title: Purification and characterization of a novel intracellular acid proteinase from the plasmodia of a true slime mold, Physarum polycephalum.
abstract: An acid proteinase was purified to apparent homogeneity from the plasmodia of a slime mold, Physarum polycephalum, by a combination of detergent extraction, acid precipitation, and column chromatographies on DEAE-Sephadex, hydroxylapatite, CM-Sephadex, and Sephadex G-100. The enzyme was shown to be composed of two polypeptide chains (a 31-kDa heavy chain and a 23-kDa light chain) cross-linked by disulfide bond(s). The NH2-terminal amino acid sequence of the heavy chain was determined to be Ala-Gly-Val- Asp-Gly-Tyr-Ile-Val-Pro-Tyr-Val-Ile-Phe-Asp-Leu-Tyr-Gly-Ile-Pro-Tyr and that of the light chain to be Ala-Glu-Pro-Pro-Ile. The heavy chain contained carbohydrate moiety composed of mannose, glucosamine, fucose, and glucose. The enzyme was optimally active at pH 1.7 toward hemoglobin as a substrate. Among the proteinase inhibitors tested only diazoacetyl-D,L-norleucine methyl ester, a typical aspartic proteinase inhibitor, inhibited the acid proteinase in the presence of cupric ions. It was insensitive to the other typical aspartic proteinase inhibitors, pepstatin A and 1,2-epoxy-3-(p-nitrophenoxy)propane. The enzyme hydrolyzed Lys-Pro-Ile-Glu-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond, but could not hydrolyze another synthetic pepsin-substrate, N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine. The enzyme showed a unique substrate specificity toward oxidized insulin B chain. The major cleavage sites were the bonds Gly8-Ser9, Leu11-Val12, Cya19-Gly20, and Phe24-Phe25, and the Gly8-Ser9 bond was most susceptible. These results indicate that the enzyme is a novel type of intracellular acid proteinase with a unique substrate specificity.
authors: Department of Biology, Faculty of Science, Ochanomizu University, Tokyo, Japan.
authors: K
authors: K
authors: Murakami-Murofushi
authors: T
authors: T
authors: Takahashi
authors: Y
authors: Y
authors: Minowa
authors: S
authors: S
authors: Iino
authors: T
authors: T
authors: Takeuchi
authors: H
authors: H
authors: Kitagaki-Ogawa
authors: H
authors: H
authors: Murofushi
authors: K
authors: K
authors: Takahashi
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002845:Chromatography; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D010804:Physarum / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D002241:Carbohydrates; D007328:Insulin; D010455:Peptides; D011480:Protease Inhibitors; D010450:Endopeptidases