bluuebunny/crossref_metadata_2025 huggingface dataset:
A methodology for the production of carbohydrate-specific antibody
Authors: Lemieux R. U. , Baker D. A. , Bundle D. R.
Published: December 2009

8-Methoxycarbonyloctyl glycosides are recommended as precursors for the preparation of artificial antigens that possess strongly immunodominant carbohydrate antigenic determinants. The preparation and immunochemical properties of a selected number of these antigens are reported to demonstrate the high levels of hapten-specific antibodies that are normally achieved using standard immunization schedules. The method, based on glycoside synthesis, promises to make available a wide spectrum of well defined antigens and the homologous antibodies.
Acetylation and phosphorylation of nuclear proteins from growing or dividing rat ventral prostate cells
Authors: Kadohama N. , Anderson K. M.
Published: December 2009

Acetylation and phosphorylation of nuclear histone and nonhistone proteins from rat ventral prostate cells undergoing cellular hypertrophy (growth) or cellular hyperplasia (replication) were compared in minced ventral prostate incubated for 1 h with 32PO4 and [3H]acetate.Under these conditions, phosphorylation of unfractionated prostate histones from hormone-treated animals was increased, but acetylation was not. When radioactive histones from the four experimental groups were separated by polyacrylamide gel electrophoresis, evidence for differential labelling of individual histones, especially acetylated or phosphorylated histones F1 and F2a2, was obtained. This was also true of histones labelled with radioactive amino acids. These differences were more marked with histones from replicating cells.Phosphorylation of prostate nuclear nonhistone proteins from hormone-treated rats was greater than their controls. Proteins of intermediate molecular weight (20–45 × 103) were most highly phosphorylated; nuclear extracts from replicating (C7T2) cells contained additional labelled higher molecular weight proteins and fewer lower molecular weight proteins (< 20 × 103), compared with hypertrophic (C3T3) cells. Previous treatment with testosterone propionate increased the apparent acetylation of nonhistone proteins, but its marked inhibition by puromycin and resistence to hydrolysis suggests that much of this incorporation occurred during their biosynthesis, and did not represent covalent modification of previously synthesized proteins.
Mitochondrial DNA synthesis in mouse L cells temperature sensitive in nuclear DNA replication
Authors: Sheinin Rose , Darragh Pamela , Dubsky Margaret
Published: December 2009

Temperature-sensitive (ts) A1S9 mouse L cells continue to synthesize double-stranded covalently closed mitochondrial (mt) DNA at a temperature (38.5 °C) which is nonpermissive for chromosomal DNA replication. The amount of mt DNA made appears to be quantitatively linked to nuclear DNA synthesis. Nuclear DNA replication proceeds normally for 6–8 h after the cells are shifted to 38.5 °C, and then declines to reach a minimum at 20–24 h. The level of mt DNA synthesis remains high during this period and decreases once the ts lesion has been established.
α-Fetoprotein production and erythropoiesis in cell cultures of human fetal liver
Authors: Congote L. F. , Bruno F. , Solomon S.
Published: December 2009

α-Fetoprotein and the synthesis of heme associated with hemoglobin were measured simultaneously in short-term cultures of human fetal liver cells to correlate the relationship of α-fetoprotein to erythroid cell function. Both synthetic processes decreased exponentially during the first 5 days of culture. The use of media supplemented with different batches of fetal calf serum and porcine portal vein serum indicated that the optimal conditions for the production of α-fetoprotein were different from those required for the synthesis of heme associated with hemoglobin. Moreover, the α-fetoprotein-producing cells could be separated from erythroid cells after velocity sedimentation in Ficoll gradients. Although it is well known that erythropoiesis and α-fetoprotein production occur simultaneously during ontogenesis, α-fetoprotein itself (0.01–100 μg/ml) did not stimulate heme synthesis in liver erythroid cells. Erythropoietin did not stimulate α-fetoprotein production. It is concluded that there is no cause–effect relationship between α-fetoprotein production and erythroid cell function in human fetal liver cells and that the two processes occur independently in different cell types.
Hypermodified alkali-stable dinucleotide sequences in each of the high-molecular-weight (26S and 18S) ribosomal RNA species of wheat
Authors: Gray M. W. , Cunningham R. S.
Published: December 2009

Two hypermodified, alkali-stable dinucleotide sequences, each containing abase modification in addition to sugar methylation, are known to be present in wheat embryo 26S + 18S rRNA (Gray, M. W. (1974) Biochemistry 13, 5453–5463). Quantitative analysis of unfractionated 26S + 18S rRNA had suggested that each of these sequences (Cm-ψp and ψm-Ap, where Cm = O2′-methylcytidine and ψm = O2′-methylpseudouridine) was present in either the 18S or the 26S rRNA species, but not in both, at a frequency of not more than once per chain. In the study reported here, the individual 32P-labeled 18S and 26S rRNA species were isolated from viable wheat embryos germinated in the presence of [32P]orthophosphate. From analyses of phosphodiesterase and alkaline hydrolysates of the separated [32P]RNAs, we conclude that ψm-Ap is confined to wheat cytosol 18S rRNA, whereas Cm-ψp is localized in wheat cytosol 26S rRNA. The presence of ψm in the 18S rRNA of wheat stands in contrast with the situation in animal cells, where this hypermodified nucleoside is located in the 28S rRNA (Khan, M. S. N. &Maden, B. E. H. (1976) J. Mol. Biol. 101, 235–254)
The solution synthesis of sequential polypeptides as models for the double-stranded coiled-coil structure of tropomyosin
Authors: St-Pierre S. A. , Hodges R. S.
Published: December 2009

The syntheses of the three sequential polyheptapeptides [L-Leu-L-Glu-L-Ser-L-Leu-L-Glu-L-Ser-L-Lys]n, [L-Ala-L-Glu-L-Ser-L-Ala-L-Glu-L-Ser-L-Lys]n, [L-Ala-L-Glu-L-Ser-L-Leu-L-Glu-L-Ser-L-Lys]n and of the polytetrapeptide [L-Leu-L-Glu-L-Ser-L-Lys]n by solution methods are described. The syntheses were performed using a combination of a modified mixed carbonic anhydride method and an active ester coupling method. The N-hydroxysuccinimide ester was used as the active group for the polymerization of the monomers. These compounds were prepared as model polypeptides for the coiled-coil structure of tropomyosin.
Identification of β-adrenergic receptors in rat hypothalamus
Authors: Caron Marc G. , Gagne Bernard , Lean Andre De
Published: December 2009

(−)-[3H]Dihydroalprenolol((−)-[3H]DHA) binding in the rat hypothalamus appears to possess all the characteristics expected of physiologically relevant β-adrenergic receptors. Binding of (−)-[3H]DHA to the hypothalamic sites was rapid (k1 = 1.3 × 107 min−1M−1) and also rapidly reversible. Binding was saturable at low concentrations of ligand( ~ 50–100 nM). The dissociation constant (KD) of (−)-[3H]DHA binding determined by equilibrium analysis was 19 nM. Binding displayed β-adrenergic specificity. β-Adrenergic agonists inhibited binding in the following order of potency: (−)-isoproterenol [Formula: see text] (−)-epinephrine > (−)-norepinephrine. Specific β-adrenergic antagonists (−)-propranol and (−)-alprenolol inhibited binding at low concentrations (KD = 25–50 nM) whereas the α-antagonist phentolamine inhibited binding at very high concentrations (KD = 42 μM). Interactions of both agonists and antagonists with the sites showed stereoselectivity. The (−)-isomers of all β-adrenergic agents tested were more potent than their respective (+)-isomers. These results suggest that specific receptor sites for β-adrenergic catecholamines are present in rat hypothalamus.
A thermal denaturation study of chromatin and nuclease-produced chromatin fragments
Authors: Lewis Peter N.
Published: December 2009

Calf thymus chromatin and nuclease-produced chromatin fragments have been examined by thermal denaturation measurements. Native chromatin gave a series of distinct melting transitions at 64, 73, 79, and 85 °C in 0.25 mM EDTA pH 8. Treatments such as dialysis, mechanical shearing, or sulfhydryl oxidation of histone H3 carried out on native chromatin significantly altered the derivative melting profiles by blurring the distinct transitions and shifting the highest melting transition to a lower temperature. Derivative melting profiles for electrophoretically purified chromatin fragments, monomer through hexamer, all resembled that obtained from dialyzed chromatin. These results suggest that higher order structures exist in chromatin that are easily disrupted. Since the products of micrococcal nuclease (EC 3.1.4.7) digestion of the altered chromatins did not exhibit any major electrophoretic differences from those obtained from nuclei, then most likely the primary arrangements of histones along the DNA are the main determinant for cleavage sites.
Human pituitary thyrotropin. The primary structure of the α and β subunits
Authors: Sairam M. R. , Li Choh Hao
Published: December 2009

The amino acid sequence of the α and β subunits of human pituitary thyrotropin has been proposed based on the data derived from the tryptic and chymotryptic peptides. The α subunit consists of 89 amino acid residues with two carbohydrate moieties, presumably linked to asparagines in position 49 and 75, and its amino acid sequence is identical to that of lutropin α. The β subunit has 112 residues with a single carbohydrate moiety attached to asparagine at position 23. Comparison of the primary structure of human thyrotropin β with that of the bovine hormone reveals that only 11 residue positions have been found to be different.
A method for the simultaneous estimation of free and ketosidically bound sialic acids
Authors: Culling C. F. A. , Reid P. E. , Ramey C. W. , Dunn W. L. , Clay M. G.
Published: December 2009

Various methods for the estimation of free and ketosidically bound sialic acid were investigated for accuracy and specificity. It was found that oxidation with 0.025 M sodium metaperiodate in pH 7.0 phosphate buffer at room temperature for 20 min provided a simple, rapid, sensitive method whereby both the free and the ketosidically bound acid present in a mixture could be quantitatively analyzed. On completion of the oxidation step, the bound sialic acid is estimated with resorcinol reagent and the free sialic acid with thiobarbituric acid reagent. Oxidation under these conditions permitted a facile analysis of mixtures of bovine submaxillary mucin and free N-acetylneuraminic acid, whereas the Warren thiobarbituric acid procedure gave an erroneous value for free sialic acid.
Electron transfer properties of melanin. Optimum conditions and the effects of various chemical treatments
Authors: Menon I. A. , Leu S. L. , Haberman H. F.
Published: December 2009

The optimum conditions for three oxidation–reduction reactions of melanin, viz. the oxidation of NADH, the reduction of ferricyanide, and a coupled reaction involving the oxidation NADH and the reduction of ferricyanide, have been studied. Experiments in which the pH of the reaction medium was varied showed that the rate of oxidation of NADH slightly increased when the pH was varied from 6.05 to 8.5, the reduction of ferricyanide markedly increased when the pH was increased from 5.5 to 8.5, and the oxidation of NADH in the coupled reaction increased when the pH was increased from 5.5 to 8.5 and the reduction of ferricyanide increased from pH 5.5 to 7.2. Experiments using varying temperatures showed that the rates of all the above reactions increased to varying degrees when the temperature was increased from 23 to 45 °C; the activation energies for the reactions were calculated. The effects of various treatments of melanin upon the above reactions were studied. Treatment of melanin with oxidizing agents such as KMnO4, benzoquinone, iodine, FeCl3, etc. increased the oxidation of NADH by melanin. Treatment of melanin with the reducing agent ascorbate decreased the oxidation of NADH by melanin, and treatment of melanin with KMnO4 decreased the reduction of ferricyanide by melanin. These results support the possibility that the oxidized and reduced forms of melanin may be participating in the reducing and oxidizing activities of melanin.
The diguanosine nucleotides: do they exist in aquatic fungi?
Authors: Warner A. H. , Thomas D. des S. , Shridhar V. , McCurdy H. D.
Published: December 2009

The aquatic fungi Achlya ambisexualis and Blastocladiella emersonii were grown in the presence of 32Pi and the 32P-labeled acid-soluble nucleotide fractions were analyzed by ion-exchange chromatography on DEAE-cellulose. Selected column fractions containing diguanosine tri- and tetra-phosphates (Gp3G and Gp4G) added as chromatographic markers were analyzed further for 32P by chromatography and (or) enzyme hydrolysis. The results of these experiments clearly indicate that neither Gp4G nor Gp3G is synthesized during vegetative growth of these organisms and cast doubt on the hypothesis that diguanosine nucleotides are important metabolic regulators in fungi.
Studies on phosphoglyceromutase from chicken breast muscle: chemical modification of lysyl residues
Authors: Carne T. J. , Flynn T. G.
Published: December 2009

To examine the role of lysyl residues in the activity of the enzyme, phosphoglyceromutase (PGM) from chicken breast muscle was chemically modified with trinitrobenzenesulfonate (TNBS) and pyridoxal 5′-phosphate. Trinitrophenylation resulted in modification of about nine lysines per mole of PGM with almost complete activity loss. Substrate (3-PGA) offered some protection to TNBS inactivation but cofactor (2,3-DPGA) did not. Reduction of the Schiff s base complex between pyridoxal 5′-phosphate and PGM gave irreversible inactivation of the enzyme. Inactivation was due to incorporation of 1 mol of pyridoxal 5′-phosphate per mole of PGM dimer through the ε-amino group of a lysyl residue. The effect of pyridoxal 5′-phosphate was specific for intact native enzyme and reaction with only one lysine per dimer was not due to induced conformational changes nor to dissociation of the reacted enzyme. 3-PGA prevented much of the reaction with pyridoxal 5′-phosphate with preservation of 70% of the activity and was a competitive inhibitor of the active site directed reagent. Cofactor (2,3-DPGA) acting noncompetitively, reduced the rate at which inactivation occurred with pyridoxal 5′-phosphate. Incorporation of 2,3-[32P]DPGA into PGM irreversibly inactivated with pyridoxal 5′-phosphate and NaBH4 was incomplete indicating hindrance to phosphorylation in the modified enzyme.The results indicate that a lysyl residue is located at or near the active site of PGM and that it is probably involved in the binding of 3-PGA.
Folic acid compounds in romaine lettuce
Authors: Batra K. K. , Wagner J. R. , Stokstad E. L. R.
Published: December 2009

The composition of folate coenzymes in romaine lettuce was studied. Lettuce extract was purified on QAE-Sephadex A-25 and folate compounds were separated into a monoglutamate fraction and a polyglutamate fraction by chromatography on Sephadex G-15. Both the mono- and poly-glutamate fractions were resolved on DEAE-cellulose. Positive identification of DEAE peaks was made by further cochromatography with high specific activity radioactive marker folate compounds and with differential microbiological assay. The distribution of folate compounds in lettuce is as follows: 32% 5-CH3-H4PteGlu; 1% 5-CHO-H4PteGlu; 3% 5-CHO-H4PteGlu4; 9% 5-CH3-H4PteGlu4; 13% 5-CHO-H4PteGlu5; and 31% 5-CH3-H4PteGlu5.
Preparation and galactosyltransferase acceptor activity of derivatives of antifreeze glycoproteins of an Antarctic fish
Authors: Shier W. Thomas , Roloson Gary J.
Published: December 2009

A series of 12 closely related glycoproteins containing α-linked N-acetyl-D-galactosamine (GalNAc) as the sole carbohydrate moiety have been prepared by degradation of the antifreeze glycoproteins from the serum of the Antarctic fish Trematomus borchgrevinki. The polypeptide moieties of these glycoproteins contain substitutions in the normal -Ala-Ala-Thr- repeating tripeptide sequence which introduce alterations in the amount of α-helical structure and the density of acceptor sites, and theoretically also in the amount of rigidity, polarity, and hydrophobicity of the polypeptide. Of these alterations only density of acceptor sites has a statistically significant effect on the ability of the [Formula: see text] moiety to act as a substrate for galactosyl-transferase (EC 2.4.1.22) activity solubilized from rat liver microsomes. This result suggests that in the biosynthesis of rat liver glycoproteins these structural features of the polypeptide moiety of glycoproteins are not part of the substrate specificity of the galactosyltransferase activity that transfers the second monosaccharide. Hence, these structural features do not play a major role in determining the structure of the threonine-linked oligosaccharide after its synthesis has been initiated.
Phosphonolipids. XXVII. Synthesis of phosphonic acid analogs of saturated and unsaturated L-α-lecithins
Authors: Robinson Ranga , Baer Erich
Published: December 2009

A procedure for the synthesis of saturated and unsaturated phosphonolecithin from the cadmium chloride complex of sn-glycerol-3-(2-trimethylammoniumethyl)-phosphonate is reported. The phosphonolecithins are obtained by acylating sn-glycerol-3(2-trimethylammoniumethyl)-phosphonate – cadmium chloride complex with fatty acid chloride and pyridine at low temperature. The preparation of the distearoyl, dipalmitoyl, dimyristoyl, and dioleoyl phosphonolecithins by this procedure is described.
Some biochemical effects of 4-deoxy-4-fluoro-D-glucose on Escherichia coli
Authors: Taylor N. F. , Louie Li-Yu
Published: December 2009

The uptake of 4-deoxy-4-fluoro-D-glucose (4FG), without subsequent catabolism, by resting cells of Escherichia coli (ATCC 11775) is 0.06 mg/mg dry weight. In frozen–thawed cells of this organism, 4FG is a substrate for the phosphoenolpyruvate phosphotransferase system with a rate of phosphorylation twice that found for the isomeric 3-deoxy-3-fluoro-D-glucose. 4FG is not a carbon source for growth of this organism and it inhibits the extent of growth of cells in the presence of glucose. The inhibition of growth of E. coli K12 on lactose by 4FG is also observed and this is considered to be consistent with the fact that 4FG is an uncompetitive inhibitor of β-galactosidase (EC 3.2.1.23) activity and that 4FG or 4-deoxy-4-fluoro-D-glucose-6-phosphate repress β-galactosidase synthesis. These results support the view that catabolite repression may be produced by compounds which are not necessarily metabolised further than hexose-6-phosphates.
Glucagon from avian pancreatic islets: radioreceptor studies
Authors: Tung Anthony K. , Rosenzweig Steven A. , Foa Piero P.
Published: December 2009

Glucagon extracted from isolated islets of the pigeon was studied by means of Sephadex gel filtration. Radioreceptor assay, using rat liver plasma membranes and radioiodinated porcine glucagon, showed that the bulk of the activity eluted with glucagon (molecular weight 3500). Avian glucagon appeared to be less effective than porcine glucagon in inhibiting the binding of labeled porcine glucagon to rat plasma membranes.
Fructose-1,6-diphosphatase activity in brown adipose tissue of the developing rat
Authors: Seccombe David W. , Hahn Peter , Skála Josef P.
Published: December 2009

An enzyme activity capable of converting fructose-1,6-diphosphate to fructose-6-phosphate was demonstrated to be present in crude tissue extracts from brown adipose tissue of the rat. Mg2+ was essential for the expression of activity. EDTA (0.5 mM) increased the activity by 30%. Fructose-1,6-diphosphate in concentrations of 1 and 10 mM inhibits activity by 30% and 60% respectively. A 65% inhibition was observed in the presence of 0.2 μM 5′AMP. The activity of the enzyme was measured in rat brown adipose tissue at different stages of development. It rises sharply between day 2 and day 6 and continues to increase reaching a maximum between 6 and 11 days. Thereafter the activity gradually declines to values observed prenatally. The normal developmental rise in activity could be prevented by chemical sympathectomy on day 2. This procedure had no effect when carried out on day 9. There was a significant increase in enzyme activity after cold adaptation. The possible physiological significance of this enzyme in brown adipose tissue is discussed.
The interaction of human glycophorin with 8-anilino-1-naphthalene sulfonate
Authors: Verpoorte Jacob A.
Published: December 2009

Both the sialoglycoprotein of human erythrocyte membranes, glycophorin, and the sialic acid free protein, obtained by treatment of glycophorin with neuraminidase (EC 3.2.1.18), increase the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS). Binding of ANS to glycophorin is weak compared with the binding to bovine serum albumin (BSA). Equilibrium dialysis gives an apparent binding constant of about 4 × 103 M−1 at neutral pH, but Ka increases 1.75 times when NaCl or CaCl2 are added and 10-fold when the pH is lowered to 3.0. Sialic acid groups do not significantly affect ANS binding, although they have some effect at low ionic strength and neutral pH.Fluorescence studies indicate only one to two binding sites for ANS, with apparent pK = 3.8 ± 0.2. and located close to aromatic residues in glycophorin.Polarization and quantum efficiency of the fluorescence of ANS associated with glycophorin fail to indicate changes in the vicinity of the binding site when the pH is lowered.
Affinity chromatography of myosin, heavy meromyosin, and heavy meromyosin subfragment one on F-actin columns stabilized by phalloidin
Authors: Grandmont-Leblanc Andrée , Gruda Julian
Published: December 2009

A method of affinity chromatography based on the trapping of actin filaments within agarose gel beads is described. This method can be used for the purification of myosin and its active proteolytic subfragments, as well as for studies on the interaction between actin and these proteins. Actin columns stabilized by phalloidin bind myosin, heavy meromyosin (HMM), and heavy meromyosin subfragment 1 (HMM-S1) specifically and reversibly. The effect of pyrophosphate and KCl on the dissociation of actomyosin, acto-HMM, or acto-HMM-S1 complex is reported. We also describe the single-step purification of myosin from a crude rabbit psoas muscle extract.
Purification and partial characterization of cysteine-glutamate transaminase from rat liver
Authors: Ip M. P. C. , Thibert R. J. , Schmidt Jr. D. E.
Published: December 2009

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.
Isolation and partial characterization of a protein synthesis inhibitor from brine shrimp embryos
Authors: Warner A. H. , Shridhar V. , Finamore F. J.
Published: December 2009

Encysted embryos of the brine shrimp, Artemia salina, contain an inhibitor of protein synthesis that appears to be important in translational control. In cyst homogenates, the inhibitor appears to be partitioned almost equally between the cytosol and ribosome fractions and it has been purified from both fractions to near homogeneity. In a cell-free protein-synthesizing system derived from Artemia cysts, with poly(U) as messenger, the protein inhibits polyphenylalanine synthesis proportional to inhibitor concentration up to about 75% inhibition, and the primary site of action appears to be at the elongation step. The inhibitor activity is not altered by 50–150 mM KCl in the reaction mixture, but it is slightly more effective at 5 mM MgCl2 than at 10 mM MgCl2. The inhibitor is a heat-labile protein of 130 000 molecular weight and is devoid of hydrolase activity. Our data indicate that the inhibitor is not elongation factor EF-1 or EF-2, but we are studying the possibility that it may be a modified form of elongation factor EF-2.
Mannose-binding hemagglutinins in extracts of Pseudomonas aeruginosa
Authors: Gilboa-Garber N. , Mizrahi L. , Garber N.
Published: December 2009

Mannose-binding hemagglutinins were found in the extracts of a pyocyanin-forming Pseudomonas aeruginosa, which contain galactose-specific hemagglutinins. They were purified simultaneously with the latter proteins by heating to 70 °C, precipitating with ammonium sulfate, application to a Sepharose 4B column, and elution from it by 0.05 M mannose.The mannose-specific hemagglutinins were shown to be similar to the galactophilic ones in (a) being glycoproteins of very low molecular weight (about 11 000 by SDS gel electrophoresis), (b) their tendency to aggregate, and (c) their ability to effect stronger agglutination of erythrocytes treated with papain than of untreated ones. They were found to resemble them also in their reaction with simple sugars and interactions with divalent cations, which are essential for their activity. In these properties, as well as in their relative resistance to heat and to proteolytic enzymes, these two types of bacterial hemagglutinins are like most of the plant, contrasted with the animal, hemagglutinins.The reactions with mannose and mannose-bearing compounds (yeast mannan, horseradish peroxidase (EC 1.11.1.7), and serum globulins), which are not shared with the galactophilic Pseudomonas hemagglutinins, indicate a relationship of the mannose-binding protein of Pseudomonas to the plant lectin concanavalin A. The mannose-binding hemagglutinins do not exhibit identical cell-agglutinating spectra owing to difference in profiles of sugar specificity and relative affinity to mannose derivatives compared with free mannose.
Apparent saturability of a 4S dihydrotestosterone-binding protein in rat muscle cytosol: role of 3α-hydroxysteroid dehydrogenase and albumin
Authors: Dionne France T. , Dube Jean Y. , Tremblay Roland R.
Published: December 2009

In our studies of the binding of steroids in rat skeletal muscles, we have shown the existence of a saturable '4S' dihydrotestosterone-binding protein of low affinity (Kd = 1.16 × 10−6 M). Competition studies led us to believe that the binding of dihydrotesterone (DHT) was due to 3α-hydroxysteroid dehydrogenase (3α-OHSD) enzyme responsible for the conversion of DHT to androstanediol (Adiol). Indeed, both the binding and the enzymatic activity are inhibited by various 3-keto steroids. In addition, the dissociation constant (Kd) and the Michaelis constant (Km) of the enzyme are similar. However, experiments with ammonium sulfate fractions of the cytosol have shown a partial separation of the binding and of the enzymatic activity. On the other hand, we have established that DHT (3 μM) is almost completely metabolized to Adiol during a 2-h incubation at 0 °C even in the absence of added coenzymes. Furthermore the '4S' protein binds Adiol more strongly than DHT and this binding is not saturable. Finally the binding behaviour of both DHT and Adiol with either muscle cytosol or rat albumin is similar when subjected to ammonium sulfate fractionation and sucrose density gradient centrifugation. In conclusion, the skeletal muscle 3α-OHSD rapidly metabolizes DHT into Adiol which then binds strongly to a nonspecific binding protein, presumably rat serum albumin. Thus it can be said that the observed saturability of DHT binding is only apparent.
Effect of neomycin and ionophore A23187 on ATP levels and turnover of polyphosphoinositides in human erythrocytes
Authors: Lang Verna , Pryhitka Glen , Buckley J. Thomas
Published: December 2009

The relationship of polyphosphoinositide metabolism to erythrocyte ATP levels was examined. Turnover of polyphosphoinositides was not as closely dependent on ATP as it is reported to be in yeast. Neomycin increased 32P incorporation into diphosphoinositide and to a lesser extent into triphosphoinositide without affecting intracellular ATP. Treatment of intact cells with ionophore A23187 resulted in a decrease of at least 80% in polyphosphoinositide levels which followed a decrease in cellular ATP and an increase in phosphatidate. The results indicate that polyphosphoinositide turnover does not regulate energy charge in the erythrocyte. However some of the events which follow treatment of erythrocytes with metabolic inhibitors or calcium and ionophore may be related to the accompanying decrease in polyphosphoinositide levels.
The binding site of rabbit skeletal α-tropomyosin on troponin-T
Authors: Pearlstone Joyce R. , Smillie Lawrence B.
Published: December 2009

The binding site for rabbit skeletal α-tropomyosin on troponin-T has been localized to the cyanogen bromide fragment, CB2 (residues 71–151), using affinity chromatography. The entire fragment is required to bring about the correct secondary structure conducive to binding. CB2 contains about 80% α-helix and accounts for most of the helical content found in troponin-T (35%). The molecular weight of CB2 obtained by sedimentation equilibrium (9700) agrees closely with the value calculated from sequence analysis (9850). Circular dichroism and sedimentation velocity experiments indicate that the helix is stable and not affected by salt concentrations of 0.1 to 1.6 M KC1 norby composition of the buffer. The helical content is unaffected by pH from 3.3 to 9.1 but decreases at pH 10–11. Temperature denaturation studies of CB2 and troponin-T show that both are similarly heat labile, with loss of 50% of the ellipticity at 39 °C. Binding of CB2 to α-tropomyosin occurs in the pH range of 5.0 to 9.1, but not at pH 3–4 or 10–11. It is concluded that the helical region of CB2, and perhaps the carboxyl side chains of aspartic and glutamic acids, may be involved in binding over a limited surface area of the double-stranded coiled coil of α-tropomyosin.
Wheat embryo ribonucleates. IX. Generation of N2-dimethylguanylate when bulk wheat embryo tRNA is used as substrate for wheat embryo S-adenosylmethionine-tRNA methyltransferases, in vitro
Authors: Kennedy T. D. , Kwong T. C. , Lane B. G.
Published: December 2009

Although the cellular ribonucleates in normally growing cells are virtually saturated with respect to their customary complement of methyl substituents, it has often been reported that 'marginal' levels of (homologous) methylation can be detected when ribonucleates and enzymes from the same source material are incubated, together with S-adenosylmethionine, in vitro. Experiments were designed to acquire new insights that might be useful for circumscribing the number of possible interpretations that could be advanced to account for the introduction of 'supernumerary' methyl groups during (homologous) methylation of wheat RNA by wheat enzymes, in vitro. For a large fraction of the supernumerary methyl groups that can be introduced into wheat RNA, in vitro, it was not possible to adduce convincing evidence in support of the view that any appreciable quantity of methyl groups is ever introduced at these same sites, in vivo. The possibility that these supernumerary methyl groups might have transient existence, in vivo, and the potential physiological significance of any such occurrence are dealt with as part of a more general discussion of the experimental findings.
Preferential in vivo accumulation of sn-2,3-diacylglycerols in postheparin plasma of rats
Authors: Morley N. , Kuksis A. , Hoffman A. G. D. , Kakis G.
Published: December 2009

The stereochemical course of in vivo hydrolysis of triacylglycerols by lipoprotein lipase was investigated by determining the structure of diacylglycerol intermediates in postheparin plasma of rats which had been fed [3H]glycerol-labeled Intralipid 2 h before an injection of heparin or had been given an injection of a mixture of [3H]glycerol-Intralipid and heparin. During the clearance of both the natural chylomicrons and the artificial emulsion, sn-2,3-diacylglycerols (60–80%) were found to be the dominant enantiomers. Similar results were obtained when the contribution of the hepatic lipase was altered, either by tying off the mesentery artery and portal vein before injection of heparin, or by injection of heparin directly into the portal vein. These findings are consistent with a preferential release of the acyl group from the sn-1 position of the triacylglycerol molecule as demonstrated previously in vitro. A preferential orientation of the substrate in the enzyme–substrate complex or at the oil–water interface is discussed as a possible basis for these effects.
Biosynthesis of chlorophyll b
Authors: Aronoff S. , Kwok E.
Published: December 2009

Using [14C]chlorophyll a derived from a mutant of wheat lacking chlorophyll b, spinach thylakoids from very young leaves and cucumber thylakoids of irradiated, etiolated cotyledons convert chlorophyll a to b with apparent yields of 19% and 5%, respectively.
Purine metabolism in isolated rat hepatocytes
Authors: Smith Camilla M. , Rovamo Liisa M. , Kekomäki Martti P. , Raivio Kari O.
Published: December 2009

The metabolism of adenine, hypoxanthine, guanine, and adenosine was studied in rat liver cell suspensions, prepared by collagenase perfusion. Oxygen supply was a critical variable in the preparation and subsequent incubation of the cells, as judged on the basis of the ratio of radioactivity in ATP to that in ADP after incubation with [14C]adenine. This ratio is suggested as an additional criterion of cell function. Adenine nucleotides synthesized from [14C]adenine were slowly catabolized to allantoin, with little incorporation of radioactivity into other purine compounds. [14C]Adenine is thus suitable for prelabelling the adenine nucleotide pool. [14C]Guanine and [14C]hypoxanthine were rapidly catabolized to allantoin, whereas nucleotide synthesis was low. [14C]Adenosine was initially phosphorylated and deaminated at about equal rates, but with continued incubation catabolic products predominated. Isolated hepatocytes were found suitable for studies of purine metabolism, in which the liver has important functions for the whole organism.
Protein content and enzyme levels of cultured chick embryo cells treated with camptothecin and actinomycin D
Authors: Morais Réjean
Published: December 2009

The alkaloid camptothecin uncouples the growth and division of chick embryo cells. At a moderate dose (0.5 μg/ml) it inhibits the incorporation of thymidine but not of uridine and leucine and the cell protein content increases and reaches twice that of control after 4 days of treatment. Twelve hours after addition of the drug, the activities per cell of the mitochondrial enzymes poly A hydrolase (EC 3.1.4.21), cytochrome c oxidase (EC 1.9.3.1), and succinate dehydrogenase (EC 1.3.99.1) are greater than that of the control and keep increasing for at least 96 h. The increase in the activities of the mitochondrial enzymes precede that of NADPH-cytochrome c reductase (EC 1.6.2.4) and cytidine triphosphatase (EC 3.6.1.15), which are microsomal and plasma membranes enzymes respectively. Actinomycin D (0.01 μg/ml) also inhibits the multiplication of the chick cells and the synthesis of DNA. The protein content of the actinomycin D treated cells decreases to 70% of the control by day 2. Nevertheless, the activities of the mitochondrial enzymes increase over that of the control but to a smaller extent than with camptothecin. The activities of the enzymes of the other organelles are not stimulated. Camptothecin at a higher dose (5.0 μg/ml) induces effects similar to those of actinomycin D.
Increased lipoprotein lipase activity of skeletal muscle in cold-acclimated rats
Authors: Bégin-Heick N. , Heick H. M. C.
Published: December 2009

The activity of lipoprotein lipase (LPL) in the heart, diaphragm, and soleus muscles was markedly increased in cold-acclimated rats and it was even greater in rats treated with oxytetracycline (OTC) while exposed to cold. Other skeletal muscles studied had low and variable activities which were not significantly increased by cold acclimation or by cold plus OTC treatment. It appears therefore that, apart from the heart and the muscles involved in respiratory movements, LPL activity is primarily associated with those muscles which contain a predominance of slow-twitch oxidative fibers, and that the enzyme in muscle, heart, and diaphragm responds to cold acclimation and cold plus OTC treatment in a parallel fashion in these tissues.
Interaction of sodium with the sarcolemmal calcium system
Authors: Morcos N. C. , Jacobson A. L.
Published: December 2009

Sarcolemma isolated from guinea pig heart binds calcium in an ATP-dependent manner. Sodium ions decrease the total amount of calcium bound by the membranes. ATP-dependent calcium binding is more sensitive to sodium than the non-ATP-dependent calcium binding. The ATPase active during calcium binding is affected by sodium ions to the same extent as the ATP-dependent calcium binding process. The inhibition of the calcium binding process and of ATPase activity by sodium was more pronounced when the membranes were preincubated with sodium. The effect of sodium on calcium binding is dependent on both the time of contact between sodium and the membranes and the concentration of sodium. It is suggested that the effect of sodium on the calcium binding system in the sarcolemma may be a link between the inhibition of Na+K+-ATPase (EC 3.6.1.3) by cardiac glycosides and the subsequent increase in intracellular calcium.
Structural studies of staphylococcal protease. III. Binding of anions to the spin-labeled enzyme
Authors: Dugas Hermann , Gaudet Fernand , Leduc Paul
Published: December 2009

The staphylococcal protease was coupled at the active-site serine residue with a spin-labeled analog of diisopropyl fluorophosphonate and the interaction of competitive inhibitors such as chloride and acetate anions, as well as N-carbobenzoxy-L-glutamic acid (Z-L-Glu), was investigated by electron paramagnetic resonance spectroscopy. It was observed that the addition of chloride ions to the spin-labeled enzyme increased the freedom of motion of the spin label while the presence of acetate ions and Z-L-Glu resulted in an increase in the immobilization of the spin label. These results suggest that these ions bind to the active site region in different ways.
Studies on the use of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline for the synthesis of acylamino acid anilides and p-nitroanilides
Authors: Chen Francis M. F. , Benoiton N. Leo
Published: December 2009

The use of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) for coupling acyl-L-amino acids with aniline and p-nitroaniline has been investigated. Optically pure anilides are obtained in good yield but partial racemization occurred in one case. p-Nitroanilides cannot be obtained under similar conditions but if the reaction solvent is removed and the residue left for several days, coupling occurs giving a racemic product in moderate yields. Reaction mechanisms are proposed on the basis of isolated and characterized intermediates. It has been found that N-benzoylamino acid anilides and p-nitroanilides are cleaved within a few minutes by hydrogen bromide in acetic acid to give the N-benzoylamino acid and the amine.
Purification and properties of β-xylosidase from Penicillium wortmanni
Authors: Deleyn F. , Claeyssens M. , Beeumen J. Van , Bruyne C. K. De
Published: December 2009

A purification method for an extracellular β-xylosidase (β-D-xyloside xylohydrolase, EC 3.2.1.37) induced in Penicillium wortmanni is described. It includes diafiltration, acetone precipitation, and hydroxylapatite chromatography. The enzyme has a molecular weight of about 100 000. Its pH optimum is at pH 3.3–4.0 and it is most stable at pH 5.0–6.0. Its isoelectric point is at pH 5.0. Sulfhydryl and histidine reagents are not inhibitory. The influence of added cations and anions is negligible. N-Bromosuccinimide oxidation of two to three tryptophan residues per molecule entails rapid inactivation. Glycon-specificity studies indicate strict requirements at C-2, C-3, C-4, and C-5, although α-L-arabinopyranosides are substrates. As the enzyme seems to hydrolyse xylooligosaccharides endwise, with retention of configuration in the reaction product, the enzyme is a true glycosidase, probably operating by a double-inversion mechanism.
Multiple isoacceptor forms of several transfer ribonucleic acids in a mutant yeast strain
Authors: Bell J. B. , Jacobson K. Bruce , Shugart Lee R.
Published: December 2009

By use of reverse phase 5 chromatography, a strain of Saccharomyces cerevisiae (XB109-5B) has been shown to exhibit multiple isoaccepting forms for several of the transfer ribonucleic acids (tRNAs). This is in contrast with a standard wild-type strain where only one acceptor is found for each tRNA studied. Multiple peaks for tRNATyr, tRNAPhe, tRNASer, and tRNAVal have been detected for strain XB109-5B. However, the observation of multiple isoacceptors cannot be extended to all tRNAs in this strain since tRNAAsp appears as a single form that is the same as in the wild type. The appearance of multiple peaks was found to depend on the growth conditions of the cells. The tRNA profiles of XB109-5B that was grown rapidly with vigorous aeration differed the most from profiles of comparably grown wild-type yeast, whereas tRNA from this mutant, grown without shaking or supplementary aeration, appeared the same as the wild type. The minor nucleoside composition of the isoacceptors of tRNAPhe was obtained.
Saturable and nonsaturable hexose uptake in cultured human skin fibroblasts
Authors: Germinario Ralph J. , Oliveira Maureen , Leung Hyman
Published: December 2009

The saturable transport of 2-deoxy-D-glucose across the cell membrane of cultured human skin fibroblasts was measured in sparse and confluent cultures. The contribution of nonsaturable sugar uptake to total sugar uptake was monitored by determining L-glucose uptake. The uptake of 2-deoxy-D-glucose was studied as a function of time and substrate concentration. Greater than 70% of transported 2-deoxy-D-glucose was phosphorylated after incubation for 2 min or less at all substrate concentrations employed (0.1 to 3.0 mM), and phosphorylation paralleled sugar uptake at these time intervals. Experiments with cytochalasin B demonstrated that an inhibition of transport was always paralleled by an equal inhibition of sugar phosphorylation.The kinetic constants for the uptake and phosphorylation of 2-deoxy-D-glucose and the inhibition of transport by competing sugars and cytochalasin B were calculated from Line-weaver-Burk plots. The Km and Vmax for saturable sugar uptake were calculated for sparse and confluent cultures after subtracting the contribution of nonsaturable sugar uptake. The resulting Km values for sugar uptake in the sparse and confluent cultures were 1.21 ± 0.04 and 0.88 ± 0.2 mM respectively. The corresponding Vmax values were 15.5 ± 1 nmol/mg protein∙min−1 for the sparse cultures and 10.1 ± 1 nmol/mg protein∙min−1 for the confluent cultures. In both sparse and confluent cultures, the Ki values for the competitive inhibition of sugar transport by D-glucose and 3-O-methyl-D-glucose were 0.8 and 2.7 mM respectively; the Ki value for the noncompetitive inhibition of sugar transport by cytochalasin B was 0.5 μM. The Km values for sugar phosphorylation by cell-free homogenates of sparse and confluent cultures were 0.57 ± 0.1 and 0.6 ± 0.1 mM respectively, while their respective Vmax values were 160 ± 53 and 139 ± 43 nmol/mg protein∙min−1.The data are in agreement with the concept that in cultured human skin fibroblasts sugar transport is the rate-limiting step in 2-deoxy-D-glucose metabolism and that phosphorylation is distinct from transport.
Precursor role of 15α-hydroxyestradiol and 15α-hydroxyandrostenedione in the formation of estetrol
Authors: Schut H. A. J. , Bowman J. M. , Solomon S.
Published: December 2009

Studies were designed to elucidate the origin of estetrol (15α-hydroxyestriol (estra-1,3,5(10)-triene-3,15α,17β-tetrol) or E4) during late human pregnancy. 3H-Labelled 15α-hydroxy-estradiol (3,15α-dihydroxyestra-1,3,5(10)-trien-17-one or 15E2) and 14C-labelled 17β-estradiol (estra-1,3,5(10)-triene- 3,17β-diol or E2) were infused into the fetus during transfusion in utero for erythroblastosis fetalis, and in another study the same substrates were injected intravenously into the maternal circulation. In a third study, 3H-labelled 15α-hydroxyandrostenedione (15α-hydroxyandrost-4-ene-3,17-dione or 15Δ4) and 14C-labelled E2 were infused into the fetus. Maternal urine was collected for 5-6 days, and after Glusulase hydrolysis, the following metabolites were isolated: estriol (estra-1,3,5(10)- triene-3,16α,17β-triol or E3) containing 14C only and 15α-hydroxyestrone (3,15α-dihydroxyestra-1,3,5(10)-trien-17-one or 15E1), 15E2, and E4, all containing both labels. From the isotope content of these metabolites, it was concluded that E4 was derived from both fetal E2 and 15Δ4 and only partially via 15E2. When administered to the fetus, E2 and 15Δ4 contributed approximately equal amounts to urinary E4. The yield of 15α-hydroxylated estrogens from E2 injected into the mother was very low indicating the predominantly fetal origin of the 15α-hydroxylase. 15Δ4 was a better precursor than E2 for urinary 15E2.
The purification and characterization of a DNA nicking–closing enzyme from Bacillus megaterium
Authors: Burrington M. G. , Morgan A. Richard
Published: December 2009

Although several eucaryote DNA nicking–closing (N–C) enzymes have been characterized, only the Escherichia coli enzyme has been extensively studied amongst procaryotes. The latter enzyme is distinctly different from the eucaryotic enzymes and we have therefore purified the N–C enzyme from Bacillus megaterium to determine if procaryotes form a distinctive class of N–C enzymes. The purified B. megaterium N–C enzyme has a molecular weight of 120 000, only partly relaxes negative supercoils, does not affect positive supercoils, requires Mg2+, and is inhibited by 0.2 M KCl. The enzyme is also inhibited by 1 mM nalidixic or oxolinic acids but unaffected by novobiocin. A crude N–C enzyme preparation from Micrococcus luteus shows very similar properties.
Synthesis of thymidine nucleotides in Bacillus subtilis
Authors: Rima B. K. , Takahashi I.
Published: December 2009

To investigate the synthesis of thymidine nucleotides in Bacillus subtilis, mutants that carried various combinations of thyA, thyB, and other mutations affecting pyrimidine metabolism were isolated. It was found that exogenously supplied deoxycytidine was converted to thymidine nucleotides. The present data suggest that deoxycytidine nucleotides are first deaminated to yield deoxyuridine nucleotides which can serve as substrates for both thyA- and thyB-coded synthetases. A deaminase activity for dCDP was found in crude extracts of B. subtilis. A mutant lacking the deaminase activity was unable to convert deoxycytidine nucleotides to thymidine nucleotides.