bluuebunny/crossref_metadata_2025_split huggingface dataset:
Relations between Structure and Function in Cytoplasmic Membrane Vesicles Isolated from an Escherichia coli Fatty‐Acid Auxotroph
Authors: SHECHTER Emanuel , LETELLIER Lucienne , GULIK‐KRZYWICKI Tadeusz 
Published: March 2005

The results presented in this paper establish relationships between structural, morphological and functional properties of cytoplasmic membrane vesicles isolated from an Escherichia coli unsaturated fatty acid auxotroph. The membranes were isolated from cells grown in the presence of either oleic, linoleic, linolenic or elaidic acids.High‐angle X‐ray diffraction studies show that the order‐disorder transitions induced by temperature variations and associated with the paraffin chains of the lipids are a function of the fatty acid composition of the membranes. In some cases “cocrystallization” of various lipid species takes place within a single type of ordered domains. In other cases there is segregation of various lipid species into more than one distinct type of ordered domain.The various order‐disorder transitions observed induce morphological changes in the hydrophobic core of the membranes which can be detected by freeze‐etch electron microscopy. A random distribution of particles on the fracture faces is observed when the paraffin chains of the lipids are disordered. Upon ordering of the paraffin chains, particles are excluded from the ordered domains and as a consequence, smooth areas and areas with densely packed particles are observed. The ratio of the smooth surface to particulated surface is proportional to the amount of ordered paraffin chains present. Moreover, the size of the smooth domains is a function of the fatty acid composition of the membranes.Discontinuities in the rate of d‐lactate‐dependent proline uptake as a function of temperature correlate with the order‐disorder transitions observed. The high energies of activation at low temperatures are attributed to decreased mobility of the carrier proteins upon aggregation. In contrast, phosphoenolpyruvate‐dependent vectorial phosphorylation does not respond to the ordering of the paraffin chains.
The Effect of Inhibitors of Protein Synthesis on Cholesterol Side‐Chain Cleavage in the Mitochondria of Luteinized Rat Ovaries
Authors: ARTHUR John R. , BOYD George S. 
Published: March 2005

Conversion of cholesterol to pregnenolone, termed cholesterol side‐chain cleavage, has been studied in isolated mitochondria from luteinized rat ovaries.
Cholesterol in these mitochondria in the presence of cyanoketone is converted to one product pregnenolone on addition of a suitable electron donor. The percentage conversion of total mitochondrial cholesterol to pregnenolone is comparable to that measured by the percentage conversion of [4‐14C]cholesterol to [4‐14C]pregnenolone when the label has been preincubated at 29°C for 10 min with the mitochondria.
In mitochondrial incubations the depletion of endogenous cholesterol follows the production of pregnenolone. The initial rate of production of pregnenolone (0.8 nmol × mg protein−1× min−1) was maintained in incubations for 5–6 min. Addition of free cholesterol to incubations allowed the production of pregnenolone to continue at the initial rate for at least 30 min.
Treatment of rats with cycloheximide 20 min prior to killing, caused a 58% inhibition of luteal mitochondrial cholesterol side‐chain cleavage and a small but significant rise in the cholesterol content of isolated mitochondria. The cycloheximide treatment did not affect the total luteal mitochondrial cytochrome P‐450 but caused a 56% decrease in the inverted type I pregnenolone‐binding spectrum, suggesting cholesterol binding to cytochrome P‐450 had been reduced.
Rats treated with chloramphenicol 3 and 10 h before killing exhibited no differences in total luteal mitochondrial cytochrome P‐450 or mitochondrial cholesterol metabolism when contrasted with saline‐injected animals.
Puromycin was shown to bind to the rat luteal mitochondrial cytochrome P‐450 to produce a type I1 difference spectrum and to inhibit luteal mitochondrial cholesterol side‐chain cleavage in vitro.
The results are discussed in terms of a protein which is rapidly turning over and which may be synthesised extra‐mitochondrially and in some way affect the binding of cholesterol to luteal mitochondrial cytochrome P‐450.
New Inhibitors of RNA Synthesis in vitro Isolated from Escherichia coli
Authors: KAGAWA Haruo , EGBERTS Egbert 
Published: March 2005

Three inhibitor proteins (I1, I2, and I3) were isolated from Escherichia coli. They depress the transcription of E. coli and T3 phage DNA by DNA‐dependent RNA polymerase from E. coli and show the following characteristics.

The inhibitors I1 and I3 consist of subunits; I2, behaves as a single protein in dodecylsulfate‐polyacrylamide gel electrophoresis.
Neither the initiation of the transcription of E. coli DNA nor the size of the RNA synthesized is influenced by the inhibitors.
In an E. coli DNA‐dependent system reinitiation of RNA synthesis at high salt concentration seems to be blocked by the inhibitors.
When inactivated, the inhibitors stimulate RNA synthesis and behave like termination factors giving rise to the production of RNA molecules of discrete size, shorter than those of control experiments.
It is suggested by hybridization competition experiments that the inhibitors I1 and I3 preferentially inhibit ribosomal RNA synthesis.
The inhibitors also depress the transcription of T3 phage DNA by E. coli RNA polymerase resulting in the synthesis of discrete RNA species smaller than those of control experiments without KCl as well as with 0.16 M KCl.
Phospholipid Metabolism in Embryonic Rat Fibroblasts following Stimulation by a Combination of the Serum Proteins S1 and S2
Authors: HOFFMANN Rainer , RISTOW Hans‐Jürgen , PACHOWSKY Heinz , FRANK Werner 
Published: March 2005

Phospholipid metabolism has been investigated in embryonic rat fibroblasts which have been incubated in serum‐free medium and subsequently stimulated by the growth‐stimulating serum proteins S1 and S2:

Incorporation of 32P into phosphatidylinositol iancreases rapidly within a few minutes of stimulation; after 30 min it is 7.4‐fold higher in stimulated cells when compared with the controls. Phosphatidylcholine synthesis and uptake of 32P into the acid‐soluble pool is not significantly enhanced during this time. Labelling of phosphatidylserine, phosphatidylethanolamine and sphingomyelin during this period is just above the background level.
Incorporation of 32P into phosphatidylinositol and phosphatidylethanolamine is strongly inhibited in the presence of dibutyryl‐adenosine 3′: 5′‐monophosphate and theophylline whereas the metabolism of phosphatidylcholine is not influenced.As demonstrated with [3H] inositol, the inhibition of phosphatidylinositol synthesis is restricted to dibutyryladenosine 3′:5′‐monophosphate; adenosine 3′:5′‐monophosphate; guanosine 3′:5′‐monophosphate and dibutyryl guanosine 3′:5′‐monophosphate are ineffective.
The growth‐stimulating serum factors must be present continuously for activation of phosphatidylinositol synthesis; within 1 h after serum deprivation the incorporation of 32P into phosphatidylinositol decreases to about 20% of the original value; in contrast, phosphatidylcholine biosynthesis is not influenced significantly during this time period.
Influence of Calf Serum on the Transport of 2‐Aminoisobutyric Acid in Embryonic Rat Fibroblasts
Authors: FRANK Werner , RISTOW Hans‐Jürgen 
Published: March 2005

Transport of 2‐aminoisobutyric acid in embryonic rat fibroblasts is mediated by a carrier system, Km, app.= 8 × 10−3 M. It was also shown by competition experiments with naturally occurring amino acids that the alanine carrier is responsible for 2‐aminoisobutyric transportation.With cells incubated for 24 h in serum‐free medium, a final Km, app. cannot be demonstrated indicating that under these conditions uptake occurs only by diffusion. After stimulation of the cells by the growth‐stimulating serum factors S1 and S2 no increase in accumulation of 2‐aminoisobutyric acid was found during the first hour, but a Km, app. value can be measured after about 2 h.Efflux of 2‐aminoisobutyric acid is also brought about by an active mechanism which is strongly dependent on the temperature and potassium concentration of the culture medium, and which is also influenced by ouabain.In the permanently growing transformed rat cell lines which have been examined, transport of 2‐aminoisobutyric acid appears to be independent of the presence of serum factors, nearly identical Km, app. values being found within cells grown with and without serum.
Polyol Metabolism in Monkey‐Kidney Epithelial‐Cell Cultures
Authors: HUTTON John C. , WILLIAMS John F. , SCHOFIELD Philip J. , HOLLOWS Frederick C. 
Published: March 2005

An investigation of polyol synthesis by monolayer cultures of monkey kidney epithelium cells was undertaken as a consequence of the observation that these cells accumulated high concentrations of acyclic hexitols (200–600 μmol/g protein) when incubated in media containing elevated glucose [590 mg‐% (w/v)] concentrations.
It was established that the acyclic hexitol which accumulated when monkey kidney epithelial cell cultures were incubated in high glucose medium was sorbitol. The involvement of the polyol pathway of metabolism in this phenomenon was implicated from radioactivity incorporation experiments using [U‐14C]glucose substrate (the specific radioactivity of the sorbitol product was 92 % of the specific radioactivity of the added glucose) and from studies involving the use of the aldose reductase inhibitor 3,3′‐tetramethylene glutaric acid, which caused marked inhibition of polyol synthesis.
The intracellular glucose concentration was found to be an important factor regulating the steady state concentrations of the pathway intermediates sorbitol and fructose in monkey kidney epithelial cell cultures. Changes in the oxygen tension in the incubation medium or the presence of the hormones vasopressin, insulin or epinephrine were shown not to affect the concentrations of sorbitol or fructose in monkey kidney epithelial cell cultures incubated in media containing a high glucose concentration.
Cultures of HeLa Cells, goldfish testes fibroblasts, or ascites tumor cells did not exhibit the same capacity as monkey kidney epithelial cell cultures for sorbitol synthesis. To our knowledge, the present findings constitute the first reported example of polyol pathway activity in cells in monolayer culture.
The Interaction of Liver Phosphorylase a with Glucose and AMP
Authors: STALMANS Willy , LALOUX Monique , HERS Henri‐Géry 
Published: March 2005

The effects of glucose and of AMP on several properties of purified dog liver phosphorylase a have been investigated.Glucose stimulated several‐fold the conversion of phosphorylase a into b, catalyzed by purified phosphorylase phosphatase. The reaction was strongly inhibited by AMP in the absence of glucose, and much less in the presence of glucose. Conversely, the concentration of glucose that gave half‐maximal stimulation was increased several‐fold by the nucleotide. Similar effects were observed when the phosphorylase phosphatase was studied in crude liver preparations. 1,5‐Anhydroglucitol, α‐methylglucoside and 2‐deoxyglucose, quoted in order of decreasing efficiency, also stimulated the phosphorylase phosphatase reaction. Similar results were obtained when the conversion of phosphorylase a into b′ by trypsin was studied.The effect of glucose on the kinetics of purified liver phosphorylase a was mostly to decrease affinity for glucose 1‐phosphate and to increase cooperativity between glucose 1‐phosphate binding sites. AMP had the opposite effect and each of the two ligands antagonized the binding of the other. Glucose derivatives, in the order of efficiency given above, mimicked the effects of glucose. Glucose and the same glucose analogues, as well as AMP, protected phosphorylase a against denaturation at 55°C.These results indicate that the stimulation of the phosphorylase phosphatase reaction by glucose is secondary to the binding of the hexose to phosphorylase a, and that this binding is modulated by the concentration of AMP. The physiological implication of these findings is discussed.
Studies of Glutamate Dehydrogenase
Authors: GAUPER Friedrich Peter , MARKAU Klaus , SUND Horst 
Published: March 2005

The association‐dissociation equilibrium of beef‐liver glutamate dehydrogenase was investigated in phosphate buffer of different ionic strengths and in the presence of the organic solvents ethylene glycol and dioxane. For some of the solvents the temperature dependence of the equilibrium constant was analyzed. The dependence of the apparent weight‐average molecular weight on enzyme concentration shows always the same general shape as that found earlier in 0.067 M phosphate buffer at pH 7.6. Each curve can be fitted assuming an open association‐dissociation equilibrium with one single equilibrium constant for all steps and with one single value for the second virial coefficient. Within the limit of the experimental error, the second virial coefficient is found to be the same for all systems (8–9 nmol × 1 × g‐2), whereas the equilibrium constants vary by orders of magnitude.At low ionic strength a very small temperature dependence was found with a reduced association at higher temperatures. At high ionic strength a temperature maximum of the equilibrium constant is observed. Preferential phosphate binding is indicated by the high values of the refractive index increment.The organic solvents investigated here (ethylene glycol, dioxane) cause a dissociation into the unimers (oligomers). The equilibrium constant decreases from 9 × 105 M‐1 in aqueous phosphate buffer to 5.2 × 103 M−1 in 6 M ethylene glycol and to 2.4 × 105 M−1 in 2 M ethylene glycol. The effects obtained with 5% dioxane (0.57 M) are similar to that with 2 M ethylene glycol. In both cases a sharp decrease of the association with increasing temperature is observed.The reaction enthalpy and the reaction entropy of the association reaction decrease with decreasing temperature in all solvents, the enthalpy being positive in the absence and negative in the presence of organic solvents. The free enthalpy of reaction, however, increases with increasing temperature in buffers of high and low ionic strengths but decreases in the presence of organic solvents. The association reaction at high ionic strength appears mainly to be entropy‐driven whereas in the presence of the organic solvents the reaction seems to be energy‐driven at all temperatures. It is suggested that the organic solvents interfere with the hydrophobic interactions between the unimers. The positive reaction enthalpy in the presence of high salt concentrations indicates that the attractive electrostatic forces are largely shielded, whereas shielding is imperfect for the repulsive forces which even seem to be enhanced. This could be due to a preferential binding of anions which would tend to increase the negative net charge and could explain the difference observed between the effects of phosphate and chloride solutions of identical ionic strength.
Purification and Specificity of Aspergillus sojae DNase
Authors: SUZUKI Masaru , SAKAGUCHI Kenji 
Published: March 2005

An endo‐type deoxyribonuclease isolated from autolysate of Aspergillus sojae was purified 175‐fold. The purified enzyme migrated as a single band during electrophoresis on polyacrylamide gel and hydrolysed only deoxyribonucleic acids. Optimal hydrolysis of DNA by the enzyme was observed at pH 9.4. The enzyme was strongly activated by Mg2+. Mn2+ and Zn2+ also enhanced the enzymatic activity, but to a lesser extent. Heat‐denatured DNA was found to be a better substrate than native DNA. The molecular weight of the enzyme determined by gel filtration on Sephadex G‐100 was 15600.A limited amount of the enzyme preferentially splits the phosphodiester bonds of purine nucleotides in DNA. The mononucleotides liberated are P‐5′‐dGuo (major) and P‐5′‐dAdo (minor). The oligonucleotides are terminated mostly either with P‐5′‐dGuo or P‐5′‐dAdo and partly with P‐5′‐dCyd at the 5′‐phosphate termini, and with P‐5′‐dGuo and P‐5′‐dAdo at the 3′‐hydroxyl termini, respectively.
Structure and Synthesis of a Lipid‐Containing Bacteriophage Preparation of Virus and Localization of the Structural Proteins
Authors: HINNEN Rosmarie , SCHÄFER Rolf , FRANKLIN Richard M. 
Published: March 2005

The four structural proteins of bacteriophage PM2 have been related to the morphological structures in the virion. Protein I can be selectively removed from the virus by degradation with bromelain, a proteolytic enzyme. From electron microscopic studies and from experiments showing that the virus cannot attach to its host cell after bromelain treatment, we conclude that protein I forms the outer spikes at the vertices of the icosahedron. Protein II can be labeled with [35S]sulfanilic acid diazonium salt. As shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, proteins I and II can be removed if the virus is treated with 1 M urea. From the above studies and from packing considerations, we conclude that protein II forms the outer shell of the virus.The dissociation of the virus in 1 M urea leads to: (a) a nucleocapsid core containing viral lipid, DNA, protein III and IV, (b) a second fraction which consists of the same elements obtained by treatment of the virus with bromelain. Both particles have been identified by electron microscopy. The dissociation of the virus in 1 M urea plus 0.5% Triton X‐100, a nonionic detergent, leads to a third fraction representing an empty shell which contains all of the lipid, proteins II and III, and about 50% of protein IV, but no DNA. We have also been able to isolate a second type of nucleocapsid core by dissociation of the virus in the presence of 4.5 M urea. This core, which has an approximate s20,w, = 133 S and which has been identified in the electron microscope as a core with icosahedral shape, contains proteins III and IV, as well as the DNA, but not phospholipid. In the presence of 4.5 M urea this core is resistant to Triton X‐100 concentrations up to 0.1%. Higher concentrations of the nonionic detergent dissociate the core into DNA and the solubilized proteins 111 and IV. In the absence or in the presence of low concentrations of urea (1 M) the core is resistant to Triton X‐100 concentrations up to 0.5%. The DNA in the core is digestible by pancreatic DNAase I and the core structure is broken down after DNAase treatment. Protein IV interacts with the DNA as shown by transformation of non‐covalent interactions between DNA and protein into covalent bonds induced by a nucleophilic reagent. In conclusion, protein I forms the spikes, protein II the outer icosahedral shell and protein III the inner icosahedral shell around the DNA, which is closely associated with protein IV. The phospholipid bilayer is sandwiched between these two shells. Protein III may also form the protein bridges to protein I which connect the inner and outer shells through the bilayer.
New Sialic Acids
Authors: BUSCHER Hans‐Peter , CASALS‐STENZEL Jorge , SCHAUER Roland 
Published: March 2005

Sialic acids were released by dilute acids or by neuraminidase from glycoproteins of bovine and equine submandibular glands, from glycoproteins of equine blood plasma and from equine erythrocyte membranes. They were purified by ion‐exchange chromatography and fractionated on cellulose.
The following methods have been adapted to the structural analysis of sialic acids with regard to the nature and position of the N‐acyl and O‐acyl groups.a) Thin‐layer chromatography in two dimensions with intermediate ammonia treatment to remove the O‐acyl groups leading to the identification of the basic N‐acylneuraminic acids.b) Thin‐layer chromatography in two dimensions with intermediate hydroxylamine treatment allowing the identification of the O‐acyl groups of sialic acids as hydroxamates.c) Gas‐liquid chromatography for identification of the different N‐acylated and O‐acylated sialic acids.
The structure of the following new sialic acids have been established:a) N‐glycoloyl‐8‐O‐acetylneuraminic acid isolated from glycoproteins of bovine submandibular glands;b) N‐acetyl‐4‐O‐glycoloylneuraminic acid isolated from glycoproteins of equine submandibular glands and also occurring in equine blood plasma and equine erythrocyte membranes;c) N‐acetyl‐7 or 8‐O‐glycoloylneuraminic acid detected in glycoproteins from bovine submandibular glands.
The O‐acetylated N‐glycoloylneuraminic acid first detected in horse erythrocyte membranes by Hakomori and Saito and in horse serum by Pepper, was determined to be N‐glycoloyl‐4‐O‐acetyl‐neuraminic acid; it was also found to occur in submandibular glands of horse.
The sialic acid patterns from the different biological materials investigated are compared.
Isolation and Characterization of α‐Galactosidase from Lens esculanta
Authors: DEY Prakash M. , WALLENFELS Kurt 
Published: March 2005

α‐Galactosidase from Lens esculanta (red lentils) was purified 4090‐fold through a multistep process involving acidification, ammonium sulphate fractionation, Sephadex gel filtration, DEAE‐cellulose and CM‐cellulose chromatography. The purified enzyme is stable at 4°C for nearly six months with only a small loss of activity. It showed a pH optimum of 5.5 with raffinose as the substrate and 6.0 with p‐nitrophenyl‐α‐d‐galactoside. The Km for the former substrate was 40 mM and for the latter, 0.26 mM.The enzyme failed to liberate galactose from guar gum, tara gum and locust bean gum galactomannans. A study of the hydrolyzability of α‐d‐galactoside, α‐d‐fucoside and β‐l‐arabinoside showed that the presence of primary alcoholic group in the sugar was important for faster hydrolysis and a stronger binding. The substrate specificity studies indicated the absence of any related glycosidase activity in the α‐galactosidase preparation.
Substituent Effects on the Pre‐Steady‐State Kinetics of Oxidation of Benzyl Alcohols by Liver Alcohol Dehydrogenase
Authors: HARDMAN Michael J. , BLACKWELL Leonard F. , BOSWELL Colin R. , BUCKLEY Paul D. 
Published: March 2005

The oxidation of benzyl alcohols by liver alcohol dehydrogenase under conditions [S]0≫ [E]0 is a biphasic process with exponential rise to a steady state. The rate constants for the transient phase were determined by a curve‐fitting procedure.
A large isotope effect was obtained for p‐methoxybenzyl alcohol, indicating that hydrogen transfer is rate‐determining for the transient phase. Rate constants for the hydrogen transfer step, k3, were obtained for the various alcohols and correlated with the Hammett σ constant by plotting log k3 against σ. The slope of this line (ϱ=–0.76) is consistent with rate‐limiting hydride transfer; a mechanism is proposed to account for the low magnitude of ϱ.
The pre‐steady‐state rate constants, kb, for all benzyl alcohols (< 20 s−1) are lower than that for ethanol. 2‐Methylpropan‐1‐ol and cyclohexylmethanol have pre‐steady‐state rate constants of about 150 s−1, indicating that the lower kb values for benzyl alcohols are due to the electron‐withdrawing effect of the benzene ring, rather than to steric effects.