bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Properties of d‐Gluconate Dehydratase from Clostridium pasteurianum
Authors: Bender Rudolf , Gottschalk Gerhard
Published: March 2005

d‐Gluconate dehydratase from Clostridium pasteurianum has been purified by heat treatment, chromatography on DEAE‐Sephadex and gel filtration to about 80% homogeneity. The overall procedure resulted in a 33‐fold purification with a yield of 16%. Further purification by electrophoresis yielded preparations homogeneous by electrophoretic and ultracentrifugal criteria. Analysis of the enzyme by sedimentation equilibrium experiments, gel‐filtration and sucrosegradient centrifugation gave a molecular weight of 131 000, 130 000 and 118 000, respectively. Treatment with sodium dodecylsulfate and mercaptoethanol dissociated the enzyme into two subunits (Mr= 64000).

The enzyme is inactivated by exposure to oxygen and the activity can be restored by incubation with sulfhydryl compounds such as mercaptoethanol, dithiothreitol, reduced glutathione and cysteine in conjunction with ferrous ions for 2 h at 35°C.

The reactivated enzyme dehydrates gluconate in the presence of divalent metal ions only. Ferrous ions (2 mM) are most effective but can be partially replaced by Mn2+, Co2+ and Mg2+. EDTA inhibits the reaction completely.

d‐Gluconate dehydratase exhibits maximum activity in the pH region of 7.3 to 7.8 Among several compounds tested only d‐gluconate is dehydrated (Km value = 5.5 mM).
Human α2‐Macroglobulin, Characterization and Trypsin
Authors: Hamberg Ulla , Stelwagen Patrick , Ervast Hanna‐Stina
Published: March 2005

The trypsin binding was compared with preparations of α2‐macroglobulin obtained from human plasma, serum or Cohn's fraction III by ammonium sulphate precipitation. Determination of the binding ratio at various purification steps showed close to 1:1 molecular proportion, regardless of the degree of purity of the preparations. The α‐macroglobulin · trypsin complex was separated by gel chromatography on Sephadex G‐200, using standardized conditions and estimating the bound and free typsin in the eluate fractions. It was found that activated plasmin interfered with trypsin binding of α2‐macroglobulin from Cohn's fraction III.

Immunologically pure α2‐macroglobulin was obtained in six preparative steps from whole plasma after applying zone electrophoresis in polyvinylchloride. The protein was characterzed by sedimentation rate, immunochemical analyses, isoelectric focusing, amino acid analysis and trypsin binding.

Decreased binding capacity towards trypsin and plasmin could be detected after lyophylization of α2‐macroglobulin. Comparison with low‐temperature denaturation in sedimentation studies suggests that the functionally intact protein was not restored in spite of 90% reaggregation of the 11–12‐S subunit. The structure dependence of the enzyme binding is discussed.
Solvent Effects on Luteinizing‐ and Follicle‐Stimulating‐Hormone Releasing Factor Polymorphism Studied by Circular Dichroism
Authors: Marche Pierre , Morgat Jean‐Louis , Fromageot Pierre
Published: March 2005

Luteinizing‐ and follicle‐stimulating‐hormone releasing factor has been studied by circular dichroism and compared with two of its peptidic fragments. Aqueous solutions of the decapeptide have been examined as a function of pH and temperature. The hormone seems to exist under a random conformation in acid medium. Increasing the pH or heating in neutral medium, favor a shift towards ordered structures. Dissolving the hormone solutions with different ratios of trifluoroethanol/water or in hexafluoroisopropanol lead to a more dramatic change in the spectra: ordered conformations are predominant for high trifluoroethanol concentrations. From our observations, it is suggested that two types of peptidic chromophore interactions are responsible for the hormone structures in the alcoholic solvents mentioned above. The role of the environment upon the conformations(s) adopted by the decapeptide is also discussed.
Proteins of Invertebrate Photoreceptor Membranes
Authors: Paulsen Reinhard , Schwemer Joachim
Published: March 2005

Polyarcylamide‐gel electrophoresis on digitonin and dodecylsulphate gels has been used to characterize visual pigment preparations obtained from rhabdomeric photoreceptors. The rhodopsin of the cephalopod Eledone moschata (P470) and the ultraviolet‐sensitive visual pigment of the insect Ascalaphus macaronius (P345) were separated from non‐rhodopsin proteins by electrophoresis on digitonin gels. Visual pigment bands in the digitonin gel were located by their absorbances and photoreactions. The mobility of visual pigment protein on sodium dodecylsulphate‐polyacrylamide gels run in parallel with marker proteins indicated a polypeptide chain with a molecular weight of approximately 43 000 for P470 and 35 000 for P345. When digitonin extracts of P470 were stored before use, a decrease in the electrophoretic mobility of the dodecyl‐sulphate‐denatured pigment was observed, raising the apparent molecular weight to about 51 000. The data indicate that the protein components of vertebrate and invertebrate visual pigments are similar in size.
Chick‐Erythrocyte Chromatin as a Template for RNA Synthesis in vitro
Authors: Bolund Lars , Johns Ernest W.
Published: March 2005

The template activity of chick erythrocyte chromatin was measured with bacterial RNA polymerase and found to be low in comparison with that of calf thymus chromatin. The chromatins were precipitated in a standardized way from very dilute solutions prior to the assay in order to minimize differences in solubility or physical state between the templates which might affect their ability to support transcription.

Selective and quantitative removal of the unique histone F2C from chick erythrocyte chromatin dramatically increased its template activity. Calf thymus chromatin devoid of histone F1 was, however, stil a better template than chick erythrocyte chromatin depleted of both F2C and F1. Reconstitution of the depleted chromatins with the adequate amount of histone restored the original template activites whether erythrocyte F2C or calf thymus F1 was used. The results argue against a unique functional role of histone F2C as a super‐repressor.

Exchange of the majority of proteins between chick erythrocyte and calf thymus chromatin caused an exchange of template properties. Non‐histone proteins extracted from calf thymus chromatin with 0.35M NaCl did not “activate” the chick erythrocyte chromatin although they were found to bind to it.
The Interaction of Chaotropic Anions with Proteins at Acid pH
Authors: Aviram Irit
Published: March 2005

The activity of pepsin on small substrates, as well as its autodigestibility, fluorescence and circular dichroism were unaffected by the presence of “disordering” anions.

Pepsin activity on hemoglobin and bovine serum albumin was inhibited by the same anions, while the hydrolysis of lysozyme was enhanced.

The anions also altered the fluorescence and circular dichroism spectra of bovine serum albumin. These findings are discussed in relation to conformational changes induced by disordering ions at acid pH.
A Comparison of Mitochondrially Synthesized Proteins from Whole Mitochondria and Cytochrome Oxidase in Neurospora
Authors: Rowe Mark J. , Lansman Robert A. , Woodward Dow O.
Published: March 2005

The four mitochondrial proteins of Neurospora crassa labeled by amino acid incorporation in vivo in the presence of cycloheximide have been characterized with regard to their solubility properties in acidic chloroform‐methanol. The labeled proteins are present in pulse‐labeled whole mitochondria, pulse‐chase labeled whole mitochondria, and a purified preparation of cytochrome oxidase. The labeled proteins in each of these preparations are similar with respect to molecular weight, as determined by dodecylsulfate · polyacrylamide‐gel electrophoresis. In addition, proteins of similar molecular weight in each preparation exhibit identical solubility properties in acidic chloroform‐methanol. The possibility of the mitochondrially synthesized subunits of cyto‐chrome oxidase being representative of total mitochondrial protein synthesis is discussed.
Cytochrome b in Neurospora crassa Mitochondria
Authors: Weiss Hanns , Ziganke Barbara
Published: March 2005

Cytochrome b was purified from Neurospora crassa mitochondria by means of chromatography on oleyl‐polymethacrylic acid resin, chromatography on DEAE‐cellulose and recycling gel filtration on Sephadex G‐75, all steps being performed in a medium containing bile acids and salts.

The heme content of the preparation was calculated on the basis of incorporated 59Fe and [3H]‐leucine to be 33–40 μmol/g protein, corresponding to a minimum molecular weight of 25000 to 30000.

Upon gel filtration on Sephadex G‐75 in a bile acid and KCl medium the preparation migrated like a 55000‐molecular‐weight protein. Upon gel electrophoresis on polyacrylamide or upon gel filtration on Sephadex G‐100 in a dodecylsulfate medium the protein part of the preparation migrated as one band of the apparent molecular weight of approximately 30000. These findings suggest that the purified cytochrome b is a dimeric heme protein.

In vivo incorporation of [3H]leucine into the protein part of the purified cytochrome b was insensitive to cycloheximide but sensitive to chloramphenicol. This indicates that the apoprotein of cytochrome b is translated on mitochondrial ribosomes.
A Simplified Procedure for the Assay of Adenosine 3′:5′‐monophosphate by the Activation of Liver Phosphorylase
Authors: van de Werve Gérald , van den Berghe Georges , Hers Henri‐Géry
Published: March 2005

The method devised by Butcher et al. (1965) for the determination of adenosine 3′:5′‐monophosphate (cyclic AMP) and based on the ability of the nucleotide to enhance the rate of activation of liver phosphrylase has been greatly simplified. The laborious purifications of enzymes included in the initial procedure were found unnecessary, since a crude liver extract could be used as a source of phosphorylase b and of reactivating system. The method requires minimal purification of tissue samples and allows the precise determination of 1 to 20 picomoles of cyclic AMP.
Hormonal Regulation of Rat Liver Cells Cultured in Chemically Defined Medium
Authors: Gerschenson Lazaro E. , Davidson Mayer B. , Andersson Myrna
Published: March 2005

The specific activity of the enzyme tyrosine 2‐oxoglutarate transaminase from an established rat liver‐cell line grown in chemically defined medium, increases under the addition of the synthetic glucocorticoid dexamethasone sodium phosphate. Insulin also induces an enzyme increase but only in cells exposed previously to dexamethasone. Lower and ineffective glucocorticoid concentrations are, however, effective per se in making the cells insulin‐sensitive. Optimal concentrations of the glucocorticoid increase the enzyme activity 3 to 4‐fold during the first 8–9 h of incubation, however the cells are not insulin‐sensitive until the tenth hour of incubation. These findings suggest that dexamethasone induces a factor(s) necessary for the insulin effects only on the enzyme since insulin increased the rate of labeled amino‐acids incorporation in trichloroacetic acid precipitates without need for cell pretreatment with the other hormone. Dexamethasone was found to increase the rate of labeled uridine incorporation into trichloroacetic acid preeipitates. Experiments using RNA and protein‐synthesis inhibitors indicate that dexamethasone operates at a transcriptional level, while insulin works at a translational level. Actinomycin does not “superinduce” the enzyme, however when the cells are preincubated with medium plus serum the above mentioned phenomenon reappears. At least two forms (I and II) of the enzyme have been observed in the cells by hydroxyapatite column chromatography using a phosphate step gradient. Both forms are inducible by dexamethasone. Form I is extremely heat‐sensitive (65 °C) while form II is completely heat‐resistant in the same condition. Incubation of the cells with dexamethasone does not change the heat stability of form II but increases significantly the resistance of form I to heat treatment. Insulin appears not to affect the heat stability of the enzyme.
Light‐Induced Absorbance Changes of the High‐Potential Cytochrome b559 in Chloroplasts
Authors: Ben‐Hayyim Gozal
Published: March 2005

Light‐induced absorption changes of cytochrome b559 have been studied in isolated chloroplast preparations at room temperature.

It was shown that cytochrome b559 could undergo both photooxidation and photoreduction, both these reactions occurring to its high‐potential form.

In the presence of FCCP and DCMU, a reversible photoreduction could be detected which was not sensitive to the presence of oxygen and which was probably a result of a cyclic electron flow around photosystem 2.

It is suggested that the high‐potential form of cytochrome b559, which is the native form, is mainly photooxidized by photosystem 2. The photoreduction is also mediated by photosystem 2 and can be detected only when electrons cannot flow from photosystem 2 to photosystem 1, i.e. in the presence of DCMU, or when the carriers are fully reduced in the absence of an electron acceptor.
The Interaction of Lysine Vasopressin with Free and Agarose‐Bound Bovine Neurophysin II
Authors: Pliška Vladimír , Sachs Howard
Published: March 2005

The interactions of tritiated lysine vasopressin with free and agarose‐coupled bovine neurophysin II were studied. Neurophysin II obtained by extraction of acetone‐dried hypophyses with strong acid contains at least two discrete populations of binding sites for lysine vasopressin, with association constants of 106 to 107 M−1 and 3×104 mM−1, respectively. The first population has a Hill coefficient higher than unity and accounts for 7–30% of the total binding sites in different batches of agarose‐bound neurophysin. With free neurophysin in solution, the size of this population was strongly dependent on the neurophysin concentration and decreased from about 17% at 0.05 mg/ml to less than 0.05% at 1.4 mg/ml. A somewhat lower Hill coefficient was found for the second class of binding sites. It is suggested that these two classes of binding sites are associated with polymerization equilibria of neurophysin in solution, the high‐affinity species being the monomeric, the low affinity species a polymeric form. For the agarose‐bound neurophysin, the pH optimum for binding was found to be 5.6–5.9 (vasopressin concentrations in the range of the first association constant). This binding was not affected by calcium chloride up to a concentration of 10 mM.
Purification and Properties of DNA Polymerases from Chick Embryo
Authors: Brun Gilbert , Rougeon François , Lauber Marc , Chapeville François
Published: March 2005

The main DNA polymerases present in cytoplasmic and nuclear soluble extracts of seven‐day‐old chick embryos have been purified. One of them, DNA polymerase I, mainly present in the cytoplasm has been obtained about 50–60% pure. Its molecular weight is 148 000 by Sephadex filtration and its s value is about 7.5S. This DNA polymerase is devoid of DNAase and RNAase activity. DNA polymerase II, of low molecular weight (50 000), sedimenting at 3 S, is the main DNA polymerase present in the nucleus and has been obtained 90% pure. The DNA polymerase II activity is also present in the cytoplasm. The purified cytoplasmic DNA polymerase II has a molecular weight of 27 000 and appears as a monomer of nuclear DNA polymerase II. The DNA polymerase II preparations, from either nucleus or cytoplasm, are devoid of nuclease activity and exhibit the same primer specificity; both are able to copy RNA strands in rAn· dTn hybrids whereas DNA polymerase I only copies the DNA strands in such hybrids.
Comparative Studies on the Template‐Initiator Requirements of the Chick‐Embryo DNA Polymerase I and the Avian‐Myeloblastosis‐Virus DNA Polymerase
Authors: Rougeon François , Brun Gilbert , Chapeville François
Published: March 2005

The template requirements of chick embryo DNA polymerase I devoid of nuclease activity, have been investigated. This enzyme catalyzes a repair reaction in vitro. Active templates were obtained by treatment of native DNA by DNAase I or exonuclease III. The DNA polymerase I activity requires divalent cations and is inhibited by high salt concentrations and sulfhydryl group blocking reagents.

Homopolydeoxynucleotides dAn, dTn, dCn, are efficient templates in the presence of hydrogen‐bound initiator. The high efficiency of hybrid structures rAn· dTn, dAn· rUn and rIn· dCn in which RNA acts as‐initiator and DNA as template suggests an essential role of this enzyme in DNA replication. The enzyme does not catalyze RNA‐directed DNA synthesis.

DNA polymerase I of chick embryo and the avian myeloblastosis virus DNA polymerase were compared on the basis of their activities in the presence of various synthetic primers. Using synthetic DNA · RNA hybrid such as rAn· dTn, the cellular enzyme elongates the RNA strand by copying the DNA template whereas the viral enzyme mainly copies the RNA strand and less efficiently the DNA strand. With polymers of the (I + C) or (G + C) series, both enzymes are equally efficient which dCn as template; the viral enzyme activity can be distinguished from cellular enzymatic activity principally by its capacity to transcribe rCn and less efficiently rIn. The relationship between the responses of these enzymes in vitro to synthetic primers and the assumed physiological function of the cellular and viral DNA polymerases are discussed.
Affinity Chromatography on Immobilised Adenosine 5′‐monophosphate
Authors: Harvey Michael J. , Craven David B. , Dean Peter D. G. , Lowe Christopher R.
Published: March 2005

The binding of various enzymes to two chemically defined affinity adsorbents, P1‐(6‐aminohexyl)‐P2‐(5′‐adenosine)‐pyrophosphate‐Sepharose and N6‐(6‐aminohexyl)‐5′‐AMP‐Sepharose, has been compared. The results have been interpreted in terms of the enzyme‐nucleotide interaction. The effect of ligand concentration on the nature and strength of enzyme binding to immobilised 5′‐AMP has been investigated and shown to be a critical paramcter. Dilution of the immobilised ligand with Sepharose or preparation of low ligand concentration adsorbents, can be utilised to facilitate elution of tightly bound enzymes. Conversely, significant increases in the binding and capacity of affinity adsorbents can be achieved by increasing the ligand concentration. The binding of lactate dehydrogenase in response to increasing ligand concentrations was not a simple function. The data have been interpreted in the light of current concepts on affinity chromatography.
Animal DNA‐Dependent RNA Polymerases
Authors: Mandel Jean‐Louis , Chambon Pierre
Published: March 2005

The RNA synthesized in vitro on superhelical Simian virus 40 (SV40) DNA (form I) by calf thymus and rat liver RNA polymerases B and by calf thymus RNA polymerase AI was characterized. Both 32P‐labeled ATP and GTP were found at the 5′ end of the RNAs synthesized by AI and B enzymes indicating that each type of enzyme possesses at least two initiation sites on SV40 DNA form I. Competition experiments between AI and B enzymes for initiation sites on SV40 DNA form I suggest that some of these sites could be different for the two enzymes. The chain growth rates of RNA chains synthesized by calf thymus AI and B enzymes on SV40 DNA form I were determined. Both calf thymus RNA polymerase AI and calf thymus and rat liver RNA polymerases B transcribed symmetrically SV40 DNA form I under a variety of incubation conditions. Under similar conditions Escherichia coli RNA polymerase holoenzyme transcribed asymmetrically SV40 DNA form I, while symmetrical transcription was observed with E. coli core enzyme. The maximum size of the RNAs synthesized by calf thymus enzyme AI was several times the length of the SV40 genome, indicating that this enzyme can pass several times over its initiation site and transcribe SV40 DNA form I over the entire genome. In contrast, RNAs made by enzymes B had a more definite maximum size (18–20 S), indicating differences between AI and B enzymes at the level of chain termination. However, this shorter size of RNA's synthesized by enzymes B does not correspond to a selective transcription of some part of the viral genome, since our results indicate that most, if not all, of the SV40 genome was transcribed by enzymes B. The arrest of elongation of RNA chains synthesized by enzymes B was related to the low ionic strength incubation conditions and did not reflect a true termination step. It appears therefore that, when transcribing SV40 DNA form I in vitro, mammalian RNA polymerases AI and B do not exhibit the two main characteristics of the prokaryotic transcription, namely asymetry and selectivity. Our results are discussed in the light of the present knowledge of the transcription of SV40 DNA in vivo during the lytic cycle.
Animal DNA‐Dependent RNA Polymerases
Authors: Hossenlopp Paul , Oudet Pierre , Chambon Pierre
Published: March 2005

The binding of purified mammalian RNA polymerases AI and B to Simian virus 40 (SV40) DNA was studied under various conditions (ionic strength, divalent cation, temperature) by two methods, electron microscopy and retention of enzyme · DNA complexes on nitrocellulose filters. Our studies demonstrate that there are multiple binding sites for mammalian RNA polymerases on SV40 DNA form I and that highly stable enzyme · DNA‐form‐I complexes are formed. However, in contrast to the bacterial RNA polymerase holoenzyme, the purified mammalian enzymes are unable to form a highly stable complex with a linear double‐stranded DNA (SV40 DNA form III). Our results strongly suggest that the formation of highly stable complexes between mammalian RNA polymerases and SV 40 DNA form I is due to their binding to the unpaired regions which are present in the superhelical DNA.
Fractionation of the DD‐Carboxypeptidase‐Transpeptidase Activities Solubilized from Membranes of Escherichia coli K12, Strain 44
Authors: Pollock Jerry J. , Nguyen‐Distèche Martine , Ghuysen Jean‐Marie , Coyette Jacques , Linder Regina , Salton Milton R. J. , Kim Kwang S. , Perkins Harold R. , Reynolds Peter
Published: March 2005

A transpeptidase activity in Escherichia coli was measured independently of other enzymes involved in peptidoglycan synthesis by quantitating the formation of UDP‐N‐acetylmuramyl‐l‐alanyl‐y‐d‐glutamyl‐(l)‐meso‐diaminopimelyl‐(l)‐d‐alanyl‐[14C]glycine when UDP‐N‐acetylmuramyl‐l‐alanyl‐y‐d‐glutamyl‐(l)‐meso‐diaminopimelyl‐(l‐d‐alanyl‐d‐alanine was used as donor substrate and [14C]glycine as acceptor in a transfer reaction. After extraction of membrane envelopes with Brij‐36T and subsequent ammonium sulfate precipitation, DEAE‐cellulose chromatography revealed two major fractions; one not adsorbed to the ion‐exchange resin and the other adsorbed. The fraction which was bound to DEAE‐cellulose was bound to and could be eluted from an ampicillin affinity chromatography system while the fraction not bound to DEAE‐cellulose was also not bound to the ampicillin column. Both unbound and bound ampicillin fractions exhibited dd‐carboxypeptidase and transpeptidase activities although for equivalent dd‐carboxypeptidase activity, the bound ampicillin fraction required about five times more glycine acceptor to achieve the same amount of transpeptidation as the unbound ampicillin fraction.
Raman Study on the Two Quaternary States of Unligated Hemoglobin
Authors: Sussner Heinrich , Mayer Adalbert , Brunner Horst , Fasold Hugo
Published: March 2005

To decide whether the change in the quaternary state of unligated hemoglobin affects the electronic and structural properties of the heme group unligated hemoglobins modified with bis‐(N‐maleimidomethyl)ether and 2, 2′‐dicarboxy‐4, 4′‐diiodoacetamidoazobenzol, which are in the high‐affinity form, have been compared with unligated normal deoxyhemoglobin A by absorption spectroscopy and Raman scattering. The absorption spectra show no difference in the frequency of the bands in the visible and near ultraviolet region but differences were found in peak height and shape of the Soret band. The Raman resonance spectra recorded between 300 and 1700 cm−1 show no difference at all. These spectral observations indicate that the change of the quaternary structure of unligated hemoglobin slightly influences the excited electronic states of the heme but does not alter the electronic ground state. Along with other spectroscopic evidence from Mössbauer and nuclear magnetic resonance spectroscopy this suggests that only a minor fraction of the free energy difference of ligand binding in the two quaternary states may originate from differences in the electronic properties of the heme in unligated hemoglobin.
The Reactive Sulfhydryl Groups of Cytoplasmic Aspartate Aminotransferase
Authors: Wilson Kenneth J. , Birchmeier Walter , Christen Philipp
Published: March 2005

The two readily reacting sulfhydryl groups out of a total of five per subunit of cytoplasmic aspartate aminotransferase have been identified as those in positions 45 and 82 of the primary structure. Cys45 exhibits approximately a 20‐fold higher reactivity toward 5,5′‐dithiobis‐(2‐nitrobenzoate) and tetranitromethane than Cys22. The alterations in enzymatic activity observed following blockage of these two thiol groups by N‐ethylmaleimide and tetranitromethane, 125% and 60% of the initial enzymatic activity, respectively [Birchmeier, W., Wilson, K. J. & Christen, P. (1973) J. Biol. Chem. 248, 1751] are due to the modification of the faster‐reacting Cys45. The rate of modification at both thiol groups is independent of the presence of the substrates. The fluorescence properties of N‐dansylcysteine substituents at both Cys45 and Cys82 indicate a polar environment corresponding to their localization on the exterior of the enzyme molecule.
Studies with Analogues of Nicotinamide Adenine Dinucleotide
Authors: Biellmann Jean‐François , Hirth Christian G. , Jung Michel J. , Rosenheimer Nicole , Wrixon Anthony D.
Published: March 2005

Through a series of correlative studies, the stereochemistry of the enzymatic processes with the coenzyme analogues of NAD+, 3‐acetylpyridine‐adenine dinucleotide, thionicotinamide‐adenine dinucleotide and 3‐cyanopyridine‐adenine dinucleotide, have been examined for horse liver and yeast alcohol dehydrogenase (both A‐specific for NAD+) and glutamate dehydrogenase (B‐specific for NAD+). In each case the stereochemistry of the process with respect to substrate and coenzyme remains identical to that for NAD+. The implications for this are examined in view of the different geometry of the substituents at the 3‐position of the pyridine ring. A ready conversion of the thionicotinamide analogue of NADH to NADH is also described.
Energetic and Spectroscopic Studies on the Interaction between Thiamine Diphosphate and Apotransketolase
Authors: Heinrich Claus P. , Schmidt Dieter , Noack Klaus
Published: March 2005

Molecular complexes found between indoleacetate and various thiamine derivatives have been studied as a model system for the coenzyme‐apoenzyme interaction in transketolase from baker's yeast. Association constants, ΔH and ΔS values for these complexes were determined by ultraviolet and proton magnetic resonance spectroscopy. No remarkable differences of these constants were found for the various thiamine derivatives, except tetrahydrothiamine and 4‐methyl‐5‐(2‐hydroxyethyl)thiazole, which did not give a complex with indoleacetate. These findings prove that only the positively charged thiazolium ring system is able to form a charge transfer complex with indoleacetate. If indoleacetate was replaced by tryptophan, charge transfer complex formation with the thiamine derivatives could also be demonstrated by circular dichroism measurements, where a new band around 320 nm appeared.

A negative Cotton effect around 320 nm is also observed upon addition of thiamine diphosphate to apotransketolase. The circular dichroism spectra of transketolase reconstituted with the diphosphates of thiamine, oxythiamine, pyrithiamine and tetrahydrothiamine indicate different kinds of coenzyme binding. A ΔH of 7.4 kcal/mol was found for the enthalpy of formation of holotransketolase from its components.
Fluorescence Time‐Resolved Spectroscopy and Fluorescence Anisotropy Decay of the Staphylococcus aureus Endonuclease
Authors: Brochon Jean‐Claude , Wahl Philippe , Auchet Jean‐Claude
Published: March 2005

The intrinsic fluorescence of Staphylococcus aureus endonuclease has been studied by the time‐resolved spectroscopy method. The tyrosine emission, with a decay time of 1.8 ns, is separated from the tryptophan emission with a decay time of 5.5 ns.

The binding of an inhibitor, thymidine 3′,5′‐bisphosphate (pdTp), decreases the intensity of the tyrosine spectrum.

The molecular correlation time determined by the anisotropy decay measurement of the tryptophanyl residue is 8.9 ns. This value corresponds to a strongly hydrated molecule.

The fixation of pdTp does not change this correlation time, showing that no gross structural modifications are induced by the inhibitor.
Studies of Glutamate Dehydrogenase
Authors: Rasched Ihab , Sund Horst , Jörnvall Hans
Published: March 2005

Glutamate dehydrogenase was reacted with glyoxal and NaB3H4 and the labelled protein digested with trypsin. The radioactive peptides were isolated and identified in the primary structure of the enzyme.

Many peptides contained small amounts of radioactivity which is probably due to unspecific reactions with glyoxal. The amino group of one lysine residue was labelled to a greater extent than any other residue and accounted for about half of the total label. This amino group is involved in the binding of the substrate 2‐oxoglutarate and was identified as lysine‐27. The lysine‐126 which reacts with pyridoxal phosphate and 4‐iodoacetamidosalicylate is not modified with glyoxal but also involved in the binding of the substrate. This leads to the conclusion that both these lysine residues, although separated by 99 amino acids in the primary structure, may be at the active site of the enzyme and hence positioned close together in the tertiary structure.
Enzymatic and Chemical Synthesis of 3‐Sulfinopropionic Acid, an Analog of Succinic Acid
Authors: JollÉS‐BERGERET Bernadette
Published: March 2005

3‐Sulfinopropionic acid, the product of transamination and dehydrogenation of homohypotaurine (3‐aminopropanesulfinic acid), was isolated as the barium salt from incubated extracts of Pseudomonas fluorescens. The sodium and barium salts of the same compound were chemically synthetized from acrylic acid and formaldehyde sulfoxylate with the intermediary formation of bis(2‐carboxyethyl)sulfone. The identity of the compounds prepared either by enzymatic or by chemical synthesis has been demonstrated.

Some physicochemical properties of 3‐sulfinopropionic acid are deseribed.
Evidence that Citrate Synthase Operates by an Ordered Ternary‐Complex Mechanism
Authors: Johansson Carl‐Johan , Pettersson Gösta
Published: March 2005

Gel equilibrium‐diffusion measurements show that acetyl‐CoA binds to two indistinguishable sites in citrate synthase with a dissociation constant of 0.13 mM, which exceeds Km for acetyl‐CoA by a factor of about 20. This observation is inconsistent with previous suggestions that the citrate synthase‐catalyzed condensation reaction between acetyl‐CoA and oxaloacetate proceeds by a rapid‐equilibrium random‐order mechanism, and provides strong evidence that the mechanism is effectively ordered with oxaloacetate adding first to the enzyme, which makes it possible to calculate on and off constants for the binding of oxaloacetate (k2= 4 × 109 min−1 M−1 and k−2= 2 × 104 min−1). Confirmatory evidence for this interpretation of the experimental data is given by the product‐inhibition pattern observed in the presence of citrate.
Purification and Some Physical Properties of a Chymotrypsin‐Like Protease of the Larva of the Hornet, Vespa orientalis
Authors: Jany Klaus‐Dieter , Pfleiderer Gerhard , Molitoris H. Peter
Published: March 2005

A chymotrypsin‐like endopeptidase has been purified by ion‐exchange chromatography, affinity chromatography, and gel filtration. The enzyme preparation is homogeneous in the ultracentrifuge and disc electrophoresis. The enzyme is proved to be free from any other proteolytic activities. The molecular weight of the proteinase as determined with several techniques (ultracentrifugation, gel filtration and electrophoresis without dodecylsulfate) is in the range between 13000–14000; however, by dodecylsulfate gel electrophoresis a molecular weight of 23000 was obtained. The obtained physical constants of hornet chymotrypsin are: sedimentation coefficient s020,w= 1.96 × 10−13 S, diffusion coefficient D020,w= 131 μm2s−1, partial specific volume v̄= 0.737 ml g−1, frictional ratio f/fmin= 1.04, and degree of hydration = 0.1 g per g protein.
Isolation and Properties of Hydroxycinnamate:CoA Ligase from Lignifying Tissue of Forsthia
Authors: Gross Georg G. , Zenk Meinhart H.
Published: March 2005

From young lignifyin stems of Forsythia an enzyme specifically catalyzing the formation of CoA thiol esters of hydroxylated cinnamic acids was isolated and partially purified. This enzyme is most active with o‐, m‐, p‐coumarate, caffeate, ferulate and isoferulate. CoA, ATP, Mg2+ and a reduced thiol are required as cofactors; ATP is cleaved in this reaction to AMP and PPi. The enzyme has a molecular weight of approx. 55000 and shows a pH optimum at pH 7.3. In the presence only of cinnamic acids, the enzyme catalyzes an exchange reaction between PPi and ATP, which is drastically inhibited by CoA. A large number of benzoic acids as well as aliphatic and α‐amino acids is inactive in this assay. Feruloyl‐AMP could be demonstrated to participate as an activated intermediate in the formation of the CoA thiol esters.
Properties of Soluble and Bound Protein Kinases Isolated from Swine Kidney
Authors: Abou‐Issa Hussein , Kratowich Nancy , Mendicino Joseph
Published: March 2005

Several protein kinases which catalyze the phosphorylation of glyeogen synthetase, histones, protamine and proteins present in isolated plasma membranes, microsomes and nuclei have been extensively purified from swine kidney homogenates. About 60% of the protein kinase activity in kidney was recovered in the soluble 100000 × g supernatant fraction following differential centrifugation. The remaining 40% of the activity was found to be associated with the plasma membrane, nuclear and microsomal subcellular fractions. The activity of the protein kinases which were bound to the particulate structures was not enhanced by adenosine 3′:5′ ‐mono‐phosphate (cyclic AMP) when kidney glycogen synthetase or histone was used as the protein substrate, however the rate of phosphorylation of these substrates by the soluble enzymes was increased 50 to 100% by the addition of 0.5 μM cyclic AMP. The bound protein kinase activity was dissociated from the particulate structures by treatment with 0.7 M NaCl. The soluble and bound protein kinase were purified by a procedure involving treatment with alumina Cγ, adsorption to calcium phosphate gel, and chromatography on diethylaminoethylcellulose. Both protein kinase preparations were separated into two fractions by the last procedure and their elution profiles were very similar. The apparent Km for ATP in the presence of 10 mM MgCl2 varied from 5 to 9 μM When either glycogen synthetase or histone was the substrate. All four preparations showed a very broad specificity for protein substrates. They phosphorylated histones, protamine, casein and glycogen synthetases. The molecular weight of the four kidney protein kinase preparations calculated from data obtained by sucrose‐density centrifugation and chromatography on Sephadex G‐200 was 170000 ± 10000. Upon polyacrylamide‐gel electrophoresis in 1% sodium dodecylsulfate all of the samples showed two major components.

The kinetic properties of the bound form of protein kinase was examined with a sonicated preparation of kidney plasma membranes. The molecular weight of the active fragment was greater than 1000000. Proteins present in the membrane were labeled with 32P when the preparation was incubated with magnesium ion and [γ‐32P] ATP. This preparation also phosphorylated glycogen synthetase and histone. However, the apparent Km of the bound form of the enzyme for ATP was 2.8 mM when glycogen synthetase was used as the protein substrate.
Purification and Some Properties of an Endocellular Sucrase from a Constitutive Mutant of Bacillus subtilis Marburg 168
Authors: Kunst Frank , Pascal Martial , Lepesant Jean‐Antoine , Walle Josette , Dedonder Raymond
Published: March 2005

In Bacillus subtilis Marburg 168 an endocellular invertase‐like sucrase can be induced by sucrose. Sucrose, glucose and glycerol exert a catabolite repression on sucrase synthesis in both the inducible strain 168 and a constitutive derivative isolated in this laboratory. Derepressed cultures of this constitutive mutant grown on glutamate and succinate procedure high levels of sucrase and are utilized as starting material for the purification process.

The purified preparation gives one single protein band after disc electrophoresis on polyacrylamide gel in the absence or in the presence of dodecylsulphate. This enzyme has a molecular weight of about 55000.

Sucrase is not a glycoprotein, it is activated by potassium ions and inhibited by p‐hydroxymercuribenzoate. Arguments are presented in favour of the hypothesis that, in vivo, 6G‐phosphorylsucrose (6‐O‐phosphoryl‐α‐D‐glucopyranosyl‐1,2‐β‐d‐fructof uranoside) rather sucrose serves as a substrate for this enzyme.
Régulation du métabolisme des hexuronates chez Escherichia coli K12
Authors: Robert‐Baudouy Janine M. , Portalier Raymond C. , Stoeber François R.
Published: March 2005

Kinetics of induction of the five enzymes catalyzing hexuronate (d‐galacturonate and d‐glucuronate) to 2‐keto‐3‐deoxy‐d‐gluconate catabolism in Escherichia coli K12 have been studied.

In wild type organism, two patterns of hexuronate system induction have been identified. The first is limited, observed with galacturonate or tagaturonate and characterized by only inducing the three galacturonate degradative enzymes: uronic isomerase, altronate: NAD+ oxidoreductase and altronic hydrolyase.

The second one is general, induced by glucuronate or fructuronate and characterized by induction of the five enzymes of the system: uronic isomerase, altronate : NAD+ oxidoreductase and hydrolyase, mannonate : NAD+ oxidoreductase and hydrolyase.

In both cases, differential rates of synthesis of enzymes have been measured when the nature or the concentration of the inducer is varied. Induction effects of d‐mannonic amide have also been examined: this compound induces all the hexuronate system under conditions of gratuity. Hexuronate system induction was subjected to catabolite repression.

Induction occurred without significant delay and differential rates of synthesis remained constant during two or three doublings of cell mass after addition of the inducer. The process is extremely specific: metabolites of the hexuronate pathway and some very closely related substances only showed inducing power.

Using a set of single mutants carrying mutations in structural genes of enzyme of the hexuronate system, it has been possible to establish that limited and general inductions were indirect processes. In the former situation, tagaturonate acted as true inducer, in the latter case, fructuronate and perhaps mannonate acted as well. Uronic isomerase and altronic hydrolase on the one hand, mannonate: NAD+ oxidoreductase and mannonic hydrolyase on the other hand were coordinately induced. These results agree very well with independent genetic studies and strongly suggest the existence of two corresponding operons.

In agreement with the isolated location of its structural gene, the induction of altronate: NAD+ oxidoreductase was completely decoordinated with the induction of all four other enzymes of the system, which indicates the existence of a corresponding independent operon.

Physiological properties of various regulatory mutants suggest an unique regulatory mechanism for controlling differential induction of hexuronate system.
Sulfhydryl Group Reactivity of d‐Galactose Dehydrogenase from Pseudomonas saccharophila
Authors: Wengenmayer Friedrich , UeberschÄR Karl‐Heinz , Kurz Gerhart
Published: March 2005

Native d‐galactose dehydrogenase from Pseudomonas saccharophila contains 12 SH‐groups and no disulfide bonds. Storage of enzyme causes a decrease of enzymatic activity, a loss of SH‐groups, and the formation of disulfide bonds. An approximately linear relationship exists between specific activity and the number of SH‐groups per mole enzyme for eight of the 12 SH‐groups.

On the basis of their reactivity against 5,5′‐dithio‐bis(2‐nitrobenzoic acid), the SH‐groups of the enzyme can be differentiated in three classes, each containing four SH‐groups.

The catalytic activity is completely lost upon stoichiometric modification of the first four SH‐groups, which react readily with 5,5′‐dithio‐bis(2‐nitrobenzoic acid). The inactivation follows pseudo‐first‐order kinetics with a second‐order rate constant of k= 1.3 mM‐1 s‐1, and is completely reversible upon the addition of reducing agents, such as 2‐mercaptoethanol or 1,4‐dithioerythritol.

Evidence is presented that the modification of these first four SH‐groups does not occur with the enzyme · NAD(H) complex; only the free enzyme is subject to the reaction with Nbs2. The presence of d‐galactose or l‐arabinose has no influence on the rate of inactivation.

In contrast to the SH‐groups of the second and third class, the first four are generally not involved in intra‐ and intermolecular disulfide bridges of the enzyme.
On the Nature of argR Mutations in Saccharomyces cerevisiae
Authors: Hoet Philippe P. , Waime Jean‐Marie
Published: March 2005

Ornithine, citrulline and homoarginine having a regulatory role on arginine biosynthetic enzymes in wild‐type, but not in argR mutants, do not interfere with aminoacylation of tRNAArgin vitro or in vivo. The same aminoacylation reaction does not seem to be modified in argR mutations, strengthening the hypothesis of a mutation, involving the apopressor.
Integrative Suppression of Temperature‐Sensitive Mutants with a Lesion in the Initiation of DNA Replication
Authors: Goebel Werner
Published: March 2005

A number of F‐like transmissible plasmids (R1drd16, F'lac and Hly‐PM152), but bot the non‐transmissible plasmids ColE1 and ColE2, can suppress the defect in initiation of DNA replication of a group of temperature‐sensitive dnaA mutants of Escherichia coli (Hfr‐120/6, CRT‐46, Hfr‐T46) by integration into the bacterial chromosome. Temperature‐sensitive Hfr‐strains of these mutants which do not already carry additional autonomous plasmids capable of causing integrative suppression also give rise to such revertants. The reversion rate of the dnaA Hfr strains is inhibited by an additional autonomous F‐like or I‐like transfer factor.

Examination of the plasmid DNA of mutants suppressed by the integration of either the R1drd16 factor, the F'lac factor or the Hly‐PM152 factor demonstrates that R1 DNA and F'lac DNA are completely integrated into the chromosome. The Hly‐PM152 factor, however, is often only partially integrated, with the rest of this plasmid being left in a stable autonomous state.

The dnaA mutants suppressed by the integration of a plasmid can support replication of the autonomous ColE1 plasmid, at elevated temperature. However, the number of copies of this plasmid maintained in cells at 43°C is lower than at 30°C.
Biological and Immunological Activity of Fructose 1,6‐bisphosphatase
Authors: Kolb Helmut J.
Published: March 2005

The relation between the catalytic activity in the bioassay and the immunologic activity in the immuno‐displacement assay of fructose 1,6‐bisphosphatase was investigated. After biological inactivation by long‐time exposure to room temperature or by treatment with 1% acetic acid, the enzyme could still be quantitatively determined by the immunoassay. Biological inactivation and structural changes were demonstrated after treatment with 4 M and 8 M urea as well as 5 M guanidine hydrochloride solution. The sedimentation coefficient dropped from 7.5 S to about 2 S, and high‐speed sedimentation equilibrium runs yielded a molecular weight of 35000 and 33000 for the enzyme denatured in 8 M urea and 5 M guanidine hydrochloride, respectively, which shows that the enzyme is dissociated into subunits prior to the incubation with the antiserum. Measurements of optical rotatory dispersion revealed a pronounced change of the absorption trough at 233 nm compared to the native enzyme. In the radioimmunoassay fructose 1,6‐bisphosphatase denatured by urea and guanidine hydrochloride followed the standard curve of the native enzyme very closely. In contrast, that inactivated and denatured by 5% sodium dodecylsulfate did not displace 135I‐labelled fructose 1,6‐bisphosphatase from the antibody.

A sensitive standard curve for the bisphosphatase was also obtained in the presence of 4 M urea in the assay medium, while higher concentrations interfered.

The data indicate that there is no correlation between the catalytic activity and the capability to displace 126I‐labelled fructose 1,6‐bisphosphatase from the antibody. It can be concluded that catalytically inactive enzymes can be quantitatively determined by a specific radioimmuno assay; however, this technique is insensitive to detect conformational changes in the structure of the enzyme.
Immunochemical Homologies among Vertebrate Lactate‐Dehydrogenase Isozymes
Authors: Holmes Roger S. , Scopes Robert K.
Published: March 2005

Lactate dehydrogenase A4, from possum, chicken, and sardine muscle extracts and lactate dehydrogenase B4, from chicken heart and sardine muscle extracts have been purified. Specific activity determinations confirmed previous studies showing the A4, isoenzyme to be approximately twice as efficient as the B4, isozyme and fish lactate dehydrogenase A4, to be considerably more active than the corresponding enzyme in warm‐blooded animals. Spectrophotometric analyses demonstrated striking similarities in ɛ2801 mg/ml values, which reflected a uniform tryptophan content (5 residues lactate dehydrogenase subunit) in the enzymes analysed.

Sheep antisera were prepared against each isoenzyme and used in immunochemical titration experiments to measure the extent of inhibition, precipitation and interaction of the lactate dehydrogenase isozymes by the antisera. Lactate dehydrogenase antibodies differed in their immunochemical specificities according to the nature and source of the antigen. For example, the relative affinities of antiserum to possum A4, isozyme for the enzymes was in the order, possum A4 >, chicken A4 > sardine A4, ≫, sardine B4 >, chicken B4, which compared with the affinities for antichicken B4, chicken B4 >, sardine B4≫ sardine, chicken and possum A4. In contrast to the results of other workers, isozymes A4, and B4, were shown to be immunochemically related. This observation is attributed to the higher degree of sensitivity in recognizing enzyme‐antibody interactions using gel zymogram/immunochemical technique.

The antisera were also used in investigating the immunochemical properties of isozymes A′4, A′4, B4, B′4, and E4, from trout, and isozymes A4, B4 and C4 from pigeon, kangaroo and rat‐tissue extracts. These vertebrate lactate dehydrogenase isozymes shared common antigenic determinants which reflected differing degrees of sequence homology and a common evolutionary origin.