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Modification of diamine oxidase activity in vitro by metabolites of asparagine and differences in asparagine decarboxylation in normal and virus-transformed baby hamster kidney cells.

ABSTRACT: 1. The oxidation of putrescine in vitro by pig kidney diamine oxidase (EC 1.4.3.6) was increased in the presence of 2-oxosuccinamic acid and malonamic acid. 2. It was inhibited by 3-aminopropionamide, oxaloacetate and pyruvate. 3. 2-Oxosuccinamate was derived from asparagine in virus-transformed baby hamster kidney (BHK) cells growing in tissue culture. 4. Asparagine was decarboxylated more efficiently by transformed than by normal BHK cells. 5. In BHK cells transformed by polyoma virus (Py BHK), 2-oxosuccinamate is the most likely immediate precursor of the 14CO2 arising from [U-14C]asparagine, and there was some evidence for its formation in an asparagine-dependent clone of BHK cells before and after their transformation by hamster sarcoma virus (respectively Asn- and HSV Asn-). 6. The relationship between 2-oxosuccinamate and pyruvate and the possible roles of these two substances in controlling cellular diamine oxidase activity are discussed.

SUBMITTER: Quash G

PROVIDER: S-EPMC1163901 | biostudies-other | 1976 Sep

REPOSITORIES: biostudies-other

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Diamine oxidase and polyamine oxidase activities in normal and transformed cells.

Project description:1. The activity of diamine oxidase (EC 1.4.3.6) in normal rat kidney cells and in normal rat kidney cells transformed by avian sarcoma virus (B77 strain) growing in tissue culture varies with the stage of growth. There is an initial stimulation of activity by 24h after seeding, followed by a steep decline during exponential growth (48-72h). Enzyme activity decreases even further as the cells reach saturation density (confluence) after 4 days in culture when the activity in normal rat kidney cells is twice as high as that in transformed cells. 2. Differences of about the same order of magnitude are observed between transformed human cells HeLa, HEp2 (a human epithelioid carcinoma) and normal human fibroblasts, in chicken cells between normal myeloblasts and leukaemic myeloblasts, and in rats between biopsy material from normal mammary tissue and 9,10-dimethylbenz[a]anthracene-induced mammary tumours. 3. Polyamine oxidase activity also varies with the growth of transformed rat kidney cells, but shows no significant variation with the growth of normal rat kidney cells between 24 and 96h after seeding. The activity in cells at confluence is from 3- to 5-fold lower in the transformed than in the normal rat kidney cells. 4. A similar 5-10-fold decrease in activity has been found in 9,10-dimethylbenz[a]anthracene-induced mammary tumours in rats and in human oesophageal tumours. 5. Possible reasons for these observations and the contribution of these two enzymes to cellular putrescine concentrations are discussed.

| S-EPMC1186366 | biostudies-other

A comparison of polyamine metabolism in normal and transformed baby-hamster-kidney cells.

Project description:Transformed baby-hamster-kidney cells contain higher intracellular concentrations of polyamines than do normal cells. The difference is greater in high-density confluent cultures. Transformed cells incorporate exogenous putrescine into the cells at a faster rate than do normal cells. They also show a marked increase in the rate of spermine biosynthesis compared with normal cells. Transformed cells grown to high cell densities released about 10% of their polyamines into the culture medium in a non-specific manner. In contrast, normal cells, under the same culture conditions, release up to 50% of their intracellular polyamines into the medium almost exclusively as free or conjugated spermidine. The elevated levels of polyamines found in transformed cells therefore appear to be the result of altered transport of polyamines across the cell membrane and of increased rates of biosynthesis.

| S-EPMC1158176 | biostudies-other

Cell-surface mucosubstances from trypsin diaggregation of normal and virus-transformed lines of baby-hamster kidney cells.

Project description:Cell disaggregation by trypsin solubilizes significantly less mucosubstance from the surface of polyoma-virus-transformed baby-hamster kidney cells than from the same non-transformed cell line. The mucosubstance, which consists of both acid mucopolysaccharides and mucoproteins, also differs qualitatively in the two cell lines.

| S-EPMC1177922 | biostudies-other

Structure and inhibition of human diamine oxidase.

Project description:Humans have three functioning genes that encode copper-containing amine oxidases. The product of the AOC1 gene is a so-called diamine oxidase (hDAO), named for its substrate preference for diamines, particularly histamine. hDAO has been cloned and expressed in insect cells and the structure of the native enzyme determined by X-ray crystallography to a resolution of 1.8 A. The homodimeric structure has the archetypal amine oxidase fold. Two active sites, one in each subunit, are characterized by the presence of a copper ion and a topaquinone residue formed by the post-translational modification of a tyrosine. Although hDAO shares 37.9% sequence identity with another human copper amine oxidase, semicarbazide sensitive amine oxidase or vascular adhesion protein-1, its substrate binding pocket and entry channel are distinctly different in accord with the different substrate specificities. The structures of two inhibitor complexes of hDAO, berenil and pentamidine, have been refined to resolutions of 2.1 and 2.2 A, respectively. They bind noncovalently in the active-site channel. The inhibitor binding suggests that an aspartic acid residue, conserved in all diamine oxidases but absent from other amine oxidases, is responsible for the diamine specificity by interacting with the second amino group of preferred diamine substrates.

| S-EPMC2791411 | biostudies-literature

Transcriptomic Analysis on Oncogene STYK1/NOK transfected Baby Hamster Kidney BHK21 Cells

Project description:RNAseq analysis identified 837 differential gene expressions (DEGs) with 423 up-regulated and 414 down-regulated after transfecting STYK1/NOK into BHK21 cells. GEO and KEGG enrichment analysis indicated that several entries such as ribosome, aging related diseases, oxidative phosphorylation and apoptosis played major roles during this cellular process. PPI analysis showed that four top down-regulated proteins (TOMM7, Uba52, Rpl28 and XP_005077076.1 (ribosomal protein S29)) localized within the highly interaction region might play key roles.

2021-04-15 | GSE172045 | GEO

A new crystal form of human diamine oxidase.

Project description:Copper amine oxidases (CAOs) are ubiquitous in nature and catalyse the oxidative deamination of primary amines to the corresponding aldehydes. Humans have three viable CAO genes (AOC1-3). AOC1 encodes human diamine oxidase (hDAO), which is the frontline enzyme for histamine metabolism. hDAO is unique among CAOs in that it has a distinct substrate preference for diamines. The structure of hDAO in space group P2(1)2(1)2(1) with two molecules in the asymmetric unit has recently been reported. Here, the structure of hDAO refined to 2.1 A resolution in space group C222(1) with one molecule in the asymmetric unit is reported.

| S-EPMC2815678 | biostudies-literature

Role of diamine oxidase during the treatment of tumour-bearing mice with combinations of polyamine anti-metabolites.

Project description:Treatment of mice bearing L1210 leukaemia with 2-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase (EC 4.1.1.17), produced a profound depletion of putrescine and spermidine in the tumour cells. Sequential combination of methylglyoxal bis(guanylhydrazone), an inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), with difluoromethylornithine largely reversed the polyamine depletion and led to a marked accumulation of cadaverine in the tumour cells. Experiments carried out with the combination of difluoromethylornithine and aminoguanidine, a potent inhibitor of diamine oxidase (EC 1.4.3.6), indicated that the methylglyoxal bis(guanylhydrazone)-induced reversal of polyamine depletion was mediated by the known inhibition of diamine oxidase by the diguanidine. In spite of the normalization of the tumour cell polyamine pattern upon administration of methylglyoxal bis(guanylhydrazone) to difluoromethylornithine-treated animals, the combination of these two drugs produced a growth-inhibitory effect not achievable with either of the compounds alone.

| S-EPMC1153170 | biostudies-other

Asparagine Hydroxylation is a Reversible Post-translational Modification.

Project description:Amino acid hydroxylation is a common post-translational modification, which generally regulates protein interactions or adds a functional group that can be further modified. Such hydroxylation is currently considered irreversible, necessitating the degradation and re-synthesis of the entire protein to reset the modification. Here we present evidence that the cellular machinery can reverse FIH-mediated asparagine hydroxylation on intact proteins. These data suggest that asparagine hydroxylation is a flexible and dynamic post-translational modification akin to modifications involved in regulating signaling networks, such as phosphorylation, methylation and ubiquitylation.

| S-EPMC7664127 | biostudies-literature

Differential segregation of human and hamster cathepsin D in transfected baby-hamster kidney cells.

Project description:The segregation of human cathepsin D, studied in baby-hamster kidney cells (BHK) transfected with human cathepsin D cDNA and compared with that of hamster cathepsin D in the same cells, showed that, in cells that expressed human cathepsin D at a low rate, most of the enzyme remained intracellular. In contrast, when the enzyme was expressed at a high rate, most was secreted. The segregation was examined with an anti-(human cathepsin D) antibody that reacted with the human enzyme exclusively and an anti-(rat cathepsin D) antibody that reacted with both enzymes. In one protocol the cells were metabolically labelled and the two antibodies were used in sequence to precipitate the enzymes from extracts of cells and medium. High expression of the human enzyme did not interfere with the segregation of hamster cathepsin D. In another protocol the activity of cathepsin D in cells and medium was measured before and after titration with anti-(human cathepsin D) antiserum. Human cathepsin D was found predominantly in the medium, and hamster cathepsin D mainly in the cells. In the presence of 10 mM-NH4Cl the intracellular segregation of hamster cathepsin D was strongly inhibited, while the segregation of human cathepsin D was only slightly diminished. In BHK cells, at least two systems participate in the sorting of the two cathepsins, one of them being rather insensitive to NH4Cl.

| S-EPMC1149854 | biostudies-other

Mapping choline metabolites in normal and transformed cells.

Project description:IntroductionCholine is an essential human nutrient that is particular important for proliferating cells, and altered choline metabolism has been associated with cancer transformation. Yet, the various metabolic fates of choline in proliferating cells have not been investigated systematically.ObjectivesThis study aims to map the metabolic products of choline in normal and cancerous proliferating cells.MethodsWe performed 13C-choline tracing followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis of metabolic products in normal and in vitro-transformed (tumor-forming) epithelial cells, and also in tumor-derived cancer cell lines. Selected metabolites were quantified by internal standards.ResultsUntargeted analysis revealed 121 LCMS peaks that were 13C-labeled from choline, including various phospholipid species, but also previously unknown products such as monomethyl- and dimethyl-ethanolamines. Interestingly, we observed formation of betaine from choline specifically in tumor-derived cells. Expression of choline dehydrogenase (CHDH), which catalyzes the first step of betaine synthesis, correlated with betaine synthesis across the cell lines studied. RNAi silencing of CHDH did not affect cell proliferation, although we observed an increased fraction of G2M phase cells with some RNAi sequences, suggesting that CHDH and its product betaine may play a role in cell cycle progression. Betaine cell concentration was around 10 µM, arguing against an osmotic function, and was not used as a methyl donor. The function of betaine in these tumor-derived cells is presently unknown.ConclusionThis study identifies novel metabolites of choline in cancer and normal cell lines, and reveals altered choline metabolism in cancer cells.

| S-EPMC7701132 | biostudies-literature

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