bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of Rat Liver Lactate Dehydrogenase and Characterization of Its mRNA
Authors: EISENMANN Andreas , SCHMELZER Elmon , NORTHEMANN Wolfgang , PHILLIPS John , KAISER Clemens , WITT Irene , DECKER Karl , HEINRICH Peter C.
Published: March 2005

Rat liver lactate dehydrogenase (LDH) has been synthesized in hepatocytes and in a cell‐free translation system. The subunit synthesized in both systems displayed the same electrophoretic mobility upon sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. Sequence analysis of the subunits translated in vitro and synthesized in vivo indicated N‐acetyl‐alanyl‐alanine for both N termini. Thus, the newly synthesized subunit does not exhibit an amino‐terminal extension.

The mRNA for the lactate dehydrogenase subunit was exclusively found in free polysomes. A size of 2120 ± 240 nucleotides was estimated for the mRNA. Only about 50% of these nucleotides are needed to code for the polypeptide chain of the enzyme.
The Simian‐Virus‐40 Large‐Tumor Antigen in Replicating Viral Chromatin
Authors: STAHL Hans , BAUER Michael , KNIPPERS Rolf
Published: March 2005

The large tumor antigen (T antigen) is a genome regulation protein, coded by simian virus 40, that binds with high affinity to specific binding sites on viral DNA. The specifically bound T antigen is released from these sites in 0.2–0.3 M NaCl. Immunoprecipitation techniques were used to show that T antigen also dissociates in 0.2–0.3 M NaCl from mature viral chromatin but not from replicating viral chromatin. In fact, a considerable fraction of T antigen remains associated with replicating chromatin at NaCl concentrations as high as 1.2 M NaCl when most chromatin proteins, including histones, dissociate. However, T antigen binding to both replicating DNA and mature DNA is sensitive to intercalating drugs such as caffeine and ethidium bromide.

We consider the possibility that the unexpectedly tight binding of T antigen to replicating DNA is related to the function that T antigen performs during viral DNA replication.
Proline Dehydrogenase from Escherichia coli K12
Authors: ABRAHAMSON John L. A. , BAKER L. Gail , STEPHENSON James T. , WOOD Janet M.
Published: March 2005

We have examined the oxidative activities of inverted cytoplasmic membrane preparations from Escherichia coli bearing proline dehydrogenase. Our measurements include both direct substrate: 2,6‐dichloroindophenol and substrate: O2 oxidoreductase assays and the 9‐aminoacridine fluorescence assay for proton translocation, employing succinate and NADH dehydrogenases as comparative standards. Our data show the following. (a) Membranes prepared in a new buffer system bear proline dehydrogenase that is stable in both activity and membrane association. This membrane‐associated enzyme shows an apparent Km for proline 20‐fold lower than that estimated from the solubilized and purified enzyme. (b) Electrons are transferred from proline to O2 via the respiratory chain since proline utilization requires porphyrin synthesis and it is coupled to trans‐membrane proton translocation. (c) Patterns of inhibition by 5‐ethyl‐5‐isopentyl barbituric acid (Amytal) and 2‐heptyl‐4‐hydroxyquinoline‐N‐oxide (HpHOQnO) suggest that parallel pathways of electron flux from NADH and proline converge at a cyanide‐sensitive terminal oxidase. Succinate: O2 and succinate: DCIP oxidoreductase activities are stimulated by HpHOQnO and Amytal, and the former is inhibited by cyanide in this system. (d) Amytal is a non‐competitive inhibitor of proline dehydrogenase. (e) Analysis of our fluorescence data suggests that Amytal and HpHOQnO dissipate ΔpH at concentration as low as 5 mM and 8.5 μM, respectively, in this system.
Heme P460 of Hydroxylamine Oxidoreductase of Nitrosomonas
Authors: HOOPER Alan B. , DEBEY Pascale , ANDERSSON Kristoffer K. , BALNY Claude
Published: March 2005

Hydroxylamine oxidoreductase (HAO) of Nitrosomonas catalyzes the dehydrogenation of NH2OH and subsequent addition of oxygen to form nitrite. HAO contains c hemes and the CO‐binding heme P460 in a 7:1 ratio; dehydrogenation of NH2OH involves passage of electrons to P460 and then c hemes. We now report that electrons rapidly pass from c hemes of HAO to the P460 center and then to H2O2. This conclusion is supported by (a) inhibition of c heme oxidation with CO and (b) loss of H2O2‐oxidizability of ferrous c hemes following specific destruction of heme P460. Reaction of ferrous P460 with H2O2 is rate‐limiting. Activation of dioxygen for N‐oxidation by ferrous HAO may involve the two‐electron reduction of O2 by P460. The reaction of ferrous HAO with H2O2 was studied as it may reveal aspects of the mechanism of activation of dioxygen.

Reaction of ferrous heme P460 with CO is slow and with low affinity as compared with other hemoproteins. Values for reaction of CO with enzyme were: k1, 1.1 × 10−3 M−1 s−1 and Kd, 12μM.
The Glucose Effect in Bacillus subtilis
Authors: PRICE Virginia L. , GALLANT Jonathan A.
Published: March 2005

An analysis of the glucose downshift mechanism in Bacillus subtilis has shown that the derepression of catabolic enzymes characteristic of the ‘glucose effect’ includes isocitate dehydrogenase and glucose‐6‐phosphate dehydrogenase. Additionally, phosphofructokinase undergoes what appears to be a reversible modification regulated by glucose transport.
Fourth‐Derivative Spectrophotometry Analysis of Tryptophan Environment in Proteins
Authors: DUNACH Mireia , SABÉS Manuel , PADRÓS Esteve
Published: March 2005

Fourth‐derivative spectrophotometry is applied to the analysis of solvent effects on the spectral transitions of N‐acetyl‐l‐tryptophan amide, as well as of its mixtures with N‐acetyl‐l‐tyrosine ethyl ester. These compounds were analyzed in different media. It was found that the position of the longest‐wavelength minimum of the fourth‐derivative spectrum is mainly determined by the nature of the Trp environment, with a minor contribution from that of Tyr. A geometrical parameter is also defined, which depends on both Trp and Tyr environments. The simultaneous consideration of both parameters allows an estimation to be made of the Trp environment. The method is applied to the study of conformational changes of three well‐characterized proteins: melittin from bee venom, cytochrome c from horse heart and bacteriorhodopsin from Halobacterium halobium. The results obtained are found to be in accordance with the known conformational properties of these proteins. In addition, the fourth‐derivative operation completely eliminates the interference from the near‐ultraviolet bands of the prosthetic groups.
Proteinases in Human Polymorphonuclear Leukocytes
Authors: RANTALA‐RYHÄNEN Sirpa , RYHÄNEN Lasse , NOWAK Felicia V. , UITTO Jouni
Published: March 2005

Polymorphonuclear leukocytes have been shown to contain proteolytic enzymes which are capable of degrading connective tissue proteins such as native collagen. In this study, proteolytic enzymes were extracted from human polymorphonuclear leukocytes and a neutral proteinase was extensively purified and characterized. The activity of this enzyme was monitored by degradation of denatured [3H]proline‐labeled type I collagen or by cleavage of a synthetic dinitrophenylated peptide with a Gly‐Ile sequence. The enzyme was readily separated from leukocyte collagenase by concanavalin–A–Sepharose affinity chromatography and further purified by QAE‐Sephadex ion‐exchange chromatography and gel filtration on Sephacryl S‐200. The purified enzyme had a molecular weight of approximately 105 000, its pH optimum was about 7.8, and it was inhibited by Na2EDTA and dithiothreitol, but not by fetal calf serum. The enzyme degraded genetically distinct type I, II, III, IV and V collagens, when in a non‐helical form, but not when in native triple‐helical conformation. Dansyl‐monitored end‐group analyses, combined with digestion by carboxypeptidase A, indicated that the enzyme cleaved denaturated type I collagen at Gly‐Xaa sequences, in which Xaa can be leucine, isoleucine, valine, phenylalanine, lysine, or methionine. Thus, the purified enzyme referred to here as Gly‐Xaa proteinase, is a neutral proteinase, which may be of importance in inflammatory disease processes by degrading further collagen peptides which have been rendered non‐helical as a result of collagenase cleavage.
Characterization of a Chinese Hamster Ovary Cell Line Resistant to Uncouplers
Authors: FREEMAN Karl B. , YATSCOFF Randall W. , MASON Jeremy R. , PATEL Hasmukh V. , BUCKLE Malcolm
Published: March 2005

The chemiosmotic theory of oxidative phosphorylation and the action of uncouplers was examined by characterizing a clone, UH5, of Chinese hamster ovary (CHO TK−) cells resistant to 5‐chloro‐3‐tert‐butyl‐2′‐chloro‐4′‐nitrosalicylanilide (S‐13), a potent uncoupler of oxidative phosphorylation. About 9‐times and 4‐times more S‐13 was required to effect growth and respiration respectively of UH5 cells compared to the parental CHO TK− cells. UH5 cells were cross‐resistant to the uncouplers SF‐6847 (3,5‐di‐tert‐butyl‐4‐hydroxy‐benzylidenemalononitrile), carbonylcyanide p‐trifluoromethoxyphenylhydrazone and 2,4‐dinitrophenol but not to oligomycin, venturicidin or Tevenel. Size, chromosome number and DNA content indicated that the UH5 cell line was probably pseudotetraploid compared to the parental pseudodiploid CHO TK− cells. Hybrid and cybrid cells formed from crosses of UH5 cells and cytoplasts, respectively, with an uncoupler‐sensitive cell line were sensitive to S‐13 indicating that resistance is probably nuclear‐determined. UH5 cell mitochondria had increased cytochrome oxidase and decreased H+‐ATPase activities. A fivefold resistance of oxidative phosphorylation to uncouplers was found at the mitochondrial level with respiration driven by either succinate or ascorbate/N,N,N′,N′‐tertramethyl‐p‐phenylenediamine. In contrast, no difference in sensitivity was found to valinomycin between mitochondria from UH5 and CHO TK− cells. The oligomycin‐sensitive H+‐ATPase activity of UH5 and CHO TK− cell mitochondria was equally stimulated by the uncoupler S‐13. Uncoupler‐resistant mitochondria would not be expected on the basis of the chemiosmotic theory, and the relation of the results to other modes of coupling is considered.
Hypodermin B, a Trypsin‐Related Enzyme from the Insect Hypoderma lineatum
Authors: LECROISEY Anne , TONG Nguyen Thanh , KEIL Borivoj
Published: March 2005

Hypodermin B, a serine proteinase with a molecular weight of 23000, was purified to homogeneity from the larvae Hypoderma lineatum. It is stoichiometrically inhibited by diisopropylfluorophosphate and fully inactivated by N‐tosyllysine chloromethyl ketone and soya bean and bovine pancreatic trypsin inhibitors. N‐Tosylphenyalanine chloromethyl ketone and ovomucoid are without effect on its activity. Hypodermin B hydrolyses both amide and ester substrates of trypsin but does not display any chymotryptic activity on synthetic substrates. Its specificity on the B chain of insulin is slightly broader than that of bovine trypsin. Its amino acid composition and N‐terminal sequence suggest structural homology with serine proteinases of the trypsin family and with two other serine proteinases, hypodermin A and Hypoderma collagenase, previously isolated from the same larave. Hypodermins A and B are very similar with respect to their inhibition and specificity, they differ however strongly from Hypoderma collagenase.
On the Transhydrogenase Activity of Baker's Yeast Flavocytochrome b2
Authors: URBAN Philippe , LEDERER Florence , ALLIEL Patrick M.
Published: March 2005

It is shown that, when baker's yeast flavocytochrome b2 is incubated with bromopyruvate in the presence of excess lactate, a transhydrogenation reaction takes place which produces bromolactate and pyruvate. The heme remains reduced during the reaction.

It is further shown that reduced flavocytochrome b2 can catalyze the reduction of a number of other keto acids like pyruvate (the product of the physiological reaction) and other halogenopyruvates. Determinations of forward and reverse reaction rates, as well as of the redox potentials of the halogenolactate/halogenopyruvate couples lead to the conclusion that the transhydrogenation reaction is under thermodynamic control.

Determinations of the steady‐state deuterium isotope effect show that the rate‐limiting step in the oxidation of halogenolactates is abstraction of the α‐hydrogen (probably as a proton), as is the case for lactate itself. According to the principle of microscopic reversibility, the rate‐limiting step in the reverse reaction must be protonation of the putative carbanion.
Somatic Antigens of Pseudomonas aeruginosa
Authors: KNIREL Yuriy A. , VINOGRADOV Evgeny V. , SHASHKOV Alexander A. , DMITRIEV Boris A. , KOCHETKOV Nikolay K. , STANISLAVSKY Evgeny S. , MASHILOVA Galina M.
Published: March 2005

On mild acid degradation of Pseudomonas aeruginosa O:3(a),c, O:3a,d,e and O:3(a),d,f lipopolysaccharides O‐specific polysaccharides were isolated. The trisaccharide repeating units of O:3(a),c and O:3a,d,e polysaccharides contained 2‐acetamido‐2,6‐dideoxy‐d‐galactose and 2,3‐(1‐acetyl‐2‐methyl‐2‐imidazolino‐5,4)‐2,3‐dideoxy‐d‐mannuronic acid, which were identified previously as the constituents of P. aeruginosa O:3a,b and O:3a,d O‐specific polysaccharides, as well as 2,3‐diacetamido‐2,3‐dideoxy‐l‐guluronic acid, which has never before been found in nature. The last monosaccharide was identified without being isolated in the free state, by means of 13C nuclear magnetic resonance spectroscopy.

The structures of O:3(a),c and O:3a,d,e polysaccharides were established by selective hydrogen fluoride solvolysis followed by modification of the trisaccharides obtained and analysis of the 13C nuclear magnetic resonance spectra at each modification stage. The polysaccharides possessed similar structures of repeating units differing from each other in the anomeric configuration of the N‐acetylfucosamine residue only:→4)DManImU(β1→4)LGul(NAc)2U(α1→3)DFucNAc(β1‐ and →4)DManImU(β1→4)LGul(NAc)2U(α1→3)DFucNAc(α1‐,where DManImU = 2,3‐(1‐acetyl‐2‐methyl‐2‐imidazolino‐5,4)‐2,3‐dideoxy‐d‐mannuronic acid, LGul(NAc)2U = 2,3‐diacetamido‐2,3‐dideoxy‐l‐guluronic acid, DFucNAc = 2‐acetamido‐2,6‐dideoxy‐d‐galactose.

The same components were detected in the O:3(a),d,f polysaccharide but not in the same stoichiometric ratio; this polysaccharide possessed no regular structure.

The immunochemical study of lipopolysaccharides of all five P. aeruginosa O:3 serotypes showed a definite interrelation between their serological properties and structures of the corresponding O‐specific polysaccharides.
Interactions of Clathrin Coat with Model Lipid Membranes
Authors: RALSTON Evelyn , ROBINSON Jane , FINSY Robert , ENGELBORGHS Yves
Published: March 2005

The interaction of coat proteins from coated vesicles with model lipid membranes was examined using small unilamellar vesicles of dipalmitoylglycerophosphocholine as model membranes. Changes in membrane permeability were measured by the leakage of entrapped fluorescent dye, carboxyfluorescein. Both clathrin and the 55000‐Da protein were found to be active. Density gradient centrifugation showed the formation of an irreversible protein‐lipid complex. Dynamic light‐scattering measurements showed that this complex is significantly larger than the original vesicles, suggesting that fusion is induced. The effects of pH, urea, Tris and ionic strength were studied and the possible biological relevance of the results is discussed.
Immunochemical Properties of Vertebrate α‐Crystallins
Authors: PURI Nirdosh , AUGSTEYN Robert C. , OWEN Elisabeth A. , SIEZEN Roland J.
Published: March 2005

A competitive radioimmunoassay was used to determine the reactivities of α‐crystallins from 13 species with antibodies directed toward calf α‐crystallin. The results indicate that species as diverse as human and dogfish share the same number of crossreacting antigenic determinants. The various α‐crystallins can be distinguished only on the basis of their differing affinities for the antiserum.

Hydrophilicity profiles for αA and αB polypeptides of all species were found to be remarkably similar. On the basis of these, four major sequential determinants could be predicted for each polypeptide. The location and sequence of these determinants were found to be essentially conserved in all α‐crystallins examined. These results are in agreement with the observed crossreactivities. However, there was little obvious correlation between substitutions in determinants and observed variations in respective α‐crystallin/antibody affinities. Conservation of antigenic determinants over such a wide evolutionary range may reflect stringent constraints on the overall surface and three‐dimensional structure of vertebrate α‐crystallins.
Mitochondrial Carbamoyl Phosphate Synthetase Activity in the Absence of N‐Acetyl‐l‐glutamate
Authors: RUBIO Vicente , BRITTON Hubert Greenslade , GRISOLIA Santiago
Published: March 2005

Rat liver carbamoyl‐phosphate synthetase I is shown to have synthetase and ATPase activity in the absence of acetylglutamate. Km values for ATP, Mg2+ and K+ are greatly increased, the Km for HCO−3 is not changed much, and the Km for NH+4 is markedly reduced. Vmax for the synthetase reaction is < 20% of that of the acetylglutamate‐activated enzyme whereas Vmax for the ATPase activity is > 40% of that with acetylglutamate. Pulse‐chase experiments with H14CO−3 show formation of less “active CO2” (the central intermediate) than with acetylglutamate; ATPase activity is reduced in proportion, but the synthetase activity is much smaller. Binding of one ATP molecule with high affinity (Kd=20–30μM) is shown in the absence of acetylglutamate. This appears to be the molecule of ATPB (ATPB provides the phosphoryl group of carbamoyl phosphate). In contrast, the affinity for ATPA (ATPA yields Pi) is much reduced. Initial velocity measurements without acetylglutamate show a time lag before reaching a constant velocity. At 50 μM acetylglutamate the lag is much longer, but at 10 μM acetylglutamate it is shorter. Activation by acetylglutamate requires ATP at concentrations sufficient to occupy the ATPA and the ATPB binding sites. Preincubation with 10 mM acetylglutamate alone shortens the activation time.

From these findings we propose an allosteric model for activation of carbamoyl‐phosphate synthetase in which there are two active states, R and R · AcGlu. Binding of ATPA is associated with the conversion of T to R. R · AcGlu differs from R in that transfer to carbamate of the γ‐phosphoryl group of ATPB appears to be facilitated.
Inhibition of Ns‐Protein‐Stimulated Human‐Platelet Adenylate Cyclase by Epinephrine and Stable GTP Analogs
Authors: JAKOBS Karl H. , SCHULTZ Günter , GAUGLER Bernhard , PFEUFFER Thomas
Published: March 2005

The influence of purified, preactivated Ns protein, mediating adenylate cyclase stimulation by hormones and guanine nucleotides, was studied on adenylate cyclase activity in membranes of human platelets. The preactivated coupling protein caused a large increase in platelet enzyme activity. The activation occurred after a short lag phase and exhibited saturation. Ns protein increased the V of the platelet adenylate cyclase without change in the enzyme's affinity for the substrate, MgATP, whereas the apparent affinity for Mg2+ was increased by more than one order of magnitude. The Ns‐protein‐activated human platelet adenylate cyclase was inhibited by the hormone, epinephrine, and by the stable GTP analogs, guanosine 5′‐[β,γ‐imido]triphosphate and guanosine 5′‐[γ‐thio]triphosphate. The stable GTP analog or hormone‐induced adenylate cyclase inhibition was not competitive with regard to the concentration of Ns protein. Inhibition of Ns‐protein‐stimulated platelet adenylate cyclase caused by stable GTP analogs appeared to be persistent, was amplified by epinephrine and was accompanied by a large decrease in the enzyme's apparent affinity for Mg2+. The data suggest that the hormone‐sensitive adenylate cyclase is regulated by two guanine‐nucleotide‐binding sites, Ns and Ni, mediating enzyme stimulation and inhibition, respectively, by hormones and guanine nucleotides, and that the two coupling components interact in a non‐competitive manner with the adenylate cyclase, exhibiting high and low affinity for Mg2+ when affected by Ns and Ni, respectively.
Characterization of the epidermal‐growth‐factor‐dependent phosphorylation system from normal mouse‐liver sinusoidal plasma membranes
Authors: EHRHART Jean‐Claude , ROLLET Emmanuelle , KOMANO Odile , CREUZET Claudine , LOEB Jacques
Published: March 2005

Blood sinusoidal plasma membrane subfractions were isolated from normal mouse liver in the presence of the proteinase inhibitors PhMeSO2F and iodoacetamide. They were purified from smooth microsomal and Golgi vesicle contaminants.

The phosphorylation reaction was studied at 33°C, in the presence of 2 mM MnCl2. Addition of epidermal growth factor (EGF) to the preparations stimulated 32P incorporation from [γ‐32P]ATP or [γ‐32P]GTP essentially into one 170000 Mr protein. Some incorporation was observed in a minor 120000‐Mr component which appears to be a degradation product of the 170000‐Mr component. No EGF‐dependent phosphorylation of other membrane proteins or various exogenous proteins could be detected in vitro.

The dephosphorylation of the 170000‐Mr component was observed after 4 min of incubation at 33°C. This dephosphorylation reaction was inhibited by addition of 5 mM p‐nitrophenyl phosphate but not by addition of micromolar Zn2+, Be2+ or orthovanadate.

The 170000‐Mr protein specifically bound 125I‐labeled EGF and thus appeared to be the hepatic EGF receptor.

The EGF stimulatable kinase activity considerably enhances incorporation of 32P into tyrosine residues of the 170000‐Mr EGF receptor at 33°C. Tryptic peptide maps of the 32P‐labeled 170000‐Mr protein revealed a multiplicity of phosphorylated sites. Seven 32P‐labeled phosphopeptides were observed after EGF stimulation, three of them being largely prominent. Tryptic peptide maps of the 170000‐Mr protein after it was covalently linked to 125I‐labeled EGF showed only one 125I‐labeled peptide, the migration of which appeared different from that of 32P‐labeled phosphopeptides. These findings were confirmed by V8 protease unidimensional peptide mapping of the 170000‐Mr protein, labeled with 32P or 125I‐EGF.
Solubilization and characterization of an angiotensin II binding protein from liver
Authors: SEN Indira , JIM Kam F. , SOFFER Richard L.
Published: March 2005

Binding sites with high affinity for angiotensin II were solubilized from hepatic membranes by treatment with digitonin. Binding of radioiodinated angiotensin II was assayed by gel filtration and independently by a technique exploiting the failure of activated charcoal to adsorb the bound ligand. The binding protein was partially purified using ammonium sulfate fractionation followed by gel filtration, and in the presence of protease inhibitors, the isolated binding protein preparation did not catalyze degradation of the angiotensin II.

Binding to the membranes as well as to the solubilized preparation was specific and saturable. The membranes exhibited a single set of high‐affinity binding sites with a Kd of 0.5 nM. The solubilized preparation, also showed the presence of a single class of high‐affinity binding sites (Kd= 10.5 nM). Displacement studies using angiotensin I as well as various fragments, agonists and antagonists of angiotensin II disclosed a structure‐activity profile similar to that found with intact membranes. Dissociation of angiotensin II from the soluble macromolecular complex was slow but was enhanced at non‐physiological pH values or in the presence of 4.5 M urea, or 1% sodium dodecyl sulfate. Covalent binding of the radioiodinated angiotensin II to a single, specific macromolecular component was achieved by treatment with disuccinimidyl suberate. The apparent molecular weight of this reduced, denatured radioactive protein was estimated at about 68000 by polyacrylamide gel electrophoresis.
Inhibition of ADP‐induced platelet aggregation by reduced factor Xa
Authors: SINHA Asru K. , COLMAN Robert W.
Published: March 2005

Treatment of blood coagulation factor Xa with insolubilized hexyl‐agarose derivative of prostaglandin E1 (PGE1) results in the generation of two sulfhydryl groups in the protein molecule. The reduced factor Xa was found to be a potent inhibitor of platelet aggregation and thromboxane A2 synthesis induced by ADP. In contrast to the inhibition of thromboxane formation, the reduced factor Xa had no effect on the formation of PGE2 indicating that thromboxane synthetase might be selectively inhibited by the reduced factor Xa. Incubation with oxidized glutathione reversed the inhibitory activity of factor Xa previously exposed to the insolubilized hormone. Soluble PGE1 also reduces factor Xa, but more slowly than the insolubilized PGE1. PGE1 also exhibits reducing ability as tested with redox dyes.

Reduction of factor Xa by dithiothreitol also transformed the coagulation factor into an inhibitor of platelet aggregation and thromboxane A2 formation. These experiments indicate that reduction of factor Xa leads to a reversible alteration of the molecule which inhibits platelet aggregation induced by ADP. This effect of reduced factor Xa is probably mediated through the inhibition of thromboxane A2 synthesis.
High‐field phosphorus NMR studies of the stoichiometry of the lactate/proton carrier in Streptococcus faecalis
Authors: SIMPSON Stephen J. , BENDALL M. Robin , EGAN Aubrey F. , VINK Robert , ROGERS Peter J.
Published: March 2005

Highfield 31P‐NMR studies of whole cells of Steptococcus faecalis have shown that ΔpH can be formed by ATP hydrolysis and also by lactate transport. We have used 31P‐NMR to measure the pH dependence of the variable stoichiometry of the proton/lactate carrier. At low external pH (pH ∼ 6.5) the influx stoichiometry was 1.1 H+/lactate, while at high pH (7.5) the ratio was almost 2; the apparent midpoint pH of this variable stoichiometry is 7. ΔΨ measurements support the electrogenic nature of lactate transport at high pH; the variable rate of membrane depolarization caused by lactate transport also had a midpoint near pH 7.0. The data is consistent with a symmetrical carrier operating with variable stoichiometry as proposed by Michels et al. [(1979) FEMS Microbiol. Lett. 5, 357–364].
A high‐affinity, calmodulin‐dependent Ca2+ pump in the basal‐lateral plasma membranes of kidney cortex
Authors: GMAJ Piotr , ZURINI Mauro , MURER Heini , CARAFOLI Ernesto
Published: March 2005

A purified preparation of kidney basolateral membrane vesicles is capable of ATP‐dependent Ca2+ uptake. The reaction has high affinity for Ca2+ (Km about 0.1 μM) and a V of 5.8 nmol Ca2+× mg−1 protein × min−1 in the predominantly right‐side‐out vesicular preparation used. It is inhibited by vanadate (K0.5 about 5 μM) and by anticalmodulin drugs. A stimulatory effect of calmodulin is visible in membranes depleted of the activator. Exposure of basolateral membranes to 125I‐azido‐modified calmodulin results in the specific labeling of a membrane protein of Mr 141 000, which is tentatively suggested to be the Ca2+‐pumping ATPase.
Protein synthesis in differentiated and dedifferentiated hepatoma cell lines
Authors: BÖSZE Zsuzsanna , ARÁNYI Péter , VENETIANER Anikó
Published: March 2005

The pattern of protein synthesis in hepatoma cell clones was analysed by two‐dimensional separation of [35S]methionine‐labelled proteins. The clones were derived from the differentiated Reuber H35 hepatoma and showed differences in the expression of a number of liver‐specific functions and the resistance to the growth‐inhibitory effect of glucocorticoids.

Five protein spots were observed in the extracts of the differentiated Faza 967 cells that were absent from the electrophoretogram of the dedifferentiated H56 cells. This clone, on the other hand, displayed six spots absent from Faza 967 cells. The growth of both Faza 967 and H 56 cells was strongly inhibited by 1 γM dexamethasone.

The dexamethasone‐resistant clone 2, a dedifferentiated derivative of Faza 967 cells, synthesized two polypeptidies that were not present in Faza 967 or H 56 cells and produced four polypeptides at a lower level than Faza 967 cells. The examination of the short‐term effect of dexamethasone on protein synthesis in Faza 967 cells revealed nine induced and one repressed protein spots, which appeared to be in good agreement with earlier published data. It is concluded that dedifferentiation, although bringing about marked changes in certain liver‐specific functions, such as enzyme activities or protein secretion, affects only a relatively small fraction of the genes expressed.
Affinity labeling of succinyl‐CoA synthetase from Escherichia coli by the 2′,3′‐dialdehyde derivative of adenosine 5′‐diphosphate
Authors: NISHIMURA Jonathan S. , MITCHELL Theresa , COLLIER Glen E. , MATULA J. Michael , BALL Dorothy J.
Published: March 2005

The 2′,3′‐dialdehyde of adenosine 5′‐diphosphate, oADP, exhibited the properties of an affinity label with Escherichia coli succinyl‐CoA synthetase. Inactivation of this synthetase by oADP followed pseudo‐first‐order kinetics and was competitively blocked by ADP. The stoichiometry of labeling of the synthetase was 1 mol/mol αβ or, extrapolated, 2 mol/mol inactive α2β2 molecule. oADP also exhibited the properties of a substrate, bringing about rapid dephosphorylation of the enzyme. Further specificity of oADP was demonstrated in partially inactivated succinyl‐CoA synthetase by selective inhibition of the succinate ↔ succinyl‐CoA exchange reaction, in comparison to the CoA ↔ succinyl‐CoA exchange reaction. Modification of the synthetase by oADP resulted in cross‐linking of the enzyme, casting uncertainty over the subunit binding site for ADP. Modification of the synthetase by ADP‐2′‐semialdehyde occurred at a faster rate than that by oADP but exhibited biphasic inhibitor concentration dependence and did not exhibit saturability.
Aglycon specificity of fetal calf liver and ovine and porcine submaxillary gland α‐N‐acetylgalactosaminide α2→6 sialyltransferase
Authors: BERGH Michel L. E. , VAN DEN EIJNDEN Dirk H.
Published: March 2005

The specificity of fetal calf liver and ovine and porcine submaxillary gland N‐acetylgalactosaminide α2→6 sialyltransferase was investigated with acceptors of low and high molecular weight containing O‐glycosidically linked carbohydrate chains. It appeared that fetal calf liver microsomes were able to transfer sialic acid to C‐6 of GalNAc in NeuAc(α2→3)Gal(β1→3)GalNAc‐R, in which the aglycon could be protein as well as p‐nitrophenol (Nph). The substrates Gal(β1→3)GalNAc‐R and GalNAc‐R were inactive as acceptor with this enzyme source. Ovine and porcine submaxillary gland microsomes were both active with GalNAc‐protein, Gal(β1→3)GalNAc‐protein, NeuAc(α2→3)Gal(β1→3)GalNAc‐protein and NeuAc(α2→3)Gal(β1→3)GalNAcα‐Nph, but not with GalNAcα‐Nph and Gal(β1→3)GalNAcα‐Nph. The N‐acetylgalactosaminide α2→6 sialyltransferase which had been purified to homogeneity from porcine submaxillary gland [Sadler, J. E., Rearick, J. I. and Hill, R. L. (1979) J. Biol. Chem. 254, 5934–5941], was able to sialylate all three protein acceptors, but was virtually inactive with each of the three p‐nitrophenyl glycosides.

Our studies indicate that two N‐acetylgalactosaminide α2→6 sialtransferases exist acting on O‐glycosidically linked carbohydrate chains. The first enzyme, present in fetal calf liver, has a narrow specificity with regard to the oligosaccharide structure, but shows no specificity for the aglycon. Based on its specificity this enzyme can be designated as an [α‐N‐acetylneuraminosyl2→3‐β‐galactosyl 1→3]‐α‐N‐acetylgalactosaminide α2→6 sialyltransferase. The second enzyme, present in porcine submaxillary gland, has an absolute requirement for protein as the aglycon. Once this condition is fulfilled, the enzyme is able to transfer sialic acid to each of the three oligosaccharide chains and thus the enzyme is an α‐N‐acetylgalactosaminylprotein α2→6 sialyltransferase. The data also seem to suggest that ovine and porcine submaxillary gland microsomes contain, in addition to the latter enzyme activity, the α2→6 sialyltransferase with the strict oligosaccharide specificity.
Inhibition of metalloendopeptidases by 2‐mercaptoacetyl‐dipeptides
Authors: BLUMBERG Shmaryahu , TAUBER Zofia
Published: March 2005

A series of 2‐mercaptoacetyl‐dipeptides, a potential group of metalloendopeptidase inhibitors, has been synthesized by coupling the N‐hydroxysuccinimide ester of S‐acetyl‐2‐mercaptoacetic acid with hydrophobic dipeptide methyl ester hydrochlorides, followed by hydrolysis with NaOH in aqueous methanol and acidification with HCl. Thus, the 2‐mercaptoacetyl derivatives of L‐phenylalanyl‐L‐leucine, L‐leucyl‐L‐phenylalanine and L‐leucyl‐D‐phenylalanine were prepared. The first two compounds inhibit effectively thermolysin from Bacillus thermoproteolyticus and a metalloendopeptidase isolated from Streptomyces griseus, with Ki values in the micromolar range or below. The third compound inhibits the two enzymes only poorly, showing the stereospecificity of the inhibition process. These inhibitors should provide a useful tool for the study of bacterial and mammalian metalloendopeptidases (or dipeptidyl carboxypeptidases) and for the assessment of their physiological role.
Evidence for an essential histidine residue in glucose dehydrogenase from Bacillus megaterium and sequence analysis of the peptides labeled with bromoacetyl pyridine
Authors: ULMER Wolfgang , FRÖSCHLE Marion , JANY Klaus‐Dieter
Published: March 2005

Bromoacetylpyridine acts as an active‐site‐directed inhibitor on glucose dehydrogenase from Bacillus megaterium. The inactivation is irreversible with a Ki of 7.7 mM. The coenzyme NAD but not the substrate glucose protects the enzyme from the inactivation. It is proposed that bromoacetylpyridine modifies a residue at or nearby the active site.

The inactivation is correlated with the modification of a single histidine residue.

Modification of the enzyme with 3‐(2‐bromo[carbonyl‐14C]acetyl)‐pyridine and partial acid hydrolysis of the protein yielded one labeled fragment. From the arginine restricted tryptic cleavage of this fragment four radioactively labeled peptides were purified. Comparison of the specific radioactivity leads to the conclusion that the active site histidine residue must be located in the 58‐residue fragment AH2‐TA3. Sequence analysis showed that only one residue is modified in this fragment and the sequence around the labeled histidine residue is ‐Met‐Ser‐Ser‐Val‐His‐Glu‐Trp‐Lys‐Ile‐Pro‐Trp‐Pro‐.

The minor labeled arginine fragments, comprising 86, 20 and 13 residues, were also sequenced. Only lysine residues are modified in these peptides. The modification of the individual residues does not exceed 10%.
Structural studies on the non‐toxic lipid A from Rhodopseudomonas sphaeroides ATCC 17023
Authors: SALIMATH Paramahans V. , WECKESSER Jürgen , STRITTMATTER Wolfgang , MAYER Hubert
Published: March 2005

The lipid A isolated from Rhodopseudomonas sphaeroides ATCC 17023 has been investigated. A sequence of analyses indicated β‐D‐glucosaminyl‐1,6‐D‐glucosamine as the sugar backbone carrying phosphate groups at C‐1 of the reducing glucosamine and C‐4 of the non‐reducing glucosamine. Both 3‐(7‐tetradecenoyl)oxytetradecanoic and 3‐oxotetradecanoic acid are linked to the NH2 groups. Two residues of 3‐hydroxydecanoic acid are linked to hydroxy groups at C‐3 of both reducing and non‐reducing glucosamine, and the hydroxy group of 3‐hydroxydecanoic acid is free. In free lipid A, the hydroxy group at C‐4 of the reducing glucosamine and at C‐6 of the non‐reducing glucosamine are unsubstituted. The latter probably arises from a 3‐deoxy‐D‐manno‐octulosonate linkage.

The structural similarities and dissimilarities existing between lipid A of R. sphaeroides, which is non‐toxic, and lipid A types such as those from Salmonella, which are toxic, are discussed.
Electron paramagnetic resonance and other properties of hydrogenases isolated from Desulfovibrio vulgaris (strain Hildenborough) and Megasphaera elsdenii
Authors: GRANDE Hans J. , DUNHAM William R. , AVERILL Bruce , VAN DIJK Cees , SANDS Richard H.
Published: March 2005

The hydrogenases of Desulfovibrio vulgaris and Megasphaera elsdenii are compared with respect to some of their physical properties. In addition to Fe the only metal ions that are present in significant amounts are Ni and Cu. From cluster extrusion experiments it follows that the D. vulgaris enzyme contains three 4 Fe‐4 S clusters, while M. elsdenii hydrogenase only releases part of its Fe‐S clusters. The resting D. vulgaris enzyme shows only a small 3 Fe‐xS type of EPR signal (maximum 5% electron equivalent). This amount can be increased to approximately 25% by treatment with ferricyanide, with a concomitant large decrease in activity. The M. elsdenii enzyme shows in its oxidized state a normal Hipip (high‐potential iron‐sulphur protein) type of EPR spectrum. After a reduction/oxidation cycle the D. vulgaris enzyme also shows a weak Hipip type of EPR spectrum. In the reduced state both enzymes show complex spectra. By integration of those spectra it is shown that 1.5 electron equivalents are present. The complex spectra do not arise from nuclear hyperfine interactions but are partially due to electron spin interactions. It is proposed that the spectrum of reduced D. vulgaris hydrogenase consists of a sum of three different ferredoxin‐like spectra.
A potent new dipeptide inhibitor of cell sickling and haemoglobin S gelation
Authors: FRANKLIN Ian M. , COTTER Rosalind I. , CHEETHAM Rosemary C. , PARDON John F. , HALE Arthur J. , HUEHNS Ernst R.
Published: March 2005

A dipeptide l‐lysine‐l‐phenylalanine is shown to inhibit both cell sickling and the gelation of solutions of sicklecell haemoglobin. The effect on deoxyhaemoglobin solutions and gels was followed by centrifugation; a progressive inhibition of gelation was observed up to 30 mM Lys‐Phe. The haemoglobin concentration at the plateau (26 g/dl) suggests that an effect might be seen in vivo if suitable concentrations of Lys‐Phe (about 20 mM) can be maintained in the blood stream. Additional studies of lag time before onset of gelation support this. Oxygen dissociation curves of sickle cells showed an effect of Lys‐Phe only after incubation for 3 h before measurement, the P50 decreasing from 51 mmHg (6.8 MPa) to 41 mmHg (5.5 MPa) for cells depleted of 2,3‐bisphosphoglycerate.

The effect of Lys‐Phe on intact sickle cells was more rapid. A marked increase in the number of unsickled cells in the presence of Lys‐Phe was observed after only 15 min incubation. This result, together with measurements of uptake both into the cell and onto the cell membrane shows that the compound produces a membrane‐mediated anti‐sickling effect in addition to the effect on haemoglobin in solution within the cell. The membrane effect is not due to a change in cell volume.

The properties of this dipeptide may be of value in the treatment of impending and early sickle crisis.
Correlation between calmodulin‐dependent increase in the rate of calcium transport and calmodulin‐dependent phosphorylation of cardiac sarcoplasmic reticulum
Authors: PLANK Brigitte , PIFL Christian , HELLMANN Gertrude , WYSKOVSKY Wolfgang , HOFFMANN Ruth , SUKO Josef
Published: March 2005

The aim of the present study was to prove a correlation between the calmodulin‐dependent increase in the rate of calcium transport by dog cardiac sarcoplasmic reticulum and calmodulin‐dependent phosphorylation.

The dependence of phosphorylation on the total calmodulin concentration at 75 μM and 1 μM free calcium gave apparent calmodulin half‐saturation constants Km(CaM) of 9.4 nM and 181 nM, respectively, whilst the apparent Km(CaM) for the rate of calmodulin‐stimulated calcium transport carried out at 1 μM calcium, but phosphorylated prior to the calcium uptake at 75 μM or 1 μM calcium, were 12.5 nM and 127 nM, respectively. A positive correlation was obtained between calmodulin‐dependent increase in the rate of calcium transport and hydroxylamine‐insensitive phosphoester formed by the calcium/calmodulin‐regulated, membrane‐bound protein kinase. More than 90% of incorporated [32P]phosphate is confined to a 26–28‐kDa or 9–11‐kDa protein as determined by polyacrylamide gel electrophoresis following solubilization in sodium dodecyl sulfate at 37°C and at 100°C, respectively, similar to the results obtained by phosphorylation with cAMP‐dependent protein kinase. The data indicate that calmodulin‐dependent phosphorylation of the above protein(s) is causally related to the stimulation of the rate of calcium transport by cardiac sarcoplasmic reticulum, which is at least partially due to a shift in the calcium dependence of the rate of calcium transport to lower free calcium concentrations, K(Ca), of 1.25 μM and 0.61 μM in controls and calmodulin‐dependent phosphorylation, respectively.

Activation of calmodulin‐dependent phosphorylation by free calcium at total calmodulin concentrations of 300 nM, 100 nM and 30 nM gave apparent K(Ca) values of 0.83 μM, 1.44 μM and 2.3 μM and Hill coefficients of 4.13, 3.76 and 3.79, respectively, indicating that all four calcium binding sites of calmodulin have to be saturated to obtain activation of the calcium/calmodulin‐regulated protein kinase. The calmodulin‐dependent modulation of calcium transport in vivo is, therefore, determined to great extent by the total calmodulin concentration present in the sarcoplasm.
The primary structure of Escherichia coli glutaredoxin
Authors: HÖÖG Jan‐Olov , JÖRNVALL Hans , HOLMGREN Arne , CARLQUIST Mats , PERSSON Magnus
Published: March 2005

An immunosorbent method using antiglutaredoxin‐Sepharose was developed for purification of glutaredoxin in high yield from a mutant strain of Escherichia coli K 12 lacking thioredoxin reductase (C 10–17). The primary structure of the protein was determined by analyses of [14C]carboxymethylated glutaredoxin and its proteolytic fragments obtained by digestions with trypsin, clostripain, chymotrypsin and staphylococcal Glu‐specific extracellular protease.

The single active‐center disulfide has the structure , with the half‐cystine residues located at positions 11 and 14 in the polypeptide chain. In total the protein was deduced to have 85 residues corresponding to a molecular weight of 9674 for the reduced form of glutaredoxin, making it one of the smallest known enzymes (a glutathione–disulfide transhydrogenase).

The half‐cystines are identically spaced and similarly positioned in the N‐terminal part of the protein when compared with a corresponding functionally active disulfide/dithiol in thioredoxins. Glutaredoxin is also distantly homologous with thioredoxins from phage T4 and E. coli, but extensive differences, even around the redox‐active disulfide, distinguish glutaredoxin from the thioredoxins. Allowing for deletions in the glutaredoxin sequence (or insertions in the T4 thioredoxin sequence) at four places, there are identical residues at 25 positions of the 77 compared (= 32% identity). The results establish that glutaredoxin belongs to the same superfamily of small redox proteins as the thioredoxins. The structures are, however, subject to large changes, only four positions have residues identical among all presently analyzed forms.

The fluorescence of reduced and oxidized glutaredoxin demonstrates an increase in the quantum yield of the tyrosine emission upon reduction with dithiothreitol. Differences in the spectra support the presence of tyrosine adjacent to the redox‐active disulfide bridge. They also confirm that glutaredoxin lacks the disulfide‐adjacent tryptophan residues of E. coli thioredoxin.

There are known to be great differences between the bacterial E. coli and phage T4 forms of thioredoxin. The glutaredoxin structure is most similar to the phage type, both with respect to size of the polypeptide chain and to actual sequence. From the structural results and the previously known functional similarities it appears possible that the phage thioredoxin may have evolved from an early glutaredoxin gene. The mixed properties are compatible with this conclusion, the superfamily assignment, and the differences in biological activity.
Gene fusions using the ompA gene coding for a major outer‐membrane protein of Escherichia coli K12
Authors: HENNING Ulf , COLE Stewart T. , BREMER Erhard , HINDENNACH Ingrid , SCHALLER Heinz
Published: March 2005

It has been shown previously that fragments of the Escherichia coli major outer membrane protein OmpA lacking CO2H‐terminal parts can be incorporated into this membrane in vivo [Bremer et al. (1982) Eur. J. Biochem. 122, 223–231]. The possibility that these fragments can be used, via gene fusions, as vehicles to transport other proteins to the outer membrane has been investigated.

To test whether fragments of a certain size were optimal for this purpose a set of plasmids was prepared encoding 160, 193, 228, 274, and 280 NH2‐terminal amino acids of the 325‐residue OmpA protein. The 160‐residue fragment was not assembled into the outer membrane whereas the others were all incorporated with equal efficiencies. Thus, if any kind of OmpA‐associated stop transfer is required during export the corresponding signal might be present between residues 160 and 193 but not CO2H‐terminal to 193.

The ompA gene was fused to the gene (tet) specifying tetracycline resistance and the gene for the major antigen (vpl) of foot‐and‐mouth disease virus. In the former case a 584‐residue chimeric protein is encoded consisting NH2‐terminally of 228 OmpA residues followed by 356 CO2H‐terminal residues of the 396‐residue ‘tetracycline resistance protein’. In the other case the same part of OmpA is followed by 250 CO2H‐terminal residues of the 213‐residue Vp1 plus 107 residues partly derived from another viral protein and from the vector. Full expression of both hybrids proved to be lethal.

Lipophilic sequences bordered by basic residues, present in the non‐OmpA parts of both hybrids were considered as candidates for the lethal effect. A plasmid was constructed which codes for 280 OmpA residues followed by a 31‐residue tail containing the sequence: ‐Phe‐Val‐Ile‐Met‐Val‐Ile‐Ala‐Val‐Ser‐Cys‐Lys‐. Expression of this hybrid gene was lethal but by changing the reading frame for the tail to encode another, 30‐residue sequence the deleterious effect was abolished. It is possible that the sequence incriminated acts as a stop signal for transfer through the plasma membrane thereby jamming export sites for other proteins and causing lethality. If so, OmpA appears to cross the plasma membrane completely during export.
Purification of the elongation factor Tu (EF‐Tu) from the cyanobacterium Spirulina platensis
Authors: TIBONI Orsola , CIFERRI Orio
Published: March 2005

Elongation factor Tu (EF‐Tu) has been purified from the cyanobacterium Spirulina platensis. By gel electrophoresis the Mr of the purified protein appears to be 49000, a value close to that reported for the EF‐Tu isolated from a number of bacteria but higher than that reported for the protein isolated from Escherichia coli (43000). Functionally, however, S. platensis EF‐Tu may replace the E. coli protein in a protein‐synthesizing system in vitro. In addition, its activity is affected by kirromycin, an antibiotic that specifically interacts with eubacterial EF‐Tu.