The complete primary structure of the purple membrane protein bacteriorhodopsin, which contains 248 amino acid residues, has been determined. Methods used for separation of the hydrophobic fragments included gel permeation and reverse-phase high-pressure liquid chromatography in organic solvents. The amino acid sequence was determined by a combination of automatic Edman degradation and mass spectrometric methods. The total sequence was derived by ordering of the CNBr fragments on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry and by analysis of N-bromosuccinimide fragments containing overlaps between CNBr fragments. The present sequence differs from that recently reported by Ovchinnikov and coworkers with respect to an additional tryptophan (position 138) and several amino acid assignments.The purple membrane of a number of extremely halophilic bacteria-e.g., Halobacterium halobium-functions as a light-driven proton pump (1, 2). It contains a single protein, bacteriorhodopsin (Mr 26,000) with one molecule of retinaldehyde covalently bound to a lysine residue (3, 4). Bacteriorhodopsin forms a continuum of seven a helices, each of which spans the membrane and is largely embedded in it (5). Knowledge of the primary structure of bacteriorhodopsin is a prerequisite to studies of the mechanism of the proton pump and of the biogenesis of this interesting protein. In a recent paper, we reported (6) on the sequence of the first 102 amino acids and of 39 amino acids from the COOH terminus of this membrane protein. The present paper reports on its complete amino acid sequence (Fig. 1). Although no experimental details have appeared, the complete sequence has also been deduced by Ovchinnikov and coworkers (7,8), and partial sequences have been reported by other laboratories (3, 9, 10). The present sequence differs from that reported by Ovchinnikov and coworkers with respect to an additional tryptophan (position 138) and amino acid assignments at positions 105, 111, 117, 146, and 206. Of the total 248 amino acids present in bacteriorhodopsin, 70% are hydrophobic, and there is significant clustering of the hydrophobic as well as of the hydrophilic amino acids. MATERIALS AND METHODSMaterials. N-Bromosuccinimide (NBS), fluorescamine, and isothiocyanatophenylthiocarbamoylaminopropyl (IPTAP) glass were purchased from Pierce, and trypsin treated with L-(tosylamido-2-phenyl)ethyl chloromethyl ketone, clostripain, and elastase were from Worthington Biochemicals. ['4CISuc-cinic anhydride was from New England Nuclear. Tetraethyltetraamino (TETA) and aminoethylaminopropyl (AEAP) glass were gifts from Mark Horn of Sequemat, Watertown, MA. Other materials were as described previously.Preparation of Chymotryptic and CNBr Fragments. Purple membrane was isolated from H. halobium and apomembrane was prepared as described (6). Digestion of the apomembrane with chymotrypsin gave two fragments, C-1 and C-2, which were collected by centrifugation and separated on a Sephadex LH-60 column ...

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Four novel peptides were isolated from the venoms of the solitary eumenine wasps Eumenes rubrofemoratus and Eumenes fraterculus. Their sequences were determined by MALDI-TOF/TOF (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) analysis, Edman degradation and solid-phase synthesis. Two of them, eumenitin-R (LNLKGLIKKVASLLN) and eumenitin-F (LNLKGLFKKVASLLT), are highly homologous to eumenitin, an antimicrobial peptide from a solitary eumenine wasp, whereas the other two, EMP-ER (FDIMGLIKKVAGAL-NH(2)) and EMP-EF (FDVMGIIKKIAGAL-NH(2)), are similar to eumenine mastoparan-AF (EMP-AF), a mast cell degranulating peptide from a solitary eumenine wasp. These sequences have the characteristic features of linear cationic cytolytic peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, they can be predicted to adopt an amphipathic α-helix secondary structure. In fact, the CD (circular dichroism) spectra of these peptides showed significant α-helical conformation content in the presence of TFE (trifluoroethanol), SDS (sodium dodecylsulfate) and asolectin vesicles. In the biological evaluation, all the peptides exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity.

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