Clupeine amino acid sequence

AOT/isooctane Protamines from sperm head nucleoproteins of fish Conformational studies on model polypeptides with amino acid sequences similar to clupeine and salmine were carried out in RMs [286]... 

The Tokyo group, making suitable use of these results, overcame the difficulties which had hampered structural studies of protamines and was able to obtain homogeneous components of clupeine and determine their complete amino-acid sequences (Ando et al. 1962 Ando and Suzuki, 1966, 1967 Ando et al.y 1967). Thus this question was finally settled about 100 years after the term protamine was coined by Friedrich Miescher. Our studies have established a general means of determining the primary structure of each component of a protamine. 

In spite of some differences in the species of fish used and the methods employed, the fractions designated i, ii, and iii in Table VII-1 correspond to one another as regards their amino-acid content. However, there are differences in the N-terminal residues. Fraction iii, which has the simplest amino acid composition with N-terminal alanine, was the first to be purified and have its complete amino-acid sequence elucidated (Ando et aL, 1962). New methods for the fractionation of clupeine have recently been developed and are described below (this chapter. Section B). 

The complete amino-acid sequence of clupeine Z was determined by the Tokyo group (Ando et aLy 1962 Ando, 1964 Azegami et al,y 1970 Iwai et aLy 1971). 

Determination of the Complete Amino-Acid Sequence of Clupeine 2... 

Employing a procedure essentially similar to that used for the structural study of unfractionated clupeine (Ando et al. 1959 Ishii et aL 1967), 16 peptides and arginine were identified and determined quantitatively in a tryptic digest of clupeine Z as shown in Fig. VIII-1. Analyses of these tryptic peptides are shown in Table VIII-2. The kinds and amounts of partial amino-acid sequences present in clupeine Z are summarized in Table VIII-3. 

Table VIII-3. Partial amino-acid sequences found in clupeine 2 (10.1 //moles)...

The sequence of fragments I—IV in a clupeine Z chain can be inferred as indicated in the last column of Table VIII-4, by considering the N-(Ala) and C-(Arg) terminals of the protamine together with the amino-acid sequences of tryptic peptides, especially the hexapeptide (H-Ala Ser-Arg Pro-Val Arg-OH). Thus the complete amino-acid sequence of clupeine Z was worked out as shown in Chart VIII-2. 

Chart VIII-2. The complete amino-acid sequence of clupeine Z (A = Arg). Modes of digestion by leucine aminopeptidase (LAPase) and carboxypeptidases (CPases) B and A are... 

The clupeine Z molecule thus proved to have a peculiar amino-acid sequence which would not have been expected from the simple repeating structures hitherto... 

The amino-acid sequence of clupeine YII was determined in 1966 [Ando and Suzuki, 1966 Suzuki and Ando, 1972 (1)]. 

By the DNP and PTC (phenylthiocarbamyl) methods, the N-terminal amino acid was determined to be proline and the N-terminal amino acid sequence to be H-Pro Arg Arg . The alternate action of carboxypeptidases B and A on clupeine YII followed by hydrazinolysis indicated that the amino-acid sequence at the C-terminal region was Pro Arg2 (Val, Ser) Arg4 Ala Arg4-OH. 

Chart VTTT-4 sets out the complete amino-acid sequence of clupeine YII as concluded from these results, combined with the information derived from the tryptic peptides. 

The existence of two sets of overlapping peptides resulting from digestion with trypsin and thermolysin, combined with the results of the action of carboxypeptidases B and A used alternately, made it possible to deduce the total amino-acid sequence of clupeine YI as shown in Chart VIII-5. 

Neither the exhaustive studies of the Tokyo group (1964 and 1969) as described in the next section (Chap. VIII. E) nor the partial study by Felix and Hashimoto (1963) have revealed any differences in the amino-acid sequences of the corresponding components of clupeine from Norwegian Sea and North Sea (Clupea harengus) and Pacific (Clupea pallasii) herring. 

E. The Amino-Acid Sequences of the Three Components (Y l, Y ll and Z ) of Clupeine from North Sea Herring... 

However, all these results were obtained by analysis of unfractionated clupeine. The complete amino-acid sequences recently determined for the three components of clupeine from Pacific herring (Clupea pallasii) (Ando et al,y 1962 Ando and Suzuki, 1966, 1967) confirmed the presence of an amino-acid residue, glycine, as a constituent and of the N-terminal alanine in addition to the N-terminal proline in some of the clupeine molecules. At that time no complete fractionation of clupeine from Clupea harengus into a number of homogeneous components had been accomplished. 

More recently the Japanese group (Chang, 1969 Chang, Nakahara and Ando, to be published) fractionated clupeine from North Sea herring into the components clupeine YT, YTI and Z, by the method described in Chap. VII. B. 4 for the fractionation of clupeine from Pacific herring (Ando and Watanabe, 1969). The complete amino-acid sequences of the components Y l, Y ll and Z were obtained from the results of N- and C-terminal sequence analysis and analysis of the thermolytic peptides, and from data on the structure of the tryptic peptides of unfractionated clupeine from Clupea harengus (see p. 37 ref. 7). They are illustrated in Chart VIII-8. 

Chart VIII-8. The complete amino-acid sequences deduced for clupeine Y l, Yll and Z with illustration of several degradations. (Structures of tryptic peptides obtained from whole clupeine from Clupea harengus were also referred to for this deduction. See text) (A = Arg) (Chang, 1969 Chang, Nakahara, and Ando, to be published)... 

Ando, T., Suzuki, Ko. The amino acid sequence of the second component of clupeine. 

Iwai, K., Nakahara, C., Ando, T. Studies on protamine. XV. The complete amino acid sequence of the Z component of clupeine. Application of N -> O acyl rearrangement and selective hydrolysis in sequence determination. J. Biochem. (Tokyo) 69, 493—509 (1971). Jaques, L. B. a study of the toxicity of the protamine, salmine. Brit. J. Pharmacol. 4, 135—144 (1949). 

Suzuki, Ko., Ando, T. (2) Studies on protamines. XVII. The complete amino acid sequence of clupeine YI. J. Biochem. (Tokyo) 72, 1433—1446 (1972). 

On the basis of his experimental results, Felix postulated that all components of clupeine (/.., several molecular species present as a mixture in a heterogeneous protamine like clupeine see Chap. VII) have the same N-terminal sequence of 6—8 amino-acid residues and that the differences among the components must occur along the chains towards the C-terminals. He also stated that tri- or tetrapeptide sequences of monoamino acids may occur in a chain of components. Such a chain should contain a hexa- or octapeptide of arginine in order to maintain the ratio of arginine residues to monoamino acid residues at 2 1. 

The sequence of sixteen amino-acid residues in the C-terminal region of TNP-clupeine YI forms more than half of the total chain this was determined by the alternate action of carboxypeptidases B and A, followed finally by hydrazinolysis. 

In 1963, Felix and Hashimoto revised their earlier results on the N-terminals and the amino-acid composition of unfractionated clupeine from Clupea harengus so that there was no longer any essential difference in material from the two herring species. A sample of unfractionated clupeine from North Sea herring sperm heads was prepared again in the late Prof. Felix s laboratory in Frankfurt am Main in March 1963 and sent to Prof. Ando s laboratory in Tokyo for use in structural studies. This sample was found to be not different from unfractionated clupeine from Pacific herring as regards amino-acid composition, N-terminal amino acids (proline, 36% and alanine, 64%), N-terminal sequences (Pro-Arg and Ala Arg-Arg ), and tryptic peptides (Thr.Thr 0.95, Thr Arg 0.84, Val Ser Arg 1.7, Pro Val Arg 0.33, Pro He Arg 0.12, Ala Gly Arg 1.03, Ser Arg 0.52, Ala Ser Arg 0.27, Ala Arg 3.54, Ser-Ser-Ser-Arg-Pro-He-Arg 0.7g, Pro-Arg O.87, Ala Ser Arg Pro-Val-Arg I.O2, Arg 8.16, Arg-Pro-Arg 1.5, Ala-Arg-Arg 0.5, Arg-Arg 10.2 moles/3 moles clupeine) (Nukushina, 1964 Nukushina 1964). 

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