The heavy chain of a human myeloma protein (Vin) belonging to the gamma4 subclass was subjected to tryptic digestion after reduction and carboxymethylation. Cyanogen bromide fragments were also prepared and all 19 tryptic peptides that account for one of them (the Fc-like fragment) were studied. Selected peptic peptides were isolated and provided evidence for the order of 15 of the tryptic peptides. In addition the sequence of two large peptic peptides derived from two sections of the molecule including all the interchain bridges is presented. Comparison with published data on other chains allows us to propose a sequence of gamma4 chains that extends from just before the presumed starting point of the invariable region (at about residue 113) to the C-terminal end of the chain (approx. residue 446), except for a section of about 50 residues. The results of the comparison suggest that the immunoglobulin subclasses have a recent independent evolutionary origin in different species. Implications for complement fixation and for the evolutionary origin of antibody diversity are also discussed.

PLATE LXXXII KUROTCHKIN (1937-38) and others have reported the formation of polysaccharide by Mycoplasma mycoides. Buttery and Plackett (1960) isolated, from washed organisms, a haptenic polysaccharide in a state of relative purity and characterised it as a galactan. Immunodiffusion studies (Plackett, Buttery and Cottew, 1963) revealed the presence in such preparations of several minor components in addition to the main component (P).Large amounts of polysaccharide were found also in culture media after growth of the organisms. A method for the recovery of this material from culture supernatants has been devised. The product behaves like P in serological tests, only a trace of a single minor component being detectable by immunodiffusion. For simplicity, we shall refer to galactan prepared from culture supernatant as galactan F, and to that prepared from washed ceUs as galactan C. Although P is the main component of both kinds of preparation, galactan C contains relatively more of the minor antigenic components detectable by immunodiffusion, whilst galactan F contains some non-specific material derived from constituents of the medium (Buttery, unpublished).During the acute phase of contagious bovine pleuropneumonia, component P is often present in the blood at concentrations high enough to cause the '' eclipse " of agglutination reactions, as described by Turner (1962). Galactan and other antigens, as yet unidentified, were found in body fluids and in urine by Gourlay (1962Gourlay ( , 1964. Two mycoplasms, L2917 and N29, isolated from cases of bovine arthritis, produce a different polysaccharide, a glucan (Plackett et a[.), which also may appear in the blood during infection. This paper describes some of the consequences of the parenteral injection of galactan F into cattle and rabbits, and, in particular, the effect that it has on the course of an infection produced by the subcutaneous inoculation of Mycoplasma mycoides. Some observations with glucan prepared from a culture of mycoplasm L2917 are included for comparison. MATERIALS AND METHODS Strains of mycoplasmIn this work, two strains of Mycoplasma mycoides and a mycoplasm isolated in Queensland from cattle affected with arthritis, and referred to as L2917, were used.The strains of M. mycoides were VS, of a virulence usually sufficient to provoke some local swelling when injected subcutaneously into susceptible cattle, and KH3J, a strain of African origin so low in virulence that no such local swelling is produced (Hudson, 1965). J. PATH. BACT.-VOL. 94 (1967) 257 258 J. R. HUDSON, S. BUTTERY A N D G. S. COTTEW Preparation of galactan F and glucan Cultures of mycoplasms grown in 1 5-litre batches of supplemented BVF-OS medium (Plackett et al.) were centrifuged in a steam-driven Sharples supercentrifuge.The supernatant was heated at 100°C for 45 min., 150 ml. glacial acetic acid was added to bring the pH to 5.5, and heating at 100°C was continued for 45 min. The mixture was filtered and 2 volumes of ethanol were added to the filtrate. The resulting precipit...

The galactan preparations were made as described in detail for galactan F by Hudson et al. (1967) except that the Sephadex G200 column formerly used (80 x 270 mm) was replaced with one 50 x 1000 mm, and that a pneumatically-driven Padberg supercentrifuge was used instead of a steam-driven Sharples supercentrifuge. Three batches of galactan F were made; these were identified as LS30, LS31, LS32. In the case of LS31 the supernate of the culture was heated before and not, as with LS30 and LS32, after acidification with glacial acetic acid; in other respects the procedures for preparation of the three batches were identical.Control solutions. Uninoculated culture medium (BVFOS) of the same type as that used in the preparation of galactan F was subjected to the same extraction procedures as those used for preparation of the galactan. This provided one of the control solutions injected intravenously into calves. The other control solution used was the 0.85% (w/v) NaCl in which freeze-dried galactan preparations were dissolved for intravenous inject ion.Testing for pyrogenic activity. This was done in conscious rabbits (2-6-3.3 kg) by the method described in the British Pharmacopoeia (1968) except that two rabbits were used for each preparation and rectal temperatures were monitored continuously with a thermistor probe (Yellow Springs Instrument Co., Yellow Springs, Ohio).Chemical analyses. Quantitative amino-acid analyses of the galactan preparations were made after hydroiodic acid hydrolysis (Inglis, Nicholls and Roxburgh, 1971). The method has been found suitable for preparations of high carbohydrate content (Nicholls and Inglis, personal communication). Total percentage of protein was calculated as the sum of the individual amino-acid contents of the preparation.Animal experiments. Most of the experiments were done on 41 calves that were 10-12 weeks old, and weighed 30-90 kg. They were of the Jersey, Hereford and Friesian breeds and crosses of the Jersey and Friesian breeds; the majority were females. All had been taken from their mothers 4 weeks before the experiments and were being maintained on reconstituted skim-milk powder and calf-concentrate pellets. All had begun ruminating. Experiments were undertaken both in conscious and anaesthetised calves.The experiments on conscious calves were done with the animals standing in a stall, or restrained in lateral recumbency on a table to minimise movement artefacts in electrocardiograms. A venous cannula (Bard, International Ltd, Sunderland) was introduced into one or both jugular veins, and in some experiments a polythene tube was introduced into the pulmonary artery, being passed from the right jugular vein via the right atrium and ventricle. The pressure profiles recorded from these saline-filled tubes on pressure transducers were used to identify the final sites of the tips of the cannulae and verification was obtained at necropsy. Standard limb leads were used, and recordings made of either lead I, I1 or I11 of the electrocardiogram (ECG). Respiratory movements ...