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Annotated Information


The emopamil-binding protein(EBP) acts as a high affinity acceptor for several antiischemic drugs and thus represents a potential common molecular target for antiischemic drug action. Degenerate oligonucleotides were synthesized according to the N-terminal amino acid sequence of purified EBP and used to amplify a guinea pig cDNA with reverse transcriptase-polymerase chain reaction and to clone full-length cDNAs from guinea pig and human liver cDNA libraries. The phenylalkylamine Ca antagonist emopamil labels a high affinity binding protein (emopamil-binding protein, EBP) for a variety of structurally different compounds in guinea pig liver. Some of these drugs such as emopamil, ifenprodil, opipramol, trifluoperazine, and chlorpromazine exert antiischemic effects in animal models of stroke, but their neuroprotective action is not fully understood at the molecular level. Since the possibility exists that EBP is involved in ischemia-related cellular events biochemical studies were undertaken to investigate the physiological function of EBP.


Guinea pig and human EBP cDNAs were subcloned into the yeast episomal plasmid YEp351ADC1. Truncated cDNAs carrying a 5′-HindIII restriction site and AAA triplet before the initial ATG(human and guinea pig EBP) and a 3′-NotI restriction site behind the stop codon (human EBP) were generated with PCR. PCR products were cloned into pBluescript IISK+ and sequenced before subloning into YEp351ADC1.


The open reading frame of guinea pig and human cDNAs coded for 229 and 230 amino acid residues corresponding to a molecular mass of 26.683 and 26.356 Da, respectively. The polyadenylation signal AATAAA of the human EBP cDNA occurred 239-244 nucleotides downstream from the stop codon of the putative EBP and 13 nucleotides upstream from a putative poly(A) tail. Although the 5′ non-translated regions of both cDNAs (nucleotides 1-114 of human EBP) were 64% homologous the first methionine codon was flanked by a consensus sequence for the initiation of translation indicating that it was indeed the start codon. The N-terminal amino acid sequence of the purified EBP determined by Edman degradation only lacked the initial methionine residue. The identity and similarity of the amino acid sequences between human and guinea pig EBP were 73 and 85%, respectively. Sequence comparison in protein and DNA databases showed no significant homology (>20%) with known sequences. Both EBPs contained potential phosphorylation sites for protein kinases A and C. The C termini of both proteins were heterologous. They contained a polylysine motif (KVMKSKGK in guinea pig and KAKSKKN in human) known to mediate the retrieval of type I integral membrane proteins into the endoplasmic reticulum. Hydropathy plots according to Kyte and Doolittle computed with a window of 19 amino acid residues predicted four transmembrane segments (TMS). The connecting loop between TMS3 and TMS4 was also highly hydrophobic. The TMS2 and TMS3 contained two glutamate residues conserved in human and guinea pig EBP whereas the TMS1 and TMS4 contained no charged residues. All cysteine residues were localized in the TMS2 and 3. A high content of aromatic amino acid residues in the transmembrane segments was determined as described in and was 28 and 23% for guinea pig and human EBP, respectively. The topology model of EBP predicts that most of the protein is buried in the lipid bilayer. From the endoplasmic reticulum retrieval sequence for type I integral membrane proteins, we conclude that the N and C termini face the cytoplasm. In this model the potential phosphorylation site for cAMP-dependent protein kinase faces the lumen of the endoplasmic reticulum. Protein kinase A did not phosphorylate the detergent-purified EBP.

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Structured Information