bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycoprotein biosynthesis during the acute-phase response to inflammation
Authors: Jamieson J. C. , Kaplan H. A. , Woloski B. M. R. N. J. , Hellman M. , Ham K.
Published: December 2009

Inflammation results in an increase in the levels of a variety of glycoproteins in serum. The glycoproteins that respond in this way are usually referred to as acute-phase reactants. Studies on the acute-phase response of rat α1-acid glycoprotein showed that there was an increase in the liver levels of this glycoprotein at 12 h after turpentine inflammation. This was followed by increased serum levels at 48–72 h after inflammation, suggesting a precursor–product relationship between liver and serum α1-acid glycoprotein. Incorporation studies coupled with measurements of synthesis rates of α1-acid glycoprotein showed that increased synthesis was responsible for the acute-phase response of this protein to inflammation. These studies also showed that albumin was a negative acute-phase reactant. The acute-phase response of α1-acid glycoprotein was accompanied by increased liver pools of UDP–N-acetylglucosamine (UDP–GlcNAc) and UDP–N-acetylgalactosamine (UDP–GalNAc) and increased liver activities of glucosamine-6-phosphate synthase and UDP–GlcNAc 2-epimerase. Activities of galactosyl and sialyl transferases in liver were also elevated and serum sialyl transferase was increased substantially in inflammation, suggesting that it may also be an acute-phase reactant. Liver activities of β-N-acetylhexosaminidase and β-galactosidase declined by about 50% at 24 h after inflammation; there was evidence that serum levels of these enzymes increased at 24–72 h after inflammation, suggesting that the lysosomal glycosidases may be released from liver during inflammation. Inflammation resulted in elevated serum Cortisol, insulin, and adrenocorticotropic hormone and induced increased glycogenosis; liver cAMP levels were also increased during inflammation. Preliminary studies are presented to show that leukocyte-derived factors may be involved in the acute-phase response of α1-acid glycoprotein to inflammation.
The relation of three-dimensional structure to biosynthesis in the N-linked oligosaccharides
Authors: Brisson Jean-Robert , Carver Jeremy P.
Published: December 2009

The three-dimensional structures are presented for representative N-linked oligosaccharides drawn from four major structural classes. The nuclear magnetic resonance evidence leading to these structures is summarized and the conclusions are compared with those obtained from potential energy calculations and from the crystal structures of fragments. The major finding is that the Manα1-3 arm structure is invariant throughout the series, whereas the Manα1-6 arm has two possible orientations in some structures and a single orientation in others. Finally, the three-dimensional structures permit a rationalization of the substrate specificity of several key enzymes in the biosynthetic pathway.
Antibodies to indolealkylamines II: site of conjugation of melatonin to protein using formaldehyde
Authors: Grota L. J. , Snieckus V. , Silva S. O. de , Brown G. M.
Published: December 2009

The site of coupling on the hapten was investigated for melatonin, formaldehyde, and protein conjugates. Model reactions of glycine ethyl ester and piperidine with formaldehyde and melatonin suggested that coupling first occurred through the indole nitrogen and then a stable bond was formed at C-2. Cross-reaction studies of antisera stimulated by melatonin, formaldehyde, and protein conjugates support the hypothesis that C-2 is the site of conjugation of melatonin to protein.
Immunoquantitation of liver glucose-6-phosphate dehydrogenase and malic enzyme in normally and prematurely weaned rats
Authors: Back Donald W. , Angel Joseph F.
Published: December 2009

Rat liver glucose-6-phosphate dehydrogenase and malic enzyme were purified and rabbit serum antibodies were prepared against each enzyme. The activities and quantities of both enzymes in the livers of infant rats were subsequently determined during the weaning period. Glucose-6-phosphate dehydrogenase was present and active in the liver of spontaneously weaned rats on postnatal day 17 and increased from postnatal day 21 onwards. Malic enzyme and its activity were undetectable on postnatal day 17. The latter enzyme was detected on postnatal day 21 and increased rapidly afterwards. These changes occurred sooner and were more pronounced when the rats were weaned prematurely on postnatal day 17, especially when the diet contained sucrose. The activities of both enzymes were highly correlated with the amounts of enzyme protein present throughout the experiment. It appeared that the activities of both enzymes in infant rats were likely to be regulated by altering their synthesis and (or) degradation, rather than by activation of existing proteins, assuming that the latter can be detected by the antibodies employed.
Energy transfer in photosynthetic bacteria as examined by photoacoustic spectroscopy
Authors: Boucher François , Lavoie Lysanne , Antippa Adel F. , Leblanc Roger M.
Published: December 2009

Antenna carotenoids are investigated by photoacoustic spectroscopy in the photosynthetic bacteria Rhodospirillum rubrum and Rhodopseudomonas sphaeroides. Energy storage in these organisms is revealed by the photoacoustic spectra. A simple method is proposed to evaluate the efficiency of energy transfer between carotenoids and bacteriochlorophyll.
Guinea-pig α1-fetoprotein: purification, characterization, developmental and hormonal regulation, and behavior in diethylnitrosamine hepatocarcinogenesis
Authors: Gourdeau Henriette , Bélanger Luc
Published: December 2009

Physicochemical and immunological procedures were developed for the purification of guinea-pig α1-fetoprotein (AFP). A double-antibody radioimmunoassay was developed which can detect 0.1 ng of AFP. The [Formula: see text] of guinea-pig AFP is 6.6. Its molecular weight is 73 000. Its amino acid composition is similar to other AFP's, but it has a higher carbohydrate content (8.5%), owing to larger amounts of mannose and N-acetylglucosamine. Guinea-pig AFP has a slower electrophoretic mobility than other AFP's, owing to its more basic ionic structure. It separates into three electrophoretic variants in nondenaturing polyacrylamide gels; these are charge variants with isoelectric points of 5.0, 5.12, and 5.54. Changes in electrophoretic mobility after neuraminidase treatment indicate that charge heterogeneity is due to a variable content in sialic acid. Guinea-pig AFP has fatty-acid- and lectin-binding properties, but no affinity for sex hormones. Its biological half-life is 2.1 days. Its sites of synthesis are the liver and the yolk sac, with minor contributions by the upper gastrointestinal tract. AFP production appears to cease synchronously in the liver and yolk sac during the 8th week of gestation. From 4 to 8 weeks of gestation, AFP levels are at 2.5–3.5 mg/mL in fetal serum and 0.1–0.2 mg/mL in amniotic fluid. The proportion of electrophoretic and lectin-reactive AFP variants changes during development, reflecting the changing glycosylation of the protein. The gestational levels of AFP in the maternal compartment are indicative of a fetomaternal equilibration through transamniotic exchange. Serum AFP levels in normal adult guinea pigs are remarkably high, from 400 to 2000 ng/mL and occasionally up to over 5000 ng/mL. The administration of dexamethasone suppresses serum AFP levels. During liver carcinogenesis induced by diethylnitrosamine, serum AFP levels increase, moderately, when tumors develop.
Activities of the phosphatidylcholine biosynthetic enzymes in rat liver during development
Authors: Pelech Steven L. , Power Ellen , Vance Dennis E.
Published: December 2009

The activities of the enzymes of rat hepatic phosphatidylcholine biosynthesis have been measured as a function of development in the rat (term, 23 days). During the last 5 days of gestation, the specific activity of choline kinase was elevated almost fivefold (p < 0.05). After parturition, choline kinase activity was reduced to adult values by the 5th postnatal day. Over 75% of the total CTP:phosphocholine cytidylyltransferase protein in prenatal liver was detected in the cytosolic fraction. On the day of birth, most of the cytidylyltransferase translocated to the microsomes so that the microsomal specific enzyme activity was 3.3-fold higher (p < 0.01) and the cytosolic specific enzyme activity (measured in the presence of phospholipid) was 68% lower (p < 0.001) than the day before parturition. CDPcholine:diacylglycerol cholinephosphotransferase activity (measured in the presence of diacylglycerol) increased 130-fold (p < 0.001) during the last 5 days of gestation. On the 10th postnatal day, cholinephosphotransferase activity was 1.7-fold higher (p < 0.001) than immediately after birth, but declined to adult values by the 19th day. Between the 5th day prior to parturition and the 10th postnatal day, phosphatidylethanolamine N-methyltransferase activity steadily increased 16-fold (p < 0.001). The results are in agreement with the hypothesis that the increase in phosphatidylcholine in rat liver during the perinatal period is due to an increased synthesis of CDPcholine, which is a consequence of the translocation of the cytidylyltransferase from cytosol to the endoplasmic reticulum.
Salvage of circulating hypoxanthine by tissues of the mouse
Authors: Moyer James D. , Henderson J. Frank
Published: December 2009

Hypoxanthine is an inefficient precursor of purine nucleotides in mouse tissues. In vitro, mouse erythrocytes salvage <10% of hypoxanthine (10 μM) added to whole blood in 30 min of incubation at 37 °C. In vivo, circulating hypoxanthine is rapidly degraded (>90% in 10 min) to allantoin and uric acid. All tissues examined (other than erythrocytes) converted small amounts of hypoxanthine to nucleotides, with kidney and lung being the most active tissues examined. It is estimated that <2% of circulating hypoxanthine is salvaged in the mouse; the remainder is catabolized.
Methylenetetrahydrofolate dehydrogenase – methenyltetrahydrofolate cyclohydrolase –formyltetrahydrofolate synthetase from porcine liver: evidence to support a common dehydrogenase–cyclohydrolase site
Authors: Smith D. Drummond S. , MacKenzie Robert E.
Published: December 2009

The cyclohydrolase activity of the trifunctional enzyme methylenetetrahydrofolate dehydrogenase – methenyltetrahydrofolate cyclohydrolase – formyltetrahydrofolate synthetase is inhibited by NADP+, a substrate of the dehydogenase. This uncompetitive inhibition, shown also by 3-aminopyridine adenine dinucleotide phosphate (AADP), indicates formation of dead-end complexes consisting of enzyme–nucleotide–methenyltetrahydrofolate. Chemical modification with diethylpyrocarbonate inactivates the dehydrogenase and cyclohydrolase but not the synthetase. Folate, but neither NADP+ nor AADP, protects both activities against modification. However, NADP+ potentiates the protection by folate by decreasing the apparent Kd for that ligand approximately sixfold. Chemical modification with phenylglyoxal also inactivates both the dehydrogenase and cyclohydrolase activities. Neither activity was protected by NADP+ or folate alone; however, the combination of NADP+ and folate protected both activities. These results are consistent with a model in which the dehydrogenase and cyclohydrolase activities share a common folate binding site.
Changes in gene expression patterns for specific proteins when vegetatively growing cells of the fungus Achlya are induced to differentiate by starvation
Authors: LéJohn Herb B.
Published: December 2009

Within 6 h after Achlya cells growing vegetatively in a defined medium were transferred to a nutrient-free medium (starvation), they synchronously developed asexual sporangia and sporulated. This response, however, depended on the vegetative age of the cells prior to starvation. If the cells had grown for less than 6 h, then only about 10% of them developed sporangia within 6 h of starvation; these are referred to as reproductively incompetent cells. If the cells had grown for more than 12 h, then more than 90% devloped sporangia within 6 h of starvation; these are referred to as reproductively competent cells. The pattern of protein synthesis in competent and incompetent starving cells was analyzed by electrophoresis; it was similar except that a few proteins of molecular weight (MW) 85 000–35 000 were synthesized at a significantly diminished rate in competent cells compared with incompetent ones. By contrast, vegetatively growing and starving competent cells displayed somewhat different patterns of protein synthesis. On the one hand, a protein of MW 83 000 was actively synthesized in growing cells, but not in starving ones. A variety of growth inhibitory conditions such as thermal shift from 24 to 42 °C, supply of actinomycin D (12.5 μg/mL), or supply of tunicamycin (25 μg/mL) drastically reduced synthesis of the 83 000 MW protein in contrast to other cell proteins. On the other hand, a group of polypeptides of MW 40 000 was actively synthesized by starving cells, but not by vegetatively growing ones. L-Glutamine arrested sporangial development in reproductively competent starving cells and altered their protein synthesis pattern in the following ways: it reduced synthesis of the 40 000 MW polypeptides, maintained an active synthesis of the 83 000 MW protein for a period, and generally elicited a vegetative cell pattern of polypeptide labelling.
Properties of a catalase from a peroxide-resistant mutant of Proteus mirabilis
Authors: Jouve Hélène M. , Lasauniere Christiane , Pelmont Jean
Published: December 2009

A catalase (EC 1.11.1.6) from Proteus mirabilis PR, a mutant with strong resistance to hydrogen peroxide, was purified to homogeneity and compared with catalase from wild-type P. mirabilis. In crude extracts from the mutant, catalase was present as two different entities called A and B, that could be resolved by ion-exchange chromatography. The B form was transformed into A. The pure catalase preparation contained the A form only. This catalase was not found to be different from the wild-type enzyme, considering its molecular weight, subunit composition, isoelectric pH, and reactivity to specific antibodies. Partial proteolytic cleavage of the two bacterial enzymes with four different proteases proceeded at the same rate and produced identical patterns. However, pure catalase from the mutant had a specific activity against H2O2 of 2.7 × 107M−1∙s−1, and its purity index (A406/A280) was 1.12. These values were higher than previously determined for the wild-type enzyme. Furthermore, the mutant catalase was more stable to heat. The results suggest that the purified catalase (A form) differs from the wild-type enzyme and appears to be a more efficient catalase against H2O2. Both enzymes were found to be much more resistant than beef liver catalase to the classically used catalase inhibitor 3-amino-1,2,4-triazole.
The growth-related 28-kilodalton protein in germinating wheat. Use of peptide mapping to identify cryptic forms in cell-free extracts and protein-synthesizing systems
Authors: Grzelczak Zbyszko F. , Lane Byron G.
Published: December 2009

Characteristic peptide maps have been prepared and used to identify cryptic forms of the methionine-labeled 28-kilodalton (28-kd) protein (the G protein) which marks onset of growth in germinating wheat embryos. Peptide mapping of material eluted from areas detected as "bands" in autoradiograms of one-dimensional Laemmli-type gels and from areas detected as "spots" in autoradiograms of two-dimensional O'Farrell-type gels has led to identification of a counterpart to G among methionine-labeled products of cell-free synthesis. Identification of its cell-free counterpart has made it possible to show that translatable mRNA for the G protein, like G itself, only begins to appear between 5 and 10 h after onset of water uptake by dry wheat embryos. Peptide mapping of methionine- or proline-labeled material eluted from an area detected as a conspicuous "band" in autoradiograms of one-dimensional Laemmli-type gels has uncovered the existence of an unusual (apparently complexed) form of G in extracts of germinating but not dry wheat embryos. Unique among the hundreds of prominent proteins made by germinating wheat embryos, G exists as part of a thermolabile "150-kd complex" which is refractory to dismutation in a reducing environment containing sodium dodecyl sulphate (SDS). Presence or absence of the G complex constitutes the only conspicuous difference between SDS–polyacrylamide distributions of newly synthesized proteins derived from unheated as opposed to heated extracts of germinating embryos. Analysis of extracts prepared at different times after onset of water imbibition by dry wheat embryos shows that time course of emergence of the G complex, detected in unheated extracts, and of the G protein, detected in heated extracts, is the same. By fractional electroelution and silver staining of protein, it has been possible to show that G may be the principal if not the only protein component of the G complex.
Studies of the biosynthesis and metabolism of rat testicular galactoglycerolipids
Authors: Hsu Liu-Hsin , Narasimhan Rajagopalan , Levine Mark , Norwich Kenneth H. , Murray Robert K.
Published: December 2009

1-O-Alkyl-2-O-acyl-3-O-β-D-(3′-sulfatoxygalactopyranosyl)-sn-glycerol (SGG) and its nonsulfated analog (GG) are the major glycolipids of rat testis. Aspects of the biosynthesis and metabolism of these two lipids have been investigated by determining their specific activities at various times after injection of [16-14C]palmitic acid, [1-14C]cetyl alcohol, and D-[1-14C]galactose into the testes of the adult rats. Evidence was obtained from studies with each of these three radioactive compounds that is consistent with the interpretation that GG exhibits a precursor relationship to SGG in vivo. The turnover time of GG, as estimated from the use of each of the three precursors, ranged between 21 and 69 h. In contrast, a slow increase of radioactivity in SGG was observed following injection of each of the three precursors, a plateau value being reached between 72 and 168 h. The radioactivity in the acyl, alkyl, and galactosyl moieties of SGG thereafter remained quite constant for another 21 days. Small amounts of monoalkylmonoacylglycerol were detected in rat testis. Radioactive studies indicated that this compound could be a precursor of GG and (or) monoalkyldiacylglycerol, another lipid that was also detected in rat testis. The results are consistent with the concept that the synthesis of SGG occurs primarily at an early stage of spermatogenesis and that the various moieties of this lipid exhibit almost complete metabolic stability during the subsequent complex stages of this process.
Phosphatidylinositol movement between isolated microsomal and mitochondrial membranes
Authors: Chudzik J. , Stanacev N. Z.
Published: December 2009

Transfer of membrane-bound phosphatidyl-[2′-3H]inositol from microsomal to unlabelled mitochondrial and from mitochondrial to unlabelled microsomal membranes was studied using partially purified cytosol proteins isolated from guinea pig liver cytosol. In the absence and presence of these proteins the amounts of phosphatidylinositol transfer from microsomal to mitochondrial membranes were approximately 21 and 33%, respectively, and the amounts from mitochondrial to microsomal membranes were approximately 31 and 39%, respectively. The release of phosphatidyl-[2′-3H]inositol from microsomal membranes in the absence of mitochondria was dependent on concentration of cytosol proteins. Two mechanisms for movement between membranes are proposed. In cytosol-protein-independent movement of phosphatidyl-[2′-3H]inositol from microsomal to mitochondrial membranes, a direct contact between membranes is required, since phosphatidyl-[2′-3H]inositol was not detected in the reaction medium. In the cytosol-protein-catalyzed transfer, formation of phosphatidyl-[2′-3H]inositol – cytosol protein complex is postulated, since phosphatidyl-[2′-3H]inositol was released into the reaction medium and its movement proceeded from mitochondrial to microsomal membranes in the presence of partially purified cytosol proteins. Thus, contact between the two membranes is probably not necessary for this transfer. Implications for the movement of phospholipids between biological membranes are discussed.
Developmental modulation of protein synthetic patterns by the human malarial parasite Plasmodium falciparum
Authors: Allred David R. , Sherman Irwin W.
Published: December 2009

Under conditions of in vitro culture, Plasmodium falciparum incorporated amino acids into particulate (membrane) and soluble proteins in a pattern which changed sequentially and which was dependent upon the stage of parasite maturation. Synchronized cultures pulse labeled with a mixture of 15 14C-labeled amino acids or [14C]histidine alone displayed stage-related patterns of polypeptide biosynthesis. Certain plasmodial proteins were associated with both particulate (membrane) and soluble fractions, whereas others appeared to be specific to a given fraction. Proteolysis of intact infected cells with pronase under conditions which removed 97 ± 2.2% of the endogenous red cell acetylcholinesterase activity did not cause the apparent removal of any radiolabeled proteins; this suggests the absence of externally exposed, parasite-synthesized proteins in the infected red cell membrane. Such a result was consistent whether the radiolabel was [14C]histidine or the 14C-labeled amino acid mixture. These results indicate that specific modulation of parasite biosynthetic patterns occurs during the asexual reproductive cycle and is probably one mechanism whereby parasite differentiation occurs. Despite the formation of surface excrescences on infected red cells containing mature parasites, results of surface digestion experiments failed to demonstrate the presence of surface-exposed plasmodial proteins.
Identification and physical characterization of a Col E1 hybrid plasmid containing a catalase gene of Escherichia coli
Authors: Loewen Peter C. , Triggs Barbara L. , Klassen Glen R. , Weiner Joel H.
Published: December 2009

A hybrid Escherichia coli: Col E1 plasmid, pLC36-19, containing a catalase gene has been identified in the Clarke and Carbon colony bank. Catalase activity was amplified two- to three-fold in the pLC36-19-containing strain relative to other hybrid-plasmid-containing strains and this activity could be induced three- or four-fold by hydrogen peroxide or ascorbic acid. The plasmid was transferred to a strain chromosomally deficient in catalase synthesis, resulting in a strain with high and inducible levels of catalase. The plasmid was also transferred to a minicell-producing strain and minicells harbouring the plasmid were found to synthesize a labelled protein with a molecular weight of 84 000 characteristic of catalase from E. coli. A catalase activity was also synthesized by the plasmid-containing minicells. Two catalase activities with associated peroxidase activities coded for by the plasmid were separable by polyacrylamide gel electrophoresis and migrated coincident with chromosomally encoded catalase–peroxidase activities. A third catalase activity which did not have an associated peroxidase activity was not coded for by the plasmid. A physical map of the 25.5-kilobase pair plasmid was constructed by restriction nuclease analysis and the relative positions of 38 restriction sites were defined.
Rat intestinal maltase–glucoamylase. Purification of the detergent-solubilized enzyme in the presence of protease inhibitors: properties and identification of a protease-sensitive subunit
Authors: Lee Lilian , Forstner Gordon
Published: December 2009

Failure to develop clear-cut, distinguishing characteristics for hydrophobic and hydrophilic forms of maltase–glucoamylase led us to attempt the purification of the detergent-extracted enzyme in the continuous presence of protease inhibitors (phenylmethylsulfonyl fluoride and N-ethylmaleimide). The enzyme was purified by molecular exclusion, anion-exchange, and affinity column chromatography to a final specific maltase activity of 80 U/mg protein, comparable to previously solubilized enzymes. Both detergent (d-maltase) and proteolytically (p-maltase) solubilized enzymes had identical Km's for maltose and similar glycogenase activity. d-Maltase was clearly amphipathic. Whereas 95% of p-maltase was eluted with aqueous buffer from an octyl-Sepharose CL-4B column, the elution of d-maltase required solutions containing Triton X-100 and ethylene glycol. On density gradient centrifugation and sodium dodecyl sulfate (SDS) – polyacrylamide gels, p-maltase migrated as one high molecular weight species of 500 000. In contrast d-maltase migrated heterogeneously and the smallest maltase-active forms delineated by these two techniques, as well as by high pressure liquid chromatography, had molecular weights which ranged from 120 000 to 150 000. Both p- and d-maltase were dissociated by heat in SDS, forming five prominent species as we have previously described. In contrast to p-maltase, in which the smallest species, band 1, equalled 36.7% of the total mass, band 1 of d-maltase accounted for 66.5%. Band 1 was separable when smaller amounts of enzyme were applied to slab gels and stained with silver, into two proteins of 130 000 and 145 000 daltons. The 145 000 dalton protein was absent in p-maltase and was replaced by a faint band of 140 000 daltons. The 140 000 dalton band, plus a new N-terminal glycine, were also generated from d-maltase during prolonged storage at −20 °C. These data suggest that rat intestinal maltase–glucoamylase contains two monomeric proteins. The largest monomer contains an additional peptide segment at the N-terminus, which is removed by proteolysis and presumably anchors the enzyme to the microvillus membrane. After removal from the membrane, the two monomers of the d-enzyme are apparently partially dissociated to account for maltase activity within the 120 000 to 150 000 dalton range. Conversely, removal of the anchor segment favours a polymeric structure.
Relationship between cell surface asparagine-linked glycoproteins and myoblast differentiation. Analysis of wheat germ agglutinin-resistant mutants
Authors: Gilfix Brian M. , Sanwal Bishnu D.
Published: December 2009

Alterations in the oligosaccharides of complex surface glycoproteins have been studied in wild-type rat skeletal myoblasts (L6-9/1) and two wheat germ agglutinin resistant mutants, selected for either high level of resistance (WGARI) or low level of resistance (WGARII). All three lines possessed high mannose oligosaccharides of the structure Mann GlcNAc, where n = 6, 7, or 8. L6-9/1 had complex oligosaccharides of the type (±NeuNAcα2-3GalβGlcNAcβ)nMan3GlcNAc(±Fuc)GlcNAc where n = 3 or 4, in addition to small amounts of biantennary and higher order structures. WGARII, the mutant with low resistance to wheat germ agglutinin, possessed similar complex oligosaccharides except that they had undergone some base-sensitive modification which rendered them resistant to Clostridium perfringens sialidase. WGARI, mutant of high resistance to wheat germ agglutinin, possessed complex oligosaccharides of the type (GlcNAcβ)nMan3GlcNAc(±Fuc)GlcNAc, with n = 2,3, or 4. Since the WGARI mutants were unaltered in differentiation, it is concluded that the terminal sialic acid and galactose residues on the protein-bound oligosaccharides are not required for differentiation.
Regulation of transcription termination in Escherichia coli
Authors: Greenblatt Jack
Published: December 2009

Experiments are summarized that deal with the mechanisms of action of the N gene transcriptional antitermination protein of bacteriophage lambda and the nusA gene transcriptional termination protein of Escherichia coli. Models are presented for the regulation of chain elongation during bacterial transcription. Current evidence suggests that chain elongation during transcription may be regulated by a "regulosome" that consists of RNA and protein and is bound to transcribing RNA polymerase.
Structure of the polysaccharide chain of Pasteurella haemolytica (serotype 4) lipopolysaccharide
Authors: Perry Malcolm B. , Babiuk Lorne A.
Published: December 2009

The antigenic polysaccharide side chain of the cell wall lipopolysaccharide of Pasteurella haemolytica (serotype 4) was investigated by methylation, periodate oxidation, partial hydrolysis, and 13C and 1H nuclear magnetic resonance analysis methods and was found to be a simple unbranched linear polymer composed of a disaccharide repeating unit having the structure —3)-α-D-Galp-(1—3)-β-D-Galf-(1—.
Evaluation of the fatty acid selectivity of a phosphatidylinositol-specific cytosolic phospholipase C from pig and human platelets
Authors: Holub Bruce J. , Celi Bernadette
Published: December 2009

The fatty acid selectivity of cytosolic phospholipase C (phosphatidylinositol phosphodiesterase) from pig and human platelets towards phosphatidylinositol was evaluated. For this purpose, the relative conversion of rat liver phosphatidylinositol (enriched in stearate and arachidonate) and sheep liver phosphatidylinositol (enriched in stearate plus oleate and containing linoleate and arachidonate) was compared and, in addition, the fatty acid compositions of the diacylglycerol products were determined by gas–liquid chromatography. The cytosolic enzyme exhibited essentially complete specificity for phosphatidylinositol when choline-, ethanolamine-, serine-, or inositol-containing phospholipids labelled with [14C]stearate were tested as substrates. Similar percentage conversions of rat and sheep liver phosphatidylinositols to 1,2-diacylglycerol were found with phospholipase C from either pig or human platelets. Furthermore, the newly formed diacylglycerols and the unreacted phospholipid had fatty acid compositions which were very similar to the corresponding substrates. These results suggest that the phospholipase C from isolated platelet cytosol is highly selective towards phosphatidylinositol, but not with respect to the fatty acid composition of naturally occurring phosphatidylinositol. They also suggest that any preferential release of arachidonoyl diacylglycerol in stimulated human platelets is more likely controlled by compartmentation of the corresponding phosphatidylinositol precursor within platelet membranes and its availability, rather than directly by a marked enzyme preference for arachidonate-containing species.
Studies on the effects of magnesium ion and propranolol on iris muscle phosphatidate phosphohydrolase
Authors: Abdel-Latif Ata A. , Smith Jack P.
Published: December 2009

The properties, subcellular distribution, and the effects of Mg2+ and propranolol on phosphatidate phosphohydrolase (EC 3.1.3.4) from rabbit iris smooth muscle have been investigated. The particulate and soluble (0–30% (NH4)2SO4 fraction) enzymes were assayed using aqueous phosphatidate dispersions and membrane-bound phosphatidate as substrates, respectively. When measured with aqueous substrate, activity was detected in both the particulate and soluble fractions, with the highest relative specific activity found in the microsomal fraction. Maximum dephosphorylation by the microsomal enzyme was about 1100 nmol of inorganic phosphate released/h per milligram protein and occurred at pH 7.0–7.5. In general Mg2+ inhibited the phosphohydrolase activity of the microsomal fraction and stimulated that of the soluble fraction, and the effects of the divalent cation on both of these activities were reversed by propranolol. The microsomal enzyme was slightly stimulated by deoxycholate and inhibited by the divalent cations Mg2+, Ca2+, and Mn2+ at concentrations > 0.25 mM. In contrast, the soluble enzyme was stimulated by Mg2+. Inhibition of the microsomal enzyme by Mg2+ (0.5 mM) was reversed by both EDTA, which also stimulated at higher concentrations (1 mM), and propranolol (0.1–0.2 mM). The inhibitory effect of Ca2+ on the enzyme was not reversed by propranolol. In the absence of Mg2+, the microsomal enzyme was inhibited by propranolol in a dose-dependent manner, and both in the absence and presence of the divalent cation the soluble enzyme was inhibited by the drug in a similar manner. These data suggest that the cationic moiety of propranolol may act by competing at the Mg2+-binding sites. Addition of propranolol (0.2 mM) to iris muscle prelabelled with [14C]arachidonic acid increased accumulation of [14C]phosphatidic acid at all time intervals (2.5–90 min) and brought about a corresponding initial decrease in the formation of [14C]diacylglycerol at short time intervals (2.5 min), thus implicating the phosphohydrolase as a possible site of action of the drug on glycerolipid metabolism in this tissue. In addition to reporting on the characteristics and distribution of phosphatidate phosphohydrolase in the iris smooth muscle, the data presented add further support to our hypothesis that propranolol redirects glycerolipid metabolism in the iris by exerting multiple effects on the enzymes involved in their biosynthesis.
Activation of hexose transport by antibody
Authors: Lo Theodore C. Y. , Duronio Vincent
Published: December 2009

In our attempts to study alterations in cell behaviour by membrane perturbations, the effects of specific antisera on membrane functions were examined. The present communication reports the nature and kinetics of the changes in hexose transport as a result of treatment of L6 rat myoblast cells with specific antibodies. Exposure to complement-inactivated rabbit anti-myoblast antiserum resulted in an almost immediate two- to three-fold increase in hexose transport; morphological changes were observed only after prolonged incubation with the antiserum. Similar stimulation of hexose transport was observed upon exposure of cells to immunoglobulin G (IgG) from both sheep anti-myoblast and rabbit anti-myoblast plasma membrane sera. It seems that the interaction of specific IgG with cell surface components is sufficient to elicit this response. We have established that over 90% of 2-deoxyglucose is phosphorylated upon entrance into rat myoblasts and that exposure of cells to specific antibody does not cause changes in cell volume or general leakiness of the cell membrane. The antibody-stimulated hexose transport system resembles its normal counterpart in its substrate affinity and specificity; it differs from that of the control cells only in the transport capacity. This increase in hexose capacity is brought about by processes independent of protein synthesis and is not likely the result of extensive reorganization of membrane components.
Improved procedures for the conjugation of oligosaccharides to protein by reductive amination
Authors: Roy René , Katzenellenbogen Ewa , Jennings Harold J.
Published: December 2009

The rate of coupling of oligosaccharides having aldose end groups to protein by reductive amination was significantly increased by changing the temperature and pH of the reaction, and even more significantly by the addition of borate ions. Under optimized conditions half of the lysine residues of bovine serum albumin could be derivatized by lactose in 7 h and their complete derivatization was achieved in approximately 24 h. All attempts to carry out similar reductive amination procedures using oligosaccharides having ketose (D-fructose, 3-deoxy-D-manno-octulosonic acid (KDO), and sialic acid) failed owing to the slowness of the reaction. Model studies on the coupling of D-fructose and KDO to glycine indicate that any coupling procedure based on reductive amination of ketose residues would of necessity require the prior introduction of a small functionalized spacer molecule.
Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver
Authors: Lin C. H. , Chung W. , Strickland K. P. , Hudson A. J.
Published: December 2009

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.
The ribosomal 5.8S RNA: eukaryotic adaptation or processing variant?
Authors: Nazar Ross N.
Published: December 2009

A striking difference between the cytoplasmic ribosomes of eukaryotes and prokaryotes is the presence (in eukaryotes) of one additional RNA component, the 5.8S rRNA. This RNA, which is hydrogen bonded to the cognate high molecular weight RNA of the large subunit, is about 160 nucleotides in length, and is cotranscribed with the high molecular weight rRNA as part of a much larger precursor molecule, the nucleolar 36S–45S rRNA. Because of its relatively small size the 5.8S RNA is considered a good model for studies on rRNA structure, synthesis, maturation, ribosomal integration, function, and even evolution. Over the last decade numerous studies have examined these questions with many interesting results, including the probability that this RNA sequence may actually be present in all ribosomes, although not necessarily as a separate RNA component. Their findings are summarized in this review.
Altered phospholipid metabolism in thrombin-stimulated human platelets
Authors: Holub Bruce J.
Published: December 2009

Extensive research during the past few years has indicated that dramatic alterations in phospholipid metabolism and composition represent early and important biochemical events in the response of human platelets to thrombin stimulation. The individual enzyme-catalyzed steps which provide for the release of free arachidonic acid for thromboxane A2 formation via the initial degradation of phosphatidylcholine and phosphatidylinositol have been studied. Their importance in this regard is influenced by the molecular species composition of the corresponding phospholipid precursors. A role for stimulated phosphatidylinositol 4,5-bisphosphate degradation in the phosphatidylinositol response and inositol triphosphate release associated with calcium mobilization has also been proposed. The 1,2-diacylglycerol released by the action of phospholipase C on phosphatidylinositol and its 4,5-bisphosphate derivative has been implicated as an activator of protein phosphorylation; the derived phosphatidic acid has been proposed as a mediator for promoting an intracellular flux of calcium associated with platelet responses.
Glycosphingolipids and plasma lipoproteins: a review
Authors: Chatterjee Subroto , Kwiterovich Jr. Peter O.
Published: December 2009

Glycosphingolipids (GSL) are complex, sugar-containing lipids that are transported in plasma on lipoproteins, particularly low density (beta) lipoproteins (LDL). LDL are taken up and metabolized by cells through a LDL receptor-mediated pathway. Cells from receptor-negative familial hypercholesterolemic (FH) homozygotes lack a functional LDL receptor and the cellular uptake of LDL occurs through a LDL receptor-independent pathway. We have studied the relation between LDL and GSL metabolism using an in vitro model of cultured human fibroblasts and an in vivo model of renal tubular cells shed in the urine. Using biochemical, morphological, and immunocytological techniques, we have demonstrated that LDL taken up through the LDL receptor-independent pathway results in the accumulation of GSL in cells in vitro and in vivo. The storage of GSL is localized to intracytoplasmic vesicles and such storage can be modulated by changing the concentration of LDL in the culture medium or in plasma. The possible effect of LDL on certain steps in the intracellular metabolism of GSL in normal and receptor-negative homozygous FH cells is reviewed.
The C1q receptor site on human immunoglobulin G
Authors: Painter Robert H.
Published: December 2009

Of the many functional properties of the immunoglobulin G (IgG) molecule, only antigen binding and the interaction with the C1q component of complement have been shown to be uniquely associated with the individual compact domains which make up this immunoglobulin. The chemical and biological evidence for the exclusive association of the C1q-binding site with the CH2 domain is reviewed, affirming that the site probably is centered on the residues 279–295 which are located on the outside of the molecule and contain, in particular, three positively charged residues which are thought to be vital to the interaction. An alternative site (residues 316–338), having similar exposure and charge characteristics, is discussed and arguments are presented indicating why the former is currently favoured.
Luminescence and circular-dichroism analysis of terbium binding by pig intestinal calcium-binding protein (relative mass = 9000)
Authors: O'Neil Joe D. J. , Dorrington Keith J. , Hofmann Theo
Published: December 2009

The structure and conformations of pig intestinal Ca-binding protein (CaBP) have been studied by terbium luminescence enhancement and circular dichroism. The two cation-binding sites bind Tb3+ sequentially; the affinity of the first site is > 107 M−1 and the affinity of the second site is approximately 105 M−1. Filling of the first site enhances the fluorescence of the single tyrosine residue, whereas Tb3+ in the second site quenches the fluorescence. Excitation spectra of the Tb3+-bound forms of CaBP show that considerable energy transfer takes place from phenylalanine residues to the bound Tb3+, although some transfer from tyrosine is also detected. The sequence in which the sites are filled was deduced from these results and the published three-dimensional structure of the cow intestinal CaBP. Tb3+ bound approximately 20 Å (1 Å = 0.1 nm) from the tyrosine induced a large increase in the optical activity of this residue. We argue that a potentially important conformational change is induced in CaBP by cation binding.
Comparative studies of manganese(II)-, nickel(II)-, zinc(II)-, copper(II)-, cadmium(II)-, and iron(III)-binding components in human cord and adult sera
Authors: Lau Show-Jy , Sarkar Bibudhendra
Published: December 2009

The binding of six trace metals, Mn(II), Ni(II), Zn(II), Cu(II), Cd(II) and Fe(III), to human cord serum has been studied by Sephadex G-100 gel filtration at physiological pH, using radioisotopes as tracers. The results are compared with those obtained from adult serum. In both cord and adult sera, extensive amounts of the metals are bound to high molecular weight proteins. Among them, Fe(III) is mostly bound to transferrin; Ni(II), Zn(II), Cu(II), and Cd(II) are bound to albumin and other macro-molecules. The binding of Mn(II) either to transferrin or albumin is not resolved. Small fractions of Zn(II), Cu(II), and Cd(II) and large fractions of Mn(II) and Ni(II) are found to be associated with low molecular weight components of both sera. The distribution varies from metal to metal. However, the low molecular weight component of the size 1500 – 10 000 is present in all the metals studied. Further purification of this component was attempted by DEAE-cellulose ion-exchange chromatography. The possible identity as well as the biological role played by this particular component of serum in the transport of metals in blood and across membranes is discussed.
The biochemistry of preneoplastic liver: a common metabolic pattern in hepatocyte nodules
Authors: Farber Emmanuel
Published: December 2009

The biochemical properties of putative preneoplastic hepatocyte nodules as they relate to the metabolism of xenobiotics have been reviewed briefly. A common pattern with low phase I components and elevated phase Il components appears evident. The phase I components included microsomal cytochromes P-450 in composite and four different mixed function oxygenase activities. The activities in the nodules were 50% or less of the control values. The phase II components included glutathione, glutathione S-transferases and UDP-glucuronyl transferase 1 and showed two- to five-fold elevations. In addition, activities of microsomal epoxide hydrolase, cytosolic DT-diaphorase, and γ-glutamyl transferase were all elevated in nodules. The possible significance of this biochemical pattern in analyzing the diversity of biochemical expressions of cancer, in the mechanism of cancer development, and in understanding the suggested role of physiological adaptation in carcinogenesis is discussed.
A highly thermostable proline:tRNA ligase from Thermus aquations. Purification and enzyme-tRNA recognition at elevated temperatures
Authors: Rivera Jorge A. , Wang Qi-Song , Wong J. Tze-Fei
Published: December 2009

Proline:tRNA ligase from Thermus aquaticus was purified to homogeneity and characterized. Its molecular weight was found to be 127 000, consisting of two identical subunits. It catalysed the prolylation of tRNAPro from Escherichia coli with a bell-shaped pH dependence peaking at about pH 7 and exhibited extreme thermostability. The Vm/Km ratios of steady-state kinetics for proline and ATP as well as tRNAPro were not extensively diminished even at 85 °C, but prolylation became insignificant at 90 °C. Since the melting of tRNAPro was in progress, yet incomplete, at 85 °C, these findings suggest that some threshold level of conformational integrity of tRNAPro, rather than the entire unmelted conformation of the molecule, is essential to effective recognition by the proline:tRNA ligase.
Dipeptidyl tetrapeptide hydrolase, a new enzyme with dipeptidyl ligase activity
Authors: Eng Francisco W. Hong Tai
Published: December 2009

Dipeptidyl tetrapeptide hydrolase, a new enzyme with dipeptidyl ligase activity, partially purified from cabbage (Brassica oleracea) extract by means of ammonium sulfate fractionation and DEAE-Sephadex A-25 column chromatography, was shown to catalyze the syntheses of Ala-Gly-Ala-Gly, Ala-Ala-Ala-Ala, and Gly-Ala-Gly-Ala from Ala-Gly, Ala-Ala, and Gly-Ala, respectively, by a direct condensation mechanism without prior activation of the carboxyl group of one molecule of substrate coupled to an energy-yielding reaction. The enzyme has a pH optimum of 7.2, a strict requirement for a reduced sulfhydryl group for catalytic activity, and a substrate specificity for a dipeptide with either two Ala residues or one Ala and one Gly residue. The enzyme substrate-binding site is estimated to be about 15 Å (1 Å = 0.1 nm) long with four subsites, each of which recognizes Ala. Only substrates with the L-isomer of Ala and with an amino group in the alpha position are active. The energy gained in the synthesis of the internal peptide bond as estimated from the equilibrium constant for the synthesis of Ala-Gly-Ala-Gly from Ala-Gly is 1861 cal (1 cal = 4.1868 J). This energy requirement is 2269 cal lower than the energy required to synthesize the peptide bond in DL-Ala-Gly from DL-Ala and Gly. When the purified enzymatically synthesized tetrapeptide product was incubated with the enzyme, it was hydrolyzed to Ala-Gly.