Biochemical and morphological changes in the subcellular fractions during myelination of rat brain.

Encephalitogenic basic protein and the enzyme nucleoside 2' :3'-cyclic monophosphate 3'-phosphodiesterase (EC 3.1.4.16) are recognized markers for myelin. Phosphodiesterase activity can be detected in the brains of newborn rats even though myelin cannot be seen by light or electron microscope. The determination of phosphodiesterase activity and the detection of basic protein by Coomassie-Blue staining in polyacrylamide gels (Sabri et al., 1974) has proved to be a very sensitive method of detecting myelin components in the subcellular fractions of immature rat brain, particularly during the early stages of myelination. Subcellular fractions of the rat brain (age &20 days) were prepared as described by Sabri et al. (1974) and minor modifications used for the preparation of purified myelin membranes (Norton & Poduslo, 1973). The distribution of myelinmarker enzyme activity and the electrophoretic separation of proteins in sodium dodecyl sulphate-polyacrylamide gels in subcellular fractions show that in the early stages of postnatal brain development (0-5 days after birth) phosphodiesterase (Table 1) and the encephalitogenic basic protein are localized in the microsomal and the myelinlike membrane fractions. This suggests that the microsomal fraction contains precursor membrane fragments at an early stage in the sequence of myelination from myelin-like membranes to compact myelin. This hypothesis was tested in metabolic studies, by using [3H]lysine injected intraperitoneally into 20-day-old rats (1 pCi/g body wt.) and by fractionating the brain at different intervals (5 h-51 days) after the injection. Myelin and myelin-like fractions were prepared and specific radioactivity was determined; 5 h after