Vitamin K dependent carboxylase: subcellular location of the carboxylase and enzymes involved in vitamin K metabolism in rat liver.
Vitamin K dependent carboxylation of an exogenous peptide substrate and endogenous protein substrates, vitamin K epoxidation, and reduction of vitamin K epoxide were measured in subcellular fractions from rat liver. The rough microsomal fraction was highly enriched in all four activities; lower levels were found in smooth microsomes. Mitochondria, nuclei, and cytosol had negligible activities. The addition of 0.2% Triton X-100 to intact microsomes resulted in a 10-20-fold stimulation in carboxylation of a peptide substrate. This marked latency suggests that the active site of the carboxylase may be accessible only from the lumen of the microsomal membrane. A lumen-facing orientation of the carboxylase was also supported by its inaccessibility to trypsin in intact microsomes contrasted with marked inhibition by trypsin in detergent-permeabilized microsomes. Vitamin K epoxidase and epoxide reductase activities were also inhibited by trypsin much more effectively in permeabilized than in intact microsomes, although some degree of exposure at the cytosolic surface was also indicated. These data suggest that carboxylation is an early event in prothrombin synthesis occurring primarily on the lumen side of the rough endoplasmic reticulum membrane. The location of the vitamin K epoxidation-reduction cycle enzymes is consistent with their possible role in the carboxylation reaction.
D000818:Animals; D019735:Carbon-Carbon Ligases; D005952:Glucose-6-Phosphatase / Q000032:analysis; D008025:Ligases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008297:Male; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D014357:Trypsin / Q000494:pharmacology; D014808:Vitamin D Deficiency / Q000201:enzymology*; D014812:Vitamin K / Q000378:metabolism*
D014812:Vitamin K; D011092:Polyethylene Glycols; D017830:Octoxynol; D005952:Glucose-6-Phosphatase; D014357:Trypsin; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase