Proposed biosynthetic pathway for rosmarinic acid in cell cultures of Coleus blumei Benth

A biosynthetic pathway for rosmarinic acid is proposed. This pathway is deduced from studies of the enzymes detectable in preparations from suspension cells of Coleus blumei. Phenylalanine is transformed to 4-coumaroyl-CoA by the enzymes of the general phenylpropanoid pathway: phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase (EC 1.14.13.11) and hydroxycinnamic acid:CoA ligase (EC 6.2.1.12). Tyrosine is metabolized to 4-hydroxyphenyllactate by tyrosine aminotransferase (EC 2.6.1.5) and hydroxyphenylpyruvate reductase. The ester can be formed from 4-coumaroyl-CoA and 4-hydroxyphenyllactate by the catalytic activity of rosmarinic acid synthase with concomitant release of CoA. Microsomal hydroxylase activities introduce the hydroxyl groups at positions 3 and 3 of the aromatic rings of the ester 4-coumaroyl-4-hydroxyphenyllactate giving rise to rosmarinic acid.Abbreviations Caf-pHPL caffeoyl-4-hydroxyphenyllactate - DHPL 3,4-dihydroxyphenyllactic acid - pC-DHPL 4-coumaryl-3,4-dihydroxyphenyllactate - pC-pHPL 4-coumaryl-4-hydroxyphenyllactate - pHPL 4-hydroxyphenyllactic acid - RA rosmarinic acid The financial support of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie is gratefully acknowledged.     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

Ethylene biosynthesis in Fusarium oxysporum f. sp. tulipae proceeds from glutamate/2-oxoglutarate and requires oxygen and ferrous ions in vivo

Liquid cultures of the deuteromycete, Fusarium oxysporum f. sp. tulipae, a tulip pathogen, produced high amounts of ethylene during stationary phase. 1-Aminocyclopropane-1-carboxylic acid, the direct precursor of ethylene in plants, was not present in the fungus. Radioactivity from [3,4-³H]glutamate as well as [U-¹⁴C]glutamate was incorporated into ethylene, indicating that it was derived from C3 and C4 of glutamate or 2-oxoglutarate. Ferrous ions markedly stimulated the rate of ethylene formation in vivo, whereas Fe³⁺, Cu²⁺ or Zn²⁺ had little or no effect. Ethylene biosynthesis was strongly inhibited by the heavy metal chelator ,-dipyridine. The effect of ,-dipyridine was fully reversed by Fe²⁺ ions and partially by Cu²⁺ and Zn²⁺ ions but not by the supply of glutamate or 2-oxoglutarate, suggesting that a step in the ethylene biosynthetic pathway downstream of 2-oxoglutarate is dependent on Fe²⁺. When stationary phase cultures were supplied with arginine, ornithine, or proline, ethylene production increased dramatically while addition of glutamate or 2-oxoglutarate had little effect. Tracer studies were performed to test the possibility that an intermediate in the catabolism of arginine to glutamate was the direct precursor of ethylene. In cultures supplied with [U-¹⁴C]arginine or [U-¹⁴C]glutamate, the specific radioactivity of ethylene was closely similar to the specific radioactivity of the endogenous glutamate pool, indicating that glutamate was on the pathway between arginine and ethylene. An enzyme system converting 2-oxoglutarate to ethylene in a reaction dependent on oxygen, ferrous ions and arginine has previously been described in extracts from Penicillium digitatum (Fukuda et al. 1986). The present results suggest that a similar enzyme system catalyzes the final step of ethylene biosynthesis in F. oxysporum.Non-standard abbreviations AdoMet S-adenosyl methionine - ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene forming enzyme     

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     

A translocation associated, loss-of-function mutation in the nitrogen metabolite repression regulatory gene of Aspergillus nidulans can revert intracistronically

Summary The areA ^r -18 mutation is a loss-of-function mutation in areA, the positive acting regulatory gene mediating nitrogen metabolite repression in Aspergillus nidulans. It results from a reciprocal translocation which splits the coding region into 5 and 3 moieties. Surprisingly, we have selected rare intracistronic revertants of areA ^r -18. From crosses heterozygous for areA ^r -18 revertant alleles, duplication-deficiency progeny containing two copies of a substantial portion of chromosome IV but lacking part of chromosome III, including the 5 moiety of areA, have been obtained. For all four revertants analysed genetically, growth properties of these duplication-deficiency strains indicate that the reversion events involve the 3 portion of areA and that the 5 portion of areA is unnecessary for the revertant phenotype. This conclusion was directly confirmed for one revertant using Southern blotting. As all four reversion events involve additional chromosomal rearrangements, they probably fuse functional promoters, ribosome binding sites and in frame initiation codons to the 3 portion of the gene. In the course of characterisation of these mutations, new mapping data for a large region of chromosome IV have been generated, and a new reciprocal translocation activating the cryptic regulatory gene areB, whose product can substitute for that of areA, has been identified.     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Biosynthesis and accumulation of rosmarinic acid in suspension cultures ofColeus blumei

This communication reviews data on the accumulation and biosynthesis of rosmarinic acid in cell suspension cultures ofColeus blumei. The influence of the medium, mainly the carbohydrate source on growth and rosmarinic acid production in these cell cultures is described. The biosynthetic pathway of rosmarinic acid was elucidated inColeus blumei cell cultures: eight enzymatic activities are involved in the transformation of the precursors phenylalanine and tyrosine to the end product rosmarinic acid.Abbreviations CAH cinnamic acid 4-hydroxylase - 4CL 4-coumarate:CoA ligase - HPPR hydroxyphenylpyruvate reductase - 3-H hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase - 3-H hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase - PAL phenylalanine ammonia-lyase - RAS rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyl transferase) - TAT tyrosine aminotransferase     

IP3-and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish

Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP₃ or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP₃ were blocked by ruthenium red, a blocker of an IP₃-gated Ca²⁺-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP₃ in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP₃ or cAMP when the buffering capacity for Ca²⁺ of the pipette solution was increased, when extracellular Ca²⁺ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca _i ] that opened Ca²⁺-activated K⁺ channels. The results suggest that different conductances modulated by either IP₃ or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca²⁺-activated K⁺ channels contribute to the termination of the IP₃ and cAMP responses.Abbreviations ATP adenosine 5-triphosphate - BAPTA (bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid - cAMP adenosine cyclic 3,5-monophosphate - cGMP guanosine cyclic 3,5-monophosphate - CTX charybdotoxin - DCB 3,4-dichlorobenzamil - EDTA ethylenediaminetetraacetic acid - EGTA ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IP₃ inositol-1,4,5-triphosphate - NMDG N-methyl-d-glucamine We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825.     

Adenosine-5′-phosphosulfate (APS) as sulfate donor for assimilatory sulfate reduction in Rhodospirillum rubrum

Crude extracts of Rhodospirillum rubrum catalyzed the formation of acid-volatile radioactivity from (³⁵S) sulfate, (³⁵S) adenosine-5-phosphosulfate, and (³⁵S) 3-phosphoadenosine-5-phosphosulfate. An enzyme fraction similar to APS-sulfotransferases from plant sources was purified 228-fold from Rhodospirillum rubrum. It is suggested here that this enzyme is specific for adenosine-5-phosphosulfate, because the purified enzyme fraction metabolized adenosine-5-phosphosulfate, however, only at a rate of 1/10 of that with adenosine-5-phosphosulfate. Further, the reaction with 3-phosphoadenosine-5-phosphosulfate was inhibited with 3-phosphoadenosine-5-phosphate whereas this nucleotide had no effect on the reaction with adenosine-5-phosphosulfate. For this activity with adenosine-5-phosphosulfate the name APS-sulfotransferase is suggested. This APS-sulfotransferase needs thiols for activity; good rates were obtained with either dithioerythritol or reduced glutathione; other thiols like cysteine, 2-3-dimercaptopropanol or mercaptoethanol are less effective. The electron donor methylviologen did not catalyze this reaction. The pH-optimum was about 9.0; the apparent K _m for adenosine-5-phosphosulfate was determined to be 0.05 mM with this so far purified enzyme fraction. Enzyme activity was increased with K₂SO₄ and Na₂SO₄ and was inhibited by 5-AMP. These properties are similar to assimilatory APS-sulfotransferases from spinach and Chlorella.Abbreviations APS adenosine-5-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - 5-AMP adenosine-5-monophosphate - 3-AMP adenosine-3-monophosphate - 3-5-ADP 3-phosphoadenosine-5-phosphate (PAP) - DTE dithiorythritol - GSH reduced glutathione - BAL 2-3-dimercaptopropanol     

Genetics and gibberellin production inGibberella fujikuroi

Gibberella fujikuroi (Fusarium moniliforme) is a complex group of plant pathogens. Some strains produce gibberellic acid and other gibberellins that promote growth and regulate various stages in plant development.The paper describes the research effort directed to development of genetic tools for this species. Furthermore the main features of the gibberellin biosynthetic pathway as established in Gibberella are described.Abbreviations AMO 1618 2-isopropyl-4-(trimethylammonium chloride)-5-methylphenylpiperidine-1-carboxylate - hydroxykaurenoic acid ent-kaur-16-en-7-ol-19-oic acid - kaurenal ent-kaur-16-en-19-al - kaurene ent-kaur-16-ene - kaurenoic acid ent-kaur-16-en-19-oic acid - kaurenol ent-kaur-16-en-19-ol - paclobutrazol 1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-lyl)pentan-3-ol - pefurazoate pent-4-enyl-N-furfuryl-N-imidazol-1-ylcarbonyl-DL-homoa laninate - tetcyclacis 5-(4-chlorophenyl)-3,4,5,9,10-pentaazatetracyclo-5,4,10^2.6,O^8.11-dodeca-3,9-diene - triarimol -(2,4-dichlorophenyl)--phenyl-5-pyrimidine methyl alcohol     

The relationship between guanosine tetraphosphate,polysomes and RNA synthesis in amino acid starved escherichia coli

ArelA⁺ strain ofE. coli with four amino acid requirements was starved separately for each amino acid, after which the levels of polysomes, guanosine-5-diphosphate-3-diphosphate and the residual net synthesis of RNA were determined. The polysome level and guanosine-5-diphosphate-3-diphosphate production were coordinately affected by starvation for the different amino acids, whereas no correlation was found between these two parameters and residual RNA synthesis. The main conclusion stemming from these results is that guanosine-5-diphosphate-3-diphosphate cannot act as the sole effector molecule in stringent control of RNA synthesis.     

Development of a selective pseudosubstrate-based peptide inhibitor of pp60c-src protein tyrosine kinase

Summary Using a combinatorial peptide library method, we identified YIYGSFK as an efficient and specific peptide substrate for pp60^c-src protein tyrosine kinase (PTK) [Lam et al., Int. J. Pept. Protein Res., 45 (1995) 587]. Employing YIYGSFK as a template, we synthesized and evaluated a series of pseudosubstrate-based inhibitors for pp60^c-src. We found that the efficiency of a given inhibitor was highly dependent on the specific tyrosine analog used at the phosphorylation site of the substrate. One of these pseudosubstrate inhibitors, YI(2-Nal)GSFK, selectively inhibited the kinase activity of pp60^c-src, with a K_i of 24 M. This peptide inhibitor exhibited selectivity for pp60^c-src as compared to other PTKs tested, such as c-Abl and Bcr-Abl. Our results suggest that selective inhibitors for a specific PTK can be developed when the structure of a specific and efficient small peptide substrate for this PTK can be used as a template for structure modification.Abbreviations 1-Nal l-1-naphthylalanine - 2-Nal l-2-naphthylalanine - BOP benzotriazolyl-N-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate - BSA bovine serum albumin - cAPK cyclic AMP-dependent protein kinase - DIEA diisopropylethylamine - EGFR epidermal growth factor receptor - Fmoc fluorenylmethoxycarbonyl - HOBt 1-hydroxybenzotriazole - MES 2-[N-morpholino]ethanesulfonic acid - PBS phosphate-buffered salts - pCl l-p-chlorophenylalanine - pF l-p-fluorophenylalanine - PTK protein tyrosine kinase - TLC thin-layer chromatography     

Observation of a distinct transition in the mode of interconversion of ring pucker conformers in non-crystalline d-ribose-2'-d from 2H NMR spin-alignment

Internal motions of d-ribose selectively ²H-labeled at the 2 position were measured using solid state ²H NMR experiments. A sample of d-ribose-2 -d was prepared in a hydrated, non-crystalline state to eliminate effects of crystal-packing. Between temperatures of –74 and –60°C the C2–H2 bond was observed to undergo two kinds of motions which were similar to those of C2–H2/H2 found previously in crystalline deoxythymidine (Hiyama et al. (1989) J. Am. Chem. Soc., 111, 8609–8613): (1) Nanosecond motion of small angular displacement with an apparent activation energy of 3.6 ± 0.7 kcal mol^–1, and (2) millisecond to microsecond motion of large amplitude with an apparent activation energy 4 kcal mol^–1. At –74°C, the slow, large-amplitude motion was best characterized as a two-site jump with a correlation time on the millisecond time scale, whereas at –60°C it was diffusive on the microsecond time scale. The slow, large-amplitude motions of the C2–H2 bond are most likely from interconversions between C2-endo and C3-endo by way of the O4-endo conformation, whereas the fast, small-amplitude motions are probably librations of the C2–H2 bond within the C2-endo and C3-endo potential energy minima.     

Mechanistic possibilities in prebiotic thiophosphate chemistry

Summary Two types of reactivities of thiophosphates have been demonstrated: one being nucleophilic displacement by the P-S moiety of nucleoside phosphorothioates and the other, phosphorylation via P-S cleavage as the driving force. We have designed a system where both displacement on carbon and P-S cleavage are possible. Adenosine derivatives have been synthesized with 5-deoxy-5-chloro and 5-O-tosyl substitutions as leaving groups utilizing the 3-O-phosphorothioate as the biphilic center. The main products of cyclization were 5-O-tosyl and 5-chloroadenosine 2:3-cyclic phosphate. Formation of 3:5-S-phosphorothioate was slow even using an excellent leaving group. This is possibly due to hydrogen bonding between the 2-OH and the neighboring P-O.^– KOH hydrolysis of the cyclic phosphorothioate yielded 2(3) phosphorothioates in a 1:1 ratio. The 2 and 3 isomers were separated and used to study the relative rates of cyclization. The cyclization via P-S cleavage of 2(3)-O-phosphorothioates showed that the 2 isomer was more reactive. This is the first report of superior reactivity of the 3-OH of a ribonucleoside.     

The investigation of the hcn derivative diiminosuccinonitrile as a prebiotic condensing agent. The formation of phosphate esters

Diiminosuccinonitrile (DISN) is formed readily by the Fe³⁺ oxidation of diaminomaleonitrile, a tetramer of HCN. DISN effects the phosphorylation of uridine in 13% yield to a mixture of the isomers of UMP when the reaction is performed in dimethylformamide solution. A 4% yield of the UMP isomers is obtained in neutral aqueous solution using 2 times the DISN concentration and 7 times the phosphate concentration used in DMF. DISN did not effect the conversion of adenosine to AMP or 5-AMP to 5-ADP in aqueous solution. The cyclization of 3-AMP and 3-UMP to the corresponding 2,3-cyclic phosphates proceeds in yields as high as 40–50% at 60°C in pH 6 aqueous solutions in the presence of divalent metal ions. Lower yields of the cyclic phosphate are observed when 2-AMP is the starting material. Substitution of acetate buffer for imidazole buffer results in a decrease in the yield of cyclic phosphate, the extent of which depends on the metal ion used in the reaction. No 3,5-cyclic AMP was detected as a reaction product with either 5-AMP or 3-AMP as the starting material except for a 2.4% yield from 3-AMP in the presence of Zn²⁺. BrCN effects the conversion of 3-AMP to the 2,3-cyclic AMP in 37–65% yield depending on the divalent cation used as catalyst. A mechanism has been proposed for these cyclization reactions and their potential significance to the prebiotic synthesis of ribonucleic acid derivatives is discussed.Chemical Evolution 41. For previous paper see Ferris, J.P., Hagan, W.J., Jr., Alwis, K. W., and McCrea, J.: 1982,J. Mol. Evol. 18, 304–309.Presented at the 7th International Conference on The Origins of Life, Mainz, F.R.G., 1983.     

Sugar conformation of a stereospecific 2′-R or 2′-S deuterium-labeled DNA decamer studied with proton-proton J coupling constants

The sugar conformation of a DNA decamer was studied with proton-proton ³J coupling constants. Two samples, one comprising stereospecifically labeled 2-R-²H for all residues and the other 2-S-²H, were prepared by the method of Kawashima et al. [J. Org. Chem. (1995) 60, 6980–6986; Nucleosides Nucleotides (1995) 14, 333–336], the deuterium labeling being highly stereospecific 99% for all 2-²H, 98% for 2-²H of A, C, and T, and 93% for 2-²H of G). The ³J values of all H1-H2 and H1-H2 pairs, and several H2-H3 and H2-H3 pairs were determined by line fitting of 1D spectra with 0.1–0.2 Hz precision. The observed J coupling constants were explained by the rigid sugar conformation model, and the sugar conformations were found to be between C3-exo and C2-endo with _m values of 26° to 44°, except for the second and 3 terminal residues C2 and C10. For the C2 and C10 residues, the lower fraction of S-type conformation was estimated from J_H1H2 and J_H1H2 values. For C10, the N–S two-site jump model or Gaussian distribution of the torsion angle model could explain the observed J values, and 68% S-type conformation or C1-exo conformation with 27° distribution was obtained, respectively. The differences between these two motional models are discussed based on a simple simulation of J-coupling constants.     

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     

Gibberellin-photoaffinity labeling of wild oat (Avena fatua L.) aleurone protoplasts

Aleurone protoplasts of wild oat (Avena fatua L.), and subcellular fractions isolated from them, were photoaffinity labeled using the synthetic gibberellin (GA) derivative GA₄-17-yl-1-(1-thia)propan-3-ol-4-azido-5-[¹²⁵I]iodosalicylate. Labeled polypeptides were identified by electrophoresis under denaturing conditions followed by autoradiography. GA-photoaffinity labeling of both intact protoplasts and isolated subcellular fractions led to the covalent attachment of the reagent to many polypeptides. A 50 kD polypeptide in the soluble fraction of homogenates of aleurone protoplasts GA-photoaffinity labeled in vivo showed specific binding. The biologically active GA₁, GA₄ and GA₄-17-yl-1(1-thia)propan-3-ol-4-azidosalicylate completed for binding whereas the biologically inactive GA₈ and GA₃₄ did not. The GA-photoaffinity labeling characteristics of this polypeptide suggested that it might interact specifically with biologically active GAs in vivo. Attempts to detect specific GA-binding in in vitro GA-photoaffinity labeling experiments met with only limited success perhaps indicating the labile nature of specific binding observed in vivo. The potential of GA-photoaffinity labeling for identifying GA-binding proteins in aleurone and other GA-responsive tissues is discussed.Abbreviations azido IAA = 5-azido-7-[³H]indole-3-acetic acid - azido NPA = 5-azido-[3,6-³H]1-N-napthylpthalamic acid - BTP = 1,3-bis(Tris(hydroxymethyl)methylamino)-propane - GA₄-O-ASA = GA₄-17-yl-1-(1-thia)propane-3-ol-4-azidosalicylate - [¹²⁵I]GA₄-O-ASA = GA₄-17-yl-1-(1-thia)propan-3-ol-4-azido-5-[¹²⁵I]iodosalicylate - NPA = 1-Naphthylphthalmic acid - PAGE = Polyacrylamide gel electrophoresis - PMSF = phenylmethylsulfonyl fluoride - SDS = Sodium dodecyl sulphate - TLCK = L-1-Chloro-3-(4-tosylamido)-7-amino-2-heptanone-HCl     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Oligonucleotide formation catalyzed by divalent metal ions. The uniqueness of the ribosyl system

Summary Polymerization of various nucleoside-5-phosphorimidazolides has been conducted in neutral aqueous solution using divalent metal ions as catalysts. Oligonucleotide formation took place from each of the ribonucleoside-5-phosphorimidazolides, ImpC, ImpU, ImpA, ImpG, and ImpI. The yields and distributions of the resulting oligonucleotides varied depending on the difference of the nucleic acid base and the metal ions used. The catalytic effect of divalent metal ions on the formation of oligocytidylates occurred in the following order: Pb²⁺>Zn²⁺>Co²⁺, Mn²⁺>Cd²⁺>Cu²⁺>Ni²⁺>Ca²⁺, Mg²⁺, none >Hg²⁺. The order changes slightly for other types of oligoribonucleotide formation. Oligoribonucleotides up to hexamers were obtained in 35–55% overall yield, when Pb²⁺ ion was used as a catalyst. Zn²⁺ ions yielded oligoribonucleotides up to tetramers in 10–20% overall yield. The resulting oligonucleotides contained mainly 2–5 internucleotide linkages.Little or no oligonucleotide was obtained from nucleoside-5-phosphorimidazolides modified in the sugars, Imp(3-dA), Imp(2-dA), Imp(Ara), Imp(Aris), and Imp(Nep). The results indicate that a ribosyl system is required for the metal ion-catalyzed synthesis of oligonucleotides. Abbreviations. EDTA, ethylenediaminetetraacetic acid; Versenol,N-hydroxyethylethylenediaminetriacetic acid; Tris, tris-(hydroxymethyl)aminomethane; pN (N is A, C, G, U, I, 3-dA, 2-dA, AraA, Aris, or Nep), nucleoside-5-phosphate; Np, nucleoside-2(3)-phosphate; I, inosine; 3-dA, 3-deoxyadenosine; 2-dA, 2-deoxyadenosine; AraA, arabinosyladenine; Aris, aristeromycin; Nep, neplanocin A; ImpN, nucleoside-5-phosphorimidazolide; NppN, P₁,P₂-dinucleoside-5,5-pyrophosphate; (pN)_n (n=2, 3, ...), oligomers of pN, numbers given between a nucleoside and a phosphate indicate the type of internucleotide linkage, e.g., pC₂ p₅C is 5-phosphorylcytidyl-(2–5)-cytidine; , cyclic dimers of pN; BAP, bacterial alkaline phosphatase; N.Pl, nuclease Pl; VPDase, venom phosphodiesterase; HPLC, high pressure liquid chromatography