Measurement of medium lysyl oxidase activity in aorta smooth muscle cells. Effects of multiple medium changes and inhibition of protein synthesis.
Cultures of rabbit aortic smooth muscle (RSM) cells are a valuable model system for studying production and metabolism of connective tissue components. This report describes various assay procedures for lysyl oxidase, the enzyme responsible for deaminating lysine residues to give aldehyde cross-link precursors, in culture medium from these cells. Studies of the medium enzyme from second-passage RSM cells indicate that approximately 40% of the total enzyme activity in the flask of cells is in the medium. The medium enzyme levels are replenished quite rapidly following refeeding, and enzyme levels in the medium appear to be feedback controlled. The mechanism for this control is unknown at present. Multiple refeeding experiments in which the medium was changed every 2-4 h for up to 40 h indicate that these cells are cap]able of producing large amounts of enzyme and are capable of altering enzyme production and secretion quite rapidly in response to changes in their environment. Protein synthesis inhibitor studies with cycloheximide suggest that the major portion of the enzyme released into the medium following refeeding is newly synthesized although a pool of latent enzyme is also present. As in intact tissue, extraction of the enzyme from the cell layer requires strong denaturing reagents such as 4 M urea. These results suggest that the production of lysyl oxidase is closely regulated and is very responsive to changes in the external environment of the cells. This cell culture system appears to be an excellent one to study the production of lysyl oxidase and its role in connective tissue fibrillogenesis.
D000594:Amino Acid Oxidoreductases / Q000032:analysis*; D000818:Animals; D001011:Aorta / Q000201:enzymology*; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003584:Cytological Techniques; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D008249:Protein-Lysine 6-Oxidase / Q000032:analysis*; D011817:Rabbits; D014158:Transcription, Genetic / Q000187:drug effects*
D003470:Culture Media; D003513:Cycloheximide; D000594:Amino Acid Oxidoreductases; D008249:Protein-Lysine 6-Oxidase