THE ISOLATION FROM BRAIN TISSUE OF A TRYPSIN‐RESISTANT PROTEIN FRACTION CONTAINING COMBINED INOSITOL, AND ITS RELATION TO NEUROKERATIN *

INTRODUCTION IT has been well established that meso-inositol exists in brain in both free and combined states. More than fifty years ago, THUDICHUM (1901) reported the presence of free inositol, and this observation has since been amply confirmed. More recent work (FOLCH and WOOLLEY, 1942) demonstrated the OcCuITence of inositol combined in brain lipides, and resulted in the isolation from brain of diphosphoinositide (FOLCH, 1949), a phosphatide which contains inositol metadiphosphate as a constituent. Results obtained in this laboratory in the course of work on the nature of neurokeratin have established the presence in brain tissue of a second form of combined inositol, namely, that of a phosphoinositide combined to protein. This paper reports these findings and some other observations bearing on the nature of neurokeratin. Preliminary reports of this work have already appeared (FOLCH and LEBARON, 195 1 , 1953). Neurokeratin is a fraction of brain proteins first isolated by EWALD and K u m (1877) which can be summarily described as a proteolytic enzyme-resistant protein residue obtained primarily from nerve tissue myelin, and which has traditionally been considered to be associated with the protein network of the myelin sheath. The classical procedure for preparing neurokeratin consists essentially of two major operations; namely, the quantitative removal of lipides with organic solvents, and the subsequent treatment of the proteins thus obtained, with digestive enzymes. The procedure takes several months, and involves Such drastic steps as continuous extraction with hot ethanol for four weeks, digestion with pepsin in dilute hydrochloric acid for three weeks, and, in some of its versions, treatment with hot alkali. The product is a protein residue insoluble in water and in dilute acids and bases, rich in sulphur, and resistant to the action of proteolytic enzymes. The preparations obtained by various workers differed somewhat in composition; and BLOCK (1951) concluded that neurokeratin is most likely an artifact, derived from a definite group of substances specific to nervous tissue. The work which led to the isolation of the protein fraction containing combined inositol was an attempt to develop a method of preparation of neurokeratin less