Cytochrome System in Oyster Tissues

1468 May 24, 1958 NATUR E whether or not the enzymes investigated by Slein and by us are identical. This work was supported by a. research grant from the Deutsche Forschungsge meinschaft. FRIEDRICH H. BRUNS ERNST N OLTMANN Institut fiir Physiologisch e Chemie der Medizinische n Akademie Duesseldorf; and Division of Experimenta l Radiology, University of Rochester School of Medicine and Dentistry, Rochester, N.Y. March 11. 1 Roe, J. H., J. Biol. 0/i,,m., 107, 15 (1934) . 'Bruns, F. H., and Vahlhaus, E., NoJ,urwi.,,., 43, 180 (1956). 3 Kornberg, A., J . B iol . CMm., 182, 805 (1950). • Lehnlnger, A. L., Ph118iol. Rel,. , 30, 393 (1950). • Malmstrom, B. G. , Dissertation, Upsala , Sweden (1966). • Vallee, B. L., "Adv. Prot. Chem.", 10,317 (1955). 'Bruns, F . H ., and Okada, S. , NoJ,ure, 177, 87 (1956). • Topper, Y . J., and Stetten, jun. , DeW., J . Biol. Chem. , 189, 191 (1951). Topper, Y. J. , ibid., 226, 419 (1957). • Sleln, M. W . , J . Biol. Chem., 188, 753 (1950). In Colowlck, S. P., and Kaplan , N . 0 ., " Methods In Enzymology", 1, 299 (Acad. Press, Inc., New York, 1955). voL. ,a1 Table 1 Oxygen uptake (µ!.)• 90 per cent nltro- 90 per cent carbon gen + 10 per cent monoxide + 10 per cent oxygen oxygent Tissue Time (min.) Mantle slices, l00mgm. wet weight 0--40 40--80 80-120 120-160 31 29 28 26 11, dark 26, light 14, dark 26, light 0--50 50-100 84 31 18, dark; 34,llght 80,light ; 17, dark Gill pieces, 60mgm. • The oxygen uptake was measured in Warburg manometers, with veaaele about 9 ml. capacity, at 25° C. The vessels contained 1 ·6 ml. of 0·0S M glycine or glycylglyclne-bul fered sea water (pH 8·0) and 60 or 100 mgm. of tl88ue pieces. t There was no dl.ft'erence between oxygen uptake In this mixture and in air. of cytochrome c was detected in any tissue examined, or of cytochrome a in certain tissues of the oyster, may be attributed to the low concentration . Details of this work will be published elsewhere. Kzyozo KAWAI Department of Zoology, University of Kyoto. March 13. Humphrey, G. F . , J. Bzp. Biol., IM, 862 (1947). • Jodrey, L. H., and Wilbur, K. M. , Biol. Bull. , 108, 346 (1955} . • Keilin, D., and Hartree, E. F . , Nol,ure, 184, 254 (1949). 1 Cytochrom e System in Oyster Tissues some experiments with tissue extra.eta of the oyster have proved the presence of an oxidase very similar to the cytochrome oxidase of higher animals 1 , 1 , it is still obscure whether the cytochrome system functions as terminal oxidase in the intact tissues of oysters, because even cytochrome has not been observed spectroscopic ally. Recently, Jodrey and Wilbur• suggested that the cytochrome system may not play a major part in the oxidative metabolism of the oyster mantle (Grasaostrea virginica), since methylene blue (l ·4 x 10- 6 M) did not reverse the cyanide inhibition of the tissue. In the present investigation , absorption bands of the cytochromes were observed with the tissue homogenate of the oyster (Graaaoatrea gigaa), when reduced with hydrosulphite , by applying the liquid-air method of K eilin and Hartree 3 • The absorption bands, however, were very faint compared with those of baker's yeast. The band correspondin g to cytochrome b (560-565 mµ. at room temp., 560 mµ. at liquid-air temp.) was det£cted in all tissues examined, namely, gill, mantle, adductor muscle and digestive diverticula.. However, the band a (around 600 mµ) was observed only in the gill and the mantle, and no trace of cytochrome c was detected. A very strong pyridine hremochromo gen band (550-560 mµ) was readily obtained with all tissues. In a. gas mixture of 90 per cent nitrogen and 10 per cent oxygen, the respiration of both the Illfill.tle and the gill was inhibited by a.bout 50 per cent in the dark, and this inhibition was reversed almost completely by illumination with a 500-wa.tt lamp (Table 1). The respiration of both the mantle and the gill was also reduced by 0·001 M cyanide to a.bout 18 per cent of the control value. In the presence of a nearly saturated solution of methylene blue in sea. water (6 x 10-a M), the inhibition was reversed to about 40 per cent ; it was confirmed that a lower concentration of methylene blue (1 x 10- 6 M) did not reverse the cyanide inhibition. From these experiments it is evident that the respiration of the oyster tissues is largely dependent on metabolic pathways involving the cytochromeoytochrome oxidase system. The fact that no band ALTHOUGH Structure of Sperm- and Sei-Whale Insulins and their Breakdown by Whale Pepsin As described in previous papers1 , our experimental results, using the method of Moore and Stein•, have shown that the amino-acid composition of whale (sei -) insulin differs from that reported by Sanger et al.•. Furthermore , we have recently re-examined the amino-acid analyses of sperm- and sei-whale insulins (whole insulin and both glycyl (A) and phenylalanyl (B) cha.ins of oxidized insulin, respectively). These experiments have confirmed that the amino-acid content of sperm insulin is similar to that obtained by Sanger et al. ; but sei-wha.le insulin contains an ala.nine instead of an iaoleucine residue in the A chain. The amino-acid sequences in both sperm- and seiwhale insulins were then compared with the sequence reported by Sanger et al. The B cha.ins were found to have the same sequence as Sanger's, but there were species differences in positions 8-10 of the A cha.in : in sperm insulin, Thr.Ser.Ileu (identical with Sanger), and in sei-whale insulin, Ala.Ser.Thr. Crystalline sperm- and sei-whale insulins', and crystalline sei-whale pepsin6 , which we have previously prepared and described, were recrystallized 4 and 7 times respectively. Crystalline swine pepsin was obtained from the Worthington Chemical Laboratory and recrystallized four times. Pepsin was used in a glycerol solution containing 10 mgm. per ml. The oxidation and fractionation of insulin, and the partial hydrolyses of each fraction with both pepsins or hydrochloric acid were carried out under the conditions described by Sanger et al. 1 • The hydrolyaates were fractionated by paper ionophoresis at pH 3·2 or 6 ·5 (pyridine-ace tate buffer), and pa.per chromatography with phenol/ammo nia and also n-butanol/ace tic acid solvent mixtures. The peptides were eluted and the eluates hydrolysed with 6 N hydrochloric acid and the constituent amino-acids identified by paper chromatogra phy. From a comparison between the amino-acid composition of these peptides and the known sequence of amino-acids in the A and B cha.ins of beef insulin, it was possible to assign positions to © 1958 Nature Publishing Group  (...truncated)