bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
Immunochemistry of scorpion toxins
Authors: BAHRAOUI Elmostafa , EL AYEB Mohamed , GRANIER Claude , ROCHAT Hervé
Published: March 2005

Peptide 19–28, a model of an antigenic region of Androctonus australis Hector toxin II, has a rigid α‐helix structure in the native protein. It was used as immunogen either in the free form, or bound to bovine serum albumin (BSA) or linked to its synthesis support (macroporous polyacrylamide resin). Only the anti‐(peptide‐BSA) and the anti‐(peptide‐resin) antibodies recognized the native toxin. The use of short synthetic analogues of peptide 19–28 suggests a specificity difference in the two antipeptides. Anti‐(peptide‐BSA) recognizes probably two determinants localized at the C and N terminals of the peptide chain. Anti‐(peptide‐resin) preferentially recognizes the N‐terminal extremity. Finally we showed that the α‐helix region remains accessible to antipeptide 19–28 when the toxin is bound to its receptor.
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.
Role of the essential thiol group in the thiol‐activated cytolysin from Clostridium perfringens
Authors: IWAMOTO Machiko , OHNO‐IWASHITA Yoshiko , ANDO Susumu
Published: March 2005

A hemolysin, θ‐toxin, produced by Clostridium perfringens has one cysteinyl residue in the free thiol form which is essential for its hemolytic activity. The cysteinyl residue was shown to be located at a position about 5 kDa from the C terminus of the molecule by the method of cysteine‐specific chemical cleavage. Modification of the residue with a thiol‐blocking agent, 5,5′‐dithiobis(2‐nitrobenzoic acid), reduced the binding affinity of the toxin to sheep erythrocytes to 1/100 that of intact toxin, resulting in a failure of binding at low cell concentrations (0.5%). Thus the failure of hemolysis at low cell concentrations is primarily ascribed to a decreased affinity of the toxin for erythrocytes. Effects of the modification on the lytic processes were examined using high cell concentrations where considerable amounts of modified toxin bound to the cells. The modified toxin hemolyzes erythrocytes once it binds to them; however, the efficiency of hemolysis is reduced by the modification. These, and additional results indicating that modification alters the sensitivity of toxin molecules to protease digestion, show that thiol‐modification inactivates the toxin by affecting both binding and the subsequent lytic processes, probably through a conformational change introduced in the toxin molecules.
Sequence‐specific 1H‐NMR assignments in rat‐liver metallothionein‐2
Authors: WÖRGÖTTER Erich , WAGNER Gerhard , VAŠAK Milan , KÄGI Jeremias H. R. , WÜTHRICH Kurt
Published: March 2005

As a basis for the determination of the metal‐coordination topology and the three‐dimensional fold of the polypeptide chain, sequence‐specific assignments were obtained for the 1H nuclear magnetic resonance (NMR) spectrum of metallothionein‐2 from rat liver. The 1H spin systems of the 20 metal‐bound cysteines were identified from comparison of two metal‐homogeneous protein preparations obtained by reconstitution of the apometallothionein‐2 with 113Cd2+ and 112Cd2+, respectively. The identification of the spin systems for the remaining amino acid residues and sequential assignments were then obtained with two‐dimensional 1H‐NMR experiments at 500 MHz. The assignments are complete except for two backbone amide protons, which were not observed, and the side‐chain hydrogen atoms beyond βCH2 for all eight lysines. The chemical shifts are presented at pH 7.0 and 25°C.
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.
The early light‐inducible proteins of Barley °
Authors: GRIMM Bernhard , KLOPPSTECH Klaus
Published: March 2005

Within 1–2 h of illumination of etiolated barley plants the mRNAs of seven nuclear‐coded proteins are transiently induced. It is proposed that at least some of these proteins are precursors to chloroplast membrane proteins since after posttranslational transport 2‐h‐specific bands of 18.5 kDa, 18 kDa and 13.5 kDa have been found bound to thylakoid membranes. cDNA clones for these early light‐inducible proteins (ELIPs) have been isolated. Hybrid‐release translation shows that part of their information must be homologous since the complete set of early light‐inducible translation products is obtained with all investigated clones although the proportions of the translated bands vary for individual clones. From hybridization data it is concluded that two ELIP families of high (24–27 kDa) and of low (16–18 kDa) molecular mass exist which are induced in parallel. Induction of ELIPs occurs even at very low light intensities and is saturated at about 1000 lx. Therefore, ELIPs are not considered to represent light stress proteins but to play a regulatory role during development.
Complete purification of the pseudorabies virus protein kinase
Authors: PURVES Frances C. , KATAN Matilda , LEADER David P.
Published: March 2005

The recently described pseudorabies virus protein kinase has been purified from infected hamster fibroblasts by a combination of anion‐exchange, hydrophobic‐interaction and affinity chromatography. The purification resulted in enzyme with a specific activity in excess of 1000 nmol phosphate mg−1 min−1 in relatively high yield. Gel electrophoresis of the purified enzyme under denaturing conditions revealed a single stained band at a position of migration corresponding to a Mr 38000. Incubation of the purified enzyme with [γ‐32P]ATP in the absence of added substrate resulted in incorporation of 32P into this protein band, consistent with the 38‐kDa protein being a protein kinase with a capacity for autophosphorylation. The phosphorylated form of the protein has an isoelectric point of approximately 4.9. Gel permeation chromatography of the purified enzyme indicated a native Mr 70000, suggesting that the protein kinase has a homodimeric structure.
Reaction of tryptophanyl‐tRNA synthetase from beef pancreas with periodate‐oxidized ATP
Authors: FOURNIER Michel , LABOUESSE Bernard , LABOUESSE Julie
Published: March 2005

Tryptophanyl‐tRNA synthetase from beef pancreas reacts with periodate‐oxidized ATP according to biphasic kinetics. A rapid phase involves two groups of the protein, presumably lysine side‐chains. The slow phase corresponds to the reaction of a larger number of groups. The time‐course of the partial losses of the ATP–PPi isotopic exchange and of the aminoacylation activities of the enzyme follow the labelling of the two fast‐reacting groups. However, the ability of the enzyme to form a bis(tryptophanyladenylate)‐enzyme complex is not lost after reaction of these two groups with the reagent. The affinity for ATP is also unaffected by this initial labelling of the protein, as seen from the Km values of this substrate in the ATP–PPi isotopic exchange reaction. These data suggest that, in this fast initial reaction, oxidized ATP reacts neither with specific ATP‐binding groups of the enzyme nor with any major catalytic residue of the tryptophan‐activation site. In contrast with this first step, the further slow labelling of lysine residues leads to a disappearance of the aminoacylation ability of the enzyme, while it does not further affect the ATP–PPi exchange activity. The behaviour of beef tryptophanyl‐tRNA synthetase during derivatization with oxidized ATP is therefore at variance with that which has been described for the homologous E. coli enzyme.
Chlorophyll‐protein composition of the thylakoid membrane from Prochlorothrix hollandica, a prokaryote containing chlorophyll b
Authors: BULLERJAHN George S. , MATTHUS Hans C. P. , MUR Luuc R. , SHERMAN Louis A.
Published: March 2005

The chlorophyll‐protein complexes of the thylakoid membrane from Prochlorothrix hollandica were identified following electrophoresis under nondenaturing conditions. Five complexes, CP1 – CP5, were resolved and these green bands were analyzed by spectroscopic and immunological methods. CP1 contains the photosystem I (PSI) reaction center, as this complex quenched fluorescence at room temperature, and had a 77 K fluorescence emission peak at 717 nm. CP4 contains the major chlorophyll‐a‐binding proteins of the photosystem II (PSII) core, because this complex contained polypeptides which cross‐reacted to antibodies raised against Chlamydomonas PSII proteins 5 and 6. Furthermore, fluorescence excitation studies at 77 K indicated that only Chl a is bound to CP4. Complexes CP2, CP3 and CP5 contained functionally bound Chl a and b as judged by absorption spectroscopy at 20°C and fluorescence excitation spectra at 77 K. CP2, CP3 and CP5 all contain polypeptides of 30 – 33 kDa which are immunologically distinct from the LHC‐II complex of higher plant thylakoids.
Characterization of three constituent chains of collagen type VI by peptide sequences and cDNA clones
Authors: CHU Mon‐Li , MANN Karlheinz , DEUTZMANN Rainer , PRIBULA‐CONWAY Dorothy , HSU‐CHEN Chuen‐Chin , BERNARD Michael P. , TIMPL Rupert
Published: March 2005

Pepsin‐solubilized collagen VI was prepared from human placenta and used to separate three constituent chains for determining partial amino acid sequences. Antibodies raised against the chains assisted in the identification and purification of several cDNA clones from three expression λgt11 libraries. Most of the clones hybridized to either a 3.5‐kb or 4.2‐kb mRNA species which by matching peptide and nucleotide sequences could be identified as coding for the α2(VI) or α1(VI) chain, respectively. Other clones hybridized to either an 8.5‐kb mRNA which very likely encodes the α3(VI) chain or to an unknown 2.0‐kb mRNA. Northern blots revealed a considerable variation in the mRNA levels for each collagen VI chain in both skin and cornea fibroblasts and in several tumor cell lines. Limited sequence data generated from peptides and cDNA clones demonstrated a characteristic cysteine pattern at the junction between N‐terminal globular domain and triple helix in all three chains. In addition, the data showed occasional interruptions of triplet sequences within the triple‐helical domain and the presence of two Arg‐Gly‐Asp sequences which are potential cell‐binding structures.
Structure and evolution of the repetitive gene encoding streptococcal protein G
Authors: OLSSON Anders , ELIASSON Margareta , GUSS Bengt , NILSSON Björn , HELLMAN Ulf , LINDBERG Martin , UHLÉN Mathias
Published: March 2005

The complete sequence of the structural gene encoding the immunoglobulin G binding protein from Streptococcus G148 has been determined, as well as its 5′ and 3′ flanking sequences. The sequence reveals an open reading frame encoding a putative preprotein with a relative molecular mass of 63294. N‐Terminal sequencing of the mature protein, spontaneously released from streptococcal cells, demonstrates that the signal peptide consists of 33 amino acids. The DNA sequence reveals extensive internal homologies similar to other cell‐wall‐bound receptors from gram‐positive bacteria. Comparisons with a related gene previously isolated from another strain of streptococci revealed large differences in size, due to variations in the number of internal repeats. The structure of the gene suggests an evolution through multiple duplications.
Spatial relationship between the nucleotide‐binding site, Lys‐61 and Cys‐374 in actin and a conformational change induced by myosin subfragment‐1 binding
Authors: MIKI Masao , dos REMEDIOS Cristobal G. , BARDEN Julian A.
Published: March 2005

The spatial relationship between Lys‐61, the nucleotide binding site and Cys‐374 was studied. Lys‐61 was labelled with fluorescein‐5‐isothiocyanate as a resonance energy acceptor, the nucleotide‐binding site was labelled with the fluorescent ATP analogues ɛATP or formycin‐A 5′‐triphosphate (FTP) and Cys‐374 was labelled with 5‐{2‐[(iodoacetyl)amino]ethyl}aminonaphthalene‐1‐sulfonic acid (1,5‐IAEDANS) as a resonance energy donor. The distances between the nucleotide binding site and Lys‐61 or between Lys‐61 and Cys‐374 were calculated to be 3.5 ± 0.3 nm and 4.60 ± 0.03 nm, respectively. (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) On the other hand, when doubly‐labelled actin with 1,5‐IAEDANS at Cys‐374 and FITC at Lys‐61 was polymerized in the presence of a twofold molar excess of phalloidin [Miki, M. (1987) Eur. J. Biochem. 164, 229–235], the fluorescence of 1,5‐IAEDANS bound to actin was quenched significantly. This could be attributed to inter‐monomer energy transfer. The inter‐monomer distance between FITC attached to Lys‐61 in a monomer and 1,5‐IAEDANS attached to Cys‐374 in its nearest‐neighbour monomer in an F‐actin filament was calculated to be 3.34 ± 0.06 nm, assuming that the likely change in the intra‐monomer distance does not change during polymerization by more than 0.4 nm. One possible spatial relationship between Lys‐61, Cys‐374 and the nucleotide binding site in an F‐actin filament is proposed.

The effect of myosin subfragment‐1 (S1) binding on the energy transfer efficiency was studied. The fluorescence intensity of AEDANS‐FITC‐actin decreased by 30% upon interaction with S1. The fluorescence intensity of AEDANS‐FITC‐actin polymer in the presence of phalloidin increased by 21% upon interaction with S1. The addition of ATP led to the fluorescence intensity returning to the initial level. Assuming that the change of fluorescence intensity can be attributed to a conformational change in the actin molecule induced by S1 binding, the intra‐monomer distance was reduced by 0.4 nm and the inter‐monomer distance was increased by 0.2 nm.
The structure of endothelial cell thrombospondin
Authors: DARDIK Rima , LAHAV Judith
Published: March 2005

The glycoprotein thrombospondin is distributed between the extracellular matrix and the platelet‐sequestered pool in the resting state and it undergoes redistribution upon platelet stimulation. It is believed to play a role in matrix structure and in coagulation. We have studied the structural domains of endothelial cell (EC) thrombospondin by use of the serine proteases thrombin, trypsin and chymotrypsin and have characterized the heparin‐binding domains of this molecule. For this purpose we used purified thrombospondin synthesized and secreted by bovine aortic endothelial cells grown in the presence of radiolabeled methionine. We find that the susceptibility of EC thrombospondin to proteolysis is five‐fold smaller than that of platelet thrombospondin. In the presence of 2 mM Ca ions the molecule is cleaved by 20 U/ml thrombin at a single locus, to yield fragments of 160 kDa and 35 kDa. Trypsin digestion for 5 min at room temperature at an enzyme‐to‐substrate ratio of 1:20 produces a stable fragment of 140 kDa but not the 30‐kDa fragment observed in platelet thrombospondin. Chymotrypsin, under identical conditions to those used for trypsin, cleaves EC thrombospondin into four stable fragments of 160 kDa, 140 kDa, 27 kDa and 18 kDa. Chelation of Ca by EDTA increases susceptibility of the molecule to proteolysis. Under the conditions used a cryptic thrombin‐cleavage site, not hitherto observed in platelet thrombospondin, was observed in EC thrombospondin. The location of this site is near a chymotrypsin‐susceptible site, which has been observed in the long connecting arm, which is particularly Ca‐stabilized. Heparinbinding capacity of EC thrombospondin was observed in at least two separate loci. Both thrombin and chymotrypsin produced small fragments (35 kDa and 27 kDa respectively) which bound to heparin with high affinity, and large fragments (160 kDa for thrombin and 140 kDa for chymotrypsin) which had low affinity. Chelation of Ca substantially decreased the low‐affinity binding of the large fragments but not the high‐affinity binding of the small fragments. Two‐dimensional gel electrophoresis of the chymotryptic heparin‐binding fragments shows that each molecule gave rise to a heterogeneous array of fragments of high molecular mass bound by disulfide bonds, indicating that there is a difference in the rate of cleavage between the three subunits of EC thrombospondin. Trypsin, despite its limited degradation, completely eliminated the heparin‐binding capacity of both high and low‐affinity loci, in contrast to platelet thrombospondin where the high affinity remains intact. Our data suggest that EC thrombospondin is less susceptible to degradation and removal than platelet thrombospondin and could, therefore, function under harsher conditions as perhaps exist at the platelet – matrix interface following activation of coagulation.
Activation of a ribosomal protein S6 kinase in mouse fibroblasts during infection with herpesvirus
Authors: JAKUBOWICZ Teresa , LEADER David P.
Published: March 2005

If confluent fibroblasts are infected with the swine α‐herpes virus, pseudorabies virus, ribosomal protein S6 becomes phosphorylated after a lag of approximately 2 h. When cell‐free extracts were prepared from such cells in the presence of glycerol 2‐phosphate and EGTA, a ribosomal protein S6 kinase activity was found to appear at approximately the same time as the phosphorylation in vivo. This protein kinase was similar to that activated in the same cells by replenishing the nutrient medium, and in other quiescent cells by the action of growth factors and mitogens. It was distinct from the previously described pseudorabies virus protein kinase, which is unique to infected cells. When medium from cells infected with pseudorabies virus was freed of virus and added to confluent fibroblasts, rapid activation of the ribosomal protein S6 kinase activity occurred. A similar, although more limited, effect could be seen when the pH of the medium was increased. These results suggest that the phosphorylation of ribosomal protein S6 in cells infected with herpes virus is a consequence of the production of a factor which initiates the metabolic programme for cellular growth. The possible function of this effect in the infective strategy of herpes viruses is discussed in relation to requirements for the replication of viral DNA.
Purification of phosphoinositide‐specific phospholipase C from a particulate fraction of bovine brain
Authors: KATAN Matilda , PARKER Peter J.
Published: March 2005

The coupling of various agonist receptors to the hydrolysis of phosphoinositides has generated much interest in the nature of the phospholipase C that is activated. Here we report the purification of a bovine brain phospholipase C derived from the particulate fraction. A 1000‐fold purification was achieved by a combination of heparin‐Sepharose, DEAE‐cellulose and gel‐permeation chromatography. The purified enzyme appears to be monomeric and under denaturing conditions shows a single staining major polypeptide of molecular mass 154 kDa in SDS gels. The enzyme is specific for phosphoinositides although it shows a marked preference for the polyphosphoinositides. With phosphatidylinositol 4,5‐bisphosphate as substrate the enzyme expresses a specific activity of > 100 μmol min−1 mg−1. The phospholipase C is activated by Ca2+ (0.1 – 10 μM). The behaviour of this particulate enzyme is discussed in the context of agonist‐induced phosphatidylinositol hydrolysis.
Characterization of the Na+‐stimulated ATPase of Propionigenium modestum as an enzyme of the F1F0 type
Authors: LAUBINGER Werner , DIMROTH Peter
Published: March 2005

The ATP‐hydrolyzing activity of Propionigenium modestum was extracted from the membranes with Triton X‐100 or by incubation with EDTA at low ionic strength. The ATPase in the Triton extract was highly sensitive to dicyclohexylcarbodiimide but not to vanadate. These properties are characteristic for enzymes of the F1F0 type. The ATPase was specifically activated by Na+ ions yielding a 15‐fold increase in catalytic activity at 5 mM Na+ concentration. The additional presence of 1% Triton X‐100 caused a further 1.5–fold activation. In the absence of Na+ Triton stimulated the ATPase about 13‐fold. The Triton‐stimulated ATPase was further activated about 1.5–2‐fold by Na+ addition.

The ATPase extracted by the low‐ionic‐strength treatment was purified to homogeneity by fractionation with poly(ethylene glycol) and gel chromatography. The enzyme had the characteristic F1‐ATPase subunit structure with Mr values of 58000 (α), 56000 (β), 37600 (γ), 22700 (δ), and 14000 (ɛ). The F1‐ATPase was not stimulated by Na+ ions. The membrane‐bound ATPase was reconstituted from the purified F1 part and F1‐depleted membranes, thus further indicating an F1F0 structure for the ATPase of P. modestum. Upon reconstitution the ATPase recovered its stimulation by Na+ ions, suggesting that the binding site for Na+ is localized on the membrane‐bound F0 part of the enzyme complex.
Specificity and other properties of three ribonucleases of Tetrahymena pyriformis
Authors: MAOURI Anastasia , GEORGATSOS John G.
Published: March 2005

Three ribonucleases, RNase I, RNase II and RNase III, were purified from the 109000 ×g supernate of detergent‐treated Tetrahymena pyriformis strain W. RNases I and II act optimally at pH 5.5–6.0 and are inhibited by increasing concentrations of salts of monovalent cations. RNase III acts optimally at pH 7.5 and is activated 1.5‐fold by millimolar concentrations of ZnSO4 and 5‐fold by 50 mM KCl. RNases II and III are activated approximately 100% in the presence of 3 M and 5 M urea respectively. All enzymes are heat‐sensitive and acid‐resistant. They are endonucleases forming 2′,3′‐cyclic products. Their base specificity, as tested against ribosomal RNAs of known sequence, is as follows: RNase I hydrolyzes preferentially YpN and secondarily GpN bonds, RNase II is highly specific for RpN bonds, though the preparation can also hydrolyze the UpU sequence. Finally the principal targets of RNase III are YpR sequences and secondarily YpY sequences. A shorthand visualization of base specificity of nucleases in the form of right isosceles triangles is presented. The triangles are constructed by subdividing each of the two perpendicular sides in as many units as the maximum number of times the most abundant dinucleotide appears in all substrates employed and plotting the frequency of hydrolysis of each dinucleotide sequence by the enzyme under study. The proximity of each dinucleotide sequence to the hypotenuse or to one of the perpendicular sides is indicative of its susceptibility or resistance to the enzyme's action.
Construction of a model for the aggregation and cross‐linking region (7S domain) of type IV collagen based upon an evaluation of the primary structure of the α1 and α2 chains in this region
Authors: SIEBOLD Bernhard , QIAN Rui‐qing , GLANVILLE Robert W. , HOFMANN Hans , DEUTZMANN Rainer , KÄHN Klaus
Published: March 2005

The amino acid sequence of the 212‐residues‐long N‐terminal aggregation and cross‐linking region of the α2(IV) chain of human basement membrane collagen is presented. Comparing this with the primary structure of α1(IV)7S [Glanville et al. (1985) Eur. J. Biochem. 152, 213–219] revealed a high degree of similar subdivisions in three functional regions. These are the 21‐residue‐long N‐terminal non‐triple‐helical regions (NH1) containing cysteine and lysine residues which are putative cross‐linking sites, a 117‐residue‐long triple‐helical region (TH1) responsible for the aggregation of four molecules to form the 7S domain and which also possess cross‐linking sites, and finally a 10‐residues‐long non‐triple‐helical region (NH2) which introduces the first of many flexible areas into the triple helical body of the molecule [Hofmann et al. (1984) J. Mol. Biol. 172, 325–343].

Computer calculations of interaction scores between parallel and antiparallelly aligned triple‐helical regions (TH1) of the 7S domain allowed the prediction of a detailed model for the structure of the 7S complex which agreed well with models based primarily on electron micrographs of rotary shadowed type IV collagen tetramers. The results indicated that the assembly of the 7S domain is directed by hydrophobic interactions and is self‐limiting to a tetramer. The most favourable chain configuration is α2‐α1‐α1.
Brain pyridoxal kinase dissociation of the dimeric structure and catalytic activity of the monomeric species
Authors: KWOK Francis , SCHOLZ Glen , CHURCHICH Jorge E.
Published: March 2005

Reversible dissociation of the dimeric structure of brain pyridoxal kinase into subunits was attained by addition of guanidinium · HCl (2 M). The molecular mass of the subunits (40 kDa) was determined by HPLC chromatography.

Separation of the processes of refolding and association of the monomeric species was achieved by attaching the protein subunits to a rigid matrix (Affi‐gel 15). The matrix‐bound monomer is catalytically competent. The reaction of the crosslinking reagent 4,4′‐dimaleimidestilbene 2,2′‐disulfonate (DMDS), a derivatized stilbene, with the dimeric structure of pyridoxal kinase resulted in the formation of an oligomeric species of 80 kDa detectable by SDS‐PAGE. The crosslinked subunits exhibit the same catalytic parameters as the native enzyme. The presence of two nucleotide‐binding sites per dimer was determined by fluorimetric titrations using pyridoxyl‐ATP, a strong competitive inhibitor with respect to ATP.

The ATP analog binds with a Kd= 5 μM to each nucleotide site of the dimeric enzyme. The mode of binding pyridoxyl‐ATP to the kinase is discussed in reference to a model which assumes the presence of two binding domains per subunit.
Monitoring the purification by high‐performance liquid chromatography of cardiotoxins from Naja mossambica mossambica using phase‐sensitive two‐dimensional nuclear magnetic resonance
Authors: OTTING Gottfried , MARCHOT Pascale , BOUGIS Pierre E. , ROCHAT Hervé , WÜTHRICH Kurt
Published: March 2005

High‐resolution phase‐sensitive two‐dimensional proton nuclear magnetic resonance was used to monitor the preparation by high‐performance liquid chromatography of homogeneous proteins from the venom of Naja mossambica mossambica. This resulted in the characterization of a heterogeneous protein preparation VII2, which had been used in earlier structural studies by NMR, as well as a homogeneous protein CTXIIb and a nearly homogeneous protein fraction CTXIIa, which are now both subject to further investigations of their solution conformations.
Effect of bacteriophage M13 infection on phosphorylation of dnaK protein and other Escherichia coli proteins
Authors: RIEUL Corinne , CORTAY Jean‐Claude , BLEICHER Françoise , COZZONE Alain J.
Published: March 2005

The effects of infection with the filamentous phage M13 on the phosphorylation of Escherichia coli proteins were studied. Phosphorylated proteins were labeled with [32P]orthophosphate and analyzed by the O'Farrell two‐dimensional gel technique and autoradiography.

Phage infection was shown to induce significant changes in the pattern of protein phosphorylation. At least eight different proteins were found to be phosphorylated to a larger extent while seven others were, by contrast, much less labeled than in uninfected bacteria.

Labeling experiments with [35S]methionine demonstrated that these quantitative changes in protein phosphorylation were not connected, in any case, with changes in the amount of protein synthesized. They rather seemed to result from a variation of the phosphorylating capacity of the relevant protein kinase(s).

The individual proteins, whose phosphorylation was affected by phage infection, were characterized by both their molecular mass and isoelectric point. One of them, whose phosphorylation was increased by a factor of 7, was identified as the dnaK protein which is necessary for both cellular and phage DNA replication.

The chemical analysis of the phosphorylated moiety of dnaK protein showed that it was modified exclusively at serine residues during normal growth of cells, and mostly at threonine residues after phage infection. These results were discussed in terms of stimulation of the protein activity by phosphorylation.
Biochemical characterization of the soluble form of the junctional plaque protein, plakoglobin, from different cell types
Authors: KAPPRELL Hans‐Peter , COWIN Pamela , FRANKE Werner W.
Published: March 2005

A polypeptide of identical molecular mass (Mr 83000) and charge to desmosomal plakoglobin from bovine snout epidermis was identified in soluble and pelletable fractions from diverse tissues and cells of different mammalian species, including cells and tissues devoid of desmosomes (e.g. endothelial, retinal, lenticular cells, fibroblasts). The protein, however, was not detected in erythrocytes and platelets and in myeloma cells, nor in smooth muscle tissue. In all cells examined, the plakoglobin soluble upon cell lysis in buffers of near‐physiological pH and ionic strength (21–31% of the total plakoglobin in the different cell types) was found to exist in a distinct molecular form. On sucrose gradient centrifugation it appeared at about 7 S and gel filtration chromatography revealed a Stokes radius of about 5.0 nm, from which an Mr of about 170000 was estimated. By using isoelectric focusing under non‐denaturing conditions, soluble ∼ 7‐S plakoglobin had an isoelectric point at about pH 5.3. The plaque‐bound and the soluble form of plakoglobin were indistinguishable by electrical charge and molecular mass, regardless of the source, indicating molecular identity. Cross‐linking of soluble proteins with cupric 1,10‐phenanthroline resulted in the formation of a cross‐linked product of plakoglobin with similar physical properties as the native ∼ 7‐S particle, which is compatible with the interpretation that the soluble plakoglobin particle is a dimer. While a major proportion of the plakoglobin in the desmosomal plaque was resistant to various extraction procedures, plakoglobin present in the plaques of non‐desmosome‐containing cells and tissues was readily extractable under low and high salt conditions. This indicates that differences exist in the binding of plakoglobin to desmosomal plaques and the plaques of non‐desmosomal junctions.
Processing of a plant vacuolar protein precursor in vitro
Authors: HATTORI Tsukaho , NAKAMURA Kenzo , ICHIHARA Shigeyuki
Published: March 2005

A precursor for sporamin A, the storage protein of the tuberous roots of sweet potato deposited in the vacuole, is synthesized on membrane‐bound polysomes and has an extra peptide of 37 amino acids at the N‐terminus of the mature form, which can be divided into an N‐terminal putative signal peptide sequence (residues −37 to −17) and a segment enriched with charged amino acids (residues −16 to −1) [Hattori, T., et al. (1985) Plant Mol. Biol. 5, 313–320]. We examined the in vitro processing of the sporamin A precursor using a messenger RNA derived from a full‐length cDNA by the SP6 transcription system. When the in vitro translation in a wheat germ cell‐free system was carried out in the presence of dog pancreas microsomal membranes, the precursor polypeptide (Mr= 24000) was processed into an intermediate form still larger than the mature polypeptide (Mr= 20 000). The processed intermediate form was also produced by addition of microsomal membranes from sweet potato and potato in the translation reaction, although less efficiently compared to dog membranes. Moreover, Escherichia coli cells expressing sporamin precursor accumulated a polypeptide with the same electrophoretic mobility as the intermediate form produced in vitro.

The processing by dog membranes is accompanied by translocation of the polypeptide across the membranes as assayed by resistance to externally added proteases. The N‐terminal amino acid sequencing analysis of [3H]leucine‐labelled intermediate form produced in vitro by dog membranes indicated that co‐translational processing of the sporamin precursor by endoplasmic reticulum membranes removes only the signal peptide segment from the extra peptide, and suggested that the charged segment following the signal peptide is removed post‐translationally during the transport of sporamin into vacuole. The significance of two‐step processing of plant vacuolar protein precursor is discussed in relation to the two‐step processing of precursors for yeast vacuolar proteins and animal lysosomal proteins.
Structure and expression of genes coding for xylan‐degrading enzymes of Bacillus pumilus
Authors: MORIYAMA Hideaki , FUKUSAKI Eiichiro , CABRERA CRESPO Joaquin , SHINMYO Atsuhiko , OKADA Hirosuke
Published: March 2005

The complete nucleotide sequence of the β‐xylosidase gene (xynB) of Bacillus pumilus IPO and its flanking regions was established. A 1617‐bp open reading frame for β‐xylosidase, a homodimer enzyme, was observed. The amino acid sequence of the N‐terminal region and the molecular mass (62607 Da) of the β‐xylosidase subunit, deduced from the DNA sequence, agreed with the result obtained with the purified enzyme. The Shine‐Dalgarno sequence was found 8 bp upstream of the initiation codon, ATG.

The xylanase gene (xynA) of the same strain was 4.6 kbp downstream of the 3′ end of xynB, and its DNA sequence was reported in our previous paper [Fukusaki, E., Panbangred, W., Shinmyo, A. & Okada, H. (1984) FEBS Lett. 171, 197–201]. The results of the Northérn hybridization suggested that the mRNA of xynA and xynB were produced separately. The 5′ and 3′ ends of the xynA and xynB gene were mapped with nuclease S1. The'‐10′ regions for promoter sequences of both genes were similar to the consensus sequence for B. subtilis RNA polymerases, the'‐35′ regions were different from all the known promoters for B. subtilis RNA polymerases.
Kinetic characterization of cytochrome c oxidase from Bacillus subtilis
Authors: de VRIJ Wim , KONINGS Wil N.
Published: March 2005

Bacillus subtilis aa
3‐type cytochrome c oxidase is capable of oxidizing cytochrome c from different origins. The kinetic properties of the enzyme are influenced by ionic strength. The affinity for Saccharomyces cerevisiae cytochrome c declines with increasing ionic strength whereas the Vmax remains almost constant. An increase of Vmax is observed when the enzyme is incorporated in artificial membranes. Negatively charged phospholipids allow high turnover rates of the aa3‐type oxidase. The effect of ionic strength on oxidation of horse heart cytochrome c results in significant changes of both Km and Vmax. These effects can be explained by disturbances of enzyme‐substrate interactions and are not related to changes in the aggregation state of the enzyme. The respiration control index of the enzyme reconstituted in artificial membranes appeared to be dependent on phospholipid composition, protein/lipid ratios and also on the external pH. The action of the ionophores nigericin and valinomycin, at various pH values, on the enzyme activity and proton‐permeability measurements of the membranes indicate that both components of the proton‐motive force, the membrane potential and the pH gradient, can in principle regulate enzyme activity in the reconstituted state.