64 ° SHORT COMMUNICATIONS

benzpyrene, pyrene and phenanthrene. These were found, from the negative values of A e, to be oriented parallel to the bases of DNA. The second group contained 2o- methylcholanthrene, tetracene, pentacene and coronene; from the positive value of Ae, these were found to be oriented perpendicular to the bases of DNA. Pinacyanol, which has a positive value of A e in DNA solution, corresponds to the second group of aromatic hydrocarbons. However, it may be anticipated that the dipole moments which contribute to the dichroism of pinacyanol are less perpendicular to the bases of DNA than that for 2o-methylcholanthrene, because the values of Ae/e for 2o- methylcholanthrene and tetracene were 0.74 and 0.87, respectively, while the value for pinacyanol is much smaller.

A mutagenic effect of pinacyanol has recently been discovered in yeast6; in the presence of this dye, a respiratory-deficient mutant was produced as readily as with other acridine dyes. The s tudy of mutagenesis by pinacyanol is an at tractive problem because of the fact that it has been found to be not intercalated between bases of DNA.

Biophysics Division, M. KODAMA National Cancer Center Research Institute, Y. TAGASHIRA Tokyo (Japan) C. N_~.C~ATA

I L. S. I.ER~tAN, J. Mol. Biol., 3 (1961) 18. 2 L. S. LERMAN, Proc. Natl. Acad. Sci., 49 (1963) 94- 3 S. BRENNER, L. BARNETT, V. n . C. CRICK AND A. ORGIEL, J. Mol. Biol., 3 (1961) 121. 4 :\. ~vVADA AND S. KOZAWA, J. Polymer. Sci., Az (1964) 853. 5 C. NAGATA, M. KODAMA, Y. TAGASHIRA AND A. IMAMIJRA, Biopolymers, 4 (1966) 4o9 . 6 T. SUGIMURA AND K. OKABE, to be published.

Received March 4th, 1966 Revised manuscript received August 26th, 1966

Biochim. Biophys. Acta, 129 (1966) 638-64 °

BBA 93178

[14C] Leucine incorporation into the diabetic rat-adrenal protein: response to insulin

Stimulation of the incorporation of amino acids into protein in vitro by insulin has been shown in various tissues 1-4. The precise mechanism by which insulin influences the protein biosynthesis is not yet clear. However, this action of insulin seems to be independent of the effects of the hormone on the transport of glucose into the cells 5-6. In muscle, a st imulatory effect of insulin on RNA synthesis has also been shown 7-8. So far the study of the effects of insulin on protein synthesis in endocrines has a t t rac ted relatively less attention. BRANSOME AND REDDY 9 reported a stimulating effect of insulin on the incorporation of amino acids into normal rat-adrenal protein in vitro; though there was no significant effect on the incorporation of glycine carbon into adrenal RNA, the role of nucleic acid metabolism in insulin-enhanced amino

Biochim. Biophys. Acta, xz9 (1966) 64o-642

SHORT COMMUNICATIONS 641

acid incorporation into adrenal protein was not ruled out. In this communication, we report findings which show that the incorporation of amino acids into adrenal protein was sub-normal in the alloxan-diabetic rats and t reatment of the diabetic rats with insulin improved the adrenal protein synthesis to almost normal levels. Furthermore, insulin-mediated stimulation of amino acid incorporation into protein of normal rat adrenals was not affected by actinomycin D.

Male rats of Long-Evans strain weighing between 18o--22o g were starved for 18 h and injected intravenously with one shot of 45 mg purified alloxan (4.5 g °/o solution in sterile normal saline) per kg body weight. Only those rats were con- sidered diabetic which had a blood glucose value of more than 300 mg/Ioo ml, 4 weeks after the administration of alloxan. Immediately after sacrificing the rats by decapitation, adrenals were removed and freed of adhering materials. Adrenals were quartered and incubated for I h at 37 ° in 2 ml Krebs-Ringer bicarbonate buffer (pH 7.4) containing ~14C6]leucine and other additions as specified in Table I.

T A B L E I

L-I14C6!LEUCINE INCORPORATION INTO ADRENAL PROTEINS OF THE RAT

Incuba t i on was carr ied ou t for I h a t 37 ° in 2 ml of Krebs--Ringer b icarbonate buffer (pH 7.4), equi l ibra ted wi th 95 % Oz-5 % CO2, conta in ing 2 pC of [14Ct]leucine specific ac t iv i ty 222.8 mC/ mmole; 2 #mole unlabeled leucine and addi t ions as shown. Resul ts are expressed as mean ± S.E. The figures in paren theses indicate the number of animals.

Group Additions Counts/rain per mg protein

Normal (7) None 434 4- 29 Normal (5) Insul in (0.2 unit) 936 :t: I 18 Normal (4) Insul in (o.2 unit) + lO2O+ 193

ac t inomycin D (2o pg) Diabet ic (5) None 228:t: 24 Diabet ic (4) Insul in (o.2 unit) 36o4- 34 Diabetic, t r ea ted wi th None 453+ 8o

insulin (4)

Additions of insulin and actinomycin D were made in o.I ml normal saline. Stock solution of insulin was prepared by dissolving 5 mg insulin in a minimum volume of 0.003 M HC1 and 5 mg of actinomycin D was dissolved in I ml of ethylene glycol. These solutions were diluted to the required strength in normal saline. The reaction was stopped by the addition of an equal volume of chilled IO °/o trichloroacetic acid. Tissues were homogenized and washed with 5 ~o trichloroacetic acid containing un- labeled leucine. Protein was isolated, processed 1° and plated infinitely thin for counting in a gas-flow counter.

The results are presented in Table I. Addition of insulin to the incubating me- dium resulted in a significant stimulation of [~4C6]leucine incorporation into normal rat-adrenal protein. Similar findings have also been reported by BRANSOME AND REDDY 9. Our data show a depression in the protein synthesis of alloxan-diabetic rat adrenals and both short-term insulin t reatment in vivo (carried out for 3 days preceding the experiment by injecting 4 units of insulin/day per rat along with glucose feeding) and addition of insulin to the incubating mixture, restored the incorporation of [14Ce]leucine into the proteins of diabetic rats to the normal level.

Biochim. Biophys. Acta, 129 (I966) 64o-642

642 SHORT COMMUNICATIONS

These findings, therefore, show that protein synthesis in adrenals is influenced to a great extent by the availability of insulin. Addition of actinomycin D to the incubation mix ture did not affect the st imulatory effect of insulin on the incorpo- ration of [14CG]leucine into the proteins of normal rat-adrenal slices. Actinomycin D is known to inhibit DNA-dependent RNA synthesis n-~s. Thus the above findings suggest that the action of insulin on protein synthesis in adrenals is not dependent on RNA synthesis. However, a firm conclusion is not warranted at this stage as it has been shown, in certain tissues including adrenals, that RNA synthesis is not fully blocked by the presence of actinomycin D in the incubation medium ~4.

Department o/ Physiology, University o~ Cali/ornia, Berkeley, Cali/. (U.S.A.)

MAHENDRA KUMAR V. N. SINGH

I. L. CHAIKOFF*

I S. KIT AND D. M. GREENBURG, Cancer Res., 1I (1951 ) 495- 2 M. G. HERRERA AND A. E. I~ENOLD, Biochim. Biophys. Acla, 44 (196o) 167. 3 M. E. IX2RAHL, The Action o] Insul in on Cells, Academic Press, New York, 1961. 4 1. G. WOOL, in G. LITWACK AND D. I~RITCHEVSKY, Action o/Hormones on Molecular Processes,

Wiley, New York, 1964, p. 422. .5 K. L. MANCHESTER AND F. G. YOUNG, Biochem. J., 7 ° (1958) 353. 6 I. Cr. WOOL AND M. E. KRAHL, .dill. J. Physiol., 196 (1959) 961. 7 I. G. WOOL, Am. J. Physiol., 199 (196o) 719 . 8 1. G. WOOL, Biochim. Biophys. Acta, 68 (1963) 28. 9 E. D. BRANSOME JR. AND W. J. REDDY, Biochim. Biophys. Acta, 76 (1963) 641.

IO I. (~. WOOL AND A. N. MOYER, Biochim. Biophys. Acta, 91 (1964) 248. I~ J. HURWlTZ, J. J. FURTH, M. MALAMY AND M. ALEXANDER, Proc. Natl. Acad. Sci., U.S., 48

(19(~2) 1222. 12 1. It. GOLDBERG AND M. I~ABINOWITZ, Science, 136 (1962) 315 . 13 E. REICH, I. H. GOLDBERG AND M. RABINOWlTZ, Nature, 196 (1962) 743. 14 J. j . FERGUSON AND Y. MORITA, Bioehim. Biophys. Acta, 87 (1964) 348.

Received July ISth, 1966

* Deceased J a n u a r y 25, 1966.

]3ioehim. Biophys. Acta, 129 (1966) 64o-642

BBA 93170

Inhibition of protein synthesis in reticulocytes by ontibiotics III. Mechanism of oction of sporsomycin

Sparsomycin, an antibiotic isolated from Streptomyces sparsogens 1, has been reported to inhibit protein synthesis in epidermoid carcinoma cells 1 and in a cell-free system prepared from rabbit reticulocytes 2. Sparsomycin does not inhibit the incorporation of amino acids into protein in intact reticulocytes 2, whereas it inhibits protein synthesis by 50 % in KB human epidermoid carcinoma cells in vilro at a concentration of 0.05/tg/ml (I.D.50) (ref. I). The I.D.s0 in the reticulocyte cell-free system was o.I/~g/ml (ref. 2).

The action of sparsomycin was preliminarily examined in the cell-free system 2, where it was found that, in its presence, tile polysomes of reticulocvtes do not

!~ioe:d~J~. 13iophys. ,4e/a, z29 ( I 9 6 6 ) 6 4 2 645