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Purification and Characterization of a Novel Intracellular Acid Proteinase From the Plasmodia of a True Slime Mold, Physarum Polycephalum

Purification and Characterization of a Novel Intracellular Acid Proteinase From the Plasmodia of a True Slime Mold, Physarum Polycephalum

K Murakami-Murofushi, T Takahashi, Y Minowa, S Iino, T Takeuchi, H Kitagaki-Ogawa, H Murofushi, K Takahashi

J Biol Chem. 1990 Nov 15;265(32):19898-903.

PMID: 2246266

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR4249422	2-Aminobenzoyl-Ala-Phe-Ala-Phe-Asp-Val-Phe-Tyr(NO2)-Asp			Price

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