4. A. Lang, Naturwissenschaften 43, 257, 284 (1956). 7. S. P. Johnson, W. C. Hall, J. L. Liverman, 8. 9. unpublished experiments. , Phystol. Plantarum 9, 389 (1956). The gibbereilins used were kindly supplied by the following: Reed A. Gray, division of microbiological research, Merck and Co., Rahway, N.J.; Frank H. Stodola, Northern Uctilization Research Branch, U.S. Department of Agriculture, Peoria, Il.; and P. C. Marth, Horticultural Crops Research Branch, U.S. Department of Agriculture, Beltsville, Md. 10. This investigation was supported in part by grant No. G-1165 from the National Science Foundation to J, L. Liverman. 14 February 1957 direct effects of itodoacctate and iodoacetamide on the aerobic oxidation of pyruvate and cycle intermediates ‘by mitochondria would provide more information on their effects on respiration and give a basis for the judicious use of a particular concentration of these inhibitors to inhibit specifically the glycolytic pathway. ric measurement of oxygen uptake were distinct inhibition with all substrates, the strongest inhibition being observed in the oxidation of pyruvate and a-keto- «Iodoacetate and iodoacetamide have glutarate, which may indicate the sensitivity of systems involving coenzyme A and lipoic acid. However, the lower con- the site of inhibition being at the triosephosphate dehydrogenase. Early reports bitions which cannot be ignored in respiratory studies. It may be noted that iodoacctate was generally more effective been used as specific inhibitors of the Embden-Meyerhof pathwayof glycolysis, (1) indicated that iodoacetate at low concentrations inhibited anaerobic glycolysis and respiration with glucose but not the oxygen uptake induced by addition of pyruvate or lactate. More recent studies (2) have shown that the oxidation of pyruvate may be reasonably sensitive to iodoacetate. A study of the Table 1. Effects of iodoacetate and iodoacetamide on the mitochondrial oxidation of various substrates. The reaction medium contained 121 mM KCl, 20 mM potas- sium phosphate buffer (#H 6.8), 0.01 mf cytochrome c, 5 mAf MgCh, 1mM adenosine monophosphate, 0.5 mM adenosine triphosphate, and 5 mM substrate. The temperature was 37°C, The mitochondrial suspension was incubated for 10 minutes with the inhibitors in the medium, and the oxygen uptake was determined over a period of 1 hour. centrations also produced definite inhi- than iodoacetamide. In order to produce complete inhibition of triose-phosphate dehydrogenase and glycolysis, concentrations of 0.2 to 0.5 mdf must be used ‘in most cases, and thus the present results indicate that a complete inhibition of glycolysis is usually accompanied with some effect on respiration (4). Wintiam C. YANG Department of Pharmacology, School of Medicine, University of Southern California, Los Angeles References and Notes 1. H. A. Krebs, Brockem. Z. 234. 278 (1931); O. Meyerhof and E. Boyland, ibid. 237, 406 (1931). 2, P. J. Heald, Biochem. jf, (London) 55, 625 (1953); J. L. Webb, P. R. Saunders, C. H. Thienes, Arch, Biochem. and Biophys., 22, 458 (1949); G. G. Laties, ibid. 20, 284 (1949). 3. C. M. Montgomery and J. L. Webb, J. Biol. Chem. 221, 359 (1956). 4. This work was supported by the Life Insurance Medical Research Fund and aided by facilities supplied by the Allan Hancock Foundation. 18 February 1957 Change (%) at various 0.01 mAL 0.10 mM Todoacetate a-Ketoglutarate Malate Pyruvate + malate Succinate Citrate Isocitrate —- 6.3 -33.3 -— 75.6 - 4.3 - 38 + 46 415.0 -43.0 - 8.0 - 81 -15.3 — 85.6 -61.2 -34.6 —35.0 - 7.4 —20.0 Todoacetamide a-Ketoglutarate Malate Pyruvate + malate Succinate Citrate Isocitrate 31 MAY 1957 1.0 mM ~- 63.9 - 64 -17.2 — 76.3 - 94 - 25 -16.1 -12.6 -17.0 -14.9 —79.7 -—43.1 -—44.1 - £0 -12.0 -21.0 -.7,3 -35.3 -29.1 Carompound bon : (Coune’mun (atom/ suet mole) Found TheLactose 12 Maltol 6 Formic acid Furfuryl alcohol © Naphthyl i Lac- tose/ prod- 8.7 353 14 cn ory ratio 104 1/0.51 17.4 5 O82 20.9 urethane 16 3,9-Dinitrobenzoate 12 0.0 6.5 0.0 8.7 1/0.81 1/0.04+ * Based on molar transfer of 1 atom of C44, atoms 2 through 6 in the glucose moiety of lactose (3). Maltol results rather uniquely from the heat-induced interac- tion of reducing disaccharides with amino compounds (2). It has been detected in evaporated milk, baked cereals, bread crust, and roasted malt, among other places (4). The three compoundsin question were recovered and purified from heated (121°C for 4 hours) condensed skim milk (30 percent total solids) to which lactose-1-C14 (National Bureau of Standards) had been added. Steam distillation was used to isolate the com- pounds from the heated milk. Maltol and furfuryl alcohol were recovered from this distillate by ethyl ether extraction and were purified as described clsewhcre (3, 5). Formic acid was recovered by neutralizing a portion of the distillate to pH 7.5 and evaporating the solution to dryness under vacuum (6). The crude formate wasselectively converted to CO, by the method of Osburn e¢ al. (7). This CO,, samples of furfuryl alcohol and its derivatives, maltol and lactose, the latter from the unheated product, were con- concentrations Substrate C chondrial suspension and the manomet- summarized in Table |. Both inhibitors at a concentration of 1.0 mM produced Uptake of Heart Mitochondria Activity of BaCos The preparation of the rat heart mito- made according to the methods of Montgomery and Webb (3). The results are Effect of Iodoacetate and Todoacetamide on Oxygen Table 1. Levels of C™ activity found some heat-degradation products of ski milk containinglastose-1-C™. Carbon-14 Activity of Some Heat-Degradation Products of Milk Containing Lactose-1-C!* The course of heat-induced lactoseprotein interaction in milk has been followed with the aid of lactose-1-C1* (7). Use of labeled lactose also appeared at- tractive for investigation of the sugar’s decomposition under these conditions. Of the many fragments known to be formed (2), formic acid, furfuryl alcohol, and maltol (3-hydroxy-2-methylpyronc-4) were evaluated in these experiments. It has been proposed that formic acid is derived from carbon atom No. ! and furfurvl alcohol from carbon verted to BaCO, (8). Radioactivity in these preparations was determined with a windowless flow gas Geiger-Miiller counter and decade scaling unit. The data thus secured (Table 1} re- veal that carbon atom No. | of lactose is involved in the formic acid and maltol, but not in the furfuryl alcohol. A preliminary experiment yielded essentially the same findings with the exception that some activity was detected in the furfuryl alcohol (9). Further investigation of the alcohol and two carefully authenticated derivatives of it, as shown in Table |, revealed that it had no activity. Under the rigorous heating conditions emploved in these experiments, a number of carbon sources could contribute to formate; however, carbon 1 of lactose 1087