Pho85 kinase, a yeast cyclin-dependent kinase, regulates the expression of UGP1 encoding UDP-glucose pyrophosphorylase.

The PHO85 gene is a negative regulator of the PHO system in the yeast Saccharomyces cerevisiae and encodes a protein kinase (Pho85) highly homologous to the Cdc28 kinase (Cdc28). Ten cyclin-like proteins are known to interact with Pho85, and combination with different cyclins is believed to be responsible for distinct Pho85 functions, including phosphate metabolism, carbon source utilization and cell cycle regulation. However, only a limited number of substrates of Pho85 kinase, including Pho4, Gsy2 and Sicl, have so far been identified. To search for more targets of Pho85 and to clarify the genetic control mechanisms by Pho85 kinase in these cellular functions, we carried out a genome-wide analysis of the effect of a pho85Delta mutation on gene expression. We found that expression of various genes involved in carbon metabolism are affected by the mutation and that among them, UGP1 promoter activity was increased in the absence of Pho85 kinase. This increase in the promoter activity was not observed in a pho4Delta mutant or with a mutant UGP1 promoter that is devoid of putative Pho4 and Bas2 binding sites, suggesting that UGP1 expression is modulated by Pho85 through Pho4. We also found that expression of several Pho85-cyclin genes were altered by the carbon source, the growth phase and Pho85 kinase itself.

Up-regulation of genes encoding glycosylphosphatidylinositol (GPI)-attached proteins in response to cell wall damage caused by disruption of FKS1 in Saccharomyces cerevisiae.

FKS1 and FKS2 encode alternative catalytic subunits of the glucan synthases that are responsible for synthesis of beta-1,3-glucan in the Saccharomyces cerevisiae cell wall. Disruption of FKS1 reduces the glucan content of the cell wall, increases chitin content and activates the expression of CWP1, which encodes a glycosylphosphatidylinositol (GPI)-dependent cell wall protein. These cellular responses have been regarded as compensating for cell wall damage in order to maintain cell wall integrity. Here, we report the identification, by genome-wide screening, of 22 genes that are transcriptionally up-regulated in fks1delta cells. Among them, five genes were found to encode GPI-attached proteins, three of which are covalently associated with the cell wall. Deletion and replacement analysis of the promoter regions identified Rlm1-binding sequences as being responsible for the up-regulation following disruption of FKS1. Using the rlm1delta tetOp-FKS1 strain, in which the expression of FKS1 can be repressed by doxycycline, we examined the requirement for Rlm1 for the transcriptional up-regulation of these five genes. Three of the five genes were not up-regulated by doxycycline, indicating that Rlm1 mediates their up-regulation when FKS1 is inactivated. The remaining two genes were up-regulated by doxycycline, suggesting that a transcription factor other than Rlm1 is involved in their response to disruption of FKS1.

Expression and activity of dehydroepiandrosterone sulfotransferase in human gastric mucosa.

Dehydroepiandrosterone sulfotransferase (DHEA-ST) is a key enzyme in the formation of Dehydroepiandrosterone sulfate (DHEAS) and is thought to be involved in the conversion of various substances such as bile acids and cholesterol. The existence of DHEA-ST in the small intestine in addition to the adrenal gland and liver in adult humans was recently reported. As the sulfotransferases can act on toxic or potentially toxic substances to reduce their biological activity, we attempted to clarify the significance of DHEA-ST in gastrointestinal tract. We examined surgically resected human stomach for the presence of DHEA-ST and attempted to determine its possible biological significance. DHEA-ST activity ranged widely from 6 to 84 pmoles/mg protein/90 min in 7 cases. Immunoblotting revealed one single band of a 35-kDa protein corresponding to the moleculr weight of DHEA-ST. Both DHEA-ST immunoreactivity and mRNA hybridization signals were localized in parietal cells of the gastric glands. The results of our present study demonstrated that the sulfation of DHEA by DHEA-ST occurs in the gastric glands. The localization of DHEA-ST in parietal cells suggests that this enzyme is correlated to mucosal function in the human stomach in addition to detoxification of exogenous substances.

Application of homogeneous time-resolved fluorescence (HTRFTM) to monitor poly-ubiquitination of wild-type p53.

Rapid degradation of wild-type p53 in the human uterine cervix is induced by the infection of high-risk human papilloma virus (HPV) types 16 and 18. HPV-E6 protein plays a critical role in the poly-ubiquitination of wild-type p53 by mediating the association of p53 with E6-associated protein (E6AP). As a result, the poly-ubiquitinated p53 is rapidly and selectively degraded by the 26S proteasome. We have established a high throughput assay system to monitor poly-ubiquitination of wild-type p53 using a new fluorescence homogeneous technology known as Homogeneous Time-Resolved Fluorescence (HTRFTM). The Europium Cryptate [Eu(K)]-labeled ubiquitins are incorporated into poly-ubiquitin chains conjugated with the biotinylated p53. In the HTRF assay, Europium cryptate-labeled ubiquitin and streptavidin-labeled allophycocyanin (XL665) are used as the fluorescence donor and acceptor, respectively. The biotinylated p53 is ubiquitinated by ubiquitination enzymes, then by the addition of streptavidin-labeled XL665, the donor and acceptor molecules are brought in close proximity, thereby generating fluorescent signals. This time-resolved fluorescence assay system shows a sufficient signal for its application in synthetic compound screening and having almost the same level of sensitivity as that monitored by the scintillation proximity assay (SPA) using 125I-labeled ubiquitin. The detection of poly-ubiquitination of wild-type p53 by using the HTRFTM or SPA systems described here is much easier and quicker than by using conventional methods. Therefore, these new systems would be appropriate for high throughput screening of compounds for the discovery of new inhibitors of poly-ubiquitination of wild-type p53.

Yolk sac tumor of the stomach with an adenocarcinomatous component: a case report with immunohistochemical analysis.

A 56-year-old male treated for a gastric yolk sac tumor with an adenocarcinomatous component is described. A mixed area of reticular and glandular neoplastic components was morphologically identified in this tumor. Immunohistochemically, the yolk sac tumor expressed alpha-fetoprotein (AFP), placental alkaline phosphatase (PLAP), and cytokeratin, but was negative for carcinoembryonic antigen (CEA). The adenocarcinoma was positive for CEA and cytokeratin, partially positive for PLAP, and negative for AFP. In the mixed area, the tumor cells were positive for cytokeratin, weakly expressed AFP and PLAP, and sporadically stained for CEA both in the reticular and glandular components. This area was identified as a transitional area of the yolk sac tumor and adenocarcinoma. These findings demonstrate that the yolk sac and adenocarcinomatous components are closely related. It also suggests that the tumor arose from multipotential neoplastic mucosal epithelial cells with both yolk sac and gastric mucosal phenotypes.

Amino acid residues in the omega-minus region participate in cellular localization of yeast glycosylphosphatidylinositol-attached proteins.

The final destination of glycosylphosphatidylinositol (GPI)-attached proteins in Saccharomyces cerevisiae is the plasma membrane or the cell wall. Two kinds of signals have been proposed for their cellular localization: (i) the specific amino acid residues V, I, or L at the site 4 or 5 amino acids upstream of the GPI attachment site (the omega site) and Y or N at the site 2 amino acids upstream of the omega site for cell wall localization and (ii) dibasic residues in the region upstream of the omega site (the omega-minus region) for plasma membrane localization. The relationships between these amino acid residues and efficiencies of cell wall incorporation were examined by constructing fusion reporter proteins from open reading frames encoding putative GPI-attached proteins. The levels of incorporation were high in the constructs containing the specific amino acid residues and quite low in those containing two basic amino acid residues in the omega-minus region. With constructs that contained neither specific residues nor two basic residues, levels of incorporation were moderate. These correlations clearly suggest that GPI-attached proteins have two different signals which act positively or negatively in cell wall incorporation for their cellular localization.

Immunohistochemical and two-parameter flow cytometric studies of DNA topoisomerase II alpha in human epithelial ovarian carcinoma and germ cell tumor.

DNA topoisomerase II alpha (topo II alpha) is associated with active cell proliferation and is a target for chemotherapeutic agents administered to patients with ovarian cancer. To evaluate the biologic significance of topo II alpha expression in human ovarian carcinomas, we examined the expression of this protein immunohistochemically in tissue sections from 99 patients with ovarian cancer (85 common epithelial carcinomas, 14 germ cell tumors). We also measured topo II alpha and nuclear DNA content by two-parameter flow cytometry in 29 cases to evaluate possible qualitative changes of topo II alpha in the cell cycle of ovarian cancer cells. We observed a significant correlation of the labeling indices (LIs) of topo II alpha and Ki-67. The topo II alpha-to-Ki-67 ratio in germ cell tumors significantly exceeded that in common epithelial ovarian carcinomas (P = .038). Among the latter, the topo II alpha-to-Ki-67 ratio was significantly higher in serous cystadenocarcinomas than in mucinous cystadenocarcinomas. Two-parameter flow cytometric analysis revealed that topo II alpha expression was mainly observed in cells at the S to G2/M phases of the cell cycle, but, in some cases, topo II alpha positivity was detected in cells at G1. A significantly higher topo II alpha-to-Ki-67 ratio was detected in tumors with topo II alpha-positive cells at the G1 than in tumors in which topo II alpha-positive cells were not at G1. Results indicated that quantitative as well as quantitative changes in topo II alpha occur in human ovarian carcinomas.

Human ubiquitin-protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin-conjugating enzymes.

BACKGROUND: Nedd4 is a ubiquitin-protein ligase containing a calcium/lipid-binding domain, multiple WW domains and a C-terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin-conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport. RESULTS: Three mRNA species for human Nedd4 were found to be 6.4-, 7.8- and 9.5-kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue- and cell-specificities of Nedd4 expression. The Nedd4 protein, approximately 120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down-regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N-terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full-length Nedd4 and E2 enzyme revealed the both proteins mostly colocalized in the cytoplasmic periphery, while the N-terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme. CONCLUSION: Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.

Immunohistochemistry of Caspase3/CPP32 in human stomach and its correlation with cell proliferation and apoptosis.

Caspase3/CPP32 is a member of the interleukin-1 beta-converting enzyme (ICE) or cell death effector (CED)-3 family, which is involved in the induction of apoptosis and has been considered to be correlated with apoptosis because of the most downstream enzyme in their apoptosis inducing pathway. We immunolocalized Caspase3/CPP32 in both normal and neoplastic human gastric mucosa. We then correlated the findings with cell proliferation studied by Ki67 immunostaining and apoptosis, which was tested for by DNA fragmentation in situ using TdT-mediated dUTP biotin nick end labeling (TUNEL) method in order to examine possible biological significance in cell turnover of normal and pathological human gastric tissues. Caspase3/CPP32 immunoreactivity was detected in both the cytoplasm and nuclei of glandular epithelial cells, predominantly in the Ki67 positive proliferative zone and TUNEL positive foveolar epithelium of normal non-neoplastic gastric mucosa (n = 10) and tumor cells of both adenoma (n = 17) and carcinoma (n = 33). We determined the labeling index (LI) of Ki67, Caspase3/CPP32 and TUNEL positive cells by evaluating the number of positive cells in the same areas of serial tissue sections using computer-assisted image analysis. Ki67 LI in adenocarcinoma (78.6 +/- 12.6%) was significantly [p < 0.0001] higher than that of adenoma (43.8 +/- 8.9%) and non-neoplastic gastric mucosa (24.2 +/- 9.0%). Caspase3/CPP32 LI in adenocarcinoma (17.1 +/- 10.3%) was significantly lower [p < 0.0001] than that of gastric adenoma (33.1 +/- 19.8%) and non-neoplastic gastric mucosa (42.4 +/- 15.8%). TUNEL LI in adenocarcinoma (1.9 +/- 2.1%) was significantly [p < 0.0001] lower than that of non-neoplastic gastric mucosa (6.0 +/- 3.5%), but not significantly different from that of adenoma (3.0 +/- 2.9%). These results indicate that gastric adenocarcinoma is associated with an inhibition of apoptosis and the augmentation of proliferative activity of tumor cells compared to non-neoplastic gastric mucosa. There was a tendency to a positive correlation between the Caspase3/CPP32 and TUNEL LI and an inverse correlation between the Caspase3/CPP32 and Ki67 LI, when evaluating all the specimens, although the correlation did not reach statistical significance. These results also suggest that Caspase3/CPP32 is involved in the development or regulation of apoptotic cell death in cell turnover of normal and neoplastic mucosa of the human stomach.

DNA topoisomerase II alpha and Ki-67 in human adrenocortical neoplasms: a possible marker of differentiation between adenomas and carcinomas.

Cell kinetic information is valuable in evaluating the diagnosis and/or biologic behavior of various human neoplasms. Monoclonal antibody Ki-67 recognizes the cells other than G0 of the cell cycle. A cell cycle-related intranuclear protein, topoisomerase II alpha (topoII alpha), separates chromosomes at the end of mitosis. Its expression is mostly limited to the S to G2/M phases of the cell cycle. We studied cell proliferative activity in adrenocortical adenomas (n = 28), carcinomas (n = 17), and normal adrenal glands (n = 6) by immunohistochemical analysis of Ki-67 and topoII alpha to evaluate their value in the diagnosis of adrenocortical malignancy. We detected Ki-67 and topoII alpha immunohistoreactivity in the nuclei of each case we examined. There was a significant positive correlation (r = 0.927) between the Ki-67 and topoII alpha labeling indexes (LIs), the percentage of positive cells. In normal adrenal cortex and adenoma, the LIs for Ki-67 and topoII alpha were 0.48 +/- 0.16 and 0.44 +/- 0.15 for normal and 0.64 +/- 0.11 and 0.72 +/- 0.12 for adenoma, respectively, with no significant differences in the LIs of adenomas and normal adrenals. The Ki-67 and topoII alpha LIs in the carcinomas were 5.84 +/- 1.33 and 6.13 +/0 1.65, respectively; these LIs were significantly higher than the LIs of adenomas. Eleven of 17 carcinomas demonstrated topoII alpha and Ki-67 LIs of more than 2.5, whereas none of the adenomas did. The topoII alpha and Ki-67 LIs in carcinomas with metastasis (11.21 +/- 3.15 and 9.75 +/- 2.31 respectively; n = 7) were significantly higher than in those without metastasis (2.58 +/- 0.61 and 3.12 +/- 0.90, respectively; n = 10). This indicates that immunohistochemical analysis of Ki-67 and topoII alpha could help to differentiate carcinoma from adenoma in resected adrenocortical neoplasms and might predict aggressive biologic behavior in carcinomas.

Analysis of cell damage and proliferation in Helicobacter pylori-infected human gastric mucosa from patients with gastric adenocarcinoma.

Helicobacter pylori (HP) infection, a cause of multifocal atrophic gastritis, is considered an important factor related to the evolution of the human gastric mucosa from normal to intestinal-type adenocarcinoma. We examined cell proliferation and both double and single strand DNA damage in situ in 35 patients undergoing gastrectomy for adenocarcinoma with HP-infected gastric mucosa by immunolocalization of Ki-67, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, and in situ nick translation. We also studied the distribution of intraepithelial neutrophils by elastase immunolocalization. HP infection was confirmed in all cases by serum anti-HP antibodies, ureas testing, and histopathological examination. HP-infected gastric mucosa was classified according to the degree of inflammation and intestinal metaplasia. Ki-67, terminal deoxynucleotidyl transferase-mediated labeling, in situ nick translation, and intraepithelial neutrophil indices all increased with the progression of gastritis and were highest in glands with incomplete intestinal metaplasia. All indices were lowest in gastric glands with complete intestinal metaplasia. Significant positive correlations were observed among these markers. Increased proliferative activity in HP-associated chronic gastritis in response to cell damage or injury was clearly demonstrated, suggesting that both HP-associated toxins and intraepithelial neutrophils are important in HP-related gastric epithelial injury. Increased cell turnover associated with incomplete intestinal metaplasia may result in DNA instability and subsequent development of intestinal-type gastric adenocarcinoma in HP-infected mucosa.

Immunohistochemical study of Ki-67 and DNA topoisomerase II in human endometrium.

Topoisomerase II (topo II) separates the chromosomes at the end of mitosis, and its expression is limited mostly to the S-to-G2/M phases of the normal cell cycle. We examined the expression of topo II immunohistochemically in 56 specimens of the human endometrium that were retrieved from surgical pathology files. Specimens included proliferative phase mucosa (n = 7), secretory phase mucosa (n = 5), nonatypical adenomatous hyperplasia (n = 7), atypical adenomatous hyperplasia (n = 7) and endometrioid adenocarcinoma (n = 30). We calculated the labeling index (LI) for topo II and correlated the findings with the LI for Ki-67. A significant positive correlation was obtained between the Ki-67 and topo II LIs in all of the specimens examined. The levels of the topo II and Ki-67 LIs in secretory phase endometrium were each significantly lower than in the other cases examined. The levels of the topo II and Ki-67 LIs in adenocarcinoma were significantly higher than those in proliferative phase endometrium and nonatypical hyperplasia. There were no significant differences between atypical hyperplasia and adenocarcinoma in the Ki-67 and topo II LIs. The level of the topo II LI in atypical hyperplasia was significantly higher than that in nonatypical hyperplasia, whereas the Ki-67 LI did not differ between atypical and nonatypical hyperplasia. The levels of the Ki-67 LI were significantly higher than those of the topo II LI in proliferative phase endometrium and in nonatypical hyperplasia, but no significant differences were observed between the LIs in atypical hyperplasia and adenocarcinoma. Topo II immunostaining can identify proliferative cells in routinely processed surgical pathology specimens of human endometrium. The relative overexpression of topo II as compared with Ki-67 in adenocarcinoma suggests a dysregulation or qualitative alteration in topo II associated with malignancy, as reported in other tissues. Such over-expression in atypical hyperplasia might reflect the possible premalignant nature of this type of endometrial hyperplasia.

A novel arachidonate-preferring acyl-CoA synthetase is present in steroidogenic cells of the rat adrenal, ovary, and testis.

We report herein the cDNA cloning of a novel rat acyl-CoA synthetase (ACS) that preferentially uses arachidonate and eicosapentaenoate. This newly identified ACS (designated ACS4) contains 670 amino acids and is 68% identical to rat ACS3, a previously characterized ACS that is highly expressed in brain. ACS4 was overproduced in Escherichia coli and the resulting enzyme was purified to homogeneity. The purified enzyme utilizes arachidonate and eicosapentaenoate most preferentially among C8-C22 saturated fatty acids and C14-C22 unsaturated fatty acids. Kinetic analyses revealed that the enzyme has a high affinity for arachidonate and eicosapentaenoate and low affinity for palmitate. ACS4 transcripts are detectable in a wide range of tissues, with the highest level in adrenal gland. Immunoreactivity to ACS4 was detected in the zona fasciculata and reticularis of adrenal gland, in the corpus luteum and stromal luteinized cells in ovary, and in the Leydig cells of testis.

Immunohistochemical study of DNA topoisomerase II in human gastric disorders.

Topoisomerase II (topo II) separates chromosomes at the end of mitosis and is also the target for various chemotherapeutic agents. Expression of this enzyme has been demonstrated to increase rapidly at the end of the S to G2/M phase and decrease after the completion of mitosis. We immunolocalized topo II in specimens of both normal and neoplastic human gastric mucosas to evaluate expression of this enzyme. Three different antibodies were used for the immunostaining of topo II (anti-topo II alpha isoform, anti-topo II beta isoform and anti-topo II alpha and -beta isoforms). There were no significant differences in topo II labeling index (LI) between frozen and paraffin-embedded tissue sections obtained from the same cases. Topo II LI was significantly correlated with Ki67 LI in all of the specimens examined. The area of cells positive for Topo II was much narrower than that of Ki67 in the normal gastric glands, and the pattern of Topo II immunolocalization in both adenomas and adenocarcinomas was also essentially the same as that of Ki67. The topo II LI values (positive cells/1000 cells) for normal gastric gland, adenoma, intestinal-type adenocarcinoma, and diffuse-type adenocarcinoma were 114.7 +/- 2.2, 266.7 +/- 18.8, 277.6 +/- 19.2, and 324.5 +/- 5.3, respectively. Significant differences in topo II LI and topo II/Ki67 index were observed between normal and neoplastic mucosas (P < 0.0001) and between adenomas or intestinal-type adenocarcinoma and diffuse-type adenocarcinoma (P < 0.001 and P < 0.01, respectively). Simultaneous measurement of topo II alpha and nuclear DNA content by two-parameter flow cytometry revealed that the Jurkat cell line established from acute lymphocytic leukemia cells expressed the enzyme in cells at other than S and G2/M phases of the cell cycle whereas topo-II alpha-positive cells were predominantly observed in S and G2/M phases of the cell cycle in the cells from normal lymph nodes. These findings suggest that dys-regulation or qualitative changes of topo II alpha expression are associated with malignancy. Topo II immunostaining can thus detect proliferating cells in routinely processed tissue sections and can indicate the altered topo II alpha expression in human cancers, which may be related to the sensitivity to topo-II-targeted chemotherapeutic agents.

In situ analysis of tissue dynamics and p53 expression in human gastric mucosa.

In situ tissue dynamics were studied in 12 cases of human gastric mucosa, including normal gastric body mucosa and gastric glands with intestinal metaplasia, obtained from gastrectomy specimens of adenocarcinoma. Cell proliferation was determined by Ki67 immunoreactivity. DNA fragmentation was studied in situ by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In addition, p53 expression was examined by both immunohistochemistry and mRNA in situ hybridization. In the oxyntic gastric glands, Ki67 immunoreactivity was observed exclusively in the proliferative zone and TUNEL-positive cells were present predominantly in the surface foveolar epithelium. In the gastric glands with complete intestinal metaplasia, Ki67-positive cells were present in the lower portion of the glands and TUNEL-positive cells in the superficial epithelium. In the gastric glands with incomplete intestinal metaplasia, TUNEL-positive cells were detected in the lower gastric glands adjacent to cells immunoreactive for Ki67; the proportion of these gastric glands with TUNEL-positive cells (40 out of 108 glands) was significantly higher than for oxyntic glands (94 out of 620 glands) or for glands with complete metaplasia (31 out of 254 glands). Relatively strong p53 immunoreactivity and mRNA hybridization were also observed in the proliferative and apoptotic areas of gastric glands with incomplete intestinal metaplasia. These results indicate that incomplete intestinal metaplasia is associated with increased cell turnover and p53 overexpression, possibly in response to various noxious or DNA-damaging stimuli.

Catabolism of adenine nucleotides in suspension-cultured plant cells.

Profiles of the catabolism of adenine nucleotides in cultured plant cells were investigated. Adenine nucleotides, prelabelled by incubation of suspension-cultured Catharantus roseus cells with [8-14C]adenosine, were catabolized rapidly and most of the radioactivity appeared in 14CO2. Allantoin and allantoic acid, intermediates of the oxidative catabolic pathway of purines, were temporarily labelled. When the cells, prelabelled with [8-14C]adenosine, were incubated with high concentrations of adenosine, the rate of catabolism of adenine nucleotides increased. The results suggest that the relative rate of catabolism of adenine nucleotides is strongly dependent on the concentration of adenine nucleotides in the cells. Studies using allopurinol, coformycin and tiazofurin, inhibitors of enzymes involved in purine metabolism, suggest that participation of AMP deaminase and xanthine oxidoreductase in the catabolism of adenine nucleotides in plant cells. AMP deaminase was found in extracts from C. roseus cells and its activity increased significantly in the presence of ATP. In contrast, no adenosine deaminase or adenine deaminase activity was detected. Qualitative differences in the catabolic activity of AMP were observed between suspension-cultured cells from different species of plants.

A high-performance liquid chromatography method for separation of purine bases, nucleosides and ureides: application to studies on purine catabolism in higher plants.

Mixtures of purine nucleotides, nucleosides, nucleobases, uric acid, allantoin and allantoic acid were fractionated by high-performance liquid chromatography on a polyvinyl alcohol gel column, Asahipak GS-320H, with isocratic elution by sodium phosphate. Application of this system to the determination of the sizes of cellular pools of purine derivatives in plant cells and of the activity of related enzymes, as well as to the purification of enzymatically synthesized radioactive compounds, is described.