bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis of poly(ethylene glycol)‐bound NADP by selective modification at the 6‐amino group of NADP
Authors: OKUDA Keiko , URABE Itaru , OKADA Hirosuke 
Published: March 2005

The N‐1 position of the adenine ring of NADP was selectively aklylated by the reaction of 2′,3′‐cyclic NADP with 3‐propiolactone to yield 2′,3′‐cyclic 1‐(2‐carboxyethyl)‐NADP (I). Derivative I was converted to a mixture of the isomers of N6‐(2‐carboxyethyl)‐NADP with their phosphate groups at the 2′ or 3′ position (IIa and IIb) by chemical reduction, alkaline rearrangement and chemical reoxidation. Carbodiimide coupling of the mixture of IIa and IIb to α,ω‐diaminopoly(ethylene glycol) gave the 2′,3′‐cyclic derivative of poly(ethylene glycol)‐bound NADP (III), which was enzymically hydrolyzed to yield poly(ethylene glycol)‐bound NADP (PEG‐NADP).PEG‐NADP has good cofactor activity (16–100% of that of NADP) for NADP‐specific and NAD(P)‐specific dehydrogenases except isocitrate and glucose dehydrogenases. For NAd‐specific enzymes. PEG‐NADP has higher cofactor activity than NADP: for horse liver alcohol dehydrogenase, the cofactor activity of PEG‐NADP is 40 times that of NADP and 14% of that of NAD. Kinetic studies show that for most of enzymes tested, Km values for PEG‐NADP are larger than those for NADP and V values for PEG‐NADP are similar to those for NADP. PEG‐NADP proved to be applicable in a continuous enzyme reactor, in which reactions of glutamate dehydrogenase and glucos‐6‐phosphate dehydrogenase were coupled by the recycling of PEG‐NADP.
Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle
Authors: KURET Jeff , WOODGETT James R. , COHEN Philip 
Published: March 2005

A casein kinase was highly purified from rabbit skeletal muscle whose substrate specificity and enzymatic properties were virtually identical to those of casein kinase‐I from rabbit reticulocytes. Prolonged incubation of glycogen synthase with high concentrations of skeletal muscle casein kinase‐I and Mg‐ATP resulted in the incorporation of >6 mol phosphate/mol subunit and decreased the activity ratio (∓ glucose‐6P) from 0.8 to <0.02. The sites phosphorylated by casein kinase‐I were all located in the N and C‐terminal cyanogen bromide peptides, termed CB‐1 and CB‐2. At an incorporatio of 6 mol phosphate/mol subunit, ∼2 mol/mol was present in CB‐1 and ∼4 mol/mol in CB‐2. Within CB‐1, casein kinase‐I phosphorylated the serines that were 3, 7 and 10 residues from the N‐terminus of glycogen synthase, with minor phosphorylation at threonine‐5. Within CB‐2, ∼9% of the phosphate incorporated was located between residues 28 and 53, and at least five of the seven serine residues in this region were phosphorylated. The remaining 10% of phosphate incorporated into CB‐2 was located between residues 98 and 123, mainly at a serine residue(s).Two of the major sites labelled by casein kinase‐I (serine‐3 and serine‐10 of CB‐1) are not phosphorylated by any other protein kinase. This will enable the role of casein kinase‐I as a glycogen synthase kinase in vivo to be evaluated.
Carbon‐13 and nitrogen‐15 nuclear‐magnetic‐resonance investigation on Desulfovibrio vulgaris flavodoxin
Authors: VERVOORT Jacques , MÜLLER Franz , LeGALL Jean , BACHER Adelbert , SEDLMAIER Helmut 
Published: March 2005

Desulfovibrio vulgaris apoflavodoxin has been reconstituted with 15N and 13C‐enriched riboflavin 5′‐phosphate. For the first time all carbon atoms of the isoalloxazine ring of the protein‐bound prosthetic group have been investigated. The reconstituted protein was studied in the oxidized and in the two‐electron‐reduced state. The results are interpreted in terms of specific interactions between the apoprotein and the prosthetic group, and the chemical structure of protein‐bound FMN.In the oxidized state weak hydrogen bonds exist between the apoprotein and the N(5), N(3) and O(4α) atoms of FMN. The N(1) and O(2α) atoms of FMN form strong hydrogen bonds. The isoalloxazine ring of FMN is strongly polarized and the N(10) atom shows an increased sp2 hybridisation compared to that of free FMN in aqueous solution. The N(3)‐H group is not accessible to bulk solvent, as deduced from the coupling constant of the N(3)‐H group.In the reduced state the hydrogen bond pattern is similar to that in the oxidized state and in addition a strong hydrogen bond is observed between the N(5)‐H group of FMN and the apoprotein. The reduced prosthetic group possesses a coplanar structure and is ionized. The N(3)‐H and N(5)‐H groups are not accessible to solvent water. Two‐electron reduction of the protein leads to a large electron density increase in the benzene subnucleus of bound FMN compared to that in free FMN.The results are discussed in relation to the published crystallographic data on the protein.
Inactivation of Clostridium botulinum type A neurotoxin by trypsin and purification of two tryptic fragments
Authors: SHONE Clifford C. , HAMBLETON Peter , MELLING Jack 
Published: March 2005

Limited treatment of Clostridium botulinum type A neurotoxin with trypsin resulted in the cleavage of the heavy (95 000 Da) subunit at approximately the mid‐position and a loss of toxic activity. The rate of toxicity loss was considerably faster than that of mid‐chain cleavage; thus a loss of toxicity in excess of 90% was accompanied by only 30–35% mid‐chain cleavage of the heavy subunit.
A study of the binding of125I‐labelled neurotoxin to rat brain synaptosomes suggesting the presence of at least two sites of tryptic action on the 95 000‐Da binding subunit.
Prolonged treatment of the neurotoxin with trypsin resulted in the complete digestion of a 46 000‐Da fragment of the heavy subunit, leaving intact a soluble fragment of approximately 105 000 Da containing the light subunit linked to the remaining (49 000‐Da) protion of the heavy subunit. This fragment exhibited less than 0.01% of the original toxicity and gave immunoprecipitation reactions indistinguishable from the native toxin.
The 49 000‐Da portion of the heavy chain was purified from the 105 000‐Da fragment of the toxin and the sequence of the first 35 amino acids determined. The sequence of the first 10 residues was found to be identical to that previously reported for the heavy subunit showing that the 49 000‐Da fragment represents the NH2‐terminal portion of the heavy chain and that this region is resistant to tryptic action.
It is suggested that the primary site(s) of tryptic action on the heavy subunit of botulinum type A neurotoxin is close to the COOH terminus and that cleavage of the polypeptide chain in this region results in a closs of toxic activity mediated by the destruction of the neurotoxin‐binding site.
Long‐term effects of physiological oxygen concentrations on glycolysis and gluconeogenesis in hepatocyte cultures
Authors: WÖLFLE Detlef , JUNGERMANN Kurt 
Published: March 2005

Primary cultures of adult rat hepatocytes were kept for 46 h with either insulin (‘insulin cells’) or glucagon (‘glucagon cells’) as the dominant hormone under different oxygen concentrations with 13% (v/v) O2 mimicking arterial and 4% hepatovenous levels. Thereafter metabolic rates were measured for a 2 h period under the same (‘overall long‐term O2 effects’) or a different (‘short‐term O2 effects’) oxygen concentration. From the differences of the two effects the ‘intrinsic long‐term O2 effects’ were derived.Glycolysis, as measured in ‘insulin‐cells’, was stimulated by low O2 levels. It was about threefold faster in cells cultured and tested under 4% O2 as compared to cells cultured and tested under 13% O2, indicating the overall long‐term effect. Glycolysis was about twofold faster in cells cultured and tested under 4% O2 as compared to cells cultured under 4% O2 but tested under 13% O2, demonstrating the short‐term effect. Glycolysis was about 1.5‐fold faster in cells cultured and tested under 4% O2 as compared to cells cultured under 13% O2 but tested under 4% O2, showing the intrinsic long‐term effect. This difference was roughly parallel to the difference in levels of glucokinase and pyruvate kinaseGluconeogenesis, as measured in ‘glucagon cells’, was stimulated by high O2 levels. Similar to glycolysis overall long‐term, short‐term and intrinsic long‐term effects could be distinguished. The intrinsic long‐term effects determined under 13% O2 corresponded to a 1.5‐fold stimulation and paralleled the difference in phosphoenolpyruvate carboxykinase levels.The present results show that physiological oxygen concentrations also modulate hepatic carbohydrate metabolism by long‐term effects and that the O2 gradient over the liver parenchyma thus contributes to the metabolic differences between periportal and perivenous hepatocytes in vivo.
Bovine fatty acid binding proteins
Authors: JAGSCHIES Günter , REERS Martin , UNTERBERG Christian , SPENER Friedrich 
Published: March 2005

When a 100000 ×g supernatant from bovine heart was incubated with [1‐14C]oleic acid and subjected to isoelectric focusing, two fatty acid binding proteins (FABPs) with isoelectric points at 4.9 and 5.1 were detected.The proteins were purified on a large scale first by heat and acid precipitation of a postmitochondrial supernatant, as well as fractionation with ammonium sulfate, then by alternate application of ion‐exchange and gel chromatography. The procedure afforded around 60 mg pure proteins from 1.5 kg fresh heart muscle.Relative molecular masses of 15 300 ± 1600 for both proteins were derived from sodium dodecyl sulfate/polyacrylamide gel electrophoresis, gel chromatography, sedimentation velocity as well as from amino acid analysis. Up to 50% of the proteins' secondary structures consisted of β‐sheet. N‐termini of the peptide chains were blocked; the amino acid compositions of the two proteins were similar, but differed considerably from those of the two FABPs isolated from bovine liver [Haunerland et al. (1984) Hoppe Seyler's Z. Physiol. Chem. 365, 365–376]. Whereas hepatic FABPs changed their pI upon binding fatty acids, cardiac FABPs did not. Cardiac FABPs were immunologically identical, but did not cross‐react with hepatic proteins.A reversible, concentration‐dependent self‐association reported for FABP from pig heart [Fournier et al. (1983) Biochemistry 22, 1863–1872] was not observed for FABP from bovine heart. Changes of concentration did not alter secondary structure, intrinsic fluorescence or the sedimentation coefficient of the protein.
Selective synthesis of the hexaenoic molecular species of ether‐linked glycerophosphoethanolamine of Ehrlich ascites tumor cells
Authors: NAKAGAWA Yasuhito , WAKU Keizo 
Published: March 2005

In a previous study [Waku, K. and Nakazawa, Y. (1978) Eur. J. Biochem. 88, 489–494], we observed the rapid turnover rate of the molecular species of alkylacyl glycerophosphoethanolamine (Gro‐P‐Etn) containing docosahexaenoic acid and the high selectivity for this molecular species of ethanolamine phosphotransferase was suggested. To clarify this point, the incorporation of [14C]ethanolamine and [14C]CDP‐ethanolamine into the individual molecular species of alkenylacyl, alkylacyl and diacyl Gro‐P‐Etn has been determined in Ehrlich ascites tumor cells.[14C]Ethanolamine was highly incorporated into the pentaenoic + hexaenoic species of alkenylacyl, alkylacyl and diacyl Gro‐P‐Etn, whereas incorporation of [14C]ethanolamine into molecular species other than the pentaenoic + hexaenoic species was quite low. The selectivity of ethanolamine phosphotransferase to form the molecular species of alkylacyl and diacyl Gro‐P‐Etn was examined by incubation of [14C]CDP‐ethanolamine and microsomes of Ehrlich ascites tumor cells. The incorporation of [14C]CDP‐ethanolamine was found to occur most into the pentaenoic + hexaenoic species of both alkylacyl and diacyl Gro‐P‐Etn. The present results suggest that the pentaenoic + hexaenoic species are preferentially synthesized among the various kinds of molecular species of alkylacyl and diacyl Gro‐P‐Etn by the ethanolamine phosphotransferase in Ehrlich ascites tumor cells.
Nuclear magnetic resonance studies of the old yellow enzyme
Authors: BEINERT Wolf‐Dieter , RÜTERJANS Heinz , MÜLLER Franz 
Published: March 2005

The apoenzyme of NADPH oxidoreductase, ‘old yellow enzyme’, was reconstituted with specifically 15N‐labeled flavin mononucleotide and investigated by 15N NMR spectroscopy in the oxidized and reduced state. The results indicate that in the oxidized state a hydrogen bond is formed between the N(5) atom and the apoprotein. In addition, hydrogen bonds exist between the N(1) and N(3) atoms of FMN and the apoprotein. The resonance position of N(10) indicates that this atom is somewhat sp3‐hybridized, i.e. lifted out of the molecular plane of the isoalloxazine ring system.In the reduced state the N(1) atom is negatively charged and the N(3) atom forms a hydrogen bond with the apoprotein. The N(10) atom in protein‐bound FMN exhibits about the same hybridization state as in free anionic reduced FMN, i.e. it is located in the plane of the isoalloxazine ring. The chemical shift of the N(5) resonance indicates that this atom is almost completely sp3‐hybridized. This interpretation can also be derived from the 15N(5)‐1H coupling constant. Among the flavoproteins thus far studied by NMR techniques, old yellow enzyme is the only protein that shows a conformation of the reduced prosthetic group with the N(5) atom lifted out of the molecular plane.The isoelectric focussing properties of old yellow enzyme and a new easy method for the preparation of the apoprotein are also reported.
Photoaffinity labelling of the renal V2 vasopressin receptor
Authors: FAHRENHOLZ Falk , BOER Rainer , CRAUSE Peter , TÓTH Mihály V. 
Published: March 2005

To identify renal vasopressin receptor proteins, analogues of 1‐deamino‐vasopressin i.e. {[1‐(2‐mercapto)propionic acid]vasopressin, [Mpa1]VP} with photoreactive aryl‐azido groups in position 4 and 8 of the vasopressin sequence were prepared. In the absence of ultraviolet light, these ligands exhibit a high binding affinity for the V2 vasopressin receptor in plasma membranes from bovine and rat kidney medulla (apparent dissociation constants 1.8 × 10−9 M to 1.7 × 10−8 M); the photoreactive analogues stimulate the renal vasopressin‐sensitive adenylate cyclase.In photoaffinity labelling experiments with tritium‐labelled ligands (34–50 Ci/mmol), a membrane protein from bovine kidney or rat kidney medulla with an apparent relative molecular mass (Mr) of 30000 was preferentially and specifically labelled. The labelling of the 30000‐Mr protein was completely inhibited by a 10–100‐fold molar excess of vasopressin; in contrast, angiotensin II, bradykinin or low‐affinity analogues of vasopressin did not suppress the incorporation of the reactive ligands into this protein. The highest specific labelling yield and only a low amount of unspecific labelling was obtained with the analogue [Mpa1,Lys(N6‐4‐azidobenzoyl)8]VP. Preparative sodium dodecyl sulfate gel electrophoresis of bovine kidney membranes photolabelled with this analogue resulted in a 20–30‐fold enrichment of the 30000‐Mr vasopressin‐binding protein. Our results suggest that this photoreactive analogue of [1‐deamino, 8‐lysine]vasopressin is a suitable tool for further purification of the renal V2 vasopressin receptor binding subunit.
Activation of phosphatidylcholine–sterol acyltransferase by human apolipoprotein E isoforms
Authors: STEINMETZ Armin , KAFFARNIK Hans , UTERMANN Gerd 
Published: March 2005

The reaction catalysed by phosphatidylcholine–sterol acyltransferase (EC 2.3.1.43) is believed to be the major source of cholesteryl ester in human plasma; the enzyme requires a protien activator. Several human apolipoproteins were found to exhibit an activator function, the major one being apolipoprotein A‐I. Human apolipoprotein E exists in the population mainly in three different genetic isoforms; apolipoprotein E‐2, E‐3 and E‐4. These isopeptides were isolated from subjects homozygous for one of the isoforms, incorporated into phospholipid/cholesterol/[14C]cholesterol complexes by the cholate dialysis procedure and used to measure capacity to activate phosphatidylcholine–sterol acyltransferase in comparison to apolipoprotein A‐I lipid substrate particles prepared by the same procedure. Acyltransferase activity was measured by the formation of [14C]cholesteryl ester from [14C]cholesterol using purified enzyme.With egg yolk phosphatidylcholine as acyl donor, apo E was 15–19% as efficient as apolipoprotein A‐I for activation of the acyltransferase. Apo‐E‐stimulated cholesteryl ester formation by the enzyme was enhanced when 1‐oleoyl‐2‐palmitoyl‐glycerophosphocholine was used as a substrate phospholipid (45% of apo A‐I/phosphatidylcholine control) and most pronounced with dimyristoylglycerophosphocholine (75% of apo A‐I/phosphatidylcholine control). No significant difference in activation was found between apo E isoforms.It is concluded that apolipoprotein E activates phosphatidylcholine–sterol acyltransferase in vitro and that apolipoprotein E isoforms are similarly effective.
Copper ions as inhibitors of protein C of soluble methane monooxygenase of Methylococcus capsulatus (Bath)
Authors: GREEN Jeffrey , PRIOR Stephen D. , DALTON Howard 
Published: March 2005

Copper(I), copper(II) and silver ions have been shown to be potent inhibitors of purified soluble methane monooxygenase (MMO) of Methylococcus capsulatus (Bath). A weaker inhibition has been observed with zinc and cadmium ions. Proteins A and B of soluble MMO are unaffected by copper but protein C is rapidly and irreversibly inhibited. The site of copper inhibition has been shown to be primarily at the iron‐sulphur centre of protein C with a secondary effect at the FAD centre when the copper(II): protein C ratio is high. Copper appears to bring about the inhibition of soluble MMO by interacting with protein C to disrupt the protein structure causing, firstly, the loss of the iron‐sulphur centre, preventing the transfer of electrons from protein C to protein A, and secondly, the loss of FAD preventing the protein from accepting electrons from NADH. Inhibition and spectral data are provided to support this thesis. The inactivation of protein C is associated with the tight binding of four Cu atoms to each protein C molecule.These data extend our knowledge of how copper, which is known to have a key role in the cellular location of MMO, interacts with and rapidly and irreversibly inactivates the soluble form of this enzyme.
Location of the binding region for 23S ribosomal RNA on ribosomal protein L2 from Bacillus stearothermophilus
Authors: WATANABE Keiichi , KIMURA Makoto 
Published: March 2005

Tryptic and chymotryptic digestions of a complex between ribosomal protein L2 and the 23S RNA of Bacillus stearothermophilus produced two protected fragments (designated TF and CF). These protein fragments remained tightly bound to the RNA and were able to rebind specifically to 23S RNA. Amino acid sequence analysis of the isolated fragments revealed that both fragments TF and CF originated from the central half of the protein L2 (positions 60–206 and 58–201) with molecular masses of 15918 Da and 15690 Da respectively.From these results it is concluded that the protected central half is the primary RNA‐binding region of the protein L2. A combination of these and other results suggests that the N and/or C‐terminal (but not the central) regions may be involved in the peptidyltransferase activity of the 50S subunit.
The amino acid sequence of snapping turtle (Chelydra serpentina) ribonuclease
Authors: BEINTEMA Jaap J. , BROOS Jaap , MEULENBERG Janneke , SCHÜLLER Cornelis 
Published: March 2005

Snapping turtle (Chelydra serpentina) ribonuclease was isolated from pancreatic tissue. Turtle ribonuclease binds much more weakly to the affinity chromatography matrix used than mammalian ribonucleases. The amino acid sequence was determined from overlapping peptides obtained from three different digests. The N‐terminal amino acid sequence of the protein determined by others [E. A. Barnard, M. S. Cohen, M. H. Gold J.‐K. Kim (1972) Nature (Lond.) 240, 395‐398] and homology were used as additional evidence for several overlaps.The polypeptide chain consists of 119 amino acid residues. Compared to most ribonucleases the N‐terminal residue, three residues in the loop near residue 71 and two residues in the loop near residue 114 are deleted, and there is one additional residue in the loop near residue 23. The half‐cystines at positions 65 and 72, which form a disulfide bond in mammalian ribonucleases, are not present in turtle ribonuclease. Turtle ribonuclease differs from bovine ribonuclease at 70 of the 118 positions where both proteins have amino acid residues.Turtle ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn‐Ala‐Ser (positions 76‐78).
Identification of amino acid substitutions differentiatiating actin isoforms in their interaction with myosin
Authors: MOSSAKOWSKA Malgorzata , STRZELECKA‐GOLASZEWSKA Hanna 
Published: March 2005

Various aspects of actin ‐ myosin interaction were studied with actin preparations from two types of smooth muscle: bovine aorta and chicken gizzard, and from two types of sarcomeric muscle: bovine cardiac and rabbit skeletal. All four preparations activated the Mg2+ ‐ATPase activity of skeletal muscle myosin to the same Vmax. but the Kapp for the smooth muscle preparations was higher. At low KCl, pH 8.0 and millimolar substrate concentrations the Kapp values differed by a factor of 2.5. This differential behaviour of the four actin preparations correlates with amino acid substitutions at positions 17 and 89 of actin polypeptide chain, differentiating the smooth‐muscle‐specific γ and α isomers from cardiac and skeletal‐muscle‐specific α isomers. This correlation provides evidence for involvement of the NH2‐terminal portion of the actin polypeptide chain in the interaction with myosin.The differences in the activation of myosin ATPase by various actins were sensitive to changes in the substrate and KCl concentration and pH of the assay medium. Addition of myosin subfragment‐1 or heavy meromyosin in the absence of nucleotide produced similar changes in the fluorescence of‐a fluorescent reagent N‐(1‐pyrenyl)‐iodoacetamide, attached at Cys‐374, or 1,N6‐ethenoadenosine 5′‐diphosphate substituted for the bound ADP in actin protomers in gizzard and skeletal muscle F‐actin. The results are consistent with an influence of the amino acid substitutions on ionic interactions leading to complex formation between actin and myosin intermediates in the ATPase cycle but not on the associated states.
Interaction of human α‐thrombin and γ‐thrombin with antithrombin III, protein C and thrombomodulin
Authors: BEZEAUD Annie , DENNINGER Marie‐Hélène , GUILLIN Marie‐Claude 
Published: March 2005

Conversion of human α‐thrombin to γ‐thrombin by limited proteolysis resulted in a decrease in the inactivation rate of the enzyme by antithrombin III. The second‐order rate constants were similar but significantly different: 11 ± 1.7 × 103 and 7 ± 0.5 × 103 M−1 s−1 for α‐ and γ‐thrombin respectively. This difference is probably related to a slight change in reactivity of the catalytic site, rather than to a structural alteration of the recognition site for antithrombin III.The rate of protein C activation, measured in the absence of thrombomodulin, was greatly reduced by conversion of α‐thrombin to γ‐thrombin. In addition, γ‐thrombin failed to displace α‐thrombin from its complex with thrombomodulin, as demonstrated by measuring either the rate of protein C activation by thrombin‐thrombomodulin, or the fibrinogen clotting activity of thrombin‐thrombomodulin, in the presence of competing diisopropylphospho‐thrombin. It is concluded that the recognition sites involved in protein‐C–thrombin and thrombomodulin–thrombin interactions are both dramatically affected by the loss of peptide material occurring during the conversion of α‐thrombin to γ‐thrombin and/or by the resulting conformational changes.