Immunoreactive Basic Fibroblast Growth Factor‐Like Proteins in Chromaffin Granules

To the Editors: Westermann et al. ( 1990) have reported the presence of immunoreactive basic fibroblast growth factor (bFGF)-like material in bovine chromaffin granules by using immuno-electronmicroscopy. Western blot analysis identified four major immunoreactive bFGFlike molecules (ir-bFGFs) in the extracts from bovine adrenal cortex, medulla, and isolated chromaffin granules. These molecules had molecular masses of approximately 18, 24, 30, and 46 kDa. In addition, the 46-kDa ir-bFGF was reported to be enriched in the extract from chromaffin granules compared to medulla and cortex. On this basis, the authors suggested that bFGF is stored in adrenal chromaffin granules and that the 46-kDa ir-bFGF might represent a novel precursor of the lower molecular weight ir-bFGFs. Because all the forms of bFGF identified so far are devoid of a classic signal sequence (Florkiewicz and Sommer, 1989), these findings raise the interesting possibility that the 46-kDa ir-bFGF might have the properties of a secretory protein. Recently, we too have attempted the characterization of ir-bFGF(s) in bovine adrenal gland. Some discrepancies between our findings and the above data have been found. Four ir-bFGFs with molecular masses of approximately 18, 22, 24, and 42 kDa were detectable when 100-400 pg of the postnuclear supernatant of a bovine adrenal medulla extract was probed in a Western blot with a polyclonal antiserum raised against recombinant human bFGF. The 18-, 22-, and 24-kDa ir-bFGFs possibly correspond to the high molecular weight bFGFs already described (Florkiewicz and Sommer, 1989), whereas the 42-kDa ir-bFGF likely corresponds to the 46-kDa protein identified by Westermann et al. (1990). Since no forms of bFGF with molecular masses higher than 25 kDa have yet been characterized, a further investigation of the 42-kDa irbFGF was attempted. Unlike other bFGFs (Moscatelli et al., 1987), this molecule does not bind to heparinSepharose beads, does not stimulate plasminogen activator production in endothelial cells, and does not compete with '251-bFGF for binding to the bFGF cell membrane receptor after separation from the other irbFGFs by gel filtration FPLC chromatography. Finally, the 42-kDa ir-bFGF was not recognized by affinitypurified anti-bFGF antibodies in a Western blot. Indeed, when the bFGF antiserum was immunopurified on a recombinant bFGF column, the 42-kDa protein cross-reacted with the irrelevant immunoglobulins present in the flow through of the column but not with the affinity-purified antibodies. Thus, our data suggest that the 42-kDa ir-bFGF may not represent a form of bFGF. We have also investigated the localization of irbFGFs in adrenal medulla by subcellular fractionation (Smith and Winkler, 1967). At variance with the results of Westermann et al. (1 990) is the fact that when 100400 pg of a chromaffin granule extract were probed with the anti-bFGF antiserum, only the 18-, 22-, and 24-kDa ir-bFGFs, but not the 42-kDa ir-bFGF, could be detected. The 42-kDa ir-bFGF was quantitatively recovered in the cytosolic fraction (100,000 g supernatant) of the medulla extract. Thus, while our data confirm the finding that some ir-bFGFs are possibly associated with chromaffin granules in bovine adrenal medulla, they failed to confirm an association of the 42-kDa ir-bFGF with these organelles. The discrepancy between our findings and those of Westermann et al. may derive from the very low recovery of ir-bFGF material achieved by these authors during the isolation of the chromaffin granules and a possible significant contamination by cytosolic 42-kDa ir-bFGF. Indeed, this molecule appears to be present in extracts of adrenal medulla at concentrations at least 10-fold higher than the other ir-bFGFs. In conclusion, our experiments suggest that the 42kDa ir-bFGF is a cytosolic protein not related to bFGF. We believe that the important issues raised by the work of Westermann and colleagues on the presence, characterization, and mechanism of entry of ir-bFGF molecules in chromaffin granules deserve further investigation.