bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of formation of the primary compound (compound I) from hydrogen peroxide and turnip peroxidases
Authors: Job Dominique , Ricard Jacques , Dunford H. Brian
Published: December 2009

A kinetic study of the reaction of two turnip peroxidases (P1 and P7) with hydrogen peroxide to form the primary oxidized compound (compound I) has been carried out over the pH range from 2,4 to 10.8. In the neutral and acidic pH regions, the rates depend linearly on hydrogen peroxide concentration whereas at alkaline pH values the rates display saturation kinetics. A comparison is made with the cyanide binding reaction to peroxidases since the two reactions are influenced in the same manner by ionization of groups on the native enzymes. Two different ionization processes of peroxidase P1 with pKa values of 3.9 and 10 are required to explain the rate pH profile for the reaction with H2O2. Protonation of the former group and ionization of the latter causes a decrease in the rate of reaction of the enzyme with H2O2. In the case of peroxidase P7 a minimum model involves three ionizable groups with pKa values of 2.5, 4, and 9. Protonation of the former two groups and ionization of the latter lowers the reaction rate. In the pH-independent region, the rate of formation of compound I was measured as a function of temperature. From the Arrhenius plots the activation energy for the reaction was calculated to be 2.9 ± 0.1 kcal/mol for P1 and 5.4 ± 0.3 kcal/mol for P7. However, the rates are independent of viscosity in glycerol–water mixtures up to 30% glycerol.
Wheat embryo ribonucleates. XII. Formal characterization of terminal and penultimate nucleoside residues at the 5′-ends of 'capped' RNA from imbibing wheat embryos
Authors: Haffner M. H. , Chin M. B. , Lane B. G.
Published: December 2009

(1) N2,N2,7-Trimethylguanosine, not previously detected as a component of plant RNA, is shown to be present in the RNA which is isotopically labelled when dry wheat embryos imbibe water in a medium that contains [methyl-3H]methionine.(2) N2,N2,7-Trimethylguanosine and 7-methylguanosine are released as part of 'capped' oligonucleotides when the isotopically labelled RNA from imbibing wheat embryos is subjected to hydrolysis by RNase T2.(3) By way of contrast with the 'capped' RNA of animal cells, 5′-terminal 'cap' structures (m7Gppp- and m32,2,7Gppp-) in the 'capped' RNA from the higher plant organism are not bonded to penultimate O2′-methylnucleoside constituents.(4) In an allied study, it has been found that recovery of poly(A)-rich RNA from dry wheat embryos depends on the inclusion of sodium dodecyl sulphate (SDS) in phosphate-buffered (pH 6.8) phenolic emulsions. By way of contrast, recovery of poly(A)-rich RNA from dry wheat embryos does not depend on the inclusion of SDS in Tris(hydroxymethyl)aminomethane buffered (pH 9.0) phenolic emulsions.
Identification of the 5S RNA binding site in intermolecular complexes of wheat embryo ribosomal 5S and 18S RNA
Authors: Nichols J. L. , Wijesinghe W.
Published: December 2009

The nucleotide sequence in wheat embryo ribosomal 5S RNA that binds by complementary base pairing to ribosomal 18S RNA has been identified. The 5S–18S RNA complex was formed in vitro and the interacting regions of the two RNA chains were isolated by a combination of nuclease digestion and electrophoresis on polyacrylamide gels. The binding site is close to the 3′-end of the 5S RNA molecule and contains a maximum of 24 nucleotides.
Cell-free synthesis of cod preproinsulin
Authors: Hew Choy L.
Published: December 2009

Poly(A)-containing ribonucleic acid from cod islet greatly stimulated the incorporation of radioactive amino acids into proteins when assayed in a wheat germ translation system. The translation products were examined by specific immunoprecipitation with guinea pig anti cod insulin antibodies and by extraction with acid–ethanol. These measurements revealed at least a fivefold increase in incorporation of labelled amino acid over the nonprogrammed system. Sodium dodecyl sulfate disc gel electrophoresis and gel filtration chromatography showed a product of molecular weight 12 500, a size considerably larger than cod proinsulin (9000). It is concluded that cod proinsulin is synthesized via a larger precursor, preproinsulin.
Investigation of polyene macrolide antibiotic-induced permeability changes in vesicles by 31P nuclear magnetic resonance
Authors: Pierce Jr. H. D. , Unrau A. M. , Oehlschlager A. C.
Published: December 2009

The permeability of egg yolk lecithin (EYL) vesicles to Pr3+ has been measured by 31P nuclear magnetic resonance (nmr) spectroscopy. Measurable Pr3+ leakage into the internal aqueous compartment of EYL vesicles at ambient (21 °C) temperature required the presence of small (7–10 mol%) amounts of dicetyl phosphate (DCP). The permeability of DCP-containing vesicles is decreased by incorporation of sterol (cholesterol > ergosterol ≈ 5,6-dihydroergosterol > zymosterol) into the lipid bilayer.Addition of the polyene macrolide antibiotic, nystatin, to DCP-containing EYL vesicles with and without sterol resulted in increased Pr3+ permeability at the three temperatures studied (21–37.5 °C). Permeability changes observed upon addition of nystatin to sterol-impregnated, DCP-containing vesicles varied with sterol structure: ergosterol ≈ 5,6-dihydroergosterol > cholesterol ≈ zymosterol. These results are compared with other polyene macrolide induced permeability changes on model and natural membrane systems. Permeability changes induced by nystatin in sterol-free EYL vesicles were generally greater than for comparable sterol-containing vesicles. This is attributed to a nonspecific interaction of the antibiotic with the latter vesicles.
Analysis of plant genomes. IV. Isolation and characterization of satellite DNA components from two dicotyledons cucumber (Cucumis sativus) and radish (Raphanus sativus)
Authors: Ranjekar P. K. , Pallotta D. , Lafontaine J. G.
Published: December 2009

Satellite DNA fractions from cucumber and radish, two plants having low DNA contents and relatively small chromosomes, were isolated and characterized. Reassociation studies of satellite and total nuclear DNA showed that the satellite fractions in these two plants contain most of the rapidly reassociating DNA. Cucumber satellite I was found to contain one major component (70% of the total satellite) having a density of 1.706 g/cm3 and a Tm of 90.5 °C and a minor component with a density of 1.712 g/cm3 and a Tm of 93.5 °C. The complexity of the major component was estimated to be 3.8 × 105 daltons while that of the minor one was 12.9 × 107 daltons. Although cucumber satellite II banded as a single peak at adensity of 1.700 g/cm3 in neutral CsCl gradients, it was observed to have a rather broad denaturation profile with a Tm of 86.5 °C. Its Cot curve was also broader than that of satellite I and one of its components (40% of the total) had a complexity of 5.8 × 105 daltons. Two satellite fractions were also observed in the case of radish DNA but only satellite I was isolated in a pure form and characterized. This radish satellite formed a sharp, symmetrical peak at a density of 1.698 g/cm3 in neutral CsCl gradients and underwent denaturation in a narrow temperature range of 6 to 7 °C. An analysis of the optical reassociation kinetics showed that this satellite contained a major and a minor component. The major component, which comprised 80% of the satellite, had a complexity of 12.9 × 105 daltons. Hybridization experiments revealed that the ribosomal DNA was present in satellite II.
Actin-like and tubulin-like proteins in synaptic junctional complexes
Authors: Mushynski Walter E. , Glen Sylvia , Thérien Hélène-Marie
Published: December 2009

Synaptic junctional complexes (SJCs), isolated by a procedure which preserves presynaptic dense projections (PDPs) contain as their major component a polypeptide (P55) which comigrates with tubulin on sodium dodecyl sulfate - polyacrylamide gels and another major polypeptide (P45) which comigrates with muscle actin. We report here the characterization of P55 and P45 by two-dimensional polyacrylamide gel electrophoresis and by peptide mapping of the radioiodinated polypeptides. Our results indicate that P55 and P45 are homologous with tubulin and actin respectively, in terms of molecular weight, protein charge, and primary structure. Comparison of the sodium dodecyl sulfate - polyacrylamide gel electrophoresis patterns of SJCs and postsynaptic densities (PSDs) indicates that P55 and P45 are associated mainly with PDPs but are also found in the PSD.
Phosphofructokinase from Escherichia coli: Further evidence for identical subunits
Authors: Thornburgh Bruce N. , Wu Licia Liu , Griffin Charles C.
Published: December 2009

A procedure for the purification of Escherichia coli phosphofructokinase by affinity chromatography is described. The results of amino acid analyses of the purified protein and tryptic peptide mapping suggest that the tetrameric phosphofructokinase is composed of chemically identical subunits. In addition, the reaction product, ADP, was observed to bind to 4.1 ± 0.1 equal and independent sites on the enzyme.
Phosphorylation of adenosine 5′-monophosphate by a dialyzed cell-free extract from Escherichia coli
Authors: Chalykoff Penu , Yamazaki Hiroshi
Published: December 2009

A cell-free extract of Escherichia coli, even after exhaustive dialysis, was found capable of phosphorylating adenosine 5′-monophosphate (AMP) to adenosine 5′-diphosphate (ADP) and adenosine 5′-triphosphate (ATP). Centrifugation at 100 000 g for 3 h sedimented most of the capacity to phosphorylate AMP to ATP, while the supernatant retained a significant capacity to phosphorylate AMP to ADP. The pellet contained a greater amount of phosphate polymers (which were neither DNA, RNA, nor proteins) than did the supernatant. The addition of authentic inorganic polyphosphates to the supernatant restored the phosphorylating capacity of the original extracts. It is concluded that the observed phosphorylation is partly due to inorganic polyphosphate.
Effect of calcium on [125I]insulin binding to rat adipocytes
Authors: Desai Kappu S. , Zinman Bernard , Steiner George , Hollenberg Charles H.
Published: December 2009

The specific binding of [125I]insulin to rat adipocytes was reduced in the absence of extracellular calcium. The addition of calcium to the calcium-free medium during incubation restored [125I]insulin binding towards normal. The specific binding of insulin was significantly increased with calcium concentrations as low as 0.5 mM and maximal binding occurred with 5 mM calcium. Scatchard analysis of the data suggests two major binding sites, one a high-affinity low-capacity site (Kd, 1.5 × 1010 M−1) and the other a lower-affinity high-capacity site (Kd, 4.7 × 109 M−1). There was a 50% decrease in the number of high-affinity sites in absence of extracellular calcium. The dissociation curve of receptor-bound insulin was nonlinear both in the absence and presence of extracellular calcium suggesting receptor heterogeneity. The dissociation rate of receptor-bound insulin was greater when insulin was bound in the absence of extracellular calcium than in its presence. These results indicate that extracellular calcium, by increasing the number of high-affinity receptor sites, can alter the the ratio of high-affinity to low-affinity receptors for insulin in adipocytes.
Purification of mouse α-foetoprotein by ampholyte displacement chromatography
Authors: Pagé M. , Belles-Isles M.
Published: December 2009

Mouse α-foetoprotein (αFP) was isolated from H4 hepatoma tissue using Con A - Sepharose salt gradient ion exchange chromatography and ampholyte displacement chromatography, the latter being a new method for a sharp separation of proteins based on their different isoelectric point. The purity of the αFP was demonstrated by (i) the absence of contaminant on sodium dodecyl sulphate polyacrylamide gel electrophetic gels, (ii) Ouchterlony's immunodiffusion against monospecific antimouse αFP and the absence of precipitation against a polyvalent antinormal mouse serum, (iii) the production of a monospecific antiserum in a rabbit after injection of the purified antigen, and (iv) immunological unreactivity of the produced antiserum against normal hepatic tissue.
Further studies on the replication of adenovirus type 2 DNA
Authors: Raptis Leda , Delbecchi Louis , Bourgaux Pierre
Published: December 2009

Monolayer cultures of KB cells infected with adenovirus type 2 (Ad2) were subjected to short pulses of tritiated thymidine, at the time of maximal viral DNA synthesis. Nascent viral DNA was selectively extracted from the cells, purified, and fractionated into partly single-stranded (or replicating) molecules and double-stranded (or completed) molecules. Completed molecules were cleaved by the restriction endonucleases from either Escherichia coli (EcoRI), Haemophilus influenzae (Hind III), Haemophilus parainfluenzae (Hpa I), or by the latter two enzymes in succession, and the resulting fragments were separated by electrophoresis through agarose–acrylamide slab gels. In the case of replicating molecules, the double-stranded fragments generated after treatment with the same enzymes were first separated from the partly single-stranded fragments by chromatography on benzoylated–naphthoylated DEAE (BND) cellulose before being subjected to electrophoresis. The relative yields of tritium of the various fragments resolved by the gels were then determined. As already described in several reports, the analysis of the completed molecules obtained after the shorter pulses revealed a strong preferential labeling of the fragments derived from the molecular ends, as expected if both of these ends contained termini for replication. The analysis of double-stranded fragments from replicating molecules revealed a predominant labeling of the central portion of the genome which could be reconciled with initiation occurring either centrally or at both molecular ends. In addition, two gradients of labeling, ascending toward each molecular end and encompassing the outer quarters of the molecule, were noted which slowly receded when the pulse length was increased. The latter finding, which is in agreement with our earlier results, could reflect an overrepresentation of molecules in the late stages of replication in the pool of nascent DNA, such as one would expect if replication slows down when it nears the termination site(s). This interpretation is consistent with the kinetics of labeling of the various portions of the genome observed in the pool of completed molecules. Because of this complication, however, the distribution of label in replicating molecules cannot be used to ascertain unambiguously the location of the origin(s) of replication on the Ad2 DNA molecule.
Localization of fumarylacetoacetate fumarylhydrolase in rat liver
Authors: Doshi Kirit S. , Schmidt Jr. Donald E.
Published: December 2009

The intracellular location of fumarylacetoacetate fumarylhydrolase (EC 3.7.1.2) has been demonstrated in rat liver tissue. Two fractionation procedures involving homogenization and differential centrifugation were adopted. The first fractionation procedure isolated the nuclear fraction while the second gave the mitochondrial, microsomal, and soluble phase fractions. The hydrolase is localized in the soluble phase of the rat liver tissue. The enzyme also showed a high relative specific activity in the soluble phase fraction. Fractionation efficiency was checked by microscopic studies and by determining the distribution of a number of marker enzymes.
Protein kinases in brown adipose tissue of developing rats: substrate specificities, separation, and changes in activity during development
Authors: Skala Josef P. , Knight Brian L.
Published: December 2009

Protein kinase activity in the 100 000 × g supernatant fraction of brown adipose tissue was assayed with various proteins as substrates. Greatest activity was obtained with histone subfraction f2b and the lowest activity with protamine. cAMP stimulated the phosphorylation of histones but not that of the other proteins. The specific activity of protein kinase in brown fat of rats changed during infancy, and the pattern of changes was different with different protein substrates.Electrophoresis of the tissue-soluble extract gave nine major bands of protein kinase activity. These were assayed on the gels under the optimal conditions for each substrate. Five of the bands were histone kinases and gave little activity with nonhistone proteins. Two bands gave their greatest activity with phosvitin or casein. Protamine and arginine-rich histone were particularly good substrates for the two remaining bands. The activity in each band exhibited a different and distinct pattern of ontogenic development, which was not affected by the nature of the protein used for the assay. There was a reasonable similarity between the overall developmental profile for the rate of phosphorylation of each substrate calculated from the sum of activities determined on the individual bands and the profile directly determined in the whole soluble fraction.Both the direct assay and the electrophoretic results indicate that brown fat contains a number of protein kinase activities that can be distinguished by their specificities for protein substrate and by the pattern of changes in their activities during development. There were three main patterns of ontogenic changes in activity: one decreased progressively from high values in late foetuses; the second showed a marked fall in activity during the 3rd week after birth; and in the third, the activity rose after birth and then remained constant. The greatest changes in kinase activity thus occur during the periods when there are pronounced changes in the rates of proliferation and differentiation in brown fat, in its capacity to produce heat, and in the diet of the animal. It seems possible that the different types of protein kinase carry out phosphorylations involved in the regulation of different processes in brown fat.
Metabolic responses of Sertoli cells in culture to various concentrations of follicle stimulating hormone and cholera toxin
Authors: Fritz Irving B. , Griswold Michael D. , Louis B. Gregory , Dorrington Jennifer H.
Published: December 2009

The concentration of cholera toxin required for half-maximal stimulation of cAMP production by Sertoli cell enriched cultures (4.48 × 10−2 μg/ml) is greater than that required for half-maximal stimulation of 17β-estradiol synthesis from testosterone (2.34 × 10−4 μg/ml), [3H]thymidine incorporation into DNA (1.48 × 10−5μg/ml), or androgen binding protein production (2.43 × 10−6 μg/ml). The same relative dose response hierarchy was obtained with respect to stimulation of Sertoli cells with follicle stimulating hormone (FSH) preparations. Again, highest concentrations were required to elicit maximal cAMP production. The data are discussed in relation to an apparent paradox: If cAMP is the mediating 'second messenger' following stimulation by FSH or cholera toxin, why should highest concentrations of these agents be required to elicit 50% of maximal cAMP levels?
Response of human lymphocytes to mitogen: At what stage is there a requirement for Ca2+?
Authors: Bard E. , ColWill R. , L'Anglais R. , Kaplan J. G.
Published: December 2009

In order for significant DNA synthesis to be observed in the case of human lymphocytes stimulated for 36 h in presence of phytohemagglutinin (PHA), Ca2+ must be present in the medium continuously for at least 20 h; access to Ca2+ for 10-h periods during the first 30 h was not sufficient to permit DNA synthesis to occur. Addition of the chelator EGTA from 0 to 60 h after stimulation caused severe inhibition of incorporation of labelled thymidine when this was measured after 36 to 144 h of culture. Equimolar calcium reversed the inhibition caused by EGTA. Incorporation of labelled uridine and leucine showed a temporal pattern of dependence on the presence of Ca2+ in the medium similar to that of thymidine. Ca2+ appears not to be required in the medium during the last half (i.e.,20–36 h) of the presynthetic G1 phase nor during S phase since removal of Ca2+ from the medium after 20 h did not prevent a subpopulation of lymphocytes from entering S phase 16 h later.
Interspecific variations in proteins synthesized by mammalian mitochondria
Authors: Yatscoff Randall W. , Aujume Luke , Freeman Karl B. , Goldstein Samuel
Published: December 2009

The products of mitochondrial protein synthesis in established cell lines of various mammalian species were labelled with [35S]methionine and their number and apparent molecular weights determined by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis and fluorography. Proteins synthesized by isolated rat liver mitochondria were labelled with [3H]valine and similarly characterized. Each species had a distinctive pattern of from 10 to 13 mitochondrially synthesized proteins with apparent molecular weights between 10 000 and 50 000. No differences were detected in the number or electrophoretic mobility of the mitochondrially synthesized proteins of SV-40-transformed and nontransformed WI-38 cells.
Oxytocin binding sites in lactating rabbit mammary gland
Authors: Markle H. V. , Warr J. L. , Branda L. A.
Published: December 2009

Material which specifically binds oxytocin was prepared from a crude preparation of lactating rabbit mammary gland by purification on a sucrose density gradient. On examination of activities of enzyme markers and the molar ratio of cholesterol to phospholipid, this material was considered to be a highly purified plasma membrane fraction. For the determination of specificity and time course of oxytocin binding, a Scatchard plot analysis was carried out for the crude and purified fractions. Dissociation constant (Kd) and binding capacity values were found to be as follows: crude, Kd equals 1.83 × 10−9 M, capacity equals 670 fmol/mg protein; purified, Kd equals 2.8 × 10−9 M, capacity equals 1700 fmol/mg protein. Treatment of the purified material with different detergents resulted in loss of all [3H]oxytocin binding capacity. However, preincubation of this material with [3H]oxytocin prior to detergent treatment resulted in solubilization of a receptor–hormone complex. This complex remained in the supernatant even after centrifugation at 210 000 × g for 30 min. Using oxytocin analogs, we have shown this solubilized complex to be oxytocin specific.
Blood replacement in dogs by dextran–hemoglobin
Authors: Tam Siu-Cheung , Blumenstein Jan , Wong J. Tze-Fei
Published: December 2009

Exchange transfusions in dogs were performed with a solution of either dextran or a covalent complex between dextran and human hemoglobin. Dogs transfused with dextran alone died when their hematocrit was lowered to 6–10%. Dogs transfused with the dextran–hemoglobin complex, however, survived a reduction of their hematocrit to 2% or below. In the latter animals, the dextran–hemoglobin complex disappeared from the circulation with an average half-life of 2.4 days. Correcting for oxidation of the hemoglobin moiety to methemoglobin, the half-life of functional unoxidized dextran–hemoglobin in the circulation was 1.9 days. In compensation for the loss of dextran–hemoglobin, vigorous erythropoiesis was observed at a rate of close to 5% hematocrit per day over the first 2 days following the exchange transfusion. As a result, the total hemoglobin concentration in blood was maintained at 5–6% during this period, and the animals went on to complete recovery in room air without the need for further transfusion with dextran–hemoglobin.
A comparison of the in vitro metabolism of biphenyl and 4-chlorobiphenyl by rat liver microsomes
Authors: Wyndham C. , Safe S.
Published: December 2009

The comparative metabolism of the hydrocarbons, biphenyl and 4-chlorobiphenyl, was investigated using two different preparations of rat hepatic microsomes. The assay was designed to account for all the metabolic products which included the ether soluble lipophilic metabolites, low molecular weight conjugates, and macromolecular adducts, and to determine the effects of induction with Aroclor 1254 and 1248, two commercial polychlorinated biphenyl (PCB) preparations. 4-Chlorobiphenyl was the more metabolically active substrate with the induced and control enzymes. In most metabolic fractions biphenyl was less inducible by the PCB's, with the exception of the 2-biphenylol metabolite which was induced ca. 18-fold. Preincubation of the microsomes with carcinogens did not enhance biphenyl 2-hydroxylation. Instead, a general inhibition of metabolic activity was observed for both biphenyl and 4-chlorobiphenyl substrates. Preincubation with phenobarbitone, a noncarcinogen, did not change the microsome-mediated metabolism of biphenyl or 4-chlorobiphenyl. The substitution of a single halogen atom on the biphenyl nucleus altered both the reactivity and pattern of metabolites for these substrates.
Chromium (III) thiamin diphosphate: a new probe of enzyme coenzyme–metal ion binding. Interaction with wheat germ pyruvate decarboxylase
Authors: Kluger Ronald , Smyth Timothy
Published: December 2009

Chromium (III) thiamin diphosphate (CrTDP) is a substitutionally inert complex which is a physical and kinetic probe of the binding mode of metal ion and coenzyme in thiamin diphosphate (TDP) dependent enzymes. CrTDP is prepared by reaction of aquated Cr (III) and TDP and purified by ion-exchange chromatography. CrTDP binds to the apoenzyme of wheat germ pyruvate decarboxylase, giving an inactive holoenzyme. Chromium (III) ion binds to the apoenzyme in a manner that suggests, in light of the results with CrTDP, that there are two metal ion binding sites. Extension of the use of CrTDP to other enzymes can give specific information about binding and can introduce an active site reporter group.
The viscosity and lipid composition of the plasma membrane of multiple drug resistant and sensitive yeast strains
Authors: Rank G. H. , Robertson A. J. , Bussey H.
Published: December 2009

Four different plasma membrane preparations were isolated from multiple drug resistant and sensitive isolates of two isogenic groups of Saccharomyces cerevisiae strains: zymolyase ghosts, concanavalin A ghosts, pH 4 nonaggregated vesicles, and sucrose-gradient purified vesicles. The viscosities of these preparations were determined by the use of a fluorescence polarization technique with 1,6-diphenyl-1,3,5-hexatriene.The viscosities of all four membrane preparations within an isogenic set were the same for resistant and sensitive strains. A comparison of the viscosity of zymolyase ghost liposomes showed that zymolyase ghost (glyco) proteins of resistant and sensitive strains had the same effect on viscosity.There was no difference between resistant and sensitive isolates in the mole concentration of the following lipid classes extracted from zymolyase ghosts: phospholipid, sterol, sterol ester, triglyceride, diglyceride, and free fatty acid. The fatty acid distribution of esterified and free fatty acids and the distribution of nine phospholipids was the same in zymolyase ghosts from sensitive and resistant strains.It was concluded that multiple drug resistance does not result from an alteration in plasma membrane viscosity or lipid composition.
Hydroxyl radical production by free and DNA-bound aminoquinone antibiotics and its role in DNA degradation. Electron spin resonance detection of hydroxyl radicals by spin trapping
Authors: Lown J. William , Sim Soo-Khoon , Chen Hsiao-Hsiung
Published: December 2009

The reduced antitumor antibiotic mitomycin C in aqueous solution exposed to air gives a 36-line electron spin resonance spectrum of the semiquinone identified by computer simulation. Incubation of this radical with the spin trap N-tert-butyl-α-phenylnitrone (PBN) gives the PBN∙OH nitroxide radical identified by independent generation. This nitroxide radical is also formed from similar treatment of a DNA to which mitomycin C is covalently attached. Incubation of the semiquinone from mitomycin C, mitomycin B, or streptonigrin (SN) with catalase or with superoxide dismutase inhibits the generation of OH∙, implying the intermediacy of H2O2 and [Formula: see text] in its formation. The formation of the spin-trapped nitroxide radical is similarly inhibited by EDTA, suggesting the intermediacy of trace metal ions in the generation of hydroxyl radicals from SN. The results are consistent with the generation by the aminoquinone antibiotics in vivo of OH∙ already implicated in the degradation of DNA.
A graphical method for extracting rate constants from some enzyme-catalyzed reactions not monitored to completion
Authors: Glick Bernard R. , Brubacher Lewis J. , Leggett David J.
Published: December 2009

A large number of enzyme-catalyzed reactions can be described by the equation y = At−B(1−e−kt) where y is the amount of product formed, A is the slope of the linear portion of the curve, and B is a constant dependent on the mechanism of the reaction. The methods which are generally used to extract the rate constant, k, from absorbance–time data described by this equation require that the reaction be monitored for some 10 to 15 half-lives. We show herein that the rate constant k is readily obtained from a plot of (y″−y′) vs. (y′−y0) where y0, y′, and y″ are the values of y at times t, t + Δt, and t + 2Δt. This graphical method is simple, reliable, and requires that the reaction be monitored for only three to five half-lives of the exponential phase of the reaction.We have used this method to measure the rate of activation of a mixed disulfide of papain and 2-nitro-5-mercaptobenzoic acid in the presence of substrate.
Arylalkylamines in the adrenal medulla
Authors: Philips S. R. , Juorio A. V.
Published: December 2009

The trace amines p-tyramine, m-tyramine, tryptamine, and phenylethylamine have been determined in the bovine adrenal medulla and the rat adrenal gland by the mass spectrometric integrated ion current technique, using the corresponding deuterated amines as internal standards. In the bovine adrenal medulla, these amines were present at levels ranging from 21.7 ng/g for tryptamine to 37.3 ng/g for phenylethylamine, while in the rat adrenal, they occurred in amounts ranging from 5.2 ng/g for m-tyramine to 11.4 ng/g for tryptamine. In the rat, intraperitoneal administration of pargyline (100 mg/kg) increased the adrenal levels of each of the trace amines. Adrenaline, noradrenaline, and p-tyramine were significantly depleted by re serpine (3 or 10 mg/kg), but the level of m-tyramine was unaffected. It is suggested that the mode of uptake of the tyramine isomers into the adrenal medullary granules may depend on the position of the hydroxyl group relative to the side chain on the aromatic nucleus.
Mechanism of glucose transfer to steroids by rabbit liver microsomes
Authors: Comerton Aileen M. , Layne Donald S. , Williamson Denis G.
Published: December 2009

The formation of the 3-glucosides of estrone, 17α-estradiol, or 17β-estradiol by washed rabbit liver microsomes in the absence of UDP glucose (UDPG) was increased by the addition of crude lipid extracts of rabbit or pig liver and of dolichol or retinol. The reaction was stimulated by Mn2+, Mg2+, and Ca2+. Direct transfer of glucose to the steroid from added UDPG also occurred by a mechanism which was inhibited by metal ions. The two transferases can be separated from each other into the 105 000 g pellet and supernatant respectively by treatment with detergents.
Horseradish peroxidase. XXXII. pH dependence of the oxidation of L-(−)-tyrosine by compound I
Authors: Ralston I. , Dunford H. B.
Published: December 2009

The rate of oxidation of L-(−)-tyrosine by horseradish peroxidase compound I has been studied as a function of pH at 25 °C and ionic strength 0.11. Over the pH range of 3.20–11.23 major effects of three ionizations were observed. The pKa values of the phenolic (pKa = 10.10) and amino (pKa = 9.21) dissociations of tyrosine and a single enzyme ionization (pKa = 5.42) were determined from nonlinear least squares analysis of the log rate versus pH profile. It was noted that the less acidic form of the enzyme was most reactive; hence, the reaction is described as base catalyzed. The rate of tyrosine oxidation falls rapidly with the deprotonation of the phenolic group.
A new recirculation reactor system for kinetic studies of immobilized enzymes
Authors: Toro Luigi , Zazzarelli Alfonso
Published: December 2009

A new recirculation reactor for kinetic studies of immobilized enzyme is proposed. The reactor consists of two tubes in the same thermostatting jacket. One of the tubes is filled with the immobilized enzyme, the other is filled only with the support. During the approach to steady-state temperature, the circulation is carried out only in the inactive tube. Subsequently, when temperature is stable, the active reactor is used and the solution is not recirculated.
Acetylcholinesterase: characterization of native and proteolytically derived forms and identification of structural protein components
Authors: Webb Geoffrey
Published: December 2009

The asymmetric 18S and 14S forms of acetylcholinesterase (EC 3.1.1.7) from Electrophorus electricus purified by affinity chromatography on N-methylacridinium Sepharose 2B were subjected to trypsin or collagenase proteolysis and changes in the enzyme composition and structure were monitored by sucrose gradient sedimentation, gel chromatography, and sodium dodecyl sulphate – polyacrylamide gel electrophoresis. A distinction between autolytic and tryptic degradation products is described and the generation of two new forms of acetylcholinesterase from the 18S and 14S enzyme by collagenase proteolysis is reported. The species derived from the 18S form of acetylcholinesterase has a sedimentation coefficient of 21.1S and a Stokes radius of 12.9 nm while the 14S form gives rise to a 17.3S species with a Stokes radius of 11.1 nm. The proteolytically sensitive component ('tail') of the asymmetric forms of acetylcholinesterase is identified with a subunit of 45 000 daltons on sodium dodecyl sulphate – polyacrylamide electrophoresis gels.
Equilibrium and kinetic studies of the interaction of site-specific ligands with acetylcholinesterase from Electrophorus electricus
Authors: Tomlinson George , Mutus Bulent , Rutherford W. John
Published: December 2009

The interactions of edrophonium chloride, gallamine triiodide, and propidium diiodide with affinity-purified acetylcholinesterase from Electrophorus electricus have been examined under conditions of low ionic strength (0.001 M Tris, pH 8.0) using kinetic and fluorescence titration techniques. Edrophonium is a competitive inhibitor of the steady-state hydrolysis of acetylthiocholine, with an inhibition constant, Kcomp, of 1.2 × 10−8 M. Double reciprocal plots in the presence of either gallamine or propidium are nonlinear. Similarly, the pre-steady-state carbamoylation of the enzyme by 7-(dimethylcarbamoyloxy)-N-methyl quinolinium iodide is competitively inhibited by edrophonium, whereas the intercepts of the double reciprocal plots of pseudo-first-order rate constant of carbamoylation versus substrate concentration are displaced downwards in the presence of gallamine or propidium. These results, and those of equilibrium binding studies utilizing the fluorescence properties of bound propidium, suggest that gallamine and propidium compete for a peripheral class of anionic sites on the enzyme, whereas edrophonium binds to the anionic subsite of the catalytic site. The characteristics of propidium binding to the eel enzyme differ from those previously observed with enzyme isolated from Torpedo californica. Whereas the tetrameric Torpedo enzyme possesses four binding sites of equal affinity for propidium, the eel enzyme appears to have two classes of propidium binding site. One set of approximately two sites per tetramer is characterized by a dissociation constant of approximately 2–5 × 10−8 M; a second set of two sites bind propidium with a dissociation constant of 4 × 10−6 M. Possible reasons for these differences are discussed.
Inhibition of cell-free protein synthesis by analogues of aurintricarboxylic acid
Authors: Kreamer B. L. , Anderson J. , Liu D. S. H. , Sparks M. B. , Richardson A.
Published: December 2009

Aurintricarboxylic acid is a potent inhibitor of cell-free protein synthesis by the post-mitochondrial supernatant of chick brain and the translation of mRNA by wheat germ lysate. Comparison of commercially available and chemically synthesized analogues of aurintricarboxylic acid indicates that the unique aurin triphenyl methane ring system and the carboxylic acid groups are both necessary for inhibition of cell-free protein synthesis in both systems.
Organization and dynamics of membrane lipids as determined by magnetic resonance spectroscopy
Authors: Smith Ian C. P.
Published: December 2009

not available
The utility of the nitrotyrosine chromophore as a spectroscopic probe in troponin C and modulator protein
Authors: McCubbin William D. , Hincke Maxwell T. , Kay Cyril M.
Published: December 2009

Rabbit skeletal and bovine cardiac troponin C (TN-C) and bovine brain protein modulator have been nitrated by treatment with tetranitromethane. The resulting derivatives have been characterized by calcium titration employing circular dichroism and by absorption difference spectroscopy. It was found that the nitrated proteins did not differ significantly in secondary structure from their native unmodified counterparts. Their response to calcium ions, as measured by the binding parameters n and K, is also comparable. Introduction of the nitrotyrosyl chromophore at specific residues in the protein chain has allowed the performance of additional calcium titrations by the circular dichroism method. For each of the three proteins the ellipticity at 275 nm, a point of maximum change for this chromophore, was monitored as a function of calcium addition. The binding parameters obtained indicated that in each case a high affinity site was being probed. This is consistent with the known locations of the tyrosine residues in these polypeptide chains. Calcium-induced absorption difference spectra were also generated for these three proteins in the wavelength range 550–250 nm. In the 320- to 550-nm region all three spectra were quite similar, suggesting a comparable change in the environment of the chromophoric moiety upon Ca2+ binding in all cases. Below 320 nm the spectra differed considerably. For skeletal TN-C, the large contribution from phenylalanine residues which has been noted previously for the native protein was still evident. For fully nitrated cardiac TN-C the contribution from nitrotyrosine at 280 nm is comparable with the skeletal case. For the partially nitrated sample the spectrum more closely resembles that of native cardiac TN-C, the major contribution coming from the 2.6 mol of unreacted tyrosine. The particularly large value of Δε290 nm of 600 M−1 cm−1 for the protein modulator has been interpreted in terms of subtle interactions between the nitrotyrosine residues located at the third and fourth Ca2+-binding sites producing an electronic enhancing effect. These two techniques have demonstrated the sensitivity of the nitrotyrosyl chromophore to subtle changes in its microenvironment, making it a useful conformational probe in the continuing study of the molecular changes elicited when calcium binds to TN-C and homologous molecules.
Isolation of a human B lymphocyte membrane protein with Ia-like properties
Authors: Sullivan A. K. , Jerry L. M. , Ikeman R. L. , Maccari R. J. , Thi H. Li , Sylvester C.
Published: December 2009

A rapid method for isolation of a major surface membrane glycoprotein from whole, unfractionated cultured human B lymphoblasts is described. After detergent solubilization the method uses gel filtration followed by affinity chromatography on Sepharose Con A and then alkaline acrylamide gel electrophoresis. Specific, high-titre, rabbit antisera to the isolated protein reacted with cultured and normal peripheral blood B lymphocytes, as well as peritoneal macrophages from a renal dialysis patient. The antisera selectively inhibited the mixed lymphocyte reaction at high dilution. The protein reacted with a heterologous antiserum to HL-B antigens and contained subunits of MW 33 000 and 27 000. Resolution of the subunits, however, required a discontinuous SDS gel system. These properties indicate its similarity to murine Ia antigens. The protein was not associated with β2 microglobulin and showed no structural or antigenic similarity to the major erythocyte glycoprotein, glycophorin. Antisera to the protein failed to precipitate surface-radioiodinated components from similarly treated extracts of cultured human T lymphoblasts. This method now makes available a reference membrane glycoprotein from a differentiated, nucleated human cell in sufficient purity and quantity for kinetic and biosynthetic studies.
Heterogeneous catalysis by phospholipase A2: formulation of a kinetic description of surface effects
Authors: Tinker David O. , Wei Jane
Published: December 2009

The kinetics of hydrolysis of aqueous dispersions of long-chain, saturated phosphatidylcholines (PC) catalysed by Crotalus atrox phospholipase A2 (PLA) have been analysed, and a reaction mechanism proposed which takes surface effects into account. PLA is proposed to form an enzyme–substrate complex with surface substrate molecules, thereby undergoing a conformational change which exposes sites that interact with the lipid surface. After a hydrolytic event, the enzyme can either desorb from the surface (path 1), or diffuse along the surface to an adjacent substrate molecule (path 2). The path 1 dominated mechanism leads to Michaelis–Menten steady-state kinetics, and characterizes hydrolysis of gel phase PC. Evidence for saturation of the surface with PLA was obtained at high enzyme concentrations. The path 2 mechanism dominates when the desorption rate is very small; this mechanism describes hydrolysis of liquid crystalline phase PC and is characterized by an initial burst of hydrolysis followed by a very slow reaction. The velocities in these two phases of the reaction are independent of bulk PC concentration. When gel and liquid crystalline PC phases coexist, as in mixtures of dimyristoyl- and distearoyl-PC, the liquid crystalline phase is preferentially hydrolysed. Products of the reaction (lyso-PC and fatty acid) stimulate hydrolysis, apparently by stimulating desorption of PLA. The desorption rate constant appears to be a linear function of the surface concentrations of lyso-PC and fatty acid. The proposed model describes hydrolysis progress curves extremely well and is consistent with current ideas on the mechanism of catalysis by this enzyme.
The localization of honey bee thorax trehalase
Authors: Brandt N. R. , Huber R. E.
Published: December 2009

Differential and sucrose gradient centrifugation of honey bee thoraces, disrupted by gentle methods and using mannitol–triethanolamine–EDTA buffer at pH 6.5, showed that in the honey bee thorax 92–94.8% of the trehalase was mitochondrial. Since only 92–95% of the cytochrome c oxidase, a known mitochondrial enzyme, was found in the mitochondrial fraction by these methods, it was concluded that honey bee trehalase is totally mitochondrial. Significant amounts of 'microsomal' or 'soluble' trehalase were formed only by harsh methods of thorax disruption and similar 'microsomal' or 'soluble' trehalases were also formed by harsh treatment of purified whole mitochondria. They thus seem to be artifacts of the isolation procedure. Studies (using marker enzymes) with purified intact mitochondria which were dispersed by various chemical, enzymatic, and physical methods showed that the trehalase in the mitochondria was membrane bound and that it was bound to either the outside of the inner membrane or to one of the sides of the outer membrane.
Purification of dihydropterin reductase using immobilized Cibacron Blue
Authors: Chauvin Michael M. , Korri Karl K. , Tirpak Anne , Simpson Robert C. , Scrimgeour K. G.
Published: December 2009

Chromatography on columns of immobilized Cibacron Blue (Blue Dextran–agarose) can be used as a major step in the purification of quinonoid dihydropterin reductase. The reductase has been isolated from fractions of beef kidney by selective binding to the immobilized Cibacron in the presence of tetrahydropterin. The binding of the reductase to Blue Dextran and its specific elution from columns of Blue Dextran – agarose indicate that the reductase possesses the dinucleotide (NAD+) binding domain. The results of kinetic experiments give validity to both our affinity chromatography of the reductase and to an ordered mechanism for the formation of tetrahydropterin. Chromatography on Blue Dextran – agarose has been used to show that folate or amethopterin can compete with Cibacron Blue for the dinucleotide domain of the reductase. The p-aminobenzoylglutamate moiety of the folates competes with Cibacron Blue for the NADH site of the reductase. A stable binary complex of dihydropterin reductase with NADH has been detected by gel electrophoresis.
Purification of UDP-N-acetylenolpyruvoylglucosamine reductase from Escherichia coli by affinity chromatography, its subunit structure and the absence of flavin as the prosthetic group
Authors: Anwar Rashid A. , Vlaovic Mariana
Published: December 2009

The enzyme UDP-N-acetylenolpyruvoylglucosamine reductase (EC 1.1.1.158) was purified to homogeneity from Escherichia coli by affinity chromatography on a NADP-agarose column. The evidence suggests that the enzyme (molecular weight 35 000) is composed of two nonidentical subunits of molecular weight 21 500 and 13 500, respectively. The absorption spectrum of the purified enzyme shows no absorption band around 450 nm and thus does not support the previous suggestions that the enzyme is a flavoprotein. However, the A280: A260 ratio gives a value of 0.86 which suggests the presence of tightly bound nucleotide. A quantitative transfer of tritium from 1,4-[4-3H]NADPH to UDP-N-acetylenolpyruvoylglucosamine to form UDP-N-[3H]acetylmuramic acid was also observed, which clearly shows that the enzyme is not a flavoprotein.
Azasterol inhibitors in yeast. Inhibition of the Δ24-sterol methyltransferase and the 24-methylene sterol Δ24(28)-reductase in sterol mutants of Saccharomyces cerevisiae
Authors: Pierce A. M. , Unrau A. M. , Oehlschlager A. C. , Woods R. A.
Published: December 2009

The effects of several monoazasterols on sterol biosynthesis were examined in the ergosterol deficient mutants erg 2, erg 3, and erg 5 of Saccharomyces cerevisiae. When the mutants were aerobically cultured in the presence of 1 μM 23-azacholesterol, the 24-methylene sterol Δ24(28)-reductase was essentially blocked and the immediate Δ24(28)-unsaturated precursor of the final sterol metabolite in each respective erg strain was found to accumulate. Total sterol production was enhanced in the cultures grown in the presence of 1 μM 23-azacholesterol. In cultures which were grown in the presence of 1 μM 25-azacholesterol which effectively blocked the Δ24-sterol methyltransferase, all three erg strains accumulated zymosterol as the major sterol component with lesser quantities of predicted terminal sterols. Mutant erg 2 (block at Δ8 → Δ7 isomerase) grew poorly in the presence of 1 μM 25-azacholesterol and produced low levels of cholesta-5,8,24-trienol and cholesta-5,8,22,24-tetraenol, which were isolated and characterized for the first time. Compared with controls, erg 2 treated with 1 μM 23-azacholesterol produced increased amounts of ergosta-5,8,22,24(28)-tetraenol, which was hitherto unidentified as a yeast sterol. In erg 5 (block at Δ22-dehydrogenase) treatment with 1 μM 25-azacholestanol effectively blocked the Δ24-sterol methyltransferase and resulted in increased total sterol production. Cholesta-5,7,24-trienol accounted for 27–29% of the sterol pool in 25-azasterol inhibited erg 5 cultures. The 25-azasteroi inhibited erg 5 mutant thus provides a source of cholesta-5,7,24-trienol, a potential provitamin D3 substitute.