J. Mol. Evol. 1 3 , 3 0 5 - 3 1 6 (1979)

Journal of Molecular Evolution © by Springer-VerIag.1979

Primary Structure of Pronghorn Pancreatic Ribonuclease: Close Relationship between Giraffe and Pronghorn

Jaap J. Beintema, Wim Gaastra, and Jan Munniksma Bioehernisch Laboratorium, Rijksuniversiteit Groningen, The Netherlands

Summary. Pancreatic ribonuclease from pronghorn (Antilocapra americana) was isolated and its amino acid sequence was determined from a tryptic digest of the performic acid~oxidized protein. Peptides were positioned by homology with other ribonucleases. Only peptides that differed in amino acid composition from the corresponding peptides of ox or goat ribonucleases were sequenced. In a most parsimonious tree of pancreatic ribonucteases, pronghorn and giraffe were placed together and these two were placed with the bovids, leaving the deer as a taxon separate from the other ruminants. The amino acid replacements that determine this tree topology are three rarely occurring replacements shared by pronghorn and giraffe. Notwithstanding their close phylogenetic relationship, both ribonucleases differ strongly in extent of glycosidation, net charge and antigenic properties. Key words: Ribonuclease - Amino acid sequence - Pronghorn - Giraffe Bovids - Pecora -- Ruminants Introduction

The position of the Giraffidae in the phylogeny of the Pecora or true ruminants is still uncertain. Generally they are ordered with the Cervidae (Simpson, 1945 ; Viret, 1961 ; Romer, 1966). However, more recent evidence indicates that placement with the Bovidae is more correct (R. Hamilton, personal communication). Amino acid sequences of proteins provide new information for biological classification and probably can be used to settle the question. Amino acid sequences of pancreatic ribonucleases from eleven bovid species, five deer species and giraffe (Beintema et al., 1977 and unpublished observations) are known to date; they point to a closer relationship of the giraffe with the bovids than with the deer (Gaastra et al., 1974). Here we present the amino acid sequence of the ribonuclease of the pronghorn (Antilocapra americana), the sole extant species of the family A ntilocapridae. Evidence is presented for a close relationship of the Giraffidae with the Antilocapridae. 0 0 2 2 - 2 8 4 4 / 7 9 / 0 0 1 3 / 0 3 0 5 / $ 02.40

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Materials and Methods

Most materials and methods were as described by Gaastra et al. (1978) and by Kuper and Beintema (1976). Rihonuclease (130 rag) was isolated in a yield of about 60% from combined pancreatic tissue (530 g) from 35 pronghorns by extraction with 0.125 M sulfuric acid, ammonium sulfate fractionation (50-90% saturation) and affinity chromatography (Wierenga et al., 1973). The amino acid sequence was determined by automatic Edman degradation of the native protein and by analysis of peptides obtained by digestion of 20 mg of the per~ formic acid-oxidized protein with 0.2 mg trypsin in 2 ml 0.1 M NH4HCO3, pH 8.0, for 2 h at 37°C. The tryptic peptides are denoted with the prefix T. The digest was fractionated by gel filtration on a column of Sephadex G-25 (0.6 x 100 cm) (Kuper and Beintema, 1976). A mixture of four large peptides (T3, T6, T8 and T13) which eluted together in the first peak was digested with 0.3 mg chymotrypsin in 0.5 ml 0.1 M NH4HCO3, pH 8.0, for 2 h at 37°C and subjected to gel filtration on the same column of Sephadex G-25. These peptides are prefixed with T..C. The last purification step for all peptides was preparative paper electrophoresis at pH 3.5. Peptide T6C2 (50 nmol) was digested with 50 ~g thermolysin in 0.2 M ammonium acetate, pH 8.5, overnight at 37°C. The resulting peptides are prefixed with T6C2H. Peptide T13C1 (75 nmol) was digested with 50 ~g Staphylococcus aureus proteinase in 0.3 ml 0.05 M NH4HCO3, pH 8.0, overnight at 37°C and subjected to preparative paper electrophoresis at pH 6.5 to isolate peptide T13CIE2. S-peptide and S-protein were prepared by digestion with subtilisin as described by Welling et al. (1974a). Amino acid sequences of peptides were determined as described by Hartley (1970) and charges of peptides, as described by Offord (1966). Additional information about amide positions was provided by evidence from automatic Edman degradation (phenylthiohydantoins of amino acids) and by the specificity of the Staphylococcus aureus proteinase (Glu-enzyme). Results and Discussion Fig. 1 shows the combination of analytical steps that have led to the proposed sequence of pronghorn ribonuclease. Fig. 2 summarizes these data in a conventional notation that distinguishes between degrees of reliability according to which different parts of the sequence were derived (Dayhoff, 1972). The amino acid compositions of the peptides are given in Table 1. We followed the recommendations by Ambler (1971) for sequencing homologous proteins, that differ less than 10% in their amino acid sequences. Therefore, only peptides differing in amino acid composition from their homologous counterparts in ox and goat ribonuclease were subjected to dansyl-Edman degradation. The peptides were positioned by homology with other pecoran pancreatic ribonucleases. Each aspartic and glumatic acid was checked for the presence of an amide group. The properties of peptides during gel filtration, paper electrophoresis at pH 3.5 and 6.5 and with several staining reagents were equal to those of identical peptides from other pecoran ribonucleases (e.g. Kuper and Beintema, 1976;Welling et al., 1974b; Gaastra et al., 1974). The sequence 11-25 could not be derived unambiguously

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