0020-71 I x/80~0801-043330? 00,o
Inr J hchcnt.. Vol 12. pp. 433 to 431 Q Pergamon Press Ltd 1980 Prmed m Great Brtlain
PHOSPHOFRUCTOKINASE PREPUTIAL GLAND
A MOUSE ESR-586
ARIEGEURSEN and MURRAYR. GRIGOR University of Biochemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand (Received 9
Abstract-l. Phosphofructokinase from both the mouse preputial gland tumour and the gland itself have electrophoretic properties similar to the enzyme from mouse brain. 2. The tumour enzyme showed allosteric kinetics with respect to both substrates and was similar to the brain in the response to a number of known effecters of phosphofructokinase. 3. The tumour enzyme was however less sensitive to AMP activation and more sensitive to 2,3-diphosphoglycerate inhibition than the brain enzyme. 4. Phosphofructokinase from the preputial gland was similar to the enzyme from brain in its response to AMP, but was intermediate between those of the tumour and brain in its sensitivity to 2,3-diphosphoglycerate.
(EC 188.8.131.52) is a key regulatory
enzyme of glycolysis (Mansour, 1972). The existence of isoenzymes of phosphofructokinase is well established. These can be distinguished by both their physical properties and their differential ability to respond to various allosteric effecters. The enzyme is a tetramer made up of one, or a combination of two, out of three different subunits. Thus, the enzyme from brain can be distinguished from those from muscle and liver (Kemp, 1971; Tsai & Kemp, 1973, 1974). Each of the enzymes has evolved to respond to a different set of allosteric effecters in such a way as to best satisfy the metabolic requirements of that tissue. This report describes the properties of phosphofructokinase from a mouse preputial gland tumour
(Fekete 8c Kent, 1955) and compares them with those of the enzyme from other mouse tissues. Although this tumour grows rapidly it exhibits a high degree of cellular differentiation (Prutkins et al., 1975). This is apparent also in its lipid composition as the tumour contains many lipid components normally found only in sebaceous secretions (Kandutsch & Russell, 196Oa, 1960b; Grigor, 1976). It appears that the tumour may provide a suitable model for studying the metabolism of holocrine sebaceous glands and already the tumour has been used as a source of material for studying the synthesis of many of these lipids (Kandutsch & Russell, 1960b; Snyder et al., 1970, Grigor et al., 1972; Grigor & Harris, 1977). Little is known about the control of metabolism in holocrine sebaceous cells. For this reason we have also studied some of the properties of phosphofructokinase from the preputial gland.
The following chemicals were purchased from the Sigma Chemical Co., St. Louis, MO: ADP, cyclic AMP, AMP, ATP, fructose-6-P (F6P). 2,3-diphosphogylcerate, NADH,
NAD+, nitroblue tetrazolium, phenazine methosulphate and phosphoenolpyruvate. Other chemicals used were purchased from a variety of sources and only analytical grade reagents were used. Crystalline suspensions of aldolase, a-glycerol-phosphate glyceraldehyde-3-phosphate dehydrogenase, dehydrogenase, glucose-6-phosphate dehydrogenase and triose phosphate isomerase, all purified from rabbit muscle, were also purchased from the Sigma Chemical Co. Strain C57BL mice were obtained from the University of Otago Animal Breeding Station and were allowed to feed ad libitum. The mouse preputial gland tumour (ESR 586) was maintained by transplantation (Grigor, 1976). Stable preparations of phosphofructokinase were prepared from the tumours and other mouse tissues by homogenizing fresh tissue in two volumes of cold 2.5 mM glycylglycine buffer, pH 7.5 containing 2.5 mM fi-glycerophosphate, 1 mM EDTA, 0.1 mM dithiothreitof and 30mM KF. ATP (0.2 mM) was added to the buRer in all preparations except those used in kinetic experiments. Electrophoresis of the enzyme extracts was carried out on 1 x 4in. Titan III cellulose acetate strips supplied by Helena Laboratories, Beaumont, TX, using the buffer and staining medium of Kemp (1971). Phosphofructokinase activity was assayed spectrophotometrically in a Unicam SP1800 spectrophotometer by coupling fructose-1,6-diphosphate formation to the reactions catalyzed by aldolase, triose phosphate isomerase. cc-glycerol phosphate dehydrogenase and measuring the rate of NADH oxidation at 340nm. The assay conditions for the unregulated activity (V,..J were those of Tsai & Kemp (1973). The kinetic properties of phosphofructokinase were assayed in a 50 mM imidazole buffer, pH 7.4, containing 1 mM EDTA, 6 mM MgCl*, 0.17 mg/ml NADH. 1 mM dithiothreitol and approximately 1.2 units/ml aldolase. 0.6 units/ml triose phosphate isomerase and 0.6 units/ml 3~glycerol phosphate dehydrogenase. The auxilliary enzymes were extensively dialyzed to remove ammonium sulphate. When potential effecters were studied, these were added to the assay 4-5 min prior to the reaction being started by the addition of F6P. The reaction was carried out at 25°C and rate measurements were made over a period of 3-Imin after an initial lag period. One unit of phosphofructokinase was defined in both assays as the amount of phosphofructokinase required to oxidize 2 pm01 of NADH per min.
ARIEGEURSENand MURRAYR. GRIG~R
A B C
Fig. 1. Electrophoresis of phosphofructokinase from preputial gland tumour and mouse tissues. Extracts were electrophoresed and stained as described in Methods. A, E. preputial gland tumour; B. liver; C, brain; D, muscle; and F. preputial gland. The band nearer the origin in B stained in the absence of fructose-6-P and is not phosphofructokinase. RESULTS
The total phosphofructokinase activity determined in tumours at various stages of growth was 4.0 + 0.9 units/g wet wt and there was no significant variation of this activity with respect to the stage of growth of the tumour. The total phosphofructokinase activity in the preputial gland was 2.0 &-0.2 units/g wet wt. Three isozymes of phosphofructokinase were identified in mouse tissues after electrophoresis; one in hind leg muscle extract, another in brain extract and a third in liver extract (Fig. 1). Electrophoresis of extracts from the preputial gland tumour and the preputial gland showed that these each have a single band of activity with electrophoretic properties similar to the brain isozyme. The enzymes from these tissues could also be distinguished by their solubility in solutions of increasing concentrations of ammonium sulphate. Most of the muscle activity precipitated between 45 and 55% saturation, the liver between 30 and 45% saturation and the tumour, gland and brain activities between 40 and 50% saturation. When the velocity of phosphofructokinase from the preputial gland tumour was determined at pH 7.4 (and in the absence of ammonium sulphate) at a given level of ATP and varying the levels of F6P a sigmoid response curve was obtained (Fig. 2). The apparent K, (K,,,) for F6P was determined as the concentration which gives half the measured V,,,.= The values ranged from 1.3 to 3.3 mM as the concentration of ATP was varied from 1 to 5 mM. When phosphofructokinase was assayed varying the concentration of ATP (Fig. 3), the enzyme reached saturation at very low concentrations of ATP. As the concentration at ATP was increased further the enzyme was strongly inhibited. The concentration required to inhibit the enzyme activity by SO%, Kico.s,, ranged from
Fig. 2. Phosphofructokinase from the preputial gland tumour: kinetics with respect to fructose-6-P. Phosphofructokinase was assayed as described in Methods at the following concentration of ATP: H, l.OmM; 0, 2SmM and q, 5.0 mM.
2 to 8 mM ATP as the concentration of F6P was varied from 1 to 5 mM. Phosphofructokinase activity is influenced by a large number of effecters. For the study of potential inhibitors of phosphofructokinase concentrations of 1 mM ATP and 2 mM F6P were chosen since, under these conditions, the tumour enzyme is near maximal activity (see Fig. 3). Citrate, phosphoenolpyruvate and 2,3-diphosphoglycerate inhibited phosphofructokinase from all the mouse tissues investigated. The Ki(o.5, values of each isozyme for these effecters are summarized in Table 1. For citrate and phosphoenolpyruvate the Kico.s, values of the tumour phosphofructokinase was strongly inhibited by 2,3-diphosphoglycerate with the degree of inhibition being similar to that of phosphofructokinase from muscle (Fig. 4). Phosphofructokinase from the mouse preputial gland is also inhibited by 2,3_diphosphoglycerate with a K,,,,, intermediate between that of the brain and tumour enzyme (Table 1). Mono- and diphosphoadenine nucleotides relieve ATP inhibition of phosphofructokinase (Fig. 5). For the study of potential activators concentrations of 1 mM F6P and 5 mM ATP were used since, under these conditions, phosphofructokinase from the tumour was approximately 90”//, inhibited by ATP (Fig. 3). Cyclic AMP, ADP and AMP all relieve this inhibition of the phosphofructokinase from all the tissues studied. The concentrations of each effector required to achieve 50% of the maximum activity with that effector, KacO,Sjrare summarized in Table 1. The K at0,5j values for cyclic AMP and ATP for the tumour and brain enzymes are similar although the phosphofructokinase of the tumour was deinhibited by ATP to a greater extent than that from brain. However, the Kat0,5, values of the tumour and brain enzymes for AMP do differ and in this respect the phosphofructokinase from the gland resembles the brain rather than the tumour enzyme (Table 1). The
Preputial tumour phosphofructokinase Table 1. Etfectors of phosphofructokinase studied in extracts of the preputial gland tumor and other mouse tissues Effector
ADP AMP Cyclic AMP
0.15 0.30 0.10
0.15 0.15 0.10
Citrate Phosphoenolpyruvate 2,3-Diphosphogiycerate
2.5 >9 0.15
2.8 >9 4.0
K d0.s, and GO.,, are defined as the concentration half maximum effect.
* same kmettc values, Kafo,st, were obtained for tumour phosphofructokin~e in the presence of a denatured brain preparation and for brain phosphofructokinase in the presence of a denatured tumour preparation, indicating that the difference in response to AMP was not due to a small molecule present in either preparation. DISCUSSION
Only one isozyme of phosphofructokinase was detected by electrophoresis in crude preparations of each of the tissues investigated. The electrophoretic and solubility properties of enzymes from the tumour and the preputial gland resemble that from mouse brain. This is reflected also in the response to the allosteric effecters investigated except for two cases. 2,34Xphosphoglycerate inhibited the tumour enzyme more effectively than it does either the brain or gland enzyme. In fact, in the presence of 2,3-diphosphoglycerate the tumour phosphofructokinase is similar to that from muscle. The other exception is AMP. Here the affinity of the tumour enzyme for AMP is
Muscle &O.&W -.0.08 -%.s, (mM) 1.5 4.0 0.20
of activator and inhibitor to produce
less than that of either the brain or gland enzyme. The enzyme from muscle, however, has an even lower EC&,,,,for AMP than does that from brain. Three other glycolytic enzymes have also been investigated. Both the tumour and the gland contain types I and II hexokinase in comparable proportions (Geursen & Grigor, 1976). They also contain the same isozyme of aldolase which is identical to that in brain as judged by its electrophoretic mobility and in ability to cleave F6P (Geursen, 1977). The tumour pyruvate kinase is electrophoretically similar to the pyruvate kinases from the preputial gland and mouse brain, but differs in its ability to be inhibited in crude prep arations by the amino acids alanine and phenylalanine (Geursen, 1977). This suggests that, as for phosphofructokinase, there are minor differences between the pyruvate kinases of the tumour and the preputial gland. Little is known about the control of metabolism in either holocrine cells or indeed, tumours themselves, both capable of high rates of glycolysis. The question arises as to whether the rate of glycolysis in the preputial gland tumour is controlled by phosphofructokinase. In order to do so phosphofructokinase must
Fig. 3. Phosphofructokinase from the preputial gland tumour: kinetics with respect to ATP. Phosphofructokinase was assayed as described in Methods at the following concentrations of fructose-6-P: B, 1.0 mM; 0, 2.5 mM and Cl, 5.0 mM. V,,, is the rate obtained using the assay for unregulated enzyme activity L3J.
2 3 2,3-dlphosphoglycerote
Fig. 4. The inhibition by 2.3-diphosphogiycerate of phosphofructokinase from the preputial gland tumour and other mouse tissues. Phosphofructokinase was assayed in the presence of 1 mM ATP and 2 mM fructose-6-P as described in Methods. V,,,,, is as defined in Fig. 2. 0. tumour phosphofructokinase: O. brain phosphofructokinase: e. muscle phosphofructokinase:
l , liver phosphofructokinase.
ARIE GEURSENand MURRAY R. GRIGOR
0.6 _ 0.5 -
2 0.2 _
1.5 2.0 2.5 3.0 Concentration lmM1
Fig. 5. The deinhibition of phosphofructokinase from the preputial gland tumour and mouse brain by ADP and AMP. Phosphofructokinase was assayed in the presence of 5 mM ATP and 1 mM fructose-6-P. V0 is defined as the velocity of the enzyme in the absence of the effector while Vrndl is as defined in Fig. 3. 0, response to tumour phosphofructokinase to ADP; n, response to brain phosphofructokinase to ADP; 0, response of tumour phosphofructokinase to AMP; 0, response of brain phosphofructokinase to AMP.
The electrophoretic and kinetic properties of phosphofructokinase from a mouse preputial gland tumour have been compared with phosphofructokinases from other mouse tissues. Phosphofructokinase from the tumour and preputial gland had similar electrophoretic properties to that of mouse brain. The tumour enzyme showed allosteric kenetics with respect to both substrates (fructose-6-P and ATP) and was similar to the brain enzyme in its response to a number of known effecters of phosphofructokinase with the exception of AMP and 2,3-diphosphoglycerate. The tumour enzyme was less sensitive to AMP activation but more sensitive to 2,3-diphosphoglycerate inhibition than the brain enzyme. Phosphofructokinase from the preputial gland itself resembled the enzyme from brain in its response to AMP. but the sensitivity to 2,3_diphosphoglycerate was intermediate between that of the tumour and brain enzymes.
AcknowlPdgernent-This work was supported by a project grant from the Medical Research Council of New Zealand.
be the rate limiting enzyme. Other enzymes which catalyze unidirectional reactions in glycolysis are hexokinase and pyruvate kinase. In the tumour the maximum activity of these enzymes are 1.6 units/g wet wt and 90 units/g wet wt respectively (Geursen, 1977), compared with phosphofructokinase at 4.0 units/g wet wt while the corresponding activities of the three enzymes in the preputial gland are 1.0, 19, and 2.0 units/g wet wt. However, it is unlikely that phosphofructokinase in the tumour is ever fully active. ATP, if present at concentrations found in normal mouse tissues (Lowry & Passonneau. 1964; Goldberg et a[., 1966) is likely to be severely inhibitory to the enzyme although ADP may partially relieve this inhibition. None of the other effecters investigated is likely to be present in concentrations sufficient to alter the activity of the enzyme. Unfortunately, it was not possible to measure the concentration of either the substrates or effector molecules because of varying amounts of necrotic tissue in the tumour. However, the maximum rate of glucose uptake by slices of the tumour has been found to be close to 1 pmol/g wet wt/min.* This is less than the activity of the hexokinase and suggests that some other factor, possibly the phosphofructokinase activity, is controlling glucose use. It is of interest to note that the phosphofructokinase from the tumour is similar to that of brain and both these tissues prefer glucose as the substrate. There is evidence that glycolysis in the brain is controlled by the activity of phosphofructokinase (Lowry er al., 1964; Ruderman et al.. 1974).
*JONES R. L., THOMPSONM. P. and GRIGOR M. R., unpublished results.
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