Predominant low-molecular-weight proteins in isolated brain capillaries are histones.
Blood-brain barrier (BBB) function is endowed by the expression of unique proteins within the brain capillary endothelium. In the absence of knowing the function of BBB-specific proteins, one strategy for identification of these proteins is the purification and amino acid sequencing of proteins within the brain capillary that are not found in other cells. Earlier studies have shown that a 16-18K triplet of low-molecular-weight proteins in isolated brain capillaries is not found in either erythrocytes or in capillary-free preparations of synaptosomal proteins. Therefore, the present studies describe the purification of the 16-18K triplet of proteins as well as a 14K protein in isolated brain capillaries using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and C4 reverse-phase HPLC. Amino acid sequencing of the N-terminus of the 14K, 17K, and 18K proteins and of two tryptic peptides of the 16K protein showed that these proteins are alpha-globin, histone 2B, histone 3, and histone 2A, respectively. SDS-PAGE of subcellular fractions of bovine brain capillaries demonstrated that the 16-18K triplet of histone proteins migrated in the nuclear fraction. In addition, a 34K doublet and a 200K protein were localized in the nuclear pellet. Therefore, the present studies demonstrate that the predominant 14-18K proteins seen on SDS-PAGE of isolated brain capillaries are known proteins and provide a general scheme for purification of brain capillary proteins isolated following SDS-PAGE.
Department of Medicine, UCLA School of Medicine 90024.
W M
WM
Pardridge
D M
DM
Nowlin
J
J
Calaycay
J E
JE
Shively
D000595:Amino Acid Sequence; D000818:Animals; D002196:Capillaries / Q000032:analysis*; D002417:Cattle; D002560:Cerebrovascular Circulation; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight
D006657:Histones