Applications of tritium NMR to macromolecules: A study of two nucleic acid molecules

Summary We have tritium labeled two nucleic acid molecules, an 8 kDa DNA oligomer and a 20 kDa hammerhead RNA for tritium NMR investigations. The DNA sequence studied has been previously used in homonuclear studies of DNA-bound water molecules and tritium NMR was expected to facilitate these investigations by eliminating the need to suppress the water resonance in tritium-detected ³H-¹H NOESY experiments. We observed the anticipated through-space interactions found in B-form DNA in the NOESY experiments and an unexpected antiphase cross-peak at the water frequency. T₁ measurements on the tritiated DNA molecule indicated that relaxation rates were also accelerated for tritium and protons. Tritium NMR spectra of the hammerhead RNA molecule indicated conformational dynamics in the conserved region of the molecule in the absence of Mg²⁺ and spermine, two components necessary for cleavage. The dynamics were also investigated by ¹⁵N-correlated ¹H spectroscopy and persisted after the addition of Mg²⁺ and spermine.     

Loss of Hydrogen from Carbon 5 of d-Glucose during Conversion of d-[5-H,6-C]Glucose to l-Ascorbic Acid in Pelargonium crispum (L.) L'Hér

Conversion of d-[5-³H,6-¹⁴C]glucose to l-ascorbic acid in detached apices of Pelargonium crispum (L.) L'Hér cv Prince Rupert (lemon geranium) was accompanied by complete loss of tritium in the product. Chemical degradation of d-glucose which was recovered from the labeled apices yielded d-glyceric acid (corresponding to carbons 4, 5, and 6 of glucose) with a ³H:¹⁴C ratio of 4 to be compared with 9, the ratio in d-[5-³H,6-¹⁴C]glucose initially. Conversion of d-[6-³H,6-¹⁴C]glucose in the same tissue was accompanied by retention of tritium in l-ascorbic acid with a ³H:¹⁴C ratio comparable to that of compounds from the hexose pool. Results indicate that during l-ascorbic acid biosynthesis from glucose in Pelargonium crispum hydrogen at carbon 5 undergoes exchange with the medium, suggesting an epimerization at this carbon atom.     

Binding of radioactively labeled saxitoxin to the squid giant axon

Summary The binding of saxitoxin, a specific inhibitor of the sodium conductance in excitable membranes, has been measured in giant axons from the squid,Loligo pealei. Binding was studied by labeling saxitoxin with tritium, using a solvent-exchange technique, and measuring the toxin uptake by liquid scintillation counting. Total toxin binding is the sum of a saturable, hyperbolic binding component, with a dissociation constant at 2–4°C of 4.3±1.7nm (meanse), and a linear, nonsaturable component. The density of saturable binding sites is 166±20.4 m^–2. From this density and published values of the maximum sodium conductance, the conductance per toxin site is estimated to be about 7 pS, assuming sequential activation and inactivation processes (F. Bezanilla & C.M. Armstrong, 1977,J. Gen. Physiol. 70: 549). This single site conductance value of 7 pS is in close agreement with estimates of the conductance of one open sodium channel from measurements of gating currents and of noise on squid giant axons, and is consistent with the hypothesis that one saxitoxin molecule binds to one sodium channel.     

Is the concentration of γ-aminobutyric acid in the nerve terminal regulated via product inhibition of glutamic acid decarboxylase?

Fractions of synaptosomes were used to study the regulation of -aminobutyric acid (GABA) synthesis. The isolated synaptosomes were superfused in media of various compositions. [³H]GABA and GABA released into the medium or remaining in the synaptosomes were analyzed by liquid scintillation and HPLC techniques. Different conditions, designed to increase the GABA efflux rate were used: the rate of superfusion was varied and the concentrations of K⁺ and Ca²⁺ were altered. Stimulation of GABA efflux was paralleled with an increased synthesis of GABA, since, in spite of the increased GABA efflux, a relatively constant intraterminal level was found. The findings suggest that the intraterminal concentration of GABA and thus also its synthesis is regulated via product inhibition. In addition, [³H]GABA, exogenous, and GABA, endogenous, responded to external stimulae (Ca²⁺, veretradine, various GABA concentrations and the glutaminase inhibitor diazo-nor-leucine) in a way which was compatible with them being localized in and/or released from different compartments.     

Incorporation of 3-deoxy-3-fluoro-D-glucose into glycogen and trehalose in fat body and flight muscle inLocusta migratoria

Flight muscle and fat body extracts fromLocusta migratoria were incubated with D-[U-¹⁴C]-glucose or D-[3-³H]-3-deoxy-3-fluoroglucose and the products were analyzed. In the case of the latter compound, radio-chromatographic analysis yielded glycogen and trehalose fractions that were shown by¹⁹F nuclear magnetic resonance to contain fluorine. Acid hydrolysis of these fractions liberated tritium labelled 3-deoxy-3-fluoro-D-glucose. In addition to the formation of fluoroglycogen and fluorotrehalose in these tissue extracts, there was an accumulation of tritium labelled fructose.     

Presynaptic Modulation of K+-Evoked [3H]Dopamine Release in Striatal and Frontal Cortical Synaptosomes of Normotensive and Spontaneous-Hypertensive Rats

Regional differences in presynaptic [³H]dopamine ([³H]DA) release and its modulation by D₂ DA-receptors between the frontal cortex and striatum obtained from Wystar-Kyoto (WKY) and spontaneous-hypertensive rats (SHR) have been evaluated using superfused synaptosomes. Synaptosomal tritium content was significantly lower in the frontal cortex than in the striatum in both SHR and WKY (45% and 48%, respectively), but no differences in tritium content were obtained between strains. However, the 15 mM K⁺-evoked [³H]DA overflow was lower in the SHR as compared to WKY rats in both brain regions (striatum 23%, frontal cortex 21). Concentration-response curves for quinpirole (1nM-10 M)-mediated inhibition of 15mM K⁺-evoked [³H]DA release showed no differences between SHR and WKY. These results suggest that SHR has less ability to release [³H]DA as compared to WKY rats, but SHR did not show differences in the autoregulation of such release in both the frontal cortex and striatum.     

Effect of ibogaine on serotonergic and dopaminergic interactions in striatum from mice and rats

The effect of ibogaine (Endabuse, NIH 10567) on serotonin uptake and release, and on serotonergic modulation of dopamine release, was measured in striatal tissue from rats and mice. Two hours after treatment in vivo with ibogaine (40 mg/kg i.p.), the uptake of labeled [³H]serotonin and [³H]dopamine uptake in striatal tissue was similar in the ibogaine-treated animal to that in the control. The 5HT_1B agonist CGS-12066A (10^–5 M) had no effect on stimulation-evoked tritium release from mouse or rat striatal tissue preloaded with [³H]serotonin; however, it elevated tritium efflux from striatal tissue preloaded with [³H]dopamine. This increase was not seen in mice treated with ibogaine 2 or 18 hours previously, or in rats treated 2 hours before. Dopamine autoreceptor responses were not affected by ibogaine pretreatment in either mouse or rat striatal tissue; sulpiride increased stimulation-evoked release of tritium from tissue preloaded with [³H]dopamine. The long-lasting effect of ibogaine on serotonergic functioning, in particular, its blocking of the 5HT_1B agonist-mediated increase in dopamine efflux, may have significance in the mediation of its anti-addictive properties.Special issue dedicated to Dr. Sidney Ochs.     

Net taurine transport and its inhibition by a taurine antagonist

P₂-fractions were isolated from rat brain, and used to study net taurine transport. The fractions were incubated in increasing concentrations of [³H]taurine and the intraterminal concentration measured by liquid scintillation and amino acid analysis. The membrane potential of the isolated fractions was estimated using⁸⁶Rb⁺ as a marker for intracellular K⁺. Taurine was synthesized in the P₂-fraction when incubated in taurine free medium. At external taurine concentrations below 370 M a significant amount of the endogenous taurine was released to the incubation medium. Net taurine uptake into the P₂-fraction was achieved at external taurine concentrations exceeding 370 M. The taurine antagonist 6-aminomethyl-3-methyl-4H, 1, 2, 4-benzothiadiazine-1, 1-dioxide (TAG) competitively inhibited taurine and [³H]taurine transport into the P₂-fraction. As the external concentration of taurine was increased, the accumulation of⁸⁶Rb⁺ into the P₂-fraction was facilitated. This indicated an increasing hyperpolarization of the neuronal membrane as taurine transport shifted from release towards uptake. TAG reduced the hyperpolarization that paralleled taurine accumulation, in a dose dependent manner. Our results indicate that relatively low transmembranal gradients of taurine may be maintained by an electrogenic taurine transporter having a large transport capacity. Such a transporter may well serve the needs of osmotic regulation, i.e. to transport large amounts of taurine in any direction across the neuronal membrane.     

Increased scintillation counting of 3H-amino acids bound to transfer RNA.

Approximately a threefold higher tritium scintillation counting efficiency was observed for the ³H-labeled amino acids covalently bound to tRNA, compared to the nonbound ³H-amino acids used as counting efficiency standards.     

Effect of progesterone on protein synthesis and secretion by cultured epithelial cells from guinea-pig endometrium

Summary The pattern of proteins synthesized and secreted in response to progesterone by guinea-pig endometrial epithelial cell cultured with estradiol-17 was investigated. Glandular epithelial cells were maintained in culture for 3 days on growth medium, then washed three times with a steroid-free medium. After this period, 2 × 10^-8 M estradiol-17 or 2 × 10^-8 M estradiol-17 plus 5 × 10^-7 M progesterone were added to the medium for 48 h. To study biochemical changes, the proteins were labeled by a 6 h pulse of ³⁵S-methionine. The proteins in medium and in cells were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. The addition of progesterone to estradiol-17 in the culture medium caused a change in the patterns of cellular and secreted proteins: one-dimensional gel electrophoresis showed that variation of 8 cellular proteins and 12 secreted proteins was caused by progesterone. Induction of individual proteins by progesterone treatment was observed by two-dimensional gel electrophoresis: one cellular protein (Mr 49000; pI 5.90) and one secreted protein (Mr 14300; pI 4.80) were specifically induced and might serve as markers of progesterone action.     

Die Tritiumabspaltung aus festem Adenin-2-T und Adenin-8-T unter der Einwirkung von60Co-γ-Strahlung

Zusammenfassung Im Anschluß an eine frühere Arbeit (Merwitz, 1971), welche die Dosiseffektkurven für die strahleninduzierte Tritiumabspaltung aus spezifisch markierten Thyminen beschreibt, wurde die Tritiumabspaltung aus Adenin-2-T und Adenin-8-T untersucht. Die graphisch ermittelten Schwellenwertdosen für die Tritiumabspaltung liegen bei 3·10⁵ rad für Adenin-8-T und bei 2·10⁷ rad für Adenin-2-T. Der Vergleich der Dosiseffektkurven zeigt, daß die Tritiumabspaltung aus den spezifisch markierten Adeninen im Gegensatz zu den Thyminen erst bei hohen Dosen einsetzt. Die unterschiedliche Strahlenresistenz von Thymin und Adenin wird diskutiert.

---

The cleavage of tritium from solid adenine-2-t and adenine-8-t induced by⁶⁰co--radiation

Summary In continuation to our previous studies (Merwitz, 1971) specifying the dose effect curves for the radiation-induced tritium cleavage from specifically labeled thymines, tritium cleavage from adenine-2-T and adenine-8-T has been investigated. The graphically determined threshold doses for the cleavage of tritium are in the range of 3·10⁵ rad for adenine-8-T and in the range of 2·10⁷ rad for adenine-2-T. Comparison of the dose-effect curves proves that, in contrast to the thymines, tritium cleavage from specifically labeled adenines requires high doses. The divergent radioresistances of thymine and adenine are being discussed.

---

---

Ich danke Fräulein Chr. Kunze für die technische Assistenz und Herrn H. Scgnudt für die Durchführung der Bestrahlungsversuche.     

Incorporation of Tritium from Thymidine-methyl-H3 into DNA,RNA, Lipids and Protein in Logarithmic and Stationary Phase Tetrahymena pyriformis,Strain W*

SYNOPSIS. Numerous reports may be found in the literature on cytoplasmic and non-DNA utilization of tritium from H³-thymidine. Such reports underscore the need to clarify the metabolic fate of H³-thymidine. This investigation outlines the fate of thymidinemethyl-H³ (TMH³) in logarithmic phase and stationary phase Tetrahymena pyriformis, strain W. Isotope identification by liquid scintillation spectrometry in chemically derived fractions of log phase cultures grown thruout the initial 48 hours of population growth with TMH³ revealed the majority of the radioactivity (90% of intracellular recovery) to be in the DNA fraction. The remainder of the intracellular label was recovered in the acid soluble fraction, lipid fraction, and a small amount in the RNA and cell residue. On chromatographs, tritium appeared only in the thymine moiety of the nucleic acid derivatives. Hence in dividing cells, thymidine-methyl-H³ is “essentially” specific for DNA at the dosage used although some incorporation into other compounds was detected. Fractionation of the lipid extract from the above experiment on a florisil column localized most of the label to the triglyceride and phospholipid fractions with some recovery in the cholesterol-esters. Similar scintillation counting of the various fractions of early stationary phase cells incubated for the last 48 hours of culture with TMH³ revealed limited tritium distribution in all fractions.     

Measurement of diameters of estuarine bacteria and particulates in natural water samples by use of a submicron particle analyzer

Desulfovibrio vulgaris strain Madison outcompetedMethanobacterium strain ivanov for hydrogen when sulfate was in excess because of higher cell yield and growth rate and a greater affinity for hydrogen as a consequence of a lower Km and higher Vmax for in vivo hydrogenase activity.Desulfovibrio vulgaris displayed a growth yield of 1.1 g/mol H₂, a Km for tritium exchange of 4 M, and a specific in vivo hydrogenase activity of 2.17 DPM³H₂O×10³/g cell protein/h; whereasMethanobacterium strain ivanov had a yield of 0.6 g/mol H₂, a Km for tritium exchange of 14 M, and a specific in vivo hydrogenase activity of 0.38 DPM³H₂O×10³/g cell protein/h. Under these physiological conditions, the Gibbs free-energy change associated with methanogenesis and sulfidogenesis from H₂ was calculated to be-47.4 kJ/mol and-62.9 kJ/mol, respectively. When sulfidogenesis was limited by sulfate concentration, the methanogen was able to successfully compete with the sulfidogen for hydrogen. Competition between methanogens and sulfidogens for hydrogen is explained in terms of thermodynamic, kinetic, and other important considerations not discussed in the previous literature.     

On the mechanism of 11-hydroxylation of lauric acid by rat liver microsomes

11-²H₂-Lauric acid with a randomly distributed tritium label was synthesized and the rate of 11-hydroxylation of this compound by rat liver microsomes was compared with the rate of 11-hydroxylation of 1-¹⁴C-lauric acid. The 11-²H₂-label was found to decrease the V_max of the reaction with a factor of about 3.6 and the K_m with a factor of about 1.5. The results support the previous suggestion that the rate-limiting step in microsomal 11-hydroxylation of lauric acid is the rate of cleavage of the CH bond.     

Release of [3H]noradrenaline by α1-adrenoceptor agonists

Mouse isolated vas deferens preincubated with [³-H]noradrenaline was superfused and the effect of ₁-adrenoceptor agonists was studied on the release of total radioactivity ([³H]noradrenaline +³H-metabolites) and [³H]noradrenaline. Reverse phase high pressure liquid chromatography (HPLC) combined with scintillation spectrometry was used to separate [³H]noradrenaline from its metabolites. Among the ₁-adrenoceptor agonists (1-phenylephrine, ST-587(2-(2-chloro-5-trifluoromethyl phenylimino)-imidazole), (–)-amidephrine, methoxamine, cirazoline and l-noradrenaline) studied l-phenylephrine, ST-587 and l-noradrenaline were capable of releasing³H-noradrenaline. The effect of noradrenaline was stereospecific. As determined by HPLC combined with scintillation spectrometry the release of total radioactivity in response to l-noradrenaline is mainly due to [³H]noradrenaline. It is suggested that l-noradrenaline, l-phenylephrine, and ST-587 in addition to their direct effect on different receptors they also have indirect action through the release of noradrenaline which might be partly involved in the pharmacological responses. The mechanisms whereby l-noradrenaline and l-phenylephrine release noradrenaline would appear to involve a saturable Ca-independent and a cocaine and temperature sensitive process. On the basis of our findings among the ₁-adrenoceptor agonist studied (–)-amidephrine, methoxamine and cirazoline is a better choice than l-phenylephrine or ST-587 for selective stimulation of postjunctional ₁-adrenoceptor, they do not release noradrenaline.     

Δ5 Desaturase activity in rat kidney microsomes

Rat kidney microsomal fraction is able to catalyze the enzymatic desaturation of eicosatrienoic acid (20:3n-6) to arachidonic acid (20:4n-6) by the 5 desaturase pathway, in the presence of reduced nicotinamide adenine dinucleotide (NADH), adenosinetriphosphate (ATP) and coenzyme A (CoA). The substrate of the reaction [1-¹⁴C]eicosa-8,11,14trienoic acid (20:3n-6), was separated from the product [1-¹⁴C]eicosa-5,8,11,14-tetraenoic acid (20:4n-6) by reverse phase high-pressure liquid chromatography (RP-HPLC). These fatty acids were individually collected by monitoring the eluent at 205 nm and their radioactivity was measured by liquid scintillation counting. The 5 desaturase activity in kidney microsomes increased linearly with the substrate concentration up to 20 M. Enzymatic activity was sensitive to pH with the maximum at 7.0 and was proportional with incubation time up to 10 min. The apparent Km and Vmax of 5 desaturase were 56 M and 60 pmoles·min^–1·mg^–1 microsomal protein, respectively. Neither the cytosolic renal fraction nor the cytosolic liver fraction enhanced the 5 desaturase activity. Contrary to a report but in accordance to others, the present results suggest that rat kidneys can synthesize arachidonic acid at least to satisfy partially their needs for eicosanoid production.     

Micropropagation of sour cherry (Prunus cerasus L.) cv. Šumadinka

Sour cherry cv. umadinka (Prunus cerasus L.) is the leading Yugoslav cultivar for production orchards. A method of micropropagation has been developed for the purpose of growing umadinka on its own roots and for rapid multiplication.Aseptic cultures were initiated from shoot explants 1–2 mm long on Murashige & Skoog medium with (in mgl^-1) 6-benzylaminopurine (BAP): 1, indole-3-yl butyric acid (IBA): 1 and gibberellic acid (GA₃): 0–1.The best medium for proliferation was MS with (in mgl^-1): BAP 0.5, IBA 0.1, GA₃ 0.1, but media with (in mgl^-1): BAP 0.5, NAA 0.1, GA₃ 0.1 and BAP 1, NAA 0.1 and GA₃ 0.1 were also shown to be good. A higher degree of proliferation obtained with some media did not necessarily result in a better quality of plantlets produced.For rooting the best combination of culture medium was achieved with pretreatment 10 days in MS 1/2 with 1 mgl^-1 IBA, followed by transfer to a hormone-free medium after 5–10 days, resulting in 88% success.The rooted plants were planted in containers and acclimatized under mist, with over 90% of plants surviving transplantation.