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Twokinds of nucleotide-specific phosphatases were separated chromatographicaIIv on a DEAEToyopearl 650 M column, and designated as NSP-I and -II. NSP-II was further purified to apparent homogeneity using both affinity and gel-filtration chromatography, hut NSP-I purified by the same procedures was not homogeneous on polyacrylamide gel electrophoresis. NSP-I and -II had many similar features in molecular size, optimum pH, heat stability, and substrate specificity. The molecular weight of each enzymeprotein was estimated to be approximately 45,000 by molecular sieve chromatography. The optimum pH of each enzyme was 6.5 and showed activity in the wide pH range of 5~9. Neither enzyme hydrolyzed bis(/>-nitrophenyl)phosphate, adenosine 2':3'-cyclic-monophosphate, or sugar phosphate, but both showed a marked preference for 5'-mononucleotides as substrate over />-nitrophenylphosphate.

A classification of fatty acid esterases includes "esterases acting on substrates in solution (esterase proper) and esterases acting predominantly on undissolved substrates (lipase-type esterase)". J) For the study of esterolytic enzymes, a reliable method for discrimination of esterases and lipases is required. The earliest report in the literature of investigations on soybean lipase appears to be that by Gorbach.2) The work on the esterase of soybean seeds was that of Payne and Koszykowski.3) The authors confirmed the presence of two types ofesterases in dormant soybean seeds. Our work wasdirected toward a further understanding of the soybean esterase which is most active toward triacetin. Soybean seeds (Glycine max, L. var. Tsurunoko) were soaked in water at 4°C overnight and homogenized with three volumes of 0.01m CaCl2. The homogenate was centrifuged at 10,000 rpm for 30min, and the supernatant was brought to 40% saturation by gradual addition of solid ammoniumsulfate. After standing for 1 hr, the suspension was centrifuged at 10,000 rpm for 30 min, and the precipitate was discarded. Solid ammoniumsulfate was further added to 80%saturation. After standing for 1 hr, the precipitate was collected by centrifugation at 10,000rpm for 30min, and dissolved in 0.01 m Tris-HCl buffer, pH 7.0. The resulting solution was dialyzed against the same buffer, and the inner solution was taken as the enzymeextract. The enxyme solution wasput on a column of DEAE-cellulose (5.4 x 40cm) equilibrated with 0.01 m Tris-HCl buffer, pH 7.0. The enzyme was eluted by the buffer in a linear gradient of0 to 1 m NaCl concentration. The active fractions were collected and concentrated, and put on a CM-Toyopearl 650M column (4.0x36cm) equilibrated with 0.01 m Tris-HCl buffer, pH 7.0, and eluted by the buffer in a linear gradient of0 to 0.5 m NaCl concentration. The fraction having esterase activity was collected and concentrated, and put on a Toyopearl HW-55 column (2 x 87cm) equilibrated with 0.01 m Tris-HCl buffer, pH 7.0 containing 0.1m NaCl. The enzyme was eluted by the buffer containing 0.1m NaCl, and the condensed enzyme solution was put on a DEAEToyopearl 650M column (I x 57cm) equilibrated with 0.01 m Tris-HCl buffer, pH 7.0. The enzyme was eluted by the buffer containing 0.1 m NaCl. All the procedures were carried out at 4°C. Protein was measured by the method of Lowry et al,A) using bovine serum albumin as the standard, or by the absorbance at 280nm. DEAEcellulose (DE 32) was purchased from WhatmanLtd., and DEAE-, CM-Toyopearl 650M, and Toyopearl HW-55were obtained from Toyo Soda Mfg. Co. The other reagents were of the highest commercial grade available. The standard assay ofesterase was done in 14ml of0.24m triacetin in water, at 37°C, pH 7.0, maintained by the addition of 0.01n sodium hydroxide, using a pH-stat (HSM-10A, Toa Electronic Ltd.). Correction was done for the uptake of base due to nonenzymatic hydrolysis of triacetin. The specific activity of the enzyme increased about 1,100-fold after DEAE-Toyopearl 650M column chro...

This study focuses on conditions and planning negotiation systems of landscape ordinance in Tokyo 23 wards. And we show problems of the landscape ordinances to make use of future landscape polices. In conclusion, we found that there are many measures not to be implemented, such as landscape agreements and Landscape controlled areas. Also planning negotiation systems play a key role in landscape ordinances, and they are operated in all wards. This paper makes clear that planning negotiation systems are different from each ordinance, but all systems have two steps of negotiations and these careful negotiations bring about some results of landscape consideration to buildings

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