A GLUCOSEPHOSPHATE ISOMERASE INHIBITOR OF SEASONAL OCCURRENCE IN COD (GADUS MORHUA) AND OTHER FISH

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1 J. mar. biol. Ass. U.K. (969) 49, Printed in Great Britain A GLUCOSEPHOSPHATE ISOMERASE INHIBITOR OF SEASONAL OCCURRENCE IN COD (GADUS MORHUA) AND OTHER FISH By P. R. DANDO The Plymouth Laboratory (Text-figs -3) A heat-stable inhibitor of glucosephosphate isomerase (D-glucose-6- phosphate ketol-isomerase, E.C ) has been found in tissues of the cod (Gadus morhua L.). The inhibitor occurred in cod in the Plymouth area from February to mid-april in 967 and from mid-march to mid-april in 968. Gel filtration studies indicate that the inhibitory component of cod muscle has a molecular weight of less than 500. A similar inhibitor has been found in red mullet {Mullus surmuletus L.), megrim (Lepidorhombus whiff-iagonis Walbaum) and greater forkbeard (Urophycis blennoides Briinnich). The inhibitor is of a seasonal nature in these fish also and may be linked with the spawning cycle. INTRODUCTION Dastugue, Bastide & Chevalier (962) noted that the activity of glucosephosphate isomerase in mammalian and avian haemolysates increased on dilution of the haemolysates. These authors suggested that the phenomenon might be due to the dilution of an inhibitor of the enzyme. This paper reports the occurrence of a similar phenomenon in cod muscle and gives evidence for the presence of an inhibitor, of seasonal occurrence, of glucosephosphate isomerase in fish tissues. MATERIALS AND METHODS Animals The fish were caught by otter-trawl in the English Channel between Start Point and the Scillies. Assay of enzymes All enzyme assays were carried out at 0 ± 0-5 C and ph 7*4. Glucosephosphate isomerase. Method A. Tubes contained 00/tmoles tris (tris (hydroxymethyl) aminomethane) buffer adjusted to ph 7-4 (io ) with HC, 8 /imoles disodium glucose-6-phosphate and aliquots of enzyme in a final volume of ml. The reaction was started by the addition of the glucose-6-phosphate and stopped after a 5 min incubation by the addition of the acidic colour-development reagent. Fructose-

2 448 P. R. DANDO 6-phosphate was determined colorimetrically as described by Roe, Epstein & Goldstein (949), but with a heating time of 5 min at 75 C (Hers, Beaufays & de Duve, 953)3 for colour development. Fructose-6-phosphate was found to give a colour yield equal to that given by an equivalent amount of fructose under these conditions. Enzyme activity was found to be linear with time, up to at least 0 min, provided that not more than 350 /wnoles of fructose-6-phosphate were produced. Method B. Reaction cuvettes contained 5 /tmoles fructose-6-phosphate, 0-5 /*mole nicotinamide adeninedinucleotide phosphate, 2-5 /tmoles magnesium sulphate, 00 /tmoles tris buffer and -5 fig of yeast glucose-6-phosphate dehydrogenase (Type X, Sigma Chemical Co., St Louis, Mo., U.S.A.) in a final volume of I ml. After 0 min. preincubation at 0 C the reaction was started by the addition of 5 /A. of suitably diluted muscle homogenate. The increase in absorbance at 340 m/t in a I cm light path semi-micro cuvette was measured in a Unicam SP 800 spectrophotometer. Fructose-6-phosphate, essentially free from glucose-6-phosphate, was obtained by hydrolysing 200 mg batches of trisodium fructose-,6-diphosphate pentahydrate (Wessex Biochemicals Ltd., Bournemouth, Hants.) in 2 ml. i-on HC in a boiling water-bath for 20 min, followed by cooling and neutralization with sodium hydroxide. Glucose-6-phosphate dehydrogenase (E.C....49) was assayed in a I ml. system containing 4 /tmoles glucose-6-phosphate, 0-5 /tmole nicotinamide adeninedinucleotide phosphate, 6 /tmoles magnesium sulphate and 00 /wnoles tris buffer ph 7-4. Aldolase (E.C. 4.i.2.b) was assayed using the coupled system suggested by Racker (947). Cuvettes contained 0 /«noles fructose-i,6-diphosphate, ^0-3 /Mnole NADH, i8o/<moles tris buffer ph 7-4, 40 units triosephosphate isomerase and 20 units glycerolphosphate dehydrogenase in a final volume of 2 ml. Triosephosphate isomerase (E.C ) was determined, in a similar manner to aldolase, as described by Beisenherz (955). Phosphoglycerate kinase (E.C ) was assayed by the procedure of Biicher (955)- Inhibitor detection and determination The glucosephosphate isomerase assay method A was used in the seasonal survey of the cod inhibitor. White muscle was removed from the fish within 4 h of death and homogenized with 7 volumes of ice-cold o-i M tris buffer, ph 7-4, for min in a Polytron homogenizer and centrifuged at 3000 g at. for 20 min at 2 C. The activity of glucosephosphate isomerase was determined using a range of dilutions of the supernatant which converted from 0 to 60 m/tmoles of glucose-6-phosphate/min to fructose-6-phosphate. If the activity was not linear with supernatant concentration, presence of inhibitor was confirmed in the following manner: aliquots of heated supernatant, which had been placed in a Pyrex test-tube in a boiling water-bath for 5 min, were added, after cooling, to a dilution of the unheated homogenate. The latter having sufficient enzyme activity to isomerise approximately 20 m/tmoles glucose-6-phosphate/min. Any inhibition of the enzyme resulting from the addition of the heated supernatant was noted. In later work a quantitative estimation of the inhibitor was used which was based on the determination of the enzyme by method B. An inhibitor unit was defined as the amount of inhibitor which caused a 50% decrease in the activity of a fixed amount of glucosephosphate isomerase. This fixed amount of enzyme gave an absorbance increase of o-02/min at 340 m/t under the defined assay conditions. The inhibitor had no effect on the activity of the yeast glucose-6-phosphate dehydrogenase used in the assay. The inhibitory component of red mullet and megrim muscle was unstable on

3 A GLUCOSEPHOSPHATE ISOMERASE INHIBITOR IN FISH 449 storage and disappeared from refrigerated and frozen muscle samples and muscle extracts within 24 h. However, the inhibitor could still be isolated from cod muscle samples frozen at 20 C for a month. Cod RESULTS The activity of glucosephosphate isomerase in homogenates of cod muscle assayed in February 967 increased on dilution of the homogenate (Fig. ). Increasing the substrate concentration did not increase the activity. In May 967 dilution did not affect the activity. so - May 967 OFeb Cod muscle homogenate (/A.) Fig.. The effect of cod muscle homogenate concentration on glucosephosphate isomerase activity. Assays were performed, using method A, at glucose-6-phosphate concentrations of 32 mm (O O) or 6 mm ( and A). An inhibitory factor could be separated from the enzyme by heating the homogenate for 5 min in a boiling water-bath. The enzyme was denatured by this treatment, leaving the inhibitor in solution. Isolation of the inhibitor could also be achieved by ultrafiltration of the homogenate through a

4 450 P. R. DANDO Sartorius LSG 60 filter which retained the enzyme but not the inhibitor. Addition of the ultrafiltrate or heated homogenate to dilute homogenates of cod muscle caused inhibition of the glucosephosphate isomerase (Fig. 2). Inhibition occurred using either glucose-6-phosphate or fructose-6-phosphate as substrate. 00 r Ultrafiltrate (al.) Fig. 2. The effect of increasing amounts of cod-muscle homogenate ultrafiltrate on glucose-phosphate isomerase activity. Assay method B was used. 500 Passage of a cod muscle homogenate through a -5x35 cm column of Sephadex G 50, equilibrated with o-i M tris buffer ph 7-4, also separated the inhibitor from the enzyme (Fig. 3). The phosphoglucose isomerase peak probably consisted of free enzyme and enzyme-inhibitor complex since the activity of the enzyme still increased slightly on dilution after passage through the column. A concentrated heated supernatant from cod muscle was passed through a Sephadex G 5 column and eluted with water. The major inhibitory component was eluted shortly after the void volume, indicating that the inhibitor has a molecular weight in the range. A seasonal survey of inhibitor occurrence in cod muscle showed that the inhibitor, which was discovered in cod muscle at the beginning of February 967, disappeared in mid-april 967 and did not reappear, with the single

5 A GLUCOSEPHOSPHATE ISOMERASE INHIBITOR IN FISH 45 exception of one of the cod sampled in July, until March 968. The inhibitor was again absent from cod muscle after mid-april 968. During the few weeks in which the inhibitor was present in cod it was found in fish of both sexes irrespective of gonad maturity. In both years the cod caught off Plymouth spawned during the period mid-february to mid-april. P/min/ml. 800 \\l\, O--O -C 'O--O ul 400 V o e 200 / - 0 i 8 2 Fraction number t i l! -! Fig. 3. Gel filtration of 2 ml. cod muscle homogenate on Sephadex G-50. Fractions of approximately 2 ml. were collected, starting after the void volume had passed. Glucosephosphate isomerase activity ( ) was determined by assay method A. Inhibitor activity (O O), given as percentage inhibition, was detected by adding 25 /i\. of each fraction to diluted muscle homogenate and determining the enzyme activity by method A, as described in the Methods section. A study of the inhibitor concentrations, determined on heated supernatants, from the tissues of one cod sampled at the beginning of April showed that most of the inhibitor was concentrated in the muscle and liver (Table ). This cod was sampled towards the end of the 'inhibitor season' and had a low concentration of the inhibitory component in the white muscle. A concentrated heated supernatant of cod muscle extract, containing the glucosephosphate isomerase inhibitor, was tested for its ability to inhibit several other enzymes in cod muscle homogenates (Table 2). The inhibitor was added at a concentration 500-fold greater than that necessary to cause 50 % inhibition in the glucosephosphate isomerase assay. Only triose phosphate isomerase was slightly inhibited at this concentration.

6 452 P. R. DANDO TABLE. INHIBITOR CONCENTRATIONS IN THE TISSUES OF A COD SAMPLED ON 5 APRIL 968 Tissue Inhibitor units/g White muscle i8-o (mid-dorsal region) Liver 8-3 Heart 3-5 Kidney 3-8 Spleen -7 TABLE 2. EFFECT OF INHIBITOR ON SOME ENZYMES PRESENT IN COD MUSCLE HOMOGENATE Activity (nytmoles substrate utilized/min/ml. reaction mixture) No Inhibitor Difference Enzyme inhibitor added (%) Glucose-6-phosphate o dehydrogenase Aldolase Triosephosphate isomerase Phosphoglycerate kinase -7 7 o Inhibitor occurrence in other fish Similar inhibitory phenomena have been found in red mullet (Mullus surmuletus), megrim (Lepidorhombus whijf-iagonis) and greater forkbeard (Urophycis blennoides). In the first two fish the inhibitory component was destroyed on heating the muscle homogenate in a boiling water-bath but could be isolated by ultrafiltration. The inhibitor was found to be of seasonal occurrence in these fish also. Three red mullet caught at the end of May 968, just before spawning, were found to have high concentrations of inhibitor in their muscle ( inhibitor units/g). Little inhibitor, less than 0 units/g, could be detected in muscle samples taken from mullet later in the year after spawning. In megrim the inhibitory component was also associated with the spawning season, being isolated from the muscle of one fish in May 967 and from several spent females during the last week of May and the first week of June 968 but not during other months. A heat-stable inhibitory component was found in a muscle sample from one greater forkbeard caught in May 967 but not from another specimen examined in March 968. DISCUSSION There is, at present, no evidence that the seasonal glucosephosphate isomerase inhibitor in cod muscle inhibits this enzyme in vivo. It is possible that the inhibitor may act on some other enzyme in the fish. The function of this

7 A GLUCOSEPHOSPHATE ISOMERASE INHIBITOR IN FISH 453 compound is not clear, but it may be associated with the spawning cycle. Since the inhibitor appears in immature as well as mature fish, its appearance cannot be initiated by changes in gonad maturity. REFERENCES BEISENHERZ, C, 955. In Methods in Enzymology, Ed. S. P. Colowick and N. O. Kaplan. Vol., pp London: Academic Press. BUCHER, T., 955. In Methods in Enzymology, Ed. S. P. Colowick and N. O. Kaplan. Vol., pp London: Academic Press. DASTUGUE, G., BASTIDE, P. & CHEVALIER, M. C, 962. De quelques activities enzymatiques de differents hemolysats en fonction de la dilution. C. r. Seanc. Soc. Biol., T. 56, pp HERS, H. G., BEAUFAYS, H. & DE DUVE, C, 953. L'analyse simultanee des hexoses des trioses et de leurs esters phosphores. Biochim. biophys. Acta, Vol. n, pp RACKER, E., 947. Spectrophotometric measurement of hexokinase and phosphohexokinase activity. J. biol. Chem., Vol. 67, pp ROE, J. H., EPSTEIN, J. H. & GOLDSTEIN, N. P., 949. A photometric method for the determination of inulin in plasma and urine. J. biol. Chem., Vol. 78, pp