Glucose-6-phosphate dehydrogenases of echinoderms

Comp. Biochem. PhysioLVol. 98B, No. 4, pp. 539-542, 1991 Printed in Great Britain

0305-0491/91 $3.00+ 0.00 © 1991 PergamonPress pie

GLUCOSE-6-PHOSPHATE DEHYDROGENASES OF ECHINODERMS NORIMASAMATSUOKA Department of Biology, Faculty of Science, Hirosaki University, Hirosaki 036, Japan (Received 3 September 1990)

Abstract--l. Glucose-6-phosphate dehydrogenase (G6PD) was partially purified from the brittle-star, Ophioplocus japonicas by the affinity chromatography of Biue-Sepharose CL6B. 2. The kinetic properties of some parameters were examined and compared with G6PDs from other echinoderms and various animal groups. 3. The results showed that echinoderm G6PDs varied in some kinetic parameters among groups, though they were the ordinary G6PD.

INTRODUCTION Echinoderms and vertebrates have two isozyraes of giucose-6-phosphate dehydrogenase (G6PD): one is G6PD in the strict sense (the first step enzyme of the pentose phosphate shunt, E.C. 1.1.1.49) that has narrow substrate specificity, and the other is the enzyme called glucose dehydrogenase or hexose-6phosphate dehydrogenase (H6PD, E.C. 1.1.1.47) that has broad substrate specificity. In regard to their evolution, we predict that H6PD and G6PD shared a common ancestral molecule and that their divergence probably occurred at the time of or before the echinoderm evolution. This prediction is based on the close similarity in their catalytic properties (Hori et al., 1975; Mochizuki and Hori, 1976; Sado and Hori, 1976; Matsuoka et al., 1977), the phylogenetic distribution of the two isozymes in the animal kingdom [G6PD occurs in a wide variety of animals, while H6PD is found in echinoderms and vertebrates (Mochizuki and Hori, 1973; Ohnishi and Hori, 1977; Hori et al., 1977; K a m a d a et al., 1978)], the immunological relatedness of the two isozymes (Matsuoka and Hori, 1980), and the close similarity of the amino acid composition of the two isozymes (Matsuoka et aL, 1983). However, the presence of the two isozymes and their catalytic properties in echinoderms have not yet been examined sufficiently. Although the two isozymes have been found in starfish, sea-cucumber and sea-urchin, it still remains unclear whether other echinoderms such as brittle-stars and sea-lilies have the two isozymes, and what catalytic properties these isozymes have. In order to further clarify the evolution of the two G6PD isozymes, an extensive survey of the two G6PD isozymes and the close examination of their catalytic properties in various echinoderms would be required. In previous papers, I .have reported on the catalytic properties of G6PDs from the starfish and sea-urchin and H6PD from the starfish (Matsuoka et al., 1977; Matsuoka and Hori, 1980; Matsuoka, 1983). Such kinetic investigations are necessary to obtain more precise information on the evolution of the two G6PD isozymes. CgPB 98/4--H

In the present study, with the background noted above, I have attempted to examine the catalytic properties of brittle-star Cr6PD using several kinetic parameters and to compare them with the properties of G6PDs from other echinoderms and various other animal groups. MATERIAI~ AND METHODS Brittle-star The brittle-star, Ophioplocasjaponicus, was collected from the coast near the Fukaura Marine Biological Laboratory of Hirosaki University by snorkelling. After collection, the matured gonads were cut off from live specimens, and exhaustively washed in sea-water. They were then stored at - 2 0 ° C until they were analysed. Reagents Glucose-6-phosphate (G6P), nicotinamide adenine dinucleotide phosphate (NADP) and nicotinamide adenine dinueleotide (NAD) were obtained from Kyowa Hakko Kogyo Co.; gal~tose-6-phosphate (GaI6P) and 2-deoxygiucose-6phosphate (dG6P) from Sigma Chemical Co., and BlueSepharose CL6B from Pharmacia Fine Chemicals. All other reagents used were commercial products of the highest grade as far as was available. Enzyme assay and kinetic studies The enzyme assay and the examinations of the effects of PCMB (p-chloromereuribenzoate), DEA (dehydroepiandrosterone), MgC12 and heat on enzyme activity were carried out by methods described previously (Matsuoka and Hori, 1980). One unit of activity was defined as the amount of enzyme that reduced 1 #tool of NADP per rain. The spec. act. of enzyme was expressed as units/rag protein. Protein was assayed according to the method of Lowry et al. 0951). Km and V values were determined from Lineweaver-Burk plots of 1/v vs 1/s. Partial purification of G6PD from the brittle-star Gonads were homogenized with 20 vols of 20 mM phosphate buffer, pH 6.4, containing 0.1 M KC1, I mM EDTA and 7 raM 2-mercaptcethanol using a Potter-Elvehjem glass homogenizer in an ice-water bath. After centrifugation at 90,000 g for 40 min, the supematant was diluted with I vol of the same buffer, and then applied to a 20 × 25 ram column of Blue-Sepharose CL6B equilibrated with the same buffer. The column was exhaustively washed with the buffer

539

540

NORIMASAMATSUOKA 100 Brittle-

100

RESULTS AND DISCUSSION

5O

50 ._> m rr

0

7 8 9 10 11 pH 100

100

50

7 8 9 10 11 Sea-cucumber

501

~

7 8 91011

7 8 91011

Fig. I. Effect of pH on the activity of echinoderm G6PDs. Activity was expressed as a percentage of the peak activity with G6P and NADP. The pH curves of the starfish, sea-urchin and sea-cucumber are taken from Matsuoka and Hori (1980), Matsuoka (1983) and Mochizuki and Hori (1976), respectively. The species used in this assay were Asterias amurensis for starfish, Ophioplocus japonicus for brittle-star, Temnopleurus hardwickii for sea-urchin, and Stichopus japonicus for sea-cucumber. O, G6P; O, Gal6P; A, dG6P. and the enzyme was eluted at a flow rate of 40 ml/hr with 0.5raM NADP in 20mM phosphate buffer, pH8.7, containing 0.1 M KCI, 1 mM EDTA and 7 mM 2-mercaptoethanol. Fractions of 10 ml were collected and those with high enzyme activity were pooled and concentrated by means of a collodione bag (Sartorius Membrane Filter). This method resulted in a 48-fold purification with 72% yield. The spec. act. was 9.59 when assayed with G6P and NADP at pH 10.0. The enzyme sample thus obtained was used for the kinetic study.

Species Echinoderms 1. Brittle-star

Effects of pH on G6PD activity are shown in Fig. 1 which also includes for comparison those of three other echinoderms (the sea-urchin, starfish and seacucumber). The profiles of pH-activity curves obtained with the three substrates in the presence of N A D P were essentially similar among the four echinoderms. Namely, the pH optimum of G6PD activity was in the range of 9.0-10.5, and the activity on GaI6P or dG6P was much lower than that on G6P in the whole range of pH tested. In particular, the activity on dG6P was lowest. Ohnishi and Hori (1977) found that G6PDs of hydra, sea-anemone and squid showed the high activities on GaI6P in the presence of N A D P as coenzyme. However, echinoderm G6PDs did not show such unusual substrate specificity. The Km values for three substrates and one coenzyme at the physiological pH (7.5) are shown in Table I. Table 1 also includes for comparison the values from other echinoderms, 11 species of invertebrates and six species of vertebrates. As evident from this table, the Kms for G6P and N A D P were very low and fell within a narrow range. In contrast with these, Kms for Gal6P and dG6P, which may well be of no physiological significance, were considerably larger than the Km for G6P. Furthermore, they varied among animal species and that for dG6P particularly so. The conservation of Km values for physiological substrates among species has been interpreted in terms of a mechanism for insuring the maintenance of proper catalytic and regulatory functions of enzymes (Atkinson, 1977; Fersht, 1977). Many intracellular substrate concentrations are near or below the Km values (Fersht, 1977; Yancey and Somero, 1978). At these subsaturating concentrations of substrate, drastic changes in Km induced by mutations would produce large alterations in reaction velocity. Therefore, such mutations might have been eliminated by natural selection, while those affecting the affinity to other substrates could not be selected unless the mutations affected the affinity to the primary substrates. The Km values for G6P, Gal6P and N A D P in the brittle-star were comparable to those in G6PDs from other echinoderms and other animal groups, but the Km for dG6P was much higher than the values in the other species.

Table 1. K= values for three substratesand one ¢oenzyme G6P (#M) GaI6P(mM) dG6P(raM) NADP(#M)

Ophioplocus japonicus

21

7.14

7.14

11

14

3.21

0.33

15

32

5.90

0.33

5

48 34 16 45 29

9.43 7.69 8.33 7.14 9.09

0,85 2,63 0.27 0.77 0.80

12 9 7 18 10

2. Starfish* Asterias amurensis

3. Sea-cucumbert Stichopus japonicus

4. Sea-urchin~ Glyptocidaris crenularis Temnopleurus hardwickii Strongylocentrotus intermedius Anthocidaris crassispina Echinometra mathaei

Average of 11 invertebrate species(mean + SE)~

27 + 9

5.4 ±

1.4

2.3 + 0.8

10 ± 4

Average of six vertebrate species (mean + SE~j

29 ± 3

4.4 _+ 1.0

1.8 + 0.3

8_ 2

*Data of Matsuoka and Hori (1980); tData of Mochizuki and Hori (1976); :[:Data of Matsuoka (1983); §Data of Sado and Hori (1976) and Ohnishi and Hori (1977)

541

Glucose-6-phosphate dehydrogenases of echinoderms

Species Echinoderms 1. Bdnle-star

Table 2. Effects of chemicalsubstancesand of heat on G6PDs PCMB DEA MgCI2 (1 mM) (70/JM) (10raM)

Ophioplocus japonicus

0

84

106

Heat

(50°C,5 rain) 37

2. Starfish* Asterias amurensis

3. Sea-cucumbert Stichopus japonicus

0

94

112

2

53

79

118

--

63 86 21 50 77

51 41 60 58 52

100 112 108 119 105

13 0 0 0 16

4. Sca-urchin:~ Glyptocidaris crenularis Temnopleurus hardwickii Strongylocentrotus intermedius Anthocidaris crassispina Echinometra mathaei

Average of 11 invertebrate species (mean+ SE)~ 33 + 8 69 + 9 113 + 3 21 + 10 Average of six vertebrate species (mean+ SE)~ 29 + 10 38 + 7 109 + 3 -The valuesare presentedas percentageof control. *Data of Matsuokaand Hori (1980);"f'Dataof Mochizukiand Hod (1976); :~Dataof Matsuoka (1983); §Data of Sado and Hori (1976) and Ohnishiand Hori (1977)

Among a number of genetic variants of human red blood cell G6PD, several have unusual substrate specificity; G6PD Markham, a common variant found in a certain New Guinean population, can use N A D as a coenzyme better than NADP and it can efficiently oxidize both dG6P and G a l 6 P (Kirkman et al., 1968); G6PD Union and G6PD Benevento CYoshida et al., 1970; Yoshida, 1975) can also oxidize them, though they cannot use NAD as coenzyme; G6PD Munum (Yoshida et al., 1974) can use NAD, but cannot oxidize dG6P efficiently. In all these mutant molecules, G6P dehydrogenating activity was remarkably lower than that of the wild molecules (Yoshida, 1975). Enzymes such as the human G6PD variants have not yet been found in echinoderms, other invertebrates and some vertebrates as far as we know. Effects of PCMB, DEA, MgCI2 and heating on G6PDs are shown in Table 2. G6PD is one of the SH enzymes, and it has the remarkable characteristic that the enzyme activity is inhibited by SH-reagents such as PCMB. Activity of the echinoderm G6PDs was inhibited by PCMB, but the degree of inhibition varied markedly among groups. Namely, the enzymes of brittle-star and starfish were inhibited 100%, while those of sea-urchin and sea-cucumber were resistant to the inhibitor. This may suggest close affinity between G6PDs of brittle-star and starfish among four echinoderm groups in the protein structure. The variation on the effect of PCMB is not restricted only to the echinoderm groups. A similar phenomenon has also been observed in the animal kingdom. For example, the activities of G6PDs from frog and crayfish are resistant, but those from rockfish, lamprey, sipunculid, planaria and sea-anemone are inhibited about 100% (Sado and Hori, 1976; Ohnishi and Hori, 1977). The variation was probably produced by random mutation at molecular level during evolution in the animal kingdom, and therefore it is not concerned with the evolutionary relationships of these various animals. Since all G6PDs from these animals show similar substrate specificity, it may be that the degree of dependence on SH groups, possibly on the number of SH groups in the active site, does not

necessarily correlate with the substrate specificity in these enzymes. DEA has been known as an allosteric inhibitor of animal G6PD (Levy, 1963; Tsutsui et al., 1962; Marks and Banks, 1960; Oertel and Rebelein, 1970; Hori and Sado, 1974; Ohnishi and Hori, 1977; Matsuoka and Hori, 1980). As already reported by Matsuoka and Hori (1980), G6PDs from invertebrates and lamprey are generally characterized by a structure resistant to aUosteric inhibition by DEA as compared with vertebrate G6PDs. Echinoderm G6PDs were generally resistant to DEA, but the enzymes from the three echinoderms (starfish, brittlestar and sea-cucumber) showed higher resistance than those from various sea-urchin species. It has therefore been suggested that the binding site of G6PD to DEA has undergone some structural changes during the echinoderm evolution. As reported by Sado and Hori (1976) and Ohnishi and Hori (1977), Mg :+ ions slightly stimulate the activities of animal G6PDs (0-25%). Similar effects were also observed in the echinoderm G6PDs. However, the degree of activation is so small that the activation by Mg 2+ ions might not be of physiological significance. The brittle-star G6PD was more resistant to heat than those of other echinoderms. Judging from the kinetic data presented here, it may be concluded that echinoderm G6PDs are the ordinary G6PD as far as the listed parameters are concerned, though some differences were observed among echinoderm groups. It is important to further investigate whether the kinetic differences observed among enzymes from four different echinoderm groups reflect functional or physiological adaptation to different environments or their evolutionary and phylogenetic relationships. Such investigation may give an insight into fine-scale molecular adaptation and the evolution of echinoderms. Acknowledgement~Tl'fis study was supported in part by a

Grant-in-Aid (Grant No. 02640576) from the Ministry of Education, Science and Culture of Japan.

542

NORIMASAMATSUOKA REFERENCES

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