Van Dijken P, Lammers A A, Ozaki S, Potter B V, Erneux C, Van Haastert P J
Department of Biochemistry, University of Groningen, The Netherlands.
Eur J Biochem. 1994 Dec 1;226(2):561-6. doi: 10.1111/j.1432-1033.1994.tb20081.x.
A series of 32P-labeled D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] analogues was enzymically prepared from the corresponding D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] analogues using recombinant rat brain Ins(1,4,5)P3 3-kinase and [gamma-32P]ATP. Ins(1,4,5)P3 analogues with bulky groups at the 2-OH position, substitutions of phosphates by thiophosphates and D-6-deoxy-myo-Ins(1,4,5)P3 were tested. Using [3H]Ins(1,4,5)P3 and ATP gamma S, a [3H]Ins(1,3,4,5)P4 analogue with a thiophosphate at the D-3 position was prepared. The D-4 and/or D-5 phosphate group seemed to be important for 3-kinase activity, while the OH group at position 6 was not crucial. The addition of bulky groups at the 2-OH position did not prevent phosphorylation. The labeled Ins(1,3,4,5)P4 analogues were purified and their degradation by type-I Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase was compared with the degradation of Ins(1,3,4,5)P4. Substitution of the phosphate group at positions 1 or 3 by a thiophosphate, or the addition of bulky groups at the 2-OH position did not prevent degradation. D-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate could not be degraded by the 5-phosphatase, indicating the importance of the 6-OH group for 5-phosphatase action. D-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate could be an important tool in elucidating the cellular functions of Ins(1,3,4,5)P4.
使用重组大鼠脑肌醇-1,4,5-三磷酸[Ins(1,4,5)P3]激酶和[γ-32P]ATP，从相应的D-肌醇-1,4,5-三磷酸[Ins(1,4,5)P3]类似物酶促制备了一系列32P标记的D-肌醇-1,3,4,5-四磷酸[Ins(1,3,4,5)P4]类似物。测试了在2-OH位置带有庞大基团的Ins(1,4,5)P3类似物、用硫代磷酸酯取代磷酸酯以及D-6-脱氧-肌醇-Ins(1,4,5)P3。使用[3H]Ins(1,4,5)P3和ATPγS，制备了在D-3位置带有硫代磷酸酯的[3H]Ins(1,3,4,5)P4类似物。D-4和/或D-5磷酸基团似乎对3-激酶活性很重要，而6位的OH基团并非关键。在2-OH位置添加庞大基团并不妨碍磷酸化。纯化了标记的Ins(1,3,4,5)P4类似物，并将其被I型Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-磷酸酶降解的情况与Ins(1,3,4,5)P4的降解情况进行了比较。用硫代磷酸酯取代1或3位的磷酸基团，或在2-OH位置添加庞大基团并不妨碍降解。D-6-脱氧-肌醇-1,3,4,5-四磷酸不能被5-磷酸酶降解，表明6-OH基团对5-磷酸酶作用很重要。D-6-脱氧-肌醇-1,3,4,5-四磷酸可能是阐明Ins(1,3,4,5)P4细胞功能的重要工具。
