Retardation of leaf senescence by urea cytokinins in Raphanus sativus

Approximately 500 urea derivatives and related compounds were tested for ability to retard leaf senescence as measured by chlorophyll retention in radish (Raphanus sativus) leaf discs. Of the 90 compounds found to be active, some had activity at 10^?6 M of the same order as kinetin. There was a high correlation between ability to promote chlorophyll retention and initiation of cell division. Highly active compounds had a planar ring and a HNCONH bridge; substitution with a HNCSNH bridge reduced activity and all other tested arrangements of the bridge gave inactive compounds. Substitution of both amino hydrogen atoms on one or both sides of the bridge reduced or removed activity. Some N′-substituted phenyl ureas were highly active. Introduction of a N-phenyl ring to a N-phenyl urea increased activity except where one ring was substituted in the para position with chloro, bromo or iodo. The activities of symmetrical disubstituted ureas were generally less than the corresponding N-monosubstituted derivative. The results suggest that the receptor site for cytokinin activity is the same for senescence retadation and cell division initiation.     

Influence of alterations in the purine ring on biological activity of cytokinins

The 1-deaza-, 3-deaza-, 8-aza-1-deaza- and 8-aza-3-deaza-analogs of kinetin and 6-(3-methyl-2-butenylamino)purine and some of their ribosides were synthesized and their growth-promoting activities in the tobacco bioassay were determined and compared with those of the parent compounds. The replacement of nitrogen by carbon in the 1 -position of the purine ring decreases cytokinin activity 15-fold for kinetin and 2-fold for 6-(3-methyl-2-butenylamino)purine (IPA); however, the replacement of nitrogen by carbon in the 3-position decreases the activity 2000 times for kinetin and 1000 times for 6-(3-methyl-2-butenylamino)-purine. The activity of 8-aza-1-deaza-analogs appears to be of the same order of somewhat lower than the corresponding 1-deaza-analogs. The corresponding 8-aza-3-deaza-analogs are less active than kinetin (400 times and 6-(3-methyl-2-butenylamino)purine (40 times). However, they are more active than the corresponding 3-deaza-analogs. The concentration range in which the ribosides show activity is nearly the same as for the corresponding free bases, but the maximum yield of tobacco-callus for the riboside of the 3-deaza-analog of 6-(3-methyl-2-butenylamino)purine is very low.     

The synthesis, transport and metabolism of endogenous cytokinins

Abstract Present evidence indicates that only the root systems of plants have been shown conclusively to synthesize cytokinins. Although most of these compounds are apparently exported to the shoot via the xylem, there are indications that more attention should be given to the possibility of translocation through the phloem. Within mature leaves the cytokinins derived from the roots are converted to inactive or storage forms by means of glucosylation. While it would appear that glucosylation could occur in all living plant cells whenever the cytokinins are no longer required for active growth, and could provide the plant with a potential reservoir of cytokinins, very little is known with regard to the transport and reutilisation of these compounds.     

Distribution and metabolism of root-applied cytokinins in Pisum sativum

When N ⁶ [8–¹⁴C] furfuryladenine was applied to the intact root system of Pisum sativum L. cv. Meteor seedlings it was almost completely metabolised to other compounds within 24 h. Of the total activity recovered from the plants 94.5% was retained in the root system itself. ¹⁴C was recovered in a number of ethanol-soluble compounds and in ribonucleic acid, deoxyribonucleic acid and protein fractions of roots, stems, leaves and axillary buds. In rapidly growing axillary buds released from apical dominance by removal of the shoot apex the combined nucleic acid fractions accounted for 63.3% of the total ¹⁴C recovered from these organs. Xylem exudate collected from decapitated plants 0 to 12 h after supplying N ⁵[8–¹⁴C]furfuryladenine to the roots consistently contained a single major ¹⁴C-labelled compound which, in three different solvent systems, had the same Rf values as a major endogenous cytokinin isolated from the xylem of unlabelled plants. The content of N ⁶ [8–¹⁴C] furfuryladenine itself in the xylem exudate was always low and in some experiments it could not be detected.
It is suggested that part of the label from N ⁶ [8- ¹⁴CJfurfuryladenine taken up by the intact root system may have become incorporated in an endogenous cylokinin before export to the shoot.     

Computer simulation of purine metabolism

A computer model of purine metabolism, including catabolism, salvage pathways and interconversion among nucleotides, is given. Steady-state rate equations corresponding to metabolic enzymes are written based on information from the literature about their kinetic behaviour. Numerical integration of this set of equations is performed employing selected parameters taken from the literature. After stabilization of purine compound concentrations is reached, simulation of enzyme deficit and enzyme overproduction is carried out. The latter is calculated by varying specified maximum velocities in the numerical integration. A pattern of intermediate metabolite concentrations is found. These results form a basis for the comparison of normal patterns or patterns reflecting the effects of inborn errors of metabolism. The aim of this paper is to demonstrate the usefulness of this computer simulation method in complex metabolism pathways.     

Inborn errors of purine and pyrimidyne metabolism

Inborn errors of purine and pyrimidine metabolism (P/P) manifest themselves by a variety of clinical picture. They may be recognized at any age and may affect any system--immunological, hematological, neurological, musculoskeletal, and because of the relative insolubility of purine bases, renal as well. At present, a total of 30 defects have been described. Fifteen of them can have serious clinical consequences. Analysis of prevalence estimated by comparing the number of detected P/P patients in Poland and the number of newborns as well as delay of diagnosis, point at insufficient degree of detectability of these defects in our country. It is necessary to improve the education among physicians as well as to popularize screening methods for these defects.     

Mitochondrial purine and pyrimidine metabolism and beyond

ABSTRACT

Carefully balanced deoxynucleoside triphosphate (dNTP) pools are essential for both nuclear and mitochondrial genome replication and repair. Two synthetic pathways operate in cells to produce dNTPs, e.g., the de novo and the salvage pathways. The key regulatory enzymes for de novo synthesis are ribonucleotide reductase (RNR) and thymidylate synthase (TS), and this process is considered to be cytosolic. The salvage pathway operates both in the cytosol (TK1 and dCK) and the mitochondria (TK2 and dGK). Mitochondrial dNTP pools are separated from the cytosolic ones owing to the double membrane structure of the mitochondria, and are formed by the salvage enzymes TK2 and dGK together with NMPKs and NDPK in postmitotic tissues, while in proliferating cells the mitochondrial dNTPs are mainly imported from the cytosol produced by the cytosolic pathways. Imbalanced mitochondrial dNTP pools lead to mtDNA depletion and/or deletions resulting in serious mitochondrial diseases. The mtDNA depletion syndrome is caused by deficiencies not only in enzymes in dNTP synthesis (TK2, dGK, p53R2, and TP) and mtDNA replication (mtDNA polymerase and twinkle helicase), but also in enzymes in other metabolic pathways such as SUCLA2 and SUCLG1, ABAT and MPV17. Basic questions are why defects in these enzymes affect dNTP synthesis and how important is mitochondrial nucleotide synthesis in the whole cell/organism perspective? This review will focus on recent studies on purine and pyrimidine metabolism, which have revealed several important links that connect mitochondrial nucleotide metabolism with amino acids, glucose, and fatty acid metabolism.     

Altered purine and pyrimidine metabolism in erythrocytes with purine nucleoside phosphorylase deficiency

Purine and pyrimidine metabolism was compared in erythrocytes from three patients from two families with purine nucleoside phosphorylase deficiency and T-cell immunodeficiency, one heterozygote subject for this enzyme deficiency, one patient with a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase, and two normal subjects. The erythrocytes from the heterozygote subject were indistinguishable from the normal erythrocytes. The purine nucleoside phosphorylase deficient erythrocytes had a block in the conversion of inosine to hypoxanthine. The erythrocytes with 0.07% of normal purine nucleoside phosphorylase activity resembled erythrocytes with hypoxanthine-guanine phosphoribosyltransferase deficiency by having an elevated intracellular concentration of PP-ribose-P, increased synthesis of PP-ribose-P, and an elevated rate of carbon dioxide release from orotic acid during its conversion to UMP. Two hypotheses to account for the associated immunodeficiency—that the enzyme deficiency leads to a block of PP-ribose-P synthesis or inhibition of pyrimidine synthesis—could not be supported by observations in erythrocytes from both enzyme-deficient families.This work was supported by U.S. Public Health Service Grant AM 19674 and 5 M01 RR 42 and by a Grant-In-Aid from American Heart Association (77-849) and with funds contributed in part by the Michigan Heart Association. N.L.E. is a Rheumatology Fellow from the Rackman Arthritis Research Unit supported by Training Grant USPHS AM 07080.     

Regulation of purine metabolism in lymphocytes

Three general questions regarding nucleosides and lymphocytes are discussed: (a) Why are so many measurements being made of adenosine deaminase activity, what do the results mean, and why is there still disagreement about some of the conclusions; (b) what do we understand about nucleosides and lymphocyte death; and (c) to what extent do we really understand nucleoside and nucleotide metabolism in lymphocytes? Experimental studies show that treatment of mice with deoxycoformycin, to produce accumulation of deoxyadenosine, leads to rapid thymus involution, elevated dATP concentrations in thymus and liver, and inhibition of adenosylhomocysteine hydrolase in these tissues. Deoxyguanosine inhibits the growth of mouse lymphoma L5178Y cells, and this toxicity is prevented by deoxycytidine plus adenine. In cells treated with deoxyguanosine, concentrations of both GTP and dGTP are elevated, and this is not affected by deoxycytidine. Adenine, however, reduces GTP concentrations to normal, and prevents most of the elevation in dGTP concentrations. Contrary to previous belief, it has been demonstrated that lymphocytes and nucleated bone marrow cells will synthesize purine nucleotides de novo if incubated in an appropriate medium; carbon dioxide is particularly important for this process.     

The purine metabolism of human erythrocytes

This review summarizes currently available information about a crucial part of erythrocyte metabolism, that is, purine nucleotide conversions and their relationships with other conversion pathways. We describe the cellular resynthesis, interconversion, and degradation of purine compounds, and also the regulatory mechanisms in the conversion pathways. We also mention purine metabolism disorders and their clinical consequences. The literature is fragmentary because studies have concentrated only on selected aspects of purine metabolism; hence the need for a synthetic approach. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 581–591.     

Short term cadmium intoxication of the shrimp Palaemon serratus: Effect on adenylate metabolism

• 1.1. Shrimps were exposed for 96 hr to various concentrations of cadmium under laboratory conditions. The LC₅₀ was around 4 ppm Cd in water, which corresponded to 0.180 μg/g wet weight of cadmium in tail muscles.
• 2.2. The effect of various concentrations of cadmium on adenylates was analyzed in tail muscles: At subletal cadmium concentrations, no variation of ATP, ADP and of the adenylate sum occurred, while the AMP concentration began to decrease from 0.06 ppm.

At the LC₅₀, the ATP, ADP and AMP concentrations dropped acutely, the ATP/ADP ratio increased acutely.The apparent equilibrium constant of the adenylate kinase reaction increased significantly from 2 ppm Cd, indicating an impairment in energetic metabolism.Cadmium intoxication did not influence the value of the adenylate energy charge.     

The purine and pyrimidine metabolism of Tetrahymena pyriformis

The metabolism of purines and pyrimidines by the ciliated protozoan Tetrahymena was investigated with the use of enzymatic assays and radioactive tracers. A survey of enzymes involved in purine metabolism revealed that the activities of inosine and guanosine phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, E.C. 2.4.2.1) were high, but adenosine phosphorylase activity could not be demonstrated. The apparent K_m for guanosine in the system catalyzing its phosphorolysis was 4.1 ± 0.6 × 10^?3 M. Pyrophosphorylase activities for IMP and GMP (GMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.8), AMP (AMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.7), and 6-mercaptopurine ribonucleotide were also found in this organism; but a number of purine and pyrimidine analogs did not function as substrates for these enzymes. The metabolism of labeled guanine and hypoxanthine by intact cells was consistent with the presence of the phosphorylases and pyrophosphorylases of purine metabolism found by enzymatic studies. Assays for adenosine kinase (ATP: adenosine 5'-phosphotransferase, E.C. 2.7.1.20) inosine kinase, guanosine kinase, xanthine oxidase (xanthine: O₂ oxidoreductase, E.C. 1.2.3.2), and GMP reductase (reduced-NADP: GMP oxidoreductase [deaminating], E.C. 1.6.6.8) were all negative. In pyrimidine metabolism, cytidine-deoxycytidine deaminase (cytidine aminohydrolase, E.C. 3.5.4.5), thymidine phosphorylase (thymidine: orthophosphate ribosyltransferase, E.C. 2.4.2.4), and uridine-deoxyuridine phosphorylase (uridine: orthophosphate ribosyltransferase, E.C. 2.4.2.3) were active; but cytidine kinase, uridine kinase (ATP: uridine 5'-phosphotransferase, E.C. 2.7.1.48), and CMP pyrophosphorylase could not be demonstrated.     

Activity and metabolism of 9-substituted cytokinins during lettuce seed germination

The activities of N⁶-benzyladenine (BA) and its 9-substituted methyl, methoxymethyl, tetrahydropyranyl, cyclopentyl, and cyclohexyl analogs were determined for the promotion of lettuce seed (Lactuca sativa L. cv. Grand Rapids) germination. Cytokinin concentrations used were 10^?4, 10^?5, 10^?6 and 10^?7M. All seeds were incubated under total dark conditions at 28 ± 1°C. After 48 h the percentage of germination was recorded. A comparison of means based on Duncan's Multiple Range Test allowed for a ranking of cytokinin activities for the promotion of lettuce seed germination. The activities were: BA = 9-tetrahydropyranyl BA > 9-methyl BA > 9-methoxymethyl BA > 9-cyclopentyl BA > 9-cyclohexyl BA. The results were significant at the 95% confidence level as determined by analysis of variance. In order to study the metabolism of a cytokinin, lettuce seeds were incubated with 9-methyl-BA-methylene-¹⁴C. The labeled cytokinin was prepared by refluxing benzylamine hydrochloride (methylene-¹⁴C) with an equal molar ratio of 6-chloro-9-methylpurine. Final cytokinin concentration was 10^?5M. Incubation periods were 2, 4, 8, 12, 16 and 20 h at 28 ± 1°C under total dark conditions. At the end of the various time periods the seeds were extracted with 70 percent methanol. The resulting extracts were purified and radioactive metabolites identified by solvent fractionation, Sephadex LH-20 column chromatography, and paper chromatography. Co-chromatography with authentic standards in the appropriate solvent system revealed that the metabolites were 9-methyl BA, N⁶-benzyladenosine-5′-monophosphate, and N⁶-benzyladenosine. The results lend support to the theory that the cytokinin ribonucleotide serves as a storage form which is converted to the active ribonucleoside as needed during lettuce seed germination.     

Short term physiological implications of NBPT application on the N metabolism of Pisum sativum and Spinacea oleracea

The application of urease inhibitors in conjunction with urea fertilizers as a means of reducing N loss due to ammonia volatilization requires an in-depth study of the physiological effects of these inhibitors on plants. The aim of this study was to determine how the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) affects N metabolism in pea and spinach. Plants were cultivated in pure hydroponic culture with urea as the sole N source. After 2 weeks of growth for pea, and 3 weeks for spinach, half of the plants received NBPT in their nutrient solution. Urease activity, urea and ammonium content, free amino acid composition and soluble protein were determined in leaves and roots at days 0, 1, 2, 4, 7 and 9, and the NBPT content in these tissues was determined 48 h after inhibitor application. The results suggest that the effects of NBPT on spinach and pea urease activity differ, with pea being most affected by this treatment, and that the NBPT absorbed by the plant caused a clear inhibition of the urease activity in pea leaf and roots. The high urea concentration observed in leaves was associated with the development of necrotic leaf margins, and was further evidence of NBPT inhibition in these plants. A decrease in the ammonium content in roots, where N assimilation mainly takes place, was also observed. Consequently, total amino acid contents were drastically reduced upon NBPT treatment, indicating a strong alteration of the N metabolism. Furthermore, the amino acid profile showed that amidic amino acids were major components of the reduced pool of amino acids. In contrast, NBPT was absorbed to a much lesser degree by spinach plants than pea plants (35% less) and did not produce a clear inhibition of urease activity in this species.