Mass-spectrometric analysis of ADP-ribosylation factors from bovine brain: identification and evidence for homogeneous acylation with the C14:0 fatty acid (myristate).

The two proteins from bovine brain previously shown to be required for the guanosine 5'-[gamma-thio]triphosphate-dependent inhibition of a well-characterized intra-Golgi transport assay, termed GGBF and GGBF, have been definitively identified as members of the ADP-ribosylation factor (ARF) family by electrospray MS analysis of the intact proteins, and of their tryptic fragments. Extensive protein-sequence information obtained from this analysis identified GGBF and GGBF as bovine ARF1 and ARF3 respectively. The sequence of bovine ARF3, which had not previously been determined, appears identical to that predicted from the rat and human ARF3 cDNAs. Further analysis of the N-terminal tryptic fragments of both bovine ARFs demonstrates N-terminal acylation solely with the C14:0 fatty acid (myristate). This finding establishes that the previously reported specific-activity difference between ARF1 and ARF3 in the intra-Golgi transport assay is not due to lipid heterogeneity at the N-terminus. This finding also indicates that the heterogeneity of N-terminal fatty-acyl groups previously observed on other myristoylated proteins is not universal.