Immunoblotting Analysis of Plasma Protein Processing in the Secretory Pathway of Rat liver: Identification of Proteolytic Conversion Sites of Complement Pro-C3 and Prohaptoglobin1

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Abstract
Using an immunoblotting technique, we have studied the processing of plasma proteins in subcellular fractions of rat liver including rough and smooth micro-somes and the Golgi subtractions. Each subcellular fraction was directly subjected to SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies against α1-protease inhibitor, haptoglobin, and the third component of complement (C3) in combination with 125I-protein A or 125I-rabbit anti-(goat IgG)-IgG. The results demonstrated that proteolytic processing of precursors of complement C3 and haptoglobin occurs in different compartments along the secretory pathway; conversion of prohaptoglobin takes place in the endoplasmic reticulum, while that of pro-C3 occurs in the Golgi complex. The processing in oligosaccharide chains of glycoproteins was also analyzed. The Golgi fraction was characterized by the presence of the mature 56 kDa α1-protease inhibitor, which was indistinguishable from the serum α1-protease inhibitor in SDS-polyacrylamide gel electrophoresis. In contrast, the immature 51 kDa form was the only form of α1-protease inhibitor found in the microsomal fraction. Similar results were obtained for the β subunit of haptoglobin; the immature 33 kDa form was detected in the microsomal fraction, while the mature 36 kDa form was found in the Golgi fraction. Taken together, these results identified the intracellular sites where these plasma proteins are modified by selective proteolysis and/or glycosylation.

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