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Transcript
164 BIOCHEMICAL SOCIETY TRANSACTIONS The sequences proposed in Fig. 1 for the Ps. aerirgirioJaand Ps.strrtzeri proteins have been confirmed by the isolation and characterization of the expected peptides from tryptic digests of the haeni-free proteins. From each of these two proteins a second tryptic peptide was isolated that contained two residues of cysteine and a residue of histidine. The amino acid composition was such that the peptide could not have been derived from the region shown in Fig. 1. The N-terminal heptapeptide sequence of the cytochromes c4 is very similar to the N-terminus of Rhodospirillirm rubrum cytochrome c2 (Dus et a/., 1968). A similar N terminal sequence, Tyr-A?p-Ala-Ala-Ala-Gly-Lys-, has also been detected in another monohaem cytochrome c from a photosynthetic bacterium, the cytochrome 12-555 of Chlorobium thiostilfatophilurn (Gibson, 1961 ; R. P. Ambler & T. Meyer, unpublished work). The very close similarity between the cytochromes c4 of A. vinelandii and the pseudonomads was unexpected. The organisms are very different in metabolism and in subcellular structure, and in the current classification (Breed et al., 1957) are placed in different orders. However, they all have G I C contents of their DNA in the range 56-66% (Hill, 1966). The deiiitrifying pseudomonads contain a wide range of haem-c-containing proteins. In addition to cytochromes c-551, c4 and c5 they contain cytochrome c peroxidase (Kodama & Mori, 1969; Ellfolk & Soininen, 1970) and cytochrome c oxidase (Horio et al., 1961). The interrelation of these components is still obscure (Horio & Kamen, 1970). We thank Dr. T. Kodama for the gift of Ps. stutzeri cytochrome c4 [‘cytochrome c-552 (II)’] and Dr. T. Meyer for the Rsp. rubrum cytochrome c2. The sequencer was purchased by the Medical Research Council, who support this part of the work. Breed, R. S., Murray, E. G. D. &Smith, N. R. (1957) Bergey’J Mar7rtalofDeterrizinarit.e Bacteriology, 7th edn., p p . 89, 281, Livingston, Edinburgh Dent, C. E. (1947) Biochem. J. 41,240-253 Dus, K., Sletten, K. & Kamen, M. D. (1968) J. Bid. Chern. 243, 5507-5518 Edman, P. & Begg, G. (1967) Eur. J. Biochem. 1, 80-91 Ellfolk, N. & Soininen, R. (1970) Acta Chem. Scand. 24, 2126-2136 Gibson, J. (1961) Biochetti. J. 79, 151-158 Hill, L. R. (1966) J. Gen. Microbiol. 44,419-437 Horio, T. & Kamen, M. D. (1970) Annu. Reu. Microbiol. 24,399-428 Horio, T., Higashi, T., Yamanaka, T., Matsubara, H. &Okunuki, J. (1961)J. Bid. Chem. 236, 944-951 Kodarna, T. & Mori, T. (1969) J. Biochem. (Tokyo) 65, 621-628 Kodama, T. & Shidara, S. (1969) J . Biochem. (Tokyo) 65, 351-360 Neumann, N. P. & Burris, R. H. (1959) J . Biol. Chenz. 234, 3286-3290 Swank, R. T. & Burris, R. H. (1969) Biochim. Biophys. Acta 180, 473-489 Tissieres, A. (1956) Biorheni. J . 64, 582-589 The Amino Acid Sequence of Chlorella fusca Plastocyanin JANICE KELLY and R. P. AMBLER Department of Molecirlur B;olog3>,Unii7ersityof Editibnrgh, Edinbrrrgh EH 9 3JR, U.K . Plastocyanin is a blue copper protein of low molecular weight. It was first found by Katoh (1960) in Chlorella ellipsoida, and has since been shown to be present in the chloroplasts of green algae and higher plants, where it functions in photosynthetic electron transport (see, e.g., Arnon e t a / . , 1970). For the present study the protein was isolated from spray-dried cells of Chlorellaji~sca, by a development of the method of Gorman & Levine (1966), in yields of about 0.7mg of pure protein/g of dry cells. The amino acid sequence of the protein was investigated by 1973 531 st MEETING, LANCASTER 108 109 110 1 1 1 Azurins 165 112 W- 80 81 132 114 115 116 117 - ~ ~ ~ - P h e - P r ~ - G l y -.H.(1.7-14 i-. r e n i d n e c ) CO2Ii -Tgr-XY:.-F1le-Php-Cy, 79 113 ~ 85 84 83 C.firrcnplastocyanin -Tyr-Gly-Tyr-PI~?-Cys - all1 - i'rn 86 - I{<?. . .(12 rPzid~ier) -C@*H Fig. 2. Amino acidsequences around the single thiolgroups of azurins (Ambler, 1971) and C. jirsca plastocyariiii XXXrepresents a site that is filled by five different amino acids in the nine azurins studied. isolation and characterization of the peptides obtained from chymotrypsin and thermolysin digests of the protein. Since the protein contains no arginine and little lysine (4 residues) and has many bonds sensitive to cleavage by pseudotrypsin (Keil-Dlouha et a/., 1971), tryptic digestion has proved to be of little use. The N-terminal sequence of the molecule was checked by using an automatic Sequenator (Beckman model 890; Edman & Begg, 1967); results were obtained as far as residue 36, and were in complete agreement with the sequence deduced by conventional methods. The structure deduced was a single polypeptide chain of 98 residues (Fig. 1). Preliminary analyses indicate the presence of small amounts of carbohydrate, and electrophoretic mobilities suggest that a labile group may be associated with residue 9 or 10. Incomplete experiments with plastocyanin from spinach (Brussica oleracea), C/ilorella pyrenoidosa and Scenedesmus obliquus have shown very considerable similarities in sequence. The sequence of the plastocyanin from French bean (Pliaseohrs vulgaris) is currently under investigation by Milne & Wells (1970). Katoh et al. (1961) suggested that the thiol groups of plastocyanin contributed to the binding of the copper. The proposed sequence contains only a single cysteine residue (position 83), clustered around by several aromatic residues. In the bacterial azurins, which are copper proteins that are believed to be of structure and function comparable with those of plastocyanin (Malkin & Malmstrom, 1970), there is a similar concentration of aromatic residues in the vicinity of the single thiol group (Fig. 2) (Ambler & Brown, 1967; Ambler, 1971). The sequence similarities are not sufficient to provide convincing evidence for homology between the two classes of proteins, but are striking enough to suggest at least a functional similarity. In both cases the residues concerned are close to the C-terminus of the molecule. Further studies [e.g. of the plastocyanin reported to be present in the blue-green alga Anahaena variahilis (Lightbody & Krognian, 1967)] may clarify the relationship. We are very grateful to Dr. B. Prokes and the staff of the Department of Applied Algology, Trebon, Czechoslovakia, for their supply of the algal cells and for their interest in the work. Vol. 1 166 BTOCH F M TCAL SOCIETY TR ANSACTJONS Ambler, R . P. ( I 971) in Rrccrit Dere/optncnfs in thc Clreiiiic~ulStudy of Proteiir Structures (Previcro, A,, Pechcre, J.-I-'. & Coletti-Previero,M.-A., eds.), pp. 289-305, Inserm, Paris Ambler, R. P. & Brown, L. H. (1967) Biochctn. J. 104, 784-825 Arnon, D. I., Chain, R. K., McSwain, B. D., Tsujimoto, H. Y . & Knaff, D. B. (1970) Proc. Not. Acud. Sci. U.S. 67, 1404-1409 Edrnan, P. & Begg, G. (1967) Errr. J . Biocheni. 1, 80-91 Gorman, D. S. & Levine, R. P. (1966) Plant Plrysiol. 41, 1637-1642 Katoh, S. (1960) Nature (London) 186, 533-534 kdtoh, S.,Suga, I.,Shiratori, I. &Takaniiya,A.(1961) Arch. Bioclienr. Biophys. 94,136-141 Keil-Dlouha,V., Zylber, N. & Keil, B. (1971) in Recent Decelopnients in the Chemical Study of Protein Structures (Previero, A,, Pechere, J.-F. & Coletti-Previero,M.-A., eds.), pp. 133-144, Inserm Paris Lightbody, J. J. & Krogman, D. W. (1967) Biochim. Biophys. Actu 131, 508-515 Malkin, R. & Malmstrom, B. G. ( I 970) Adcan. Enzyinol. Relat. Areas Mo/.Bid. 33,177-244 Milne, P. R. &Wells, J. R. E. (1970)J. Biol. Cheni. 245, 1566-IS74 Amino Acid Sequence of Cytochrome c5 from Pseudomonas mendocina R. P. AMBLER and ELIZABETH TAYLOR Department of Molecular Biology, University of Ediiibiirgh, Edinbnrgh EH9 3JK, U.K . Many bacteria contain several soluble c-type cytochromes. Both pseudomonads (Horio, 1958) and Azotobacter (cytochrome cs; Tissieres, 1956) contain proteins with a-band maxima at about 555nm. In studies of other cytochromes from pseudomonads, we have frequently met with cytochromes of the cs type, but chroniatographic properties and yields have been very erratic. All purifications used have involved exposure to low pH values (pH4-5; Ambler, 1963a), and Swank & Burris (1969) have shown instability of Azotobacter cytochrome cs at acid pH values. However, it has been found possible to isolate an adequately homogeneous cytochrome of this type from Pseudomonas mendocina CH-110 (Palleroni et a/., 1970), in good yield. The growth of the organism and the general procedures for the isolation of the protein were similar to those that have been described for Pseudomonas cytochrome c-551 (Ambler, 1963~).The major cytochrome present (c-551) was eluted from CM-cellulose at pH4.45, and a mixture of the cytochrome c5 and cytochronie c4 (Ambler & Murray, 1973) was eluted at pH4.75. The latter components were then separated by chromatography on DEAE-cellulose (at p H 8 . 5 in 12m~-tris-HCl,with an NaCl gradient; cytochrome cs was eluted at about 32ni~-NaCl,ahead ofcytochrome c4), and further purified by (NH&SO, precipitation (7G95 % saturation) and gel filtration through Sephadex G-75 (superfine grade; in 50m~-ammoniumacetate, pH5.1). The yield of cytochrome cs was about 2.2pmo1/100g of acetone-dried cells (compared with about 8pmol of cytochrome c-551 and 0.12pmol of cytochrome c4). This cytochrome cs was homogeneous by gel electrophoresis, and had a haerii content (from visible spectrum) of about I residue/10000 daltons of amino acids. The behaviour of the protein on Sephadex (3-75 was indistinguishable from that of the 9000-dalton cytochronie c-551. Amino acid analysis showed the complete absence of tyrosine and phenylalaiiine (<0.03 renidue/haem group), and a valine content of 1.1 residue/haem group. Nevertheless N-terminal group analysis by the dansyl method showed both alanine and serine. After removal of the haem (Hg2Cl2-0.l M-HC1-8M-urea, 3 7 T , 24h), the apoprotein was digested with trypsin or chymotrypsin, and the peptides formed were isolated and characterized by standard methods (Ambler & Brown, 1967; Gray, 1972). The peptides could all be fitted together to form the unique sequence shown in Fig. 1 , which agrees very well with the amino acid analysis results for the whole protein, and with the results of C-terminal analysis with carboxypeptidase A. However, at least four different tryptic peptides were derived from the N-terminal region of the molecule, and all the available 1973