Guanine Deaminase Activity Research Articles

Retracing the Rapid Evolution of an Herbicide-Degrading Enzyme by Protein Engineering.

The mechanisms underlying the rapid evolution of novel enzymatic activities from promiscuous side activities are poorly understood. Recently emerged enzymes catalyzing the catabolic degradation of xenobiotic substances that have been spread out into the environment during the last decades provide an exquisite opportunity to study these mechanisms. A prominent example is the herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine), which is degraded through a number of enzymatic reactions constituting the Atz pathway. Here, we analyzed the evolution of the hydroxyatrazine ethylaminohydrolase AtzB, a Zn(II)-dependent metalloenzyme that adopts the amidohydrolase fold and catalyzes the second step of the Atz pathway. We searched for promiscuous side activities of AtzB, which might point to the identity of its progenitor. These investigations revealed that AtzB has low promiscuous guanine deaminase activity. Furthermore, we found that the two closest AtzB homologues, which have not been functionally annotated up to now, are guanine deaminases with modest promiscuous hydroxyatrazine hydrolase activity. Based on sequence comparisons with the closest AtzB homologues, the guanine deaminase activity of AtzB could be increased by three orders of magnitude through the introduction of only four active site mutations. Interestingly, introducing the inverse four mutations into the AtzB homologues significantly enhanced their hydroxyatrazine hydrolase activity, and in one case is even equivalent to that of wild-type AtzB. Molecular dynamics simulations elucidated the structural and molecular basis for the mutation-induced activity changes. The example of AtzB highlights how novel enzymes with high catalytic proficiency can evolve from low promiscuous side activities by only few mutational events within a short period of time.

Stage-Related Neurotoxicity of BPA in the Development of Zebrafish Embryos

Bisphenol A (BPA) is one of the most widely produced chemicals in the world used in the production of epoxy resins and polycarbonate plastics. BPA is easily migrated from the outer packaging to the contents. Due to the lipophilic property, BPA is easily accumulated in organisms. Perinatal low-dose BPA exposure alters brain neural development in later generations. In this study, after BPA treatment, the spontaneous movement of zebrafish larvae from the cleavage period to the segmentation period (1-24 hpf) was significantly decreased, with speed decreasing by 18.97% and distance decreasing between 18.4 and 29.7% compared to controls. Transcriptomics analysis showed that 131 genes were significantly differentially expressed in the exposed group during the 1-24 hpf period, among which 39 genes were significantly upregulated and 92 genes were significantly downregulated. The GO enrichment analysis, gene function analysis and real-time quantitative PCR of differentially expressed genes showed that the mRNA level of guanine deaminase (cypin) decreased significantly in the 1-24 hpf period. Moreover, during the 1-24 hpf period, BPA exposure reduced guanine deaminase activity. Therefore, we confirmed that cypin is a key sensitive gene for BPA during this period. Finally, the cypin mRNA microinjection verified that the cypin level of zebrafish larvae was restored, leading to the restoration of the locomotor activity. Taken together, the current results show that the sensitive period of BPA to zebrafish embryos is from the cleavage period to the segmentation period (1-24 hpf), and cypin is a potential target for BPA-induced neurodevelopmental toxicity. This study provides a potential sensitive period and a potential target for the deep understanding of neurodevelopmental toxicity mechanisms caused by BPA.

Real-Time Monitoring of Human Guanine Deaminase Activity by an Emissive Guanine Analog

Guanine deaminase (GDA) deaminates guanine to xanthine. Despite its significance, the study of human GDA remains limited compared to other metabolic deaminases. As a result, its substrate and inhibitor repertoire are limited, and effective real-time activity, inhibitory, and discovery assays are missing. Herein, we explore two emissive heterocyclic cores, based on thieno[3,4-d]pyrimidine (thN) and isothiazole[4,3-d]pyrimidine (tzN), as surrogate GDA substrates. We demonstrate that, unlike the thieno analog, thGN, the isothiazolo guanine surrogate, tzGN, does undergo effective enzymatic deamination by GDA and yields the spectroscopically distinct xanthine analog, tzXN. Further, we showcase the potential of this fluorescent nucleobase surrogate to provide a visible spectral window for a real-time study of GDA and its inhibition.

Cypin: A novel target for traumatic brain injury

Cytosolic PSD-95 interactor (cypin), the primary guanine deaminase in the brain, plays key roles in shaping neuronal circuits and regulating neuronal survival. Despite this pervasive role in neuronal function, the ability for cypin activity to affect recovery from acute brain injury is unknown. A key barrier in identifying the role of cypin in neurological recovery is the absence of pharmacological tools to manipulate cypin activity in vivo. Here, we use a small molecule screen to identify two activators and one inhibitor of cypin's guanine deaminase activity. The primary screen identified compounds that change the initial rate of guanine deamination using a colorimetric assay, and secondary screens included the ability of the compounds to protect neurons from NMDA-induced injury and NMDA-induced decreases in frequency and amplitude of miniature excitatory postsynaptic currents. Hippocampal neurons pretreated with activators preserved electrophysiological function and survival after NMDA-induced injury in vitro, while pretreatment with the inhibitor did not. The effects of the activators were abolished when cypin was knocked down. Administering either cypin activator directly into the brain one hour after traumatic brain injury significantly reduced fear conditioning deficits 5 days after injury, while delivering the cypin inhibitor did not improve outcome after TBI. Together, these data demonstrate that cypin activation is a novel approach for improving outcome after TBI and may provide a new pathway for reducing the deficits associated with TBI in patients.

A Novel Short Isoform of Cytosolic PSD-95 Interactor (Cypin) Regulates Neuronal Development.

The guanine deaminase cypin (cytosolic PSD-95 interactor) binds to PSD-95 (postsynaptic density protein 95) and regulates dendrite branching by promoting microtubule polymerization. Here, we identify a novel short isoform of cypin, termed cypinS, which is expressed in mouse and human, but not rat, tissues. Cypin and cypinS mRNA and protein levels peak at P7 and P14 in the mouse brain, suggesting a role for these isoforms during development. Interestingly, although cypinS lacks guanine deaminase activity, overexpression of cypinS increases dendrite branching. This increase occurs further away from soma than do increases resulting from overexpression of cypin. In contrast, overexpression of cypin, but not cypinS, decreases dendritic spine density and maturity. This suggests that changes to spines, but not to dendrites, may be dependent on guanine deaminase activity. Furthermore, overexpression of either cypin or cypinS increases miniature excitatory postsynaptic current (mEPSC) frequency, pointing to a presynaptic role for both isoforms. Interestingly, overexpression of cypinS results in a significantly greater increase in frequency than does overexpression of cypin. Thus, cypin and cypinS play distinct roles in neuronal development.

Transcriptional Regulation of the Gene Cluster Encoding Allantoinase and Guanine Deaminase in Klebsiella pneumoniae

Purines can be used as the sole source of nitrogen by several strains of K. pneumoniae under aerobic conditions. The genes responsible for the assimilation of purine nitrogens are distributed in three separated clusters in the K. pneumoniae genome. Here, we characterize the cluster encompassing genes KPN_01787 to KPN_01791, which is involved in the conversion of allantoin into allantoate and in the deamination of guanine to xanthine. These genes are organized in three transcriptional units, hpxSAB, hpxC, and guaD. Gene hpxS encodes a regulatory protein of the GntR family that mediates regulation of this system by growth on allantoin. Proteins encoded by hpxB and guaD display allantoinase and guanine deaminase activity, respectively. In this cluster, hpxSAB is the most tightly regulated unit. This operon was activated by growth on allantoin as a nitrogen source; however, addition of allantoin to nitrogen excess cultures did not result in hpxSAB induction. Neither guaD nor hpxC was induced by allantoin. Expression of guaD is mainly regulated by nitrogen availability through the action of NtrC. Full induction of hpxSAB by allantoin requires both HpxS and NAC. HpxS may have a dual role, acting as a repressor in the absence of allantoin and as an activator in its presence. HpxS binds to tandem sites, S1 and S2, overlapping the -10 and -35 sequences of the hpxSAB promoter, respectively. The NAC binding site is located between S1 and S2 and partially overlaps S2. In the presence of allantoin, interplay between NAC and HpxS is proposed.

Structural characterization of the zinc binding domain in cytosolic PSD‐95 interactor (cypin): Role of zinc binding in guanine deamination and dendrite branching

Dendrite morphology regulates how a postsynaptic neuron receives information from presynaptic neurons. The specific patterning of dendrite branches is promoted by extrinsic and intrinsic factors that trigger the activation of functional signaling pathways. However, most of the regulating factors and the biochemical mechanisms involved in regulating dendrite branching are unknown. Our laboratory previously reported that cypin (cytosolic PSD-95 interactor) plays an active role in regulating dendrite branching in hippocampal neurons. Cypin-promoted increases in dendrite number are dependent on guanine deaminase activity. In order to identify the specific structural role of zinc-binding in cypin-mediated dendrite branching and guanine deaminase activity, we employed computational homology modeling techniques to construct a three dimensional structural model of cypin. Analysis of the protein-ion sequestration scaffold of this model identified several histidines and aspartic acid residues responsible for zinc binding. Single substitution mutations in these specific sites completely disrupted the guanine deaminase enzymatic activity and rendered cypin unable to promote dendrite branching in rat hippocampal neurons. The specific zinc ion-binding function of each residue in the protein scaffold was also confirmed by Inductively Coupled Plasma-Optic Emission Spectrometry. Inspection of our structural model confirmed that His82 and His84 coordinate with the zinc ion, together with His240, His279, and Asp330, residues that until now were unknown to play a role in this regard. Furthermore, promotion of dendrite branching by cypin is zinc-dependent.

Cypin regulates dendrite patterning in hippocampal neurons by promoting microtubule assembly.

Dendrite branching has an important role in normal brain function. Here we report that overexpression of cypin, a protein that has guanine deaminase activity and is expressed in developing processes in rat hippocampal neurons, results in increased dendrite branching in primary culture. Mutant cypin proteins that lack guanine deaminase activity act in a dominant-negative manner when expressed in primary neurons. Furthermore, we knocked down cypin protein levels using a new strategy: expressing a 5' end-mutated U1 small nuclear RNA (snRNA) to inhibit maturation of cypin mRNA. Neurons that express this mutant snRNA show little or no detectable cypin protein and fewer dendrites than normal. In addition, we found that cypin binds directly to tubulin heterodimers and promotes microtubule polymerization. Thus, our results demonstrate a new pathway by which dendrite patterning is regulated, and we also introduce a new method for decreasing endogenous protein expression in neurons.

Cloning and characterization of guanine deaminase from mouse and rat brain

A search for genes differentially expressed in the rat striatum revealed a gene fragment with a ventral to dorsal striatal expression pattern. The sequence of the fragment was used to isolate mouse and rat clones that upon sequencing were identified as homologous to human guanine deaminase. Here we report the distribution of guanine deaminase in the rodent brain. In situ hybridization localization of the encoding mRNA showed a distribution primarily in forebrain areas including cortical pyramidal neurons, ventral striatal medium spiny neurons, hippocampal pyramidal neurons in CA3–CA1 and granule cells in the dentate gyrus, and neurons of the amygdala. Immunohistochemistry using antibodies raised against peptide fragments derived from the guanine deaminase protein sequence showed localization of guanine deaminase in areas predicted by the mRNA distribution. In addition to immunolabeling of neurons in the cerebral cortex, hippocampus, striatum and amygdala there was also labeling in the terminal fields of these neurons including the thalamus, globus pallidum and substantia nigra. A functional histochemical assay that demonstrates the site of guanine deamination shows guanine deaminase activity in a pattern that matched the immunohistochemical localization. The cellular distribution of guanine deaminase to distal areas of the cell including terminals and dendrites was additionally demonstrated by the expression of recombinant guanine deaminase in transformed cortical neurons in culture. In summary we have described the isolation and characterization of mouse and rat guanine deaminase. The expression of guanine deaminase is primarily restricted to forebrain neurons. A histochemical assay was used to localize guanine deaminase activity to the dendrites and axons of neurons expressing guanine deaminase.

Human guanine deaminase: cloning, expression and characterisation.

Guanine deaminase activity may contribute significantly to overall urate production in mammals. In xanthine dehydrogenase deficiency, the mean urine excretion ratio for xanthine: hypoxanthine is approximately 4:11, suggesting that the conversion of guanine to xanthine may be quantitatively more important than that of hypoxanthine to xanthine, albeit the potential recycling of hypoxanthine to IMP would be expected to reduce the hypoxanthine level. In another study the relative amount of xanthine formation via guanine was estimated to be approximately 5-fold greater than via hypoxanthine2. In a porcine model, lead induced inactivation of guanine deaminase activity, resulted in the deposition of guanine crystals in the epiphyseal plate of the femoral head3. A full term infant who presented with acute anoxia and died from respiratory distress at day 2 was reported to be deficient in brain guanine deaminase activity4 .

Purine pathway enzymes in a cyst forming strain of Toxoplasma gondii

The activities of purine salvage enzymes in tachyzoites from a cyst-forming strain of Toxoplasma gondii were determined using HPLC. Six enzymes were assayed both in vitro and in vivo: adenosine deaminase, guanine deaminase, purine nucleoside phosphorylase, xanthine oxidase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase. In vitro, the tachyzoites were cultured in the human myelomonocytic cell line THP-1, for 24 h to 96 h. Neither guanine deaminase nor hypoxanthine-guanine phosphoribosyltransferase activity was detected in 24 and 96 h cultures. In vivo, in controls and infected animals, the purine nucleoside phosphorylase and adenosine deaminase activities were the most important activities both in sera and cerebral tissue in comparison with the other activities. It was also noted that the infection modified the enzymatic activities of this purine salvage pathway, in particular, the guanine deaminase cerebral activity of infected mice was 20-fold lower than the value of controls. The treatment of mice with 2′,3′-dideoxyinosine, a purine analog, at the dose of 100 mg.kg −1.d for 30 days, induced an important increase of all enzymatic activities in the brains in comparison with control animals. These data suggest that one target of 2′,3′-dideoxyinosine is the purine metabolism.

Cloning and Characterization of Human Guanine Deaminase: PURIFICATION AND PARTIAL AMINO ACID SEQUENCE OF THE MOUSE PROTEIN

Mouse erythrocyte guanine deaminase has been purified to homogeneity. The native enzyme was dimeric, being comprised of two identical subunits of approximately 50,000 Da. The protein sequence was obtained from five cyanogen bromide cleavage products giving sequences ranging from 12 to 25 amino acids in length and corresponding to 99 residues. Basic Local Alignment Search Tool (BLAST) analysis of expressed sequence databases enabled the retrieval of a human expressed sequence tag cDNA clone highly homologous to one of the mouse peptide sequences. The presumed coding region of this clone was used to screen a human kidney cDNA library and secondarily to polymerase chain reaction-amplify the full-length coding sequence of the human brain cDNA corresponding to an open reading frame of 1365 nucleotides and encoding a protein of 51,040 Da. Comparison of the mouse peptide sequences with the inferred human protein sequence revealed 88 of 99 residues to be identical. The human coding sequence of the putative enzyme was subcloned into the bacterial expression vector pMAL-c2, expressed, purified, and characterized as having guanine deaminase activity with a Km for guanine of 9.5 +/- 1.7 microM. The protein shares a 9-residue motif with other aminohydrolases and amidohydrolases (PGX[VI]DXH[TVI]H) that has been shown to be ligated with heavy metal ions, commonly zinc. The purified recombinant guanine deaminase was found to contain approximately 1 atom of zinc per 51-kDa monomer.

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines.

THE METABOLISM OF GUANINE BY THE DIATOM PHAEODACYLUM TRICONUTUM BOHLIN1

ABSTRACTGuanine taken up by intact cells of Phaeodactylum tricornutum Bohlin was rapidly converted to allantoin which accumulated in the cells; the earleir view that the compound which accumulated was a methylahypoxanthine is shown to be erroneous. In contrast, cells of P. tricornutum, after premeabilisation with toluene, converted guanine only to xanthine, the reaction presumably being catalysed by guanine diaminase. Freshly harvested N‐replete cells contained substantial guanine deaminase activity (ca. 200 nmol (108 cells h)−1); this activity doubled during 5 hours of N‐deprivation. During the same period, the ability to take up guanine, which was initially low, increased by about 25x.

血清Ｇｕａｎａｓｅ活性値による輸血後非Ａ非Ｂ型肝炎発症予防

Total of 102 patients who received blood transfusion for their cardiovascular surgery were the materials of this investigation.The incidence of post-transfusion hepatitis in correlation to guanase (guanine deaminase) activity was studied.Diagnosis of post-transfusion hepatitis was made when a patient showed elevation of s-GPT over 100 units in three weeks after surgery. The follow up periods were ranging from three to six months.13 patients were developed post-transfusion hepatitis which accounted 12.7% of incidence. Patients were divided into two groups according to the level of guanase activity. Significantry high incidence was observed in the groups of patients who received transfusion with guanase activity over 2.6IU/L (12.7% VS 64.3%, χ20=18.06>χ20 (1, 0.001)=10.83)The guanase activity in transfusion blood may be feasible as a parameter for the prevention of posttransfusion non-A, non-B type hepatitis.

Guanine deaminase in serum as an indicator of survival probability in severe shock patients

To find ways of predicting survival or death in cases of severe shock, arterial blood pH and gases, vital signs, and the half-life of activity in the enzymes guanine deaminase, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were studied in 24 patients. The mean arterial blood pH (± SD) in survivors was 7.325 ± 0.092 while that in non-survivors was 7.108 ± 0.251 ( p < 0.05). The mean half-life of guanine deaminase in survivors was 19.8 ± 3.3 hours, while that in non-survivors was 58.6 ± 11.8 hours ( p < 0.001). When the screening values were set at 7.190 for the arterial blood pH and 36 hours for the half-life of guanine deaminase activity, the ‘validity” (sensitivity plus specificity) for the combination of the two tests was 1.88. These values may be useful for the prognosis of survival in severe shock patients.

Enzymatic activities for interconversion of purines in spirochetes.

Enzymatic activities that catalyze the interconversion of purines and purine derivatives were detected in cell extracts of Spirochaeta aurantia, Spirochaeta stenostrepta, Treponema succinifaciens, and Treponema denticola. Phosphoribosyltransferase activities present in cell extracts of each of the four spirochete species functioned in the conversion of adenine, hypoxanthine, and guanine to AMP, IMP, and GMP, respectively. Nucleotidase activities in the extracts mediated the formation of nucleosides from nucleotides. The conversion of adenosine, inosine, and guanosine to the respective purine bases was catalyzed by nucleoside phosphorylase and, in some instances, by nucleoside hydrolase activities. Guanine deaminase activity was found in both S. aurantia and S. stenostrepta, whereas adenosine deaminase activity was detected only in S. aurantia. Adenine deaminase activity in T. succinifaciens extracts was sensitive to O2 and was relatively resistant to heating. Our results indicate that the four species of spirochetes studied possess a broad spectrum of purine interconversion enzymes. It is suggested that these enzymes may function in metabolic processes important for the survival of spirochetes in nutrient-poor natural environments.

Activities of purine-metabolizing enzymes in human colon carcinoma cell lines and xenograft tumors

The activities of eleven enzymes of purine metabolism have been determined in extracts of four human colon carcinoma cell lines (clones A and D of the heterogeneous DLD-1 line, HCT-15 and DLD-2) in culture and as xenografts in nude mice. Activities of the enzyme adenine phosphoribosyltransferase (APRT), hypoxanthine phosphoribosyltransferase (HPRT), adenosine kinase, 5'-deoxy-5'-methylthioadenosine (MTA) phosphorylase, adenylate (AMP) kinase, guanylate (GMP) kinase, and nucleoside diphosphokinase (NDP kinase) changed little from line to line in vitro. Adenosine deaminase (ADA) activity varied more than 5-fold between HCT-15 and clone A cells. Purine nucleoside phosphorylase (PNP) activity was about 3-fold higher in DLD-2 cells than in the other three lines. Guanine deaminase (guanase) activity was 4- to 6-fold higher in clone A and D cells than in HCT-15 and DLD-2 cells. Xanthine oxidase was not detected in any of the lines grown in vitro or in vivo. The activity of APRT was highest in clone D xenografts. HPRT was lowest in DLD-2 tumors. ADA activity in DLD-2 tumors was more than 2-fold elevated over that for clone A and HCT-15 xenografts. PNP activity in clone A and HCT-15 xenografts was lower than that for clone D or DLD-2 tumors. Guanase was lower in HCT-15 and DLD-2 tumors than in those of clones A and D. Clone D tumor GMP kinase activity was elevated more than 4-fold above that for the other three tumors. The activity of NDP kinase was highest in clone D tumors. MTA phosphorylase activity was similar in all four xenografts. In general, the activity of a given enzyme was similar in cells growing in monolayer culture and as xenografts in nude mice (37/44 comparisons). Enzyme activities were also measured in normal human colon and compared to those in the xenografts. Where significant differences were seen, the values for normal tissue were almost always lower than those of the tumors. The striking similarities between the in vitro and in vivo enzymic profiles for each of these four human colon carcinoma lines indicate that, at least on this basis, responses of these cells to purines and purine analogs in the two-dimensional monolayer culture system should be predictive of in vivo responses.

Specific resistance to 8-azaguanine in cells with normal hypoxanthine phosphoribosyltransferase (HPRT) activity: the role of guanine deaminase.

The role of guanine deaminase in selective cellular resistance to 8-azaguanine was examined, using eight mammalian cell lines and their subclonal derivatives isolated on the basis of increasing resistance to this drug. 8-Azaguanine and 6-thioguanine are synthetic analogs of guanine and are lethal to cells with normal hypoxanthine phosphoribosyltransferase (HPRT) activity. In principle, however, HPRT-positive cells could become selectively resistant to 8-azaguanine if, by any mechanism, the cells expressed higher levels of guanine deaminase. This is because 8-azaguanine, but not 6-thioguanine, is converted by this enzyme to a noncytotoxic metabolite, 8-azaxanthine. Our study shows that HPRT-positive cells inherently resistant to relatively high levels of 8-azaguanine contain high levels of guanine deaminase. In general, guanine deaminase activity was higher in 8-azaguanine-resistant cells, regardless of their HPRT activity. Our results support the view that elevated guanine deaminase activity constitutes a potential mechanism of selective 8-azaguanine resistance in cells with normal HPRT activity. Guanine deaminase levels were significantly elevated in HPRT-positive cells briefly exposed to sublethal concentrations of 8-azaguanine, but this elevation was transient. Long-term exposure of cells to increasingly higher levels of the drug did not lead to high stable levels of guanine deaminase, indicating that 8-azaguanine is not an inducer of guanine deaminase in the cells examined.

Biochemical commitment to replication in cancer cells

The elucidation of the metabolic imbalance that underlies the biochemical commitment to continued replication of cancer cells is essential in the rational targeting of selective anti-cancer chemotherapy. The metabolic strategy of cancer cells entails an ordered pattern of imbalance in the activities of key enzymes and metabolic pathways and in the concentrations of intermediary metabolites, ribonucleotides and deoxyribonucleotides. A meaningful metabolic program was identified which is displayed in neoplastic cells as a result of a reprogramming of gene expression that is manifested in a transformation- and progression-linked fashion. The transition of the non-proliferating, resting cell culture into the proliferating phase provides a particularly suitable biological system of altered gene expression where it is possible to pinpoint the display of enzymic programs that illuminates the biochemical commitment of cancer cells to replication. In the proliferating hepatoma 3924A cells the activity of the pyrimidine synthetic enzymes, ribonucleotide reductase, thymidine kinase, CTP synthetase, uracil phosphoribosyltransferase, OMP decarboxylase, orotate phosphoribosyltransferase and uridine kinase were increased 23-, 13-, 4.9-, 3.1-, 3.5-, 3.6- and 2.1-fold over those of the resting phase cells. Concurrently, the activity of the rate-limiting catabolic enzyme, dihydrothymine dehydrogenase, was decreased to 42%. In consequence, the ratio of the activities of the synthetic to catabolic enzymes of thymidine metabolism increased 31-fold. In purine metabolism, the activity of the rate-limiting enzyme of de novo purine biosynthesis, glutamine PRPP amidotransferase, was increased 1.5-fold and that of the rate-limiting catabolic enzyme, xanthine oxidase, decreased to 75%. In consequence, the ratio of the synthetic to the catabolic enzyme was elevated 2-fold. The activities of the key enzymes of pentose phosphate synthesis and of carbohydrate catabolism rose 1.4- and 1.3-fold, respectively. The extensive rise in the activities of enzymes of pyrimidine and purine biosynthesis indicates that the behavior of these enzymes is more stringently linked with replication than that of other enzymes. The results are in line with earlier ones in this Laboratory on solid tumors indicating an integrated imbalance of the key enzymes of pyrimidine, purine and carbohydrate metabolism that should confer selective advantages to cancer cells. In studying the behavior of purine enzymes that may reveal a relationship with the commitment of cancer cells to replication, the behavior of inosine phosphorylase and guanine deaminase provided such examples. Inosine phosphorylase activity was decreased in all the hepatomas examined irrespective of growth rates of these neoplasms; thus, the decrease of this enzymic activity was transformation-linked. By contrast, the guanine deaminase activity fluctuated in the various lines of the hepatoma spectrum indicating that the behavior of the activity of this enzyme was coincidental to transformation or progression in liver tumors. The relationship of 4 key enzymes with the progression-linked increase in the deoxynucleoside triphosphate pools in hepatomas was examined. The activities of CTP synthetase, thymidine kinase, IMP dehydrogenase and ribonucleotide reductase markedly increased in parallel with tumor growth rates and with the enlargement of the deoxyribonucleotide pools. The applicability of alterations of enzymic programs to 14 different tumor types was shown. There is a shared enzymic program that applies to rodent, avian and human neoplasms. The pattern was observed in carcinomas, sarcomas, leukemias and in human colon tumor xenografts carried in the nude mouse. The biochemical programs displayed were independent from the carcinogenic agent that caused the neoplasms because there was a shared enzymic program in chemically-induced, transplantable rat and mouse tumors, in viral-induced, transplantable avian neoplasms and in primary liver, kidney, lung and colon tumors in human. These observations indicate that strategic aspects of the biochemical commitment of the cancer cells to replication have now been identified. These novel insights should assist in the targeting of selective chemotherapy against cancer cells.