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The inhibition of some glycolytic enzymes by chlorambucil
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638
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I-K,. I, ‘The efi’cctof chl~~r~unhuc:ilon hexokirlasc. The tinul assay mixtures contained 447pg hex<+ kinasc; .:.‘., control hexokinasc: 0, hcxokinasc prcincubated with 2 IO .: M chlorambucil; ,, hexokinase preincubated with 4 ,. IO-:’ M chlorambucil inhthits i.~spir~Iti(~tlof yeast more signiti~~~ntly with glucose as an energy sotrrce I ban dots citrnle (ii Luccinate.” and lowers the glycogcn content of lymphocytes,,’ / il5 effect on bcvcral glycctlytic enzyme> was investigated in order 10 correlate the observed action on whole cells and organisms. * Preliminary investigations were carried out at the Division of Pharmacology, Food and Drug Administration, Washington, D.C. The chlorambucil was kindly supplied by Dr. hl. V. Nadkarni, Cancer Chemotherapy National Service Center, National Institutes of Health, Bethesda, Md.
639
Short communications
Crystalline yeast hexokinase, rabbit muscle lactic dehydrogenase, aldolase, and enolase were treated in the manner described by Dixon and Needham. The enzymes were preincubated at 0” with 2O’j/, ethanol or ethanolic chlorambucil for up to 1 IO-min periods, and assayed for enzyme activity.i-u’ Determination of the concentration of hexokinase was performed by measurement of the absorption at 278 w in the Beckman DU spectrophotometer. Lactic dehydrogenase and aldolase concentrations were determined by measurement of the absorption at 280 w. Enolase concentrations were estimated by the method of Lowry et al,” with crystalline bovine albumin as a standard. Chlorambucil at a concentration of 4 ,’ IO-” M effects a 96 7; inhibition of hexokinasc activity, whereas 2 d 10-a M chlorambucil exerts a 57 “/, inhibition (Fig. 1). Lactic dehydrogenase is fully inhibited by chlorambucil at a concentration of 5 ’ lo-” M. It is IO-” M chlorambucil and is not ntfccted by I IO ’ hl ininhibited to the extent of 47 “/6by 2 hibitor (Fig. 2). /
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Fro. 2. The effect of chlorambucil on lactic dehydrogenase (LDH). The final assay mixtures contained IO. I pg LDH ; 2,. control LDH ; x , LDH preincubated with 5 .’ 1OWrM chlorambucil; 0, LDH preIO ‘I M chlorambucil: I?. LDH preincubated with I x lo-” M chlorambucil. incubated with 2
0
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7 TIME
8
9
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12
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14
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I6
17
, MINUTES
Fro. 3. The effect of chlorambucil on aldolase. The final assay mixtures contained 14.95 pg aldolase; i>,control aldolase; 0, aldolasepreincubated with 1 J 10-s M chlorambucil; A,aldolnsepreincubnted with 2 ‘_ IO-” M chlorambucil.
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I-K,. I, ‘The efi’cctof chl~~r~unhuc:ilon hexokirlasc. The tinul assay mixtures contained 447pg hex<+ kinasc; .:.‘., control hexokinasc: 0, hcxokinasc prcincubated with 2 IO .: M chlorambucil; ,, hexokinase preincubated with 4 ,. IO-:’ M chlorambucil inhthits i.~spir~Iti(~tlof yeast more signiti~~~ntly with glucose as an energy sotrrce I ban dots citrnle (ii Luccinate.” and lowers the glycogcn content of lymphocytes,,’ / il5 effect on bcvcral glycctlytic enzyme> was investigated in order 10 correlate the observed action on whole cells and organisms. * Preliminary investigations were carried out at the Division of Pharmacology, Food and Drug Administration, Washington, D.C. The chlorambucil was kindly supplied by Dr. hl. V. Nadkarni, Cancer Chemotherapy National Service Center, National Institutes of Health, Bethesda, Md.
639
Short communications
Crystalline yeast hexokinase, rabbit muscle lactic dehydrogenase, aldolase, and enolase were treated in the manner described by Dixon and Needham. The enzymes were preincubated at 0” with 2O’j/, ethanol or ethanolic chlorambucil for up to 1 IO-min periods, and assayed for enzyme activity.i-u’ Determination of the concentration of hexokinase was performed by measurement of the absorption at 278 w in the Beckman DU spectrophotometer. Lactic dehydrogenase and aldolase concentrations were determined by measurement of the absorption at 280 w. Enolase concentrations were estimated by the method of Lowry et al,” with crystalline bovine albumin as a standard. Chlorambucil at a concentration of 4 ,’ IO-” M effects a 96 7; inhibition of hexokinasc activity, whereas 2 d 10-a M chlorambucil exerts a 57 “/, inhibition (Fig. 1). Lactic dehydrogenase is fully inhibited by chlorambucil at a concentration of 5 ’ lo-” M. It is IO-” M chlorambucil and is not ntfccted by I IO ’ hl ininhibited to the extent of 47 “/6by 2 hibitor (Fig. 2). /
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2 TIME
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Fro. 2. The effect of chlorambucil on lactic dehydrogenase (LDH). The final assay mixtures contained IO. I pg LDH ; 2,. control LDH ; x , LDH preincubated with 5 .’ 1OWrM chlorambucil; 0, LDH preIO ‘I M chlorambucil: I?. LDH preincubated with I x lo-” M chlorambucil. incubated with 2
0
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2
3
4
5
6
7 TIME
8
9
IO
I,
12
13
14
I5
I6
17
, MINUTES
Fro. 3. The effect of chlorambucil on aldolase. The final assay mixtures contained 14.95 pg aldolase; i>,control aldolase; 0, aldolasepreincubated with 1 J 10-s M chlorambucil; A,aldolnsepreincubnted with 2 ‘_ IO-” M chlorambucil.
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