title: Effects of ATP, vanadate, and molybdate on cathepsin D-catalyzed proteolysis.
abstract: The effects of ATP, vanadate, and molybdate on cathepsin D-catalyzed hydrolysis of proteins and peptides were examined. Hydrolysis of bovine serum albumin, hemoglobin, parathyroid hormone, and a synthetic octapeptide was activated by ATP. Degradation of the protein substrates all had similar ATP concentration dependence, but the magnitude of the activation varied. Kinetic constants for ATP activation were obtained with a synthetic substrate. ATP increased kcat from 0.4 to 2 s-1 but did not change KM. Kact for ATP was 800 microM. Studies with pepstatin-Sepharose confirm that ATP does not alter the substrate binding site on cathepsin D. Pepsin, a homologous aspartate protease, was not activated by ATP. It was also found that vanadate and molybdate inhibit cathepsin D-catalyzed proteolysis. However, this inhibition was dramatically dependent on substrate concentration and was eliminated at high substrate. Hydrolysis of the synthetic peptide was not inhibited at concentrations of molybdate below 50 microM, and above this concentration the peptide precipitated. Protein substrates were also found to precipitate in the presence of molybdate. The ATP dependence of the enzyme was not altered by molybdate or vanadate. These results suggest that inhibition by vanadate and molybdate is related to interactions with the substrate rather than with cathepsin D. It is concluded that ATP activation of cathepsin D may play a physiological role in regulation of proteolysis in lysosomes, but that vanadate and molybdate inhibition of lysosomal proteolysis does not establish ATP dependence.
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002402:Cathepsin D / Q000378:metabolism*; D004789:Enzyme Activation; D006454:Hemoglobins / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008982:Molybdenum / Q000494:pharmacology*; D010281:Parathyroid Hormone / Q000378:metabolism; D010436:Pepstatins / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology*
chemical_list: D006454:Hemoglobins; D010281:Parathyroid Hormone; D010436:Pepstatins; D014639:Vanadium; C014195:Streptomyces pepsin inhibitor; C044659:molybdate; D012710:Serum Albumin, Bovine; D014638:Vanadates; D008982:Molybdenum; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D002402:Cathepsin D; C031375:pepstatin