Purification and characterization of intestinal adenosine deaminase from mice

Adenosine deaminase (ADA) was isolated from small intestine of mice and purified to utmost homogeneity. SDS-PAGE of purified ADA gave a molecular weight of 41 kDa. Western blot analyses gave a single reactive band at 41 kDa and the other band was an associated ADA binding protein. The purified enzyme was more stable in the alkaline pH. The optimum pH and the pI values were about 7.0 and 4.96, respectively. Km values of the small intestinal ADA for adenosine and 2-deoxyadenosine were 23 and 16M, respectively. Purine riboside was a competitive inhibitor with Ki of 5 M, whereas 2-3-o-isopropylidene adenosine acted as an uncompetitive inhibitor (Ki 66 M). Activity of ADA was inhibited by the presence of theophylline (-40%), caffeine (-30%), and L-cysteine (-50%). Significantly, Hg²⁺ (100 M) inhibited 98% of the initial ADA activity. In addition, various purine analogs such as inosine, purine, -adenosine and adenine showed variable inhibitions on the activity of ADA. Relative ADA activity towards 3-deoxyadenosine and 6-chloropurine riboside was lower by 30% and 40%, respectively. However, the activity towards 2-o-methyl adenosine was higher (30%) compared to the activity obtained using adenosine.     

Expression of a Synthetic Porcine α-Lactalbumin Gene in the Kernels of Transgenic Maize

The main nutritional limitation of maize used for feed is the content of protein that is digestible, bioavailable and contains an amino acid balance that matches the requirements of animals. In contrast, milk protein has good digestibility, bioavailability and amino acid balance. As an initial effort to create maize optimized as a source of swine nutrition, a codon-adjusted version of a gene encoding the milk protein porcine -lactalbumin was synthesized. Maize expression vectors containing this gene under the control of the Ubi-1 promoter and nos 3 terminator were constructed. These vectors were used to transform maize callus lines that were regenerated into fertile plants. The -lactalbumin transgenes were transmitted through meiosis to the sexual progeny of the regenerated plants. Porcine -lactalbumin was detected in callus and kernels from transgenic maize lines that were transformed by two constructs containing the 27-kDa maize gamma-zein signal sequence at the 5 end of the synthetic porcine -lactalbumin coding sequence. One of these constructs contained an ER retention signal and the other did not. Expression was not observed in kernels or callus from transgenic maize lines that were transformed by a construct that does not contain an exogenous protein-targeting signal. This suggests that the signal peptide might play an important role in porcine -lactalbumin accumulation in transgenic maize kernels.     

Erythrocytenenzyme der Primaten

Zusammenfassung Die Adenosindesaminasen der Primaten zeigen eine genetisch determinierte Variabilität. Bei der Untersuchung von 327 subhumanen Primaten (289 Simiae der Alten Welt, 38 Prosimiae) konnten wir neun Adenosindesaminase-Varianten nachweisen, die auf Grund ihrer unterschiedlichen elektrophoretischen Wandergeschwindigkeit als ADA 6, ADA 4, ADA 2, ADA 2, ADA 1, ADA 3, ADA 5, ADA 5 und ADA 7 bezeichnet werden. Die Verteilung der verschiedenen Phänotypen wurde ermittelt.

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Red cell enzymes of primatesAdenosine deamisnase (EC: 3.5.4.4.)

Summary The polymorphism of adenosine deaminase has been investigated in 327 subhuman Primates (289 Old World Monkeys and 38 Prosimians). Nine adenosine deaminase variants were found to be present, which on the basis of their different electrophoretic mobilities were designated ADA 6, ADA 4, ADA 2, ADA 2, ADA 1, ADA 3, ADA 5, ADA 5 and ADA 7. The distribution of these various phenotypes has been estimated.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Template-directed reactions of aminonucleosides

Summary We have studied the reactions between adenosine 5-phosphorimidazolide and 9-(2-amino-2-deoxyxylofuranosyl) adenine (I) or 3-methylamino-3-deoxyadenosine (II), both with and without a poly (U) template. We find that both amino compounds react much more rapidly than does adenosine, in the absence of a template. The rate of reaction is greatly enhanced by a poly (U) template in the case of I, but the enhancement is slight in the case of II.Abbreviations A adenosine - xylo A_NH2 9-(2-amino-2-deoxy--D-xylofuranosyl) adenine - A_NHMe 3-methylamino-3-deoxyadenosine - ImpA adenosine 5-phosphorimidazolide - A³ pA adenylyl-[35]-adenosine - A² pA adenylyl-[25]-adenosine - U_NPA adenylyl-[52]-2-amino-2-deoxyuridine - xylo A_NPA 9-[adenylyl-(52)-2-amino-2-deoxy--D-xylofuranosyl]adenine - A_(NMe)pA adenylyl-[53]-3-methylamino-3-deoxyadenosine - pA adenosine 5phosphate - AppA P₁, P₂-diadenosine 5pyrophosphate - (pA)_n n = 2, 3 [2-5]-linked oligomers of pA - A² pA² pA [2-5]-linked trinucleoside diphosphate of A - poly (U) polyuridylic acid     

Regulators of cell division in plant tissues

Summary [³H] Zeatin was supplied to Zea mays L. seedlings with roots excised; the metabolites identified were adenosine-5-phosphate, adenosine, adenine, and 7-glucosylzeatin (a minor metabolite). The principal metabolites formed from zeatin by the roots of intact Z. mays seedlings were adenosine-5-phosphate, zeatin riboside-5-phosphate, zeatin riboside, adenine, adenosine and an unknown compound termed Y. This was isolated and identified as 9-glucosylzeatin. This glucoside also appeared to form from zeatin in cultured embryonic tissue of Z. mays.Part XVII: Gordon et al. (1973)     

Effect of nucleosides and nucleotides and the relationship between cellular adenosine 3′∶5′-cyclic monophosphate (cyclic AMP) and germ tube formation in Candida albicans

A yeast-mycelium (Y-M) transition in Candida albicans was induced by exogenous yeast extract, adenosine, adenosine 5-monophosphate (AMP), adenosine 5-diphosphate (ADP), adenosine 35 cyclic monophosphate (cAMP) and its analogue N⁶, O²-dibutyryl adenosine 35-cyclic monophosphate (dbcAMP) in defined liquid medium at 25°C. Adenosine 5-triphosphate (ATP) was found to delay germ tube formation in yeast cells, whereas the cAMP phosphodiesterase inhibitors, theophylline and caffeine, induced a Y-M transition. Intracellular and extracellular cyclic AMP levels increased during the yeast-mycelium transition and maximum levels of intracellular cyclic AMP coincided with maximum germ tube formation. Of the many inducers and inhibitors of germ tube and mycelium formation in C. albicans tested, including incubation at 37°C or in the presence of 1.5mM CaCl₂, the calmodulin inhibitor calmidazolium (R24571) added together with CaCl₂ induced the highest intra- and extracellular cyclic AMP levels. These results confirm the involvement of cyclic AMP in the yeast-mycelium transition of C. albicans.     

Purinergic regulation of basal and arginine vasopressin-stimulated hydraulic conductivity in rabbit cortical collecting tubule

Summary An extracellular adenosine responsive site that stimulates adenylate cyclase activity has been identified in several tissues. There is limited information on the presence and physiologic significance of adenosine receptors in well-defined segments of the mammalian nephron. We therefore examined the effect of adenosine and selected analogues on basal hydraulic conductivity in rabbit cortical collecting tubules (CCT) perfused in vitro. Adenosine and analogues with an intact ribose moiety produced a significant, sustained increase in hydraulic conductivity. No increase in hydraulic conductivity was seen in either time control CCT's or CCT's exposed to an adenosine analogue with an altered ribose moiety. These experiments are compatible with the presence of a functional adenosine receptor which requires an intact ribose moiety and acts to increase hydraulic conductivity in the mammalian CCT.An intracellular adenosine responsive site, termed the P site, which inhibits adenylate cyclase activity, has also been described in several tissues. We therefore examined the effect of aP site agonist on hydraulic conductivity responses to arginine vasopressin, forskolin and cAMP.P site stimulation with 25 dideoxyadenosine inhibited the effect of AVP and of forskolin but not of cAMP to increase hydraulic conductivity. These results are compatible with a functionalP site in the rabbit CCT which acts at the catalytic subunit of adenylate cyclase to inhibit hydraulic conductivity. Together, these results demonstrate purinergic modulation of basal and arginine vasopressin-stimulated water flux in the mammalian collecting tubule.     

Regio-selective preparation of vinyl adenosine ester catalyzed by alkaline protease

The transesterification of divinyladipate with adenosine in DMF containing 20% (v/v) DMSO was catalyzed by Streptomyces sp. alkaline protease and esterification occurred exclusively at the 3-position of hydroxyl group of ribofuranose in adenosine to give 3-O-vinyladipoyl adenosine without other products.     

Occurrence of pppApp-synthesizing activity in actinomycetes and isolation of purine nucleotide pyrophosphotransferase

The occurrence of adenosine 5-triphosphate-3-diphosphate-synthesizing activity was detected in five strains of actinomycetes; Streptomyces morookaensis, Streptomyces aspergilloides, Streptomyces hachijoensis, Actinomyces violascens and Streptoverticillium septatum, out of 825 strains of actinomycetes, bacteria, fungi and imperfecti. Purine nucleotide pyrophosphotransferase were extracellularly excreted associating with the cell growth, and were purified partially or to apparent homogeniety from the culture filtrate. The enzymes are a monomeric protein with a molecular weight of 18000–26000 and synthesize adenosine, guanosine and inosine 5-phosphate (mono, di or tri)-3-diphosphate such as pApp, ppApp, pppApp, pGpp, ppGpp, pppGpp and pppIpp by transferring a pyrophosphoryl group from the 5-position of ATP, dATP and pppApp to the 3-position of purine nucleotides in the presence of a divalent cation and in alkaline state.Abbreviations pppApp adenosine 5-triphosphate 3-diphosphate - ppApp adenosine 5-diphosphate 3-diphosphate - pApp adenosine 5-monophosphate 3-diphosphate - pppGpp guanosine 5-triphosphate 3-diphosphate     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Properties and regulation of adenosine 5′-phosphosulfate sulfotransferase from suspension cultures ofNicotiana sylvestris

The properties and the regulation of adenosine 5-phosphosulfate sulfotransferase extracted from cell suspension cultures ofNicotiana sylvestris was investigated. Optimal adenosine 5-phosphosulfate sulfotransferase activity was obtained from the cells by extraction with 0.1 M tris-HCl, pH8.0, containing 2 M MgSO₄ and 10 mM dithioerythritol. The K _m for adenosine 5-phosphosulfate in the sulfotransferase reaction was about 11 M. Adenosine 5-phosphosulfate in concentrations above 50 M were inhibitory. The extratable adenosine 5-phosphosulfate sulfotransferase activity decreased during cultivation with sulfate as the sole sulfur source, but after about 3 days it reached a constant level (50 to 100 nmol activated sulfate transferred h^-1 mg^-1 protein) which was maintained for at least 24 h. Addition of 0.5 mM cysteine to the culture medium decreased the extractable adenosine 5-phosphosulfate sulfotransferase activity and blocked growth completely. With 0.1 mM cysteine an enzyme level of about 10% of the initial value was reached within 6 to 12 h without significant inhibition of growth. The added cysteine was absorbed rapidly and after 24 h cysteine could no longer be detected in the medium. Before the cysteine was completely depleted, the activity of adenosine 5-phosphosulfate sulfotransferase started to increase, reaching ultimately a level which was comparable to the initial value.Abbreviations APS Adenosine 5-phosphosulfate - APSSTase adenosine 5-phosphosulfate sulfotransferase - DTE dithioerythritol - PAPS adenosine 3-phosphate 5-phosphosulfate - 2,4-D 2,4-di-chlorophenoxyacetic acid - BAP benzyladenine This paper is no. 10 in the series Regulation of Sulfate Assimilation in Plants.     

Metabolism of ribosylzeatin-5′-monophosphate by soybean callus

Using cytokinin dependent soybean callus and HPLC analysis it was shown that soybean callus rapidly metabolises ribosylzeatin-5-monophosphate to biologically active compounds which co-chromatographed with trans-ribosylzeatin and trans-zeatin.Abbreviations Z zeatin - RZ ribosylzeatin - RZMP ribosylzeatin-5-monophosphate     

The effect of G2-treatments with 2′-deoxyadenosine on the frequency of chromatid aberrations in human lymphocytes depends on the type of culture

The effect of G₂-treatments with 2-deoxyadenosine (dAdo) on the frequency of chromatid aberrations in X-irradiated and unirradiated human lymphocytes depends on the method of culture. In whole-blood cultures dAdo alone produced very few if any aberrations, but in the presence of inhibitors of adenosine deaminase (ADA), such as EHNA or coformycin, a high frequency of chromatid gaps, chromatid breaks, and isochromatid breaks were produced. In cultures of purified lymphocytes, dAdo produced aberrations even in the absence of an ADA inhibitor. Apparently the lymphocytes are protected against the chromosome-damaging effect of dAdo by the ADA activity of the erythrocytes. — When given as a post-treatment, dAdo also enhances the frequency of chromatid aberrations induced by X-rays in G₂. In whole-blood cultures this effect is obtained even in the absence of an ADA inhibitor, although the concentration required to produce enhancement is about twenty times higher than in the presence of the inhibitor.     

Localization of adenosine 5′-phosphosulfate sulfotransferase in spinach leaves

Roots of spinach (Spinacia oleracea L.) seedlings contained only a very low activity of adenosine 5-phosphosulfate sulfotransferase compared to the cotyledons. Adenosine 5-phosphosulfate sulfotransferase activity increased about tenfold in cotyledons during greening. Preparation of organelle fractions from spinach leaves by a combination of differential and isopycnic density gradient centrifugation showed that adenosine 5-phosphosulfate sulfotransferase banded with NADP-glyceraldehyde-3-phosphate dehydrogenase, a marker enzyme for intact chloroplasts. In the fractions of peroxisomes, mitochondria and broken chloroplasts virtually no adenosine 5-phosphosulfate sulfotransferase activity was measured. Comparison with the chloroplast enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase indicates that in spinach, adenosine 5-phosphosulfate sulfotransferase is localized almost exclusively in the chloroplasts.Abbreviations APS Adenosine 5-phosphosulfate - APSSTase Adenosine 5-phosphosulfate sulfotransferase - BSA Bovine serum albumin - BRIJ58 Polyethylene glycolmonostearylether - DTE Dithioerythritol - DTT Dithiothreitol - EDTA Ethylenediaminetetraacetic acid - ME 2-Mercaptoethanol - NADP-GPD NADP-linked glyceraldehyde-3-phosphate dehydrogenase - PAPS Adenosine 3-phosphate 5-phosphate 5-phosphosulfate - POPOP 1,4 Di [2-(5-phenyloxazolyl)]-benzene - PPO 2,5-Diphenyloxazol The results presented in this paper are taken from the Ph. D. thesis of H.F.     

Nucleic acid metabolism in yeast VI. Utilisation of exogenous dAMP

Summary It is shown that mutants of Saccharomyces cerevisiae able to efficiently utilise exogenous dTMP can also utilise exogenous dAMP. Under extracellular conditions permissive for dTMP uptake label stemming from offered [8-³H]dAMP is incorporated preferentially into alkali-resistant, high molecular weight material (putative DNA): only about 30% of high molecular weight cell-bound dAMP label was found to be sensitive towards mild alkali hydrolysis. This putative RNA label can be minimised to practically zero when mM Ade is employed in a dAMP labelling assay. Exogenous dAMP at 10 M was found to be cytostatic similarly to M dTMP and similarly to inhibit effectively import of exogenous P_i. We conclude from our results that there exists a yeast cytoplasmic membrane permease able to import dAMP. A model of this hypothetical permease system is presented.Abbreviations S. cerevisiae Saccharomyces cerevisiae - TIP yeast cytoplasmic membrane permease importing dTMP under permisive conditions - AIP hypothetical yeast cytoplasmic membrane permease importing dAMP under permissive conditions - tlr symbol for the recessive genetic trait to utilise effeciently exogenous dTMP - tmp symbol for the recessive genetic trait leading to dTMP auxotrophy - dTMP 2-deoxythymidine 5-monophosphate - dAMP 2-deoxyadenosine 5-monophosphate - (d)NMP (deoxy)ribonucleoside 5-monophosphate - dThd 2-deoxythymidine - Thy thymine - dAdo 2-deoxyadenosine - Ado adenosine - Ade adenine - Ura uracil - P_i inorganic phosphate Apart from discrete abbreviations we followed the rules of nomenclature as recommended by the IUPAC-IUB commission of biochemical nomenclature (CBN)     

The malate synthase gene of cucumber

The complete sequences of a full-length cDNA clone and a genomic clone encoding the Cucumis sativus glyoxysomal enzyme malate synthase, have been determined. The sequences have enabled us to identify putative control regions at the 5 end of the gene, three introns, and possible alternative polyadenylation sites at the 3 end. The deduced amino acid sequence predicts a polypeptide of 64961 molecular weight, which has 48% identity with that of Escherichia coli. Comparison of the sequence of malate synthase from cucumber with that from E. coli and with other glyoxysomal and peroxisomal enzymes, shows that a conserved C-terminal tripeptide is a common feature of those enzymes imported into microbodies.     

Formation of nucleoside 5′-phosphoramidates under potentially prebiological conditions

Summary Adenosine 5-phosphoramidates form when solutions containing adenosine 5-polyphosphates p_nA (n 3) or P₁, P₂-diadenosine 5-diphosphate and amines are allowed to dry out. Mg ions catalyze these reactions. We have studied systems containing ammonia, imidazole, glycine, ethylenediamine and histamine. The yields of adenosine 5-phosphoramidates range from 10–50 % based on the nucleotide. The prebiotic significance of the reactions is discussed.Abbreviations Im imidazole - hist histamine - gly glycine - en ethylenediamine - CDI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride - EDTA ethylenediaminetetraacetic acid - A adenosine - P_n (n = 1, 2 ) linear polyphosphate containing n phosphate residues - p_nA adenosine 5-polyphosphate containing n phosphate residues - ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - AppA P₁, P₂-diadenosine 5-diphosphate - gly-pA adenylyl-(5N)-glycine - ImpA adenosine 5-phosphorimidazolide - NH₂-pA adenosine 5-phosphoramidate - en-pA adenylyl-(5N)-ethylenediamine - hist (NH) - pA adenosine 5-phospho-[2-(4-imidazolyl)-ethylamide] - hist(Im)-pA adenosine 5-phospho-[4-(2-aminoethyl)-imidazolide] - enP_1,2 phosphoramidates of ethylenediamine derived from H₃PO₄ and H₄P₂O₇     

Association of the 5′ HS4 sequence of the chicken β‐globin locus control region with human EF1α gene promoter induces ubiquitous and high expression of human CD55 and CD59 cDNAs in transgenic rabbits

Whatever its field of application, animal transgenesis aims at a high level of reproducible and stable transgene expression. In the case of xenotransplantation, prevention of hyperacute rejection of grafts of animal origin requires the use of organs expressing human inhibitors of complement activation such as CD55 (DAF) and CD59. Pigs transgenic for these molecules have been produced, but with low and variable levels of expression. In order to improve cDNA expression, a vector containing the 5HS4 region from the LCR of the chicken globin locus and the promoter and the first intron from the human EF1 gene, was used to coexpress human CD55 and CD59 cDNAs in transgenic rabbits. The transgenic lines with the 5HS4 region displayed dramatically enhanced CD55 and CD59 mRNA concentrations in brain, heart, kidney, liver, lung, muscle, spleen and aortic endothelial cells in comparison with the transgenic lines without the 5HS4 region. In the absence of the 5HS4 region, only some of the transgenic lines displayed specific mRNAs and at low levels. Human CD55 and CD59 proteins were detectable in mononuclear cells from transgenic rabbits although at a lower level than in human mononuclear cells. On the other hand, primary aortic endothelial cells from a bitransgenic line were very efficiently protected in vitro against human complementdependent lysis. Transgenic rabbits harbouring the two human inhibitors of complement activation, CD55 and CD59, can therefore be used as new models in xenotransplantation. Moreover, the vector containing the 5HS4 region from the LCR of the chicken globin locus seems appropriate not only for xenotransplantation but also for any other studies involving transgenic animals in which cDNAs have to be expressed at a high level in all cell types.     

Monomers for Oligonucleotide Synthesis with Linkers Carrying Reactive Residues: I. The Synthesis of Deoxynucleoside Derivatives with Methoxyoxalamide Groups in Heterocyclic Bases

A number of monomers for the standard phosphoamidite oligodeoxynucleotide synthesis that carry reactive methoxyoxalamide groups attached to the thymidine, 2-deoxycytidine, and 2-deoxyadenosine heterocyclic bases were prepared.     

Immunohistochemical localization of cyclic AMP and ultrastructural demonstration of adenylate cyclase activity in the testis of Esox lucius at time of spermiation

Summary In the testis of Esox lucius at the time of spermiation, activity of cyclic adenosine 3,5-monophosphate (cAMP) was immunocytochemically localized at the level of the Sertoli cells. In these cells adenylate cyclase activity was also ultracytochemically demonstrated by using adenylyl imidodiphosphate as a substrate. Reaction products of adenylate cyclase were primarily detectable on the basal and adluminal plasma membranes and on the surface of protrusions of the cell body into the lumen.