bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metal Activation of Galactosylhydroxylysyl Glucosyltransferase, an Intracellular Enzyme of Collagen Biosynthesis
Authors: MYLLYLÄ Raili , ANTTINEN Henrik , KIVIRIKKO Kari I. 
Published: March 2005

Metal activation of galactosylhydroxylysyl glucosyltransferase was studied with EDTA‐treated highly purified enzyme from chick embryos and EDTA‐treated citrate‐soluble collagen substrate. Mn2+ activated the transferase at much lower concentrations than thought previously, and high Mn2+ concentrations were more effective than could be expected from the Michaelis‐Menten equation. The data suggest that the enzyme can bind at least two Mn2+ ions, the first having a Kd of 3–5 μM (site I) and the second 50–70 μM (site II). The binding of the second Mn2+ decreases the Km of UDP‐glucose from 25 μM to 6–7 μM, whereas the V at a saturating concentration of UDP‐glucose is unaffected. The EDTA‐treated enzyme possessed some activity even without added Mn2+, suggesting that either the transferase can be activated in the absence of any metal or that it has one additional metal binding site with high affinity. The kinetic data would be consistent with a reaction scheme involving an ordered binding of Mn2+, UDP‐glucose and the collagen substrate at low Mn2+ and an ordered binding of Mn2+, Mn2+, UDP‐glucose and the collagen substrate at high Mn2+ concentrations. In both cases the binding of the Mn2+, which occurs before that of UDP‐glucose, is at thermodynamic equilibrium. Zn2+, Cu2+, Cd2+, Ni2+ and Ca2+ are inhibitors of the Mn2+‐activated enzyme.Fe2+ (Kd 5–7 μM) and Co2+ (Kd 30 μM) also activate the enzyme at low concentrations, whereas no further activation, but instead an inhibition, was found at high concentrations. Ca2+ and Mg2+, which have previously been reported to serve as metal co‐factors, did not activate the enzyme alone and did not stimulate the glucosyltransferase activated at low Mn2+ concentrations. The Kd values for Mn2+ at site II and for Co2+ are probably much higher than the physiological concentrations, whereas the activation by Mn2+ at site I and by Fe2+ may be of physiological significance.
Purification and Properties of Hydrogenase fromMegasphaera elsdenii
Authors: VAN DIJK Cees , MAYHEW Stephen G. , GRANDE Hans J. , VEEGER Cees 
Published: March 2005

A hydrogenase has been purified to homogeneity from the soluble fraction of the rumen bacteriumMegasphaera elsdenii, the overall purification is 200 times with a yield of 14%. The pure enzyme consists of a single polypeptide chain withMr∼ 50000 which contains 12 atoms of non‐haem iron and 12 atoms of acid‐labile sulphide. The enzyme is rapidly inactivated by O2and it is therefore purified under nitrogen and in the presence of sodium dithionite. The optical spectrum of the enzyme, after removal of the dithionite with air, shows a peak at 275 nm (ε275 nm= 143 mM−1cm−1) and a shoulder between 350 nm and 400 nm (ε400 nm= 46 mM−1cm−1).The enzyme catalyses hydrogen production from sodium dithionite at a low rate. The rate is greatly enhanced by addition of the electron donors flavodoxin, ferredoxin and methyl viologen. The kinetic data with these three electron donors suggest co‐operativity, but no indication of self‐association of the enzyme was obtained. Sodium chloride enhances the rate of hydrogen production with methyl viologen semiquinone and changes the kinetic behaviour of the enzyme with this electron donor, but causes inhibition of the reactions mediated by ferredoxin and flavodoxin.Two kinetic models were developed which are consistent with the kinetic data of the three electron donors tested. The apparent co‐operativity for the hydrogen production can be fitted with the mathematical form of those models. The identical kinetic behaviour of the hydrogenase with the one‐electron donors flavodoxin and methyl viologen semiquinone monomer and the two‐electron donor ferredoxin indicates that the hydrogenase accepts two electrons in two separate, independent steps and further indicates that the two (4Fe‐4s) clusters of the donor ferredoxin are independent. The interpretation of the kinetic data with methyl viologen semiquinone is complicated by the fact that the semiquinone dimerises, and that the formation of the dimer is enhanced by salt. Taking into account the association of this donor, the activity of the enzyme with methyl viologen semiquinone can be described by the sum of the activities of the enzyme with methyl viologen monomer and methyl viologen dimer.The enzyme catalyses the oxidation of hydrogen gas with methyl and benzyl viologen as electron acceptors to their semiquinone forms; both electron acceptors show Michaelis‐Menten kinetics. The hydrogen oxidation activity with both electron acceptors is stimulated by addition of sodium chloride. The kinetic data of the oxidation of hydrogen with the two‐electron acceptors used are consistent with the proposed models, if it is assumed that the pathway followed is compulsory.At this moment no choice can be made between the models proposed.
Characterization of Preribosomal Ribonucleoprotein Particles from Tetrahymena pyriformis
Authors: RODRIGUES‐POUSADA Claudina , CYRNE Maria Luisa , HAYES Donal 
Published: March 2005

Ribonucleoprotein particles present in extracts of nuclei prepared from Tetrahymena pyriformis labelled for 1, 2.5, 5 and 10 min with [3H]uridine during exponential growth were analysed by sedimentation through linear 10–30% sucrose gradients.After 1 min of labelling, the early ribosomal RNA precursor (36‐S) is found to be associated with slowly sedimenting particles which form a broad peak centred at approximately 50 S. Other kinds of particles sedimenting at 80 S, 66 S, 60 S and 44 S are observed when labelling is carried out for longer periods (2.5, 5 and 10 min).The 80‐S particle contains 29‐S and 18‐S RNA species together with traces of 36‐S RNA; the 60‐S and 44‐S particles contain 26‐S and 17‐S RNAs respectively. Similar results were obtained when [Me‐3H]methionine was used for labelling in place of [3H]uridine. Methylation of the RNA present in slowly sedimenting nuclear components (30–70‐S) is rapid, reaching a plateau at 5 min while that of the faster sedimenting (70–90‐S) components is still increasing after 10 min.Only three types of ribonucleoprotein particles (80‐S, 66‐S, and 44‐S) were observed when the cells were labelled after prolonged starvation.A scheme of ribosome biogenesis based on these results is presented.
HeLa Cell Cytoplasmic mRNA Contains Three Classes of Sequences Predominantly Poly(A)‐Free, Predominantly Poly(A)‐Containing and Bimorphic
Authors: MILCAREK Christine 
Published: March 2005

The mRNA species which exist in the HeLa cell polyribosomes in a form devoid of A sequences longer than 8 nucleotides constitute the poly(A)‐free class of mRNA. The rapidly labelled component of this mRNA class shares no measurable sequence homology with poly(A)‐containing RNA. If poly(A)‐free mRNA larger than 12 S labelled for 2 h in vivo is hybridized with total cellular DNA, it hybridizes primarily with single‐copy DNA. When a large excess of steady poly(A)‐containing RNA is added before hybridization of labelled poly(A)‐free RNA, no inhibition of hybridization occurs. This indicates the existence of a class of poly(A)‐free mRNA with no poly(A)‐containing counterpart.Some mRNA species can exist solely as poly(A)‐containing mRNAs. These mRNAs in HeLa cells are found almost exclusively in the mRNA species present only a few times per cell (scarce sequences). Some mRNA species can exist in two forms, poly(A)containing and lacking, as evidenced by the translation data in vitro of Kaufmann et al. [Proc. Natl Acad. Sci. U.S.A. 74, 4801–4805 (1977)]. In addition, if cDNA to total poly(A)‐containing mRNA is fractionated into abundant and scarce classes, 47 % of the scarce class cDNA can be readily hybridized with poly(A)‐free mRNA. 10 of the abundant cDNA to poly(A)‐containing mRNA will hybridize with poly(A)‐free sequences very rapidly while the other 90% hybridize 160 times more slowly, indicating two very different frequency distributions. The cytoplasmic metabolism of these three distinct mRNA classes is discussed.
Structural Features of the Carbohydrate Units of Plasma Glycoproteins
Authors: FINNE Jukka , KRUSIUS Tom 
Published: March 2005

For the characterization of the type and relative abundance of different carbohydrate structures in plasma glycoproteins, rat whole plasma was subjected to exhaustive proteolytic digestion and the resulting glycopeptides were fractionated into structurally district groups by chromatography on concanavalin‐A‐Sepharose followed by mild NaOH/NaBH4 treatment and gel filtration. The most abundant fraction was found to be the biantennary transferrin‐type glycopeptides (71% of recovered carbohydrate) followed by triantennary fetuin‐type structures and a smaller proportion of more branched structures (together 24%), neutral mannose‐rich chains (2%) and O‐glycosidic chains (3%). Structural similarity of the two major fractions to the transferrin and fetuin glycopeptides was supported by similar elution volumes in gel filtration, similar behaviour in concanavalin A affinity chromatography and similar sugar composition and substitution pattern of sugars in methylation analysis.The glycopeptide pattern differs significantly from that observed previously for brain, kidney, liver or erythrocytes, where the more branched structures predominate, and which also contain higher amounts of the neutral mannose‐rich chains. As indicated by methylation analysis, the plasma glycoproteins are characterized by carbohydrate units terminating in N‐acetylneuraminic acid. The level of terminal galactose, N‐acetylglucosamine and fucose residues was low. This finding is in correlation with the described occurrence of binding‐activities in liver and other tissues which remove glycoproteins having these sugar terminals from the circulation.
Sialic‐Acid Content of Low‐Density Lipoproteins Controls Their Binding and Uptake by Cultured Cells
Authors: FILIPOVIC Ivan , SCHWARZMANN Günter , MRAZ Wilfried , WIEGANDT Herbert , BUDDECKE Eckhart 
Published: March 2005

The (high‐affinity receptor)‐mediated uptake of homologous low‐density (low‐ϱ) lipoproteins by cultured human arterial smooth muscle cells or human skin fibroblasts is controlled by the sialic acid content of low‐ϱ lipoprotein particles. This conclusion is derived from the following results.1. Gangliosides incubated with native low‐ϱ lipoproteins associate with low‐ϱ lipoprotein particles. Low‐ϱ lipoproteins modified by associated GLac1, GGtet1, and GGtet2b + GGtet3 gangliosides are internalized by arterial smooth muscle cells at a rate up to 80% lower than native low‐ϱ lipoproteins or those preincubated with desialized gangliosides.2. The inhibitory effect of gangliosides is specific for high affinity uptake and not detectable on skin fibroblasts deficient in low‐ϱ lipoprotein receptor.3. Desialyzed low‐ϱ lipoproteins are internalized by smooth muscle cells up to 100% faster than native low‐ϱ lipoproteins, the enhancement of uptake corresponding to the degree of desialization.
Yeast tRNALeuUAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods
Authors: RANDERATH Erika , GUPTA Ramesh C. , CHIA Li‐Li S. Y. , Simon H. CHANG  , RANDERATH Kurt 
Published: March 2005

A second major species of leucine tRNA, tRNALeuUAG (formerly designated tRNALeuCUA) was purified from baker's yeast in a three‐step procedure entailing BD‐cellulose chromatography in the presence and absence of Mg2+ and Sephadex G‐100 gel filtration. Results of aminoacylation and partial RNase T1 digestion experiments showed that this tRNA retains a native conformation under conditions that denature yeast tRNALeum5CAA (tRNALeu3).The primary structure of baker's yeast tRNALeuUAG was elucidated by application of sensitive radioactive isotope derivative (‘postlabeling’) methods. Complete RNase T1 and A and partial RNase U2 fragments, prepared from non‐radioactive tRNA and 5′‐half and 3′‐half molecules, were separated by two‐dimensional polyethyleneimine‐cellulose anion‐exchange thin‐layer chromatography and isolated by a novel micropreparative procedure affording high yields of these compounds in sufficient purity for subsequent tritium derivative analysis. Base composition and sequence of oligonucleotides were analyzed by tritium derivative methods. Molar ratios of the fragments were determined from the radioactivity of 3H‐labeled nucleoside trialcohols in combination with base analysis. 2′‐O‐Methylated guanosine was characterized using the [γ‐32P]ATP/polynucleotide kinase reaction. The analysis of classical complete and partial RNase digests by the tritium derivative methods yielded the complete nucleotide sequence of the RNA. A total of about 20 A260 units of the RNA was used for analysis, i.e. considerably less material than required for conventional spectrophotometric analysis.A different sequencing approach, consisting of a combination of ‘readout sequencing’ with tritium sequencing of complete RNase T1 and A fragments, was applied to the 3′‐half molecule. The 3′‐half molecule was labeled with 32P at its 5′ terminus, partially degraded with RNases T1, U2, and Phy1 and with alkali, and subjected to polyacrylamide gel electrophoresis. The sequence was read off the gel on the basis of cleavage patterns and size of the fragments. While the readout procedure provided only the positions of A, U, C, and G residues in the chain, additional information from tritium derivative analysis was utilized to define the positions of the modified nucleosides. The readout sequencing procedure was found to require less than 0.01 A260 unit of RNA and the analysis of the complete fragments about 6 A260 units.Interesting structural features of tRNALeuUAG are (a) the location of unique, leucine tRNA isoacceptor‐specific sequences next to U‐8, a constant nucleotide participating in synthetase recognition, (b) the occurrence of 1‐methyladenosine in the T loop, a modification not present in the structurally related tRNALeum5CAA, and (c) the unusual presence of an unmodified uridine in the first position of the anticodon, which may be related to the unusual coding properties reported for this tRNA.
Fluctuations in Proline and Other Free Amino Acids during the Mitotic Cycle of the Myxomycete Physarum polycephalum
Authors: THRELFALL Richard J. , THOMAS Alan J. 
Published: March 2005

1. During synchronous growth of the acellular slime mould Physarum polycephalum the free amino pool had two maxima, one of 650 units [nmol/plasmodium dry weight (mg)] at metaphase and the other of 780 units in mid G2 with minima of 550 units before and after mitosis.2. Proline formed 20–25% of the total pool with aspartic acid, glutamic acid, threonine, valine, leucine, lysine and arginine making up 55% of the pool.3. The fluctuation of proline during the mitotic cycle was quite different from that of the other amino acids and was transiently very low during telophase.
Location and Encapsidation of the Coat Protein Cistron of Tobacco Mosaic Virus
Authors: LOMONOSSOFF George P. , BUTLER P. Jonathan G. 
Published: March 2005

The coat protein cistron of tobacco mosaic virus has been located on the viral RNA starting between 975 and 1050 nucleotides from the 3′‐hydroxyl end. This locates its 5′ end close to the origin for virus assembly, where the first protein disk interacts with the RNA. It also means that the coat protein mRNA must have a short 5′‐untranslated tail and a long (over 500 nucleotides) 3′ one.The recovery of characteristic oligonucleotides in nuclease‐protected rods during the growth from RNA and a protein disk preparation shows that elongation of the nucleated rods proceeds independently in both directions though, on average, much more rapidly along the longer 5′ tail than the shorter 3′ tail. protected RNA of length equal to that in the complete virion is first seen within 6 min, showing that the most rapidly elongated particles are substantially complete by this time.
Brain Pyridoxal Kinase. Mobility of the Substrate Pyridoxal and Binding of Inhibitors to the Nucleotide Site
Authors: KWOK Francis , CHURCHICH Jorge E. 
Published: March 2005

Pyridoxal kinase has been purified 2000‐fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. The interactions of the substrate pyridoxal and the inhibitor N‐dansyl‐2‐oxopyrrolidine (dansyl = 5‐dimethylaminonaph‐thalene‐1‐sulfonyl) with the catalytic site were examined by means of fluorescence spectroscopy. The increase in emission anisotropy that follows the binding of pyridoxal to the kinase was used to determine the equilibrium dissociation constant. Pyridoxal kinase binds one molecule of substrate with a Kd= 11 μM at pH 6. The emission anisotropy spectrum of bound pyridoxal reveals that the substrate is not rigidly trapped by the protein matrix. N‐Dansyl‐2‐oxopyrrolidine is a competitive inhibitor with respect to ATP at saturating concentrations of pyridoxal. It binds to the enzyme with a dissociation constant of 6 μM. N‐Dansyl‐2‐oxopyrrolidine is immobilized by strong interactions with the enzyme, but it is displaced from the catalytic site by ATP. The results are consistent with the hypothesis that N‐dansyl‐2‐oxopyrrolidine binds at the nucleotide binding site of pyridoxal kinase.
DNA Polymerases of Anucleated Cells
Authors: TARRAGO‐LITVAK Laura , DESGRANGES Claude , ARAYA Alejandro , LITVAK Simon 
Published: March 2005

Two different DNA polymerases have been purified and characterized from human platelets. In the mitochondrial fraction a unique activity of the polymerase γ type has been found. The same enzyme is found in the extramitochondrial supernatant. A second DNA polymerase, called ‘cytoplasmic’ DNA polymerase has been found in the 10000 ×g supernatant of human platelets.The following properties of the latter DNA polymerase from human platelets are identical to those of DNA polymerase α from normal cells: DEAE‐cellulose and phosphocellulose chromatography, size, thermal stability, phosphonoacetic acid and ethidium bromide inhibition. However, some of its properties, like high resistance to N‐ethylmaleimide and the lack of DNA polymerization using synthetic RNA primers, are those of DNA polymerase β.
Taurocyamine Kinase from Body‐Wall Musculature of the Lugworm Arenicola marina
Authors: SURHOLT Bernhard 
Published: March 2005

ATP: taurocyamine phosphotransferase was extracted from body‐wall musculature of the lugworm Arenicola marina and purified by sequential ammonium sulfate precipitation, Sephadex G‐25, DEAE‐cellulose and hydroxylapatite column chromatography to polyacrylamide discelectrophoretic homogeneity. The enzyme was enriched 860‐fold to a specific activity of 1200–1300 μmol×min−1× mg protein−1.For direct measurement of the enzyme activity of forward as well as of reverse reaction one spectrophotometrical assay was evolved.The purified enzyme has a molecular weight of approximately 60000 (assayed by means of sucrose‐density‐gradient centrifugation and by gel‐sieve chromatography on a Sephadex G‐100 column). Dodecylsulfate/polyacrylamide gel electrophoresis revealed three bands: at 22000, 13000–14000, and 11000.Temperature optimum of the taurocyamine kinase activity was found to be 33°C. The pH optimum of the reaction velocity of the forward reaction is at pH 8.0, that of the reverse reaction at pH 7.2. K
m values for the substrates of the forward reaction are 1.0 × 10−4 M for taurocyamine and 3.3 × 10−3 M for ATP, and for those of the reverse reaction 8.3 × 10−4 M for phosphotaurocyamine and 1.2 × 10−3 M for ADP. V value of the forward reaction is 240 μmol×min−1× mg protein−1, and that of the reverse 1340 μmol×min−1× mg protein−1.Phosphocreatine and phosphoarginine are inactive. To become active the taurocyamine kinase needs Mg2+ (10 mM).Isoelectric focusing of the purified enzyme showed a pattern of about 8 bands between pH 6.1 and 7.8, the main band lying at pH 7.3.Within the cells of the body‐wall musculature 92–94% of the taurocyamine kinase activity were found to be in the cytosol, the mitochondria, in all probability, have 6–8% of the total activity.
Purification and Characterization of the Hypoxanthine‐Guanine Phosphoribosyltransferase from Saccharomyces cerevisiae
Authors: SCHMIDT Rainer , WIEGAND Helga , REICHERT Uwe 
Published: March 2005

1. Hypoxanthine‐guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400‐fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation.2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G‐100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51000.3. The enzyme requires Mg2+ and has its pH optimum at 8.5.4. Isoelectric focussing as well gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1.5. The enzyme displays Michaelis‐Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 μM, 18 μM, and 50 μM respectively.
Site of Interaction between Phenazine Methosulphate and the Respiratory Chain of Bacillus subtilis
Authors: BISSCHOP Arnold , BERGSMA Jack , KONINGS Wil N. 
Published: March 2005

Membrane vesicles from Bacillus subtilis W23 and from the menaquinone‐deficient Bacillus subtilis aroD were incubated with phenazine methosulphate and excess soluble phenazine methosulphate was subsequently removed by washing (preincubated vesicles). Phenazine methosulphate which remains attached to the membrane is accessible from the outer surface of the membrane because it can be reduced chemically by NADH or ascorbate and the reduced forms can mediate electrons to horse heart cytochrome c in the external medium of the membrane vesicles.The oxidation rates of ascorbate in preincubated vesicles of B. subtilis W23 and B. subtilis aroD and of NADH in preincubated vesicles of B. subtilis aroD increased with increasing amounts of phenazine methosulphate attached per mg membrane protein. Electrons from membrane‐attached reduced phenazine methosulphate are essentially all mediated to the terminal part of the respiratory chain before cytochrome a601 because the oxidation of reduced phenazine methosulphate is almost completely inhibited by cyanide and only to a small extent by 2‐heptyl‐4‐hydroxy‐quinoline‐N‐oxide while NADH oxidase is strongly inhibited by both compounds. Furthermore, reduced phenazine methosulphate reduces completely cytochrome a601 and not all cytochromes b560 and c553.Active transport of amino acids is at least ten‐fold more effectively stimulated by NADH or ascorbate oxidation via phenazine methosulphate than by NADH oxidation via NADH dehydrogenase. Possible explanations are discussed.
A 13C Nuclear‐Magnetic‐Resonance Study of CO2‐HCO3− Exchange Catalyzed by Human Carbonic Anhydrase C at Chemical Equilibrium
Authors: SIMONSSON Ingvar , JONSSON Bengt‐Harald , LINDSKOG Sven 
Published: March 2005

The effects of human carbonic anhydrase C on the 13C nuclear magnetic resonance spectra of equilibrium mixtures of 13CO2 and NaH13CO3 were measured at 67.89 MHz. Enzyme‐catalyzed CO2‐HCO3− exchange rates were estimated from the linewidths of the resonances. The results show that: (a) the maximal exchange rates are larger than the maximal turnover rates; (b) the exchange is equally rapid with 1H2O or with 2H2O as solvents; (c) the exchange is equally rapid in the presence or in the absence of added buffers; (d) the apparent substrate binding is weaker than predicted if steady‐state Km values are assumed to represent substrate dissociation constants.The main conclusion concerning the catalytic mechanism of the enzyme is that the proton‐transfer processes which limit turnover rates in the steady state are not directly involved in CO2‐HCO3− exchange. In addition, the results suggest that CO2‐HCO3− interconversion takes place by a nucleophilic mechanism, such as a reversible reaction of zinc‐coordinated OH− with CO2.