bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibodies to dog heart sarcoplasmic reticulum
Authors: LEVITSKY Dmitri O. , SYRBU Sergei I. , CHEREPAKHIN Vladimir V. , ROKHLIN Oskar V.
Published: March 2005

Purified sarcoplasmic reticulum (SR) vesicles from dog heart were used as an antigen to produce monclonal antibodies (mAbs) to the Ca2+‐ATPase. Nine of twelve clones of hybridoma cells produce mAbs which cross‐react with seven SR preparation isolated from cardiac and skeletal muscles of various species. Three mAbs of IgM type interact with the 45‐kDa tryptic fragment of rabbit skeletal muscle Ca2+‐ATPase and markedly inhibit Ca2+ uptake (by 95%) and ATPase activity (by 80%) and decrease (by 30–50%) the steady‐state level of the Ca2+‐ATPase phosphoenzyme. The ATPase activity could be completely blocked by one of these mAbs if the incubation medium was supplemented with 2 μM orthovanadate. On the other hand, when SR vesicles were treated with increasing concentrations of a nonioinic detergent C12E8, the inhibiting effect of mAb 4B4 is diminished. It is concluded that the mAbs inhibit the Ca2+‐ATPase only if the enzyme exists in an oligomeric form. The inhibition of the SR activities is due to an effect of the mAbs on the whole active center of the enzyme, rather than on a single partial reaction.
Characteristics of the redox‐linked proton ejection in beef‐heart cytochrome c oxidase reconstituted in liposomes
Authors: PAPA Sergio , CAPITANIO Nazzareno , De NITTO Emanuele
Published: March 2005

In this paper a study is presented of the characteristics of redox‐linked proton ejection exhibited by isolated beef‐heart cytochrome c oxidase incorporated in asolectin vesicles. The enzyme was 90% oriented ‘right‐side out’ as in the mitochondrial membrane. The effects on the H+/e− stoichiometry of the modalities of activation of electron flow, the pH of the medium and its ionic composition were investigated. The results obtained show that, whilst ferrocytochrome c pulses of the aerobic oxidase vesicles at neutral pH and in the presence of saturating concentrations of valinomycin and K+ to ensure charge compensation produced H+/e− ratios around 1 (as has been shown previously), oxygen pulses of reduced anaerobic vesicles supplemented with cytochrome c, gave H+/e− ratios around 0.3.

The H+/e− ratios exhibited, with both reductant and oxidant pulses, a marked pH dependence. Maximum values were observed at pH 7.0–7.7, which decreased to negligible values at acidic pH with apparent pKa of 6.7–6.3.

Mg2+ and Ca2+ caused a marked depression of the H+/e− ratio, which in the presence of these cations and after a few ferrocytochrome pulses, became negligible.

Analysis of cytochrome c oxidation showed that the modalities of activation of electron flow and divalent cations exerted profound effects on the kinetics of cytochrome c oxidation by oxidase vesicles. The observations presented seem to provide interesting clues for the nature and mechanism of redox‐linked proton ejection in reconstituted cytochrome c oxidase.
Identical mRNA for preproglucagon in pancreas and gut
Authors: NOVAK Ulrike , WILKS Andrew , BUELL Gary , McEWEN Stephen
Published: March 2005

The structure of the human glucagon gene and the sequence around the putative promoter region and transcriptional start site are shown. Cloning and sequencing of the cDNAs, coding for preproglucagon from human pancreas and colon, as well as primer extension analysis of RNA from pancreas, ileum and colon point to an identical transcriptional start site in all tissues, about 3 × 103 base pairs upstream from the first coding exon as defined by Bell et al.[1].
Equilibrium constants of several reactions involved in the fermentation of glutamate
Authors: BUCKEL Wolfgang , MILLER Stanley L.
Published: March 2005

The equilibrium constants of the reactions catalysed by (S)‐citramalate lyase and (R)‐2‐hydroxyglutarate dehydrogenase were determined using the purified enzymes from Clostridium tetanomorphum and Acidaminococcus fermentans, respectively. The former constant had to be determined at high ionic strength (I). Therefore it was corrected to I= 0.1 M by applying single‐ion activity coefficients estimated from literature data. The result (Kapp= 4.31 ± 0.07 M−1; direction of citramalate formation) agreed very well with the constant of the (2R,3S)‐2,3‐dimethylmalate lyase equilibrium when all optical isomers were taken into account. From these and other data values for the free energies of formation (ΔG°f) of (2S,3S)‐3‐methylaspartate, mesaconate and (S)‐citramalate were calculated. The constant of the (R)‐2‐hydroxyglutarate dehydrogenase equilibrium [Kapp= (1.47 ± 0.12)10−12 M, direction of 2‐oxoglutarate formation, I= 0.1 M] was shown to lie between those for malate and lactate dehydrogenases as expected
Light‐dependent accumulation and localization of photosystem II proteins in maize
Authors: SUTTON Ann , SIEBURTH Leslie E. , BENNETT John
Published: March 2005

We have raised antibodies against several major components of photosystem II. These antisera, which are directed against the apoproteins of two chlorophyll‐binding proteins (CPa‐1 and CPa‐2), the apoprotein of lightharvesting complex II and the 33‐kDa extrinsic protein of the oxygen‐evolving complex, were used to examine the light regulation of photosystem II assembly in maize. The principal findings of this study are as follows.

1. The 33‐kDa protein is present in dark‐grown maize and the content increases 5–10‐fold upon illumination.

2. The level of the protein is mediated at least in part by phytochrome and is independent of the accumulation of chlorophyll.

3. In contrast, none of the three chlorophyll‐binding proteins examined was detectable in leaves of maize grown in darkness or under other light regimes where chlorophyll does not accumulate.

4. Even in the absence of photosystem II assembly, the 33‐kDa protein is properly transported across the thylakoid into the lumen. However, the protein does not attach in the normal way to the inner surface of the membrane under these conditions.
Subunit 4 of ATP synthase (F0F1) from yeast mitochondria
Authors: VELOURS Jean , ARSELIN de CHATEAUBODEAU Geneviève , GALANTE Monique , GUÉRIN Bernard
Published: March 2005

One subunit of the membrane portion of yeast ATP synthase was purified. Structural data are reported. This subunit (subunit 4) is the fourth polypeptide of the complex when classifying subunits in order of decreasing molecular mass. Its apparent relative molecular mass is about 25000. The polypeptide was extracted from the complex with a mixture of chloroform/methanol (1/1) and 0.5 M pyridinium acetate pH 6.0. Purification was performed with a combination of gel permeation chromatography on Sephadex G‐75 and high‐performance gel permeation chromatography with aqueous solvents containing 5% sodium dodecyl sulfate. The amino acid composition is reported here. The following sequence of the NH2‐terminal ten residues was determined: Met‐Ser‐Ser‐Thr‐Pro‐Glu‐Lys‐Gln‐Thr‐Asp.
Glucagon antagonists
Authors: ANDREU David , MERRIFIELD Robert B.
Published: March 2005

In an effort to find analogs of glucagon that would bind to the glucagon receptor of the rat liver membrane but would not activate membrane‐bound adenyl cyclase, several hybrid molecules were synthesized which contained sequences from both glucagon and secretin. [Asp3, Glu9]Glucagon and [Asp3, Glu9, Arg12]glucagon were inactive in the adenyl cyclase assay even at high concentrations but retained some binding affinity for the receptor. They were able to displace 125I‐glucagon completely from its receptor and could completely inhibit the activation of adenyl cyclase by natural or synthetic glucagon. The inhibition index [I/A]50 was approximately 110 for both analogs. [Asp3]Glucagon, [Glu3]glucagon and [Asp3, Lys17,18, Glu21]glucagon were weak partial agonists, while [Asp3, Glu21]glucagon was inactive and a poor inhibitor. The peptides were synthesized by solid‐phase methods and purified to homogeneity by reverse‐phase high‐performance liquid chromatography on C18 silica columns. These are the first fully synthetic competitive glucagon antagonists to be reported.
Nitrogen catabolite regulation of proline permease in Saccharomyces cerevisiae
Authors: JAUNIAUX Jean‐Claude , VANDENBOL Micheline , VISSERS Stephan , BROMAN Kathleen , GRENSON Marcelle
Published: March 2005

The proline permease gene PUT4 has been cloned. Nitrogen‐source regulation (‘ammonia sensitivity’) of this and at least two other amino‐acid permeases is believed to occur at two distinct levels, i.e. permease synthesis and permease activity. Therefore, PUT4 transcription/messenger stability was examined in the ammonia‐and proline‐grown wild type as well as in mutant strains supposedly affected at only one or at both of these levels. We report transcript‐level repression of proline permease synthesis in ammonia‐grown cells. Repression is lifted at this level in gdhCR, glnlts and gdhA mutants which exhibit pleiotropically derepressed permease and catabolic enzyme activities.

On the other hand, the npil and npi2 mutations, formerly called mut2 and mut4, relieve an inactivation process which seems only to affect permeases. These mutations do not affect the detected PUT4 RNA level. The only known positive factor in proline permease regulation, the nitrogen permease reactivator protein Npr1, is believed to counteract the inactivation process on derepressing media. This protein appears to have an additional, indirect effect on PUT4 transcription/messenger stability: it would actually mediate repression via its activating effect on ammonia uptake.
Isolation of the NPRI gene responsible for the reactivatiion of ammonia‐sensitive amino‐acid permeases in Saccharomyces cerevisiae
Authors: VANDENBOL Micheline , JAUNIAUX Jean‐Claude , VISSERS Stephan , GRENSON Marcelle
Published: March 2005

The NPR1 gene codes for a protein, called the nitrogen permease reactivator protein or Npr1, which appears to promote the activity of several permeases for nitrogenous substances under conditions of nitrogen catabolite derepression, but fails to do so in the presence of ammonium ions. This gene has been cloned. Its transcription seems unaffected by growth on ammonia, so any ammonia regulation of Npr1 function most likely occurs at another level. In order to elucidate further the mechanism of permease inactivation, which requires an intact NPI1 gene product (NPI1 for nitrogen permease inactivator gene, formerly termed MUT2) and the role of Npr1 in counteracting this process, we have studied the effects of NPR1 and NPI1 gene dosage on general amino‐acid permease activity. On nitrogen‐derepressing media, NPR1 gene dose can be increased from 1 copy in a diploid to 16 plasmid‐borne copies in a haploid strain without altering general amino‐acid permease activity. On minimal ammonia medium, the plasmid‐bearing haploid cells exhibit low but increased general amino‐acid permease activity with respect to non‐transformed cells. The adverse effect of the NPI1 gene product on general amino‐acid permease activity is reduced when NPI1 gene dose is decreased to 1 gene copy in a diploid strain, regardless of the nitrogen source. We hypothesize that this product inactivates the permease by stoichiometric binding and that the Npr1 protein or a product of its catalytic action opposes this binding under conditions of nitrogen derepression.
Studies on the effect of serine protease inhibitors on activated contact factors
Authors: TANS Guido , JANSSEN‐CLAESSEN Truus , ROSING Jan , GRIFFIN John H.
Published: March 2005

Amidolytic assays have been developed to determine factor XIIa, factor XIa and plasma kallikrein in mixtures containing variable amounts of each enzyme. The commercially available chromogenic p‐nitroanilide substrates Pro‐Phe‐Arg‐NH‐Np (S2302 or chromozym PK), Glp‐Pro‐Arg‐NH‐Np (S2366), Ile‐Glu‐(piperidyl)‐Gly‐Arg‐NH‐Np (S2337), and Ile‐Glu‐Gly‐Arg‐NH‐Np (S2222) were tested for their suitability as substrates in these assays. The kinetic parameters for the conversion of S2302, S2222, S2337 and S2366 by β factor XIIa, factor XIa and plasma kallikrein indicate that each active enzyme exhibits considerable activity towards a number of these substrates. This precludes direct quantification of the individual enzymes when large amounts of other activated contact factors are present. Several serine protease inhibitors have been tested for their ability to inhibit those contact factors selectively that may interfere with the factor tested for. Soybean trypsin inhibitor very efficiently inhibited kallikrein, inhibited factor XIa at moderate concentrations, but did not affect the amidolytic activity of factor XIIa. Therefore, this inhibitor can be used to abolish a kallikrein and factor XIa contribution in a factor XIIa assay. We also report the rate constants of inhibition of contact activation factors by three different chloromethyl ketones. D‐Phe‐Pro‐Arg‐CH2Cl was moderately active against contact factors (k= 2.2 × 103 M−1 s−1 at pH 8.3) but showed no differences in specifity. D‐Phe‐Arg‐CH2Cl was a very efficient inhibitor of plasma kallikrein (k= 1.2 × 105 M−1 s−1 at pH 8.3) whereas it slowly inhibited factor XIIa (k= 1.4 × 103 M−1 s−1) and factor XIa (k= 0.11 × 103 M−1 s−1). Also Dns‐Glu‐Gly‐Arg‐CH2Cl was more reactive towards kallikrein (k= 1.6 × 104 M−1 s−1) than towards factor XIIa (k= 4.6 × 102 M−1 s−1) and factor XIa (k= 0.6 × 102 M−1 s−1). Since Phe‐Phe‐Arg‐CH2Cl is highly specific for plasma kallikrien it can be used in a factor XIa assay slectively to inhibit kallikrein. Based on the catalytic efficiencies of chromogenic substrate conversion and the inhibition characteristics of serine protease inhibitors and chloromethly ketones we were able to develop quantitative assays for factor XIIa, factor XIa and kallikrein in mixtures of contact activation factors.
The uncoupling protein from brown‐adipose‐tissue mitochondria
Authors: FERNÁNDEZ Margarita , NICHOLLS David G. , RIAL Eduardo
Published: March 2005

Mitoplasts prepared from brown adipose tissue mitochondria were treated with chymotrypsin and the fragments derived from the 32‐kDa uncoupling protein identified by immunoblotting. Extensive proteolysis of the uncoupling protein occurred, the polypeptide pattern being affected by binding of the inhibitory nucleotide GDP. Chymotrypsin modifies the nucleotide binding site, lowering its affinity from 1.7 μM to 21 μM but without decreasing its binding capacity. Nucleotide bound to the modified site can still inhibit the permeation of H+ and Cl− through the protein. The ion conducting pathway itself is also sensitive to chymotrypsin, Cl− and H+ transport being partially inhibited in parallel. The ability of fatty acids to increase the H+ permeability of the protein is also inhibited in parallel with the basal H+ permeability. The results confirm that the transport of H+ and Cl−, and the fatty acid regulation of H+ permeation all share a common structural element within the 32‐kDa protein.
The titre of juvenile hormone during the pupal and adult stages of the life cycle of Drosophila melanogaster
Authors: BOWNES Mary , REMBOLD Heinz
Published: March 2005

Using combined gas chromatography/selected‐ion mass spectroscopy, the titer of juvenile hormone was determined for whole‐body extracts at various morphologically defined stages of the life cycle of Drosophila melanogaster. Only juvenile hormone III (JH‐III) was detected.

JH‐III is present in early metamorphosis but by mid‐metamorphosis it is below the level of detection (0.05 pmol/g). It then increases as the pharate adult matures and rises dramatically, beginning just prior to eclosion and reaching 5–7 pmol/g shortly after eclosion. The titer then begins to fall again as the adults mature in both males and females, though the decrease is more rapid in females. Preliminary studies show that low levels of JH‐III are present during all the larval instars but are absent from eggs.
The chromosomal gene structure for murine granulocyte colony‐stimulating factor
Authors: TSUCHIYA Masayuki , KAZIRO Yoshito , NAGATA Shigekazu
Published: March 2005

The chromosomal gene for murine granulocyte colony‐stimulating factor (G‐CSF) has been isolated from a mouse genomic library. The nucleotide sequence analysis of the gene and its flanking region has revealed that the murine G‐CSF gene is composed of four introns and five exons, which is similar to the human G‐CSF gene. The S1 mapping analysis of murine G‐CSF mRNA has identified the major transcription initiation site for the murine G‐CSF gene at 36 base pairs upstream of the ATG initiation codon. In the flanking region, there are sequences of about 300 base pairs which are highly conserved between human and murine G‐CSF genes. The decanucleotide GAGRTTCCAC, which was found at the 5′‐flanking region of genes for other hemopoietic growth factors, was also present within the corresponding regions of human and murine G‐CSF gene. Unlike the human G‐CSF gene, no alternative splicing was observed for the murine G‐CSF gene.
Substrate specificity of 3‐methyladenine‐DNA glycosylase from calf thymus
Authors: MALE Rune , HAUKANES Bjørn Ivar , HELLAND Dag E. , KLEPPE Kjell
Published: March 2005

3‐Methyladenine—DNA glycosylase from calf thymus recognizes both 3‐methyladenine (3‐mAde), 7‐methylguanine (7‐mGua) and 3‐methylguanine (3‐mGua) residues in calf thymus DNA; the rate of release of 3‐mAde is approximately eightfold higher than that for 7‐mGua. The best DNA polymer substrates appeared to be those having an A‐type helical conformation such as d(A‐T)n and d(G‐C)n. The Km values for release of 3‐mAde and 7‐mGua were approximately the same for the above mentioned two substrates whereas the Vmax for excision of 3‐mAde was threefold higher than that of 7‐mGua. The rate of hydrolysis of 7‐mGua residues in d(G‐C)n was similar to that found for the excision of 3‐mAde in calf thymus DNA. The polymer d(G)n· d(C)m, which possesses a B‐type helical conformation, was a poor substrate and the rate of excision here was approximately the same as with calf thymus DNA having the B‐type structure. Polyamines greatly influenced the activity and at low concentrations a 50–100% increase in the release of 7‐mGua, but not 3‐mAde, was observed. With higher concentrations the rate of excision of both bases decreased sharply.

The sequence specificity of the DNA glycosylase on naturally occurring DNA was studied using methylated DNA fragments from the plasmid pUC18. The results revealed that some 3‐mAde as well as 7‐mGua residues were seldom attacked. These 3‐mAde residues were positioned either 5′ to another Ade residue or in a stretch of pyrimidines, and the 7‐mGua residue 3′ to another Gua residue. The 3‐mAde residue most frequently recognized was situated 3′ to another Ade residue, and in the case of 7‐mGua it was the central Gua residue in the sequence ‐G‐G‐G‐.
CpG deficiency, dinucleotide distributions and nucleosome positioning
Authors: ADAMS Roger L. P. , DAVIS Terence , RINALDI Angela , EASON Robert
Published: March 2005

The dinucleotide CpG is deficient in (A + T)‐rich regions of vertebrate DNA in both coding and non‐coding sequences and there is a corresponding increase above expectation in the occurrence of TpG and CpA. By contrast in (G + C)‐rich regions no deficiency of CpG is found. Such (G + C)‐rich sequences, containing the expected number of CpG dinucleotides, alternate along the genome with (A + T)‐rich sequences which have a lower than expected CpG content. The G + C content of vertebrate DNA can oscillate with a period of 150–200 bp and this may be a factor in positioning nucleosomes. The role of mutagenesis in loss of CpG and increase of A + T, particularly in non‐coding regions, is discussed.
Cytoplasmic gels from macrophages
Authors: PACAUD Michèle , MOLLA Annie
Published: March 2005

A method has been devised to study the influence of Ca2+ on the in vitro formation of actin gel networks. Under appropriate conditions low‐Ca2+ cytosolic extracts (< 1 nM) from macrophages rapidly formed a macromolecular complex composed of actin, filamin, α‐actinin and two new proteins of 70 kDa and 55 kDa. [Pacaud, M. (1986) Eur. J. Biochem. 156, 521–530]. Increasing concentrations of free Ca2+ to 1–2 μM resulted in complete inhibition of the association of 70‐kDa protein, a protein which associates actin filaments into parallel arrays. Concentrations of Ca2+ greater than 3 μM caused incorporation of two additional proteins, gelsolin and a 18‐kDa polypeptide, with no change in either the actin or α‐actinin content of the cytoskeletal structures. Use of a polyacrylamide gel overlay technique with 125I‐calmodulin revealed that a high‐Mr calmodulin‐binding protein analogous to spectrin was also associated with these structures when micromolar Ca2+ was present. Similar assays with 45CaCl2 indicated that the 70‐kDa protein binds Ca2+ with high affinity. It is thus suggested that Ca2+ might regulate the dynamic assembly of microfilaments through several target proteins, gelsolin, the 70‐kDa protein and calmodulin.
Electrostatic effects of bound NADH and NAD+ on ionizing groups in liver alcohol dehydrogenase
Authors: PETTERSSON Gösta , EKLUND Hans
Published: March 2005

A simple point charge model has been used to estimate the effect of electrostatic interactions of bound NADH and NAD+ on ionizing groups in liver alcohol dehydrogenase. The results provide clear evidence that the much‐discussed effects of pH on coenzyme binding in the pH range 7–10 are predominantly of electrostatic origin and attributable to ionization of the water molecule which is bound at the active‐site zinc ion. Ionization of Lys‐228 is proposed to account for the proton‐dependent destabilization of the enzyme · NADH and enzyme · NAD+ complexes which has been reported to occur above a pKa of 10.4.
Signal sequence and DNA‐mediated expression of human lysosomal α‐galactosidase A
Authors: TSUJI Shoji , MARTIN Brian M. , KASLOW David C. , MIGEON Barbara R. , CHOUDARY Prabhakara V. , STUBBLEFLIED Barbara K. , MAYOR June A. , MURRAY Gary J. , BARRANGER John A. , GINNS Edward I.
Published: March 2005

Twelve complementary DNA clones for human lysosomal α‐galactosidase A were isolated from an Okayama‐Berg library constructed from SV40‐transformed human fibroblasts. The identity of these clones was confirmed by complete colinearity of the nucleotide‐deduced amino acid sequence with that determined by direct chemical sequencing of human placental α‐galactosidase A. Hybridization of the α‐galactosidase A cDNA to genomic DNA from individuals with varying numbers of X chromosomes as well as from interspecies somatic‐cell hybrids showed only a single locus in the genome at Xq 13.1 – Xq 22. One cDNA clone (pcD‐AG210) contained the complete coding sequence for both the signal peptide and mature α‐galactosidase A. The signal peptide of 31 amino acids contains the expected hydrophobic domains consisting of Leu‐Gly‐Cys‐Ala‐Leu‐Ala‐Leu and Phe‐Leu‐Ala‐Leu‐Val and has Ala at the signal peptidase cleavage site. Twelve out of fifteen G residues flanking the 5′ end of the cDNA in pcD‐AG210 were removed and the truncated fragment was ligated into the original vector. This construct, pcD‐AG502, encoded enzymatically active human α‐galactosidase A in monkey COS cells.
Molecular characterization of transposable‐element‐associated mutations that lead to constitutive l‐ornithine aminotransferase expression in Saccharomyces cerevisiae
Authors: DEGOLS Geneviéve , JAUNIAUX Jean‐Claude , WIAME Jean‐Marie
Published: March 2005

The carg B or CAR2 gene, coding for ornithine aminotransferase, was isolated by functional complementation of a carg B mutation in Saccharomyces cerevisiae. It was used as a hybridization probe to analyse RNA and chromosomal DNA from four strains bearing cis‐dominant regulatory mutations leading to constitutive, mating‐type‐dependent, ornithine aminotransferase synthesis. The four mutations appear to be insertions. Their size and restriction pattern suggested that they were transposable elements, Ty1. All were inserted in the same orientation with respect to the carg B gene. We cloned the carg B gene with its associated insertion from two constitutive mutants (carg B+Oh‐1 and carg B+Oh‐2). We determined the sequence of the carg B 5′ region from the wild‐type gene and from the two mutated genes. The DNA sequences of the extremities of the two insertions were very homologous but not identical and were similar to previously reported Ty1 element direct repeats (δ). The same five‐base‐pair sequence, ATATA, was found at both ends of both Ty1 elements, indicating that both Ty1 were transposed to the same site. This site is located 115 base pairs upstream from the putative carg B coding region. The 5′ end of carg B transcript as determined by S1 mapping was the same in the wild‐type strain and in the four mutants. The carg B transcript was not detected in the wild‐type strain grown under non‐induced conditions, while under the same conditions it was present in all four mutants.
Structure, DNasel hypersensitivity and expression of integrated papilloma virus in the genome of HeLa cells
Authors: LAZO Pedro A.
Published: March 2005

Three integrated copies of human papilloma virus‐18 (HPV‐18) have been identified in HeLa DNA as Hind III bands. HPV‐18 has no Hind III restriction site in its‐genome. The three segments: A, 8.4 kb; B, 7.9 kb and C, 5.8 kb, have an incomplete viral genome. All of them have most of the 1.1‐kb BamH1 non‐coding fragment of HPV‐18, which seems to contain the viral origin of replication and regulatory elements. Two of the segments (A and B) have a common 5′‐end break‐point in the viral genome within the L2 open reading frame (ORF). In both segments the second early transcriptional unit of the virus (E6, E7 and E1) is structurally conserved in a new environment. The 3′‐end break‐point for segments A and B is within the E2 ORF. Segment C has the L2 and L1 ORF but none of the genes of the early region. Segments A and B have a specific DNaseI‐hypersensitive site located in the E7/E1 region. The nucleotide sequence of this region has twelve papova virus enhancer‐like consensus sequence (5′‐TACCACANTA‐3′) and a double inverted repeat with fixed spacing capable of forming a hairpin loop. Two viral RNA transcripts of 4.8 kb and 1.7 kb have been detected in poly(A)‐rich RNA. The larger transcripts hybridizes to E6, E7 and E1 ORFs as well and has 2.4 kb of host sequence in its 3′ end. The smaller transcript hybridizes with E6 and E7 ORFs and the beginnig of E1, the final 0.7 kb of E1 are not detectable. No transcripts have been detected carrying E2, E4, E5, L2 and L1 ORF sequences. The transcripts are derived from segments A or B. Segment C is not transcriptionally competent.
Nucleotide sequence of the gene encoding the 11‐kDa subunit of the ubiquinol – cytochrome‐c oxidoreductase in Saccharomyces cerevisiae
Authors: MAARSE Ammy C. , GRIVELL Leslie A.
Published: March 2005

The nucleotide sequence of the gene encoding the 11‐kDa subunit VIII of the ubiquinol‐cytochrome‐c oxidoreductase in. Saccharomyces cerevisiae has been determined. The coding sequence has a length of 330 bp and is preceded at a distance of 361 bp by another reading frame, coding for a protein of as yet unknown function. The 11‐kDa gene is transcribed independently of the URFx gene and transcription of both is sensitive to catabolite repression. Multiple 5′ and 3′ termini of transcripts of the gene for the 11‐kDa subunit were identified by S1 nuclease protection analysis of DNA. RNA hybrids. The 5′ termini map 52 ± 2 and 60 ± 2 nucleotides upstream of the initiation codon whereas the 3′ termini map 336 ± 2 and 350 ± 2 nucleotides downstream of the stop codon. The subunit VIII reading frame encodes a protein with a molecular mass of 12.4 kDa and a polarity of 37.6%. It is predicted to contain a high content of β‐sheet segments, which may be capable of forming a barrel‐like structure in a lipid bilayer. A comparison of the sequence with those of the small subunits of the beef heart complex reveals similarity with the 9.5‐kDa subunit VII (core‐linked protein) characterized by Borchart et al. (1986) FEBS Lett. 200, 81–86. The significance of this is discussed.
Nucleotide sequence and characterization of the yeast PSS gene encoding phosphatidylserine synthase
Authors: NIKAWA Jun‐ichi , TSUKAGOSHI Yuko , KODAKI Tsutomu , YAMASHITA Satoshi
Published: March 2005

1. A yeast chromosomal DNA which contains the structural gene for phosphatidylserine synthase (PSS) was isolated by genetic complementation from a wild‐type yeast genomic library. The PSS gene was subcloned into a 1.1‐kb fragment of the yeast DNA on the YEp13 vector.

2. The PSS gene on the multicopy plasmid caused the fourfold over‐production of the enzyme and fully restored the phosphatidylserine content of the transformant. The phospholipid composition of the transformant was similar to that of the wild type.

3. Sequence analysis showed that this DNA fragment contains an open reading frame capable of encoding 276 amino acid residues with a calculated relative molecular mass of 30 804. Northern blot analysis of poly(A)‐rich RNA of the wild‐type yeast indicated that this DNA segment is transcribed into a single mRNA species.

4. The DNA sequence contained two putative transcriptional initiation signals, each followed by the ATG initiator codon. Deletion experiments indicated that the 5′‐proximal ATG codon is essential for the synthesis of the functional phosphatidylserine synthase.
The elucidation of the microheterogeneity of highly purified p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens by various biochemical techniques
Authors: VAN BERKEL Willem J. H. , MÜLLER Franz
Published: March 2005

Highly purified p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens can be separated into at least five fractions by anion‐exchange chromatography. All fractions exhibit the same specific activity and the enzyme exists mainly in the dimeric form in solution. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of a mixture of the different fractions reveals two apparent forms of enzyme molecules, while isoelectric focusing experiments, on the other hand, reveal six apparently different forms of enzyme molecules. It is shown that the different forms of enzyme molecules are due to the (partial) oxidation of Cys‐116 in the sequence of the enzyme. This interpretation of the data is supported by kinetic measurements of the formation of hybrid dimeric molecules monitored by fast protein liquid chromatography, using purified enzyme containing Cys‐116 either in the native and or the fully oxidized (sulfonic acid) state. By chemical modification studies using maleimide derivatives, 5,5′‐dithiobis(2‐nitrobenzoate) and H2O2, it is shown that sulfenic, sulfenic and sulfonic acid derivatives of Cys‐116 are products of oxidation. The results are briefly discussed with respect to the possibility that this isolation artifact might also be partially responsible for the appearance of multiple forms of enzyme molecules in other biochemical preparations.
Characterization of cDNA coding for the complete light meromyosin portion of a rabbit fast skeletal muscle myosin heavy chain
Authors: MAEDA Kayo , SCZAKIEL Georg , WITTINGHOFER Alfred
Published: March 2005

Myosin‐heavy‐chain‐specific cDNA clones have been isolated from a cDNA library prepared from hind leg muscle of a 14‐day‐old rabbit. According to restriction enzyme analysis these can be grouped into at least two, probably three different classes. RNA dot‐blot hybridization shows that all of these clones correspond to mRNAs expressed in fast skeletal muscle. The clones of the most abundant form, class I, can be aligned to cover the complete light meromyosin portion of myosin heavy chain. The sequence of the coding and the 3′‐untranslated region, together comprising 2143 base pairs, has been determined. The class I clone detects a multigene family of 8–12 members on a Southern blot of rabbit genomic DNA.
Reactions of nucleic acid bases with α‐acetylenic esters
Authors: ROQUES Pierre , OLOMUCKI Martin
Published: March 2005

The reaction of chlorotetrolic (4‐chloro‐2‐butynoic) esters with adenine and cytosine derivatives, in which a new heterocycle bearing an alkylating chloromethyl side chain is fused to the purine or pyrimidine ring, was extended to poly(A) and poly(C) used as models of nucleic acids. The derivatization proceeds under mild conditions and its extent can be controlled by the reaction time. The additional rings can exist in two isomeric forms and the nature of the isomer formed depends on steric factors in the vicinity of the reacting base. The reaction with chlorotetrolic esters discriminates between the single‐stranded (reactive) and double‐stranded (unreactive) forms, between the exposed an hidden adenine and cytosine bases and even between the exposed and sterically hindered fragments of the base moiety and thus allows structural investigations of these nucleic acids. The chloromethyl group of the derivatized nucleobases can be used to bind the modified polymers to other molecules.
The 165‐KDa peptide of the purified skeletal muscle dihydropyridine receptor contains the known regulatory sites of the calcium channel
Authors: SIEBER Manfred , NASTAINCZYK Wolfgang , ZUBOR Vladimir , WERNET Wolfgang , HOFMANN Franz
Published: March 2005

The dihydropyridine receptor purified from rabbit skeletal muscle yields in the presence of dithiothreitol and sodium dodecyl sulfate on polyacrylamide gels bands of apparent molecular mass 165 ± 5, 130 ± 5, 55 ± 3, 32 ± 2 and 28 ± 1 kDa (x± SEM, n= 12). Under nonreducing conditions, the 130‐kDa and 28‐kDa peptides migrate as a single peptide of 165 kDa. These peptides were separated on a HPLC size‐exclusion column. The specific absorption coefficients of the isolated peptides were determined. From these a stoichiometry of 1:1.7 ± 0.2:1.4 ± 0.3 (x± SEM of 12 experiments with three different preparations) was calculated for the 165‐kDa, 55‐kDa and 32‐kDa peptides. The relative amount of the 130/28‐kDa peptide varied with different preparations. Tryptic, chymotryptic and V‐8 protease peptides of the isolated proteins suggested that the 130/28‐kDa peptide was not related to the 165‐kDa peptide. The dihydropyridine photoaffinity analog (±)‐azidopine was specifically incorporated only into the 165‐kDa peptide with an efficiency of about 2.4%. The azido analog of desmethoxyverapamil, LU 49888, was specifically incorporated into the same peptide with an efficiency of 1.5%.

These results suggest that only the 165‐kDa peptide contains the regulatory sites detected so far in the voltage‐operated L‐type calcium channel. They suggest further that the 130/28‐kDa peptide, which migrates as a 165‐kDa peptide under nonreducing conditions, does not contain high‐affinity binding sites for the calcium channel blockers.
A 50‐kDa fragment from the NH2‐terminus of the heavy subunit of Clostridium botulinum type A neurotoxin forms channels in lipid vesicles
Authors: SHONE Clifford C. , HAMBLETON Peter , MELLING Jack
Published: March 2005

1. A 50‐kDa fragment representing the NH2‐terminus of the heavy subunit of botulinum type A neurotoxin was found, at low pH, to evoke the release of K+ from lipid vesicles loaded with potassium phosphate. Similar K+ release was also observed with the intact neurotoxin, its heavy chain and a fragment consisting of the light subunit linked the 50‐kDa NH2‐terminal heavy chain fragment. The light subunit alone, however, was inactive.

2. In addition to K+, the channels formed in lipid bilayers by botulinum neurotoxin type A or the NH2‐terminal heavy chain fragment were found to be large enough to permit the release of NAD (Mr 665).

3. The optimum pH for the release of K+ was found to be 4.5. Above this value K+ release rapidly decreased and was undetectable above pH 6.0.

4. The binding of radiolabelled botulinum toxin to a variety of phospholipids was assessed. High levels of toxin binding were only observed to lipid vesicles with an overall negative charge; much weaker binding occurred to lipid vesicles composed of electrically neutral phospholipids.

5. A positive correlation between the efficiency of toxin binding and the efficiency of K+ release from lipid vesicles was not observed. Whereas lipid vesicles containing the lipids cardiolipin or dicetyl phosphate bound the highest levels of neurotoxin, the toxin‐evoked release of K+ was low compared to vesicles containing either phosphatidyl glycerol, phosphatidyl serine or phosphatidyl inositol.

6. The implications of these observations to the mechanism by which the toxin molecule is translocated into the nerve ending are discussed.
Characteristics of alcohol/polyol dehydrogenases
Authors: JÖRNVALL Hans , PERSSON Bengt , JEFFERY Jonathan
Published: March 2005

Sixteen characterized alcohol dehydrogenases and one sorbitol dehydrogenase have been aligned. The proteins represent two formally different enzyme activities (EC 1.1.1.1 and EC 1.1.1.14), three different types of molecule (dimeric alcohol dehydrogenase, tetrameric alcohol dehydrogenase, tetrameric sorbitol dehydrogenase), metalloproteins with different zinc contents (1 or 2 atoms per subunit), and polypeptide chains from different kingdoms and orders (mammals, higher plants, fungus, yeasts). Present comparisons utilizing all 17 forms reveal extensive variations in alcohol dehydrogenase, but with evolutionary changes that are of the same order in different branches and at different times. They emphasize the general importance of particular residues, suggesting related overall functional constraints in the molecules. The comparisons also define a few coincidences between intron positions in the genes and gap positions in the gene products. Only 22 residues are strictly conserved; half of these are Gly, and most of the remaining ones are Pro or acidic residues. No basic residues, no straight‐chain hydrophobic residues, no aromatic residues, and essentially no branched‐chain or polar neutral residues are invariable. Tentative consensus sequences were calculated, defining 13 additional residues likely to be typical of but not invariant among the alcohol dehydrogenases. These show a predominance of Val, charged residues, and Gly. Combined, the comparisons, which are particularly relevant to the data base for protein engineering, illustrate the requirements for functionally important binding interactions, and the extent of space restrictions in proteins with related overall conformations and functions.
The plasma membrane ATPase of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius
Authors: LÜBBEN Mathias , LÜNSDORF Heinrich , SCHÄFER Günter
Published: March 2005

The plasma‐membrane‐associated ATPase of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius characterized in a previous work [M. Lübben & G. Schäfer (1987) Eur. J. Biochem. 164, 533–540] has been solubilized. It can be easily removed from the membrane by mild treatment with zwitterionic detergents, therefore it appears to be a peripheral membrane protein analogous to the soluble F1‐ATPase of eubacteria and eukaryotes. Further purification has been achieved by subsequent gel permeation and ion‐exchange chromatography. The final purity is greater than 70% as judged by staining intensities after SDS/polyacrylamide gel electrophoresis. The ATPase consists of two major polypeptides of 65 kDa (α) and 51 kDa (β) in comparable quantities; a minor band (20 kDa) is assumed to be a contaminant or a constitutive part of the enzyme, possibly copurified in substoichiometric amount. The native molecular mass of the solubilized ATPase determined by gel permeation is 430 kDa. Considering the precision of these methods, it remains open whether a 3:3 stoichiometry reflects the contribution of α and β subunits to the quaternary structure, in analogy to known F1‐ATPases.

The catalytic properties resemble those of the membrane‐bound state. There are two pH optima at 5.3 and 8.0 in the absence and only one optimum at 6.5 in the presence of the activating anion sulfite.

Activity is strictly dependent on the divalent cations Mg2+ or Mn2+. ATP and dATP are hydrolyzed with highest rates; also other purine and pyrimidine nucleotides are cleaved significantly, but not ADP, pyrophosphate and p‐nitrophenyl phosphate. The ATPase is insensitive to azide or vanadate but is inhibited by relatively low concentrations of nitrate.

Polyclonal antisera have been raised against the β subunit of the Sulfolobus ATPase. Cross‐reactivities with cellular or membrane extracts of a number of archaebacteria, eubacteria and chloroplasts have been analyzed by means of Western blotting and immunodecoration. A strong cross‐reactivity with other genera of the Sulfolobales is observed, also with Methanobacterium, Methanosarcina, Methanolobus and Halobacterium. Even membranes of the eubacterium Escherichia coli and of eukaryotic chloroplast react with the antibodies. With one exception, in all cases the molecular mass of the cross‐reacting polypeptide falls in the range of 51–56 kDa. Only in Halobacterium halobium, bands at 66 and 68 kDa have been detected.

In order to identify the cross‐reacting polypeptides, the purified F1‐ATPases of E. coli, chloroplasts and beef heart mitochondria have been tested. Unequivocally the β subunits of these coupling factors are labeled by the Sulfolobus anti‐β antibody. Our data support the idea that the Sulfolobus ATPase is at least closely related or even affiliated to the group of eubacterial and eukaryotic F0F1‐ATPases.

In addition, the ATPase could be localized as a constituent of the plasma membrane by immuno‐electron microscopy.
Cytochalasin B selectively releases ovalbumin mRNA precursors but not the mature ovalbumin mRNA from hen oviduct nuclear matrix
Authors: SCHRÖDER Heinz C. , TRÖLLTSCH Dieter , WENGER Rosemarie , BACHMANN Michael , DIEHL‐SEIFERT Bärbel , MÜLLER Werner E. G.
Published: March 2005

Hen oviduct nuclear matrix‐bound mature ovalbumin mRNA is released from the matrix in the presence of ATP, while the ovalbumin mRNA precursors remain bound to this structure. Detachment of the mature mRNA from the matrix by ATP as well as ATP‐dependent efflux of mRNA from isolated nuclei were found to be inhibited by cytochalasin B. On the other hand, in the absence of ATP, cytochalasin B exclusively caused the release (and nucleocytoplasmic efflux) of the ovalbumin messenger precursors, but not of the mature mRNA. After cytochalasin B treatment, actin could be detected in the matrix supernatant. Phalloidin which stabilizes actin filaments did not cause RNA liberation in the absence of ATP, but inhibited the ATP‐induced detachment of mature mRNA. RNA release was also achieved with a monoclonal antibody against actin but not with monoclonal antibodies against tubulin and intermediate filaments. These results suggest that actin‐containing filaments are involved in the restriction of immature messengers to the cell nucleus.
Comparison of binding sites in DNA for berenil, netropsin and distamycin
Authors: PORTUGAL José , WARING Michael J.
Published: March 2005

Techniques of DNase I and micrococcal nuclease footprinting have been used to compare the binding sites for berenil, netropsin and distamycin on two different DNA fragments. Each ligand binds to the A + T‐rich zones which contain clusters of at least four A + T base pairs. Neither guanosine nor cytidine nucleotides appear to be allowed within the A + T‐rich runs which constitute the preferred binding sites, although they are sometimes protected from DNase I cleavage in neighbouring regions. Berenil and netropsin share with distamycin the property of causing enhanced rates of cleavage at certain sequences flanking their binding sites. There are significant differences in the concentrations of each ligand required to produce defined patterns of protection, seemingly dependent upon the nature (and possibly the gross base composition) of the piece of DNA being used in the experiment.
Amino acid substitutions in subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae
Authors: WILLSON Tracy A. , NAGLEY Phillip
Published: March 2005

This work concerns a biochemical genetic study of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Subunit 9, encoded by the mitochondrial olil gene, contains a hydrophilic loop connecting two transmembrane stems. In one particular olil mit− mutant 2422, the substitution of a positively charged amino acid in this loop (Arg39 → Met) renders the ATPase complex non‐functional.

A series of 20 revertants, selected for their ability to grow on nonfermentable substrates, has been isolated from mutant 2422. The results of DNA sequence analysis of the olil gene in each revertant have led to the recognition of three groups of revertants. Class I revertants have undergone a same‐site reversion event: the mutant Met39 is replaced either by arginine (as in wild‐type) or lysine. Class II revertants maintain the mutant Met39 residue, but have undergone a second‐site reversion event (Asn35 → Lys). Two revertants showing an oligomycin‐resistant phenotype carry this same second‐site reversion in the loop region together with a further amino acid substitution in either of the two membrane‐spanning segments of subunit 9 (either Gly23 → Ser or Leu53 → Phe). Class III revertants contain subunit 9 with the original mutant 2422 sequence, and additionally carry a recessive nuclear suppressor, demonstrated to represent a single gene.

The results on the revertants in classes I and II indicate that there is a strict requirement for a positively charged residue in the hydrophilic loop close to the boundary of the lipid bilayer. The precise location of this positive charge is less stringent; in functional ATPase complexes it can be found at either residue 39 or 35. This charged residue is possibly required to interact with some other component of the mitochondrial ATPase complex. These findings, together with hydropathy plots of subunit 9 polypeptides from normal, mutant and revertant strains, led to the conclusion that the hydrophilic loop in normal subunit 9 extends further than previously suggested, with the boundary of the N‐terminal membrane‐embedded stem lying at residue 34.

The possibility is raised that the observed suppression of the 2422 mutant phenotype in class III revertants is manifested through an accommodating change in a nuclear‐encoded subunit of the ATPase complex.
Construction and stability of a sixfold repeated artificial gene
Authors: HOFER Bernd
Published: March 2005

The concatenation of an artificial gene consisting of 69 base pairs is described. A simple and versatile procedure was employed which is applicable to any DNA segment bordered by different restriction sites. If applied in the ‘continuous duplication mode’, it does not necessitate the isolation of any DNA fragment. The addition of adaptor oligonucleotides to one terminus of the segment is required to create complementary ends. The ligation of single‐stranded oligonucleotides to single‐stranded DNA termini was analyzed and optimized. A simple procedure is described for the separation of excess adaptor molecules. The in vivo stability of constructs containing up to six directly repeated genes was investigated. Up to four copies of the gene appeared stable during transformation of strain HB101. With six copies exact excision of one copy was occasionally observed after transformations of rec+, recA, and recBC host cells. During long‐term growth the hexameric construct appeared stable in all three strains.