bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
An Approach for the Determination of Equilibrium Constant of Structural Motility
Authors: Vas Mária , Boross László 
Published: March 2005

A kinetic approach is presented for the determination of equilibrium constant of a local conformational fluctuation of a protein molecule. As a result of this fluctuation a distinct side chain becomes temporarily accessible to a specific reagent. Determination of fluctuation constant, Kf, is based on the kinetic analysis of chemical modification of the side chain with a selective reagent which reacts faster than the conformational changes occur.The use of p‐mercuribenzoate‐mercaptide of 2‐nitro‐5‐mercaptobenzoate as an–SH reagent is described to determine Kf of d‐glyceraldehyde‐3‐phosphate dehydrogenase around residue Cys‐153, when the hyper‐reactive side‐chain Cys‐149 was previously carboxymethylated (CM‐enzyme). The values of Kf are 0.034 ± 0.005 and 0.014 ± 0.003 in the case of CM‐apoenzyme and holoenzyme, respectively, in Tris‐HCl buffer, I= 0.05, pH 7.5, at 5 ° C. The free enthalpy change (ΔG) accompanying the conformational change which makes Cys‐153 accessible to the reagent, is 1.86 ± 0.08 and 2.34 ± 0.11 kcal/mol for CM‐apoenzyme and holoenzyme, respectively. The enthalpy change (ΔH) is + 3.9 ± 0.7 kcal/mol both in the absence and presence of coenzyme. The entropy change (ΔS) is + 7.6 ± 1.3 and + 6.3 ± 1.1 cal × degree−1× mol−1 for CM‐apoenzyme and holoenzyme, respectively.Based on the value of Kt and our earlier kinetic data for mercaptide formation of Cys‐153 one can characterize the structural motility: the rate constants of exposure and masking of residue Cys‐153 of the apoenzyme are k+1, = 2.2 ± 0.2 min−1 and k−1= 65 ± 16 min−1. respectively, under the experimental conditions.
Control of Lipid Metabolism in Adipose‐Tissue Hornogenates of Fasted Refed Rats
Authors: Winand Jacques , Furnelle Jacques , Hebbelinck Marcel , Christophe Jean 
Published: March 2005

The 700 ×g fat‐poor internatant from epididymal adipose tissue homogenate of fasted refed rats was incubated at pH 6.5 with 0.5 mM [1‐14C]acetate in the presence of cofactors. After incubation, six lipid classes were separated by thin‐layer chromatography and their fatty acids analyzed by radio gas‐liquid chromatography. The incorporation of [1‐14C]acetate into glycerides was limited by the activity of acetyl‐CoA carboxylase, and was stimulated two to three‐fold by the addition of 0.1 mM unlabelled malonyl‐CoA.
The chain‐length of fatty acids labelled in vitro was controlled by the relative concentrations of malonyl‐CoA and acetyl‐CoA. Addition of unlabelled malonyl‐CoA influenced the operation of fatty acid synthetase in such a way that the ratio of radioactive palmitate to radioactive myristate increased markedly.
sn‐Glycerol 3‐phosphate stimulated the esterification of fatty acids into triglycerides. Under these conditions whereas the specific activity of free fatty acids was much higher than that observed in triglycerides, the specific activity of diglycerides was low, when compared to the same triglycerides. Newly synthesized monounsaturated fatty acids were preferentially esterified into triglycerides.
0.5 mM [1‐14C]palmitate was efficiently incorporated into triglycerides but not into diglycerides.
Inhibition of 3‐sn‐Phosphatidylcholine Biosynthesis in Baby‐Hamster Kidney‐21 Cells Infected with Semliki Forest Virus
Authors: Vance Dennis E. , Burke Derek C. 
Published: March 2005

The infection of baby hamster kidney cells with Semliki Forest virus results in the inhibition of [Me‐3H]choline incorporation into 3‐sn‐phosphatidylcholine of the cytoplasmic membranes of intact cells. This inhibition was also observed in purified plasma membrane and endoplasmic reticulum. The major decrease of incorporation of label occurred between 4 and 6 h after infection.Microsomes from baby hamster kidney cells infected with Semliki Forest virus exhibit approximately a 50% decrease in total activity and a 40% decrease in specific activity of the enzyme CDP‐choline: 1,2‐diglyceride cholinephosphotransferase. This decrease in enzymatic activity occurred between 4 and 6 h post‐infection. When compared to mock‐infected cells, the enzymatic activity from infected cells had a similar apparent Km for CDP‐choline and a similar half‐maximal stimulation value for 1,2‐diacyl‐sn‐glycerol. Experiments with cycloheximide and actinomycin D suggest that viral inhibition of host protein and RNA synthesis would not account for the depressed cholinephosphotransferase activity. However, since cycloheximide stimulates the choline‐phosphotransferase activity of mock‐infected cells, the results of these experiments must be interpreted with caution. A freely diffusable inhibitor was not detected in the microsomes from infected cells.Infection of baby hamster kidney cells with a picornavirus (bovine enterovirus) did not inhibit or stimulate [Me‐3H]choline incorporation into 3‐sn‐phosphatidylcholine nor did it affect the activity of CDP‐choline : 1,2‐diglyceride cholinephosphotransferase.
Structure and Antigenicity of Hen Egg‐White Lysozyme Fragments
Authors: Matthyssens Gaston E. , Giebens Gonda , Kanarek Louis 
Published: March 2005

Under controlled conditions of pepsin digestion of hen egg‐white lysozyme, large fragments with composition identical to sequence 57—107 and sequence (57—83) + (91—107), have been obtained.Inhibition of lysozyme‐anti‐lysozyme precipitation by these fragments, showed that section 84—90 is important for the antigenic specificity of fragment 57—107. The position of one lysozyme antigenic region was suggested, namely section 85—90 combined with a region close to methionine 105, reacting with about 10% of total anti‐lysozyme antibodies. Structure evaluation of the purified peptides through circular dichroism, revealed that only 30% of the “native” alpha‐helicity amount (ie. the amount of helicity this fragment possesses when integrated in the lysozyme molecule) remained. We concluded that in the case of lysozyme, mainly long‐range interactions stabilize the native configuration. Secondly, that differences in antigenic specificity among related peptides are not due to differences in structure. Fragment‐antibody interaction could be explained by assuming the existence of a simple equilibrium Fr⇌ Fn between the completely random and the native fragment, or, through an “induced fint” mechaism.
Purification of Neurophysins by Affinity Chromatography
Authors: Frénoy Jean‐Pierre , Ménard Joël , Pesquiès Pierre , Corvol Pierre 
Published: March 2005

(8‐Lysine)‐vasopressin was coupled to activated Sepharose by cyanogen bromide with 65%, yield. Lysine‐vasopressin antibodies were specifically retained on a lysine‐vasopressin Sepharose column and purified in a single step. They kept their ability to bind lysine‐vasopressin.Pig neurophysins prepared from fresh porcine posterior pituitaries by 0.1 M HCOOH extraction and by Sephadex gel filtration, were applied to the lysine‐vasopressin‐Sepharose column. A major protein peak was retained and eluted by 0.1 M HCOOH. Polyacrylamide gel electrophoresis of this peak revealed two major protein bands and six minor bands. These proteins have been called neurophysins for several reasons: (1) they bind lysine‐vasopressin coupled to Sepharose, (2) they show no defined absorption at 275—300 nm, (3) they have a molecular weight on dodecylsulfate‐polyacrylamide gel electrophoresis of 11 000 and 15500 which is close to those already reported for neurophysins I and II, (4) the whole amino‐acid composition is similar to that already given for the purified neurophysins. This study demonstrates the usefulness of affinity chromatography for the purification of lysine‐vasopressin‐binding proteins. It shows the heterogeneity of porcine neurophysins.
The Intracellular Location of Pyruvate Carboxylase in Rat Liver
Authors: Weiss Gerhard , Lamartiniere Coral A. , Müller‐ohly Brigitte , Seubert Werner 
Published: March 2005

Because of reported extramitochondrial location of pyruvate carboxylase in rat liver concluded from extraction studies and the importance of these findings for the concept of the regulation of gluconeogenesis, the intracellular location of this enzyme in rat liver was reinvestigated. The following results were obtained.

Reported differences in extraction properties between pyruvate carboxylase and glutamate dehydrogenase in rat liver are due to secondary binding of the latter enzyme to nuclei, cell debris, mitochondria and microsomes.
Rat‐liver pyruvate carboxylase is located exclusively in mitochondria, a location in another subcellular component is excluded.
Rat liver mitochondria are heterogeneous with respect to their enzyme content. Rate‐zonal centrifugation reveals asymmetric distribution of mitochondrial matrix enzymes: Ornithine aminotransferase is shifted towards slower‐sedimenting mitochondria, pyruvate carboxylase, malate dehydrogenase and aspartate aminotransferase in faster‐sedimenting mitochondria. Glutamate dehydrogenase as well as monoamine oxidase (outer membrane) and adenylate kinase (inter‐membrane space) are located in an intermediate position.
Electronmicrographs show that the mitochondria retain their intact outer membrane and most of them are in condensed conformation.

The biological significance of the observed heterogeneities with respect to the regulation of gluconeogenesis is discussed.
Modulation of Mitochondrial Adenylate Kinase by Citric‐Acid‐Cycle Intermediates
Authors: Pradhan Tapas K. , Criss Wayne E. 
Published: March 2005

Modulation of rat liver mitochondrial adenylate kinase by citric acid cycle intermediates is reported.Activation of adenylate kinase in the presence of Mg2+ was dependent upon the following. (1) It was activated by citrate, cis‐aconitate, isocitrate, fumarate, and malate but not by 2‐oxo‐glutarate, succinate, or oxaloacetate. (2) Protonated forms of the intermediates were most effective. (3) Citrate caused a decrease from 6.9 to 4.1 in the Km for ATP. (4) The activation was not a simple citrate‐Mg2+ chelation phenomenon. (5) The Ka for citrate was 90 μM. (6) Activation was very rapid. (7) Citrate caused a decrease from 2.1 to 1.3 in the slopes of Hill plots with increasing ATP. (8) Measurements in differential absorbancies in the presence and absence of citrate and Mg2+ indicated structural changes in adenylate kinase upon modulator binding. (9) Non‐additive effects of saturating concentrations of several citric acid cycle intermediates could be interpreted as a common binding site for each of the intermediates. It is postulated that the citrate modulation of adenylate kinase involves an alteration in the structure of the enzyme.