Steinberg et al.5 and many other workers6-7 previously demonstrated that synthetically prepared amino-acid analogues like the ammo-acids themselves could be incorporated into proteins and they can thus compete with the natural ammo -acids for the latter's incorporation into proteins. That ACPC might act as an amino-acid antagonist was first mentioned by Ross3. Mickelson8, using bacterial systems, failed to demonstrate any such effect of ACPC. In order to elucidate the mechanism by which this unnatural amino-acid exerts its antitumour effect, the following experiments were performed. A 20 per cent rat liver homogenate was prepared in a Dounce homogenizer at 0 C. in a medium containing 0-35 M sucrose, 0-25 M potassium chloride, 0-035 M potassium bicarbonate and 0-004 M magnesium chloride, and centrifuged at 2,3000 in the cold. 0'05 ml. of the supernatant fraction containing the microsomes and cell sap was incubated at 37 C. for 30 min. with 14C-ACPC (1-5 x 105 counts/min.) or with 14C-valine (0-5 x 105 counts/min,) in the presence of 3 [jtmoles of ACPC, 5 pimoles of Mg++, 2 (/moles of ATP, 5 mgm. of creatine phosphate and 50 (jLgm. (protein) of purified creatine kinase. The reaction was terminated by adding 5 ml. of 5 per cent TCA. The TCA precipitable material was washed, plated and counted according to the procedures previously described by Devi et al,9.Table 1. INCORPORATION OF "C-VALINE INTO PROTEINS IN THE ABSENCE AND PRESENCE OF I-AMINO-CJ/CZOPENTANE CARBOXYLIC ACID c.p.m./mgm. TCA-precipitable material Range ofvalues in Percentage Source of microsome- ISTo. of Range of presence ofcell sap preparation Expts. values of ACPC inhibition Liver of normal rat 5 142-160 84-96 40 Liver of tumour bearingrat (Walker) 4 130-146 86-98 32 Walker tumour 4 150-166 96-108 36For in vivo experiments, five rats weighing 120-140 gm. each received an injection of 20 mgm. of ACPC 48 hr. prior to the administration of 14C-valine (1"2 x 106 counts/min.); in another set of experiments 14C-valine was injected after 3 hr, of the administration of ACPC. A similar number of rats received only 14C-valine, which served as controls. The animals were killed 3 hr. after injection of carbon-14. 1 gm. of liver from each rat was homogenized in 5 ml. of 0-25 M sucrose and to 1 ml. of such homogenates 5 ml. of 5 per cent TCA added. The washings, plating and counting of the TCA-precipitable material were similar to those described before. Similar experiments were also performed with tumour (Walker)-bearing rats, where 1 gm. of the tumour tissue was homogenized, precipitated and the precipitate was washed, plated and assayed for its radioactivity. The results can be summarized as follows. When the microsome-cell sap fraction was incubated with 14C-valine and ACPC, the incorporation of carbon-14 into proteins was reduced to varying degrees depending upon the amount of ACPC present. The maximum inhibition noted was 41-47 per cent in the presence of 10 ^moles of A.CPC. This effect of ACPC to inhibit the incorporation of 14C-valine was also noted when microsome-cell sap preparation obtained from Walker tumour was used. When 14C-ACPC was used instead of 14C-valine no incorporation of carbon-14 into proteins could be demonstrated.In in vivo experiments where ACPC (1-56 X 107 counts) was injected 48 hr. prior to the injection of 14C-valine, the extent of the latter's incorporation into proteins was reduced by 32-40 per cent. Somewhat less (25-28 per cent) inhibition was noted when ACPC was administered only 3 hr. prior to the injection of 14C-valine. When 14C-ACPC alone was injected, a very small but definitely measurable amount of it was found to be incorporated into proteins. Table 2. INHIBITION OF INCORPORATION OF "C-VAIINE, in vivo, INTO PROTEINS BY I-AMINO-C^OPBNTANE CARBOXYMC ACID c.p.m,/mgm, TCA-precipitable materialACPC ACPC administered 3 hr.Source of homogenate Controls Liver of normal rat 90 Liver of tumour bearing rat 82 Tumour 102 prior to U-C injection6658 75 administered 48 hr. Percentage prior Percentageinhibition 2727 to "-C injection48 44 60of inhibition 4746 41 Each value is an average of four such determinations.The incorporation of 14C-uridine into RNA was not inhibited by ACPC whether the incorporation of 14C-uridine was carried out in vitro or in. vivo. The procedure for these experiments is similar to those of Devi and Sarkar10. ACPC did, however, prevent the incorporation of 14C-valine into transfer RNA by 40-50 per cent in the experiment where 14C-valine was incubated with high-speed supernatant fraction in the presence of ACPC, Mg++ and ATP. It follows from the foregoing results that ACPC does inhibit the incorporation of 14C-valine into proteins to an extent of 40-50 per cent.The initial stages of protein synthesis, as we know them to-day, are the activation of amino-acids by specific ammo-acid-activating enzymes, followed by their incorporation (attachment) into specific soluble UNA's. In the second stage, the amino-acids fromRKA-aa's complexes are transferred to template RNA's whereby some hitherto unknown mechanism, protein with proper amino-acid sequences, is formed and released11. The fact that ACPC does inhibit the incorporation of 14C-valine by 40-50 per cent suggests that this unnatural amino-acid perhaps exerts its antitumour effect by suppressing protein synthesis although no significant amount of it has been found to be incorporated into proteins. This is not unexpected as several analogues have been recently shown to inhibit protein synthesis without being incorporated into proteins12'13. Our results also indicate that the most likely site of its action is at the initial stage of protein synthesis because in the presence of ACPC the incorporation (attachment) of 14C-valine into-HNA is reduced by 40-50 per cent. This work has been supported by grants received from the National Cancer Institute of Canada to two of us (L. B. and N. K. S.).