bluuebunny/crossref_metadata_2025 huggingface dataset:
Lateral diffusion of phospholipids in the lipid surface of human low‐density lipoprotein measured with a pyrenyl phospholipid probe
Authors: VAUHKONEN Matti , SASSAROLI Massimo , SOMERHARJU Pentti , EISINGER Josef
Published: March 2005

Human low‐density lipoprotein (LDL) was labelled with the excimeric fluorescent phospholipid analogue 1‐palmitoyl‐2‐(1′‐pyreneoctanoyl)‐sn‐glycero‐3‐phosphocholine by using phosphatidylcholine‐specific transfer protein for the probe insertion. The lateral diffusivity of the probe in the phospholipid/cholesterol surface monolayer of LDL was determined from the measured dependence of the pyrene monomer fluorescence yield on probe concentration. The data were analyzed by the milling‐crowd model [J. Eisinger et al. (1986) Biophys. J. 49, 987–1001] to obtain the short‐range lateral diffusivity of the probe. The lateral mobility of the probe in LDL was compared to that in model lipid systems, i.e. in protein‐free LDL‐like lipid particles and in small unilamellar vesicles, with a phospholipid/cholesterol composition characteristic of LDL. This analysis with the probability PE= 1 for excimer production between nearest‐neighbour probes gives the lower limits for f, the frequency of translational lipid–lipid exchanges of the probe of 0.62 × 108, 0.19 × 108 and 0.19 × 108 s−1 in LDL, LDL‐like lipid particles, and small unilamellar vesicles, respectively. The lower limits for the corresponding lateral diffusion constants are 16, 5 and 5 μm2 s−1. The results suggest that the translational mobility of phospholipid molecules in the lipid–protein surface of LDL is not constrained by the apolipoprotein B‐100 moiety or the neutral lipid core of the lipoprotein. Instead, the protein moiety may perturb the lipid order with the lipid–associating peptide domains and thus fluidize the amphiphilic surface monolayer of LDL relative to the protein‐free model systems. In general, lateral diffusivity of the pyrenyl phospholipid probe in LDL and the model lipid systems is comparable to the lateral mobility of lipid analogue probes in a variety of model and biological membranes.
Thiol‐directed immobilization of recombinant IgG‐binding receptors
Authors: LJUNGQUIST Charlotta , JANSSON Birger , MOKS Tomas , UHLÉN Mathias
Published: March 2005

A genetic engineering approach is described for directed immobilization of IgG‐binding receptors to a thiolcontaining matrix using a single cysteine residue. The cysteine residue is introduced into the C‐terminal part of receptors based on staphylococcal protein A. Receptors containing one, two or five IgG‐binding domains were produced in Escherichia coli and subsequently immobilized on thiopropyl‐Sepharose. A high amount, 5 μmol/ml gel (45 mg/ml), of recombinant receptor could be rapidly immobilized to the solid support and both the gel and the immobilized receptor could be regenerated by reduction and oxidation reactions. The gel was used for affinity purification of human IgG and analysis of IgG‐binding capacity at different amounts of immobilized recombinant protein show the same maximal IgG‐binding capacity (20–25 mg/ml) for all three immobilized receptors. However, at low substitution grade of receptors, the immobilized receptor molecules were shown to bind one (Z‐Cys) and two (ZZ‐Cys) IgG molecules, respectively. These results demonstrate that the immobilized protein molecules are in a functionally active form and that a two‐domain receptor can bind two molecules of IgG without steric hindrance. Interestingly, the five‐domain receptor (ZV‐Cys), with a structure similar to native protein A, can only bind approximately two IgG molecules, despite the five‐domain structure of the molecule.
Immobilization and affinity purification of recombinant proteins using histidine peptide fusions
Authors: LJUNGQUIST Charlotta , BREITHOLTZ Anna , BRINK‐NILSSON Helena , MOKS Tomas , UHLÉN Mathias , NILSSON Björn
Published: March 2005

A gene fusion approach to simplify protein immobilization and purification is described. A gene encoding the protein of interest is fused to a gene fragment encoding the affinity peptide Ala‐His‐Gly‐His‐Arg‐Pro. The expressed fusion proteins can be purified using immobilized metal affinity chromatography. A vector, designed to ensure obligate head‐to‐tail polymerization of oligonucleotide linkers was constructed by in vitro mutagenesis. A linker encoding the affinity peptide, was synthesized and polymerized to two, four and eight copies. These linkers were fused to the 3′ end of a structural gene encoding a two‐domain protein A molecule, ZZ, and to the 5′ end of a gene encoding β‐galactosidase. Fusion proteins, of both types, with zero or two copies of the linker showed little or no binding to immobilized Zn2+, while a relatively strong interaction could be observed for the fusions based on four or eight copies of the linker. Using a pH gradient, the ZZ fusions were found to be eluted from the resin at different pHs depending on the number of the affinity peptide. These results demonstrate that genetic engineering can be used to facilitate purification and immobilization of proteins to immobilized Zn2+ and that the multiplicity of the affinity peptide is an important factor determining the binding characteristics.
Kinetics of the reaction of compound II of horseradish peroxidase with hydrogen peroxide to form compound III
Authors: ADEDIRAN Suara A. , LAMBEIR Anne‐Marie
Published: March 2005

The kinetics of the reaction of H2O2 with compound II of horseradish peroxidase were studied as a function of pH at 25°C and constant ionic strength of 0.11 M. The reaction of H2O2 with compound II involves the transient formation of ferric peroxidase and superoxide anion as the first step followed by the reaction of the intermediate species with H2O2 to form compound III. Both reactions are also observed with peracetic acid as substrate, though the amplitude of the first step was too small for the rate to be measured. Observation of the first reaction was not possible below pH 8.5 under the conditions of this investigation. It tends to occur faster at lower pH so an increasing fraction is lost in the dead time of the stopped‐flow apparatus. The rate constants for the second reaction, leading to compound III formation, are small at all pH values, with a maximum of 20 M−1 s−1 at pH 7.0. Groups on the enzyme intermediate species with pKa values of 4.2 and 9.1 appear to be involved in this reaction. Compound III formation is accompanied by oxidation of aromatic amino acid groups on the protein. The compound III formed from horseradish peroxidase compound II and hydrogen peroxide has bands with molar absorption coefficients in excellent agreement with those obtained by flash photolysis of aerated carbonmonoxyperoxidase [Wittenberg, J. B., Noble, R. W., Wittenberg, B. A., Antonini, E., Brunori, M. and Wyman, J. (1967) J. Biol. Chem. 242, 626–634]. Attempts to use m‐chloroperbenzoic acid as oxidant resulted in the destruction of compound II.
Poly(A) polymerase from Vigna unguiculata seedlings
Authors: TARUI Yutaka , MINAMIKAWA Takao
Published: March 2005

Poly(A)‐specific ribonuclease was co‐purified with poly(A) polymerase from Vigna unguiculata seedlings. Both activities were separated into two forms (enzymes I and II) by a final hydrophobic column chromatography. The enzyme I preparation, which was homogeneous as examined by SDS/PAGE, had both poly(A) polymerase and poly(A)‐specific ribonuclease activities. The antibody raised to the enzyme I preparation precipitated both enzyme activities. These indicate that a single polypeptide (Mr 63 000) is responsible for both poly(A)‐polymerizing and poly(A)‐hydrolyzing activities. The poly(A)‐specific ribonuclease was a 3′‐exonuclease specific to single‐stranded poly(A), forming 5′AMP as the sole reaction product. The hydrolytic activity required either Mn2+ or Mg2+ with different optimum concentrations, whereas the polymerizing activity required Mn2+ but not Mg2+. ATP and PPi had little or no effect on the poly(A)‐specific ribonuclease activity.
Effects of lipopolysaccharide chemotype structure on binding and inactivation of hen egg lysozyme
Authors: OHNO Naohito , MORRISON David C.
Published: March 2005

The structural features of lipopolysaccharide [LPS] which influence the binding and inactivation of lysozyme have been examined. Binding of polysaccharide‐containing LPS (S‐LPS) and Ra – Rc‐LPS preparations was independent of temperature between 37 – 50°C; in contrast, binding of Rd‐LPS, Re‐LPS and lipid A was temperature‐dependent. The binding of lysozyme to Rd‐LPS and Re‐LPS was increased by treatment with mild alkali, which has little detectable effect on binding of lysozyme to S‐LPS and Ra – Rc‐LPS preparations. Competitive binding experiments using dansylated lysozyme and/or dansylated polymyxin B indicated independent binding sites on the LPS for lysozyme and polymyxin B. These results indicate significant differences between most LPS preparations and lipid A and glycolipid LPS in their interaction with proteins of mammalian origin.
Lipopolysaccharide interactions with lysozyme differentially affect lipopolysaccharide immunostimulatory activity
Authors: OHNO Naohito , MORRISON David C.
Published: March 2005

The effect of complex formation between lysozyme and lipopolysaccharide (LPS) on the immunostimulatory activities of LPS have been investigated in vitro. Three prototype immunostimulatory activities were examined: B‐lymphocyte proliferation, B‐lymphocyte differentiation and macrophage production of lymphocyte‐activating factor activity. Different effects of lysozyme were noted, depending upon the structure of the LPS, even though previous studies have established that all LPS preparations readily bind lysozyme. Both Re‐LPS‐ and lipid‐A‐dependent immunostimulatory activities were readily inhibited by lysozyme in a dose‐dependent fashion. In contrast, S‐LPS and Ra‐LPS were unaffected in their immunostimulatory activities by lysozyme. These differences were not the result of quantitative differences in LPS binding of lysozyme, or effects of lysozyme on overall binding of LPS to target cells. These data suggest that the factors which dictate the initial interactions between LPS and lymphoreticular cells may not be identical for all LPS preparations and/or purified lipid A.
Hydroxyurea increases the phosphorylation of 3′‐fluorothymidine and 3′‐azidothymidine in CEM cells
Authors: KARLSSON Anna , REICHARD Peter , ECKSTEIN Fritz
Published: March 2005

The triphosphates of the nucleoside analogues 3′‐azidothymidine and 3′‐fluorothymidine inhibit reverse transcriptase and are of therapeutic interest for the treatment of retrovirus infections. At equimolar concentrations 3′‐fluorothymidine was more effectively transformed to the triphosphate by human CEM cells than azidothymidine which mainly accumulates as the monophosphate. Hydroxyurea, a drug that inhibits de novo synthesis of deoxyribonucleotides, considerably increased the ability of cells to phosphorylate both analogues. Addition of as little as 50 μM hydroxyurea decreased the amount of dideoxynucleoside required to attain a given intracellular concentration of its triphosphate by an order of magnitude. Hydroxyurea is known to shift the balance of substrate cycles between natural deoxynucleosides and their 5′‐phosphates in the direction of synthesis and thereby to increase the import and intracellular phosphorylation of the nucleoside. The present results demonstrate a similar effect for the two analogues and raise the possibility of using this effect in therapy.
13C‐NMR study of acetate assimilation in Thermoproteus neutrophilus
Authors: SCHÄFER Sigrid , PAALME Toomas , VILU Raivo , FUCHS Georg
Published: March 2005

Acetate assimilation into amino acids and the functioning of central biosynthetic pathways in the extremely thermophilic anaerobic archaebacterium Thermoproteus neutrophilus was investigated using 13C NMR as the method for determination of the labelling patterns. Acetate was assimilated via reductive carboxylation of acetyl‐CoA to pyruvate and pyruvate conversion to phosphoenolpyruvate which was further carboxylated to oxaloacetate. 2‐Oxoglutarate was mainly formed via citrate. However, the labelling patterns of glutamic acid and alanine were in agreement with the concurrent synthesis of about 15% 2‐oxoglutarate and 5% pyruvate through the reductive citric acid cycle. A scrambling phenomenon occurring in aspartate and all amino acids derived through oxaloacetate was observed. The labelling patterns of amino acids were in agreement with their standard biosynthetic pathways, with two remarkable exceptions: isoleucine was synthesized via the citramalate pathway and lysine was synthesized via the 2‐aminoadipate pathway which has previously been reported only in eukaryotic microorganisms.
Regulation of protein phosphatase‐1G from rabbit skeletal muscle
Authors: HUBBARD Michael J. , COHEN Philip
Published: March 2005

The glycogen‐associated form of protein phosphatase‐1 (PP‐1G) comprises a 37‐kDa catalytic (C) subunit and a 161‐kDa glycogen‐binding (G) subunit. In the preceding paper in this issue of the journal we showed that the C subunit is released from PP‐1G in response to phosphorylation of the G subunit by cAMP‐dependent protein kinase. We now show that at 0.15–02 M KCl the phosphorylase phosphatase activity of glycogen‐bound PP‐1G is 5–8 times higher than that of released C subunit or unbound PP‐1G, which are strongly inhibited at these ionic strengths. The activity of glycogen‐bound PP‐1G towards glycogen synthase was about 5‐fold higher than that of released C subunit at 0.15M KCl. Studies with glycogen‐bound substrates and myosin P‐light chain (which does not interact with glycogen) indicated that PP‐1G activity is only enhanced compared to free C subunit at near physiological ionic strength and when both PP‐1G and substrate are glycogen‐associated.

The inhibition by increasing ionic strength and enhanced activity upon binding to glycogen reflected changes in K′m, but not Vmax. From the determined specificity constant, k′cat/K′m∼ 4 × 106 s−1 M−1, it was calculated that at physiological levels of glycogen‐bound PP‐1G (200 nM) and phosphorylase (70 μM). dephosphorylation of the latter could occur with a half time of 15 s, sufficient to account for inactivation rates in vivo.

The much higher catalytic efficiency of glycogen‐bound PP‐1G toward the glycogen‐metabolising enzymes at physiological ionic strength compared to free C subunit substantiates the role of PP‐1G in the regulation of these substrates, and establishes a novel mechanism for selectively regulating their phosphorylation states in response to adrenalin and other factors affecting phosphorylation of the G subunit.
Folding of thermolysin fragments
Authors: VITA Claudio , FONTANA Angelo , JAENICKE Rainer
Published: March 2005

Sedimentation analysis in the analytical ultracentrifuge has been used to characterize the size and shape of thermolysin and a number of its fragments obtained by chemical or enzymatic cleavage of the protein. Four fragments (121–316, 206–316, 225/226–316 and 255–316) originate from the C‐terminal domain, and two (1–155 and 1–205) from the N‐terminal domain of the intact molecule. In aqueous solution at neutral pH the hydrodynamic properties of the C‐terminal fragments, except 255–316, are consistent with compact homogeneous monomers. Fragment 255–316 is a monomeric species below 0.08 mg/ml concentration and forms a dimer above this concentration. Dimerization does not lead to changes in fragment conformation, as determined by farultraviolet circular dichroic measurements, but to an increase of 5.6°C (to 68.2°C at 1.0 mg/ml) in the temperature for thermal unfolding and a corresponding increase of 4.6 kJ/mol in the free energy of unfolding. Fragments derived from the N‐terminal domain show a strong tendency to form high‐molecular‐mass aggregates. Previous experiments utilizing circular dichroic measurements and antibody binding data suggested that the C‐terminal fragments listed above are able to refold in aqueous solution at neutral pH into a stable conformation of native‐like characteristics [Dalzoppo, D., Vita, C. & Fontana, A. (1985) J. Mol. Biol. 182, 331–340] (and references cited therein). Present data establish that all these C‐terminal fragments are globular monomeric species in solution (at concentrations ∼ 0.1 mg/ml) and thus represent ‘isolated’ domains (or subdomains) with intrinsic conformational stability typical of small globular proteins.
Structure of the human lysosomal acid phosphatase gene
Authors: GEIER Carola , von FIGURA Kurt , POHLMANN Regina
Published: March 2005

We have isolated a 12‐kb genomic clone, which encodes human lysosomal acid phosphatase (LAP), a lysosomal membrane glycoprotein. The human LAP gene has a size of about 9 kb and contains 11 exons (83–947 bp in size). The signal sequence and the first eight amino acids of the LAP protein are encoded by exon 1, the remaining luminal domain by exons 2–10 and the transmembrane and cytoplasmic domains, as well as the 3′‐untranslated region, by exon 11. The sequence of the LAP gene confirmed the sequence deduced from the cDNA clone except for nucleotide 1917 in the 3′‐untranslated region, where T is changed to C. The 5′‐flanking sequence shows promoter activity, as analysed by coupling to bacterial chloramphenicol acetyltransferase. S1‐nuclease‐protection and primer‐extension analysis demonstrate transcription initiation at multiple sites clustering within 23 bp upstream of the translation‐initation codon. Sequences characteristic for promoter regions like TATA‐box and CAAT‐box sequences could not be identified at typical positions. The absence of these sequences, the high GC content (63.5%), two GC boxes and a region complying with the properties of a CpG island, indicate that LAP is a housekeeping gene.
Two isoforms of smooth muscle myosin regulatory light chain in chicken gizzard
Authors: INOUE Akihiro , YANAGISAWA Masashi , TAKANO‐OHMURO Hiromi , MASAKI Tomoh
Published: March 2005

We isolated a cDNA clone for a new isoform of chicken smooth muscle myosin regulatory light chain (MRLC) from a cDNA library of embryonic chicken gizzard. The deduced amino acid sequence was different in 10 amino acid residues from the previously reported polypeptide sequences of chicken gizzard MRLC. The in vitro transcription/translation product from the cDNA comigrated with a minor isoform of chicken gizzard MRLC (L20‐B) in a two‐dimensional gel electrophoresis. This isoform was detected only in the embryonic gizzard and was slightly more acidic than the predominant isoform (L20‐A). The partial polypeptide sequence of L20‐A was confirmed to be identical to the previously reported MRLC sequence. Nevertheless, Northern blot analysis showed that L20‐B‐related mRNAs were present in both the embryonic and adult gizzard. Non‐denaturing pyrophosphate polyacrylamide gel electrophoresis showed that the in vitro transcription/translation product could be associated with native myosin when mixed and coprecipitated in a low‐ionic‐strength buffer with adult chicken gizzard myosin. Moreover, the coprecipitated translation product was phosphorylated in vitro by chicken gizzard myosin light chain kinase apparently more rapidly than L20‐A on the native myosin heavy chain. From these findings, we concluded that at least two isoforms of smooth muscle MRLC exist in chicken gizzard and that their expression may be regulated translationally depending on the developmental stage.
Glycogen synthesis in the obliquely striated muscle ofAscaris suum
Authors: GHOSH Paritosh , HEATH A. Chris , DONAHUE Manus J. , MASARACCHIA Ruthann A.
Published: March 2005

A glycogen synthase, designated GS II, which occurs in a protein/carbohydrate complex has been purified from Ascaris suum muscle. The purified GS‐II complex which is eluted from concanavalin‐A–Sepharose contains proteins with Mr 140000 and 66000 and a glycoprotein with a carbohydrate/protein mass ratio of 3:1 GS II activity was totally dependent on glucose 6‐phosphate, but exogenous glycogen was not required for polysaccharide synthesis. The GS‐II complex was not phosphorylated by cyclic‐AMP‐dependent protein kinase, and antibodies to the protein and carbohydrate components of GS II did not cross react with the purified cyclic‐AMP‐regulated glycogen synthase (GS I) from A. suum muscle. Polysaccharide which was synthesized de novo by the complex was added to the large‐molecular‐mass glycoprotein in GS II. The glycogen‐like character of the newly synthesized polysaccharide was confirmed by the observation that glycogen phosphorylase utilized the polymer as substrate in both the synthesis and degradation reactions. A model is discussed in which a core glycoprotein serves as the substrate for a glycogen synthase which is distinctly different from GS I.
cDNA cloning of cytochrome P‐450 related to P‐450p‐2 from the cDNA library of human placenta
Authors: YOKOTANI Noboru , SOGAWA Kazuhiro , MATSUBARA Shyuichiro , GOTOH Osamu , KUSUNOSE Emi , KUSUNOSE Masamichi , FUJII‐KURIYAMA Yoshiaki
Published: March 2005

We have isolated and analyzed cDNA (designated P‐450HP cDNA) clones from a human placenta cDNA library, using the cDNA for rabbit pulmonary cytochrome P‐450P‐2, a prostaglandin ω‐hydroxylase, as a hybridization probe. The cDNA obtained encoded a polypeptide comprising 511 amino acids with a calculated molecular mass of 58987 Da, and the amino acid sequence similarity with P‐450P‐2 and rat liver laurate ω‐hydroxylase (P‐450LAω) was only about 50%. RNA blot analysis showed that the mRNA hybridizable with the human P‐450HP cDNA was inducibly expressed 3 – 5‐fold in rabbit small intestine and lung by gestation, but the expression remained constant in rabbit liver and kidney. This mode of expression was quite different from that of P‐450P‐2 and P‐450LAω. Interestingly, the mRNA hybridized with the cDNA of P‐450HP was found to be expressed in all the human tumor tissues so far examined, in sharp contrast with the facts that almost all the other species of P‐450s are known to disappear in the tumor tissues. Taken together, the deduced hemoprotein termed P‐450HP dose not seem to be the human counterpart of rabbit P‐450P‐2 or rat P‐450LAω, and is presumably a new member of the P‐450 family including P‐450P‐2 and P‐450LAω. Furthermore, the corresponding genomic DNA was also cloned and analyzed. The gene of P‐450HP spanned 18.8 kb and was separated into 11 exons by 10 introns whose locations were completely different from those of P‐450 genes so far determined.
Enzyme kinetics in reversed micelles
Authors: VERHAERT Raymond M. D. , TYRAKOWSKA Bożena , HILHORST Riet , SCHAAFSMA Tjeerd J. , VEEGER Cees
Published: March 2005

Enoate reductase (EC 1.3.1.31) can stereospecificially reduce a variety of α,β‐unsaturated carboxylates. Its use was extended to apolar media by incorporating the enzyme into a reversed micellar medium. The kinetics of the enzyme in such a medium have been investigated using 2‐methylbutenoic acid as substrate and NADH as a cofactor and compared with the reaction rates in aqueous solution.

In aqueous solution the enzyme obeys a ping pong mechanism [Bühler et al. (1982) Hoppe‐Seyler's Z. Physiol. Chem 363, 609–625]. In 50 mM Hepes pH = 7.0 with ionic strength of 0.05 M the Michaelis constants for NADH and 2‐methylbutenoic acid are 20 μM and 6.0 mM respectively. In reversed micelles the kinetics of the reaction (Michaelis constant, maximum velocity as well as inhibitory effects) were markedly different. The rate of the enzymatic reaction of enoate reductase was studied using various concentrations of 2‐methylbutenoic acid and various NADH concentrations. In reversed micelles composed of the anionic detergent sodium di(ethylhexyl)sulphosuccinate, the enzymatic reaction deviates substantially from the values in aqueous solution. Using our model (see preceding paper in this issue of the journal), all kinetics could be explained as evolving from enclosure in reversed micelles without any change in the intrinsic rate parameters of the enzyme. So the enzyme itself is unaffected by incorporation in reversed micelles, but the rate of intermicellar exchange as well as the microheterogeneity of the medium, resulting in very high local concentrations of the substrate, are the most important factors altering the reaction pattern. The effect of the composition of the reversed micellar medium was also investigated using either a nonionic or a cationic surfactant. In these solutions too, exchange and microheterogeneity of the medium proved to be the most important parameters influencing the enzymatic reaction.

In all reversed micellar solutions inhibition by the enoate was observed at an overall concentration of 0.5–5 mM, implying that a concentration of substrate equal to the Km value in aqueous solution may already cause inhibition in reversed micelles. At this level no inhibition by NADH was observed. The microheterogeneity of the medium also explains this inhibition of the enzyme at relatively low 2‐methylbutenoic acid concentrations.
Mn(II)‐EPR measurements of cation binding by aequorin
Authors: KEMPLE Marvin D. , LOVEJOY Michael L. , RAY Bruce D. , PRENDERGAST Franklyn G. , NAGESWARA RAO B. D.
Published: March 2005

Cation binding at 5°C by aequorin, a bioluminescent protein from the jellyfish Aequorea victoria, was examined by means of Mn(II) EPR. The bioluminescence of aequorin is triggered by Ca(II), as well as by trivalent lanthanides, and is inhibited by Mg(II) and Mn(II). Three EF‐hand Ca(II)‐binding domains have been identified in the aequorin amino acid sequence. In the work reported here, active native aequorin was found to have a single tight binding site for Mn(II) with an association constant of 0.566 μM−1. Ca(II) and La(III) competed for the Mn(II) site with association constants of 1.92 μM−1 and 1.38 μM−1, respectively. The affinity of Ca(II) and La(III) for their two other (presumed) sites on aequorin was an order of magnitude less than their affinity for the Mn(II) site. Mg(II) competed for the Mn(II) site as well but with a much smaller association constant of 0.0109 μM−1. Ca(II)‐independent discharged aequorin did not bind Mn(II) to a significant degree. Conjectures on the location of the Mn(II) site in the aequorin amino acid sequence and on the relationship between the binding parameters of the cations and their influence on aequorin activity are given.
Resonance energy transfer between points in a reconstituted skeletal muscle thin filament
Authors: MIKI Masao
Published: March 2005

The spatial relationships between Lys‐61, Cys‐374 on actin or SH1 on myosin subfragment‐1 (S1) and Cys‐190 on tropomyosin or Cys‐133 on troponin‐I (TnI) in a reconstituted thin filament were studied by fluorescence resonance energy transfer. 5‐(2‐Iodoacetylaminoethyl)aminonaphthalene 1‐sulfonic acid (IAEDANS) attached to Lys‐190 on tropomyosin or to Cys‐133 on TnI was used as a donor. Fluorescein 5‐isothiocyanate (FITC) attached to Lys‐61 or 5‐(iodoacetoamido)fluorescein (IAF) attached to Cys‐374 on actin and 4‐dimethylaminophenyl‐azophenyl 4′‐maleimide (DABMI) attached to SH1 on S1 were used as an acceptor. The transfer efficiency between AEDANS attached to Cys‐190 on tropomyosin and FITC attached to Lys‐61 on actin was 0.42 in the absence of troponin, 0.46 in the presence of troponin and Ca2+ and 0.55 in the presence of troponin and absence of Ca2+. The corresponding distances between the probes were calculated to be 4.7 nm, 4.6 nm and 4.3 nm respectively, assuming a random orientation factor K2= 2/3. A large difference in the transfer efficiency from AEDANS attached to Cys‐133 on TnI to FITC attached to Lys‐61 on actin was observed between in the presence (0.52) and absence (0.70) of Ca2+. The corresponding distances between the probes were calculated to be 4.5 nm in the presence of Ca2+ and 3.9 nm in the absence of Ca2+.

The distance between Cys‐190 on tropomyosin and Cys‐374 on actin was measured to be 5.1 nm and the transfer efficiency (0.35) did not change upon addition of troponin whether Ca2+ is present or not, in agreement with the previous report [Tao, T., Lamkin, M. & Lehrer, S. S. (1983) Biochemistry 22, 3059–3064]. The distance between Cys‐133 on TnI and Cys‐374 on actin was measured to be 4.4 nm. No detectable change in transfer efficiency (0.58) was observed between values in the presence and absence of Ca2+. These results suggest that a relative movement of the two domains of actin monomer in a reconstituted thin filament occurs in response to a change in Ca2+ concentration.

The transfer efficiencies between DABMI attached to SH1 on S1 and AEDANS attached to Cys‐190 on tropomyosin or Cys‐133 on TnI were too small (less than 2%) for an accurate estimation of the distances, suggesting the distances are longer than 7.3 nm.
Characterization of the S′‐subsite specificity of bovine pancreatic α‐chymotrypsin via acyl transfer to added nucleophiles
Authors: SCHELLENBERGER Volker , SCHELLENBERGER Ute , MITIN Yuri V. , JAKUBKE Hans‐Dieter
Published: March 2005

The S′‐subsite specificity of bovine pancreatic α‐chymotrypsin was investigated by acyl transfer reactions using a series of amino‐acid‐ and peptide‐derived nucleophiles. The nucleophilic efficiency covers a range of more than three orders of magnitude, reflecting the specificity of the acyl transfer process. Positively charged H‐Arg‐NH2 was the most efficient nucleophile of the series while peptides with free carboxyl groups show poor nucleophilic behaviour. This is explained by electrostatic interactions with the residues Asp35 and Asp64 of the enzyme. These negatively charged groups, which are localized near the appropriate S′ binding sites, repel carboxylate groups of the nucleophiles. There is a good correlation between the nucleophile efficiencies found for different acyl enzymes. An investigation of a series of 14 water‐soluble acyl donor esters, differing both in the P1 residue and in the number of amino acids, revealed that the nature of the acyl group affected the acyl‐enzyme partitioning between water and added nucleophile in the range of one order of magnitude.
Glucagon activates mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase in vivo by decreasing the extent of succinylation of the enzyme
Authors: QUANT Patti A. , TUBBS Philip K. , BRAND Martin D.
Published: March 2005

1. 3‐Hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) synthase (EC 4.1.3.5) in extracts of rat liver mitochondria can be inactivated by succinyl‐CoA and activated by incubation in a medium designed to cause desuccinylation (‘desuccinylation medium’). 2. The enzyme is less active in extracts of whole liver from control rats than from rats treated with glucagon or mannoheptulose. Incubation in desuccinylation medium raises the activity in extracts from control rats to the same value as treated rats, suggesting that the extent of succinylation in vivo is greater in controls than in hormone‐treated animals. 3. This result is also obtained in liver homogenates and in isolated mitochondria. 4. Increasing the succinyl‐CoA content of mitochondria to the same high level lowers the enzyme activity to the same value in mitochondria isolated from control or treated rats. In each case subsequent incubation of the lysates in desuccinylation medium raises the enzyme activity by the same extent. 5. Measurement of the incorporation of radiolabel from 2‐oxo[5‐14C]glutarate into protein is consistent with the proposal that all these changes in activity in isolated mitochondria may be explained by changes in the extent of succinylation of the enzyme. 6. From these data and our earlier work we conclude that, in vivo, mitochondrial HMG‐CoA synthase in fed rats is normally substantially succinylated (about 40%) and inactivated, and that glucagon increases the activity of HMG‐CoA synthase by lowering the concentration of succinyl‐CoA and thus decreasing the extent of succinylation of the enzyme (to less than 10%). This may be an important control mechanism in ketogenesis.
Primary stimuli of icosanoid release inhibit arachidonoyl‐CoA synthetase and lysophospholipid acyltransferase
Authors: HORNBERGER Wilfried , PATSCHEKE Heinrich
Published: March 2005

Icosanoid formation in platelets depends on the concentration of free arachidonate that is mainly liberated from membrane phospholipids by phospholipase A2. The concentration of free arachidonate is also controlled by the activities of the reacylating enzymes arachidonoyl‐CoA synthetase and lysophospholipid acyltransferase. In human platelet microsomes we determined the high enzyme activities of 5.9 nmol · min−1· (109 platelets)−1 for the arachidonoyl‐CoA synthetase and 37 nmol · min−1· (109 platelets)−1 for the lysophospholipid acyltransferase. The activities of these reacylating enzymes were strongly reduced by hydrogen peroxide (H2O2) and methyl mercury that are primary stimuli of arachidonate release in intact platelets. H2O2 inhibited the arachidonoyl‐CoA synthetase with an IC50 of 3.3 nmol/1 without affecting the lysophospholipid acyltransferase. Sulfhydryl group protection by 3‐mercapto‐1,2‐propanediol did not overcome the inhibition but glutathione prevented the inhibition of the arachidonoyl‐CoA synthetase by H2O2. This suggests that glutathione by virtue of the glutathione peroxidase reduces H2O2 rather than that it protects free sulfhydryl groups of the arachidonoyl‐CoA synthetase. Methyl mercury left the arachidonoyl‐CoA synthetase activity unaffected but inhibited the lysophospholipid acyltransferase activity with an IC50 of 3.4 · mol/l. The inhibition is probably evoked by the blockade of sulfhydryl groups of the lysophospholipid acyltransferase because it disappeared when 3‐mercapto‐1,2‐propanediol was added at a concentration higher than that of methyl mercury. Thrombin as a physiological full agonist, Ca2+· 1 mmol/1, the calcium ionophore A23187 and phorbol 12‐myristate 13‐acetate (TPA) and 1‐oleoyl‐2‐acetyl‐glycerol as model stimuli of protein kinase C neither influenced arachidonoyl‐CoA synthetase nor lysophospholipid acyltransferase. It is concluded that the inhibitory effect of H2O2 and methyl mercury on the arachidonate‐reacylating enzymes arachidonoyl‐CoA synthetase or lysophospholipid acyltransferase, respectively, are responsible for their capacity to stimulate icosanoid release in intact cells. Thrombin and its intracellular messengers Ca2+ and diacylglycerol do not directly affect arachidonoyl‐CoA synthetase and lysophospholipid acyltransferase.
Location and function of three sites phosphorylated on rat acetyl‐CoA carboxylase by the AMP‐activated protein kinase
Authors: DAVIES Stephen P. , SIM Alastair T. R. , HARDIE D. Grahame
Published: March 2005

We have sequenced two tryptic/chymotryptic peptides (TC3 and TC3a) containing a third site phosphorylated on rat acetyl‐CoA carboxylase by the AMP‐activated protein kinase. Comparison with the complete sequence of rat acetyl‐CoA carboxylase predicted from the cDNA sequence [López‐Casillas et al. (1988) Proc. Natl Acad. Sci. USA 85, 5784–5788] shows that this site corresponds to Ser1215.

Comparison of the cDNA sequence with previous amino acid sequence data identifies the other two sites for the AMP‐activated protein kinase as Ser79 and Ser1200. A total of eight serine residues phosphorylated in vitro by six protein kinases can now be identified: six of these (Ser23, Ser25, Ser29, Ser77, Ser79 and Ser95) are clustered in the amino terminal region, while two (Ser1200 and Ser1215) are located in the central region.

Prior phosphorylation of Ser77 and Ser1200 by cyclic‐AMP‐dependent protein kinase prevents subsequent phosphorylation of Ser79 and Ser1200, but not Ser1215, by the AMP‐activated protein kinase. Phosphorylation of Ser1215 under these conditions is not associated with a change in enzyme activity.

Limited trypsin treatment of native acetyl‐CoA carboxylase selectively cleaves off the highly phosphorylated amino‐terminal region containing Ser79.

Phosphorylation at Ser79 and Ser1200 by the AMP‐activated protein kinase dramatically decreases Vmax and increases the A0.5 for citrate. Phosphorylation at Ser77 and Ser1200 by cyclic‐AMP‐dependent protein kinase causes more modest changes in the A0.5 for citrate and the Vmax. Dephosphorylation, or removal of the amino‐terminal region containing Ser77/79 using trypsin, reverses all of these effects.

These results suggest that the effects of the AMP‐activated protein kinase on acetyl‐CoA carboxylase activity are mediated entirely by phosphorylation of Ser79, and not Ser1200 and Ser1215. The smaller effects of cyclic‐AMP‐dependent protein kinase are mediated by phosphorylation of Ser77.
Diacylglycerols modulate phosphorylation of the insulin receptor from human mononuclear cells
Authors: GRUNBERGER George , LEVY Joseph
Published: March 2005

It has been found that 1,2‐ but not 1,3‐diacylglycerols stimulated phosphorylation of the insulin receptor of cultured human monocyte‐like (U‐937) and lymphoblastoid (IM‐9) cells both in the intact‐ and broken‐cell systems. The stimulation of the receptor's β‐subunit phosphorylation was dose‐dependent, with optimal effect at 100 μg/ml of diacylglycerol. The effects of insulin and 1,2‐diacylglycerols on the phosphorylation of partially purified insulin receptors were additive. Phosphoamino acid analysis showed a major effect of diacylglycerols on phosphorylation of tyrosine residues. The diacylglycerols also stimulated tyrosine kinase activity of the partially purified U‐937 and IM‐9 insulin receptors 2.5–3.5‐fold when measured by phosphorylation of an exogenous substrate, poly(Glu80Tyr20) in the absence of any added insulin, calcium or phospholipid. Since this diacylglycerol effect could not be reproduced under conditions optimal for protein kinase C activation and the purified protein kinase C did not stimulate phosphorylation of the β‐subunit of the insulin receptor in this system, it is unlikely that the diacylglycerol effect was mediated by protein kinase C. Since these exogenous 1,2‐diacylglycerols at the same high concentration also inhibited 125I‐insulin binding to the insulin receptor of the intact U‐937 and IM‐9 cells, diacylglycerols could modulate the function of the insulin receptor and insulin action in human mononuclear cells.
Roles of the AMP‐activated and cyclic‐AMP‐dependent protein kinases in the adrenaline‐induced inactivation of acetyl‐CoA carboxylase in rat adipocytes
Authors: HAYSTEAD Timothy A. J. , MOORE Frances , COHEN Philip , HARDIE D. Grahame
Published: March 2005

In isolated rat adipocytes, acetyl‐CoA carboxylase is inactivated by treatment of the cells with adrenaline or the β‐agonist isoproterenol, but not by the α‐agonist phenylephine. The inactivation is stable during purification in the presence of protein phosphatase inhibitors, and is associated with a 30–40% increase in the labelling of enzyme isolated from 32P‐labelled cells.

Increased phosphorylation occurs within peptide T1, which was identified by sequencing to be the peptide Ser‐Ser77‐Met‐Ser79‐Gly‐Leu‐His‐Leu‐Val‐Lys, containing Ser‐77 (phosphorylated by cyclic‐AMP‐dependent protein kinase) and Ser‐79 (phosphorylated by the AMP‐activated protein kinase). Analysis of the release of radioactivity as free phosphate during Edman degradation of peptide T1 revealed that all of the phosphate was in Ser‐79 in both basal and hormone‐ or agonist‐stimulated cells. Treatment of adipocytes with various agents which activate cyclic‐AMP‐dependent protein kinase by receptor‐independent mechanisms (forskolin, cyclic AMP analogues, isobutylmethylxanthine) also produced inactivation of acetyl‐CoA carboxylase and increased phosphorylation at Ser‐79.

The (Rp)‐[thio]phosphate analogue of cyclic AMP, which is an antagonist of binding of cyclic AMP to the regulatory subunit of cyclic‐AMP‐dependent protein kinase, opposes the effect of adrenaline on phosphorylation and inactivation of acetyl‐CoA carboxylase. Together with the effects of isobutylmethylxanthine and the stimulatory cyclic AMP analogues, this strongly indicates that cyclic‐AMP‐dependent protein kinase is an essential component of the signal transduction pathway, although clearly it does not directly phosphorylate acetyl‐CoA carboxylase.

As shown by okadaic acid inhibition, > 95% of the acetyl‐CoA carboxylase phosphatase activity in extracts of rat adipocytes or liver is accounted for by protein phosphatase‐2A, with <5% attributable to protein phosphatase‐1. Inhibition of protein phosphatase‐1 via phosphorylation of inhibitor‐1 is therefore unlikely to be the mechanism by which cyclic‐AMP‐dependent protein kinase indirectly increases phosphorylation of acetyl‐CoA carboxylase. Various other potential mechanisms are discussed.
The 2‐oxoglutarate dehydrogenase complex from Azotobacter vinelandii
Authors: WESTPHAL Adrie H. , de KOK Arie
Published: March 2005

The nucleotide sequence encoding the succinyltransferase component (E2o) of the 2‐oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined. Previously the cloning in Escherichia coli of the gene encoding lipoamide dehydrogenase from A. vinelandii was reported [Westphal, A. H. & de Kok, A. (1988) Eur. J. Biochem. 172, 299–305]. The 3.2‐kb fragment used for the sequence determination contained the main part of the gene encoding succinyltransferase. The complete E2o gene, as well as the gene encoding the 2‐oxoglutarate dehydrogenase component, resided on a 14.7‐kb fragment from which the 3.2‐kb fragment was subcloned.

The protein‐coding sequence of the gene consists of 1200 bp (400 codons, including the AUG start codon and the UGA stop codon). It is separated from the gene encoding the 2‐oxoglutarate dehydrogenase component by 42 bp. No E. coli‐like promoter sequence was found. A putative ribosome‐binding site is located 9–15 bp upstream from the start codon. No terminator sequences were found downstream of the stop codon. This makes it likely that the three genes of the oxoglutarate dehydrogenase complex are transcribed as a single mRNA transcript analogous to the pyruvate dehydrogenase complex in E. coli. The intact gene was subcloned from the 14.7‐kb fragment and brought to high expression under the influence of the vector‐encoded lacZ promotor.

The similarity with the E. coli enzyme is high with 63% identity. Like the enzyme from E. coli, it consists of a single lipoyl‐binding domain, a putative E1‐and E3‐binding domain and a catalytic domain. The main difference is found in a 31‐residue sequence rich in alanine and proline located between the lipoyl domain and the putative E1‐ and E3‐binding domain. This sequence, usually found in acetyltransferases and there identified as a highly mobile region by 1H‐NMR, is replaced by a more polar, charged region in the E. coli enzyme.
Isolation and structure elucidation of a novel 5‐kDa peptide from neurohaemal lobes of the corpora cardiaca of Locusta migratoria (Insecta, Orthoptera)
Authors: HIETTER Hélène , Van DORSSELAER Alain , GREEN Brian , DENOROY Luc , HOFFMANN Jules , LUU Bang
Published: March 2005

Two predominant peptides have been isolated from neurohaemal lobes of corpora cardiaca of 8000 adults of Locusta migratoria. Both peptides have been unambiguously characterized by automated peptide microsequencing and liquid secondary‐ion mass spectrometry as a 50‐residue peptide (5K peptide) and a 48‐residue isologue (5K′ peptide). Computer search of sequence data banks did not reveal any significant similarity with other identified proteins. The 5K peptides are remarkably rich in alanine residues (25%) and contain a stretch of five consecutive alanines. This structure suggests that these molecules could correspond to spacer peptides. This assumption is corroborated in the accompanying paper [Lagueux et al. (1990) Eur. J. Biochem. 187, 249–254] on the molecular cloning of the precursor protein which attributes to the 5K peptides a role analogous to that of the C peptides of insulins.
cDNAs from neurosecretory cells of brains of Locusta migratoria (Insecta, Orthoptera) encoding a novel member of the superfamily of insulins
Authors: LAGUEUX Marie , LWOFF Laurence , MEISTER Marie , GOLTZENÉ Francine , HOFFMANN Jules A.
Published: March 2005

From neurohaemal lobes of corpora cardiaca of Locusta migratoria a 5‐kDa peptide has been isolated and its sequence established [see the accompanying paper, by Hietter et al. (1990) Eur. J. Biochem. 187, 241–247]. We have designed oligonucleotide probes from the peptide sequence of this molecule and screened a library prepared from mRNA of the neurosecretory cell region of the brain of this insect. Several positive cDNAs were isolated, the combined nucleotide sequences of which predict a large precursor of 145 residues (15770 Da) containing the newly isolated 5‐kDa peptide. The peptide is flanked by regions homologous to the A and B chains of the superfamily of insulins. The overall organization of the precursor is as follows: signal peptide/domain homologous to the B chain of insulins/C (connecting)‐peptide (corresponding to the newly isolated 5‐kDa peptide)/domain homologous to the A chain of insulins. The numbers and relative positions of the cysteines of the Locusta peptide are equivalent to those of the other members of the insulin superfamily and most of the hydrophobic core residues are conserved.
Identification of two essential histidine residues of ribonuclease T2 from Aspergillus oryzae
Authors: KAWATA Yasushi , SAKIYAMA Fumio , HAYASHI Fumiaki , KYOGOKU Yoshimasa
Published: March 2005

Ribonuclease (RNase) T2 from Aspergillus oryzae was modified by diethyl pyrocarbonate and iodoacetic acid. RNase T2 was rapidly inactivated by diethyl pyrocarbonate above pH 6.0 and by incorporation of a carboxymethyl group. No inactivation occurred in the presence of 3′AMP. 1H‐NMR titration and photo‐chemically induced dynamic nuclear polarization experiments demonstrated that two histidine residues were involved in the active site of RNase T2. Furthermore, analysis of inactive carboxymethylated RNase T2 showed that both His53 and His115 were partially modified to yield a total of one mole of Nτ‐carboxymethylhistidine/mole enzyme. The results indicate that the two histidine residues in the active site of RNase T2 are essential for catalysis and that modification of either His53 or His115 inactivates the enzyme.
Rapid purification of homodimer and heterodimer HIV‐1 reverse transcriptase by metal chelate affinity chromatography
Authors: Le GRICE Stuart F. J. , GRÜNINGER‐LEITCH Fiona
Published: March 2005

We have modified an Escherichia coli vector expressing 66‐kDa HIV‐1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol‐coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over‐expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse‐transcriptase‐coding sequence (His‐p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His‐p66 and His‐p66/His‐p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.