Himasthla quissetensis: uptake and utilization of glucose by rediae as determined by autoradiography and respirometry

Daughter rediae of Himasthla quissetensis removed from the digestive gland of Nassarius obsoletus were placed in sterilized seawater fortified with antibiotics. When [³H]-glucose was added to this medium and autoradiographs were made after 3, 9, and 24 hr of exposure, labeling was observed associated with the redial walls and developing germ balls and cercariae within the brood chambers. Respirometric determinations on starved rediae suspended in the seawater medium with and without glucose revealed the rate of oxygen utilization by rediae exposed to exogenous glucose is significantly elevated. These results are interpreted to mean that the daughter rediae of H. quissetensis can take up and utilize glucose.     

Determination of the accessible surface of globular proteins by means of tritium planigraphy

Results are presented for proteins with known three-dimensional structure (lysozyme, myoglobin, ribonuclease), which show that the probability of label incorporation upon bombardment by hot tritium atoms may be quantitatively linked with the surface area of the protein accessible to water molecules. Possible deviations from simple linear dependency caused by particular mechanisms of label introduction are discussed. The data obtained in experiments with model systems were used to determine the accessible surface area of human serum albumin, for which structural data is not sufficiently accurate to allow estimation of accessible surface area. Experimental data correlate reasonably well with estimations based on conventional concepts of the relationship between accessible surface area and molecular weight for globular proteins. Correspondence to: A. V. Volynskaya     

Characterization of [3H]Met-Enkephalin-Arg6-Phe7 Binding to Opioid Receptors in Frog Brain Membrane Preparations

Abstract: A tritiated heptapeptide, [³H]Tyr-Gly-Gly-Phe-Met-Arg-Phe ([³H]Met-enkephalin-Arg⁶-Phe⁷), with high specific radioactivity has been synthesized in order to characterize its opioid binding activity to frog brain membrane fractions. The apparent K _D value of the radioligand calculated from homologous displacement experiments was 3.4 n M , and the maximal number of specific binding sites was 630 fmol/mg of protein. The K _D determined from equilibrium saturation binding studies was found to be 3.6 n M . However, the Hill coefficient was far below unity ( n _H = 0.43), which suggests the presence of a second, lower affinity binding site. The presence of this binding component is strengthened by the displacement experiments performed with levorphanol and some other ligands. It is assumed that the lower affinity site has no opiate character. The rank order of potency of the applied ligands in competing reversibly with [³H]Met-enkephalin-Arg⁶-Phe⁷ binding reflects a κ₂- and/or δ-subtype specificity of the heptapeptide. Binding to a κ₁ and/or μ site of opioid receptors is excluded, but the existence of a novel endogenous opiate receptor subtype for Met-enkephalin-Arg⁶-Phe⁷ in frogs cannot be ruled out. The [³H]Met-enkephalin-Arg⁶-Phe⁷ binding was inhibited by both sodium ions and GppNHp, which suggests the opioid agonist character of the heptapeptide.     

[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1

Summary The synthesis of [2-³H]ATP with specific activity high enough to use for ³H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single ³H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes.The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg²⁺, S1 converts [2-³H]ATP to [2-³H]ADP and the complex S1.Mg[2-³H]ADP has ADP bound in the active site. At 0°C, 1D ³H NMR spectra of S1.Mg[2-³H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-³H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-³H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (V_i) to produce S1.Mg[2-³H]ADP.V_i shifts both peaks slightly more upfield without chaning their widths or relative areas.     

Tritium NMR studies of the human carbonic anhydrase I–benzenesulfonamide complex

Tritium NMR spectroscopy has been used to examine the complexformed by [4-³H]benzenesulfon-amide and human carbonicanhydrase I. The results show that in solution the inhibitor forms a 1:1complex with the enzyme. A 100-spin computational model of the system,constructed with reference to crystallographic results, was used tointerpret tritium relaxation behavior and ³H{¹H}NOEs. The analysis shows that the rate of dissociation of theenzyme–sulfonamide complex is 0.35 s^–1 and thatthe aromatic ring of the inhibitor undergoes rapid rotation while complexed.     

Compartmentation of cytosolic dehydrogenases studied by transfer of tritium from labelled substrates into lactate in rat hepatocytes

This paper describes the transfer of tritium from [2-³H]xylitol or (1R)-[1-³H]ethanol into lactate in cells from fed rats either untreated or triiodothyronine-treated. The labelling pattern of lactate during the metabolism of [2-³H]xylitol or (1R)-[1-³H]ethanol follows the equation $\text{L = K(1?e}{}^{\mathrm{<mtext>?</mtext><mtext>t</mtext><mtext>\tau </mtext>}}\text{)}$ (μmol tritium/μmol lactate). The yield in lactate together with the minimum value of the total flux of reducing equivalents are used to estimate the specific radioactivity of NADH. We have calculated the lactate dehydrogenase-catalysed oxidation rate of NADH from the experimental values of lactate labelling and the specific radioactivity of NADH. We found the calculated flux of reducing equivalents from NADH to pyruvate to be of the same order of magnitude whether labelled ethanol or labelled xylitol was metabolized. We found the flux to be only a few percent of the maximal activity of lactate dehydrogenase. The results obtained suggest that the cytoplasm can be regarded as one compartment, containing a single pool of NAD(H).     

Synthesis of R and S tritiated reduced beta-nicotinamide adenine dinucleotide 2' phosphate

Nicotinamides are ubiquitous cofactors used by many biological systems as redox agents. Stereospecifically labeled cofactors are useful in many studies of nicotinamide-dependent enzymes. Enzyme-directed synthesis of these cofactors is rather common but their stability imposes significant challenges on yield, purity, and preservation. This paper presents the stereospecific synthesis of reduced R- and S-[4-3H] beta-nicotinamide adenine dinucleotide 2' phosphate (NADPH). The method of Valera et al. [Biochem. Biophys. Res. Commun. 148 (1987) 515] was modified to a synthetic procedure that produces both isotopic diastereomers within 2h with an improved yield of 75-90% after purification and lyophilization. In the synthesis, [4-3H]NADP+ was generated as an intermediate (which can be isolated if desired). The specific radioactivities reported here are 2.7 and 1.1 Ci/mmol for the S and R diastereomers, respectively. Specific radioactivities ranging from carrier-free to trace labeling can be achieved with a minor change to the procedure.     

Ribonucleic acid of Echinococcus granulosus protoscolices

Echinococcus granulosus protoscolices RNA has been characterized in terms of its nucleotide composition, its behavior in sucrose density gradients and methylated albuminkieselguhr columns, its template activity, its biosynthesis kinetics, and the effect of actinomycin D.     

黄鳝二价体上随机引物原位DNA合成的探讨

采用随机引物,在黄鳝二价体上建立了氚标记引物原位DNA合成技术,并详细探索了该技术的实验条件。其结果显示黄鳝二价体上DNA延伸效果十分明显,银颗粒基本上是非随机性的相间排列,而且主要特征是基本稳定的,据此可以进行染色体识别。此外,还讨论了建立该技术的重要意义。 Abstract:We have developed a technique of random-primed tritium labeled DNA extension on the pachytene bivalents of rice-field eels.The bivalents were not homogeneously labeled with this technique and the extension signals were very apparent and relatively stable,and these can be applied to identify the individual bivalent.The significance to develop this technique has also been discussed in detail.     

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.