Search

Presents upcoming research papers on biochemistry to be published in "European Journal of Biochemistry." Discussion of the molecular cloning and sequencing of a cDNA encoding the acyl carrier protein; Exploration of the primary structure of human thyroglobulin; Examination of the reversible activation of hydrogenase from Escherichia coli.

Presents a list of forthcoming papers to be published in 1980 issues of the "European Journal of Biochemistry." Subjects; Authors.

Lists forthcoming papers to be featured in the "European Journal of Biochemistry."

Presents a list of papers scheduled to be published in the "European Journal of Biochemistry," as of August 15, 1992. Topics; Authors.

Lists the forthcoming papers to be published in the "European Journal of Biochemistry."

Lists research papers scheduled for publication in the "European Journal of Biochemistry," in 1989. Topics; Authors; Data presented.

Lists the forthcoming papers to be published in the "European Journal of Biochemistry."

Presents a list of forthcoming papers scheduled to be pubilshed in the "European Journal of Biochemistry," in 1988. Subjects; Authorship.

Presents a list of forthcoming papers scheduled for publication in the "European Journal of Biochemistry," in 1988. Topics; Authors; Studies and data to be presented.

Lists the forthcoming papers to be published in the "European Journal of Biochemistry."

The authors reflect on the important changes to the board and content of the "Journal of Physiology" in 2011. He says that several new initiatives were announced throughout the year including the journal's new look in April 2011 and the publication of a series of dedicated neuroscience issues in September 2011. He also discusses the evolving content of the journal and the symposium issues published throughout the year.

Lists titles of research papers to be published in the "European Journal of Biochemistry."

Presents a list of forthcoming papers on biochemistry which appeared in the January 1983 issue of the "European Journal of Biochemistry."

The deoxyoctanucleotide (5′-3′)d(A-A-G-G-A-G-G-T), which is complementary to the 3′ end of 16-S RNA, inhibits the formation of the complex between the 30-S subunit and MS2 RNA described in the preceding paper. If the complex is preformed, the octanucleotide cannot prevent entry of the complex into the ribosome cycle upon supplementation with the components for protein synthesis. The subunit · MS2-RNA complex is unable to bind the octanucleotide. It is concluded that in the subunit · phage-RNA initiation precursor the 16-S terminus is base-paired with a complementary MS2 RNA sequence. Edeine, aurintricarboxylic acid and antibodies against ribosomal protein S1 prevent the association of phage RNA with 30-S subunits. These compounds do not, however, inhibit the binding of (5′-3′)d(A-A-G-G-A-G-G-T) to 30-S subunits. It is concluded that the formation of the complex between MS2 RNA and 30-S subunits does not depend solely on the Shine and Dalgarno base-pairing reaction. [ABSTRACT FROM AUTHOR]

Adult bovine aortic endothelial (ABAE) cells, exposed to serum-free medium, specifically bind 125I-labeled human high-density lipoprotein (125I-HDL). Addition of human lipoprotein-deficient serum (LPDS) reduces the specific binding of 125I-HDL in a concentration-dependent manner, such that LPDS at a concentration of 6 mg protein/ml almost completely inhibits the specific binding of 125I-HDL. ABAE cultures exposed to 125I-labeled LPDS (125I-LPDS) specifically bind two peptides, which appear as minor iodinated components in 125I-LPDS. The binding of these two components is abolished in the presence of excess amounts of unlabeled LPDS or HDL. Preincubation of ABAE cells with 25-hydroxycholesterol (25-HC) results in an increase in the binding of the two 125I-LPDS components, similar to the increase observed in 125I-HDL binding in the presence of 25-HC. These two LPDS components comigrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) with apolipoproteins A-I and A-IV of molecular masses 28 kDa and 43 kDa respectively. Furthermore, these two proteins were transfered from the SDS gel to nitrocellulose paper and interacted specifically with anti-(A-I) and anti-(A-IV) sera respectively. When ABAE cultures, pretreated with 25-HC in the presence of LPDS, are subjected to cell-surface iodination, the A-IV appears as one of the major proteins on the cell surface accessible to iodination. The interaction of A-IV with the cell surface of 25-HC-treated cells is not specific to ABAE cells and appears also in human skin fibroblasts. Analysis of the relative amounts of various apolipoproteins in the 125I-HDL bound to ABAE cells demonstrates a decrease in the relative amount of iodinated A-II concomitant with increase in the relative amounts of the other iodinated apolipoproteins, when compared to the composition of the native 125I-HDL. These changes are similar whether the binding is done in the presence or absence of LPDS. It indicates that the decrease in 125I-HDL binding in the presence of LPDS is not due to displacement of the iodinated apolipoproteins A-I and A-IV in the 125I-HDL by unlabeled A-I and A-IV present in LPDS. The results indicate that free apolipoproteins A-I and A-IV, present in LPDS, can displace HDL on the cell surface of ABAE cells. Thus, free A-I and A-IV, present in plasma, control the binding of HDL to endothelial cells and may regulate the process of cholesterol removal from the cells performed by HDL. [ABSTRACT FROM AUTHOR]

ORAL PRESENTATION ABSTRACTS Lamb1Dendra2 - a new mouse model to study basement membrane dynamics in vivo J. Morgner SP * sp ; K. Hahn SP * sp ; C. L. Iglesias SP ‡ sp ; L. Kroese SP † sp ; C. Pritchard SP † sp ; P. Peters SP ‡ sp ; I. Huijbers SP † sp ; J. van Rheenen SP * sp I SP * sp Department of Molecular Pathology, Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands; SP † sp Mouse Clinic for Cancer and Aging, The Netherlands Cancer Institute, Amsterdam, The Netherlands; SP ‡ sp The Maastricht Multimodal Molecular Imaging Institute, Maastricht University, Maastricht, The Netherlands i B Introduction: b The basement membrane (BM) maintains tissue architecture by separating epithelia and blood vessels from the surrounding tissue. POSTER ABSTRACTS Production of soluble basement membrane proteins in the cytoplasm of E. coli A. A. Sohail; M. Gaikwad; L. W. Ruddock I University of Oulu, Finland i B Introduction: b Basement membranes (BM) are thin layers of extracellular matrix deposition which surrounds cells of epithelial, muscle, adipose and the endothelium lining. From target identification to disease-modifying therapies for a basement membrane disease A. Nyström I Department of Dermatology, Medical Faculty, Medical Center - University of Freiburg, Hauptstrasse 7, D-79104, Germany i In skin, collagen VII enables firm attachment of the epidermal basement membrane to the superficial papillary dermal extracellular matrix. Report by The 2020 BSMB Autumn meeting was planned as an in-person get-together of matrix biologists with a focus on the important role of basement membranes in health and disease. [Extracted from the article]

B Abstract: b Plasticity of cancer invasion and metastasis depends on the ability of cancer cells to switch between collective and single cell dissemination, under the control of cadherin mediated cell-cell junctions and the organization of the extracellular matrix. Using spatially defined organotypic culture, intravital microscopy in breast cancer in mice and in silico modeling, we here identify cell jamming by 3D tissue boundaries as dominant physical mechanism which supports collective invasion irrespective of the composition and stability of cell-cell junctions. These data reveal that steric hinderance by 3D tissue can substitute for cadherin-dependent cell-cell cooperation and dictates cell jamming and unjamming transitions in complex environments. B Results: b We demonstrate the effect of size (10 nm - 100 nm), coating (+/- polymer) and concentration (up to 50 g/mL) of silver nanoparticles with respect to cytotoxicity (MTT), immunogenicity (ELISA; IL-6), cell motility (scratch assays) and cell proliferation (clonogenic) in human skin fibroblasts and keratinocytes cells in 2D. Upregulation of LaNt 31 in tumour cells did not influence invasion into collagen I, however it did influence the way in which MDA-231 cells invaded into matrigel; instead of the characteristic multi-cellular streaming of control cells, LaNt 31 overexpressing cells invaded as individuals. [Extracted from the article]

Local analgesics are the most commonly used group drugs in dental practice. However, due to their frequent use and high margin of safety, often dental practitioners neglect to properly understand the biology and pharmacology of these drugs. This article reviews the basic concepts of pain, pain pathways, the mode of action of local analgesics and factors which affect their usage. Specific details and properties of some currently available solutions are also outlined. A greater understanding of the biology and pharmacology of local anaesthetics will ultimately lead to safer and more effective use in everyday clinical practice. [ABSTRACT FROM AUTHOR]

A mathematical description was made of an autocatalytic zymogen activation mechanism involving both intra- and intermolecular routes. The reversible formation of an active intermediary enzyme–zymogen complex was included in the intermolecular activation route, thus allowing a Michaelis–Menten constant to be defined for the activation of the zymogen towards the active enzyme. Time–concentration equations describing the evolution of the species involved in the system were obtained. In addition, we have derived the corresponding kinetic equations for particular cases of the general model studied. Experimental design and kinetic data analysis procedures to evaluate the kinetic parameters, based on the derived kinetic equations, are suggested. The validity of the results obtained were checked by using simulated progress curves of the species involved. The model is generally good enough to be applied to the experimental kinetic study of the activation of different zymogens of physiological interest. The system is illustrated by following the transformation kinetics of pepsinogen into pepsin. [ABSTRACT FROM AUTHOR]

Why are there so many dimeric proteins and enzymes? While for heterodimers a functional explanation seems quite reasonable, the case of homodimers is more puzzling. The number of homodimers found in all living organisms is rapidly increasing. A thorough inspection of the structural data from the available literature and stability (measured from denaturation–renaturation experiments) allows one to suggest that homodimers can be divided into three main types according to their mass and the presence of a (relatively) stable monomeric intermediate in the folding–unfolding pathway. Among otherexplanations, we propose that an essential advantage for a protein being dimeric may be the proper and rapid assembly in the cellular milieu. [ABSTRACT FROM AUTHOR]

Cell lines resistant to growth inhibition by 10 μM DL-p-fiuorophenylalanine (PFP) have been isolated from a stock cell suspension culture line of Acer pseudoplatanus L. One of the lines (Xs) retained its resistance when serially subcultured in the absence of PFP. X[SUB8] could not be distinguished from the parent line in growth rate and biomass yield but in mixed cultures of the two lines, X8 cells progressively declined as a fraction of the cell population during serial subculture in the absence of PFP. The X[SUB8] line could be recovered from such mixed cultures by subculture to medium containing 10 μM PFP. Line X[SUB8] resembled the parent cell line in phenylalanine (PA) pool size and in showing no evidence of discrimination between PA and PFP in protein synthesis. It differed from the parent line in exhibiting lower uptake of PFP and PA; at an appropriate level PA almost completely suppressed PFP uptake. X[SUB8] cells had a higher content of phenols and higher extractable activity of phenylalanine ammonia lyase (PAL); they actively degraded absorbed PFP. The problem of isolation of biochemical mutants from plant cell cultures is discussed. [ABSTRACT FROM AUTHOR]

Improved methodology was used to establish that the phosphorylation of a serine located 10 residues from the N-terminus of glycogen synthase (N10) increases from 0.12 mol · mol-1 to 0.54 mol · mol-1 in vivo in response to adrenalin. The only ‘N10 kinase’ detected in muscle extracts was casein kinase-1 (CK1), although its activity was unaffected by injection of adrenalin in vivo or by incubation with cyclic-AMP-dependent protein kinase and MgATP in vitro. Prior phosphorylation of the serine residue N7 by phosphorylase kinase increased sixfold the rate of phosphorylation of glycogen synthase by CK1, and altered the specificity of CK1 so that it phosphorylated the serine residue N10 specifically. Stoichiometric phosphorylation of N7 decreased the activity ratio (± glucose 6-phosphate) of glycogen synthase from 0.80 to 0.45, and subsequent phosphorylation of N10 to 0.8 mol · mol-1 produced a further decrease to 0.17, demonstrating that N10 phosphorylation inhibits glycogen synthase. The major ‘N10 phosphatase’ in skeletal muscle extracts was identified as the glycogen-associated form of protein phosphatase-1 (PP1G), accounting for approximately 75% of the N10 phosphatase activity in the extracts and about 90% of the activity in isolated glycogen particles. Phosphorylation of N10, after prior phosphorylation of N7, decreased the rate of dephosphorylation of N7. These results, in conjunction with previous findings, establish that adrenalin inhibits glycogen synthase by increasing the phosphorylation of N7, N10 and three further serines located 30,4 and 38 residues from the start of the C-terminal CNBr peptide (termed the region C30-C38). They also indicate that increased phosphorylation of N10, the region C30-C38, and perhaps N7, is initiated through the inhibition of PP1G by adrenalin, which results from phosphorylation of its glycogen-targetting subunit by cyclic-AMP-dependent protein kinase [Hubbard, M. J. & Cohen, P. (1989) Eur. J. Biochem. 186, 711–716]. The conclusion that direct phosphorylation of glycogen synthase by cyclic-AMP-dependent protein kinase makes little contribution to inhibition by adrenalin, is at variance with the teachings of the major textbooks of biochemistry. [ABSTRACT FROM AUTHOR]

Calmodulin contains several binding sites for hydrophobic compounds. The apparent specificity of various ‘calmodulin antagonists’ for these sites was investigated. The Ki values for the inhibition of calmodulin-activated cyclic-nucleotide phosphodiesterase and myosin light-chain kinase was determined. In addition, the Kd values of the same compounds for binding to calmodulin were measured. The compounds could be separated into four. groups. Group I and II compounds inhibited competitively the activation of the phosphodiesterase and myosin light-chain kinase by calmodulin. Group I compounds inhibited the activation of the phosphodiesterase and myosin light-chain kinase at identical concentrations. In contrast, group II compounds inhibited the activation of the phosphodiesterase at 5–10-fold lower concentrations than that of myosin light-chain kinase. Group III compounds inhibited the activation of these enzymes by an uncompetitive mechanism. Group IV compounds inhibited the activation of the phosphodiesterase with Ki values above 10 μM and did not affect the activation of myosin light-chain kinase. Binding of [3H]bepridil to calmodulin under equilibrium conditions yielded one high-affinity site (apparent Kd 0.4 μM) and four low affinity sites (apparent Kd 44 μM). Group I compounds interfered with the binding of bepridil to the high and low-affinity sites in a competitive manner. Group II compounds interfered in a non-competitive manner with the high-affinity site and apparently competed only with one of the low-affinity sites. Group III compounds did not compete with any of the bepridil-binding sites. Nimodipine, a group III compound, bound to one site on calmodulin with a Kd value of 1.1 μM. Other dihydropyridines competed with [3H]nimodipine for this site. The group I and II compounds, trifluoperazine and prenylamine, did not affect the binding of [3H]nimodipine. These data show that ‘calmodulin antagonists’ can be differentiated into at least three distinct groups. Kinetic and binding data suggest that the three groups bind to at least three different sites on calmodulin. Selective occupation of these sites may inhibit specifically the activation of distinct enzymes. [ABSTRACT FROM AUTHOR]

Changes in the concentration of several metabolites and enzymes related to carbohydrate metabolism were measured during the growth of Saccharomyces cerevisiae on a mineral medium containing glucose as the limiting nutrient. When about 50% of the original glucose was used the exponential phase ended and the culture entered a ‘transition’ phase before the complete exhaustion of glucose. In this transition phase several metabolic changes occurred. cAMP, that decreased along growth, reached a constant value of about 0.7 nmol/g dry weight. A pronounced drop in fructose-6-phosphate-2-kinase activity and in the concentration of fructose 2,6-bisphosphate and fructose 1,6-bisphosphate was observed accompanied by a less marked decrease in hexose monophosphates. Trehalase activity also dropped and reached a minimal value at the onset of the stationary phase when synthesis of trehalose began. Glycogen concentration and glycogen synthase activity increased sharply during the transition phase. Plasma membrane ATPase began to increase at the middle of the exponential phase and then, coincident with the glucose exhaustion, a 90% decrease in the measurable activity was observed. [ABSTRACT FROM AUTHOR]

We have monitored the thermotropic behavior of mixed dimyristoylglycerophosphoserine (Myr2GroPSer)/dimyristoylglycerophosphocholine (Myr2GroPCho) and Myr2GroPSer/dipalmitoylglycerophosphocholine (Pam2GroPCho) vesicles in the presence of blood-clotting factor Va, using 1,6-diphenyl-1,3,5-hexatriene as a lipid probe. The Ca2+-independent interaction of factor Va with these vesicles caused a small increase (1-2&geg;C) in the phase transition temperature, regardless of whether Myr2GroPChe was the lower or higher-melting component of the mixed vesicles. The major effect of factor Va was to increase the polarization of diphenylhexatriene when the mixed vesicles were in the liquid crystalline phase. The protein did not change the anisotropy in the bilayer gel state. The increase in the polarization value above the transition temperature closely correlated with the amount of phospholipid-bound factor Va, as verified by a direct binding technique. In addition, we found that the affinity of factor Va for Myr2GroPSer/Myr2GroPCho and Myr2GroPSer/Pam2GroPCho greatly increased at temperatures above the transition temperatures. Time-dependent fluorescence anisotropy measurements of diphenylhexatriene embedded in vesicles in the liquid crystalline state give fluorescence decay curves which can best be fitted by two exponential functions with two rotational correlation times and a constant term. Vesicles composed of Myr2GroPSer exhibit more ordering than Myr2GroPCho vesicles. However, the order parameter of mixed vesicles composed of 40% Myr2GroPSer and 60% Myr2GroPCho (mol/mol) approached that of Myr2GroPCho. Factor Va dramatically increased the longer rotational correlation time of diphenylhexatriene embedded in mixed vesicles in the liquid crystalline state from 3.7 ns to about 17 ns. The second rank-order parameter increased only slightly, but the calculated steady-state auisotropy increased by twofold. These results indicate that the acidic phospholipid-dependent binding of factor Va to mixed vesicles has an ordering effect on the acyl chains of the acidic phospholipids in the outer layer, but leaves the bulk of the phospholipids, mainly phosphatidylcholine, unaltered. None of the factor-Va-induced alterations in the anisotropy parameters point to the occurrence of lateral phase separation. [ABSTRACT FROM AUTHOR]

A specific ribonuclease was detected and purified to homogeneity from six-day-old larvae of the insect Ceratitis capitata and its homogeneity was checked by analysis in polyacrylamide gels in the presence of sodium dodecyl sulfate. The nuclease specifically degrades poly(U) and poly(C) whilst it fails to do so with other single-stranded homopolyrihonucleotides. The enzyme has a pH optimum in the region 7–9 and relative molecular mass of about 25000. The effect of this ribonuclease on the integrity of RNAs isolated from six-day-old larvae or rat liver was also studied. [ABSTRACT FROM AUTHOR]

Hyoscyamine 6β-hydroxylase, a 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of l-hyoscyamine to 6β-hydroxyhyoscyamine in the biosynthetic pathway leading to scopolamine [Hashimoto, T. & Yamada, Y, (1986) Plant Physiol. 81, 619–625] was purified 310-fold from root cultures of Hyoscyamus niger L. The enzyme has an average Mr of 4l000 as determined by gel filtration on Superose 12 and exhibited maximum activity at pH 7.8. l-Hyoscyamine and 2-oxoglutarate are required for the enzyme activity, with respective Km values of 35 μM and 43 μM. Fe2+, catalase and a reductant such as ascorbate significantly activated the enzyme. 2-Oxoglutarate was not replaced by any of ten other oxo acids tested, nor was Fe2+ by nine other divalent cations tested. The enzyme was inhibited moderately by EDTA, Tiron and various oxo acids and aliphatic dicarboxylic adds, and strongly by nitroblue tetrazolium and divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Several pyridine dicarboxylates and o-dihydroxyphenyl derivatives inhibited the hydroxylase. Pyridine 2,4-dicarboxylate and 3,4-dihydroxybenzoate are competitive inhibitors with respect to 2-oxoglutarate with the respective Ki values of 9 μM and 90 μM. Several alkaloids with structures similar to l-hyoscyamine were hydroxylated by the enzyme at the C-6 position of the tropane moiety. The enzyme preparation also epoxidized 6,7-dehydrohyoscyamine, a hypothetical precursor of scopolamine, to scopolamine (Km 10 μM). This epoxidation reaction required the same co-factors as the hydroxylation reaction and the epoxidase activities were found in the same fractions with the hydroxylase activities during purification. Two possible pathways for scopolamine biosynthesis are discussed in the light of the hydroxylase and epoxidase activities found in the partially purified preparation of hyoscyamine 6β-hydroxylase. [ABSTRACT FROM AUTHOR]

The Helical structures of d(C-G-C-A-m5C-G-T-G-mSC-G), d(m5C-G-C-A-m5C-G-T-G-C-G) and d(C- 2aminoA-C-G-T-G) were studied in aqueous solution at various salt concentrations and temperatures by 1H-NMR spectroscopy. In 0.1 M NaCl solution only the B form was evidenced for these DNA fragments whereas in 4 M NaCI both B and Z forms, in slow exchange on the NMR time scale, were observed. Under these conditions the Z form accounted for less than 60% of the decamer conformation; conversely d(C-G)3 hexamers containing methylated cytidines were predominantly in the Z form (> 90%) [Tran-Dinh et al. (1984) Biochemistry 23, 1362: Cavailles et al. (1984) J. Biomol. Struct. Dyn. I. 1347–1371]. On the other hand, d(C-2aminoA-C-G-T-G) in which the d(2ammoA) · dT base pair forms three hydrogen bonds, was found to adopt the Z conformation in 4M NaCI solution which was not the case for d(C-A-C-G-T-G) (unpublished results). The present study shows that the B ⇄ Z transition in solution is highly sequence-dependent and that correlation exists between the stability of the duplexes (essentially governed by the number of hydrogen bonds between complementary bases) and their ability to adopt the Z conformation. [ABSTRACT FROM AUTHOR]

Basic fibroblast growth factor (bFGF), a potent mitogen for arterial smooth muscle cells has been shown to play a fundamental role in the pathogenesis of arteriosclerosis and restenisos by stimulating the proliferation of vascular smooth muscle cells. We found that partially phosphorothioate-modified 15-redisue antisense oligodeoxynucleotides complementary to bFGF mRNA at 0.1-2.0 μM clock growth and division of cultured human and bovine coronary smooth muscle cells I a dose-dependent manner. The effect is sequence specific at low (0.1-0.5 μM) nontoxic concentrations. It is associated with inhibition of expression of pericellular and intracellular bFGF, with a decreased de novo synthesis of bFGF and is partly reversible by the addition of exogenous (recombinant) bFGF. The antisense effect lasts 48-72h and diminishes therafter. If the antisense oligodeoxynucleotide medium is replaced by an oligonucleotide-free medium after 24 h. the [³H]thymidine incorporation rate returns to control levels. Under the same conditions, the corresponding sense oligodeoxynucleotide exerts negligible nonspecific inhibitoty actions. The antiproliferative potency of the 15-residue antisense oligodeoxynucleotide is markedly enhanced by adding 3-4 nobase-pairing guanosine residues at the 5'- and 3'-termini of the 15-residue antisense oligonucleotide. [ABSTRACT FROM AUTHOR]

A human monocyte-activating CC chemokine has been identified based on sequences in an expressed sequence tag (EST) cDNA database. The protein shows highest sequence identity to th macrophage inflammatory protein (MIP) group of chemokines, particularly MIP-3 (76.7$) and MIP-1α (75.4%), and has been named MIP-5. Model building confirms that the protein has a similar three dimensional structure to other chemokines, but has an additional third disulphide bond. Northern blot analysis and reverse-transcriptase PCR show that the mRNA for MIP-5 is expressed at a high levels in liver, intestine and in lung leukocytes. MIP-5 induces chemotaxis of human monocytes. T-lymphocytes and, to a lesser degree, eosinophilsat nanomolar concentrations; it has no effect on neutophil migration. In receptor-binding assays. MIP-5 shows IC50 values of 12 mM for competition with 125I-MIP-1α for binding to CC-chemokine receptor (CCR)1, and 2.5 nM for competition with 125I-MCP-3 for inding to CCR3. It shows no ability to compete with ligand for binding to the two interleukin (IL)-8 receptors (CXC-chemokine receptors 1 and 2) or to CCR2, CCR4 or CCR5. Consistent with this binding data, MIP-5 wa sonly able to induce calcium fluxes in CHO cells stably transfected with CCR1 or CCR3. [ABSTRACT FROM AUTHOR]

The interaction of recombinant ascorbate peroxidase (APX) with its physiological substrate, ascorbate. has been studied by electronic and NMR spectroscopies, and by phenylhydrazine-modification experiments. The binding interaction for the cyanide-bound derivative (APX-CN) is consistent with a 1:1 stoichiometry and is characterised by an equilibrium dissociation binding constant. Kd, of 11.6.&plusmnl;0,4 μM (pH 7.002, μ = 0.10 M, 25.0°C). Individual distances between the non-exchangeable substrate protons of APX-CN and the haem iron were determined by paramagnetic-relaxation NMR measurements, and the data indicate that the ascorbate binds 0.90-1.12 nm from the haem iron. The reaction of ferric APX with the suicide substrate phenylhydrazine yields predominantly (60%) a covalent haem adduct which is modified at the C20 carbon, indicating that substrate binding and oxidation is close to the exposed C20 position of the haem. as observed for other classical peroxidases. Molecular-modelling studies, using the NNMderived distance restraints in conjunction with the crystal structure of the enzyme [Patterson. W. R, & Poulos. T L, (1995) Biochemistry 34, 4331-4341], are consistent with binding of the substrate close to the C20 position and a possible functional role for alanine 134 (proline in other class-III peroxidases) is implicated. [ABSTRACT FROM AUTHOR]

In the present study, we synthesized 14C-labelled 5-nitro-l.2.4-triazol-3-one (NTO) and investigated its hepatic metabolism by dexamethasone-induced murine hepatic microsomes. Under the nitrogen atmosphere. 5-amino-l.2.4-triazol-3-one was the only detected metabolite of NTO. The microsomal nitroreductase activity was dependent on NADPH, totally inhibited by carbon monoxide and partially inhibited by oxygen. In aerobic conditions, beside a low amount of amine, the major metabolite formed is the 5-hydroxy-triazolone, urazole. This compound resulted from the oxidative denitrification of NTO, which produced equivalent amount of nitrite. This reaction, like the nitroreductase activity, was dependent on NADPH and totally inhibited by carbon monoxide. Both nitroreduction and oxidative denitrification were inhibited by imidazole-related inhibitors: miconazole and methimazole, and to a less extent by N-octylamine. The microsomal denitrification was induced by the treatment of mrs with dexamethasone and phenobarbital. The microsomal reductase activity is present in untreated rat microsomes, and recovered with various inducers. The results of this study indicate the role played by cytochrome P-450 in the metabolism of NTO. supported by its transformation with reconstituted cytochrome P-450 systems. [ABSTRACT FROM AUTHOR]

The fatty acid specificity of phospholipase D purified from germinating sunflower seeds was studied using mixed micelles with variable detergent/phospholipid ratios. The main advantage of this approach is that since the substrate is integrated in the detergent micelles, comparisons can be made between the kinetic constants of a wide range of phosphatidylcholine (PtdCho) compounds with various fatty acid contents. Phospholipase D is subject to interracial activation as it is most active on water-insoluble substrates. It is not active on sphingomyelin and only slightly on lysophosphatidylcholine. By fitting the curves based on the experimental kinetic data, the interfacial dissociation constant of phospholipase D. the maximum hydrolysis rate Vm and the kinetic constant Kbm were determined with the micellar substrate. The specificity of various substrates was examined by comparing the Vm/KKbm values, and it was noted that sunflower phospholipase D is most active on medium-chain fatty PtdCho compounds. With long-chain natural phospholipids, the specificity of phospholipase D was slightly dependent on the level of fatty acid unsaturation. The pure enzyme was able to hydrolyse the sunflower phospholipids present m mixed detergent micelles but not the phospholipids integrated in the natural sunflower oil body structure. We concluded, however, that during the germination of sunflower seeds, phospholipase D might be involved in the degradation of oil bodies, since other factors present in crude seed extracts may make phospholipids accessible to the enzyme. [ABSTRACT FROM AUTHOR]

Protein kinase CK2 is a ubiquitous pleiotropic serine/threonine protein kinase whose holoenzyme is comprised of two catalytic (α and/or α') and two non-catalytic, β-subunits. The β-subunit possesses antagonist functions that can be physically dissected by generating synthetic fragments encompassing its N-terminal and C-terminal domains. Here we show that by mutating basic residues in the 74-77 and in the 191- 198 regions of the α-subunit, the negative regulation by the β-subunit and by its N-terminal synthetic fragment CK2β-(41-77), which is observable using calmodulin as a substrate for phosphorylation, is drastically reduced. In contrast, the positive regulation by a C-terminal CK2β-(155-215)-peptide is unaffected or even increased. Moreover, the basal activity of α mutants K74-77A, K79R80K83A. and R191R195K198A toward specific peptide substrates is stimulated by the β-subunit many fold more than rival of α wild type, while extrastimulation by β mutant D55L56E57A, observable with α wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of β is mediated by basic residues in the 74-83 and in the 191-198, sequences of the α-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the β subunit operates as a pseudosubstrate. In contrast, another CK2α mutant, V66A, is more sensitive to inhibition by either β-subunit or its N-terminal. CK2β(1 - 77)-peptide, while its stimulation by the C-terminal peptide, C K 2β-(155- 2l5 ), is comparable to that of a wild type. These observations suggest an indirect rote of Val66, in conferring to the α-subunit a conformation less sensitive to down regulation by β-subunit. [ABSTRACT FROM AUTHOR]

(1) In the presence of suitable phyllosphere bacteria, aqueous washings of the foliage of Camelina satica contain allelochemicals. (2) Investigations into the nature of these chemicals and their precursors, and the essential role of bacteria in the production of allelochemicals are discussed. (3) Benzylamine is identified as an allelochemical influencing the association of C. sativa with Linum usitatissimum. [ABSTRACT FROM AUTHOR]

A new reagent, ethyl 4-azidobenzoylaminoacetimidate, was prepared in a four-step synthesis starting from 4-aminobenzoic acid. This compound was used to cross-link RNA with proteins within the Escherichia coli 30-S ribosomal subunits. Following the reaction of the imidoester function with protein NH2 groups, photoactivation of the azide binds the other end of the reagent to RNA. The cross-linked proteins were labelled with 125I and identified by bidimensional gel electrophoresis. Proteins S3, S4, S5, S7, S9, S17, S18 and, in a lower and more variable yield, S12, S13, S14 and S16 were bound to 16-S RNA. These results were confirmed by isolating cross-linked protein-oligonucleotide complexes from 30-S subunits containing 32p-labelled RNA. [ABSTRACT FROM AUTHOR]

30-S ribosomal subunits of Escherichia coli form a stable complex with MS2 RNA or Qβ RNA at 37βC in the absence of initiator tRNA. The complex functions as a precursor of initiation since it can enter the ribosome cycle in the presence of inhibitors of de novo initiation. [ABSTRACT FROM AUTHOR]

The aim of the present work was to determine whether the accumulation of Ig mRNA in myeloma cells is due to a high rate of production or to a high stability of these molecules. Specific mRNAs for the light and heavy polypeptide chains of IgG were isolated from the murine MPC-11 myeloma tumor cells by immune precipitation of polysomes which synthesize these chains. It was found that the immune-precipitated polysomes were enriched 10–30-fold in the γ and [This symbol cannot be presented in ASCII format] mRNA sequences respectively. In the wheat germ cell-free system the [This symbol cannot be presented in ASCII format] mRNA preparation was translated mainly into three polypeptides of Mr 25000, 18000, and 15000. The method of immune precipitation of polysomes was also used to characterize three variant clones of MPC-11 myeloma. It was found that little if any γ-chain polysomes are present in the L-chain producer and non-producer clones, while a substantial amount of [This symbol cannot be presented in ASCII format]-chain polysomes was present in the non-producer clone. This may be due to the presence in the non-producer cells of the constant region [This symbol cannot be presented in ASCII format]-chain fragment. In order to determine the relative synthesis rate of [This symbol cannot be presented in ASCII format] and γ mRNAs, pulse-labeled polysomes were immune precipitated using antibodies to [This symbol cannot be presented in ASCII format] and γ chains. It was found that [This symbol cannot be presented in ASCII format] and γ mRNA molecules are produced at a very high relative rate each accounting for 10–15% of the total labeled mRNA after 1 h of labeling. These values are higher than the steady-state pool size of [This symbol cannot be presented in ASCII format] and γ mRNA, which was 5–6%, and indicates that the half-life of these molecules is not unusually high. It is concluded that the amplified synthesis of immunoglobulin chains in myeloma cells is mainly due to a high rate of production of translatable [This symbol cannot be presented in ASCII format] and γ mRNAs. [ABSTRACT FROM AUTHOR]

Highly active photosystem-II particles were rapidly isolated using detergents and obtained in good yield from a mutant of the green alga Chlamydomonas reinhardtii. The particles are completely devoid of reaction centers of photosystem I, and of the secondary electron acceptor to photosystem II. They show: (a) a specific activity (4A of Csso/unit chlorophyll) 4–7-times that of the starting material and of spinach chloroplasts; (b) an antenna size of 40 to 50 chlorophyll molecules containing little light-harvesting chlorophyll a/b complex (chlorophyll a/chlorophyll b = 4–6.4); (c) a ratio of variable to dark-adapted fluorescence yield of up to 3. Further treatment of these particles by ion-exchange chromatography largely removes five proteins and further decreases the antenna size with little loss in primary photoactivity. [ABSTRACT FROM AUTHOR]

Whole neurointermediate lobes were used to study the processing of the common precursor to β-endorphin and α-melanotropin (melanotropin) during pulse and pulse-chase incubations with various radioactive amino acids. After a 30-min pulse with radioactive amino acids, two major radioactive protein bands with apparent molecular weights of 34000 and 36000 were observed on a 10–30% acrylamide/sodium dodecylsulfate slab gel electrophoretogram. After a 2-h chase, most of the radioactivity associated with these two protein bands had disappeared and was recovered in smaller peptides. Analysis of the tryptic fragments from the 34000-Mr and 36000-Mr proteins showed that both contained amino acid sequences characteristic of adrenocorticotropin (corticotropin) and β-lipotropin. Processing of the two precursor forms involves several proteolytic steps. The first occurring between 30 min and I h after the start of the incubation, releases the β-lipotropin sequence from the rest of the molecule, leaving two polypeptides with apparent molecular weights of 25000 and 27000. These two peptides are thought to have identical or closely related amino acid sequences differing only in the number of carbohydrate side chains. Analysis of the tryptic peptides of these [3H]phenylalanine-labeled peptides showed that they contained fragments characteristic of corticotropin plus at least another fragment from the non-corticotropin, non-β-lipotropin part of the precursor molecule. Further maturation of the β-lipotropin and the two high-molecular-weight forms of corticotropin occurred between 1 h and 2 h after the start of incubation. β-Lipotropin served as a precursor for β-endorphin while α-melanotropin was generated by cleavage of the two high-molecular-weight forms of corticotropin. Peptides with apparent molecular weights of 17000 and 19000 were also recovered as major and stable end products of the maturation process. Tryptic peptide analysis showed that they were related to the portion of the precursor which does not contain either corticotropin or β-lipotropin. When these peptides, labeled with [3H]leucine or [3H]serine, were analyzed by automated Edman degradation, it was shown that their sequence was identical in both cases to the N-terminal sequence of the initial precursor molecule prepared during the 30-min pulse incubation. From these data, It clearly appears that the 19000-Mr and 17000-Mr peptides are related to the N-terminal fragment of the original precursor molecule. [ABSTRACT FROM AUTHOR]

Individual lysates from human erythrocyte suspensions, completely deprived of leucocytes and platelets, were assayed for a number of proteolytic activities using both naturally occurring and synthetic substrates. Removal of hemoglobin by batchwise DEAE-cellulose chromatography did not modify the complement of the various proteolytic activities which were then fractionated by means of chromatography on a column of DEAE-cellulose, followed by conventional techniques such as gel chromatography and preparative electrophoresis. This procedure allowed a number of proteinases to be identified in the erythrocyte cytosol while providing a tool for their selective though partial separation. The following peptidases were found to be present in the soluble fraction of mature human erythrocytes: (a) a neutral endopeptidase having an approximate molecular weight of 110000; (b) three acidic endopeptidases, with pH optima between 2.5 and 3.5, showing molecular and functional properties almost identical with those of the three proteinases previously purified from solubilized erythrocyte membranes [Pontremoli et al. (1979) Biochem. J. 181, 559–568]; (c) two dipeptidylaminopeptidases whose molecular weights are around 80000 and tentatively identified as dipeptidyl aminopeptidases II and III, respectively, on the basis of their substrate specificities and pH optima; (d) presumably two aminopeptidases, having an approximate molecular weight of 80000 and classified as an aminopeptidase with broad substrate specificity and an aminopeptidase B, respectively. No evidence for any carboxypeptidase activity was found in the cytosolic compartment of mature human erythrocytes. [ABSTRACT FROM AUTHOR]

Two nucleases active on alkylated-depurinated DNA have been extracted from rat liver chromatin with 1 M KCl. The major enzyme was purified to near homogeneity; it has a molecular weight of 12500 (although some dimerization might occur), needs Mg2+ or Mn2+ for activity. The endonuclease activity is specific for apurinic/apyrimidinic sites in DNA; the enzyme has no associated exonuclease activity. [ABSTRACT FROM AUTHOR]

In the presence of fluoroglucose, an inhibitor of formation of mannosylphosphoryl and glucosylphosphoryl-dolichol, lipid-dependent glycosylation of influenza virus glycoproteins is strongly, but not completely inhibited. The oligosaccharides that were transferred to protein in the presence of fluoroglucose came directly from dolichol-linked intermediates. However, they were smaller than the normal high-mannose oligosaccharides and, furthermore, resistant towards digestion with endo-β-N-acetylglucosaminidase H. By excluding mannosylphosphoryl-dolichol, similar dolichylpyrophosphate-linked intermediates were synthesized in vitro by membranes from fluoroglucose-treated cells and they were shown to glycosylate protein. [ABSTRACT FROM AUTHOR]

The effect of inorganic phosphate on Ca2+ retention has been investigated using phosphate-depleted liver mitochondria. Phosphate induces the release of Ca2+ through an efflux route insensitive to ruthenium red. This effect is not due to functional or structural damage, since mitochondria maintain their membrane potential during phosphate-induced Ca2+ efflux. Direct enzymatic measurement of mitochondrial pyridine nucleotides has established that changes in their redox state (i.e. increased oxidation) do not play a role in the phosphate-effect. The phosphate-induced Ca2+ efflux requires transport of phosphate out of mitochondria. However, the fluxes of Ca2+ and phosphate do not coincide: the release of phosphate preceeds that of Ca2+. [ABSTRACT FROM AUTHOR]