Identification and characterization of a cathepsin B-like protease in Physarum sclerotium.
In response to dry stress the plasmodium of a true slime mold, Physarum polycephalum, undergoes formation of sclerotium, which is a dormant body resistant to desiccation. The sclerotium can germinate within several hours after addition of water, followed by generation of the plasmodium. In the early phase of the germination many enzymes and other proteins of the sclerotium are required for formation of the plasmodium. As dehydration of proteins often leads to destruction of their structure or reduction in their activity, it is important to elucidate whether the dehydrated enzymes are present as the intact in the sclerotium. In this study three peaks of protease activity were detected with anion exchange column chromatography of the extract from the sclerotia. From among them, an acid protease was purified to homogeneity by gel filtration column chromatography, hydroxyapatite column chromatography, acid treatment, and cation-exchange column chromatography. Treatment of the protease fractions with pH 4.0 resulted in approximately 20-fold activation of the activity. The purified protease was a monomer with a molecular mass of 35 kDa. The optimum pH and temperature were 6.3 and 40 degrees C, respectively. Beta-casein, histone H1, and H2B were degraded by the 35 kDa protease, but human hemoglobin and human serum albumin were very poor substrates. In addition, the enzyme was sensitive to the cysteine protease inhibitors chymostatin, E-64, and leupeptin. These results indicate that, in the sclerotium, a premature form of a cathepsin B-like protease remains non-denatured under dehydrated conditions.
Furuhashi
Kiyoshi
K
Department of Biology, Faculty of Education, Shiga University, Hiratsu 2-5-1, Otsu, Shiga 520-0862, Japan. furuhasi@sue.shiga-u.ac.jp
D000818:Animals; D002401:Cathepsin B / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008970:Molecular Weight; D010804:Physarum / Q000201:enzymology*; D013379:Substrate Specificity
D002401:Cathepsin B