ISOLATION AND PARTIAL CHARACTERIZATION OF BASIC PROTEINASES FROM STEM BROMELAIN
ISOLATION AND PARTIAL CHARACTERIZATION OF BASIC PROTEINASES FROM STEM BROMELAIN
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DOI：
10.1007/bf01902843
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发表时间：
1995-01-01
期刊：
影响因子：
--
通讯作者：
MAURER, HR
中科院分区：
文献类型：
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作者：
HARRACH, T;ECKERT, K;MAURER, HR
采用两步高效液相色谱-阳离子交换色谱法对菠萝茎中菠萝蛋白酶粗提物进行分离。至少检测到8种基本的蛋白水解活性成分。两个主要组分F4和F5以及最具活性的蛋白酶组分F9通过SDS-PAGE、质谱、多区阴极电泳、部分氨基酸序列和单糖组成分析进行表征。F9占总蛋白质的约2%，并且对底物L-焦谷氨酰基-1-苯基分析-1-亮氨酸-对硝基苯胺（PFLNA）的比活性比主要组分F4高15倍。通过质谱法测定F4、F5和F9的分子量分别为24，397、24，472和23，427。部分N-末端氨基酸序列分析（20个氨基酸）显示，F9与F4和F5的测定序列的不同之处在于第10位（酪氨酸->丝氨酸）和第20位（天冬酰胺->甘氨酸）的交换。F4和F5含有比例为1.0：2.0：1.0：2.0的岩藻糖、N-乙酰葡糖胺、木糖和甘露糖，但只有50%的蛋白质似乎是糖基化的，而F9被发现是非糖基化的。针对F9的多克隆抗体（IgG）检测到反应性降低十倍的F4和F5。F4和F5的最适pH值在4.0 ~ 4.5之间，F9的最适pH值接近中性。（K-m 2.30 mM，k（cat）0.87 sec（-1））和F5（K（m）2.42 mM，k（cat）0.68 sec（-1）），并且与F9（K-m 0.40 mM，k（cat）3.94 sec（-1））差异很大。
Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substrate L-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosine --> serine) and position 20 (asparagine --> glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.0:2.0:1.0:2.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. The pH optimum of F4 and F5 was between pH 4.0 and 4.5 and for F9 close to neutral pH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (K-m 2.30 mM, k(cat) 0.87 sec(-1) and F5 (K(m)2.42mM, k(cat) 0.68 sec(-1)), and differed greatly from F9 (K-m 0.40mM, k(cat) 3.94 sec(-1)).