Weidanz J A, Campbell P, Moore D, DeLucas L J, Rodén L, Thompson J N, Vezza A C
Department of Microbiology, University of Alabama at Birmingham 35294-2170, USA.
Br J Haematol. 1996 Dec;95(4):645-53. doi: 10.1046/j.1365-2141.1996.d01-1955.x.
The major pathways of glucose metabolism in the malaria parasite, Plasmodium falciparum, have now been elucidated, and the structures and properties of parasite-specific enzymes are presently being investigated. Little is known, however, about the enzymes catalysing monosaccharide interconversions in the parasite. In the present investigation we have examined the pathway of N-acetylglucosamine catabolism which, in higher organisms, involves the following reaction sequence: N-acetylglucosamine -->N-acetylglucosamine 6-phosphate-->glucosamine 6-phosphate-->fructose 6-phosphate. Assay of the specific kinase (E.C. 2.7.1.59) catalysing the phosphorylation of the sugar showed that the enzyme is present in Plasmodium extracts as well as in normal human erythrocytes; specific activities of 7.2 and 5.3 nmol/h/mg protein were measured for the parasite and erythrocyte extracts, respectively, N-Acetylglucosamine 6-phosphate deacetylase (E.C. 3.5.1.25), catalysing the second reaction, was also detected in both normal and Plasmodium-infected erythrocytes. At 75% parasitaemia, the deacetylase activity was close to 3 times higher than that of normal control cells. The erythrocyte deacetylase was purified approximately 16,000-fold by chromatography on DE52 cellulose, chromatofocusing, and size exclusion chromatography. Attempts to purify the parasite enzyme by the same procedures were unsuccessful due to loss of activity. A partially purified erythrocyte deacetylase preparation (eluted from DE52 cellulose) had a pH optimum of 7.5, a pI of 6.0, as indicated by chromatofocusing, and a K(m) of 29 microM. In conjunction with previous investigations, the present study indicated that all three enzymes required for N-acetylglucosamine utilization are present in Plasmodium parasites as well as in normal erythrocytes.
恶性疟原虫这一疟原虫中葡萄糖代谢的主要途径现已阐明，目前正在研究寄生虫特异性酶的结构和特性。然而，对于催化该寄生虫中单糖相互转化的酶却知之甚少。在本研究中，我们研究了N - 乙酰葡糖胺的分解代谢途径，在高等生物中，该途径涉及以下反应序列：N - 乙酰葡糖胺→N - 乙酰葡糖胺6 - 磷酸→葡糖胺6 - 磷酸→果糖6 - 磷酸。对催化该糖磷酸化的特异性激酶（E.C. 2.7.1.59）的测定表明，该酶存在于疟原虫提取物以及正常人红细胞中；分别测定出寄生虫和红细胞提取物的比活性为7.2和5.3 nmol/h/mg蛋白质。催化第二步反应的N - 乙酰葡糖胺6 - 磷酸脱乙酰酶（E.C. 3.5.1.25）在正常和感染疟原虫的红细胞中也均被检测到。在疟原虫血症为75%时，脱乙酰酶活性比正常对照细胞高近3倍。红细胞脱乙酰酶通过在DE52纤维素上进行色谱分离、色谱聚焦和尺寸排阻色谱法纯化了约16,000倍。由于活性丧失，采用相同程序纯化寄生虫酶未获成功。经色谱聚焦表明，从DE52纤维素上洗脱下来的部分纯化的红细胞脱乙酰酶制剂的最适pH为7.5，等电点为6.0，米氏常数（K(m)）为29 microM。结合先前的研究，本研究表明疟原虫寄生虫以及正常红细胞中均存在利用N - 乙酰葡糖胺所需的所有三种酶。
