[CANCER RESEARCH 45,6376-6378, December 1985]
Anomeric Specificity of Hexokinase in Rat, Human, and Murine Tumor Cells1
W. J. Malaisse,2 M-H. Giroix, S. P. Dufrane, F. Malaisse-Lagae, and A. Sener
Laboratory of Experimental Medicine, Brussels Free University, Brussels, Belgium
ABSTRACT equilibrium (n = 4 in each case). Likewise, the conversion of /3-o-glucose (1.0 mM) to a-o-glucose amounted to only 0.04 ±0.01 HIM after 60 min In tumoral cells derived from the insulin-producing rat cell line of incubation at 7°C, as distinct from 0.35 ± 0.04 mM at anomeric RINmSF, both low- and high-Km glucose-phosphorylating en- equilibrium (n = 4 in each case). Virtually identical results were obtained zymic activities were present. The hexokinase-like enzyme was in the presence of the islet cell homogenate, no mutarotase activity being inhibited by glucose 6-phosphate and displayed a greater affinity detected under the present experimental conditions. for but lower maximal velocity with a-D-glucose than /3-o-glucose. The calculation of Kms for a- and /3-o-glucose was performed as described elsewhere (2, 3). All results are expressed as the mean ±SE A comparable anomeric behavior of hexokinase was observed together with the number of individual observations (¡nparentheses). in breast cancer (MCF-7) and lymphocytic leukemia (P388) cells. Thus, the anomeric specificity of hexokinase in tumoral cells was not different from that recently characterized in normal mam RESULTS malian cells. Insulin-producing Cells. In the first series of experiments, the phosphorylation rate of D-[l/-14C]glucose, in anomeric equilib INTRODUCTION rium, was measured in homogenates of insulin-producing tu- moral cells incubated at 30°C(Chart 1). The results obtained at The anomeric specificity of glucose metabolism in intact tu- the five lowest concentrations of glucose (10-100 U.M)yielded a moral cells may differ from that in nontumoral cells. For instance, straight line corresponding to a hexokinase-like enzyme with a at low-glucose concentrations (2.8-5.6 mivi), normal islet cells Km for glucose close to 57 /¿Manda V,â„¢,,close to 0.74 pmol/ utilize a-D-glucose at a higher rate than /8-o-glucose, as judged min/103 cells (Chart 1). At higher glucose concentrations (250 from the conversion of o-[5-3H]glucose to 3H2O, whereas no /iM-20 mM), however, the results indicated the presence of low- anomeric difference in o-glucose utilization is observed in tumoral affinity enzyme(s). At low glucose concentrations (20 ÃÃM-1.0 insulin-producing cells exposed to the same concentration of the mM), glucose 6-phosphate caused a dose-related decrease in hexose (1). It was recently reported (2, 3) that the anomeric reaction velocity (Chart 1, inset; Table 1), with 50% inhibition at specificity of mammalian hexokinase represents a mirror image a concentration of glucose 6-phosphate close to 0.1 mM. of that of yeast hexokinase (4), in terms of both affinity and Since the present study aimed at investigating the anomeric maximal velocity. The difference in the anomeric specificity of specificity of hexokinase, further experiments in the insulin- glucose metabolism in tumoral and nontumoral cells, respec producing cells were conducted at 6-7°C at glucose concentra tively, and the knowledge that hexokinase underwent a phylo- tions not exceeding 1.0 mM. As illustrated in Table 2, unlabeled genetic evolution led us to investigate, in the present study, the a-D-glucose inhibited more than /8-D-glucose the phosphorylation anomeric specificity of hexokinase in three types of tumoral cells. of D-[(A14C]glucose in anomeric equilibrium, indicating a greater affinity of hexokinase for the a- than 0-anomer. From these data, it was calculated that the Km for a-D-glucose was one-half that MATERIALS AND METHODS for 0-D-glucose, averaging 18.0 ± 2.3 and 33.3 ± 2.1 /*M, Tumoral cells were derived from the insulin-producing rat cell line respectively (Table 3). The close-to-maximal velocity of hexoki RINmSF (5), the MCF-7 breast cancer cell line (6), and lymphocytic nase, as measured at a glucose concentration of 1.0 mM, was leukemia P388 cells propagated in DBA/2 mice (7) by methods described higher, however, with |8-D-[l/-14C]glucose than a-o-[U-14C]glu- in the cited references. The cells were homogenized in KCI (150 ÃŒTÃŒM).cose (Table 3). The unlabeled anomers of o-glucose were purchased from Sigma Breast Cancer and Leukemic Cells. The results obtained in Chemical Co. (St. Louis, MO). The labeled anomers were prepared from a solution of D-[U-"C]glucose in anomeric equilibrium by a technique the breast cancer and leukemic cells were similar to those obtained in the insulin-producing cells. Thus, the phosphorylation described elsewhere (8). of D-[U-14C]glucose in anomeric equilibrium was inhibited by The methods used to measure the phosphorylation of glucose by glucose 6-phosphate (Table 1) and more markedly decreased by either a radioisotopic (9) or a nonisotopic (10) procedure were previously described. All experiments were performed over 30-60 min of incubation unlabeled a- than 0-D-glucose (Table 2). The absolute values of at 6-7°C in a triethanolamine-HCI buffer (50 ITIM,pH 7.4) containing KCI the Kmfor a- and /8-D-glucose were higher, however, in the breast (150 HIM). MgCI2 (6 ITIM),cysteine (2 HIM), EDTA (1 HIM), bovine albumin cancer or leukemic cells than in the insulin-producing cells (Table (0.1 mg/ml), and ATP (5 ITIM). As judged by measurements performed 3). The maximal velocity, which was measured by a nonradio- with the glucose oxidase method (11), the generation of 0-D-glucose isotopic procedure in the presence of 1.0 mM of the unlabeled from a-D-glucose (1.0 ITIM)did not exceed 0.06 ±0.02 mM after 60 min anomers, was higher with ß-than a-D-glucose (Table 3). of incubation at 7°C, as distinct from 0.68 ± 0.03 mM at anomeric
Received 1/28/85; revised 8/7/85; accepted 8/14/85. 1This work was supported by grants from the Belgian Foundation for Scientific DISCUSSION Medical Research and Belgian Ministry of Scientific Policy. 2 To whom requests for reprints should be addressed, at Laboratory of Experi The present results indicate that, in three distinct types of mental Medicine. 115, Boulevard de Waterloo. B-1000 Brussels. Belgium. tumoral cells, the hexokinase-like enzyme displays a greater
CANCER RESEARCH VOL. 45 DECEMBER 1985 6376
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100
1.5 Ov*/
• K u O 50 o. (A O LO S. a e l «A25 O o U E O o 33 66 100 33O g GLUCOSE 6-PHOSPHATE (jjM) JJJ 0.5
O 2 4 6 8 10 12 VELOCITY/[GLUCOSEJ(pmol/min per103cells.mM-1) Chart 1. Hofstee plot for the phosphorylationof o-[t/-14C]glucose,in anomericequilibrium,in homogenatesof insulin-producingtumor cells incubated at 30°C.Points, mean of 6-30 individual measurements;bars, SE. Inset, effect of increasingconcentrations of glucose 6-phosphate (logarithmicscale)upon the phosphorylationof D-[U- "CJglucose (1.0 mM)at 30°C;meanvalues refer to four individual measurements and are expressed in the percentage of the control value found in the absence of glucose 6-phosphate, such a control value averaging 1.05 ±0.02pmol/min/103cells.
Table 1 Table2 Effect of glucose 6-phosphateupon the phosphorylation of o-glucose Effect of unlabeled o-glucose anomersupon the phosphorylation of D-[U-"C]glucose in anomeric equilibrium
typeNo Cell producing39 cancer736 leukemia177 D-[ü-14C]Glucosephosphorylation (%)" glucose 6-phosphate ±1a'" ±7 ±3 cose(,M, Glucose 6-phosphate, 8±14±1Breast143±836±1Lymphocytic68±220 0.3 mM Glucose 6-phosphate, ±1 ce7.4 producing100.0cancer100.0 leukemia100.0 1.0 mMInsulin ±2.o" ±0.9 ±2.0 aThe phosphorylation of o-[(P4C]glucose (20 I¿M),inanomericequilibrium,was 7.4 12.6 51.7 ±1.0 62.3 ±0.6 63.2 ±1.3 measured at 6°C,andthe results were expressed as fmol/min/103cells. 7.4 12.6 65.5 ±0.7 86.9 ±1.2 80.1 ±1.6 6 Mean ±SEof 3-4 individualmeasurements. 7.47.4ß12.612.6 80.0ß30.0 26.4 ±0.5 38.2 ±1.5 35.9 ±1.0 12.6or30.0 80.0Insulin44.7 ±0.7Breast59.8 ±0.8Lymphocytic59.7 ±1.0 affinity for a- and ß-D-glucose,but a lower maximal velocity with ' The phosphorylationof o-[U-14C]glucose(20iiu), in anomericequilibrium,was measuredat 6°C,andthe results were expressed in the percentageof the control the oc-than ß-anomer.These results are qualitatively identical to value (first line; see Table 1 for absolute values). those recently obtained in islet cells, erythrocytes, and parotid Mean ±SEof 3-4 individualmeasurements. glands removed from normal rats (2). Thus, the anomeric speci ficity of hexokinase appears similar in normal and tumoral mam overlooked since conflicting data have been published on the malian cells. presence in insulin-producing cells of a glucokinase-like enzy It should be stressed that, in the insulin-producing cells, our matic activity (12, 13). data were restricted to the low-Km hexokinase-like enzyme. The We have recently observed (1) that, in insulin-producing and kinetics of glucose phosphorylation at high glucose concentra lymphocytic leukemia tumoral cells, the anomeric specificity of tions (>1.0 HIM) and the anomeric specificity of the high-Km glucose metabolism differs from that previously found in nontu- enzyme(s) were not analyzed. Nevertheless, the presence of the moral cells (10, 14). The present data indicate that such a latter enzyme(s), as documented in Chart 1, should not be disparity cannot be accounted for by any obvious difference in
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Table 3 in normal and tumoral cells. Biochim. Biophys. Acta, in press, 1985. Michaelis constant
CANCER RESEARCH VOL. 45 DECEMBER 1985 6378
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W. J. Malaisse, M-H. Giroix, S. P. Dufrane, et al.
Cancer Res 1985;45:6376-6378.
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