Tsirka S A, Kyriakidis D A
Faculty of Chemistry, School of Science, Aristotelian University of Thessaloniki, Greece.
Mol Cell Biochem. 1990 Jun 1;95(1):77-87. doi: 10.1007/BF00219533.
Most of L-asparaginase activity of Tetrahymena pyriformis was found to be present in microsomal membranes from which it has been purified to homogeneity (Tsirka, S.A.E. and Kyriakidis, D.A. Mol. Cell. Biochem. 83: 147-155, 1988). The native enzyme has a relative molecular weight of approximately 200 kDa, while under denaturing conditions the enzyme exhibits a subunit size of 39 kDa. Aminoacid analysis and an oligopeptide from N-terminal sequence have been determined. Dephosphorylation of L-asparaginase by alkaline phosphatase results in an activation of its catalytic activity. This enzyme also exhibits intrinsic phosphorylation activity with a Km value for ATP of 0.5 mM. Autophosphorylation with [gamma-32P] ATP of purified L-asparaginase results in the phosphorylation of tyrosine residues as well as in loss of its activity. Mg2+ and Ca2+ added together act synergistically to stimulate the kinase activity by more than 160%. The polyamines putrescine, spermidine and spermine activate the kinase approximately 100%, while neither cAMP or cGMP have any effect. These results indicate that this membrane protein with dual L-asparaginase/kinase activity must play an important role in regulating the intracellular levels of L-asparagine in Tetrahymena pyriformis.
梨形四膜虫的大多数L-天冬酰胺酶活性存在于微粒体膜中，已从该膜中将其纯化至同质（Tsirka, S.A.E.和Kyriakidis, D.A.《分子与细胞生物化学》83: 147 - 155, 1988）。天然酶的相对分子质量约为200 kDa，而在变性条件下，该酶呈现出39 kDa的亚基大小。已确定氨基酸分析和N端序列的一个寡肽。碱性磷酸酶对L-天冬酰胺酶进行去磷酸化会导致其催化活性激活。该酶还表现出内在的磷酸化活性，对ATP的Km值为0.5 mM。用[γ-32P]ATP对纯化的L-天冬酰胺酶进行自磷酸化会导致酪氨酸残基磷酸化以及其活性丧失。一起添加的Mg2+和Ca2+协同作用，使激酶活性刺激超过160%。多胺腐胺、亚精胺和精胺使激酶激活约100%，而cAMP或cGMP均无任何作用。这些结果表明，这种具有双重L-天冬酰胺酶/激酶活性的膜蛋白必定在调节梨形四膜虫细胞内L-天冬酰胺水平方面发挥重要作用。
