Determination of butaperazine in plasma and red blood cells by fluorometry

A simple and sensitive fluorometric method is described for the determination of butaperazine in plasma and red blood cells. This method is specific for butaperazine and reproducible over a wide range of drug levels in the blood. The sensitivity of the method is 8 ng/ml of blood, but could be increased for determining much lower levels.     

Evidence for three enzymatic activities in one electrophoretic band of 3-phosphoglycerate mutase from red cells.

Electrophoresis of 3-phosphoglycerate mutase from erythrocytes of man and several animal species has been performed on cellulose acetate strips. In most cases the electrophoretic pattern of this enzymatic activity shows three bands. 2,3-diphosphoglycerate phosphatase and diphosphoglycerate mutase from erythrocytes of the same species have been revealed after migration during the same electrophoresis. We found that the band of 2,3-diphosphoglycerate phosphatase and the band of diphosphoglycerate mutase activities migrate at the same level as one of the bands corresponding to 3-phosphoglycerate mutase. Here, we discuss the possible existence of a single molecule carrying three enzymatic activities.     

Interaction between heparin and human pregnancy-associated plasma protein A (PAPP-A): a simple purification procedure

Human pregnancy-associated plasma protein A (PAPP-A) binds to heparin-Sepharose. This affinity chromatography preceded by molecular sieve chromatography provides a simple two-step purification procedure of PAPP-A from late pregnancy plasma. One hundred percent of the applied PAPP-A was recovered, with more than 40% being electrophoretically homogeneous after the two procedures. The remaining PAPP-A could be purified by negative affinity chromatography on anti-total human serum immobilized on agarose.     

Effect of hypoxic cell radiosensitizers on glutathione level and related enzyme activities in isolated rat hepatocytes

A comparative study of the effect of misonidazole and novel radiosensitizers on glutathione (GSH) levels and related enzyme activities in isolated rat hepatocytes was performed. Incubation of hepatocytes with 5 mM radiosensitizers led to a decrease in the intracellular GSH level. The most pronounced decrease in cellular GSH was evoked by 2,4-dinitroimidazole-1-ethanol (DNIE); after incubation for only 15 min, GSH was hardly detected. DNIE-mediated GSH loss was dependent upon its concentration. DNIE reacted with GSH nonenzymatically as well as with diethylmaleate, while misonidazole and 1-methyl-2-methyl-sulfinyl-5-methoxycarbonylimidazole (KIH-3) did not. Addition of partially purified glutathione S-transferase (GST) did not enhance DNIE-mediated GSH loss in a cell-free system. DNIE inhibited glutathione peroxidase (GSH-Px), GST, and glutathione reductase (GSSG-R) activities in hepatocytes, while misonidazole and KIH-3 did not. GSH-Px activity assayed with H2O2 as substrate was the most inhibited. Inhibition of GSH-Px activity assayed with cumene hydroperoxide as substrate and GST was less than that of GSH-Px assayed with H2O2 as substrate. GSSG-R activity was decreased by DNIE, but not significantly. Incubation of purified GSH-Px with DNIE resulted in a little change in the activity when assayed with H2O2 as substrate.     

Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with n_H = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mm). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (K_i = 120 μm). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 μmol of glucose/h/ml red blood cells (RBC) compared with values of 0.56–2.85 μmol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mm against a plasma level of 5 mm). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.     

A method for measuring anion transfer across red cell membranes by continuous monitoring of fluorescence

A method was developed for the identification of Ca²⁺-binding proteins after electrophoresis on polyacrylamide gels. The method involves equilibration of the gel with ⁴⁵Ca either during or after electrophoresis, followed by visualization of the ⁴⁵Ca-binding proteins by autoradiography.     

Enhanced chondrocytic differentiation in chick limb bud cell cultures by inhibitors of poly(ADP-ribose) synthetase

Inhibitors of poly(ADP-ribose) synthetase, namely nicotinamide, benzamide, m-methoxybenzamide and 3-aminobenzamide, augmented chondrocytic differentiation chick embryo limb bud mesenchymal cells, in culture. These inhibitors stimulated early appearance and massive formation of cartilage nodules in micromass cultures stage 23-24 chick embryos. They also induced nodule formation in micromass and cartilage colonies at micromass plating densities from stage 18-19 embryo Benzamide, however, did not prevent differentiated chondrocytes from undergoing a pleiotypic change in cell type. These results are compatible with the putative regulatory function of poly(ADP-ribose) on cell differentiation.     

New steroids with antiprogestational and antiglucocorticoid activities

A number of 11-substituted 19-norsteroids with inverse configuration at C-13 were synthesized. 11 beta-Aryl compounds in this series were found to possess antiprogestational and antiglucocorticoid activities.     

A mathematical model describing the effect of temperature and substrate concentration on the activities of M4 and H4 lactate dehydrogenase from the big brown bat (Eptesicus fuscus)

The effect of temperature on the activities of M₄ and H₄ lactate dehydrogenases (LDH, EC 1.1.1.27) isolated from the big brown bat (Eptesicus fuscus) was examined. Temperature effects were dependent on the concentrations of all four LDH substrates, pyruvate, lactate, NADH, and NAD. Arrhenius plots of In v_i vs reciprocal of absolute temperature were linear for all but the lowest substrate concentrations. The slopes of these Arrhenius plots were used to calculate the temperature effect parameter (μ). Substrate-dependent temperature effects for M₄ and H₄ LDH were described by an equation for a rectangular hyperbola, $\text{μ =}\text{[E}{}_{\mathrm{\beta }}\text{S + E}{}_{\mathrm{\alpha }}\text{K}{}_{\mathrm{t}}\text{]}\text{[K}{}_{\mathrm{t}}\text{+ S]}$ proposed by G. R. Harbison and J. R. Fisher (1974, Comp. Biochem. Physiol.47B, 27–32) for adenosine deaminase. The parameters E_α (μ at infinitely low substrate concentration), E_β (μ at infinitely high substrate concentration), and K_t (the concentration of substrate when $\text{μ =}\text{[E}{}_{\mathrm{\alpha }}\text{+ E}{}_{\mathrm{\beta }}\text{]}\text{2}$) can be used to describe the temperature dependence of LDH activity at any substrate concentration and to compare the substrate-dependent temperature effects on the two isoenzymes. Significantly different E_β and K_t values for pyruvate-dependent temperature effects and different E_β, E_α, K_t, and E_β ? E_α (the range of possible μ values) for lactate-dependent temperature effects were found between M₄ and H₄ LDH isoenzymes. High lactate concentrations inhibited bat H₄ LDH activity to a greater degree at low temperatures than at high temperatures. Thus substrate inhibition plays an important role in the effect of temperature on the activity of H-type LDH at high lactate concentrations. Substrate-dependent temperature effects on bat LDH activity were the result of temperature effects on the apparent K_m value of the respective substrate. Since both the apparent K_m for pyruvate and the K_i for the competitive inhibitor oxamate decreased with decreasing temperature, the substrate-dependent temperature effects observed for pyruvate probably resulted from an increased affinity between pyruvate and the LDH-NADH complex with decreasing temperature.     

Inhibition by ultraviolet light of pole cell formation in Smittia sp (Chironomidae,Diptera): Action spectrum and photoreversibility

The formation of pole cells (primordial germ cells) in Smittia sp can be inhibited by ultraviolet (uv) irradiation without causing significant mortality. Until 70 min after egg deposition, pole cells are suppressed by low uv doses applied to the posterior pole region. Microbeam irradiation of a target area including the oosome inhibits pole cell formation; this is not observed after irradiation of other target areas. The action spectrum for uv inhibition of pole cells shows a distinct peak at 260 nm; its shape suggests that a nucleic acid or nucleic acid-protein complex acts as an effective target. Independent evidence for the involvement of a nucleic acid moiety is derived from the fact that uv inhibition of pole cell formation is photoreversible. The results are discussed in the context of pole cell determination by localized cytoplasmic components.     

The interaction of cyanocobalamin and some of its analogs with manganese(II) and gadolinium(III)

The influence of the paramagnetic ions Mn²⁺ and Gd³⁺ on the proton-decoupled ¹³C nuclear magnetic resonance spectra of three cyanocobalamin-monocarboxylic acids has been observed. The paramagnetic ions bind preferentially to the free carboxylate groups and cause the broadening of specific carbon resonances adjacent to these groups. These specific line-broadening effects have been used to assign the carbonyl carbons of the a-, f-, and g-acetamide side chains and have allowed us to confirm and/or correct the assignments of several carbon resonances that were assigned tentatively before.     

Quantitative resolution of Cu,Zn- and Mn-superoxide dismutase activities

Assays of superoxide dismutase on total soluble extracts of eucaryotic cells generally result in the sum of activities of the Cu,Zn- and Mn-enzymes. Accurate quantitative resolution of the total activity on the basis of inhibition of the Cu,Zn-enzyme with cyanide may require that a correction is introduced for incomplete saturation with inhibitor at the finite concentration of cyanide used. This correction is calculated using the apparent dissociation constant of one to one complex formation with cyanide, K′, under the conditions of the assay. A linear relationship describing the activity in the presence of graded concentrations of cyanide enables the determination of K′ on soluble extracts without isolation of the Cu,Zn-enzyme. An evaluation of the precision, based on a propagation of error analysis, of different methods for the quantitative resolution of the two dismutases is presented. This reveals most notably that the random error in the resolution in terms of McCord and Fridovich activity units is reduced by carrying out the resolution at high pH (where a lower K′ for cyanide applies) followed by the conversion of the result to pH 7.8.     

Crystal and molecular structure of [Cu(5'-inosine monophosphate) (dipyridylamine) (H2O)]2.4H2O.

The ternary complex [Cu(5′-IMP)(dpa)(H₂O)]₂ has been prepared and its structure analyzed by x-ray diffraction. It has a dimeric structure in which the 5′-IMP ligands coordinate solely through their phosphate groups. This geometry is in marked contrast to that of another $\text{Cu}\text{5′-}\text{IMP}$ ternary complex, [Cu(5′-IMPH)(bipy)(H₂O)₂]⁺, which shows metal binding through the purine base rather than the phosphate group.     

X-ray crystal and molecular structures of the Ni(II) and Co(II) complexes of 2'-deoxyguanosine-5'-monophosphate.

The structures of [Ni(5′-dGMP)(H₂O)₅] and [Co(5′-dGMP)(H₂O)₅] have been solved by single-crystal x-ray diffraction techniques. Their common geometry consists of a metal ion octahedrally coordinated to the N₇ atom of guanine and five water ligands. The phosphate group of the nucleotide is hydrogenbonded to two of the coordinated water molecules.     

Multiple deoxyribonuclease activities in nuclei of HeLa cells

Extracts of purified HeLa nuclei contain DNase activities which can be separated by CM-Sephadex column chromatography into four distinct enzymes. These DNases differ in their resistance to thermal inactivation and relative activity on different substrates. Each of these DNases can increase the template-primer activity of native DNA for DNA polymerase action.     

Fluorometric assay of kininase activities in rat tissues

A simple method for the assay of bradykinin (BK)-degrading enzymes was investigated. The procedure of the method includes enzymatic degradation of BK, separation of the residual BK on a small P-cellulose column (0.6 X 3 cm), and its fluorometrical determination based on the reaction with fluorescamine. BK was separated completely from its fragments produced during enzymatic reaction by the column chromatography. The recovery rate of BK was 96 +/- 3%. Quantitative determinations could be carried out on 0.2 nmol of BK, at least in the fluorometry. This method was available for the assay of the enzymes in tissue homogenates as well as in purified preparations, and its usefulness for the study of the enzymes is presented.     

Effect of nitration on prolactin activities.

The seven tyrosines of ovine prolactin were modified with tetranitromethane and the resulting products were assayed for α-lactalbumin production in a mouse mammary gland explant assay system, for binding activity in a radioreceptor assay, and by radioimmunoassay. The five tyrosines which were exposed can be nitrated without loss of activity. The two remaining tyrosines can be nitrated only under denaturing conditions, a reaction that caused a loss of binding and biological activities. The loss of activity was not a consequence of the denaturants but was due to modification of either or both of the two relatively unreactive tyrosines. It is postulated that this activity loss is the result of alterations of conformation rather than the modification of a tyrosine which is in contact with the prolactin receptor.