bluuebunny/crossref_metadata_2025_split huggingface dataset:
NMR studies of mobility within protein structure
Authors: WILLIAMS Robert J. P. 
Published: March 2005

NMR studies of dynamics within structure have revealed that a quite new approach to protein structure and its relation to function is necessary. This approach requires the consideration in detail of the following:

Local movements of groups and small segments to allow fast recognition and fitting. The motion concerns on/off rates as well as binding. The observations affect surface/surface recognition, e.g. of antigen/antibody as well as of substrate and protein.
Somewhat larger interdomain or N‐ and C‐terminal segments which allow rearrangement. Cases in point are the movement of segments in blood‐clotting proteins or in histones.
Relative motion of helices in hinges. These actions are likely in such enzymes as kinases and P‐450 cytochromes.
Relative motion of helices within domains (relative to other helices or sheets) in mechanical devices (triggers) e.g. in calmodulin.
General motion in random proteins. Examples extend from rubber‐like proteins (entropy sensors), some glycoproteins, to proteins carrying peptide hormones to be generated only after hydrolysis.
Order → disorder transitions locally as in osteocalcin and metallothionine.
Swinging arm motions associated with special sequences such as (Ala‐Pro)n.
Of great interest is the power of NMR to look at proteins which are relatively large, up to 50 k Da proteins, and to isolate certain zones of interest. This needs careful temperature dependent studies and analysis of separated domains [72] as well as the use of a great variety of pulse sequences [15] and of nuclei other than protons.
In this article I have illustrated the different possibilities using work in my own group. This is done to lessen the burden of extensive review. I fully realise that the range of examples is now large. I would stress though that the production of the necessary technology was the endeavour of several of us within the Oxford Enzyme Group from 1970 to 1985, i.e. from 270–600 MHz Fourier‐transform NMR spectroscopy.
While all of these features have been demonstrated by NMR methods there are parallel developments both using X‐ray diffraction methods and theoretical approaches. All these procedures are changing the view of protein structure to one which incorporates dynamics all the way from conventional vibronic/rotational coupling to the disordered motions characteristic of random polymers. It is the understanding of dynamics that leads to an appreciation of function.
The core region of Proteus mirabilis R110/1959 lipopolysaccharide
Authors: RADZIEJEWSKA‐LEBRECHT Joanna , MAYER Hubert 
Published: March 2005

The complete core structure present in the lipopolysaccharide of the R mutant R110/1959 from Proteus mirabilis (Proteus type II core) was investigated using methylation analysis and a number of degradation methods such as Smith degradation and β‐elimination. These studies combined with earlier work on a Rc‐type mutant of P. mirabilis O28 (R4/O28) which shares the same inner core region, allowed formulation of the complete core structure of the Proteus type II core as shown in Scheme 1.

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[Structural proposal of the core region of P. mirabilis R110/1959 (Proteus type II core). Incomplete substitutions are indicated by broken arrows.]A characteristic feature of the Proteus core of type II is the presence of two units of D‐galacturonic acid (DGalA); one in terminal, the other one in a chain‐linked position. In addition, the presence of the two isomers of glycero‐D‐manno‐heptose (LDHep and DDHep) and the lack of galactose are conspicuous. DDHep occupies a terminal position in the external core region, whereas the three units of LDHep in addition to dOclA form, as in other enterobacterial core types, the internal core region.The taxonomic significance of the presence of DGalA in the Proteus type II core, but also in all R cores of other Proteeae investigated so far, will be discussed.
The Na+ ‐motive terminal oxidase activity in an alkalo‐ and halo‐tolerant Bacillus
Authors: SEMEYKINA Anna L. , SKULACHEV Vladimir P. , VERKHOVSKAYA Marina L. , BULYGINA Evgenia S. , CHUMAKOV Konstantin M. 
Published: March 2005

An alkalo‐ and halo‐tolerant aerobic microorganism has been isolated which, according to microbiological analysis data and the ribosomal 5S RNA sequence, is a Bacillus similar, but not identical, to B. licheniformis and B. subtilis. The microorganism, called Bacillus FTU, proved to be resistant to the protonophorous uncoupler carbonylcyanide m‐chlorophenylhydrazone (CCCP). The fast growth of Bacillus FTU in the presence of CCCP was shown to require a high Na+ concentration in the medium. A procedure was developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only when an exogenous respiratory substrate was added. The exhausted cells were found to oxidize ascorbate in the presence of N,N,N′,N′‐tetramethyl‐p‐phenylenediamine (TMPD) in a cyanide‐sensitive fashion. The ascorbate oxidation was coupled to the uphill Na+ extrusion which was stimulated by CCCP and a penetrating weak base, diethylamine, as well as by valinomycin with or without diethylamine.Operation of the Bacillus FTU terminal oxidase resulted in the generation of a ΔΨ which, in the Na+ medium, was slightly decreased by CCCP and strongly decreased by CCCP + diethylamine. In the K+ medium. CCCP discharged ΔΨ even without diethylamine. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na+ medium and sensitive to CCCP in the K+ medium as if Na+ ‐ and H+ ‐coupled oxidative phosphorylations were operative in the Na+ and K+ media, respectively.Inside‐out subcellular vesicles of Bacillus FTU were found to be competent in the Na+ uptake supported by oxidation of ascorbate + TMPD or diaminodurene. CCCP or valinomycin + K+ increased the Na+ uptake very strongly. The process was completely inhibited by cyanide or monensin, the former, but not the latter. being inhibitory for respiration.The data obtained indicate that in Bacillus FTU there is not only H+ ‐motive but also Na+ ‐motive terminal oxidase activity.
Molecular forms of acetylcholinesterase in two sublines of human erythroleukemia K562 cells
Authors: TOUTANT Jean‐Pierre , RICHARDS Michael K. , KRALL Jennifer A. , ROSENBERRY Terrone L. 
Published: March 2005

Acetylcholinesterase (AChE) in K562 cells exists in two molecular forms. The major form, an amphiphilic dimer (G2a) which sediments at 5.3 S, and the minor form, an amphiphilic monomer (G1a) which sediments at 3.5 S. Extraction in the presence of the sulfhydryl alkylating agent N‐ethylmaleimide was required to preserve the G2a form.In Triton X‐100 extracts of the subline K562‐243, phosphatidylinositol‐specific phospholipase C (PtdIns‐PLC) from Bacillus thuringiensis converted most of the G2a AChE into a hydrophilic dimer (G2h), indicating that the G2a form possessed a hydrophobic glycoinositol phospholipid that mediated its attachment to the membrane. Treatment of intact K562‐243 cells with PtdIns‐PLC released approximately 60% of the total AChE activity and provided an estimate of the externally exposed AChE. The direct conversion from an amphiphilic to a hydrophilic dimeric form by PtdIns‐PLC was not obtained in extracts or intact cells of the subline K562‐48. Instead, pretreatment with alkaline hydroxylamine was necessary to render the amphiphilic G2 form of this subline susceptible to digestion by the phospholipase. In this respect, the amphiphilic dimer of K562‐48 AChE resembles the G2a form of human erythrocyte AChE, which is resistant to PtdIns‐PLC because of the direct palmitoylation of an inositol hydroxyl group in the anchor [Roberts et al. (1988) J. Biol. Chem. 263, 18766–18775]. Release of this acyl chain by hydroxylamine renders the enzyme susceptible to PtdIns‐PLC [Toutant et al. (1989) Eur. J. Biochem. 180, 503–508].In both K562 sublines, sialidase decreased the migration of the G2a form but not of the G1a form of AChE. G1a forms thus appear to represent an intracellular pool of newly synthesized molecules residing in a compartment proximal to the trans‐Golgi apparatus. The sialidase‐resistant G1a molecules were also resistant to PtdIns‐PLC digestion; possible explanations for this resistance are presented.
Affinity labelling of endothelin receptor and characterization of solubilized endothelin–endothelin‐receptor complex
Authors: MIYAZAKI Hitoshi , KONDOH Motohiro , WATANABE Hirotoshi , MASUDA Yasushi , MURAKAMI Kazuo , TAKAHASHI Masami , YANAGISAWA Masashi , KIMURA Sadao , GOTO Katsutoshi , MASAKI Tomoh 
Published: March 2005

Chick cardiac membranes were affinity labelled by cross‐linking to membrane‐bound 125I‐endothelin‐1 with disuccinimidyl tartarate. SDS/PAGE and autoradiographic analysis of the 125I‐endothelin‐1‐labelled material in the presence or absence of 2‐mercaptoethanol revealed one major labelled band, corresponding to a molecular mass of 53 kDa, whose appearance was dose‐dependently inhibited by the addition of unlabelled endothelin‐1 (1–100 nM). Subtracting the molecular mass of 125I‐endothelin‐1 and disuccinimidyl tartarate, the binding protein appeared to have a molecular mass of 50 kDa.To investigate further the molecular properties of endothelin receptor, the 125I‐endothelin‐1–endothelin‐receptor complex was solubilized from chick cardiac membranes using the detergent digitonin. Sucrose gradient sedimentation of the solubilized complex indicated a sedimentation coefficient of 13 S, whereas the complex of (+)‐[3H]PN200–110, a dihydropyridine derivative, and dihydropyridine‐sensitive Ca2+ channels sedimented at 22 S. A monoclonal antibody raised against dihydropyridine‐sensitive Ca2+ channels from the chick brain did not immunoprecipitate the 125I‐endothelin‐1–endothelin‐receptor complex. These data suggest that endothelin receptor is clearly distinct from dihydropyridine‐sensitive Ca2+ channels and endothelin has its own specific 50‐kDa receptor.
Soluble hydrogenase of Anabaena cylindrica
Authors: EWART Gary D. , REED Ken C. , SMITH Geoffrey D. 
Published: March 2005

A gene potentially encoding a subunit of the soluble hydrogenase of Anabaena cylindrica was isolated from a genomic library by screening with a set of redundant oligonucleotides, the sequence of which was deduced from the amino acid sequence of the purified hydrogenase subunit that catalyses tritium exchange. The nucleotide sequence of the potential gene was determined from two overlapping DNA fragments spanning 7237 bp of the A. cylindrica genome. The region sequenced contained an open reading frame encoding a protein of 383 amino acids with a predicted molecular mass of 41 108 Da. The NH2‐terminal amino acid sequence of the purified enzyme, determined by Edman degradation, corresponds exactly with that deduced from the nucleic acid sequence.No significant amino acid or nucleotide similarity is evident between this gene and the periplasmic hydrogenases from three species of Desulfovibrio (D. vulgaris, D. baculatus and D. gigas), or with the membrane‐bound ‘uptake’ hydrogenases of Bradyrhizobium japonicum and Rhodobacter capsulatus. This suggests that the soluble enzyme from cyanobacteria represents a discrete class of hydrogenase.The gene encoding the second subunit (m= 50 kDa) of the soluble hydrogenase, which is required for the catalysis of hydrogen production from dithionite‐reduced methyl viologen [Ewart, G. D. & Smith, G. D. (1989) Arch. Biochem. Biophys. 268, 327–337], apparently comprises a separate transcription unit since it appears not to be located adjacent to that for the 42‐kDa subunit.
Antigenic sites on cytochrome c2 from Rhodospirillum rubrum
Authors: SAAD Bashar , BOSSHARD Hans Rudolf 
Published: March 2005

The antigenic determinants for three monoclonal antibodies against cytochrome c2 from Rhodospirillum rubrum were partially characterized by differential chemical modification of free and antibody‐bound cytochrome c2 and by cross‐reactivity analysis with different antigens. Circular dichroism spectroscopy was used to probe the effect of antibody binding on the conformation of cytochrome c2. The binding of two antibodies was strongly dependent on the native folding of the antigen. The first antibody bound to a determinant around the exposed heme edge on the ‘front side’ of the molecule which is not antigenic in mitochondrial cytochrome c2. Binding of this antibody to cytochrome c increased the induced CD of the ferric heme in a manner similar to that observed previously when mitochondrial cytochrome‐c oxidase bound to the front side of cytochrome c. This observation points to a subtle conformational adaptation of the antigen induced by the antibody. The determinant for the second antibody, which also affected the heme CD spectrum of the antigen, was on a polypeptide loop where cytochrome c2 differs from mitochondrial cytochrome c by an eight‐residue insertion. The third antibody, which did not induce a change in CD, bound to a sequential determinant near the amino end of cytochrome c2. Only this antibody cross‐reacted with isolated cytochrome‐c‐derived peptides and with apo‐cytochrome c2.A preliminary analysis of the polyclonal immune response of five rats against cytochrome c2 indicates that, unlike in eukaryotic cytochrome c, antigenic determinants are distributed over the whole polypeptide chain of the prokaryotic immunogen.
The ribosomal domain of the bacterial release factors
Authors: TATE Warren P. , KASTNER Berthold , EDGAR Christina D. , McCAUGHAN Kim K. , TIMMS Kirsten M. , TROTMAN Clive N. A. , STOFFLER‐MEILICKE Marina , STOFFLER Georg , NAG Bishwajit , TRAUT Robert R. 
Published: March 2005

Polyclonal antibodies (pAb 1–73 and pAb 26–120) have been raised against both an N‐terminal fragment of Escherichia coli ribosomal protein L7/L12 (amino acids 1–73), and a fragment lacking part of the N‐terminal domain (amino acids 26–120).
Only pAb 26–120 inhibited release‐factor‐dependent in vitro termination functions on the ribosome. This antibody binds over the length of the stalk of the large subunit of the ribosome as determined by immune electron microscopy, thereby not distinguishing between the C‐terminal domains of the two L7/L12 dimers, those in the stalk or those in the body of the subunit.
A monoclonal antibody against an epitope of the C‐terminal two thirds of the protein (mAb 74–120), which binds both to the distal tip of the stalk as well as to a region at its base, reflecting the positions of the two dimers is strongly inhibitory of release factor function.
A monoclonal antibody against an epitope of the N‐terminal fragment of L7/L12 (mAb 1–73), previously shown to remove the dimer of L7/L12 in the 50S subunit stalk but still bind to the body of the particle, partially inhibited release‐factor‐mediated events.
The mAb 74–120 inhibited in vitro termination with a similar profile when the stalk dimer of L7/L12 was removed with mAb 1–73, indicating that the body L7/L12 dimer, and in particular its C‐terminal domains, are important for release factor/ribosome interaction.
The two release factors have subtle differences in their binding domains with respect to L7/L12.
Mapping of glucose and glucose‐6‐phosphate binding sites on bovine brain hexokinase
Authors: JARORI Gotam K. , IYER Sujata B. , KASTURI Sitapati R. , KENKARE Umakant W. 
Published: March 2005

Inhibition of bovine brain hexokinase by its product, glucose 6‐phosphate, is considered to be a major regulatory step in controlling the glycolytic flux in the brain. Investigations on the molecular basis of this regulation, i.e. allosteric or product inhibition, have led to various proposals. Here, we attempt to resolve this issue by ascertaining the location of the binding sites for glucose and glucose 6‐phosphate on the enzyme with respect to a divalent‐cation‐binding site characterized previously[Jarori, G. K., Kasturi, S. R. & Kenkare, U. W.(1981) Arch. Biochem. Biophys. 211, 258–268]. The paramagnetic effect of enzyme‐bound Mn(II) on the spinlattice relaxation rates (T−11) of ligand nuclei (1H and31P)in E. Mn(II). Gle and E. Mn(II). Glc66 complexes have been measured. The paramagnetic effect of Mn(II) on the proton relaxation rates of C1‐Hα, C1‐Hβ and C2‐Hβ of glucose in the E · Mn(II) · Glc complex was measured at 270 MHz and 500 MHz. The temperature dependence of these rates was also studied in the range of 5–30°C at 500 MHz. The ligand nuclear relaxation rates in E · Mn(II) · Glc are field‐dependent and the Arrhenius plot yields an activation energy (ΔE) of 16.7–20.9 kJ/mol. Similar measurements have also been carried out on C1‐Hα, C1‐Hβ and C6‐31P at 270 MHz (1H and 202.5 MHz (31P) for the E · Mn(II) · Glc6P complex. The temperature dependence of 31P relaxation rates in this complex was measured in the range 5–30°C, which yielded Δ= 9.2 kJ/mol. The electron‐nuclear dipolar correlation time (τc), determined from the field‐dependent measurements of proton relaxation rates in the E · Mn(II)· Gle complex, is 0.22–1.27 ns. The distances determined between Mn(II) and C1‐H of glucose and glucose 6‐phosphate are ∼ 1.1 nm and ∼ 0.8 nm, respectively. These data, considered together with our recent results [Mehta, A., Jarori, G. K. & Kenkare. U. M.(1988) J. Biol. Chem. 263, 15492–15498], suggest that glucose and glucose 6‐phosphate may bind to very nearly the same region of the enzyme.The structure of the binary Glc6P· Mn(II) complex has also been determined. The phosphoryl group of the sugar phosphate forms a first co‐ordination complex with the cation. However, on the enzyme, the phosphoryl group is located at a distance of ∼ 0.5–0.6 nm from the cation.
Identification of a cholesterol‐regulated 180‐kDA microsomal protein in rat hepatocytes
Authors: HARO Diego , MARRERO Pedro F. , AYTÉ Jose , HEGARDT Fausto G. 
Published: March 2005

The immunoprecipitation by antibodies to 3‐hydroxy‐3‐methylglutry‐coenzyme A (HMG‐CoA) reductase of extracts of [35S]methionine‐pulse‐labelled isolated hepatocytes, followed by electrophoresis and fluorography, showed the presence not only of 97‐kDa HMG‐CoA reductase, but also of another protein of 180 kDa. Boiling the immunoprecipitates both in the presence and in the absence of 2‐mercaptoethanol, followed by SDS/polyacrylamide gel electrophoresis both in the presence and in the absence of 8 M urea, was not found to change the ratio of 180‐kDa/97‐kDa proteins. These facts suggest that the 180‐kDa protein is not an aggregated form of HMG‐CoA reductase. A different batch of antibodies obtained from a newly purified HMG‐CoA reductase fully titrated the reductase activity, but did not immunoprecipitate the 180‐kDa protein, showing that there is no cross‐reactivity between these proteins. The 180‐kDa polypeptide is a glycoprotein of N‐linked high‐mannose oligosaccharide chains, which is not processed on the Golgi system. The apparent molecular mass of the carbohydrate is 16 kDa.The incubation of rat hepatocytes with sterols produces, on the one hand, a decrease in the rate of synthesis, and on the other hand, an acceleration in the turnover rate of the 180‐kDa protein. In addition, mevalonate is known to decrease its rate of synthesis. The carbohydrate‐free 164‐kDa protein was found to degrade only a tenth as fast as the glycoprotein and, furthermore, the degradation was no longer accelerated by sterols. These results support the notion that the 180‐kDa protein is not a modified form of 97‐kDa reductase, but probably a different protein related to cholesterol metabolism. and also that the N‐linked, high‐mannose chains. Which are bound to the glycoprotein, are required for rapid and controlled degradation of the protein.
Two subcellular locations for peripheral‐type benzodiazepine acceptors in rat liver
Authors: O'BEIRNE Gerard B. , WOODS Margaret J. , WILLIAMS D. Clive 
Published: March 2005

Subcellular fractionation of rat liver by differential centrifugation showed the mitochondrial fractions to have the greatest enrichment of ‘peripheral‐type’ benzodiazepine acceptor. Two peaks of acceptor sites were found on isopycnic density‐gradient centrifugation, one peak (ϱ= 1.19 g/ml) corresponding to the peak of mitochondria as judged by marker enzyme distribution and by transmission electron microscopy, and the other peak (ϱ= 1.17 g/ml) which is not mitochondrial as judged by the lack of mitochondrial enzyme markers. Whereas the density of the mitochondrial acceptor was sensitive to sonication and was shown to have an outer‐membrane location, the density of the non‐mitochondrial acceptor was insensitive to sonication.The non‐mitochondrial acceptor was shown not to be associated with Golgi, lysosomes, rough endoplasmic reticular microsomes, peroxisomes, or some type of plasma membranes, as jugged by differences in the distribution of marker activities. No enrichment of benzodiazepine acceptor was found in the purified nuclear fraction.Both acceptors were shown to be peripheral‐type high‐affinity acceptors as judged by ligand specificities and by photoaffinity labelling.
Purification and properties of two oxidoreductases catalyzing the enantioselective reduction of diacetyl and other diketones from baker's yeast
Authors: HEIDLAS Jürgen , TRESSL Roland 
Published: March 2005

The NADPH‐linked diacetyl reductase system from the cytosolic fraction of Saccharomyces cerevisiae has been resolved into two oxidoreductases catalyzing irreversibly the enationselective reduction of diacetyl (2,3‐butanedione) to (S)‐ and (R)‐acetoin (3‐hydroxy‐2‐butanone) [so‐called (S)‐ and (R)‐diacetyl reductases] (EC 1.1.1.5) which have been isolated to apparent electrophoretical purity.The clean‐up procedures comprising streptomycin sulfate treatment, Sephadex G‐25 filtration, DEAE‐Sepharose CL‐6B columm chromatography, affinity chromatography on Matrex Gel Red A and Superose 6 prep grade filtration led to 120‐fold and 368‐fold purifications, respectively.The relative molecular mass of the (R)‐diacetyl reductase, estimated by means of HPLC filtration on Zorbax GF 250 and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was 36000, The (R)‐enzyme was most active at pH 6.4 and accepted in addition to diacetyl C5‐, C6‐2,3‐diketones, 1,2‐cyclohexanedione, 2‐oxo aldehydes and short‐chain 2‐ and 3‐oxo esters as substrates. The enzyme was characterized by high enantioselectivity and regiospecificity. The Km values for diacetyl and 2,3‐pentanedione were determined as 2.0 mM.The Mr of the (S)‐diacetyl reductase was determined as 75000 by means of HPLC filtration on Zorbax GF 250. The enzyme decomposed into subunits of Mr 48000 and 24000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The optimum pH was 6.9. the purified (S)‐enzyme reduced stereospecifically a broad spectrum of substrates, comprising 2,3‐, 2,4‐ and 2.5‐diketones, 2‐oxo aldehydes, 1,2‐cyclohexanedione and methyl ketones as well as 3‐, 4‐ and 5‐oxo esters. The 2.3‐ and 2,4‐diketones are transformed to the corresponding (S)‐2‐hydroxy ketones; 2,5‐hexanedione, however, was reduced to (S,S)‐2,5‐hexanediol. The Km values for diacetyl and 2,3‐pentanedione were estimated as 2.3 and 1.5 mM, respectively. Further characterization of the (S)‐diacetyl reductase revealed that it is identical with the so‐called ‘(S)‐enzyme’, involved in the enantioselective reduction of 3‐, 4‐ and 5‐oxo esters in baker's yeast.