The heterogeneity of the trypsin‐resistant protein residue from brain white matter

TRYPsIN-resistant brain proteins have been classified into two categories : (1) the proteolipids, which are extracted from the tissue by chloroform-methanol (FOLCH and LEES, 1951), and (2) a trypsinresistant protein residue (TRPR) which is isolated from the chloroform-methanol-insoluble residue as a fraction insoluble in either aqueous or organic solvents (LEBARON and FOLCH, 1956). This lack of solubility has hampered previous attempts to characterize TRPR. Recently, however, we noted that TRPR became soluble in an aqueous medium upon reduction of disulphide linkages and alkylation of its sulphydryl groups (LESTON and LEES, 1969). Consequently, further studieson the composition of TRPR became feasible. In this communication, we present evidence that TRPR is a mixture consisting mainly of denatured proteolipid protein but contaminated with (1) collagen, (2) other insoluble protein or peptide material and (3) nucleic acids. TRPR is described in the literature as a specific entity and continues to be discussed as such (WOLMAN, 1969). Our findings support a previous suggestion that TRPR is a heterogeneous fraction (WOLFGRAM and ROSE, 1962). Data demonstrating the heterogeneous nature of the TRPR obtained from CNS white matter, however, have not previously been published. TRPR was prepared by the method of LEBARON and FOLCH (1956) except that digestion was carried out for 4 days at pH 8.5 with use of crude trypsin (Sigma Chemical Co., St. Louis, Mo.). No further release of free amino groups occurs after that period of time (LEBARON, 1951 ; LEES, unpublished). A total of four additions of trypsin was made; the pH was adjusted with NaOH twice a day for the first 2 days and daily thereafter. Various preparations contained 1.0-1.7 per cent P, most of which was identified as a mixture of phosphatidyl inositols by TLC. The major portion of the phosphorus could be removed in the form of a phosphatidopeptide fraction by extraction with acidified chloroform-methanol (CHC13-CH30H<onc. HCI, 200: 100: 1, by vol.). Some 85-95 percent of the TRPR could be dissolved in 1 %(w/v)aqueous sodium dodecyl sulphate (SDS) containing mercaptoethanol buffered to pH 9 with triethylamine (TEA). The small amount of material which always remained insoluble was collagen-like in nature: it contained hydroxyproline and hydroxylysine, and its amino acid composition was essentially identical to that of collagen (Table 1). A comparable residue was obtained after solution of the major portion of TRPR in 80% (w/v) phenol. Although the early experiments suggested that reduction was a prerequisite for dissolving TRPR (LESTON and LEES, 1969), we later found that the TEA buffer which provided the alkaline medium for reduction was, by itself, an effective solvent. The amount of TRPR which dissolved was a function of the concentration of TEA; as the TEA concentration was increased, more TRPR went into solution (Table 2). However, in the presence of a specific amount of TEA (3.5 x mol per 5 ml of H,O) the percentage of TRPR (57 per cent f 6; n = 8) which dissolved was independent of protein concentration over an eight-fold range (5-40 mg of TRPR), an observation suggesting that the material was heterogeneous. The solubilization by TEA was not attributable to its alkaline pH, since NaOH at a comparable pH dissolved only minimal amounts of TRPR. The high extinction exhibited by TEA in the U.V. region precluded investigation of the spectrum of TRPR in TEA. We observed considerable variability in both yield and behaviour of different TRPR preparations. Certain preparations dissolved to a considerable extent in 1 % (w/v) SDS. The U.V. spectra of these preparations exhibited a peak at 260 nm that suggested the presence of nucleic acids. After treatment of the sample by procedures for the removal of nucleic acids (5 % (w/v) TCA at 100°C for 15 min or 1.6 N-perchloric acid at 37°C overnight), the extinction peak was shifted to 280 nm. The treatment with acid also extracted most of the phosphatidopeptide phosphorus.