Biogenesis of chromaffin granules
Kilpatrick, Lynn Agnes
Bovine adrenal medullary messenger RNA was isolated and translated in optimised reticulocyte lysate and wheatgerm cell-free translation systems. Reticulocyte lysate was found to be the superior translation system. An attempt was made to characterize the primary polypeptide precursors to the major chromaffin granule secretory protein, chromogranin A, and to the two major chromaffin granule membrane proteins, dopamine β-hydroxylase and cytochrome b561 using previously prepared and well-characterized antisera raised against these proteins. Two similar polypeptides of 70,000 and pI of about 5.2 were immunoprecipitated from the translation products by the antiserum against chromogranin A. When reticulocyte lysate was supplemented with dog pancreas microsomes, one polypeptide of slightly lower which was translocated into the lumen of the microsomal vesicles as determined by alkaline washing, was immunoprecipitated by this antiserum. Precursors to chromogranin A were subsequently identified from the polypeptide products when reticulocyte lysate was supplemented with adrenal medullary bound polysomes or rough microsomes. The effects of post-translational processing on chromogranin A were observed during the cellular synthesis of chromogranin A, during which chromogranin A becomes more heterogeneous with respect to pi and it is concluded that the smaller members of the chromogranin A family result from the action of intragranular proteolysis on chromogranin A during the maturation and storage of the granules. Two polypeptides of 72,000 and 46,000, were immunoprecipitated from translation products by the antiserum raised against the soluble form of dopamine β-hydroxylase. The 46,000 dalton polypeptide is most likely a breakdown product of the 72,000 dalton polypeptide. When reticulocyte lysate was supplemented with dog pancreas microsomes, a polypeptide of 67,000 daltons was immunoprecipitated by the antiserum to dopamine β-hydroxylase. This polypeptide was translocated into the lumen of the microsomal vesicles as determined by phase separation of the microsomes with Triton X—114. Thus, the soluble form of dopamine β-hydroxylase would appear to be synthesized as a precursor of 72,000 daltons which, on removal of its signal sequence, is reduced to a 67,000 dalton polypeptide. Antisera were prepared against various chemically and enzvmatically modified forms of cytochrome b561 in an attempt to immunoprecipitate the polypeptide precursor to this protein from translation products. However, all attempts to identify the precursor to this protein were unsuccessful. An extensively-labelled small acidic translation product was tentatively identified as calmodulin.