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BBA 33128

Characterization of fibrinopeptides A and B from a drill (Mandrillus leucophaeus)

Comparative studies on fibrinopeptides have been of interest primarily because of the high degree of interspecies variability manifest in these portions of the verte- brate fibrinogen molecule1, 2. As such, these efforts are beginning to prove useful in what might be called "fine structure taxonomy ''3. In the case of the order primates, the amino acid sequences of the fibrinopeptides A and B of human 4 and two old-world monkeys a have previously been reported. The two monkey genera had identical fibrinopeptides A, the sequence differing in only I residue (of 16) from the human peptide A. In the case of the fibrinopeptide B, the monkey peptides had only 9 resi- dues, in contrast to the 14 found in the human B. In addition to this purported deletion, there were three amino acid interchanges between any pair of the three species, i.e., between the two monkey sequences as well as between either one and the human sequence, indicating a significantly greater degree of variability for primate B peptides than for their fibrinopeptides A.

In this report we present a characterization of tile fibrinopeptides from another old-world monkey, the drill (Mandrillus leucophaeus). Its fibrinopeptide A differs from the human amino acid sequence in at least 4 of the 16 positions. On the other hand, its fibrinopeptide B is the same length as the human B (14 residues) ; the two B peptides differ at 4 positions. The evidence suggests that the short B peptides previously reported for old-world monkeys resulted from the rupture of a particularly sensitive peptide bond either in vivo or during the isolation process.

Blood from a single male drill was obtained by the staff at the San Diego Zoo. Fibrinogen was prepared from about ioo ml of plasma by a modified Cohn ethanol fractionation6; it was clotted with bovine thrombin, and the clot liquor passed over a short (2.5 cm × 25 cm) Dowex 5o-X2 column at pH 3.0 (o.I M ammonium formate). The fibrinopeptides were eluted with 0.2 M ammonium acetate, pH 5.5 (ref. 7). After pooling and freeze-drying, the peptide material was redissolved and subjected to preparat ive paper electrophoresis at pH 2.0 (8% acetic acid-2% formic acid), at 300 V for 3 h. Guide strips were stained with an arginine-specific stain s. Three peptides were readily distinguished, and these were eluted with o.oi M ammonia. Aliquots were subjected to total acid hydrolysis and analysis on a modified (long light path) Model I2oB Spinco amino acid analyzer.

The most predominant peptide corresponded to a fibrinopeptide A in its amino acid composition; it had the same electrophoretic mobility as human A. The other two corresponded, respectively, to a human peptide B and to the nonapeptides previ- ously reported as monkey B. The nonapeptide was present in less than half the amount of the I4-residue material (fibrinopeptide B) and was designated B*.

Chymotrypsin digestion of the drill A failed to produce fragments; sheep fibrinopeptide A controls were readily split by the treatment. Thermolysin 9, however, produced several peptides from the drill A, the two biggest of which correspond to the amino- and carboxy- halves of the molecule, respectively. The carboxy-terminal dipeptide (Val-Arg) was also recovered from the digest. Resolution of the enzymatic digestions was obtained at pH 4.1, pyridine-acetic acid buffer, 4 h at 300 V. Guide

Biochim. Biophys. Acla, 175 (1969) 217 219

218 SHORT COMMUNICATIONS

str ips were s ta ined with the arginine reagent , n inhydr in , and/or b y the chlorine gas me thod ~°. Chymot ryps in spli t the dril l B pep t ide into two pieces. One of these was a n inhydr in -nega t ive , arginine-negat ive , ondecapept ide . The lack of reac t ion with nin- hydr in was a t t r i b u t e d to a pyr ro l idone residue at the amino- te rmina l , as is thought to occur in the human B (ref. 4). The q u a n t i t a t i v e amino acid composi t ions of the var ious f ragments , including B*, suggest the a r r angemen t of amino acids dep ic ted in Fig. I.

Human A Rhesus monkey A Green monkey A Drill A

Human B Rhesus monkey B Green monkey B Drill B

16 ~5 14 13 i2 1i 1o 9 8 7 (~ 5 4 3 -'

Ala Asp-Ser Gly Glu Gly-Asp Phe-Leu -Ala Glu Gly-Gly-Gly Val-Arg Ala Asp-Thr Gly Glu Gly-Asp Phe Leu-Ala Glu Gly-Gly-Gly Val Arg Ala Asp-Thr Gly Glu Gly Asp Phe-Leu--Ala Glu Gly-Gly-Gly Val-Arg

(Ala, Asp, Thr, Gly, Asp, Gly, Asp, Phe) [le (Thr, Glu, GIy, Gly, Gly) Val-Arg

Th Th Pyr-Gly Val Asn Asp Asn Glu-Glu-Gly Phe Phe-Ser Ala Arg

(< ?- --- --~)Asn Glu Glu-Ser Pro Phe Ser-Gly-Arg Pyr (Gly, Val, Asx, Gly) Asn Glu-Glu Gly Leu-Phe Gly Gly Arg Pyr (Gly, Val, Asx, Gly)(Asx, Glu, Glu, Gly, Leu) Phe-Gly-Gly-Arg

WI. Ch

Fig. L. Proposed amino acid sequences of fibrinopeptides A and B from an individual drill (,~la~z- drillus leucophaeus) based on quantitative amino acid analyses of enzymatically derived frag- ments and homology with structures reported for other primates4, a. Th, thermolysin attack; Ch, chymotrypsin attack; WL, "weak link", proposed labile bond; Pyr, pyrrolidone carboxylic acid residue.

In thei r original repor t BLOMBACK et al. ~ noted tha t , in the case of thei r green monkey prepara t ions , t hey had found a small amoun t of pep t ide ma te r i a l corre- sponding to the I4-res idue B type found in humans. Because they were working with pooled ma te r i a l f rom m a n y individuals , t hey caut ious ly refra ined from render ing an i n t e rp re t a t i on of i ts significance. Unfor tuna te ly , all subsequent rendi t ions of the p r ima te f ibr inopept ide B sequences have included only the nonapep t ide version 2, giving rise to the not ion t ha t a dele t ion has occurred in the genet ic mater ia l . Our results on an ind iv idua l old-world monkey (drill) indica te tha t , genet ica l ly speaking, monkey f ibr inopept ides B have 14 residues. The in terchange of one amino acid, however, has ev iden t ly produced a very sensi t ive pept ide bond (presumably glycyl- asparagine) , since human B pept ides do not exhibi t this behavior . We repor ted a s imilar s i tua t ion for the pronghorn f ibr inopept ide A, in which case a subs tan t i a l amoun t of ma te r i a l lacking the t e rmina l t r ipep t ide was found a. Tile bond thought to be broken in t ha t case is glycylserine.

I t is no tab le t ha t two of the changes r epor ted for the dril l A pept ide have occurred at exac t ly the same locat ions in o ther an imal groups. The change a lanine to threonine at A 7 has occurred i ndependen t ly among the bovids and among the deer a. Similar ly, isoleucine is found a t A8 in donkeys (the horse has leucine), among the carnivores, and in the ra t and r abb i t u. A m a p of the changes occurring in the fibrino- pep t ides dur ing the evolut ion of these 4 p r ima tes is dep ic ted in Fig. 2. The fact t ha t the dril l B pep t ide is a p p a r e n t l y ident ica l wi th tha t of the green monkey (Cercopithecus aethiops), whereas the i r A pept ides differ in 3 of 16 amino acids, tends to discount the not ion t ha t p r ima te B pept ides have changed more than A pept ides during recent evolut ion.

Blockish. Biophvs..4cta, I75 (19(,9) 2i 7 219

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Numan

Green monkey

B5 Leu ~ Pro

A14 Se r /Th r B2 AIO/Gty B5 Phe/Leu B10Asp/G[y

DritL

A7 Alcl ~Th r ' A8 Leu ~ I [ e A12G u ~Asp

B3 S e r ~ G l y

Fig. 2. Schemat ic dep ic t ion of amino acid r ep l acemen t s in f ibr inopept ides A and B du r ing p r i m a t e evolu t ion . Green m o n k e y Cercopithecus aethiops; dri l l -- Mandrillus leucophaeus; the maca- ques referred to are rhesus m o n k e y s and were classified by the or ig inal au tho r s 5 as bo th Rhesus m a c a q u e and Cynomologous macaque . According to the classi f icat ion of WALKER n they would be Macaca mullala and Macaca cynomologous, respect ive ly .

We are grateful to Dr. STAFFAN MAGNUSSON for his gift of bovine thrombin and to Dr. HIROSHI MATSUBARA for the thermolysin. This work was supported by American Heart Association Grant No. 67 624 and National Science Foundation GB-46I 9 and U.S. Public Health Service Trg. lO45.

Department of Chemistry and Biology, University of California, San Diego, La Jolla, Calif. 92o37 (U.S.A.)

RUSSELL F. DOOLITTLE

CAROL GLASGOW

GEORGE n . MROSS

t 1{. F. DOOLITTLE AND 13. BLOMB~_CK, Nature, 202 (1964) 147. 2 t~{. BLOMB'/'-CK, M. BLOMBACK, N. J. GRONDAHL AND E. HOLMBERG, Arkiv h'emi, 25 (1966) 411. 3 G. A. MROSS AND 1R. F. DOOLITTLE, Arch. Biochem. Biophys., 12z (1967) 674. 4 B. BLOMBACK, M. BLOMBACK, P. EDMAN AND B. HESSEL, Biochim. Biophys. Acta, 115 (1966)

371 • 5 g . BLOMBACK, M. BLOMBACK, ~7~-. J. GRONDAHL, C. GUTHRIE AND M. HINTON, Acta Chem.

Stand., 19 (1965) 1788. 6 R. F. DOOLITTLE, D. SCHUBERT AND S. A. SCHVVARTZ, Arch. Biochem. Biophys., I t 8 (1967)

456. 7 B. BLOMBACK ANn A. VESTERMARK, Arkiv Kemi, 12 (1958) 173. 8 S. YAMADA AND H. A. ITANO, Biochim. Biophys. Acta, 13o (1966) 538. 9 H. MATSUBARA, A. SINGER, R. SASAKI AND T. H. JUKES, Biochem. Biophys. Res. Commun., 21

(1965) 242. io F. REINDEL AND W. HOPPE, Chem. Ber., 87 (1954) 11o3. I 1 E. P. V~TALKER, Mammals of the World, Vol. I, The Johns Hopk i ns Press, Ba l t imore , 1964,

p. 448-449.

Received October 2nd, 1968

Biochim. Biophys. Acta, 175 (1969) 217-219