Complete Amino Acid Sequences of Bovine and Human Endozepines HOMOLOGY WITH RAT DIAZEPAM BINDING INHIBITOR*

The complete amino acid sequences of bovine and human brain endozepines have been determined. The amino-terminal serine of both endozepines is acylated. Assignment of the first 7 residues was achieved through Edman degradation after acid-induced rearrangement and subsequent acid hydrolysis of the amino-terminal blocking group. Cleavage of endozepine by chemical and enzymatic techniques established all the fragments in an unambiguous sequence. Bovine and human endozepines are single-chain polypeptides of 86 residues, with calculated molecular weights of 9913, displaying 93% homology. A comparison be- tween the sequences of bovine and human endozepines with the partial sequences of the functionally related diazepam binding inhibitor from rat brain reveals sig- nificant sequence homology. The reported results sug-gest that bovine and human endozepines as well as rat diazepam binding inhibitor belong to a new family of polypeptides which presumably take part in the mod- ulation of y-aminobutyric acid-ergic transmission.

The amino acid sequence of bovine endozepine (bEP3), as presented in Fig. 1, was determined by automated Edman degradation of selected fragments using standard enzymatic and chemical cleavage techniques. The separation of complex peptide mixtures over a broad range of fragment sizes was achieved through a single rpHPLC chromatography step. The use of trifluoroacetic acid in both solvents appeared to maintain the solubility of even large fragments at high acetonitrile concentrations. In addition, the buffer system used is completely volatile, which enabled subsequent chemical characterization of separated peptides.
The amino-terminal serine residue of bEP is acylated. Acidinduced rearrangement and subsequent acid hydrolysis of the 0-acyl derivative of the amino-terminal heptapeptide were used to establish an unambiguous assignment of the first 7 residues. The nature of the amino-terminal blocking group was not identified. The hydrophobic nature of the blocking group in E P was, however, recognized by rpHPLC. The observed retention time of endoproteinase Lys-C peptide 1-7 was slightly increased when compared with the retention time predicted by the sequence of the unmodified peptide (4), suggesting an N-acetyl blocking group contributing to the increased hydrophobicity.
The sequence of the amino-terminal heptapeptide and Edman degradation of selected fragments derived from bEP through cleavage with the endoproteinase Lys-C and Staphylococcus aureus V8 protease accounted for all the residues in the amino acid composition of the protein. Overlap information between glutamic acid residue 61 and aspartic acid residue 68 was provided by the sequence of chymotryptic peptide 62-73. Additional overlap information between glutamic acid I 9 and leucine 80 was provided by the sequences of chymotryptic peptide 74-80 and cyanogen bromide peptide 11-86 which established all the fragments in an unambiguous sequence of 86 residues. The amino acid composition calculated from the complete sequence is in agreement with the compo-  TABLE I   35   40 45 5 0 55 60 T y r~L y s~G t n~A l a~T h r~V a I -G l y -A s p -I l e~A s n~T h r~G l u~A r g -P r o -G l y -M e t -L e u -A s~~P h e L y s -G l y -L y s -A l o -L y s~T r p~A s p -A I a -T r p -A s n~G l u Le~-iys-Gly-Thr-Ser~Lys~Glu~Asp-Ala-Mel-Lys.Ala-Tyr-Ile-Asp-Lys-Val-Glu-Glu-Leu-Lys~Lys-Lys-Tyr-Gly-Ile  Table I The amino acid sequence of hEP is presented in Fig. 2 blocked, possibly through N-acetylation'at serine. Treatment of endoproteinase Lys-C peptide 1-7 with hydrochloric acid partially deblocked the amino terminus and Edman degradation established the first 7 residues. No overlap information between lysine 32 and glutamine 33, glutamic acid 67 and aspartic acid 68, glutamic acid 79 and leucine 80 was provided by the experiments described. However, the amino acid composition of hEP, as presented in Table I, is in agreement with the proposed structure. In addition, the extensive sequence homology between bEP and hEP lends credence to the proposed structure. The calculated molecular weight of 9913 for hEP from the amino acid sequence is in agreement with the determined apparent molecular weight.  for 86 amino acids to the carboxyl-terminal isoleucine, followed by a stop codon.

Y K Q A T V G D I N T E R P G M L D F T G K A K W D A W N E
A comparison of the amino acid sequences of bEP and hEP is presented in Fig. 3. The data indicate that bEP and hEP differ from each other by only 6 conservative amino acid substitutions: 80 out of 86 residues, or 93%, are identical. Each of these amino acid substitutions is compatible with a single base change at the DNA level. The amino acid sequences of bEP and hEP are homologous to the recently published partial sequences of DBI isolated from rat brain (1, 3), as shown in Fig. 3. Forty out of 50 residues, or 80%, are identical when the sequences of bEP and rDBI are compared. A comparison of the amino acid sequence of hEP with the partial sequences of rDBI shows 38 identities out of 50 comparisons, or 76% sequence homology. The authors, however, have concluded (2) that rDBI contains 104 amino acid resi- natant, the mixture was stirred for 1 h and allowed to separate into two phases.
The upper aqueous phase was removed, dialyzed against 0.1 H acetic acid and subsequently concentrated by lyophilization. Each endorepine was quantitated in a radioreceptor assay (7) based on receptor cross-reactivity with benzodiazepine to synaptosomal membranes. The purification was achieved by gel permeation chromatography on a Bio-SiL TSK-250 column (2.1 x 60 cm. Bio Rad Laboratories) in 0.1% trifluoroacetic acid containing 40% acetonitrile. followed by reversed phase HPLC OD piBondapak C18 support (Waters) using a linear gradient of acetonitrile in 0.1% trifluoroacetic acid, essentially a s described (1) for the purification of rat DBI.
Sequencer reagents were obtained from Applied Biosystems. Solvents for HPLC were from Burdick and Jackson. CNBr was from Kodak; all other chemicals were reagent grade. Endoproteinase Lys-C was from Boehringer Mannheim; CDI obtained from Miles Laboratories. qd Human Endozepines 9729 dues, a carboxyl-terminal tyrosine, and two identical copies of an octadecapeptide, comprising tryptic peptide 33-50. Rat DBI has been shown to act as an inverse antagonist of benzodiazepines in vitro and in vivo (1). Benzodiazepines are thought to act by modulating 7-aminobutyric acid-ergic transmission. The completion of the primary structure of E P might permit direct examination of its proposed role as a precursor of small signal-transducing peptides (3). Isolation of complementary DNAs encoding the bovine and human forms of the proposed endogenous ligand should allow studies of its biosynthesis and the molecular mechanisms of its actions. Peptide-containing fractions were pooled. Aliquots were taken for sequence analyses, and for amino acid analyses when necessary (Tables SI and  SII) (Table SII).