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Catalysis of Pentose Phosphate Pathway Reactions by Cytoplasmic

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Catalysis of Pentose Phosphate Pathway Reactions by Cytoplasmic Biochem. J. (1974) 138, 71-76 71 Printed in Great Britain Catalysis of Pentose Phosphate Pathway Reactions by Cytoplasmic Fractions from Muscle, Uterus and Liver of the Rat, and the Presence of a Reduced Nicotinamide-Adenine Dinucleotide Phosphate-Triose Phosphate Oxidoreductase in Rat Muscle By TERRY WOOD* Department ofBiochemistry, McGill University, Montreal, Que., Canada (Received 9 July 1973) 1. The enzymes of the pentose phosphate pathway were assayed in supernatant fractions from rat muscle, liver and uterus. 2. On incubation of ribose 5-phosphate with uterus and liver supernatants, triose phosphate, sedoheptulose 7-phosphate and hexose monophosphate accumulated. 3. When a muscle supernatant was used, glycerol 3-phos- phate instead of triose phosphate appeared and there was a negligible accumulation of hexose monophosphate. 4. Hexose monophosphate production from ribose 5-phosphate was also followed bymeasuring NADP+ reduction in the presence ofan excess ofphospho- glucose isomerase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydro- genase. 5. With a muscle supernatant, NADPH was reoxidized as rapidly as it was formed owing to the presence of a NADPH-triose phosphate oxidoreductase. 6. A modification of the pentose phosphate pathway in skeletal muscle incorporating this enzyme is proposed. As part of an investigation of the pentose mixture of D-ribose 5-phosphate and D-ribulose phosphate pathway in smooth and striated muscle, 5-phosphate were prepared as described previously the activities of the enzymes of the pathway in a (Kiely et al., 1973). cytoplasmic fraction from rat skeletal muscle, liver and uterus were measured and compared with Preparation of tissue extracts the abilities of fractions from these tissues to convert ribose 5-phosphate into hexose monophosphate. Adult Wistar rats of body weight 250-360g were Discrepancies between the nature and amounts ofthe used. Water and Purina rat chow (Ralston Purina intermediates that accumulated and those expected of Canada, Woodstock, Ont., Canada) were allowed on the basis of the pathway as usually formulated, ad lib. Skeletal muscle was taken from male rats, and indicated the presence of a NADPH-triose phosphate uterus and liver from virgin female rats. Muscle and oxidoreductase in rat skeletal muscle and suggested liver were homogenized in 4ml of ice-cold 50mM- that a modified pathway may be operative in this triethanolamine-HCl buffer, (pH7.4)/g in the 50ml tissue. stainless-steel container of a Lourdes Multi-Mixer (Lourdes Instrument Corp., Old Bethpage, N.Y., Materials and Methods U.S.A.) cooled in ice. Uterus was homogenized in the same buffer at 5°C by using the Eberbach micro- Materials container attachment to a Waring Blendor (Eberbach Corp., Ann Arbor, Mich., U.S.A.). Homogenates Sugar and triose phosphates, coenzymes and were subsequently centrifuged for 2h at 1000OOg enzymes were obtained either from Sigma Chemical at 4°C in a Spinco model L preparative ultracentri- Co., St. Louis, Mo., U.S.A., or from Boehringer fuge and the clear supernatants were collected. Mannheim Corp., New York, N.Y., U.S.A. Diaflo PM-30 membranes were from Amicon Corp., Cambridge, Mass., U.S.A. D-Erythrose 4-phosphate Enzyme assays was prepared by oxidation of D-glucose 6-phosphate All assays were performed at 37°C, pH7.4, in a and precipitated as the barium salt of the hydrazone final volume of 2.00ml with a lcm-light-path cell. (Sieben et al., 1966). D-Xylulose 5-phosphate and a Absorbance changes were measured with a Beckman * Present address: Department of Chemistry, Njala DB spectrophotometer connected to a Varicord 43 University College, University of Sierra Leone, Private linear-log recorder (Photovolt Corp., New York, Mail Bag, Freetown, Sierra Leone. N.Y., U.S.A.). Enzyme activities were calculated in Vol. 138 72 T. WOOD units/g of tissue as the mean ±S.E.M. for six animals. membrane to halt the reaction. Ultrafiltration took D-Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) between 5 and 8min. Triose phosphates and di- and 6-phosphogluconatedehydrogenase(EC 1.1.1.44) phosphates were determined immediately. Other were measured as described by Glock & McLean intermediates were measured after storage of the (1953) and Shonk & Boxer (1964) respectively. ultrafiltrates at -20°C. D-Ribose 5-phosphate ketol-isomerase (EC 5.3.1.6) and D-ribulose 5-phosphate 3-epimerase (EC 5.1.3.1) were assayed at 290nm by previously published Spectrophotometric experiments procedures (Wood, 1970; Kiely et al., 1973). Trans- The reduction of NADP+ was followed directly ketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2) at 340nm and 37°C, over a 60min period, in a Beck- were assayed as described previously (Tan & Wood, man spectrophotometer connected to a recorder. 1969). The cuvette contained, in a final volume of 2.00ml, NADPH-triose phosphate oxidoreductase activity. 50mM-triethanolamine-HCl buffer, pH7.4, 1 mM- This was measured in 50mM-triethanolamine-HCl dithiothreitol, 0.1 mM-thiamin pyrophosphate, 1 mM- buffer, pH 7.4, containing 1 mM-dithiothreitol and NADP+, IOmM-D-ribose 5-phosphate, 0.3 unit of 0.1 mM-NADPH. The rate of NADPH oxidation in glucose 6-phosphate dehydrogenase, 0.1 unit of the absence of triose phosphate was first recorded 6-phosphogluconate dehydrogenase, 2.1units of from the E340, then DL-glyceraldehyde 3-phosphate phosphoglucose isomerase, and the same propor- was added to 0.4mm and the rate measured again. tions of the tissue supernatants as were used in the The activity was calculated from the difference incubation experiments. The observed rates were between the two rates. corrected for any reaction occurring in the absence of ribose 5-phosphate. Assay ofmetabolic intermediates Results Triose phosphate was measured as described by Bucher & Hohorst (1965) and glycerol 3-phosphate Enzyme assays as described by Hohorst (1965). Sedoheptulose 7-phosphate was determined by using ATP and fruc- The activities of the enzymes in rat liver and uterus tose 6-phosphate kinase as described by Wood & were in accord with the values reported by Novello & Poon (1970). A correction was made for fructose McLean (1968) and Baquer & McLean (1972), 6-phosphate in the mixture by assuming that 30% except for uterus transaldolase, which was higher at of the hexose monophosphate present was the fruc- 1.70±0.38 units/g oftissue. The activities in muscle of tose derivative. The reaction, repeated in the absence ribose 5-phosphate ketol-isomerase (6.4+0.5units/g) of fructose 6-phosphate kinase and ATP, gave the and ribulose 5-phosphate 3-epimerase (16.0±0.5 sum of fructose diphosphate and sedoheptulose units/g) were higher than reported previously (Tan diphosphate and a correction was made for the & Wood, 1969), owing to the use of saturating con- contribution ofthese compounds inthe sedoheptulose centrations of substrate and improved assay 7-phosphate assay. Hexose monophosphate was procedures. measured fluorimetrically as described previously (Wood, 1972). Erythrose 4-phosphate was deter- Spectrophotometric experiments mined with transaldolase (Racker, 1965), andribulose 5-phosphate and xylulose 5-phosphate were measured The formation of hexose monophosphate was with transketolase and ribulose 5-phosphate 3- measured directly in a spectrophotometer cuvette epimerase (Wood, 1974). by observing NADP+ reduction in the presence of auxiliary hexose monophosphate dehydrogenases and phosphoglucose isomerase. Ribose 5-phosphate Incubation experiments was the substrate. With supernatants from liver and uterus, in the absence of substrate, the rates were These were performed at 37°C and pH7.4 in relatively low, but in the presence of 10mM-ribose 50mm-triethanolamine-HCl buffer containing 1 mm- 5-phosphate the rates of reduction gradually acceler- dithiothreitol and 0.1 mM-thiamin pyrophosphate. ated as intermediates built up, until, between 50 and The concentration ofD-ribose 5-phosphate was 10mM 60min later, they had attained constant values and the mixtures contained, in 15 ml, 0.1vol. of a comparable with the rates of hexose monophosphate fresh muscle or uterus supernatant fraction, or accumulation in the incubation experiments discussed 0.01vol. of a liver supernatant. Controls contained below. all components except ribose 5-phosphate. Samples When the same experiment was carried out with (5ml) were removed at 20, 40 and 60min and a muscle supernatant, there was a steady rate of ultrafiltered at room temperature through a Diaflo increase in E340 in the absence ofribose 5-phosphate, 1974 PENTOSE PHOSPHATE CYCLE 73 but addition of this compound to a 10mm concen- 00 ^ tration did not increase this rate, and on occasion even decreased it to zero. A model system containing CU4 I+1 +1 +1 o - N k purified shunt enzymes in the amounts present in e1i en~ +I +I +I +I 0.2ml of muscle supernatant responded with a 0 4)) ready reduction of NADP+ when ribose 5-phosphate iri, 04 was added at concentrations between 0.5 and 5mM, 000en was the failure of a muscle and it concluded that c 0 0 supernatant to behave similarly was due to interfer- 4) was 4), ing enzyme reactions. When pure NADPH 0 N added to the system it was not oxidized in the ° ++10 +1 oo became oxidized absence of ribose 5-phosphate, but 0000 -O when this substance was added. The oxidation was +C o not inhibited by 0.1 mM-NaCN, a concentration that I"0, Cl4 Cl4 en inhibited the direct oxidation of NADH by the +l +1 +1 +1 muscle supernatant, so a transfer of reducing equi- \0 en 00 O'i tt ^ 00 valents to NAD+ with a subsequent oxidation of _I~ c 1- Cd NADH could not explain the observed behaviour. cis0 that when NADPH ) Further experiments revealed +1 +1 +1 I-Y 0 1- o C '.d t was added, or accumulated in the system, to a Cln greater than 0.03mm, it was oxidized 0, concentration 8* +lI+ +I when ribose 5-phosphate, fructose diphosphate, 0V 00 0 dihydroxyacetone phosphate, or 0.2mM-ATP in 0 m +1 +1 +1 +1 o75 5 the presence of ribose 5-phosphate, were added.
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