bluuebunny/crossref_metadata_2025 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.
Leucyl‐tRNA and lysyl‐tRNA synthetases, derived from the high‐Mr complex of sheep liver, are hydrophobic proteins
Authors: ÇIRAKOḠLU Beyazit , WALLER Jean‐Pierre
Published: March 2005

The leucyl‐tRNA and lysyl‐tRNA synthetase components of the multienzyme complex from sheep liver were selectively dissociated by hydrophobic interaction chromatography on hexyl‐agarose and purified to homogeneity. Conservation of activities during the purification required the presence of Triton X‐100. The homogeneous enzymes corresponded to a monomer of Mr 129 000 and a dimer of Mr 2 × 79 000, respectively. Both were strongly adsorbed to the hydrophobic support phenyl‐Sepharose, in conditions where the corresponding purified enzymes from yeast the and Escherichia coli were not bound. Moreover, like the corresponding enzymes from yeast but unlike those of prokaryotic origin, the purified leucyl‐tRNA synthetases derived from the complex displayed affinity for polyanionic supports. It is shown that proteolytic conversion of lysyl‐tRNA synthetase to a fully active dimer of Mr 2 × 64000, leads to loss of both the hydrophobic and the polyanion‐binding properties. These results support the view that each subunit of lysyl‐tRNA synthetase is composed of a major catalytic domain, similar in size to the subunit of the prokaryotic enzyme, contiguous to a chain extension which carries both cationic charges and hydrophobic residues. The implications of these findings on the structural organization of the complex are discussed in relation to its other known properties.
Amino acid substitutions that reduce the affinity of penicillin‐binding protein 3 of Escherichia coli for cephalexin
Authors: HEDGE Philip J. , SPRATT Brian G.
Published: March 2005

The location of amino acid substitutions that allow an enzyme to discriminate between the binding of its normal substrate and a substrate analogue may be used to identify regions of the polypeptide that fold to form the substrate binding site. We have isolated a large number of cephalexin‐resistant mutants of Escherichia coli in which the resistance is due to the production of altered forms of penicillin‐binding protein 3 that have reduced affinity for the antibiotic. Using three mutagens, and a variety of selection procedures, we obtained only five classes of mutants which could be distinguished by their patterns of cross‐resistance to other β‐lactam antibiotics. The three classes of mutants that showed the highest levels of resistance to cephalexin were cross‐resistant to several other cephalosporins but not to penicillins or to the monobactam, aztreonam. The penicillin‐binding protein 3 gene from 46 independent mutants was cloned and sequenced. Each member of the five classes of cephalexin‐resistant mutants had the same amino acid substitution in penicillin‐binding protein 3. The mutants that showed the highest levels of resistance to cephalexin had alterations of either Thr‐308 to Pro, Val‐344 to Gly, or Asn‐361 to Ser. The Thr‐308 to Pro substitution had occurred within the β‐lactam‐binding site since the adjacent residue(SEr‐307) has been shown to be acylated by benzylpenicillin. The Asn‐361 to Ser change occurred in a region that showed substantial similarity to regions in both penicillin‐binding protein 1A and 1B and may also define a residue that is located within the β‐lactam‐binding site in the three‐dimensional structure of the enzyme.
Multiple sulfatase deficiency: Degradation of arylsulfatase A and B after endocytosis in fibroblasts
Authors: STECKEL Friedhelm , HASILIK Andrej , VON FIGURA Kurt
Published: March 2005

Multiple sulfatase deficiency can be classified into group I with severe and group II with moderate deficiencies in sulfatases. In fibroblasts in both groups the stability of arylsulfatase A and of the 47 000‐Mr form of arylsulfatase B is decreased [F. Steckel, A. Hasilik, & K. von Figura (1985) Eur. J. Biochem. 151, 141–145]. After endocytosis in control fibroblasts or those from multiple sulfatase deficiency, arylsulfatase A and B derived from the latter were subjected to enhanced degradation in both types of recipient cells. The degradation was closely linked in time to endocytosis. Whereas instability of arylsulfatase A derived from different cell lines from multiple sulfatase deficiency was comparable, a marked heterogeneity was observed for the instability of the 47 000‐Mr polypeptide of arylsufatase B. Each of the cell lines from multiple sulfatase deficiency synthesized arylsulfatase A and B polypeptides with normal and with decreased stability. Treatment with benzyloxycarbonyl‐Phe‐Ala‐CHN2, an inhibitor of cysteine proteinases, stabilized arylsulfatase A polypeptides polypeptides and partially restored arylsulfatase A activity in group II fibroblasts. The inhibitor had no protective effect on the 47 000‐Mr polypeptide or the activity of arylsulfatase B. The bearing to these findings on the yet unknown primary defect in multiple sulfatase deficiency is discussed.
Membrane‐linked energy transductions. Bioenergetic functions of sodium: H+ is not unique as a coupling ion
Authors: SKULACHEV Vladimir P.
Published: March 2005

The concept is developed according to which Na+, like H+, can play the role of a coupling ion in energy‐transducing biomembranes. This idea is based on observations that (i) Na+ can be extruded from the cell by primary pumps (Na‐motive NADH‐quinone reductase, decarboxylase or ATPase), and (ii) the downhill Na+ flux into the cell can be coupled with the performance of all the three types of membrane‐linked work i.e. chemical (ATP synthesis), osmotic (accumulation of solutes) and mechanical (motility). Marine alkalotolerant Vibrio alginolyticus represents the first example of such a complete sodium cycle pattern. Simplified versions of the sodium cycle or some of its constituents are found in the cytoplasmic membrane of a great variety of taxa including anaerobic, aerobic and photosynthetic bacteria, cyanobacteria and animals; this fact indicates that Na+ energetics should be regarded as a common case, rather than a rare exception applyed to some natural niches only.
The mechanism of ion selectivity of OmpF‐porin pores of Escherichia coli
Authors: KOBAYASHI Yoriko , NAKAE Taiji
Published: March 2005

The OmpF porin from the outer membrane of Escherichia coli acts as a lightly cation‐selective pore, allowing the diffusion of small cations and cationic molecules, whose Mr are a little larger than the threshold exclusion limit. To ascertain the mechanism of this cation selectivity, we have examined a possible influence of cationic solutes on the fluorescence emission and the circular dichroic spectrum of tryptophan residues of the porin trimer, searching for conformational change(s). The diffusion of cationic solutes was determined with the native and the amidated porins in the presence or the absence of the effector cations. The following results were obtained. (a) Cations, e.g. spermidine, caused fluoresence quenching in the native trimer, with a half‐maximum fluorescence quenching at 11–18 μM. A change in the circular dichroic spectrum was also recorded at around 280 nm. (b) The dissociation constant of spermidine to the native trimer was calculated to be 16 μM as determined by the method of equilibrium dialysis. (c) The cation‐caused fluorescence quenching was reversed when the carboxyl groups of the trimer were modified by the amidation reaction, though amidation of the trimer resulted in no significant change in the fluorescence intensity. (d) The diffusion rate of N‐benzyloxycarbonyl‐glycyl‐l‐prolyl‐l‐arginine p‐nitroanilide through the native and the amidated porins was lowered in the presence and the absence, respectively, of cations. Both the extent of fluorescence quenching in the presence of cation and the rate of cation diffusion were inversely proportional to the number of amidated carboxyl residues. The relative fluorescence quenching of the porin trimer (the amidated versus the native) in the presence of cations was linearly related to the relative solute diffusion via the porin (the amidated versus the native). These results suggested that cations caused a conformational change in the trimer, resulting in an easier diffusion of the solutes. The results suggested further that a limited number of carboxyl groups in the pore interior are involved in the cation selectivity of OmpF‐porin pores.
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 kinase

Gluconeogenesis, 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.
Investigation of the preferred Mg(II)‐adenine‐nucleotide complex at the active site of ectonucleotidases in intact vascular cells using phosphorothioate analogues of ADP and ATP
Authors: PEARSON Jeremy D. , CUSACK Noel J.
Published: March 2005

In the presence of Mg2+ the ecto‐(nucleoside diphosphatase) on intact vascular endothelial or smooth muscle cells in culture selectively catabolises the PS diastereoisomer of adenosine 5′‐[α‐thio]diphosphate, (PS)‐ADP[αS], and the ecto‐(nucleoside triphosphatase) selectively catabolises the PS isomer of adenosine 5′[ß‐thio]triphosphate, (PR)‐ATP[ßS], but exhibits no selectivity towards ATP[αS] isomers. In the presence of Cd2+ selectivity to ADP[αS] and to ATP[ßS] isomers is reversed; in the presence of Co2+, selectivity is lost. We conclude that each enzyme preferentially recognises the ′ (screw‐sense) bidentate Mg(II)‐nucleotide complex at its active site.
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.
Photoreaction of 8‐methoxypsoralen with yeast‐tRNAPhe
Authors: NIELSEN Peter Eigil , LEICK Vagn
Published: March 2005

Yeast tRNAPhe was photoreacted with [3H]8‐methoxypsoralen and the product was digested with ribonuclease T1, ribonuclease A or a combination of the two or cleaved with sodium borohydride/aniline. The oligonucleotides from these digestions were analyzed by polyacrylamide gel electrophoresis or high‐pressure liquid chromatography and the psoralen‐containing fragments were identified. The results indicate that one major and two minor photoreaction sites for 8‐methoxypsoralen exist in yeast tRNAPhe. The major site (containing about 55% of the label) was determined as U50 in the TΨ arm of the tRNA molecule while the minor sites were assigned to U59 (30% of the label) and C70 (15%) respectively. Our results suggest that psoralens may be used as photoprobes for studying conformational changes in tRNA molecules.
Analysis of secondary structures in M13mp8 (+) single‐stranded DNA by the pausing of DNA polymerase α
Authors: RECKMANN Bernd , GROSSE Frank , URBANKE Claus , FRANK Ronald , BLÖCKER Helmut , KRAUSS Gerhard
Published: March 2005

The pausing of DNA replication has been used as a tool for analyzing secondary structures in a single‐stranded DNA. M13mp8 (+) single‐stranded DNA was replicated in vitro by the DNA polymerase α from calf thymus. The positions of pausing were determined from DNA sequencing gels. All experimentally observed pausing sites could be correlated with computer‐predicted secondary structures of the M13 single‐stranded DNA. In the computer calculations of the secondary structures, long‐range base‐pairing, G · T mispairs and loop‐out of bases were allowed. By using six different primers, the pausing site pattern and the corresponding secondary structure map of a region comprising 1400 nucleotides of the M13 genome has been established. Our experiments indicate that the M13 DNA is highly structured. Most of the stable structures are clustered around the origin of replication. With fragments of the M13 DNA, we show that long‐range base‐pairing exists in the M13 single‐stranded genome and we present evidence for tertiary structure interactions. Furthermore we observe structures that form newly during the course of replication. The Escherichia coli single‐stranded DNA‐binding protein facilitates replication through the barriers.
Analysis of modification‐dependent structural alterations in the anticodon loop of Escherichia coli tRNAArg and their effects on the translation of MS2 RNA
Authors: BAUMANN Ulrich , FISCHER Wolfgang , SPRINZL Mathias
Published: March 2005

The conformation of the anticodon loop of Escherichia coli tRNAArg was investigated. It is shown that the structure of the anticodon loop is influenced by the base composition of the anticodon stem, and the natural modification of the nucleoside residue 32 in the anticodon loop. The structural effects detected by analysis of the accessibility of the anticodon loop to nuclease S1 could be correlated with the ability of different Arg‐tRNAArg species to suppress frame‐shifting during translation of MS2 RNA.
Biosynthesis, isolation and properties of NAD‐dependent formate dehydrogenase from the yeast Candida methylica
Authors: AVILOVA Tatyana V. , EGOROVA Olga A. , IOANESYAN Lidia S. , EGOROV Aleksey M.
Published: March 2005

NAD‐dependent formate dehydrogenase (EC 1.2.1.2), was isolated from the methanol‐utilizing yeast Candida methylica. Two purification techniques for the enzyme from the crude yeast extract have been developed: a two‐step procedure, involving a sequential application of DEAE‐cellulose ion‐exchange chromatography and Sephadex G‐200 gel filtration, and a single‐step procedure, preparative isoelectric focusing in a granulated gel layer. The enzyme proved to be electrophoretically homogeneous. It consisted of two identical subunits with a relative molecular mass of 46000, each containing one –SH group related to manifestation of the catalytic activity. The Michaelis constant was 1 · 10−4 M for NAD and 1.3 · 10−2 M for formate. Formate dehydrogenase was inhibited with p‐chlormercuribenzoate, iodoacetamide, dithionitrobenzoate, cyanide and azide.
Purification and tissue‐specific expression of casein kinase from the lactating guinea‐pig mammary gland
Authors: MOORE Alison , BOULTON Anthony P. , HEID Hans W. , JARASCH Ernst‐Dieter , CRAIG Roger K.
Published: March 2005

A serine‐specific casein kinase, an integral membrane protein of the lactating guinea‐pig mammary gland, has been purified from a Golgi‐enriched membrane fraction, using a combination of surcrose gradient centrifugation and chromatography on ATP‐agarose. The enzyme comprises a polypeptide of estimated Mr 74000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, compared with a monomer Mr of 50 000 as determined by sucrose gradient centrifugation in the presence of 500 mM NaCl and 0.1% Triton X‐100. Kinetic studies show that the purified enzyme exhibits kinetic constants distinctly different from the rabbit reticulocyte casein kinases I and II, whilst polyclonal antisera raised against the mammary gland enzyme did not cross‐react with soluble liver or reticulocyte protein kinase activities. Immunoblotting and immunocytochemical analyses demonstrate the mammary gland enzyme's apparently unique location in lactating mammary gland tissue. Comparative studies with polyclonal antisera raised against bovine galactosyltransferase, show that casein kinase and galactosyltransferase have a similar intracellular localisation in the lactating mammary gland as judged by immunocytochemistry at the light level, but that casein kinase was unique to mammary gland whereas galactosyltransferase could be found in other tissues. The results extended our earlier observations which suggest a Golgi location for casein kinase, and demonstrate that future studies using this enzyme may well prove advantageous for the study of intracellular mechanisms involved in the biogenesis of organelles, in this instance the Golgi apparatus.
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.
Bacteriophage CP‐T1 of Vibrio cholerae
Authors: GUIDOLIN Angelo , MANNING Paul A.
Published: March 2005

The attachment site on the cell surface of Vibrio cholerae for the bacteriophage CP‐T1 has been determined. Purified lipopolysaccharide from the Inaba and Ogawa serotypes, and of both the Classical and El Tor biotype of strains of V. cholerae show equal phage‐inactivating capacities. Lipopolysaccharide extracted from a CP‐T1‐resistant mutant has no phage‐inactivating capacity. Such mutants lack O‐antigen as demonstrated by bactericidal assays utilizing a monoclonal antibody directed against O‐antigen side chain of V. cholerae lipopolysaccharide. Radiolabelling of lipopolysaccharide with 33p and analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis also revealed the absence of O‐antigen in phage‐resistant strains. A number of V. cholerae typing phage show cross‐resistance with phage CP‐T1.
Orientation of rat liver cytochrome c oxidase subunits investigated with subunit‐specific antisera
Authors: KUHN‐NENTWIG Lucia , KADENBACH Bernhard
Published: March 2005

The orientation of rat liver cytochrome c oxidase subunits in the inner mitochondrial membrane was investigated with monospecific antisera against subunit II and nine nuclear‐coded subunits. Mitoplasts were incubated with the antisera and the amount of bound antibodies was determined either directly with fluorescein‐conjugated protein A or indirectly by back‐titration of unbound antibodies with a nitrocellulose immunoassay. All subunits were found oriented to the cytosolic side, except subunits VIb and VIIc which did not react with their corresponding antisera. Antisera against subunits I, III and Vb were not available.
The effect of mevalonate on 3‐hydroxy‐3‐methylglutaryl‐CoA reductase activity and the absolute rate of cholesterol biosynthesis in human monocyte‐derived macrophages
Authors: PATEL Dilip D. , KNIGHT Brian L.
Published: March 2005

Normal monocyte‐derived macrophages maintained in medium containing 20% whole serum exhibited a high activity of 3‐hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) reductase while expressing appreciable, but not maximal receptor‐mediated uptake and degradation of low‐density lipoprotein (LDL). Addition of extracellular mevalonate led to a concentration‐dependent fall in both processes, although a higher concentration was required to produce the same effect on LDL degradation as on HMG‐CoA reductase activity. The true rate of cholesterol synthesis by the cells was already high without mevalonate and was not further increased when the intracellular pool of mevalonate was expanded. After preincubation with lipoprotein‐deficient serum receptor‐mediated degradation of LDL was increased but HMG‐CoA reductase activity was decreased. Both were more sensitive to extracellular mevalonate and a given concentration of mevalonate now produced the same percentage fall in LDL degradation and reductase activity. The amount of cholesterol synthesized by the cells was markedly reduced during incubation with lipoprotein‐deficient serum, probably as a result of the decreased reductase activity compounded by a lack of endogenous substrate. If these were circumvented by adding mevalonate, cholesterol synthesis was restored to normal. These results suggest that the fall in HMG‐CoA reductase activity during incubation with lipoprotein‐deficient serum resulted from repression by some non‐sterol product of mevalonate metabolism. They imply that the need for this product by cells growing in whole serum leads to a particularly high HMG‐CoA reductase activity and could account for their apparent preference for endogenous synthesis of cholesterol rather than for receptor‐mediated uptake of LDL.
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.
Substrate specificity and stereospecificity of calf spleen phosphodiesterase towards deoxyribonucleosidyl 3′‐(4‐nitrophenyl phosphates) and phosphorothioates
Authors: NIEWIAROWSKI Wojciech , UZNANSKI Bogdan
Published: March 2005

Phosphodiesterase from calf spleen exhibits nucleotidyltransferase activity when incubated with either the (PR) or the (PS) diastereomer of thymidyl 3′‐(4‐nitrophenyl phosphorothioate). Thymidylyl(3′‐5′)thymidyl phosphorothioate 3′‐(4‐nitrophenyl phosphorothioate) was identified as the main product of the enzyme‐catalyzed reaction and the absolute configuration at the internucleotide phosphorus atom of the product was determined. The nucleotidyltransferase reaction is shown to proceed with retention of configuration at phosphorus, implying involvement of a double displacement mechanism with the formation of a nucleotidylated enzyme intermediate. To study the substrate specificity of spleen phosphodiesterase a series of deoxyribonucleosidyl 3′‐(4‐nitrophenyl phosphates) and phosphorothioates were synthesized, and Km and V parameters for each substrate were measured. The results obtained show virtually no specificity for substrates with different nucleosidyl moieties, while about a 20–30‐fold drop in V and a slight increase in Km values is observed for phosphorothioate analogues as compared with corresponding phosphates. The enzyme showed no significant stereoselectivity towards phosphorothioates of opposite configurations at phosphorus.
Identification of a 150‐kDa membrane component which is modulated by parathyroid hormone
Authors: WEINSHANK Richard L. , LUBEN Richard A.
Published: March 2005

Monoclonal antibodies have been produced against primary bone cells obtained from the collagenase digestion of mouse cranial bone. Antibodies were selected on the basis of their immunoglobulin class and those which were identified as IgG were further screened for their ability to inhibit cAMP accumulation in response to sub‐maximal doses of the 1–34 amino‐terminal peptide of bovine parathyroid hormone, bPTH(1–34). Nine hybridoma clones were subsequently characterized as inhibitory with respect to parathyroid hormone (PTH) responses in intact mouse cranial bone and which also identified a variety of membrane components from detergent extracts of surface‐labeled primary bone cells. Five of these antibodies immunoprecipitated a membrane component with Mr of 80000 that appeared to be a major component of the extract susceptible to surface‐labeling with 125I. All nine monoclonal antibodies were shown to bind to a suspended‐cell preparation of primary bone cells with 2–3 orders of magnitude greater binding than that of control antibodies. Using this assay, one clone, designated 3G12 IgG, was observed to exhibit desensitization effects at the binding level with a time course and dose dependency for PTH pre‐incubation that was similar to the establishment of the refractory state in other systems. In addition. the desensitization effect occurred at 37°C but not at 4°C. This antibody was shown to bind saturably to both intact mouse cranial bone and primary bone cells with an apparent affinity constant (Ka) in the range of 109 M. Inhibition of bone cAMP accumulation in response to 2.5 nM bPTH(1–34) was directly correlated to the binding of 3G12 IgG to intact mouse calvariae. A maximum inhibition of approximately 85% was observed. 3G12 IgG immunoprecipitated a single membrane component, Mr 150000, from NP‐40 detergent extracts of 125I‐labeled primary mouse bone cells. The molecular mass of this component was also 150000 daltons when run on polyacrylamide gel slabs under non‐reducing conditions. Control and PTH‐pre‐treated bone cells were surface‐labeled, detergent‐solubilized and immunoprecipitated with 3G12 IgG in order to investigate further the desensitization effect at the molecular level. Incubation of bone cells with 1 μg/ml bPTH(1–34) for 45 min at 37°C caused an increased susceptibility to surface‐labeling with 125I that was approximately three‐fold higher in specific activity than that of control cells. Under conditions of quantitative immunoprecipitation, 3G12 IgG precipitated less of the 150‐kDa membrane component from the PTH pre‐treated bone cell extract, indicating a decreased availability of this membrane component under conditions of desensitization. We propose that the 150‐kDa protein from mouse bone cells may be the PTH receptor, a component of the receptor or a membrane protein which is directly involved in the PTH responsiveness of these cells.
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.
Synthesis and expression of a human growth hormone (somatotropin) gene mutated to change cysteine‐165 to alanine
Authors: TOKUNAGA Tomoko , TANAKA Toshiki , IKEHARA Morio , OHTSUKA Eiko
Published: March 2005

We have mutated a synthetic human growth hormone (hGH) gene specifically at the codon for Cys‐165 to a codon for Ala by replacement of synthetic deoxyoligonucleotides corresponding to this site. This modification prevented the formation of a disulfide bond between Cys‐53 and Cys‐165 in the hGH molecule. This mutated protein, [Ala165]hGH was expressed at the same level as the intact hGH, 4x105 molecules per cell under the control of the tryptophan promoter in Escherichia coli, and retained similar immunological activity to intact hGH. The limited digestion pattern of the mutated protein with human plasmin suggests that the tertiary structure of [Ala165]hGH resembles to that of the intact hGH molecule. [Ala165]hGH revealed full biological activity as examined by the body weight increase of hypophysectomized rats.
A novel, highly phosphorylated protein, of the high‐mobility group type, present in a variety of proliferating and non‐proliferating mammalian cells
Authors: ØSTVOLD Anne C. , HOLTLUND Jostein , LALAND Søren G.
Published: March 2005

The present work describes a perchloric‐acid‐soluble high‐mobility‐group (HMG)‐like protein present in HeLa and Ehrlich ascites cells, rat and calf liver. The protein is designated P1 and has, depending on the source, a molecular mass 48–53 kDa and an amino acid composition which, like the HMG proteins, is characterized by a high content of acidic and basic residues and of proline. The protein contains about 10 mol serine 100 mol amino acid residues, is highly phosphorylated and has, in contrast to the known HMG proteins, an acidic isoelectric point of 5.0. An estimate suggests that protein P1 in HeLa interphase cells contains 25–30 residues of phosphate. Like HMG 1 and 2 it is distributed between the nucleus and the cytoplasm. In HeLa metaphase cells P1 is further modified, resulting in an increase in apparent molecular mass from 53 kDa to 56 kDa.