bluuebunny/crossref_metadata_2025 huggingface dataset:
Incorporation of [3H]Thymidine into Nuclear and Mitochondrial DNA in Synchronized Mammalian Cells
Authors: Koch J. , Stokstad E. L. R.
Published: March 2005

Human liver cells (Chang) were synchronized by cold shock. The second generation was scanned with two‐hour pulses of [3H]thymidine. The incorporation of [3H]thymidine into nuclear and mitochondrial DNA during each pulse was determined. Maximal incorporation into the nuclear DNA occurred during S‐phase (6 hours long) and to a smaller extent during and shortly after cytokinesis. The mitochondrial DNA was most highly labeled between S‐phase and cytokinesis. The nuclear DNA's had a buoyant density in caesium chloride of 1.699 g/ml, whereas the mitochondrial DNA banded at a density of 1.688 g/ml.
The Synthesis of Phosphoenolpyruvate in the Gluconeogenesis
Authors: Felicioli R. A. , Gabrielli F. , Rossi C. A.
Published: March 2005

Livers of chick embryos at different stages of incubation have been fractionated into mitochondrial and “soluble” fractions by centrifugation.

The pyruvate carboxylase and phosphoenolpyruvate carboxykinase activities, which constitute the major pathway for the synthesis of phosphoenolpyruvate, have been measured in both preparations.

The pyruvate carboxylase activity occurs only in the mitochondria and parallels the gluconeogenetic ability.

The bulk of phosphoenolpyruvate carboxykinase activity appears in the mitochondria. Only traces can be detected in the “soluble” fraction. The level of the mitochondrial phosphoenolpyruvate carboxykinase activity varies during the incubation period in accordance with its presumed role as a major source of phosphoenolpyruvate in the gluconeogenesis.

The hypothesis that the pathway of synthesis of phosphoenolpyruvate in the process of gluconeogenesis is mainly located in the mitochondria of chick embryo liver is proposed.
A Fraction of Newly Synthesized DNA of HeLa Cells
Authors: Levis A. G. , Krsmanovic V. , Miller‐Faures A. , Errera M.
Published: March 2005

HeLa cells were labelled for 10 or 30 minutes with [3H]thymidine and the DNA fractionated with chloroform‐isoamylalcohol; almost 90% of the labelled DNA remained in the interphase whilst about 60% of total DNA (bulk) was extracted in the aqueous phase.

When added to a column of methylated serum albumin adsorbed on kieselguhr the extracted DNA was eluted as a major labelled peak with 0.8 M NaCl but a small fraction could only be eluted at higher NaCl concentrations. This “tail” contained both bulk and recently labelled DNA.

After a short pulse the specific activity of the DNA eluted at higher salt concentrations increased; this was confirmed when pooled fractions were concentrated on a caesium sulphate gradient.

Electron microscopy and sucrose gradient centrifugation of fractions recovered on a Cs2SO4 gradient shows that the eluted DNA was polydisperse (peak at about 18 S) with molecular dimensions of a few to almost 20 μ.

When [3H]uridine was used as a label, both labelled DNA and rapidly labelled RNA could be studied simultaneously in the same way: these experiments indicated that in the fractions analysed, the radioactivity due to RNA and to DNA was not carried on the same molecules, suggesting the mutual exclusion of replication and transcription.
Studies of the Esterase Activity and the Anion Inhibition of Bovine Zinc and Cobalt Carbonic Anhydrases
Authors: Thorslund A. , Lindskog S.
Published: March 2005

The rates of hydrolysis of nitrophenyl esters, catalyzed by bovine carbonic anhydrase, depend on the position of the nitro group and on the size of the acyl residue. The most rapidly hydrolyzed substrate of those investigated is p‐nitrophenyl acetate. The catalyzed rates are proportional to both enzyme and ester concentrations. Only in the case of m‐nitrophenyl acetate could a value of the Michaelis constant, Km, be estimated, approximately 10 mM. Product inhibition by o‐nitrophenol occurs during the enzyme‐catalyzed hydrolysis of o‐nitrophenyl acetate.

The metal specificity of the enzyme seems to be independent of the substrate. Zn(II) and Co(II) are about equally effective in restoring esterase activity to the apoenzyme while all other metal ions studied do not activate or have a very small effect.

The pH‐dependence of the esterase activity of both Zn(II)‐ and Co(II)‐carbonic anhydrase is very similar to that of the CO2 hydration activity, and the basic form of a group with a pK near 7 is required for both reactions. Anionic inhibitors are strongly bound to the enzyme when this group is in its acidic form, but the inhibition is almost abolished at alkaline pH.

The inhibitory powers of cyanide and sulfide decrease at acid as well as at alkaline pH. The results agree with the assumption that, formally, CN− and HS− do not bind to the basic form of the enzyme, while HCN and H2S do not bind to the acidic form.
Reactivity of Sulfhydryl Groups in Micelles
Authors: Heitmann P.
Published: March 2005

In order to get some insight into the factors determining the different reactivities of protein SH groups, the reactions of a micelle‐forming cysteine derivative, the N‐dodecanoyl‐DL‐cysteinate anion, with iodoacetamide, chloroacetamide, and p‐nitrophenyl acetate have been studied kinetically in aqueous solution at 25°. The incorporation of the mercapto compound into an anionic micelle, consisting of N‐dodecanoyl‐DL‐cysteinate alone or in mixture with N‐dodecanoyl‐glycinate, is accompanied by a “masking” of the sulfhydryl group. On the other hand, when the model compound is incorporated into a cationic micelle, formed by hexadecyltrimethyl‐ammonium, bromide, the true second‐order rate constants for the reactions of the RS− species with chloroacetamide, iodoacetamide, and p‐nitrophenyl acetate increase by factors of 5–7, 60–100, and 100–200, respectively, in comparison to the corresponding constants in the bulk phase. The observed effects on the apparent second‐order rate constants related to the total concentration of mercapto compound are even higher, since the degree of SH ionization is greater in the eationic micelle than in the bulk phase. The magnitude of the latter effect decreases strongly with addition of other electrolytes (NaBr) to the medium, due to a decrease of the surface potential of the micelle. The reactions of N‐acetyl‐L‐cysteinate and L‐cysteine with iodoacetamide and p‐nitrophenyl acetate are also strongly accelerated in the presence of cationic micelles under the condition that the test solution contains no or only small amounts of other electrolytes. It is assumed that the catalytic effect of the cationic micelle is mainly caused by the participation of a positively charged head group spatially close to the RS− group in the activated complexes of the reactions. The “masking” of the SH group in the anionic micelle could also be related, at least in part, to electrostatic effects. In agreement with this assumption is the fact, that the addition of a nonionic micelle‐forming surfactant, Brij 35 (polyoxyethylene dodecyl ether), has only a relatively small effect on the reaction rates.
The Reaction Pathway of Pig Brain Mitochondrial Monoamine Oxidase
Authors: Tipton K. F.
Published: March 2005

Initial rate measurements of the oxidation of tyramine were carried out with purified pig brain mitochondrial monoamine oxidase. The reciprocal plots obtained when the concentration of either substrate was varied at a series of fixed concentrations of the other gave families of parallel lines consistent with a kinetic mechanism in which a ternary complex is not involved. This mechanism was supported by product inhibition studies and the demonstration of a half‐reaction in the absence of oxygen.
The Stereospecificity of the Citrate Synthase in Sulfate‐Reducing and Photosynthetic Bacteria
Authors: Gottschalk G.
Published: March 2005

The stereospecificity of the citrate synthase of a number of sulfate‐reducing and photosynthetic bacteria was determined by the following method: radioactive citrate was synthesized from [4‐14C]oxaloacetate and acetylcoenzyme A by cell‐free extracts. The resulting citrate was then cleaved to acetate and malate by citrate lyase plus malate dehydrogenase. In the case that the usual citrate synthase was present in the cell‐free extracts, the citrate cleavage yielded radioactive malate and unlabeled acetate. The presence of the Clostridium kluyveri type of citrate synthase led to the formation of radioactive acetate and unlabeled malate.

It was found that four species of the Athiorhodaceae, Desultfovibrio gigas, D. sale xigens and Desulfotomaculum ruminis contain the usual citrate synthase. Two strains of D. desulfuricans and two strains of D. vulgaris contain the C. kluyveri type of citrate synthase, which is now referred to as (R)‐citrate synthase.

The presence of the (R)‐citrate synthase in D. desulfuricans and D. vulgaris was confirmed by an in‐vivo experiment. When grown on pyruvate and radioactive carbon dioxide, the protein of these two species contains [5‐14C]glutamate, whereas the protein of D. gigas contains [1‐14C]‐glutamate.

The presence of the (R)‐citrate synthase in certain strictly anaerobic bacteria is discussed from an evolutionary point of view.
Lysolecithin‐Stoffwechsel in Erythrocytenmembranen
Authors: Ferber E. , Munder P. G. , Kohlschütter A. , Fischer H.
Published: March 2005

Human erythrocytes have a life span of about 120 days. One can easily separate young and aged cells by density centrifugation. For these reasons they are especially suited for studying ageing processes.

There is general agreement that the activity of several glycolytic enzymes decreases during ageing. However, nothing is known about typical membrane enzymes in connection with ageing, and since it was felt that membrane alterations might be of primary importance, a comparative study of phospholipid‐metabolizing enzymes was undertaken.

The enzymes studied in this investigation were:

a) the structure‐bound acid: CoA ligase (AMP) and acyl‐CoA: Iysolecithin‐O‐acyltransferase which together incorporate lysolecithin and fatty acid into membrane bound lecithin.

b) lysophospholipase (lysolecithin acyl‐hydrolase) which degrades lysolecithin into glycerophosphorylcholine plus fatty acid.

Human erythrocytes from healthy donors were separated by simple ultracentrifugation and by albumin density centrifugation according to age. The mean volume of the young erythrocytes was 109 μ3, while that of the aged cells was 74 μ3. The enzyme activities of freeze‐thawed cells were determined and corrected for cell numbers. Furthermore, the enzyme activities‐in hemoglobin‐free stroma were measured.

[14C]‐labelled substrates were used to determine the enzyme activities: [1‐14C]oleic acid for the acylation of lysolecithin and [1‐acyl‐14C]lysolecithin for the lysophospholipase reaction.

The results showed that the activities of both enzymic reactions as measured with old cells were 40 to 50% lower than the activities found in young cells. In hemolysates from young and old cells the following enzyme activities were determined: the acylating activity decreased from 117 to 67 MolX10−20/cell×min, and the lysophospholipase activity from 185 to 123 Mol×10−20/cellXmin.

The ATP content of old cells was only slightly lower than that of young cells.

Comparative analyses of erythrocytes stored in acid citric dextrose for two months at 4° gave different results: these cells which had been “aged in vitro” had an extremely low ATP content, while the activities of the lysolecithin metabolizing enzymes remained unchanged.

These findings suggest that the lysolecithin metabolism has a significance in cell ageing processes. It remains to be shown, however, that lysolecithin produced by macrophages lining the RES sinuses is the decisive factor responsible for the death and removal of aged cells.
The Effect of Piericidin A on Energy‐Linked Processes in Submitochondrial Particles
Authors: Vallin I. , Löw H.
Published: March 2005

Piericidin A inhibits the oxidative phosphorylation capacity of the NADH oxidase in submitochondrial systems at concentrations of 30 pmoles/mg protein, that of the succinic oxidase at 6 nmoles/mg protein and the phosphorylation in the cytochrome oxidase region at concentrations of 5 μmoles/mg protein.

The energy dependent NAD+ reductions are 10 times more sensitive to piericidin A than the NADH oxidase reaction. There is a correlation between the oxidation reduction state of the respiratory chain components, presumably of coenzyme Q and the extent of sensivity to the inhibitor.

Piericidin A and guanidine compounds showed an additive or more than additive inhibitory effect when added together.

p‐Chloromercuribenzoate and piericidin have a synergistic effect when added simultaneously. 2, 3‐Dimercaptopropanol (BAL) could partly release the piericidin inhibition, in contrast to glutathione, indicating an involvement of proximal –SH‐groups in the piericidin sensitive site.

Some discrepancies between the piericidin action and the inhibitory action of rotenone and amytal are noted.

A hypothesis is put forward for the possible interaction of a non‐heme iron component, structurally arranged by S‐bonds and coenzyme Q and the relevance of this chelate for the generation of a primary high energy complex.
Biosynthesis of Phosphatidylcholines in Rat Liver
Authors: Arvidson G. A. E.
Published: March 2005

The incorporation of [1,2‐14C2]choline and methyl groups from L‐[Me‐14C]methionine into subfractions of phosphatidylcholines of liver and blood plasma in the rat has been studied. Fractionation by thin‐layer chromatography on AgNO3‐impregnated plates followed by reversedphase partition thin‐layer chromatography permitted the isolation of 8 different molecular types of phosphatidylcholines. In the liver, incorporation of [1,2‐14C2]choline occurred most rapidly in palmitoyl‐oleoyl and palmitoyl‐linoleoyl phosphatidylcholine but was very slow in the stearoyl‐arachidonoyl species. Initially, after injection of [1,2‐14C2]choline, the distribution of isotope in the subfractions of different degree of unsaturation of the phosphatidylcholines in blood plasma was very similar to that of the phosphatidylcholines in the liver. Approximately 80% of the incorporated isotope was found in the mono‐ and dienoic fractions. After administration of L‐[Me‐14C]methionine the tetra and hexaenoic liver phosphatidylcholines contained 75 to 85% of the total [14C]phosphatidylcholine. The proportion of isotope in these fractions of blood plasma was lower. Among the polyunsaturated phosphatidylcholines in liver those containing palmitic and docosahexaenoic acid were found to incorporate isotope from L‐[Me‐14C]methionine to a greater extent than would be expected from a stoichiometric methylation of all types of phosphatidylethanolamines. The stearoyl‐arachidonoyl subfraction exhibited the lowest metabolic reactivity. In both male and female rats the amounts per whole liver of the mono‐ and dienoic fractions were greatly reduced during fasting.
Effects of Ultraviolet Irradiation (2537 Å) on the Structure of Human Thyroglobulin
Authors: Vecchio G. , Salvatore G. , Dose K.
Published: March 2005

The effect of in vitro ultraviolet irradiation at 2537 Å on the structure of highly purified human thyroglobulin has been investigated by following the ultracentrifugal pattern and the appearance of free sulfhydryl groups in the irradiated protein.

Thyroglobulin (19 S) dissociates into two 12 S subunits when it is irradiated with doses of a few hundred Einsteins per mole. The quantum yields of dissociation vary between 0.5 × 10−3 and 2 × 10−3, depending on the experimental conditions. At higher ultraviolet doses, the protein is partially aggregated and denatured.

The extent of dissociation is largely dependent on the ionic strength and the pH of the medium. Doses up to 1,500 Einsteins per mole cause only a partial (30%) splitting of the 19 S molecules, at neutral pH in 0.1 M KCl, whereas at pH 10 and low salt (Γ/2=0.0042), the same dose produces almost complete dissociation into 12 S subunits.

Ultraviolet light also causes the rupture of some of the disulfide bonds of native thyroglobulin. On irradiation with several hundred Einsteins per mole protein, as many as 21 –SH groups per molecule can be titrated. The quantum yields for –SH production and destruction of cystine residues are both 0.05.

No direct correlation has been found between the extent of dissociation of thyroglobulin and the number of –S–S– bonds cleaved, nor between the reassociation of the 12 S subunits and the reoxidation of –SH groups. It is concluded that two 12 S units present in human thyroglobulin are not linked by interchain disulfide bridges; the disulfides cleaved by ultraviolet irradiation, however, may play an important role in maintaining the subunit organization of the protein molecule.
Carotenoids in the Carapace of the Orconectes limosus (Raf.), Crustacea: Decapoda
Authors: Czeczuga B. , Czerpak R.
Published: March 2005

The presence of carotenoids in Decapoda‐Orconectes limosus (Raf.) was studied.

In extracts from carapace Orconectes limosus1 separated by means of column and thin‐layer chromatography, the following carotenoids were identified: β‐carotene, echinenone, lutein (epoxide and ester), astaxanthin (free and ester) and one unknown xanthophyll.
Substrate Specificity of Adenine‐Specific Transfer RNA Methylase in Normal and Leukemic Tissues
Authors: Baguley B. C. , Staehelin M.
Published: March 2005

High speed supernatant enzyme fractions derived from each of three tissues (livers and spleens of normal rats, and spleens of rats with the Dunning leukemia) were compared in their ability to transfer methyl groups from S‐adenosyl‐methionine to Escherichia coli transfer‐RNA (tRNA). In comparison to the supernatant fractions from the normal tissues, that from leukemic spleen had a six‐fold higher tRNA methylase activity and methylated E. coli tRNA to a much higher extent. A tRNA methylase which methylated adenine in the 1‐position was isolated from each high speed supernatant fraction by DEAE‐cellulose and Sephadex G‐200 chromatography. Although the purified leukemic spleen enzyme contained six‐fold higher methylase activity, it methylated E. coli, yeast, and mammalian tRNA to the same extent as did the corresponding enzyme from normal liver or spleen. The normal and leukemic tissue enzyme preparations appeared to methylate the same sequences in E. coli tRNA, and could methylate this tRNA to an extent of about one methyl group per molecule. The results suggest that in comparison to that of normal tissue, 1‐adenine methylase in the leukemic spleen tissue is increased in activity but possesses identical substrate specificity.
Metabolism of Prostaglandins in Rat Liver Mitochondria
Authors: Hamberg M.
Published: March 2005

A series of prostaglandins has been incubated with the β‐oxidizing enzyme system of rat liver. All of the compounds tested were oxidized. Prostaglandin E1, prostaglandin B1, prostaglandin F1α and prostaglandin F1β were converted into the corresponding C18 homologues. Prostaglandin A1, 11α,15‐dihydroxy‐9‐ketoprostanoic acid and 11α‐hydroxy‐9,15‐diketoprostanoic acid yielded mixtures of the C16 and C18 homologues. Nor‐prostaglandin F1α and homo‐prostaglandin F1α were both converted into trinor‐prostaglandin F1α.
On the Absolute Configuration of 19‐Hydroxy‐Prostaglandin B1
Authors: Hamberg M.
Published: March 2005

19‐Hydroxy‐prostaglandin B1, isolated from human seminal plasma, was treated with tritiumlabeled acetic anhydride and degraded by oxidation with permanganate in acetone. Tritiumlabeled 3‐acetoxybutanoic acid was isolated from the oxidation product and diluted with unlabeled dl‐3‐acetoxybutanoic acid. The morphine salt of the acid was resolved. These experiments showed that the tritium‐labeled 3‐acetoxybutanoic acid had the d‐configuration and consequently the parent 19‐hydroxy‐prostaglandin B1 has the d‐configuration at C‐19. The configuration at C‐15 is discussed.
Sedimentation Coefficient and Molecular Weight of Beef Liver Glutamate Dehydrogenase at the Microgram and the Milligram Level
Authors: Sund H. , Burchard W.
Published: March 2005

The molecular weight and the sedimentation behaviour of beef liver glutamate dehydrogenase was investigated in the range of protein concentration between 25 μg/ml and 8 mg/ml. sO20,ω was found to be 13.0 S. Light scattering experiments (at 20° and 4360 Å) and sedimentation equilibrium experiments performed in M/15 potassium sodium phosphate buffer, pH 7.6, show that the molecular weight of the smallest enzymatically active subunit is 280,000 and that the associated enzyme molecule (molecular weight about 2.2 millions) is formed by association of eight subunits. From the dependence of the apparent molecular weight of the protein concentration and from the comparison with calculated curves it follows that the associated enzyme molecule is formed in a stepwise association‐dissociation equilibrium and not in a closed equilibrium between monomers and the octamer without any intermediate steps.
Bovine Liver Homogentisicase: Apo‐ and Reconstituted Holoenzymes
Authors: Takemori S. , Furuya E. , Mihara K. , Katagiri M.
Published: March 2005

Bovine liver homogentisicase can exist in two forms which are interpreted as being apo‐and holoenzymes. It is obtained in an unenriched form (apoenzyme) from bovine liver by acetone and ammonium sulfate fractionations and chromatography on hydroxylapatite column. This form of the enzyme can be enriched to an active form (holoenzyme) by incubation with ferrous ion under anaerobic conditions. The extent of activation is completely time dependent and the rate is extremely sensitive to the pH. Reducing substances do not affect the restoration of activity of the apoenzyme under anaerobic conditions. Properties of the apo‐ and holoenzymes are compared with respect to absorption spectrum, effects of reducing agents and pH on activity (or on reconstitution), stability, and molecular weight.

The activity of homogentisicase is strongly inhibited by various diphenolic compounds as well as chelating agents specific for ferrous ion. However, the reconstitution of homogentisicase from the apoenzyme with ferrous ion is strongly prevented only by the substrate, homogentisate or 2,5‐dihydroxybenzoate, a compound structurally similar to the substrate.
Biosynthesis of Two New Steroids in the Human Foetal Liver, 1 β‐ and 2β‐Hydroxytestosterone
Authors: Lisboa B. P. , Gustafsson J.‐Å.
Published: March 2005

Two new C19‐steroids with a 3‐oxo‐Δ4‐structure have been isolated and identified after incubation of testosterone with 105,000 × g microsomes of human foetal liver. These two ultraviolet positive metabolites were isolated by ascending one‐dimensional thin‐layer chromatography. The isolated compounds were identified as 1β‐ and 2β‐hydroxytestosterone by means of gas chromatography‐mass spectrometry. They were formed in approximately the same amount, each one representing about 15 % of the amount of 6β‐hydroxytestosterone formed.
Molecular Classes and Properties of Antibodies in Cattle Serum and Colostrum Synthesized during the Primary and Secondary Response to Protein Antigens
Authors: Hammer D. K. , Kickhöfen B. , Henning G.
Published: March 2005

The kinetics of the antibody response in cattle have been analyzed and the following results were obtained:

A single administration of proteins to cattle given intravenously or as alum‐precipitated antigen evoked a concurrent synthesis of γM‐ and γG‐antibodies as reflected in the serum 7 days following immunization. Employing the antigen binding capacity method for assaying antibody activity, the γG‐antibody collected 7 days after immunization exceeded by several times that of the γM‐class.

The γG‐antibody response as reflected in serum and colostrum three weeks after primary stimulation showed a marked increase when compared with their respective antibody levels formed earlier. The colostral 19 S antibody, composed of 19 S γG‐globulin and the easily discernible γM‐component was also increased and surpassed by several times the γM‐antibody level in a 3‐weeks serum.

When colostrum was fed to the newborn calf, the γG‐antibody appeared to be absorbed quantitatively by the intestinal tract, whereas the high molecular weight globulins (19 S) were transmitted much less efficiently. This might indicate that some sort of selective absorption occurred between different classes of immunoglobulins.

Kinetic studies of the secondary response demonstrated that in cattle serum an anamnestic reaction exists for γM‐antibody as well as for γG‐antibody.

The dissociation of the 19 S molecules from serum and colostrum by reduction and alkylation produces little effect on the antigen binding capacity of the monomer subunits obtained.

The kinetic characteristics of serum and colostral 19 S‐ and γG‐antibodies were studied by measurement of the association rates. γM‐Antibodies present in serum 7 days following antigenic challenge had a higher affinity for protein antigens than γG‐antibody molecules. There is considerable evidence, however, that the affinity of the γG‐antibody for the antigenic determinants increased progressively with time after immunization.
Comparative Studies on the Inhibition of Trypsin, Plasmin, and Thrombin by Derivatives of Benzylamine and Benzamidine
Authors: Markwardt F. , Landmann H. , Walsmann P.
Published: March 2005

Derivatives of benzylamine and benzamidine were found to be competitive inhibitors of the proteolytic enzymes trypsin, plasmin, and thrombin. The inhibitor activity of these compounds was determined by the inhibition of the hydrolysis of N‐α‐benzoyl‐dl‐arginine‐4‐nitroanilide, catalyzed by the three enzymes. Inhibitor constants (Ki) were ascertained. Relations be‐between the chemical structure and the activity against trypsin, plasmin, and thrombin were deduced by comparing the inhibitor constants. Generally the inhibition of these enzymes by the investigated compounds runs parallel but there exist also some differences especially among the benzylamine derivatives. 4‐Chlorobenzylamine, a strong thrombin inhibitor, is only of low effectiveness against trypsin and plasmin. Derivatives of benzamidine are stronger inhibitors than derivatives of benzylamine. The following compounds proved to be the most potent inhibitors: 4‐amidinophenylpyruvic acid, 4‐aminobenzamidine, and 4‐amidinobenzoic acid benzyl ester. These compounds exhibited also an anticoagulant and antifibrinolytic effect.
Immunochemical Studies on Salmonella
Authors: Fuller N. A. , Etievant M. , Staub A.‐M.
Published: March 2005

The wild strain of Salmonella cholerae suis carries the O‐antigen specificities 62 and 7. The phage converted strain has specificities 62, (7) and 14.

Four serologically active oligosaccharides can be isolated from the O‐antigenic polysaccharide of the phage converted strain of S. cholerae suis 62, (7) 14; their general formula is (Man)n→ Man → GlcNAc ↑ Glc where n ↑ 0, 1, 2 or 3. Data from precipitation inhibition studies using rabbit antisera indicate that there are several fractions of antibodies (anti 62, 7, 14) all directed against the same terminal (= immunodominant) sugar trait, i. e.d‐glucose. Chemical analysis shows that there is only one terminal non‐reducing glucose residue per “repeat unit”, both in the wild and converted strains. The position of the glucose residue, however, on the oligomannose chain is different in the wild and converted strains. It is established therefore that the change in antigenic specificity (appearance of factor 14) brought about by phage conversion of S. cholerae suis is correlated with the new and definite position of d‐glucose on the oligomannose chain.
The Properties of Molecular Fragments Obtained on Treating Calfskin Collagen with Collagenase from Clostridium histolyticum
Authors: Stark M. , Kühn K.
Published: March 2005

Collagenase was found to attack the collagen molecule at both ends at 10°. Six molecular fragments of different lengths were observed and could be correlated with the intact molecule as long‐spacing segment fragments. It was possible to isolate the 2,600,1,250 and 780 Å fragments by fractional ammonium sulphate precipitation.

The sharp helix‐coil transition on denaturation shows that the fragments have an intact triple helix. It was possible to separate the two shorter fragments into three components (the α1, α2 and α3 chain fragments) by chromatography on CM cellulose. The α1 and α3 chains have very similar amino acid compositions and peptide maps for both these fragments. This suggests that the differences between these two components are probably due to telopeptides and not to the amino acid sequence of the main chain.
Molecular Fragments of Rat Skin Collagen Obtained on Collagenase Digestion
Authors: Stark M. , Kühn K.
Published: March 2005

The behaviour of rat skin collagen on collagenase treatment is analogous to that of calf skin collagen. Fragments are also formed, but differ to a certain extent in length and amount from those by calf skin collagen. All molecular fragments consist of only two electrophoretically separable chains, namely α1 and α2.
Metal‐Combining Properties of Human Lactoferrin (Red Milk Protein)
Authors: Masson P. L. , Heremans J. F.
Published: March 2005

Human lactoferrin, the red iron‐binding protein from milk, was prepared by chromatography on DEAE‐cellulose of the milk proteins soluble in 2 M ammonium sulfate. Several preparations differing in iron content were obtained. Apolactoferrin was prepared by dialyzing the protein against 0.1 M citric acid.

Saturation experiments with iron ascorbate indicated the iron: protein ratio at saturation to be 0.025 micromoles of metal per mg of protein. Assuming 80,000 to be the molecular weight of lactoferrin, this would correspond to a ratio of two iron atoms per molecule. With copper, a similar molar proportion at saturation was obtained. Gasometrie measurements were used to calculate the amount of carbon dioxide liberated upon dissociation of the metal‐protein complexes by acidification. One mole of CO2 was freed per atom of iron or copper.
An Analysis of Perturbations in the Ultraviolet Absorption Spectra of Proteins and Model Compounds
Authors: Bailey J. E. , Beaven G. H. , Chignell D. A. , Gratzer W. B.
Published: March 2005

It is shown that the perturbation of the ultraviolet absorption spectra of the tyrosine and tryptophan chromophores in aqueous solutions by addition of solvents of lower polarity leads not only to red shifts but also to changes in peak intensity, and oscillator strength. The effect is much greater in tyrosine than in tryptophan, and can be as large as 30% (transfer from water to ethanol). It is shown that observed difference spectra can be reasonably reproduced by the addition of two calculated curves, one corresponding to a shift, the other to a change in intensity, i.e. an admixture of shift difference spectrum and absolute spectrum. Similar intensity changes are seen to accompany the denaturation of several proteins. The changes of the characteristic parameters of difference spectra with the concentration of perturbant are analyzed. The relation between the difference spectra generated by aromatic chromophores near 230 mμ and those in the 280 mμ region are considered, and their relative magnitudes have been estimated for a variety of perturbants. It is shown that in denaturation of a number of proteins, the difference spectra near 230 mμ cannot be satisfactorily accounted for solely in terms of the model compounds containing aromatic chromophores. Possible contributions to difference spectra of proteins in this region are considered.
Rubredoxin from a Nitrogen‐Fixing Variety of Desulfovibrio desulfuricans
Authors: Newman D. J. , Postgate J. R.
Published: March 2005

Rubredoxin from Desulfovibrio desulfuricans var. azotovorans, with a molecular weight of 6,500 ± 60, has an amino acid composition different from those of other rubredoxins. Ultraviolet, optical rotatory dispersion and circular dichroism spectra indicate chemical similarities to other rubredoxins: including stability to guanidine denaturation, relative inaccessibility of the chromophoric iron to cyanide and certain chelating agents. The presence of four –SH groups, relatively inaccessible to –SH reagents, and one Fe per molecule, the latter in an environment of low asymmetry when reduced, is consistent with a structure proposed by Bachmayer et al. in 1968 for the rubredoxin of Micrococcus aerogenes.
Isoenzymes of Malate Dehydrogenase in Saccharomyces cerevisiae
Authors: Atzpodien W. , Gancedo J. M. , Duntze W. , Holzer H.
Published: March 2005

Extracts from yeast cells grown under different conditions and from mitochondria were subjected to agar gel electrophoresis. Three bands showing malate dehydrogenase activity were found in extracts from aerobic derepressed cells (c‐MDH I, c‐MDH II, and m‐MDH III), whereas generally only one band was present in extracts from anaerobic repressed cells (c‐MDH I) and another one in extracts from mitochondria (m‐MDH III) of aerobic derepressed cells.

The different malate dehydrogenases showed dissimilar regulatory and physical properties, e. g. sensitivity to inactivation by heat, inhibition by oxaloacetate and by p‐chloromercuribenzoate, activation by Pi, Michaelis constants etc. A separation of malate dehydrogenases by DEAE‐cellulose chromatography was performed giving only two malate dehydrogenase forms. These two forms were subjected to agar gel electrophoresis and gave one band each, corresponding to the cytoplasmic c‐MDH I and c‐MDH II. The possible metabolic significance of the three malate dehydrogenase isoenzymes is discussed.
Oxidation of n‐Hexadecane by Subcellular Preparations of Guinea Pig Small Intestine
Authors: Mitchell M. P. , Hübscher G.
Published: March 2005

A method is described for the quantitative assay of the conversion of [1‐14C]n‐hexadecane to palmitic acid using gas‐liquid chromatography with [1‐14C]heptadecanoic acid as internal standard.

Subcellular preparations of the mucosa of guinea pig small intestine were found to convert n‐hexadecane to palmitic acid. The oxidation system was located mainly in the microsomal fraction (8,400,000 ×g× min) but there was definite activity also in the high‐speed supernatant (8,400,000 ×g× min). The possible involvement of microbial enzymes from the flora of the small intestine was excluded.

Factors affecting the conversion were studied with the microsomal fraction and with a low‐speed supernatant (377,000 ×g× min). Substantial conversions were obtained in the presence of NAD or an NADPH‐generating system separately, but maximum conversion was only obtained when these were present together. When air was replaced by carbon monoxide the conversion was inhibited by 42%.

Labelled n‐hexadecanol was detected as an intermediate and was also shown to be oxidised to palmitic acid.

The role of the enzyme system is discussed in relation to the occurrence of n‐alkanes in the diet and their absorption from the small intestine.
Fractionation on DEAE‐Sephadex Columns of some Oligonucleotides from Pancreatic Ribonuclease Digests of RNA from Bacteriophage μ2
Authors: Matthews H. R.
Published: March 2005

The hepta‐ octa‐ and nonanucleotides in a complete pancreatic ribonuclease digest of μ2 phage RNA were fractionated according to nucleotide composition. Together with the deca‐ and undecanucleotides this gave 19 separated components each with a unique nucleotide composition. Some are mixtures of sequence isomers, others may be homogeneous. The fractionation depended quantitatively on the net charge on the oligonucleotide between charge −1 and −4 at pH 2.7 in 7 M urea.
Amino Acid Control of RNA Synthesis in T4‐Infected Escherichia coli
Authors: Olsnes S. , Hauge J. G.
Published: March 2005

A comparison has been made of incorporation of [5‐3H]uracil after T4‐infection in several strains of Escherichia coli that show stringent control of RNA synthesis in the uninfected state. Some of the strains exhibited a greatly reduced initial rate of [3H]uracil incorporation when a required amino acid was removed. The nature of the reduced [3H]uracil incorporation was investigated for one strain and shown by hybridization competition to reflect a reduced rate of T4 mRNA synthesis. In other strains, [3H]uracil incorporation was not appreciably affected by amino acid starvation. RNA synthesized under these conditions entered polysomes at a high rate in spite of a low rate of protein synthesis.
Kinetics of Reversible Denaturation of Trypsin in Water and Water–Ethanol Mixtures
Authors: Pohl F. M.
Published: March 2005

The kinetics of the reversible denaturation of trypsin at pH 1–3 have been measured by following the change in absorption and optical rotation after temperature‐jumps. This structure‐change can be described by an equilibrium between two states of the protein. A kinetic mechanism, involving the binding of two protons to identical sites with differing pK in the two states, can account for the results. The temperature‐dependence of ΔH° is due to the renaturation step as indicated by the corresponding activation‐energy. Measurements in ethanol–water mixtures suggest that this might be caused by a change of solvation of non‐polar groups with temperature. Reasons for using this denaturation of trypsin as a model‐system for studying the kinetics of structure‐changes in proteins are discussed.
Preliminary X‐Ray Diffraction Study of the Wild‐Type and a Mutationally‐Altered Tryptophan Synthetase α Subunit
Authors: Schulz G. E. , Creighton T. E.
Published: March 2005

Single crystals of the tryptophan synthetase α subunit of Escherichia coli suitable for X‐ray diffraction analysis have been obtained. The diffraction pattern of a mutationally‐altered α subunit (α‐33) has been compared with that of the wild‐type subunit. No significant differences in unit cell dimensions or in relative intensities of individual reflections at low diffraction angles were apparent. It is concluded that the mutational replacement of the glutamic acid residue at position 48 by methionine causes no more than a small alteration of the subunit's conformation, although the enzymatic activity of the subunit has been abolished.
Sur la biosynthèse de la piloquinone, o‐phénanthrène‐quinone produite par Streptomyces pilosus
Authors: Zylber J. , Zissmann E. , Polonsky J. , Lederer E.
Published: March 2005

Piloquinone (m.p. 178°, C21 H20 O5), the principal pigment produced by Streptomyces pilosus had been isolated in 1963 in our laboratory and its structur determined [1, 2]. It is a 2‐methyl‐3‐iso‐caproyl‐1, 8 dihydroxy‐9, 10 phenanthrene quinone (I).

Quinones of this type are very rare in nature; for one of these: denticulatol (II) Birch has proposed a biosynthetic pathway from polycetate.

For piloquinone two biosynthetic schemes had been proposed [2]:

a) isocaproyl‐ or isobutyryl‐CoA could condens with respectively eight or nine acetate units to give precursor VIII, which would be transformed into piloquinone (I) by decarboxylation, cyclisation and appropriate modifications of the oxidation level;

b) propionyl‐CoA could condense with nine units of acetate to give the precursor IX which, after C‐methylation, would lead again to VIII. This latter hypothesis had been proposed because ɛ‐pyrromycinone (X) is fromed by way of the same intermediate (IX).

The following precursors were added to growing cultures of Streptomyces pilosus: [1‐14C]‐acetic acid, [1‐14C]propionic acid, [1‐14C]isobutyric acid, [U‐14C]valine, [14C‐methyl]methionine and [2‐14C]mevalonic acid.

Piloquinone (I) incorporated radioactivity from acetic acid, isobutyric acid, isocaproic acid and valine, but did not incorporate radioactivity from methionine, propionic acid, or mevalonate.

These results confirm hypothesis (a) and seem to exclude pathway (b).

In view of localizing the radioactivity in piloquinone isolated from cultures grown in presence of isocaproic acid, isobutyric acid or valine, the leuco‐acetate derivative of piloquinone was submitted to a Baeyer‐Villiger oxidation giving the tetraacetate‐ester XII. After hydrolysis, radioactive isocaproic acid was isolated as its p‐phenyl‐phenacylate.

Table 2 shows that it contains 11.8% of the radioactivity of piloquinone when [1‐14C]acetate was the precursor, and 93 or 98% when [U‐14C] valine or [1‐14C]isobutyrate were the precursors. These results show that isobutyrate (as such, or formed from valine) is the “starter” of the side chain of piloquinone whereas the rest of the carbon skeleton comes from polyacetate.

It seems that our work is the first experimental proof showing that phenanthrene derivatives can also be synthetised in vivo from a polyacetate precursor.
Spectroscopic Differentiation of the Electron‐Accepting Sites in Fungal Laccase
Authors: Malmström B. G. , Vänngård T. , Malkin R.
Published: March 2005

The difference specrum between oxidized and reduced fungal laccase at pH 5.5 shows an absorption band at 330 nm in addition to the visible absorption band at 610 nm.

The 610 nm band and the 330 nm band are reduced together in a linear fashion with the addition of 3.5 electron equivalents of ascorbate.

Titration of laccase in the presence of 3 mM fluoride leads to a differentiation of the electron‐accepting site in the molecule. The 610 nm band is found to be associated with a single electron‐accepting site and the 330 nm band is associated with a two electron‐accepting site. Electron paramagnetic resonance analysis during the titration indicates that neither the Type 1 Cu2+(the “blue” Cu2+) nor the Type 2 Cu2+ (the “non‐blue” Cu2+) are associated with the 330 nm band.

Titration of laccase at pH 8.3 with quinol also associates the 330 nm band with a two electron‐accepting site.

Preliminary stopped‐flow kinetic measurements on the absorbance changes at 330 nm and 610 nm are described and the results suggest an involvement of the component responsible for the 330 nm band in the catalytic reaction.

The nature of the component responsible for the 330 nm band is discussed in the light of these results and our previous titration results. The findings are consistent with the 330 nm band being associated with the two additional copper atoms in the molecule and agree with our previous suggestion that these copper atoms exist as an electron‐accepting Cu2+ ‐Cu2+ pair in the oxidized protein.
RNA Metabolism in Mammalian Cells at Elevated Temperature
Authors: Warocquier R. , Scherrer K.
Published: March 2005

RNA synthesis and metabolism were investigated in Hela cells heated to 42°. At this temperature celluar growth is arrested. After prolongued periods of preheating the initial rate of uridine incorporation into RNA is only slightly affected but no new stable RNA accumulates. After a shift down to 37° the normal pattern of RNA formation resumes.

45 S precursor to rRNA is still formed but its normal metabolism to functional rRNA is blocked. No new rRNA appears in cytoplasmic ribosomes. On the contrary, the synthesis of nascent messenger‐like RNA is only slightly affected and non‐ribosomal RNA appears in the cytoplasm at a normal rate. The temperature sensitive event seems thus to be selectively located in the metabolic chain producing functional rRNA.
Nucleotide Sequences from Specific Areas of the 16S and 23S Ribosomal RNAs of E. coli
Authors: Fellner P.
Published: March 2005

The primary structures of the 16S and 23S ribosomal RNAs of Escherichia coli have been studied. Nucleotide sequences have been determined which occur in specific areas close to the small numbers of methylated nucleotides which are present in these molecules. Fragments containing these methylated nucleotides were obtained by digestion of radioactive RNA substrates with T1 ribonuclease. Such digestion products were fractionated by high‐voltage electrophoresis in two dimensions.

The 23S RNA yielded predominantly 11 methylated oligonucleotides on digestion, and the 16S RNA gave rise to 6 methylated components. Nucleotide sequence analysis of these products was carried out, and the nature of the modified nucleotides occurring in each product was also examined. It was found that some of the sequences contained several modified components positioned close to each other. Quantitative studies indicate that each of the methylated sequences is probably present twice in a molecule of the 23S RNA, so that these sequences encompass about 5% of the nucleotides present in the entire molecule. It is postulated that extensive repetition of nucleotide sequences occurs within the 23S RNA.
Role of RNA Polymerase Stimulating Factor on Chain Initiation
Authors: Darlix J. L. , Sentenac A. , Ruet A. , Fromageot P.
Published: March 2005

Using T4 DNA as template, it has been shown that RNA polymerase purified by phospho‐cellulose chromatography (phospho‐cellulose enzyme) binds to DNA but does not initiate RNA chains. The stimulating factor (σ) released during chromatography does not bind to DNA, but associates readily to phospho‐cellulose enzyme and σ exhibits all the properties of undissociated RNA polymerase with respect to initiation and rate of RNA synthesis, as well as release of long RNA chains.
Heterogeneity of Erythrocyte Catalase
Authors: Mörikofer‐Zwez S. , Cantz M. , Kaufmann H. , von Wartburg J. P. , Aebi H.
Published: March 2005

Human and horse erythrocyte catalase is separated into three fractions (A, B and C) upon chromatography on DEAE‐cellulose. Fractions B and C are formed out of fraction A by oxidation of sulfhydryl groups during preparation.

A purification method, including chromatographies on DEAE‐ and CM‐cellulose was devised. The whole procedure was carried out under nitrogen and in presence of EDTA, yielding the pure native enzyme (fraction A).

Both the human and horse enzyme contain 16 cysteine residues per molecule, determined by titration with p‐chloromercuribenzoate and amino acid analysis of the fully carboxymethylated or the performic acid oxidized protein.

The transition of the chromatographic fraction A to fraction C is characterized either by a conformational change or by an irreversible oxidation of sulfhydryl groups to higher oxidation products.
Yeast Alcohol Dehydrogenase: –SH Groups, Disulfide Groups, Quaternary Structure, and Reactivation by Reductive Cleavage of Disulfide Groups
Authors: Bühner M. , Sund H.
Published: March 2005

Storage of yeast alcohol dehydrogenase in crystal suspension causes a decrease of the enzymatic activity which is strongly related to the number of free –SH groups. With a rate slower than the rate of inactivation the enzyme molecule (molecular weight reinvestigated from hydrodynamic measurements to be 141000) dissociates into four polypeptide chains (molecular weight 35000) which are enzymatically inactive. By sulfitolysis in 4 M urea and amperometric titration it was shown that free –SH groups are oxidized mainly to disulfide groups during inactivation.

The formation of disulfide bonds does not occur between –SH groups of different polypeptide chains. It is assumed that the inactivation process from the fully active enzyme molecule to the inactive polypeptide chains is not an all‐or‐none process but passes through three stages: 141000 (fully active) → 141000 (partially active) → 141000 (inactive) → 4 → 35000 (inactive). The more inactive the alcohol dehydrogenase, the more sensitive it is to heat denaturation and peptidase attack.

By treatment with mercaptoethanol or Cleland's reagent the disulfide bonds could be reduced and the enzymatic activity was recovered partially. The undissociated inactive molecules could be fully reactivated. With one of the investigated enzyme preparations which contains a peptidase it was also possible to reactivate and reassociate alcohol dehydrogenase from the polypeptide chains.
Elektronenspinresonanz‐Untersuchungen an Hämin‐Mercaptan‐Komplexen
Authors: Röder A. , Bayer E.
Published: March 2005

The oxidized cytochrome P‐450 shows an ESR‐spectrum with g‐values at 2.41, 2.26 and 1.91.

An attempt has been made to simulate these properties by studying the products of the reaction of a series of thiols with heminechloride in various solvents. g‐values have been obtained around g3= 2.4, g2= 22 and g1= 1.9, depending on the chemical nature of the thiol and the solvent.

The structure of the heme site in the oxidized cytochrome is discussed.