Structure of Clupeine

These figures show that thermolysin hydrolyzes no arginylarginine bonds at all, and furthermore, no bonds between basic amino acids (Hayashi, Iwai, and Ando, unpublished data). Thermolysin is thus an excellent new tool for sequence deter- 

Chart VIII-5. The amino-acid sequence of clupeine YI with demonstration of peptides obtained by hydrolysis with carboxypeptidases B and A, trypsin and thermolysin [Ando and Suzuki, 1967 Suzuki and Ando, 1972 (2)] 

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. 

The primary and secondary structures of all the molecular species of clupeine (from Pacific herring, Clupea pallasii) YI, YII and Z, have been elucidated [see Chart VIII-6 for the primary structures, and Suzuki and Ando, 1968 (1), for the secondary structures]. This is the first and only instance at present of structure determination and elucidation of the number of molecular species present in the 

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The structure of clupeine YII thus proved to be quite similar to that of clupeine Z, except for the substitution of the 6 residues at the N-terminal region (H-Ala-Arg -Ser-) in the Z-chain by the 5 (H-Pro Arg3 Thr-) in the Yll-chain. 

Studies on the secondary structure of clupeine [Suzuki and Ando, 1968 (1) see also Chap. IX. B] indicate that the three component molecules have no helical parts in their chains and that their conformation in physiological condition is completely random. Therefore, not only their primary structures but also their secondary structures must be essentially the same. The significance of the presence of these three similar molecules in clupeine and the question whether each plays a different and specific role in biological activity are matters of fundamental interest which await further study. 

Chang, W. J. Studies on the chemical structure of clupeine from North European herring. Dissert, for a degree of M. Sci., Univ. Tokyo, Feb. 1969. 

Tobita, T., Yamasaki, M., Ando, T. Studies on protamines. XII. Determination of the carboxyl-terminal structure of clupeine and salmine using enzymatic procedures. J. Biochem. (Tokyo) 63,119—126 (1968). 

Further results obtained with clupein may be shortly mentioned, which throw some light on the connection between structure of the anion and its affinity for the ionogenic groups. 

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. 

Although clupeine and salmine are known to be resistant to the action of pepsin, they are readily hydrolyzed by pancreatic enzymes. Waldschmidt-Leitz et al. [1929 (1), 1931, 1933, 1935 (1)] proposed the following structure for clupeine based on their finding of peptides of the types M A, A M, (A-M A)2, A-Pro-A, and A A in the hydrolyzates of clupeine with various pancreatic enzymes. The structure is of the same type as proposed by Kossel]... 

A rechromatographed specimen of clupeine Z obtained by column chromatography on alumina was subjected to structural analyses. Table VIII-1 shows the... 

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. 

Peaks obtained by column chromatographic separation (Fig. VIII-1) of tiyptic peptides in a 20-h digest of clupeine Z were analyzed for their amino-acid composition, amino termini, etc. to determine the structure and amounts of peptides involved... 

It is concluded on the basis of the chemical structure determined for the protamine that the N — O acyl rearrangement reaction of clupeine Z and the selective chemical degradation proceed as shown in Chart VIII-3. 

Chart VIII-6. The total primary structures of 3 main components of clupeine (A = Arg)... 

Clupeine obtained from sperm cell nuclei of Clupea harengus has been studied for about 50 years, since the time of Kossel, by European protein researchers, and facts have accumulated on some features of the chemical structure of unfractionated (whole) clupeine. Over 10 years ago, some differences were noted in the amino-acid composition and the N-terminal amino acid residue of clupeine specimens prepared from Norwegian Sea (Felix et al.y 1950 Waldschmidt-Leitz et aLy 1951) and Pacific [Ando et al.y 1952,1953,1957 (1,2)] herring. It was considered by both the German and the Japanese workers that such differences were due to the species specificity of the materials used. 

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. 

There is hardly any difference in the primary structure found for the corresponding components of clupeine from North Sea and Pacific herring. It is thus concluded that... 

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., Iwai, K., Ishii, S., Azegami, M., Nakahara, C. The chemical structure of one component of clupeine. Biochim. biophys. Acta (Amst.) 56, 628—630 (1962). 

Crick, and Vand for the form factor for helical structures requires that the orders of Bessel functions for the successive layer lines from 0 to 8 be 0, 3, 6, 9, 12, 9, 6, 3, and 0. The layer-line intensities agree satisfactorily with this prediction, in the region from layer line 4 to layer line 8. There is an unexplained blackening near the meridian for layer lines 2 to 4, which, however, differs in nature for sodium thymonucleate and clupein thymo-nucleate, and which probably is to be attributed to material between the polynucleotide chains. 

From these results Felix assumed a structure which comprised repeating sequences of MAA and A AM for clupeine (Felix and Mager, 1937), particularly taking into account the isolation of tetraarginine (Felix et al.y 1933) ... 

After World War II, a large number of peptides were obtained and identified in partial hydrolyzates of several protamines by various research groups using the modern techniques developed in protein chemistry. These are summarized in Table VI-2. As can be seen from the upper part of the table, Felix considered the N-ter-minal sequence to be H-Pro Ala Arg-, which was slightly different from the previous structure. He finally proposed the following general formula for clupeine (from... 

Quite similar results have also been obtained recently with whole clupeine from Clupea harengus (Nukushina, 1964 Nukushina et al, 1964), whole salmine from Oncorhjnchus keta and iridine from Salmo irideus (Watanabe, 1969 Ando and Wata-NABE, 1969 Watanabe and Ando, to be published). Thus, a few components having similar but not identical structures are expected to be present in each of these unfractionated protamines, clupeine, salmine, and iridine. 

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