Effect Of Phosphorylation On Activity Of Transaldolase In Human SMMC-7721 Cell Lines

Transaldolase (TAL) is an essential enzyme of the non-oxidative part of the pentose phosphate pathway(PPP) which produces reductive potential in the form of NADPH as well as ribose 5-phosphate for incorporation into nucleotides, RNA and DNA . NAPDH is not only a reductor to keep glutathione reducted, but also H+ supplier for many metabolic pathways. Recent studies have shown that Transaldolase plays important roles in the development of Multiple Sclerosis, malignant tumor, Liver Cirrhosis, organ development and embryogenesis, and also functions in some factors induced apoptosis.Previous studies of Heinrich's lab had demonstrated that in the liver tumors, transaldolase activity increased 1.5- to 3.4-fold over the activities observed in normal control rat liver, while it showed no change in the regenerating liver. Meanwhile, studies of Lachaise's group showed that Transaldolase function was related to the malignant tumor development. Previous studies of our lab had demonstrated that the increase of the TAL's activity caused by hCG may be independent of the level of the transcription and translation, Which indicated that hCG may promote post-translational modification of TAL in mouse K9 cells.Due to the high conservation of Transaldolase gene and the homology of Transaldolase protein structure in different creature, we are trying to study the potential role of Transaldolase on human hepatocarcinogenesis.The study was divided into two parts: the first part was to study the activity of Transaldolase and expression level of TAL protein and mRNA in the hepatocellular carcinoma cells of different proliferation state after cell synchronization. The second part was to determine the relationship between phosphorylation and TAL activity by phosphotase digestion, in order to investigate whether Transaldolase has any isoforms.and research on the relationship between Transaldolase activity and post-translational modification. We set out to investigate roles of Transaldolase in the molecular mechanism of tumor development.1 Activity of Transaldolase in the hepatocellular carcinoma cells of different proliferation stateWe measured the activity of Transaldolase in the hepatocellular carcinoma cells of different proliferation state by the method described by Lachaise, Using RT-PCR, SDS-PAGE and Western blotting, Transaldolase expression in mRNA level and protein level were determined. Our results showed that TAL activity in human SMMC-7721 cell lines cultured in 10% fetal serum was significantly higher than those cultured in 1 % fetal serum at 2, 4, 8h (P<0.05). but the expression levels of both messenger and protein are no significant difference. It was consistent with our previous results at tissue level. It suggested that the regulation of TAL activity mainly took place through post-translational modification, while there was no relation between TAL activity variation and protein or mRNA expression.2 To study the effect of phosphorylation on activity of transaldolase by phosphotase digestion. The expression of Transaldolase in the hepacellular carcinoma.By phosphotase digestion, the relationship between phosphorylation and TAL activity was determined. After phosphotase digestion, TAL activity in human SMMC-7721 cell lines decreased dramatically. This is a discovery which has not been reported in human carcinoma. We did 2-Dimentional electriphoresis Western blotting to check the protein level of Transaldolase in the hepatocellular carcinoma tissue, two TAL isoforms were detected (named a and b), isoform a in the hepatocellular carcinoma tissue was separated in seven spots and isoform b was separated in six spots. In Pericancerous tissues, isoform a was separated in seven spots and isoform b was separated in five spots, comparison of TAL protein spots in both groups indicated that there is an appearance of a new protein spot b6 and tending to the acidic end. It indicated the change of activity may be associated with post-translational modification.These results show that Transaldolase is a key enzyme which maintains the well-balanced cellular function. In addition, The change of transaldolase activity may play a crucial role in pathological changes and may be an initiating agent. It is a question for discussion to research for other function of the well-informed enzyme in carbohydrate metabolism.