Purification and characterization of extracellular cysteine protease inhibitor, ECPI-2, from Chlorella sp.
An extracellular cysteine protease inhibitor (ECPI-2) was purified to homogeneity from the culture filtrate of Chlorella sp. 4533 by the combination of various column chromatographies. The molecular mass of the inhibitor was estimated to be 340 kDa by SDS-PAGE. The inhibitor was extremely heat-stable under acidic or neutral condition. ECPI-2 exhibited an inhibitory activity against the proteolytic activity of papain, ficin, or chymopapain, but not against stem bromelain or cathepsin B. The inhibitor showed no inhibitory activity against trypsin, alpha-chymotrypsin or thermolysin. ECPI-2 contains 33.6% carbohydrate residues by weight and inhibits papain at a molar ratio of 1:2. The proteolysis of the inhibitor by trypsin or alpha-chymotrypsin was apparent, but the inhibitory activity of ECPI-2 was unaffected by these enzymes. The alpha-chymotrypsin hydrolysis product from ECPI-2 was further separated into six fractions by gel filtration. From these results, it is suggested that ECPI-2 has several reactive sites for papain.
Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Okinawa 903-0213, Japan. mishiha@agr.u-ryukyu.ac.jp
Masanobu
M
Ishihara
Tomoaki
T
Shiroma
Toki
T
Taira
Shinkichi
S
Tawata
D020418:Algal Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D001963:Bromelains / Q000037:antagonists & inhibitors; D002401:Cathepsin B / Q000037:antagonists & inhibitors; D002708:Chlorella / Q000737:chemistry*; D002917:Chymopapain / Q000037:antagonists & inhibitors; D002918:Chymotrypsin / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D005361:Ficain / Q000037:antagonists & inhibitors; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D010206:Papain / Q000037:antagonists & inhibitors; D020539:Sequence Analysis, Protein; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D020418:Algal Proteins; D015853:Cysteine Proteinase Inhibitors; D001963:Bromelains; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D014357:Trypsin; D002401:Cathepsin B; D010206:Papain; D005361:Ficain; C091760:stem bromelain; D002917:Chymopapain