Processing of prothrombin in the secretory pathway.
Antibodies raised against plasma prothrombin and the prothrombin propeptide were used to identify prothrombin precursors in rough and smooth microsomes and in the Golgi apparatus. The data demonstrate that the propeptide is part of the prothrombin molecule when undergoing a variety of modifications in the Golgi apparatus. It is shown that these modifications result in an increase in the apparent molecular mass of the prothrombin precursor from 78 kDa in early processing to 83 kDa in late processing. The 83 kDa prothrombin precursor was not recognized by the anti-propeptide antiserum and most likely represents the final product of the precursor in the secretory pathway. Evidence is presented that the propeptide is released from the parent molecule in the Golgi apparatus by a membrane-bound Ca(2+)-dependent serine proteinase(s) with characteristics similar to those of the proalbumin-to-albumin-converting enzyme. Vitamin K-dependent carboxylase activity was measured in membrane fragments obtained from the Golgi apparatus preparation. Sucrose-density-gradient centrifugation and the use of marker enzymes showed that carboxylase activity was highest in fractions enriched in cis-Golgi cisternae. Two different synthetic peptides were used as substrates for the carboxylase. These peptides were from the N-terminal and the C-terminal part of the gamma-carboxyglutamic acid (Gla) region of prothrombin. It is shown that the N-terminal and the C-terminal peptides were preferred as substrates for the carboxylase in the microsomal and the Golgi apparatus preparations respectively. It is also shown that the prothrombin precursor acquires negative charges in the Golgi apparatus that do not result from addition of sugars in late processing. These negative charges could be eliminated by thermal decarboxylation, suggesting that Gla residues may also be synthesized in late processing.
Stanton
C
C
Department of Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27103.
Taylor
R
R
Wallin
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D006056:Golgi Apparatus / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011516:Prothrombin / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D010455:Peptides; D011516:Prothrombin