDTA, 23 mM MgCI2, 0.20 mM NADP, 3.3 U G6PDH and varying concentrations of F6P.

755

Brown trout PGI It is possible that these stocks are fixed for a null allele at Pgl-2, although many different mutations may result in a null allele, it would not necessarily be the same one as reported here. The Pgi-2(n) allele has so far only been recorded from one population out of over 200 examined in Britain and Ireland. The population with the Pgi-2(n) allele is a migratory (sea-trout) one yet the allele is absent from similar populations in rivers flowing into the same sea bay some 2 km distant. This observation together with the relatively high frequency of 0.4 is suggestive of a rapid increase in the frequency of this allele possibly under strong selective pressure, Current population estimates and the overall level of genetic variability suggest that the population is and has been in the past, sufficiently large for genetic drift to be discounted. However, if positive selection is involved the absence of the allele from adjacent river populations is puzzling especially as some gene flow would be expected. Allendorf et al. (1984), in considering various models for the loss of duplicate gene expression in salmonids, suggest that selection against null alleles occurs in most cases. Kinetic comparisons o f isozymes

The much higher Km(F6P) values for the "muscle" (PGI-1 and PGI-2) isozymes compared to the "liver" (PGI-3) isozyme of brown trout may be explained by the different physiological roles of these tissues. Liver may be regarded as a highly aerobic tissue where all the major metabolic processes (e.g. glycolysis, gluconeogenesis, the tricarboxylic acid cycle and oxidative phosphorylation) occur. One of its functions during oxygen stress, when muscle cells become primarily "anaerobic" is to remove lactate from the blood and convert it to glucose via gluconeogenesis. The glucose thus produced is shuttled to muscle where it is used for energy production through glycolysis. Evidence for a giuconeogenic role for liver and kidney tissue of salmonids has been reported by Cowey et al. (1977), Knox et al. (1980) and Walton and Cowey (1982). In contrast it has been suggested that fish white skeletal muscle lacks gluconeogenesis (Newsholme and Start, 1973). This is supported by the finding that the activities of the key gluconeogenic enzymes are very low in rainbow trout white muscle tissue (Cowey et al., 1977). In brown trout, the presence of only the PGI-3 isozyme in liver and kidney and the higher affinity (i.e. lower K~) of PGI-3 for F6P, compared to PGI-1 and PGI-2, suggests that this isozyme may be a more efficient gluconeogenic enzyme. In muscle tissue a higher concentration of F6P would be expected as PGI supplies substrate to the key regulatory enzyme phosphofructokinase. Therefore, an enzyme having a higher Km(F6P) would appear to be more suited to the metabolic environment in white skeletal muscle. For most enzymes which have been studied Km values are of the order of physiological substrate concentrations (Fersht, 1977; Somero, 1978). Indeed it has been suggested that natural selection would favour such a relationship between K~, an evolutionarily variable parameter and substrate concentration, an environmental condition (Cornish-Bowden, 1976). Hence in the brown trout if the F6P concentration was consistently higher in muscle tissue than in liver

and kidney, this could provide the selective pressure for the evolution and maintenance of separate PGI isozymes. Thus the/(~ values can be viewed as an evolutionary response to accommodate different concentrations of F6P in different tissues. The Km(F6P) differences reported here support the suggestion by Dando (1974, 1980) that the "muscle-type" PGI of teleosts acts as a glycolytic or catabolic enzyme while the "liver-type" PGI acts as a gluconeogenic or anabolic enzyme. Kinetic comparisons o f allozymes The kinetic studies revealed mean /~(F6P) differences for both PGI-2 and PGI-3 aUozymes. If a high Km(F6P) is advantageous for "muscle" PGI, as argued above, then the PGI-2(100/135) and PGI2(135/135) allozymes would appear to be at a selective advantage compared to the PGI-2(100/100) one. This may explain the moderate to high frequencies of the Pgl-2(135) allele which are found in those populations which possess it. If a low Km(F6P) is selectively advantageous in liver tissue then the PGI3(100/100) allozyme, by virtue of having a significantly lower mean Km(F6P) than POI-3(ll0/ll0), would appear to be at a selective advantage. This again may account for the low frequencies of the Pgi-3(110) allele observed in almost all populations.

REFERENCES

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fish to mammals by gene duplication. Hereditas 59, 169-187. Palumbi S. R., SideU B. D., Van Ikneden R., Smith G. D. and Powers D. A. (1980) Glueosephosphate isomeras¢ (GPI) of the teleost Fundulus heteroclitus (Linnaeus): isozymes allozymes and their physiological roles. J. compo Physiol. 138, 49-57. Place A. R. and Powers D. A. (1979) Genetic variation and relative catalytic efficiencies: lactate dehydrogenase B allozymes of Fundulus heteroclitus. Proc. natn. Acad. Sci. USA 76, 2354-2358. Place A. R. and Powers D. A. (1984) Kinetic characterization of the lactate dehydrogenase (LDH-B4) allozymes of Fundulus heteroclitus. J. biol. Chem. 259, 1309-1318. Ridgway G. J., Sherburne S. W. and Lewis R. D. (1970) Polymorphism in the esterases of Atlantic herring. Trans. Am. Fish. Soc. 99, 147-151. Roberts D. V. (1977) Enzyme Kinetics. Cambridge University Press, Cambridge, UK. Schmidtke J., Dunkhase G. and Engel W. (1975) Genetic variation of phosphoglu¢ose isomerase isoenzymes in fish of the orders Ostariophysi and Isospondyli. Comp. BIOchem. Physiol. 50B, 395-398. Somero G. N. (1978) Temperature adaptation of enzymes: biological optimization through structure-function compromises. A. Rev. Ecol. Syst. 9, 1-29. Somero G. N. and Childress J. J. (1980) A violation of the metabolism-size scaling paradigm: activities of glycolytic enzymes in muscle increase in larger-size fish. Physiol. Zool. 53, 322-337. Taggart J. B. and Ferguson A. (1984a) An electrophoretically-detectable genetic tag for hatchery-reared brown trout (Salmo trutta L.). Aquaculture 41, 119-130. Taggart J. B. and Ferguson A. (1984b) Allozyme variation in the brown trout (Salmo trutta L.): single locus and joint segregation inheritance studies. Heredity 53, 339-359. Taggart J. B., Ferguson A. and Mason F. M. (1981) Genetic variation in Irish populations of brown trout (Salmo trutta L.): electrophoretic analysis of allozymes. Comp. Biochem. Physiol. 69B, 393-412. Walton M. J. and Cowey C. B. (1982) Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol. 73B, 59-79.