Charles A. Vernon

The disulphide bridges of apamin.

It has been known for some years that bee venom contains an easily dialysable substance which has central excitatory properties [l] . The substance, which has been given the name apamin, was first isolated by Habermann and his colleagues, and shown to be a peptide containing eighteen residues and two disulphjde bridges [2a,b]. Recently [3], we found the aminoacid sequence of apamin is: ties was made by an investigation of some of the pep tides arising from partial acid hydrolysis of apamin, using conditions similar to those described by Ryle and Sanger [S] . Apamin (25 mg) was dissolved in a mixture (2.5 ml) of sulphuric acid, acetic acid and water (5 : 3 : 2 v/v) containing thioglycollic acid (0.05%) and heated for 45 minutes at 100°. The hydrolysate was freed from sulphuric acid by passage

The primary sequence of phospholipase‐A from bee venom

We recently reported [ 1 ] the purification and characterization of phospholipase-A from the venom of the common European honey bee (&is melZifica). We now wish to report the primary (peptide) sequence of the enzyme. Most experiments were carried out using reduced and carboxymethylated enzyme. A short digestion with trypsin gave 18 peptides, cleavage being observed at 6 arginine, 9 lysine, 1 tyrosine and 1 phenylalanine residues. The peptides were separated on Sephadex G-25 (superfine) and then by paper electrophoresis at pH 6.5 or pH 3.5 in solvent cooled tanks. Paper chromatography was used as an additional step where necessary. The amino acid sequence of the peptides was determined by the Dansyl-Edman procedure [2]. Similar experiments were carried out using the peptides derived from digestion with chymotrypsin, pepsin and elastase. Some of the peptides obtained by tryptic digestion of enzyme which had been reduced, carboxymethylated and maleylated were also examined. The C-terminal sequence was identified as X-LysTyr-COOH by treatment with carboxypeptidase-A where X was not liberated by the enzyme. The N-terminal sequence was readily found by using the Edman procedure on the native enzyme. The results of these experiments lead to the sequence shown in table 1. The positions of cleavage produced by trypsin and chymotrypsin are shown by the symbols T and C respectively. All overlaps were satisfactorily established except for positions Tyr(76)-Phe(77). This particular peptide bond was cleaved by all the enzymes used and under all experimental conditions examined. It is, therefore formally possible that the sequence given is incomplete in that a fragment lying between residues (76) and (77) has escaped detection. That this is not the case was established by examining all the peptides produced by separate digestion with chymotrypsin, trypsin and pepsin. Furthermore, the stoichiometric amino acid composition calculated from the amino acid analysis [ 1 ] and from the sequence given in table 1 are in close agreement (A and B respectively).

New methods of isolating bee venom peptides

Abstract The three major peptides of bee venom, melittin, apamin, and peptide 401 (MCD peptide), can be readily separated on columns of Heparin Sepharose CL-6B at pH 6.8 (phosphate buffer) using a linear salt gradient. Contamination by melittin of the other two components can be assessed by fluorescence spectrophotometry.

Immunological properties of nerve growth factors

Antisera were raised against nerve growth factors isolated from mouse salivary gland and five venoms representative of the three main families of poisonous snakes. Immunochemical cross-reactivity was assessed from the ability of the antisera to inhibit the biological activities of the heterologous antigens. The high and low molecular weight forms of the salivary gland factors were found to be immunologically identical but distinct from the snake venom factors. The snake venom factors, on the other hand, were immunologically closely related to each other but not identical.

Nerve growth factor has anti-inflammatory activity in the rat hindpaw oedema test

Nerve growth factor (NGF) affects the development of sympathetic neurons in neonatal rodents and, at very much lower levels, stimulates fibre growth from certain neurones in culture. At levels comparable to those used in culture and found in mammalian sera, NGF has recently been shown to suppress the hindpaw oedema induced in adult rats by injection of carrageenin. On a weight basis, NGF is ten times more active in this test than dexamethasone, and a thousand times more active than the non-steroidal anti-inflammatory drug, indomethacin. It is equally effective given locally and systemically.

Reassessment of the role of ATP in vivo

Abstract In 1941 Lipmann introduced the term “high energy phosphate bond”. This concept has had profound effects on the development of biochemical thinking and has led to a considerable preoccupation with free energy changes, either actual or standard, associated with metabolic reactions and, in particular, with the hydrolysis of ATP. In this paper we wish to propose the view that the original concept put forward by Lipmann was ill-founded and that its effect is to divert attention from the genuine problem of the mechanism of events in which ATP takes part. We shall show that (a) the hydrolysis of ATP is a forbidden reaction in intermediary metabolism, (b) that the Lipmann concept would be appropriate for a closed system containing energy-linked reactions (of which there are no known examples in biochemistry) and (c), most importantly, that since real organisms are open and not closed systems even the direction of flow of matter through a particular unit step cannot be predicted from the associated standard free energy change but only from the properties of the whole system making up the steady state. In other words simple thermodynamic parameters are irrelevant in discussing whole organisms: these must be understood in kinetic and mechanistic terms. We shall illustrate these ideas by considering some metabolic processes and, in particular, we shall show that for oxidative phosphorylation the exclusion of simple thermodynamic considerations enormously simplifies the discussion and leads naturally to a plausible mechanism for ATP formation. We shall conclude by considering the factors which may have been important in the selection of ATP for its metabolic role. Although the values of the standard free energies of hydrolysis of phosphate esters are irrelevant in vivo , a critical account is given in the Appendix of the experimental data on which the accepted values are based. Brief accounts of these views have already been published.

The preparation of nerve growth factor from the prostate of the guinea-pig and isolation of immunogenically pure material from the mouse submandibular gland

It was recently discovered [ 1] in these laboratories that the prostate of the guinea-pig is (like the submandibular gland of the adult male mouse) a rich source of nerve growth factor (NGF). We now report, by a modification of the method in [2], the total purification of the factor from this source and describe some of its properties. A similar purification method can also be used to prepare the p-subunit of NGF from mouse submandibular gland free of immunogenic contaminants. We have shown [3] that such contaminants are extremely widespread and may seriously interfere with the interpretation of biochemical and biological experiments.

The primary structure of aspartate aminotransferase from pig heart muscle determined in part using a protease with specificity for lysine

We have recently reported [1] partial amino acid sequences of the cytoplasmic aspartate aminotransferase from pig heart muscle, based on studies of peptides produced by digestion of the protein with pepsin and trypsin [2] and with thermolysin and elastase [ 1]. This work gave ten major fragments containing 395 amino acid residues. We now present the complete structure of the molecule which consists of a chain of 412 amino acid residues. The structure given here is essentially in agreement with that of Ovchinnikov et al. [3]. However, the authors gave no indication of the data which was used to overlap their previously reported 21 partial sequences [4]. We consider it important, for this reason and due to the large size of the molecule, to confirm the correctness of the published structure and to show how the structure was derived from our earlier data. An important tool used in the present work was a newly characterized protease isolated from the fruiting body of the basidiomycete Armillaria mellea [5]. The enzyme has been shown to be an endopeptidase cleaving proteins such as fibrinogen and casein N-terminal to lysine residues. Our results confirm this specificity and provide further information about the type of peptide bond hydrolysed by the enzyme.

Anti-inflammatory activity of bee venom peptide 401 (mast cell degranulating peptide) and compound 48/80 results from mast cell degranulation in vivo.

1 The relationship between the anti‐inflammatory activity of the bee venom peptide 401 in the carrageenin‐induced oedema of the rat hind paw and its mast cell degranulating activity has been reinvestigated. 2 Mast cell degranulation caused by compound 48/80 (10 mg kg−1) or by allergen challenge in rats sensitized to Nippostrongylus brasiliensis also suppressed rat hind paw oedema in the same test. 3 The anti‐inflammatory activities of peptide 401 and compound 48/80 were partially suppressed by pretreatment of rats with mepyramine and methysergide, at doses (2.5 mg kg−1) that completely suppressed skin reactions to these mast cell‐derived amines. Pretreatment of rats with compound 48/80 also suppressed the apparent anti‐inflammatory actions of peptide 401 and of compound 48/80. 4 Injection of peptide 401 together with carrageenin increased the inflammatory response in the rat hind paw. 5 The anti‐inflammatory activity of peptide 401 and of compound 48/80 in the carrageenin‐induced swelling of the rat hind paw arises from mast cell degranulation in vivo.

Some studies on the mechanism of action of antibodies to nerve growth factor

Abstract The effects of antibodies to the nerve growth factor from mouse salivary gland were examined in vitro and in vivo. Treatment of explants of receptive ganglia with antibody and complement did not produce cell damage as judged by the ability of the tissue to respond to nerve growth factor. New-born mice experimentally depleted of or genetically deficient in key complement components were susceptible to the action of the antiserum. These results show that the effect of the antibody is independent of complement and are consistent with the view that it acts by neutralization of endogenous nerve growth factor.