Purification and characterization of a cytosol protease from dormant cysts of the brine shrimp Artemia.
A thiol protease has been isolated and purified from the postribosomal fraction of encysted embryos of the brine shrimp Artemia using a six-step procedure. The purified enzyme has a molecular weight of 55,000 +/- 4,200 and is composed of subunits of Mr 31,500 +/- 559 and 25,867 +/- 1,087. Isoelectric focusing revealed two discrete bands, one at pH 4.6 and the other at pH 5.1. The protease appears to be a member of the thiol group of proteases based on its inhibition by leupeptin, antipain, chymostatin, Ep-475, and several other thiol protease inhibitors. The enzyme was stimulated by heavy metal chelators and thiol reagents. At pH 3.5-4.0 the thiol protease hydrolyzed a wide range of proteins including bovine serum albumin, hemoglobin, Artemia embryo soluble proteins, Artemia lipovitelline, and protamine, whereas at pH 6.0-6.5 the enzyme showed a high degree of specificity for Artemia elongation factor 2 and lipovitelline alpha 1. The total amount of protease activity in crude homogenates of Artemia embryos decreased by about 50% during the first 24 h of development, while the amount of free, active enzyme decreased proportionally for 9 h of development then remained constant during the next 26-27 h of development. These changes in protease activity appear to reflect changing levels of an endogenous protease inhibitor during development.
D000818:Animals; D001156:Artemia / Q000201:enzymology*; D003546:Cysteine Endopeptidases; D003600:Cytosol / Q000201:enzymology; D004527:Egg Proteins / Q000378:metabolism; D015387:Egg Proteins, Dietary; D010450:Endopeptidases / Q000302:isolation & purification*; D006454:Hemoglobins / Q000378:metabolism; D007526:Isoelectric Point; D008970:Molecular Weight; D011479:Protamines / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism
D004527:Egg Proteins; D015387:Egg Proteins, Dietary; D006454:Hemoglobins; D011479:Protamines; D012710:Serum Albumin, Bovine; C001549:lipovitellin; D010450:Endopeptidases; D003546:Cysteine Endopeptidases