bluuebunny/crossref_metadata_2025_split huggingface dataset:
Presteady‐state and steady‐state kinetic properties of human cytochrome c oxidase
Authors: Van KUILENBURG André B. P. , GORREN Anton C. F. , DEKKER Henk L. , NIEBOER Popko , Van GELDER Bob F. , MUIJSERS Anton O. 
Published: March 2005

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady‐state and steady‐state kinetic properties of the human cytochrome c oxidase preparations with either human cytochrome c or horse cytochrome c were studied spectrophotometrically and compared with those of bovine heart cytochrome c oxidase.The interaction between human cytochrome c and human cytochrome c oxidase proved to be highly specific. It is proposed that for efficient electron transfer to occur, a conformational change in the complex is required, thereby shifting the initially unfavourable redox equilibrium. The very slow presteady‐state reaction between human cytochrome c oxidase and horse cytochrome c suggests that, in this case, the conformational change does not occur.The proposed model was also used to explain the steady‐state kinetic parameters under various conditions. At high ionic strength (I= 200 mM, pH 7.4), the kcat was highly dependent on the type of oxidase and it is proposed that the internal electron transfer is the rate‐limiting step.The kcat value of the ‘high‐affinity’ phase, observed at low ionic strength (I= 18 mM, pH 7.4), was determined by the cytochrome c/cytochrome c oxidase combination applied, whereas the Km was highly dependent only on the type of cytochrome c used. Our results suggest that, depending on the cytochrome c/cytochrome c oxidase combination, either the dissociation of ferricytochrome c or the internal electron transfer is the rate‐limiting step in the ‘high‐affinity’ phase at low ionic strength.The ‘low‐affinity’kcat value was not only determined by the type of oxidase used, but also by the type of cytochrome c. It is proposed that the internal electron‐transfer rate of the ‘low‐affinity’ reaction is enhanced by the binding of a second molecule of cytochrome c.
Eukaryotic DNA primase appears to act as oligomer in DNA‐polymerase‐α–primase complex
Authors: PODUST Vladimir N. , VLADIMIROVA Olga V. , MANAKOVA Elena N. , LAVRIK Olga I. 
Published: March 2005

Human placenta and calf thymus DNA‐polymerase‐α–primases were analyzed using native gradient‐polyacrylamide‐gel electrophoresis followed by overlay assays of polymerase and primase activities. The human enzyme contained three catalytically active native forms of 330, 440 and 560 kDa and the bovine enzyme five forms of 330, 440, 500, 590 and kDa. Of the various DNA polymerase forms. only the largest (560 kDa for human DNA polymerase and 590 kDa and 660 kDa for bovine DNA polymerase) contained primase activity. Titration of human DNA‐polymerase‐α–primase with DNA‐polymerase‐free primase caused the conversion of the 440‐kDa to the 560‐kDa form. The data favour the idea that primase binds to DNA polymerase α as an oligomer of 3 primases/polymerase core.In addition, the ability of primase to utilize oligoriboadenylates containing (prA)n or pp(prA)n was investigated. The primase elongated pp(prA)2 – 7 up to nanoadenylates or decaadenylates, but did not add 9 or 10 mononucleotides to a preexistent primer. In contrast to pp(prA)n < 10, (prA)n < 10 were rather poor primers for the primase. Both pp(prA)8,9 and (prA)n > 10 were elongated by primase, producing characteristic multimeric oligonucleotides. The possible connection of the structure of the DNA‐polymerase‐α–primase complex with the catalytical properties of primase is discussed.
Properties of yeast debranching enzyme and its specificity toward branched cyclodextrins
Authors: TABATA Shiro , HIZUKURI Susumu 
Published: March 2005

Debranching enzyme was purified from Saccharomyces cerevisiae by DEAE‐cellulose, ω‐aminobutyl agarose and hydroxyapatite column chromatography. The activity of the eluent was monitored by the iodine‐staining method which detects both the direct and indirect debranching enzymes. The elution profiles at every step showed a single peak with no shoulder. The crude and the purified enzyme preparations gave a single activity band with the same mobility on PAGE. The crude product produced 80% glucose compared to reducing sugar from glycogen‐phosphorylase‐limited dextrin while the partially purified and purified preparations produced 100% glucose.The activity of the purified enzyme was characterized and compared with that of the rabbit muscle enzyme by using various branched cyclodextrins as substrates. Both enzymes hydrolyzed 6‐O‐α‐d‐glucosyl cyclodextrins to glucose and cyclodextrins, but did not act on 6‐O‐α‐maltosyl cyclomaltoheptaose. The yeast enzyme gave rise to glucose as a sole reducing sugar from 6‐O‐α‐maltotriosyl cyclomaltoheptaose and 6‐O‐α‐maltotraosyl cyclomaltoheptaose, indicating that maltosyl and maltotriosyl transfers, respectively, had occurred, prior to the action of amylo‐1,6‐glucosidase.6‐O‐α‐d‐Glucosyl cyclomaltoheptaose and 6‐O‐α‐d‐glucosyl cyclomalto‐octaose, respectively, were better substrates than glycogen‐phosphorylase‐limited dextrin for the yeast and muscle enzymes. The yeast enzyme released glucose at a similar rate from 6‐O‐α‐maltotriosyl cyclomaltoheptaose as from 6‐O‐α‐maltotetraosyl cyclomaltoheptaose, but considerably lower rates than that from limit dextrin.The yeast debranching enzyme appears to be exclusively oligo‐1,4→1,4‐glucantransferase‐amylo‐1,6‐glucosidase and does not have isoamylase.
Frontier orbital study on the 4‐hydroxybenzoate‐3‐hydroxylase‐dependent activity with benzoate derivatives
Authors: VERVOORT Jacques , RIETJENS Ivonne M. C. M. , van BERKEL Willem J. H. , VEEGER Cees 
Published: March 2005

Based on molecular orbital computer calculations the present paper provides a new hypothesis for catalytic characteristics of 4‐hydroxybenzoate‐3‐hydroxylase (EC 1.14.13.2). A clear correlation between In kcat for the conversion of a series of 4‐hydroxylated substrates and their E(HOMO) leads to the hypothesis that Frontier orbital HOMO characteristics [E(HOMO) and HOMO density on C3] of the substrates are the predominant factor in regulating the fate of a benzoate derivative at the active site of the enzyme. The HOMO characteristics can be used to explain whether a compound will be converted by the enzyme or merely acts as an effector. Furthermore the hypothesis provides quantitative theoretical support for a catalytic mechanism in which the substrate reacts in its dianionic form and for a mechanism in which the electrophilic attack of the C(4a)‐peroxyflavin, or of the hydroxyl radical derived from it, on the benzoate dianion is the rate limiting step in catalysis at pH 8, 25°C.Finally, it is demonstrated that the hypothesis can be used as a basis for the formulation of working hypotheses in future research, investigating the conversion and regioselective orientation of the various possible substrates in the active site of the wild‐type 4‐hydroxybenzoate‐3‐hydroxylase, its mutants as well as of various other flavin‐dependent aromatic hydroxylases, such as for example 3‐hydroxybenzoate‐4‐hydroxylase (EC 1.14.13.23), 3‐hydroxybenzoate‐6‐hydroxylase (EC 1.14.13.24) and phenol hydroxylase (EC 1.14.13.7).
Mammalian thymidine kinase 2
Authors: JANSSON Olof , BOHMAN Christina , MUNCH‐PETERSEN Birgitte , ERIKSSON Staffan 
Published: March 2005

Thymidine kinase 2 (TK2), also called mitochondrial thymidine kinase, is a pyrimidine deoxyribonucleoside kinase expressed in all cells and tissues. It was recently purified to apparent homogeneity from human leukemic spleen and the active enzyme was shown to be a monomer of a 29‐kDa polypeptide. The enzyme is feedback‐inhibited by both end products, dCTP and dTTP. Here we show that TK2 purified from several different sources, including purified beef heart mitochondria, could be directly photoaffinity labeled with radioactive dTTP (approximately 18% of all TK2 molecules were cross‐linked to dTTP after 20 min of ultraviolet irradiation) or to a lower extent with dCTP.Photo‐incorporation was inhibited by the presence of the other effector but also the phosphate donor ATP blocked photolabeling, with dTTP. Addition of nucleoside substrates gave only a marginal inhibition of photo‐incorporation. There were no detectable difference in the molecular size of photolabeled TK2 isolated from human spleen, brain or placenta, monkey liver, beef heart and beef heart mitochondria. Nor was there any significant differences in the enzyme kinetic properties of these enzymes. Cleavage of labeled TK2 with cyanogen bromide showed that dTTP was incorporated into a single 3‐kDa peptide.TK2 was the only pyrimidine deoxynucleoside kinase expressed in liver, heart and brain. A detailed characterization of the subunit structure and substrate specificity of this enzyme is of importance for the design of new antiviral and cytostatic therapies based on nucleoside analogs.
Changes in membrane fluidity and Na+/K+‐ATPase activity during human trophoblast cell culture
Authors: MAZZANTI Laura , RABINI Rosa Anna , BIAGINI Graziella , PUGNALONI Armanda , DE PIRRO Roberto , FALOIA Emanuela , MANCINI Valentina , ROMANINI Carlo , CESTER Nelvio 
Published: March 2005

The human placenta plays an essential role in embryo development, in particular regulating the transport of ions, nutrients and immunoglobulins from the maternal to the fetal circulation. Trophoblast organization into a syncytial layer involves structural and functional steps that may be monitored and elucidated by in vitro studies. The structural stages by which the syncytial trophoblast is formed are not yet understood. In order to clarify the mechanism of trophoblast development, we studied the morphological characteristics of the syncytial trophoblast formation in culture and the functional changes (transport properties and membrane microviscosity) accompanying the structural modifications.By using both 5‐nitroxystearate and 16‐nitroxystearate as spin labels, we observed an initial increase in membrane order over 0–24 h of culture, which can be associated with two events: recovery of cell membranes from trypsin and initial aggregation of cytotrophoblasts. The similar behaviour of the order parameters determined with both probes indicates that membrane order changes both inside and in the outer part of the lipid bilayer. The subsequent decrease in membrane order observed at 36–48 h might be related to the process of cellular fusion.The increase in sodium/potassium pump activity in the first 24 h of culture might be an expression of cell recovery following trypsin treatment. The subsequent decrease might represent an adaptive mechanism by which metabolic energy is mainly used for morphogenetic changes.
Activation of ATPase activity of 14S dynein from Tetrahymena cilia by microtubules
Authors: SHIMIZU Takashi , HOSOYA Natsumi , HISANAGA Shin‐ichi , MARCHESE‐RAGONA Silvio P. , PRATT Melanie M. 
Published: March 2005

The ATPase activity of 14S dynein was activated by the presence of microtubule‐associated‐protein‐free microtubules. The activation was 2.5–3.5 fold at 10 mg microtubule/ml, and the activity increased further with increasing microtubule concentration. The microtubule–14S‐dynein complex, microtubule bundles with 14S dynein, was treated with a zero‐length chemical cross‐linker, 1‐ethyl‐3‐[3‐(dimethylamino)propyl]carbodiimide (EDC). The ATPase activity of the complex responded to EDC in a biphasic, concentration‐dependent manner and, at most, it was enhanced 5–10 fold. The complex treated with EDC was no longer unbundled by addition of ATP, as revealed by electron‐microscopic observation.Several ATP analogues, which support in vitro microtubule translocation mediated by 14S dynein, were turned over faster by this mechanochemical enzyme in the presence of microtubules than in their absence. However, some ATP analogues which do not support the translocation were also turned over faster in the presence of microtubules. Thus, microtubule‐dynein motility and substrate‐turnover activation are not tightly coupled, which indicates that all three major motor systems, actin–heavy‐meromyosin, microtubule‐kinesin [Shimizu, T., Furusawa, K., Ohashi, S., Toyoshima, Y. Y., Okuno, M., Malik, F. & Vale, R. D. (1991) J. Cell Biol. 112, 1189–1197] and microtubule‐dynein, have this characteristic property in common.
Yeast actin is relatively well behaved
Authors: NEFSKY Bradley , BRETSCHER Anthony 
Published: March 2005

Actin from yeast has been reported previously to have unusual polymerization properties. Here we report a simple sensitive spot assay for actin and use it to develop a high‐yield procedure for the purification of actin from the yeast Saccharomyces cerevisiae. The polymerization properties of purified yeast actin are quantitatively similar to all other characterized actins. We have characterized this actin with respect to its ability to interact with yeast profilin and tropomyosin, the only yeast actin‐binding proteins so far purified and characterized. Yeast profilin can sequester yeast actin monomers and thereby reduce the ability of yeast actin to polymerize, whereas it has little effect on the degree of polymerization of rabbit skeletal muscle actin. By contrast, there is no apparent difference between the binding of yeast or smooth muscle tropomyosin to yeast or rabbit skeletal muscle actin. The availability of purified yeast actin should facilitate a detailed examination of its interaction with recently discovered yeast actin‐binding proteins.Greer and Schekman (1982) [Greer, C. & Scheckman, R. (1982), Mol. Cell Biol. 2, 1279–1286] reported that an intrinsic property of yeast actin is a Ca2+ dependent increase in critical concentration with the formation of 15–50‐nm particles. Our purified actin does not have this property. By modifying the purification protocol, we can obtain a preparation having a Ca2+‐dependent change in polymerization properties. The Ca2+‐dependent effect results in a slower polymerization rate as well as the formation of shorter filaments. Since this effect could be mediated by a protein present at a very low stoichiometry to actin, and we do not see any contaminating peptides, we have not pursued this effect further. We suggest that the Ca2+‐dependent properties of the Greer and Schekman preparation are most likely due to a minor contaminant.
Lipomide dehydrogenase from Azotobacter vinelandii: site‐directed mutagenesis of the His450‐Glu455 diad
Authors: BENEN Jacques , van BERKEL Willem , DIETEREN Nicole , ARSCOTT David , WILLIAMS Charles , VEEGER Cees , de KOK Arie 
Published: March 2005

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced with other residues. His450, the active‐site base, was replaced with Ser, Tyr or Phe. Pro451, from X‐ray analysis found to be in cis conformation positioning the backbone carbonyl of His450 close to N3 of the flavin, was changed to Ala. Glu455, from X‐ray analysis expected to be involved in modulating the pKa of the base (His450), was replaced with Asp and Gln. The general conclusion is that mutation of the His‐Glu diad impairs intramolecular electron transfer between the disulfide/dithiol and the FADH−/FAD.The wild‐type enzyme functions according to a ping‐pong mechanism in the physiological reaction in which the formation of NADH is rate‐limiting. Above pH 8.0 the enzyme is strongly inhibited by the product NADH. The pH dependence of the steady‐state kinetics using the NAD+ analog 3‐acetylpyridine adenine dinucleotide (AcPyAde+) reveals a pKa of 8.1 in the pKm AcPyAde+ plot indicating that this pKa is related to the deprotonation of His450 [Benen, J., Berkel van, W., Zak, Z., Visser, T., Veeger, C. & Kok de, A. (1991) Eur. J. Biochem. 202, 863–872] and to the inhibition by NADH.The mutations considerably affect turnover. Enzymes with the mutations Pro451 → Ala, His450 → Phe and His450 → Tyr appear to be almost inactive in both directions. Enzymes His450 → Ser is minimally active, V at the pH optimum being 0.5% of wild‐type activity in the physiological reaction. Rapid reaction kinetics show that for the His450‐mutated enzymes the reductive half reaction using reducted 6,8‐thioctic acid amide [Lip(SH)2] is rate‐limiting and extremely slow when compared to the wild‐type enzyme. For enzyme Pro451 Ala it is concluded that the loss of activity is due to over‐reduction by Lip(SH)2 and NADH. The Glu455‐mutated enzymes are catalytically competent but show strong inhibition by the product NADH (enzyme Glu455 → Asp more than Glu455 → Gln). The inhibition can largely be overcome by using AcPyAde+ instead of NAD+ in the physiological reaction.The rapid reaction kinetics obtained for enzymes Glu455 → Asp and Glu455 → Gln deviate from the wild‐type enzyme. It is concluded that this difference is due to cooperativity between the active sites in this dimeric enzyme. Rapid reaction kinetics of enzymes His450 → Ser and Glu455 → Gln show the existence of two intermediates at the two‐electron reduced level: a species with the NAD+ bound, the flavin reduced and the disulfide intact (oxidized) and a species with NAD+ bound, the disulfide reduced and the flavin oxidized. No spectral evidence is obtained for the participation of a proposed flavin C4a adduct intermediate [Thorpe, C. & Williams, C. H. Jr (1981) Biochemistry 20, 1507–1513] in the reaction mechanism of enzymes His450 → Ser and Glu455 → Gln.
The essentail active‐site lysines of clostridial glutamate dehydrogenase
Authors: LILLEY Kathryn S. , ENGEL Paul C. 
Published: March 2005

Glutamate dehydrogenase (GDH) of Clostridium symbiosum, like GDH from other species, is inactivated by pyridoxal 5′‐phosphate (pyridoxal‐P). This inactivation follows a similar pattern to that for beed liver GDH, in which a non‐covalent GDH‐pyridoxal‐P complex reacts slowly to form a covalent complex in which pyridoxal‐P is in a Schiff's‐base linkage to lysine residues.

image
The equilibrium constant of this first‐order reaction on the enzyme surface determines the final extent of inactivation observed [S. S. Chen and P. C. Engel (1975) Biochem. J. 147, 351–358]. For clostridial GDH, the maximal inactivaton obtained was about 70%, reached after 10 min with 7mM pyridoxal‐P pH 7. In keeping with the model,

inactivation became irreversible after reduction with NaBH4.
The NaBH4‐reudce enzyme showed a new absorption peak at 325 nm.
K
m values for NAD+ and glutamate were unalterd, although Vmax values were decreased by 70%.

Kinectic analysis of the inactivation gave values of 0.81 ± 0.34 min−1 for K3 and 3.61 ± 0.95 mM for K2/K1. Thelineat plot of 1/(1‐R) against 1/[pyridoxal‐P], where R is the limiting residual activity reached in an inactivation reaction, gave a slightly higher valus for K2/K1 of 4.8 ± 0.47 mM and K4 of 0.16 ± 0.01 min−1.NADH, NAD+, 2‐oxoglutrate, glutarate and succinate gave partial protection against inactivation, the biggest effect being that of 40 mM succinate (68% activity compared with 33% in the control). Paired combinations of glutarate or 2‐oxoglutarate and NAD+ gave slightly better protection than the separate components, but the most effective combination was 40mM 2‐oxoglutarate with 1 mM NADH (85% activity at equilibrium). 70% inactivated enzyme showed an incorporation of 0.7 mM pyridoxal‐P/mol subunit, estimated spectrophotometrically after NaBH4 reduction, in keeping with the 1:1 stoichiometry for the inactivation. In a sample protected with 2‐oxoglutarate and NADH, however, incorporation was 0.45mol/mol, as against 0.15mol/mol expected (85% active).Tryptic peptides of the enzyme, modified with and without protection, were purified by HPLC. Two major peaks containing phosphopyridoxyllysine were unique to the unprotected enzyme. These peaks yielded three peptide sequences clearly homologous to sequences of other GDH species. In each case, a gap at which no obvious phenythiohydantion‐amino‐acid was detected, matched a tryptic cleavage. Modification of these three lysines, corresponding to Lys92, Lys116 and Lys128 in Escherichia coli GSH, appears to be mutually exclusive and modification of any one of them abolishes activity. Comparison with the cystal structure indicated that these lysines are clustered in the activesite cleft and likely to interact with the dicarboxylate substrate.
Kinetics of daunorubicin transport by P‐glycoprotein of intact cancer cells
Authors: SPOELSTRA Ellen C. , WESTERHOFF Hans V. , DEKKER Henk , LANKELMA Jan 
Published: March 2005

Drug permeation across the plasma membrane of multidrug‐resistant cells depends on the kinetics of the P‐glycoprotein‐mediated pump activity as well as well as on the passive permeation of the drug. We here demonstrate a method to characterize kinetically the pump in intact cells.To this purpose, we examined the membrane‐transport properties of daunorubicin in various sensitive cancer cell lines and in their multidurg resistant (MDR) counterparts. First, we determined the passive permeability coefficient for daunorubicin. Then, using a flow‐through system, the drug flux into the cell was measured after inhibition of the P‐glycoprotein‐mediated efflux pump. Combining the two results allowed us to calculate the intracellular free concentration of the drug. In the steady‐state, the pump rate must equal the net rate of passive diffusion of the drug and, therefore, the same experiments gave us the pumping rate of daunorubicin. These experiments were then repeated at various extracellular durg concentrations. By plotting the pumping rate versus the intracellular durg concentration, we then characterized the P‐glycoprotein kinetically.Four independent methods were used to measure the passive permeability coefficient for the cell line A2780. Similar values were obtained. Maximal pump rates (Vmax) showed a good correlation with the amount of P‐glycoprotein in the cell lines used. We obtained saturation curves for the variation of the pump rates the intracellular daunorubicin concentration. These curves were typical for positive cooperativity, which provides evidence that at least two binding sites for daunorubicin are present on the active transport system of daunorubicin. The apparent Km values for P‐glycoprotein‐mediated transport, the intracellular free cytosolic daunorubicin conccntrations at half‐maximal velocity for the cell lines used, were approximately 1.5μM. Except for the cell lines with the highest amount of P‐glycoprotein, the passive efflux rate of daunorubicin proved to be a substantial part of the total daunorubicin efflux rate for the cell lines used. In cell lines with relatively low levels of P‐glycoprotein, passive daunorubicin efflux was even the main route of daunorubicin transport from the cell, determining the intracellular steady‐state concentrations of daunorubicin.