Guanosine 5', alpha-beta-methylene, triphosphate, a novel GTP analog, causes persistent activation of adenylate cyclase: evidence against pyrophosphorylation mechanism.

To test the hypothesis that guanine nucleotides activate adenylate cyclase by a covalent mechanism involving pyrophosphorylation of the enzyme, we studied the effect of a novel GTP analog, guanosine 5′, α-β-methylene triphosphate (Gp(CH₂)pp), with a methylene bond in the α-β-position that is stable to enzymatic hydrolysis. Gp(CH₂)pp was as effective as GTP in stimulating rat reticulocyte adenylate cyclase in the presence of isoproterenol. Previously only guanine nucleotides with modified terminal phosphates such as guanylyl 5′-imidodiphosphate (Gpp(NH)p) were thought capable of causing persistent activation of adenylate cyclase. Gp(CH₂)pp, however, caused persistent activation of rat reticulocyte and turkey erythrocyte adenylate cyclase. We conclude that guanine nucleotides do not activate adenylate cyclase by a pyrophosphorylation mechanism and that a modified γ-phosphate is not essential in guanine nucleotides for generation of the irreversibly-activated enzyme state.     

A new method for oligo(ADP-ribose) fractionation according to chain length

A new method was developed to separate mono- and oligo-(ADP-ribose) with chain lengths below 11 ADP-ribose units by size difference of one ADP-ribose residue. The separation was performed on a DEAE-cellulose column by elution with a NaCl gradient (0–0.3 $\text{M}$) in the presence of 7 $\text{M}$ urea at pH 7.6. Using this method, the chain length distribution of oligo(ADP-ribose) molecules attached to histones by incubation of isolated nuclei with radioactive NAD was determined. The average chain length estimated from this distribution coincided exactly with the value obtained by the phosphodiesterase digestion method, suggesting that the oligomers were synthesized directly on histones and not elongated from pre-existing ADP-ribose.     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na⁺, K⁺, Mg²⁺ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1)$\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$, (2) $\text{Mg}{}^{\mathrm{2+}}$, $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}$ and none, (3) $\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{,}\text{Na}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}$ and $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$, (4) $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}\text{,}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}$ and $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}\text{, (5)}\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ and ATP. The highest rate was obtained in the presence of Na⁺, Mg²⁺ and ATP. The apparent concentrations of Na⁺, Mg²⁺ and ATP for half-maximum stimulation of inhibition ($\text{K}{}_{0.5}{}^{\mathrm{<mtext>s</mtext>}}$) were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na⁺ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E₁, E₂, ES, E₁-P and E₂-P, i.e., E₂ has higher reactivity with showdomycin than E₁, while E₂-P has almost the same reactivity as E₁-P. We conclude that the reaction of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ proceeds via at least four kinds of enzyme form (E₁, E₂, E₁ · nucleotide and EP), which all have different conformations.     

Picosecond time-resolved study of MgCl2-induced chlorophyll fluorescence yield changes from chloroplasts

The MgCl₂-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3′,4′-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a picosecond time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp $\text{?At}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, where A was found to be 0.052 $\text{ps}{}^{\mathrm{<mtext>?1</mtext><mtext>2</mtext>}}$, and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl₂ produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence decay law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.     

Nucleotide activation of glycogen phosphorylase b occurs only when the nucleotide phosphate is in a dianionic form

Adenosine monophosphofluoridate has been synthesised and purified to remove all contaminating AMP. This AMP analogue fails to activate glycogen phosphorylase $\text{b}$, even at high concentration, but inhibits the AMP activation with a Ki value of 3 mM. Activation of phosphorylase $\text{b}$ by adenosine phosphoramidate has been re-investigated in the light of these findings and a purified sample of this nucleotide analogue has been shown to produce little or no activation of the enzyme. These findings are interpreted in terms of an absolute requirement of the nucleotide activatorsite in phosphorylase for a nucleotide with a dianionic phosphate. The implications of this for the role of the phosphate moiety in the proposed mechanism of activation are discussed.     

Guanine nucleotides modulate cell surface cAMP-binding sites in membranes from Dictyostelium discoideum

D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (K_d = 15 nM, $\text{t}\text{1}\text{2}\text{= 15 s}$). A second class is fast dissociating ($\text{t}\text{1}\text{2}$ about 1 s) and is composed of high affinity binding sites H (K_d ≈ 60 nM), and low affinity binding sites L (K_d = ≈ 450 nM) which interconvert during the binding reaction. Guanine nucleotides affect these three binding types in membranes prepared by shearing D.discoideum cells through Nucleopore filters. The affinity of S for cAMP is reduced by guanine nucleotides from 13 nM to 25 nM, and the number of S-sites is reduced about 50%. The number of fast dissociating sites is not altered by guanine nucleotides, but these sites are mainly in the low affinity state. Half-maximal effects are obtained at about 1 μM GTP, 2 μM GDP and 10 μM Gpp(NH)p(guanyl-5′-yl-imidodiphosphate); ATP and ADP are without effect up to 1 mM. These results indicate that D.discoideum cells have a functionally active guanine nucleotide binding protein involved in the transduction of extracellular cAMP signals via cell surface cAMP receptors.     

The metabolism of formycin B in Leishmaniadonovani

Formycin B, a pyrazolo(4,3-d)pyrimidine C-nucleoside, inhibited the growth of $\text{Leishmania}\text{donovani}$ promastigotes in culture with an ED₉₀ of 0.2 μg/ml. Promastigotes incubated for 24 hrs with Formycin B at 10 μg/ml were found to convert it to the ribonucleotide, formycin B 5′-monophosphate. The parasites were also capable of aminating formycin B 5′-monophosphate as evidenced by the appearance of formycin A di- and triphosphate. The RNA contained the formycin A moiety in 3′,5′-polynucleotide linkage. Succino-AMP synthetase from these parasites was able to use formycin B 5′-monophosphate as an alternate-substrate with a K'm of 26 μM and a V'm of about 1% the V'm IMP. Formycin B 5′-monophosphate was also a substrate for mammalian succino-AMP synthetase with a V_m' of 40% the V_m' of IMP.     

Errata

Optimal conditions for activation of adenylate cyclase in membrane particles were studied. Enzyme activation with serotonin (5-hydroxytryptamine), NaF, and guanosine $\text{5′-(3-O-}\text{thio}\text{)-}\text{triphosphate}$ (GTPγS) was time- and temperature-dependent. Mg²⁺ was required for enzyme activation. Adenylate cyclase that was activated by NaF or GTPγS was gradually inhibited by $\text{N-}\text{methylmaleimide}$ while enzyme activated with serotonin and GTP responded faster to inhibition by the same sulfhydryl reagent. The enzyme responded in a similar fashion to a spin-labeled $\text{N-}\text{methylmaleimide}$ analog 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrolidinyloxyl (i.e., $\text{N-}\text{methylmaleimide}$ nitroxide). Binding of the spin label was enhanced following enzyme activation by serotonin, NaF, or GTPγS in the presence of Mg²⁺. Activation of the enzyme was accompanied by an increase in the strong immobilization peaks in the EPR spectra. Both effects, the increase in binding and in the strong immobilization peaks, can be induced by Mg²⁺ alone. The results indicate that a general conformational change induced by Mg²⁺ may be essential for adenylate cyclase activation.     

A flash photolysis ESR study of Photosystem II Signal IIvf, the physiological donor to P-680+

In flash-illuminated, oxygen-evolving spinach chloroplasts and green algae, a free radical transient has been observed with spectral parameters similar to those of Signal II ($\text{g ≈ 2.0045}$, $\text{ΔH}{}_{\mathrm{<mtext>pp</mtext>}}\text{≈ 19}\text{G}$). However, in contrast with ESR Signal II, the transient radical does not readily saturate even at microwave power levels of 200 mW. This species is formed most efficiently with “red” illumination ($\text{λ < 680}\text{nm}$ and occurs stoichiometrically in a 1 : 1 ratio with $\text{P-700}{}^{\mathrm{+}}$. The Photosystem II transient is formed in less than 100 μs and decays via first-order kinetics with a halftime of 400–900 μs. Additionally, the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for radical decay is temperature independent between 20 and 4 °C; however, below 4 °C the transient signal exhibits Arrhenius behavior with an activation energy of approx. 10 kcal · mol^?1. Inhibition of electron transport through Photosystem II by $\text{o-}\text{phenanthroline}$, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or reduced $\text{2,5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isopropyl}\text{-p-}\text{benzoquinone}$ suppresses the formation of the light-induced transient. At low concentrations (0.2 mM), $\text{2,5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isopropyl}\text{-p-}\text{benzoquinone}$ partially inhibits the free radical formation, however, the decay kinetics are unaltered. High concentrations of $\text{2,5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isopropyl}\text{-p-}\text{benzoquinone}$ (1–5 mM) restore both the transient signal and electron flow through Photosystem II. These findings suggest that this “quinoidal” type ESR transient functions as the physiological donor to the oxidized reaction center chlorophyll, $\text{P-680}{}^{\mathrm{+}}$.     

Lactones,flavonoids and benzophenones from Garcinia conrauana and Garcinia mannii

Two novel lactones have been isolated from the stem barks of Garcinia conrauana and G. mannii. The major component of the bark of G. conrauana was identified as 3-(3$\text{,}\text{?}$ 3″-dimethylallyl)-conrauanalactone [4-hydroxy-3-(3″, 3″-dimethylallyl)-6-pentadecylpyran- 2-one] by comparison of spectral data of the isolated compound and two methylethers with that obtained for the previously isolated conrauanalactone. A minor component of the bark of G. mannii was tentatively identified as 3-α-hydroxy-5-(heptadec-8′-enyl)-tetrahydro- furan-2-one on the basis of spectral data from the isolated compound and its monoacetate. The distributions of biflavonoids and related compounds and benzophenones in the stem bark, heartwood, seeds and leaves of the two species are reported.     

Kinetics of reduction of the oxidized primary electron donor of Photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (ΔA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures.In the microsecond time range the difference spectrum of ΔA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P⁺?700; it decays in a polyphasic manner with half-times of 17 μs, 210 μs and over 1 ms. The oxidized primary donor of Photosystem II (P⁺_II) is not detected with a time resolution of 3 μs. After treatment with 3–10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P⁺_II is observed and decays biphasically (a major phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 20–40 μ}\text{s}$, and a minor phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 200 μ}\text{s}$), probably by reduction by an accessory electron donor.In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P⁺_II is reduced with a half-time of 25–45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.     

Effects of lanthanum on calcium-dependent phenomena in human red cells

Lanthanum (0.25 mM) does not penetrate into fresh or Mg²⁺-depleted cells, whereas it does into ATP-depleted or $\text{ATP + 2,3-diphosphoglycerate-depleted cells}$, into cells containing more than 3 mM calcium, or cells stored for more than 4 weeks in acid/citrate/dextrose solution. In fresh cells loaded with calcium, extracellular lanthanum blocks the active Ca²⁺-efflux completely and inhibits $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) activity to about 50%. In Mg²⁺-depleted cells Ca²⁺-Ca²⁺ exchange is inhibited by lanthanum. Ca²⁺-leak is unaffected by lanthanum up to 0.25 mM concentration; higher lanthanum concentrations reduce leak rate. In NaCl medium Ca²⁺-leak ± S.D. amounts to $\text{0.28 ± 0.08 μ}\text{mol/l}$ of cells per min, whereas in KCl medium to $\text{0.15 ± 0.04 μ}\text{mol/l}$ of cells per min at 2.5 mM [Ca²⁺]_e and 0.25 mM [La³⁺]_e pH 7.1.Lanthanum inhibits Ca²⁺-dependent rapid K⁺ transport in ATP-depleted and propranolol-treated red cells, i.e. whenever intracellular calcium is below a critical level. The inhibition of the rapid K⁺ transport can be attributed to protein-lanthanum interactions on the cell surface, since lanthanum is effectively detached from the membrane lipids by propranolol.Lanthanum at 0.2–0.25 mM concentration has no direct effect on the morphology of red cells. The shape regeneration of Ca²⁺-loaded cells, however, is blocked by lanthanum owing to Ca²⁺-pump inhibition. Using lanthanum the transition in cell shape can be quantitatively correlated to intracellular Ca²⁺ concentrations.     

Primary structure of E. coli alanine transfer RNA: relation to the yeast phenylalanyl tRNA synthetase recognition site

Nucleotide sequence comparison of tRNAs aminoacylated by yeast phenylalanyl tRNA synthetase (PRS) have lead to the proposal that the specific nucleotides of the dihydrouridine (diHU) stem region and adenosine at the fourth position from the 3′ end are involved in the PRS recognition site. Kinetic analysis and enzymatic methylation have shown that the size of the diHU loop and the methylation of guanine at position 10 from the 5′ end both directly affect the PRS aminoacylation kinetics. $\text{E. coli}$ tRNA₁^A1a, which is aminoacylated by PRS, should therefore have 1- the specific nucleotides of the diHU stem region and, 2- adenosine at position 4 from the 3′ end. The PRS aminoacylation kinetics of this tRNA indicates that this molecule 3- has a diHU loop of 8 nucleotides and 4- has an unmethylated guanine at position 10 from the 5′ end. We report here the complete sequence of $\text{E. coli}$ tRNA₁^A1a and confirmation of each of these four predictions.     

A simultaneous protein-binding assay for adenosine 3':5'-monophosphate in biological materials.

Adenosine 3′:5′-monophosphate (cyclic AMP) and guanosine 3′:5′-monophosphate (cyclic GMP) have been determined simultaneously by combining individual protein binding assays using different isotopically labeled cyclic nucleotides. Preparations of cyclic AMP-binding protein from beef adrenal cortex and cyclic GMP-binding protein from the fat body of silkworm pupae ($\text{Bombyx mori}$) have been used for the assay. The method allows the analysis of cyclic AMP and cyclic GMP levels in crude extracts without any purification. The assay has been applied to hormone-stimulated Mouse liver and phorbol ester-treated Rat embryo cells.     

Phospholipid metabolism of stimulated lymphocytes: Comparison of the activation of acyl-CoA: Lysolecithin acyltransferase with the binding of concanavalin A to thymocytes

Calf thymocytes were isolated and incubated with concanavalin A. The effect of the mitogen on the enzyme activity of membrane-bound lysolecithin acyltransferase (acyl-CoA: $\text{1-acylglycero-3-phosphorylcholine}\text{-O-}\text{acyltransferase}$, EC 2.3.1.23) was determined as also the binding of ¹²⁵I-labelled concanavalin A to intact cells and isolated membranes.The lysolecithin acyltransferase was found to be activated three times in microsomal membranes. The activation occurred directly after binding of concanavalin A and was temperature independent, since similar activities were found in cells treated with concanavalin A at 0 and 37 °C.The acyltransferase activation using increasing concentrations of concanavalin A revealed a different behaviour, as compared to the binding of concanavalin A. While the binding of concanavalin A to intact cells expressed a normal hyperbolic saturation function the activation process of the acyltransferase described a sigmoidal relationship. Corespondingly, the interaction coefficients for both functions were different (Sips coefficient for binding = 1.0 and Hill coefficient of the enzyme activation = 1.8).These results indicate that the acyltransferase activation is due to a cooperative interaction between the ligand-receptor complex and the enzyme.     

Interaction of oncodazole (R 17934), a new antitumoral drug, with rat brain tubulin.

Oncodazole (R 17934), methyl [5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl] carbamate (I), a new synthetic drug with anti-tumoral activity, inhibits the polymerization of rat brain tubulin $\text{in vitro}$. It has no depolymerizing effect on preformed microtubules $\text{in vitro}$. Binding studies by means of molecular sieving and equilibrium dialysis indicates that the drug binds to purified rat brain tubulin in a mole to mole ratio. Finally the drug competitively inhibits colchicine binding to purified rat brain tubulin. From these results the conclusion may be drawn that oncodazole is a true microtubule inhibitor.     

The site of inhibition of photosystem II by 3-(3,4-dichlorophenyl)-N-N'-dimethylurea in thylakoids of the cyanobacterium Anabaena cylindrica.

Thylakoids isolated from the cyanobacterium $\text{Anabaena}\text{cylindrica}$ exhibit Photosystem II activity. Photosynthetic electron transfer from water to ferricyanide and to 2,6-dichlorophenolindophenol is inhibited by 3-(3,4-dichlorophenyl)-N-N′-dimethyl urea. Diphenylcarbazide stimulates ferricyanide and 2,6-dichlorphenolindophenol photoreduction, whilst inhibiting oxygen evolution. Diphenylcarbazide-supported Photosystem II activity is completely insensitive to 3-(3,4-dichlorophenyl)-N-N′-dimethyl urea, indicating that the site of action of this inhibitor lies on the donor side of Photosystem II in $\text{A}\text{.}\text{cylindrica}$, before the site of electron donation by diphenylcarbazide.     

Enzymatic synthesis of 2-O-α-d-mannopyranosyl-methyl-α-d-mannopyranoside by a cell-free particulate system of mycobacterium smegmatis

A cell-free particulate enzyme preparation of Mycobacterium smegmatis ATCC 607 catalyzed the transfer of labeled mannose from GDP[¹⁴C]mannose to methyl-α-mannopyranoside (an exogenously added acceptor) to form a product that was characterized to be $\text{2-O-α-}\text{d}\text{-[}{}^{14}\text{C}\text{]}\text{mannopyranosyl-methyl}\text{-α-}\text{D-mannopyranoside}$. This tranmannosylase activity was specific for both the sugar nucleotide donor and methyl monosaccharide acceptor. The reaction was stimulated by the addition of various metal ions and had a pH optimum of 6.0. The apparent K_m of this transmannosylase reaction for methyl-α-d-mannopyranoside was 35 mM.The possible relationship between this “artificial” mannosyl-transfer system and the “natural” system which leads to the formation of the oligomannosides and glycoproteins is discussed.