Polyol Dehydrogenase of the Silkworm by P
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374 E. REID AND B. M. STEVENS I958 Richert, D. A. & Westerfeld, W. W. (1957). Fed. Proc. 16, Stevens, B. M. & Reid, E. (1956). Biochem. J. 64, 238. 735. Romanoff, E. B. & Hunt, C. A. (1954). Amer. J. Physiol. Umbreit, W. W. & Tonhazy, N. E. (1951a). J. biol. Chem. 179, 15. 191, 257. Schneider, W. C. & Hogeboom, G. H. (1952). J. biol. Chem. Umbreit, W. W. & Tonhazy, N. E. (1951 b). J. biol. Chem. 195, 161. 191, 249. Sonnenschein, N. & Kopac, M. J. (1955). J. cell. comp. Williams, J. N. Feigelson, P. & Elvehjem, C. A. (1950). Ph.iol. 45, 361. J. biol. Chem. 185, 887. Polyol Dehydrogenase of the Silkworm BY P. FAULKNER Laboratory of In8ect Pathology, Sault Ste Marie, Ontario, Canada (Received 24 June 1957) It is becoming increasingly evident that a number glucose 6- and ribose 5-phosphates (Schwarz Laboratories of important metabolic events take place in the Inc., New York, U.S.A.); reduced triphosphopyridine haemolymph of insects. Examples of the reactions nucleotide (TPNH) and reduced diphosphopyridine nucleo- catalysed by enzymes of insect haemolymph are tide (DPNH) (Sigma Chemical Co., St Louis, Mo., U.S.A.); D-galactose, D-mannose (Difco Inc., Detroit, Mich., U.S.A.); hydrolysis of carbohydrates, fat and proteins, the and methylglyoxal (Bios Chemical Co., New York, U.S.A.); oxidation of tyrosine and the decomposition of the remaining chemicals were purchased from Fisher hydrogen peroxide (see review by Buck, 1953). Scientific Co., Toronto, Ontario, Canada. Transaminase has also been detected (Bheemeswar The sugar acids supplied as lactones were converted & Sreenivasaya, 1952), as well as a specific phos- into the sodium salts by heating with dil. NaOH. Materials phatase (Faulkner, 1955). used as test substrates were neutralized by addition of HC1 Recent studies from this laboratory have indi- or NaOH. cated that the haemolymph of the silkworm, Spectrophotometric detrmination of dehydrogena8e. The mori L., contains triphosphopyridine standard test system used in the assay of dehydrogenase Bombyx activity contained the following in a final volume of 2-5 ml.: nucleotide-linked dehydrogenases which oxidize 2-amino-2-hydroxymethylpropane-1:3-diol (tris) buffer, L-malate and reduce sugar phosphates (Faulkner, pH 7*5 (18 mM); MgSO4 (4 mM); TPNH (0.1 mm); sub- 1956a, b). It has now been found that hydroxy- strate and enzyme. Cuvettes (1 cm. light path) containing aldehydes and other carbonyl compounds are all the components except the substrate were incubated at reduced by the same enzyme preparation and an room temperature (20°) in a Beckman DU spectrophoto- account of these studies is given here. meter. Optical-density readings were taken at 340 m,u and after a 2 min. equilibration periodthe substrate (final concn. 2 mm) was added and readings were taken every 30 sec. The MATERIALS AND METHODS blank cell used in the assay did not contain substrate or Dialysed silkworm haemolymph. This was obtained from TPNH. Optical density was plotted against time of incuba- fifth-instar larvae as described previously (Faulkner, tion and the enzyme activity was calculated from the slope 1956a). of the initial linear portion of the curve. One unit of Extras of silkworm t8sue8. These were obtained by enzyme was defined as the amount necessary to reduce the grinding the tissue (0.3-1 g.) with 3 ml. of water at 00 in a optical density by 0-01/min. Incubations did not normally motor-driven glass homogenizer. The mixture was centri- continue more than 5 min. after the addition of substrate fuged at 10 000 g for 5 min. at 0° and the supernatant was and during this period no appreciable heating of the collected. Enzyme activities of the extracts were expressed samples was observed. as units/mg. of protein. Protein was determined by the method of Lowry, Chemicals. Glycolaldehyde was prepared by the pro- Rosebrough, Farr & Randall (1951), and polyhydric cedure of Powers, Tabakoglu & Sable (1955), glyoxylic acid alcohols by the method of West & Rapoport (1949) with according to Weinhouse & Friedmann (1951) and D- and L- the modifications reported previously (Faulkner, 1956b). glyceraldehyde by the method of Perlin & Brice (1956). D-Erythrose and D-threose were gifts from Dr A. S. Perlin, EXPERIMENTAL AND RESULTS and L-erythrulose from Dr N. Yattrie, both of the Prairie Regional Laboratories, Saskatoon. Other chemicals were Reduction of DL-glyceraldehyde purchased as follows: pentoses, sugar acids and uronic acids (Pfanstiehl Chemical Co., Waukegan, Ill., U.S.A.); di- In the presence of DL-glyceraldehyde and buffer hydroxyacetone, DL-glyceraldehyde and L-sorbose (Nutri- (pH 7.5) dialysed silkworm haemolymph catalyses tional Biochemicals Corp., Cleveland, Ohio, U.S.A.); the oxidation of TPNH. The reaction does not Vol. 68 POLYOL DEHYDROGENASE OF THE SILKWORM 375 occur in the absence of dialysed haemolymph nor Substrate specificity when the latter is replaced by heated enzyme, nor does it take place when DPNH is substituted for A number of sugars and other carbonyl com- TPNH. The rate of oxidation of TPNH in the pounds were tested to determine whether they standard test system is proportional to the concen- are reduced by a triphosphopyridine nucleotide tration of enzyme (Fig. 1). These observations (TPN)-linked enzyme in silkworm haemolymph. indicate that a TPN-linked dehydrogenase which The incubation mixture contained TPNH, Mg2+ reduces DL-glyceraldehyde is present in the ions and buffer (pH 7-5) in addition to the enzyme haemolymph of the silkworm. The enzyme, which and test substrate. The results are expressed as the is active in the range pH 4 0-9 5, has no pronounced rate at which the test substrate was reduced com- maximum between these pH limits. pared with that for D-glyceraldehyde, which was Although magnesium was included in the arbitrarily fixed at 100. The concentration of test standard incubation mixture used to study the substrate and of D-glyceraldehyde was 4 mm. reduction of DL-glyceraldehyde, an absolute Comparative reduction rates with a number of requirement for the cation could not be shown with sugars and sugar acids are given in Table 1. It will either dialysed haemolymph or haemolymph that be seen that glycolaldehyde, D- and L-glyceralde- had been fractionated by treatment with am- hyde and the tetroses are all readily reduced. In monium sulphate and calcium phosphate. Sodium contrast, the pentoses are reduced only slowly and ethylenediaminetetra-acetate (10 mM) had no the hexoses not at all. The rate with L-glyceralde- effect on the rate of reduction of DL-glyceralde- hyde is about half that obtained with D-glyceralde- hyde in the standard test system with dialysed hyde. The enzyme has a higher affinity for D- than haemolymph. for L-glyceraldehyde since the Michaelis constant It is possible to estimate the amount of DL- glyceraldehyde that can be reduced in 1 hr. by 1 ml. of silkworm haemolymph containing 30 mg. Table 1. Rates of reduction of sugars compared of protein/ml. The specific activity of the glycer- with the rate for D-glyceraldehyde aldehyde-reducing enzyme in whole or dialysed haemolymph is about 1-3 units/mg. of protein. If The standard test conditions were used with dialysed value of 6-22 cm.2/mole is taken for the haemolymph and with the substrates present at 4 mm. the x 106 For details see text. extinction coefficient of TPNH (Horecker & Relative Kornberg, 1948), it may be calculated that 1 ml. of rate of haemolymph could catalyse the reduction of Class Test substrate reduction 0.85 mg. of DL-glyceraldehyde/hr. under the Diose Glycolaldehyde 47 standard test conditions. Trioses D-Glyceraldehyde 100 L-Glyceraldehyde 49 DL-Glyceraldehyde 100 Dihydroxyacetone 5 Tetroses D-Erythrose 131 D-Threose 104 L-Erythrulose 19 Pentoses D-Arabinose 5 L-Arabinose 12 D-Ribose 9 D-Lyxose 2 t)c D-Xylose 19 E c Hexoses D-Glucose 0 N D-Galactose 0 C LU D-Mannose 0 D-Fructose 0 L-Sorbose 0 Sugar acids Gluconic 0 Galactonic 0 Gulonic 0 1.1 2-2 3.3 44 Uronic acids Glucuronic 89 Protein (mg.) Galacturonic 50 Sugar phosphates Ribose 5-phosphate 107 Fig. 1. Relationship between rate of reduction of DL- Glyceraldehyde 3-phosphate 0 glyceraldehyde and enzyme concentration. The standard + dihydroxyacetone test system was used, with DL-glyceraldehyde (2 mM) as phosphate mixture* substrate and dialysed haemolymph as enzyme. The * Obtained by treatment of hexose diphosphate with total volume incubated was 2-5 ml. aldolase. 376 P. FAULKNER I958 for D-glyceraldehyde is 0-066 mm and for L- applied to Whatman no. 1 papers for ascending chromato- glyceraldehyde is 2 7 mm in dialysed haemolymph, graphy. Papers were developed for 18 hr. in the following as determined by the method of Lineweaver & solvents: (i) n-butanol-acetic acid-water (25:6:25, by Burk (1934). vol., upper layer; Woiwod, 1949); (ii) n-butanol-ethanol- water (4:1:5, by vol., upper phase; Hackman & Trikojus, The rate of reduction of dihydroxyacetone is 1952); and (iii) phenol (80 %, w/v). After development, only 5 % of that obtained with D-glyceraldehyde, polyhydric alcohols were detected by spraying the paper which indicates that the insect enzyme is quite with periodate followed by a benzidine reagent (test 1, distinct from the glycerol dehydrogenases of Cifonelli & Smith, 1954). Escherichia coli (Asnis & Brodie, 1953), Aerobacter The incubated mixture was found to contain a substance aerogene8 (Burton & Kaplan, 1953; Burton, 1955) having the same R. as glycerol in all three developing and Pfeudomona8 8alinaria (Baxter & Gibbons, solvents. No glycerol was found in the control. It is con- 1954) with which the reduction of dihydroxy- cluded that glycerol is formed when TPNH reduces DL- acetone is the faster reaction. glyceraldehyde in the presence of dialysed haemolymph.