Abstract
Human myelin basic protein was fractionated into its various charge isomers by CM52 cation exchange chromatography. Approximately 25-30% of the total charge applied to the column appeared in the void volume. This material termed "C-8," was further purified by reversed phase high performance liquid chromatography. Amino acid analyses of C-8 revealed low Arg (7 residue % in C-8 compared to 11-12 residue % in C-1) and increased Glx residues. The low Arg was accounted for by a corresponding amount of citrulline. Sequence analysis after chemical fragmentation (cyanogen bromide and BNPS-skatole) and enzymatic (cathepsin D and carboxypeptidase S-1) digestion localized the citrulline at residues 25, 31, 122, 130, 159, and 170 of the amino acid sequence. The effect of this loss of positive charge on the ability of the protein to aggregate lipid vesicles was demonstrated with vesicles composed of phosphatidylcholine (92.2 mol %) and phosphatidylserine (7.8 mol %). C-1 was the most effective charge isomer, and C-8 was the least effective. The ability of these charge isomers to aggregate vesicles correlated with the net positive charge on each. Vesicles composed of phosphatidylcholine alone were not aggregated by lipophilin or any of the charge isomers. However, when lipophilin was incorporated into phosphatidylcholine vesicles (50% w/w), small, optically clear suspensions of vesicles were formed. None of C-1, C-2, or C-3 aggregated these vesicles, but C-8 produced rapid vesicle aggregation. Since the substitution of citrulline for Arg would generate several relatively long apolar sequences, these would enhance the ability of C-8 to interact with the hydrophobic lipophilin molecule, promoting vesicle aggregation by hydrophobic interactions. The mechanism by which citrulline is generated in myelin is not known, although enzymatic conversion has been described in other systems. Studies are underway to elucidate the mechanism by which this post-translational modification is generated.
Highlights
Sequence analysis after chemical fragmentation and enzymaticdigestion localized the citruIlineat residues 25,31,122,130,159, and 170 of the amino acid sequence
The mechanism by which citruliine is generated in myelin is not known, enzymatic conversion has been described in other systems
The myelin sheath is a multibilayer structure which surrounds axons in thecentral nervous system and i s composed of lipids and proteins in a ratio of about 3:l (1).The protein composition is relatively simple since two proteins, the basic
Summary
P r e ~ r a t ~of nProtein-Basic protein was isolated from normal human brain (age 46 years) white matter as described by Cheifetz et ai. (26). The microheteromers, components of BP, were prepared by added 0-100 pg of the protein in a final volume of 500 gl. After 10 min at room temperature, the turbidity of the solution was measured at 450 nm. The BP was dissolved in a urea-glycine buffer, p H 9.6; and applied on to a. 0-300 gg of B P components were added to vesicles containing 100pg of PC and 100 pgof lipophilin in a final volume of 500 J.The. CM52cellulosecation exchange column equilibrated in a urea-glycine turbidity of the solutions were measured at 450 nm after 10 min at buffer, pH 10.6. The components were eluted from the column using room temperature. A NaCl gradient (0-0.20 M) and desalted on a Bio-Gel P-2 column in 0.01 N HCI.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
