Characterization of two phosphodiesterase components in an extract of larch forest soil

Abstract

A phosphodiesterase-active extract obtained from a larch forest soil by phosphate buffer extraction was fractionated by DEAE-cellulose column and affinity column chromatography. 2′,3′-Cyclic phosphodiesterase was detected as the main component of the bis-(p-nitrophenyl)-phosphatase (BPNPase) in the subfractions 1 and 2 which were obtained from fraction 5 of the main fraction in the course of DEAE-cellulose column chromatography. The properties of the phosphodiesterase in subfractions 1 and 2 were characterized as follows. Subfractions 1 and 2, which exhibited a shoulder at about 280 nm in the UV absorption spectrum, hydrolyzed 2′,3′-cyclic nucleotides, adenylyl-(3′ → 5′)adenosine, uridylyl-(3′ → 5′)adenosine, thymidine 3‱-p-nitrophenyl phosphate and bis-(p-nitrophenyl)phosphate (BPNP). The most important substrates were 2′,3′-cyclic-nucleotides. Subfraction 2 hydrolyzed BPNP into 2 mol p-nitrophenol and 1 mol inorganic phosphate, whereas, in the case of subfraction 1, the molar ratio of p-nitrophenol to inorganic phosphate was higher than that of subfraction 2, suggesting that the phosphodiesterase activity in subfraction 1 was at least, associated with more than two types of phosphodiesterases. Apparent molecular weight (MW) of the phosphodiesterase was estimated to be 19,000, 45,000, and 230,000 in subfraction 1 and 29,000 and 50,000 in subtraction 2. BPNPases with a MW of 45,000 in subfraction 1 and MW of 50,000 in subfraction 2 were the main components of the phosphodiesterase, respectively. The BPNPase activities of subfractions 1 and 2 were sensitive to Hg²⁺ and inhibited in a mixed type by various nucleotides. These BPNPases displayed a higher affinity to adenosine 3′- and 5′-mononucleotide, whereas the p-nitrophenyl phosphatase activities in these fractions showed lower affinity to phosphodiesters than phosphomonoesters.

Key Words:

• bis-(p-nitrophenyl)phosphatase
• phosphodiesterase
• forest soil extract
• cyclic nucleotide.