bluuebunny/crossref_metadata_2025 huggingface dataset:
Wheat embryo ribonucleates. XIV. Mass isolation of mRNA from wheat germ and comparison of its translational capacity with that of mRNA from imbibing wheat embryos
Authors: Cuming A. C. , Kennedy T. D. , Lane B. G.
Published: December 2009

Commercially milled wheat germ is shown to be a convenient source material for facile recovery of mass (milligram) quantities of highly purified poly(A)-rich RNA. This poly(A)-rich RNA is efficiently translated in a nuclease-treated extract of rabbit reticulocytes. By sucrose density gradient fractionation of bulk poly(A)-rich RNA from wheat germ, it has been possible to show that there is a direct relationship between the molecular weights of the polypeptide products of cell-free synthesis and the molecular weights of the wheat mRNA molecules which program their synthesis. As assessed by SDS – polyacrylamide gel electrophoresis, the same array of polypeptides is synthesized when nuclease-treated reticulocyte extract is programmed by poly(A)-rich RNA from either commercially supplied or laboratory-prepared wheat embryos. Significantly, there are gross quantitative if not qualitative differences between the translational capacities of poly(A)-rich RNA from dry and imbibing wheat embryos, and the possible importance of these differences for interpreting a changing pattern of polypeptide synthesis in imbibing wheat embryos is the subject of a brief discussion.
Nonspecific inhibition of dehydrogenases by folates: an artifact
Authors: Scrimgeour K. G. , Tirpak Anne
Published: December 2009

The previous reports of inhibition of alcohol dehydrogenase and lactate dehydrogenase by the vitamin folic acid and its analogues are in error. The high absorbance of solutions containing folate causes distortion of the measurements of reaction velocities, leading to apparent inhibitions. When cuvettes of sufficiently short optical path length are used, no inhibition by folate can be observed. Similarly, the reported inhibition of ribonuclease by folate is an artifact. Glutamate dehydrogenase and dihydropterin reductase actually are inhibited by folate. The reported nonspecific inhibitions of over a dozen enzymes by folate, though, must be regarded as erroneous.
Erratum: Translocation of isomeric spin-labelled radioactive cytidinediphosphodiglycerides from isolated guinea pig liver microsomal to mitochondrial membranes
Authors: Stuhne-Sekalec L. , Sanacev N. S.
Published: December 2009

not available
Chromatography of heparin on Sepharose–lysine: molecular size fractionation and its relevance to thrombin and antithrombin III binding
Authors: Hatton M. W. C. , Berry L. R. , Kaur H. , Koj A. , Regoeczi E.
Published: December 2009

Batches of Sepharose–lysine, which varied in lysine content from 35 to 430 μmol/g of dry gel, were prepared by varying the quantity of CNBr in the activation reaction. The batches were tested for heparin binding by using a controlled chromatographic procedure. Sepharose–lysine, containing < 150 μmol of lysine/g, did not significantly bind heparin whereas conjugates with > 400 μmol/g retained the entire heparin load. For intermediate batches of Sepharose–lysine (150–400 μmol/g) the quantity of heparin bound largely paralleled the lysine content. Thus, Sepharose–lysine of an intermediate lysine content separated heparin into an unretained fraction and a bound fraction which was recovered from the column by eluting with 1 M NaCl. On testing for anticoagulant activity by factor Xa inhibition assay, no significant difference in specific anticoagulant activity was observed between these heparin fractions and the heparin load. However, from gel filtration studies, a substantial difference in molecular size was noted. An unretained heparin fraction from Sepharose–lysine was of a lower average molecular weight than the parent heparin. In contrast, a retained heparin peak was of a higher average molecular weight compared with the parent heparin. These observations were confirmed by studying the chromatographic properties of low (10 000) and high (23 000) molecular weight heparin samples on various Sepharose–lysine batches. A model is proposed to explain this discriminating property of Sepharose–lysine. For a conjugate containing 400 μmol/g, the mean lysine spacing is calculated at 47 Å (1 Å = 0.1 nm), which is approximately equivalent to five to six disaccharide units in heparin.The property of Sepharose–lysine to bind heparin was compared with the affinities of the mucopolysaccharide for both thrombin and antithrombin III. Evidence has been proposed for the involvement of lysine residues of antithrombin III in this process. Our investigations suggest that lysine, in addition to arginine, groups of thrombin are also involved in heparin binding. By specifically modifying two to four lysine residues using nitrous acid, the heparin-binding capacity of the enzyme and its plasma clotting activity were largely destroyed, although the esterase activity was retained.
Kinetics of acetylcholinesterase immobilized on polyethylene tubing
Authors: Ngo T. T. , Laidler K. J. , Yam C. F.
Published: December 2009

Acetylcholinesterase was covalently attached to the inner surface of polyethylene tubing. Initial oxidation generated surface carboxylic groups which, on reaction with thionyl chloride, produced acid chloride groups; these were caused to react with excess ethylenediamine. The amine groups on the surface were linked to glutaraldehyde, and acetylcholinesterase was then attached to the surface. Various kinetic tests showed the catalysis of the hydrolysis of acetylthiocholine iodide to be diffusion controlled. The apparent Michaelis constants were strongly dependent on flow rate and were much larger than the value for the free enzyme. Rate measurements over the temperature range 6–42 °C showed changes in activation energies consistent with diffusion control.
The cyclic adenosine monophosphate phosphodiesterases of myoblasts, fibroblasts, and their somatic cell hybrids
Authors: Ball E. H. , Narindrasorasak S. , Sanwal B. D.
Published: December 2009

Three forms of cAMP phosphodiesterases are found in mouse L cells (fibroblasts) and rat skeletal myoblasts. The myoblast enzymes can be resolved by chromatography on DEAE-cellulose and the fibroblast enzymes by chromatography on DEAE-Biogel. The myoblast enzymes are "high affinity" cAMP specific forms and have different molecular weights, while all L-cell enzymes have an apparent molecular weight of 450 000. Only one of the L-cell enzymes is able to hydrolyze both cyclic guanosine monophosphate (cGMP) and cAMP. Hydrolysis of the latter is stimulated by micromolar amounts of cGMP. The myoblast × L cell hybrids possess at least five phosphodiesterases, three of which can be identified as being of myoblast or fibroblast origin. One of the fibroblast enzymes appears to be modified in hybrids. The entire phosphodiesterase regulatory system of the myoblasts is active in the hybrids.
Structure of ribosomes
Authors: Wittmann H. G.
Published: December 2009

Ribosomes are multicomponent particles on which biosynthesis of proteins occurs in all organisms. The best known ribosome, namely that of Escherichia coli, consists of three RNAs and 53 different proteins. All proteins have been isolated and characterized by chemical, physical, and immunological methods. The primary sequences of 47 E. coli ribosomal proteins have so far been determined. Studies of the shape, as well as the secondary and tertiary structure, of the proteins are in progress.Various techniques (e.g. immune electron microscopy and cross-linking of neighbouring components in situ) give information about the architecture of the ribosomal particle. The first technique resulted in illustrative and detailed knowledge not only on the shape of the ribosomal subunits but also about the location of many proteins on the surface of the particles. The analysis of cross-links between ribosomal proteins and (or) RNAs has in several cases been pursued to the level of elucidating which amino acids and (or) nucleotides are cross-linked together in situ. Reconstitution of a fully active E. coli 50S ribosomal subunit from its isolated RNA and protein components can be accomplished by means of a two-step incubation procedure. From the analysis of the intermediates occurring during the reconstitution process it has been concluded that the in vitro reconstitution process resembles the in vivo assembly of 50S subunits in many respects. Escherichia coli mutants with alterations in almost all ribosomal proteins have been isolated. Their biochemical and genetic analyses are very useful tools for obtaining information about the structure, function, and biosynthesis of ribosomes as well as about the location of the genes for these proteins on the chromosome. From comparative electrophoretic, immunological, protein–chemical, and reconstitution studies on ribosomes from various species it has become clear that there is little homology between ribosomes from prokaryotes and those from eukaryotes. This finding is surprising since there is no essential difference in the way in which pro- and eu-karyotic ribosomes function in protein biosynthesis.
2,3-Butanedione as a photosensitizing agents: application to α-amino acids and α-chymotrypsin
Authors: Fliss Henry , Viswanatha Thammaiah
Published: December 2009

2,3-Butanedione sensitized the rapid photodestruction of free α-amino acids, and the photoinactivation of α-chymotrypsin, in the presence of ultraviolet light and oxygen. These reactions showed "pseudo-first-order" kinetics at 2,3-butanedione concentrations approximating those employed for the chemical modification of arginine residues in proteins. The photoreactions were inhibited in anoxic media or in the presence of azide; findings were consistent with a singlet oxygen mechanism for these reactions. No enhancement in the rate of reaction was observed in D2O. The rate of 2,3-butanedione-sensitized photodestruction of free amino acids increased with increasing pH. However, the rate constants for the photosensitized inactivation of α-chymotrypsin, as well as those for the photodestruction of the tryptophan residues of this enzyme, decreased linearly with increasing pH.
In vivo preparation of [32P]cAMP and thin-layer chromatography of cAMP-related compounds
Authors: Yamazaki Hiroshi , Leung Kwok-Luen , Feaser Ann D. E.
Published: December 2009

A simple and rapid method for preparing [32P]adenosine 3′,5′-cyclic monophosphate (cAMP) is described. A culture of an Escherichia coli mutant which excretes cAMP about 150 times faster than does a wild-type strain was incubated overnight with [82P]orthophosphate of high specific activity (e.g., 4000 Ci/mol (1 Ci = 37 GBq)). The [32P]cAMP which accumulated extracellularly was then purified to 99.9% radiochemical purity in less than 4 h by adsorption to charcoal and alumina column chromatography. A two-dimensional chromatography system using a PEI-cellulose plate is also described which should prove useful for studying cAMP metabolism with 32P- or 3H-labeled cAMP or ATP.
Purification of a basic somatomedin, from human plasma Cohn fraction IV-1, with physicochemical and radioimmunoassay similarity to somatomedin-C and insulin-like growth factor
Authors: Bala R. Marvin , Bhaumick B.
Published: December 2009

A basic somatomedin (SM) was purified from human plasma Cohn fraction IV-1 using an initial acid–ethanol–acetone extraction procedure followed by alternating molecular size or charge protein separation techniques. The final recovery of SM bioactivity was approximately 2% of that present in the starting Cohn fraction. The purified SM has an approximate molecular weight of 7500, pI 8.6, 4000 SM bioactivity units per milligram (as measured by a hypophysectomized rat bioassay) and a parallel approximately equipotent radioimmunoassay dose–response curve to SM-C and insulin-like growth factor-I (IGF-I). Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of this purified SM revealed a single protein band. The preliminary determination of the amino acid sequence of the N terminus suggested that this SM preparation was over 75% pure and the first five N-terminal amino acids were identical with those of IGF-I.
Metabolism of oligo(A) tracts and association of oligo(A)(+)RNA with polysomes in molluscan embryos
Authors: Gerdes Pamela A. , Kidder Gerald M.
Published: December 2009

Six-day preveliger embryos of the marine gastropod Nassarius (Ilyanassa) obsoletus were labeled with [3H]adenosine, and RNA was extracted and then digested with DNase I, RNase A, and RNase T1 under conditions which leave adenosine homopolymers intact. Nuclease-resistant material was characterized as poly(A) and oligo(A) by analysis of size distribution, affinity chromatography on oligo(dT)-cellulose, and sensitivity to RNase T2. Oligo(A) tracts, less than 30 nucleotides long, were identified which incorporate adenosine but not uridine, and are resistant to RNases A and T1 but sensitive to T2; those tracts less than 20 nucleotides long were not retained on oligo(dT)-cellulose and could then be obtained free of poly(A). After a 6-h labeling period, about 80% of nuclease-resistant label from postnuclear supernatant fractions was in oligo(A). Alkaline hydrolysates of Ilyanassa poly(A) had the adenosine content expected of "3′-poly(A)," whereas oligo(A) hydrolysates contained little adenosine, indicating their location elsewhere in RNA molecules. Of the two principle size classes of oligo(A), 8 and 18 nucleotides, the first was distributed among subcellular fractions, including polyribosomes, in a fashion similar to poly(A). Oligo(A)18, in contrast, was largely localized in molecules or molecular complexes smaller than ribosomal subunits, although it, too, appeared in association with polysomes. Whereas the labeled poly(A) of ribosome pellet fractions underwent turnover, the labeled oligo(A) was stable for at least 10 h. Oligo(A)8 and oligo(A)18 are thus metabolically distinct from each other and from poly(A), and are a prominent feature of cytoplasmic RNA in this system.
Acylglycerol structure of mustard seed oil and of cardiac lipids of rats during dietary lipidosis
Authors: Myher J. J. , Kuksis A. , Vasdev S. C. , Kako K. J.
Published: December 2009

Stereospecific degradation and combined gas chromatographic – mass spectrometric (gc/ms) analysis were employed in a detailed investigation of the triacylglycerol structure of mustard seed oil and of the triacylglycerols transiently accumulating in the hearts of young rats receiving the oil in their diet. It was shown that feeding of mustard seed oil at 40% of the daily caloric requirement resulted in a deposition of cardiac triacylglycerols containing a high proportion of enantiomers of a positional distribution and molecular association of fatty acids which were closely similar to those found in the dietary oil. Complete structures were derived for a total of 88 species representing 75 to 85% of the triacylglycerols. About 90%, of the accumulated triacylglycerol contained at least one long-chain (C20–C22) monounsaturated fatty acid per molecule. The long-chain acids were confined mainly to the primary positions and preferentially to the sn-3-position of the glycerol molecule. The dietary lipidosis is, therefore, accompanied by little or no accumulation of the normal rat tissue triacylglycerols containing C16 and C18 fatty acids. It is suggested that the deposition and eventual clearance of the enantiomeric long-chain triacylglycerols in the rat heart during mustard seed oil feeding may be largely a result of a gradual change in specificity of the cardiac lipases.
Hybrid plasmids complement a putP mutation in Escherichia coli K12
Authors: Wood Janet M. , Zadworny David , Lohmeier Elke , Weiner Joel H.
Published: December 2009

Clarke and Carbon have prepared a colony bank of 2000 Escherichia coli strains each containing a random segment of the Escherichia coli chromosome inserted into the EcoR1 restriction site of the plasmid ColE1. We have screened the colony bank by conjugation and have identified three strains bearing hybrid plasmids that complement a defective putP gene. Each of these strains shows increased L-proline uptake activity in comparison with the unmodified host or with the host bearing noncomplementing hybrid plasmids. However, CS520, the DNA source strain employed in constructing the hybrid plasmids, is a putP mutant. Since Escherichia coli possesses two L-proline porters, a variety of possible complementation mechanisms are discussed.
The effect of protein modification reagents on the chemotactic response in Salmonella typhimurium
Authors: Clarke Steven , Koshland Jr. D. E.
Published: December 2009

Several classes of reagents that covalently modify proteins have been shown to inhibit the ability of Salmonella typhimurium to reverse the direction of its flagellar rotation. Such reversal normally allows the bacterium to tumble and reorient its movement in a more favorable direction. These reagents include those that react with amino, guanidino, sulfhydryl, and disulfide groups on proteins. At high concentrations, most of these compounds also cause the paralysis of flagellar rotation.Tumbling in bacterial chemotaxis has been shown to be dependent on the methylation of a class of membrane proteins. The effects of these reagents in an in vitro methylation system have been studied. The results obtained suggest that most of these compounds are probably not acting on intact cells by inhibiting the activities of the cheR methyltransferase or the methyl-accepting proteins.
A rapid technique for measuring calcium uptake in mitogen-induced T And B Lymphocytes
Authors: Freedman M. H. , Khan N. R.
Published: December 2009

The procedures for lymphocyte activation and for removing the cells from the radioactive loading solution in incubation medium were modified to routinely obtain significant and reproducible 45Ca2+ uptakes in mitogen-induced mouse T and B lymphocytes. Factors such as mouse strain, lymphocyte origin, and media pH were not critical to the 45Ca2+ uptake measurements. In contrast, factors such as lymphocyte cell concentration during mitogenic activation, filtering the 45Ca2+:3H2O mixtures, and the nature and purity of the B-cell mitogens were critical for obtaining maximal and reproducible 45Ca2+ uptakes. Centrifugation through silicone oil into sucrose was an efficient and rapid procedure for separating the cells from the radioactive loading solution in the incubation medium. Using optimal conditions, an approximate twofold increase in 45Ca2+ uptake (representing an influx of ~ 97 amol per lymphocyte and an increase in average cellular Ca2+ of ~ 0.72 mM) was routinely obtained with purified mouse lymphocytes activated with a variety of T- and B-cell mitogens (using concentrations resulting in maximal [3H]thymidine incorporation). A larger 45Ca2+ uptake was routinely obtained with mitogenic concentrations of A23187, a divalent cation ionophore stimulating T cells. Experiments employing [14C]sucrose and [14C]inulin with control and mitogen-induced lymphocytes showed that the trapped extracellular fluid measurements in the cell pellets should be used to correct the magnitude of the 45Ca2+ uptake measurements.
The deacylation of phosphatidylinositol by rat brain preparations
Authors: Shum T. Y. P. , Gray N. C. C. , Strickland K. P.
Published: December 2009

The deacylation of phosphatidylinositol (PI) in rat brain was studied in vitro. Using 1-acyl, 2-[1-14C-oleoyl] sn-glycerol-3-phosphoinositol and [U-14C]phosphatidylinositol as substrates, a release of 14C-free fatty acid was found when incubations were conducted with the PI labelled in the 2 position, both in the 12 000 – 106 000 g pellet (microsomal) and in the 106 000 g supernate prepared from rat brain homogenate. With the 106 000 g pellet the deacylation activity was linear with time up to 15 min and was directly proportional to the amount of protein added. Two pH optima were observed, one in the region of pH 7.5 which constituted the major activity and the other in the region of pH 6.0. The apparent Km for the enzyme activity at pH 7.5 was found to be 6.2 × 10−4 M and the Vmax was 1.24 nmol of [14C]oleic acid released per minute per milligram of protein. The Ca2+ ion stimulated the activity maximally at 5 mM while other divalent cations such as Mg2+, Mn2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Ni2+, Pb2+, and Zn2+ either partially or completely inhibited the activity. The nonionic detergent, Triton X-100, stimulated the deacylation more than twofold at a concentration of 0.01%. The sulfydryl reagents, p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetamide showed partial inhibition of the reaction which was reversed by the addition of dithiothreitol. The deacylation activity in the 106 000 g supernate from rat brain was found to be directly proportional to the amount of protein added, and to the time (up to 15 min). A pH optimum was observed in the region of pH 6.0. Substrate concentration studies showed that the apparent Km was 5.0 × 10−4 M and the Vmax was 3.93 nmol of [14C]oleic acid released per minute per milligram of protein. "lyso-PI," diacylglycerol, and fatty acid were formed at pH 6.0 and pH 7.5. The data obtained indicate that from 54 (pH 7.5) to 70% (pH 6.0) of the altered PI is due to phospholipase A2 activity, 24 (pH 6.0) to 28% (pH 7.5) is due to phospholipase "C-like" activity, and from 2 (pH 6.0) to 22% (pH 7.5) may be due to phospholipase A1 activity. These results provide evidence for the deacylation component of a deacylation–reacylation cycle for the generation of specific molecular species of PI.
Stability of microtubule protein over the pH ranges 6.9–9.5
Authors: Galella G. , Smith David B.
Published: December 2009

The pH stability range of a microtubule protein preparation has been investigated between 6.9 and 9.5. Microtubule protein was exposed to various pH values in this range and then returned to pH 6.9. The appearance of microtubules as verified by electron microscopy and sedimentation analysis under polymerizing conditions was taken as an indication of a conformationally stable protein. Between pH 6.9 and pH 8.0 the loss in the ability to form microtubules was found to be reversible, at pH 8.2 it was partially reversible, above pH 8.2 it was irreversible. Tubulin and the microtubule-associated protein fraction were separately exposed to high pH. It was observed that tubulin exposed to high pH can still form microtubules in the presence of untreated microtubule-associated protein. On the other hand, microtubule-associated protein exposed to high pH could not initiate microtubule assembly with untreated tubulin. It was concluded from these observations that the loss in the ability of a microtubule protein preparation to assemble at high pH is due to a change in the microtubule-associated protein fraction and that tubulin is conformationally stable even after exposure to pH 9.5.
The action of tributyltin chloride on energy-dependent transhydrogenation of NADP+ by NADH in membranes of Escherichia coli
Authors: Singh A. P. , Bragg P. D.
Published: December 2009

Respiration- and ATP-dependent transhydrogenation of NADP+ by NADH in everted membrane vesicles from Escherichia coli is inhibited by nigericin but is relatively insensitive to valinomycin. The sensitivity to nigericin is enhanced 30-fold in the presence of valinomycin. It is concluded that both the transmembrane pH difference and the membrane potential constitute the driving force for energy-dependent transhydrogenation.Respiration- and ATP-dependent transhydrogenation are inhibited by tributyltin chloride. Although effects on the energization system have not been excluded, the inhibitor appears to react with a sulfhydryl group on the transhydrogenase enzyme. This inhibition is not dependent on the presence of a permeant anion and can be reversed by mono- and particularly di-thiol compounds. The transhydrogenase is also inhibited by 5,5′-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, p-chloromercuriphenyl sulfonic acid, and Zn2+, but these reagents are less effective than tributyltin chloride. Energy-independent transhydrogenation is inhibited at high concentrations (20 mM) of cysteine. The reason for this is unknown.
Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli. Purification to homogeneity and some properties
Authors: Waygood E. B. , Steeves T.
Published: December 2009

Enzyme I of the phosphoenolpyruvate – sugar phosphotransferase system (PTS) has been purified to homogeneity from Escherichia coli. A merodiploid strain P650 which had an extra copy of the gene for enzyme I resulting in a twofold increase in the amount of activity was used. The enzyme is a dimer of 67 000 ± 5000 molecular weight subunits. At low protein concentration and 4 °C the monomer predominates, while at room temperature the dimer predominates. At higher protein concentrations (2 to 10 mg) this reversible temperature-dependent association-dissociation is not found. Enzyme I has a pH optimum of pH 7.2, a Km for HPr of 9 ± 3 μM, a Km for phosphoenolpyruvate of 0.18 ± 0.04 mM, and kinetics that are consistent with a bi bi Ping-Pong mechanism. No allosteric regulation of kinetic activity has been found. The amino acid composition has been determined and the ε1%280 nm is 4.4. Evidence suggests that the phosphorylated form of enzyme I is more stable.
Specificity of xanthine oxidase for nitrogen heteroaromatic cation substrates
Authors: Bunting John W. , Laderoute Keith R. , Norris Donald J.
Published: December 2009

A variety of pyridinium, quinolinium, and benzoquinolinium cations have been investigated as potential substrates for milk xanthine oxidase at pH 9.9 and (or) pH 10.6. Steady-state kinetic parameters (kc, Km and (or) kc/Km) have been evaluated for all substrates which are enzymically oxidized. Simple N-alkyl pyridinium cations are neither substrates nor inhibitors, although N-aryl pyridinium cations are slowly oxidized to the 4-pyridinones. N-Methylpyridinium cations bearing 3-CONH2, 3-CONHCH3, 3-COCH3, 3-CO2− or 3-CN substituents are readily oxidized at C-6 and this suggests an important hydrogen-bonding interaction between an enzyme donor and the C-3 carbonyl substituent. A variety of N-methylquinolinium cations bearing C-6 substituents are enzymically oxidized at C-2. Analogous substituent effects on kc/Km for these 6-substituted 1-methylquinolinium cations and the corresponding 1-(substituted phenyl)-pyridinium cations is suggestive of the relative productive binding orientations of these two classes of substrate in the active site. N-Methyl benzoquinolinium and 1,10-phenanthrolinium cations are the best cationic substrates found to date, and suggest a relatively large active-site region for the reducing substrate, and important hydrophobic interactions between enzyme and substrate. The overall enzymic specificity observed for these cationic substrates allows a mapping of the general features of the reducing substrate binding site of this enzyme
Lipid involvement in oleoyl CoA desaturase activity of Fusarium oxysporum microsomes
Authors: Wilson Alan C. , Adams Wyman C. , Miller Richard W.
Published: December 2009

Desaturation of oleoyl CoA by the microsomal fraction of Fusarium oxysporum hyphal cells required O2, NADPH, MgCl2,and the addition of either bovine serum albumin or the 105 000 g supernatant fraction. In the absence of reduced nucleotide, L14C]oleoyl CoA was rapidly incorporated into phospholipid and triacylglycerol and hydrolyzed to free fatty acids. Alter addition of NADPH, oleate was desaturated at the normal rate. Analysis of the distribution of [14C]oleate and [14C]linoleate between different lipid classes revealed that phosphatidylcholine and phosphatidylethanolamine were labeled with [14C]linoleate before any other lipid class. These results are consistent with oleoyl phospholipid being a direct intermediate in the desaturation of oleoyl CoA. The preference of the oleoyl-desaturase for NADPH, the relatively high pH optimum of 8.2, and the sensitivity to thenoyltrifluoroacetone inhibition suggest that some components of the microsomal electron-transport chain are common to both the oleoyl desaturase and stearoyl CoA desaturase systems in this fungus.
Structural determination and serology of the native polysaccharide antigen of type-III group B Streptococcus
Authors: Jennings Harold J. , Rosell Karl-Gunnar , Kasper Dennis L.
Published: December 2009

The native polysaccharide antigen isolated from type-III group B Streptococcus contains D-galactose (Gal), D-glucose (Glc), 2-acetamido-2-deoxy-D-glucose (GlcNAc), and sialic acid (NeuNAc) in the molar ratio of 2:1:1:1 and its structure can be represented by the following repeating unit:[Formula: see text]By cleavage of all the labile sialic acid end groups the incomplete type-III antigen is obtained which is structurally identical to the S. pneumoniae type-14 polysaccharide. Thus, the native type-III polysaccharide is serologically distinct from the incomplete type-III antigen by virtue of the former having determinants terminating in sialic acid and the latter in β-D-galactopyranose units. None of these latter determinants could be detected in streptococcal organisms grown under pH-controlled conditions (pH 7.0) or in rabbit antiserum made to these pH-controlled organisms. However, in antisera produced in rabbits to the same organisms grown without pH control (Lancefield procedures), antibodies to both types of determinant could be detected. This can be attributed to the removal of some of the masking sialic acid residues from the cell-associated native polysaccharide by degradative procedures which occur during these latter conditions.
The R-type lipopolysaccharides of Neisseria meningitidis
Authors: Jennings Harold J. , Bhattacharjee Apurba K. , Kenne Lennart , Kenny C. Paul , Calver Gerald
Published: December 2009

The lipopolysaccharides of all the different serogroups of Neisseria meningitidis are of the "R" type despite the morphologically smooth appearance and the demonstrated virulence of the organisms from which they were derived. This was confirmed when each of the lipopolysaccharides was found to be devoid of detectable O-antigen side chains, giving only a low "molecular" weight core oligosaccharide when subjected to mild acid hydrolysis. The cores were modified by dephosphorylation and subjected to sugar and methylation analysis by gas–liquid chromatography. Although all the different cores contained identical components (glucose, galactose, glucosamine, heptose, and 2-keto-3-deoxyoctonate) they could be separated into three distinct categories according to their galactose:glucose ratios. These categories are typified by the cores obtained from groups A, C, and 29-e which have galactose:glucose ratios of 1:2, 2:2, and 2:1, respectively. The modified cores were methylated and analyzed by gas chromatography – mass spectrometry and on the basis of differences in the derived methylated sugars the cores could again be divided into the same three categories as above. This structural diversity also results in some serological specificity as demonstrated by the complete serogroup specificity of the group A lipopolysaccharide.
Synthesis of spin-labelled 2-(16′-(N-oxyl-4″,4″-dimethyloxazolidine)stearoyl)-phosphatidylcholine
Authors: Vaughan D. J. , Stanacev N. Z.
Published: December 2009

Chemical synthesis of spin-labelled lecithin 2-(16′-(N-oxyl-4″,4″-dimethyloxazolidine)-stearoyl)-phosphatidylcholine was achieved by reaction of equimolar amounts of purified egg yolk lyso-lecithin with commercially available 16-(N-oxyl-4′,4′-dimethyloxazolidine)stearic acid activated with N,N-carbonyldiimidazole. After purification by silicic acid column chromatography, spin-labelled lecithin was obtained in 52.2% yield and had an ester:phosphorus:spin molar ratio of 2.0:1.1:1.0. When dimyristoyl-phosphatidylcholine and dimyristoyl-phosphatidylcholine:cholesterol (10:1) liposomes, containing 2-(16′-(N-oxyl-4″,4″-dimethyloxazolidine)stearoyl)-phosphatidylcholine as a probe, were examined for thermotropic changes monitored by electron spin resonance spectroscopy, transition temperatures of 24.0 and 24.2 °C, respectively, were obtained in a very good agreement with previously reported values obtained with different probes and by different techniques, and with our own differential scanning calorimetry measurements. The potential usefulness of synthetic 2-(16′-(N-oxyl-4″,4″-dimethyloxazolidine)stearoyl)-phosphatidylcholine as a spin-label probe in studies of lipid–protein interactions in biological membranes was discussed.
The variability, arrangement, and rearrangement of immunoglobulin genes
Authors: Rabbitts T. H. , Hamlyn P. H. , Matthyssens G. , Roe B. A.
Published: December 2009

The multiplicity of heavy-chain variable-region (VH) genes in mouse and human DNA has been estimated using a mouse heavy- (H) chain cDNA clone. We found about 10 hybridization components in mouse DNA and about 20 components in human DNA. Cross-hybridization studies of variable region (V) genes indicate that these components represent the numbers of genes within the VH subgroups of each of these species. The arrangement and rearrangement of the H-chain γ subclasses have been studied in order to assess possible mechanisms of the H-chain switch. Evidence has been found for rearrangement events involving the γ2a and γ2b constant-region (CH) genes in DNA from cells making IgG2a and IgG2b respectively. In addition we found that cells making IgG2a lack detectable genes for γ1 and γ2b. Both sets of observations are discussed in relation to H-chain diversity and the switch.
13C and 31P nuclear magnetic resonance of lipid dispersions from human aorta
Authors: Cushley Robert J. , Forrest Bruce J. , Grover Ashok K. , Wassall Stephen R.
Published: December 2009

Coarse ("multilamellar") and sonicated ("unilamellar") dispersions of lipid constituents of human aorta have been prepared. 31P nuclear magnetic resonance (nmr) spectra of the coarse dispersions show an unusual pattern compared with pure phospholipid bilayers. The reconstituted membrane is suggested to have considerable amounts of nonbilayer phase, possibly in the form of regions of hexagonal phase with a short major axis, or regions of cubic or rhombic phase admixed with the bilayer phase. High resolution 13C nmr spectra of the sonicated dispersions give narrow signals, indicative of lipids in a fluid environment, and are due mostly to cholesteryl esters and triglycerides.
Poly(A)-containing RNA in the livers of ethionine-treated rats
Authors: Dennis James , Kisilevsky Robert
Published: December 2009

Ethionine intoxication in nonfasted rats leads to a rapid change in the levels of poly(A)+ RNA. Prolonged fasting causes a decrease in poly(A)+ RNA which is not aggravated further by ethionine. The size distribution of poly(A) tracts at the 3′ end of mRNA is not affected by ethionine. However, ethionine induces a change in the metabolism of the poly(A) sequence relative to the remainder of the mRNA.
Poly(A) polymerase activity in ethionine-intoxicated rats: the relative effectiveness of disaggregated and intact polyribosomes as primers for poly(A) polymerase
Authors: Dennis James , Kisilevsky Robert
Published: December 2009

Ethionine intoxication causes a change in the metabolism of poly(A) sequences on the 3′ OH terminus of mRNA in rat liver in vivo. In an attempt to determine the factors responsible for these changes, nuclear and cytoplasmic poly(A) polymerase activities and the state of the primer were examined in vitro. Requirements for optimal enzyme activities were determined. The nuclear and cytoplasmic enzymes had different K+, Mn2+, and poly(A) primer optima. The levels of nuclear and cytoplasmic poly(A) polymerase activity were shown to decrease following ethionine intoxication. Poly(A)+ RNA isolated from the livers of saline- and ethionine-treated rats served equally well as primers for the cytoplasmic poly(A) polymerase. Disaggregated polysomes were seven times more effective as primers than were intact polysomes. The results suggest that the mRNP particle which is released from polysomes as a result of ethionine intoxication functions better as a poly(A) polymerase primer than does the intact polysome.
Properties of a phosphoprotein phosphatase from rat epididymal fat pads: deactivation of hormone-sensitive triglyceride lipase and activation of glycogen synthase in adipose tissue
Authors: Severson David L. , Sloan Shellie
Published: December 2009

A phosphoprotein phosphatase has been partially purified from rat epididymal fat pads by a procedure utilizing ammonium sulfate and ethanol precipitations and chromatography on DEAE-Sephadex A-50. The phosphatase was eluted from Sephadex G-75 columns with an apparent molecular weight of 28 000. The phosphoprotein phosphatase catalyzed the reversible deactivation of protein kinase activated chicken adipose tissue hormone-sensitive triglyceride lipase. Phosphatase activity measured with activated triglyceride lipase as substrate was completely dependent upon the presence of metal ions (Mg2+, Ca2+, or Mn2+) and was inhibited by inorganic phosphate and adenine nucleotides. The fat pad phosphatase increased the rate of activation of glycogen synthase in rat adipose tissue infranatant fractions from fed and 24-h-fasted rats but had little or no effect on synthase activity in infranatant fractions from rats fasted for 48 h. Fasting had no effect on rat fat pad phosphatase activity measured with triglyceride lipase as substrate, but phosphatase activity was decreased in preparations from diabetic rats.
Synthesis of peptides by the solid-phase method. V. Substance P and analogs
Authors: Fournier A. , Couture R. , Magnan J. , Gendreau M. , Regoli D. , St-Pierre S.
Published: December 2009

We have synthesized a series of 12 analogs of the undecapeptide substance P in order to perform a structure–activity study of this peptide. In the present work, each residue was substituted by L-alanine, and the C-terminal amide was replaced by the free carboxyl in order to pinpoint biologically important side chains and functional groups. The synthesis of the analogs was carried out by the automatic solid-phase method. Couplings were performed by the symmetrical anhydride procedure. After cleavage with liquid HF, the peptides were purified by gel filtration and ion-exchange chromatography. Their purity was assessed by thin-layer chromatography, paper electrophoresis, amino acid and elemental analyses, and high pressure liquid chromatography. They were tested for biological activity in vitro on the ileum of the guinea pig, the mesenteric vein of the rabbit, and the vas deferens of the rat, and in vivo by measuring their effect on the blood pressure of the rat.
Calmodulin-dependent myosin light chain kinases from cardiac and smooth muscle: a comparative study
Authors: Walsh Michael P. , Cavadore Jean-Claude , Vallet Bernard , Demaille Jacques G.
Published: December 2009

Various properties of cardiac and smooth muscle calmodulin-dependent myosin light chain kinases (MLCKs) have been compared. The enzymes exhibit the same isoelectric point (6.5) but differ markedly in molecular weight (Mr = 72 000 for both canine and bovine cardiac MLCK, and Mr = 130 000 for smooth muscle MLCK). Comparison of the tryptic peptide maps of bovine cardiac and turkey gizzard MLCKs indicates that the cardiac enzyme is a fragment of a protein homologous to the smooth muscle kinase. While the smooth muscle kinase can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, the same is not true for either bovine or canine cardiac MLCK. Controlled tryptic hydrolysis of phosphorylated smooth muscle MLCK, followed by affinity chromatography on a column of calmodulin–Sepharose, enables separation of a phosphopeptide (Mr = 22 000) from a mixture of peptides of Mr = 50 000 and 24 000 which are bound to the column in the presence of Ca2+ and eluted with ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The phosphorylation site, therefore, is distinct from the calmodulin-binding site. It appears that cardiac MLCK is proteolyzed during the isolation procedure. The purified cardiac enzyme represents a proteolytic fragment which retains Ca2+ and calmodulin dependence but only a fraction of the specific activity of the native enzyme, and has lost the site of phosphorylation by cAMP-dependent protein kinase. A protease is shown to exist in myocardium which is capable of digesting smooth muscle MLCK rapidly at low temperature, and which is resistant to classical antiproteases.
Identification and partial characterization of an Escherichia coli mutant with altered hydrogenase activity
Authors: Glick Bernard R. , Wang Patrick Y. , Schneider Henry , Martin William G.
Published: December 2009

An Escherichia coli mutant strain with altered hydrogenase activity was isolated using a filter paper assay. This assay depends on the ability of hydrogenase-containing microorganisms to reduce methyl viologen impregnated in filter paper, producing purple-colored colonies in the presence of hydrogen. Membrane-bound and cytoplasmic hydrogenase activities of wild-type and mutant strains were compared by amperometric measurement of hydrogen production. The cytoplasmic activities of mutant and wild type were comparable. The membrane-bound activity was lower in the mutant than in the wild type. Upon addition of detergent to the membrane fraction the specific activity of the enzyme from the mutant strain increased so that it equalled that of the wild type. The mutant requires an exogenous electron acceptor for anaerobic growth providing evidence for the function of the hydrogenase in anaerobic growth.
The pH dependence of xanthine oxidase catalysis in basic solution
Authors: Bunting John W. , Laderoute Keith R. , Norris Donald J.
Published: December 2009

The steady-state kinetics of the oxidation of the following six heteroaromatic substrates by xanthine oxidase have been investigated over the range pH 9.0–11.1 at 25 °C, ionic strength 0.1: 1-methylquinolinium, 6-methoxy-1-methylquinolinium, 1-methylnicotinamide, 3-acetyl-1-methylpyridinium, and 1-(4-methoxyphenyl)pyridinium cations and 1-methylnicotinate zwitterion. For the first four of these species, kc and Km were evaluated as a function of pH while only kc/Km was accessible in the latter two cases. Where available, kc is pH independent, whereas plots of log (kc/Km) vs. pH are linear with slopes in the range 0.54–1.17.The rates of enzymic oxidation of the 1-methylquinolinium cation and its 2-deuterio derivative were investigated and kinetic isotope effects were calculated at pH 9.8 and 10.6: kcH/kcD = 1.7 and KmH/KmD = 0.4 at each pH. Detailed comparisons of the oxidation of heteroaromatic cations and xanthine-derived substrates indicate that similar rate-determining steps control the enzymic oxidations of these two classes of substrate.
Yeast, rye, and calf histones. Similarities and differences detected by electrophoretic and immunological methods
Authors: Tessier Anne , Roland Birgitte , Gauthier Claude , Anderson William A. , Pallotta Dominick
Published: December 2009

Yeast histones H2A, H2B, and H3 were purified using the standard histone purification procedures of differential solubility and exclusion chromatography. Yeast histone H4 was isolated by the same methods in a fraction containing one other major protein component. The four yeast core histones were identified by their reactions with antisera against rye and (or) calf histone fractions as well as by their electrophoretic, chromatographic, and solubility properties. The immunological distances between yeast H2B and rye and calf H2B fractions are substantial, as is the rye–calf distance for H2B. The immunological distance between yeast H2A and rye H2A is also large and is similar to the rye H2A – calf H2A distance. On the other hand, the immunological distance between yeast H3 and rye and calf H3 is much greater than that between rye H3 and calf H3. These and other results indicate that yeast H3 differs appreciably from the H3 of higher eucaryotes.
Kinetic study of the interaction between rat haptoglobin and rat liver cathepsin B
Authors: Pagano M. , Engler R. , Gelin M. , Jayle M. F.
Published: December 2009

One of the roles of the acute phase reactants (APR), according to Koj, is to regulate the action of tissue proteinases released during the inflammatory reaction. To study this phenomenon in vitro, rat liver cathepsin B (EC 3.4.22.1), a lysosomal proteinase, and a typical APR, rat serum haptoglobin, were purified. By kinetic study, haptoglobin was found to inhibit cathepsin B and the inhibitory activity of a competitive type was increased with the molecular weight of the substrate. When 125I-labelled denatured bovine serum albumin (BSA) was used as a substrate, the apparent Michaelis constant (Km app.) for cathepsin B was 5 ± 0.4 × 10−6 M. When haptoglobin was added, the apparent inhibition constant (Ki app.) was 3.5 ± 1.5 × 10−8 M. Native haptoglobin was not catabolized by cathepsin B while under the same conditions denatured haptoglobin was degraded by the enzyme.
Passive diffusion of nucleosides into Micrococcus sodonensis membrane vesicles
Authors: Pickard M. A.
Published: December 2009

Nucleoside entry into isolated membrane vesicles of Micrococcus sodonensis (luteus) was studied using 14C-labelled nucleosides: adenosine, inosine, cytidine, uridine, guanosine, and thymidine. All nucleosides were recovered unmetabolized from the vesicles except adenosine and cytidine which were partly deaminated by membrane-bound enzymes. Vesicle preparations actively transported proline but no energy source was found capable of supporting concentrative nucleoside uptake. The entry of nucleosides into M. sodonensis vesicles was not saturable, nor was there competition between the nucleosides studied for entry. It was concluded that nucleoside entry into M. sodonensis vesicles occurs by passive diffusion.
Jack bean urease (EC 3.5.1.5). III. The involvement of active-site nickel ion in inhibition by β-mercaptoethanol, phosphoramidate, and fluoride
Authors: Dixon Nicholas E. , Blakeley Robert L. , Zerner Burt
Published: December 2009

Interaction of β-mercaptoethanol with urease produces large, rapid and fully reversible spectral changes in that part of the electronic absorption spectrum which is associated with the tightly bound nickel ions. The spectrophotometrically determined value of the dissociation constant of the β-mercaptoethanol–urease complex (0.95 ± 0.05 mM at pH 7.12 and 25 °C) is in agreement with the Ki (0.72 ± 0.26 mM) for β-mercaptoethanol acting as a competitive inhibitor in the hydrolysis of urea. This constitutes direct evidence that the nickel in jack bean urease is at the active site. Inhibition of urease by phosphoramidate is slowly achieved and slowly reversed, and upon reactivation of the isolated phosphoramidate–urease complex, phosphoramidate is regenerated in good yield. Spectrophotometric experiments indicate that phosphoramidate binds to nickel ion in urease. Competition with β-mercaptoethanol was used to determine a dissociation constant (1.23 ± 0.10 mM at pH 7.12 and 25 °C) for a fluoride–urease complex in which fluoride ion also coordinates with an active-site nickel ion. Kinetic evidence is presented which indicates that in the presence of urea, a ternary complex (fluoride–urea–urease) is formed.
Purification of a base-specific ribonuclease Ru from Rhizopus niveus
Authors: Horitsu Hiroyuki , Takihi Noriyuki , Sugiura Masahisa , Tomoyeda Mikio
Published: December 2009

A base-specific ribonuclease (RNase) Ru (EC 3.1.27.5) was isolated and purified from Rhizopus niveus in a yield of 17% by the procedures of acetone precipitation, column chromatography on Duolite A-2, DEAE-cellulose, CM-cellulose, and 2′(3′)-aminohexyl-5′-UMP-agarose. The enzyme was shown to be homogeneous by polyacrylamide disc electrophoresis. The amino- and carboxyl-terminal amino acids of the enzyme were determined to be an arginine and an aspartic acid, respectively. The enzyme has a base specificity: it released only 3′-UMP from yeast RNA or poly(U) and, in addition, small amounts of 3′-CMP from poly(C).