Active cytokinins: photoaffinity labeling agents to detect binding

Four series of azidopurines have been synthesized and tested for cytokinin activity in the tobacco callus bioassay: 2- and 8-azido-N⁶-benzyladenines, -N⁶-(Δ²-isopentenyl)adenines, and -zeatins, and N⁶-(2- and 4-azidobenzyl)adenines. The compounds having 2-azido substitution on the adenine ring are as active as the corresponding parent compounds, while those with 8-azido substitution are about 10 or more times as active. The 8-azidozeatin, which is the most active cytokinin observed, exhibited higher than minimal detectable activity at 1.2 × 10⁻⁵ micromolar, the lowest concentration tested. The shape of the growth curve indicates that even a concentration as low as 5 × 10⁻⁶ micromolar would probably be effective. By comparison, the lowest active concentration ever reported for zeatin has been 5 × 10⁻⁵ micromolar, representing a sensitivity rarely attained.     

The synthesis and biological properties of 8-azido-N 6-benzyladenine,a potential photoaffinity reagent for cytokinin

A cytokinin photoaffinity reagent, 8-azido-N ⁶-benzyladenine (8N₃BA), was synthesized from 8-bromoadenosine via azide replacement, benzylation at N–1, rearrangement to the N-6-benzyl derivative and acid hydrolysis. The compound thus obtained was found to have full cytokinin activity in the moss and tobacco cell-suspension bioassays. Photolysis of 8N₃BA was accomplished with long and short-wavelength ultraviolet light and produced compounds which had very little or no biological activity in the two bioassays. In-vivo photolysis of 8N₃BA caused loss of the cytokinin activity of this compound in moss protonemata. This result was similar to earlier ones where the biological response of moss protonemata to benzyladenine was reversed following removal of the hormone by a short rinse with water.Abbreviations BA N ⁶-benzyladenine - 8N₃BA 8-azido-N ⁶-benzyladenine - PMR proton magnetic resonance - TLC thin-layer chromatography - UV ultraviolet In partial fulfillment of requirements for the Ph.D. degree at Michigan State University     

Nucleosides. 140. Stereoselectivity of the Reaction of 1-(2,3-Anhydro-5-O-Benzoyl-β-D-Lyxofuranosyl)Uracil with Ammonium Azide. Isolation and Characterization of 2-Azido-XYLO-Nucleoside Derivatives.1

Abstract

The composition of the products of reaction of 1-(2,3-anhydro-5-O-benzoyl-β-D-lyxofuranosyl)uracil (1) with NH₄N₃ was studied by a reverse-phase HPLC system which was found to separate the 3-azido-arabino 2 and 2-azido-xylo 3 isomers that were formed. The use of a 10:1 ratio of NH₄N₃ to 1 in refluxing EtOH was found to minimize ring opening at C-2 (7%). The higher stereoselectivity of ring opening produced by using a large excess of NH₄N₃ was suppressed by conducting the reaction in DMF. Preventing the escape of the NH₃ by-product only resulted in debenzoylation. The isolation of pure, crystalline 3 was achieved by reverse-phase preparative HPLC. Separation from the arabino isomer was also effected by debenzoylation and selective acetonide formation with the xylo isomer, which allowed facile isolation of the latter by normal phase chromatography. Hydrolysis of the acetonide 7 provided unprotected 2-azido-xylo nucleoside 6, which was also obtained by NaOMe treatment of 3. The mechanistic basis for the stereo-selectivity of epoxide opening is discussed.     

5'-Azido-N-1-Napthylphthalamic Acid, a Photolabile Analog of N-1-Naphthylphthalamic Acid : Synthesis and Binding Properties in Curcurbita pepo L

A photolabile analog of N-1-naphthylphthalamic acid (NPA), 5′-azido-N-1-naphthylphthalamic acid (Az-NPA), has been synthesized and characterized. This potential photoaffinity label for the plasma membrane NPA binding protein competes with [³H]NPA for binding sites on Curcurbita pepo L. (zucchini) hypocotyl cell membranes with K_0.5 = 2.8 × 10⁻⁷ molar. The K_0.5 for NPA under these conditions is 2 × 10⁻⁸ molar, indicating that the affinity of Az-NPA for the membranes is only 14-fold lower than NPA. While the binding of Az-NPA to NPA binding sites is reversible in the dark, exposure of the Az-NPA treated membranes to light results in a 30% loss in [³H]NPA binding ability. Pretreatment of the membranes with NPA protects the membranes against photodestruction of [³H]NPA binding sites by Az-NPA supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment.     

The synthesis of oligoribonucleotides containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines via post-synthetic modification of precursor oligomers

The N⁶-alkyladenosines and 2-methylthio-N⁶-alkyladenosines are the most common modified adenosine nucleosides and transfer ribonucleic acids (tRNA) are particularly rich in these modified nucleosides. They are present at position 37 of the anticodon arm and the contribution of these hypermodified nucleosides to codon–anticodon interactions, as well as translation, are significant, although not fully understood. Herein we described a new chemical synthesis method of the oligoribonucleotides containing N⁶-alkyladenosines and 2-methylthio-N⁶-alkyladenosines via post-synthetic modifications of precursor oligoribonucleotides. To obtain oligoribonucleotides containing N⁶-alkyladenosines, the precursor oligoribonucleotide carrying 6-methylthiopurine riboside residue was used, whereas for the synthesis of oligoribonucleotides containing 2-methylthio-N⁶-alkyladenosines the precursor oligoribonucleotide carrying the 2-methylthio-6-chloropurine riboside was applied. Among the modified oligoribonucleotides of different length and secondary structures, there were several containing naturally occurring modified nucleosides such as: N⁶-isopentenyladenosine (i⁶A), N⁶-methyladenosine (m⁶A), 2-methylthio-N⁶-isopentenyladenosine (ms²i⁶A), and 2-methylthio-N⁶-methyladenosine (ms²m⁶A), as well as several unnaturally modified adenosine derivatives.     

Functional replacement of the endogenous tyrosyl-tRNA synthetase–tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase–tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)–tRNA^Tyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS–tRNA^Tyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA^Tyr. The endogenous TyrRS and tRNA^Tyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS–tRNA^Tyr pair. In this engineered strain, 3-iodo-l-tyrosine and 3-azido-l-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-l-tyrosine and was also found to recognize 3-azido-l-tyrosine. The structural basis for the 3-azido-l-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.     

The use of a tritium release assay to measure 6-N-trimethyl-L-lysine hydroxylase activity: synthesis of 6-N-[3-3H]trimethyl-DL-lysine

6-N-[3-³H]Trimethyl-dl-lysine was synthesized from 6-N-acetyl-l-lysine by the following chemical scheme: 6-N-acetyl-l-lysine → 2-keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid oxime → 6-N-[3-³H]acetyl-dl-lysine → dl-[3-³H]lysine → 2-N-[3-³H]formyl-dl-lysine → 2-[3-³H]formyl-6-N-trimethyl-dl-lysine → 6-N-[3-³H]trimethyl-dl-lysine. Using a 70% ammonium sulfate fraction obtained from a high-speed rat kidney supernatant, the cosubstrate and cofactor requirements for 6-N-trimethyl-l-lysine hydroxylase activity as measured by tritium release from 6-N-[3-³H]trimethyl-dl-lysine were: α-ketoglutarate, ferrous ions, l-ascorbate, and oxygen, with added catalase showing a slight but distinct stimulatory effect. On incubation with the crude rat kidney preparation, the release of tritium from 6-N-[3-³H]trimethyl-dl-lysine was linear with both time of incubation and protein concentration. Hydroxylation of 6-N-trimethyl-l-lysine, as measured by tritium release from the labeled substrate, was examined in rat kidney, heart, liver, and skeletal muscle tissues, and found to be most active in the kidney.     

The thermodynamic stability of RNA duplexes and hairpins containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines

The N⁶-alkyladenosines and 2-methylthio-N⁶-alkyladenosines make up over half of the population of all naturally modified adenosines and they are present in the transfer ribonucleic acids (tRNA) at position 37. We measured effects of N⁶-alkyladenosines and 2-methylthio-N⁶-alkyladenosines on the thermodynamic stability of RNA duplexes containing a U-A^Mod base pair at internal and terminal duplex positions, as well as containing modified adenosines as a 3′-terminal unpaired nucleotide. Beside naturally modified adenosines such as N⁶-isopentenyladenosine (i⁶A), N⁶-methyladenosine (m⁶A), 2-methylthio-N⁶-isopentenyladenosine (ms²i⁶A) and 2-methylthio-N⁶-methyladenosine (ms²m⁶A), we studied several artificial modifications to evaluate the steric and electronic effects of N⁶-alkyl substituents. Moreover, some N⁶-alkyladenosines and 2-methylthio-N⁶-alkyladenosines were placed in hairpins at positions corresponding to nucleotide 37 of the tRNA anticodon arm, and the thermodynamic stability of those hairpins was studied. The stability of the modified RNA hairpins was measured in standard melting buffer containing 1 M sodium chloride as well as in physiological buffer containing 10 mM magnesium chloride and 150 mM potassium chloride. The results obtained indicate that the nature of the adenosine modification and the position of U-A^Mod base pairs within the duplex influence the thermodynamic stability of RNA duplexes. For most of the modification, the destabilization of duplexes was observed. Moreover, we found that the buffer composition and the structure of the modified adenosine very significantly affect the thermodynamic stability of RNA.     

Nucleosides. 140. Stereoselectivity of the Reaction of 1-(2,3-Anhydro-5-O-Benzoyl-β-D-Lyxofuranosyl) Uracil with Ammonium Azide. Isolation and Characterization of 2-Azido-Xylo-Nucleoside Derivatives

Abstract

The composition of the products of reaction of 1-(2,3-anhydro-5-O-benzoyl-β-D-lyxofuranosyl)uracil (1) with NH₄N₃ was studied by a reverse-phase HPLC system which was found to separate the 3-azido-arabino 2 and 2-azido-xylo 3 isomers that were formed. The use of a 10:1 ratio of NH ₄ N ₃ to 1 in refluxing EtOH was found to minimize ring opening at C-2 (7%). The higher stereoselectivity of ring opening produced by using a large excess of NH ₄ N ₃ was suppressed by conducting the reaction in DMF. Preventing the escape of the NH ₃ by-product only resulted in debenzoylation. The isolation of pure, crystalline 3 was achieved by reverse-phase preparative HPLC. Separation from the arabino isomer was also effected by debenzoylation and selective acetonide formation with the xylo isomer, which allowed facile isolation of the latter by normal phase chromatography. Hydrolysis of the acetonide 7 provided unprotected 2-azido-xylo nucleoside 6, which was also obtained by NaOMe treatment of 3. The mechanistic basis for the stereoselectivity of epoxide opening is discussed.     

Isolation and Estimation of Cytokinins and Cytokinin-Containing Transfer RNAs from Cucumis sativus L. var. Guntur Seedlings

N⁶-(Δ²-Isopentenyl) adenosine antibodies were used for the isolation of free cytokinins and cytokinin-containing tRNAs from parts of Cucumis sativus L. var. Guntur seedlings and for the estimation of cytokinins in them. Immobilized N⁶-(Δ²-isopentenyl) adenosine antibodies retained tRNAs containing N⁶-(Δ²-isopentenyl) adenosine and N⁶-(4-hydroxy-3-methylbut-2-enyl) adenosine with equal efficiencies. There were at least five cytokinins in the free form in cucumber seedlings. N⁶-(4-Hydroxy-3-methylbut-2-enyl) adenosine, N⁶-(Δ²-isopentenyl) adenosine, and N⁶-(Δ²-isopentenyl) adenine were present at least to the extent of 80, 23, and 9 nanograms, respectively, in the cotyledons and 40, 6, and 3 nanograms, respectively, in the decotyledonated seedlings per gram of tissue. Only two cytokinins were found in the tRNAs of cucumber cotyledons, namely N⁶-(Δ²-isopentenyl) adenosine and N⁶-(4-hydroxy-3-methylbut-2-enyl) adenosine in amounts of 12 and 318 nanograms, respectively, per gram of tissue. Immunoaffinity chromatographic analysis of radiolabeled aminoacyl tRNAs from cucumber cotyledons showed that tRNA^Phe and tRNA^Tyr contained cytokinins whereas tRNA^Ala did not.     

Two novel dATP analogs for DNA photoaffinity labeling

Two new photoreactive dATP analogs, N⁶-[4-azidobenzoyl–(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (AB-dATP) and N⁶-[4-[3-(trifluoromethyl)-diazirin-3-yl]benzoyl-(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (DB-dATP), were synthesized from 2′-deoxyadenosine-5′-monophosphate in a six step procedure. Synthesis starts with aminoethylation of dAMP and continues with rearrangement of N¹-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate to N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate (N⁶-dAMP). Next, N⁶-dAMP is converted into the triphosphate form by first protecting the N-6 primary amino group before coupling the pyrophosphate. After pyrophosphorylation, the material is deprotected to yield N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-triphosphate (N⁶-dATP). The N-6 amino group is subsequently used to attach either a phenylazide or phenyldiazirine and the photoreactive nucleotide is then enzymatically incorporated into DNA. N⁶-dATP and its photoreactive analogs AB-dATP and DB-dATP were successfully incorporated into DNA using the exonuclease-free Klenow fragment of DNA polymerase I in a primer extension reaction. UV irradiation of the primer extension reaction with AB-dATP or DB-dATP showed specific photocrosslinking of DNA polymerase I to DNA.     

Methylation of the nucleobases in RNA oligonucleotides mediates duplex-hairpin conversion

We have systematically investigated the duplex to hairpin conversion of oligoribonucleotides under the aspect of nucleobase methylation. The first part of our study refers to the self-complementary sequence rCGCGAAUUCGCGA, which forms a stable Watson–Crick base paired duplex under various buffer conditions. It is shown that this sequence is forced to adopt a hairpin conformation if one of the central 6 nt is replaced by the corresponding methylated nucleotide, such as 1-methylguanosine N²,N²-dimethylguanosine, N⁶,N⁶-dimethyladenosine (m⁶₂A) or 3-methyluridine. On the other hand, the duplex structure is retained and even stabilized by replacement of a central nucleotide with N²-methylguanosine (m²G) or N⁴-methylcytidine. A borderline case is represented by N⁶-methyladenosine (m⁶A). Although generally a duplex-preserving modification, our data indicate that m⁶A in specific strand positions and at low strand concentrations is able to effectuate duplex–hairpin conversion. Our studies also include the ssu ribosomal helix 45 sequence motif, rGACCm²GGm⁶₂Am⁶₂AGGUC. In analogy, it is demonstrated that the tandem m⁶₂A nucleobases of this oligoribonucleotide prevent duplex formation with complementary strands. Therefore, it can be concluded that nucleobase methylations at the Watson–Crick base pairing site provide the potential not only to modulate but to substantially affect RNA structure by formation of different secondary structure motifs.     

Synthesis and characterization of oligodeoxyribonucleotides containing a site-specifically incorporated N6-carboxymethyl-2′-deoxyadenosine or N4-carboxymethyl-2′-deoxycytidine

Humans are exposed to both endogenous and exogenous N-nitroso compounds (NOCs), and many NOCs can be metabolically activated to generate a highly reactive species, diazoacetate, which is capable of inducing carboxymethylation of nucleobases in DNA. Here we report, for the first time, the chemical syntheses of authentic N⁶-carboxymethyl-2′-deoxyadenosine (N⁶-CMdA) and N⁴-carboxymethyl-2′-deoxycytidine (N⁴-CMdC), liquid chromatography–ESI tandem MS confirmation of their formation in calf thymus DNA upon diazoacetate exposure, and the preparation of oligodeoxyribonucleotides containing a site-specifically incorporated N⁶-CMdA or N⁴-CMdC. Additionally, thermodynamic studies showed that the substitutions of a dA with N⁶-CMdA and dC with N⁴-CMdC in a 12-mer duplex increased Gibbs free energy for duplex formation at 25°C by 5.3 and 6.8 kcal/mol, respectively. Moreover, primer extension assay revealed that N⁴-CMdC was a stronger blockade to Klenow fragment-mediated primer extension than N⁶-CMdA. The polymerase displayed substantial frequency of misincorporation of dAMP opposite N⁶-CMdA and, to a lesser extent, misinsertion of dAMP and dTMP opposite N⁴-CMdC. The formation and the mutagenic potential of N⁶-CMdA and N⁴-CMdC suggest that these lesions may bear important implications in the etiology of NOC-induced tumor development.     

Comparison of cytokinin activities of the base,ribonucleoside and 5′- and cyclic-3′,5′-monophosphate ribonucleotides of N6-isopentenyl-, N6-benzyl or 8-bromo-adenine

The cytokinin activities of adenosine 3′,5′-monophosphate, N⁶,O^2″-dibutyryladenosine 3′,5−'monophosphate, 8-bromoadenosine 3′,5′-monophosphate, N⁶-(Δ²-isopentenyl)adenosine 3′,5′-monophosphate, and N⁶-benzyladenosine 3′,5′-monophosphate were determined in the tobacco bioassay and compared with the activities of the corresponding non-cyclic nucleotides, nucleosides and bases of the N⁶-isopentenyl-substituted, N⁶-benzyl-substituted, 8-bromo-substituted, and unsubstituted adenine series. In each of these series the cytokinin activities in decreasing order were: bases ⪢ nucleosides ⪖ nucleotides > cyclic nucleotides. All members of the N⁶-isopentenyl- substituted and N⁶-benzyl-substituted series were highly active cytokinins, reaching maximum activity at concentrations of 1 μM or less, whereas, as expected, all members of the unmodified adenine series were inactive in the tested concentration ranges of up to 180 and 200 μM for adenosine and adenine, and 40 μM for the adenine nucleotides. Members of the 8-bromo-substituted adenine series were much weaker cytokinins than the N⁶-substituted adenine derivatives but showed activity in the same sequence starting at a concentration of about 5 μM. Thus, in the cases of 8-bromoadenosine 3′,5′-monophosphate and N⁶,O^2′-dibutyryl-adenosine 3′,5′-monophosphate, both of which have been reported to promote cell division and growth of plant tissues, the cytokinin activity is related to the 8-bromo substituent and to the N⁶-butyryl substituent, respectively, rather than to the 3′,5′-cyclic monophosphate moiety.     

Metabolism of cytokinin: ribosylation of cytokinin bases by adenosine phosphorylase from wheat germ

As part of the study of cytokinin metabolic pathways, an enzyme, adenosine phosphorylase (EC 2.4.2.-), which catalyzed the ribosylation of N⁶-(Δ²-isopentenyl)adenine, N⁶-furfuryladenine, and adenine to form the corresponding nucleosides, was partially purified from wheat (Triticum aestivum) germ. The pH optimum for the ribosylation of the cytokinins and adenine was from 6.5 to 7.8; for guanine and hypoxanthine it was from 7.0 to 8.5 At pH 7.2 (63 millimolar N-2-hydroxyethyl piperazine-N′-ethanesulfonic acid) and 37 C the K_m for N⁶-(Δ²-isopentenyl)adenine was 57.1 micromolar; N⁶-furfuryladenine, 46.5 micromolar; adenine, 32.2 micromolar; and the V_max for N⁶-(Δ²-isopentenyl)adenine, N⁶-furfuryladenine, and adenine were 134.7, 137.1, and 193.1 nanomoles per milligram protein per minute, respectively. The equilibrium constants of the phosphorolysis of N⁶-(Δ²-isopentenyl)adenosine and adenosine by this enzyme indicated that the reaction strongly favored nucleoside formation. This enzyme was shown to be distinct from inosine-guanosine phosphorylase based on the differences in the Sephadex G-100 gel filtration behaviors, pH optima, and the product and p-hydroxymercuribenzoate inhibitor studies. These results suggest that adenosine phosphorylase may play a significant role in the regulation of cytokinin metabolism.     

Cytokinin oxidase from wheat: partial purification and general properties

As part of the study of the possible role(s) of CBF-1, a cytokinin-binding protein abundant in wheat embryo, a cytokinin oxidase was found in wheat (Triticum aestivum L.) germ and partially purified by conventional purification techniques and high performance chromatofocusing. This preparation catalyzes conversion of N⁶-(Δ²-isopentenyl)adenosine to adenosine at a V_max of 0.4 nanomol per milligram protein per minute at 30°C and pH 7.5, the K_m being 0.3 micromolar. This high affinity and the apparent molecular weight of 40,000 estimated by high performance gel permeation on a Spherogel TSK-3000 SW column indicate that this enzyme is different from other cytokinin oxidases previously reported. Oxygen is required for the reaction, as for other cytokinin oxidases already described. N⁶-(Δ²-isopentenyl)adenine and zeatin riboside are also degraded, but N⁶-(Δ²-isopentenyl)adenosine-5′-monophosphate is apparently not a substrate. Benzyladenine is degraded, but to a small extent, and it inhibits slightly the degradation of N⁶-(Δ²-isopentenyl)adenosine. The degradation of N⁶-(Δ²-isopentenyl)adenosine is strongly inhibited by diphenylurea and its highly active derivative N-(2-chloro-4-pyridyl)-N′-phenylurea.     

Cytokinin structure-activity relationships and the metabolism of N-(delta-isopentenyl)adenosine-8-C in phaseolus callus tissues

The activities of the free base and ribonucleoside forms of cytokinins bearing saturated and unsaturated N⁶-isoprenoid side chains have been examined in callus cultures derived from Phaseolus vulgaris cv. Great Northern, P. lunatus cv. Kingston, and the interspecific hybrid Great Northern × Kingston. In callus of cv. Great Northern, cytokinins bearing saturated side chains (N⁶-isopentyladenine, N⁶-isopentyladenosine, dihydrozeatin, and ribosyldihydrozeatin) were always more active than the corresponding unsaturated analogs (N⁶-[Δ²-isopentenyl]adenine, N⁶-[Δ²-isopentenyl]adenosine, zeatin, and ribosylzeatin). In callus of cv. Kinston, the cytokinins bearing unsaturated side chains were either more active or equally as active as the saturated compounds. These differences in cytokinin structure-activity relationships were correlated with differences in the metabolism of ¹⁴C-N⁶-(Δ²-isopentenyl)adenosine. In Great Northern tissues, this cytokinin was rapidly degraded to adenosine; in Kingston tissues, the major metabolite was the corresponding nucleotide. The growth responses of callus of the interspecific hybrid were intermediate between the parental tissues, and the metabolism of ¹⁴C-N⁶-(Δ²-isopentenyl)adenosine by the hybrid callus exhibited characteristics of both parental tissues. The results are consistent with the hypothesis that the weak activity of cytokinins with unsaturated side chains in promoting the growth of Great Northern callus is due to the rapid conversion of these cytokinins to inactive metabolites.     

Aeribactin synthesis in a cell-free system of Aerobacter Aerogenes 62-1

The supernatant fraction of the cell-free system of Aerobacter aerogenes 62-1 was found to contain enzyme(s) capable of synthesising aerobactin from citrate and N⁶-acetyl-N⁶-hydroxylysine. Studies with partially purified enzyme systems revealed that aerobactin production was dependent on Mg²⁺ and ATP, the latter being used for the activation of both the precursors, citrate and N⁶-acethyl-N⁶-hydroxylysine. The generation of ADP in the activation process was consistent with the formation of phosphorylated precursor intermediates in the reactions leading to the peptide bond formation.     

Differential cytokinin structure-activity relationships in phaseolus

The activities of eight cytokinins in promoting callus growth were tested in two Phaseolus genotypes, P. vulgaris L. var. Great Northern, and P. lunatus L. var. Kingston. The structural feature which contributes to the major genotypic difference in cytokinin structure-activity relationships is the presence or absence of a double bond at the 2,3-position of the isoprenoid N⁶ side chain. In Kingston, trans-zeatin was 3-fold more active than dihydrozeatin and 30-fold more active than cis-zeatin. The activities of N⁶-(Δ²-isopentenyl)adenine and N⁶-isopentyladenine were nearly the same. In Great Northern, however, dihydrozeatin was at least 30-fold more active than both trans-zeatin and cis-zeatin, and N⁶-isopentyladenine was 100-fold more active than N⁶-(Δ²-isopentenyl)adenine. The results suggest the possibility of employing cytokinin structure-activity relationships in distinguishing genotypic differences in cytokinin function and metabolism.     

Effects of growth hormone on ATPase and fluorescence of isolated liver membranes utilizing the fluorescent substrate, 1,N6-etheno-adenosine triphosphate

The influence of bovine growth hormone on Mg²⁺-ATPase (EC 3.6.1.4) in isolated liver plasma membranes of hypophysectomized rats has been investigated in vitro by means of spectrofluorescence measurements in parallel with Mg²⁺-ATPase assays, using 1, N⁶-etheno-ATP as substrate and fluorescence probe.Bovine growth hormone, at concentrations of 10^?14m and above, enhanced significantly Mg²⁺-ATPase activity in the presence of GTP at concentrations from 10^?6m to 10^?10m. Moreover, bovine growth hormone decreased fluorescence intensity of membrane protein at the peak at 330 nm and of 1, N⁶-etheno-ATP at its peak at 395 nm as well. The greatest decrease in fluorescence intensity of 1, N⁶-etheno-ATP was observed in the presence of 5 mm MgCl₂ and 10^?8m GTP, consistent with the stimulating effect of bovine growth hormone on Mg²⁺-ATPase activity. In addition, bovine growth hormone caused a small decrease in fluorescence intensity of 1, N⁶-etheno-ADP, but not the corresponding fluorescent analogs of AMP, cyclic AMP, and adenosine. The decrease in fluorescence intensity of 1, N⁶-etheno-ATP by bovine growth hormone was completely eliminated by addition of ATP, ADP, and cyclic AMP at concentrations five times that of 1, N⁶-etheno-ATP. Neither AMP nor adenosine exerted any effects.Bovine growth hormone also increased the fluorescence polarization of 1, N⁶-etheno-ATP from 0.177 ± 0.006 to 0.212 ± 0.010 at 300 nm under the same conditions employed for the Mg²⁺-ATPase assay.These observations suggest that bovine growth hormone produced changes in tertiary structure of membrane proteins in general and probably Mg²⁺-ATPase in particular with consequent enhanced enzyme activity.