bluuebunny/crossref_metadata_2025 huggingface dataset:
The Properties of the tRNA · protein Complex of the Escherichia coli Ribosome
Authors: MATSPALU Ene , USTAV Mart , VILLEMS Richard
Published: July 2010

A complex between immobilized tRNA and Escherichia coli 50‐S ribosomal subunit proteins provides binding sites for several other components of the protein biosynthesis system. We found that this complex binds 5‐S RNA, tRNA and 30‐S ribosomal subunits. The binding of 5‐S RNA and tRNA takes place at equilibrium; in 10 mM Tris/HCl buffer, pH 7.5, containing 10 mM MgC12 and 100 mM KCI at 4°C it is characterized by the dissociation constants 1.7 ×10−7 M and 4 ×10−7 M respectively. We show that the perfornicd complex between tRNA and 50‐s ribosomal subunit protein can bind these two RNAs simultaneously by forining a subribosoinal domain consisting of two molecules of tRNA, 5‐S RNA and the riboSomal proteins L2. L15, L16, L17, L18, L22, L33 and L34. The preformed tRNA. protein complex interacts also with the 30‐S ribosomal subunit, suggesting that it is located at the ribosomal interface.
Polynucleotide · Ribosomal‐Protein Complexes and Their Decoding Properties
Authors: SARAPUU Tago , VILLEMS Richard
Published: July 2010

Polyadenylic acid, polycytidylic acid, polyuridylic acid or phage MS2 RNA, immobiliLed on Sepharose, form a complex with Escherichiu coli ribosomal proteins. Regardless of their particular nucleotide composition, all four polynucleotides bind an invariable set of proteins consisting of S1, S3, S4, S5, S9. S13, L2 and L17. We found that these polynucleotide. protein complexes bind tRNA. Furthermore, it was possible to show that the poly(A). protein and poly(U). protein complexes select efficiently their cognate tRNAs, tRNALys and tRNAPhe respectively. This important functional property of the polynucleotide. protein complexes suggests that these ribosomal proteins belong in the ribosome to a functional domain responsible for the decoding of mRNA.
The Interactions of Androgen Receptor with Poly(A)‐Containing RNA and Polyribonucleotides
Authors: LIN Syr‐yaung , OHNO Susumu
Published: July 2010

The androgen receptor from mouse kidney cytosol binds not only to DNA but also to RNA as assayed by competition with DNA‐cellulose centrifugation or by direct binding with agarose‐polynucleotides. The dihydrotestosterone‐receptor complex interacts more strongly to poly(A)‐containing mRNA than to native natural DNA; it binds much more tightly to synthetic poly(G) than to natural DNA and other homopoly‐ ribonucleotides such as poly(U), poly(A), or poly(C). Poly(U) is moderately effective in competing for the complex, whereas poly(A) and poly(C) are not effective. Competition studies with heteropolyribonucleotides show that polymers containing G are most effective on binding to the androgen receptor, whereas those containing U are moderately effective, and those containing only A and C are least effective. Several analogues of poly(G), such as poly(X), poly(I), and poly(m7G) interact very well with the dihydrotestosterone‐receptor with some differences in the bindings. A double‐stranded polymer, poly(1). poly(C) competes poorly for the androgen receptor. The observations that the androgen receptor binds to RNA and interacts with polyribonucleotides selectively suggest that androgen receptor‐RNA interaction could play important roles in gene regulation.
1H NMR Studies of the Electron Exchange Between Cytochrome c and Iron Hexacyanides
Authors: ELLY Crispin G. S. , MOORE Geoffrey R. , WILLIAMS Glyn , WILLIAMS Robert J. P.
Published: July 2010

Binding of [Fe(CN)6]3−, [Cr(CN)6]3−, [Co(CN)6]3− and [Cr(C2O4)3]3− to horse, tuna and Candida krusi cytochromes c has been studied by high‐resolution 1H NMR spectroscopy. All the reagents bind at the same sites. There are at least two binding sites, and probably three, on horse cytochrome c at pH 7. One of the sites is only a weak binding site and is far from the haem group, whereas the other site(s) is(are) at the haem crevice. Ka for binding of [Fe(CN)6]3− to trimethyllysine‐72 of C. krusei ferricytochrome c is 140±15 M1 at 27 C and pH 7.
Characterization of a Ca2+‐Dependent Guanylate Cyclase in the Excitable Ciliary Membrane from Paramecium
Authors: KLUMPP Susanne , SCHULTZ Joachim E.
Published: July 2010

The membraneous guanylate cyclase of cilia from Paramecium tetraurelia used MgGTP and MnGTP as substrate with Michaelis constants for GTP of 71.5 μM and 36 μM, respectively. A linear Arrhenius plot indicated that a single enzyme entity exists not sensitive to possible phase transitions of membrane lipids. Guanylate cyclase is activated by low concentrations (< 100 μM) and inhibited by high concentrations (> 100 μM) of calcium, half‐maximal effects were obtained with 8 μM and 500 μM Ca2+, respectively. Only strontium ions displayed partial activating and inhibiting potency, all other divalent cations tested, Ba2+, Fe2+, Co2+, Mn2+, Sn2+ and Ni2+ had no effect on guanylate cyclase activity. Ca2+ activation increased V; Km remained identical. The Ca2+. Stimulated activity was not inhibited by trifluoperazine, tentatively suggesting that the stimulation may not be mediated by calmodulin. Ca2+ inhibition was due to a single binding site of Ca2+ at the guanylate cyclase as evidence by a Hill coefficient h=−1 and was noncompetitive. The lanthanides La3+, Ce3+ and Tb3+ were powerful inhibitors of guanylate cyclase, with La3+ the half‐maximal effect was obtained with 0.6 μM, it was kinetically a mixed‐type inhibition. La3+ and Ca2+ competed for the same binding site on the guanylate cyclase as determined by detailed kinetic analysis. Addition of EGTA reversed the activation and inhibition by Ca2+ and the inhibition by La3+. It is discussed that guanylate cyclase may be the initial target enzyme in the cilia for the calcium transient of the calcium/polassium action potential of Paramecium.
Photoincorporation of Tetracycline into Rat‐Liver Ribosomes and Subunits
Authors: REBOUD Anne‐Marie , DUBOST Simone , REBOUD Jean‐Paul
Published: July 2010

[3H]Tetracycline was covalently incorporated into rat liver ribosomes and isolated 40‐S and 60‐S subunits on irradiation at 254 nm. The antibiotic was almost exclusively incorporated into ribosomal proteins. At least some of these proteins are assumed to be involved in ribosomal function, since photoincorporated tetracycline was found to inhibit the activity of 40‐S and 60‐S subunits in the poly(U)‐directed protein‐synthesizing system as well as that of the 40‐S subunit in the poly(U)‐mediated [14C]Phe‐tRNA binding. The results from simultaneous one‐dimensional and two‐dimensional gel electrophoreses showed a small distribution of label among ribosomal proteins in 60‐S subunits and in 80‐S ribosomes, L10 being the most radioactive protein. As non‐acylated tRNA partly competed with this labeling, it is likely that tetracycline interaction with these proteins occurred at a functional site. L10 has already been found to interact with puromycin [Reboud, A. M., Dubost, S., Buisson, M. & Reboud, J. P. (1981) Biochemistry, 20, 5281–5288]. In the case of free 40‐S subunits the label distribution was wider among ribosomal proteins. No particular role has yet been found for the most labeled protein, S12, but protein S3a, which was also highly labeled, has already been reported to be involved in subunit function.
Biological Activity of Chemically Synthesized Analogues of Lipid A
Authors: YASUDA Tatsuji , KANEGASAKI Shiro , TSUMITA Toru , TADAKUMA Takushi , HOMMA J. Yuzuru , INAGE Masaru , KUSUMOTO Shoichi , SHlBA Tetsuo
Published: July 2010

The adjuvant effect of synthetic analogues of lipid A was investigated in tnice in vivo. Two cbf the five synthetic lipid A analogues showed adjuvant activity when they were incorporated into dipalmitoylglyceerophosphocholine‐cholesterol‐dipalmitoylglycerophosphate liposomes sensitized with trinitrophenylaminocaprcylglycerophospho‐ethanolamine. The minimum structure for an adjuvant effect in the liposomal immunogen system was l‐mono‐phosphate of 6‐O‐(2‐deoxy‐2‐tetradecanoylamino‐6‐O‐tetradecanoyl‐D‐glucopyranosyl)‐2‐deoxy‐2‐tetradecanoyl‐amino‐3,4‐di‐O‐tetradecanoyl‐D‐glucopyranose.
Amino‐Acid Sequence of the α‐Subunit of Taipoxin, an Extremely Potent Presynaptic Neurotoxin from the Australian Snake Taipan (Oxyurunus s. scutellatus)
Authors: LIND Peter , EAKER David
Published: July 2010

The amino acid sequence of the α‐subunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan has been determined. The very basic protein, by itself a moderately neurotoxic phospholipase, consists of a single polypeptide chain of 119 amino acids. The main fragmentation of the reduced and S‐carboxymethylated derivative was accomplished by cleavage with Staphylocoeus aureus V8 pro‐ tease and trypsin. Chymotryptic peptides and cyanogen bromide fragments were used to align and complete the sequence, which was determined by automated Edman degradation. The taipoxin γ‐subunit is closely homologous to the other taipoxin subunits and to other elapid snake venom phospholipases A2.
New Mutants Resistant to Glucose Repression Affected in the Regulation of the NADH Reoxidation
Authors: GUERIN Martine , CAMOUGRAND Nadine , VELOURS Giséle , GUÉRIN Bernard
Published: July 2010

Spontaneous mutants resistant to vanadate, arsenate or thiophosphate were isolated from a haploid strain of Saccharomyces cerevisiae. These three anions have an inhibitory effect on some mitochondrial functions and at the level of glyceraldehyde 3‐phosphate dehydrogenase, a glycolysis enzyme. All the selected mutants had the same phenotype: they were deficient in alcohol dehydrogenase I, the terminal enzyme of the glycolysis, and possessed a high content of cytochrome c oxidase, the terminal enzyme of the respiratory chain. Moreover, cytochrome c oxidase biosynthesis had become insensitive to the catabolite repression. while the biosynthesis of the other enzymes sensitive to this phenomenon were always inhibited by glucose.

Metabolic effects of this pleiotropic mutation manifested themselves in the following ways.

1. Growth rate and final cell mass were enhanced, compared to the wild type, when cells were grown on glucose or on glycerol, but not on lactate or ethanol.

2. Growth under anaerobiosis was nil and mutants did not ferment.

3. Mitochondria1 respiration of the mutant strains was identical to the wild type with succinate or 2‐oxoglutarate as substrate, and weak with ethanol. But with added NADH, respiration rate of the mutants was higher than that of the wild type and partially insensitive to antimycin, even when cells were grown in repression conditions.

It is postulated that in mutants strains, NADH produced at the level of glyceialdehyde 3‐phosphate dehydrogenase, failing to be reoxidized via alcohol dehydrogenase, could be reoxidized with a high turnover owing to the enhancement of the amount of cytochrome c oxidase. Since NADH reoxidation is partially insensitive to antimycin, a secondary pathway going from external NADH dehydrogenase to cytochrome c oxidase is suggested.
Partial P1 Nuclease Digestion as a Probe of tRNA Structure
Authors: AULTMAN Kathryn S. , CHANG Simon H.
Published: July 2010

Partial P1 nuclease digestion of 5′‐32P‐labeled tRNAs, followed by polyacrylamide gel electrophoresis and autoradiography. results in a series of bands of unequal intensities. Comparison of the digestion profiles of two conformers of yeast tRNALeu3 shows that P1 nuclease is sensitive to structural features of its substrate, Examination of the digestion pattern of yeast tRNAPhe in light of its known three‐dimensional conformation shows that the enzyme's activity reflects the primary, secondary and tertiary levels of tRNA structure.
Macromolecular Complex of Aminoacyl‐tRNA Synthetases from Sheep Liver
Authors: BREVET Annie , GEFFROTIN Claudine , KELLERMANN Odile
Published: July 2010

Both the tRNA aminoacylation and aminoacid‐dependent ATP‐PPi exchange activities of monomeric trypsin modified methionyl‐tRNA synthetase from sheep liver are lost upon incubation with oxidized initiator tRNAmet. The inactivation, which reflects the formation of a Schiff's base between the 5′‐terminal adenosine of tRNA and a lysine within the catalytic site of the enzyme, is accompanied by the covalent attachment of one tRNA molecule per enzyme molecule. The affinity labeling method is applied to the sheep liver complex of Mt106 carrying seven aminoacyl‐tRNA synthetase activities, from which the monomeric trypsin‐modified methinnyl‐tRNA synthetase (Mt 68000) was derived. Upon incubation with oxidized initiator tRNAMet, the methionyl‐tRNA synthetase activity of the complex is lost. Of the eleven polypeptidechains composing the high‐molecular‐weight complex, only one polypeptide chain with Mt 103000 reacts with the modified tRNAMet The blocking by periodate‐treated tRNA of the methionyl‐tRNA synthetase activity in the complex has no effect on the other aminoacyl‐tRNA synthetase activities. This strongly argues in favor of the independent parallel functioning of the seven aminoacyl‐tRNA syn‐ thetases associated in a high‐molecular‐weight complex.
Maturation of Exported Proteins in Escherichia coli
Authors: PAGES Jean Marie , LAZDUNSKI Claude
Published: July 2010

Precursor forms of periplasmic and outer membrane proteins were accumulated in phenethyl‐alcohol‐treated cells in membrane fractions. After removal of phenethyl alcohol, maturation occurred in the absence but not in the presence of carbonylcyanide m‐chlorophenylhydrazone (30 μM). The site and kinetics of processing were investigated for OmpA, LamB and OmpF proteins and for maltose binding protein and TEM β‐lactamase. With regard to sites of processing, no fundamental difference between outer membrane and periplasmic proteins was observed. For maltose binding protein and TEM β‐lactamase, maturation, like that of outer membrane protein precursors, occurred in membrane fractions. Processing of pro‐OmpA protein was about as fast as that of pro‐ LamB protein whereas pro‐OmpF protein appeared to mature more slowly. While carbonylcyanide m‐chlorophenylhydrazone (30 μM) prevented processing of all precursor forms, arsenate, which alters formation of ATP even when it was used at 1 mM, did not totally prevent maturation occurring. These results are discussed with regard to the biosynthesis and assembly of exported proteins.
Rate of Translation and Kinetics of Processing of Newly Synthesized Molecules of Two Major Outer‐Membrane Proteins, the OmpA and OmpF Proteins, of Escherichia coli K12
Authors: CROWLESMITH Ian , GAMON Konrad
Published: July 2010

The rate of synthesis of the OmpA and OmpF proteins, two of the major outer membrane proteins of Escherichia coli K12, was determined. At 25°C both proteins were translated at 6.5 amino acids/s, and the OmpF protein was translated at 15 amino acids/s at 37°C. The former rate corresponded to a synthesis time of just over 50 s for both proteins, which is significantly faster than their reported rates of assembly into the outer membrane at 25°C. The kinetics of processing of the pro‐OmpF protein were also investigated in detail, and the pro‐OmpF half‐life estimated to be 3–5 s at 25°C. However a fraction of the precursor was processed more slowly, which may explain the discrepancy between these data and our earlier published estimate of 30 s. Pro‐OnipA protein was processed with similar kinetics. These results demonstrate that the rate‐limiting step in the assembly of both proteins into the outer membrane is post‐translational and follows the processing step.
A Rapid Method for Acid Hydrolysis of Protein with a Mixture of Trifluoroacetic Acid and Hydrochloric Acid
Authors: TSUGITA Akira , SCHEFFLER Jean‐Jacques
Published: July 2010

Proteins have regions which resist hydrolysis with mineral acid. The presence of a strong organic acid was found to be efficient for hydrolysis of a hydrophobic peptide bond. The proposed condition, a 2: 1 (by vol.) mixture of concentrated hydrochloric acid and trifluoroacetic acid at 166°C for 25 min was observed to be equivalent to the conventional conditions (6 M HCl at 110°C for more than 24 h) without significant decomposition of amino acids. The method was shown to be superior to the conventional conditions, especially for hydrophobic proteins. The present method destroys tryptophan, as the conventional acid hydrolysis does.
Temperature Dependence of ADP/ATP Translocation in Mitochondria
Authors: KLINGENBERG Martin , GREBE Karin , APPEL Maria
Published: July 2010

The temperature dependence of the adenine nucleotide exchange in mitochondria has been determined by employing a rapid mixing, quenching and sampling apparatus and the inhibitor quench‐back exchange method. Thus the exchange is resolved down to 0.1 s. Rates are evaluated from accumulating the time‐dependent progress at about 10 points.

The exchange rate in liver mitochondria was determined from –10°C to +10°C in the presence of 20% glycol, from 0°C to 25°C, and from 20°C to 40°C under partial inhibition by carboxyatractylate. The total range between –10°C to + 40°C has only one temperature break at 13°C. From the Arrhenius plot between –10°C to +13°C, EA∼ 140 kJ and above 13°C, EA∼ 56 kJ is evaluated, corresponding to a Q10 of 8 and 2 respectively.

In beef heart mitochondria the exchange rate was measured between 0°C and 20°C, and between 15°C and 30°C under partial inhibition with carboxyatractylate. There is a temperature break around 14°C with EA∼ 143 kJ between 0°C and 14°C and EA∼ 60 kJ from 15°C to 30°C.

The extrapolated translocation rates at 37°C are 500 and 1800 μmol min−1 (g protein)−1 for rat liver and for beef heart mitochondria respectively.

The temperature break is suggested to reflect a conformation change since there is no reversed break at low temperature, the temperature break changes in sonic particles and no lipid phase transition at 14°C in mitochondria has been reported.
Induction in Primary Culture of ‘Gluconeogenic’ and ‘Glycolytic’ Hepatocytes Resembling Periportal and Perivenous Cells
Authors: PROBST Irmelin , SCHWARTZ Peter , JUNGERMANN Kurt
Published: July 2010

Adult rat hepatocytes were kept in primary culture for 48 h under different hormonal conditions to induce an enzyme pattern which with respect to carbohydrate metabolism approximated that of periportal and perivenous hepatocytes in vivo.

Glucagon‐treated cells compared with control cells possessed a lower activity of glucokinase, a 4.5‐fold higher activity of phosphoenolpyruvate carboxykinase and unchanged levels of glucose‐6‐phosphatase, phosphofructokinase, fructose‐bisphosphatase and pyruvate kinase; they resembled in a first approximation the periportal cell type and are called for simplicity ‘periportal’. Inversely, insulin‐treated cells compared with control cells contained a 2.2‐fold higher activity of glucokinase, a slightly decreased activity of phosphoenolpyruvate carboxykinase, increased activities of phosphofructokinase and pyruvate kinase and unaltered levels of glucose‐6‐phosphatase and fructose‐bisphosphatase; they resembled perivenous cells and are called simply ‘perivenous’. Gluconeogenesis and glycolysis were studied under various substrate and hormone concentrations.

Physiological concentrations of glucose (5 mM) and lactate (2 mM) gave about 80% saturation of gluconeogenesis from lactate and less than 15% saturation of glycolysis at a simultaneous 40% inhibition of the glycolytic rate by lactate.

Comparison of the two cell types showed that under identical assay conditions (5 mM glucose, 2 mM lactate, 0.5 nM insulin, 0.1 μM dexamethasone) gluconeogenesis was 1.5‐fold faster in the ‘periportal’ cells and glycolysis was 2.4‐fold faster in the ‘perivenous’ cells.

Metabolic rates were under short‐term hormonal control. Insulin increased glycolysis three fold in both cell types with a half‐maximal effect at about 0.4 nM, but did not influence the gluconeogenic rate. Glucagon inhibited glycolysis by 70% with a half‐maximal effect at about 0.1 nM. Gluconeogenesis was stimulated by glucagon (half‐maximal dose: 0.5 nM) 1.8‐fold only in ‘periportal’ cells containing high phosphoenolpyruvate carboxykinase activity, not in the ‘perivenous’ cells with a low level of this enzyme.

A comparison of the two cell types showed that with maximally stimulating hormone concentrations gluconeogenesis was threefold faster in ‘periportal’ cells and glycolysis was eightfold faster in ‘perivenous’ cells.

The results support the view that periportal and perivenous hepatocytes in vivo catalyse gluconeogenesis and glycolysis at inverse rates.
Functions of the Peptide Antibiotics Tyrocidine and Gramicidin
Authors: HANSEN Jutta , PSCHORN Wolfgang , RISTOW Hansjürgen
Published: July 2010

The peptide antibiotic tyrocidine which is produced by Bacillus brevis and is probably involved in sporogenesis, unwinds superhelical plasmids in vitro at low peptide: DNA ratios, as found by gel electrophoresis. At higher peptide concentrations, the DNA is packed tightly leading to apparent nuclease stability of the complex and inhibition of RNA synthesis. The addition of the linear gramicidin, another peptide antibiotic synthesized by the same bacterial strain, partially restores transcription by breaking down the tightly packed DNA · peptide complex. The complexed DNA, after nuclease digestion, is retained on a nitrocellulose filter, but loses its affinity for the filter in the presence of gramicidin. The results are discussed with respect to possible functions of the two peptides within in the cell.
Distinct α‐Tropomyosin mRNA Sequences in Chicken Skeletal Muscle
Authors: MacLEOD Alexander R.
Published: July 2010

Recombinant plasmids have been isolated which contain sequences complementary to two distinct α‐tropomyosin mRNA species present in chicken leg muscle. The proteins coded for by these different mRNAs could be distinguished by their electrophoretic behaviour in the presence of 3.5 M urea. The properties of the minor α‐tropomyosin of chicken leg muscle were similar to those reported for the α‐tropomyosin of slow twitch chicken skeletal muscle. Sequence analysis of available plasmids showed that the deduced protein sequences of both types of α‐tropomyosin were very similar and closely related to the known protein sequence of rabbit α‐tropomyosin. However considerable variation in nucleotide coding sequence of the two α‐tropomyosin mRNAs was found.
Optical Characteristics of All Individual Proteins from the Small Subunit of Escherichia coli Ribosomes
Authors: VENYAMINOV Sergei Yu. , GOGIA Zurab V.
Published: July 2010

The procedure of isolation and renaturation of all ribosomal proteins from the 30‐S subunit of Escherichia coli ribosomes is described. Absorption spectra of these proteins in the near‐ultraviolet region have been measured and molar absorption coefficients have been determined on the basis of nitrogen content. Molar absorption coefficients have been calculated for 20 proteins with a known amino acid sequence and the calculated values have been compared with the experimentally determined ones. The absorption spectra obtained allow an easy, precise and highly reproducible spectrophotometric determination of the concentration of individual ribosomal proteins.

Circular dichroic spectra of 21 individual proteins from the 30‐S subunit of E. coli ribosomes were measured in the range 184–310 nm. The secondary structure of the proteins studied was calculated from the spectra in the range 190–240 nm. Almost all proteins (except proteins S12, S17, S18 and S19) are characterized by a high content of secondary structure. Circular dichroic spectra in the near‐ultraviolet region (240–310 nm) indicate that the side groups of aromatic amino acids are fixed in the tertiary structure of the proteins studied.

Some internal characteristics (independent of the measurement conditions) of the circular dichroic spectrum in the far‐ultraviolet region were proposed as a measure of the resemblance to the native state of ribosomal proteins; these characteristics may be useful for comparison of protein preparations obtained by different methods in different laboratories.
Inhibition of Free and Bound Trypsin‐Like Enzymes
Authors: STEVEN Frank S. , GRIFFIN Margaret M. , ITZHAKI Shaul
Published: July 2010

The inhibition of β‐naphthylamidase activity of free trypsin, trypsin‐Sepharose and a trypsin‐like neutral protease on the surface of tumour cells have been studied in independent systems and with mixtures of free trypsin plus surface‐bound trypsin.

Kinetic data have demonstrated that high‐molecular‐weight (protein) inhibitors of free trypsin are less effective inhibitors of trypsin‐Sepharose and fail to inhibit the cell surface neutral protease.

Inhibition of mixtures of free trypsin plus trypsin‐Sepharose follows independent kinetic plots for each component. The free trypsin is reacted before any Sepharose‐bound trypsin reacts with high‐molecular‐weight inhibitors.

Low‐molecular‐weight inhibitors of trypsin also inhibit bound trypsin equally well.

Papain‐derived peptides from high‐molecular‐weight inhibitors of trypsin inhibit free trypsin, trypsin‐Sepharose and the cell‐surface neutral protease almost equally well.

Fluorescence microscopy has shown that a high‐molecular‐weight inhibitor of trypsin does not bind to the tumour cell‐surface neutral protease, but it does bind to trypsin‐Sepharose.

The cell‐surface neutral protease has been shown to be capable of activation of latent β‐naphthylamidase activity in the presence of excess extracellular inhibitors of free trypsin.

The mechanism by which trypsin‐Sepharose remains partially active in the presence of excess inhibitor necessary to inhibit an equivalent quantity of free trypsin has been discussed.

These studies indicate that a search for inhibitors which are selectively active against the cell‐surface neutral protease and have no action on trypsin‐like enzymes in free solution must take into account the modifying effects of the cell surface on neutral protease activity.
Solubilization and Characterization of the Initial Enzymes of the Dolichol Pathway from Yeast
Authors: SHARMA Chandra B. , LEHLE Ludwig , TANNER Widmar
Published: July 2010

Preparation and purification of substrate amounts of radioactive as well as non‐radioactive dolichyl diphosphate N‐acetylglucosamine and dolichyl diphosphate chitobiose made it possible to test and characterize tentatively the first three reactions of the dolichol pathway (enzyme I–III). The test conditions are described in detail.

All three enzymes were solubilized from yeast membranes with detergents. Enzyme II and III were purified to give a purification factor of 35‐fold and 70‐fold, respectively. The reactions required divalent metal ions with an optimum concentration of 10 mM Mg2+. Enzyme II was stimulated almost to the same extent also by Ca2+.

The Km values for UDP‐N‐acetylglucosamine for enzyme I and II were 15 and 10 μM, respectively, and for GDP‐mannose (enzyme III) 7 μM. The apparent Km values for the lipophilic acceptor was 180 μM for enzyme I (dolichyl phosphate), 40 μM for enzyme II (dolichyl diphosphate N‐acetylglucosamine) and 17 μM for enzyme III (dolichyl diphosphate chitobiose). The corresponding V values were approximately 1, 10, and 50 nmol × h−1× mg protein−1. All reactions were inhibited by nucleoside diphosphates.
The Application of 13C‐NMR Spectroscopy to Study Lipid A from Yersinia pseudotuberculosis Lipopolysaccharide
Authors: KRASIKOVA Inna N. , GORBACH Vladimir I. , ISAKOV Vladimir V. , SOLOV'EVA Tamara F. , OVODOV Yury S.
Published: July 2010

The use of 13C‐NMR spectroscopy in the establishment of lipid A backbone structure from lipopolysaccharide of Yersinia pseudotuberculosis has been described. The 13C‐NMR spectra of degraded lipid A and its N‐acetate were obtained. The assignment of signals was made by comparison with the chemical shifts for 13C‐NMR spectra of glucosaminitol, N‐acetyl‐glucosaminitol and their β‐1,4 and β‐1,6 disaccharides. It was shown that lipid A backbone of the lipopolysaccharide in question consists of β‐1,6‐linked glucosamine disaccharide.
Self‐Inactivation by 13‐Hydroperoxylinoleic Acid and Lipohydroperoxidase Activity of the Reticulocyte Lipoxygenase
Authors: HÄRTEL Brigitte , LUDWIG Peter , SCHEWE Tankred , RAPOPORT Samuel M.
Published: July 2010

The self‐inactivation of lipoxygenase from rabbit reticulocytes with linoleic acid at 37°C is caused by the product 13‐hydroperoxylinoleic acid. This inactivation is promoted by either oxygen or linoleic acid.

Lipohydroperoxidase activity was demonstrated with 13‐hydroperoxylinoleic acid plus linoleic acid as hydrogen donor under anaerobic conditions at 2°C. The products were 13‐hydroxylinoleic acid, oxodienes and compounds of non‐diene structure similar to those produced by soybean lipoxygenase‐1.

13‐Hydroperoxylinoleic acid also changed the absorbance and fluorescence properties of reticulocyte lipoxygenase. The results indicate that one equivalent of 13‐hydroperoxylinoleic acid converts the enzyme from the ferrous state into the ferric state as described for soybean lipoxygenase‐1. The spectral changes were reversed by sodium borohydride at 2°C, but not at 37°C; it is assumed that the ferric form of reticulocyte lipoxygenase suffers inactivation.
Fluorescence of Intramolecular and Intermolecular Interactions of Aminonaphthyl‐sulfonate with Nucleotides
Authors: FICK Jutta , LAWACZECK Rüdiger , SCHNEIDER Friedemann W.
Published: July 2010

Fluorescence studies of the intramolecular and intermolecular interactions between aminonaphthylsulfonate and nucleotides of uracil or adenine are described. The fluorescence originates solely from the naphthyl moiety and is intramolecularly quenched by the base, uracil being more effective than adenine. The enzymatic splitting of the molecule into a nucleoside monophosphate and the pyrophosphate product of the aminonaphthylsulfonate removes the intramolecular quenching and, especially in the case of uracil, a drastic increase of the fluorescence intensity results. The intact molecule exists predominantly in the folded form except in cases where electrostatic repulsion exceeds the stacking attraction. This is borne out by the pH dependence and the existence of a pronounced solvent‐isotope effect of the fluorescence quantum yield for the uracil derivative at basic pH. At pH values above the pK of the enol proton of the uracil base the fluorescent properties of the intact and phosphodiesterase‐digested molecules are very similar. The intermolecular interactions between 1‐aminonaphthalene‐5‐sulfonate with AMP and UMP can be explained on the basis of dynamic quenching (collisional quenching) without any significant participation of ground‐state complexes (static quenching). The interaction of the pyrophosphate adduct of 1‐aminonaphthalene‐5‐sulfonate with UMP can best be explained by invoking two interacting nucleotide species: the free nucleotide and a sodium‐nucleotide complex.
An Alteration in Phosphofructokinase 2 of Escherichia coli which Impairs Gluconeogenic Growth and Improves Growth on Sugars
Authors: DALDAL Fevzi , BABUL Jorge , GUIXÉ Victoria , FRAENKEL Dan G.
Published: July 2010

Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkBl mutation is known to increase the amount of phosphofructokinase 2 and allow growth on sugars of mutants lacking phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e., ‘gluconeogenic growth’). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkBl*. We show here that strains with pfkBl* contain an altered form of phosphofructokinase 2, called phosphofructokinase 2*, which has been purified. Phosphofructokinase 2* is cold labile and has slightly different kinetic characteristics from phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1,6‐bisphosphate.

The Km for fructose 6‐phosphate is low (about 5 × 10−5 M) in both phosphofructokinase 2 and phosphofructokinase 2*. However, in strains lacking phosphofructokinase 1, a high level of phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with phosphofructokinase 2* instead of phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal in phosphofructokinase 2 strains, while the impaired growth of phosphofructokinase 2* strains is associated with high levels of fructose 2,6‐bisphosphate and very low levels of hexose monophosphates.

These results show that phosphofructokinase 2, as studied in vitro, should no longer be regarded as a ‘non‐allosteric’ protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 117, 569–574 (1981)]. The fact that mutational alteration of phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1,6‐bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment.
The Subcellular Localization of DNA Components from Cyanophora paradoxa, a Flagellate Containing Endosymbiotic Cyanelles
Authors: BOHNERT Hans J. , CROUSE Edwin J. , POUYET Jean , MUCKE Herrmann , LÖFFELHARDT Wolfgang
Published: July 2010

Cyanophora paradoxa, a unicellular flagellate, contains cyanelles which are supposed to be cyanobacterial origin. DNA was isolated from subcellular fractions and separated according to density components in CsCl density gradients.

The main DNA component, comprising more than 90% of the total DNA, has a buoyant density of 1.724 g · cm−3. Several subsfractions in the range from 1.718 g · cm−3 to 1.735 g · cm−3 are contained in this component. This DNA of high complexity was considered to be host nuclear DNA.

The DNA from the endosymbiotic cyanelles, which were isolated, treated with DNase, and purified by sucrose density gradient centrifugation exhibited a buoyant density of 1.692 g · cm−3 in one strain and 1.695 g · cm−3 in a second strain. Both cyanelle DNAs (cyDNA) have a complexity of approximately 126 · 103 base pairs and comprise about 5% of the total cellular DNA content.

Two additional DNA components of low complexity were isolated from crude cyanelle pellets obtained without DNase treatment. The larger of these, approximately 48 × 103 base pairs in size, had a density of approximately 1.688 g · cm−3. The second component, about 15 × 103 base pairs in size, banded in the density range between 1.710 g · cm−3 and 1.720 g · cm−3. The latter is associated with nuclear DNA. The 48 × 10−3 base‐pair component was located in the cytosol and could be obtained after CsCl/ethidium bromide density gradient centrifugation at the position of covalently closed circular DNA. Both these components amounted to approximately 0.5–1% of total DNA.

A further DNA component with a complexity of more than 150 × 103 base pairs, enriched in fractions where mitochondria are expected, was not characterized further. The density was intermediate between cyDNA and nuclear DNA (1.710–1.720 g · cm−3) and it amounted to 1–2% of the total DNA.

Our results indicate that the DNA from cyanelles, believed to be endosymbiotic cyanobacteria, is not more complex than higher plant chloroplast DNAs.
Nuclear Magnetic Resonance of Protamines
Authors: FERRARA Luciano , NAPOLITANO Roberto , PAOLILLO Livio , WURZBURGER Silvana , ANDINI Salvatore , TONIOLO Claudio , BONORA Gian M.
Published: July 2010

The three main fractions of clupeine, the protamine extracted from herring sperm, have been investigated by 13C nuclear magnetic resonance techniques.

The dynamic behaviour, examined through the evaluation of the spin lattic relaxation times (T1) of individual carbon resonances assigned to both backbone and side chains, reveals interesting features. The relaxation times of backbone α‐carbons, interpreted on the basis of an axially symmetric ellipsoid, point to the clupeine fractions as being essentially extended in aqueous solution. These times remain constant along the polypeptide chain and are of the order of 0.16 ± 0.02 s. Conversely, the side chains show different flexibilities in the presence of monophosphate counterions, thus demonstrating a diverging behaviour which may be biologically relevant. In particular, the side‐chain flexibilities of fraction YI decrease, while those of fractions Z and YII are either constant or increase.

Comparison of these data with the viscosity measurements helps in explaining the viscosity changes observed in the presence of phosphate.
Differential Activation of Two Protease‐Activated Protein Kinases from Reticulocytes by a Ca2+‐Stimulated Protease and Identification of Phosphorylated Translational Components
Authors: TAHARA Stanley M. , TRAUGH Jolinda A.
Published: July 2010

Two protein kinases have been partially purified from rabbit reticulocytes and shown to be activated by limited proteolysis with trypsin [S. M. Tahara and J. A. Traugh (1981) J. Biol. Chem. 256, 11558–11564; P. T. Tuazon, W. C. Merrick, and J. A. Traugh (1980) J. Biol. Chem. 255, 10954–10958]. Reticulocyte lysate was examined for protease activities which might be involved in activation of the protein kinases in vivo. Two neutral proteases, differentially activated by Fe2+ and Ca2+, were identified and partially purified. The Ca2+‐stimulated protease specifically activated protease‐activated kinase II; no effect was observed on protease‐activated kinase I. The Fe2+‐stimulated protease was not active on either protein kinase. The protease‐activated kinases were examined using initiation factors (eIF) and 40‐S ribosomal subunits as substrate. Protease‐activated kinase I phosphorylated one subunit of eIF‐3 (Mr 130000), eIF‐4B and 40‐S ribosomal protein S10. Protease‐activated kinase II modified the β subunit of eIF‐2 (Mr, 53000) and 40‐S ribosomal protein S6. The substrate specificities are unique when compared with other cAMP‐dependent and cAMP‐independent protein kinases from reticulocytes.
Blood Group A and H Determinants in Polyglycosyl Peptides of A1 and A2 Erythrocytes
Authors: VIITALA Juha , FINNE Jukka , KRUSIUS Tom
Published: July 2010

Polyglycosyl peptides were isolated from delipidated erythrocyte membranes of human blood‐group A1 and A2 erythrocytes by extensive pronase digestion and gel filtration. As estimated by the amounts of N‐acetyl‐galactosamine and 2‐O‐substituted galactose residues about 85% of the possible acceptor sites (H determinants) were saturated with A determinants in A1 polyglycosyl peptides whereas only 25% of H sites were filled in A2 glycopeptides. The distribution of A and H determinants in the glycopeptides was studied by affinity chromatography with Sepharose‐bound Bandeiraea simplicifolia I‐lectin (binds blood‐group A and B determinants) and Ulex europaeus I‐lectin (binds blood‐group H determinants). About 55% of the polyglycosyl peptides contained A, H, or A and H determinants in both A1 and A2 blood subgroups. 48% of the polyglycosyl peptides of blood group A1 and 10% of A2 bound to Bs I‐lectin. 25% of the polyglycosyl peptides in A1 and 53% in A2 carried H determinants.

The molecular size, monosaccharide composition and the substitution pattern of the monosaccharides in the Bs‐I‐bound polyglycosyl peptides were very similar in both A1 and A2 blood groups. The only difference was the amount of N‐acetylgalactosamine which was on the average 3.7 mol/mol in A1 and 2.5 mol/mol in A2. The active fraction was found to be heterogeneous with respect to the amount of A determinants, which varied from 1 to 6 per glycopeptide in A1 and A2 polyglycosyl peptides. The findings do not indicate a structural difference between blood‐group A1 and A2 polyglycosyl peptides and state chemically that A1 glycopeptides contain more A determinants than A2 glycopeptides.
Phosphorylation of the Type‐II Regulatory Subunit of Cyclic‐AMP‐Dependent Protein Kinase by Glycogen Synthase Kinase 3 and Glycogen Synthase Kinase 5
Authors: HEMMINGS Brian A. , AITKEN Alastair , COHEN Philip , RYMOND Michael , HOFMANN Franz
Published: July 2010

The regulatory (RII) subunit of type‐II cyclic‐AMP‐dependent protein kinase from bovine heart is phosphorylated at a significant rate in vitro by glycogen synthase kinase 3 and glycogen synthase 5, but not by glycogen synthase kinase 4 or phosphorylase kinase. The regulatory (RI) subunit of type‐I cyclic‐AMP‐dependent protein kinase from rabbit skeletal muscle is not phosphorylated by any of these four protein kinases.

Glycogen synthase kinase 3 phosphorylates two serine residues on the RII subunit located 44 and 47 amino acids from the N terminus of the polypeptide chain. Glycogen synthase kinase 5 phosphorylates serine‐74 and serine‐76. These sites are distinct from the residue phosphorylated by the catalytic subunit of cyclic‐AMP‐dependent protein kinase (serine‐95).

The RII subunit, as normally isolated, contains 1.5–1.8 mol alkali‐labile phosphate per subunit. At least 80% of this phosphate (∼ 1.3 mol/subunit) is located in the thermolytic peptide containing serine‐74 and serine‐76, demonstrating that phosphorylation of the RII subunit by glycogen synthase kinase 5 occurs in vivo. Only small amounts of phosphate (∼ 0.1 mol/subunit) are associated with the thermolytic peptides containing serine‐44/serine‐47 and serine‐95.

The phosphorylation sites on the RII subunit are organised in a strikingly similar manner to those of glycogen synthase, the amino acid sequences in the immediate vicinity of the phosphorylation sites showing a particular resemblance. These include the presence of a number of proline residues near the sites phosphorylated by glycogen synthase kinase 3, five consecutive acidic residues C‐terminal to the sites phosphorylated by glycogen synthase kinase 5, and two adjacent arginine residues just N‐terminal to the sites phosphorylated by the catalytic subunit of cyclic‐AMP‐dependent protein kinase.

Glycogen synthase kinase 5 is very similar or identical to the enzyme that has been variously termed casein kinase TS, casein kinase 2, casein kinase G, casein kinase N‐II or troponin‐T kinase. The biological role of this enzyme is reviewed.
Ligand‐Binding Studies on Heavy Riboflavin Synthase of Bacillus subtilis
Authors: BACHER Adelbert , LUDWIG Heide Carla
Published: July 2010

Heavy riboflavin synthase is a complex enzyme consisting of three α subunits and approximately 60 β subunits. Ligand‐binding studies were performed with a variety of substrate and product analogues by analytical ultracentrifugation and by equilibrium dialysis. Nonlinear binding curves indicate the involvement of non‐equivalent binding sites which could be assigned to the α and β subunits by comparison with light riboflavin synthase (subunit composition α3) and with aggregates of isolated β subunits. The β subunit binding site shows a high degree of stereospecificity. Tightly binding ligands must have a ribityl side chain and a pyrimidine or pteridine moiety with polar substituents.
Molecular Basis of the Permeabilization of Mammalian Cells by Ionophores
Authors: ALONSO Miguel A. , CARRASCO Luis
Published: July 2010

Mammalian cells become permeable to low‐molecular‐weight compounds and proteins when they are treated with nigericin. Concentrations of this ionophore that have no effect on protein synthesis induce the entry of the translation inhibitors hygromycin B (Mr 550) and χ‐sarcin (Mr 16800). Treatment of HeLa cells with low concentrations of nigericin stimulate the (Na+/K+) ATPase and, as a consequence, increase the plasma membrane potential. A correlation is observed between the increase in membrane potential and the entry of antibiotics and protein toxins into cells. These results suggest a relationship between the two phenomena and help an understanding of the molecular mechanism for the passage of charged molecules and proteins into mammalian cells. This permeabilization is specific for certain ionophores, such as nigericin and monensin, and does not occur on treatment with amphotericin B, valinomycin or A23187. These compounds depolarize the plasma membrane and drastically block protein synthesis in HeLa cells under these conditions.
Accelerated Turnover of Hepatic Glucokinase in Starved and Streptozotocin‐Diabetic Rat
Authors: SIBROWSKI Walter , STAEGEMANN Ulrich , SEITZ Hans J.
Published: July 2010

Rat liver glucokinase synthesis and degradation was estimated in fasted/fed and diabetic/diabetic‐insulin‐treated rats by the radioimmunological technique. Starvation and Streptozotocin‐diabetes led to basal rates of synthesis and, consequently, to low levels in enzyme activity. In addition, a decrease in the apparent half‐life from about 19 h in the fed or diabetic‐insulin substituted to about 11 h in the starved or diabetic rat, respectively, was observed. Injection of Bt2cAMP into glucose‐fed animals reduced glucokinase synthesis to basal levels within 90 min, without affecting enzyme activity.

It is concluded that in metabolic states associated with elevated levels in tissue cAMP glucokinase synthesis is reduced to basal values and, in addition, its rate of degradation is significantly enhanced.
Structure of Ribosomal Protein L6 from Escherichia coli
Authors: STEINHÄUSER Klaus G. , WOOLLEY Paul , EPE Bernd , DIJK Jan
Published: July 2010

Protein L6 from the 50‐S ribosomal subunit has been investigated using fluorimetric techniques. The intrinsic fluorophore Trp‐61 and fluorescent labels (acetylaminoethyl‐dansyl and acetylaminofluorescein) attached to the residue Cys‐124 were used. It proved possible to incorporate fluorescence‐labelled L6 into the 50‐S ribosome.

Trp‐61 is exposed to solvent, as shown by its emission wavelength and by quenching experiments; the latter also show that it lies in a pocket with a high positive charge due to the basic residues in the N‐terminal fragment. Cys‐124 lies in a less strongly positive region. Upon incorporation into the 50‐S subunit, the label on Cys‐124 becomes less accessible for quenching but its positive potential rises, showing the absence of direct contact with 23‐S RNA.

Analysis of anisotropy data indicates a considerable degree of asphericity of free L6. Energy transfer between Trp‐61 and the dansyl label on Cys‐124, measured by donor quenching and acceptor enhancement, reveals a separation of 3.5 ± 0.4 nm (35 ± 4Å) between fluorophores.
A Hypothesis for the Role of Dithiol‐Disulfide Interchange in Solute Transport and Energy‐Transducing Processes
Authors: ROBILLARD George T. , KONINGS Wil N.
Published: July 2010

We have recently shown that the physical mechanism for Δ+ ‐driven changes in the Km for three different transport systems is an oxidation‐reduction reaction involving a dithiol‐disulfide interconversion [Robillard, G. T. and Konings, W. N. (1981) Biochemistry, 20, 5025–5032; Konings, W. N. and Robillard, G. T. (1982) Proc. Natl Acad. Sci. USA, in the press]. Based on the similarities between the data from these three systems and published data from other systems, we now propose that dithiol‐disulfide interchange may play a general role in membrane‐related processes such as transport, energy transduction and hormone‐receptor interactions.

We propose that the affinities of the substrate‐binding sites are regulated by a dithiol and a disulfide situated at different depths in the membrane. In addition we propose that the oxidation states of these two redox centers are coupled by dithiol‐disulfide interchange such that, when one is oxidized, the other is reduced. Since a transmembrane electrical potential, Δ;ψ, or a pH gradient, ΔpH, can alter the redox state, it can change the affinity of the substrate‐binding sites. The Δ;H+ ‐induced changes in affinity are sufficient to drive active transport (symport or antiport) and energy‐transducing processes. A similar mechanism can be applied to transport systems driven by phosphorylated enzyme intermediates instead of Δ;H+.

Changes of the redox potential in a given compartment during metabolism could also control the affinity of ligand binding even in the absence of a Δ;H+. The ligand‐binding affinities of facilitated diffusion transport systems and receptor proteins may be regulated in this manner.