bluuebunny/crossref_metadata_2025 huggingface dataset:
The Purification and Properties of Cyclohexanone Oxygenase from Nocardia globerula CL1 and Acinetobacter NCIB 9871
Authors: DONOGHUE Nuala A. , NORRIS David B. , TRUDGILL Peter W.
Published: March 2005

Cyclohexanone oxygenases from Nocardia globerula CL1 and Acinetobacter NCIB 9571 have been purified 12‐fold and 35‐fold respectively and each gives a single symmetrical sedimentation peak in the ultracentrifuge and a single protein band on 2.25 nm average pore radius polyacrylamide gels.

The enzyme from N. globerula has a molecular weight of 53000 while that from Acinetobacter has a molecular weight of about 59000. Each is a single polypeptide chain with one mole of bound FAD per mole of protein that does not dissociate during purification. Acidification of the Acinetobacter enzyme in the presence of (NH4)2SO4 releases the bound FAD and yields native apoenzyme from which the active holoenzyme can be reconstituted. The apparent dissociation constant for the FAD is 40 nM.

The near unitary stoichiometry of cyclohexanone, NADPH and oxygen consumption is typical of mixed function oxygenases with external electron donors. The oxygenated product has been identified as 1‐oxa‐2‐oxocycloheptane thus placing these enzymes in the small group of lactone and ester‐forming oxygenases. Their correct systematic name is cyclohexanone. NADPH: oxygen oxidoreductase (1,2‐lactonizing) (EC 1.14.13.‐).

A functionally essential sulfhydryl group is present at the catalytic centre of both enzymes but there is no reliable indication from inhibitor studies that they contain any functional metal ion. The three titratable sulfhydryl groups of the Acinetobacter enzyme are not equivalent since reaction with one of them selectively inhibits catalytic activity. Protection against sulfhydryl active agents is afforded by NADPH but not by cyclohexanone.

The N. globerula enzyme has a pH optimum of 8.4, apparent Km values of 1.56 μM and 31.3 μM for cyclohexanone and NADPH respectively and a catalytic centre activity of 1018 ml substrate transformed × mol enzyme−1× min−1. The Acinetobacter enzyme has a pH optimum of 9.0, apparent Km values of 6.9 tM and 17.8 μM and a catalytic centre activity of 1390 mol × mol enzyme−1× min−1. Both enzymes display absolute specificity for electron donor which contrasts with the broad specificity for ketone substrate.

An enzyme‐cyclohexanone complex has been detected by difference spectroscopy only in the case of the Nocardia enzyme. Rapid reduction of the enzyme‐bound FAD occurs upon addition of NADPH in the absence of cyclohexanone. Titration of enzyme with NADPH under anaerobic conditions and anaerobic photoreduction in the presence of EDTA have not revealed the formation of any stable flavin semiquinones.

These enzymes bear a strong resemblance to several of the monooxygenases that hydroxylate aromatic compounds.
Structure of Flavin Adducts with Acetylenic Substrates
Authors: GÄRTNER Bernd , HEMMERICH Peter , ZELLER E. Albert
Published: March 2005

The photoreaction of flavoquinones (lumiflavin, riboflavin, FMN etc. and their 3‐alkylated derivatives) with propargylamine‐type acetylenic substrates, R4–Cα≡ Cβ–CγHR3–NR2R1, yields a mixture of two adducts, which result from covalent Cα fixation of the Cγ‐deprotonated substrate to either position C(4a) or N(5) in the flavin nucleus.

The N(5) adduct is a dihydroflavin‐5‐trimethine‐cyanine with very intense (ɛ > 20000 M−1 cm−1) absorption maxima in the region 380–450 nm depending on the R1, R2· This absorption allows recognition of minute amounts of this species of flavocyanine even in complex mixtures. Flavocyanines can be reconverted to starting flavin by base. Its spectral properties are identical with those obtained for the pargyline‐flavin inhibitor complex from bovine kidney or pig liver monoamine oxidase.

The C(4a) adduct is a 4a: 5‐propenylidene‐dihydroflavin, the cyclisation probably arising through secondary attack of the Cγ carbon at N(5). It cannot be reconverted to starting flavin in relevant yields. Its chromophor (σmax∼ 360 nm) is identical with that of lactate‐oxidase inhibitor complex. If N(3) is alkylated, these cyclic adducts undergo cleavage of the 4–4a bond upon treatment with base, followed by aromatization of the substrate substituent to yield novel pyrroloquinoxal‐4‐ones. Upon treatment with LiAlH4, however, the adducts are hydrogenated at the N(1)=C(10a) double bond, which by concomitant base hydrolysis yields 1,2‐dihydroquinoxaline derivatives. Their autoxidation yields a pyrrolo‐quinoitaiinium cation which exhibits spectral identity with the product isolated from the α‐hydroxybutynoate‐flavin inhibitor complex of Mycobacterium smegmatis lactate oxidase, after borohydride treatment.

The scope and limitations of covalent adduct formation in flavin‐dependent biocatalysis will be discussed.
Specific Irreversible Inhibition of Enzymes Concomitant to the Oxidation of Carbanionic Enzyme · Substrate Intermediates by Hexacyanoferrate(III)
Authors: CHRISTEN Philipp , COGOLI‐GREUTER Marianne , HEALY Michael J. , LUBINI Ditmar
Published: March 2005

Four different enzymes, class I fructose‐1,6‐bisphosphate aldolase from rabbit muscle, class II fructose‐l,6‐bisphosphate aldolase from yeast, transaldolase, and transketolase, are inactivated progressively in the presence of their specific substrates and hexacyanoferrate(III). The inactivation is strictly linked to the oxidation of the carbanionic enzyme · substrate intermediates of these enzymes reported previously [Healy, M. J. and Christen, P. (1973) Biochemistry, 12, 35]. However, the loss of activity is not due to the products of this oxidation, i.e. to hexacyanoferrate(II), or to the oxidation product of the substrate such as hydroxypyruvaldehyde phosphate in the case of aldolase [Healy, M. J. and Christen, P. (1972) J. Am. Chem. Soc. 94, 7911]. The inactivation is not reversed on removal of low‐molecular‐weight compounds by gel filtration or extensive dialysis indicating a covalent modification of the enzyme. The rate of inactivation obeys saturation kinetics with respect to substrate concentration. Hence, the modifying agent is a transiently reactive intermediate formed during the oxidation of the carbanionic enzyme · substrate intermediate by hexacyanoferrate(III). The rate of inactivation is independent of the concentration of the enzyme, suggesting that the inactivation results from an intramolecular reaction, i.e. the inactivating agent produced by oxidation of the enzyme · substrate intermediate is not released from the active site but rather reacts in situ with the enzyme protein. Accordingly, addition of fructose 1,6‐bisphosphate to a mixture of aldolase and transaldolase (or transketolase) with hexacyanoferrate(III) initiates the exclusive inactivation of aldolase while the addition of fructose 6‐phosphate induces the exclusive inactivation of transaldolase (or transketolase respectively).

The combination of natural substrate with hexacyanoferrate(III) thus constitutes a binary system for the chemical modification of enzymes that form oxidizable carbanionic intermediates. The modification by oxidative activation of enzyme · substrate intermediates is (a) highly specific for a given enzyme (the degree of specificity is determined by the binding and kinetic specificity of the enzyme for its natural substrate) and (b) results in a modification of groups at or near the enzyme's active site (reaction of the oxidatively activated substrate in situ).
Nuclear‐Magnetic‐Resonance Study of the Active‐Site Structure of Yeast Phosphoglycerate Kinase
Authors: TANSWELL Paul , WESTHEAD Edward W. , WILLIAMS Robert J. P.
Published: March 2005

The enzyme 3‐phosphoglycerate kinase from yeast has been studied by observation of the proton nuclear magnetic resonance spectrum at 270 MHz using Fourier transform techniques. Difference spectroscopy was used to enhance the resolution and to identify specific ligand binding effects and conformational changes. Perturbations involving single protons of amino‐acid residues could thus be detected despite the relatively high molecular weight of the protein (47000), particularly in the aromatic (6–9 ppm) and methylene (2ndash;3 ppm) regions of the spectrum.

A. pH titration enabled assignment of the C‐2 and C‐4 imidazole protons of each of three histidine residues remote from the active site, and the determination of their pKa values as 8.20 and 7.05 (two) respectively. The former histidine is deduced to lie buried within the polypeptide chain whilst the latter pair lie in practically identical environments on the surface of the enzyme exposed to solvent.

Conformation changes were monitored on binding ADP and ATP both with and without Mg2+. A single site was observed for Mg · ADP, Mg · ATP, La · ATP and the uncomplexed nucleotides.

Using paramagnetic difference spectroscopy, the line‐broadening inhibitor Gd · ATP and the substrate Mn · ATP were found to induce identical perturbations of the enzyme resonances, showing that these species bind identically. The shift probes Pr · ATP and Eu · ATP could then be applied to map geometrically the enzyme active site. Three non‐titratable (with pH) histidine resonances, three non‐histidine aromatic resonances and one resonance corresponding to a methylene group of a polar amino acid were located vectorially relative to the ATP unit. No group is close enough to bind directly in the first coordination sphere of the metal. The conformation of lanthanide‐ATP in the active site was effectively unchanged with respect to free lanthanide‐ATP.

The conformation changes induced by the nucleotides were further resolved by analysis of the separate effects of adenosine and of lanthanide‐pyrophosphate; the latter caused no conformation change but bound specifically at the nucleotide phosphate binding site. Sulphate anions (which are often used as a medium in growing crystals for X‐ray structure analysis) were shown also to compete for the phosphate groups of the nucleotides.

A conformation change was observed on binding 3‐phospho‐D‐glycerate, and a study of the quaternary complexes E · ADP · P‐glycerate · Gd and E · ATP · P‐glycerate · Gd indicated that P‐glycerate, although binding close to the nucleotides, did not significantly perturb the E · metal · nucleotide conformations.
An Extracellular Aminopeptidase from Clostridium histolyticum
Authors: KESSLER Efrat , YARON Arieh
Published: March 2005

An aminopeptidase was isolated from the culture filtrate of Clostridium histolyticum and purified to homogeneity. Absence of endopeptidase activity in the purified preparation was demonstrated. Gel filtration on a calibrated column indicates an apparent molecular weight of 340000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of dodecylsulfate in constant acrylamide concentration and in a concentration gradient, resulted in the appearance of a single component for which a molecular weight of 51000 and 59000 respectively, was calculated. From mobilities of crosslinked and denatured protein species a molecular weight of 56000 was obtained for the monomer. Specificity studies show that the enzyme cleaves all types of N‐terminal amino acid residues including proline and hydroxyproline from small peptides and from polypeptides. The peptide bond formed between an N‐terminal amino acid residue and proline is not cleaved by the enzyme. The combined action of aminopeptidase‐P and clostridial aminopeptidase leads to complete hydrolysis of the proline‐rich nonapeptide bradykinin. Low rate of hydrolysis was observed for charged residues, and amides of amino acids. Kinetic studies with five tripeptides of the general structure X‐Gly‐Gly, where X stands for Leu, Phe, Val, Ala or Pro, show a decrease in Km with the increasing size of the hydrophobic side chain of X. The highest kCat values are observed with proline and alanine. In the series Pro‐Gly, Pro‐Gly‐Pro, Pro‐Gly‐Pro‐Pro, the last peptide is the best substrate, indicating an active site size complementary to at least four amino acid residues. The enzymic activity is dependent on the presence of divalent cations, maximal activation being reached with Mn2+ and Co2+. The optimal, pH for the Mn2+ and Co2+ activated enzyme is 8.6 and 8.2, respectively. The optimal temperature is 40 °C. Inhibition of the aminopeptidase was achieved with Zn2+, Cu2+ and p‐mercuribenzoate; but not with diisopropylphosphofluoridate.
Synthesis of Albumin via a Precursor Protein in Cell Suspensions from Rat Liver
Authors: EDWARDS Kaylene , SCHREIBER Gerhard , DRYBURGH Heide , URBAN Jörg , INGLIS Adam S.
Published: March 2005

The mechanism of the biosynthesis of albumin was studied in cell suspensions from rat liver. The cells were prepared by continuous perfusion of the liver in situ with 0.05% collagenase and 0.10% hyaluronidase and incubated under conditions optilnized for the incorporation of amino acids into protein. Seven minutes after starting the incubation l‐[1‐14C]leucine was added, followed after 25 min by a 15 or 30‐min chase with an 830‐fold excess of non‐radioactive l‐leucine. Total protein, an albumin‐like protein, and albumin were isolated from samples withdrawn immediately after addition of non‐radioactive leucine and after the 15 and 30‐min chases. The specific radioactivity of total protein was found to remain constant after addition of the non‐radioactive l‐leucine, whereas that of the albumin‐like protein decreased and that of albumin increased with incubation time. The increase in albumin radioactivity accounted for the decrease in radioactivity of the albuminlike protein, suggesting that the latter is a precursor of albumin. The precursor protein differed from albumin by an oligopeptide extension at the N‐terminal end.
Specific Inhibition of Translocation by Tubulosine in Eukaryotic Polysomes
Authors: CARRASCO Luis , JIMENEZ Antonio , VÁZQUEZ David
Published: March 2005

The alkaloid tubulosine inhibits the process of peptide chain elongation by eukaryotic polysomes by, specifically preventing the elongation‐factor‐2‐dependent step of translocation. Tubulosine does not affect either the elongation‐factor‐l‐dependent binding of aminoacyl‐tRNA or peptide bond formation.

The site of action of tubulosine appears to be independent from the ribosomal site involved in cycloheximide action, since the alkaloid is active in blocking polymerization and enzymic translocation by polysomes from a yeast mutant resistant to cycloheximide. Furthermore tubulosine does not affect the non‐enzymic translocation which takes place in the presence of high potassium ion concentrations, whereas this reaction is strongly inhibited by cycloheximide. The different steps of translocation are discussed to explain the reactions blocked by tubulosine and cycloheximide.
Two Forms of Serine Transhydroxymethylase, One Absent in a Thymidylate‐less Mutant in Saccharomyces cerevisiae
Authors: ZELIKSON Ruth , LUZZATI Mario
Published: March 2005

Two forms of serine transhydroxymethylase, separable by TEAE‐cellulose chromatography, are present in wild‐type extracts of Saccharomyces cerevisiae. One of these forms is mitochondrial and the other one cytoplasmic. The mitochondrial form is absent in the mutant tmp3.
Translation of the mRNA for Rabbit Uteroglobin in Cell‐Free Systems
Authors: BEATO Miguel , NIETO Antonio
Published: March 2005

Uteroglobin, an hormonally induced protein composed of two similar subunits, represents around 50% of the proteins synthesized and secreted into the uterine lumen of rabbits treated sequentially with estradiol and progesterone. The endometrium of these animals was used as a source for the isolation of the mRNA for uteroglobin. Poly(A)‐rich RNA, extracted from purified polysomes with phenol chloroform and isolated on oligo(dT)‐cellulose columns, contains one fourth of the total protein coding activity of the endometrium. Between 20–25% of the polypeptides synthesized by this RNA in cell‐free systems derived from Krebs II ascites cells or wheat germs react with a monospecific antiserum prepared in guinea pigs against uteroglobin. The material bound to the antibody was identified as a precursor of uteroglobin according to the following criteria.

The product synthesized in vitro can be displaced from the complex with the specific immunoglobulin by purified uteroglobin.

Analysis of the immunoprecipitate an polyacrylamide gels containing urea and dodecylsulfate demonstrate the existence of a single labelled polypeptide with an apparent molecular weight larger than the uteroglobin subunits.

The tryptic digest of this polypeptide, labelled in vitro with [3H]lysine, shares seven peptides with mature uteroglobin labelled with the same amino acid in perfused uteri, and exhibits an additional peptide not present in uteroglobin.

Injection of the same mRNA preparation into Xenopus oocytes results in the production ol uteroglobin.

The endometrium of intact animals treated with estradiol alone also contains the same mRNA bound to polysomes but in a smaller proportion, indicating that the progesterone‐induced synthcsis of uteroglobin is accompanied by an accumulation of the specitic mRNA in the polysomes
Conformation of Escherichia coli Glutamic Acid tRNA II as Studied by Hydrogen‐Tritium Exchange Catalyzed by Cysteine Methyl Ester
Authors: WATAYA Yusuke , LIDA Shigeru , KUDO Ichiro , HAYATSU Hikoya , OHASHI Ziro , NISHIMURA Susumu , SUGA Kazuo , TAKAGI Hideyuki , YOKOSHIMA Tetsuyoshi
Published: March 2005

Incubation of CMP in 2H2O with 0.5 M cysteine methyl ester at p2H 5 and 37 °C for 24 h resulted in 43% exchange of 5‐H to 5‐2H. No deamination of the cytosine nucleus was noted during this treatment. Native and denatured DNA samples from calf thymus were treated in 3H2O with cysteine methyl ester at pH 5 and 37 ‘C for 24h and incorporation of tritium into Bach DNA base was determined by enzymic digestion of the treated DNA.

The order of the specific radioactivity found was cytosine ≫ guanine > adenine ≫ thymine for denatured DNA and guanine > adenine ∼ cytosine > thymine for native DNA. The ratio of radioactivity for denatured/native was 11.6 for cytosine, 1.5 for guanine, 1.8 for adenine and 1.1 for thymine. Hence the incorporation in cytosine under the reaction conditions is preferential for single‐stranded, nonhelical regions of DNA.

Escherichia coli glutamic acid tRNA II was treated in 3H20 with 1.24 M cysteine methyl ester at pH 5 and 37 C. The 24‐h‐treated tRNA was digested with ribonuclease T1 and the fragments were fractionated. Each fragment was then digested with ribonuclease T2 into mononucleotides and the radioactivity distribution among the bases was determined. The average radioactivity found for each of the bases of the four major nucieotides was cytosine ∼ guanine ∼ adenine > uracil. The radioactivity in cytosine varied greatly among the RNasc T1 fragments, the ratio of the highest to the lowest radioactivity boing 18.7. The corresponding valuc for guanine was 11.1, for adenine 4.73 and for uracil 3.64. Based on the data obtained, it was deduced that in this tRNA the anticodon loop, the dihydrouridinc loop and the extra loop were ‘exposed’ under the conditions employed for the labeling. The 5′‐terminal cytosine of the anticodon loop was in a ‘non‐exposed’ state, a situation similar to that previously reported for E. coli tyrosine tRNA [Cashmore, A. R., Brown, D. M. & Smith, J. D. (1971) J. Mol. Biol. 59, 359‐373] and for E. coli formylmethionine tRNA [Goddard, J. P. & Schulman, L. H. (1972) J. Biol. Chem. 247, 3864‐3867]. Both cytosine 48, located at the 3′‐terminal of the extra loop, and guanine 15 in the dihydrouridine loop were in an ‘exposed’ state. This finding does not agree with a tRNA model in which this pair of cytosine and guanine, commonly found in tRNA sequences, forms hydrogen bondings. Positions 30–32, 61–64 and 71, which are located in the stems, were found to be strongly ‘buried’
Purification and Properties of d‐Glyceraldehyde‐3‐Phosphate Dehydrogenase from an Extreme Thermophile, Thermus thermophilus Strain HB 8
Authors: FUJITA Shinobu C. , OSHIMA Tairo , IMAHORI Kazutomo
Published: March 2005

d‐Glyceraldehyde‐3‐phosphate dehydrogenase from an extreme thermophile, T. thermophilus strain HB8, was purified and crystallized.

The enzyme was found to possess remarkable heat stability, being slowly inactivated at 900C.

Basic kinetic constants and pH profile are reported. The enzyme was activated 25‐fold by 90 mM NH4CI, and also by ethanol up to 5‐fold at 30 °C.

The enzyme was found to be far more resistant to urea or sodium dodecylsulfate than the rabbit enzyme.

The enzyme was shown to be a tetramer of molecular weight 130000‐ 135000. Amino acid composition analysis revealed no unusual features. Circular dichroic spectra suggested that the contents of the ordered structure of the thermophile enzyme are similar to those of the rabbit enzyme.

The other catalytic properties of the thermophile enzyme are discussed in comparison with those of the enzymes from other sources.
The Structure of Kinetoplast DNA
Authors: KLEISEN Coen M. , BORST Piet , WEIJERS Peter J.
Published: March 2005

We have analysed limit digests of mini‐circles from kinetoplast DNA of Crithidia luciliae by gel electrophoresis. Endonucleases HapII and AluI cut the circles into at least 37 and 21 fragments, respectively, and leave no circles intact. In both cases the added molecular weights of the fragments, estimated from mobility in gels, exceeds 18 × 106, i.e. more than 12 times the molecular weight of the mini‐circle DNA. Endonucleases HindII + III, EcoRI and HpaI cut only part of the circles. These results show that the mini‐circles are heterogeneous in base sequence. Different sequence classes are present in different amounts.

DNA‐DNA renaturation analysis of mini‐circle DNA yields a complexity of about 3 × 106, i.e. twice the molecular weight of one mini‐circle. The Δtm of native and renatured duplexes is about 1 °C, showing that the sequence heterogeneity is a micro‐heterogeneity.

Electron microscopy, gel electrophoresis and sedimentation analysis show that the circles that are not cut by endonucleases HindII + III remain catenated in very large associations. These associations lack the ‘rosette’ structures and the long edge loops characteristic of intact kinetoplast DNA. This suggests that the mini‐circle classes cut by endonucleases HindII + III are present throughout the network and that the maxi‐circle component of the network (see accompanying paper) is not essential to hold the network together.

Prolonged electrophoresis on 1.5% or 2% agarose gels resolves the open mini‐circles into three and linearized mini‐circles into four bands, present in different amounts. We conclude that the minicircles are also heterogeneous in size, the difference in size between the two extreme size classes being 4% of the contour length. Digestion with endonuclease HapII shows that at least three out of these four bands differ in sequence.

Possible mechanisms that could account for the micro‐heterogeneity in sequence of minicircles are discussed.
Simultaneous Translation of Structural and Nonstructural Proteins from Semliki‐Forest‐Virus RNA in Two Eukaryotic Systems in vitro
Authors: GLANVILLE Niall , MORSER John , UOMALA Pertti , KÄÄRIÄINEN Leevi
Published: March 2005

The Semliki Forest virus genome, 42‐S RNA, and the virus‐specific intracellular 26‐S RNA were translated in two cell‐free protein‐synthesising systems, the wheat germ extract, and a partially purified system from mammalian tissues.

The 26‐S RNA directed the synthesis of structural proteins only, as revealed by tryptic peptide mapping. About 75–80% of the radioactivity in the products comigrated with capsid and about 4–8% with envelope protein peptides. All the capsid peptides and the full‐sized capsid protein were found in the products in vitro, whereas no complete envelope protein was formed and fewer than half of the envelope peptides were detected. This result is consistent with reports that there is only one initiation site for the translation ofvirus structural proteins, and that the capsid protein is N‐terminal in the polyprotein followed by envelope proteins.

The systems programmed with 42‐S RNA yielded virtually the same structural peptides. However, the bulk of the radioactivity was in peptides which did not comigrate with the structural ones. These peptides were mostly associated with relatively small‐sized products. This shows that Semliki Forest virus 42‐S RNA has at least two initiation sites, one for the structural proteins and the other(s) for the nonstructural proteins.
Uptake of Bromosulfophthalein byIsolated Liver Cells
Authors: SCHWENK Michael , BURR Reinhold , SCHWARZ Leslie , PFAFF Erich
Published: March 2005

Uptake of the hepatodiagnostic dye bromosulfophthalein into isolated hepatocytes was studied with special regard to the kinetics of transport. The following results were obtained.

The uptake of bromosulfophthalein follows Michaelis‐Menten kinetics only at low substrate concentrations with an apparent Km= 7 ± 2 μM and V= 2.6 ± 1.7 nmol × mg protein−1× min−1′. At higher bromosulfophthalein concentrations a second mechanism of uptake is observed as indicated by the deviation from linearity in the Lineweaver‐Burk plot.

The activation energy of uptake was found to be 11 kcal/mol at 10 μM bromosulfophthalein.

Uptake is independent of metabolic energy and of the Na+ gradient across the membrane.

Taurocholate does not inhibit uptake while indocyanine green inhibits competitively at low bromosulfophthalein concentrations and activates uptake at high bromosulfophthalein concentrations (> 201 μM).

Amino acid reagents, such as dinitrofluorobenzene, mersalyl, N‐ethylmaleimide, and dithionitrobenzene, which modify specific functional groups, did not affect uptake at a concentration of 100 μM.

No pH optimum for bromosulfophthalein uptake was observed in the physiological pH range.

Adsorption of bromosulfophthalein to the liver cell membrane has two distinguishable site, with affinities K1= 5.7 × 10−6 M and K2=7 × 10−5 M and binding capacities n1= 1.2 nmol/mg protein and n2= 7 nmol/mg protein. Adsorption is inhibited by indocyanine green.

The results do not indicate the mediation of bromosulfophthalein uptake by a carrier protein and are consistent with the hypothesis that bromosulfophthalein is bound in an energy‐consuming process to a translocating site, possibly in the undissociated form or as ion pair. The consecutive transfer across the membrane appears to require little additional energy.
Studies on the Control of Development
Authors: RHAESE Hans‐Jürgen. , GRADE Reinhardt , DICHTELMÜLLER Herbert
Published: March 2005

Unusual highly phosphorylated nucleotides are found in sporulating cells of Bacillus subtilis. Adenosine 3′(2′)‐diphosphate 5′‐diphosphate, ppApp (highly phosphorylated nucleotide I), and adenosine 3′(2′)‐diphosphate 5′‐triphosphate, pppApp (highly phosphorylated nucleotide II), are detectable in vivo when cells are starved for carbon and nitrogen sources. These nucleotides are correlated with sporulation because only ribosomes from sporulating but not vegetative cells are able to synthesize ppApp and pppApp in vitro. Two other nucleotides, adenosine 3′(2′)‐triphosphate 5′‐triphosphate; pppAppp (highly phosphorylated nucleotide IV), and a nucleotide with a tentative structure of, ppZpUp (highly phosphorylated nucleotide III), where Z is an undetermined sugar, also seem to be involved in regulation of sporulation, especially initiation of sporulation. Sporulation can be initiated even in the presence of amino acids, salts, vitamins etc. in logarithmically growing or stationary‐phase cells when carbon sources, i.g. glucose, are used up or artifically removed from the medium. A drastic increase in spore titer is observed 4–5 h later. Also, carbon starvation causes accumulation of the highly phosphorylated nucleotides pppAppp and ppZpUp. On the other hand, sporulation is prevented under the same conditions when excess glucose is maintained in the medium. Correlated with this inhibition of sporulation is the inhibition of formation of highly phosphorylated nucleotides, pppAppp and ppZpUp. Since synthesis of these nucleotides is closely related to sporulation, we anticipate that these substances can cause initiation of development in B. subtilis.

Further evidence for our hypothesis on initiation of sporulation by highly phosphorylated nucleotides is that phosphate starvation also causes sporulation with prior accumulation of pppAppp and ppZpUp. Apparently, as long as phosphate is present to synthesize phosphorylated metabolites of glucose, formation of highly phosphorylated nucleotides is repressed. Derepression occurs when either lack of glucose or phosphate or both prevents synthesis of phosphorylated metabolites of glucose allowing synthesis of highly phosphorylated nucleotides. These nucleotides, representing the signal lack of glucose or phosphate′, then somehow cause changes in gene activity, initiating the complex process of sporulation. Whether or not pppAppp alone or together with ppZpUp or even further substances (nucleotides, proteins etc.) is necessary for the above described processes will be answered with the help of suitable mutants lacking the ability to synthesize either one or both regulatory nucleotides.

Guanosine 3′(2′)‐diphosphate 5′‐diphosphate, ppGpp, and guanosine 3′(2′)‐diphosphate 5′triphosphate, pppGpp, are not involved in regulation of development as is shown by using a normally sporulating mutant of B. subtilis, unable to synthesize these nucleotides.
Snake Venom Toxins
Authors: JOUBERT François J.
Published: March 2005

Three toxins (CM‐8, CM‐11, and CM‐13a) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex G‐50 and by ion‐exchange chromatography on CM‐cellulose. Whereas toxin CM‐8 and CM‐11 comprise 60 amino acid residues, toxin CM‐13a contains 61 residues. All three toxins are cross‐linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have been elucidated. The reduced and S‐carboxymethylated toxins were digested with trypsin and chymotrypsin and the peptides purified by ion‐exchange chromatography, gel filtration and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequencer or by manual manipulation, was employed to obtain the sequence of the intact toxins and the pure peptides. The chymotryptic digests provided the necessary overlapping peptides which allowed the alignment of the tryptic peptides. The properties of the three toxins were compared with those of the cytotoxin group. The toxicities the serological properties, the sequences and the invariant amino acid residues of toxin CM‐8 and CM‐11 resemble the corresponding properties of the cytotoxin group. The sequence and serological properties of toxin CM‐13a show that it is related to the cytotoxin group, but its toxicity is much lower than those encountered in the cytotoxin group.
The Influence of Insulin on Various Enzyme Activities in Human and Rat Hepatoma Cells
Authors: GRIMM Johanna
Published: March 2005

Incubation of human and rat hepatoma cells with insulin (1 mU/106 cells) decreases their content of adenosine 3′:5′‐monophosphate by more than half after 1 h and by about a quarter after 4 h.

The activities of the ATP‐metabolising enzymes, adenylate kinase and Mg2+‐adenosine triphosphatase are significantly increased by insulin within I h and after 4 h. Activity of succinate dehydrogenase. and lactic dehydrogenase showed no change at either time interval.

Insulin markedly stimulated glucose 6‐phosphate dehydrogenase activity within I h but by 4 h the increase was less apparent. Glutamate dehydrogenase activity by contrast was not increased by 1,h but was elevated at 4 h.
Acetic Anhydride: an Intermediate. Analogue in the Acyl‐Exchange Reaction of Citramalate Lyase
Authors: BUCKEL Wolfgang
Published: March 2005

Reactivation of deacetyl citramalate lyase by acetic anhydride proceeds through an enzymeanhydride complex prior to actual acetylation. The reaction is inhibited by citramalate which is competitive with acetic anhydride.

A corresponding complex is an intermediate in the carboxymethylation of deacetyl enzyme by iodoacetate. However, the inhibition of this reaction by S‐citramalate appears to be non‐competitive with iodoacetate.

The results lead to the conclusion that acetic anhydride can be regarded as a structural analogue of citramalic acetic anhydride, the proposed intermediate in the acyl exchange reaction on citramalate lease.

The formation of 6‐citryl thiolester from the 1‐thiolester via the cyclic citric anhydride provides a chemical model for enzymic acyl exchange.

The data suggest that anhydrides are of general importance in acyl exchange reactions of thiolesters.
Small‐Angle X‐Ray and Light‐Scattering Study of Native and Trypsin‐Modified Methionyl‐tRNA Synthetase from Escherichia coli
Authors: GULIK Annette , MONTEILHET Claude , DESSEN Philippe , FAYAT Guy
Published: March 2005

Small‐angle X‐ray scattering experiments were performed on an absolute scale on solutions of methionyl‐tRNA synthetase from Escherichia coli in its native and trypsin‐modified forms. A light‐scattering study was performed on the same solutions to verify monodispersity.

The structural parameters for the trypsin‐modified enzyme, radius of gyration (2.48 nm), volume (90 nm3), surface/volume (1.5 nm−1) and the distribution of chords can account for an equivalent prolate ellipsoid of revolution having an axial ratio 2.3 and a maximum length of 9 nm, with a creviced surface.

The results obtained for the native enzyme [i.e. radius of gyration (4.3 nm), volume (244 nm3), distribution of the scattering intensity and distribution of chords] exclude the possibility of a very compact quaternary structure and suggest that the enzyme consists of at least two globular parts, probably the two protomers, linked together by interactions involving a limited region of the structure.
Control of Fatty‐Acid Synthetase Levels by Exogeneous Long‐Chain Fatty Acids in the Yeasts Candida lipolytica and Saccharomyces cerevisiae
Authors: MEYER Karl‐Heinz , SCHWEIZER Eckhart
Published: March 2005

Endogeneous fatty acid biosynthesis in the two yeast species, Saccharomyces cerevisiae and Candida lipolytica is completely repressed by the addition of long‐chain fatty acids to the growth medium. In Candida lipolytica, this repression is accompanied by a corresponding loss of fatty acid synthetase activity in the cell homogenate, when the cells were grown on fatty acids as the sole carbon source. The activity of the Saccharomyces cerevisiae fatty acid synthetase, however, remains unaffected by the addition of fatty acids to a glucose‐containing growth medium. From fattyacid‐grown Candida lipolytica cells no fatty acid synthetase complex can be isolated, nor is there any immunologically cross‐reacting fatty acid synthetase protein detectable in the crude cell extract. From this it is concluded that Candida lipolytica, but not Saccharomyces cerevisiae, is able to adapt to the growth on fatty acids either by repression of fatty acid synthetase biosynthesis or by a fatty‐acid‐induced proteolytic degradation of the multienzyme complex. Similarly, the fatty acid synthetase complex disappears rapidly from stationary phase Candida lipolytica cells even after growth in fatty‐acid‐free medium. Finally, it was found that the fatty acid synthetase complexes from Saccharomyces cerevisiae and Candida lipolytica, though very similar in size and subunit composition, were immunologically different and had no common antigenic determinants.
The Initiation of Globin Synthesis in Differentiating Rabbit‐Bone‐Marrow Erythroid Cells
Authors: STEWART Andrew G. , CLISSOLD Patricia M. , ARNSTEIN Henry R. V.
Published: March 2005

At a particular stage of erythropoiesis, the immature erythroid cell commences synthesis of the messenger RNA for globin. Experiments were performed to determine whether this mRNA is immediately translated into globin or whether there is any substantial delay and accumulation of mRNA awaiting a further stage of differentiation. Rabbit bone marrow erythroid cells were separated: according to their degree of maturity by velocity sedimentation on a sucrose gradient. Their relative abilities to synthesise globin were measured by radioactive labelling in culture and the relative proportions of globin mRNA in cellular RNA were determined by hybridisation to DNA complementary to globin mRNA. The increasing ability to synthesise globin closely parallelled the concentration of globin m RNA at all stages of differentiation, thus suggesting that there is no significant delay between transcription and translation.
Studies on the Role and Mode of Operation of the Very‐Lysine‐Rich Histones in Eukaryote Chromatin
Authors: PUIGDOMÉNECH Pedro , MARTÍNEZ Paz , CABRÉ Oriol , PALAU Jaime , BRADBURY E. M. , CRANE‐ROBINSON Colyn
Published: March 2005

Proton magnetic resonance and other measurements have been carried out in order to study the behaviour of the lysine‐rich histones Ψ 1 in the sperm chromatin of certain marine invertebrates. Well defined particles (12 S) have been obtained from this chromatin by nuclease treatment. Chromatin solubility as a function of ionic strength shows a relaxation at salt concentrations higher than in the case of calf thymus nucleoprotein. Nuclear magnetic resonance (NMR) studies show that the release of histone from DNA occurs both in chromatin and in the reconstituted complexes at practically the same ionic strength as solubility relaxation. The higher the arginine content of a given Ψ 1, the higher the ionic strength at which both effects take place. The NMR results demonstrate that arginine residues are bound more strongly than lysine residues. The data overall show that the Ψ 1 histones play a role in the contraction mechanism of sperm chromatin similar to that of H 1 histone in calf thymüs chromatin. The highly contracted state of sperm chromatin is directly related to the increased arginine content of the Ψ 1 histone.
Interaction of RNA Polymerase from Escherichia coli with DNA
Authors: DAUSSE Jean‐Pierre , SENTENAC André , FROMAGEOT Pierre
Published: March 2005

Factors influencing promoter site selection by Escherichia coli RNA polymerase were investigated using T7 DNA as template. The utilization of the three major early promoters A1 A2 and A3, was followed by processing the RNA transcripts with RNAase III, which generates the three corresponding initiator RNA fragments. The three promoters proved to be functionally distinct. A strong differential effect of temperature and ionic strength on promoter selection was observed. The transition temperature of promoters A1 A2 and A3 was measured directly by preincubating, at different temperatures, RNA polymerase and T7 DNA, with primer‐substrate combinations which selected each site independently. The transition temperature of the three sites was markedly different. Promoter A3 was used predominantly at low temperature, whereas A1 or A2 promoters were gradually activated by increasing the temperature. The temperature response curves were strongly dependent upon the salt concentration. On the other hand, challenge experiments with rifampicin or poly (inosinic acid) showed that, once preinitiation complexes are formed by incubating RNA polymerase with DNA at 37 °C, the three sites A1 A2 and A3 promote chain initiation with equal efficiency and at the same rate.
Studies of Glutamate Dehydrogenase. Regulation of Glutamate Dehydrogenase from Candida utilis by a pH and Temperature‐Dependent Conformational Transition
Authors: NEUMANN Peter , MARKAU Klaus , SUND Horst
Published: March 2005

Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH and low temperature. This conformational transition can also be followed by fluorescence measurements.

The temperature‐dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 °C and a ΔH value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 °C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure.

The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2‐oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme.

The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s−1.

Preincubation experiments with the substrate 2‐oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition.

The sedimentation coefficient (s°20, W) and the molecular weight were found to be 11.0 S and 276000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47000.
A Spectroscopic Investigation of S‐Trifluoroethylthiopapain
Authors: BENDALL M. Robin , LOWE Gordon
Published: March 2005

The pH dependence of the 19F chemical shift and the fluorescence spectrum of S‐2,2,2‐trifluoro1,1‐dideuteroethyl‐thio‐papain are analysed in terms of dependence on the ionisation of aspartic‐acid158 and histidine‐159. The 19F probe causes negative cooperativity between these groups, and does not detected any ionisation at high pH. The intermediate chemical exchange rates for the ionisation of aspartic‐acid‐158 and histidine‐159 allow the approximate rate constants for proton transfer to be calculated. The rather low rate constants are explained in terms of the hydrophobicity of the activesite region and the net positive charge on the enzyme resulting from its high isoelectric point.
Existence, Localization and Regulation of the Biosynthesis of Aminoendopeptidase in Gram‐Negative Bacteria
Authors: MURGIER Maryse , PELISSIER Corinne , LAZDUNSKI Andrée , LAZDUNSKI Claude
Published: March 2005

An enzyme capable of hydrolyzing the substrate l‐alanine p‐nitroanilide has been found in 18 gram‐negative Enterobacteriaceae and Pseudomonas that we tested. This enzyme might be located near the cell surface. Contrastingly we have not detected this activity on colonies of the 8 grampositive Bacillus that we tested, which sugest a taxonomic value for this test. The aminoendopeptidases found in the various grain‐negative bacteria showed similar electrophoretic mobilities and immunological cross‐reactivity when tested against Escherichia coli anti‐aminoendopeptidase antiserum. The regulation of aminoendopeptidase biosynthesis by the endogenous level of inorganic phosphate that we previously demonstrated in E. coli has been found to exist in all gram‐negative organisms tested.
Baker's Yeast Flavocytochrome b2 [l‐(+)‐Lactate Dehydrogenase]
Authors: GUIARD Bernard , LEDERER Florence
Published: March 2005

Baker's yeast flavocytochrome b2 has been suggested before to be a polyglobular protein formed after a gene fusion process. It is known that one of the potential globules, the heme‐binding core, can exist in the absence of the rest of the protein. Using antibodies elicited against the whole enzyme acid against the core, we show in this paper that part of the core surface, and in particular the mouth of the heme‐binding crevice, must be exposed in the complete enzyme molecule.

Antibodies inhibit the activity of intact and proteolytically‐cleaved enzymes, which normally show a number of differences in some kinetic parameters. Interestingly, antibodies against the core induce a modification of some kinetic constants for the cleaved enzyme (in particular the Km for the substrate and Ki for d‐(−)‐lactate), bringing them back to values similar to those for the intact enzyme. These results can be interpreted as a tightening of the cleaved enzyme by anti‐core antibodies. The conformational effect is transmitted from the heme‐binding region to other parts of the molecule. This implies some intimate contacts between the core and the rest of the protein.
AMP Analogs: Their Function in the Activation of Glycogen Phosphorylase b
Authors: MORANGF Michel , GARCÍA BLANCO Francisco , VANDENBUNDER Bernard , BUC Henri
Published: March 2005

A series of AMP analogs has been selected in order to better understand the structural requirements (a) for the efficient binding of the activator molecule at the correct site on phosphorylase b from rabbit skeletal muscle and (b) for the activation which is observed. Two types of activation are known, according to Black and Wang [J. Biol. Chem. 243, 5892—5898 (1968)]: either a cooperative response with respect to the activator concentration (like the one which is obtained for AMP itself) or a non‐cooperative response observed in the case of IMP.

It is shown that the 5′‐phosphate moiety is absolutely required for the analog to bind at the correct site (adenine or adenosine bind at another enzymic site), and that the free enthalpy, ΔG, corresponding to the association process varies in a complex manner with respect to the substitution of the different positions of the AMP molecule. Moreover, the differences ΔG (analog) ‐ ΔG (AMP) =δG obtained for two types of substitution separately do not add up to the same energy difference as the one obtained when the two substitutions are made simultaneously on the AMP molecule.

It appears that all the mononucleotides which have been tested up to now may be divided into two classes. Class I (AMP class) is characterized, apart from a strong activation, by the following features: (a) one molecule of analog expels two molecules of bound glucose 6‐phosphate as it binds on the enzyme; (b) bound analog protects slowly one crucial cysteinyl residue against attack by 5.5′‐dithio‐bis(2‐nitrobenzoic acid) at 4 °C; (c) association of two molecules of dimer is strengthened at 4 °C in the presence of the analog. Class II (IMP class) is associated with a weak activation and with the following set of properties: (a) a single molecule of bound glucose 6‐phosphate is released as the first molecule of analog binds on the dimer; (b) two slowly reacting cysteinyl residues per subunit are immediately protected against 5,5′‐dithio‐bis(2‐nitrobenzoic acid) by the binding of the analog at 4 °C; (c) the analog dissociates the low amount of tetramer which is present at 4 °C in the absence of AMP into two molecules of dimer.

These results are discussed according to a plausible scheme of transconformations taking place in glycogen phosphorylase b, a model which has been derived earlier by relaxation studies.