bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermotropic mesomorphism in aqueous dispersions of 1-palmitoyl-2-oleoyl- and 1,2-dilauroyl-phosphatidylglycerols in the presence of excess Na+ or Ca2+
Authors: Fleming Blair D. , Keough Kevin M. W.
Published: December 2009

1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG) and 1,2-dilauroyl-sn-glycero-3-phospho-rac-glycerol (DLPG) have been made by the conversion of the appropriate lecithins in the presence of crude phospholipase D and excess glycerol. Aqueous dispersions of the lipids have been examined by differential scanning calorimetry and 31P-nuclear magnetic resonance spectroscopy. When dispersed in excess Na+ at pH 7, POPG and DLPG underwent gel to liquid-crystalline phase transitions at −2.0 ± 1.3 and −3.7 ± 1.9 °C, respectively. The enthalpy of transition for POPG in excess Na+ was 6.2 ± 0.3 kcal∙mol−1 (1 cal = 4.1868 J), but the value for DLPG in Na+ was variable. In the presence of excess Ca2+ at pH 7, both phospholipids displayed complex thermotropic mesomorphism. For DLPG in the presence of excess Ca2+, thermal events did not occur at the same temperatures as did those for POPG in excess Ca2+. The complexity of the observed behaviour may have arisen because of the formation of intermediate forms which slowly interconverted to more stable forms.
Fluorescence of actin-bound hydrophobic molecules
Authors: Burtnick L. D. , Chan K. W.
Published: December 2009

2-(N-methylanilino)naphthalene-6-sulfonic acid (MANS) binds to G-actin at a single high affinity hydrophobic site (Kd = 41 μM). Salt-induced polymerization of MANS–G-actin results in a general enhancement of sample emission intensities at all wavelengths. At 430 nm, KCl-induced polymerization yields a 2.3-fold enhancement, while MgCl2-induced polymerization gives a 2.0-fold increase. Polymerization of MANS–G-actin in the absence of agitation produces MANS–F-actin samples that have fluorescence polarization values at 430 nm of 0.33. Subsequent mixing or sonication of such MANS–F-actin samples results in a dramatic drop in fluorescence polarization values to 0.14. After cessation of mixing, the polarization values do not recover to their initial levels. Circular polarization of luminescence studies on MANS–actin demonstrate that agitation of MANS–F-actin samples drastically alters emission anisotropy values. 9-Anthroyl choline (9AC) binds to G-actin at a single hydrophobic site (Kd = 68 μM). The fluorescence of 9AC–actin is sensitive to salt-induced polymerization and depends upon the identity of the salt employed. KCl causes a drop in the fluorescence intensity at 490 nm to 70% of the value for 9AC–G-actin, while MgCl2 produces a 30% increase in intensity. Polarization experiments with 9AC–actin produced qualitatively the same results as did those with MANS–actin. Differences in the behaviours of MANS–actin and 9AC–actin in response to polymerization by KCl and MgCl2 and in response to the binding of deoxyribonuclease I suggest that the binding sites on actin for MANS and 9 AC do not overlap completely. The binding of MANS and 9 AC to actin may be modulated by the opposite signs of the net charge borne by each of these probes.
Adsorption of rat plasma phosphorylcholine-binding protein to synthetic lipid emulsions in the presence of divalent cations
Authors: Connelly Philip W. , Kuksis Arnis , Mookerjea Sailen , Nagpurkar Arun
Published: December 2009

Lipoproteinlike lipid emulsions were incubated with rat serum, plasma, or plasma-derived serum. Two previously unidentified components which bound to these emulsions only in the presence of divalent cations were identified as the rat plasma phosphorylcholine-binding proteins.
Heterogeneity in the collagens extracted from human embryonic calvaria
Authors: Leushner James R. A.
Published: December 2009

Collagens were obtained from decalcified human embryonic calvaria by pepsin digestion. After removal of the type I collagen, the more soluble collagens were precipitated at 1.2 M NaCl (acid pH), followed by a selective precipitation step at neutral pH, using a NaCl concentration of 4.5 M. Analysis of this latter precipitate by polyacrylamide slab gel electrophoresis and ion-exchange chromatography revealed the presence of a heterogeneous group of proteins ranging in size from approximately 10 000 daltons to over 120 000 daltons. Proteolysis, as a source for these diverse components, was ruled out both by studies employing protease inhibitors and experiments employing thrombin which indicated that no helical denaturation had occurred during extraction. A comparison with standard collagen preparations suggested that the major bands present in these samples corresponded to the α1-, α2-, and α3- chains of type V collagen. The data also showed that the major helical organization of these chains was [α1(V)]2 α2(V). Data suggesting that proteolysis can occur during the chromatographic separation of the individual α-chains are presented. This proteolysis was sensitive to inhibitors and its possible role in modifying the chain composition of type from calvaria and other tissues is discussed. Unique cyanogen bromide peptides distinguishable from those of type I and type V were present in these precipitates and suggests the presence of novel collagen types. The small amounts of these collagens precluded a determination of the exact nature of these components.
Control of branching during the biosynthesis of asparagine-linked oligosaccharides
Authors: Schachter Harry , Narasimhan Saroja , Gleeson Paul , Vella George
Published: December 2009

Many mammalian and avian complex carbohydrates (glycoproteins and glycolipids) have highly branched oligosaccharides. Although the function of complex carbohydrates is not known, there is evidence to suggest that oligosaccharide branching may be an important factor in the process by which cells recognize one another and their environment. Asparagine-linked (N-glycosyl) oligosaccharides can be subdivided into at least 12 classes according to their branching patterns. It is presently believed that these classes all stem from a common precursor oligosaccharide containing three D-glucose, nine D-mannose, and two N-acetyl-D-glucosamine residues. This precursor is incorporated into the protein backbone in the rough endoplasmic reticulum and is then processed within the endoplasmic reticulum and Golgi apparatus by a series of highly specific glycosidases and glycosyltransferases to yield the various classes of N-glycosyl oligosaccharides. The branches that occur in N-glycosyl oligosaccharides are usually initiated by the incorporation of a N-acetylglucosamine (GlcNAc) residue. Our laboratory has studied four of the N-acetylglucosaminyltransferases (GlcNAc-transferases) involved in this initiation process. We have defined various factors which determine the synthetic pathway. There are at least three types of control that are commonly found, (i) Tissues differ in the relative activities of the different glycosyltransferases and glycosidases and, therefore, competition between two or more enzymes for a common intermediate often determines the synthetic route. (ii) The incorporation of a key glycosyl residue into an oligosaccharide may convert a nonsubstrate to a substrate for either a glycosyltransferase or a glycosidase. (iii) Conversely, the incorporation of a key residue may convert a substrate into a nonsubstrate. Other controls are undoubtedly operative during glycoprotein synthesis: e.g., the effect of the polypeptide sequence on transferase specificity, the distribution of transferases along the endomembrane system, and compartmentation and the availability of substrates and cofactors. These factors have not been studied in our laboratory. However, the oligosaccharides made by the hen oviduct correlate quite well with the control factors elucidated by our approach; other tissues are presently under investigation. Recent studies on the three-dimensional structures of N-glycosyl oligosaccharides have enabled us to explain certain features of glycosyltransferase substrate specificity on the basis of steric factors.
Glucose-6-phosphatase: is activity regulated by phosphorylation–dephosphorylation?
Authors: Singh Jasbir , Martin Richard E. , Nordlie Robert C.
Published: December 2009

Incubation of rat liver microsomes with ATP and Mg2+ in the absence or presence of an exogenous protein kinase showed no changes in the activity of glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9). These observations confirm the recent findings of the Burchells and colleagues and refute on methodological grounds the earlier conclusions of Begley and Craft implicating regulation of this enzyme by protein phosphorylation–dephosphorylation. In other studies, the time-dependent inactivation of microsomal glucose-6-phosphatase by incubation with deoxycholate was used to obtain the inactive enzyme which in the presence of a protein kinase, ATP, and Mg2+ could not be restored to its original level. A number of substrates and competitive inhibitors of glucose-6-phosphatase, most notably vanadate which is the most potent inhibitor of the enzyme identified, stabilized this enzyme against its time-dependent inactivation in the presence of detergent as effectively as did fluoride and molybdate which are also effective competitive inhibitors of glucose-6-phosphatase. An alternative explanation to the involvement of a phosphoprotein phosphatase, as discussed by the Burchells, in the time-dependent inactivation of glucose-6-phosphatase is thus suggested.
Identification and optimization of the effects of endogenous free amino acids on the translational activity of the wheat germ cell-free system
Authors: Mezl Vasek A.
Published: December 2009

This study identifies factors that are responsible for unpredictable problems associated with the wheat germ cell-free translation system. It is shown that variable amounts of an inhibitor of radioisotope incorporation are routinely included in wheat germ cell-free system preparations, even though this activity can be readily reduced by discarding the appropriate fractions of the wheat germ extracts. This inhibition of the incorporation of radioactive amino acid appears to be due to the production of large amounts of some amino acids by wheat germ extracts. Since the free amino acids that occur at high concentrations are described, the results of this study allow one to predict and to minimize this problem. The results also indicate that the choice of the radioactive amino acid precursor used for a translation should be based not only on the abundance of the amino acid in the protein that is being made, but also on the concentration of this amino acid in the wheat germ preparation.
Rapid activation of cAMP phosphodiesterase in rat platelets
Authors: Hamet P. , Franks D. J. , Tremblay J. , Coquil J. F.
Published: December 2009

Incubation of intact platelets with prostaglandins (PGE1 and PGI2) and phosphodiesterase inhibitors (1-methyl-3-isobutyl-xanthine, indomethacin, dipyridamol) lead to activation of cAMP phosphodiesterase. The activation was rapid (maximal within 30 s) and stable after removal of agents and homogenization of platelets. The activation remained after DEAE-Sepharose chromatography. The effect of the two types of agents on phosphodiesterase activity was more than additive and activation did not alter the nonlinear kinetic behavior of phosphodiesterase. The mechanism of the ex vivo stimulation is unknown at the present time, however, it does not seem to be due to cellular redistribution of the enzyme. The results suggest that activation of a cAMP-dependent protein kinase is an intermediate step. The ex vivo stimulation is regulated by a calcium-dependent process, since addition of Ca2+ ions and ionophore A23187 to Ca2+ depleted platelets abolished the ex vivo stimulation by PGE1 and MIX.
Characteristics of chondroitin sulfate proteoglycans of differentiating nonchondrogenic tissues
Authors: Crawford Terry
Published: December 2009

The chondroitin 4- and 6-sulfate proteoglycan monomers from two differentiating tissues (brain and muscle of 14-day chick embryos) were compared by indirect means, including use of chondroitinase AC, with the major class of cartilage chondroitin sulfate proteoglycan monomers. In addition to slower sedimentation in sucrose density gradients, the monomers from both noncartilaginous tissues have longer chondroitin sulfate chains and exhibit little difference in the susceptibilities of their core proteins to papain and trypsin, in contrast to the cartilage monomer. However, slight differences in average sizes and polydispersity of fragments formed by enzyme treatments and β-elimination were found between the proteochondroitin sulfate monomers of embryonic brain and muscle. Although the chondroitin sulfate chains are attached to separate tryptic peptides from the majority of other sulfated glycosaminoglycans in the noncartilaginous tissues, it is still uncertain whether these units form part of separate proteoglycan monomers.
Cytoplasmic membrane of Rhizobium meliloti bacteroids. I. Alterations in lipid composition, physical properties, and respiratory proteins
Authors: Miller R. W. , Tremblay P. A.
Published: December 2009

Differentiation of Rhizobium meliloti cells in alfalfa root nodules is characterized by alteration in the lipid composition and physical properties of the bacteroid cytoplasmic membrane. Nitrogen-fixing bacteroids isolated from nodules showed an altered phospholipid and fatty acid composition and lacked aliphatic alcohols normally associated with the bacterial cell wall. Bacteroid cytoplasmic membrane lipids were more fluid, but responded specifically to the presence of calcium ion with an increase in the ordered packing of fatty acid side chains, as determined from electron spin resonance spectra of spin probes intercalated in the membrane. This cation also specifically enhanced rates of respiration of the bacteroids and rates of reduction of the spin probe. Bacteroid cytoplasmic membranes differed from those of air-grown cells, both in the amount of cytochromes b and c per cell and in the nature of the terminal oxidase. Cytochrome aa3, was present in bacteroids at less than 1/10 the level found in air-grown cells. The observed differences in the structure and function of the cytoplasmic membrane of bacteroids may be associated with requirements of the bacterial symbiont for utilization of carbon substrates and provision of reducing power in support of the nitrogenase enzyme system which is expressed in the nodule environment.
Physical studies on the ribosomal "A" protein from two archaebacteria, Halobacterium cutirubrum and Methanobacterium thermoautotrophicum
Authors: Gudkov A. T. , Venyaminov S. Yu , Matheson A. T.
Published: December 2009

Physical studies on the effect of temperature and ionic conditions on the secondary, tertiary, and quaternary structure of the ribosomal "A" protein, equivalent to L7/L12 in Escherichia coli, from two archaebacteria were performed using circular dichroism and sedimentation equilibrium measurements. The two archaebacteria investigated were Halobacterium cutirubrum, an extreme halophile, and Methanobacterium thermoautotrophicum, a thermophile which also showed properties of a moderate halophile. The changes in the secondary structure and the thermostability of these proteins were directly related to the internal salt concentrations of the two archaebacteria. At the higher salt concentrations the changes in the secondary structure resulted in changes in the tertiary and quaternary structure of these proteins.
Instability of endothelium-bound lipoprotein lipase activity in perfused rat hearts
Authors: Noël Simon-Pierre
Published: December 2009

The enzymatic activity of endothelium-bound lipoprotein lipase was measured in rat hearts perfused for 1 h at 37 °C. Viability parameters such as beating rate, flow rate, aortic pressure, and oxygen consumption were all kept strictly constant during the entire perfusion time. The lipase activity was determined by input–output difference of the triacylglycerol content in the nonrecirculating heart perfusate which contained either an artificial triolein emulsion or rat lymph chylomicrons. With either substrate, the lipase activity decreased with time: approximately 2% of initial lipase activity was lost per minute. The presence of rat serum (10%) in heart perfusate enhanced the rate at which the lipase activity disappeared. Only a small portion of the lipoprotein lipase activity, which was lost from the perfused heart, was recovered in the outflow perfusate. Our data demonstrate that, under our experimental conditions, the enzymatic activity of rat heart lipoprotein lipase does not remain constant during heart perfusion. Caution should therefore be taken by users of heart perfusion technique, especially for those who need constant lipoprotein lipase activity. The results suggest that the first step in the catabolic fate of endothelium-bound cardiac lipoprotein lipase is the loss of its catalytic activity. Whether the inactivated enzyme is released from the endothelium or remains in situ is not yet known.
Effects of erythropoietin on uridine metabolism in cell cultures of fetal calf liver
Authors: Congote L. F.
Published: December 2009

The effect of sheep plasma erythropoietin preparations on the incorporation of [5-3H]uridine into erythroid cells has been studied using cells of fetal calf liver cultured in serum-free medium. The cells were incubated for 20 h with the hormone, followed by a 1-h incubation with [3H]uridine. Erythropoietin caused a 2.5-fold increase in the incorporation of uridine into cold trichloroacetic-acid-insoluble cell extracts and a 70% increase in the incorporation of uridine into the cold acid-soluble cell extracts. The phosphorylated metabolites of labeled uridine present in the cold acid-soluble fraction were analyzed by anion-exchange high performance liquid chromatography (HPLC). Erythropoietin increased the amounts of labeled UDP and UTP per cell. However, the specific activity of UTP and the labeled amounts of UDP-glucose in erythropoietin-treated cells were not significantly different from those in control cell cultures. After chromatography of the crude erythropoietin preparations on reversed-phase and gel-permeation HPLC, there was a perfect coincidence of the fractions stimulating uridine incorporation into acid-soluble and acid-insoluble cell extracts. The protein fractions from crude erythropoetin which stimulated uridine incorporation after purification by reversed-phased HPLC were also able to stimulate globin chain synthesis in fetal calf liver cells. These experiments suggest that the multiple effects on uridine metabolism described above are due to erythropoietin, rather than other proteins contaminating the crude hormone preparations.
Effects of Ca2+ and Mg2+ on the switch-on of transcriptase function in reovirus in vitro
Authors: Sargent M. D. , Borsa J.
Published: December 2009

Effects of Ca2+ and Mg2+ on the "switch-on" of transcriptase function in reovirus intermediate subviral particles were examined. Switch-on in this system is triggered by K+ ions, whereas Na+ ions antagonize the effects of K+ ions. Once effected, switch-on is irreversible. In the absence of Ca2+ and Mg2+ ions, switch-on of transcriptase is effected by incubation at 37 °C in the presence of a particular ratio of K+/Na+ (total K+ plus Na+ being kept constant). Inclusion of Ca2+ in the activation step lowers the ratio that triggers the switch-on event. This indicates that Ca2+ destabilizes the switched-off state of the transcriptase. Inclusions of Mg2+ ions in the activation step raises the K+/Na+ ratio necessary to trigger switch-on. Thus Mg2+ tends to stabilize the switched-off state of the transcriptase to the triggering action of K+ ions. Inclusion of both Ca2+ and Mg2+ together during the activation step results in mutual inhibition of the effects expected from each divalent cation alone. These results indicate that switch-on of transcriptase function, which is triggered by K+ ions, can be modulated by interactions involving the major physiological cations (Na+, K+, Mg2+, Ca2+).
Anesthetic-membrane interaction: A 2H nuclear magnetic resonance study of the binding of specifically deuterated tetracaine and procaine to phosphatidylcholine
Authors: Kelusky Eric C. , Smith Ian C. P.
Published: December 2009

The binding of the local anesthetics tetracaine and procaine with multilamellar dispersions of egg phosphatidylcholine has been studied by 2H nuclear magnetic resonance (NMR). The 2H-NMR line shapes of specifically deuterated local anesthetics are found to be very dependent on the attainment of a true equilibrium. The equilibrium could be most properly reached by the use of repeated freeze–thaw–vortex cycles. The data for tetracaine are consistent with the three-site exchange model proposed earlier. Tetracaine is in slow exchange between a strongly bound site and a weakly bound site and in fast exchange between the weakly bound site and free in solution. The slow exchange rate is estimated, from temperature and dilution studies, to be approximately 1.5 × 103 s−1 at pH 5.5 and slightly faster at pH 9.5. Comparisons of the quadrupole splittings with those seen for our earlier work in egg phosphatidylethanolamine suggest that the location of the strongly bound site in phosphatidylcholine is dependent on the anesthetic charge. This is in contrast to egg phosphatidylethanolamine, where molecular shapes appear to be the determining factor for the location of the anesthetic. Procaine bound very weakly to the model membranes, to yield only a broad resonance and no quadrupole splitting. It appears that procaine, unlike tetracaine, is not bound by the ordered acyl chains.
Endogenous and exogenous factors affecting ribosomal RNA release from rat liver nuclei in a cell-free system
Authors: Hanausek-Walaszek M. E. , French B. T. , Schumm D. E. , Walaszek Z. , Webb T. E.
Published: December 2009

rRNA release from isolated liver nuclei has been analyzed in a reconstituted cell-free system using density-gradient analysis and hybridization to a specific recombinant DNA probe to monitor the process. The cell-free system was shown previously to be energy- and cytosol-dependent and to support the formation and release of functional ribosomal subunits. The release of rRNA is now shown to have an absolute dependence on a 70 000 dalton cytosol protein. Although in vivo studies suggest that chronic administration of thioacetamide may block formation of a protein involved in the nucleocytoplasmic transfer of ribosomes, the 70 000 dalton transport factor is not affected by the treatment. Rather the defect appears to be localized to the nucleus, since it cannot be reversed with normal cytosol from a homologous source. Early stages of nRNA processing appear to be affected by thioacetamide, although additional effects on transport are not ruled out.
An osteonectinlike protein in porcine periodontal ligament and its synthesis by periodontal ligament fibroblasts
Authors: Wasi Safia , Otsuka Kichibee , Yao Kam-Ling , Tung Pierre S. , Aubin Jane E. , Sodek Jaro , Termine John D.
Published: December 2009

Periodontal ligament, a soft connective tissue that lies between cementum and alveolar bone in the periodontium, has been shown to contain an osteonectinlike protein. The similarity between porcine ligament osteonectin and bovine bone osteonectin was evident from immunochemical studies, from migration characteristics on sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and from binding studies on hydroxyapatite. Using immunotransfer and immunodot analyses, ligament osteonectin was found to be extractable from tissues with 4 M guanidine–HCl (GuHCl) and 4 M GuHCl − 0.5 M EDTA and to comigrate with authentic bovine osteonectin on SDS–PAGE with a relative mass ~ 38 000. Furthermore, osteonectin from guanidine extracts of ligament was bound to hydroxyapatite in the presence of 4 M GuHCl. Immunofluorescence studies showed the osteonectin to be distributed throughout the extracellular matrix of the ligament and to be present within the ligament fibroblasts in a perinuclear, punctate distribution. Biosynthesis of osteonectin by ligament fibroblasts was studied following pulse-chase labelling with [35S]methionine and immunoprecipitation. The labelled osteonectin in the chased culture medium represented ~0.5% of the total labelled proteins secreted. It comigrated on SDS–PAGE with the corresponding labelled protein from pulsed cells and with the protein extracted from the tissue.
Biosynthesis of the 32-kdalton uncoupling protein in brown adipose tissue of developing rabbits
Authors: Freeman Karl B. , Patel Hasmukh V.
Published: December 2009

The biosynthesis of the 32-kdalton uncoupling protein of brown adipose tissue was examined with polysomes isolated from brown adipose tissue of developing rabbits. Polysomes were translated in a reticulocyte protein-synthesizing system and newly synthesized uncoupling protein was isolated with monospecific antibodies. The newly synthesized protein had the same molecular mass as the mature protein. Its synthesis is initiated and increases about five- to seven-fold before birth and decreases progressively after birth.
Synthesis acid phosphatase and other proteins by human prostatic tissue in vitro
Authors: Dubé J. Y. , Chapdelaine P. , Tremblay R. R. , Thabet M. , Roy R.
Published: December 2009

We have studied the synthesis of proteins by normal and hyperplastic human prostatic tissue incubated in vitro in the presence of [35S]methionine. The overall pattern of newly synthesized proteins was similar in individuals with an age ranging from 15 to 68 years. The pattern of labeled proteins was quite different from that of total proteins stained with Coomassie blue in two-dimensional polyacrylamide gels, since the major stained proteins were not labeled. Among the most heavily labeled proteins (about a dozen) were several spots representing charge isoforms with molecular weights ranging from 46 000 to 51 000, and these were the only proteins immunoprecipitated by a polyclonal antibody developed against purified acid phosphatase. The other heavily labeled proteins had molecular weights ranging from 26 000 to 72 000. These results show that tissue slices can be used to study the synthesis and processing of acid phosphatase, the major secretory product of the prostate, and of other unidentified proteins.
Evolutionary stability of mitochondrial DNA organization in Achlya
Authors: Boyd David A. , Hobman Thomas C. , Gruenke Sally A. , Klassen Glen R.
Published: December 2009

A physical map of the mtDNA (mitochondrial DNA) of Achlya klebsiana has been constructed. The molecule is circular and has a size of 50.7 ± 1.3 kilobases (kb). A large portion of genome exists in the form of an inverted repeat region, each copy being from 9.74 to 10.92 kb in length. Part of the repeat region is homologous to the yeast mitochondrial 21S rRNA gene. Two isomers of the molecule are present owing to a flip–flop rearrangement of the unique regions. Since no restriction-site differences have been detected between the two repeat regions, it is likely that the inverted repeat condition promotes continual sequence conversion, thus preserving the evolutionary advantages of gene duplication. The physical map of A. klebsiana mtDNA was compared with that of Achlya ambisexualis E-87 and it was found that the inverted repeat regions, as well as a 7-kb unique region adjacent to one of the repeat regions, were highly conserved with respect to restriction-site spacing.
Cytoplasmic membrane of Rhizobium meliloti bacteroids. II. Functional differentiation and generation of membrane potentials
Authors: Tremblay P. A. , Miller R. W.
Published: December 2009

The expression of the nitrogenase enzyme system in the alfalfa symbiont Rhizobium meliloti is associated with a process of functional differentiation of the bacterial cytoplasmic membrane. Morphological changes are complemented by an increase in cellular volume and enclosure of the bacterioids in a plant-derived peribacteroidal membrane. The average volume of bacteroids isolated in isotonic media was found to be three times that of air-grown cells and the average protein content increased by three- to four-fold. The induction of transmembrane electrical potentials was observed on oxygenation of anaerobic cells in the presence of succinate or sucrose. Membrane potentials were measured with radioactive probes in static experiments or by flow dialysis. Calcium ion enhanced bacteroid membrane potentials which were greater in magnitude (negative inside) than those of air-grown cells. Bacteroids exhibited weak proton transport activity in response to respiration and had no measurable pH gradient as determined by 31P nuclear magnetic resonance of intracellular phoshate ion. This finding contrasted with free-living R. meliloti which developed a strong, directly measurable proton pumping action on oxygenation. A fluorescent probe indicated that both air-grown cells and isolated bacteroids became energized in the same manner upon oxygenation, even though probes of membrane potential other than 86Rb+ indicated reverse polarity of the electrical potential of the former. The calcium ion effect on the bacteroid cytoplasmic membrane was also qualitatively demonstrable with the fluorescent probe. Calcium ion caused respiration-supported bacteroid membrane potentials to increase. This effect was accompanied by increases in membrane fluidity and nitrogenase activity in the absence of external reducing agents or added ATP.
Quantitative contribution of brown adipose tissue thermogenesis to overall metabolism
Authors: Foster David O.
Published: December 2009

Measurement of brown adipose tissue (BAT) blood flow coupled, when feasible, with measurement of the arteriovenous difference in oxygen across the tissue has been used to estimate the contribution of BAT thermogenesis to the metabolism of several species of laboratory, domestic, or wild mammals under various conditions: warm or cold exposure; arousal from hibernation; stimulation of metabolism by exogenous noradrenaline in warm- or cold-acclimated animals, in lean or obese animals, and in animals exhibiting high- or low-diet-induced thermogenesis. These studies have shown that in some species and under certain conditions BAT thermogenesis may account for as much as about one-third of the overall metabolic rate.
A two-dimensional immunoelectrophoretic analysis of the heat-shock response exhibited by Neurospora crassa cells
Authors: Kapoor M. , Chow A. W. L.
Published: December 2009

The heat-shock (HS) response of Neurospora crassa was studied by two-dimensional (2-D) immunoelectrophoresis, in conjunction with in vivo labelling of proteins with [35S]methionine. Antisera against extracts of normally grown and shocked cells were tested with both extracts as antigens. The resolution of normal cell proteins by interaction with homologous antisera yielded at least 35 immunoprecipitates. Using antisera to shocked cells with normal and shocked cell extracts resolved four heat-shock proteins (nhsps): new polypeptides synthesized upon HS and those with an enhanced production. Extracts of partially recovered cells showed proteins typical of normal cells, as well as those specific to the shocked state. Immunogenicity of some proteins appeared altered after heat shock. Following adsorption of HS cell extracts by normal antisera, three unique proteins were recognized in shocked cell extracts, along with a fourth displaying a markedly enhanced level. Stained bands corresponding to two abundant hsps (relative mass 68 000 and 81 000 daltons; referred to as nhsp 70 and nhsp 80) were excised from slab gels and the eluted proteins were employed as immunogens in rabbits. The resulting antibodies were used to identify corresponding proteins in autoradiographs of 2-D immunoelectrophoretograms.
Physical force considerations in model and biological membranes
Authors: Rand R. P. , Parsegian V. A.
Published: December 2009

We have emphasized the general lessons learned by measuring the forces between and within many kinds of phospholipid bilayers. Interbilayer forces within about 30 Å separation (1 Å = 0.1 nm) are dominated by a strong hydration repulsion that we believe exists for all interacting hydrophilic surfaces. This hydration can be characterized by the polarization properties of water. Its decay with distance is common to all hydrophilic surfaces, while its strength is characteristic of the specific surface groups. Interactions at distances greater than 30 Å between surfaces charged by adsorbed ions or by dissociation of surface groups qualitatively follow expectations for electrostatic double-layer forces, but quantitatively reveal an influence by the long-range hydration on electrostatic double layers. Bilayer lateral pressures and compressibilities, measured at thermodynamic equilibrium, are nonlinear and highly dependent on lipid species. Comparison with monolayers is not possible, since the latter are usually metastable states. In applying these results to contact and fusion of phospholipid vesicles, we define weak and strong regimes of interbilayer force, the former being too weak to overcome the hydration barrier. The special case of divalent cation binding with acidic phospholipids provides sufficient attraction to compete with hydration repulsion. However, such attraction will distort vesicles to rupture. Any observed fusion in such systems may be incidental to this destruction. This suggests that the control of biological membrane fusion, divalent cation antagonism, and postfusion stability is lacking in these systems. Biological fusion more likely involves focal biochemical change simultaneous with reduction of the hydration forces.
Theoretical studies of phospholipid bilayers and monolayers. Perturbing probes, monolayer phase transitions, and computer simulations of lipid–protein bilayers
Authors: Pink David A.
Published: December 2009

This paper describes some mathematical models for studying properties of lipid bilayer membranes. It is shown that there is evidence from fluorescent probe and electron spin resonance studies that some integral proteins are "randomly" distributed in the plane of a lipid bilayer for T > Tc, so that protein–protein "contacts" can occur. This implies that there is no permanent annulus of lipids around these proteins, so that there is no unique stoichiometric ratio of "boundary lipids" to protein. Computer simulation techniques are reviewed and comments are made about some recently introduced methods. Dynamical models of lipid – integral protein bilayers are outlined and it is shown that much differential scanning calorimetry, freeze-fracture, X-ray, and 2H nuclear magnetic resonance data can be understood. It is predicted that specific heat curves should show a rise at a temperature, TK < Tc, at which a protein-rich phase starts to "melt"; that dipalmitoyl phosphatidylcholine (DPPC) hydrocarbon chains in such a protein-rich phase should exhibit some static disorder down to ~ −10 °C, around which they should freeze noncooperatively; and that the crossing of phase boundaries, as protein concentration expressed as mole fraction x changes just below Tc, should be reflected in a complicated dependence of protein lateral motion upon x. Models to study the liquid condensed – liquid expanded (LC–LE) transition of phosphatidylcholine (PC) monolayers at the air–water interface are described. The rounding in the LC phase of pressure–area isotherms are understood as the melting of microscopic gel-phase "domains" into macroscopic regions of fluid-phase lipids. There is some experimental support for this. Finally, to model the difference between a monolayer and the corresponding bilayer, an interaction is introduced between the two halves of a bilayer. It is shown to be very weak in the case of PC bilayers and it is outlined in the paper how monolayer and bilayer thermodynamics can be related. It was found that the internal lateral pressure of a DPPC bilayer is ~ 30 dyn/cm (1 dyn = 10 μN).
Inhibition of microtubule polymerization by micromolar concentrations of mercury(II)
Authors: Keates Robert A. B. , Yott Brian
Published: December 2009

Polymerization of microtubule protein in vitro has been tested in the presence of Hg2+. Inhibition occurs in the presence of Hg2+ concentrations lower than the molarity of the tubulin, even in the presence of 0.5 mM ethylenebis(oxyethylene-nitrilo)tetraacetic acid. The estimated concentration of free Hg2+ under these conditions is less than 10−20 M. The extent of inhibition is approximately stoichiometric in that the molar quantity of tubulin that fails to polymerize is comparable to or slightly greater than the total Hg2+ present as chelate. The affinity of the susceptible site for Hg2+ in microtubule protein must be exceptionally high and may not be protected by natural chelating agents in the cell. Preformed microtubules are rapidly depolymerized on addition of Hg2+ chelate. In vitro, inhibition is both prevented and rapidly reversed by 2-mercaptoethanol.
The incorporation of [3H]glycerol and 1-[14C]acyl-sn-glycero-3-phosphocholine into different molecular species of phosphatidylcholine in human platelets
Authors: Mahadevappa Vhundi G. , Holub Bruce J.
Published: December 2009

The molecular species of phosphatidylcholine which are formed by de novo synthesis and by the acylation of 1-acyl-sn-glycero-3-phosphocholine were characterized and compared in human platelets. For this purpose, intact human platelets were incubated in the presence of [3H]glycerol or 1-[14C]palmitoyl-sn-glycero-3-phosphocholine and the newly formed radioactive phosphatidylcholine was isolated by thin-layer chromatography. The labelled phosphatidylcholines were converted to their 1,2-diacylglycerol acetate derivatives and fractionated into their various chemical classes (saturates, monoenes, dienes, trienes, tetraenes, and >tetraenes) by argentation thin-layer chromatography. Regardless of the precursor used, the radioactivity distributions among the various classes did not correspond closely to that of the mass. The highest percentage of the radioactivity incorporated from [3H]glycerol was found in the saturates (25% of total), followed closely by the tetraenes (21%) and monoenes (18%), with lesser amounts in the dienes (15%), pentaenes plus hexaenes (14%), and trienes (7%). These results indicate that the de novo pathway is capable of substantial synthesis of tetraenoic (1-acyl 2-arachidonoyl) phosphatidylcholine in human platelets in contrast to previous observations in rat liver. In close agreement with work in rat liver, 59% of the radioactivity in the [14C]phosphatidylcholine derived from 1-[14C]palmitoyl-sn-glycero-3-phosphocholine was found in the tetraenoic species. The present results, together with the existence of phospholipase A2 and acyltransferase activity in platelets, support the potential importance of a deacylation–acylation cycle for the enrichment of human platelet phosphatidylcholine in arachidonoyl molecular species.
Synthesis of putative microtubule-associated proteins by mouse blastocysts during early outgrowth in vitro
Authors: Bates William R. , Kidder Gerald M.
Published: December 2009

The outgrowth of mouse trophoblast in culture provides a simplified model system analogous in certain ways to blastocyst implantation in vivo. Day-four blastocysts cultured for 3 days in vitro undergo extensive changes in cell shape and motility which are likely to involve the complex cytoskeletal system of the trophoblast cells. To explore the biochemical basis of these changes, one set of cytoskeletal proteins, the microtubule-associated proteins (MAPs), was studied. Day 4 blastocysts were labeled with [35S]methionine and blastocyst outgrowths, after 3 days in culture from the blastocyst stage, were labeled with [3H] methionine. Labeled embryos were disrupted and the soluble supernatants were pooled, and newly synthesized proteins from the two stages were coassembled with taxol-stabilized brain microtubule polymer enriched for MAP-binding sites. Double-labeled coassembly proteins (putative MAPS) were then released from the microtubule polymer by treatment with 0.35 M NaCl and analyzed by one-dimensional polyacrylamide gel electrophoresis. 3H/35S dpm ratios were determined for individual protein bands to compare the relative synthesis rates for day 4 blastocyst and day 3 outgrowth MAPs. In spite of the extensive changes in cell shape and motility associated with blastocyst outgrowth, a common set of putative MAPs characterizes the two stages investigated, including several in the size range of tau factors. No synthesis of high molecular weight MAPs comparable with MAP 1 or MAP 2 from brain was detected. The synthesis rates of individual MAPs relative to each other remain constant over this period and are likely coordinated with total protein and tubulin synthesis.
Purification and characterization of trypsin from the Greenland cod (Gadus ogac). 1. Kinetic and thermodynamic characteristics
Authors: Simpson B. K. , Haard N. F.
Published: December 2009

Trypsinogen was isolated from the pyloric ceca of Greenland cod by ammonium sulfate fractionation followed by acetone precipitation, and the trypsin(ogen) thus obtained was purified by affinity chromatography on soybean trypsin inhibitor – Sepharose 4B. The purified trypsin migrated as a single zone during polyacrylamide gel electrophoresis and its identity as trypsin (EC 3,4.21.4) was established by its catalytic specificity for amide or ester bonds involving the carboxyl group of arginine, its sensitivity to serine protease inhibitors and soybean trypsin inhibitor, and its molecular weight of 23 500. With tosylarginine methyl ester (TAME) as substrate, the turnover number of the hydrolytic reaction was about three times greater for the cod trypsin than for bovine trypsin at 5 °C. The Michaelis–Menten constant (Km,app) for cod trypsin and TAME increased from 0.14 mM at 5 °C to 0.26 mM at 35 °C, while the Km,app for bovine trypsin – TAME was about 0.05 mM at all assay temperatures. The free energy of activation (ΔG*) for the hydrolysis of TAME was about 600 cal/mol (1 cal = 4.1868 J) lower for the cod trypsin than for bovine trypsin at 5 °C. The contribution of enthalpy of activation (ΔH*) and entropy of activation (ΔS*) to ΔG* differed considerably for the two enzymes. The "physiological efficiency" (Vmax/Km,app) of the two enzymes with TAME was similar at 5 °C, but was much greater for bovine trypsin than cod trypsin at warmer temperatures. With N-α-benzoylarginine-p-nitroanilide (BAPA) as substrate, the turnover number was about eight times greater for the cod trypsin at 25 °C. The Km,app for cod trypsin – BAPA increased from 1.67 mM at 25 °C to 1.84 mM at 35 °C, whereas the Km,app for bovine trypsin – BAPA decreased from 0.97 mM at 25 °C to 0.90 mM at 35 °C. The ΔG* for hydrolysis of BAPA was about 1800 cal/mol lower for cod trypsin than it was for bovine trypsin at 25 °C. Vmax/Km,app was three to four times greater for cod trypsin than for bovine trypsin at 25 and 35 °C. These results show that Greenland cod trypsin is a better catalyst than bovine trypsin at low temperatures and that catalysis by the fish trypsin is less responsive to temperature change than is catalysis by bovine trypsin.
Partial amino acid sequence of the wheat germ Ec protein. Comparison with another protein very rich in half-cystine and glycine: wheat germ agglutinin
Authors: Hofmann Theo , Kells David I. C. , Lane Byron G.
Published: December 2009

A wheat germ protein (Ec), the dominant site of cysteine incorporation during early (E) germination of isolated wheat embryos, has been partially sequenced by automated Edman degradation. The sequence of residues 1–59 is not significantly similar to the amino acid sequence known for any other protein, including wheat germ agglutinin which, like Ec, is very rich in half-cystine and glycine. The partial sequence for Ec contains an almost identical pattern of half-cystine residues in segments 6–20 and 35–48, a duplication which includes 10 of the 12 half-cystine residues in the sequence. The partial sequence of Ec may include a large part of the complete sequence, but this remains uncertain because it has not been possible to arrive at a definitve estimate of molecular weight using different physical techniques. Protein Ec can be prepared from a reticulocyte lysate in which cell-free synthesis is programmed by bulk wheat germ mRNA. Determination of the distribution of half-cystine moieties between residues 1 and 20 by Edman degradation of the [35S]cysteine-labeled product of cell-free synthesis shows that it is devoid of an N-terminal extension. Unlike wheat germ agglutinin, Ec does not seem to arise by processing of a conspicuously larger precursor protein. Unlike Ec, another wheat germ protein, Em, the most conspicuous methionine-labeled protein when cell-free protein synthesis is directed by wheat germ mRNA, is refractory to direct sequence analysis by Edman degradation. However, again unlike Ec, uncertainty about the molecular weight of Em, based on its mobility in different sodium dodecyl sulphate – polyacrylamide gel systems, has been resolved by virtue of Em being ideally suited to study by the Yphantis high-speed sedimentation equilibrium method.
Photoactivated linkage of catechol O-methyltransferase and S-adenosyl-L-methionine
Authors: Yu Peter H.
Published: December 2009

The formation of a stably linked complex of tritiated S-adenosyl-L-methionine (AdoMet) and catechol O-methyltransferase (COMT) has been achieved by irradiating the enzyme and ligand in Tris–HCl buffer (pH 7.5) with ultraviolet light at 254 nm. The reaction is specific as shown by a number of criteria. COMT inhibitors such as S-adenosylhomocysteine can block this photoactivated linkage. The [3H]AdoMet–COMT adduct has been shown to be a homogeneous protein by Sephadex gel filtration, sodium dodecyl sulfate – polyacrylamide gel electrophoresis, and isoelectric focussing. After extensive proteolysis of the [3H]AdoMet–COMT adduct with pronase P, one major labelled product was released. This fragment could be separated by paper chromatography and was shown to be chromatographically identical to that released from the [3H]AdoMet – phenylethanolamine N-methyltransferase adduct.
Surface layer virulence A-proteins from Aeromonas salmonicida strains
Authors: Kay William W. , Phipps Barry M. , Ishiguro Edward E. , Olafson Robert W. , Trust Trevor J.
Published: December 2009

Superficial surface layer proteins (A-proteins) were present on diverse isolates of Aeromonas salmonicida which differed both physiologically and in pathogenesis. Three of these proteins were purified directly from the surface of whole cells or from outer membrane preparations. These A-proteins were unusually hydrophobic (45–47%) and of similar but not identical molecular mass (49, 50, and 51 kdaltons). They were nearly identical in amino acid composition and were highly conserved, but not identical with respect to their hydrophobic N-terminal amino acid sequences. These proteins differed, however, with respect to their oligomerization properties, isoelectric forms, and chymotryptic peptide patterns. All three proteins were immunologically closely related and shared surface-exposed immunoreactive peptides with 28 separate isolates.