The phospholipase A from the venom of the common European honey bee (Apis mellifica) has been completely purified. The final product (13 g from 700 g of crude venom) readily crystallizes and is homogeneous with respect to starch gel electrophoresis at pH 8.0, isoelectric focussing in polyacrylamide gel in the pH range 3–10, and sedimentation and diffusion analysis in the ultracentrifuge. Only one N‐terminal residue, isoleucine, can be detected either by the Edman or dansyl methods. Quantitative N‐terminal analysis and gel filtration on Sephadex G‐100 give values for the molecular weight of about 19000. Ultracentrifugation studies lead to a value of about 40000: in concentrated solution the molecule exists, therefore, as a dimer. The identity of the enzyme as a phospholipase of the A2 type has been confirmed since, with 1‐oleyl‐2‐isolauroyl phosphatidyl ethanolamine as substrate, isolauric acid is liberated in 100% yield whereas no oleic acid is released. A method for the assay of the enzyme based on continuous titrimetric estimation of hexanoic acid liberated from 1,2‐dihexanoyl lecithin has been used to study various aspects of the activity of the enzyme.
The complete amino acid sequence of phospholipase A2 from the venom of the common European honey-bee (Apis mellifica) has been determined. The sequence of amino acid residues at the N-terminus was obtained by direct application of the Edman degradation technique and that at the C-terminus by digestion with carboxypeptidasesA and B. Digestion of the reduced and carboxymethylated enzyme with trypsin yielded a completely soluble peptide mixture, all the components of which were isolated and their structures determined. Overlaps of the tryptic peptides were deduced from the structures of peptides isolated after digestion of the reduced and carboxymethylated enzyme with chymotrypsin and of the reduced and aminoethylated enzyme with a protease specific for cleavage at lysine residues. Two remaining ambiguities were resolved by peptides obtained from a digest of the reduced, carboxymethylated and maleylated enzyme with trypsin. The phospholipase A2 consists of a single chain of 128 amino acid residues and contains attached carbohydrate residues. The composition and point of attachment of the carbohydrate moiety have been established. Phospholipase A2 is the name given to a family of enzymes which catalyse the hydrolysis of fatty acid ester bonds at the 2-carbon position of 3-sn-phosphoglycerides. Enzymes with this specificity have been isolated from pancreatic tissue [l], bee venom [2] and a variety of snake venoms [3]. The enzymes have certain characteristics in common, for example relatively small size, high disulphide-bridge content, heat stability and requirement for Ca2+ ions for activity. There are, however, important differences. In particular, the pancreatic enzyme appears to be active on the aggregated (micellar) form of phospholipids [4], whereas the substrate for the bee-venom enzyme has been shown to be the dispersed (monomolecular) phospholipid [2]. It is also of interest that the enzyme from pancreas is secreted as an inactive zymogen which may be activated by limited proteolysis with trypsin sequence of the zymogen and the positions of the disulphide bridges in porcine pancreatic phospholipase A2 have been reported [6,7].Our interest in phospholipase A2 from bee venom arose from the observation [2] that the enzyme crystallises readily which, combined with its relatively small size, suggests that it may be amenable to total structure determination. As a preliminary to crystallographic analysis we have determined the primary structure of the enzyme; this work has been the subject of a preliminary report [S]. A missing overlap in the earlier work has now been established with certainty and three corrections have been made to the previously published sequence. In addition, the composition and point of attachment of the carbohydrate moiety have been established. An interesting aspects of the present work has been the use of a recently characterized protease isolated from the basidiomycete Armillaria mellea [9] ; the enzyme has been shown to have specificity for cleavage of peptide bonds N-terminal to lysin...
End-group analysis has often been used together with ultracentrifugation analysis to elucidate protein sub-unit structure. It is also sometimes used to estimate the molecular weight of single polypeptide chains. However, most methods are only semi-quantitative, since handling losses occur in the isolation of derivatives and corrections have to be applied for loss of derivatives during the hydrolysis procedures. There is now considerable evidence to show that the Edman degradation with phenyl isothiocyanate proceeds quantitatively under certain conditions (Eriksson & Sjoquist, 1960; Edman & Begg, 1967). Consequently, quantitative end-group analysis is possible if errors due to handling losses of the phenylthiohydantoin derivatives can be avoided. A procedure that gives good results has been described by Eriksson & Sjoquist (1960), but it involves exacting experimental techniques. We now report the details of a relatively simple method in which handling losses are avoided by the use of radioisotope dilution. Two samples of salt-free protein (approx. 0-25/mole) are weighed out on a microanalytical balance (e.g. Cahn Gram Electro Balance). One sample is heated to constant weight at 1100 to determine moisture content. The other is dissolved in
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