Synthesis and spectral properties of cytosine nucleosides of 2-amino-2-deoxy-alpha-D-arabino-furanose and -pyranose.

Ethyl 2-deoxy-3,5-di-O-p-nitrobenzoyl-1-thio-2-(trifluoroacetamido)-beta-D-arabinofuranoside (3) was converted into the glycosyl chloride. Condensation of the latter with 2,4-dimethoxypyrimidine, followed by amination, gave 1-(2-amino-2-deoxy-alpha-D-arabinofuranosyl)cytosine (6), which was also obtained from the alpha-D anomer (4) of 3. Similarly, 1-(2-amino-2-deoxy-alpha-D-arabinopyranosyl)cytosine (12) was synthesized from ethyl 2-deoxy-3,4-di-O-p-nitrobenzoyl-1-thio-2-(trifluoroacetamido)-alpha-D-arabinopyranoside (9). The p.m.r. spectra of these nucleosides, as well as those of the 1-thioglycosides, are discussed in terms of the conformation of the sugar portion. In particular, a large change of the J1,2 coupling constants of the alpha-D-furanosides, according to the substituents at C-1 and C-2, was interpreted on the basis of conformational mobility.     

A study of the reaction of 2-phenyl-1,3,2-benzodiazaborolidine with benzoyl chloride

Reaction of 2-phenyl-1,3,2-benzodiazaborolidine (2) with benzoyl chloride afforded N,N′-dibenzoyl-o-phenylenediamine (8) regardless of the amount of the acid chloride employed. Depending on the work-up procedure, the 2-phenylboron moiety was isolated as either phenylboronic acid or phenylboronic acid anhydride. In the absence of added bases, the reaction was observed to be susceptible to solvent assistance by ethers, presumably by coordination with the empty p-orbital of boron. In the presence of added amines, the extent of benzoylation was altered considerably. With pyridine as a co-reagent, small amounts of 1,3-dibenzoyl-2-phenyl-1,3,2,-benzodiazaborolidine mono- or dihydrocholoride were isolated though this compound readily underwent hydrolysis or oxidation upon standing. Interaction of 2 with sodium hydride in tetrahydrofuran caused ionization of only one of the two hydrogens bound to nitrogen even upon extended reflux. Benzoylation of the heterocycle under these latter conditions again afforded only the dibenzoylated compound 8.     

Alteration of processive mechanism of native polynucleotide phosphorylase by fixation to solid matrix.

Native $\text{E. coli}$ polynucleotide phosphorylase can be covalently bound to BrCN activated Sepharose. The Sepharose bound enzyme retains 70 % of its initial activity in polymerisation of nucleoside diphosphate. The Km of the enzyme for the polymerisation reaction in comparison to the soluble enzyme is not affected by its linkage to a solid matrix. The phosphorolysis of an hexanucleotide by the Sepharose-bound enzyme is not affected either. However, the rate of phosphorolysis of a long chain polynucleotide is dramatically altered. The Km values for poly(A) or poly(U) are increased by two orders of magnitude. The decrease of affinity for polymeric substrate is accompanied by a significant modification of the known processive mechanism characteristic of the native soluble enzyme.     

Kinetic study of reduction of [Tc(V)OBr5]2− in concentrated HBr

Pertechnetate is rapidly reduced in concentrated (8.7 M) HBr to Tc(V). Subsequently reduction to give TcBr₆^2? is a slow process. The kinetics of this last process have been investigated. They indicated a combination of first and zero order reactions in the presence of the high HBr concentration. The first order rate constant was 4.8 × 10^?2 h^?1, and the zero order process constant was 6.0 × 10^?6 mol l^?1 h^?1.     

Glutamine synthetase from Salmonella typhimurium: manganese(II), substrate, and inhibitor interaction with the unadenylylated enzyme.

Unadenylylated glutamine synthetase (EC 6.3.1.2) was isolated and purified to homogeneity from Salmonella typhimurium. The enzyme molecule is a symmetrical aggregate of 12 subunits arranged in two hexagonal layers, as is evident from electron micrographs. The subunit molecular weight of the enzyme was found to be approximately 50,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate when compared to Escherichia coli glutamine synthetase and other protein standards. A long tube of glutamine synthetase was formed as a single-stranded coil resulting from incubation of the enzyme in a low ionic strength buffer. A study of Mn(II) binding to the unadenylylated enzyme at 25 °C was conducted as a function of pH. At pH 7.1 two classes of metal ion sites per subunit were found with K_D values of 3.7 × 10^?6 and 1.7 × 10^?4m, while at pH 6.8 these values were 1.1 × 10^?5 and 1.0 × 10^?4m, respectively. Only one set of binding sites was observed at pH 6.2 with a K_D value of 1.0 × 10^?4m. The metal ion binding sites were further investigated by monitoring proton relaxation rates (prr) and the epr spectrum of enzyme-bound Mn(II). The longitudinal prr of water protons at pH 7.1 indicate that protons interacting with enzyme-Mn(II) at the “tight” site (K_D = 3.7 × 10^?6) are de-enhanced (?_b1 = 0.42) and result from water protons beyond the inner coordination sphere. The second Mn(II) site has a value of ?_b2 = 35 for the binary enhancement, suggesting that this site probably has two to three rapidly exchanging water molecules in its coordination sphere. The epr spectrum of enzyme-bound Mn(II) at the “tight” site is isotropic and is dramatically sharpened by adding the substrate analog methionine sulfoximine. Subsequent addition of ATP or the ATP analog, AMP-PCP (adenylyl methylene diphosphate) produced anisotropic spectra that were similar, suggesting that both ATP and AMP-PCP bind similarly on the enzyme surface. However, a marked change in the Mn(II) environment from anisotropic to near cubic results from the addition of ADP to the quaternary enzyme-Mn(II)-sulfoximine- (AMP-PCP) complex, indicating that ADP displaces AMP-PCP. No change in the anisotropic spectrum due to the enzyme-Mn(II)-sulfoximine-ATP complex is seen by the addition of ADP. This experimental result supports the experimental findings of Ronzio and Meister [Proc. Nat. Acad. Sci. USA59, 164 (1968)], who established that ATP phosphorylates methionine sulfoximine, thereby producing an inactive enzyme. The allosteric effectors, AMP and Trp, have little effect on the epr spectrum of the complex formed from Mn(II), enzyme, sulfoximine, and ADP, suggesting the absence of direct coordination of AMP or Trp to the bound Mn(II). The prr and epr results reported herein with glutamine synthetase from S. typhimurium when compared to those seen for the enzyme from E. coli [Villafranca et al., Biochemistry15, 544 (1976)] demonstrate some similarities but also many substantial differences between the enzymes from these two bacterial sources.     

Effects of calcium on the permeability of isolated adult rat heart cells to sodium

The permeability of rat heart myocytes to Na increases when extracellular Ca (Ca₀) is decreased. This increased permeability is reflected in elevated $\text{Na}\text{K}$ ratios in nonenergized myocytes and in increased ouabain-sensitive lactate production in anaerobic myocytes supplemented with glucose. Myocytes treated with ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid (1 mm) maintain low $\text{Na}\text{K}$ ratios, but expend considerable glycolytic ATP in ouabain-sensitive cation cycling. The data suggest that Ca₀ bound to the sarcolemma can restrict transmembrane movement of Na via pathways that are not yet defined. The lack of significant net accumulation of Ca argues against the explanation that Ca₀ maintains low internal Na levels as a result of NaCa exchange. Both the increased uptake of Na and increased utilization of ATP in the absence of Ca₀ may be relevant to the phenomenon of “Ca-paradox” in situ.     

Effects of net charge on the hydrogen-deuterium exchange parameters of lysozyme.

Possible effects of changes in net charge on protein hydrogen exchange rates were investigated by desalting hen egg-white lysozyme, which allowed its net charge to increase with decreasing pH in the acid region. Chloride ion-binding ratios, expressed as ratios of free to total Cl^?, were measured with a chloride-specific electrode at pH 5 on a 2.4% solution of a five-time-desalted product. This ratio was used to show a 97% reduction of the 11% Cl^? present in a commercial lysozyme preparation upon three passes of the enzyme through a column of ion-retardation resin. Net charges on the purified product were assigned from a combination of electrophoretic mobility and proton titration data gathered under minimal ionic strength conditions. The net charge on the desalted product increased by 1.64 units between pH 5.0 and 3.0. Hydrogendeuterium exchange studies on the purified lysozyme in D₂O were obtained using the near-infrared region of a Cary 14R spectrophotometer. The rate-pD profile for k₂, the rate constant for the intermediate class of exchanging hydrogens, showed a decrease in the apparent pD of minimum exchange rate of 0.3 units, when compared to that obtained earlier in 0.2 m added NaCl. However, the rate of exchange at pD minimum and the number of hydrogens in the class remained largely unaffected. A similar shift was observed for the rate-pD profile of the class 1 hydrogens. Thus, the effect of an increase in net positive charge is to shift the rate-pD profile to a lower pD. Moreover, the effect extended to the interior peptide hydrogens of this globular protein. Consequently, the exchange rates of all the observable hydrogens are altered by the net charge changes, and the effect appeared uniform. The shift can be accounted for quantitatively by applying electrostatic interaction terms to the acid and base catalytic constants characterizing the exchange process. The calculated electrostatic interaction factors in minimal salt and 0.2 m added NaCl were found to be 29 and 18% lower, respectively, than those obtained theoretically. Therefore, under conditions where changes in net charge may occur for a globular protein, the effect on hydrogen exchange rates can be estimated fairly well theoretically, especially at moderate ionic strengths.     

The synthesis of 3-nonaprenyl-4-hydroxybenzoate in rat liver mitochondria: the effect of Ca2 , Mg2+, chelators, and bacitracin on the activity of p-hydroxybenzoate-polyprenyl transferase

The formation of the first intermediate in ubiquinone-9 biosynthesis, 3-nonaprenyl-4-hydroxybenzoate (NPHB), by the enzyme p-hydroxybenzoate:polyprenyl transferase, has been studied in isolated rat liver mitochondria using solanesol pyrophosphate and p-hydroxybenzoate as the substrates. Phosphate buffer (100 mm) is inhibitory but at 20 mm inhibition is not apparent compared to other buffers at the same concentration. With various buffers at low concentration (20 mm) both EDTA and Mg²⁺ stimulate formation of NPHB while Ca²⁺ inhibits. Release of Ca²⁺ inhibition can be achieved by the addition of Mg²⁺, or EDTA, or EGTA, with EGTA being less effective than EDTA. When Mg²⁺, Ca²⁺, and EDTA are present together, a two- to threefold increase in activity of the enzyme is observed. The antibiotic bacitracin inhibits the synthesis of NPHB and the inhibition is increased when divalent cations are present. EGTA is more effective than EDTA in overcoming inhibition due to bacitracin. The possibility that these effects are partially due to alteration of mitochondrial membrane conformation as well as a direct effect on the enzyme is evaluated. The possible role of polyprenylphosphates in mitochondrial membrane function is discussed.     

Leukocyte adherence inhibition response to murine sarcoma virus-induced tumors. II. Requirement for T-cell subpopulations and macrophages

Murine sarcoma virus (MSV)-immune T cells from C57BL/6 mice respond to intact RBL-5 tumor cells with the production of leukocyte adherence inhibition factor (LAIF), which mediates an adherence inhibition response of macrophages. LAIF is elaborated by isolated Lyt-2+ cells incubated with RBL-5 cells, whereas Lyt-1+ cells elaborate a substance that enhances macrophage adherence. Spleen macrophages or peritoneal exudate macrophages from MSV-immune mice when present at concentrations of 0.1% changed the response of Lyt-1+ cells from the formation of an adherence enhancing factor to the formation of an adherence inhibiting factor. Migration inhibition factor (MIF) was formed by Lyt-1+ cells, but not by Lyt-2+ cells under identical culture conditions. Addition of either spleen macrophages from mice with progressively growing tumors or tumor-infiltrating macrophages suppressed LAIF formation by both Lyt-1+ and Lyt-2+ cells. Tumor-infiltrating macrophages elicited an adherence enhancing factor from Lyt-2+ cells when present at high concentrations. The results suggest that the extent of macrophage adherence in vitro is the outcome of an interaction of macrophages with mediators that have opposing effects.     

A subnanosecond-pulse fluorometric study of the CA2+ and MG2+ induced conformational changes on S-100a protein

The fluorescence parameters of the single tryptophan residue (Trp 90 alpha) in S-100a (alpha beta) protein have been studied by steady state fluorimetry and by subnanosecond fluorescence decays excited by pulsed-picosecond laser system. At pHs 7.1 and 8.5, double exponential decays were consistently observed. At both pHs, Ca2+ and to a less extent Mg2+ ions proved to modify the percentage contribution of the two decaying species. The interest of the finding is discussed.     

Synthesis of leucine enkephalin derivatives: structure-function studies

Nucleoli isolated from livers of rats injected intraperitoneally with one dose of thioacetamide had a five-fold increase in the rate of RNA synthesis $\text{in vitro}$ when compared with livers of rats treated with saline or CCl₄. The stimulation was maximal 24 hours after treatment and decreased to control values 73 hours after treatment. The enhanced level of nucleolar activity was maintained at that level when thioacetamide was injected daily. Along with the increase in the endogenous activity there was a 7-fold increase in the “free” RNA polymerase I activity determined by blocking the bound enzyme with actinomycin D (7). The nucleoli of the thioacetamide-treated rats offer a useful model of modulation of ribosomal gene function.     

Characterization of a serum inhibitor of MLC reactions. III. Specificity: HL-A-related antibodies.

A mixed leukocyte culture (MLC)-inhibitory serum from a healthy multipara, JH, has been characterized with regard to the specificity of its inhibitory antibody. When added directly to MLC, JH serum will inhibit most combinations. However, when lymphocytes intended as responder or stimulator cells in MLC were preincubated with this serum, the specificity narrowed considerably. Four groups of lymphocyte donors were recognized, depending upon whether their lymphocytes were inhibited as responders, as stimulators, as both, or as neither. Absorptions of inhibitory activity, followed by assay of the absorbed sera in pretested MLC combinations, yielded reliable data for determining which donors' cells shared pertinent antigens. An association of MLC inhibition by JH serum with the HL-A types of the involved lymphocytes was observed and these relationships are summarized in Table 4. The three HL-A specificities identified, W19, W29, and 12, correspond with the HL-A typing of the husband of the serum donor. Various cell types absorbed relevant inhibitory activity (against responder and stimulator functions) in the following order of efficiency: LCL cells, B lymphocytes, T lymphocytes, fibroblasts, and erythrocytes. When the above three HL-A specificities were removed by absorption, the serum was no longer inhibitory in any combinations. Whether the inhibitory activity of JH serum is directly related to anti-HL-A antibodies or to antibodies against closely related MLR determinants will depend to a large extent upon the degree of linkage disequilibria found between W19, W29, and 12 antigens and the MLR locus.     

The enthalpy of oxidation of flavin mononucleotide: Temperature dependence of in vitro bacterial luciferase bioluminescence

The enthalpy of the bioluminescent reaction

$\text{FMNH}{}_2\text{+ RCHO + O}{}_2\text{→}\text{luciferase}\text{FMN + RCOO + H}{}_3\text{O}{}^{\mathrm{+}}\text{+ hv}$

has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH₂) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔH_L = ?338.9 k.J (mol FMN)^?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)^?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔG_L = ?464.8 kJ (mol FMN)^?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH₂ + O₂ ? FMN + H₂O₂; ΔH_D = ?158.5 kJ (mol FMN)^?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG₁, and intrinsic enthalpy, ΔH₁, which are functionally defined by the relations ΔG_I = ΔG_L ? uhvΔH_I = ΔH_L ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole^?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)^?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.     

A study of three vasodilating agents as selective inhibitors of thromboxane A2 biosynthesis

Three clinically efficacious vasodilatory drugs were found to be selective inhibitors of thromboxane A₂ biosynthesis. Hydralazine, dipyridamole, and diazoxide inhibited platelet aggregation at 1 × 10^?4 M, 1.75 × 10^?4 M, and 2 × 10^?3 M respectively. Their relative inhibitory potencies on thromboxane B₂ production in human platelet microsomes were examined and found to be similar to that observed for their inhibition on human platelet aggregation. At 10^?3 M, hydralazine, dipyridamole, and diazoxide inhibited thromboxane B₂ formation by 65 percent, 27 percent and 18 percent respectively. These compounds were examined in the sheep vesicular gland system, and they were shown not to be inhibitors of the cyclooxygenase enzyme. Thus, the inhibition of thromboxane A₂ biosynthesis by these three drugs in human platelet microsomes appeared to be specific at the thromboxane synthetase level.     

Butanedione treatment reduces receptor binding of a lysosomal enzyme to cells and membranes

Treatment of the lysosomal enzyme, α-L-iduronidase, with 2,3 butanedione, an arginine modifying reagent, under conditions where enzyme activity was unaffected, reduced by 50% the internalization of the enzyme into cultured human fibroblasts. The lowered rate of internalization was a result of a reduced binding of the enzyme to cell surface receptors. The butanedione treatment of α-L-iduronidase caused a 90% reduction of binding when isolated fibroblast membranes were used as the source of receptor. This marked reduction of binding was also seen when membranes from a rat chondrosarcoma were examined. Although there is ample evidence that the receptor recognizes mannose 6-phosphate residues on the enzyme, the results suggest that other structural features, such as arginine moieties, may also be important in iduronidase binding.     

Reactivity of tryptophans in rhodopsin.

Oxidation with N-bromosuccinimide detects a total of about ten tryptophan residues in detergent-solubilized bovine rhodopsin. One of these tryptophans is more reactive in bleached than in unbleached rhodopsin, suggesting its involvement in the chromophore binding site. Oxidation of this residue is accompanied by loss of the 500nm. absorbance in unbleached rhodopsin. Similar experiments with bacteriorhodopsin are inconclusive.     

Reaction of BCNU (1,3-bis (2-chloroethyl)-1-nitrosourea) with polycytidylic acid. Substitution of the cytosine ring

BCNU has been reacted with polycytidylic acid and two derivatives of CMP, 3-hydroxyethyl-CMP and 3,N⁴-ethano-CMP, have been identified in the acid hydrolysate of the polymer. Their formation accounts for some of the reaction of BCNU with nucleic acids, and may be related to the mechanism of action of this compound.     

Does carboxypeptidase Y have intrinsic endopeptidase activity?

Carboxypeptidase Y preparations from baker's yeast have been found to exhibit endopeptidase activity when assayed with oxidized insulin B-chain. Amino acid analysis and peptide isolation studies indicate that a specific internal cleavage occurs between Leu-15 and Tyr-16 in addition to the C-terminal carboxypeptidase activity. Blocking the C-terminal residue of the substrate prevents the exopeptidase activity of the enzyme, but has no effect on the endopeptidase activity. On the other hand, pepstatin A inhibits the endopeptidase but not the exopeptidase activity. These results suggest that the endopeptidase activity is due to the presence of contaminating amounts of yeast proteinase A and indicate that caution should be taken when employing carboxypeptidase Y preparations for sequence studies.