bluuebunny/crossref_metadata_2025_split huggingface dataset:
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‐.
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 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.
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.
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.
1‐Acyl‐sn‐glycerol‐3‐phosphate acyltransferase in maturing safflower seeds and its contribution to the non‐random fatty acid distribution of triacylglycerol
Authors: ICHIHARA Ken'ichi , ASAHI Takahiro , FUJII Shoji 
Published: March 2005

A 20000 ×g particulate preparation isolated from maturing safflower seeds catalyzed the acylation of 1‐acyl‐sn‐glycerol 3‐phosphate with acyl‐CoA to form phosphatidate. The specific activity of the reaction exceeded 200 nmol min−1 mg protein−1. Although this preparation was also capable of catalyzing the acylation of sn‐glycerol 3‐phosphate with acyl‐CoA, the hydrolysis of phosphatidate, and the acylation of 1,2‐diacylglycerol, phosphatidate was the only major product when the preparation was incubated with 1‐acyl‐glycerol‐3‐P and acyl‐CoA.The enzyme responsible for this phosphatidate synthesis, 1‐acyl‐glycerol‐3‐P acyltransferase, showed a strict acyl‐CoA specificity. The relative order of specificity for acyl‐CoA was linoleoyl = oleoyl > palmitoleoyl > elaidoyl > cis‐vaccenoyl > stearoyl = palmitoyl. This observation strongly suggests that the fatty acid composition of position 2 in phosphatidate synthesized in vivo primarily depends on both the acyl‐CoA specificity of the 1‐acyl‐glycerol‐3‐P acyltransferase and the fatty acid composition of the acyl‐CoA pool in the cell. Thus, the absence of saturated fatty acids at position 2 of safflower triacylglycerol may be explained in terms of the acyl‐CoA specificity of the 1‐acyl‐glycerol‐3‐P acyltransferase. The fatty acid moiety esterified at position 1 of glycerol‐3‐P also affected the effectiveness of the reaction. The 1‐acyl‐glycerol‐3‐P acyltransferase utilized 1‐acyl‐glycerol‐3‐P molecular species in the following order of effectiveness: linoleoyl = oleoyl > palmitoyl. With a rise in incubation temperature, the initial rates of acylation with unsaturated acyl‐CoA species increased more rapidly than those for saturated acyl‐CoA species. A similar tendency was observed for saturated and unsaturated acyl acceptors. These data suggest that affinity of the acyltransferase for substrates may vary in response to changes in temperature, and that 1‐acyl‐glycerol‐3‐P acyltransferase may be involved in the alteration of the individual fatty acid compositions at positions 1 and 2 of glycerolipids in tissues grown at different temperatures.Based on these findings, further metabolism of 1‐acyl‐glycerol‐3‐P acyltransferase products could be the major factor determining the non‐random distribution of fatty acids in safflower triacylglycerol.
Hybrid glycophorins from human erythrocyte membranes
Authors: BLANCHARD Dominique , DAHR Wolfgang , BEYREUTHER Konrad , MOULDS John , CARTRON Jean‐Pierre 
Published: March 2005

Human red cells from donor Pj carry the Sta blood group antigen and an unusual sialoglycoprotein of 24 kDa molecular mass tentitatively identified as a hybrid molecule of the anti‐Lepore type [Blanchard et al. (1982) Biochem. J. 203, 419–426]. This component is resistant towards proteinase treatment and was purified from trypsin‐treated and chymotrypsin‐treated Pj erythrocytes. The molecule is composed of 99 amino acid residues whose alignment was established following manual and automatic sequencing of cyanogen bromide, trypsin, chymotrypsin and V8 proteinase peptides. The polypeptide chain comprises residues 1–26/28 of glycophorin B and residues 59/61–131 of glycophorin A. The sugar composition resembles that of glycophorin B, indicating the absence of an N‐glycosidic chain. Identical sequences were obtained from analyses of the 24‐kDa component purified from unrelated St(a+) donors. These results support the hypothesis that glycoprotein Pj represents a B‐A hybrid molecule which is encoded by a new gene product resulting from an unequal crossing‐over between the genes coding for the polypeptide chains of the glycophorins A and B.The novel molecule carries both N and Sta blood group antigens. The N activity is clearly understandable from the sequence of the five N‐terminal residues (Leu and Glu at positions 1 and 5 respectively). Inhibition studies with the untreated and chemically modified hybrid glycoprotein indicate that the Sta determinant is located within residues approximately 25–30 of the molecule, which corresponds to the newly formed sequence found neither in glycophorin A nor in glycophorin B.
Protein phosphatases of the guinea‐pig parotid gland
Authors: MIESKES Gottfried , SÖLING Hans‐Dieter 
Published: March 2005

The nature of protein phosphatases of the guinea‐pig parotid gland was investigated. The protein phosphatases were characterized by (a) the use of five different 32P‐labelled substrate proteins (phosphorylase a, histone H2B, casein, and the α and β subunits of phosphorylase kinase), (b) their behaviour during ion‐exchange chromatography, (c) their relative molecular mass distribution during gel filtration, (d) their sensitivity towards inhibition by inhibitor 2, (e) their ability to be stimulated by protamine and (f) by their behaviour during freezing and thawing in the presence of 2‐mercaptoethanol. The following results were obtained.

The ‘cytosol’ (100 000 xg supernatant) contains protein phosphatases of the types 1, 2A and 2B.
On the basis of inhibition with inhibitor 2 (1.2 μg/ml) the ‘cytosolic’ phosphorylase phosphatase activity consists to about 40% of protein phosphatase 1 and to about 60% of protein phosphatase 2A.
In the cytosol about 80–90% of the protein phosphatases 1 and 2A exist in an inactive state.
A 5–10‐fold activation can be achieved by ethanol precipitation, which results in the generation of a mixture of forms of low apparent molecular mass of about 30 kDa.
Microsome‐associated phosphorylase phosphatase activities can be extracted in a highly active state by detergent (1% Triton X‐100) or by 0.8 M NaCl.
Activity measurements in the presence of inhibitor 2 (1.2 μg/ml) indicate that the microsomal activities consist to about 75% of protein phosphatase 1 and to about 25% of protein phosphatase 2A. Activities corresponding to protein phosphatases 2B and 2C could not be detected.
The ‘microsomal’ protein phosphatase activities exhibit lower apparent molecular masses (70 kDa and 30 kDa) than the ‘cytosolic’ protein phosphatases (about 260 kDa).
After ethanol treatment of the microsomal protein phosphatases only activities with apparent molecular masses of about 30 kDa can be detected. These share several similarities with the ethanol‐treated cytosolic protein phosphatases.
Both cytosolic and microsomal protein phosphatases display activity towards histone H2B and casein.
A two‐dimensional 1H‐NMR study of the dam methylase site: comparison between the hemimethylated GATC sequence, its unmethylated analogue and a hemimethylated CATG sequence
Authors: FAZAKERLEY G. Victor , QUIGNARD Etienne , TEOULE Robert , GUY André , GUSCHLBAUER Wilhelm 
Published: March 2005

We report two‐dimensional NOE (NOESY) spectra on the sequence d(GCGATCATGG) d(CCATGATCGC) which contains the unmethylated dam site. As expected the DNA adopts a B‐form conformation but appears to be distorted at the TG step of the second strand. This distorsion, probably bending, is not seen on the opposite strand. When the first strand is methylated on adenine in the GATC or CATG sequence the NOESY spectra indicate little or no change in the conformation. However the single strand‐duplex exchange is slowed down to the slow‐exchange region on a proton NMR time scale. We have assigned the exchangeable imino and cytidine amino resonances of the three duplexes. From the imino linewidths as a function of temperature, we observe that the unmethylated and the hemimethylated Gm6ATC duplexes melt normally from the ends. However, this is not so for the hemimethylated Cm6ATG duplex which, apart from the terminal base pairs, melts cooperatively and at higher temperature. In spectra recorded in H2O a second duplex is observed, for the Gm6ATC sequence, which we have not been able to identify. It is however unlikely to be a hairpin structure. Ultravioletmelting curves also indicate the presence of two transitions for this duplex.The effect of methylation upon base‐pair lifetimes has been studied by comparing the above three duplexes. Little effect is observed upon methylation in the GATC sequence but a drastic increase in the lifetimes of all base pairs is observed upon methylation in the CATG sequence.
Lipopolysaccharide‐sensitive serine‐protease zymogen (factor C) of horseshoe crab hemocytes. Identification and alignment of proteolytic fragments produced during the activation show that it is a novel type of serine protease
Authors: TOKUNAGA Fuminori , MIYATA Toshiyuki , NAKAMURA Takanori , MORITA Takashi , KUMA Kei‐ichi , MIYATA Takashi , IWANAGA Sadaaki 
Published: March 2005

The horseshoe crab clotting factor, factor C, present in the hemocytes is a serine‐protease zymogen activated with lipopolysaccharide. It is a two‐chain glycoprotein (Mr= 123 000) composed of a heavy chain (Mr= 80 000) and a light chain (Mr= 43 000) [T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511–521]. In our continued study of this zymogen, we have now also found a single‐chain form of factor C (Mr= 123 000) in the hemocyte lysate. The heavy chain had the NH2‐terminal sequence of Ser‐Gly‐Val‐Asp‐, consistent with that of the single‐chain factor C, indicating that the heavy chain is derived from the NH2‐terminal part of the molecule. The light chain had an NH2‐terminal sequence of Ser‐Ser‐Gln‐Pro‐. Incubation of the two‐chain zymogen with lipopolysaccharide resulted in the cleavage of a Phe‐Ile bond between residues 72 and 73 of the ligth chain. Concomitant with this cleavage, the A (72 amino acid residues) and B chains derived from the light chain were formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar in sequence to a family of repeats in human β2‐glycoprotein I, complement factors B, protein H, C4b‐binding protein, and coagulation factor XIII b subunit. The NH2‐terminal sequence of the B chain was Ile‐Trp‐Asn‐Gly‐. This chain contained the serine‐active site sequence‐Asp‐Ala‐Cys‐Ser‐Gly‐Asp‐Ser‐Gly‐Gly‐Pro‐.These results indicate that horseshoe crab factor C exists in the hemocytes in a single‐chain zymogen form and is converted to an active serine protease by hydrolysis of a specific Phe‐Ile peptide bond.
Nucleotide sequence and comparative analysis of the frd operon encoding the fumarate reductase of Proteus vulgaris
Authors: COLE Stewart T. 
Published: March 2005

The fumarate reductase of Escherichia coli is a bioenergetically important membrane‐bound flavoenzyme consisting of four subunits. A and B comprise a membrane‐extrinsic catalytic domain whereas C and D are hydrophobic polypeptides which link the catalytic centres to the electron‐transport chain. The nucleotide sequence of the frd operon encoding the fumarate reductase of the distantly related bacterium, Proteus vulgaris has been determined and used to predict the primary structures of the respective subunits. Extensive amino acid sequence identity (> 80%) was found between the fumarate reductase A and B subunits of P. vulgaris and E. coli. In contrast, the primary structures of the P. vulgaris and E. coli C and D proteins are much less closely related (about 60% homology) although the overall hydrophobicity of their three membrane‐spanning segments has been conserved.In most enteric bacteria, the frd operon is followed by genes, ampR and/or ampC, required for the genetic regulation and biosynthesis of a cephalosporinase. The corresponding region of the P. vulgaris genome is occupied by an operon (orf A'BCD) containing at least four genes which are clearly unrelated to the ampC system. Intriguingly the primary structures of the OrfA and OrfD proteins suggest that, like fumarate reductase, they may be components of a membrane‐bound enzyme complex involved in energy metabolism.