bluuebunny/crossref_metadata_2025 huggingface dataset:
Headgroup oligosaccharide dynamics of a transmembrane glycoprotein
Authors: Lee Pat M. , Grant Chris W. M.
Published: December 2009

Glycophorin, a major integral membrane glycoprotein of the human erythrocyte, has been spin labelled on oligosaccharide chains. Electron paramagnetic resonance studies of this glycoprotein in systems of controlled complexity have provided a degree of insight into its headgroup behaviour. (i) When glycophorin is free in solution its oligosaccharide chains exhibit uniformly high freedom of motion. This motional freedom is not attributable to the presence of N-acetylneuraminic acid residues. (ii) No evidence has been found of a finite tendency for headgroup sugars to associate with hydrophboic regions of phospholipid or glycoprotein. (iii) Headgroup oligosaccharide dynamics are essentially independent of the state of and interactions of the polypeptide hydrophobic portion (that portion which traverses the membrane), (iv) Nonspecific interaction with proteins and polysaccharides can readily reduce oligosaccharide chain mobility by some 25%, but does not alter their basic behaviour, (v) Binding of wheat germ agglutinin, dramatically immobilizes (terminal) N-acetylneuraminic acid residues, (vi) The above observations hold over the temperature range 0–40 °C. (vii) Headgroup carbohydrate mobility is at a minimum in the region of headgroup neutrality (pH 2.6–3.5) and is pH invariant over several pH units in the physiological range.
Energy coupling to K+ transport in a marine bacterium
Authors: Sedgwick Edward G. , MacLeod Robert A.
Published: December 2009

Cells of the marine bacterium Alteromonas haloplanktis 214 ATCC 19855 (previously referred to as marine pseudomonad B-16) were depleted of K+ by washing with 0.1 M MgSO4. Washing with 0.05 M MgSO4 lowered the Vmax for K+ transport compared with washing with 0.1 M but did not change the Km, while washing with lower concentrations of MgSO4 caused loss of ultraviolet-absorbing material from the cells. K+ uptake was a strictly aerobic process and was accompanied by proton release. When an anaerobic suspension of cells was added to incubation mixtures containing increasing amounts of O2, intracellular ATP concentrations increased as the O2 concentration increased and reached near maximum values before K+ transport began. The O2 concentration initiating K+ transport caused transport to proceed at its maximum rate. For these experiments A. haloplanktis was depleted of ATP by incubating under anaerobic conditions. Incubating with either N′,N′-dicyclohexyl carbodiimide (DCCD) or arsenate failed to deplete intact cells of ATP or prevent K+ transport. The inhibitory activity of DCCD for ATPase in membrane preparations was higher at 5 mM than at other MgSO4 concentrations and increased with time. Cyanide and the uncoupling agents tetrachlorosalicylanide (TCS) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented K+ uptake while TCS and FCCP though not cyanide caused K+ to be released from K+-containing cells. It is concluded that the driving force for K+ transport in these cells is likely to be the membrane potential and that K+ transport may be gated.
Uptake and transport of sterols by isolated villus cells of rat jejunum
Authors: Child P. , Kuksis A.
Published: December 2009

Cellular uptake and transport of cholesterol and β-sitosterol was studied in rat jejunal villus cells isolated by hyaluronidase dispersion. Sterol absorption was measured from micellar solutions using a Millipore filtration technique. In actively metabolizing cells both cholesterol and β-sitosterol exhibited rapid initial uptake curves which leveled off after 10 min, corresponding approximately to a 1:1 sterol:phospholipid molar ratio within the microsomal membranes of the cell, which suggested a physicochemical end point. Using various proportions of cholesterol and β-sitosterol in the incubation medium, it was shown that each sterol interfered with the absorption of the other, the one present in the highest concentration showing the relatively higher uptake. Mixtures of equimolar proportions were taken up by the cells in a ratio that slightly favoured β-sitosterol, but significantly more cholesterol than β-sitosterol was found to be transferred to the microsomes. The limited mass discrimination between cholesterol and β-sitosterol seen in the isolated cells is in contrast to the preferential absorption of cholesterol in vivo. These studies demonstrate that the molecular basis for the differential transport of cholesterol and β-sitosterol in the lymph observed in vivo must be sought at a higher level of physiological activity of the cells (e.g., chylomicron formation and secretion) than that achieved in the present experiments.
Etude conformationnelle par résonance magnétique nucléaire du carbone 13 de peptides à activité antalgique
Authors: Smolders Robert René , Coomans Roland , Jacquemotte Françoise , Haverbeke Yves Van , Maquestiau André , Muller Robert
Published: December 2009

13C Nuclear magnetic resonance studies of proline peptides with central antalgic activity (Pro-Arg, Arg-Pro, Pro-Arg-Pro, Pro-Lys-Pro) indicate a predominance of trans conformation at all pH values; but their physiological activities appear to be related to the ability of the C-terminal proline peptides to assume the cis conformation.
Subunit structure of Trypanosoma cruzi chromatin
Authors: Rubio Julieta , Rosado Yolanda , Castañeda Mario
Published: December 2009

Chromatin of Trypanosoma cruzi epimastigotes was found to be arranged in a subunit structure by the demonstration of (i) nu-bodies, (ii) monomeric and oligomeric DNA fragments, and (iii) four basic nuclear proteins which presumably correspond to the intrinsic protein component of nucleosomes. A discrete core fragment of 140 base pairs and a basic nuclear protein fraction clearly similar to most histones H1 were not detected. In contrast with vertebrate material, the nuclear preparations were physically and enzymatically fragile, the basic proteins showed different mobilities in electrophoresis, but the DNA repeat length was similar. The data are discussed in relation to histone regional charge, histone content, and chromatin condensation.
Characterization of mononucleosomes and associated glycoproteins from Ehrlich ascites tumour cells
Authors: Miki Brian L. A. , Gurd James W. , Brown Ian R.
Published: December 2009

Mononucleosomes generated by the digestion of ascites nuclei with micrococcal nuclease were resolved into three types by polyacrylamide gel electrophoresis. All three types were present in early and late digests and a precursor–product relationship was not apparent. Each mononucleosome type was associated with a unique pattern of subnucleosome DNA fragments and differences were apparent in histone and nonhistone proteins. On the basis of reactivity to 125I-labelled concanavalin A and labelling with [3H]glucosamine, glycoproteins were identified as components of two of the mononucleosome types. The principal glycoprotein species associated with mononucleosomes had an apparent molecular weight of 130 000.
Horseradish peroxidase. XLII. Oxidations of L-tyrosine and 3,5-diiodo-L-tyrosine by compound II
Authors: Ralston Isobel M. , Dunford H. Brian
Published: December 2009

The oxidations of both L-tyrosine and 3,5-diiodo-L-tyrosine by compound II of horseradish peroxidase were studied over the pH range of approximately 3 to 10 at 25 °C and at a constant ionic strength of 0.11. The rate versus pH profile for the tyrosine – compound II reaction illustrates the influences of at least two acid group ionizations. An enzyme dissociation (pKa ~ 6.2) has a small effect on the reaction rate; whereas, a second pKa of 9.2, which may be attributed to either the enzyme or substrate, has a greater influence on the rate. The oxidation of tyrosine by compound II is fastest at pH 7.6. In the case of the diiodotyrosine – compound II reaction, three acid dissociations are necessary to describe the plot of log (kapp) versus pH. These include two enzyme pKa values of 3.6 and 8.6, and one substrate pKa of 6.6. The rate optimum for the reaction occurs at pH 5.2 and deprotonation of the phenolic group of diiodotyrosine results in a dramatic decrease in kapp. Diiodotyrosine is required in only a 0.5 M equivalent for the conversion of horseradish peroxidase compound I to compound II. The diiodotyrosine pKa values were estimated as 6.4 and 9.4 for the phenolic and amino groups, respectively.
Experimental determinations of tubulin in the in vivo mitotic apparatus of sea urchin zygotes
Authors: Forer Arthur , Larson D. E. , Zimmerman A. M.
Published: December 2009

Mitotic apparatus (MA) were isolated from zygotes of a sea urchin (Strongylocentrotus purpuratus), using hexylene glycol (pH 6.4) as lysing–stabilizing agent. Protein was measured in the MA pellet and in the remainder of the cell lysate (using the Lowry procedure). Tubulin was measured in the MA pellet and in the remainder of the cell lysate (using microdensitometry of stained gels after sodium dodecyl sulphate – polyacrylamide gel electrophoresis). From these data we calculated the maximum possible amounts of tubulin in the isolated MA and in the MA in vivo; in these calculations we assumed that all the tubulin in the cell is associated with the MA, and we assumed that, as reported in the literature, the MA lose 90% of their dry matter during the isolation. We conclude that tubulin probably comprises less than 7% of the protein in the in vivo MA, and, even if there are very large errors, tubulin is considerably less than haf the protein in the MA.
Renal phosphoenolpyruvate carboxykinase during perturbations of acid–base homeostasis in rats. Immunochemical studies
Authors: Parry David M. , Brosnan John T.
Published: December 2009

The activity of phosphoenolpyruvate carboxykinase (PEPCK) increased in the rat kidney after induction of metabolic acidosis. Immunotitration of the enzyme in renal extracts from normal and acidotic rats indicate that this increase in catalytic activity correlates with the amount of immunotitrateable activity. PEPCK activity decreased rapidly during recovery from acidosis and the time required for the activity to return to normal was not altered by the administration of NaHCO3 at the start of recovery. Immunotitration of the enzyme from rats allowed to recover from metabolic acidosis revealed that the decrease in catalytic activity was paralleled by a decrease in the amount of immunotitrateable activity. It is concluded that the rise and fall in PEPCK activity during acidosis and recovery from acidosis, respectively, are due to comparable changes in enzyme protein.
The formation of bilirubin diglucuronide by rat liver microsomal preparations
Authors: Gordon Ellen R. , Goresky Carl A.
Published: December 2009

Bilirubin transformation in vitro to bilirubin conjugates in the presence of activated rat liver microsomal preparations and UDPglucuronate was assessed with a method involving isolation of the products as tetrapyrroles. The proportions of bilirubin monoglucuronide and diglucuronide formed by the microsomal bilirubin UDPglucuronosyltransferase were found to be governed by the concentration of bilirubin present and the nature of the activation of the microsomal membrane. Activation of the microsomal preparations with the nonionic detergents Triton X-100 or Emulgen 911, or with digitonin for 24 h, produced bilirubin monoglucuronide as the only product at all concentrations of bilirubin investigated. In contrast, bilirubin diglucuronide was the only conjugate formed when hepatic microsomal preparations were activated with digitonin for periods of less than 2 h and the concentration of bilirubin was 20 μM. Increasing the concentration of bilirubin utilized in this assay system changed the relative amounts of bilirubin monoglucuronide and diglucuronide formed. As the level of bilirubin was increased from 20 to 166 μM, the proportion of bilirubin diglucuronide decreased and that of bilirubin monoglucuronide increased, until at levels of 108 and 166 μM bilirubin only bilirubin monoglucuronide was formed. No evidence was found with liver plasma membranes that transglucuronidation plays a major role in the formation of bilirubin diglucuronide from bilirubin monoglucuronide.
Identification and quantitation of elongation factor EF-P in Escherichia coli cell-free extracts
Authors: An Gynheung , Glick Bernard R. , Friesen James D. , Ganoza M. Clelia
Published: December 2009

EF-P, an elongation factor that stimulates peptide bond synthesis in vitro with some aminoacyl-tRNAs, has been identified by two-dimensional gel electrophoresis and the cellular content at three points in the growth curve has been measured. The molecular weight of EF-P is estimated to be 21 000. EF-P is a slightly acidic protein whose isoelectric point is close to RNA polymerase subunit α. The amount of EF-P present in Escherichia coli is about [Formula: see text]that of EF-G and the level is independent of the stage of cell growth; there is about one EF-P per 10 ribosomes. It is also shown that a highly purified preparation of EF-P is free of all known protein synthesis factors and ribosomal proteins.
Cell interactions in concanavalin A activated cation flux and DNA synthesis of mouse lymphocytes
Authors: Owens Trevor , Kaplan J. G.
Published: December 2009

Co-culture at constant cell density of nude mouse spleen cells (by themselves unresponsive to the T-cell mitogen concanavalin A (Con A)), with congenic T-enriched lymphocyte suspensions and Con A caused anomalously high activation of K+ transport (measured by 86Rb uptake) and of incorporation of thymidine into DNA; the expected dilution of these two responses by nude spleen cells did not occur. However, if the nude splenocytes were added immediately prior to assay to the enriched T cells that had been precultured in presence of Con A, the expected dilution of the activated T-cell responses occurred; both 86Rb uptake and thymidine incorporation were reduced proportionally to the degree of dilution of the T cells by the nonresponding cells. These data indicate that during co-culture in presence of Con A there is interaction between the T cells, capable of responding to the mitogens, and the nude spleen cells. Attempts to demonstrate a diffusible factor in the supernatants of stimulated T cells were unsuccessful. The measured interaction is sufficient to explain our previous paradoxical findings that enrichment of T cells as measured by membrane markers did not cause a corresponding enrichment for either cation transport or for thymidine incorporation, and that depletion of T cells in the B-enriched cultures did not cause a corresponding decrease in these two Con A induced responses.
Jack bean urease (EC 3.5.1.5). IV. The molecular size and the mechanism of inhibition by hydroxamic acids. Spectrophotometric titration of enzymes with reversible inhibitors
Authors: Dixon Nicholas E. , Hinds John A. , Fihelly Ann K. , Gazzola Carlo , Winzor Donald J. , Blakeley Robert L. , Zerner Burt
Published: December 2009

Kinetic, spectral, and other studies establish that hydroxamic acids bind reversibly to active-site nickel ion in jack bean urease. Equilibrium ultracentrifugation studies establish that the molecular weight of native urease is 590 000 ± 30 000 while that of the subunit formed in 6 M guanidinium chloride in the presence of β-mercaptoethanol is ~95 000. Essentially the same subunit molecular weight (~93 000) is found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent to denaturation in a guanidinium chloride – β-mercaptoethanol system at various temperatures. Coupled with an equivalent weight of 96 600 for binding of the inhibitors acetohydroxamic acid and phosphoramidate, these results establish securely that urease is a hexamer with one active site per 96 600-dalton subunit. Consistent values for the equivalent weight are obtained by a routine spectrophotometric titration of the active site of freshly prepared urease with trans-cinnamoylhydroxamic acid. General equations are derived which describe spectrophotometric titrations of binding sites of any enzyme with a reversible inhibitor. These equations allow the evaluation of the difference spectrum of the protein–inhibitor complex even when the binding sites cannot readily be saturated with the inhibitor or vice versa.
A comparative proton magnetic resonance conformational study of the tRNA "wobble" nucleosides 5-carboxymethyl-, 5-methoxycarbonylmethyl-, and 5-carbamoylmethyl-uridine
Authors: Lipnick Robert L. , Fissekis John D.
Published: December 2009

The 270-MHz proton magnetic resonance spectra of 5-carboxymethyluridine, 5-methoxy-carbonylmethyluridine, and 5-carbamoylmethyluridine have been obtained, and analyzed iteratively using LAOCOON 3. A standard treatment of the obtained data reveals that in each the 5-substituent produces no significant alteration in the furanose puckers, their equilibrium state, and the relative populations of the gg, ig, and gt exocyclic hydroxymethyl rotamers relative to uridine. The analogous similarity in furanose chemical shifts suggests that these derivatives favor the anti glycosyl state. The 5-substituent may act at the polynucleotide level in modulating the conformation and interaction(s) of the anticodon loop.
The base exchange enzymes and phospholipase D of mammalian tissue
Authors: Kanfer Julian N.
Published: December 2009

The base exchange enzymes and phospholipase D represent a group of enzymes which alter the polar portion of phospholipids. The base exchange enzymes provide a mechanism of inter-converting different species of phospholipids whereas phospholipase D hydrolyzes these phospholipids to phosphatidic acid. Although the occurrence of these activities is widespread, this article is restricted to a description of the mammalian enzymes.Originally it was believed that these catalytic activities resided in a single enzyme. As a result of successes in partial purifications and separations it is clear that separate enzymes exist for the exchange of serine, for the exchange of choline, and for the exchange of ethanolamine resulting in their appearance in the corresponding phospholipid. These enzyme activities are not a reflection of phospholipase D action. Conversely, a partially purified phospholipase D from rat brain was devoid of detectable base exchange activity. However, this enzyme preparation possessed transphosphatidylation activity producing phosphatidylglycerol from glycerol and lecithin.These enzymes are presumed to be functionally significant for cellular homeostasis; however, strong evidence supporting this contention is unavailable. The base exchange enzymes appear to be concentrated in the "microsomal" fraction of tissues. The choline base exchange enzyme is located on the cytoplasmic surface while the serine and ethanolamine base exchange enzymes are located on the luminal surface of the endoplasmic reticulum of rat brain tissue. The lipid environments in which the individual enzymes reside are also distinctly different suggesting that their in situ domains within the microsomal membrane are dissimilar. Therefore it appears that the enzymes are distinctly separate catalytic entities. They also are in topographically different locations on the microsomal membrane and reside in different lipid environments as well. These observations would imply functionally distinct roles for the separate enzymes. Evidence is available which suggests that their existence might be demonstrable in vivo.
Lectin pulses as determinants of lymphocyte activation and inactivation during the first six hours of culture: sequential action of concanavalin A and complement cause cell lysis
Authors: Forsdyke D. R.
Published: December 2009

Rat lymph node cells were incubated for 6 h in medium containing bovine calf serum. Cell activation or inactivation by concanavalin A (Con A) was assessed by measuring changes in radioactive labelling with [3H]uridine. Two inactivation processes were distinguished: complement-dependent inactivation needing moderately high Con A concentrations (500 μg/mL serum) and complement-independent inactivation needing very high Con A concentrations. The times needed for cells to react with sufficient Con A to produce activation and the complement-dependent inactivation were compared by adding extra serum at different times to bind Con A and reduce the effective Con A concentration. Cells exposed to Con A prior to adding serum rapidly reacted with sufficient Con A to produce optimum activation and inactivation. Cells exposed to Con A in the presence of serum reacted with Con A at a lower rate. Initial reaction of cells with Con A was dependent on time and Con A concentration in a reciprocal manner (i.e., a high concentration of Con A could compensate for a short pulse time). Inactivation of cells was dependent on the concentrations of Con A and serum (complement) in a reciprocal manner (i.e., a high concentration of Con A compensated for a low concentration of serum). Studies with complement inhibitors added at different times of culture showed that, whereas cell activation begins early in the first hour of culture, inactivation did not begin until approximately 40 min after exposing cells to Con A. The 40-min delay in onset of inactivation was divisible into (i) an initial stage needed for cell reaction with Con A and (ii) a subsequent stage which did not need reaction of cells with more Con A molecules. The results indicate that a brief Con A pulse provides sufficient lectin to activate cells to proceed from G0 into the G1 phase of the cell cycle. Once cells have reacted with Con A there are, at least during the first 6 h of culture, no further stages dependent on reaction of cells with more Con A molecules. At high Con A concentrations in the presence of complement, an optimum activation signal is soon countermanded by an optimum inactivation signal. The delay in onset of inactivation is in keeping with previous studies suggesting a need for cell-bound Con A to aggregate cell-surface molecules thus generating receptor sites for complement components.
Multivalent interaction between asialofetuin and plasma membrane preparations from the rat liver
Authors: Debanne Maria T. , Regoeczi Erwin , Hatton Mark W. C.
Published: December 2009

Binding of bovine asialofetuin by rat liver plasma membranes was studied using different techniques for the separation of the free and bound forms of the glycoprotein and also different approaches to measure nonspecific binding. The membrane preparations had the electron microscopic appearance of a mixture of lamellae and vesicles and their lipid:protein ratios and marker enzyme profiles fell within the range of values available from the literature. The binding capacity was approximately 15 pmol of asialofetuin per milligram of membrane protein.Scatchard plots of the values obtained over a wide range of concentrations (4.8–12.6 μg asialofetuin per 30 μg membrane protein) after incubation at 22 °C showed pronounced non-linearity which, in combination with evaluations according to other theoretical models, was referable to heterogeneity of binding. In sharp contrast, after incubation at 4 °C the Scatchard plot was linear. This difference is interpreted as the expression of a functional, rather than a chemical, heterogeneity in asialofetuin binding. The underlying mechanism is thought to be competition of galactose groups for binding sites with the result that the number of bonds varies between the galactose groups of a bound asialofetuin molecule and the hepatic lectin, depending on the concentration of the glycoprotein in the incubation mixture.
Binding of the chloroplast coupling factor CF1 to lipid vesicles
Authors: Dijkmans Huguette , Daniel Anne-Marie
Published: December 2009

The binding of the chloroplast coupling factor CF1 to lipid vesicles was analyzed by gel filtration. CF1 can be bound to vesicles made of chloroplast lipids but not of lecithin. The presence in the vesicle walls of a proteolipid subunit of the hydrophobic component of the coupling factor increases the binding of CF1. The apparent binding constant and the maximum protein/lipid ratio are calculated. The Ca2+-ATPase activity of bound CF1 is markedly lower than that of dissolved CF1. It is confirmed that the proteolipid is a N,N′-dicylohexylcarbodiimide sensitive proton channel. The binding of CF1 on proteolipid vesicles decreases their proton permeability.
Nuclease sensitivity of postreplicated chromatin from Ehrlich ascites tumour cells
Authors: Miki Brian L. A. , Heikkila John J. , Brown Ian R.
Published: December 2009

Chromatin appears to undergo structural modification after replication and before integration into bulk chromatin. In ascites cells, postreplicated chromatin displays a transient resistance to digestion with micrococcal nuclease. This resistance may be correlated with a shorter DNA repeat length (178 base pairs) than that found in bulk chromatin (187 base pairs). Selective labelling or selective digestion of DNA sequence classes could not account for these observations. In both bulk and postreplicated chromatin, three electrophoretic types of mononucleosomes were found. Postreplicated mononucleosome types showed selective sensitivities to nuclease digestion whereas bulk mononucleosome types did not.
Studies on the pargyline-binding site of different types of monoamine oxidase
Authors: Yu Peter H.
Published: December 2009

[3H]Pargyline has been covalently linked to active sites of both type A and type B monoamine oxidase (MAO) obtained from various tissues. Rat heart and human placenta were chosen to represent predominantly type A MAO, pig and bovine livers to represent type B MAO, and rat liver and brain to represent mixed type A and type B MAO’s. The [3H]pargyline–MAO adducts were isolated and hydrolyzed by proteolytic enzymes, and the labelled peptides (pargyline-binding sites) separated and compared by paper chromatography and by paper electrophoresis at various pH values. Only one common pargyline peptide was obtained from all the different MAO’s. The alternative A and B sites were assessed after preincubation of rat liver MAO with the selective inhibitors deprenyl (to block the B site) and clorgyline (to block the A site). Following proteolysis of the [3H]pargyline peptides of both type A and type B MAO from this pretreated rat liver, MAO has been purified by a series of chromatographic and electrophoretic procedures. Micro-Edman degradation, followed by dansylation, revealed the amino acid sequence to be Ser-Gly-Gly-Cys(X)-Tyr. It is concluded that the primary structures immediately surrounding the pargyline-binding sites are identical for both type A and type B MAO in these tissues.
Semliki Forest virus does not inhibit phosphatidylcholine biosynthesis in BHK-21 cells
Authors: Whitehead Frederick W. , Trip Everard , Vance Dennis E.
Published: December 2009

The mechanism by which Semliki Forest virus inhibits the incorporation of [methyl-3H]- choline into phosphatidylcholine has been investigated. Decreased labeling of the lipid was not due to altered uptake of [methyl-3H]choline. The specific activities of choline kinase and CTP:phosphocholine cytidylyltransferase were unchanged. The previously observed inhibition (Vance, D. E. &Burke, D. C. (1974) Eur. J. Biochem. 43, 327–336) of CDP-choline:1,2-diacylglycerol phosphocholinetransferase was confirmed. Since the decreased activity of the phosphocholinetransferase may not have caused the reduced labeling of phosphatidylcholine, the amounts of this lipid and its precursors were measured. We observed changes in the concentration of phosphocholine (34 ± 12 and 120 ± 40 nmol∙g cells−1 in mock- and virus infected cells, respectively) and CTP (116 ± 35 and 36 ± 13 nmol∙g cells−1 in mock- and virus-infected cells, respectively). Pulse–chase studies with [methyl-3H]choline demonstrated that, initially, most of the radioactivity was in phosphocholine. As it disappeared from this compound, it appeared in phosphatidylcholine. From these results, we calculated the rate of phosphatidylcholine biosynthesis to be 0.56 and 1.23 nmol∙min−1∙g cells−1 in mock- and virus-infected BHK-21 cells, respectively. We conclude that phosphatidylcholine biosynthesisis not inhibited in Semliki Forest virus infected BHK cells, but rather is stimulated 6.75 h after infection. The decreased labeling observed during pulse studies with [methyl-3H]choline is due to dilution of the labeled choline into a pool of phosphocholine which is 3.5 times larger in the infected cells.
Rat renal γ-glutamyltransferase activity: a reaction of glutamine synthetase
Authors: Bregar Frank W. , Hughey Rebecca P. , Curthoys Norman P.
Published: December 2009

The experimental observations of Welbourne (Am. J. Physiol. 226, 544–548 (1974)) led him to propose that a γ-glutamyltransferase activity contributes significantly to basal renal ammonia production. He measured the transferase activity in vitro as γ-glutamylhydroxamate or ammonia production from glutamine and hydroxylamine. We report that in crude homogenates of rat kidney, γ-glutamyltransferase activity requires the presence of a divalent cation, arsenate, and ADP. These conditions are also required for the maximal expression of the γ-glutamyltransferase reaction catalyzed by glutamine synthetase. Glutamine synthetase and γ-glutamyltransferase activities exhibit an identical distribution during differential centrifugation. In addition, the two activities comigrate during sucrose gradient velocity centrifugation, and exhibit identical heat inactivation and L-methionine sulfoximine inhibition profiles. Further-more, both activities are found to be localized primarily in the outer stripe region of the renal medulla. Based on these observations, it is concluded that the primary γ-glutamyltransferase activity as assayed in rat renal tissue is a partial reaction of glutamine synthetase, an enzyme which does not catalyze the production of ammonia and γ-glutamyl peptides from glutamine.
Isolation and characterization of a FAD-dependent NADH diaphorase from Methanospirillum hungatei strain GP1
Authors: McKellar R. C. , Shaw K. M. , Sprott G. D.
Published: December 2009

Crude extracts of Methanospirillum hungatei strain GP1 contained NADH and NADPH diaphorase activities. After a 483-fold purification of the NADH diaphorase the enzyme was further separated from contaminating proteins by polyacrylamide disc gel electrophoresis. Two distinct activity bands were extracted from the acrylamide, each one having oxygen, 2,6-dichlorophenoiindophenol, and cytochrome c linked activities. In these preparations NADPH could not replace NADH as electron donor. During the initial purification steps all activity was lost due to the removal of a readily released cofactor. Enzyme activity was restored by either FAD or a FAD fraction isolated from M. hungatei. Oxidase activity exhibited a broad pH optimum from 7.0 to 8.5 and apparent Km values of 26 μM for NADH and 0.2 μM for FAD. Superoxide anion, formed in the presence of oxygen, accounted for all of the NADH consumed in this reaction. The molecular weight of the diaphorase was about 117 500 by sodium dodecyl sulfate gel electrophoresis. Sulfhydryl reagents and chelating agents were inhibitory. Inactivation, which occurred during storage in phosphate buffer at 4 °C, was delayed by dithiothreitol. The isolated NADH diaphorase lacked NADPH:NAD transhydrogenase and NAD reductase activities.
L-Phenylalanine induced changes of sulfhydryl reactivity in rabbit muscle pyruvate kinase
Authors: Kwan Chiu-Yin , Davis Robert C.
Published: December 2009

Reactivity of sulfhydryl groups in rabbit muscle pyruvate kinase toward 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) was studied in the presence of activating divalent metal ions, substrate, substrate analogue, and the allosteric inhibitor, L-Phe. The pattern of sulfhydryl modification in various complexes of pyruvate kinase was consistent with the extent of enzyme inactivation by DTNB under very similar conditions. The sulfhydryl reactivity of Mg(II)-, Co(II)-, and Mn(II)-substituted pyruvate kinase toward DTNB depended upon the nature of the activating divalent metal ions used in the following order of increasing potency, Mg(II) < Mn(II) < Co(II), which is inversely related to the order of catalytic efficiency of these metal ions at alkaline pH. Similar optical spectra and the patterns of sulfhydryl modification by DTNB of the metal derivatives of pyruvate kinase were observed upon the binding of the substrate, phosphoenolpyruvate (PEP), or the substrate analogue, phosphoglycolate, which also provided a complete protection against enzyme inactivation by DTNB. L-Phe, on the other hand, deprotected the enzyme from inactivation and further sulfhydryl modification by DTNB in the presence of PEP with the following order of potency depending upon the activating metal ions, Mn(II) < Co(II) < Mg(II), which parallels the order of metal dependency of L-Phe inhibition of this enzyme. L-Ala, which reverses the L-Phe inhibition of Mg(II)- or Co(II)-activated enzyme, restored the protective effect of PEP in the presence of L-Phe. The different patterns of sulfhydryl reactivity toward Mn(II)–enzyme (hyperbolic) and Mg(II)–enzyme (sigmoidal) correspond well with their kinetic patterns in the presence of L-Phe, indicating the presence of different conformational states between these two metal–enzyme complexes. These results led us to conclude that enzyme sulfhydryl reactivity toward DTNB can be used as a valid index for allosteric conformational changes of rabbit muscle pyruvate kinase.
Recovery of nucleotide levels after cell injury
Authors: Warnick C. Terry , Lazarus Harrison M.
Published: December 2009

The major pathway of purine catabolism in mouse kidney during ischemia occurs through IMP, inosine, hypoxanthine, and xanthine. Short periods of ischemia (reversible cell injury) allow a rapid return of the energy charge to control values and a rapid return of ATP and GTP to values of 60–70% of control ATP and GTP then slowly return to control levels over the next 24 h. Long periods of ischemia (irreversible cell injury; ischemic times longer than 1 h) allow a gradual return of the energy charge to control levels. ATP, GTP, or total adenine or guanine nucleotides do not return to control levels even after 24 h of reinfusion under these circumstances. We conclude that irreversibly injured kidney cells retain the ability to phosphorylate purine nucleotides, but lose the ability to restore the concentrations of the purine nucleotides to control values.
DNA binding of a nonstructural reovirus protein
Authors: Shelton Iris H. , Kasupski Jr. George J. , Oblin Colette , Hand Roger
Published: December 2009

The specific early inhibition of DNA synthesis in reovirus-infected cells suggests that the cell nucleus is a target for virus-induced damage. We have now examined the affinity of reovirus proteins for DNA, postulating that such affinity could provide a mechanism for the inhibition. Cytoplasmic and nuclear extracts of cells labeled with [35S]methionine from 6 to 8.5 h after infection at high multiplicity were subjected to chromatography on denatured DNA – cellulose columns. Fractions from both cytoplasm and nucleus eluted with 0.6 M NaCl contained a protein with the same electrophoretic mobility on polyacrylamide slab gels as the nonstructural (NS) reovirus protein of the sigma size class. The protein also exhibited affinity for native DNA – cellulose and denatured DNA – agarose. Electrophoretic analysis in tube gels of cell extracts labeled for 48 h before infection with [14C]leucine and from 6 to 8.5 h after infection with [3H]leucine showed increased 3H label in this protein indicating it is reovirus specific. Small amounts of mu proteins also had DNA affinity. Purified virus did not bind strongly to DNA, suggesting that the binding protein is not a structural protein of the sigma size class on the outer surface of the virus. Our results provide evidence that the sigma NS protein binds to DNA. This affinity could interfere with chromosome function in the infected cell.
Oxidation–reduction titration of cytochrome components in the electron transport chain of Azotobacter vinelandii
Authors: Yang Tsanyen
Published: December 2009

The multiple cytochrome components in the electron transport particle of Azotobacter vinelandii were resolved and their oxidation–reduction midpoint potentials were determined by a simultaneous potentiometric and absorption measurements under anaerobic condition with or without CO. The midpoints of the individual cytochrome component corresponding to the membrane-bound types were also determined in the solubilized fractions prepared by a differential detergent solubilization of the membrane particles of A. vinelandii. Two cytochromes of b type, one with an absorption maximum measured at 559 nm and another at 561 nm in the membrane particle, were resolved and their Em, 7.4 values determined to be −30 mV and +122 mV, respectively. Cytochrome b559 reacted with CO readily in both membrane-bound and solubilized forms, however, cytochrome b561 was inert to CO treatment. Only one cytochrome of c type (c4) measured at 575–551 nm was resolved, its midpoint potential at pH 7.4 was +322 mV in the membrane-bound form and +278 mV in the solubilized form. This c-type cytochrome had no CO reactivity. Cytochrome d, a CO-reactive component, had a midpoint of +270 mV in the membrane fraction. The midpoint of cytochrome a1 in its membrane-bound form could not be measured accurately because of its low concentration. However, in the solubilized preparations, cytochrome a1 apparently had a red shift with an absorption maximum at 613 nm, with an estimated Em, 7.4 of −45 mV, while cytochrome d was no longer detected, possibly because of denaturation.
Decreased protein synthesis in rats during a single short-term immobilization
Authors: Huszti Zsuzsanna , Kenessey Agnes
Published: December 2009

The effect of short-term immobilization on protein synthesis was investigated in various tissues of the rat. Male, Sprague-Dawley rats, weighing 140–150 g, were immobilized by wrapping them into plaster bandages for 3 h and treated with [3H]tyrosine or [14C]histidine to measure the rate of incorporation of the amino acid into tissue proteins. The labeling of proteins in various tissues of the immobilized rats, examined after the administration of radioactive amino acid, was markedly decreased. A detailed analysis of the experimental data provided evidence that the depressed incorporation of the radioactivity into proteins reflected a real and generalized decrease in protein synthesis in various tissues.
Comparison of cartilage structural glycoproteins with matrix proteins and fibronectin
Authors: Bowness J. Michael
Published: December 2009

After extraction of proteoglycans and soluble matrix proteins from canine puppy rib cartilage, with 4 M guanidine∙HCl, the insoluble collagen-containing residue has been shown to contain two collagenase-resistant structural glycoproteins, A and G. The characteristic subunits of these insoluble structural glycoproteins have been identified by solubilizing them with 50 mM dithiothreitol (DTT) in 1% sodium dodecyl sulfate (SDS) and 8 M urea and comparing the SDS disc gel patterns with those of the more readily soluble matrix proteins. Three subunit bands which did not occur in gels from the soluble matrix proteins were found in the solubilized material from both the collagen-containing residue and the structural glycoproteins. One band, of about 87 000 daltons, was found equally in both A and G glycoproteins. The other two bands formed a closely spaced doublet, of about 30 000 and 27 500 daltons, of which the lower band is present in higher concentration in G. Although none of these SDS gel bands corresponds with the 220 000 dalton band produced by pure fibronectin under the same conditions, a fraction of the solubilized glycoprotein material resembles fibronectin in showing an affinity for collagen, fibrinogen, heparin, and an antibody to plasma fibronectin. Crude fibronectin from human plasma contains minor components (including one of about 87 000 daltons) which show partial identity in immunodiffusion reactions with components of the solubilized cartilage structural glycoproteins. Solubilized A gave a stronger reaction with anti-plasma fibronectin than did G and the soluble matrix proteins gave no reaction. It is possible either that the intercellular structural glycoproteins are formed by selection of some of the partial cleavage products of fibronectin which occur in connective tissues as well as in plasma, or that cleavage products of tissue structural glycoproteins occur in plasma which cross-react with anti-plasma fibronectin.
Kinetic analysis of compound I formation and the catalatic activity of chloroperoxidase
Authors: Araiso Tsunehisa , Rutter Rick , Palcic Monica M. , Hager Lowell P. , Dunford H. Brian
Published: December 2009

When the only substrate added to a solution of chloroperoxidase is a hydroperoxide, the reactions are: ferric enzyme + ROOH → compound I + ROH and compound I + ROOH → ferric enzyme + O2 + ROH. When H2O2 is used as substrate, the rate constants for the formation and catalatic decomposition of compound 1 are 2.4 × 106 M−1∙s−1 and 3.4 × 105 M−1∙s−1 at pH 4.7 and it is predicted that a maximum of 87% of the enzyme converts to compound I in the steady state of the catalatic reaction. With methyl hydroperoxide, formation of compound I has a rate constant of 4.7 × 105 M−1∙s−1 and its decomposition 2.9 × 104 M−1∙s−1. When peracetic acid is used, compound I is formed with a rate constant of 3.8 × 106 M−1∙s−1 and a 100% yield of compound I is obtained.
A structural difference between the β-chains in hexosaminidase B and hexosaminidase A
Authors: Mahuran D. , Lowden J. A.
Published: December 2009

Hexosaminidase (Hex) BA was prepared from purified placental Hex A by treatment with merthiolate and rechromatography on DEAE Sepharose 6B-CL. The heat stability, isoelectric focussing pattern, and peptide map of this isoenzyme were compared with those of naturally occurring purified placental Hex B. Hex BA proved to be less heat stable at 60 °C, have a slightly lower pI, and have one more peptide spot than did Hex B. The lower pI of Hex BA is not caused by sialic acid residues since it was unaffected by neuraminidase treatment. The extra peptide spot in the Hex BA map cannot be explained by carbohydrate differences since no corresponding unpaired peptide spot was seen in the Hex B map. Though Hex B and BA are products of the same gene, there is evidence that both are derived from a larger precursor polypeptide chain. Our results indicate that Hex B and BA are processed differently resulting in a small peptide present in Hex BA that is not found in Hex B
Purification and partial characterization of a plasma inhibitor of tonin
Authors: Tremblay J. , Thibault G. , Gutkowska J. , Boucher R. , Genest J.
Published: December 2009

A plasma inhibitor of tonin activity in the rat, was purified by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Its purity was investigated by analytical electrophoresis on polyacrylamide gel and by ultracentrifugation sedimentation velocity. The molecular weight (360 000) of the purified inhibitor was determined by sodium dodecyl sulfate electrophoresis and its isoelectric point (4.5) by gel isoelectrofocusing. The Stokes radius (640 nm) was evaluated by gel filtration studies and a frictional ratio (f/f0) of 1.95 was calculated from the molecular weight and Stokes radius.Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by the purified inhibitor was noncompetitive and does not exceed 70%. Electrophoresis showed the same mobility for [125I]tonin bound to plasma proteins and for [125I]tonin bound to the purified inhibitor. The inhibitor may be a protein resembling half of the dimeric protease inhibitor rat α1-macroglobulin or human α2-macroglobulin
The in vivo effects of 3-deoxy-3-fluoro-D-glucose metabolism on respiration in Locusta migratoria
Authors: Romaschin A. , Taylor N. F.
Published: December 2009

The basis of the toxicity of 3-deoxy-3-fluoro-D-glucose (3FG) in adult Locusta migratoria is examined in vivo by a radiorespirometric analysis of 14CO2 from the locust after injections of 3FG prior to injections of D-[1-14C]glucose, D-[6-14C]glucose, or [1-14C]acetate. The results indicate that 3FG metabolism irreversibly inhibits glycolysis and not the hexose monophosphate pathway or the tricarboxylic acid cycle. It is also established that during metabolism of 3FG fluoride ion is released. Evidence for the metabolism of 3FG in the whole insect as far as triosephosphate isomerase is based on 3H2O release after injections of D-[3-3H]3FG. Further support for the metabolism of 3FG to fluorinated sugar phosphates is provided by chromatographic and 19F NMR analysis of 3FG poisoned locust tissue extracts. Based on these results a biochemical mode of toxicity of 3FG in locusts is discussed.
Binding of protoporphyrin to melanin and oxidation–reduction properties of melanin–protoporphyrin complex
Authors: Persad S. , Haberman H. F. , Menon I. A.
Published: December 2009

The binding of protoporphyrin to melanin and the effect of the binding of protoporphyrin to melanin on the oxidation–reduction properties of melanin were studied. Various concentrations of protoporphyrin were incubated with melanin suspensions. The amounts of protoporphyrin remaining in the supernatant after sedimentation of the melanin were determined. The amount of bound protoporphyrin was determined by extraction with 3 M HCl. The results showed that when the initial concentration of protoporphyrin was varied, the amounts of protoporphyrin bound were in accordance with Langmuir's isotherm. From these results it was calculated that at infinite concentration of protoporphyrin 7.62 ± 0.38 μg protoporphyrin would be bound to 1 mg melanin. The activity to catalyze the coupled oxidation of NADH and reduction of ferricyanide was less in the case of the melanin–protoporphyrin complex than the free melanin. It is suggested that the binding of a naturally occurring photosensitizer such as protoporphyrin to melanin may have significant biological effects viz (i) photosensitivity due to protoporphyrin, (ii) the chemical reactivity of melanin in internal organs, and (iii) protective action of melanin in skin against ultraviolet radiation.
Extracellular alkaline phosphatase from alveolar secretions of patients with pulmonary alveolar proteinosis
Authors: Nadeau Denis , Reasor Mark J. , Hook Gary E. R.
Published: December 2009

Alkaline phosphatases in alveolar secretions from the lungs of patients with pulmonary alveolar proteinosis have been studied. A soluble form of alkaline phosphatase was isolated from the secretions and characterized. The extracellular enzyme had a pH optimum at 9.95; was stimulated by Mg2+, Ni2+, and Mn2+; was inhibited by Zn2+, Be2+, Cu2+, and low concentrations (8 mM) of L-homoarginine and imidazole; and was heat-stable at 55 °C. The soluble phosphatase existed primarily as a high molecular weight complex (excluded from Sepharose 4B) and could be dispersed into low molecular weight forms (205 000 – 285 000) by treatment with n-butanol. Following butanol treatment, the thermostability of the enzyme was markedly decreased but the kinetic properties such as the Km values, activation energies, and responses to various inhibitors were unchanged.The alkaline phosphatase may originate from unusual type 2 cells present in the alveoli of patients with pulmonary alveolar proteinosis.
Inhibition of anterior pituitary prostaglandin-stimulated adenylyl cyclase activity by somatostatin
Authors: Rouleau Dominique , Barden Nicholas
Published: December 2009

Rat anterior pituitary gland adenylyl cyclase activity is stimulated by prostaglandins with an order of potency of [Formula: see text] which is the same for cAMP accumulation and growth hormone release. Somatostatin inhibited prostaglandin-stimulated adenylyl cyclase with an ID50 of 1.48 × 10−8 ± 0.27 × 10−8 M. GTP also stimulated adenylyl cyclase activity at concentrations greater than 10−6 M but did not prevent inhibition by somatostatin. These results indicate that at least one action of somatostatin is exerted directly on cAMP formation.