Oligonucleotide conformations. (5) NMR and relaxation studies on GpU and UpG at neutral pH.

The average conformation of GpU and UpG in neutral aqueous solutions has been investigated by proton chemical shifts and coupling measurements as well as T1 relaxation time experiments. The proportion of the N and S pseudorotational conformers of the ribose ring has been derived from the vicinal coupling constants. The relaxation data provide information about the syn--anti equilibrium of the orientation of the base about the glycosidic bond. This orientation is predominantly syn for the Guo base in both dinucleoside phosphates, that of Urd is anti in the case of GpU and shows an almost equivalent syn and anti character for UpG.     

Kinetics of nucleoside transport in human erythrocytes. Alterations during blood preservation

The transmembrane equilibration of radiolabeled uridine was measured by rapid kinetic techniques in human erythrocytes from freshly drawn blood and in the same cells during conventional storage of the blood as well as in cells from outdated blood. Our results confirm earlier reports that the maximum velocity of uridine equilibrium exchange (Vee) at 25 degrees C is about 30% lower in outdated than fresh red cells, whereas the opposite is the case for the Michaelis-Menten constant for equilibrium exchange (Kee), and that maximum zero-trans efflux (Vzt21) is about 4-times greater than maximum zero-trans influx (Vzt12) in outdated cells (directional asymmetry), whereas they are about the same in fresh red cells. At 25 degrees C, the nucleoside-loaded carrier of fresh cells moves on the average 6-times more rapidly than the empty carrier, whereas the differential mobility of loaded and empty carrier from outdated cells is about 15-fold. Our results also show that greater efflux than influx in outdated cells is not due to a general leakiness of outdated cells, that the differences in kinetic properties of the transporter developed during the first two weeks of blood storage and that the differences are greatly amplified when transport is measured at 5 degrees C rather than 25 degrees C. At 5 degrees C, the loaded carrier from outdated red cells moves about 325-times more rapidly than the empty carrier and maximum zero-trans efflux exceeds maximum zero-trans influx about 14-times, whereas the transport of fresh cells exhibits directional symmetry just as at 25 degrees C. The changes in kinetic properties of transport induced by temperature and storage are probably related to structural alterations in the plasma membrane and suggest that the operation of carrier is subject to modification by the membrane environment. Other results show that the kinetics of the sugar transport of human red cells is not affected in the same manner by blood storage as those of the nucleoside transporter.     

On the mechanism of the acid-catalyzed hydrolysis of uridine to uracil. Evidence for 6-hydroxy-5,6-dihydrouridine intermediates

In acidic media, the 5,6-double bond of uridine is rapidly hydrated to give a small amount of 6-hydroxy-5,6-dihydrouridine (Urd-H2O), the mechanism of which is known from studies of the acid-catalyzed dehydration of Urd-H2O (Prior, J. J., Maley, J., and Santi, D. V. (1984) J. Biol. Chem. 258, 2422-2428). In addition to dehydration, Urd-H2O also undergoes direct hydrolysis of the N-glycosidic bond in acidic solution. The kinetics of the above reaction demonstrates that Urd-H2O, or an intermediate in the pathway leading from Urd to Urd-H2O, is kinetically competent to account for the hydrolysis of the N-glycosidic bond of Urd. The hydrolysis of (1'-2H)Urd proceeds with an alpha-secondary deuterium isotope effect of kH/kD of 1.11 at 25 degrees C. This isotope effect is sufficiently large to implicate carbonium ion character at the 1'-carbon during hydrolysis but, since it is not the maximal value expected, suggests that N-glycoside cleavage is rate-determining with a transition state intermediate between reactant and products. Importantly, the hydrolysis of [6-3H]Urd proceeds with a substantial inverse secondary isotope effect of kT/kH = 1.15 at 25 degrees C which indicates some degree of sp2 to sp3 rehybridization of C-6 of the pyrimidine moiety during hydrolysis. From the data available, it appears that an important pathway in the hydrolysis of the N-glycoside bond of Urd involves either spontaneous cleavage of Urd which is protonated at the 5-carbon or a protonated species of Urd-H2O. The studies described here, together with the known susceptibility of the 6-position of pyrimidine heterocycles toward nucleophiles, permits the proposal of chemically reasonable mechanisms for enzyme-catalyzed cleavage of N-glycosidic bonds of pyrimidines.     

Copper(II) complexes with uridine,uridine 5'-monophosphate,spermidine, or spermine in aqueous solution

Molecular complexes of the types (Urd)H(x)(PA) and (UMP)H(x)(PA) are formed in the uridine (Urd) or uridine 5'-monophosphate (UMP) plus spermidine or spermine systems, as shown by the results of equilibrium and spectral studies. Overall stability constants of the adducts and equilibrium constants of their formation have been determined. An increase in the efficiency of the reaction between the bioligands is observed with increasing length of the polyamine. The pH range of adduct formation is found to coincide with that in which the polyamine is protonated while uridine or its monophosphate is deprotonated. The -NH(x)(+) groups from PA and the N(3) atom of the purine base as well as phosphate groups from the nucleotides have been identified as the significant centres of non-covalent interactions. Compared to cytidine, the pH range of Urd adduct formation is shifted significantly higher due to differences in the protonation constants of the endocyclic N(3) donor atoms of particular nucleosides. Overall stability constants of the Cu(II) complexes with uridine and uridine 5'-monophosphate in ternary systems with spermidine or spermine have been determined. It has been found from spectral data that in the Cu(II) ternary complexes with nucleosides and polyamines the reaction of metallation involves mainly N(3) atoms from the pyrimidine bases, as well as the amine groups of PA. This unexpected type of interaction has been evidenced in the coordination mode of the complexes forming in the Cu-UMP systems including spermidine or spermine. Results of spectral and equilibrium studies indicate that the phosphate groups taking part in metallation are at the same time involved in non-covalent interaction with the protonated polyamine.     

Uridine phosphorylase from Schistosoma mansoni

Uridine phosphorylase is the only pyrimidine nucleoside cleaving activity that can be detected in extracts of Schistosoma mansoni. The enzyme is distinct from the two purine nucleoside phosphorylases contained in this parasite. Although Urd is the preferred substrate, uridine phosphorylase can also catalyze the reversible phosphorolysis of dUrd and dThd, but not Cyd, dCyd, or orotidine. The enzyme was purified 170-fold to a specific activity of 2.76 nmol/min/mg of protein with a 16% yield. It has a Mr of 56,000 as determined by molecular sieving on Sephadex G-100. The mechanism of uridine phosphorylase is sequential. When Urd was the substrate, the KUrd = 13 microM and the KPi = 533 +/- 78 microM. When dThd was used as a substrate, the KdThd = 54 microM and the KPi = 762 +/- 297 microM. The Vmax with dThd was 53 +/- 9.8% that of Urd. dThd was a competitive inhibitor when Urd was used as a substrate. The enzyme showed substrate inhibition by Urd, dThd (greater than 0.125 mM) and phosphate (greater than 10 mM). 5-(Benzyloxybenzyloxybenzyl)acyclouridine was identified as a potent and specific inhibitor of parasite (Ki = 0.98 microM) but not host uridine phosphorylase. Structure-activity relationship studies suggest that uridine phosphorylase from S. mansoni has a hydrophobic pocket adjacent to the 5-position of the pyrimidine ring and indicate differences between the binding sites of the mammalian and parasite enzymes. These differences may be useful in designing specific inhibitors for schistosomal uridine phosphorylase which will interfere selectively with nucleic acids synthesis in this parasite.     

Temperature- and time-dependent inactivation of pyrroline-5-carboxylate synthase: suggestive evidence for an allosteric regulation of the enzyme

When pyrroline-5-carboxylate (PC) synthase activity in the membrane of mitochondria of rat small intestine mucosa was assayed in the presence of 0.5 mM ornithine, the time course of inactivation showed that the activity disappeared entirely by about 8 min at 30 degrees C, whereas there was no decrease in the activity at 15 degrees C. A prior incubation of the enzyme with ornithine at 30 or 37 degrees C in the presence of 50% sorbitol as a thermal stabilizer resulted in a marked loss of the activity, while that at 0 or 15 degrees C did not lose any. This suggests that PC synthase is inactivated by ornithine regardless of the presence of substrates. The inactivation at 30 degrees C proceeded gradually for about 7 h, until an equilibrium was attained. Extensive dialysis allowed the inactivated enzyme to regain about 60% of the original activity. These results suggest that the inactivation is reversible. The concentration of ornithine and the percentage of inactivation at equilibrium was correlated by the Hill equation and displayed a sigmoidicity with n = 1.47 and [S]50 = 0.036 mM. In the presence of sorbitol, the inactivation was prevented by 0.2 mM ATP or ADP. The role of the nucleotides in PC synthase regulation is discussed.     

Specific type of interactions in the quaternary system of Cu(II), adenosine 5'-triphosphate, 1,11-diamino-4,8-diazaundecane and uridine

Complexation reactions in the quaternary system Cu/ATP/3,3,3-tet/Urd have been studied. The stability constants of the complexes of the Cu(ATP)(3,3,3-tet)H(x)(Urd) type have been determined by computer analysis of the potentiometric titration. On the basis of the results of spectroscopic as well as equilibrium studies, the mode of interactions has been proposed. Metal ions coordinate phosphate groups of ATP and nitrogen atoms of polyamine. It has been established that in the conditions of the complex Cu(ATP)(3,3,3-tet) formation, uridine introduced into the Cu(II)/ATP/3,3,3-tet ternary system is involved in hydrogen bonding with the endocyclic nitrogen atoms N(1) and N(7) of the ATP purine ring and formation of the adduct Cu(ATP)(3,3,3-tet)H(Urd) is observed. Introduction of metal ions into the system changes substantially the mode of interactions between complementary base pairs relative to that proposed in the Watson and Crick model.     

Binding of NAD+ to pertussis toxin.

The equilibrium dissociation constant of NAD+ and pertussis toxin was determined by equilibrium dialysis and by the quenching of the protein's intrinsic fluorescence on titration with NAD+. A binding constant, Kd, of 24 +/- 2 microM at 30 degrees C was obtained from equilibrium dialysis, consistent with the previously determined value for the Michaelis constant, Km, of 30 +/- 5 microM for NAD+ (when the toxin is catalysing the ADP-ribosylation of water and of dithiothreitol). The intrinsic fluorescence of pertussis toxin was quenched by up to 60% on titration with NAD+, and after correction for dilution and inner filter effects, a Kd value of 27 microM at 30 degrees C was obtained, agreeing well with that found by equilibrium dialysis. The binding constants were measured at a number of temperatures using both techniques, and from this the enthalpy of binding of NAD+ to toxin was determined to be 30 kJ.mol-1, a typical value for a protein-ligand interaction. There is one binding site for NAD+ per toxin molecule.     

Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases

An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.     

Effect of temperature on kinetics and differential mobility of empty and loaded nucleoside transporter of human erythrocytes

The transmembrane equilibration of [3H]uridine was measured in human erythrocytes as a function of temperature using rapid kinetic techniques. Arrhenius plots of the maximum velocity of equilibrium exchange were continuous between 5 and 30 degrees C (Ea = 17-20 kcal/mol), but the increase in velocity with increase in temperature leveled off above 30 degrees C. This leveling off did not reflect heat inactivation of the carrier since transport activity was stable for 3 h at 37 degrees C. Transmembrane equilibration of uridine in equilibrium exchange and zero-trans modes at 5, 15, 25, and 35 degrees C conformed to appropriate integrated rate equations derived for the simple transporter. The nucleoside transporter exhibited directional symmetry, but the loaded carrier moved on the average 5 times more rapidly than the empty carrier at 15, 25, and 35 degrees C, but 25-40 times faster at 5 degrees C. This marked shift in differential mobility of loaded and empty carrier between 15 and 5 degrees C was entirely attributable to an impairment of mobility of empty carrier. The Michaelis-Menten constant for equilibrium exchange increased about 3-fold with increase in temperature between 5 and 35 degrees C. The van't Hoff plot of the values was approximately linear and yielded an estimate of the enthalpy of carrier:substrate dissociation of 7.8 kcal/mol.     

Quenching of intrinsic tryptophan fluorescence in membranes of rat pituitary cells.

The GH4C1 strain of hormone-producing rat pituitary cells has specific receptors for the tripeptide thyrotropin-releasing hormone (TRH). Membranes prepared from GH4C1 cells show intrinsic tryptophan fluorescence which was quenched by low concentrations (10--100 nM) of TRH and Ntau-methyl TRH but not by biologically inactive analogs of TRH. Membranes from GH4C1 cells were subjected to thermal denaturation. A conformational transition was noted above 40 degrees C and an irreversible denaturation was observed at 52 degrees C. TRH-induced quenching of intrinsic fluorescence was lost completely in membranes previously incubated for 10 min at 30 degrees C while loss of [3H]-TRH binding was only about 20% at this temperature. Collisional quenching by iodide revealed that about 38% of the tryptophanyl residues in GH4C1 membranes were exposed to solvent. Quenching by TRH occurred with a shift in wavelength maximum from 336 to 342 nm suggesting that few of the tryptophanyl residues quenched by the tripeptide are totally exposed. Membranes prepared from cells preincubated with 20 nM TRH for 48 h, in which TRH receptors were decreased to 30% of control values, showed no quenching of tryptophan fluorescence in response to freshly added TRH. We conclude that the TRH-receptor interaction in GH4C1 cells is associated with a change in membrane conformation that can be measured by differential spectrofluorometry of intrinsic tryptophan fluorescence.     

Partial purification and some properties of a phospholipase C from Pseudomonas sp. strain KS3.2

An extracellular phospholipase C was partially purified from Pseudomonas sp. strain KS3.2. The enzyme was composed of an approximately 18-kDa peptide. Maximal enzyme activity was found at pH 7.2 and 50 degrees C. The enzyme retained activity between pH 8 and 9, and 50% activity at about 52 degrees C for 30 min. The enzyme sample showed the highest activity on phosphatidylcholine and low activity toward other phospholipids.     

Influence of the sporulation temperature upon the heat resistance of Bacillus subtilis.

The influence of different sporulation temperatures (30, 37, 44 and 52 degrees C) upon heat resistance of Bacillus subtilis was investigated. Heat resistance was greater after higher sporulation temperatures. Relation of heat resistance and temperature of sporulation was not linear over all the range of temperatures tested. Heat resistance increased about tenfold in the range of 30-44 degrees C. Sporulation at 52 degrees C did not show any further increase in heat resistance. This effect was constant over all the range of heating temperatures tested (100-120 degrees C). z value remained constant (z = 9 degrees C). Greater heat resistances at higher temperatures of sporulation were not due to selection of more heat resistant cells by a higher sporulation temperature. Spores obtained from cells incubated at 32 or 52 degrees C always possessed heat resistances that corresponded to the sporulation temperature regardless of the incubation temperature of their vegetative cells.     

Adsorption kinetics of azinphosmethyl from aqueous solution onto pyrolyzed Horseshoe sea crab shell from the Atlantic Ocean

The adsorption behavior of azinphosmethyl on pyrolyzed Horseshoe Crab (Limulus polyphemus) outer shell, as a residue, from the Atlantic Ocean, collected along the Maine coast, USA, has been studied with regards to its kinetic and equilibrium conditions, taking into account adsorbate concentrations of 2 x 10(-3), 4 x 10(-3), 6 x 10(-3), and 8 x 10(-3), as well as temperatures of 30 degrees C, 40 degrees C, 50 degrees C, and 60 degrees C. The yield of adsorption of azinphosmethyl from aqueous solution ranged from 56.1% to 61% with temperature increasing. Kinetic studies showed that adsorption rate decreased as the initial azinphosmethyl concentration increased. It was found, that the adsorption reaction obeyed first-order kinetics. The overall rate constants were estimated for different temperatures. The activation energy for adsorption was about 1.52 kJmol(-1), which implies that azinphosmethyl mainly adsorbed physically onto Horseshoe Crab outer shell. Langmuir and Freundlich isotherms were applied to the experimental data and isotherm constants were calculated. The thermodynamic parameters DeltaG0, DeltaH0 and DeltaS0 for the adsorption reaction were evaluated based on equilibrium data and in connection with this result the thermodynamic aspects of adsorption reaction were discussed. The adsorption was found to be endothermic in nature. The adsorbent used in this study proved highly efficient for the removal of azinphosmethyl.     

Intracellular gradients of ion activities in the epithelial cells of theNecturus gallbladder recorded with ion-selective microelectrodes

In Necturus gallbladder epithelial cells the intracellular electrical potential, as recorded with microelectrodes, varied from -28 mV in the mucosal end to about -50 mV in the serosal end of the transporting cell. The Na+ activity varied concurrently from about 39 mM to between 8 and 19 mM. Thus, within the cell both the recorded electrical and chemical gradients caused Na+ to move towards the serosal end. Serosal addition of ouabain (5 X 10(-4) M) caused the intracellular Na+ activity to attain electrochemical equilibrium within 30 min. However, the intracellular electrical potential gradient was only slowly affected. In cells from animals stored at 5 degrees C, the Cl- activity varied from about 55 mM in the mucosal end to 28 mM in the serosal end, and the K+ activity from 50 mM to between 95 and 131 mM. Both ions were close to electrochemical equilibrium within the cytoplasm but were too concentrated to be in equilibrium with the mucosal solution. Bubbling CO2 through the mucosal solution caused the intracellular gradients to vanish. When Na+ in the bathing solutions was exchanged for K+, the intracellular electrical potential became roughly constant at about -5 mV. The Cl- activity became constant in 65 mM, and the K+ activity became constant at 109 mM, both close to equilibrium with the mucosal solution. The Na+ activity was reduced to about 1 mM. The ratio of cytoplasmic resistivities between cells bathed in K+-rich saline to cells bathed in Na+-rich saline was measured by means of triple-barreled electrodes and compared to the same ratio as assessed from the activity measurements. The two values were equal only if one assumes the mobility of Na+ inside the cell to be less than 1/10 of the mobility of K+ or Cl-. The same conclusion was reached by comparing the intracellular Na+ flux calculated from the gradient of electrochemical potential to that flux assess from the net solute absorption. Animals kept at 15 degrees C had lower intracellular Na+ activities, higher Cl- and K+ activities, and higher rates of absorption than animals stored at 5 degrees C. Finally, the degree to which the intracellularly recorded electrical and chemical potentials could reflect an electrode artefact is discussed.     

Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures

Cells of Salmonella typhimurium LT2 were dehydrated on hydrophobic membranes (Millipore FGLP2500) placed in a controlled atmosphere chamber held at 57% equilibrium relative humidity (ERH) and 37 degrees C. Dehydration for 48 h under the above conditions increased the heat resistance of Salm. typhimurium LT2 when measured as the surviving fraction after a heat challenge of 135 degrees C for 30 min. Results also showed that little or no death occurred during heat challenges of 1 h at temperatures of up to 100 degrees C. The survival of Salm. typhimurium LT2 was measured as the ability to form colonies on solid media tryptone soy broth plus 1.2% agar (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of 0.2% into the recovery medium, did not enhance the recovery of heated Salm. typhimurium LT2. Dehydrated cells of S. typhimurium LT2 showed a triphasic death curve. Increasing the period of dehydration from 48 h to 34 d, reduced initial numbers due to die off but did not alter the shape of the subsequent survival curve.     

Refolding rate of stability-enhanced cytochrome c is independent of thermodynamic driving force.

N52I iso-2 cytochrome c is a variant of yeast iso-2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49-56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a "global suppressor" of destabilizing mutations. The equilibrium m-value for denaturant-induced unfolding of N52I iso-2 increases by 30%, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso-2 have been measured by stopped-flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild-type protein, iso-2 cytochrome c. The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso-2 and N52I iso-2, despite the greater thermodynamic stability of N52I iso-2. Thus, there is no linear free-energy relationship between mutation-induced changes in stability and observable refolding rates. However, for N52I iso-2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso-2. The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state. Two aspects of the results are inconsistent with a two-state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two-state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes ("burst phase") are observed for both N52I iso-2 and iso-2 refolding. The "burst phase" amplitude is larger for N52I iso-2 than for iso-2, suggesting that the intermediates formed during the "burst phase" are stabilized by the N52I substitution.     

茄子光系统Ⅱ的热胁迫特性

以耐热性较弱的黑贝一号圆茄和耐热性较强的黑贝二号圆茄为试材,热胁迫处理后采用植物效率仪PEA进行快速叶绿素荧光诱导曲线及其参数测定.结果表明：当温度高于40 ℃,PSⅡ结构受热胁迫影响较为敏感,表现为初始荧光F_o缓慢上升;PSⅡ原初光化学效率F_v/F_m和ΔF/F_m′大幅度下降,且黑贝二号F_v/F_m的半衰时间T₅₀和ΔF/F_m′的半衰温度t₅₀分别大于黑贝一号.较高的热胁迫剂量（48℃处理5 min或44℃处理20～30min）下,快速荧光诱导动力学曲线呈现OKJIP型,在700μs处出现与放氧复合体失活有关的K相.黑贝一号在44 ℃下处理20 min才有K相出现,黑贝二号则晚10 min出现.与35℃相比,在48℃,特别是在52℃的较高剂量热胁迫下,Strasser能量流动模型参数中的DI_o/RC有大幅度地增加,体现了热耗散对PSⅡ的较强保护能力.随着热胁迫温度的升高和热胁迫时间的延长,两品种的无活性中心F_vi/Fv显著增加.     

99mTc-labeling of hydrazones of a hydrazinonicotinamide conjugated cyclic peptide.

Eight HYNICtide hydrazones (three with aliphatic substituents and five with aromatic groups) were studied for their potential use as the final intermediate for preparation of RP444, a new radiopharmaceutical under development for imaging thrombosis. The goal of this study is to screen various hydrazones through stability testing and radiolabeling and find those which are able to remain stable without significant degradation in the manufacturing process and at the same time are reactive to produce enough free hydrazine in situ for successful (99m)Tc-labeling. In an initial screening study, only hydrazones 6 and 8, which contain aliphatic substituents, gave satisfactory (>/=90%) yields of RP444 using 50 degrees C and 30 min of heating. However, their solution instability excludes them from being used as commercial reagents. Hydrazones 1 and 4 gave >/=90% yields when the reaction mixtures were heated at 80 degrees C for 30 min. Both hydrazone 1 and hydrazone 4 can be used as the final intermediate for preparation of RP444. The combination of 40 mg of tricine, 1-10 mg of TPPTS, 20-40 microg of hydrazone 1 or 4 for 50 mCi of [(99m)Tc]pertechnetate, 20-50 microg of stannous chloride, pH 4.5 +/- 0.5, and heating at 80 degrees C for 30 min gives the best yield for RP444. It is surprising that hydrazones 1 and 4 have both the solution stability with respect to decomposition and to reaction with aldehydes and ketones and yet are able to hydrolyze in situ to produce enough free HYNICtide for the (99m)Tc-labeling.     

Re-activation of the peptidyltransferase centre of rabbit reticulocyte ribosomes after inactivation by exposure to low concentrations of magnesium ion.

1. The larger subrivosomal particles of rabbit reticulocytes retained full activity in the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis after 4h at 0 degrees C when buffered 0.5M-NH4Cl/10-30mM-MgCl2 was the solvent. 2. Activity in the puromycin reaction was diminished to approx 10% after 15-30 min at 0 degrees C when the concentration of MgCl2 was lowered to 2mM. 3. Activity was not restored when the concentration of MgCl2 was raised from 2mM to 10-30 mM at 0 degrees C. However, activity was recovered as measured by both assay systems when the ribosome fraction was heated to 37 degrees C at the higher concentrations of MgCl2. 4. Recovery of activity was noted during the course of the polyphenylalanine synthesis in 50 mM-KCl/5mM-MgCl2/25mM-Tris/HCl, pH 7.6, at 37 degrees C. Re-activation was slow at 20 degrees C and below. 5. No more than about 5% of the protein moiety of the subparticle was lost in 0.5M-NH4Cl on decreasing MgCl2 concentration from 10mM to 2mM. No proteins were detected in the supernatant fractions by gel electrophoresis after ribosomes were separated by differential centrifugation. The supernatant fraction was not essential for the recovery of activity. However, at higher (e.g. 1M) concentrations of NH4Cl, proteins were split from the subparticle. 6. The loss and regain of activity found on lowering and restoring the concentration of MgCl2 at 0.5M-NH4Cl appears to arise from a conformational change that does not seem to be associated with a loss and regain of particular proteins. 7. A 2% decrease in E260 was noticed when the concentration of Mg2+ was restored, and the change in the spectrum indicated a net increase of approx. 100A-U base-pairs per subribosomal particle. 8. When the concentration of Mg2+ was restored, S20,W of the subparticle remained at 52+/- 1S until the sample was incubated at 37 degrees C when S20,W increased to 56 +/- 1S compared with the value of 58 +/- 1S for the subparticle as originally isolated.