A stopped-flow mixer device for a batch microcalorimeter application to NAD-NADase reaction

A new molded polypropylene, diamond-like carbon (DLC)-coated mixing cell has been developed for use in the batch microcalorimeter. Reagent volume can be varied from 25 microliters to 100 microliters. A 10 microcalorie reaction heat can be measured to 5%. Repeat reactions can be done as often as every 10 min for a fast reaction. Reactions can be started within 1 h or less after loading. A pre-equilibrator and a temperature-controlled syringe drive unit permit solutions to be stored at 4 degrees C while being run at any temperature from -20 degrees C to 40 degrees C. The kinetics and enthalpy of reaction of NAD-NADase have been measured. delta H is about 21 kcal/mol endothermic.     

Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in vivo

The water-accessible volumes, the amounts of all significant osmolytes, and the protein concentration in the cytoplasm of aerobically grown Escherichia coli K-12 have been determined as a function of the osmolarity of the minimal growth medium. The volume of cytoplasmic water (Vcyto) decreases linearly with increasing osmolarity from 2.23(+/- 0.12) microliters/mg dry weight in cells grown at 0.10 OSM to 1.18(+/- 0.06) microliters/mg dry weight at 1.02 OSM. Above 0.28 OSM, growth rate decreases linearly with increasing osmolarity. The growth rate extrapolates to zero at an osmolarity of approximately 1.8, corresponding to an estimated Vcyto of 0.5(+/- 0.2) microliters/mg dry weight. Measurements of Vcyto in titrations of non-growing cells with the plasmolyzing agent NaCl were used to obtain volumes of "bound" water (presumably water of macromolecular hydration) and cytoplasmic osmotic coefficients for cells grown in medium of low (0.10 OSM) and moderate (0.28 OSM) osmolarity. The volume of bound water Vb is similar in the two osmotic conditions (Vb = 0.40(+/- 0.04) microliters/mg dry wt), and corresponds to approximately 0.5 g H2O/g cytoplasmic macromolecule. Since Vcyto decreases with increasing osmolarity, whereas Vb appears to be independent of osmolarity, water of hydration becomes a larger fraction of Vcyto as the osmolarity of the growth medium increases. Growth appears to cease at the osmolarity where Vcyto is approximately equal to Vb. K+ and glutamate (Glu-) are the only significant cytoplasmic osmolytes in cells grown in medium of low osmolarity. The amount of K+ greatly exceeds that of Glu-. Analysis of cytoplasmic electroneutrality indicates that the cytoplasm behaves like a concentrated solution of the K+ salt of cytoplasmic polyanions, in which the amount of additional electrolyte (K+ Glu-) increases with increasing osmolarity. As the osmolarity of the growth medium becomes very low, the cytoplasm approaches an electrolyte-free K+-polyanion solution. In vivo osmotic coefficients were determined from the variation of Vcyto with external osmolarity in plasmolysis titrations of non-growing cells. The values obtained (phi = 0.54(+/- 0.06) for cells grown at 0.10 OSM and phi = 0.71(+/- 0.11) at 0.28 OSM) indicate a high degree of non-ideality of intracellular ions arising from coulombic interactions between K+ and cytoplasmic polyanions. Analysis of these osmotic coefficients using polyelectrolyte theory indicates that the thermodynamic activity of cytoplasmic K+ increases from approximately 0.14 M in cells grown at an external osmolarity of 0.10 OSM to approximately 0.76 M at 1.02 OSM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heats of carbon monoxide binding by hemoglobin M Iwate.

The heat of reaction of CO gas with the alpha2Mmetbeta2 and alpha2Mbeta2 species of the alpha-chain mutant hemoglobin M Iwate has been studied in buffers with different heats of ionization of 25degrees and in the absence of organic phosphates. For the alpha2Mmetbeta2deoxy species we find a small Bohr effect (0.12 mol of H+/mol of CO) which is in correspondence with that found in equilibrium studies. The heat of reaction, when corrected for proton reaction with buffer, is -18.4 +/- 0.3 kcal/mol of CO at pH 7.4 At pH 9 the same value is observed within experimental error. This value compares closely with heats of reaction of CO with myoglobin and with van't Hoff determinations of the heat of oxygen binding to isolated hemoglobin alpha and beta chains after correction for the heat of replacement of O2 by CO. Furthermore, an analysis of the differential heat of ligand binding as a function of the extent of reaction indicated that, within experimental error, the heat of reaction with the first beta-chain heme in alpha2Mmetbeta2deoxy is the same as the second. Since the quaternary Tleads to R transition is blocked in this mutant hemoglobin, we compared it with Hb A to estimate the enthalpic component of the allosteric T leads to R transition in Hb A. The heats of reaction with CO(g) and Hb A are -15.7 +/- 0.5 and -20.9 +/- 0.5 kcal/mol at pH 7.4 and 9.0, respectively. In going from the T to the R state we find an enthalpy of transition of 9 +/- 2.5 kcal at pH 7.4 and -12 +/- 2.5 kcal at pH 9.0. From published free energies of transsition we conclude the T leads to R transition is enthalpically controlled at p/ 7.4 but entropically controlled at pH 9.0 A near normal Bohr effect is estimated from heats of reaction of CO with alpha2Mdeoxybeta2deoxy in various buffers. A large than normal heat of reaction (-21.6 +/- 0.5 kcal/mol of CO) is attributed to the abnormal alpha chains in Hb M Iwate.     

The enigma of conditioned taste aversion learning: stimulus properties of 2-phenylethylamine derivatives

The functional aversive stimulus properties of several IP doses of (+/-)-amphetamine (1.25-10 mg.kg-1), 2-phenylethylamine (PEA, 2.5-10 mg.kg-1, following inhibition of monoamine oxidase with pargyline 50 mg.kg-1) and phenylethanolamine (6.25-50 mg.kg-1) were measured with the conditioned taste aversion (CTA) paradigm. A two-bottle choice procedure was used, water vs. 0.1 % saccharin with one conditioning trial and three retention trials. (+/-)-Amphetamine and phenylethanolamine induced a significant conditioned taste aversion but PEA did not. (+/-)-Amphetamine and PEA increased spontaneous locomotor activity but phenylethanolamine had no effects on this measure. Measurement of whole brain levels of these drugs revealed that the peak brain elevation of PEA occurred at approximately 10 min whereas the peak elevations of (+/-)-amphetamine and phenylethanolamine occurred at approximately 20 min. The present failure of PEA to elicit conditioned taste aversion learning is consistent with previous reports for this compound. The differential functional aversive stimulus effects of these three compounds are surprising since they exhibit similar discriminative stimulus properties and both (+/-)-amphetamine and PEA are self-administered by laboratory animals. The present data suggest that time to maximal brain concentrations following peripheral injection may be a determinant of the aversive stimulus properties of PEA derivatives.     

Quantitative structure toxicity relationships for catechols in isolated rat hepatocytes

One- and two-parameter quantitative structure toxicity relationship (QSTR) equations were obtained to describe the cytotoxicity of isolated rat hepatocytes induced by 23 catechols in which LD(50) represents the catechol concentration required to induce 50% cytotoxicity in 2 h. A QSTR equation logLD(50) (microM = - 0.464(+/-0.065) log P + 3.724(+/-0.114) (n = 20, r(2) = 0.740, s(y,x) = 0.372, P < 1 x 10(-6), outliers: 4-methoxycatechol, 3-methoxycatechol, L-dopa) was derived where logP represents octanol/water partitioning. Outliers were determined by adopting a statistical method to standardize the identification of outliers. When pK(a1), the first ionization constant, was considered as a contributing parameter a two-parameter QSTR equation was derived: logLD(50) (microM = - 0.343(+/-0.058) log P - 0.116(+/-0.041) pK(a1)+4.389 (+/-0.315) (n = 22, r(2) = 0.738, s(y,x) = 0.375, P < 0.01, outlier: 4-methoxycatechol). Replacing logP with logD(7.4), the partition coefficient at pH 7.4, improved the first correlation by limiting the outlier to 4-methoxycatechol: logLD(50) (microM)=-0.252(+/-0.039) logD(7.4)+3.168(+/-0.090) (n = 22, r(2) = 0.671, s(y,x) = 0.420, P < 1 x 10(-5). In this study, 4-methoxycatechol (readily autooxidizable) was found to be an outlier for all QSTR equations derived. These findings point to lipophilicity and pK(a1) as two important characteristics of catechols that can be used to predict their cytotoxicity towards isolated rat hepatocytes. The catechols with the higher lipophilicity/distribution coefficient, the lower degree of ionization and the higher pK(a(catechol)) were more toxic towards hepatocytes than the other catechols.     

A microtiter plate assay for aspartate transcarbamylase

A discontinuous, colorimetric method for the assay of aspartate transcarbamylase has been adapted for use with 96-well microtiter plates. The method is based on that of L.M. Prescott and M.E. Jones (1969 Anal. Biochem. 32, 408-419) for the detection of ureido compounds, using monoxime and antipyrine. The enzymatic reaction is carried out in a volume of 150 microliters and is stopped by the addition of 100 microliters of a color mix. After development, the absorbance at 460 nm is directly proportional to the quantity of N-carbamyl-L-aspartate up to at least 0.125 mumol and to the quantity of Escherichia coli aspartate transcarbamylase up to about 7 ng. Kinetic parameters obtained from saturation curves for L-aspartate in 50 mM Tris-acetate, pH 8.0, are indistinguishable from those previously obtained: Vmax = 26,225 mumol h-1 mg-1; S0.5 = 14.7 mmol liter-1; hill constant = 2.5.     

Development in the cyclic hamster of refractoriness to the superovulatory action of anti-LH serum

Hamsters injected s.c. on the day of ovulation (Day 1) with 100 microliters equine anti-bovine LH serum ovulated 28 eggs at the end of a 5-day cycle. When a second injection of anti-LH serum was administered 4-93 days later, the animals did not superovulate and had normal 4-day cycles. Injection of 100 microliters normal rabbit serum (NRS) on Day 1 followed 14 days later by anti-LH serum resulted in the ovulation of 32 ova whereas a priming injection of 100 microliters normal horse serum (NHS) followed by anti-LH serum resulted in the ovulation of only 18 ova. When hamsters were injected on Day 1 with anti-LH serum, NHS or NRS and then with anti-LH serum in the 4th cycle, high titres of free antibodies to LH were present on Days 2-4 only in the animals treated with NRS; these hamsters ovulated a mean of 35 ova. These experiments suggest that the hamster rapidly forms antibodies to equine immunoglobulins, thus preventing a second injection of anti-LH serum from inducing superovulation.     

The association of yeast phosphoglycerate kinase (EC 2.7.2.3).

1. A mol.wt. of 40030 +/- 830 has been estimated for phosphoglycerate kinase in concentrations less than 0.1 g/100 cm3 comparing favourably with expected values from X-ray diffraction measurements by 10% lower than the previously reported molecular weights made at higher concentrations. 2. The so20w, was estimated to be 3.12(+/-0.02)x10(-13)s and the coefficient had a low concentration dependency giving a g value (concentration-dependency) of 2.3 +/- 1.6cm3 .g-1. This agrees with previous qualitative observations. 3. By using fluctuation-intensity spectroscopy, the D20,w was estimated to be 7.4(+/-0.2)x10(-11)m2.s-1, and this was indistinguishable from the D20,w calculated from ultracentrifuge results. The water of hydration was estimated to be 0.46 g/g of protein. 4. It is inferred from the estimates that phosphoglycerate kinase associates with an interaction coefficient at 20 degrees C for monomer/dimer of between 10 and 12 cm3.g-1. 5. The ratio of molecular asymmetry (a/b) was estimated to be 2.5+/-0.2 from the values of D20,w and water of hydration. This compares favourably with the ratio from the overall dimensions estimated from X-ray diffraction measurements.     

Assay of alpha 1,3 N-acetyl-D-galactosaminyl transferase by affinity chromatography.

A high-performance liquid affinity chromatography column that contains immobilized anti-A monoclonal antibody specifically retards blood group A-active oligosaccharides and can be used to detect the product(s) of the reaction catalyzed by alpha-1,3-N-acetyl-D-galactosaminyltransferase: [formula: see text] After a brief incubation (15 min) of an assay mixture containing 1-100 microliters human serum, the sugar nucleotide donor UDP-GalNAc, and radiolabeled oligosaccharide acceptors 2'-fucosyllactose and/or lacto-N-fucpentaose I blood group A-active products are isolated and quantitated in a single affinity chromatographic step that takes less than 30 min. Kinetic studies to determine the pH optima for serum alpha-3-GalNAc transferase from individuals of blood groups A1 and A2 and the Km value for UDP-GalNAc for the A1 transferase agree with previous determinations. As monoclonal antibodies against many different complex carbohydrate antigens are now available, the method described could be adapted to give rapid, inexpensive assays for a variety of glycosyltransferases.     

A laboratory hot tub model for disinfectant efficacy evaluation

This paper describes a novel laboratory hot tub (LHT) apparatus and associated standard operating procedure (SOP) designed to reproduce the key biological, chemical, and engineering parameters associated with recreational and therapeutic hot tubs. Efficacy, as measured quantitatively by log reduction values, was determined against both biofilm and planktonic bacteria. When the LHT was run according to the SOP, with no antimicrobial treatment, a consistent level of bacterial contamination occurred. The means of log10 viable cell densities (+/- the repeatability standard deviation of log densities) were 7.2 (+/-0.31) for the bulk water (density in units of cfu ml-1), 5.3 (+/-0.56) for the coupons (density in units of cfu cm-2), and 6.6 (+/-0.50) for the filters (density in units of cfu cm-2). When control and chlorine treated LHTs were run in parallel, the log reduction increased significantly with chlorine concentration for samples of planktonic bacteria in the bulk water (p=0.016), biofilm bacteria on the coupons (p=0.09) and biofilm bacteria on the filter (p=0.005), indicating that the method was sensitive to chlorine concentration. The method also displayed sensitivity by differentiating between chlorine and bromine treatments; in every case, chlorine produced a greater log reduction than did the same concentration of bromine. The model and SOP were shown to be rugged with respect to slight changes in fluid mixing intensity, water chemistry (saturation index), inoculum size, and organic loading. The LHT and associated SOP provide a reliable second tier in a three-tiered testing process, in which the first tier is a suspension test and the final tier is a field test.     

Hofmeister effects of anions on the kinetics of partial reactions of the Na+,K+-ATPase.

The effects of lyotropic anions, particularly perchlorate, on the kinetics of partial reactions of the Na+,K+-ATPase from pig kidney were investigated by two different kinetic techniques: stopped flow in combination with the fluorescent label RH421 and a stationary electrical relaxation technique. It was found that 130 mM NaClO4 caused an increase in the Kd values of both the high- and low-affinity ATP-binding sites, from values of 7.0 (+/- 0.6) microM and 143 (+/- 17) microM in 130 mM NaCl solution to values of 42 (+/- 3) microM and 660 (+/- 100) microM in 130 mM NaClO4 (pH 7.4, 24 degrees C). The half-saturating concentration of the Na+-binding sites on the E1 conformation was found to decrease from 8-10 mM in NaCl to 2.5-3.5 mM in NaClO4 solution. The rate of equilibration of the reaction, E1P(Na+)3 left arrow over right arrow E2P + 3Na+, decreased from 393 (+/- 51) s-1 in NaCl solution to 114 (+/- 15) s-1 in NaClO4. This decrease is attributed predominantly to an inhibition of the E1P(Na+)3 --> E2P(Na+)3 transition. The effects can be explained in terms of electrostatic interactions due to perchlorate binding within the membrane and/or protein matrix of the Na+,K+-ATPase membrane fragments and alteration of the local electric field strength experienced by the protein. The kinetic results obtained support the conclusion that the conformational transition E1P(Na+)3 --> E2P(Na+)3 is a major charge translocating step of the pump cycle.     

DNA synthesis in a cell-free system from Xenopus eggs: priming and elongation on single-stranded DNA in vitro

We describe a eucaryotic in vitro system for DNA replication derived from Xenopus eggs. In this system, priming and elongation of DNA chains occurs with unusually high efficiency on single-stranded circular DNA templates. Up to 1.5 micrograms M13 DNA can be converted to a completely double-stranded form by 100 microliters egg extract in 1 hr at 22 degrees C, a rate of synthesis comparable with the fastest rates of chromosomal DNA synthesis in early embryogenesis. Initiation of DNA synthesis on double-stranded circular DNA templates was undetectable however. The enzymatic events responsible for complementary-strand synthesis in vitro resemble those presumed to act at the lagging strand of the eucaryotic replication fork in vivo in three ways. First, inhibitor studies indicate that DNA polymerase alpha is required. Second, priming of DNA synthesis by oligoribonucleotides is strongly supported by the complete dependence on ribonucleoside triphosphates in the assay, and the detection of an oligoribonucleotide terminus of 9 or possibly 10 nucleotides associated with nascent DNA chains. Third, the priming reaction is resistant to alpha-amanitin.     

Stereochemistry of catalysis by the ketoreductase activity in the first extension module of the erythromycin polyketide synthase

Multiple ketoreductase activities play a crucial role in establishing the stereochemistry of the products of modular polyketide synthases (PKSs), but there has been little systematic scrutiny of catalysis by individual ketoreductases. To allow this, a diketide synthase, consisting of the loading module, first extension module, and the chain-terminating thioesterase of the erythromycin-producing PKS of Saccharopolyspora erythraea, has been expressed and purified. The DNA encoding the ketoreductase-1 domain in this construct is flanked by unique restriction sites so that another ketoreductase domain can be readily substituted. The purified recombinant diketide synthase catalyzes, at a very low rate (k(cat) equals 2.5 x 10(-3) s(-1)), the specific production of the diketide (2S,3R)-2-methyl-3-hydroxypentanoic acid. The activity of the ketoreductase domain in this model synthase was analyzed using as a model substrate (+/-)-2-methyl-3-oxopentanoic acid N-acetylcysteaminyl (NAC) ester for which k(cat)/K(m) was 21.7 M(-1) s(-1). The NAC thioester of (2S,3R)-2-methyl-3-hydroxypentanoic acid was the major product and was strongly preferred over other stereoisomers as a substrate in the reverse reaction. The bicyclic ketone (9RS)-trans-1-decalone, a known substrate for ketoreductase in fatty acid synthase, was found also to be an effective substrate for the ketoreductase of the diketide synthase. Only the (9R)-trans-1-decalone was reduced, selectively and reversibly, to the (1S,9R)-trans-decalol. The stereochemical course of reduction and oxidation is exactly as found previously for the ketoreductase of animal fatty acid synthase, an additional indication of the close similarity of these enzymes.     

Photooxidation of pterin in aqueous solutions: biological and biomedical implications

Studies of the photochemical reactivity of pterin (= 2-aminopteridin-4(3H)-one; PT) in acidic (pH 5.0-6.0) and alkaline (pH 10.2-10.8) aqueous solutions have been performed. The photochemical reactions were followed by UV/VIS spectrophotometry, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), and an enzymatic method for H2O2 determination. PT is not light-sensitive in the absence of molecular oxygen, but it undergoes photooxidation in the presence of O2, yielding several nonpteridinic products. The quantum yields for PT disappearance were found to be 8.2 (+/-0.6) x 10(-4) and 1.2 (+/-0.2) x 10(-3) in acidic and alkaline media, respectively. H2O2 was detected and quantified in irradiated solutions of PT; and its importance from a biomedical point of view is discussed. The rate constant of the chemical reaction between singlet oxygen ((1)O2) and PT was determined to be 2.5 (+/-0.2) x 10(5) l mol(-1) s(-1) in alkaline medium, and the role of (1)O2 in the photooxidation of pterin was evaluated.     

Application of differential flow microcalorimetry for study of drug interactions in the blood system

A compact differential flow microcalorimeter has been developed to investigate biomolecular reactions, especially drug interactions in the blood system. The calorimeter is an adiabatic type and consists of a twin-cell structure, each mixing part having a volume of 60 microliters. Both the precision and accuracy of the instrument have been evaluated by dilution of sucrose solutions to be 0.1-0.5% at a heat effect of 100-10 microW. The resolution is approximately 0.5 microW (less than 10(-3) Torr). The heat produced in erythrocyte hemolysis induced by chlorpromazine hydrochloride (CPZ) and the binding heat of CPZ to human blood components viz., intact erythrocytes, erythrocyte membranes, serum albumin and plasma were measured. The heat effect of hemolysis was endothermic and related to the quantity of free hemoglobin released from erythrocytes. The overall binding of CPZ to blood components was, however, an exothermic process. The thermodynamic and binding parameters were computed directly from the calorimetric data by use of a nonlinear least squares regression method, assuming a one-class binding model, and the stoichiometry of the binding reaction was determined.     

Modulation of dopamine release by the nicotinic agonist epibatidine in the frontal cortex and the nucleus accumbens of naive and chronic nicotine treated rats

The effect of the nicotinic acetylcholine receptors (nAChRs) agonist (+/-)epibatidine on the modulation of dopamine (DA) release was investigated by microdialysis in vivo in the frontal cortex and the nucleus accumbens of naive and chronic nicotine-treated awake rats. (+/-)Epibatidine (2.5 microg/kg, s.c.), contrary to (-)nicotine (0.5 mg/kg, s.c.), decreased the extracellular concentrations of DA in the brain of naive rats. Subchronic nicotine treatment (0.45 mg/kg, s.c., twice daily for 7 days) attenuated the (+/-)epibatidine induced decrease in the DA level. The extracellular concentrations of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were elevated by (+/-)epibatidine administration in both na?ve and subchronic treated rats. The findings suggest that the decrease in DA extracellular concentrations induced by the high affinity nAChRs agonist (+/-)epibatidine might be due to inactivation of nAChRs, which can be overcome by subchronic treatment with nicotine. Different mechanisms in modulation of DA release appears to be involved in the rat brain by (+/-)epibatidine compare to (-)nicotine.     

Concentration of bronchoalveolar lavage fluid by ultrafiltration: evidence of differential protein loss and functional inactivation of proteinase inhibitors

Bronchoalveolar lavage samples were concentrated using positive-pressure ultrafiltration. The starting material, concentrates, and eluates were assayed for immunoglobulin A (IgA), albumin (Alb), alpha 1-proteinase inhibitor (alpha 1-PI), antileukoprotease (ALP), and total leukocyte elastase inhibitory capacity (LEIC). No enzyme inhibitory capacity or protein was detected in membrane eluates, confirming the selectivity of the membrane used (Mr cutoff 2000 or 500). However, the concentrated lavages showed a generated loss of protein. The proportion of each protein recovered using the 500 Mr cutoff membrane was: IgA, 50.6% (+/- 15%); albumin, 43% (+/- 8.4); alpha 1-PI, 53.6 (+/- 17.3); ALP, 43% (+/- 2.1); and LEIC, 18.4% (+/- 2.6). Similar results were obtained with the 200 Mr cutoff membrane. The alpha 1-PI/Alb and the IgA/alb ratios were higher (2P less than 0.05) in the concentrates than in the starting material, suggesting differential protein loss. Protein losses were due to binding to the membrane since the wash with saline solution improved recoveries: IgA, 80%; Alb, 56%; alpha 1-PI, 64%; ALP, 66%; LEIC, 29%. Concentration of bronchoalveolar lavage fluids therefore resulted in substantial differential losses in elastase inhibitory capacity and protein concentrations, suggesting analysis of these fluids should be performed on unconcentrated samples.     

Investigation by luminal perfusion of the transfer of compounds into the epididymis of the anaesthetized rat.

Controlled perfusion through the lumen of the distal cauda epididymidis in the anaesthetized rat has been explored as a means of examining physiological exchanges from blood across the epididymal epithelium. The mean length of the perfused, sperm-free, tubule was 14.5 cm (+/- 1.5 s.e.m., n = 9). No cholesterol, protein or sialic acid was detected in the perfusate at flow rates exceeding 10 microliters/min, but at rates of 0.4--1.2 microliters/min, protein appeared at concentrations of 0.21--0.55 mg/ml (i.e. secretion rates of 0.21--0.83 micrograms/min; 3 rats). Glucose was detected at all perfusion rates (3--27 microliters/min) at concentrations of 0.06--0.58 mM (0.8--6.8% blood levels). During intravenous infusions of 3H2O, radioactivity in the perfusate rapidly attained 87% blood plasma concentrations; no radioactivity was detected when carboxy-E114C]dextran or methoxy-[3H]inulin were infused. Radioactivity appeared in the epididymal perfusate to 1--7% of blood levels during intravenous infusions of D-E1U-1RC]glucose or 3-O-methyl[1-3H]glucose. This evidence suggests that the preparation is physiological and could be used to explore the dynamics of exchanges between blood and epididymis.     

Microquantitative determination of Pi-ATP and ADP-ATP exchange kinetics using thin-layer chromatography on silica gel

A new method for determination of 32Pi-ATP and [14C]ADP-ATP exchange rates is described. It is based upon separation of nucleotides and Pi by thin-layer chromatography on commercial aluminum or plastic sheets precoated with silica gel. The method permits avoiding special procedures for stopping of the reaction and for preparation of aliquots for thin-layer chromatography separation. It also allows to separate all adenine nucleotides and Pi in one chromatography procedure, and to work with a double label. The volume of the reaction mixture was 50-100 microliters. Aliquots (2-6 microliters) of the reaction mixture were taken at various moments without stopping the reaction and were layered immediately on a heated silica gel sheets. The nucleotides and Pi were separated in a solvent system which consisted of dioxane, isopropanol, 25% ammonia, and water (4:2:3:4, v/v). The nucleotide spots were detected in ultraviolet light, cut out, and their radioactivity was measured with a liquid scintillation counter. The method for measurement of kinetics of exchange reactions catalyzed by reconstituted into liposomes H+-ATPase complexes from beef heart mitochondria and high plant chloroplasts, is described.     

A rearranging ligand enables allosteric control of catalytic activity in copper-containing nitrite reductase

In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type-1 site was replaced (M150G) to make the copper ion accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A(460)/A(600)=0.71) differs significantly from that of the native nitrite reductase (A(460)/A(600)=1.3). The midpoint potential of the type-1 site of nitrite reductase M150G (E(M)=312(+/-5)mV versus hydrogen) is higher than that of the native enzyme (E(M)=213(+/-5)mV). M150G has a lower catalytic activity (k(cat)=133(+/-6)s(-1)) than the wild-type nitrite reductase (k(cat)=416(+/-10)s(-1)). The binding of external ligands to M150G restores spectral properties, midpoint potential (E(M)<225mV), and catalytic activity (k(cat)=374(+/-28)s(-1)). Also the M150H (A(460)/A(600)=7.7, E(M)=104(+/-5)mV, k(cat)=0.099(+/-0.006)s(-1)) and M150T (A(460)/A(600)=0.085, E(M)=340(+/-5)mV, k(cat)=126(+/-2)s(-1)) variants were characterized. Crystal structures show that the ligands act as allosteric effectors by displacing Met62, which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that removal of an endogenous ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of copper-containing redox sites.