Occurrence of Guanosine-5′-diphosphate-3′-diphosphate and Adenosine-5′-triphosphate-3′-diphosphate in Streptomyces galilaeus

Synthetic activity and existence of ppGpp and pppApp in an anthracycline-producing strain Streptomyces galilaeus were determined by radioimmunoassay and ³²P-labeling method during cultivation under both the antibiotic productive and non-productive conditions. The cellular ppGpp(pppGpp)-synthesizing activity was highest at the end of exponential growth, and 3-fold higher in the antibiotic-productive cultivation than in non-productive cultivation. The intracellular level of ppGpp determined by radioimmunoassay was high at the end of exponential growth, and afterwards its level decreased by one fifth. The low level of cellular ppGpp during the period of intense antibiotic production suggests that ppGpp consumption may play an important role in antibiotic production of S. galilaeus. The concentration of pppApp was not determined intracellularly by radioimmunoassay.     

Effect of cyclic adenosine-3′,5′-monophosphate on the simultaneous synthesis of β-galactosidase and tryptophanase inEscherichia coli

When inducing simultaneously β-galactosidase and tryptophanase in a batch culture either the synthesis of tryptophanase or of both enzymes is decreased due to an insufficient cAMP concentration. The addition of this nucleotide can overcome this decrease. In a continuous culture both enzymes are synthesized at the maximum rate, as the amount of cAMP produced during carbon limitation of growth is probably sufficient for the simultaneous synthesis of both enzymes. In the β-galactosidase hyperproduction mutant cultivated continuously the level of β-galactosidase markedly decreases when tryptophanase is simultaneously induced. Also this decrease is caused by cAMP insufficiency and can be overcome by increasing its concentration. cAMP is thus an important regulatory factor of both enzymes and becomes a limiting factor in their simultaneous synthesis; a competition for this regulatory compound apparently occurs and probably also a different mutual affinity of the regulatory complex with the promoter site of the enzyme opérons is involved.     

Induction of blastocladiella emersonii germination by cyclic adenosine-3′, 5′-monophosphate

The role of adenosine 3′,5′-monophosphate (cyclic AMP) in the control of Blastocladiella emersonii germination was studied. This differentiative transition may be induced by replacing K⁺, a classical inducer, by cyclic AMP or by competitive inhibitors of cyclic AMP phosphodiesterase activity. When zoospores are treated simultaneously with two inducers at non-effective concentrations, a synergistic effect is observed between cyclic AMP and either KCl or adenine. The calcium ionophore A23187 per se is not able to elicit germination, but the association of A23187 and sub-optimal concentrations of cyclic AMP is effective. These results suggest that germination may depend on a correlation between the intracellular mobilization of calcium and cyclic AMP levels.     

Effects of Exogenous Adenosine 5′-triphosphate 3′-diphosphate on Growth and Sporulation of Bacillus subtilis

Exogenous adenosine 5′-triphosphate 3′-diphosphate (pppApp) had interesting effects on the cell cycle of B. subtilis IFO 3027. The growth rate was reduced by the addition of 1 mm pppApp, and the vegetative cell form was significantly changed. Moreover, the sporulation frequency was increased by 100 times or more as compared with the culture without pppApp. The sporulation process seemed to be stimulated around t₀. pppGpp and ppGpp also showed the same effects as pppApp. Among these effects, depression in growth rate was restored by Mg²⁺ and Ca²⁺, and stimulation of sporulation was inhibited by Mg²⁺, Ca²⁺ and certain carbon sources, such as glucose and glycerol. On the other hand, casamino acids or monovalent cations showed no influence on the pppApp effects. pppApp was not incorporated into cells in experiments with radioactive pppApp.     

Studies on degradation of adenosine-5′-phosphosulphate (APS) and adenosine-3′-phosphate-5′-phosphosulphate (PAPS) in extracts of Anabaena cylindrica

[³⁵S]Adenosine-5′-phosphosulphate ([³⁵S]APS) and [³⁵S]adenosine-3′-phosphate-5′-phosphasulphate ([³⁵S]PAPS) were rapidly degraded by extracts of Anabaena cylindrica. The loss of radioactivity from these sulphur nucleotides resulted in a corresponding increase of free ³⁵SO₄^ in the incubation mixture. The soluble fraction of the broken cells (S₇₅) hydrolysed both PAPS and APS, whereas the pellet fractions (P₂₀ and P₇₅) hydrolysed PAPS only. The degradation of [³⁵S]PAPS was almost completely suppressed by various 5′-adenine nucleotides, 3′-AMP, nucleotide triphosphates or pyrophosphate, while glucose-6-phosphate, phosphate ions and sodium sulphite were less effective. The hydrolysis of [³⁵S]APS was prevented by sodium fluoride and 5′-AMP, but 3′-AMP was ineffective.     

Cyclic adenosine 3′,5′-monophosphate and germination of sporangiospores from the fungus Mucor

Cyclic adenosine 3,5-monophosphate (cAMP) metabolism was examined in germinating sporangiospores of Mucor genevensis and Mucor mucedo. Exogenous cAMP prevented normal hyphal development from sporangiospores. Internal pools of cAMP fluctuated profoundly during development. Spherical growth of the spores was characterized by large pools of cAMP whereas germ tube emergence and hyphal elongation were characterized by small pools of cAMP. These observations suggest a possible role for cAMP in sporangiospore germination. Adenylate cyclase activities fluctuated significantly during germination with maximum values attained during spherical growth. In contrast, cAMP phosphodiesterase activities remained constant throughout germination. Internal cAMP levels may therefore be regulated by adjustment of adenylate cyclase activities. The binding of cAMP by soluble cell proteins was measured. cAMP-binding activity changed greatly during germination. Dormant and spherically growing spores possessed the highest activities. Developing hyphae contained the lowest activities. Use of the photoaffinity label, 8-azido-[^32P]cAMP, in conjunction with sodium dodecyl sulfate-polyacrylamide-gel electrophoresis allowed the identification of a small population of morphogenetic-stage-specific proteins which bind cAMP and may be of regulatory significance to development.     

Synthesis of Guanosine-3′-diphosphate- 5′-diphosphate by Nucleotide Pyrophosphotransferase

An α-glucosidase was purified from sweet corn seeds by fractionation with ammonium sulfate, chromatographies on CM-Sepharose and Sepharose 4B, and gel filtrations on Sephadex G-100. The enzyme was homogeneous in disc electrophoretic analysis. The molecular weight was estimated to be about 9.6 × 10⁴ by SDS-disc electrophoresis.

The enzyme showed high activities toward maltose, nigerose, phenyl-α-maltoside, and maltooligosaccharides. The ratios of maximum velocity for maltose, nigerose, kojibiose, isomaltose, phenyl-α-glucoside, phenyl-α-maltoside, panose, turanose, and soluble starch were estimated to be 100 : 78 : 17 : 11 : 28 : 100 : 31 : 3.4 : 126, and the Km values for these substrates, 1.5 mM, 1.4 mM, 0.48 mM, 14 mM, 4.2 mM, 1.1 mM, 5.0 mM, 0.28 mM and 52mg/ml, respectively. The maximum velocity for soluble starch was high, but this α-glucan was not a favorable substrate because the Km value was also very high. The V_max for maltooligosaccharides were somewhat dependent on the degree of polymerization (n). The Km values for substrates having four or more glucose units increased with the increase in n.     

Coordination of magnesium with adenosine 5′-diphosphate and triphosphate

³¹P NMR chemical shifts of salts of adenosine 5′-triphosphate and diphosphate: ATPH^2?₂2(Me₄N⁺) · H₂O, ATPH^2?₂2 Na⁺ · 3.5 H₂O, ATPH^2?₂Mg²⁺ · 4 H₂O, ATPH^2?₂Ca²⁺ · 2 H₂O, ADPH^2?2(Me₄N⁺) · H₂O and ADPH^2?Mg²⁺ · 4 H₂O have been measured in 0.02 M ²H₂O solutions at 145.7 MHz (22° C) at constant p²H values (8.20 and 6.20). The results are compared with those obtained from salts of adenosine 5′-monophosphate and other simpler phosphomonoesters, e.g. AMP^2?2(Me₄N⁺), AMP^2?Mg²⁺, AMPH^?Me₄N⁺ and (AMPH^?)₂Mg²⁺. It is concluded that the effects exerted by Mg²⁺ and Ca²⁺ on the ³¹P NMR shifts of dipoly- and tripolyphosphates relative to monovalent cations are due mainly to changes in conformation of the polyphosphate chain rather than to purely electronic factors associated with the binding of divalent cations to the phospho-oxyanions. The data are consistent with the existence of the following complexes at p²H 8.20: (MgP_αP_β)ADP^? and (MgP_αP_γ)ATP^2?af (MgP_αP_β)ATP^2?af (MgP_βP_γ)ATP^2? with the latter equilibrium relatively fast in the NMR time scale. Monoprotonation of the terminal phosphate appears to weaken the Mg²⁺-polyphosphate binding, particularly at P_β of MgADPH and at P_β and P_γ of MgATPH^?. The Mg²⁺-polyphosphate binding weakens further at p²H 3.70, i.e. in MgATPH₂. Possible implications of the results in the mechanism of actomyosin Mg²⁺-ATPase in muscle contraction are discussed.     

Effect of guanosine 3′:5′-monophosphate on the hydroosmotic activity of adenosine 3′:5′-monophosphate in toad urinary bladder

Guanosine 3′:5′-monophosphate has a slight hydroosmotic effect on toad urinary bladder. Furthermore, this nucleotide strongly inhibits the responses to 3′:5′-adenosine monophosphate and oxytocin. The response to an increase in medium tonicity is not modified by the guanosine nucleotide. A role for guanosine 3′:5′-monophosphate in the regulation of water permeability in toad urinary bladder is proposed.     

Efficient chemoenzymatic synthesis of uridine 5′-diphosphate N-acetylglucosamine and uridine 5′-diphosphate N-trifluoacetyl glucosamine with three recombinant enzymes

Uridine 5′-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a natural UDP-monosaccharide donor for bacterial glycosyltransferases, while uridine 5′-diphosphate N-trifluoacetyl glucosamine (UDP-GlcNTFA) is its synthetic mimic. The chemoenzymatic synthesis of UDP-GlcNAc and UDP-GlcNTFA was attempted by three recombinant enzymes. Recombinant N-acetylhexosamine 1-kinase was used to produce GlcNAc/GlcNTFA-1-phosphate from GlcNAc/GlcNTFA. N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12 MG1655 was used to produce UDP-GlcNAc/GlcNTFA from GlcNAc/GlcNTFA-1-phosphate. Inorganic pyrophosphatase from E. coli K12 MG1655 was used to hydrolyze pyrophosphate to accelerate the reaction. The above enzymes were expressed in E. coli BL21 (DE3) and purified, respectively, and finally mixed in one-pot bioreactor. The effects of reaction conditions on the production of UDP-GlcNAc and UDP-GlcNTFA were characterized. To avoid the substrate inhibition effect on the production of UDP-GlcNAc and UDP-GlcNTFA, the reaction was performed with fed batch of substrate. Under the optimized conditions, high production of UDP-GlcNAc (59.51?g/L) and UDP-GlcNTFA (46.54?g/L) were achieved in this three-enzyme one-pot system. The present work is promising to develop an efficient scalable process for the supply of UDP-monosaccharide donors for oligosaccharide synthesis.     

The effect of glucose and manganese on adenosine-3′, 5′-monophosphate levels during growth and differentiation of Aspergillus nidulans

The role of cyclic adenosine monophosphate (cAMP) during growth and development of Aspergillus nidulans was investigated. In normal cultures the highest amount of cAMP, expressed on a dry weight basis, was found after 24 h of growth when still more than 5% glucose was present in the medium. After depletion of the medium even a slight fall in cAMP was noted. Glucose concentrations ranging from 0.5–12% resulted in a slight decrease in the amount of cAMP as measured after 24 h of growth.Cultures with manganese deficiency resulted in a low cAMP level after 24 h of growth. However, the exhaustion of glucose in the absence of manganese was connected with a sharp increase in cAMP. This indicates that manganese shortage was not a direct cause of the low cAMP level after 24 h. The amount of cAMP rose with increasing concentration of manganese in the medium until a maximum at 0.25 M. It is tempting to speculate that this rise in cAMP in the manganese deficient culture is explained by the absence of glucose, that in the control culture is derived from the breakdown of the reserve material -1,3-glucan.Addition of manganese after glucose exhaustion to a manganese deficient culture induced cleistothecium formation. However, they contained only a few ascospores indicating the importance of -1,3 glucan as a carbon and energy source for ascospore formation. The regulation of the level of cAMP by the transport of glucose into the cell or its intracellular concentration is discussed.     

Formation of nucleoside 5′-polyphosphates from nucleotides and trimetaphosphate

Summary When solutions of nucleoside 5-phosphates and trimetaphosphate are dried out at room temperature, nucleoside 5-polyphosphates are formed. The Mg⁺⁺ ion shows a superior catalytic function in this reaction when compared with other divalent metal ions. Starting with nucleoside 5-phosphates, Mg⁺⁺ and trimetaphosphate, the predominant products in the nucleoside 5-polyphosphate series p_nN are p₄N, p₇N and P₁₀N. Nucleoside 5-diphosphates yield p₅N and p₈N, nucleoside 5-triphosphates give p₆N and p₉N. The prebiological relevance of these reactions is discussed.Abbreviations P_n (n = 1,2,3,) linear polyphosphate containing n phosphate residues - P₃! trimetaphosphate - A adenosine - U uridine - dA 2-dexyadenosine - T thymidine - P_nN nucleoside 5-polyphosphate containing n phosphate residues, e.g. with N = A and n = 4 - p₄A adenosine 5-tetraphosphate     

Effect of adenosine 3′,5′ monophosphate on the mutation frequency induced by N-methyl-N′-nitro-N-nitrosoguanidine

The effect of increased cellular concentrations of adenosine 3′,5′ monophosphate (cAMP) upon mutation frequency induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was studied in V79 Chinese hamster lung cells. Incubation with either forskolin, which increased the accumulation of cAMP, or 8BrcAMP, an analogue of cAMP, resulted in an increase in the mutation frequency which was concentration-dependent, regardless of whether these agents were added before or after mutagen treatment. Increased cAMP concentrations were shown in these cells to inhibit growth; however, this does not seem to be the mechanism responsible for the increase in mutation frequency as low serum concentrations which also retard growth reduced the mutation frequency observed with MNNG.     

Effect of sodium deoxycholate on 5′-nucleotidase

The 5′-nucleotidase localized in rat liver plasma membranes was purified to a single protein, which contained phospholipid. The molecular weight and the sedimentation constant were about 150 000 and 7 S in the presence of sodium deoxycholate, while the enzyme protein was aggregated when the preparation was dialyzed thoroughly. The purified 5′-nucleotidase exhibited the same properties as the 5′-nuelcotidase in plasma membranes. The 5′-nucleotidase activity was increased by the addition of various bile salts or by the solubilization of membranes with trypsin, papain or phospholipase C. The solubilized and aggregated forms of the enzyme showed different substrate specificity for nucleotides, pH optimum, heat stability and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. The purified enzyme catalyzed an exchange reaction between AMP and adenosine, which was diminished by the addition of sodium deoxycholate.