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Three novel peptides derived from lipotropin, the C-Fragment (residues 61-91), C'-Fragment (61-87) and N-Fragment (1-38), were isolated from pig pituitary, and the C-Fragment was shown to be present in brain. The experimental procedures developed for their isolation are described. The formation of each of the fragments involves enzymic cleavage of lipotropin at consecutive basic residues, with specificity identical with that involved in the activation of known prohormones. In brain assays C-Fragment exhibits a range of biological activities related to its ability to act as an inhibitory neurotransmitter.

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The tendency to aggregate a t neutral pH exhibited by zinc insulin, insulin and some N-acyl derivatives was studied with the aid of gel filtration. On Sephadex 6-75 and at an initial concentration of 1 mgjml, zinc insulin appeared t o possess an average molecular weight of 24000 and insulin an average molecular weight in the region of 12000. I n contrast, NUA1, NaB1, NEBz9-trisuccinylinsulin and N a A l , NUB1, N8B2Q-tricarbamylinsulin appeared predominantly monomeric with a molecular weight in the region of 6000. NaAl-Monocarbamjdhsulin resembled the tricarbamyl derivative whereas NaB1-monocarbamylinsulin aggregated like insulin. The results appear to reflect conformational changes undergone by the insulin molecule as a consequence of acylation at the A1 glycine.A characteristic property of the insulin molecule is its tendency to form molecular aggregates, a phenomenon that has been demonstrated at a variety of pH values by ultracentrifugal analysis [l-31. At pH 7.5 and a concentration of 3.3mg/ml, insulin exhibits an average molecular weight in the region of 20000 (~2 0 ,~ = 2.41 S, compared with the value 1.17 S reported for dealanine-deasparagine insulin which is considered to be a monomer) ; and the tendency to form polymers is increased in the presence of zinc ion [ 1,2].The introduction of succinyl groups into a protein has been shown in a number of cases t o reduce the tendency to aggregate [a-61. It was therefore considered of interest to examine the effect of succinylation, and of carbamylation, on the aggregation of insulin. By employing the technique of gel filtration, it was possible to study the molecular size of insulin and its derivatives a t a concentration below that normally required for ultracentifugal analysis. The results obtained provide indications of the molecular forms of insulin and some acyl derivatives under conditions which would be relevant to their administration. MATERIALS AND METHODSBovine zinc insulin, once crystallised, was obtained from Burroughs Wellcome Ltd (lot no. AN 38353N, moisture content 7.090/,, zinc content 0.340/,, biological activity 23.2 U/mg assayed by mouse convulsion). Zinc-free insulin was obtained by chromatography of zinc insulin on Sephadex A-25 with 0.09 M Tris-HC1 pH 7.5 in 8 M urea [3]. N~*1,NaB~,N~~~9-Trisuccinylinsulin Zinc-free insulin (100 mg) was dissolved in 5 ml 0.5 M Tris-acetete pH 8.5 in 8 M urea. To the stirred solution was added finely powdered succinic anhydride (7.5 mg) and incubation was carried out a t room temperature for 1 h. The succinylated protein was desalted on a column (30x2 cm) of Sephadex G-25 with O.lO/o (v/v) N-ethylmorpholine as eluent and the desalted protein was lyophilized. The succinylated insulin was chromatographed on a column (20x2 cm) of Xephadex A-25 in 0.15 M Tris-HC1 pH 7.0 in 8 M urea; after 1 5 0 d a linear gradient was applied to 0.27M Tris-HC1 in 8 M urea. The eluted derivatives were located by measurement of absorbance a t 280nm. Two major fractions were obtained ; NaAl, NaBWisuccinyhulin emerged from the co...