Ullah A H, Cummins B J
Southern Regional Research Laboratory, ARS, USDA, New Orleans, Louisiana 70124.
Prep Biochem. 1988;18(1):37-65. doi: 10.1080/00327488808062512.
An extracellular acid phosphatase, pH optimum 6.0 from crude culture filtrate of Aspergillus ficuum was purified to homogeneity using cation exchange chromatography and chromatofocusing steps. SDS-PAGE of the purified enzyme exhibited two stained bands at approximately 82-KDa and 70-KDa. The mobility of the active enzyme in gel permeation chromatography indicated the molecular mass to be about 85-KDa. In the concentrated form the enzyme appeared to be purple, the visible absorption spectrum shows a lambda max at 580 nm. On the basis of molecular mass of 82-KDa, the molar extinction coefficient of the enzyme at 280 nm and 580 nm was estimated to be 1.2 x 10(5) M-1 cm-1 and 1.3 x 10(3) M-1 cm-1 respectively. Judging by chromatofocusing, the isoelectric point of the enzyme was about 4.9. The purified enzyme was unstable at 70 degrees C. The enzyme was catalytically very active from 55 degrees to 65 degrees C with a maximum activity at 63 degrees C. The Michaelis constant of the enzyme for p-nitrophenylphosphate was 200 microM with a computed Kcat of 260 per sec. Although the enzyme was insensitive to fluoride, tartrate, and N-ethylmaleimide (NEM), it was competitively inhibited by phosphomycin (Ki = 1.00 mM) and inorganic orthophosphate (Ki = 165 microM). While the enzyme was relatively insensitive to Mn++, Cu++ and Zn++ inhibited the activity 540 fold at a concentration of 100 microM. The enzyme showed positive PAS staining and hence is a glycoprotein (28% glycosylation); the sugar composition suggests the presence of N-linked high mannose-oligosaccharides and galactose. A partial N-terminal amino acid sequence up to the thirty-fourth residue was elucidated.
从泡盛曲霉粗培养滤液中分离出一种细胞外酸性磷酸酶，其最适pH值为6.0，通过阳离子交换色谱法和色谱聚焦步骤将其纯化至同质。纯化酶的SDS-PAGE显示在约82 kDa和70 kDa处有两条染色带。凝胶渗透色谱中活性酶的迁移率表明分子量约为85 kDa。浓缩形式的酶呈紫色，可见吸收光谱在580 nm处有最大吸收峰。基于82 kDa的分子量，该酶在280 nm和580 nm处的摩尔消光系数分别估计为1.2×10⁵ M⁻¹ cm⁻¹和1.3×10³ M⁻¹ cm⁻¹。通过色谱聚焦判断，该酶的等电点约为4.9。纯化后的酶在70℃不稳定。该酶在55℃至65℃具有很高的催化活性，在63℃时活性最高。该酶对对硝基苯磷酸酯的米氏常数为200 μM，计算得出的催化常数Kcat为每秒260次。虽然该酶对氟化物、酒石酸盐和N-乙基马来酰亚胺（NEM）不敏感，但它受到磷霉素（Ki = 1.00 mM）和无机正磷酸盐（Ki = 165 μM）的竞争性抑制。虽然该酶对Mn²⁺相对不敏感，但Cu²⁺和Zn²⁺在100 μM浓度下可抑制其活性达540倍。该酶显示出阳性PAS染色，因此是一种糖蛋白（糖基化程度为28%）；糖组成表明存在N-连接的高甘露糖寡糖和半乳糖。阐明了该酶直至第34个残基的部分N端氨基酸序列。
