Rat myelin basic proteins: relationship between size differences and microheterogeneity

RECENTLY we reported the separation of the basic protein of rat CNS myelin into two fractions after repeated gel filtration on Sephadex G-100 (MARTENSON, DEIBLER and Jhs, 19704. Each fraction appears to be homogeneous when examined by gel Ntration and by electrophoresis at acid pH in 5 and 15 % (w/v) polyacrylamide gels. These two fractions, with molecular weights of approximately 17,000 and 14,000 daltons (DEIBLER, MARTENSON and KIES, 1970), differ in amino acid composition and encephalitogenic activity. Earlier studies have shown that the total basic protein of myelin extracted from rat (JNS tissue could be resolved into six components by electrophoresis in polyacrylamide gel at alkaline pH in the presence of urea (MARTENSON, DEIBLER and KIEs, 19693). Furthermore, MARTENSON and GAITONDE (1969~) observed that the length of exposure of the initial homogenate of rat brain to chloroformmethanol influences the relative amounts of the individual components in the high-pH electrophoretic patterns. The possibility that this variation reflects the breakdown of larger to smaller components mediated by an incompletely denatured tissue proteinase was considered but could not be proven. Inasmuch as the total basic protein from rat myelin is heterogeneous with regard to both size and charge, we sought to establish the relationship of the several electrophoretic components resolved at neutral and alkaline pH to the larger and smaller molecular-size fractions separated by gel filtration. The basic protein of rat myelin was isolated from spinal cord pretreated with chloroform-methanol and separated on the basis of molecular size into its two fractions as previously described (MARTENSON e f ul., 1970~). Electrophoresis was carried out in 5 % (w/v) persulphate-polymerized gels containing 8 M-urea and either 1 M-acetic acid (pH 2.4) (MARTENSON et ul., 1970a), 0.05 M-ammonium acetate (pH 7.0) or 0.05 M-glycine-NaOH buffer (pH 10.6) (MARTENSON and GAITONDE, 1969~). Electrophoresis in the absence of urea was carried out in 5 % (w/v) riboflavin-photopolymerized gels with 0.05 M glycine-NaOH buffer (pH 8.6) (MARTENSON and GAITONDE, 1969~). The results of electrophoretic analyses carried out at different pH values are illustrated in Figs. 1 and 2. At acid pH in 8 M-urea (Fig. 1A) each of the fractions (designated L and S for larger and smaller molecular size) appeared to be essentially homogeneous and, when present as a mixture (T), were readily separated electrophoretically. However, at neutral pH in 8 M-urea (Fig. 1B) each fraction was resolved into multiple components. Moreover, some components of fraction L appeared to be superimposable upon those of fraction S in the electrophoretogram of the total protein (T). Somewhat better resolution within each fraction was achieved at pH 8.6, in the absence of urea, in riboflavin-photopolymerized gels (Fig. lC), providing that the electrophoresis was carried out for a sufficient length of time. In the latter case, electrophoresis was carried out in the absence of urea to preclude possible carbamylation of the protein(s) (COLE and MECHAM, 1966). Likewise, persulphate was omitted to prevent oxidation of the proteins (FANTES and FURMINGER, 1967). At high pH in 8 M-urea (Fig. 2) good resolution was achieved with a relatively short migration. Under these conditions components 1, 2 and 4 observed upon electrophoresis of T were contributed by fraction S. Components 3, 5 and 6 were contributed by fraction L. The basic protein of myelin isolated from representatives of four mammalian orders (man, ox, rabbit and guinea pig) each consists of a group of very closely related molecules (MARTENSON and GAITONDE, 1969b); MARENSON, DEIBLER and ~ E s , 1969~). Fractionation of the guinea pig protein on carboxymethylcellulose at alkaline pH yields chromatographic components identical to the Components observed upon electrophoresis at pH 10.6 (MARTENSON, DEIBLER and a s , 19706). The apparent homogeneity of the mixture when it is subjected to gel electrophoresis at acid pH can be explained either by the components having identical net charge at this pH or by the differences in net charge being too small in comparison with the total net charges on the molecules for them to be resolved. We have shown here that the basic protein of myelin isolated from the rat consists of two groups of closely related molecules. One group (L) that is composed of molecules of mol. wt. 17,000 is very similar with respect to molecular size, amino acid composition, and encephalitogenic activity to the group found in other mammals (DEIBLER et al., 1970; MARTENSON et a/ . , 1970~). Moreover, its electrophoretic pattern at high pH is indistinguishable from that of other mammals (MARTENSON et a/ . , 1970b). The other group (S) that is composed of molecules of mol. wt. 14,000