Search

We describe a sensitive immunological procedure for the detection of specific proteins in total cell extracts and for the comparison of antigenically related polypeptides. Proteins are fractionated in polyacrylamide gels and transferred electrophoretically to diazophenylthioether paper, to which they bind covalently. Specific proteins are identified by incubation with specific antibody and ⊃125 -labeled protein A from Staphylococcus aureus, followed by autoradiography. High-resolution separation of proteins prior to transfer is achieved by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate or by nonequilibrium pH gradient electrophoresis, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Further information can be obtained by limited enzymatic proteolysis of the proteins in the gel following polyacryllamide gel electrophoresis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by gel electrophoresis at right angles to the first gel. We show the application of this technique to the detection and comparison in extracts from infected cells of proteins related immunologically to the simian virus 40 capsid proteins VP1 and VP3. [ABSTRACT FROM AUTHOR]

Introduces the research papers to be published in the "European Journal of Biochemistry," succeeding the May 15, 1985. "Primer-independent abortive initiation by wheat-germ RNA polymerase B," by H. Mosig et al; "Protein structure and gene organization of mouse lactate dehydrogenase-A isozyme," S.S.-L. Li et al; "A model for intracellular complexation between gene-5 protein and bacteriophage fd DNA," by G.D. Bayer et al.

The article lists several research papers that would be published in the coming issues of the European Journal of Biochemistry. Some of the articles and topics are related to glucogen synthase, protein phosphates, DNA sequences, etc.

Presents research articles on biochemistry to be published in the "European Journal of Biochemistry." "Secondary and Tertiary Structure Characteristic of Megasphaera elsdenii Flavodoxin in the Reduced State as Determined by Two-Dimensional 1H NMR," by C. P. M. van Mierlo, F. M¨ller, and J. Vervoort; "Presence and Characterization of Glycolipid Sulfotransferase in Human Cancer Serum," by S. Gasa, M. -T. Casl, A. Makita, N. Sakakibara, T. Koyanagi, and T. Atsuta; "Interaction of Hemin With Placental Glutathione Transferase," by A. M. Caccuri; A. Aceto, F. Piemonte, C. di Ilio, N. Rosato, and G. Federici.

Lists articles about biochemistry. "Modulation of the interaction between the two halves of troponin C by the other troponin subunits," by C.-L.A. Wang and J. Gergely; "Sequence determinants of cytosolic N-terminal protein processing," by C. Flinta, B. Persson, H. Jornvall and G. von Heijne; "Effect of benzoate on the metabolism of fructose 2,6-biphosphate in yeast," by J. Francois, E. van Schaftingen and H.-G. Hers; "Isolation and properties of porcine cholesterol acyltransferase," G. Knipping; Others.

The article presents information on various research papers which will be published in the forthcoming issue of the "European Journal of Biochemistry". Some of the papers discusses are "Enzymatic Synthesis of Neolactotetraosylceramide by the N-Acctyllactosamine Synthase of Human Serum". "Rate of Transplantation and Kinetics of Processing of Newly Synthesized Molecules of Two Major Outer-Membrane Proteins, the OmpA and OmpF Proteins of Escherichia Coli," by the researchers I. Crowlesmith and K. Gamon.

Lists the titles of papers that will be published in future issues of the "European Journal of Biochemistry." "Regulation of the ontogeny of rat liver metallothionein mRNA by zinc," by G.K. Andrews et al; "Processing of a plant vacuolar protein precursor in vitro," by T. Hattori et al "Structural studies of the putatitve O-specific polysaccharide of Serratia marcescens 09," by D. Oxley and S.G. Wilkinson.

The article presents a list of forthcoming research papers including "Kinetic Studies of Proton Transfer in the Microenvironment of a Binding Site," by M. Gutman, D. Huppert and E. Nachliel," "The Substrate Binding Site of Aldehyde Reductase From Pig Liver: Stereochemical Investigations Using NADP-2-Oxodiacid Adducts As Probe," by G. Branlant and "Membranes of Protein Bodies: I. Isolation from Cotyledons of Germinating Cucumber Seeds" by U. A. K. Kara and H. Kindl.

Presents a list of research papers to be published in the upcoming issues of the "European Journal of Biochemistry." "Extraction and Translation of Collagen mRNA From Fetal Calf Skin," by R. Kaufman, A. Belayew, B. Nusgens, and C. M. Lapie&rgrave;e; "Messenger RNA For Ribosomal Proteins in Xenopus laevis Oocytes," by P. Pierandrei-Amaldi and E. Beccari; "Purification and Characterization of Esterase 2B of the House Mouse, Mus musculus," by U. Lexow, A. Ronai, and O. von Deimling.

Lists articles about biochemistry. "Impact of altered protein structures on the intracellular traffic of a mutated vasopressin precursor from Brattleboro rats," by H. Schmale et al; "Direct electron transfer of redox proteins at the bare classy carbon electrode," by W.R. Hagen; "Molecular cloning of preproacrosin and analysis of its expression pattern in spermatogenesis," by I.M. Adham; "Reduced temperature does not prevent transport of lysosomal integral membrane proteins from endoplasmic reticulum and through the Golgi system to lysosomes," by P.G. Morales; Others.

Offers information on articles for publication in future issues of the "European Journal of Biochemistry". Synthesis of two proteins in chloroplasts and messenger RNA; Types of acids from Mycobacterium thamnopheos; Use of the inhibition of enzymatic antioxidant systems in order to evaluate their physiological importance.

Presents several articles to be published in the "European Journal of Biochemistry." "Modification of Ribosomal Proteins in Sea Urchin Eggs Following Fertilization," by K. Takeshima and E. Nakano; "A Phosphotyrosyl-Protein Phosphatase Activity Associated With Acid Phosphatase From Human Prostate Gland," by H.-C. Li, J. Chernoff, L. B. Chen and A. Kirschonbaum; "Molecular Characterization of the Gene Coding for Major Outer Membrane Protein OmpA from Enterobacter Aerogenes," by G. Braun and S. T. Cole; "Nidogen: A New, Self-Aggregating Basement Membrane Protein," by R. Timpl, M. Dziadek, S. Fujiwara, H. Nowack and G. Wick; Others.

A neurodepressing hormone has been isolated and purified to homogeneity from aqueous extracts of 2000 eyestalks of the Mexican crayfish Procambarus bouvieri (Ortmann). Purification was achieved by gel filtration on Sephadex G-25 and G-15, and preparative paper electrophoresis at four pH values (1.8, 3.6, 6.0 and 10.0). The recovery of hormone activity was 85% and the specific activity was 4.0 × 104 limes that of the starting material. The hormone is a thermostable peptide of approximately 1200 molecular weight and composed of neutral amino acids. No N-terminal group could be found. From its electrophoretic behavior it is concluded that the C-terminal group is also blocked. [ABSTRACT FROM AUTHOR]

Lists papers that will published in the upcoming issues of the "European Journal of Biochemistry". "Eurabutoxin b--Initial protein refinement and sequence analysis at 0.140-nm resolution," by P.E. Bourne and colleagues; "Preferential conformation of substance P in solution," by G. Chassaing and colleagues; "Interaction of actinogelin with actin--No nucleation but high gelation activity".

After CNBr cleavage of p-hydroxybenzoate hydroxylase from Pseudornonas fluorescens, five peptides and free homoserine were isolated (see preceding paper in this journal). The amino acid sequences of the three smallest peptides, viz. CB3, CB4 and CB5, were determined by automated Edman degradation and analysis of enzymatic subdigests. These peptides form a continuous stretch of 110 residues from the N terminus: [This equation can not be represent in ASCII.TXT]. [ABSTRACT FROM AUTHOR]

The methylation of HeLa cell ribosomal proteins has been studied by paper electrophoresis separation of the peptides produced after trypsin digestion of individual ribosomal proteins. Methylation in vivo was detected by 3H labeling of peptides from cells that had been incubated with a mixture of [methyl-3H]methionine plus [35S]methionine, the incorporation of the 3H-labeled amino acid methionine being corrected by the 35S uptake. Each methylated peptide thus represented at least one methylation site (one or more methylated amino acids and their adjacent amino acid sequence). Eleven ribosomal proteins were found to be methylated. As many as five methylation sites were present in one ribosomal protein. In several ribosomal proteins some sites were methylated when ribosome synthesis was suppressed with low levels of actinomycin D, while other sites were not. Therefore, some of the sites in a given protein were apparently methylated in mature ribosomes, while other sites were methylated during ribosome processing. The methyl-group-specific labeling of some sites, but not others, was suppressed by cycloheximide, thus suggesting that the methylation of some sites was dependent on protein synthesis. A cell-free system from cultured HeLa cells is described, which reproduces the methylation pattern of ribosomal proteins in vivo in two ways: methylation was observed both in vivo and in vitro (a) in the same ribosomal proteins; and (b) in the same methylation sites of a given ribosomal protein. In addition, our data in vitro suggest that the methylation of some human ribosomal protein sites may involve a methyl donor other than S-adenosylmethionine. [ABSTRACT FROM AUTHOR]

A series of bacterial cell wall glycopeptides of low molecular weight and cell wall nucleotide precursors have been tested for their inhibitory action on the digestion by T4 lysozyme of a radioactively labeled linear uncrosslinked peptidoglycan. The disaccharide-peptides GlcNAc-MurNAc- L-Ala-D-Glu(A2pm) (C5) and GlcNAc-MurNAc-L-Ala-D-Glu(A2pm-D-Ala) (C6) as well as the monosaccharide-peptide MurNAc-L-Ala-D-Glu(A2pm) were found to be good competitive in- hibitors (with similar Ki values) whereas the disaccharide-pentapeptide GlcNAcMurNAc-L-Ala-D Glu-Gly [This symbol cannot be represented into ASCII Text]-L-Lys-D-Ala was a poor inhibitor. T4 lysozyme did not catalyse transglycosylation reactions from Escherichia coli B peptidoglycan to the disaccharide-peptide C6. No changes were seen in the circular dichroism spectra (200–250 nm) or fluorescence emission spectra upon binding of the good inhibitors. The results obtained indicate that T4 lysozyme has a small active site capable of recognizing a unit consisting of MurNAc-L-Ala-D-Glu(A2pm). [ABSTRACT FROM AUTHOR]

Procedures are described for the assay of collagen galactosyltransferase and collagen glucosyltransferase activities. The methods are based on the transfer of [14C]galactose or [14C]glucose from the corresponding radioactive UDP-glycoside to hydroxylysyl or galactosylhydroxylysyl residues in calf-skin gelatin substrate, and on the specific assay of the products of the enzymic reactions. The assay of the products includes precipitation of the protein, and removal of most of the radioactivity present in the free UDP-glycoside by several washings of the precipitate. The [14C]galactosylhydroxylysine or [14C]glucosylgalactosylhydroxylysine is then liberated by alkaline hydrolysis, and purified further by a modified Dowex chromatography and by paper electrophoresis. The procedure is entirely specific when judged by amino acid analysis, and it is possible to assay a series of 36 samples in 2 days. The sensitivity of the method permits assays in extracts from several tissues, including tissues with low transferase activities, such as liver. Preliminary characterization of the transferases from chick embryo cartilage indicated that the galactosyltransferase did not catalyze the transfer of galactose to free hydroxylysine. Dialyzable peptides were found to act as substrates for both transferases. The Km for the gelatinized collagen substrate was 150 g/1 in the reaction with the galactosyltransferase, and 14 g/1 in that with the glucosyltransferase. The Km for UDP-galactose or UDP-glucose in the reaction with the corresponding transferase was about 30 μM. Both transferases required a metal co-factor, and manganese was found to be far more effective than any other metal tested. The optimal manganese concentration for both transferases was 10 mM. No stimulation of the glucosyltransferase activity was found with folate, which has recently been reported to stimulate collagen glucosyltransferase from rat kidney. [ABSTRACT FROM AUTHOR]

Features the new semi-dry electrophoretic transfer system PhastTransfer from Pharmacia LKB Biotechnology AB. Usability of the system; Operational function; Contact information.

A new approach of predicting structural classes of protein domain sequences is presented in this paper. Besides the amino acid composition, the composition of several dipeptides, tripeptides, tetrapeptides, pentapeptides and hexapeptides are taken into account based on the stepwise discriminant analysis. The result of jackknife test shows that this new approach can lead to higher predictive sensitivity and specificity for reduced sequence similarity datasets. Considering the dataset PDB40-B constructed by Brenner and colleagues, 75.2% protein domain sequences are correctly assigned in the jackknife test for the four structural classes: all-α, all-β, α/β and α + β, which is improved by 19.4% in jackknife test and 25.5% in resubstitution test, in contrast with the component-coupled algorithm using amino acid composition alone (AAC approach) for the same dataset. In the cross-validation test with dataset PDB40-J constructed by Park and colleagues, more than 80% predictive accuracy is obtained. Furthermore, for the dataset constructed by Chou and Maggiona, the accuracy of 100% and 99.7% can be easily achieved, respectively, in the resubstitution test and in the jackknife test merely taking the composition of dipeptides into account. Therefore, this new method provides an effective tool to extract valuable information from protein sequences, which can be used for the systematic analysis of small or medium size protein sequences. The computer programs used in this paper are available on request. [ABSTRACT FROM AUTHOR]

As shown in the preceding paper [Ito, M., Ikegami, Y., Tai, T. & Yamagata, T. (1993) Eur. J. Biochem. 218, 637–643], endoglycoceramidase (EGCase: EC,3,3,1,123) was found to hydrolyze the cell-surface glycosphingolipids (GSLs) of erythrocytes without any damage to other cell membrane components. This paper represent the kinetics of EGCase action toward cell-surface GSLs of erythrocytes. Without activator or detergents, cell-surface GSLs were found to be hydrolyzed by EGCase II very slowly at pH 7.0. The initial reaction velocity of EGCase II under this condition was 0.038 pmol⋅min ¹ ⋅ mU ¹ for horse erythrocyte cell-surface GM3 and 0.032 pmol ⋅ min ¹ ⋅ mU ⋅ for guinea pig erythrocyte cell-surface Gg3Cer. The addition of activator protein (60 μM), which stimulates EGCase II in the absence of detergents, increased the initial reaction velocity of the enzyme 616-fold for cell-surface GM3 and 468-fold for Gg3Cer, while no increased hemolysis was observed with the addition of activator. However, even in the presence of the activator, the cell-surface GSLs were very resistant to hydrolysis by EGCase II compared to GSL vesicles (or micelles) under same conditions. In contrast to the activator, Triton X-100 (0.4%, mass/vol.) not only stimulated the enzyme activity but also solubilized erythrocyte GSLs into detergent micelles, inducing further increment of the enzyme activity but also solubilized erythrocyte GSLs into detergent micelles, inducing further increment of the enzyme reaction velocity. The apparent Km and Vmax values of EGCase II were calculated from the Lineweaver­Burk plot as 47 μM and 35 pmol min ¹ mU ¹ for horse erythrocyte cell-surface GM3 and 44 μM and 27 pmol ⋅ min ¹ ⋅ mU ¹ for guinea pig erythrocyte cell-surface Gg3Cer, at pH 7.0 in the presence of activator at a concentration of 60 μM. [ABSTRACT FROM AUTHOR]

The electron transfer kinetics between the hydrogenase from Desulfovibrio vulgaris (strain Hildenborough) and the mediators methyl viologen, di-(n-aminopropyl) viologen and propyl viologen sulfonate have been investigated by chronoamperometry. Second-order rate constants were calculated on basis of the theory for a simple catalytic mechanism and are compared with the results obtained before by cyclic voltammetry (preceding paper in this journal). From the ionic-strength dependence and the observed differences in the rate constants for the differently charged viologens, the existence of an electrostatic interaction between mediator and a negatively charged part of the protein is confirmed. Chronoamperometry (computer-controlled) was found to possess advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate, and better reproducibility). [ABSTRACT FROM AUTHOR]

This paper is a further study of ion binding to protein surfaces and builds on the studies of the binding of [Crk(CN)6]3- and [Fe(edta)(H2O)]- previously reported [Williams et al. (1982) FEBS Lett. 15, 293-299; Eley et al. (1982) Eur. J. Biochem. 124, 295-303]. In the present paper the binding of polyaminocarboxylate complexes of gadolinium have been studied. Eight ion-binding sites have been identified on the surface of cytochrome c. These exhibit different binding specificities which, in some cases, are not fully understood. However it is clear that simple outer-sphere interactions are not the sole determining factor for the association of metal ion complexes with proteins. The NMR paramagnetic difference spectrum method has been shown to be good at locating binding sites and revealing qualitative differences in their relative affinities for a range of complex types. However the use of relaxation probes is not a good method for the quantitative determination of binding constants; for this, isostructural shift probes must be sought. [ABSTRACT FROM AUTHOR]

Transcripts from histone H5 and α and β chicken globin genes were analysed in total RNA extracted from 2-6-day-old chick embryos Denatured RNA, separated on gels, was transferred to either aminophenylthioether (APT) paper or to nitrocellulose and was detected with 32P-labeIled recombinant DNA probes. Good resolution was achieved with methyl mercury hydroxide gels and aminophenylthioether transfer, allowing estimation of H5 and α and β globin rnRNA sizes. With glyoxal gels and nitrocellulose transfer, α and β globin mRNAs were not resolved, but an increased sensitivity of detection of at least 10-fold was achieved. Levels of H5 and α g]obin rnRNAs increased in parallel over 2-6 days. The appearance of definitive red cells in 6-day embryos was indicated by detection of adult β globin transcripts at this stage. [ABSTRACT FROM AUTHOR]

Tryptic and peptic peptides of the L-4 light chain of chicken skeletal muscle myosin were isolated. The amino acid sequences of all the tryptic peptides were determined. The alignment of the tryptic peptides in the protein was deduced from their homology with the primary structure of the A2 (L-4) light chain of rabbit skeletal muscle myosin. The compositions of the peptic peptides confirmed the alignment. Comparing the whole sequence of the L-4 light chain thus established with that of the A2 light chain of rabbit skeletal muscle myosin, 24 amino acid substitutions, were recognized. [ABSTRACT FROM AUTHOR]

The light chain fraction was separated from myosin extracted from chicken cardiac muscle. Two light chain components, L-1 and L-2 in the fraction were isolated by chromatography on a column of DEAE-cellulose (DE-52) in the presence of 4 M urea. After performic acid oxidation, the L-1 chain was digested with trypsin and the resulting peptides were isolated. The amino acid sequences of the peptides were established. The ordering of these tryptic peptides in the L-1 chain was deduced from the amino acid compositions and the partial sequences of peptic peptides from S-carboxymethylated L-1 chain. Comparing the whole sequence of the L-1 chain thus established with that of alkali light chain of rabbit skeletal muscle myosin, 67 amino acid substitutions and two insertions were recognized. [ABSTRACT FROM AUTHOR]

The covalent structures of the four highly homologous Fc-binding regions in protein A, regions D, A, B, and C, have been studied by enzymic fragmentations of previously isolated fragments originating from these regions and subsequent isolation of the generated peptides by ion-exchange chromatography, molecular-sieve chromatography, high-voltage paper electrophoresis and paper chromatography. The complete sequence of region B was elucidated by combining the results of Edman degradations on isolated fragment B peptides with the previously reported N-terminal sequence of the same fragment. Furthermore, Edman degradations of fragment D, A and C peptides differing from the region B sequence provided the structures of subregions not identical to corresponding subregions within region B. Thus, it is possible to propose a highly probable covalent structure for the N-terminal 27 000-molecular-weight portion of protein A responsible for the IgG-Fc-binding activities. However, it was not possible to assign the activities to specific structures within the regions. The sequence data indicate that not only mutual homology between the four regions exists, but also internal homologies within the regions. Furthermore, the data strongly supports the hypothesis of a stepwise gene fusion procedure being involved in the evolution of the protein. [ABSTRACT FROM AUTHOR]

A purified glycoprotein with blood-group-A specificity, was digested with insoluble pronase to give a serologically active product enriched in peptide-bonded serine and threonine residues; this was degraded with 0.25 M sulphuric in glacial acetic acid for 24 h. The resultant N-acetylgalactosaminyl-peptide were hydrolysed with 0.2 M hydrocholoric acid for 24 h at 110 °C. fractionation of the products on Sephadex G-10, followed by preparative separation on amino-acid analyzer and paper electrophoresis at pH 1.9 yielded the following homogeneous oligopeptides; one tripeptide with the N-terminal sequence Thr-Thr-Ser, three tetrapeptides with the sequences, Thr-Ser-Thr-Ser, Thr-Pro-Thr-Ser and Pro-Thr-Thr-Ser, one pentapeptide with the sequence Thr-Pro-Thr-Ser, one hexapeptide with the sequence Pro-Thr-Thr-Thr-Pro-Ser and one heptapeptide with the sequence Ala-Pro-Thr-Thr-Ser-Gly-Ser. Galactosamine was present in several of these fragments and the hexapeptide and heptapeptide both contained sufficient of this sugar for all the serine and threonine residues to be glycosylated. The following five tetrapeptides sequences were deduced from mixtures of two peptides which differed by the amino-acid substituent at only one position in the chain; Pro-Thr-Ala-ser, Thr-Thr Pro-Ser, thr-Thr-Ala-Ser, Ser-Pro-Thr-Ser, and Ser-Ala-Thr-Ser. A tripeptide mixture which differed only in the nature of the third amino acid yielded the following three tripeptide sequences: Pro-Thr-Ser, Pro-thr-Ala. These peptides do not indicate a clearly defined amino-acid sequence surrounding the glycosylated hydroxy amino acids but establish that in the human blood-group-specific glycoproteins there is close packing of the threonine in the region of the peptide moiety carrying the carbohydrate chains. [ABSTRACT FROM AUTHOR]

Arginine is a versatile additive to prevent protein aggregation. This paper shows that arginine ethylester (ArgEE) prevents heat-induced inactivation and aggregation of hen egg lysozyme more effectively than arginine or guanidine. The addition of ArgEE decreased the melting temperature of lysozyme. This data could be interpreted in terms of ArgEE binding to unfolded lysozyme, possibly through the ethylated carboxyl group, which leads to effective prevention of intermolecular interaction among aggregation-prone molecules. The data suggest that ArgEE could be used as an additive to prevent inactivation and aggregation of heat-labile proteins. [ABSTRACT FROM AUTHOR]

We extend the analysis of unbranched chains (preceding paper) to large parameter changes in branched systems using linear kinetic assumptions. More complex relationships between flux control coefficients and deviation indices are established. In particular, the deviation index in such systems depends on more than one control coefficient as well as on the magnitude of the enzyme change. Non-additivity of the indices is the general rule. Combined changes of groups of enzymes, whether co-ordinate or not, have also been formulated. Control coefficients can be estimated from a small number of independent large-change experiments. Alternatively, the amplification factors can be calculated given the knowledge of the control coefficients. A 'case study' using published data is presented. The movement of intermediate metabolites as a consequence of large parameter changes can be dealt with in a similar manner. Experimental methods for showing the admissibility of assuming the simplifying assumptions used are summarised. Some simulation studies show possible limits of the application of the approach and some aspects of the general, non-linear, case are discussed. It is concluded that, although metabolic systems are in principle non-linear, many behave, in practice, as quasi-linear systems. The relationships established between deviation indices and control coefficients therefore provide a practical way of predicting the effects of large-scale changes in parameters for many metabolic systems. [ABSTRACT FROM AUTHOR]

Hexameric hemocyanin from a spiny lobster. Panulirus japonicus, comprises three major subunits (Ib, II and lid end one minor subunit (Ia), as reported in the preceding paper in this journal. It has previously been shown that the O2 equilibria of Panulirus hemocyanin can be described by a concerted model extended to three affinity states [Makino, N. (1986) Fur. J. Biochem. 154, 49–55]. In this study the equilibrium binding of O2 to the reassociated sebunits (Ib, II and III) was examined at various pH in the presence or absence of Ca2+ in order to test the applicability of the three-state model to the homogeneous hexamers. The hexameric structure of the reassembled subunits was less stable than that of the native protein under the conditions examined. The model could be fitted to the O2-binding isotherms of the homohexamers composed of the subunits II or III, if the molecular dissociation of the protein was taken into account. It was postulated that the monomeric hemocyanin has the same ligand affinity as that of the hexamer in the intermediate-affinity state (S). The fitting of the model to the O2 binding of the subunit I was unsuccessful mainly because of the low cooperativity of the assembled subunits. [ABSTRACT FROM AUTHOR]

Two c-type cytochromes were isolated from cells of the gram-negative bacterium Aquaspirillum itersonii grown under low aeration in the presence of nitrate. The major component, cytochrome c-550, was equated with the (single) c-type cytochrome previously reported to be present in this organism [Clark-Walker, G. D. & Lascelles, J. (1970) Arch. Biochem. Biophys. 136, 153-159], although a significantly higher molecular mass was apparent in the present work. The complete amino acid sequence of this cytochrome is reported in the accompanying paper. A second soluble c-type cytochrome, designated c-556, was also isolated. The molecular mass, isoelectric point, spectrum, midpoint oxidation reduction potential and amino acid composition of this monoheam cytochrome are reported. The possible relationship of this cytochrome to other cytochromes c-556 is discussed. [ABSTRACT FROM AUTHOR]

Protein kinase C (PKC) isoenzymes are involved in diverse cellular functions, including differentiation, growth control, tumor promotion, and cell death. In recent years, evidence has began to emerge suggesting a role for PKC in cell cycle control. A paper published recently, demonstrating a functional link between PKC and cell cycle control in yeast (Marini, N. J., Meldrum, E., Buehrer, B., Hubberstey, A. V., Stone, D. E., Traynor-Kaplan A. & Reed, S. I. (1996) EMBO ,J. 15, 3040—3052), strengthens this data. Thus, the existence of cell-cycle-regulated pathways involving PKC in both yeast and mammals indicate that PKC may be a conserved regulator of cell cycle events that links signal transduction pathways and the cell-cycle machinery. In this paper, we will review current data on the cell cycle components that are targets for PKC regulation. PKC enzymes appear to operate as regulators of the cell cycle at two sites, during GI progression and G2/M transition, In GI, the overall effect of PKC activation is inhibition of the cell cycle at mid to late Gl. This cell cycle inhibition correlates with a blockage in the normal phosphorylation of the tumor suppressor retinoblastoma Rb protein, presumably through an indirect mechanism. The reduced activity of the cyclin-dependent kinase, Cdk2, appears to be the major effect of PKC activation in various cell systems. This may also underlie the inhibition of Rb phosphorylation exhibited by PKC activation. Several mechanisms were described in different studies on the regulation of Cdk2 activity by PKC; reduced Cdk-activating kinase activity, diminished expression of the Cdk2 partners cyclins E or A, and the increased expression of the cyclin-dependent inhibitors, p21WAFIand p27KIPI, which are capable of binding to cyclin/Cdk2 complexes. PKC enzymes were also shown to play a role in G2/M transition. Among the suggested mechanisms is suppression of Cdc2 activity. However, most of the published data strongly implicate PKC in lamin B phosphorylation and nuclear envelope disassembly. [ABSTRACT FROM AUTHOR]

The particulate enzyme from Bacillus subtilis AHU 1031 catalyzed the synthesis of a polysaccharide and glycolipids from UDP — N-acetylmannosaminuronic acid (UDP-ManNAcUA), UDP — N-acetylglucosamine (UDP-GlcNAc), and UDP-glucose (UDP-Glc). The polysaccharide synthesis required UDP-ManNAcUA and UDP-GlcNAc, proceeded optimally at pH 8.5 and in the presence of 5 mM MgCl2 and 2.5 mM dithiothreitol, and was stimulated by the addition of UDP-Glc. The molar ratio of ManNAcUA, GlcNAc, and Glc incorporated into polysaccharide was calculated to be 1:1:1.8 from chemical analysis involving reduction with water soluble carbodiimide; its relative molecular mass was estimated to be 12000. The analysis of Smith degradation products revealed that the polysaccharide backbone is composed of repeating trisaccharide units comprising ManNAcUA, GlcNAc, and Glc. Based on the data regarding the time course of the incorporation of glucose into the polysaccharide, extra glucose seems to be attached to the polysaccharide backbone as lateral branches. The saccharide moieties of the glycolipids were identified as GlcNAc, ManNAcUA-GlcNAc, and Glc-ManNAcUA-GlcNAc from several analytical criteria. The addition of antibiotic 24010, a tunicamycin-like antibiotic, at 10 μg/ml resulted in almost complete inhibition of the synthesis of glycolipids and polysaccharide. It is therefore concluded that the glycolipids function as intermediates in polysaccharide formation. Incubation of the ManNAcUA-GlcNAc-linked lipid. (labeled in the ManNAcUA moiety) with the particulate enzyme and UDP-Glc resulted incorporation of radioactivity into a trisaccharide-linked lipid and a polysaccharide. These results suggest that the particulate enzyme utilizes the trisaccharide moiety of the Glc-ManNAcUA-GlcNAc-linked lipid for the elongation of the main polysaccharide chain presumed to be cell wall acidic polysaccharide of this strain. [ABSTRACT FROM AUTHOR]

The study of gel-filtered rabbit reticulocyte lysates and lysates which have been passed through 2'5'ADP- Sepharose columns has shown that the presence of a sugar phosphate and a reducing system is necessary to maintain maximum rates of protein synthesis and to prevent an early inhibition. In this paper it is shown that the inhibition is due to a reduced rate of initiation of translation and a decrease in the level of methionyl-tRNA 40S- ribosomal-subunit complexes. Sugar phosphates and reducing agents are shown to act co-operatively to prevent these changes: the absence of either sugar phosphates or reducing agents leads to a decrease in polysomes and in methionyl-tRNA - 40S-subunit complexes. The requirements for reducing power can be satisfied by either dithiothreitol or by an NADPH-generating system together with a functional thioredoxin/thioredoxin reductase system: evidence is presented to show that these are required For the reduction of disulphide bonds. Incubation of lysates in the absence of a suitable reducing system leads to disulphide bond formation in a limited number of proteins present in the lysate, but no disulphide bonds could be detected in initiation factor elF-2α. the protein which catalyses the formation of methionyl-tRNA 40S-subunit complexes. On the other hand, when these lysates were incubated under conditions in which protein synthesis is inhibited, eJF-2 was phosphorylated in the smallest of its three polypeptide chains (eIF-2α). The phosphorylation of e1F-2α is shown to be controlled by the presence or absence of a suitable reducing system but not by sugar phosphates, it appears to be caused by activation of a protein kinase rather than through regulation of the rate of dephosphorylation of this protein. It is concluded that sugar phosphates do not control the phosphorylation of eIF-2α but play some role as an activating cofactor affecting the rate of initiation of protein synthesis. The presence of a suitable reducing system is required to prevent or reverse disulphide bond formation in some critical protein(s) in the lysate; the oxidation of sulphydryl groups in this protein leads to activation of an elF-2α kinase and hence to inhibition of initiation. [ABSTRACT FROM AUTHOR]

Translation of cellular and early vaccinia RNA in nuclease-treated lysates, derived from uninfected and vaccinia-virus-infected cells al the early stage, has been investigated. When using limiting amounts of RNA no discrimination of translation was observed in the infected cells lysates; This conclusion was confirmed by sensitive RNA competition experiments for translation in vitro and also when using two different fractionated systems for protein synthesis in vitro. This absence of detectable discrimination in vitro was established both by comparing incorporation of [35S]methionine into proteins and by analysis of the products thus synthesized by sodium dodecylsulfate gel electrophoresis. However, a modification of the translational machinery from vaccinia-virus-infected cells did occur since only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis of an early protein involved in translation or from a better recovery of translational factors from the infected cells., as suggested in the accompanying paper. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of turnip peroxidase TP 7, the principal isoperoxidase during winter in turnip, Brassica napus L., variety Blanc dur d'hiver, has been determined by sequence analysis of cyanogen bromide fragments and of tryptic peptides. The turnip peroxidase TP 7 enzyme is composed of 296 amino acids, one hemin group and one neutral carbohydrate side chain attached through asparagine. The molecular weight of the polypeptide part is 31 060, and including heroin and carbohydrate the molecular weight of the native enzyme is close to 33 400. The isoelectric point of turnip peroxidase TP 7 is 11.6. Comparison of turnip peroxidase TP 7 and horseradish peroxidase HRP C shows that they contain four similarly located disulfide bridges and have pyrrolidone carboxylyl N termini. Their common evolutionary origin is distant as their amino acid sequences are only 49% identical. Furthermore, turnip peroxidase TP 7 differs significantly from three other isoperoxidases of turnip root, turnip peroxidases TP 1, TP 2 and TP 3, and from horseradish peroxidase HRP C in its physico-chemical and enzymatic properties, and its pronounced season-dependent appearance. All these differences of turnip peroxidase TP 7 and of the others suggest they serve separate biological functions. [ABSTRACT FROM AUTHOR]

The effects of microtubule-associated protein binding to DNA on microtubule assembly in vitro were studied by turbidity measurements and electron microscopy. DNA inhibited microtubule assembly in a concentration-dependent manner. At DNA concentrations low enough to allow some polymerisation to occur, assembled microtubules were longer than those formed in the absence, indicating a reduction in the number of microtubule-associated proteins available for polymerisation. Also, by binding microtubule-associated proteins, DNA raised the critical con- centration of microtubule protein necessary for microtubule assembly. However, satellite DNA, which bound microtubule-associated proteins more effectively than bulk DNA, raised the critical concentration less. Similar effects were observed for the synthetic DNA polymers poly(dA) · poly(dT) and poly(dA-dT) · poly(dA-dT) compared to poly(dG) · poly(dC) and poly(dG-dC) · poly(dG-dC). Nitrocellulose filter binding assays showed that only microtubule-associated protein fractions which contained high-molecular-weight components exhibited DNA binding specifity. These high- molecular-weight polypeptides bound preferentially to eukariotic DNA as compared to phage DNA; fractions containing tau protein bound equally well to both DNA species. High-molecular-weight polypeptides also showed preferential binding to poly(dA-dT) · poly(dA-dT) compared to poly(dG-dC) · poly(dG-dC) synthetic DNA polymers. We suggest that satellite DNA acts in vivo as an initiation site for microtubule formation and that high-molecular-weight polypeptides could be the linker components of the kinetochore. [ABSTRACT FROM AUTHOR]

The DNA-binding protein HU from Escherichia coil is a heterodimer constituted of two poly- peptide chains termed HU-1 and HU-2, of 90 residues each. Their primary structures were established from structural data obtained from tryptic peptides of each monomer in addition to the structural data provided by the automated Edman degradation of the dimer and by peptides derived from cleavage of the dimer with trypsin, chymotrypsin, V8 staphylococcal protease and dilute acid. The results presented in this paper confirm the amino-terminal and carboxy-terminal sequences of the dimer HU reported previously [Laine et al. (1978) FEBS Lett. 89, 116–120]. The amino acid sequences of proteins HU-1 and HU-2 are identical to those of proteins NS-1 and NS-2 respectively, determined independently by Mende et al. [FEBS Lett. (1978) 96, 395–398]. The amino acid sequences of proteins HU-1 and HU-2 are closely related but differ by 28 residues. These proteins are characterized by their high content of hydrophobic residues represented mostly by alanine. In both proteins, half of the basic residues are scattered along the polypeptide chain and the remainder is found within two short sequences located in the carboxy-terminal part of the molecule. No sequence homology could be established between the proteins HU-1 and HU-2 and any one of the five histones from different eukaryotes. [ABSTRACT FROM AUTHOR]

The acyl carrier protein of citrate lyase contains adenine, phosphate, sugar, cysteamine, β-alanine and pantoic acid in a molar ratio of 1:2:2:1:1:1. Peptides containing these components in the same stoichiometric relationship were isolated after proteolytic digestion of acyl carrier protein. All components were linked together in a single prosthetic group. This was released from the peptide by mild alkaline hydrolysis. Under these conditions a phosphodiester bond is cleaved which links the prosthetic group to a serine residue of the peptide. Incubation of the prosthetic-group-containing peptide with phosphodiesterase I yielded 4′-phosphopantetheine and adenylic acid. The 5′-AMP was not free but was substituted by presumably an acidic sugar residue, which was released by mild acid hydrolysis yielding free 5′-AMP. It was concluded from these results that the prosthetic group of citrate lyase acyl carrier protein consists of a substituted isomeric dephospho-CoA. This is bound to the protein by the 5′-phosphate group of adenylic acid. The 4′-phosphopantetheine residue is bound by a phosphodiester linkage to the 2′ or 3′ position of ribose and the remaining hydroxyl group of ribose is substituted with presumably an acidic sugar residue. The structural similarities of this prothetic group and coenzyme A are discussed and related to the catalytic properties of citrate lyase [ABSTRACT FROM AUTHOR]

A strong synergistic response was observed between the five endo-1,4-β-glucanases and the exo-1,4-β-glucanase obtained from culture solutions of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum), when these enzymes were allowed to degrade de-waxed cotton and Avicel. No synergism was observed if Walseth cellulose, an acid-swollen cullulose, was used. If de-waxed cotton was pretreated with endo-1,4-β-glucanases, the exo-1,4-β-glucanase enzyme released much more degradation products than from an untreated cotton. This strongly supports the theory that the endo-1,4-β-glucanases, acting randomly over the cellulose chain, go in first and open up chain ends the exo-enzymes can act. In an earlier paper [Eriksson, K.-E. & Pettersson, B. (1975) Eur. J. Biochem. 51, 193-206] physicochemical differences between the endo-1,4-β-glucanases were demonstrated. Functional differences have now been found. The five endo-enzymes give rise to different degradation products from Avicel, show differences in preferred mode of attack on cellodextrins, and exhibit pronounced differences in synthetic activity. The exo-1,4-β-glucanase released its products in the α-configuration. A method for the separation of cellodextrins on a Bio-Sil A Column is also described. [ABSTRACT FROM AUTHOR]

Methods which use the scintillator PPO to record film images of ³H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between radioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by microdensitometry of the image obtained on pre-exposed film. Using pre-exposed film 300 dis. ³H/min or 30 dis. 14C/min can be detected in a band in a gel in a 24-h exposure. The Appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use. [ABSTRACT FROM AUTHOR]

1. Leghemoglobin component a from Phaseolus vulgaris (kidney bean)was digested with trypsin; 15 tryptic peptides and free lysine were purified and the amino acid sequences of the peptides determined. 2. The internal order of the tryptic peptides was determined by the bridge peptides obtained from the thermolytic digest and the dilute acid hydrolyzate of kidney bean leghemoglobin α; 12 thermolytic peptides and two acid hydrolysis peptides were purified and the sequences were partially or completely determined. 3. The complete amino acid sequence of kidney bean leghemoglobin a is compared to that of leghemoglobin a from soybean (Glycine max) and to some animal globins. As regards sequence, the kidney bean globin has 79% identity with the soybean globin and 21 % identity with human hemoglobin γ-chain. Seven of the 14 amino acid residues common to most globins are found in the kidney bean globin. Trp-15 and Tyr-145 are evolutionarily conserved in this globin, which confirms the concept of a common origin of animal and plant globins. [ABSTRACT FROM AUTHOR]

Semliki-Forest-virus 42-S virion RNA is translated in a mouse ascites cell-free system. One of the major polypeptides made in vitro has been identified as Semliki-Forest-virus capsid protein by co-electrophoresis on dodecylsulfate -- polyacrylamide gels and by tryptic peptide analysis on paper. Another major product of the cell-free system has been characterised as envelope protein by peptide analysis but it does not co-electrophorese on gels with envelope proteins from purified virions. It is possible that the nascent, non-glycosylated polypeptide is partially degraded by proleolytic enzymes present in the cell-free extract, or alternatively that fragments of envelope protein are produced by premature termination on the viral RNA. Several other polypeptides are synthesised in the cell-free system in variable amounts but these have not been identified. The genetic content and the mechanism of translation of Semliki-Forest-virus 42-S RNA are discussed. [ABSTRACT FROM AUTHOR]

We have used the circular dichroism and infrared spectra of a specially designed 50 protein database [Oberg, K.A., Ruysschaert, J.M. & Goormaghtigh, E. (2003) Protein Sci. 12, 2015–2031] in order to optimize the accuracy of spectroscopic protein secondary structure determination using multivariate statistical analysis methods. The results demonstrate that when the proteins are carefully selected for the diversity in their structure, no smaller subset of the database contains the necessary information to describe the entire set. One conclusion of the paper is therefore that large protein databases, observing stringent selection criteria, are necessary for the prediction of unknown proteins. A second important conclusion is that only the comparison of analyses run on circular dichroism and infrared spectra independently is able to identify failed solutions in the absence of known structure. Interestingly, it was also found in the course of this study that the amide II band has high information content and could be used alone for secondary structure prediction in place of amide I. [ABSTRACT FROM AUTHOR]

Mitochondrial fatty acid oxidation deficiencies are due to genetic defects in enzymes of fatty acid β-oxidation and transport proteins. Genetic defects have been identified in most of the genes where nearly all types of sequence variations (mutation types) have been associated with disease. In this paper, we will discuss the effects of the various types of sequence variations encountered and review current knowledge regarding the genotype–phenotype relationship, especially in patients with acyl-CoA dehydrogenase deficiencies where sufficient material exists for a meaningful discussion. Because mis-sense sequence variations are prevalent in these diseases, we will discuss the implications of these types of sequence variations on the processing and folding of mis-sense variant proteins. As the prevalent mis-sense variant K304E MCAD protein has been studied intensively, the investigations on biogenesis, stability and kinetic properties for this variant enzyme will be discussed in detail and used as a paradigm for the study of other mis-sense variant proteins. We conclude that the total effect of mis-sense sequence variations may comprise an invariable – sequence variation specific – effect on the catalytic parameters and a conditional effect, which is dependent on cellular, physiological and genetic factors other than the sequence variation itself. [ABSTRACT FROM AUTHOR]

This paper reports the isolation and characterization of the regulatory moiety of the multicomponent enzyme phenol hydroxylase from Acinetobacter radioresistens S13 grown on phenol as the only carbon and energy source. The whole enzyme comprises an oxygenase moiety (PHO), a reductase moiety (PHR) and a regulatory moiety (PHI). PHR contains one FAD and one iron-sulfur cluster, whose function is electron transfer from NADH to the dinuclear iron centre of the oxygenase. PHI is required for catalysis of the conversion of phenol to catechol in vitro , but is not required for PHR activity towards alternative electron acceptors such as cytochrome c and Nitro Blue Tetrazolium. The molecular mass of PHI was determined to be 10 kDa by SDS/PAGE, 8.8 kDa by MALDI-TOF spectrometry and 18 kDa by gel-permeation. This finding suggests that the protein in its native state is a homodimer. The isoelectric point is 4.1. PHI does not contain any redox cofactor and does not bind ANS, a fluorescent probe for hydrophobic sites. The N-terminal sequence is similar to those of the regulatory proteins of phenol hydroxylase from A. calcoaceticus and Pseudomonas CF 600. In the reconstituted system, optimal reaction rate was achieved when the stoichiometry of the components was 2 PHR monomers: 1 PHI dimer: 1 PHO (αβγ) dimer. PHI interacts specifically with PHR, promoting the enhancement of FAD fluorescence emission. This signal is diagnostic of a conformational change of PHR that might result in a better alignment with respect to PHO. [ABSTRACT FROM AUTHOR]

In the accompanying paper [Adams, H., Scotti, P.A., de Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Biochem. 269 , 5564–5571], we showed that the precursor of outer-membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position -10 in the signal sequence (G-10L) is targeted to the SecYEG translocon via the signal-recognition particle (SRP) route, instead of via the SecB pathway. Here, we studied the fate of the mutant precursor in a prlA4 mutant strain. prlA mutations, located in the secY gene, have been isolated as suppressors that restore the export of precursors with defective signal sequences. Remarkably, the G-10L mutant precursor, which is normally exported in a wild-type strain, accumulated strongly in a prlA4 mutant strain. In vitro cross-linking experiments revealed that the precursor is correctly targeted to the prlA4 mutant translocon. However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K-accessibility experiments in pulse-labeled cells. Furthermore, the mutant precursor was found to accumulate when expressed in a secY40 mutant, which is defective in the insertion of integral-membrane proteins but not in protein translocation. Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon. [ABSTRACT FROM AUTHOR]

In this paper we address the problem of extracting features relevant for predicting protein–protein interaction sites from the three-dimensional structures of protein complexes. Our approach is based on information about evolutionary conservation and surface disposition. We implement a neural network based system, which uses a cross validation procedure and allows the correct detection of 73% of the residues involved in protein interactions in a selected database comprising 226 heterodimers. Our analysis confirms that the chemico-physical properties of interacting surfaces are difficult to distinguish from those of the whole protein surface. However neural networks trained with a reduced representation of the interacting patch and sequence profile are sufficient to generalize over the different features of the contact patches and to predict whether a residue in the protein surface is or is not in contact. By using a blind test, we report the prediction of the surface interacting sites of three structural components of the Dnak molecular chaperone system, and find close agreement with previously published experimental results. We propose that the predictor can significantly complement results from structural and functional proteomics. [ABSTRACT FROM AUTHOR]

The D1 protein constitutes one of the reaction center subunits of photosystem II and turns over rapidly due to photooxidative damage. Here, we studied the degradation of a truncated D1 protein. A plasmid with a precise deletion in the reading frame of the psbA gene encoding D1 was introduced into the chloroplast of Chlamydomonas reinhardtii. A homoplasmic mutant containing the desired gene was able to synthesize the truncated form of the polypeptide, but could not accumulate significant levels of it. As a consequence, other central photosystem II subunits did not assemble within the thylakoid membrane. In vivo pulse–chase experiments showed that the abnormal D1 protein is rapidly degraded in the light. Degradation was delayed in the light in the presence of an uncoupler, or when cells were incubated in the dark. Pulse–chase experiments performed in vitro indicate that an ATP and metal-dependent protease is responsible for the breakdown process. The paper describes the first in vivo and in vitro functional test for ATP-dependent degradation of a defect polypeptide in chloroplasts. The possible involvement of proteases similar to those removing abnormal proteins in prokaryotic organisms is discussed on the basis of proteases recently identified in chloroplasts. [ABSTRACT FROM AUTHOR]