30P PROCEEDINGS OF THE BIOCHEMICAL SOCIETY Possible Active Sites in and heptyl-4-hydroxyquinoline N-oxide (400,ug/mg of Hydroxylamine Reductases from Vegetable protein) did not inhibit nitrite reductase or either of Marrow (Cucurbita pepo L.) the hydroxylamine reductases appreciably. did and D. M. Phenylhydrazine and semicarbazide (10mM) By D. P. HUCTLESBY,* E. J. HEwniTr not inhibit or hydroxylamine JA1Es. (Universrity of Bri8tol Research Station, Long reductase 1, but semicarbazide, hydrazine and A8hton, Bri8tol BS18 9AF, U.K.) isonicotinic acid hydrazide (5-50mM) inhibited In plants, nitrite reductase behaves as a single hydroxylamine reductase 2 by 20-36%. A flavin protein producing NH3 without involving free component in nitrite reductase (Huzisige, Satch, hydroxylamine and is dependent on one-electron Tanaka & Hyashida, 1963) was not confirmed by donors: reduced benzyl viologen or ferredoxin (see precipitation with acid (NH4)2SO4 Weak unpredict- Hewitt, Hucklesby & Betts, 1968). A closely able nitrite reductase activity may occur with associated hydroxylamine reductase (hydroxylamine FMNH2, and hydroxylamine reductase 1 is inhibited reductase 1) utilizes additionally FMNH2, FADH2 by 25-100% by 10mM-atabrine with FMNH2 as (Hewitt & Hucklesby, 1966; Hewitt et al. 1968) and electron donor. NADPH2. Another hydroxylamine reductase (hy- Both nitrite reductase-hydroxylamine reductase droxylamine reductase 2) functions at present 1 and hydroxylamine reductase 2 may be iron- only with reduced benzyl viologen or dithionite. In containing proteins with possibly haem and carbonyl fungi (see Nicholas, 1959) containing groups in the latter. iron plus copper in nitrite reductase and manganese in hydroxylamine reductase occur. In soya bean Cresswell, C. F., Hageman, R. H., Hewitt, E. J. & nitrite reductase and hydroxylamine reductase were Hucklesby, D. P. (1965). Biochem. J. 94, 40. thought to be flavoproteins activated by manganese Hewitt, E. J. & Hucklesby, D. P. (1966). Biochem. biophy8. Re8. Commun. 25, 689. (Nason, Abraham & Averbach, 1954). Experiments Hewitt, E. J., Hucklesby, D. P. & Betts, G. F. (1968). with azide, 1,1'-bipyridyl, o-phenanthroline, 8- In Recent Aspects of Nitrogen Metabolism in Plant8, hydroxyquinoline, diethyldithiocarbamate or EDTA p. 47. Ed. by Hewitt, E. J. & Cutting, C. V. London did not indicate metals in nitrite reductase or hy- and New York: Academic Press. droxylamine reductase from marrow (Cresswell, Huzisige, H., Satoh, K., Tanaka, K. & Hyashida, T. Hageman, Hewitt & Hucklesby, 1965), but cyanide (1963). PI. Cell Phy8iol., Tokyo, 4, 307. (pH7.5) inhibited nitrite reductase by 85% at 0.1mM Nason, A., Abraham, R. G. & Averbach, B. D. (1954). and hydroxylamine reductase by 55% at 0.2mM, Biochim. biophy8. Acta, 15,159. with reduced benzyl viologen as electron donor. Nicholas, D. J. D. (1959). Symp. Soc. exp. Biol. 13, 1. CO inhibits nitrite reductase and hydroxylamine reductase 1 (reduced benzyl viologen as electron donor) irreversibly in light. Inhibition of hydroxyl- Bovine Brain Creatine Phosphotransferase: amine reductase 2 by CO is reversed by light. Amino Acid Sequence around the Essential Activity of hydroxylamine reductase 2 is related to Thiol Groups light-absorption at 399nm. This peak shifts to 415 or 433nm on reduction by reduced benzyl viologen By R. S. ATHERTON, J. F. LAWS, B. J. Mms and or dithionite respectively and returns to 399nm on A. R. THOMSON. (Chemical Engineering Dsvijson, autoxidation. Pyridine, dithionite and 0.1 M-NaOH Atomic Energy Research E8tabli8hment, Harwell, induce peaks at 419, 524 and 557nm. CO with Didoot, Berk8., U.K.) dithionite produces a sharper peak at 420nm. Comparable nitrite reductase preparations do not The guanidino phosphotransferases form a closely show these peaks. related group of of similar properties and The iron chelator bathophenanthroline (4,7- function. When creatine phosphotransferases from diphenyl-1,10-phenanthroline) disulphonate inhibits muscle and brain tissues of the same species are nitrite reductase by 50% at 0.1mM and hydroxyl- compared significant differences in their Km values, amine reductase 2 by 50% at 1mM, with reduced stability, response to alkylation, amino acid composi- benzyl viologen as electron donor and 0.3mM sub- tion and peptide 'maps' are observed (Eppenberger, strate. Iron reverses the inhibition of nitrite re- Dawson & Kaplan, 1967; Dawson, Eppenberger ductase. & Kaplan, 1967; Thomson, Eveleigh, Laws & The copper chelators 2,2'-biquinolyl, 2,9-dimethyl- Miles, 1968; Atherton & Thomson, 1969). However, 1,10 - phenanthroline, 2,9 - dimethyl - 4,7 - diphenyl - there is good evidence that in the region of their 1,10-phenanthroline and biscyclohexanoneoxalyl essential thiol groups the amino acid sequences are dihydrazone at saturation and antimycin A and 2-n- similar (Thomson et al. 1968). This has been in- * Present address: Department of Agronomy, Univer- vestigated further. sity of Illinois, Urbana, Ill. 61801, U.S.A. Pure bovine brain creatine phosphotransferase was PROCEEDINGS OF THE BIOCHEMICAL SOCIETY 31P labelled with iodo[2-14Clacetate and digested with Study by Mass Spectrometry of Amino Acid trypsin (Thomson, Eveleigh & Miles, 1964). The Sequences in Peptides containing Histidine resulting peptides were purified by ion-exchange and gel-filtration chromatography and by high-voltage By J. F. G. VLIEGENTEART and L. DORLAND. electrophoresis. Chymotryptic digests of the major (Laboratory of Organic Chemistry, University of radioactive tryptic peptides were similarly treated. Utrecht, Utrecht, The Netherland8) Theamino acidsequencesofpeptidesweredetermined by the 'dansyl'-Edman method (Gray, 1967). The choice of a method for the preparation of From the sequences obtained, together with amino peptide derivatives suitable for mass spectrometry acid analysis data, it was deduced that the amino depends to a certain extent on the composition ofthe acid sequence around the reactive thiol groups of peptides. Itisnowcommon practice, besidesblocking bovine brain creatine phosphotransferase is: ofthe functional groups, to permethylate the peptide Asx-Tyr-Glx-Phe-Met(Glx,Asx,His,Leu,Gly,Tyr)Ile- bonds. The permethylation of peptides containing -Gly-Thr-Gly-Leu- histidine, however, gives rise to some problems. Leu-Thr-CMC-Pro-Ser-Asx-Leu Our procedure is the following. We prepare a Arg protected peptide by dissolution of 0.01 mmol of a where CMC represents S-carboxymethylcysteine. peptide in 2ml of water and subsequently add The sequence fromisoleucine to arginine isidentical 0.01 mmol ofdiethylpyrocarbonate/acylatable group. with that in the enzymes from rabbit and bovine The pH is kept constant at 8.0 with a pH-stat for muscle (Thomson et al. 1964, 1968) except that 30min. After acidification with 1 M-HCI the ethoxy- isoleucine substitutes for valine. Arginine phospho- carbonylated component is extracted with ethyl from lobster has a very similar sequence acetate (Kamerling, Heerma & Vliegenthart, 1968). (Thoai, 1968). Further, the sequence from phenyl- Besides the amino group, the phenolic hydroxyl alanineto arginine (exceptforisoleucine) islikelytobe group of tyrosine, the thiol group of cysteine and the identical with the corresponding sequence in muscle N-imidazole group of histidine are also ethoxy- creatine phosphotransferases. The yields of labelled carbonylated. Thepeptidederivative issubsequently peptides together with kinetic evidence suggests that permethylated with methyl iodide and methylsulph- the chemical environments of the two essential inyl carbanion as base in dimethyl sulphoxide thiol groups of the brain are identical. (Vilkas & Lederer, 1968). The application of this It is confirmed that, despite differences in primary method to histidine-containing peptides leads to the structure in other parts ofthe molecule, the sequence formation of a quaternary base, which remains in the around the reactive thiol groups of bovine brain water phase during the extraction with chloroform. creatine phosphotransferase is virtually identical We were able to circumvent this problem by cleavage with that of muscle enzyme. This part of the of the imidazole ring of histidine with an excess of sequence of guanidino phosphotransferases has been diethyl pyrocarbonate. After the reaction of the substantially conserved throughout their evolution. peptide with the initial amount of diethyl pyro- These enzymes should thus prove invaluable in un- carbonate an excess of 4mol of reagent/mol of ravelling evolutionary pathways. histidine residue is added in four 1 mol portions at at constant Mr Milsom 30min intervals pH 8.0. This operation S. rendered skilled technical assistance. splits off the C-2 atom of the imidazole ring as Atherton, R. S. & Thomson, A. R. (1969). Biochem. J. formate, and converts the remaining part of the ring 111, 797. into the 1,2-bis(ethoxycarbonylamino)ethylene Dawson, D. M., Eppenberger, H. M. & Kaplan, N. 0. group. Afteracidificationthe reaction can be (1967). J. biol. Chem. 242, 210. isolated by extraction with ethyl acetate and sub- Eppenberger, H. M., Dawson, D. M. & Kaplan, N. 0. sequently be The (1967). J. biol. Chem. 242, 204. permethylated. product is now Gray, W. R. (1967). In Method8 of Enzymology, vol. 11, soluble in chloroform and suitable for mass spectro- p. 139. Ed. by Hirs, C. H. W. New York: Academic metry. Press Inc. Histidine itself can also be cleaved by exhaustive Thoai, N. V. (1969). Abstr. FEBS 6th Meet., Madrid, treatment with diethyl pyrocarbonate at pH 8.0, no. 66. yielding 2,4,5 - tris(ethoxycarbonylamino)pent - 4 - Thomson, A. R., Eveleigh, J. W., Laws, J. F. & Miles, enoic acid. This product contains a small percentage B. J. (1968). In Biochemical Evolution, and Homologous of bis(ethoxycarbonyl)histidine. The cleavage re- Enzymes, p. 255. Ed. by Thoai, N. V. & Roche, J. action is comparable with cleavage of the imidazole New York: Gordon and Breach. in ester with Thomson, A. R., Eveleigh, J. W. & Miles, B. J. (1964). ring histidine methyl benzoyl chloride Nature, Lond., 203, 267. under Schotten-Baumann conditions (Ashley & Harington, 1930). After permethylation of 2,4,5- tris(ethoxycarbonylamino)pent-4-enoic acid, the mass spectrum showed the presence of exclusively