Harutoshi Kizaki

Increased CTP synthetase activity in cancer cells.

AN insight into the biochemical strategy of cancer cells has been achieved by the conceptual and experimental approach provided by the molecular correlation concept1,2. Studies carried out using this approach with model systems of rat hepatomas and kidney tumours of different growth rates revealed a biochemical imbalance in cancer cells, which manifested itself in progressive changes in activities of key enzymes and overall metabolic pathways that correlated with tumour growth rate1,2. Some of these biochemical alterations have been shown to apply to human primary liver3 and kidney4 carcinomas. Those alterations in gene expression that are linked to the increase in the expression of malignancy are manifested in the increased activities of key glycolytic-, purine-, pyrimidine-, DNA- and polyamine-synthesising enzymes1–8. Concurrently, decreases occur in the activities of the key enzymes of gluconeogenesis, purine and pyrimidine catabolism and of the urea cycle1–5,9,10. In addition to such a progression-linked (growth-rate-linked) metabolic imbalance, the reprogramming of gene expression in cancer cells entails transformation-linked alterations that are present in all hepatomas irrespective of growth rate or extent of differentiation1,2,11–15.Here we report that CTP synthetase (UTP:L-glutamine amido ligase, EC 6.3.4.2) increased in all the hepatomas examined, the activity being highest in the rapidly growing tumours. Thus, in liver neoplasia the activity of this enzyme is both transformation- and progression-linked. CTP synthetase activity was also markedly increased in transplantable kidney tumours in the rat and in primary renal cell carcinomas in man.

A micro-assay method for hypoxanthine-guanine and adenine phosphoribosyltransferases☆

Abstract An improved micro-assay method for hypoxanthine-guanine and adenine phosphoribosyltransferases is described. The assay relies on the isolation of isotopically labeled end-products by electrophoresis on cellulose acetate membrane using an electrophoretic apparatus which is used commonly in clinical laboratories. The assay is rapid and sensitive, and is applicable to crude tissue extracts which contain other enzymes acting on the substrates or products, since all possible reaction products, as well as the unreacted substrate, are isolated.

Purine nucleoside metabolizing enzyme activities in mouse thymocytes at different stages of differentiation and maturation

The activities of the enzymes involved in purine nucleoside metabolism, adenosine deaminase (ADA), adenosine kinase (AK), purine nucleoside phosphorylase (PNP) and deoxycytidine kinase (deoxyCRK), were determined in mouse thymocytes at various stages of differentiation and maturation, and compared with those in other tissues. The thymocytes were characterized by high ADA and deoxyCRK activities with high ADA/AK and ADA/PNP ratios and low PNP/deoxyCRK ratio. In fetal thymocytes of 16 gestational days, ADA activity was lower, and PNP, AK and deoxyCRK activities were higher than those in the adult thymocytes. During differentiation of fetal thymocytes, ADA activity increased while PNP and AK activities decreased. DeoxyCRK activity decreased after birth. In spleen T lymphocytes, ADA and deoxyCRK activities were lower and PNP activity was about 2.5-fold higher than in the thymocytes. Thus the differentiation stages of T lymphocytes may be characterized by the absolute levels and the ratios of these enzymes.

Purification and properties of CTP synthetase from Ehrlich ascites tumor cells

CTP synthetase (UTP: ammonia ligase (ADP-forming), EC 6.3.4.2) was purified 200-fold from Ehrlich ascites tumor cells with about 50% purity as judged by polyacrylamide gel electrophoresis. The molecular weight estimated to be 118 000 by gel filtration. The optimal pH was 8.6. 2-Mercaptoethanol was required for optimal activity and stabilization of the enzyme. Magnesium was essential for the reaction and other divalent cations were ineffective. Ammonia could replace glutamine as the amino donor. When other substrates were at saturating concentrations, Michaelis-Menten kinetics were observed yielding Km values for UTP, ATP and glutamine of 0.18, 0.8 and 0.13 mM, respectively. With ATP at subsaturating concentration, the double-reciprocal plot for UTP saturation was sinusoidal and the Hill plot showed an n value of 1.3. The double-reciprocal plot for ATP saturation, when UTP was at subsaturating concentration, departed from Michaelis-Menten kinetics with an n value of 1.4. These data suggest the existence of cooperative interactions between enzyme and substrates at subsaturating concentrations of ATP or UTP. GTP was not essential, but it acted as an activator on the glutamine reaction; optimal activation was observed at 1 mM GTP. The affinity for glutamine was not affected by GTP.

CTP synthetase from Ehrlich ascites tumor cells. Subunit stoichiometry and regulation of activity

CTP synthetase (UTP: glutamine ligase (ADP-forming), EC 6.3.4.2) was purified from Ehrlich ascites tumor cells to near homogeneity and found to be a dimer composed of two seemingly identical 66 kDa subunits. The formation of CTP was accompanied by the production of equivalent amounts of ADP from ATP and glutamate from glutamine. The reaction product, CTP, was a potent inhibitor generating sigmoidal kinetics as a function of UTP with an n value of 2.0. UTP and CTP pools in the ascites cells were elevated in an early period (12-16 h) following implantation into the intraperitoneal cavity of mice, whereas ATP, GTP and glutamine pools did not change. Kinetic data and analysis of the nucleotide pools in the cells growing in vivo suggested that the biosynthesis of CTP is regulated at the level of CTP synthetase by UTP and CTP.

Role of GTP in CTP synthetase from Ehrlich ascites tumor cells

Abstract GTP was nearly essential as an activator for the CTP synthetase from Ehrlich ascites tumor cells when glutamine was the nitrogen source. GTP accelerated the glutaminase activity of the enzyme about 2.5-fold in the absence of the other substrates and about 45-fold in the presence of UTP and ATP. UTP and adenylylimidodiphosphate, a competitive inhibitor for ATP (Ki=1.1 mM), did not affect the glutaminase activity. In addition to the activating effect, GTP could replace ATP as a substrate with a Km of 1.7 mM in the glutamine reaction and 2.2 mM in the ammonia reaction.

Synthesis of N4-substituted CTP by mammalian CTP synthetase.

Highly purified CTP synthetase from Ehrlich ascites tumor cells catalyzes the formation of N4-substituted CTP from UTP and hydroxylamine and its derivatives. The products with hydroxylamine and O-methylhydroxylamine were identified as N4-hydroxyCTP and N4-methoxyCTP by absorption spectra and chromatographic behavior on Dowex column, respectively. The weak nucleophilic amines such as methylamine, ethylamine or diethylamine and a less nucleophilic amine, sulfamic acid, did not react with UTP. These results suggest that the nucleophilicity and basicity of amines are important in the enzymic reaction with UTP.

PURINE PHOSPHORIBOSYLTRANSEERASE ACTIVITIES IN GUINEA PIG EPIDERMIS

Purine phosphoribosyltransferase activities in normal and experimental hyperkeratotic epidermis of guinea pig skin were demonstrated quantitatively by a new microassay method.