bluuebunny/crossref_metadata_2025 huggingface dataset:
The aerobic/anaerobic transition of glucose metabolism in Trypanosoma brucei
Authors: Eisenthal Robert , Panes Alison
Published: July 2002

The ratio of glycerol to pyruvate produced by T. brucei incubated with glucose at various oxygen tensions has been used as an index of the aerobic and anaerobic pathways of glucose metabolism. A minimal model is presented which fits the observed data. The value of the notional K of the aerobic/anaerobic transition from the model is close to that of the K
m of trypanosomal glycerophosphate oxidase. The anaerobic pathway appears to be almost completely inoperative at oxygen tensions in the range of those found in venous and arterial blood.
A calcium‐protease activator associated with brain microsomal‐insoluble elements
Authors: Takeyama Yoshihumi , Nakanishi Hiroyuki , Uratsuji Yuko , Kishimoto Akira , Nishizuka Yasutomi
Published: July 2002

A factor which markedly activates Ca2+‐dependent thiol protease (calpain) is associated with Triton X‐100‐insoluble materials, presumably structural elements such as cytoskeletons, of bovine brain microsomal fraction. This factor is extracted with 0.6 M KCl, and purified partially by sucrose density gradient centrifugation and hydroxyapatite column chromatography. The factor appears to be a heat‐stable protein with an approximate M
r, of 15000. With casein as substrate this factor activates both calpain I and calpain II severalfold up to more than 10‐ fold without alteration of their affinity to Ca2+. Calmodulin is unable to substitute for this factor. A similar factor is associated with human platelet insoluble materials.
Low‐temperature EPR and near‐infrared MCD studies of highly anisotropic low‐spin ferrihaem complexes
Authors: Gadsby Paul M.A. , Thomson Andrew J.
Published: July 2002

The low‐temperature EPR spectra and near‐infrared magnetic circular dichroism (MCD) spectra are reported of the bis complexes of imidazole, 1‐methylimidazole, 4‐methylimidazole, 1,2‐dimethylimidazole and 2‐methylimidazole with Fe(III) octaethylporphyrin (OEP) in the mixed organic solvent dichloromethane/ diethyl ether. It is shown that the latter two have highly anisotropic EPR spectra characteristic of the lowspin ferric state. The optical charge‐transfer bands have an unusually high MCD intensity, with a narrow linewidth. It is proposed that this feature is typical of bis‐histidine‐ligated haem in cytochromes with sterically strained coordination and may be used as diagnostic of such a conformation. The EPR and near‐infra‐red MCD spectra of the bis‐butylamine complex of Fe(III(OEP are also reported. It is shown that bisamine‐ligated haem may be clearly distinguished from sterically hindered bis‐imidazole (bis‐histidine) by near‐infrared MCD spectroscopy whereas the assignment is uncertain using EPR spectroscopy alone.
Phosphorylase a is an allosteric inhibitor of the glycogen and microsomal forms of rat hepatic protein phosphatase‐1
Authors: Alemany Susana , Cohen Philip
Published: July 2002

The dephosphorylation of glycogen synthase by protein phosphatase‐1 in hepatic glycogen and microsomes was inhibited by nanomolar concentrations of phosphorylase a. The I50 for phosphorylase a was 1000‐fold lower than its K
m as a substrate, while tryptic digestion increased the I50 1000‐fold without affecting K
m. Protein phosphatase‐1 from skeletal muscle and protein phosphatase‐2A from liver were only inhibited at 1000‐fold higher concentrations. Protein phosphatase‐1 became desensitized to phosphorylase a when released from hepatic microsomes, but sensititvity was partially restored by readdition of the solubilized enzyme to the microsomes. The results demonstrate that phosphorylase a is a potent allosteric inhibitor of hepatic protein phosphatase‐1 and suggest that inhibition may be conferred by a novel phosphorylase a‐binding subunit.
The susceptibility of the P‐benzoquinone‐mediated electron transport and atrazine binding to trypsin and its modification by CaCl2 in thylakoids and PS II membrane fragments
Authors: Renger G. , Hagemann R. , Fromme R.
Published: July 2002

Comparative studies in thylakoids and oxygen‐evolving Triton X‐100 PS II membrane fragments reveal: (i) There exists one high‐affinity atrazine binding site per PS II in both preparations. The affinity is reduced in PS II membrane fragments, (ii) The susceptibility to tryptic attack on atrazine binding and p‐BQ‐mediated electron transport is markedly reduced by CaCl2 in PS II membrane fragments, but no protection is observed in thylakoids. NH2OH and K3[Fe(CN)6] both reduce the binding affinity in PS II membrane fragments. The action of NH2OH is invariant to CaCl2 addition. The implications of these findings for functional studies with PS II membrane fragments are discussed.
A U3 RNA pseudogene in mouse: sequence and organization in genomic DNA
Authors: Ferrer P. , Qu L.H. , Bouche G. , Bachellerie J.P.
Published: July 2002

A mouse U3 RNA pseudogene has been identified; it corresponds to a U3B full length coding sequence with a 3'‐oligo(A) tail, precisely flanked at both ends by a pair of 15 bp direct repeats. These structural features suggest that it has arisen through an RNA‐mediated mechanism involving an insertion at staggered nicks in the genome. Sequence data indicate that this mouse specimen has been generated by a different event as compared to the recently described rat pseudogenes. It represents the first reported case, for a pseudogene of this class, to be present at more than one copy per genome.
In vitro synthesis of catalase protein in sweet potato root microbodies
Authors: Sakajo Shigeru , Asahi Tadashi
Published: July 2002

Poly(A)+ RNA prepared from sweet potato root tissue was translated in a wheat germ in vitro translation system. A translation product was immunoprecipitated with anti‐sweet potato catalase immunoglobulin G. The product was identical to the subunit of the catalase with respect to the mobility on an SDS‐polyacryl‐amide gel and the pattern of the peptide map, indicating that the catalase protein is synthesised in vitro in the same size as the mature subunit. No amino acids were released from the purified enzyme protein by Edman degradation, suggesting the occurrence of a minor modification in the N‐terminal part of the protein during the enzyme formation.
cDNA and protein sequence of the NC1 domain of the α2‐chain of collagen IV and its comparison with α1(IV)
Authors: Schwarz-Magdolen Ulla , Oberbäumer Ilse , Kühn Klaus
Published: July 2002

We present the complete cDNA and derived amino acid sequence of the non‐collagenous domain NCl of α2(IV). Comparison with the corresponding NC1 domain of α(IV) reveals a high degree of homology at the protein level in contrast to the barely homologous triple‐helical sequences of both chains.
Electrochemical titration of the S = 3/2 and S = ½ states of the iron protein of nitrogenase
Authors: Morgan T.Vance , Prince Roger C. , Mortenson Leonard E.
Published: July 2002

The iron protein of nitrogenase delivers electrons and ATP to the iron‐molybdenum protein, which in turn reduces dinitrogen to ammonia. The iron protein contains a single four‐iron, four‐sulfur prosthetic group, detectable by ESR spectroscopy m its reduced form. Until recently, spin quantitations suggested that only a portion of the reduced iron protein was being detected by ESR, but recent work in several laboratories has shown that there is a spin = signal near g = 5 in addition to the well characterized spin = signal near g = 1.94. In this paper we characterize the redox properties of both states in the presence and absence of ATP and ADP, and find that the new spin state has identical redox properties to those previously determined for the spin = state.
The modulation of cytochrome c electron self‐exchange by site‐specific chemical modification and anion binding
Authors: Concar David W. , Hill H.Allen O. , Moore Geoffrey R. , Whitford David , Williams Robert J.P.
Published: July 2002

The site‐specific chemical modification of horse heart cytochrome c at Lys‐13 and ‐72 using 4‐chloro‐3,5‐dinitrobenzoic acid (CDNB) increases the electron self‐exchange rate of the protein. In the presence of 0.24 M cacodylate (pH∗ 7.0) the electron self‐exchange rate constants, k
ex, measured by a 1H NMR saturation transfer method at 300 K, are 600, 6 × 103 and 6 × 104 M−1·s−1 for native, CDNP‐K13 and CDNP‐K72 cytochromes c respectively. Repulsive electrostatic interactions, which inhibit cytochrome c electron selfexchange, are differentially affected by modification. Measurements of 1H NMR line broadening observed with partially oxidised samples of native cytochrome c show that ATP and the redox inert multivalent anion Co(CN)6
3− catalyse electron self‐exchange. At saturation a limiting value of ~ 1.4 × 105 M−1·s−1 is observed for both anions.
Evidence for N coordination to Fe in the [2Fe‐2S] center in yeast mitochondrial complex III Comparison with similar findings for analogous bacterial [2Fe‐2S] proteins
Authors: Telser Joshua , Hoffman Brian M. , LoBrutto Russell , Ohnishi Tomoko , Tsai Ah-Lim , Simpkin David , Palmer Graham
Published: July 2002

Yeast mitochondrial complex III contains a subunit with a [2Fe‐2S] cluster (the Rieske center) that has unusual physical and chemical properties. For apparently similar centers isolated from bacteria, it has been shown by electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) measurements that these [2Fe‐2S] centers are coordinated by at least one and probably two nitrogen ligands. This work describes similar ENDOR and ESEEM studies on the intact mitochondrial complex. We find that this [2Fe‐2S] cluster exhibits ESEEM and ENDOR properties that appear to be indistinguishable from those observed with the isolated bacterial systems. Furthermore, changes in EPR lineshape that occur as complex III is progressively reduced are not accompanied by any changes in the nitrogen coupling parameters. This spectroscopic evidence for nitrogen coordination is supported by published sequence data on four Rieske iron‐sulfur subunits. It seems likely that this is a general characteristic of such [2Fe‐2S] redox active centers.
Fatty acid synthesis in mitochondria from Saccharomyces cerevisiae
Authors: Bessoule Jean-Jacques , Lessire René , Rigoulet Michel , Guerin Bernard , Cassagne Claude
Published: July 2002

The ability of purified mitochondria isolated from S. cerevisiae to synthesize fatty acids and especially very long chain fatty acids (VLCFA) has been investigated. The VLCFA synthesis requires malonyl‐CoA as the C2 unit donor and NADPH as the reducing agent. Moreover the yeast mitochondrial elongase is able to accept either exogenous long chain fatty acyl‐CoAs as substrates or elongate endogenous substrates. In the latter case, ATP is required for full activity. Besides this important VLCFA formation, the mitochondria from S. cerevisiae were also able to synthesize C16 and C18.
Phosphorylation of spinach chlorophyll‐protein complexes CPII*, but not CP29, CP27, or CP24, is phosphorylated in vitro
Authors: Dunahay Terri G. , Schuster Gadi , Staehelin L.Andrew
Published: July 2002

Previous studies have indicated that the reversible phosphorylation of a population of antenna complexes that can donate energy to PS II (‘mobile LHC II’) plays a regulatory role in the state 1—state 2 transition in thylakoid membranes. The relationship of phosphorylated LHC II to the multiple PS II‐associated chlorophyll a/b‐proteins resolvable on green gels is currently unclear. We have used a high resolution gel system to analyze thylakoids phosphorylated in vitro. The only PS II‐associated antenna complex to become phosphorylated is CPII*, indicating that this complex represents the mobile LHC II. The other putative PS II antenna complexes, CP29, CP24, and the new complex designated CP27 which comigrates with CPII, are not phosphorylated and are probably components of the bound ‘LHC II’ antenna.
Changes in the expression of N‐acetylglucosaminyltransferase III, IV, V associated with the differentiation of HL‐60 cells
Authors: Koenderman Anky H.L. , Wijermans Pierre W. , van den Eijnden Dirk H.
Published: July 2002

By use of a new assay method based on HPLC, GlcNAc‐transferase III, IV and V activities were determined in HL‐60 cells, differentiated HL‐60 cells and normal myeloid cells. Differentiation along the monocytic lineage with 1α,25‐dihydroxyvitamin D3 resulted in increased GlcNAc‐transferase IV and decreased GlcNAc‐transferase III activity. Differentiation along the myeloid lineage with retinoic acid resulted in a decrease in GlcNAc‐transferase III activity. Although differentiated HL‐60 cells show a changed GlcNAc‐transferase pattern, they do not resemble normal myeloid cells. Macrophages and granulocytes are characterized by a very low level of GlcNAc‐transferase III activity whereas differentiated HL‐60 cells still contain this activity. This is the first demonstration of GlcNAc‐transferase IV and V activity in a human cell.
Identification of a UDP‐Gal:β‐Galactoside βl→3‐galactosyltransferase in the albumen gland of the snail Lymnaea stagnalis
Authors: Joziasse David H. , Damen Hanny C.M. , de Jong-Brink Marijke , Edzes Hommo T. , Van den Eijnden Dirk H.
Published: July 2002

Detergent extracts of the albumen gland of the snail Lymnaea stagnalis contain an enzyme activity that transfers Gal from UDP‐Gal to acceptor substrates with terminal non‐reducing β‐galactose residues. The products formed with lactose (Galβl→4Glc) as the acceptor were characterized by HPLC, and subjected to 400‐MHz 1H‐NMR and methylation analysis. The main product was shown to have the structure Gal‐βl→3Galβl→4Glc. Therefore, the galactosyltransferase can be identified as a UDP‐Gal:β‐galactoside βl→3‐galactosyltransferase. In view of its linkage and acceptor specificity, the enzyme may be essential to the biosynthesis of galactogen, the main polysaccharide produced by albumen glands of L. stagnalis.
Possible involvement of eicosanoids in the actions of sympathetic hepatic nerves on carbohydrate metabolism and hemodynamics in perfused rat liver
Authors: Iwai Masaru , Jungermann Kurt
Published: July 2002

In isolated rat liver perfused at constant pressure with Krebs‐Henseleit buffer containing 5 mM glucose, 2 mM lactate, 0.2 mM pyruvate and 0.1% bovine serum albumin, perivascular nerve stimulation (20 V, 20 Hz, 2 ms) and infusion of ATP (100 μM), noradrenaline (1 μM) or arachidonic acid (100 μM) caused an increase in glucose and lactate output and a reduction of perfusion flow. The metabolic effects of nerve stimulation but not those of ATP and noradrenaline were inhibited strongly by the phospholipase A2 inhibitor bromophenacyl bromide (BPB, 20 μM) and the cyclooxygenase inhibitor indomethacin (Indo, 20 μM) and only slightly by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 20 μM). In contrast, the hemodynamic effects not only of nerve stimulation but also of ATP and noradrenaline were inhibited strongly by BPB and Indo and slightly by NDGA. The metabolic and hemodynamic actions of arachidonate were inhibited specifically by Indo. These results suggest that the effects of nerve stimulation were at least partially mediated or modulated by eicosanoids, especially by prostanoids.
Linear relationships between mitochondrial forces and cytoplasmic flows argue for the organized energy‐coupled nature of cellular metabolism
Authors: Berry M.N. , Gregory R.B. , Grivell A.R. , Henly D.C. , Phillips J.W. , Wallace P.G. , Welch G.R.
Published: July 2002

We have studied rates of formation of glucose, urea and lactate by isolated hepatocytes incubated with a variety of inhibitors of energy transduction. Linear relationships have been found between these metabolic rates and mitochondrial forces (membrane, redox and phosphorylation potentials). The findings are suggestive of extensive enzyme organization within these metabolic pathways.
On the dephosphorylation of the ATP,Mg‐dependent protein phosphatase modulator
Authors: Vandenheede Jackie R. , Abeele Carline Vanden , Merlevede Wilfried
Published: July 2002

The dephosphorylation of the modulator subunit is an essential step in the kinase FA‐mediated activation of the ATP,Mg‐dependent protein phosphatase. Mg2+ is implicated in this autocatalytic dephosphorylation which is not effected by the addition of phosphoinhibitor‐1. Dephosphorylation of free modulator by the catalytic subunit is also largely Mg2+‐dependent but can be abolished by phosphoinhibitor‐1 in concentrations comparable to the amount of modulator used as substrate (micromolar). The phosphorylase phosphatase activity of the catalytic subunit is inhibited by nanomolar concentrations of phosphoinhibitor‐1 and is completely independent of divalent cations.
Aldose reductase and ϱ‐crystallin belong to the same protein superfamily as aldehyde reductase
Authors: Carper Deborah , Nishimura Chihiro , Shinohara Toshimichi , Dietzchold Bernard , Wistow Graeme , Craft Cheryl , Kador Peter , Kinoshita Jin H.
Published: July 2002

Aldose reductase (EC 1.1.1.21) has been implicated in a variety of diabetic complications. Here we present the first primary sequence data for the rat lens enzyme, obtained by amino acid and cDNA analysis. We have found structural similarities with another NADPH‐dependent oxidoreductase: human liver aldehyde reductase (EC 1.1.1.2). The identity between these two enzymes is 50%. Both enzymes share approx. 40–50% homology with ϱ‐crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and ϱ‐crystallin are members of a superfamily of related proteins.
Hydroxyl radical footprinting of the sequence‐selective binding of netropsin and distamycin to DNA
Authors: Portugal J. , Waring M.J.
Published: July 2002

Hydroxyl radicals, generated by allowing an iron (II)·EDTA complex to react with hydrogen peroxide, have been employed to cleave the 160‐base pair tyrT DNA fragment in the presence and absence of the minor groove‐binding antibiotics netropsin and distamycin A. The control DNA cleavage pattern is practically independent of nucleotide sequence, which overcomes certain limitations of other footprinting techniques, so that additional information can be gained about the AT‐rich sequence preference of the minor groove‐binding ligands.
NADP efficiently inhibits endogenous but not pertussis toxin‐catalyzed co valent modification of membrane proteins incubated with NAD
Authors: Rosenthal W. , Binder T. , Schultz G.
Published: July 2002

Incubation of membranes of human erythrocytes and platelets but not of human neutrophils with [32P]NAD leads to covalent modification of various membrane proteins and of added albumin. In membranes of all three cell types, pertussis toxin (PT), in the presence of NAD, specifically labelled a 40 kDa peptide, i.e. the α‐subunit of a guanine nucleotide‐binding protein. This effect of PT was slightly reduced by NADP, whereas modification of other membrane proteins and of albumin was largely suppressed, independent of whether PT was present or not. Labelling of cytosolic proteins in the presence of NAD was marginal; only in neutrophil cytosol, PT modified a 40 kDa peptide. Membranes of erythrocytes and platelets exhibited NAD‐degrading activity, which was inhibited by NADP. The data suggest a high substrate specificity of PT for NAD. Inhibition of endogenous enzymes by NADP may prove useful for the evaluation of PT substrates.
Bimodal regulation of secretion by cytoplasmic Ca2+ as demonstrated by the parathyroid
Authors: Nygren Peter , Larsson Rolf , Lindh Erik , Ljunghal Sverker , Rastad Jonas , Åkerström Göran , Gylfe Erik
Published: July 2002

Bovine parathyroid cells were used to study parathyroid hormone (PTH) release and the cytoplasmic Ca2+ concentration (Ca2+
1). When the extracellular Ca2+ concentration was decreased from 3.0 to 0.5 mM, perifused cells reacted with rapid stimulation of PTH release. However, a further reductionof extracellular Ca2+ to < 10 nM resulted in prompt inhibition. Both effects were readily reversible. Using the intracellular Ca2+ indicator quin‐2 also as a buffer for calcium it was possible to control Ca2+
1 within the 20–600 nM range. PTH release was found to increase with Ca2+ up to 200 nM but was gradually suppressed above this concentration.
Isolation of NADPH:isoflavone oxidoreductase, a new enzyme of Pterocarpan phytoalexin biosynthesis in cell suspension cultures of Cicer arietinum
Authors: Tiemann Karin , Hinderer Walter , Barz Wolfgang
Published: July 2002

Chickpea Cicer arietinum L.) cell suspension cultures transferred into a medium containing yeast extract accumulate the phytoalexins medicarpin and maackiain. Concomitant with accumulation of the pterocarpans a new enzyme activity is induced which was characterized as NADPH:isoflavone oxidoreductase. Maximum enzyme activity was reached 16 h after transfer of cells and then activity rapidly declined. The soluble enzyme was partially purified and shown to catalyze the reduction of the isoflavone 2'‐hydroxyformononetin to the isoflavanone vestitone which is an intermediate in medicarpin biosynthesis. The enzyme data suggest that 2'‐hydroxylation is a prerequisite for the conversion of isoflavones to pterocarpans.
NMR study of the interaction between cytochrome b5 and cytochrome c
Authors: Hartshorn R.Timothy , Mauk A.Grant , Mauk Marcia R. , Moore Geoffrey R.
Published: July 2002

The interaction between horse cytochrome c and the tryptic fragment of bovine liver microsomal cytochrome b
5 in the absence and presence of [Cr(ethylenediamine)3]Cl3 was studied by 1H NMR spectroscopy. The protein‐protein interaction region on cytochrome b
5 was found to be different from the [Cr(en)3]3+ binding region. The solvent‐exposed propionate‐bearing edge of the haem of cytochrome b
5 is accessible to [Cr(en)3]3+ in the interprotein complex.
Two‐electron reduction is required for rapid internal electron transfer in resting, pulsed and oxygenated cytochrome c oxidase
Authors: Fabian Marian , Thörnström Per-Eric , Brzezinski Peter , Malmström Bo G.
Published: July 2002

The resting as well as the 420 nm and 428 nm forms of cytochrome oxidase have been studied in kinetic experiments with an excess of enzyme over reduced cytochrome c. No difference was found in the behavior of the two activated forms. With all three forms, a fraction of cytochrome a was reoxidized with a rate which was much lower than k
cat. This suggests that intramolecular transfer to the dioxygen‐reducing site occurs only if both cytochrome a and CuA are reduced. An initial rapid phase in the oxidation of cytochrome a in the pulsed and oxygenated enzymes is related to the presence of a three‐electron‐reduced dioxygen intermediate. The increased catalytic activity of pulsed and oxygenated oxidase can be explained on the basis of a shift in the redox equilibrium between cytochrome a and CuA.
Inhibition of Na+/H+ exchange reduces Ca2+ mobilization without affecting the initial cleavage of phosphatidylinositol 4,5‐bisphosphate in thrombin‐stimulated platelets
Authors: Siffert W. , Siffert G. , Scheid P. , Riemens T. , Gorter G. , Akkerman J.W.N.
Published: July 2002

Stimulation of human platelets increases cytoplasmic pH (pHi) via activation of Na+/H+ exchange. We have determined the effect of inhibiting Na+/H+ exchange on (i) thrombin‐induced Ca2+ mobilization and (ii) turnover of 32P‐labelled phospholipids. Blocking Na+/H+ exchange by removal of extracellular Na+ or by ethylisopropylamiloride (EIPA) inhibited Ca2+ mobilization induced by 0.2 thrombin, whereas increasing pHi by NH4C1 enhanced the thrombin‐induced increase in cytosolic free Ca2+. The effect of EIPA was bypassed after increasing pHi by moneasin. The thrombin‐induced cleavage of phosphatidylinositol 4,5‐bisphosphate (PIP2) was unaffected by treatments that blocked Na+/H+ exchange or increased pHi. It is concluded that activation of Na+/H+ exchange is a prerequisite for Ca2+ mobilization in human platelets but not for the stimulus‐induced hydrolysis of PIP2.
Enzymatic alteration of C1q, the collagen‐like subcomponent of the first component of complement, leads to cross‐reactivity with type II collagen
Authors: Heinz Hans-Peter , Brackertz Dieter , Loos Michael
Published: July 2002

Native serum C1q, the collagenous‐like subcomponent of the first component of complement, is not recognized by polyclonal anti‐collagen type II antibodies. However, when purified C1q was subjected to limited proteolysis by collagenase it showed antigenic cross‐reactivity with collagen type II. The same cross‐reactivity was observed with hemolytically active C1q in synovial fluids of patients with rheumatoid arthritis (RA), whereas C1q from synovial fluids of patients with osteoarthritis (OA), villo‐nodular synovitis and ankylosing spondylitis was not recognized by this antibody. However, incubation of synovial fluid C1q of OA patients with synovial fluid leucocytes from RA patients led to an alteration of OA‐C1q which was now recognized by the anti‐collagen type II antibody.
Synthesis and biological characterization of monocyte‐derived neutrophil chemotactic factor
Authors: Tanaka Shuji , Robinson Elizabeth A. , Yoshimura Teizo , Matsushima Kouji , Leonard Edward J. , Appella Ettore
Published: July 2002

MDNCF is a human monocyte‐derived, 72‐residue chemotactic peptide, which has sequence similarity with members of a family of pro‐inflammatory cytokines. The peptide was synthesized by the solid‐phase method, and is identical to the natural peptide in amino acid composition, sequence and chemotactic potency. MDNCF forms two loops via a neighboring pair of disulfide bridges, the probable locations of which are residues 7–34 and 9–50. Reduction and alkylation eliminated chemotactic activity. MDNCF fragments 7–37, 30–72 and 17–72 were all biologically inactive. The data suggest that the region of the clustered pair of disulfide bridges is important for biological activity.
Diacylglycerol stimulates phospholipase A2 from Swiss 3T3 fibroblasts
Authors: Burch Ronald M.
Published: July 2002

We recently demonstrated that diacylglycerol induced arachidonate release and prostaglandin E2 synthesis in 3T3 fibroblasts, and greatly augmented prostaglandin E2 synthesis in response to submaximal and maximal concentrations of bradykinin. We have now partially purified a phospholipase A2 from the cells. When phosphatidyl[3H]choline was used as substrate, several diacylglycerols augmented phospholipase A2 activity. Diacylglycerol was effective at concentrations as low as 30 nM. Protein kinase C inhibition did not affect diacylglycerol's stimulation of phospholipase A2. Diacylglycerol did not alter the calcium requirement for phospholipase A2 or its pH optimum. The present study demonstrates that the effect of diacylglycerol to augment arachidonate metabolism is at the level of phospholipase A2, itself.
31P NMR investigations on free and enzyme bound thiamine pyrophosphate
Authors: Flatau Sabine , Fischer Gunter , Kleinpeter Erich , Schellenberger Alfred
Published: July 2002

Pyruvate decar☐ylase (PDC) contains thiamine pyrophosphate (TPP) and Mg2+ as cofactors. 31P NMR studies with PDC in the presence of added Mn2+ reveal the pyrophosphate moiety of TPP to be a nonaccessible area for the external Mn2+ and thus proving the Mg‐P‐complex (taking part in the binding of the coenzyme to the protein) to be a nonaccessible area for the medium. Glyoxylic acid, acting as an inhibitor of PDC by forming a noncleavable bond with the catalytic center of TPP causes a steric immobilization of the coenzyme indicated by a line broadening of the pyrophosphate moiety.
Expression of four types of human tyrosine hydroxylase in COS cells
Authors: Kobayashi Kazuto , Kiuchi Kazutoshi , Ishii Akira , Kaneda Norio , Kurosawa Yoshikazu , Fujita Keisuke , Nagatsu Toshiharu
Published: July 2002

Alternative splicing from a single gene produces four kinds of human tyrosine hydroxylase (types 1–4) which have structural diversity only in the N‐terminal region. We attempted expression of the type 1–4 enzymes in COS cells and performed kinetic analyses. All had enzymatic activities. The K
m values of the four types for L‐tyrosine and 6‐methyl‐5,6,7,8‐tetrahydropteridine were similar, although their relative homospecific activities were clearly different. The type 1 enzyme displayed the highest activity.
The primary structure of human platelet profilin: Reinvestigation of the calf spleen profilin sequence
Authors: Ampe Christophe , Markey Francis , Lindberg Uno , Vandekerckhove Joël
Published: July 2002

The primary structure of human platelet profilin was determined by aligning the sequences of its tryptic peptides to the previously determined calf spleen profilin sequence [(1979) FEBS Lett. 101, 161–165]. Comparison of the peptide fingerprints of the two proteins suggested a higher homology than that found by direct sequence comparison. We therefore reinvestigated the sequences of the peptides from calf spleen profilin. We identified four incorrect charge assignments and a deletion of three residues. The similarity between the two vertebrate profilins amounts to 95%.
Carbohydrate as covalent crosslink in human inter‐α‐trypsin inhibitor: A novel plasma protein structure
Authors: Jessen Torben E. , Faarvang Karen L. , Ploug Michael
Published: July 2002

The primary structure of inter‐α‐trypsin inhibitor is partially elucidated, but controversy about the construction of the polypeptide backbone still exists. We present evidence suggesting that inter‐α‐trypsin inhibitor represents a novel plasma protein structure with two separate polypeptide chains covalently crosslinked only by carbohydrate (chondroitin sulphate)
The identification of T2; the phosphate/pyrophosphate transport protein of the hepatic microsomal glucose‐6‐phosphatase system
Authors: Waddell Ian D. , Lindsay J.Gordon , Burchell Ann
Published: July 2002

The phosphate/pyrophosphate translocase protein (T2) of the hepatic microsomal glucose‐6‐phosphatase system was identified and then purified using antibodies raised against the rat mitochondrial phosphate/hydroxyl ion antiport protein. The T2 protein was shown to be absent in the microsomes isolated from a patient previously diagnosed as having type 1c glycogen storage disease.
Regulation of yeast fructose‐1,6‐bisphosphatase in strains containing multicopy plasmids coding for this enzyme
Authors: de la Guerra R. , Valdés-Hevia M.D. , Gancedo J.M.
Published: July 2002

The structural gene for yeast fructose‐1,6‐bisphosphatase (FbPase) has been cloned by complementation of a strain carrying the fbp1‐1 mutation and lacking FbPase activity. By Northern analysis it could be shown that the fbp1‐1 mutation does not interfere with the transcription of the gene although no normal active enzyme is produced. Strains transformed with the cloned gene on a multicopy plasmid overproduce FbPase but are still subject to strong catabolite repression. Catabolite inactivation, however, is slowed down in such strains.
Conformational changes of a mitochondrial precursor protein on binding to phospholipid vesicles and SDS micelles A circular dichroism and fluorescence spectroscopy study
Authors: Endo Toshiya , Oya Masanao
Published: July 2002

Conformations of an artificial mitochondrial precursor protein pCox IV‐DHFR have been analyzed by CD and fluorescence spectroscopy in the presence of (cardiolipin‐rich) phospholipid vesicles or SDS micelles. Binding of pCox IV‐DHFR to phospholipid vesicles involves a conformational change, which is presequence‐dependent, accompanies alteration in the secondary structure of the DHFR moiety, but is different from total unfolding of the polypeptide chain. On the other hand, a conformational change of the fusion protein on binding to the micelles of a positively charged detergent, SDS, is not presequence‐dependent.
The TRH‐related peptide pyroglutamylglutamylprolinamide is present in human semen
Authors: Cockle Sheena M. , Aitken Alastair , Beg Fatima , Morrell Jane M. , Smyth Derek G.
Published: July 2002

We have recently identified a novel peptide in the rabbit prostate complex which cross‐reacts with an antibody to thyrotrophin‐releasing hormone (TRH) and has the structure pGlu‐Glu‐ProNH2. In the present study, high concentrations of a TRH‐related tripeptide and also a polypeptide (10–12 kDa) containing a TRH‐immunoreactive peptide at its C‐terminus were detected in human semen. The low molecular mass TRH‐like peptide and the immunoreactive fragment from the polypeptide were isolated from human semen and shown to have identical structures. Amino acid analysis suggested compositions Glx2, Pro1, and after mild acid hydrolysis, the same sequence, Glu‐Glu‐Pro, was established for the two peptides. Fast atom bombardment (FAB) mass spectrometry yielded a pseudomolecular ion (M + H)+ of 355.38 which was identical to that of the synthetic peptide pGlu‐Glu‐ProNH2. The data demonstrate that human semen contains the TRH‐like peptide pyroglutamylglutamylprolinamide and also a polypeptide terminating in the sequence Gln‐Glu‐ProNH2.
A new and general procedure for refolding mutant Bowman‐Birk‐type proteinase inhibitors on trypsin‐Sepharose as a matrix with complementary structure
Authors: Flecker Peter
Published: July 2002

The trypsin‐reactive subdomain of the Bowman‐Birk‐inhibitor of serine proteinases is shown to be highly vulnerable to perturbations induced by single amino acid replacements. Dramatic deviations from a stoichiometric 1:1 ratio to the chymotrypsin‐reactive subdomain used as an internal standard occur with all variants after renaturation in solution. A stoichiometric 1:1 ratio of subdomains is achieved via a new refolding procedure on trypsin‐Sepharose as a matrix with complementary structure.
Phosphorylation of protein kinase C by casein kinase‐1
Authors: Vila Jordi , Walker Jefrrey M. , Itarte Emilio , Weber Michael J. , Sando Julianne J.
Published: July 2002

Because phosphorylation of protein kinase C (PKC) may provide a mechanism for regulation of this enzyme, we have examined the ability of two other kinases to phosphorylate PKC. Our results show that casein kinase 1 (CK‐1), but not casein kinase 2 (CK‐2), can phosphorylate PKC in the absence of Ca2+ and phospholipids. The 32P incorporation into PKC in the presence of Ca2+ and phospholipids is also enhanced by CK‐1.
A phospholipase A2 hydrolyzing arachidonoyl‐phospholipids in mouse peritonea macrophages
Authors: Wijkander Jonny , Sundler Roger
Published: July 2002

A calcium‐dependent phospholipase A2 with half‐maximal activity at approx. 0.7 μM free Ca2+ has been identified in the cytosolic fraction from macrophages. The enzyme eluted as a 70 kDA protein upon gel chromatography and showed increased activity after 10 min pretreatment of the cells with 10 nM phorbol myristate acetate. No significant activity could be detected in the membrane fraction. The enzyme hydrolyzed arachidonic acid‐containing phosphatidylcholine and ‐ethanolamine as well as phosphatidylinositol. The release of arachidonic acid in the vitro assay was inhibited in a dose‐dependent manner by nordihydroguaiaretic acid and quercetin that are also potent inhibitors of the mobilization of arachidonic acid in intact macrophages.
Dexamethasone regulation of the expression of cytokine mRNAs induced by interleukin‐1 in the astrocytoma cell line U373MG
Authors: Nishida Tsutomu , Nakai Satoru , Kawakami Takuma , Aihara Koutoku , Nishino Naoki , Hirai Yoshikatsu
Published: July 2002

BSF‐2/IL‐6, GM‐CSF and IL‐1β mRNAs were induced by recombinant IL‐1 in human astrocytoma cell line U373MG. The induction of BSF‐2/IL‐6 and IL‐1β mRNAs did not require de novo protein synthesis while that of GM‐CSF mRNA required a newly synthesized protein. Dexamethasone inhibited the induction of these cytokine mRNAs by IL‐1. This process seems to require continued protein synthesis. These results suggest that the production of these cytokines are positively and negatively controlled by IL‐1 and glucocorticoids, respectively, in astrocytes.
Increase of glucose and lactate output and decrease of flow by human anaphylatoxin C3a but not C5a in perfused rat liver
Authors: Püschel Gerhard P. , Oppermann Martin , Muschol Waldemar , Götze Otto , Jungermann Kurt
Published: July 2002

The complement fragments C3a and C5a were purified from zymosan‐activated human serum by column chromatographic procedures after the bulk of the proteins had been removed by acidic polyethylene glycol precipitation. In the isolated in situ perfused rat liver C3a increased glucose and lactate output and reduced flow. Its effects were enhanced in the presence of the carboxypeptidase inhibitor DL‐mercaptomethyl‐3‐guanidinoethylthio‐propanoic acid (MERGETPA) and abolished by preincubation of the anaphylatoxin with carboxypeptidase B or with Fab fragments of an anti‐C3a monoclonal antibody. The C3a effects were partially inhibited by the thromboxane antagonist BM13505. C5a had no effect. It is concluded that locally but not systemically produced C3a may play an important role in the regulation of local metabolism and hemodynamics during inflammatory processes in the liver.
Maize leaf phosphoenolpyruvate carboxylase: phosphorylation of Ser15 with a mammalian cyclic AMP‐dependent protein kinase diminishes sensitivity to inhibition by malate
Authors: Terada Kazutoyo , Kai Takako , Okuno Sachiko , Fujisawa Hitoshi , Izui Katsura
Published: July 2002

The so‐called light‐activation of phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) involved in C4 photosynthesis is known to be mediated by phosphorylation. A cyclic AMP‐dependent protein kinase from bovine heart was found to be able to phosphorylate PEPC. The phosphorylation was accompanied by the changes in kinetic properties, which were very similar to the reported light activation. The phosphorylated amino acid residue was identified as Ser and the position of this Ser on the primary structure [(1988) FEBS Lett. 229, 107‐110] was determined to be Ser15.
Sequence‐specific [1H]NMR resonance assignments and secondary structure identification for 1‐ and 2‐zinc finger constructs from SWI5
Authors: Neuhaus David , Nakaseko Yukinobu , Nagai Kiyoshi , Klug Aaron
Published: July 2002

Complete [1H]NMR resonance assignments are presented for the second of the three zinc fingers from SWI5 as it appears in both 1‐ and 2‐finger constructs. Signals from finger 2 are unaffected by the presence or absence of finger 1, showing that the protein has a modular construction. The structure of finger 2 comprises a helix running from N56 to Q67, and a region approximating to an anti‐parallel β‐sheet running from Y42 to F53. These features combine to produce a hydrophobic core in the structure involving the invariant residues Y42, F53, L59 and H62.
Stimulation of the receptor for melanocyte‐stimulating hormone by retinoic acid
Authors: Chakraborty Ashok K. , Orlow Seth J. , Pawelek John M.
Published: July 2002

Treatment of Cloudman S91 melanoma cells with retinoic acid (RA) inhibits MSH‐induced tyrosinase activity and melanin formation [(1990) J. Invest. Dermatol. 94, 461‐464]. We report here, however, that in spite of inhibiting MSH‐induced pigmentation, RA treatment caused a marked increase in MSH binding capacity for both cell surface and internal MSH binding sites. The stimulation was dose‐ and time‐dependent and reversible, with half‐maximal effects seen at 2 μM RA. Stimulation of MSH binding was seen as early as 3 h after exposure of cells to RA. Cell surface and internal binding activity increased in concert. Scatchard analyses indicated that increased MSH binding resulted from a 3–4‐fold increase in the number of sites with no significant difference in their affinity for MSH. It appears that in suppressing MSH‐induced melanogenesis, RA elicited a compensatory up‐regulation of the MSH receptor system.
Isolation and RNA sequence analysis of cytochrome b mutants resistant to funiculosin, a center i inhibitor of the mitochondrial ubiquinol‐cytochrome c reductase in Saccharomyces cerevisiae
Authors: di Rago J.-P. , Perea J. , Colson A.-M.
Published: July 2002

Funiculosin is a well‐known inhibitor of the mitochondrial respiratory chain, probably acting at the ubiquinone reducing site or center i of QH2 cytochrome c reductase. We report here the isolation, mapping and RNA sequence analysis of yeast apo‐cytochrome b mutants resistant to this inhibitor. Funiculosin‐resistance was found to be conferred, in 4 independent isolates, upon replacement of a leucine residue by phenylalanine in position 198 of the cytochrome b polypeptide chain.
Light chain of botulinum neurotoxin is active in mammalian motor nerve terminals when delivered via liposomes
Authors: de Paiva Anton , Oliver Dolly J.
Published: July 2002

Liposomal encapsulation of the individual light and heavy chain of botulinum neurotoxin A was used to investigate their intra‐cellular effects on synaptic transmission at the murine neuromuscular junction. Bath‐application to phrenic nerve‐hemidiaphragms of liposomes containing heavy chain (up to 75 nM) caused no alteration in neurally‐evoked muscle tension. In contrast, liposomes with entrapped light chain (9–20 nM final concentration) gave a pre‐synaptic blockade of neuromuscular transmission that could be relieved temporarily by 4‐aminopyridine, as for the dichain toxin. Any contribution from contaminating intact toxin was excluded both by the purity and minimal toxicity in mice of the light chain preparations used, and by the lack of neuromuscular paralysis seen with liposomes containing the maximum amount of native toxin that could have been present in the light chain liposomes. As bath‐application of high concentrations of light chain in the absence of liposomes failed to affect neurotransmiter release, it is concluded that this chain alone can mimic the action of the whole toxin inside mammalian motor nerve endings, its predominant site of action. Thus, light chain could provide a more effective probe for an intra‐cellular component concerned with Ca2+‐dependent secretion.
Localization of the α‐oxoacid dehydrogenase multienzyme complexes within the mitochondrion
Authors: Maas Edith , Bisswanger Hans
Published: July 2002

Bovine kidney mitochondria were separated into matrix and membrane fractions by treatment with digitonin and Lubrol PX. While malate dehydrogenase was found essentially in the matrix fraction, both the pyruvate and the α‐oxoglutarate dehydrogenase multienzyme complexes remained bound to the inner membrane fraction and became solubilized only after repeated treatments with detergents. Thus both multienzyme complexes must be associated with the inner membrane rather than located within the matrix space.
Use of electrospray mass spectrometry to directly observe an acyl enzyme intermediate in β‐lactamase catalysis
Authors: Aplin Robin T. , Baldwin Jack E. , Schofield Christopher J. , Waley Stephen G.
Published: July 2002

Electrospray mass spectrometry was used to directly observe intact acyl enzyme complexes formed between a class C β‐lactamase (from Enterobacter cloacae P99) and four poor substrates/inhibitors. In each case the molecular weight difference between the unreacted and the reacted β‐lactamase was consistent with the formation of an acyl enzyme.
Selective solubilization of chlorosome proteins in Chloroflexus aurantiacus
Authors: Eckhardt Anne , Brunisholz René , Frank Gerhard , Zuber Herbert
Published: July 2002

Proteins were solubilized selectively from chlorosomes of Chloroflexus aurantiacus by electrophoretic gel filtration according to Griebenow et al. Whereas the 11 kDa and 18 kDa proteins were extracted almost completely, the remaining modified chlorosomes contained high amounts of pigment and c‐protein. It was concluded that the c‐protein in contradiction to the publication by Griebenow et al. is indeed localized in the interior of Chloroflexus chlorosomes.
Tissue inhibitor of metalloproteinases from human bone marrow stromal cell line KM 102 has erythroid‐potentiating activity, suggesting its possibly bifunctional role in the hematopoietic microenvironment
Authors: Hayakawa Taro , Yamashita Kyoko , Kishi Jun-ichi , Harigaya Kenichi
Published: July 2002

In this study, we demonstrated that tissue inhibitor of metalloproteinases (TIMP) produced by human bone marrow stromal cell line KM‐102 had erythroid‐potentiating activity (EPA) which stimulates the proliferation of erythroid progenitor cells. We, then, propose a scheme for the bifunctional role ofTIMP/EPA in hematopoietic microenvironment, that is, the maintenance of the integrity of bone marrow matrix and the proliferation of erythroid progenitor cells proceeding on the matrix.
Enhanced synthesis of heat shock proteins and augmented thermotolerance after induction of differentiation in HL‐60 human leukemia cells
Authors: Yufu Yuji , Nishimura Junji , Ideguchi Hiroshi , Nawata Hajime
Published: July 2002

The effects of the induction of differentiation were investigated on the expression of heat shock proteins (hsps) and thermotolerance. The synthesis of the major hsps in response to heat stress was markedly enhanced in HL‐60 human leukemia cells after differentiation. An increased amount of mRNA transcripts for hsp70 was also noted. In addition, induction of differentiation resulted in acquisition of greater resistance to heat, which may be advantageous since cells in the peripheral blood must survive many stresses.
An analysis of pseudocontact shifts and their relationship to structural features of the redox states of cytochrome b5
Authors: Veitch Nigel C. , Whitfbrd David , Williams Robert J.P.
Published: July 2002

The assignment of proton resonances inboth redox states of a heme protein is necessary for the evaluation of pseudocontact shift data. Many new assignments are presented here for cytochrome b
5 particularly in the paramagnetic oxidised state, thereby allowing both the calculation of electronic g‐tensor values with the magnetic axis orientation and a comparison of observed and calculated pseudocontact shifts utilising a computational procedure. The possible redox linked confonnational changes are found to be minimal in contrast with cytochrome c although the procedure additionally highlights aspects of the mobility of certain residues in cytochrome b
5. In this respect the residue Gly‐42 appears mobile both by this method and by the observation from NMR spectra of a major and minor conformation in this region.
A single cell observation of staurosporine effect on the Ca2+ signals in rat basophilic leukemia cells
Authors: Teshima Reiko , Ikebuchi Hideharu , Terao Tadao , Miyagawa Takehiko , Arata Yoji , Nakanishi Mamoru
Published: July 2002

A digital imaging fluorescence microscope was used to study the effect of a protein kinase inhibitor staurosporine on the antigen‐dependent calcium signals in an individual rat basophilic leukemia cell (RBL‐2H3). Although dose dependency of staurosporine was different from one cell to another, staurosporine inhibited, at low concentration, the calcium influx from the external medium into RBL‐2H3 cells. At high concentration, however, it inhibited both the removal of calcium ion from internal stores and the calcium influx from the external medium. These results indicated that staurosporine is necessary for the inhibition of the calcium influx from the external medium and that a protein kinase (possibly protein kinase C) is involved in the calcium influx from the external medium into the cytoplasm.
Flight and heat dissipation in birds
Authors: Giardina Bruno , Corda Marcella , Pellegrini Maria G. , Sanna Maria T. , Brix Ole , Clementi Maria E. , Condo Saverio G.
Published: July 2002

Birds during normal sustained flight must be able to dissipate more than 8 times as much heat as during rest in order not to be overheated. The experiments reported in this note on the hemoglobin systems from two different birds indicate the existence of a molecular mechanism by which hemoglobin is used simultaneously for oxygen transport and heat dissipation.
Localization of transcription initiation sites in the human coagulation factor IX gene
Authors: Reijnen Marleen J. , Bertina Rogier M. , Reitsma Pieter H.
Published: July 2002

The transcription start sites of the human gene coding for the coagulation factor IX have been identified. Three major transcription initiation sites within a small area of ~ 30 nucleotides were found by Sl nuclease analysis and primer extension studies.