Reactions of sugar nitro-alkenes with 3-aminocrotonic esters

3,4,5,6,7-Penta-O-acetyl-1,2-dideoxy-1-nitro-d-gluco- and -d-galacto-hept-1-enitol and 3,4,5,6-tetra-O-acetyl-1,2-dideoxy-1-nitro-d-xylo-hex-1-enitol react with 3-aminocrotonic esters, yielding mixtures of the epimeric Michael adducts. These are thermally stable, and do not cyclize to pyrroles. The structures, configurations, and conformations of these compounds were established on the basis of their spectroscopic and X-ray crystallographic data. The intramolecularly bonded, (Z) configuration was deduced for all of them. Mild hydrolysis of adducts with acid yields the corresponding 2-(nitromethylpolyacetoxyalkyl)acetoacetates.     

Stereospecific binding of 3H-phencyclidine in brain membranes

Phencyclidine (PCP) displaceable binding of ³H-PCP to glass-fiber filters was eliminated and total binding markedly reduced by initial treatment of the discs with 0.05% polyethyleneimine. Assessed with treated filters, unlabeled PCP displaced ³H-PCP in both rat and pigeon brain membranes with an EC50 of 1 μM. Of similar high inhibitory potency were dextrorphan, levorphanol, SKF 10047 and ketamine, while morphine, naloxone and etorphine had EC50 values higher then 1 mM. Using the dissociative anesthetic dexoxadrol and its inactive isomer levoxadrol as displacing agents, stereospecific binding of ³H-PCP was obtained in rat and pigeon brain membranes. The markedly higher potency of dexoxadrol, relative to levoxadrol, in displacing bound ³H-PCP is compatible with behavioral data for these enantiomers. However, they were equipotent in displacing ³H-PCP bound to glass-fiber filters in the absence of tissue. Heat denaturation, but not freezing, abolished stereospecific binding of ³H-PCP, which was also absent in rat liver membranes. The stereospecific binding component in brain displayed biphasic saturability at 60–70 nM and 300–400 nM, respectively.     

Myristoyl esters of lactose

Whereas lactose did not undergo a base-catalyzed transesterification with methyl esters of fatty acids, methyl beta-lactoside reacted under identical conditions to give mono- and di-myristates. This difference in behavior is explained in terms of the formation of an unreactive, internally chelated potassium-lactose complex. Supporting evidence for this hypothesis is the observed change in the anomeric equilibrium of lactose in the presence of potassium carbonate. The monomyristates of methyl beta-lactoside were assigned the structures of 3' and 6' derivatives, and it is concluded that the diesters are the 3',6', and 6,6' derivatives.     

Reaction of peptide thiobenzyl esters with mammalian chymotrypsinlike enzymes: A sensitive assay method

Sensitive methods for the determination of rat mast cell protease I, rat mast cell protease II, human skin chymotrypsinlike enzyme, dog skin chymotrypsinlike enzyme, human leukocyte cathepsin G, and bovine chymotrypsin A_α with peptide thiobenzyl ester substrates are reported. Kinetic constants as well as the maximum sensitivity for the hydrolysis of the peptide substrates succinyl-phenylalanyl-leucyl-phenylalanine thiobenzyl ester and succinyl-alanyl-alanyl-prolyl-phenylalanine thiobenzyl ester were determined. Hydrolysis rates were followed spectrophotometrically at 324 nm by the formation of 4-thiopyridone (? = 19,800 m^?1 cm^?1), the product of the reaction between benzylthiol, released during hydrolysis of the peptide thiobenzyl esters, and 4,4′-dithiodipyridine present in the assay mixture. Peptide thiobenzyl ester substrates were shown to be very sensitive substrates, predominantly because of the large extinction coefficient of 4-thiopyridone and the high $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ values for these compounds.     

Automatic collector of expired 14CO2 for liquid scintillation counting

A filtration technique was employed to trap ¹⁴CO₂ continuously for liquid scintillation counting. Devices for delivering scintillator and ethanolamine solutions were combined symmetrically with two fritted-glass aspirators for altenating operation. The collector was regulated by a fraction collector timer. Trial and animal tests indicated that the described method was efficient, reliable, and more convenient for frequent collection over long periods than alternative methods. The automatic collector was used for metabolic studies of [1-¹⁴C] arachidonic acid in rats kept in metabolic cages and the results were processed by multicompartmental analysis.     

Regulation of insulin binding and stimulation of sugar transport in cultured human fibroblasts by sugar levels in the culture medium

Studies were carried out on cultures of human skin fibroblasts to explore the effects of culture medium glucose levels on insulin binding and action. Cell cultures in 5.55 mm glucose-containing medium depleted their medium glucose within 3 days, and at that time exhibited elevated deoxy-d-glucose (2-DG) transport (84% greater than control cultures fed 22.2 mm glucose) and failure of insulin to stimulate 2-DG transport (an insulin:control transport ratio of 1.02). There was also a significant negative correlation between basal 2-DG transport and insulin binding (r = ?0.621; n = 29; P < 0.01), while insulin binding exhibited a significant positive correlation with insulin action (r = 0.816; n = 12; P < 0.01). Glucose starvation of cultures for 18 h resulted in several changes: (i) a 49% decrease in specific ¹²⁵I-insulin binding due to a reduction in binding capacity; (ii) elevated basal 2-DG transport; and (iii) an absence of insulin stimulation of 2-DG transport. Exposure to increasing concentrations of glucose for 18 h led to a glucose concentration-dependent increase in specific insulin binding. Additionally, the various changes in the glucose-starved group were reversed after as little as 6 h of glucose refeeding. The results indicate that basal sugar transport, and insulin binding and action can be regulated by the amount of glucose in the medium.     

Inhibition of sugar uptake by ascorbic acid in Escherichia coli

The uptake of glucose by the glucose phosphotransferase system in Escherichia coli was inhibited greater than 90% by ascorbate. The uptake of the nonmetabolizable analog of glucose, methyl-alpha-glucoside, was also inhibited to the same extent, confirming that it was the transport process that was sensitive to ascorbate. Similarly, it was the transport function of mannose phosphotransferase for which mannose and nonmetabolizable 2-deoxyglucose were substrates that was partially inhibited by ascorbate. Other phosphotransferase systems, including those for the uptake of sorbitol, fructose and N-acetylglucosamine, but not mannitol, were also inhibited to varying degrees by ascorbate. The inhibitory effect on the phosphotransferase systems was reversible, required the active oxidation of ascorbate, was sensitive to the presence of free-radical scavengers, and was insensitive to uncouplers. Because ascorbate was not taken up by E. coli, it was concluded that the active inhibitory species was the ascorbate free radical and that it was interacting reversibly with a membrane component, possibly the different enzyme IIB components of the phosphotransferase systems. Ascorbate also inhibited other transport systems causing a slight reduction in the passive diffusion of glycerol, a 50% inhibition of the shock-sensitive uptake of maltose, and a complete inhibition of the proton-symport uptake of lactose. Radical scavengers had little or no effect on the inhibition of these systems.     

Disposition of polar and nonpolar residues on outer surfaces of transmembrane helical segments of proteins involved in proton translocation

"Helical wheel" projections of transmembrane helical segments of membrane proteins involved in proton translocation were constructed. The particular proteins studied were the uncF protein subunit of the Escherichia coli proton-ATPase, the uncE protein subunit of the E. coli proton-ATPase, and cytochrome oxidase subunit III. Clear demarcation of polar and nonpolar regions on surfaces of transmembrane helical segments was seen in the uncF protein and in uncE protein helical segment two, but not in uncE protein helical segment one. The transmembrane segment of cytochrome oxidase subunit III which includes the dicyclohexylcarbodiimide (DCCD)-reactive residue was very similar to E. coli uncE protein helical segment two. The DCCD-reactive residue in both was clearly located on a nonpolar surface.     

The reaction of tertiary amino alcohols with active esters: I. Acylation and deacylation steps

The reactions of triethanolamine and four other tertiary amino alcohols with six active ester substrates were studied in the pH range 6–10 at 30°C. The reaction products were in all cases the respective O-acyl-amino alcohols. Analysis of the effects of substituents in the leaving group as well as in the acyl moiety of the substrates showed that the ester product was formed by direct attack of the nucleophilic hydroxyl group. Comparison with reactions of tertiary amines with the same substrates supports this conclusion. The reactions of tertiary amino alcohols were also compared with those of zwitterionic quaternary amino alcohols and 3-quinuclidinol, a “rigid” tertiary amino alcohol. On the basis of these comparisons, it is proposed that one of the pathways for the predominant effect of the neutral species of tertiary amino alcohols involves intramolecular general base assistance by the tertiary amino group to the nucleophilic attack of the hydroxylic oxygen on the substrate. The contribution of this pathway to the rate of reaction is evaluated.In several systems the first product of the reaction, an O-acyl-amino alcohol, undergoes relatively rapid deacylation, the overall reaction being thus hydrolysis of active esters, catalyzed by the amino alcohol via an acylation-deacylation mechanism.     

The reaction of amino alcohols with active esters: II. Catalysis of the acylation step by metal ions

The reaction of triethanolamine (TEA) with active substrates—p-nitrophenyl esters and cinnamoyl imidazole (CI)—is catalyzed by divalent heavy metal ions. With Hg²⁺, rate enhancements of 100–1000 (depending on the substrate) were observed, the overall rate constants of substrate decomposition thus exceeding those of spontaneous hydrolysis up to 100,000-fold. The predominant active species at low L:M ratio was found to be the Hg-(TEA)₂ complex. The dependence of the reaction rate upon excess of amino alcohol—at constant Hg²⁺ concentration—is attributable to formation of another active complex—Hg-(TEA)₃.The high reactivity of the system is due to the alcoholate group of metal-bound TEA, whose pK has been lowered by the proximity of the metal ion. This labile nucleophilic alcoholate attacks the substrate causing its alcoholysis and forming O-acyl-TEA. The lability of the metal-alcoholate bond can be enhanced by low concentrations of halide ions, thus causing up to 5-fold additional increase in alcoholysis rate. Higher halide ion concentrations cause inhibition, probably due to formation of inactive HgX₂ molecules.Presumably an important role of the metal ion in metalloenzymes is to affect the decrease in the pK value of a reactive group so that it can exhibit activity under physiological conditions.     

Reactions of the sulfhydryl groups of alanyl-tRNA synthetase

The six sulfhydryl groups in each subunit of the alanyl-tRNA synthetase of Escherichia coli react with sulfhydryl reagents with at least four different rates. One reacts very rapidly with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and a second reacts somewhat less rapidly with this reagent. These two groups are required for transfer activity, which is lost in proportion to the extent of derivatization. Two other groups react more slowly, with a consequent loss of exchange activity. The remaining two sulfhydryl groups do not react with DTNB until the protein is denatured. The inactivations are reversed by dithiothreitol. Two sulfhydryl groups react with N-ethylmaleimide (NEM) and with a spin-label derivative of NEM. These reactions resemble the modification of two sulfhydryl groups with DTNB, in that they also inactivate the transfer reaction but not the ATP:PP_i exchange. The two spin labels are incorporated at similar rates but are in very different environments, one highly exposed and one highly immobilized. These groups do not interact with Mn²⁺, which is bound to the enzyme in the absence of ATP.     

Induction of synthesis of bovine adrenocortical cytochromes P-450scc, P-45011 beta, P-450C21, and adrenodoxin by prostaglandins E2 and F2 alpha and cholera toxin

To further elucidate the mechanisms by which ACTH (adrenocorticotropin) exerts its long-term action to maintain normal levels of adrenocortical cytochromes P-450 and related enzymes, the abilities of cholera toxin and prostaglandins E2 and F2 alpha to induce the synthesis of cytochromes P-450scc, P-45011 beta, and P-450C21 and adrenodoxin have been examined. These effectors stimulate the production of cyclic AMP and thus steroidogenesis in the adrenal cortex. Using bovine adrenocortical cells in primary monolayer culture, we have shown that treatment with cholera toxin results in increased synthesis of cytochromes P-450scc and P-45011 beta and adrenodoxin, similar to the effect observed upon ACTH treatment. Prostaglandins E2 and F2 alpha are less effective at inducing the synthesis of the mitochondrial cytochromes P-450, and do not seem to induce the synthesis of adrenodoxin. Furthermore, cholera toxin was found to be less effective at inducing the synthesis of microsomal cytochrome P-450C21 than ACTH, and no more effective than the prostaglandins. Thus, while it appears that elevation of cyclic AMP levels is a necessary step leading to increased synthesis of adrenocortical forms of cytochrome P-450, the detailed mechanism of this induction will be found to be different for each of the different enzymes.     

ATP depletion promotes deactivation of insulin-stimulated sugar transport in rat soleus muscle

It has been reported that deactivation of insulin-stimulated sugar transport in adipocytes is an energy-dependent process (F. V. Vega, R. J. Key, J. E. Jordan, and T. Kono (1980) Arch. Biochem. Biophys. 203, 167-173). The stimulatory effect of insulin (0.1 U/ml) on the uptake of D-[U-14C]xylose by rat soleus muscle was rapidly reversed when muscle ATP was depleted by exposure to 2,4-dinitrophenol (0.5 mM). Insulin action was not completely eliminated by ATP depletion; there was a small, residual stimulatory effect of the hormone which persisted for about 30 min after muscle ATP had been lowered to an unmeasurable level. The extent of deactivation was not altered when the rate of ATP depletion was accelerated, either by increasing the 2,4-dinitrophenol concentration, or by inducing leakiness by incubating muscles for 90 min at 37 degrees C prior to the addition of the uncoupler. 2,4-Dinitrophenol lowered steady-state 125I-insulin binding. These differences between the effect of ATP depletion on insulin-stimulated sugar transport in muscle and adipose tissue may be related to the action of the uncoupler in lowering steady-state insulin binding in muscle. Such a fall in bound insulin could be expected to promote deactivation during the period of ATP depletion. However, at present the possibility that these differences may represent some more fundamental difference in deactivation between muscle and adipose tissue cannot be excluded.     

Phytohemagglutinin-lnduced acquisition of T-cell surface markers by rat bone marrow precursor cells in the absence of the thymic microenvironment

Two monoclonal antibodies specific for different rat T-cell subpopulations, the anti-helper-T-cell antibody, W3/25, and the OX8 suppressor cell antibody were used to investigate lectin-stimulated T-lymphocyte differentiation of F-344 rat bone marrow cells in culture. Cytofluorometric analysis of freshly isolated lymphocytes from thymus and spleen revealed that these tissues contained both W3/25? and OX8-positive populations but differed with respect to the number of cells and receptor density distribution. By contrast, bone marrow-derived lymphocytes exhibited negligible W3/25? or OX8-associated fluorescence. However, several days after stimulation of bone marrow lymphocytes with phytohemagglutinin (PHA), cells appeared bearing these markers. Two-parameter histogram analysis of light scatter measurements with cell surface immunoflu-orescence indicated that this phenomenon represented the appearance of a new population of cells, presumably mature T cells, bearing an increased density of marker. These findings suggest an induction of differentiation of bone marrow T precursor cells by nonthymic factors (PHA) since lymphocytes lacking mature T-cell marker expression developed this characteristic after several days in culture.     

Kinetic analysis of superoxide anion production by activated and resident murine peritoneal macrophages

Using a continuous spectrophotometric assay, we have monitored the formation of superoxide anion (O₂^?) by activated and resident murine peritoneal macrophages. Macrophages elicited by injection with Corynebacterium parvum, as well as resident macrophages from untreated mice, were kept in suspension culture overnight to eliminate short-lived, contaminating neutrophils. Cytochemical analysis of the cultured macrophages disclosed that essentially all of the activated macrophages reduced nitroblue tetrazolium (NBT) dye vigorously. In contrast, only 18% of the resident macrophages demonstrated vigorous NBT reduction; the remainder of the resident macrophages reduced NBT very weakly. Kinetic analysis of macrophage O₂^? formation revealed that activated macrophages exposed to phorbol myristate acetate (PMA) produced O₂^? at a 13-fold greater maximum rate than resident macrophages. The decline in the rate of O₂^? production with time by activated macrophages was also greater than that of resident macrophages. The data indicate that the greater O₂^? production by activated macrophage populations is due to (i) the presence of an increased percentage of macrophages that respond to PMA with vigorous O₂^? production, and (ii) an increased maximum rate of O₂^? formation by these macrophages.     

Phorbol esters inhibit LH stimulated steroidogenesis by mouse Leydig cells in vitro

The effect of phorbol esters on the stimulation of testosterone production in response to LH was studied in mouse Leydig cells incubated in vitro. The tumor promoting phorbol esters, Phorbol-12-myristate-13-acetate and Phorbol-12-13-didecanoate at nanomolar concentrations effectively inhibited testosterone production by Leydig cells in response to stimulation by LH, whereas non-tumor promoting phorbol esters were ineffective. When the cells were stimulated by 8Br-cAMP, instead of LH, the testosterone production was stimulated similarly as in the presence of LH, but phorbol esters were without any effect. This suggests that the tumor promoting phorbol esters may act in the Leydig cells by suppressing the stimulation of cAMP production in response to hormonal activation and/or by interfering with the hormone-receptor interaction.     

Catalytic action of comicelles containing imidazolyl and hydroxyl groups in the hydrolysis of enantiomeric esters

The rate constants for hydrolysis of the enantiomers of amino acid p-nitrophenyl esters catalyzed by bifunctional comicellar catalysts containing the imidazolyl and hydroxyl groups have been determined at pH 7.30, 0.02 m phosphate buffer, and 25°C. The kinetic analysis suggests a reaction scheme which involves acylation followed by deacylation at the imidazolyl group. Although no appreciable cooperative catalytic efficiencies are observed between the bifunctional groups in the acylation step, it is found that the deacylation rates are thus accelerated by surfactant hydroxyl groups, and some of the stereoselective acyl transfer reaction occurs from the imidazolyl to the hydroxyl group in optically active comicellar systems.