bluuebunny/crossref_metadata_2025 huggingface dataset:
Soybean Lipoxygenase
Authors: ALLEN J. C.
Published: March 2005

The reliability of the assay of lipoxygenase has been improved by minimising sources of error, such as the adherence of the enzyme to the glass of the containing vessel.

The electrophoretic method of preparing crystalline lipoxygenase has been improved, and a new chromatographic purification has been developed.

The shape of the pH‐activity profile of the enzyme has been shown to depend critically upon the amount of organic solvent present in the solution, as well as upon its ionic strength. These effects are discussed, and an attempt has been made to fit theoretical curves to the pH‐profiles in solutions containing no organic solvents. The dissociation constants for the ionisable groups on the enzyme extracted from these data are pKa= 6.5‐6.8 and pKb= 9.0‐9.1, in 0.2 M Tris.

Micelle formation has little effect on the Km of the enzyme. However, substrate concentration vs. activity curves show perturbations at the critical miselle concentration in certain instances, and a possible relationship between the critical micelle concentration and a substrate concentration derived by extrapolation of the activity curves is presented.
Studies on Methionyl Transfer RNA Synthetase
Authors: LEMOINE F. , WALLER J.‐P. , van RAPENBUSCH R.
Published: March 2005

Methionyl transferRNA (tRNA) synthetase fromEscherichia colistrain K‐12 was purified 675 fold from extracts of kilogram quantities of cell paste as a product that appears to be homogenous on chromatography, electrophoresis and ultracentrifugation. The activities measured by the ATP‐PPi exchange assay and by methionyl tRNA formation did not vary significantly during the purification. The final product, which catalyzed the exchange of 1,000 μmoles of pyrophosphate into ATP/mg protein in 15 min at 37°, was obtained with a yield of 50%.

Some general properties of purified methionyl tRNA synthetase are described. The enzyme has a sedimentation coefficient (s20,w) of 6.5 S, and a molecular weight of 173,000. In the presence of dissociating reagents (8M urea or 6M guanidine) it undergoes dissociation into subunits which have a molecular weight of about 43,000. Within the limits of the accuracy of the ultracentrifugation analyses, there was no indication of heterogeneity in mass among these subunits. Furthermore, acrylamide gel electrophoresis in the presence of 8 M urea failed to reveal any significant heterogeneity of the dissociated enzyme. The results indicate that methionyl tRNA synthetase fromE. coliK‐12 is composed of four, possibly identical, subunits.
The Influence of Thyroid Hormones on β‐Hydroxy‐β‐Methylglutaryl‐Coenzyme A Reductase of Rat Liver
Authors: GUDER W. , NOLTE I. , WIELAND O.
Published: March 2005

Rat liver β‐hydroxy‐β‐methylglutaryl‐CoA reductase has been measured by an isotopic assay system. In normal rats, an activity of about 70 nmoles/g liver · hour was found.

Thyroidectomy lowers this rate to less than 50%. Injection of 3,3′,5′‐tri‐iodothyronine into hypothyroid rats brings the activity up to the range of normal animals after a latent period of about 30 hours.
Studies on the Metabolism of C19‐Steroids in Rat Liver
Authors: LISBOA B. P. , GUSTAFSSON J.‐Å. , SJÖVALL J.
Published: March 2005

The formation of polar C19‐steroids with a 3‐oxo‐Δ4‐structure after incubation of testosterone with 105,000 ×g microsomes of adult male rat liver was investigated. The ultraviolet‐positive metabolites were isolated by thin‐layer chromatography. The isolated compounds were identified as 2β‐, 6β‐, 7α‐, and 16α‐hydroxytestosterone by means of gas‐liquid chromatography‐mass spectrometry.

A dihydroxytesterone was also isolated and tentatively identified as 6β,16α,17β‐tri‐hydroxyandrost‐4‐en‐3‐one.
Properties of Aqueous Dispersions of Phospholipid and Cholesterol
Authors: BRUCKDORFER K. R. , EDWARDS P. A. , GREEN C.
Published: March 2005

Very little lipid can be extracted with diethyl ether or chloroform from aqueous dispersions of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid, or from mixed dispersions of phosphatidic acid and phosphatidylethanolamine with cholesterol. Nearly all of the cholesterol and about 60% of the phosphatidylcholine can be removed from a dispersion of these two lipids in equimolar proportions but not from a dispersion of two molecules of phosphatidylcholine to one molécule of cholesterol.

NaCl and CaCl2 greatly increase the extraction of lipids from phosphatidylcholine and phosphatidylcholine/cholesterol dispersions whereas urea and dimethylsulphoxide prevent extraction.

Cholesterol molecules readily exchange between rat erythrocytes and dispersions of equimolar proportions of phosphatidylcholine and cholesterol.

At lower proportions of cholesterol, the dispersions remove cholesterol from the ghosts. Up to 60% of the sterol can be removed from the ghost membrane.

Phosphatidylcholine/cholesterol dispersions are readily attacked by phospholipase D in the absence of ether but dispersions of phosphatidylcholine alone need ether present to facilitate enzymic attack.

These properties are compared with those of soluble and structural lipoproteins.
The Incorporation of Steroid Molecules into Lecithin Sols, β‐Lipoproteins and Cellular Membranes
Authors: BRUCKDORFER K. R. , GRAHAM J. M. , GREEN C.
Published: March 2005

The dispersion of various steroids in aqueous solution with ovolecithin was investigated and the following molar ratios of lecithin to steroid obtained: cholesterol, 1:1; 7‐dehydrocholesterol, 13.3:1; ergosterol, 13.1:1; sitosterol, 2.0:1; sterophenol 4.2:1; cholesta‐4,6‐dien‐3‐one, 2.0:1; cholest‐4‐en‐3‐one, 1.7:1.

The cholesterol contents of sterol‐depleted cellular membranes can be restored to their original level by incubation with plasma or an equimolar dispersion of cholesterol and ovolecithin, in the presence of 10% dimethylsulphoxide (v/v).

Cholesta‐4,6‐dien‐3‐one, cholest‐4‐en‐3‐one, 7‐dehydrocholesterol and sterophenol can exchange readily with the cholesterol erythrocyte ghosts and can replace cholesterol molecules removed from the membrane. Ergosterol can do so only to a very limited extent.

7‐Dehydrocholesterol exchanges between rat erythrocyte ghosts and human piasma β‐lipo‐proteins in the same way as cholesterol. Cholesta‐4, 6‐dien‐3‐one and cholest‐4‐en‐3‐one also exchange but, in addition, there is a net uptake of these steroids by β‐lipoproteins. The extra steroid entering the lipoproteins is probably dissolved in the hydrophobic core of the latter.

The exchange of cholesta‐4,6‐dien‐3‐one between erythocyte ghosts and lecithin/steroid dispersions or β‐lipoproteins is much more rapid than that of cholesterol. The rate of exchange of the dienone, like that of cholesterol, is increased by the addition of a small quantity of dismethylsulphoxide to the medium.
On Collagens of Invertebrates with Special Reference to Mytilus edulis
Authors: PIKKARAINEN J. , RANTANEN J. , VASTAMÄKI M. , LAMPLIAHO K. , KARI A. , KULONEN E.
Published: March 2005

The amino acid compositions of Metridium‐, Lumbricus‐ and Mytilus‐collagens corresponded to those of the lowest vertebrates: the content of imino acids was low and that of serine and threonine high. Metridium‐collagen also contained cystine and large amounts of hydroxylysine. In Lumbricus‐collagen the hydroxylation of proline was almost complete and the total content of hydroxy amino acids thus exceptionally high.

The solubility of collagens from the invertebrates varied in wide range. In some cases, e. g. the byssus apparatus of Mytilus, the insolubility seems to depend on the stabilizing effect of the adjacent materials which is manifested also in the unique thermal shrinking temperature of 90°.

The starch‐gel electrophoretic patterns were studied on soluble collagens from the following species: octopus, Loligo, Lumbricus, Diphyllobothrium, Mytilus, and Metridium. A single α‐component pattern, resembling that from lamprey, was observed in all the cases.
Incorporation of Choline into the Outer and Inner Membranes of Isolated Rat Liver Mitochondria
Authors: KAISER W. , BYGRAVE F. L.
Published: March 2005

Separation of the inner and outer membranes following incubation of mitochondria with 14C‐labelled choline chloride revealed the specific radioactivity both per mg protein and per μg phospholipid phosphorus to be greater in the outer membrane. Under the present conditions of incubation, the incorporation into the microsome fraction was less than 10% (on a mg protein basis) of that into the isolated mitochondria.

Labelled choline is also incorporated into the membrane components of isolated mitochondrial fractions, but to a lesser extent than that incorporated into the membranes of intact mitochondria, and in different proportions.

The phospholipid phosphorus contents of the “pure” outer and “pure” inner membranes of isolated rat liver mitochondria are respectively about 18.3 and 6.1 μg phosphorus per mg protein.

The data are discussed with particular refernce to the biosynthesis of the mitochondrial membranes and to the biogenesis of mitochondria.
2‐Amino‐2, 6‐Dideoxy‐L‐Mannose (L‐Rhamnosamine) Isolated from the Lipopolysaccharide of Escherichia coli 03: K2ab(L):H2
Authors: Jaan B. , Jann K.
Published: March 2005

From the lipopolysaccharide of Escherichia coli U41/14 (serotype 03:K2ab(L):H2) an unusual amino sugar was isolated.

By sequential oxidation with periodate and hypoiodite and by oxidation with periodate, followed by detection of acetaldehyde formed, the amino sugar was characterized as a 2‐amino‐dideoxyaldose.

The hydrochloride of the amino sugar, in comparison to those of 2‐amino‐2,6‐dideoxy‐D‐glucose, 2‐amino‐2,6‐dideoxy‐L‐galactose, 2‐amino‐2,6‐dideoxy‐D‐mannose, 2‐amino‐2,6‐dideoxy‐D‐allose, and 2‐amino‐2,6‐dideoxy‐D‐gluose, was studied with aid of ion exchange column chromatography. The N‐acetylated amino sugar was compared with the N‐acetylated reference amino sugars with regard to their relative mobilities in paper chromatography. The amino sugar alcohols and reference amino sugar alcohols were studied using paper electrophoresis in molybdate buffer.

On the basis of the results obtained the unknown amino sugar was identified as 2‐amino‐2,6‐dideoxymannose (rhamnosamine). Its optical rotation showed it to be of the L‐configuration.

This is the first report on the natural occurrence of L‐rhamnosamine.
Interaction of DNA with Bifunctional Aldehydes
Authors: Brooks B. R. , Klamerth O. L.
Published: March 2005

The bifunctional aldehydes glyoxal and malondialdehyde, known to be toxic products of the radiolysis of sugar and other food constituents, interact with DNA in vitro and in vivo as demonstrated by thermal denaturation profiles, chromatographic behavior, and incomplete degradation of the reaction product by deoxyribonuclease. After irradiation of DNA in diluted radioactive glucose solution followed by incubation at slightly acid pH, the radioactivity is found in the core of the enzymic digest only. Attempts to locate the binding site proved that the radiolytic compound is associated preferentially with the guanine and cytidine moiety of the DNA core.
Some Properties of Lysyl Ribonucleic Acid Synthetase from Escherichia coli
Authors: Waldenström J.
Published: March 2005

When the lysyl‐RNA synthetase from Escherichia coli was incubated with ATP and L‐lysine in the presence of Mg++, a complex of lysyl‐RNA synthetase, AMP and lysine was formed. The complex was isolated by filtration on Sephadex G‐25. Maximum complex formation was obtained at a molar ratio of substrate to enzyme between 3 and 4. The complex was very stable at low temperatures in the absence of Mg++, while incubation at 45° for 10 minutes gave 75% breakdown of the complex. The amino acid of the complex could be transferred to transferRNA in a reversible reaction that did not require Mg++. The transfer reaction was enhanced by the monovalent cations NH4+, Na+, and K+.

The lysyl‐RNA synthetase from E. coli recognized about 70% of the lysine specific chains of yeast transferRNA, while the yeast enzyme recognized about 80% of the lysine specific chains of E. coli transferRNA. The esterification of E. coli transferRNA by the yeast enzyme proceeded at only 2% of the rate obtained with homologous transferRNA, while the E. coli enzyme esterified yeast transferRNA at about 60% of the rate obtained with E. coli transferRNA.
Zum Vorkommen von Sterinen in Bakterien
Authors: Schubert K. , Rose G. , Wachtel H. , Hörhold C. , Ikekawa N.
Published: March 2005

Investigations concerning sterols were made in one of strain each of nine species of bacteria and one representative of the basidiomycetes through adsorption chromatography, gas chromatography and mass spectrometry as well as ultraviolet and infrared spectroscopy.

Three strains contained sterols in quantities ranging from 0.0004 to 0.01% of the dry weight. In Azotobacter chroococcum six sterols were demonstrated. In six strains sterols were not detectable. The limit of detectability at present is at 0.0001% sterol related to 100 g dry cells.

From the extract of Aerobacter cloacae, which contained no sterol, a C16‐hydroxy‐ketone was isolated.
Stereospecific Alkylation with Asymmetric Reagents
Authors: Wallenfels K. , Eisele B.
Published: March 2005

When undergoing nucleophilic reactions with proteins, α‐iodopropionic acid and its amide show greater specificity for SH groups than to inodoacetic acid and its amide.

The kinetics and sterochemistry of the reactions of the D(+) and L(—) antiopodes of α‐iodopropionic acid and its amide with both eysteine and papain were investigated. The antipodes of the SH reagents reacted with these asymmetric SH compound at different rates. In the reaction with papain, the L(—) antipode of the acid reacted faster than the D(+) antipode; the stereochemical preference was inverted when the amide was used, the D(+)antipode reacting faster than the L(—)antipode.

Arguing from the differing reactivities of the SH reagents and their antipodes and the pH dependence of the reaction rates, it has been suggested, that L(—)α iodopropionic acid is specifically oriented onto the reaction center through the concerted attractive and repulsive actios of a cations and an anionic group in the protein. These have been tentatively identifid as a protonated imidazole residue and a carboxylate group respectively.

Parallels between these stereospecific alkylations and the catalytic action of the enzyme are discussed.
Nucleotide Sequence Homologies in Nuclear and Cytoplasmic Ribonucleic Acid from Rat Liver and Hepatomas
Authors: Drews J. , Brawerman G. , Morris H. P.
Published: March 2005

The RNA popoulations of rat liver and three rat hepatomas were compared by DNA‐RNA hybridization studies in the presence and absence of competing RNA. The hepatoma nuclei were found to lack hybridizable RNA species present in normal liver nuclei, while the latter appeared to contain all the hepatoma RNA species. In normal liver, the cytoplasmic RNA could compete with nuclear RNA only to the extent of 20–30 %. This indicates that a large number of nuclear RNA species are not transferred to the cytoplasm. In the case of the hepatomas nearly all the nuclear RNA species were detected in the cytoplasmic RNA preparations. A more extensive homology between nuclear and cytoplasmic RNA was also observed in regerenating liver. It is suggested that a selective mechanism governs the transfer of RNA species from the nucleus to the cytoplasm and that this mechanism is altered in the hepatomas.
The Cholesterol Side Chain Cleavage Enzymes in Immature Rat Ovary
Authors: Sulimovici S. , Boyd0 G. S.
Published: March 2005

Cholesterol side chain cleavage reaction has been studied in ovaries of immature female rats pretreated with pregnant mare serum gonadotrophin and human chorionic gonadotrophin. The cleavage enzymes were found to be in the mitochondria and to have the characteristics of a mixed function oxidase requiring NADPH and oxygen. Using [4‐14C]cholesterol as substrate the main product obtained was progesterone while pregnenolone and 20α‐hydroxypregn‐4‐en‐3‐one, were formed in smaller amounts. This shows that ovarian mitochondria contains a Δ5–3β‐hydroxysteroid‐dehydrogenase, a Δ 5–6/4–5 isomerase and a 20α‐hydroxy steroid dehydrogenase. The proposed intermediates in cholesterol side chain cleavage (20α‐hydroxy cholesterol and 20α, 22ɛ‐dihydroxy cholesterol) were not detected.

Mitochondrial production of progesterone in the presence of NADPH was further stimulated by succinate while fumarate was without effect. In the presence of NADPH, NADH was found to be inhibitory while NAD+ gave rise to an increase in pregnenolone, the overall reaction being unaffected. Excess of NADPH causes an accumulation of pregnenolone and slightly decreases the overall reaction. The action of reduced glutathione (GSH) and ascorbic acid‐like reducing agents of physiological occurrence has been studied. It was found that GSH in small amounts stimulates and in larger amounts inhibits the side chain cleavage reaction. Ascorbic acid in physiological amounts produced an inhibitory effect.

The action of adenosine 3′,5′‐cyclic‐monophosphoric acid (3′5′AMP) and other nucleotides (AMP, ADP, ATP) was tested. 3′5′AMP markedly stimulates the conversion of cholesterol to pregnenolone and decreases the synthesis of progesterone. The action of AMP shows some similarities with that of 3′5′AMP but the action of ADP and ATP was found to be different.
Untersuchungen zur Induktion der Lac‐Enzyme
Authors: Boos W. , Wallenfels K.
Published: March 2005

1‐O‐β‐d‐Galactopyranosyl‐d‐glycerol was shown in a previous publication [1] to be an abnormal inducer. It has a maximal activity even at 10−7 M [2] whereby no autocatalytic phase can be observed. On addition of galactose the quoted abnormal behaviour of the inducer galactosyl glycerol is normalized. From these data it was concluded that for this particular case galactose competes with galactosyl glycerol for a lac‐operon independent permease.

We could now show that the abnormal behaviour of the inducer galactosyl glycerol also disappears when a bacterial strain is used which lacks the gen MGP for the methyl galactoside permease. This gen maps outside the lac operon of the Escherichia coli chromosome [4] and his expression is constitutive under the conditions of the experiment. In order to prove the responsibility of the methyl galactoside permease for the abnormal behaviour of galactosyl glycerol as inducer we measured the accumulation of galactosyl glycerol in uninduced cells of E. coli K 12 W 3100 and W 3092i, MGP+‐ and MGP−‐strains respectively. It could be shown that in fact the strain W 3100 accumulates galactosyl glycerol whereas the strain W 3092i does not.

Since derivatives of galactosyl glycerol are probably the most abundant galactosides in nature it seems likely that galactosyl glycerol is the main natural galactoside substrate during the evolution of the “Lac” operon.
Identification of 22‐, 24‐ and 26‐Hydroxycholesterol in the Steroid Sulphate Fraction of Faeces from Infants
Authors: Gustafsson J.‐Å. , Sjövall J.
Published: March 2005

A fraction of solvolyzable sterol conjugates with the chromatographic properties of steryl sulphates was isolated from pooled faeces collected from infants 1–4 and 6–12 months old. The major sterols in this fraction were analyzed by gas chromatography‐mass spectrometry. The pool from the younger infants was shown to contain cholesterol and 22α‐, 24χ‐, and 26‐hydroxycholesterol and the pool from the older infants contained cholesterol, campesterol, β‐sitosterol, and 22α‐, and 24χ‐hydroxycholesterol. The concentrations of cholesteryl and 24‐hydroxy‐cholesteryl sulphates were higher in faeces from the older than from the younger subjects.
Triacetic Acid Lactone, a Derailment Product of Fatty Acid Biosynthesis
Authors: Yalpani M. , Willecke K. , Lynen F.
Published: March 2005

If no NADPH is available for reduction, fatty acid biosynthesis is blocked at the stage of the acetoacetyl‐acyl carrier protein intermediate. In this case highly purified fatty acid synthetase from baker's yeast catalyzes, in a derailment reaction, the formation of triacetic acid lactone from acetyl‐ and malonyl‐CoA. The identity of the product was shown by the enzymatic incorporation of radioactivity from [1‐14C]acetyl‐CoA and from recrystallization to constant specific radioactivity. In paper chromatography, thin layer chromatography, and ionophoresis both the chemically synthesized triacetic acid lactone and the enzymatically formed compound migrated with the same RF‐values and electrophoretic mobility. By an oxidation with chromic acid according to the procedure of Kuhn‐Roth it could be demonstrated that the radioactivity of [1‐14C]acetyl‐CoA is incorporated only into the C‐6 position of triacetic acid lactone. Free triacetic acid and tetraacetic acid lactone do not seem to be formed by fatty acid synthetase. The ratio of the malonyl‐CoA utilization for the synthesis of palmitoyl‐ and stearoyl‐CoA to that for the synthesis of triacetic acid lactone was found to be about 90:1. The significance of these findings is discussed. A chemical mechanism for the formation of triacetic acid lactone by fatty acid synthetase is proposed.
Free Ribonucleotides of Physarum polycephalum
Authors: Sachsenmaier W. , Immich H. , Grunst J. , Scholz R. , Bücher Th.
Published: March 2005

Free ribonucleotides in plasmodia of Physarum polycephalum at different stages of the synchronous mitotic cycle were determined by anion exchange chromatography. The nucleotide pattern of this myxomycete resembled that obtained from rat liver. The contents of total adenine nucleotides based on protein content were almost identical in both tissues, whereas higher contents of guanine‐, uracil‐, and cytosine‐nucleotides were found in the myxomycete. During the mitotic cycle, the levels of nucleoside triphosphates increased just prior to mitosis and decreased in the following reconstruction period. It is suggested that the accumulation of triphosphates results from a diminished demand for RNA precursors due to decreasing RNA synthesis during late interphase.
Modification of Folic Acid‐Induced Changes in Renal Nucleic Acid and Protein Synthesis by Actinomycin D and Cycloheximide
Authors: Threlfall G. , Taylor D. M.
Published: March 2005

Parenteral administration of folic acid to rats produces marked increases in the rates of synthesis of protein, RNA and DNA in the kidney. Basic nuclear protein synthesis is increased within 1 hour of folate administration with a sharper increase appearing about 18 hours and reaching a maximum about 26 hours. RNA synthesis increases to reach a maximum at 12 hours after folate before declining to normal levels at 24 hours. The increase in the rate of DNA synthesis commences at about 18 hours and reaches a maximum, about 20 times above control levels, about 32 hours after folate.

Using the increased rate of DNA synthesis normally seen 26 hours after folate as a reference point, actinomycin D was found to produce a very marked depression of DNA synthesis when administered between 0 and 4 hours after folate; at longer time intervals this depression became less marked and when actinomycin D was given at 20 hours no effect was observed. Actinomycin D, given at the same time as folic acid, also abolished the increase in the rate of RNA synthesis normally seen 12 hours after folate.

In contrast to the time dependent effect of actinomycin D, cycloheximide produced a similarly profound decrease in the rate of DNA synthesis, measured 26 hours after folate, when the drug was given at any time between 0 and 25 hours after folate.

These results suggest that the complete requirement for increased RNA synthesis has been met by the time that increased DNA synthesis begins at 18 hours after folate administration, but that there is continuous need for newly synthesised protein both before and during the period of increased DNA synthesis.
Effects of Streptomycin in Bacterial Cultures Growing at Different Rates; Interaction with Bacterial Ribosomes in vivo
Authors: Kogut M. , Harris M.
Published: March 2005

Growth‐inhibitory effects of streptomycin and dihydrostreptomycin are influenced by initial growth rates of treated cultures. Growth rates were varied by medium composition and by temperature. A given change in initial growth rate brought about by temperature change produced a larger effect on the decline in growth rate under the influence of the antibiotic, than did a similar variation in initial growth rate brought about by differences in medium composition. These effects of growth conditions on the growth‐inhibitory action of the antibiotics could not be accounted for by effects on intracellular accumulation. It is concluded that interaction of intracellular streptomycin or dihydrostreptomycin with ribosomes must be restricted to a certain stage in the cycle of ribosome function. Such restriction of effective interaction to the free 30 S ribosomal subunits would fit all observations. It suggests that the antibiotic effect of streptomycin and dihydrostreptomycin may be due to combination with specific sites on the 30 S ribosomal subunits which are not accessible during peptide synthesis by the fully functional ribosome‐messenger RNA‐transfer RNA complex.
Polynucleotide Phosphorylase: Anomalous Viscosity Changes Observed during the Polymerization of Nucleoside Diphosphates
Authors: Harvey R. A. , Mclaughlin C. S. , Grunberg‐Manago M.
Published: March 2005

Reaction mixtures containing buffer, nucleoside diphosphate, magnesium ion and polynucleotide phosphorylase of Escherichia coli exhibit a high, transient viscosity during polymer synthesis. The magnitude of this viscosity depends on the reaction conditions, particularly on the concentration of enzyme, the level of Mg++, and the presence or absence of oligonucleotides with free 3′‐hydroxyl groups. The viscosity observed during polymer formation is markedly dependent on concentration, a property not shared by solutions of isolated poly U. The number average molecular weight of poly U synthesized by polynucleotide phosphorylase changes only from approximately 58,000 to 70,000 during a time interval where there is a ten‐fold change in reduced viscosity. Binding studies using Millipore filters suggest the formation of an enzyme‐polynucleotide complex. The observed transient viscosity is considered to result from either (a) the enzymic redistribution of nucleotides from extremely long polymer chains formed initially in the reaction medium, or (b) from the formation of a slowly dissociating enzyme‐multiple polynucleotide complex.
Interactions of Glucagon and Insulin on the Metabolism of Perfused Livers from Fasted Rats
Authors: Menahan L. A. , Wieland O.
Published: March 2005

Glucagon (28 nM) stimulated gluconeogenesis, urea production and ketogenesis in perfused livers from fasted rats. Since the livers were perfused without added substrate, liver protein is the source of carbon of the glucose synthesis. Addition of N6, O2′‐dibutyryl cyclic adenosine 3′:5′‐monophosphate in concentrations of 100 and 10 μM to the perfusion medium resulted in a response similar to that of glucagon. It is suggested that cyclic adenosine 3′:5′‐monophosphate is the mediator of glucagon action in liver.

Preperfusion of the isolated livers with insulin diminished the glucagon effects. The glucagon‐stimulation of glucose and urea production was completely suppressed, while the increased ketogenesis was inhibited by 60% in the presence of insulin. Since all three glucagon responses were inhibited, this supports the concept that insulin lowers, in some way, the elevation of cyclic adenosine 3′:5′‐phosphate which occurs following glucagon administration. That insulin does not exert its effect subsequent to cyclic adenosine 3′:5′‐phosphate is suggested by the observation that insulin, under the same conditions which inhibited the response of liver to glucagon, had little or no effect on the increases in gluconeogenesis, ureogenesis, and ketogenesis following, dibutyryl cyclic adenosine‐3′:5′‐phosphate. Since an effect of insulin on cyclic adenosine 3′:5′‐phosphate phosphodiesterase seems unlikely, it is suggested that the inhibition of glucagon responses by insulin is the result of an interaction at the adenyl cyclase or one of the step(s) between binding of the hormone and the enzyme.
Mise en évidence du mécanisme d'action de l'éthacrynate de sodium sur des mitochondries de foie de rat
Authors: Foucher B. , Geyssant A. , Goldschmidt D. , Gaudemer Y.
Published: March 2005

The competition of ethacrynate with phosphate shown during oxidation of succinate by rat liver mitochondria [2] is confirmed by measuring [14C]ethacrynate incorporation into mitochondria: on increasing the phosphate present during preincubation, less ethacrynate is incorporated into the mitochondria and there is a decrease in the inhibition of succinate oxidation.

Ethacrynate exhibits different effects according to the substrate used:

a) with NAD‐linked substrates, ethacrynate inhibits the oxidation, this inhibition being insensitive to phosphate and accompanied by complete oxidation of NADH to NAD+;

b) with succinate, as already mentionned, the inhibition is sensitive to phosphate; if rotenone is present during preincubation with ethacrynate, there is oxidation, but it is an uncoupled oxidation, while, with phosphate plus ethacrynate, the oxidation is still coupled. Methylene blue added during preincubation with ethacrynate and rotenone prevents the uncoupling and reestablishes the inhibition of oxidation. As with NAD‐linked substrates, the inhibition of oxidation by ethacrynate is accompanied by complete oxidation of NADH to NAD+;

c) with TMPD + ascorbate (plus rotenone), ethacrynate completely uncouples oxidative phosphorylation; again phosphate prevents this effect. Ethacrynate inhibits the [32P]Pi‐ATP exchange reaction.

The presence of Mg++ ions is required in order to get maximal effect of ethacrynate: the more Mg++ ions present, the greater is the inhibition (succinate oxidation measurement).

The results presented in this paper are discussed on the basis that the primary action of ethacrynate is a phosphate‐sensitive uncoupling action.

The fact that inhibition of NAD‐linked substrate oxidation is not sensitive to phosphate suggests that ethacrynate interferes in another way with these substrates.

However, by depleting mitochondria of energy‐rich compounds, the penetration of these substrates (assumed to be an energy requiring process) would be inhibited by ethacrynate and, consequently, the oxidation of these substrates would be impossible.

As a thiol reagent, ethacrynate is believed to react with specific proteic thiol groups implicated in the formation of an energy‐rich intermediate, and to compete with phosphate in the formation of a phosphorylated energy‐rich compound from a non‐phosphorylated energy‐rich compound.
Effect of Hyper‐ and Hypoinsulinism on the Metabolism of Ethanol in Rat Liver
Authors: Rawat A. K.
Published: March 2005

Administration of insulin to normal fed male rats resulted in a significantly increased rate of ethanol oxidation by liver slices as compared to the control animals. The rate of ethanol oxidation by liver slices from alloxan diabetic and anti‐insulin serum treated rats was significantly lower than normal.

Administration of ethanol to normal, insulin treated and alloxan diabetic animals caused a similar relative increase in the NADH/NAD ratio as measured by 4 different extra‐ and intramitochondrial redox pairs. After ethanol administration to alloxan diabetic rats, the hepatic redox state was very much reduced.

Intravenous injection of ethanol (1.5 g/kg body wt.) after 30 min resulted in significantly increased concentration of plasma free fatty acids. The ethanol induced increase in plasma free fatty acid level, like that of alloxan diabetic and anti‐insulin serum treated animals was significantly lowered following insulin injection.

The total activities of hepatic alcohol dehydrogenase and aldehyde dehydrogenase were significantly decreased, after alloxan treatment. The enzyme activities in the insulin treated group were of the same magnitude as in control animals.

Partially purified rat liver alcohol dehydrogenase and aldehyde dehydrogenase were inhibited by free fatty acids. Laurate had a stronger inhibitory effect than palmitate and oleate. The inhibition of alcohol dehydrogenase was competitive with respect to alcohol and not with respect to NAD. The inhibition of aldehyde dehydrogenase was competitive with respect to aldehyde and not NAD.

Addition of laurate (0.8 mM) to liver slices caused a significant decrease in the ethanol oxidation rate.
Translocation of some Anions Cations and Acids in Rat Liver Mitochondria
Authors: Mitchell P. , Moyle J.
Published: March 2005

Studies of net translocation of various anions and cations and their corresponding acids and bases, using extensions of established techniques, imply that the M phase of the cristae membrane of rat liver mitochondria is very impermeable (i.e. not permitting net permeation) to the following ions: CH3COO−, H2PO4−, HPO42−, SO42−, Fe(CN)63−, Fe(CN)64−, choline+, K+, Na+ and NH4+. How‐ever, the M phase appears to be permeated rapidly by SCN− and slowly by Cl−.

It has been confirmed that CH3COO− and NH4+ probably permeate as CH3COOH and NH3 respectively, and that phosphate equilibrates across the M phase by an electrically neutral mechanism equivalent to H3PO4 translocation. The possibility that phosphate is obligatorily translocated as KH2PO4 has been eliminated.

Sulphate equilibrates by an electrically neutral mechanism equivalent to H2SO4 translocation.

The alkali metals Na+ and K+ (and possibly NH4+) equilibrate across the M phase via specific electrically neutral H+/Na+ and H+/K+ antiport, the former being the more active at neutral pH. Choline does not participate significantly in proton‐coupled antiport.
Die “freien” Lipide aus Brucella abortus Bang
Authors: Thiele O. W. , Kehr W.
Published: March 2005

Two different strains of Brucella abortus, strain 1119 and strain Scherle II, were grown on solid media; strain 1119 was harvested after approximately 70 h, strain Scherle II was harvested at different ages of the culture up to 72 h. The “free” total lipids were extracted and from these the neutral lipids were prepared and analyzed. The following results were obtained.

Strain 1119 contains 4.5% free lipids of bacterial dry weight. Strain Scherle II contains decreasing amounts of free total lipids from approximately 6% to 1.4% as the age of the culture increases.

On a weight basis the free total lipids of strain 1119 contain 8.1% neutral lipids. The neutral lipids of strain Scherle II decrease from more than 60% to approximately 24% (of free total lipids) with increasing age of the culture. The amount of phospholipids, however, remains comparatively constant (in relation to the percentage of total lipids) with increasing age of the culture of strain Scherle II. The results indicate that polar lipids other than phospholipids accumulate as the age of the culture increases, but there is no net storage of lipids.

The neutral lipids of strain 1119 contain approximately 30% (on a weight basis) of ubiquinone‐50 (corresponding to 1.2 μmoles/g bacterial dry weight). Strain Scherle II also contains ubiquinone‐50, but in amounts demonstrable by thin‐layer chromatography only after an age of 36–38 h. Ubiquinone‐50 has hitherto been found only in a few bacterial species, while most bacterial species contain lower isoprenologs of ubiquinone.

The neutral lipids of strain 1119 contain 51% (on a weight basis) of diglycerides, consisting of the 1,3‐ and the 1,2‐isomers. In strain Scherle II only 1,2‐diglycerides were found. The component fatty acids of the diglycerides of strain 1119 consist of approximately 55% (on a weight basis) of C19 cyclopropane acid (lactobacillic acid).

The lactobacillic acid/cis‐vaccenic acid ratio is fairly independent of the age of the culture of strain Scherle II.

The amount of unesterified fatty acids is lower in strain 1119 (4.9% of the neutral lipids, on a weight basis) than in strain Scherle II (49 or 67%, according to the culture conditions).

Both strain 1119 and strain Scherle II contain probably small amounts of triglycerides, monoglycerides and long‐chain hydrocarbons.

The possible significance of the results in the classification of Brucella is discussed.
Comparaison de la composition chimique d'une fraction lipopolysaccharidique et d'une fraction polysaccharidique isolées de Brucella melitensis
Authors: Lacave C. , Asselineau J. , Serre A. , Roux J.
Published: March 2005

Westphal's procedure was found the most suitable for the isolation of lipopolysaccharide fractions from Brucella melitensis cells; only the fraction obtained in the aqueous layer (LPS) is studied in this paper. Beside LPS, an extracellular polysaccharide fraction (PS) was extracted from the culture medium of the bacteria, and the chemical composition of both fractions, LPS and PS, has been compared.

In both cases, nucleic acid contaminants were eliminated either by alcohol precipitation or by use of cetavlon. These treatments also removed an acidic polysaccharide containing mainly galactosaminuronic acid, which might be Vi antigen.

The main difference between LPS and PS is the presence of a small amount of lipid A in LPS (ca. 1%). As in the case of lipid A isolated from LPS of Enterobacteria, Brucella lipid A contains glucosamine and fatty acids, but β‐hydroxymyristic acid could not be detected.

The behaviour of LPS and PS fractions on DEAE‐cellulose columns is very similar, and the small differences that could be observed may be explained by solubility changes due to the lipid moiety present in LPS.

Both fractions contain 2‐keto‐3‐hydroxy‐octulosonic acid, a mixture of d‐ and l‐glycero‐d‐mannoheptoses, a high amount of glucose, galactose, a 3,6‐dideoxyhexose (colitose or abequose), and a methylated sugar behaving as a methylated derivative of dideoxyhexose. Small amounts of rhamnose in PS and of arabinose in LPS seem to be due to contaminants.
Artspezifität Polysaccharid‐haltiger Zellmembran‐Antigene von Dictyostelium discoideum
Authors: Gerisch G. , Malchow D. , Wilhelms H. , Lüderitz O.
Published: March 2005

In the course of studies on species‐specific cell aggregation in the cellular slime molds, the specificity of heat‐stable surface antigens was investigated. With phenol–water, antigens could be extracted from freeze‐dried amoebae of Dictyostelium discoideum, the serological reactivity of which is insensitive to ribonuclease, pronase and α‐amylase; however, it is sensitive to periodate. Serological reactivity of phenol–water extracts was traced to a fraction which contains N ‐acetyl‐glucosamine, fucose, mannose and polypeptide, and behenic acid as the principal fatty acid. The polysaccharide moieties of these compounds are considered to carry the serological specificities. In comparing the two species, Dictyostelium discoideum and Polysphondylium pallidum, it was found that the non‐dialyzable fraction of the phenol–water‐extracts contain different proportions of the three sugars (mannose–fucose–N ‐acetyl‐glucosamine in D. discoideum 1:4.5:12; in P. pallidum 1:1:2). Species‐specificity of the phenol–water extracted antigens of the two species was demonstrated by precipitation with the respective antisera.

With the fluorescent antibody technique and by agglutination, species‐specific antigens which are periodate sensitive could be detected on the surface of heat‐killed (2 h; 100°) cells. As revealed by agglutination inhibition, these heat‐stable cell surface antigens are identical, at least in part, with the phenol–water extracted antigens.
L'ATP : L‐arginine phosphotransférase de poids moléculaire élevé
Authors: Lacombe G. , Thiem N. v. , Thoai N. v.
Published: March 2005

ATP:l‐arginine phosphotransferase has been isolated from Sipunculus nudus muscle; the preparation is homogeneous to analytical centrifugation, molecular sieving on Sephadex G‐100 and electrophoresis on cellogel and on polyacrylamide discs.

Treated by 8 M urea, arginine phosphokinase from Sipunculus is dissociated into two subunits having the same molecular weight whereas arginine kinase from lobster gives no dissociation evidence. Furthermore the number of peptides shown on fingerprints of the tryptic hydrolysate of the first enzyme amounts to half of its lysine plus arginine residues. The enzyme from Sipunculus is named arginine kinase type II, to be distinguished from that of molecular weight near 40 000 (arginine kinase type I) and from that of molecular weight 160 000 (arginine kinase type III).

Unlike arginine kinase type I which contains 6 total SH groups and a single reactive and essential SH, arginine kinase type II is found to have 12 total thiols and 6 SH groups reacting at the same rate with 5,5′‐dithio‐bis‐(2‐nitrobenzoic) acid or with N‐ethylmaleimide. From the numerous thiols similarly exposed results the particular autooxydisability and lability of the enzyme on study. No evidence is shown of the content of the disulfide bridges.

Like arginine kinase type I, arginine kinase type II is strictly specific. The kinetics of the reaction are the same for both: no cooperative effect is observed between guanidine and nucleotide substrates fixation on the enzymes.
The Subcellular Distribution of Acyltransferases which Catalyze the Synthesis of Phosphoglycerides
Authors: Eibl H. , Hill E. E. , Lands W. E. M.
Published: March 2005

The distribution of acyltransferase activities in rat liver homogenates is described in relation to that for five other enzymes, each known to belong to a certain subcellular particle. Five different transfer reactions were studied with 1‐acyl‐sn‐glycero‐3‐phosphorylcholine, 2‐acyl‐sn‐glycero‐3‐phosphorylcholine, 1‐acyl‐sn ‐glycero‐3‐phosphoylethanolamine, 1‐acyl‐sn‐glycero‐3‐phosphate and sn‐glycero‐3‐phosphate as substrates, and different acyl‐CoA esters served as donors of acyl groups. Glucose‐6‐phosphatase and the acyltransferase activities studied were distributed with similar relative specific activities among the subfractions.

The results indicated that:

a) Acyltransferases were located in the microsomal membranes.

b) Nuclei and mitochondria did not have acyltransferase activity under the conditions used in these experiments, which were optimal for microsomal acyltransferases.

c) Plasma membranes appeared to have only a small amount of acyltransferase activity in comparison to that in the microsomal particles.

d) All acyltransferase activities tested showed similar patterns of distribution.

e) Acyltransferase activities may be useful in the future as marker enzymes for microsomal particles from tissues which do not contain glucose‐6‐phosphatase, but do contain the acyltransferase within the microsomal membranes.
Nuclear RNA Fractions Obtained by Sequential Extraction of Rat Liver Nuclei with Salt Solutions and their Relationship to Polysome‐Associated RNA
Authors: Drews J.
Published: March 2005

Pulse‐labeled RNA from rat liver nuclei was fractionated by sequential treatment of purified nuclei with buffered salt solutions of different ionic strength in the presence of ribonuclease inhibitor prepared from rat liver cytoplasm. Two fractions of pulse‐labeled nuclear RNA were successively extracted from rat liver nuclei with 0.14 M NaCl, 0.001 M MgCl2 at pH 7.0 and pH 8.0, respectively. The labeled material isolated at pH 7.0 sediments between 6–16 S while the pulse‐labeled RNA extracted at pH 8.0 sediments in the range of 10–34 S. Both fractions are characterized by a high content in AMP. With respect to [32P]nucleotide composition, sedimentation characteristics and hybridizability, these fractions correspond closely to non‐ribosomal RNA associated with polysomes. When a third extraction is carried out at pH 8.0 in 0.3 M NaCl another rapidly labeled RNA component is obtained. This RNA sediments above 34 S, turns over more rapidly than the RNA species extracted in 0.14 M NaCl, and contains less AMP. As judged by [32P]base‐composition, sedimentation and hybridizability, this RNA component is not present in polysomes. The pulse‐labeled RNA remaining in the nuclei after three salt extractions comprises mainly ribosomal or ribosomal precursor RNA as well as DNA‐like RNA species which show little or no nucleotide sequence homology with polysome‐associated RNA.

Since nuclear RNA species clearly different from polysome‐associated cytoplasmic RNA are released from nuclei only at a relatively high ionic strength and are retained at a physiologic salt concentration it is suggested that RNA species not designated to function in cytoplasmic protein synthesis are a priori bound more tightly to nuclear structures than those types of RNA which serve as vehicles for the nucleo‐cytoplasmic transfer of genetic information.
Steroids in Germfree and Conventional Rats
Authors: Eriksson H. , Gustafsson J.‐Å. , Sjövall J.
Published: March 2005

Free steroids in faeces from germfree and conventional rats were studied by gas chromato‐graphy‐mass spectrometry after purification on carboxymethyl Sephadex LH‐20 and separation on methylated Sephadex G‐25. Only minor amounts of free steroids were found in germfree rats; these could not be identified. In contrast to this, conventional rats excreted considerable amounts of free steroids: 3α‐hydroxy‐5α,17α‐pregnan‐20‐one, 3α‐hydroxy‐5α‐pregnan‐20‐one, 3β‐hydroxy‐5α,17α‐pregnan‐20‐one (major component), 3β‐hydroxy‐5α‐pregnan‐20‐one, and 5α‐pregnane‐3β,20β‐diol. Furthermore, 5α‐pregnane‐3α,20β‐diol was tentatively identified. These findings are discussed in relation to what is known about steroid sulphates in faeces from germfree and conventional rats.
Purification and Some Properties of a Soluble DNA‐Dependent RNA Polymerase from Nuclei of Human Placenta
Authors: Mertelsmann R.
Published: March 2005

More than 75% of the RNA polymerase activity of isolated nuclei from human placental tissue was found in the supernatant (20 min at 40000Xg) after incubation of the nuclear suspension for 24 hours at 0°, followed by 15 min at 37° and a further 15 min at 37° in the presence of 120 mM ammonium sulphate.

Chromatography of this soluble preparation on DEAE cellulose (0.64 mequiv./g) was carried out using a linear gradient from 100 to 500 mM ammonium sulphate. RNA polymerase activity, eluted at 300 mM ammonium sulphate, was fully dependent on added DNA template.

Some properties of human RNA polymerase at various stages of purification are described.

The process of solubilization is discussed.
The Mechanism of Laccase‐Catalyzed Oxidations: Kinetic Evidence for the Involvement of Several Electron‐Accepting Sites in the Enzyme
Authors: Malmström B. G. , Finazzi Agrò A. , Antonini E.
Published: March 2005

The anaerobic reduction of fungal laccase by quinol and ferrocyanide has been studied in a stopped‐flow apparatus by measurements of the change in the absorbancy at 610 mμ associated with the reduction of the so‐called Type 1 Cu2+. With ferrocyanide the rate of formation of product was also measured.

The reoxidation of reduced laccase by molecular oxygen has been studied by the same technique.

The kinetics of reduction and reoxidation as well as of product formation has also been investigated in a series of experiments with both the reducing substrate and oxygen present. In addition, the effect of an inhibitor, fluoride, on the kinetics has been studied.

The results confirm earlier static experiments showing that laccase contains two electronaccepting sites other than Type 1 and Type 2 Cu2+. Type 1 Cu2+ is reduced rapidly enough to involve it in the catalytic reaction. However, it appears to be rapidly reoxidized only when the other electron‐accepting sites are also occupied. A mechanism involving the cooperation of several electron‐accepting sites is consistent with the kinetic results.