Comp. Biochem. Physiol., 1963, Vol. 8, pp. 109 to 114. PergamonPress Ltd., London. Printed in Great Britain
GLYCOLYTIC ENZYMES OF THE AQUATIC SNAIL P H Y S . 4 H A L E I LEA* CALVIN G. BEAMES, JR.t Department of Zoology, The University of Oklahoma, Norman, Oklahoma (Received 28 March 1962, in revised form 1 October 1962)
Abstract--1. The enzymes of the Embden-Meyerhof pathway and the initial reactions of the hexose monophosphate shunt were studied in cell-free preparations of the aquatic snail Physa halei Lea. 2. Both glucose-6-phosphate dehydrogenase and 6-phosphogluconic dehydrogenase were demonstrated and found to resemble the mammalian enzymes in co-factor specificity. The presence of these two enzymes suggests a functional hexose monophosphate shunt in P. hale/. 3. Measurements on the glycolytic enzymes, phosphoglucomutase, phosphoglucoisomerase, aldolase, triosephosphate isomerase, ct-glycerophosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglyceric kinase, phosphoglyceric mutase, enolase, phosphopyruvic kinase and lactic dehydrogenase are presented. Most of the enzymes have characteristics that are similar to mammalian enzymes, however, aldolase was found to be sensitive to cyanide and lactic dehydrogenase functions as well with NADP:~ as with NAD. INTRODUCTION THE presence of the E m b d e n - M e y e r h o f pathway in aquatic snails is suggested by results from several studies. Overall glycolysis has been demonstrated in a number of species of intact aquatic snails (von Brand et al., 1950; Mehlman & v o n Brand, 1951). Weinbach (1953) measured increased production of pyruvic acid upon addition of excess fructose diphosphate to the minced tissue of the snail, Australorbis glabratus. He also demonstrated inhibition of endogenous oxygen consumption and pyruvic acid formation upon addition of iodoacetamide to the minced tissue. In studies with homogenate and soluble-fraction preparations of the albumen gland of the snail L y m n a e a stagnalis both phosphate uptake and acid production have been demonstrated under anaerobic conditions (Weinbach, 1956). * Taken from a dissertation submitted to the Graduate Faculty, The University of Oklahoma, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. t Present address: Department of Physiology and Pharmacology, Oklahoma State University, Stillwater, Oklahoma. :~ Abbreviations: Adenosine diphosphate, ADP; adenosine triphosphate, ATP; fructose6-phosphate, F-6-P; glucose-l-phosphate, G-l-P; glucose-6-phosphate, G-6-P; glyceraldehyde-3-phosphate, G-3-P; iodoacetic acid, IAA; nicotinamide adenine dinucleotide (diphosphopyridine nucleotide), NAD; nicotinamide adenine dinucleotide phosphate (triphosphopyridine nucleotide), NADP; 6-phosphogluconic acid, 6-PG; 2-phosphoglyceric acid, 2-PGA; 2,3-diphosphoglyceric acid, 2,3-PGA. 109
CALVIN G. BEAMES, JR.
However, no information is available on individual enzymatic reactions of tile Embden-Meyerhof pathway of aquatic snails. In fact, as pointed out in a recent review, evidence for the presence of the enzymes of the Embden-Meverhof pathway is quite incomplete in all groups of Mollusca (Martin, 1961). This report concerns measurements on the glycolytic enzymes of the aquatic snail, Pkrsa halei Lea. MATERIALS AND METHODS All snails used in this study were laboratory-reared. The tissue was prepared for analysis by the following method. Total soft parts of the snails were removed from the shell and ground in a chilled Tenbroeck tissue grinder with sufficient homogenizing solution (0.154M KC1 made alkaline with 8 ml/1. of 0"02 M KHCOa) to make a 25 per cent (w/v) homogenate. The homogenate was centrifuged at 1600g for 20 min at 5°C and the supernatant fluid ("tissue extract") was used as the source of enzyme. Most of the enzymatic assay procedures were essentially standard methods. With measurements on pyruvic kinase, 3-phosphoglyceric mutase and enolase, however, it was necessary to add lactic dehydrogenase* to the reaction mixture. The colorimetric determination on lactic dehydrogenase was patterned after the method by Ells (1959). All spectrophotometric measurements were made in a Beckman Model DU spectrophotometer, except the pyruvate kinase determination which was measured with a Macalaster Bicknell Coenzymeter. The chemicals used in the study were commercial products of high purity. Substrates obtained as the barium salt were converted to the sodium or potassium salt by standard methods. RESULTS AND DISCUSSION Measurements on the glycolytic enzymes and initial steps of the hexose monophosphate shunt are presented in Table 1. Both G-6-P and 6-PG dehydrogenase have relative high rates of activity in P. haM and they are similar to the mammalian enzymes in their specificity for NADP. The presence of these two dehydrogenases suggests a functional hexose monophosphate shunt in this snail. Phosphoglucomutase and phosphoglucoisomerase activity was measured spectrophotometrically by coupling the reaction to G-6-P dehydrogenase. The characteristics appear to be quite similar to the enzymes of mammalian tissue. In initial measurements on P. halei aldolase, cyanide was used to fix the triose phosphates formed in the reaction. Typical results with this method are presented in Table 1, Exp. A. In each determination activity, measured as alkali-labile phosphate, was reduced rather than increased by the addition of cyanide. This is the reverse of observations made in similar experiments on mammalian tissue extracts. Since cyanide is known to inhibit the aldolase of some organisms, e.g. the aldolase of yeast (Warburg & Christian, 1942), measurements were carried out * Sigma type II lactic dehydrogenase from rabbit muscle, Sigma Chemical Co., St. Louis, Missouri, U.S.A.
GLYCOLYTIC ENZYMES OF THE AQUATIC SNAIL P H Y S A H A L E I LEA T A B L E 1 - - A C T I V I T Y OF ENZYMES OF THE E M B D E N - M E Y E R H O F PATHWAY
IN EXTRACTS OF
Complete NAD replacing NADP Minus G-6-P
Rate of reaction* Exp. A 22.8 0"0 0"0
Exp. B ----
Complete NAD replacing NADP Minus 6-PG
6"9 0"0 0"0
Complete Minus G-1-P
Complete Plus 120/zmole KCN Plus 120/~mole hydrazine
23"2 6"3 --
13.5 9"4 20'0
Glycerophosphate dehydrogenase + triosephosphate isomerase
Complete Minus G-3-P
Complete Minus G-3-P Minus cysteine Phosphate replacing arsenate Plus 10/~mole IAA
30"2 0.0 0"0 10"2 0"0
Complete Minus ATP M i n u s M g 2+ Minus 3-PGA
51.9 15"4 21"1 4"5
Complete Minus phosphopyruvate Minus ADP M i n u s M g ~+ Minus K + Minus extract
22.6 1-7 2.7 1"7 -1"0
54"8 -12.7 -26'7 --
Phosphoglyceric mutase and enolase
Complete Minus 3-PGA Minus 2,3-PGA Plus 10/~mole NaF
5'2 0"9 3"6 0"9
Complete Minus pyruvate Minus lactate NADP replacing NAD
2"4 0'7 ---
1" 5 -0"6 1'7
* / z m o l e s u b s t r a t e m e t a b o l i z e d / m i n p e r m g p r o t e i n x 10 ~. t See overleaf.
CALVIN (~. BEAMES, JR.
Glucose-6-phosphate and 6-phosphogluconic dehydrogenase : The complete system contained 300 k~mole Na phosphate, pH 7"0; 4 /zmole substrate, added immediately before taking the zero time reading; 0'3 /zmole TPN; 10/zmole IAA, pH 7"0; 0-05 ml undialyzed tissue extract (61 #g protein). Final volume 2"5 ml. Temperature 25C. Phosphoglucomutase: The complete system contained 20/~mole tris-(hydroxymethyl)aminomethane, pH 7"4; 2 /xmole MgC12; 0"3 /xmole TPN; 20 /xmole G-l-P, added immediately before taking the zero time reading; 20/zmole cysteine, pH 7'4; 10 #mole IAA, pH 7'4; 0"1 ml tissue extract (181 /zg protein). Final volume 3'0 ml. Temperature 25°C. Phosphoglucoisomerase: The complete system contained 400/mlole Na phosphate, pH 7"0; 10 /xmole F-6-P, added immediately before taking the zero time reading; 0'3 /xmole TPN; 0'05 mt tissue extract (47/xg protein). Final volume 3'0 ml. Temperature 25"C. Aldolase: The complete system for Exp. A and Exp. B contained 150 /zmole tris(hydroxymethyl)-aminoethane, pH 7"5; 10/zmole FDP, added at zero time; 0'2 ml tissue extract. Final volume 3"0 ml. Temperature 30:C. Glycerophosphate dehydrogenase and triosephosphate isomerase: The complete system contained 40 /zmole Na phosphate, pH 7"6; 0"96 /zmole G-3-P, added immediately before taking the zero time reading; 0"15 /zmole DPNH; 0"1 ml tissue extract (70 /zg protein). Final volume 2"5 ml. Temperature 25~C. Glycerald.ehyde-3-phosphate dehydrogenase: The complete system contained 60/xmole pyrophosphate, pH 8-4; 8/xmole cysteine; 0-6/~mole DPN; 40/zmole arsenate; 0'96 ktmole G-3-P, added immediately before taking the zero time reading; 0"1 ml dialyzed tissue extract (112 /~g protein). Final volume 3"0 ml. Temperature 25~'C. 3-Phosphoglyceric kinase: The complete system contained 84 /zmole tris-(hydroxymethyl)-aminomethane, pH 7"4; 40 /zmole ATP; 12 /xmole MgC12; 40 /zmole 3-PGA; 200 /~mole hydroxylamine, pH 7'4; 0'2 ml tissue extract (340 /xg protein), added at time zero. Final volume 3"0 ml. Temperature 30c'C. Pyruvic kinase: The complete system contained (Exp. A) 150/xmole K phosphate, pH 7'5, or (Exp. B) 15/xmole triethanolamine, pH 7"5; 0-3 ¢zmole DPNH; 1-5/xmole ADP; 4-5 /zmole PE, added immediately before taking the zero time reading; 24 /zmole MgC12; 0"5 /xmole KC1 with triethanolamine buffer; 0"2 ml dialyzed tissue extract (152 /xg protein); 0"2 ml lactic dehydrogenase (180,000 units). Final volume 3'0 ml. Temperature 30°C. Phosphoglyceric mutase and enolase: The complete system contained 100 /zmole triethanolamine, pH 7'5; 0"13 /zmole DPNH; 0"4 /zmole ADP; 0-03 /~mole 2,3-PGA; 24 /zmole MgC12; 10 /zmole IAA; 2-5 /zmole 3-PGA, added immediately before the zero time reading; 0"2 ml dialyzed tissue extract (160/zg protein); 0.01 ml lactic dehydrogenase (0.5 mg/ml). Final volume 3"0 ml. Temperature 25C. Lactic dehydrogenase : The complete system for (Exp. A) contained 40 #mole Na phosphate, pH 7-3; 0.67 /zmole DPNH; 10 /zmole Na pyruvate, added at time zero; 10 /zmole IAA; 0.2 ml tissue extract (286/xg protein). Final volume 3'0 ml. Temperature 25°C. The complete system for Exp. B contained 30/xmole Na phosphate, pH 7'3; 0'6 /zmole DPN or 0"3 /zmole TPN; 20 /xmole lithium lactate, added at time zero; 10 /xmole IAA; 30 /tmole KCN, pH 7'3; 0"2 mg phenazine methosulfate; 0'03 /zmole 2,6-dichlorophenolindophenol; 0-2 ml tissue extract (234/xg protein). Final volume 3"0 ml. Temperature 25C. with hydrazine replacing cyanide as the binding agent (Table 1, Exp. B). When this substitution was made the formation of alkali-labile phosphate was enhanced. I n further measurements on aldolase the formation of alkali-labile phosphate was found to follow a linear time course through a 20-min period of measurement and also to have a linear relation to the concentration of snail extract in the system. T h e cyanide sensitivity of P. ham aldolase suggests that it may be a metal-requiring enzyme but this has not been investigated in detail.
GLYCOLYTIC ENZYMES OF THE AQUATIC SNAIL P H Y S A H A L E I LEA
The ability of G-3-P to stimulate oxidation of NADH provides evidence for glycerophosphate dehydrogenase and triosephosphate isomerase. While the activity for this system is low, the rate remained linear through a 15-min period of measurement. In general, the requirements of glyceraldehyde-3-phosphate dehydrogenase of P. ham are quite similar to those for mammalian tissue enzyme. Arsenylation (or phosphorylation) is an obligatory accompaniment of oxidation of G-3-P. The requirement for cysteine, the inhibition by iodoacetic acid and the inactivity of NADP as a co-factor are other points of similarity between G-3-P dehydrogenase in P. halei and mammalian tissue. Measurements on 3-phosphoglyceric l~inase were obtained by following the reverse reaction and employing hydroxylamine as a trapping agent for the acyl phosphate formed. With this method the reaction was shown to be linked with ATP and to be stimulated by Mg 2+. In both these characteristics 3-phosphoglyceric kinase is similar in P. haM and mammalian tissue. Initially, efforts were made to study pyruvic kinase activity by coupling the reaction with lactic dehydrogenase and following the oxidation of NADH. However, lactic dehydrogenase activity in the snail extract was so low that it was impossible to obtain reliable measurements. To overcome this problem lactic dehydrogenase (see p. 110) was added to the assay mixture. The measurements in Table 1 were obtained with such a fortified system. Potassium phosphate was the buffer in Exp. A and triethanolamine was the buffer in Exp. B. The requirement for ADP, Mg 2+ and K + are characteristics that are similar with snail and mammalian tissue pyruvic kinase. Phosphogluceric mutase and enolase were demonstrated using a fortified system similar to the one used in studying pyruvic kinase. The activity was quite low; however, the rate remained linear for a 7-min period of measurement. The ability of 2,3-PGA to stimulate activity suggests that the reaction mechanism of P. ham phosphoglyceric mutase is similar to that of the mammalian enzyme. Sodium fluoride inhibited the system, presumably via the action of fluoride upon enolase. Additional evidence for enolase in extracts of P. haM was obtained by following the formation of phosphopyruvic acid spectrophotometrically at 240 m/~ in a system with excess 2-PGA. Phosphopyruvic acid was formed at a rate of 0.402/zmole/min per mg protein and NaF completely inhibited the reaction. Lactic dehydrogenase activity was measured spectrophotometrically (Exp. A) and colorimetrically (Exp. B). The activity is quite low with both methods; however, the rate of reaction remained linear through a 10-min period of measurement. In colorimetric measurements using mammalian lactic dehydrogenase no measurable activity is observed with NADP as co-factor. The snail enzyme, on the other hand, is able to utilize NADP as readily as NAD. This ability is a marked difference between snail and mammalian lactic dehydrogenase. The low lactic dehydrogenase activity in P. haM tissue may not be due to a low concentration of the enzyme. In studies with lobster heart muscle Kaplan et al. (1960) found low lactic dehydrogenase activity with NAD but a high activity with the analog 3acetyl pyridine NAD. A similar situation may well exist with snail lactic dehydrogenase. 8
CALVIN G. BEAMES, JR.
Acknowledgements--The author wishes to express his appreciation to Dr. H. Alan Ells and Dr. Harriet Harvey for their encouragement during this investigation. Thanks also are extended to Dr. Branley A. Branson, Pittsburg State Teachers College, Pittsburg, Kansas, for identifying the aquatic snail. REFERENCES BRAND T. VON, BAERSTEINH. D. & MEHLMAN B. (1950) Studies on the anaerobic metabolism and the aerobic carbohydrate consumption of some fresh water snails. Biol. Bull., Woods Hole 98, 266-276. ELLS H. A. (1959) A colorimetric method for the assay of soluble succinic dehydrogenase and pyridinenucleotide-linked dehydrogenase. Arch. Biochem. Biophys. 85, 561-562. KAPLAN N. O., CIOTTI MARGARETM., HAMOLSKYM. & BIEBERR. (1960) Molecular heterogeneity and evolution of enzymes. Science 131, 392-397. MARTIN A. W. (1961) T h e carbohydrate metabolism of the mollusca. In Comparative Physiology of Carbohydrate Metabolism in Heterothermic Animals (Edited by ~VlARTIN A . W . ) . University of Washington Press, Seattle. MEHLMAN B. & BRANDT. VON (1951) Further studies on the anaerobic metabolism of some fresh water snails. Biol. Bull., Woods Hole 100, 199-205. WARBURG O. & CHRISTIANW. (1942) Wirkungsgruppe des G~irungsferments Zymohexase. Biochem. Z. 311, 209-210. WEINBACH E. C. (1953) Studies on the intermediary metabolism of the aquatic snail, Australorbis glabratus. Arch. Biochem. Biophys. 42, 231-244. WEINBACH E. C. (1956) The influence of pentachlorophenol on oxidative and glycolytic phosphorylation in snail tissue. Arch. Biochem. Biophys. 64, 129-143.