bluuebunny/crossref_metadata_2025_split huggingface dataset:
Alkylamine derivatives of cytochrome c
Authors: WALLACE Carmichael J. A. , CORTHÉSY Blaise E. 
Published: March 2005

For investigations of the functional roles of the lysine residues of cytochrome c, analogues in which these residues are modified without charge loss are highly desirable. The 19 lysine residues of the horse heart protein have been modified by reductive alkylation. Two analogues were prepared, using formaldehyde and acetone as the dialkylating and monoalkylating reagent respectively. Modification was shown to be clean and quantitative. Characterisation of the alkylamine derivatives by physicochemical measurements and biological activity determinations was carried out. The potential of these analogues in structure/function studies of cytochrome c is discussed. It is illustrated by their use, in conjunction with other lysine‐modified derivatives, to investigate the extent to which surface charge determines redox potential, and to study the physicochemical changes that accompany rising pH. In the latter case the observed phenomena are not as closely correlated as previously thought, suggesting that there is a complex set of rearrangements of structure underlying the functional changes.The data confirm that modification of the lysine residues influences these changes. These residues participate in numerous surface intramolecular links, so the lack of correlation may be explained if each of the changing parameters were under the influence of a different set of residues. However, neither a lysine residue, nor a histidine residue directly displaces methionine from the sixth coordination position of the haem iron at alkaline pH.
Characterization of two d‐glyceraldehyde‐3‐phosphate dehydrogenases from the extremely thermophilic archaebacterium Thermoproteus tenax
Authors: HENSEL Reinhard , LAUMANN Silvia , LANG Jutta , HEUMANN Herrmann , LOTTSPEICH Friedrich 
Published: March 2005

Thermoproteus tenax possesses two different glyceraldehyde‐3‐phosphate dehydrogenases, one specific for NADP+ and the other for NAD+. NADP(H) inhibits the NAD+‐specific enzyme competetively with respect to NAD+ whereas NAD(H) virtually does not interact with the NADP+‐specific enzyme. Both enzymes represent homomeric tetramers with subunit molecular masses of 39 kDa (NADP+‐specific enzyme) and 49 kDa (NAD+ ‐specific enzyme), respectively.The NADP+‐specific enzyme shows significant homology to the known glyceraldehyde‐3‐phosphate dehydrogenases from eubacteria and eukaryotes as indicated by partial sequencing.The enzymes are thermostable, the NADP+‐specific enzyme with a half‐life of 35 min at 100°C, the NAD+ ‐specific enzyme with a half‐line of ≥20min at 100°C, depending on the protein concentration. Both enzymes show conformational and functional changes at 60 – 70°C.
Ester composition of carnitine in the perfusate of liver and in the plasma of donor rats
Authors: SANDOR Attila , KISPAL Gyula , MELEGH Bela , ALKONYI Istvan 
Published: March 2005

When the carnitine pool of fed rats was labelled with tritium, in non‐recirculating perfusate of their liver 44% of acid‐soluble 3H activity was identified as free carnitine and 47% as short‐chain acylcarnitine. Of the latter component acetylcarnitine accounted for 30% and propionylcarnitine for 10% of total acid‐soluble.In plasma the contribution of short‐chain acylcarnitines to total carnitine in fed, fasted and diabetic rats was 15.6%, 43.1% and 48.0%, respectively. Recirculating perfusion of livers from the same animals revealed that livers from fed rats released short‐chain acylcarnitines as much as 56.2% of total and this proportion did not increase further in the other two groups. At the same time, ketone bodies in the perfusate increased gradually in the fed, fasted and diabetic group, paralleling the plasma ketone levels. Although liver supplies the organism with carnitine the increment of plasma short‐chain acylcarnitines seen in ketosis is not a result of some extra output by the liver.
Developmental changes in rat hepatic casein kinases 1 and 2
Authors: PEREZ Mercè , GRANDE Jorge , ITARTE Emilio 
Published: March 2005

Cytosolic histone kinase and casein kinase activities varied considerably in the late fetal and postnatal periods of liver development. Both activities showed a maximum at day 21 of gestation and decreased at birth to values close to those of adult rats. The changes in total casein kinase activity were due to variations of casein kinase 1 and casein kinase 2. Similarly the activities of both the cyclic‐AMP‐dependent protein (histone) kinase and the cyclic‐AMP‐independent histone kinase varied during development.Besides the changes in total activity, the affinity of casein kinases 1 and 2 for casein also varied in fetal and postnatal development. The Km values of casein kinase 2 increased from day 18, reached a maximum at day 20 of gestation and then started to decrease until one day after birth. In contrast the Km values of casein kinase 1 decreased from day 18, reached its lowest value at day 21 of gestation and attained values similar to those in the adult at the day of birth. Changes in this parameter were also observed when insulin (3 IU/kg) was administered by intraperitoneal injection to one‐day‐old rats. The Km values of casein kinase 1 decreased while those of casein kinase 2 increased after administration of this hormone. On the other hand, the Km values for ATP of casein kinases 1 and 2 as well as their apparent molecular masses and sensitivity to heparin and GTP did not significantly change during ontogeny of rat liver.
Further purification and characterization of the DNA 3′‐phosphatase from rat‐liver chromatin which is also a polynuclotide 5′‐hydroxyl kinase
Authors: HABRAKEN Yvette , VERLY Walter G. 
Published: March 2005

The DNA 3′‐phosphatase activity of rat‐liver chromatin has been purified. A DNA 5′‐hydroxyl kinase activity comigrates at each step of purification. Both enzymes have the same molecular mass (79 kDa) and the same isoelectric point (8.6). It thus seems that the two activities are born by the same protein just as with the phage T4 enzyme which is, at the same time, a 5′‐hydroxyl kinase and a 3′‐phosphatase. An additional argument is that ATP, which does not influence the rate of the 3′‐phosphatase reaction but which is a cosubstrate of the 5′‐hydroxyl kinase, protects the 3′‐phosphatase activity against thermal denaturation and trypsin digestion.The two active sites must, however, be largely independent within a common support: the thermal denaturation and trypsin inactivation rates are very different for the two activities; increasing the ionic strength activates the kinase and inhibits the phosphatase; polyvalent anions inhibit the phosphatase and have little effect on the kinase. The two active sites might belong to different domains of the protein; they could not however be separated by a partial trypsin digestion.The rates of 3′‐dephosphorylation and 5′‐phosphorylation by the chromatin enzyme are the same in native and denatured DNA. The 3′‐phosphatase has no action on 3′‐monodeoxynucleotide, but it hydrolyzes the 3′‐phosphate in dinucleotides. The Km of the 3′‐phosphatase is 0.548 μM. The Km (5′‐OH) and Km (ATP) of the 5′‐hydroxyl kinase are about 3.9 μM and 0.69 μM respectively. The chromatin enzyme is unable to hydrolyze 3′‐phosphoglycolate ends in DNA.
Purification and characterization of a casein‐kinase‐II‐type enzyme from Xenopus laevis ovary
Authors: MULNER‐LORILLON Odile , MAROT Jean , CAYLA Xavier , POUHLE Robert , BELLE Robert 
Published: March 2005

A casein kinase of type II has been highly purified from Xenopus laevis ovary. A new experimental protocol has been developed for the purification, consisting in four chromatographic steps: hydrophobic on tyrosine‐agarose, ion exchange on DEAE‐Sepharose, affinity on heparin‐Sepharose and fast protein liquid on Mono Q. The purification was > 20000, taking into account an inhibitor present in the starting material which masked the activity in the crude fraction. The overall yield was >20%. Full‐grown Xenopus oocytes contain 64 milliunits per oocyte corresponding to an intracellular concentration in the nanomolar range.The enzyme shares the following features with the mammalian casein kinase II: (a) comparable subunit composition (42‐kDa doublet, 38 kDa and 26 kDa), (b) autophosphorylation of the 26‐kDa subunit, (c) ability to use GTP as well as ATP as phosphate donor, (d) inability to use Mn2+ instead of Mg2+ to support the activity, (e) phosphorylation of both threonine and serine residues of casein, (f) inhibition by low doses of heparin.Biological effects of the highly purified enzyme have been investigated upon microinjection into Xenopus full‐grown oocytes. At nanomolar concentrations (∼ 3 nM) the enzyme inhibited progesterone induction of meiotic cell division whereas it facilitates meiotic maturation at the level of maturation‐promoting factor. These results suggest a role for the kinase in the phosphorylation cascade involved during the prophase/metaphase transition of meiotic cell division, both in the mechanism of the meiotic prophase arrest and in the activity of the cytoplasmic factor maturation‐promoting factor. When microinjected into oocytes above 45 nM, the kinase provoked complex changes in the profile of the in ovo32P‐labelled proteins indicating that its targets could be other kinase/phosphatase regulatory proteins.
Immunochemical properties of elongation factors 1 of plant origin
Authors: PULIKOWSKA Jadwiga , WOJTASZEK Przemyslaw , KORCZI Aleksandra , MICHALSKI Zbigniew , CANDRESSE Thierry , TWARDOWSKI Tomasz 
Published: March 2005

Elongation factors 1 (EF‐1) have been isolated from different plants: wheat, yellow lupine, blue lupine, Chinese cabbage and Norway maple.Antibodies for EF‐1 from yellow lupine have been obtained in rabbits; antibodies for wheat EF‐1 were elicited in mice. The immunological properties of EF‐1 were assayed by the following methods: western blotting, double immunodiffusion and rocket immunoelectrophoresis. Our results suggest that one antigenic site is similar for all plant elongation binding factors tested. This epitope probably overlaps the centre of biological activity of EF‐1, as was shown for wheat EF‐1.The hypothesis concerning the potential presence of plant EF‐1 as a subunit of turnip yellow mosaic virus RNA replicase (similar to prokaryotic EF‐Tu in the Qβ RNA replicase system) has also been tested using immunotechniques as well as tests of biological activity, but has not been confirmed.
Conformational analysis of the tetranucleotides m62A‐m62A‐U‐m62A (m62A =N6‐dimethyladenosine) and U‐m62A‐U‐m62A and of the hybrid dA‐r(U‐A)
Authors: van den HOOGEN Yvonne Th. , TREURNIET Stevin J. , ROELEN Harlof C. P. F. , de VROOM Erik , van der MAREL Gijs A. , van BOOM Jacques H. , ALTONA Cornelis 
Published: March 2005

A 1H‐NMR investigation was carried out on the tetranucleotides U‐m62A‐U‐m62A and m62A‐m62A‐U‐m62A (m62=N6‐dimethyladenosine) as well as on the hybrid trinucleotide dA‐r(U‐A). An extensive comparison with m62A‐U‐m62A and other relevant compounds is made.Previous proton NMR studies on trinucleotides have shown that purine‐pyrimidine‐purine sequences prefer to adopt a mixture of states which have as a common feature that the interior pyrimidine residue bulges out, whereas the flanking purine residues stack upon each other. A stacking interaction on the 3′ side of the bulge is known to have no measurable effect on the bulge population. Chemical‐shift data, ribose ring conformational analysis and information from NOE experiments now show unambiguously that the moderate U(1)‐m62A(2) stack in U‐m62A‐U‐m62A diminishes the population of bulged‐out structures in favour of a regular stack. This tendency towards conformational transmission in the downstream 5′→3′direction is fully confirmed by the fact that the strong m62A(1)‐m62A(2) stack in the tetranucleotide m62A‐m62A‐U‐m62A virtually precludes the formation of bulged‐out structures.The conformational characteristics of dA‐r(U‐A) appear comparable with those of m62A‐U‐m62A, which indicates that the presence of a 2′‐hydroxyl group in the first purine residue is not a necessary prerequisite for the formation of a bulge.
Penicillin‐degrading activities of peptides from pneumococcal penicillin‐binding proteins
Authors: ELLERBROK Heinz , HAKENBECK Regine 
Published: March 2005

Trypsin treatment of native penicillin‐binding proteins (PBPs) 1a, 2 b and 3 from Streptococcus pneumoniae resulted in the formation of stable peptides containing the β‐lactam‐binding site with molecular masses ranging from 26 kDa to 36 kDa.Whereas the PBP 1a peptide (Ia) was enzymatically rather unstable, the PBP 2b peptide (IIb) and the PBP 3 peptide (III) were able to bind and release β‐lactams with similar rates compared to the intact PBP, the turnover rate of fragment IIb was even twice as fast as that observed with PBP 2b. Analysis of the turnover products by thin‐layer chromatography revealed that PBP 2b and 3 produced penicilloic acid as well as phenylacetylglycine. On the other hand, with the corresponding tryptic fragments only the hydrolysis product penicilloic acid was obtained.
Higher oxidation states of prostaglandin H synthase
Authors: KARTHEIN Rainer , DIETZ Reiner , NASTAINCZYK Wolfgang , RUF Hans Heinrich 
Published: March 2005

Purified prostaglandin H synthase (EC 1.14.99.1), reconstituted with hemin, was reacted with substrates of the cyclooxygenase and peroxidase reaction. The resulting EPR spectra were measured below 90 K. Arachidonic acid, added under anaerobic conditions, did not change the EPR spectrum of the native enzyme due to high‐spin ferric heme. Arachidonic acid with O2, as well as prostaglandin G2 or H2O2, decreased the spectrum of the native enzyme and concomitantly a doublet signal at g= 2.005 was formed with maximal intensity of 0.35 spins/enzyme and a half‐life of less than 20 s at ‐12 °C. From the conditions for the formation and the effect of inhibitors, this doublet signal was assigned to an enzyme intermediate of the peroxidase reaction, namely a higher oxidation state.The doublet signal with characteristic hyperfine structure was nearly identical to the signal of the tyrosyl radical in ribonucleotide reductase (EC 1.17.4.1). Hence the signal of prostaglandin H synthase was assigned to a tyrosyl radical. Electronic spectra as well as decreased power saturation of the tyrosyl radical signal indicated heme in its ferryl state which coupled to the tyrosyl radical weakly. [FeIVO(protoporphyrin IX)]…Tyr+· was suggested as the structure of this two‐electron oxidized state of the enzyme.A hypothetical role for the tyrosyl radical could be the abstraction of a hydrogen at C‐13 of arachidonic acid which is assumed to be the initial step of the cyclooxygenase reaction.
An antibody to the low‐density lipoprotein (LDL) receptor that partially inhibits the binding of LDL to cultured human fibroblasts
Authors: GAVIGAN Sean J. P. , PATEL Dilip D. , SOUTAR Anne K. , KNIGHT Brian L. 
Published: March 2005

A monoclonal antibody, raised initially against pure bovine low‐density lipoprotein (LDL) receptor, was found to bind saturably to LDL receptors on the surface of cultured human fibroblasts. It was apparently internalized by the cells but rapidly returned to the surface and was not significantly degraded. LDL did not affect the binding of antibody. However, the antibody reduced both the apparent affinity of the receptor for LDL and the amount of LDL bound. In the presence of antibody, the concentration of LDL required for one‐half maximum binding increased from 14.5 nM to 22.3 nM at 37°C and from 2.8 nM to 11.5 nM at 4°C. Maximum heparin‐releasable binding of LDL was reduced by 56% at 37°C and by 36% at 4°C. Fab fragments halved the affinity at both temperatures without affecting the maximum amount of LDL bound. At 4°C, maximum heparin‐releasable LDL binding was the same as that of antibody and of Fab fragments. At 37°C, heparin‐releasable binding of LDL and binding of Fab fragments were double that at 4°C, whereas binding of antibody remained unchanged.The results suggest that the antibody bound to a site on the receptor remote from the LDL binding region and that intact antibody recycled with the receptor. It halved LDL binding and degradation at 37°C and reduced binding at 4°C, probably through a different mechanism. The possibility that both cooling and antibody led to receptor aggregation is discussed.
The effect of thyroxine on the calmodulin‐dependent (Ca2+‐Mg2+)ATPase activity and protein phosphorylation in rabbit fast skeletal muscle sarcolemma
Authors: FAMULSKI Konrad S. , PILARSKA Maria , WRZOSEK Antoni , SARZAŁA M. Gabriela 
Published: March 2005

Enzymatic properties and the protein pattern of sarcolemma fractions isolated from three groups of rabbits: euthyroid, hyperthyroid and hypothyroid, were studied. The amount of phosphorylated intermediate formed by the calmodulin‐dependent (Ca2+‐Mg2+)ATPase and the activity of this enzyme as well as that of (Na+‐K+)ATPase were the highest in membranes isolated at the hyperthyroid state. On the other hand, sarcolemma obtained from the hypothyroid animals exhibited a decreased activity of (Na+‐K+)ATPase, while the activity of calmodulin‐dependent (Ca2+‐Mg2+)ATPase was the same as in the preparations obtained from euthyroid animals. Thyroid hormones also changed the protein pattern of muscle sarcolemma. Membranes isolated from hyperthyroid animals lacked peptides of apparent molecular masses of 41 kDa and 53 kDa, while a peptide of the apparent molecular mass of 63 kDa was enriched in the preparation from hypothyroid animals. Thyroid hormones affected endogenous cAMP‐dependent protein phosphorylation. The sarcolemma fraction obtained from hyperthyroid animals exhibited a decreased phosphorylation of peptides of apparent molecular masses of 30 kDa and 47 kDa, while the cAMP‐independent phosphorylation of several other peptides was augumented.Moreover, sarcolemma preparations isolated from hyperthyroid animals showed higher activity of cAMP‐independent protein kinase(s) and lower activity of cAMP‐dependent protein kinase when compared to the euthyroid preparations. It is proposed that thyroxine increases the content of calmodulin‐dependent (Ca2+‐Mg2+)ATPase protein and affects the activity of cAMP‐independent and cAMP‐dependent protein kinases bound to sarcolemma.
The structure of brain‐specific rat aldolase C mRNA and the evolution of aldolase isozyme genes
Authors: KUKITA Akiko , MUKAI Tsunehiro , MIYATA Takashi , HORI Katsuji 
Published: March 2005

The cDNA clones for rat aldolase C mRNA having the nearly complete length were isolated from a rat brain cDNA library and sequenced. The nucleotide sequence of pRAC2‐1, a cDNA clone having the largest cDNA insert, indicates that the cDNA is composed of a 105‐base‐pair 5′‐noncoding sequence, a 1089‐base‐pair coding‐sequence and a 382‐base‐pair 3′‐noncoding sequence. The amino acid sequence of aldolase C deduced from a possible open reading frame was composed of 362 residues having a relative molecular mass of 39164 excluding the initiating methionine, one amino acid shorter than aldolases A and B. The length of aldolase C mRNA was 1750 residues, somewhat longer than that of the aldolase A and B transcripts. The aldolase C mRNA was distributed mainly in the brain, some in ascites hepatoma and fetal liver.Comparison of the amino acid sequences of rat aldolase C with those for rat aldolase A and B [Joh et al. (1985) Gene 39, 17–24; Tsutsumi et al. (1984) J. Biol. Chem. 259, 14572–14575], which have been determined previously, shows the existence of highly conserved stretches of amino acid among the three isozymic forms throughout their sequences. The extent of the homology between aldolases A and C is 81%, while those between aldolases A and B, and B and C are 70%, respectively. The analysis of amino acid substitution among aldolases A, B and C from several species suggests that the isozyme genes diverged much earlier than animal species appeared and that the aldolase C gene has evolved from the aldolase A gene after aldolase A and B genes diverged.
T‐kinin release from T‐kininogen by rat‐submaxillary‐gland endopeptidase K
Authors: GUTMAN Ninette , MOREAU Thierry , ALHENC‐GELAS François , BAUSSANT Thierry , EL MOUJAHED Abdel , AKPONA Simon , GAUTHIER Francis 
Published: March 2005

Submaxillary gland extracts have been fractionated to characterize the enzyme responsible for the T‐kininogenase activity previously reported in this tissue [Damas, J. & Adam, A. (1985) Mol. Physiol 8, 307–316] and to know whether this activity could be of physiological relevance, since no enzyme reacting in catalytic amounts has been described so far to be able to release a vasoactive peptide from T‐kininogen.The purified enzyme, provisionally called endopeptidase K, has an apparent Mr of 27000 when not reduced prior to analysis but 21000 after reduction and an acidic pI of 4.3 ± 0.1. Antigenically, it is not related to tissue kallikrein. Upon incubation with purified T‐kininogen it may induce a complete liberation of T‐kinin from the precursor provided it is added in stoichiometric amounts. However, in parallel with the liberation of immunoreactive kinin, a proteolysis of T‐kininogen is observed which is not restricted to the site of insertion of T‐kinin as would be expected using a specific kininogenase.In agreement with these results, no change of the mean blood pressure was observed upon injection of endopeptidase K into the circulation of normal rats even if the amount of injected enzyme was up to ten times that required for tissue kallikrein to induce a significant fall in blood pressure.However, in spite of the large proteolysis induced by incubation with stoichiometric amounts of endopeptidase K, the total papain inhibiting capacity of T‐kininogen as well as the value of the apparent inhibition constant, Ki, with this proteinase remained unchanged. Proteolytic fragments which retain cysteine‐proteinase‐inhibiting activity may therefore be released from T‐kininogen by endopeptidase K more easily than immunoreactive kinin, thus emphasizing a prominent function of proteinase inhibitor or of proteinase inhibitor precursor for this molecule.
Biosynthesis of vitamin B‐12 in anaerobic bacteria
Authors: VOGT Joachim R. A. , RENZ Paul 
Published: March 2005

The pathway of vitamin B‐12 biosynthesis in anaerobic bacteria differs in several respects from the pathway found in aerobic or aerotolerant microorganisms. The aim of this investigation was to elucidate the formation of the 5,6‐dimethylbenzimidazole part and the amide groups of vitamin B‐12 in anaerobic bacteria. [15N]Ammonium chloride or l‐[amido‐15N]glutamine or a mixture of [15N]ammonium sulfate and [15N]glycine was added to fermentations with Eubacterium limosum. The vitamin B‐12 isolated from these fermentations was methylated and degraded to cobinamide and 1,5,6‐trimethylbenzimidazole. The amide groups of cobinamide were hydrolyzed and the amide nitrogen of the side chains a, b, c, d, e and g trapped as benzamide. The 15N incorporation was determined by mass spectroscopy. Thus in the experiment with [15N]ammonium chloride the benzamide and the 1,5,6‐trimethylbenzimidazole contained 9.6%15N, whereas in the experiment with l‐[amido‐15N]glutamine 37.5% of the molecules were 15N labeled. The 1H‐NMR spectrum of 1,5,6‐trimethylbenzimidazole revealed that the 15N from the ammonium salts and from glutamine was incorporated into N‐3 of the 5,6‐dimethylbenzimidazole moiety of vitamin B‐12. With a mixture of [15N]ammonium sulfate and [15N]glycine both nitrogens of 5,6‐dimethylbenzimidazole became 15N‐labeled.These experiments demonstrate that in E. limosum the amide nitrogen of glutamine is not only the precursor of the six amide groups of the corrin ring, but also of N‐3 of the 5,6‐dimethylbenzimidazole moiety of vitamin B‐12.