bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characteristic binding of human plasma apolipoprotein B to gangliotetraosylceramide and gangliotriaosylceramide
Authors: LEDVINOVA Jana , IWAMORI Masao , NAGAI Yoshitaka 
Published: March 2005

The binding of human plasma low‐density lipoproteins (LDL), freshly prepared by discontinuous ultracentrifugation, to several neutral and acidic glycosphingolipids was examined by TLC immunostaining with the anti‐[apolipoprotein B (apoB)] antibody. ApoB was found to bind characteristically to the asialogangliosides, gangliotetraosylceramide (Gg4Cer) and gangliotriaosylceramide (Gg3Cer), the former being a more potent receptor than the latter, indicating that the sequences Galβ1‐3GalNAcβ1‐4Gal and GalNAcβ1‐4Gal are involved in the binding of apoB. A weak positive reaction with fucosylgangliotetraosylceramide (IV2Fuc‐Gg4Cer), which has the same internal recognition sequence, was also observed (the binding ability was only 1/7 of that in the case of Gg4Cer). No binding to other neutral glycosphingolipids, or glycosphingolipid sulfates (I3‐SO3‐GalCer) and gangliosides, was detected, and therefore substitution of the receptor glycolipid with sialic acid was thought to inhibit the binding.The results indicate that, along with the binding of apoB to the LDL‐binding domain of the receptor glycoprotein, interaction with some carbohydrate chains in the receptor, or with glycolipids coexisting on the plasma membrane, may be important for the binding of apoB to cells.
Activation and inhibition of the calcium‐release channel of isolated skeletal muscle heavy sarcoplasmic reticulum
Authors: WYSKOVSKY Wolfgang , HOHENEGGER Martin , PLANK Brigitte , HELLMANN Gertrude , KLEIN Sabine , SUKO Josef 
Published: March 2005

Calcium‐independent calcium efflux from heavy sarcoplasmic reticulum (HSR) of skeletal muscle was found to be biphasic, with half‐times of 2–6 s and 200–400 s for the first and second phase, respectively. Calcium‐, AMP‐ and caffeine‐induced calcium efflux was triphasic, with half‐times of 0.05–0.2 s, 1–5 s and 100–400 s for the first, second and third phases, respectively. This very fast first phase is certainly due to calcium efflux via the calcium‐release channel of HSR vesicles. Both ruthenium red and neomycin inhibited the first phase of the calcium‐independent calcium efflux and the first phase of the calcium‐, AMP‐ or caffeine‐induced calcium efflux completely, whilst the second phase was fully inhibited by ruthenium red only and partially inhibited by neomycin at high concentrations, indicating that the second phase of calcium release also occurs via the calcium‐release channel.Various models for calcium efflux through the release channel have been tested by simulation. Activation and inhibition of the channel‐mediated calcium efflux from HSR cannot be explained by two states of the calcium‐release channel (open or closed), but requires the existence of at least three states. A channel with one open state and two closed states, resulting in a rapid inactivation, is the most simple model compatible with the experimental data. According to this model, activation is assumed to reduce inactivation of the channel, whilst inhibition assumes an acceleration of channel inactivation. This mechanism most likely applies to neomycin. An additional open‐blocked state has to be assumed for inhibition by ruthenium red.
Changes of the oligomeric structure of legumin from pea (Pisum sativum L.) after succinylation
Authors: SCHWENKE Klaus Dieter , ZIRWER Dietrich , GAST Klaus , GÖRNITZ Eckhard , LINOW Karl‐Joachim , GUEGUEN Jacques 
Published: March 2005

The influence of various levels of succinylation on the structure of the legumin from pea seed has been studied by the techniques of sedimentation velocity, viscometry, fluorescence and circular dichroism spectroscopy, as well as dynamic light scattering. The protein dissociates gradually into the 3S subunit forming a 7S intermediate. At a level of 75–80% succinylation, sudden unfolding of the protein occurs characterized by drastic changes in viscometric and spectroscopic properties. The fluorescence spectra point to the formation of a novel organized structure at a moderate degree of modification before the molecular unfolding takes place.The succinylated subunit was shown to have a sedimentation coefficient of 3.2S, a diffusion coefficient of 5.03 × 10−7 cm2· s−1 a Stokes' radius of 4.24 nm, a partial specific volume of 0.703 ml/g, an intrinsic viscosity of 0.13 dl/g, a molar mass of 52.2 kDa and a frictional ratio of 1.74.
Evidence that enzymes of a novel aerobic 2‐amino‐benzoate metabolism in denitrifying Pseudomonas are coded on a small plasmid
Authors: ALTENSCHMIDT Uwe , ECKERSKORN Christoph , FUCHS Georg 
Published: March 2005

A new pathway for aerobic metabolism of 2‐aminobenzoate which proceeds via anthranoyl‐CoA has recently been revealed in a Pseudomonas strain KB740. This bacterial strain was found to contain a small 8.1‐kbp plasmid pKB740 which appears to harbour the genes encoding for two key enzymes catalyzing the initial reactions of the pathway, 2‐aminobenzoate coenzyme A ligase and 2‐aminobenzoyl‐coenzyme A monooxygenase/reductase. The evidence is as follows:The plasmid content of the culture varied by a factor of ten depending on the growth substrates; it was highest when cells were grown aerobically on 2‐aminobenzoate.The plasmid pKB740 could be introduced into Escherichia coli strain JM83 by transformation. Wild‐type E. coli and E. coli JM83 are unable to metabolize 2‐aminobenzoate whereas the transformed E. coli JM83 cells could grow with this aromatic compound as sole organic substrate and oxidize it completely to CO2. The plasmid recovered from E. coli had the same restriction map as the original plasmid, but was dimerized.The two key enzyme activities were demonstrated in the transformed E. coli in sufficiently high amounts to explain growth. They appear to be regulated on the transcription level by induction; they were formed only during aerobic growth in the presence of 2‐aminobenzoate, as in the parent Pseudomonas.The N‐terminal amino acid sequence of 2‐aminobenzoyl‐CoA monooxygenase/reductase was similar to the consensus sequence of the FAD binding site of different flavoenzymes. The data also prove that the enzyme with two flavin functions is an α2 homodimer.Southern blotting of digested chromosomal and plasmid DNA and hybridization against a labelled 15‐base oligonucleotide derived from the N‐terminal amino acid sequence of 2‐aminobenzoyl‐CoA monooxygenase/ reductase revealed that the gene for this enzyme is coded on the plasmid rather than on the chromosome. The gene was localized on a 3.2‐kbp restriction fragment.The formation of 2‐aminobenzoyl‐CoA monooxygenase/reductase protein in transformed E. coli was demonstrated by Western blotting of proteins of cell extracts separated by SDS/PAGE. The enzyme protein band, which was stained by a procedure based on antibodies against 2‐aminobenzoyl‐CoA monooxygenase/reductase, was demonstrated in transformed E. coli.
Distances between functional sites of the Ca2++ Mg2+‐ATPase from sarcoplasmic reticulum using Co2+ as a spectroscopic ruler
Authors: CUENDA Ana , HENAO Fernando , GUTIERREZ‐MERINO Carlos 
Published: March 2005

Cobalt ion inhibits the Ca2++ Mg2+‐ATPase activity of sealed sarcoplasmic reticulum vesicles, of solubilized membranes and of the purified enzyme. To use Co appropriately as a spectroscopic ruler to map functional sites of the Co2++ Mg2+ ‐ATPase, we have carried out studies to obtain the kinetic parameters needed to define the experimental conditions to conduct the fluorimetric studies.

The apparent K0.5 values of inhibition of this ATPase are 1.4 mM, 4.8 mM and 9.5 mM total Co2+ at pH 8.0, 7.0 and 6.0, respectively. The inhibition by Co2+ is likely to be due to free Co binding to the enzyme. Millimolar Ca2+ can fully reverse this inhibition, and also reverses the quenching of the fluorescence of fluoresceinlabeled sacroplasmic reticulum membranes due to Co2+ binding to the Ca2++ Mg2+‐ATPase. Therefore, we conclude that Co2+ interacts with Co2+ binding sites.
Co2+· ATP can be used as a substrate by this enzyme with Vmax of 2.4 ± 0.2 p.mol ATP hydrolyzed min−1 (mg protein)−1 at 20–22°C and pH 8.0, and with a K0.5 of 0.4–0.5 mM.
Co2+ partially quenches, about 10 ± 2%, the fluorescence of fluorescein‐labeled sarcoplasmic reticulum Ca2+ Mg2+ATPase upon binding to this enzyme at pH 8.0. From the fluorescence data we have estimated an average distance between Co2+ and fluorescein in the ATPase of 1.1–1.8 nm or 1.3–2.1 run for one or two equidistant Co2+ binding sites, respectively.
Co2+· ATP quenches about 20‐25% of the fluorescence of fluorescein‐labeled Ca2++ Mg2+‐ATPase, from which we obtain a distance of 1.1–1.9 nm between Co2+ and fluorescein located at neighbouring catalytic sites.
Tooth ‘enamelins’ identified mainly as serum proteins
Authors: STRAWICH Elsa , GLIMCHER Melvin J. 
Published: March 2005

The major enamelin protein component present in EDTA or EDTA/guanidine hydrochloride extracts of developing bovine enamel has a molecular mass of about 67 kDa; it has an amino acid composition similar to that of bovine serum albumin and reacts with polyclonal and monoclonal antibodies to albumin. Two‐dimensional separation of the components in the enamelin extract by isoelectric focusing and SDS/PAGE reveal that the major ∼ 67‐kDa component and almost all of the minor Coomassie‐staining protein components of 7ap; 67 kDa, as well as many of the other minor components with different molecular masses, also react with polyclonal and monoclonal antialbumin. The ∼ 67‐kDa band eluted after SDS/PAGE, as well as the major ∼ 67‐kDa spots eluted after two‐dimensional separation, were found to have N‐terminal amino acid sequences identical to that of bovine serum albumin. Albumin accounted for at least 70–80% of the total protein content of the enamelin extract and was essentially the only protein in the ∼ 67‐kDa component. The serum proteins α‐2 HS glycoprotein, γ‐globulin and fetuin, and the proline‐rich salivary protein termed P‐B were also identified in the enamelin extract. The serum proteins and the salivary protein account for > 95% of the proteins in the enamelin extracts. Of the remaining very small amounts of non‐serum or salivary protein isolated from the enamelin extracts, three minor components were isolated which had N‐terminal amino acid sequences which were not similar to any known protein in the protein sequence data base and could therefore conceivably be true ‘enamelins’ synthesized by ameloblasts. One additional protein had the first five N‐terminal amino acids and residue 8 of amelogenin, residues 6 and 7 being different from those of amelogenin. Two other very minor protein components had amino acid compositions distinct from the amelogenins and the serum proteins, but were N‐terminally blocked on attempted sequencing. None of the components in the neutral soluble low‐ionic‐strength extract or in the 4 M guanidine hydrochloride extract, both of which consist principally of amelogenins, immunoreacted with anti‐albumin or with any of the antibodies to other serum proteins and fetuin, despite the fact that the amelogenin extracts also contain non‐amelogenin proteins.On the basis of the data presented, studies employing antibodies to the so‐called enamelin proteins and hypotheses as to their molecular conformation, their roles as evolutionary markers, or their positive role in mineralization should be reconsidered and reviewed.
Purification and characterization of an (ADP‐ribose)n glycohydrolase from human erythrocytes
Authors: TANUMA Sei‐ichi , ENDO Hiroyoshi 
Published: March 2005

An (ADP‐ribose)n, glycohydrolase from human erythrocytes was purified approximately 13000‐fold and characterized. On sodium dodecyl sulfate/polyacrylamide gel the purified enzyme appeared homogeneous and had an estimated relative molecular mass (Mr) of 59000. Amino acid analysis showed that the enzyme had a relatively high content of acidic amino acid residues and low content of basic amino acid residues. Isoelectrofocusing showed that the enzyme was an acidic protein with pI value of 5.9. The mode of hydrolysis of (ADP‐ribose)n by this enzyme was exoglycosidic, yielding ADP‐ribose as the final product. The Km value for (ADP‐ribose)n, (average chain length, n = 15) was 5.8 μM. and the maximal velocity of its hydrolysis was 21 μmol · min−1. mg protein−1. The optimum pH for enzyme activity was 7.4. KCI was more inhibitory than NaCl. The enzyme activity was inhibited by ADP‐ribose and cAMP but not the dibutyryl‐derivative (Bt2‐cAMP), cGMP or AMP. These physical and catalytic properties are similar to those of cytosolic (ADP‐ribose)n, glycohydrolase II, but not to those of nuclear (ADP‐ribose)n, glycohydrolase I purified from guinea pig liver [Tanuma, S., Kawashima, K. & Endo, H. (1986)J. Biol. Chem. 261, 965–969]. Thus, human erythrocytes contain (ADP‐ribose)n, glycohydrolase II.The kinetics of degradation of poly(ADP‐ribose) bound to histone H1 by purified erythrocyte (ADP‐ribose)n, glycohydrolase was essentially the same as that of the corresponding free poly(ADP‐ribose). In contrast, the glycohydrolase showed appreciable activity of free oligo(ADP‐ribose), much less activity on the corresponding oligo(ADP‐ribose) bound to histone H1. The enzyme had more activity on oligo(ADP‐ribose) bound to mitochon‐drial and cytosolic free mRNA ribonucleoprotein particle (mRNP) proteins than on oligo(ADP‐ribose) bound to Histone HI. It did not degrade mono(ADP‐ribosyl)‐stimulatory guanine‐nucleotide‐binding protein (Gs) and ‐inhibitory guanine‐nucleotide‐binding protein (Gi) prepared with cholera and pertussis toxins, respectively. These results suggest that cytosolic (ADP‐ribose)n, glycohydrolase II may be involved in extranuclear de(ADP‐ribosyl)n, ation, but not in membrane de‐mono(ADP‐ribosyl)ation.
Structure and biosynthesis of the xylose‐containing carbohydrate moiety of rice α‐amylase
Authors: HAYASHI Makoto , TSURU Akiko , MITSUI Toshiaki , TAKAHASHI Noriko , HANZAWA Hiroyuki , ARATA Yoji , AKAZAWA Takashi 
Published: March 2005

Suspension‐cultured cells of rice secrete α‐amylase into the culture medium. It has been shown that the mature form of the α‐amylase contains xylose‐bearing N‐linked oligosaccharide:

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We demonstrate that suspension‐cultured cells of rice secrete α‐amylase containing oligomannose‐type oligosaccharides in the presence of 1‐deoxymannojirimycin or tris(hydroxymethyl)aminomethane. On the other hand, α‐amylase purified from germinated rice seedlings contains several kinds of oligomannose‐type and N‐acetyllactosamine‐type oligosaccharides. The processing pathway of oligosaccharide moieties in rice cells is discussed on the basis of a comparison of these oligosaccharides structures.
Transcriptional control of the cysG gene of Escherichia coli K‐12 during aerobic and anaerobic growth
Authors: PEAKMAN Timothy , BUSBY Stephen , COLE Jeffrey 
Published: March 2005

The 74‐min region of the Escherichiu coli chromosome includes five open reading frames of known sequence. The first and last of these genes, nirB and cysG, are transcribed in the same direction and both are essential for NADH‐dependent nitrite reductase activity. The functions of the other genes, nirD, nirE and nirC, which are located between nirB and cysG, are unknown. The nirB gene is transcribed from a promoter which is anaerobically induced, expression being dependent on the transcription activator protein, Fnr. Here we show that the nirD, nirE, nirC and cysG genes are also expressed from the nirB promoter. After subcloning cysG, a second promoter was located less than 100 bases upstream of cysG. Two groups of transcription start points separated by 40 bases were detected in this region by S1 mapping. Rates of transcription from the isolated cysG promoter were the same during aerobic growth and anaerobic growth in the presence or absence of nitrite. However, when the nirB gene and its promoter were cloned back upstream from the cysG promoter, the rate of transcription was higher during anaerobic growth than during aerobic growth and was further induced by nitrite. These increases were totally dependent on a functional fnr gene and were shown by S1 mapping experiments to be due to transcriptional read‐ through from the Fnr‐dependent nirB promoter.No promoter activity was associated with DNA fragments between the BamHI site located within the N‐terminal coding region of the nirB gene and the cysG promoter located at the C‐terminus of nirC.
Localization of membrane‐associated (NiFe) and (NiFeSe) hydrogenases ofDesulfovibrio vulgaris using immunoelectron microscopic procedures
Authors: ROHDE Manfred , FÜRSTENAU Ursel , MAYER Frank , PRZYBYLA Alan E. , PECK Harry D. , Le GALL Jean , CHOI Eui S. , MENON Nanda K. 
Published: March 2005

The intracellular location of membrane‐associated (NiFe) and (NiFeSe) hydrogenases of Desulfovibrio vulgaris was determined using pre‐embedding and post‐embedding immunoelectron microscopic procedures. Polyclonal antisera directed against the purified (NiFe) and (NiFeSe) hydrogenases were raised in rabbits. One‐day‐old cultures of D. vulgaris, grown on a lactate/sulfate medium, were used for all experiments in these studies. For post‐embedding labeling studies cells were fixed with 0.2% glutaraldehyde and 0.3% formaldehyde, dehydrated with methanol, and embedded in the low‐temperature resin Lowicryl K4M.Our post‐embedding studies using antibody‐gold or protein‐A‐gold as electron‐dense markers revealed the location of the two hydrogenases exclusively at the cell periphery; the precise membrane location was then demonstrated by pre‐embedding labeling. Spheroplasts were incubated with the polyclonal antisera against (NiFe) and (NiFeSe) hydrogenase followed by ferritin‐linked secondary antibodies prior to embedding and sectioning. The observed labeling pattern unequivocally revealed that the antigenic reactive sites of the (NiFe) hydrogenase are located in the near vicinity of the cytoplasmic membrane facing into the periplasmic space, whereas the (NiFeSe) hydrogenase is associated with the cytoplasmic side of the cytoplasmic membrane.
How do MgATP analogues differentially modify high‐affinity and low‐affinity ATP binding sites of Na+/K+ ‐ATPase?
Authors: SERPERSU Engin Halit , BUNK Sabine , SCHONER Wilhelm 
Published: March 2005

The exchange‐inert tetra‐ammino‐chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na +/K+ ‐ATPase slowly by interacting with the high‐affinity ATP binding site. The inactivation proceeded at 37°C with an inactivation rate constant of 1.34 × 10−3 min−1 and with a dissociation constant of 0.62 μM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K+ ‐ATPase by Cr(NH3)4ATP, and its H2O‐substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4ATP was 29‐fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active‐site residues does not seem to play a significant role in the inactivation of Na+/K+ ‐ATPase by Cr(III)‐ATP complexes.Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high‐affinity ATP site with an apparent dissociation constant of 1.8 μM. The observed inactivation rate constant of 2.11 × 10‐3 min−1 became higher when Na + or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP‐inactivated enzyme.Co(NH3)4ATP inactivates Na+/K+ ‐ATPase by binding to the low‐affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)‐ATP or Rh(III)‐ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low‐affinity ATP site. Inactivation of Na+/K+ ‐ATPase was faster by the Δ isomer of Co(NH3)4ATP than by the Δ isomer. Co(NH3)4ATP, but not Cr(H2O)4ATP or adenosine 5′ ‐[β,γ‐methylene]triphosphate competitively inhibited K+ ‐activated p‐nitrophenylphosphatase activity of Na+ K+ ‐ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low‐affinity ATP binding site.
Expression of cytochrome P‐450 enzymes in cultured human hepatocytes
Authors: MOREL Fabrice , BEAUNE Philippe H. , RATANASAVANH Damrong , FLINOIS Jean‐Pierre , YANG Chung S. , GUENGERICH F. Peter , GUILLOUZO André 
Published: March 2005

Hepatocytes from adult and newborn humans were put into primary culture and exposed to phenobarbital, 3‐methylcholanthrene, or rifampicin, three well‐known inducers of cytochrome P‐450 in animals. The expression of four cytochrome P‐450 enzymes (or groups of enzymes, namely P‐450 IIIA, P‐450 IIC8/9/10, P‐450 IIE1, and P‐450 IA2) was investigated. These enzymes were found to remain expressed during the period of culture studied. Treatment with the inducers for three days resulted in different responses, depending upon the inducer and the enzyme. Phenobarbital and rifampicin increased P‐450 IIC8/9/10 mRNA transcripts and the corresponding protein, while 3‐methylcholanthrene was ineffective. Both P‐450 IIIA mRNA and protein were strongly induced by rifampicin. All of the hepatocytes were found to synthesize P‐450 IIIA in response to rifampicin, as shown by immunoperoxidase staining. P‐450 IIIA expression was not affected by phenobarbital and was decreased by 3‐methylcholanthrene. P‐450s IA2 and IIE1 decreased to 25–50% of the initial level during these cultures. P‐450 IA2 and ethoxyresorufin O‐deethylase activity (which is a monooxygenase activity related to P‐450 IA family) were increased only by 3‐methylcholanthrene and did not respond to the other inducers. P‐450 IIE1 was not induced by any of these compounds. P‐450 IIC8/9/10 and P‐450 IIIA mRNA levels were also measured in human hepatocytes from one newborn. P‐450 IIC8/9/10 was barely expressed in freshly isolated cells but increased dramatically with time in culture. P‐450 IIIA transcripts were abundant in both freshly isolated and cultured cells derived from a newborn.These results clearly demonstrate that human hepatocytes continue to express cytochrome P‐450 enzymes and respond to inducers in culture. This model system provides a useful approach for investigating the effects of drugs on maturation and expression of drug‐metabolizing enzymes in human liver.