AP30, A DIFFERENTIAL PROTEIN MARKER FOR PERILYMPH AND CEREBROSPINAL-FLUID IN MIDDLE-EAR FLUID, HAS BEEN PURIFIED AND IDENTIFIED AS HUMAN APOLIPOPROTEIN-D

Using two-dimensional (2-D) gel electrophoresis, human perilymph and c erebrospinal fluid have been shown to be highly enriched for an acidic protein with Mr 30 000, we designated it as AP30. The protein exhibit s charge heterogeneity, with at least eight isoforms visible between p I 4.5 to 5.5 on 2-D gels. Purification of the protein was carried out by ammonium sulfate precipitation, polybuffer exchanger column chromat ofocusing, and acetone fractional precipitation. The resulting prepara tion also contains eight spots in the acidic area of 2-D gels, and one broad band located at Mr 30 000 by SDS-PAGE. Digestion of AP30 with n euraminidase causes the isoforms to shift to a more basic position and to consolidate into two primary spots, indicating that AP30 is a vari ably sialylated glycoprotein. Amino acid analysis of AP30 revealed an amino acid content very similar to that of human apolipoprotein D. Att empts to determine the amino acid sequence demonstrated that the N-ter minus is blocked. Edman sequencing of two peptide fragments, generated by cyanogen bromide cleavage of AP30, both revealed sequences having 100% identity to human apolipoprotein D. Western blot analysis of AP30 with the antibody against authentic human apolipoprotein D demonstrat ed a high degree of cross-reactivity. These studies indicate that AP30 from human perilymph and cerebrospinal fluid is a member of the apoli poprotein D family. (C) 1998 Elsevier Science B.V. All rights reserved .