bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the Metabolism of C19 Steroids in Rat Liver Biosynthesis of Saturated 17‐Oxo‐C19O3 Steroids in Rat Liver Microsomes
Authors: Gustafsson Jan‐Åke , Lisboa Belisário P.
Published: March 2005

After incubation of 4‐androstene‐3,17‐dione, 5α‐androstane‐3,17‐dione, 3α‐hydroxy‐5α‐androstan‐17‐one and 3β‐hydroxy‐5α‐androstan‐17‐one with rat liver microsomes the following steroids were isolated and identified by thin‐layer chromatography and by gas chromatography‐mass spectrometry: 3α‐ and 3β, 16α‐dihydroxy‐5α‐androstan‐17‐one, 3α‐ and 3β, 7α‐dihydroxy‐5α‐androstan‐17‐one, 3β, 7β‐dihydroxy‐5α‐androstan‐17‐one and 3β, 17β‐dihydroxy‐5α‐androstan‐16‐one. The importance of the 17‐oxo and 17β‐hydroxy pathways in the metabolism of C19 steroids in rat liver is discussed.
The Active Site Cysteines of Thiolase
Authors: Harris J. Ieuan , Gehring Ulrich
Published: March 2005

Inactivation of thiolase with iodo[1‐14C]acetamide has been shown to be due to its reaction with cysteine residues in the enzyme. [1‐14C]Carbamoylmethylcysteine was identified as the only product of the reaction and a study of the radioactive peptides that were isolated from a tryptic digest of the [1‐14C]carbamoylmethyl‐enzyme has shown that the reactive cysteines occur in a unique sequence extending to at least 26 amino acid residues in the primary structure.

The enzyme‐substrate compound formed when thiolase reacts with [1‐14C]acetyl‐CoA is shown to contain [14C]acetyl groups bound in thioester linkage to at least three cysteines per mole. Moreover these residues of [14C]acetyl‐cysteine occur exclusively in a sequence that is identical to the sequence around the [14C]carbamoylmethylcysteine residues in the enzyme‐inhibitor compound, showing that the substrate and the inhibitior react with the same four cysteine residues in the enzyme.

These results provide additional evidence for the chemical identity of the four subunits of thiolase.
The Partial Dissociation of Nucleohistone by Salts
Authors: Henson Patricia , Walker Ian O.
Published: March 2005

The circular dichroism of calf thymus deoxyribonucleohistone was compared with that of samples partially depleted of histone in solutions of sodium chloride. Differences in the near ultraviolet circular dichroism of DNA and deoxyribonucleohistone were associated with the presence of the f2b, f2a2 and f3 histone fractions in the complex. The near ultraviolet circular dichroic spectra of both DNA and deoxyribonucleohistone were very sensitive to the solvent environment. Thus differences in the circular dichroic spectra of DNA and deoxyribonucleohistone cannot necessarily be associated with long‐range effects, such as supercoiling, but may be due to short‐range effects, such as might be caused by histone‐DNA interactions.

The absorbance and the circular dichroic melting profiles of deoxyribonucleohistone were biphasic. A new dichroic species was formed during the melting of the first of these steps and evidence is presented that this is a new histone‐DNA complex produced by the migration of some or all of the fractions f2b, f2a2 and f3 from denatured to native regions of the DNA during the melting process. Thus the biphasic melting profile of deoxyribonucleohistone does not necessarily imply that these fractions are arranged heterogeneously on the DNA.
Molecular Weight of Native and Dissociated Cysteamine Oxygenase
Authors: Cavallini Doriano , Cannella Carlo , Federici Giorgio , Duprè Silvestro , Fiori Anna , Del Grosso Elena
Published: March 2005

Cysteamine oxygenase has been further purified to a degree where it appeared homogeneous in disc gel electrophoresis. The purified enzyme possesses a molecular weight of 52000 when it is treated with either 8 M urea or with 0.1 % sodium dodecyl sulfate. The molecular weight of the native undissociated enzyme was 96000 when determined by dextran gel filtration and 92000 when determined by sedimentation equilibrium in the ultracentrifuge. These results are consistent with the occurrence of two similar subunits in the molecule of eysteamine oxygenase.
Metabolism of Steroids in Germfree and Conventional Rats Treated with a 3β‐Hydroxy‐δ5‐Steroid Oxidoreductase Inhibitor
Authors: Björkhem Ingemar , Gustafsson Jan‐Åke , Gustafsson Sven A.
Published: March 2005

The 3β‐hydroxy‐Δ5‐steroid oxidoreductase inhibitor 2α‐cyano‐4,4,17α‐trimethyl‐5‐androsten‐17β‐ol‐3‐one was administered intramuscularly to germfree and conventional, male and female, rats. Under the experimental conditions used, no steroids were found in faeces and urine from the male rats, but a large number of steroids were identified in excreta from female rats. The following 3β‐hydroxy‐Δ5‐steroids, none of which is present in normal rats, appeared in urine and faeces from germfree and conventional cyanoketone treated female rats (chiefly as mono‐sulphates): 5‐androstene‐3β, 17β‐diol, 3β, 7α(and 7β)‐dihydroxy‐5‐androsten‐17‐one, 5‐androstene‐3β, 7α(and 7β), 17β‐triol, 3β, 16α‐dihydroxy‐5‐androsten‐17‐one, 3β, 17β‐dihydroxy‐5‐androsten‐16‐one, 3β‐hydroxy‐5,16‐pregnadien‐20‐one, 3β‐hydroxy‐17α‐pregn‐5‐en‐20‐one, 3β‐hydroxy‐5‐pregnen‐20‐one, 3β,7α(and 7β)‐dihydroxy‐5‐pregnen‐20‐one, 3β, 17α‐dihydroxy‐5‐pregnen‐20‐one, 3β, 16β‐dihydroxy‐5‐pregnen‐20‐one, 5‐pregnene‐3β, 16α, 20α‐triol, 5‐pregnene‐3β, 17α, 20α‐triol and 3β, 21‐dihydroxy‐5‐pregnen‐20‐one. In addition, several saturated steroids were identified which were identical with steroids excreted by normal germfree and conventional female rats.

3β, 16α‐Dihydroxy‐5‐pregnen‐20‐one normally accounts for less than 1% of the total amount of 3β, 16α‐dihydroxy‐5‐pregnen‐20‐one and 3α, 16α‐dihydroxy‐5α‐pregnan‐20‐one found in urine. This percentage was found to be about 70–80 in urine from cyanoketone treated germfree as well as conventional female rats. This figure is in good agreement with the extent of inhibition (75–85%) of adrenal and gonadal (testicular and ovarian) 3β‐hydroxy‐Δ5‐steroid oxidoreductase found in experiments in vitro. It was established that the formation of saturated steroids from 3β‐hydroxy‐Δ5‐steroids in cyanoketone treated rats involved 3‐oxo‐steroids as intermediates since [3α‐3H,4‐14C]pregnenolone was converted into saturated steroids with loss of 3H. The hepatic 3β‐hydroxy‐Δ5‐steroid oxidoreductase active on C27 steroids was unaffected by treatment in vivo or in vitro with the cyanoketone.
The Enzymatic Breakdown of Pantethine to Pantothenic Acid and Cystamine
Authors: Duprè Silvestro , Graziani Maria Teresa , Rosei Maria Anna , Fabi Andrea , Del Grosso Elena
Published: March 2005

The enzyme splitting pantethine into pantothenic acid and cystamine has been purified about 3000 times starting from horse kidney. The purification procedure is reported. The enzyme, whose optimal activity lies in the pH range 4.0–5.5, requires the presence of a reduced thiol for the full activity: mercaptoethanol and dithiothreitol give the higher effect. This activation seems not to be specific. The enzyme has a Km value for the substrate of the order of 5 mM and shows inhibition by excess substrate and by products. Substrate specificity studies show that the enzyme splits only the intact substrate molecule, which is not hydrolyzed by several hydrolytic enzymes, even with a broad specificity (as bacterial pronase and subtilisin). The enzyme could be responsible for the production in vivo of cysteamine, which is known to be oxydized by a specific oxygenase, isolated recently in this laboratory.
Acid Protease from Germinated Sorghum
Authors: Garg Govind K. , Virupaksha Tumkur K.
Published: March 2005

The substrate specificity of the sorghum acid protease was elucidated using synthetic peptides and pancreatic ribonuclease A as substrates. These studies revealed that the enzyme has a rather narrow and novel specificity requirement. The protease specifically cleaves the peptide linkages involving the α‐carboxyl group of either aspartic acid or glutamic acid with the release of the acyl portion of these acidic amino acids. Further, the side chain carboxyl groups of aspartic acid and glutamic acid in the peptides should be unsubstituted for the enzyme to act on the peptide, and therefore, glutaminyl and asparaginyl peptides are not cleaved by the enzyme. Studies carried out with performate oxidized ribonuclease and ribonuclease methyl esters have further substantiated the results obtained with the synthetic substrates. These results suggest that the sorghum acid protease will be very valuable in sequence analyses of proteins.
Structural Studies on Ovine Growth Hormone
Authors: Peña Clara , Paladini Alejandro C. , Dellacha Juan M. , Santomé José A.
Published: March 2005

The cyanogen bromide fragments obtained from ovine growth hormone have been isolated and chemically characterized. Two N‐terminal peptides 4 and 5 amino acids long and a C‐terminal dodecapeptide have been identified among them. The sequence of these fragments has been established and found identical to the corresponding peptides in bovine growth hormone.

All the information obtained gives further support to the already known analogy between ovine and bovine growth hormones.
Incorporation of Inorganic Phosphate into Phospholipids by the Homogenate and by Sub‐Cellular Fractions of Rat Liver
Authors: Williams Mary L. , Bygrave Fyfe L.
Published: March 2005

Optimal incorporation of [32P]phosphate into phospholipids by rat liver homogenates requires the presence of NADH, Fru‐1,6‐P2 and of CMP. In the absence of 5 mM NADH and of 10 mM Fru‐1,6‐P2 little incorporation occurs.

Using the experimental conditions for optimal incorporation into the phospholipids by the rat liver homogenate, attempts to incorporate [32P]phosphate into the major phospholipids of mitochondria and of microsomes were undertaken. Incorporation into either microsomes or mitochondria alone was negligible. The addition of the cytosol (supernatant) to microsomes stimulated the incorporation but had little effect on the incorporation into mitochondria. Maximal incorporation of [32P]phosphate into the major phospholipids occurred only when mitochondria, microsomes and cytosol were present together in the reaction medium.

It is considered on the basis of these findings that the incorporation of the bases choline and serine into mitochondrial phospholipids observed in previous studies reflect exchange reactions and not de novo synthesis of the phospholipids by the mitochondria.
The Stereochemistry of α‐Oxidation of Fatty Acids in Leaves
Authors: Hitchcock Christopher H. S. , Morris Lindsay J.
Published: March 2005

When [U‐14C]palmitic acid is incubated with a particulate fraction obtained from young pea leaves, [14C]pentadecanal accumulates. L‐[U‐14C, 2‐3H]Palmitic acid similarly yields [14C]pentadecanal which retains the tritium (90% retention). D‐[U‐14C, 2‐3H]Palmitic acid gives [14C]‐pentadecanal with loss of tritium (13% retention). These results suggest that D‐2‐hydroxypalmitic acid is an intermediate in this reaction, but that L‐2‐hydroxypalmitic acid and 2‐ketopalmitic acid are not. The mechanism of α‐oxidation of palmitic acid in leaves is discussed.
Kinetics of Structural Transitions Induced by pH Changes
Authors: Bellon Bernard R. , Delaage Michel A.
Published: March 2005

Bovine trypsinogen, trypsin and chymotrypsinogen undergo an isomerization in acidic medium, like many other proteins. We have studied the kinetics of these isomerizations by means of pH changes in a stopped‐flow apparatus. The order of magnitude of the rate constants is 20–100 sec‐1. The model used to interpret the effect of pH on kinetics leads to an unequivocal identification of at least one carboxylate group buried in the native form of these proteins. The effects of temperature and ionic strength on the kinetics are described.
The Amino Acid Sequence of the β‐Chains of Rabbit Hemoglobin
Authors: Galizzi Allessandro
Published: March 2005

A partial formula of the primary structure of the β‐chain of rabbit hemoglobin is shown. Two variants of the β‐chain of rabbit hemoglobin have been found. One variant has β52His, β56Ser and β76Asn. The other variant has β52Asn, β56Asn and β76Ser. These variants are not strain specific.
Factors Governing the Kinetics and Steady State of the Mitochondrial Nicotinamide Nucleotide Transhydrogenase System
Authors: Rydström Jan , Teixeira da Cruz António , Ernster Lars
Published: March 2005

The mitochondrial nicotinamide nucleotide transhydrogenase catalyzes a rapid reduction of NAD+ by NADPH as compared to the reduction of NADP+ by NADH. By assuming pseudo first‐order kinetics it is shown that the rate constant of the reaction is strongly dependent on the actual [NAD+] [NADPH] / [NADP+] [NADH] ratio, giving a half‐maximal rate constant at [NAD+] [NADPH]/[NADH] [NADP+] of approx. 0.003. The altered rate constant can possibly be explained on the basis of a reversible conformational change of the enzyme between an active and an inactive state, controlled by the prevailing concentrations of oxidized and reduced nicotinamide nucleotides. Possible similarities with bacterial transhydrogenases are considered.

In the presence of an energy source the overall transhydrogenase reaction involves a strong shift of the equilibrium towards the conversion of NADH and NADP+ into NAD+ and NADPH. Evidence is presented that the final extent of the overall energy‐linked transhydrogenase reaction is strikingly dependent on the ratio of the initial rates of the energy‐linked reduction of NADP+ by NADH and the nonenergy‐linked reduction of NAD+ by NADPH. Thus, the final extent of the transhydrogenase reaction in the presence of an energy source can by explained in terms of a steady state rather than an equilibrium. The possibility of an energy‐linked conformational change of transhydrogenase, involving a conversion of the enzyme from an inactive to an active state, is considered.
Coat Protein Repression of Bacteriophage M12 RNA Directed Polysome Formation
Authors: Ward Richard , Konings Ruud N. H. , Hofschneider Peter Hans
Published: March 2005

Phage M12 RNA, serving as a messenger for protein synthesis in vitro, is incorporated into polysomal structures containing up to ten ribosomes per RNA molecule. If this RNA messenger is repressed by M12 coat protein prior to its translation, it binds a maximum of four ribosomes per RNA genome. This difference in polysome distribution is similar to the size difference between phage specific polysomes formed at early and late times of viral development in the infected cell. Such a finding indicates that the theory of translational repression, as it has been proposed from previous experiments in vitro, is also a major control mechanism of phage protein synthesis in vivo.
The tRNA Pyrophosphorylase Activity of Escherichia coli. A Study on Substrate Specificity
Authors: Gross Hans J. , Duerinck Fred R. , Fiers Walter C.
Published: March 2005

tRNA pyrophosphorylase was partially purified from Escherichia coli MRE 600 cells. Upon chromatography on a DEAE‐cellulose column, two enzymatic activity peaks were partially resolved. Gel filtration indicated a molecular weight of approx. 42000 dalton for both. Some general properties are reported. The only consistent difference between the two enzyme fractions was that the first incorporated more AMP‐residues for a given tRNA preparation, as if the second has a more restricted specificity with respect to the acceptor tRNA molecules in the adenylation reaction.

Partial degradation of tRNA by combined digestion with ribonuclease T1 and spleen exo‐nuclease indicated a rapid removal of approx. 16 nucleotides per tRNA molecule, presumably from more exposed regions. The amino acid acceptor activity was more sensitive to this nuclease pretreatment than the 3′‐terminal adenylic acid acceptor activity. Direct analysis showed that RNA fragments of chain length approx. 40 and even 10 were still functional in accepting terminal adenylic acid.
Association de RNA repidement marqué avec des structures cytoplasmiques dans les tumeurs plasmocytaires de la souris
Authors: Kempf Jules , Zahnd Jean‐Pierre , Mandel Paul
Published: March 2005

The cytoplasmic fraction of mouse plasmocytoma cells contains messenger RNA‐protein particles similar to those called “informosomes” by Spirin [1,2]. Informosomes can be found in free form or bound to ribosomes, thus forming polysomes of buoyant density in CsCl ϱ∼ 1.55. In plasmocytoma cells, polysomes can in turn build up more complex structures of buoyant density ϱ∼ 1.4, by association with low‐density components of protein or lipoprotein nature.

Evidence for the occurence of the different components in the ϱ∼ 1.4 density complexes is produced. Short‐time labelling and high sensitivity to mild ribonuclease treatment may account for the existence of messenger RNA. Presence of ribosomes and lack of membrane fragments are demonstrated by electron microscopic studies. Results obtained after detergent treatment favour the involvement of a lipoprotein fraction.

It is noteworthy that the bulk of the short‐time labelled messenger sediments with the 1.4 density material, either as free informosomes, or as informosome‐ribosome‐lipoprotein complex. Our results combined with further studies suggest that the latter complex has a specific role in protein synthesis.
Replication of the Single Stranded DNA Bacteriophage M 13
Authors: Wirtz Antje , Hofschneider Peter Hans
Published: March 2005

The intracellular flow and the transfer to progeny phages of the single stranded parental DNA of the filamentous phage M13 has been studied in Escherichia coli KMBL 42 at low multiplicities of infection (<1). The cellular bound parental DNA was found to be first converted quantitatively into replicative form DNA. Then, contrary to the events in phage φX 174 infection the parental DNA is further transferred as a complete molecule to the pool of single stranded progeny DNA and to progeny phage particles. The efficiency of transfer per infecting phage is approximately as high as 0.75 and 0.5, respectively. Evidence is presented that during transfer the parental DNA is incorporated into DNA strands having up to several times the length of single stranded phage DNA.
An Investigation of the Conformational Changes of Histone F2b by High Resolution Nuclear Magnetic Resonance
Authors: Boublík Miloslav , Bradbury E. Morton , Crane‐Robinson Colyn , Johns Ernest W.
Published: March 2005

The recent sequence determination of histone F2b has allowed a detailed nuclear magnetic resonance study of the conformational changes and interactions of this protein with (a) increasing ionic strength, (b) increasing concentration and (c) change of pH. In all cases it has been found that a specific region of the polypeptide chain is involved in the induced changes: with increasing ionic strength the changes and interaction can be located in the segment of the polypeptide chain from residues 60 to 102; part of this segment is also involved in the specific aggregation that occurs on increasing the concentrations, while for the pH induced changes the same segment together with additional residues at each end is involved in the observed changes.
Acid Phosphomonoesterase from Phaseolus mungo. Purification and Characterization
Authors: Felenbok Beatrice
Published: March 2005

Purification of acid phosphomonoesterase from Phaseolus mungo seedlings is reported here. Phosphatase homogeneity was evaluated at 85—90% by several methods. The enzyme is a single polypeptide chain of molecular weight 55000 ± 5000 daltons with serine and leucine as the N and C terminal residues, respectively. A saccharidic fraction containing 8% of protein weight as reducing sugar and 3% as amino sugars was found. Attempts to dissociate the saccharidic and protein moieties were unsuccessful. It was concluded that a single phosphatase was active toward phosphomonoesters, phosphoanhydride, and β‐D‐glycosyl 1‐phosphate.
Hormone Action on Liver Adenyl Cyclase Activity
Authors: Hepp K. Dieter , Edel Roswitha , Wieland Otto
Published: March 2005

Adenyl cyclase activity was measured by conversion of [32P]ATP to adenosine 3′:5′‐cyclic [32P]phosphate in a washed particle suspension from mouse and rat liver. The test contained an ATP regenerating system and 10 mM unlabeled Ado‐3′:5′‐P to trap the labeled Ado‐3′:5′‐P formed. Decreased activities were observed when methyl xanthines were used in place of unlabeled Ado‐3′:5′‐P. The reaction was stimulated by Mg++ which increased Vmax without changing the apparent Km towards ATP. Adenyl cyclase activity was stimulated by synthetic glucagon over the range of 0.1–20 μg/ml. Removal of Ca++ by ethylene glycol‐bis‐(β‐amino‐ethyl ether)‐N,N'‐tetraacetic acid (EGTA) strongly augmented glucagon action, while 1 mM Ca++ was inhibitory. The data suggest enhanced transmission of the hormone signal from the (hypothetical) receptor to the catalytic enzyme unit by chelation of membrane bound calcium.

In contrast to the hyperbolic dose response curve observed with glucagon, the curve with fluoride (1–10 mM) was sigmoidal in shape, the effect being inhibited by 1 mM pyrophosphate. Although inhibition by Ca++ was observed, EGTA did not augment the effect of fluoride. The effects of fluoride and glucagon at maximal concentrations were not additive, indicating stimulation of the same adenyl cyclase system.
Studies on Rabbit Immunoglobulin Heavy Chains
Authors: Florent Gerard , van der Loo Wessel , Hamers Raymond
Published: March 2005

Large peptides containing the allotypic determinants can be isolated from tryptic digests of rabbit IgG reduced with dithiothreitol.

Their antigenic activity is detected by simple screening for interference in the allotype anti‐allotype reaction.

Active peptides are obtained for A1 A2 and A3 allotypes and carry apparently the major determinants involved in the allotype anti‐allotype reaction.

Further characterisation of these peptides is in progress.
The Phosphoglycerate Kinase Reaction and its Metal Ion Specificity
Authors: Larsson‐Raźanikiewicz Märtha
Published: March 2005

The present study shows that MgATP2‐ or MnATP2‐ as substrates of phosphoglycerate kinase can be replaced by CaATP2‐, ZnATP2‐, CoATP2‐ or NiATP2‐. MnATP2‐ and NiATP2‐ are about 90% and 15%, respectively, and the other ATP4‐‐metal ion complexes roughly 70% as good substrates as MgATP2‐. No measurable activity was found with Be(II) or Fe(III). The effectiveness of the different substrate species appears to be determined by factors such as the size and polarizing capability of the metal ion as well as of the structure of the relevant metal ion complex and the rate of ligand dissociation processes.

Detailed kinetics with Zn(II), Mn(II) and Co(II) showed that: (a) Zn2+ is a strong uncompetitive inhibitor of ZnATP2‐, Ki approx. 0.02 mM. (b) Mn2+ is a competitive inhibitor of MnATP2‐ at concentrations < 0.1 mM, Ki approx. 2.3 mM. This inhibition is dependent on the 3‐P‐glycerate concentration. At about 1 mM and higher concentrations Mn2+ acts as an uncompetitive inhibitor of MnATP2‐. (c) Co2+ is a competitive inhibitor of CoATP2‐ at about 1 mM and higher concentrations, Ki approx. 3 mM. With CoATP2‐ as substrate the activity is slightly increased in the presence of free Co2+ and/or free ATP4‐ at concentrations < 0.5 mM. When the CoATP2‐ concentration is varied, the activity seems not to become constant at concentrations ten times the apparent Michaelis constant for CoATP2‐.

Equilibrium dialysis studies on the binding of Co2+ to the enzyme showed that this ion binds more strongly to the enzyme than, for example, Mn2+. This probably explains the differences in activation and inhibition observed with these two ions.

The kinetic patterns obtained with the different metal ions indicate that Ni(II) behaves as Co(II), Cd(II) as Zn(II) and Ca(II) as Mg(II), which is somewhat similar in behaviour to Mn(II).
An Enzymatic Method for the Determination of dCTP and dGTP in Picomole Amounts
Authors: Skoog Lambert
Published: March 2005

A rapid and accurate method for the measurement of picomole amounts of dCTP and dGTP is described. The method is based upon the ability of DNA‐polymerase, in the presence of poly[d(I‐C)] primer template, to incorporate dCTP and dGTP into an acid‐insoluble product. When either dCTP or dGTP is the limiting factor in the reaction, the extent of polymerization can be measured by the extent of incorporation of complementary radioactively labeled excess nucleoside triphosphate into an acid‐insoluble product. The incorporation of radioactivity was found to be directly proportional to the amount of the limiting nucleoside triphosphate. It was possible to measure as little as 0.2 pmole of either nucleoside triphosphate. The optimal conditions for the assay were determined in terms of pH, magnesium requirement, poly[d(I‐C)] concentration, and enzyme concentration.
Hydrolyse trypsique de dérivés d'amino‐acides non substitués en α
Authors: Seydoux François
Published: March 2005

The tryptic hydrolysis of l‐lysine methyl ester, l‐lysine paranitroanilide and l‐tyrosine methyl ester has been studied as a function of pH.

In addition, these reactions have been studied in the presence of methanol as a nucleophilic reagent wich competes with water in the deacylation step.

With lysine methyl ester, a bell‐shaped profile for Kcat vs pH has been observed. The optimum pH for this substrate occurs at pH 5.8. Km decreases 200‐fold when the pH is increased from pH 5 to 8.5. From the nucleophilic competition data, it can be deduced that the k2/k′3 ratio increases between pH 4.8 and 5.8 with deacylation as rate‐determining step.

With tyrosine methyl ester, the profiles of both kcat and Km vs pH are bell‐shaped, but the pH optimum in this case occurs at a pH higher than with lysine methyl ester (pH 7.5). The nucleophilic competition data indicate that the acylation step is rate‐limiting for this substrate.

With lysine paranitroanilide, kcat increases when the pH is increased from 5 to 6.5, but at higher pH, the kinetic behavior of this substrate is complicated by a non‐Michaelian saturation phenomenon, probably related to transpeptidation.

From the quantitative interpretation of our experimental data, the following conclusions could be drawn:

The conjugate acids are more reactive than their corresponding α‐amino acid derivatives.

In the acylenzyme formed with bicationic conjugate acid of lysine methyl ester, the active imidazole pK is strongly displaced from its normal value to the acidic region. (This pK is lowered from 6.90 in the free enzyme to 4.80 in this acylenzyme.)

These two first points explain the anomalous value of the pH optimum for this substrate.

The pK shift is less pronounced in the Michaelis complex formed with the conjugate acid of tyrosine methyl ester, lysine methyl ester and lysine paranitroanilide. Since this pK shift can be interpreted as resulting from a repulsive electrostatic interaction between the imidazolium group of enzyme and the α‐ammonium group of the substrate, our data suggest a change of the active site geometry (including substrate) after the acylation step.

The comparison of the kinetic parameters obtained in this work with those of corresponding α‐N‐acyl derivatives suggests that the α‐peptidic linkage is an important structural factor for the acylation step but not for the deacylation.

Finally, the effect of possible transpeptidation reaction on the kinetic data is discussed.
The Occurrence of 4‐Amino‐4‐deoxy‐l‐arabinose as a Constituent in Salmonella Lipopolysaccharide Preparations
Authors: Volak Wesley A. , Galanos Chris , Lüderitz Otto
Published: March 2005

When the lipopolysaccharides of two Salmonella R mutants were hydrolyzed in 0.5 N HC1 at 37° for 7 to 24 h, a previously undescribed sugar was liberated. This sugar has been identified as 4‐amino‐4‐deoxy‐l‐arabinose by comparison of the free amino sugar, its two reduced derivatives, its two N‐acetylated derivatives, and its methyl glycoside with the authentic amino sugar and its corresponding derivatives on thin layer, gas liquid, and column chromatography. A survey of about 50 enterobacterial lipopolysaccharides revealed that this amino sugar is present in a large percentage of the preparations. The preparation of 4‐amino‐4‐deoxyarabinose by the reduction and limited periodate oxidation of 2‐amino‐2‐deoxymannose is described.
The Cu2+‐Alkaline Phosphatase of Escherichia coli
Authors: Lazdunski Claude , Chappelet Danielle , Petitclerc Claude , Leterrier François , Douzou Pierre , Lazdunski Michel
Published: March 2005

The Cu2+‐alkaline phosphatase is considerably less active than the Zn2+‐ and Co2+‐enzymes. It also differs from the native Zn2+‐alkaline phosphatase by its sensitivity to nucleophiles and ionic strength. However, in spite of these differences, there remains an important common property, the considerable analogy between the kinetics parameters (Km and Vmax) for substrates with very different chemical structures.

The Cu2+‐enzyme, like the Zn2+‐enzyme, is phosphorylated on both sites at acidic pH with the substrate AMP or the product inorganic phosphate. No phosphorylation occurs at alkaline pH where phosphorylation of the active centers is the rate‐limiting step. Only non‐covalent complexes containing one mole of inorganic phosphate per mole of enzyme could be isolated. The binding of another phosphate on the second site appears to be very difficult and to occur only at high concentrations of inorganic phosphate. Anticooperativity, an important property of the Zn2+‐phosphatase is preserved in the Cu2+‐phosphatase.

Electron paramagnetic resonance indicates that the 4 coppers of the Cu2+‐alkaline phosphatase belong to 2 different classes. There are 2 tight metal sites of type I and 2 loose metal sites of type II. Orthophosphate binding to the Cu2+‐phosphatase alters both the visible and the electron paramagnetic resonance spectra. Electron paramagnetic resonance provides direct evidence for the participation of the coppers of type I in substrate binding. This is further evidence that each active site in the metallo‐phosphatases contains 2 metal atoms, one of type I and one of type II.
Ein Beitrag zur Isolierung und Charakterisierung des C‐Inaktivators aus Humanplasma
Authors: Haupt Heinz , Heimburger Norbert , Kranz Theodor , Schwick H. Gerhard
Published: March 2005

A simple procedure for the isolation of C‐Inactivator from fresh human ACD plasma is described consisting of the following fractionation steps:

DEAE chromatography, ammonium sulfate precipitation, Sephadex G‐150 gel filtration and zone electrophoresis. A 250‐fold concentration was achieved in good agreement with an immunologically determined C‐Inactivator concentration of 25 mg/100 ml serum.

C‐Inactivator is a glycoprotein containing 35% carbohydrates; it migrates with the α2‐globulins at pH 8.6 and in the PS‐1 zone at pH 4.4. The isoelectric point is between 2.7–2.8. It had s20,w of 3.67 and a molecular weight of 104000 according sedimentation equilibrium patterns. C‐Inactivator could not be dissoziated in subunits even not by reduction with 2‐mercaptoethanol and subsequent alkylation with iodoacetamide. 1 mg C‐Inactivator neutralizes 17 C‐Inactivator units respectively 5 Remmert and Cohen plasmin units. Only weak affinities for trypsin and chymotrypsin were found.
A Sensitive Technique for Detecting and Estimating the Peptide Hormone Angiotensin‐II‐β‐amide and its Antibodies by Using Chemically Modified Bacteriophage and Activated Sepharose
Authors: Hurwitz Esther , Dietrich Felix M. , Sela Michael
Published: March 2005

Angiotensin‐II‐β‐amide was conjugated to bacteriophage T4 using glutaraldehyde as a cross‐linking agent. The conjugated phage can be inactivated by anti‐angiotensin antibodies and this reaction may be used to detect antibodies at a concentration as low as 100 picograms/ml. Angio‐tensin II specifically inhibits the inactivation of the conjugated phage by antibodies and as little as 0.5 pg of it was detectable by this system. The purification of antibodies to angiotensin on an angiotensin‐Sepharose immunoadsorbent permitted the evaluation of the antibody concentration in the serum.
Characterization of a 72 S Mitochondrial Ribosome from Saccharomyces cerevisiae
Authors: Schmitt Henri
Published: March 2005

A 72 S ribosome can be isolated from EDTA‐washed mitochondria of Saccharomyces cerevisiae.

It easily dissociates into 38 S and 50 S subunits. Incorporation of amino acids into the protein of mitochondria purified in this way is shown to be inhibited by chloramphenicol, while cycloheximide has no effect.

When EDTA treatment of the mitochondria is omitted, the mitochondrial fraction contains in addition a special class of 80 S ribosomes, presumably membrane‐bound, which dissociate less easily than the “free” cytoplasmic ribosomes.

Incorporation of amino acids into protein of these mitochondria is only partially inhibited by either chloramphenicol or cycloheximide and is stimulated by the addition of a cytoplasmic supernatant.
Effect of Cholestyramine and Diet on Hydroxylations in the Biosynthesis and Metabolism of Bile Acids
Authors: Johansson Gunnar
Published: March 2005

The effects of biliary drainage, cholestyramine treatment, starvation and a semisynthetic diet on nine different hydroxylations in the biosynthesis and metabolism of bile acids were studied. Biliary drainage and feeding a diet containing 3% cholestyramine resulted in a five‐ to seven‐fold stimulation of the 7α‐hydroxylation of cholesterol. Biliary drainage resulted in a five‐fold stimulation of the 12α‐hydroxylation of 7α‐hydroxy‐4‐cholesten‐3‐one. The other hydroxylations as well as the formation of 7α‐hydroxy‐4‐cholesten‐3‐one from 5‐cholestene‐3β,7α‐diol were unaffected by biliary drainage and cholestyramine treatment. Starvation stimulated the 12α‐hydroxylation of 7α‐hydroxy‐4‐cholesten‐3‐one about four‐fold. Feeding a fat‐free, semisynthetic diet resulted in a decrease in the rate of all reactions.

The results support the concept that the 7α‐hydroxylation of cholesterol is a major rate‐limiting reaction in the biosynthesis of bile acids and indicate that also the 12α‐hydroxylation of 7α‐hydroxy‐4‐cholesten‐3‐one may be rate limiting to some extent.
Messenger RNA in HeLa Cells:
Authors: Spohr Georges , Granboulan Nicole , Morel Carlos , Scherrer Klaus
Published: March 2005

Messenger RNA (mRNA) and messenger like RNA (mlRNA) were investigated in the cytoplasm of HeLa cells while ribosomal RNA synthesis was arrested. Under these conditions, functional mRNA associated in polyribosomes and cytoplasmic free mlRNA are formed and can be labelled selectively to steady state.

All cytoplasmic non‐ribosomal RNA sedimenting at more than 6–7 S exists in the form of ribonucleoprotein complexes which pre‐exist in the cell, and are stable upon cell lysis, sedimentation and (after fixation) CsCl density gradient analysis. The functional, true mRNA is contained in a complex of mRNA and protein which bands in association with ribosomes (ϱ= 1.52 to 1.60 g/cm3) in CsCl density gradients or, released by EDTA, at its own intrinsic density of 1.40–1.48 g/cm3. The cytoplasmic free mlRNA bands as a particle of mlRNA‐containing ribonucleoprotein at an identical low density. The molecular weight spectrum of mRNA is identical to that of mlRNA and the sedimentation pattern of the mRNA · protein complex released from polyribosomes is similar to that of the free mlRNA · protein complex.

The physico‐chemical separation of mRNA · and mlRNA · protein complexes allowed us to follow their relative kinetics of synthesis and decay. Each type of ribonucleoprotein obeys a different, strictly time‐dependent pattern. Label enters the pool of free mlRNA · protein complexes first and may, in a pulse‐chase experiment, be partially chased into polyribosomes. At steady state (6 h) 40–60% of the labelled RNA remains in the form of free mlRNA · protein particles. These cannot be chased into polyribosomes, the kinetics of mRNA · and mlRNA · protein complexes decay following identical patterns.

These findings are in agreement with a model according to which mlRNA from the nucleus first joins the pool of free ribonucleoprotein. Then, the activated mRNA · protein complexes attach to ribosomal subunits and form polyribosomes whereas inactivated mlRNA · protein complexes remain free in the cytoplasm.

In order to further strengthen the evidence in favour of the real existence of mRNA · and mlRNA · protein complexes in the cells, the corresponding fractions from sedimentation or CsCl density gradients were observed in the electron microscope. By this method it was possible to see small cytoplasmic particles which have not before been identified. The rounded structures, with diameters ranging from 100 Å to 200 Å, seem to consist of the coiling of a 35 Å wide pearllike chain which may also be identified in polyribosomes. The frequency of these particles is highest in the mlRNA · protein band (ϱ= 1.40–1.48 g/cm3). Thus they may correspond to the mRNA · protein complex. However since they share some morphological features with other known biological structures the evidence is not conclusive.
Mammalian Cell‐Free Protein Synthesis Directed by Viral Ribonucleic Acid
Authors: Mathews Michael B. , Korner Asher
Published: March 2005

A cell‐free system from mouse Krebs II ascites cells is described which responds by increased amino acid incorporation into protein on addition of encephalomyocarditis virus (EMC‐RNA). RNA from other sources does not produce this response.

The stimulation by EMC‐RNA occurs over a narrow range of Mg2+ concentrations and is maximal at 5 mM, which is optimal for the endogenous incorporation and lower than that required in the presence of poly(U).

The EMC‐RNA‐directed product is distinguished from the endogenous products by its size and composition.

After an initial lag of 4 min, during which the nascent chains are too short to be acid‐insoluble, EMC‐RNA‐directed protein synthesis is active for more than 1 h.

EMC‐RNA‐directed synthesis is particularly sensitive to the inhibitors cycloheximide and dextran sulphate. Using the latter, it has been shown that the initiation of protein synthesis is completed during the first 15 min of incubation.

Only 20–30% of the product of the cell‐free system, whether endogenous or EMC‐RNA‐directed, is released from the ribosomes. Despite the size of the viral RNA, it did not appear to promote the formation of exceptionally large polysomes.

It is concluded that the EMC‐RNA is acting as a message for viral proteins in the ascites cell‐free system.
The Inhibitory Action of a Mammalian Viral RNA on the Initiation of Protein Synthesis in a Reticulocyte Cell‐Free System
Authors: Mathews Michael B. , Korner Asher
Published: March 2005

RNA from mouse encephalomyocarditis virus inhibited endogenous protein synthesis in reticulocyte cell‐free systems and was not, itself, translated. The inhibition was caused by interference with globin chain initiation. The inhibitory effect was observed under a variety of experimental conditions, including those where the Krebs II ascites cell system translates the viral RNA. Preincubation of the reticulocyte lysate increased the severity of the inhibition because it increased the dependence of endogenous protein synthesis upon reinitiation. Reticulocyte and ascites cell ribosomes bound encephalomyoearditis [3H]RNA to cellulose nitrate filters with equal efficiency. RNA from bacteriophage f2 and tobacco mosaic virus also appear to inhibit globin chain initiation.
Sterol Biosynthesis in Euglena gracilis Z
Authors: Brandt Roger D. , Pryce Robert J. , Anding Claude , Ourisson Guy
Published: March 2005

Free and bound (esterified and water solubilised) sterols of light grown (green) and dark grown (white) Euglena gracilis Z have been characterised by thin layer chromatography, gas chromatography and mass spectrometry. Differences in sterol metabolism by these two types of E. gracilis are reflected in profound quantitative and qualitative differences in their sterol compositions. Free sterols predominate over bound sterols in green E. gracilis whereas in white E. gracilis the reverse is true. The free sterols of green E. gracilis consist almost exclusively (98%) of Δ7‐sterols while in white E. gracilis they are a mixture containing Δ5‐ (31%) and Δ7‐sterols (47%). However, in both types of E. gracilis only Δ5‐sterols are bound in esterified and water soluble forms.

The following sterols were found in green and white E. gracilis: Δ7‐cholestenol, Δ5,7‐cholestadienol, cholesterol, episterol, chalinasterol, Δ7‐ergostenol, 5‐dihydroergosterol, Δ7‐chondrillasterol, Δ5,7‐chondrillasterol, Δ22‐dihydrobrassicasterol, clionasterol and poriferasterol. Chondrillasterol, the major sterol in green E. gracilis, was not found in white E. gracilis. Cholesterol, a minor constituent of free sterols of green (1%) and white (12%) E. gracilis, is the major constituent of their bound, water soluble sterols.
Hefe‐Phosphofruktokinase
Authors: Freyer Renate , Kubel Martin , Hofmann Eberhard
Published: March 2005

The kinetic behaviour of different preparations of yeast phosphofructokinase is described, predominantly regarding the interactions between substrate‐ and effector‐binding sites.

The substrate‐binding site for ATP reacts with ITP and UTP too, whereas the ATP‐inhibitor site possesses higher specifity: other nucleoside triphosphates do not compete with ATP for this site.

Binding of protons to the enzyme produces a decrease in cooperativity towards fructose‐6‐phosphate with ATP as well as with ITP and UTP as phosphate donors, accompanied by a lowering of the fructose‐6‐phosphate concentration necessary for half‐maximal activity. At the same time the inhibitory action of ATP is descreased.

AMP is bound to an effector site evidently different from the ATP‐inhibitor site.

With ITP as phosphate donor, AMP shows no activating effect. AMP decreases the apparent Km value for fructose‐6‐phosphate too without affecting cooperative interactions. Magnesium and ammonium ions are necessary for full enzymatic activity. The latter increases maximal activity and decreases the half‐maximal concentration of fructose‐6‐phosphate.

Free ATP seems to be a stronger inhibitor for yeast phosphofructokinase than the magnesium‐ATP‐chelate.
On the Biosynthesis of Laminitol (d‐4‐C‐Methyl‐myo‐inositol) in Porphyridium sp.
Authors: Wöber G. , Hoffmann‐Ostenhof Otto
Published: March 2005

Of all tested presumable precursors of laminitol (d‐4‐C‐methyl‐myo‐inositol) which were applied in labelled form to the alga Porphyridium sp., l‐gluco‐heptulose showed by far the highest rate of incorporation. It seems probable that in this organism l‐gluco‐heptulose is derived from l‐arabinose by C2 addition in a way similar to which it is formed in some higher plants.

The occurrence of stationary concentrations of both l‐gluco‐heptulose and 1‐deoxy‐l‐gluco‐heptulose in the algae was established.

On the basis of these results a biosynthetic pathway for laminitol which comprises as key reaction an intramolecular condensation between the carbon atoms 2 and 7 of 1‐deoxy‐l‐gluco‐heptulose or of a close derivative of this deoxysugar is suggested.
Characterization of the Heavy Form (9 S) of Mitochondrial Malate Dehydrogenase
Authors: Consiglio Eduardo , Varrone Stelio , Covelli Italo
Published: March 2005

A new, heavy form of malate dehydrogenase has been purified and characterized both from pig heart mitochondria and from a commercial source of the enzyme. The preparations is homogeneous by ultracentrifugal and electrophoretic criteria. The physico‐chemical and chemical properties of this enzyme are compared with those of the well‐known mitochondrial enzyme (5 S). The specific activity is much less than that of the 5 S form. The sedimentation constant and molecular weight are 8.7 and 138000, respectively. Although there are some similarities in the amino acid composition, the 9 S enzyme differs substantially from the lighter form by: (a) the presence of tryptophan, (b) the content of sulfhydryl groups, (c) immunochemically, and (d) in electrophoretic properties. The configuration of this new form of malate dehydrogenase appears to be that of a tetramer composed of two non‐identical subunits.
ω‐ and (ω‐1)‐Oxidation of Fatty Acids by Rat Liver Microsomes
Authors: Björkhem Ingemar , Danielsson Henry
Published: March 2005

Rat liver microsomes fortified with NADPH catalyzed the conversion of laurate, palmitate and stearate into the corresponding ω‐ and (ω‐1)‐hydroxy derivatives. In addition, small amounts of the dicarboxylic acids were formed from palmitate and stearate. The ratio between ω‐ and (ω‐1)‐oxidized products was about 1 in incubations with laurate, about 2 in those with palmitate and about 3 in those with stearate. Addition of NAD to incubations with palmitate and stearate increased the relative amount of dicarboxylic acids and also the total yield of ω‐oxidized products showing the presence in microsomes of ω‐hydroxy‐fatty acid dehydrogenases. This conclusion was confirmed by the finding that 16‐[1‐14C]hydroxypalmitic acid and 18‐[9,10‐3H2]hydroxystearic acid were converted efficiently into the corresponding dicarboxylic acids by the microsomal fraction fortified with NAD. The same reactions occurred also in the presence of the 100000 ×g supernatant fluid. The microsomal fraction fortified with NAD as well as the 100000 x g supernatant fluid were found to catalyze the oxidation of 17‐hydroxystearic acid into 17‐oxostearic acid.

Administration of phenobarbital had no significant effect on microsomal ω‐hydroxylation of laurate, whereas ω‐hydroxylation of palmitate and stearate was inhibited. (ω‐1)‐Hydroxylation of laurate, palmitate and stearate was stimulated about two to three‐fold. Carbon monoxide inhibited ω‐ as well as (ω‐l)‐hydroxylation of laurate, palmitate and stearate.
The Structure of Legumin, a Storage Protein of Broad Bean (Vicia faba) Seed
Authors: Bailey Christopher J. , Boulter Donald
Published: March 2005

Legumin from broad bean seed has been carboxymethylated and analysed. The protein contained three N‐terminal residues, leucine, glycine and threonine. Three polypeptide chains were found by polyacrylamide gel electrophoresis of the dissociated protein; their molecular weights were 56000, 42000 and 23000. 140 peptides were found on fingerprints of tryptic digests of S‐carboxymethyl‐legumin. Of these 14 were radioactive in digests of [14C]carboxymethylated legumin, and 16 were radioactive in digests of legumin prepared from material labelled in vivo with [35S]sulphate. Three new end groups were found after cyanogen bromide cleavage corresponding to Met‐Val, Met‐Phe and Met‐Pro sequences. Taken in conjunction with the amino acid analyses these peptide mapping experiments suggest that the protein contains a total sequence weight of 115000, which is in agreement with the finding of three polypeptide chains whose weights total 121000.

On the basis of dye binding studies and of the incorporation in vivo of uniformly 14C‐labelled amino acid mixture, it is suggested that the polypeptide chains of molecular weights 56000, 42000 and 23000 are present in molar ratios of 1:4:6 or 1:3:6, respectively. From consideration of experimental accuracy the 1:3:6 structure is preferred. The proposed 10 chains model for legumin has a molecular weight of 320000.
The Essential Thiol Group of Arginine Kinase from Homarus vulgaris Muscle Studied by Difference Spectrophotometry and N‐Ethyl‐[l‐14C]Maleimide Labelling
Authors: Roustan Claude , der Terrossian Elisabeth , Pradel Louise‐Anne
Published: March 2005

Interaction of N‐ethylmaleimide with the essential cysteinyl residue of ATP:l‐arginine phosphotransferase from Homarus vulgaris muscle has been studied by means of difference spectrophotometry and labelling with radioactive N‐ethyl‐[1‐14C]maleimide.

Cysteine and glutathione were used as model compounds reacting with the inhibitor. The difference spectrum at 238 nm observed with the l‐arginine‐arginine kinase complex might be ascribed to the modification of ionized – SH groups. The absorption change observed near 287 nm. produced by tyrosyl perturbation, is the same when the enzyme reacts with the inhibitor or with l‐arginine.

Labelling data give evidence of a significant protection of the essential thiol group by l‐arginine against N‐ethyl‐[1‐14C]maleimide. This cysteinyl residue is included in the tryptic peptide T1A the amino acid sequence of which, recently determined, is very similar to that to the homologous peptides of muscle and brain rabbit creatine kinases and of earth‐worm muscle lombricine kinase.