bluuebunny/crossref_metadata_2025 huggingface dataset:
Magnetic resonance studies on the interaction of metal‐ion and nucleotide ligands with brain hexokinase
Authors: JARORI Gotam K. , MEHTA Alka , KASTURI Sitapati R. , KENKARE Umakant W.
Published: March 2005

Our previous studies have shown that one manganous ion binds tightly to bovine brain hexokinase, with a Kd= 25 ± 4 μM. The characteristic proton relaxation rate (PRR) enhancement of this binary complex (ɛb) is 3.5 at 9 MHz and 23°C [Jarori, G. K. Kasturi, S. R., and Kenkare, U. W. (1981) Arch. Biochem. Biophys. 211, 258–268]. On the basis of PRR enhancement patterns, observed on the addition of nucleotides ATP and ADP to this E · Mn binary complex, we now show the formation of a nucleotide‐bridge ternary complex, enzyme · nucleotide · Mn. Addition of glucose 6‐phosphate to enzyme · ATP · Mn, results in a competitive displacement of ATP Mn from the enzyme. However, a quaternary complex E · ADP · Mn · Glc‐6‐P appears to be formed when both the products are present.

β, γ‐Bidentate Cr(III)ATP has been used to elucidate the role of direct binding of Mn(II) in catalysis, and the stoichiometry of metal‐ion interaction with the enzyme in the presence of nucleotide. Bidentate Cr(III)ATP serves as a substrate for brain hexokinase without any additional requirement for a divalent cation. However, electron‐spin resonance studies on the binding of Mn(II) to the enzyme in the presence of Cr(III)ATP suggest that, in the presence of nucleotide, two metal ions interact with hexokinase, one binding directly to the enzyme and the second interacting via the nucleotide bridge. It is this latter one which participates in catalysis.

Experiments carried out with hexokinase spin‐labeled with 3‐(2‐iodo‐acetamido)‐2,2,5,5‐tetramethyl 1‐pyrrolidinyloxyl clearly showed that the direct‐binding Mn site on the enzyme is distinctly located from its ATP Mn binding site.
The nature of the decreased activity of glycogen synthase phosphatase in the liver of the adrenalectomized starved rat
Authors: BOLLEN Mathieu , DOPERE Frans , GORIS Jozef , MERLEVEDE Wilfried , STALMANS Willy
Published: March 2005

We have investigated the nature of the decrease in synthase phosphatase activity which occurs progressively in the livers of adrenalectomized rats that are starved for 48h. No evidence could be found for the accumulation of an inhibitor. Addition of the heat‐stable deinhibitor protein, which antagonizes the effects of thermostable inhibitor proteins (inhibitor‐1 and modulator), did not affect the activity of synthase phosphatase in gel‐filtered liver extracts from normal or adrenalectomized starved rats; it did, however, increase the activity of phosphorylase phosphatase about fivefold in either condition. The restoration of synthase phosphatase activity by cortisol in vivo was prevented by actinomycin D.

Further evidence concerning the nature of the missing protein came from a comparison of synthase phosphatase activities in liver homogenates from control and adrenalectomized starved rats, with the use of three distinct synthase b substrates. The apparent loss of synthase phosphatase activity in the deficient homogenates varied between 30% and 90% according to the type of substrate. The magnitude of this decrease corresponds to the degree of dependence of these substrates on the G‐component of synthase phosphatase for efficient conversion to the a‐form. No G‐component could be isolated from livers of adrenalectomized starved rats. Cross‐combination of subcellular fractions from control and deficient livers revealed an almost total loss of G‐component, with little loss of S‐component. This specific loss of functional G‐component is identical to the deficiency previously observed in the livers of rats with severe chronic alloxan‐diabetes.
Intracellular transport of the major glycoprotein of zymogen granule membranes in the rat pancreas
Authors: HAVINGA Johan R. , STROUS Ger J. , POORT Cees
Published: March 2005

The intracellular transport and destination of the major glycoprotein associated with zymogen granule membranes in the pancreas (GP‐2) was established. In suspensions of isolated acinar cells from rat pancreas, pulse‐chase experiments were performed. The incorporation of the first newly synthesized GP‐2 molecules into zymogen granule membranes occurred at about 60 min after beginning of the pulse. We demonstrated by using two different methods that newly made GP‐2 reaches the cell surface within the same time span. After 6–8 h chase considerable more newly synthesized GP‐2 has reached the cell surface than would be expected on account of secreted newly synthesized zymogens. These observations strongly suggest that at least part of the GP‐2 molecules bypass the mature zymogen granule compartment on their way to the plasma membrane.

GP‐2 is the only protein that appears in discernable quantity in the plasma membrane during 1–4h after a pulse label. Nevertheless GP‐2 comprises only a small percentage of externally 125I‐iodinated plasma membrane proteins. We conclude that GP‐2 has a high turnover rate at the plasma membrane level.

Treatment of the acinar cells with the N‐gylcosylation inhibitor tunicamycin does not block the intracellular transport of GP‐2.
Isolation and characterization of a somatomedin‐binding protein from mid‐term human amniotic fluid
Authors: PÓVOA Guilherme , ENBERG Gösta , JÖRNVALL Hans , HALL Kerstin
Published: March 2005

Human amniotic fluid is rich in a binding protein for somatomedins. This binding protein competes with human placenta membranes for labelled somatomedin A. Consequently, the placenta radioreceptorassay for somatomedin can be used for detection of the binding protein. The protein was isolated from human amniotic fluid by a three‐step procedure: First, stepwise ammonium sulphate precipitation; second, hydrophobic chromatography (phenyl‐Sepharose); and third, anion‐exchange chromatography (fast protein liquid chromatography). The total recovery of binding protein calculated with the placenta radioreceptorassay was 50%.

Polyacrylamide gel electrophoresis under native and denaturating conditions of the isolated protein disclosed a single band. The relative molecular mass was 35000, determined by exclusion chromatography, and 32000 under denaturating conditions in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The isoelectric point was 4.3 according to chromatofocusing and the amino acid composition also disclosed a high content of acidic/amidated residues. The N‐terminal amino acid sequence was Ala‐Pro‐Trp‐Gln‐Cys‐Ala‐Pro‐Cys‐Ser‐Ala.
Purification and characterization of tomato polygalacturonase converter
Authors: PRESSEY Russell
Published: March 2005

Extracts of ripe tomatoes contain two forms of polygalacturonase (PG I and PG II). A heat‐stable component that binds PG II to produce PG I has been isolated from tomato fruit. This component has been named polygalacturonase converter (PG converter). The PG converter has been purified by gel filtration, ion‐exchange chromatography and chromatofocusing. It appears to be a protein with a relative molecular mass of 102000. It was readily inactivated by papain and pronase. The converter was labile at alkaline conditions, and treatment of PG I at pH 11 released free PG II. A similar factor with a lower molecular mass was extracted from tomato foliage.
Hexose metabolism in pancreatic islets
Authors: SENER Abdullah , GIROIX Marie‐Hélène , MALAISSE Willy J.
Published: March 2005

Fructose, like glucose, rapidly equilibrates across the plasma membrane of pancreatic islet cells, but is poorly metabolized and is a weak insulin secretagogue in rat pancreatic islets. A possible explanation for such a situation was sought by investigating the modality of fructose phosphorylation in islet homogenates. Several findings indicated that the phosphorylation of fructose is catalyzed by hexokinase, but not fructokinase. First, at variance with the situation found in liver homogenates, the phosphorylation of fructose in the islet homogenate was unaffected by K+ and inhibited by glucose, mannose, glucose 6‐phosphate or glucose 1,6‐bisphosphate. Second, the Km for fructose was much higher in islets than in liver. Third, in islet homogenates the Km and Vmax for fructose were much higher than those for glucose or mannose phosphorylation, at low aldohexose concentrations, in good agreement with the properties of purified hexokinase. In intact islets fructose augmented the islet content in glucose 6‐phosphate sufficiently to cause marked inhibition of its own rate of phosphorylation. These findings may account, in part at least, for the low rate of fructose utilization by rat pancreatic islets.
Histidine residues in elongation factor EF‐Tu from Escherichia coli protected by aminoacyl‐tRNA against photo‐oxidation
Authors: JONÁK Jiří , PETERSEN Torben E. , MELOUN Bedřich , RYCHLÍK Ivan
Published: March 2005

Complexes of Escherichia coli elongation factor EF‐Tu with GTP or GTP and aminoacyl‐tRNA were photo‐oxidized by irradiation with visible light in the presence of rose bengal dye. EF‐Tu was isolated, digested with trypsin, the resulting tryptic peptides were separated by high‐performance liquid chromatography (HPLC), and the position of most of the peptides on the chromatogram was determined. Irradiation of complexes resulted in the inactivation of the factor (as tested by its capacity to interact with aminoacyl‐tRNA) and was accompanied by the loss of its histidine residues (as revealed by amino acid analysis) and by the decrease in the amount of some tryptic peptides (as detected by HPLC).

Aminoacyl‐tRNA, bound to EF‐Tu during the irradiation, protected the protein from inactivation, from the loss of histidine residues and some of its peptides from photo‐oxidative degradation. Comparison of quantities of individual tryptic peptides recovered from the irradiated EF‐Tu GTP aminoacyl‐tRNA complex with those from the irradiated EF‐Tu GTP complex revealed that histidine‐containing peptides T12 and T15 as well as methionine‐containing peptide T14 were in the ternary complex markedly protected against the photo‐oxidative degradation. This finding suggests that their histidines, i.e. His‐66 and His‐118 respectively and at least one of the methionines (Met‐91, 98 or 112) present in peptide T14 are located near to or at the binding site of EF‐Tu for aminoacyl‐tRNA and could be involved in the interaction between aminoacyl‐tRNA and the factor.
Computer simulation of purine metabolism
Authors: FRANCO Rafael , CANELA Enrique I.
Published: March 2005

A computer model of purine metabolism, including catabolism, salvage pathways and interconversion among nucleotides, is given. Steady‐state rate equations corresponding to metabolic enzymes are written based on information from the literature about their kinetic behaviour. Numerical integration of this set of equations is performed employing selected parameters taken from the literature. After stabilization of purine compound concentrations is reached, simulation of enzyme deficit and enzyme overproduction is carried out. The latter is calculated by varying specified maximum velocities in the numerical integration. A pattern of intermediate metabolite concentrations is found. These results form a basis for the comparison of normal patterns or patterns reflecting the effects of inborn errors of metabolism.

The aim of this paper is to demonstrate the usefulness of this computer simulation method in complex metabolism pathways.
Apolipoprotein A‐IGiessen (Pro143→Arg)
Authors: UTERMANN Gerd , HAAS Jochen , STEINMETZ Armin , PAETZOLD Ralph , RALL Stanley C. , WEISGRABER Karl H. , MAHLEY Robert W.
Published: March 2005

Apolipoprotein A‐IGiessen is a variant form of apo A‐I that is displaced from the corresponding normal A‐I isoforms on isoelectric focusing gels by a single charge unit towards the cathode [Utermann et al. (1982) J. Biol. Chem. 257, 501–507]. Three subjects heterozygous for the variant were detected in one family. The percentage of the total A‐I in plasma represented by the A‐IGiessen in these subjects ranged over 25–30%. The variant and normal major A‐I isoforms from the proband (Y.J.) were purified by preparative isoelectric focusing and cleaved with CNBr. Analytical focusing of CNBr fragments demonstrated a charge difference between CB3Giessen and normal CB3. Sequence analysis of CB3Giessen revealed that a proline existing in normal A‐I was replaced by an arginine in the variant A‐I at residue 143. The ability of the mutant A‐I to activate purified lecithin:cholesterol acyltransferase was determined in vitro. The cofactor activity of [Arg143]apolipoprotein A‐I was about 60–70% of that demonstrated by control A‐I. Residue 143 is in a putative β‐turn between two of the repeating amphiphilic helices in apolipoprotein A‐I and may be a critical determinant of the protein's structure and function.
Baker's yeast flavocytochrome b2
Authors: URBAN Philippe , LEDERER Florence
Published: March 2005

It has been shown that reduced flavocytochrome b2 not only catalyzes reduction of bromopyruvate [P. Urban, P.M. Alliel and F. Lederer (1983) Eur. J. Biochem. 134, 275–281] but also transforms it into pyruvate in a reductive elimination process. The dehydrohalogenation reaction also takes place when oxidized enzyme acts on bromolactate, but the reaction is more difficult to observe under these conditions because of its low efficiency compared to the normal oxidative process. The maximal rates of pyruvate production from bromopyruvate and chloropyruvate differ by a factor of less than 10, whereas elimination from fluoropyruvate cannot be detected. These results support a mechanism in which the dehydrohalogenation reaction takes place from a carbanion intermediate of the normal reductive‐oxidative pathway.
Neuronal nuclei and glial nuclei from mammalian cerebral cortex
Authors: PEARSON Edwin C. , BATES David L. , PROSPERO Terence D. , THOMAS Jean O.
Published: March 2005

We have characterised the histone and DNA contents of neuronal and glial nuclei from ox cerebral cortex which have, respectively, repeat lengths of 162 base pairs and 201 base pairs. Although the neuronal population cannot be obtained completely free of glial nuclear contamination, the degree of contamination is easily determined by counting, and has been allowed for in all the methods used here.

By diphenylamine assay and flow cytofluorometry we find that the DNA contents of both nuclear types are essentially equal, and equivalent to the diploid value, contrary to some reports.

By quantification of the core histones in known numbers of nuclei with respect to an added external standard, we have shown that the ratio of core histone octamers in the two nuclear types, neuronal and glial, is the inverse of the ratio of repeat lengths. Thus the same proportion of DNA is associated with core histone octamers in the two nuclear types, most simply all of the DNA.

By complete radiolabelling of the lysine side chains of the histones with methyl [1‐3H]acetimidate we have determined the stoichiometry of H1 relative to the core histones. Neuronal nuclei‐have a low H1 content of 0.45 molecule H1/nucleosome on average; glial nuclei have the ‘normal’ 1 H1 molecule/nucleosome. In neuronal nuclei about half of the nucleosomes therefore probably lack H1.

Whether there is any relation between the low H1 content and the short DNA repeat length of neuronal nuclei, on the one hand, and their high transcriptional capacity (at least when assayed in vitro), on the other, remains to be established.
The properties of the large subunit of maize ribulose bisphosphate carboxylase/oxygenase synthesised in Escherichia coli
Authors: GATENBY Anthony A.
Published: March 2005

The maize chloroplast gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase has been expressed in Escherichia coli in vivo. This enables the properties of the native large‐subunit polypeptide to be examined in the absence of small‐subunit polypeptides, and avoids the use of denaturing agents. The product synthesised in bacteria is slightly larger (Mr 54300) than the form present in the chloroplast (Mr, 53300), suggesting the involvement of a precursor polypeptide. In addition several smaller polypeptides are synthesised, predominantly of molecular mass 41 and 30 kDa, but also some of 44 and 12–14 kDa. Pulse‐chase experiments with [35S]methionine indicate that all the immunoprecipitable polypeptides are stable. The smaller products are probably the result of premature termination of translation. Virtually all of the large subunits are insoluble, whether synthesised at levels of 100–200 molecules per cell, or up to 60000 molecules per cell. A small amount of the full‐length polypeptide is soluble, but the major soluble product, as determined by sucrose gradient centrifugation, is a polypeptide of molecular mass 12–14 kDa. Ribulose bisphosphate carboxylase activity was undetectable in cell extracts, and binding of a mixture of the radiolabelled transition state analogues carboxyribitol 1,5‐bisphosphate and carboxyarabinitol 1,5‐bisphosphate could not be detected. It is proposed that other components are required to prevent the large subunit from adopting an inactive, insoluble conformation after, or during, synthesis.
Preparation of red‐cell‐membrane cytoskeletal constituents and characterisation of protein 4.1
Authors: OHANIAN Vasken , GRATZER Walter
Published: March 2005

A new and rapid method is described for the preparation of protein 4.1, the protein which modulates the interaction between spectrin and actin in the membrane cytoskeleton of the red cell. The method is based on the dissociation of purified membrane cytoskeletons in concentrated Tris at neutral pH, followed by gel filtration in the same medium. This procedure also yields spectrin and actin, as well as the fourth cytoskeletal constituent, protein 4.9, in relatively pure form, and ankyrin. Protein 4.1 is monomeric under our conditions of solvent and protein concentration, with a relative molecular mass, as determined from sedimentation equilibrium, of about 78000; its sedimentation coefficient and Stokes' radius are those of a globular, though somewhat asymmetric or flexible molecule. It forms a strong complex with F‐actin and spectrin. Protein 4.9 is also recovered in active form, and will bind strongly to F‐actin.
Import and processing of cytochrome b‐c1 complex subunits in isolated hepatoma ascites cells
Authors: KOLAROV Jordan , NELSON B. Dean
Published: March 2005

The import and processing of cytochrome c1 and the iron sulfur protein of the cytochrome b‐c1 complex were studied in Zajdela hepatoma ascites cells. Both peptides were synthesized as larger percursor molecules which were approximately 2–3kDa and 5–6kDa larger than the mature forms of apocytochrome c1 and apo‐iron sulfur protein, respectively. Comparison of these precursors to those reported for functionally homologous peptides in yeast and Neurospora indicate significant size changes have occurred in mammals.

Rhodamine 6G, a specific vital stain for mitochondria, is a potent inhibitor of precursor processing in isolated hepatoma cells. Both precursor to cytochrome c1 and precursor to FeS accumulate in the soluble and paniculate fractions obtained by digitonin treatment of tumor cells treated with Rhodamine 6G. Appearance of the mature peptides was abolished. The precursors are unstable, however, and disappear from the cytosolic and membrane fractions during a 10 min chase. Comparison of the effects of Rhodamine 6G and carbonylcyanide m‐chlorophenylhydrazone on precursor processing shows that: (a) Rhodamine 6G is a more effective inhibitor of processing, (b) it has less of an inhibitory effect on cellular protein synthesis, and (c) it inhibits processing under conditions in which it appears to have little influence on coupled B. D. Nelson, Biokemiska Institutionen, Stockholms Universitet, Arrheniuslaboratoriet, Bergiusvägen 65, Frescati, S‐106‐91 Stockholm, Sweden respiration in whole cells. The data suggest that the most likely mode of action of Rhodamine 6G is on the matrix processing step.
α‐2→4‐Interlinked 3‐deoxy‐d‐manno‐octulosonic acid disaccharide
Authors: BRADE Helmut , RIETSCHEL Ernst‐Theodor
Published: March 2005

After mild acid hydrolysis, a disaccharide of 3‐deoxy‐d‐manno‐octulosonic acid (dOclA) was obtained from Re‐mutant lipopolysaccharide of Salmonella minnesota, Salmonella godesberg, Proteus mirabilis, and Escherichia coli, and from the lipopolysaccharide of an S. minnesota Rb2 mutant. Combined gas‐liquid chromatography/ mass spectrometry of the reduced and permethylated derivatives indicated that the disaccharide is interlinked by a 2 4‐glycosidic bond in all lipopolysaccharides tested. In addition, it was shown by gas‐liquid chromatography of appropriate synthetic standards and a previously characterized α2 4‐linked dOclA disaccharide (derived from lipopolysaccharide of S. godesberg) that the non‐reducing dOclA residue possesses the α configuration. In the case of lipopolysaccharide of S. minnesota Rb2 mutant, this result, together with earlier findings, suggests that it contains a linear dOclA trisaccharide of the sequence dOclA (α2–4)dOclA(α2–4)dOclA. The results show that a dOclA (α2–4)dOclA disaccharide represents a common architectural principle in enterobacterial lipopolysaccharides.
31P nuclear magnetic resonance and chemical studies of the phosphorus residues in bovine milk xanthine oxidase
Authors: DAVIS Michael D. , EDMONDSON Dale E. , MÜLLER Franz
Published: March 2005

In addition to the phosphate residues contained in the acid‐dissociable FAD and the molybdenum cofactor moieties, milk xanthine oxidase contains one mole of covalently bound phosphorus per active‐center molybdenum. Acid hydrolysis of the apoprotein moiety and subsequent analysis by high‐voltage thin‐layer electrophoresis has identified the phosphorylated amino acid residue to be phosphoserine. 31P NMR data show the phosphopeptide to be monosubstituted, in agreement with the chemical analysis. A pH‐dependent chemical shift of the phosphorus residue in the molybdenum cofactor moiety is also observed which provides unequivocal support for suggestions in the literature that this cofactor contains a monosubstituted phosphate. 31P NMR studies on the intact enzyme show phosphorus resonances at about –3 ppm, +1 ppm, +8.8 ppm and at +13.5 ppm. The resonances at + 8.8 ppm and at +13.5 ppm are assigned to those of the pyrophosphate linkage of the FAD moiety by analogy with chemical shift data of the FAD on glucose oxidase [James, T. L., Edmondson, D. E., and Husain, M. (1981) Biochemistry 20, 617] and from the absence of any resonances in this region upon examination of preparations of deflavo xanthine oxidase. The intensity and resolution of the resonance at about –3 ppm is dependent on the degree of functionality of the enzyme. This resonance has a small amplitude relative to the FAD resonances in 50–60% functional enzyme, but increases dramatically in intensity in the desulpho enzyme. This resonance is the only one exposed to solvent as it is the only one susceptible to paramagnetic line‐broadening on the addition of Mn(II) to the enzyme solution. Treatment of the enzyme with allopurinol leads to alteration of the ∼‐3‐ppm resonance, but does not significantly affect the other resonances. Formation of the stable Mo(V) ‘inhibited’ form of the enzyme with ethylene glycol results in extensive line‐broadening of the resonances at –3 ppm and +1 ppm, but has no observable affect on the FAD resonances. These data suggest that in addition to the phosphate on the molybdenum cofactor, the phosphoserine residue in xanthine oxidase is also in close proximity to the active‐site molybdenum center of this enzyme. These results are discussed with respect to possible implications on the catalytic mechanism of the enzyme.
Characterization of the transcription products of glyceraldehyde 3‐phosphate‐dehydrogenase gene in HeLa cells
Authors: DANI Christian , PIECHACZYK Marc , AUDIGIER Yves , EL SABOUTY Sanaa , CATHALA Guy , MARTY Louise , FORT Philippe , BLANCHARD Jean‐Marie , JEANTEUR Philippe
Published: March 2005

We have partially purified the messenger RNA coding for glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from HeLa cells and obtained a cDNA clone containing part of its sequence. Using this clone to probe electrophoregrams of RNA transferred to nitrocellulose, we have investigated the characteristics of nuclear and cytoplasmic transcripts in these cells.

In the cytoplasm, nature GAPDH mRNA was detected in Northern blots as an intense band, apparently unique, of approximately 1400 nucleotides. The half‐life of this mRNA was determined both from the decay kinetics, after a chase with actinomycin D, and from the labeling kinetics during an accumulation experiment. Both kinds of experiments yielded a half‐life value of about 8 h, while the accumulation experiment indicated that steady‐state GAPDH mRNA amounted to about 1.6% of cytoplasmic poly(A)‐rich RNA.

Much longer species, likely to be restricted to the nucleus, were also detected in RNA extracted from total cells. At least three discrete species of 1600,4000, 5800 and 6800 bases were observed above a trailing background extending up to about 8000 bases. This value is commensurate with a functional size of the GAPDH transcription unit in the order of 13000 bases, which we determined by measuring the size of the ultraviolet inactivation target. Until direct evidence can be obtained at the genomic level, the present results provide the first clue to the existence of introns, presumably at least four, in a GAPDH gene from a higher eucaryote.
Conformational stability of the myosin rod
Authors: CROSS Robert A. , BARDSLEY Ronald G. , LEDWARD David A. , SMALL J. Victor , SOBIESZEK Apolinary
Published: March 2005

Chymotryptic cleavage patterns of myosin rods from pig stomach, chicken gizzard, and rabbit skeletal muscle indicate that short (∼45 nm) heavy meromyosin subfragment 2 (SF2) is a consistent product of all three rods, whereas long (∼ 60 nm) SF2 is derived only from skeletal muscle myosin.

Differential scanning calorimetry was used to follow the thermally induced melting transition of the rods and certain of their subfragments. In 0.12 M KCl, sodium phosphate buffer, pH 6.2–7.6, the light meromyosin (LMM) and SF2 domains of each rod had essentially identical conformational stabilities. Temperature midpoints for the melting transitions were 54–56°C for the two smooth muscle myosin rods and 50–53°C for the skeletal muscle myosin rod. In 0.6 M KCl buffer, melting transitions for the smooth muscle myosin rods were essentially unchanged, but skeletal muscle myosin rods showed multiphase melting, with major transitions at 43°C and 52°C. The first of these was tentatively attributed to LMM, and the second to SF2. In 0.12 M KCl buffer, the LMM transition was stabilised so that it superimposed on that of SF2. No melting was observed in any of the rods at physiological temperature. These results indicate that, excluding a possible but only narrow hinge region, the entire myosin rod has essentially uniform conformational stability at physiological pH and ionic strength, and thus that the contractile and elastic properties of the cross‐bridge exist in the heavy meromyosin subfragment 1 (SF1) domains of the molecule.
pH‐induced co‐operative effects in hysteretic enzymes
Authors: RICARD Jacques , NOAT Georges , NARI Joannès
Published: March 2005

A new model which provides an explanation for pH‐induced co‐operativity of hysteretic enzymes is proposed. The essence of the model is that a region, or a domain, of the enzyme undergoes a spontaneous ‘slow’ conformational change which does not affect the geometry of the active site. The region which undergoes this spontaneous conformational transition bears an ionizable group. Kinetic co‐operativity occurs if the pK of this ionizable group changes upon this conformational transition. Thus co‐operativity does not arise from a distortion of the active site.

An interesting prediction of the model is that at ‘extreme’ pH values co‐operativity must be suppressed. Although the kinetic equation pertaining to the model is of the 2:2 type, co‐operativity is not maximum or minimum at half‐saturation of the enzyme by the substrate, as occurs with 2:2 binding isotherms. A new index of maximum or minimum kinetic co‐operativity, whether this extreme occurs at half‐saturation or not, has been proposed which allows the change of kinetic co‐operativity to be followed as a function of pH.

It is believed that this model will be useful in explaining the behaviour of enzymes attached to biological polyelectrolytes, such as membranes or cell envelopes.
pH‐induced co‐operative effects in hysteretic enzymes
Authors: NARI Joannès , NOAT Georges , RICARD Jacques
Published: March 2005

A β‐glucosyltransferase, extracted and purified from the cell walls of isolated soybean cells, displays hysteretic behaviour. The enzyme is monomeric and has a negative co‐operative between pH 5.5 and 7.5. Below and above these pH values, the enzyme follows, or approaches, classical Michaelis‐Menten kinetics.

The free enzyme and the enzyme‐glucose complex exhibit, upon pH jumps, conformational transitions which may be followed by monitoring the fluorescence of enzyme‐bound toluidinylnaphthalene sulfonate.

Taken together these results are consistent with the model of pH‐induced co‐operativity described in the preceding paper in this journal. This special type of co‐operativity relies on a change of the pK value of a strategic ionizable group located outside the active site in a region (or a domain) of the protein which undergoes the conformational transition. The result that at ‘low’ and ‘high’ pH values, the enzyme follows or approaches Michaelis‐Menten kinetics is explained by assuming that the conformational changes do not affect the active site.
Ni‐mediated inhibition of human platelet adenylate cyclase by thrombin
Authors: AKTORIES Klaus , JAKOBS Karl H.
Published: March 2005

The influence of the proteolytic enzyme, thrombin, was studied on adenylate cyclase activity in human platelet membranes. Thrombin had a biphasic inhibitory effect on the enzyme. Up to a concentration of about 1 U/ ml, thrombin inhibited the enzyme in a strictly GTP‐dependent manner by maximal 60–70%, with half‐maximal inhibition occurring at about 0.005 U/ml thrombin. At higher concentrations, thrombin‐induced inhibition of platelet adenylate cyclase was independent of GTP. The inhibitory effect of thrombin observed at low concentrations was further evaluated. The inhibition was half‐maximal and maximal at about 0.1 and 3 μM GTP, respectively, occurred without major lag phase and was observed with the unstimulated and the forskolin or prostaglandin E1‐stimulated platelet adenylate cyclase. Additionally, thrombin accelerated and potentiated the enzyme inhibition caused by the stable GTP analog, guanosine 5′‐(γ‐thio)triphosphate. Furthermore, at identical concentrations causing adenylate cyclase inhibition, thrombin effectively stimulated GTP hydrolysis in platelet membranes. Finally, the thrombin inhibition of human platelet adenylate cyclase was impaired or abolished by Mn2+ and by treatment of the platelet membranes with N‐ethylmaleimide or pertussis toxin. All of these data indicate that thrombin at low concentrations inhibits platelet adenylate cyclase by a process involving the inhibitory guanine nucleotide‐binding regulatory component, Ni, in a manner similar to hormonal factors. However, in contrast to typical hormonal inhibition, thrombin inhibition of the platelet enzyme was not reversed by washing of the platelet membranes or by subsequent addition of the thrombin inactivator, hirudin, which prevented the inhibition when added together with thrombin. These data suggest that thrombin by its proteolytic capacity causes a persistent activation of its receptor site which leads to a persistent activation of Ni with subsequent persistent adenylate cyclase inhibition.
Increased ribosomal affinity for mRNA causes resistance to edeine in a mutant of Saccharomyces cerevisiae
Authors: HERRERA Flor , MORENO Nancy , MARTÍNEZ José Antonio
Published: March 2005

The effect of edeine on the translation of mRNA or poly(U)‐directed polyphenylalanine synthesis has been studied in an edeine‐resistant mutant of Saccharomyces cerevisiae under three different experimental conditions: in the whole lysate system, in a micrococcal‐nuclease‐treated lysate, and in a high‐salt‐treated lysate. The results indicate that translation of messenger is more resistant to edeine in the whole lysate than in the depleted lysates: these observations suggest that resistance to edeine is associated with the presence of endogenous mRNA.

It is shown that 40S mutant subunits have a higher affinity for polysomal RNA than 40S wild‐type subunits. Since the mRNA binding is inhibited by 7‐methylguanosine 5′‐monophosphate, the interaction between polysomal RNA and 40S ribosomes is specific for mRNA.

The data demonstrate that in each of the depleted lysates, with edeine initially present, the formation of the 80S initiation complex is inhibited. However, edeine inhibition of [3H]methionine binding to 80S ribosomes is overcome completely in the mutant extract by preincubation of this lysate with polysomal RNA.

The results indicate that the mutant may carry a specific change in a messenger‐binding factor or in a ribosomal protein thereby permitting an increased stability of the messenger‐ribosome complex which consequently results in an increased resistance of the mutant lysate to edeine.
Developmental regulation of gene expression in wheat embryos
Authors: CUMING Andrew C.
Published: March 2005

The early‐methionine‐labelled (Em) polypeptide is the most abundant single polypeptide found in the cytosolic fraction (30000 ×g supernatant) of dry wheat embryos. Synthesis of this polypeptide is readily detectable during the earliest stages (0–3 h) of wheat embryo germination, by labelling with [35S]methionine. Thereafter, synthesis of the Empolypeptide declines rapidly.

A DNA sequence encoding a portion of this polypeptide has been isolated by the molecular cloning of DNA complementary to the messenger RNA from dry wheat embryos. The identity of this cloned sequence has been confirmed by hybrid‐selected translation of the Emmessenger, the product being analysed by one‐ and two‐dimensional polyacrylamide gel electrophoresis, and by analysis of its partial proteolytic digestion products.

Gel blot hybridisation of RNA isolated from embryos at successive stages of germination with this cloned DNA sequence confirms that the decline in the synthesis of the Empolypeptide, detected by labelling in vivo, is a consequence of the degradation of its corresponding messenger RNA.
Yeast tRNAAsp: codon and wobble codon‐anticodon interactions
Authors: GRONENBORN Angela M. , CLORE G. Marius , McLAUGHLIN Larry W. , GRAESER Erika , LORBER Bernard , GIEGÉ Richarge
Published: March 2005

The conformations of the ribotrinucleoside bisphosphates GpApC and GpApU, the codon and wobble codon for aspartic acid respectively, bound to yeast tRNAAsp in solution, have been examined by means of time‐dependent transferred nuclear Overhauser enhancement measurements to determine distances between bound ligand protons. The conformations of the two bound ribotrinucleoside bisphosphates are shown to be very similar with an overall root‐mean‐square difference in interproton distances of 0.03 nm. The ribose conformations of all the residues are 3′‐endo; the glycosidic bond torsion angles of the A and C residues of GpApC and of the A and U residues of GpApU are in the low anti range. These features are typical of an A‐RNA type structure. In contrast, the G residue of both GpApC and GpApU exists as a mixture of syn and anti conformations. The overall conformation of the two bound ribotrinucleoside bisphosphates is also similar to A‐RNA and the stability of the complexes is enhanced by extensive base‐base stacking interactions. In addition, it is shown that the binding of the codon GpApC to tRNAAsp induces self‐association into a multicomplex system consisting of four GpApC‐tRNAAsp complexes, whereas the wobble codon GpApU fails to induce any observable self‐association.
Identification using 1H NMR spectroscopy of slowly exchanging amide hydrogens of hen lysozyme in solution
Authors: DELEPIERRE Muriel , DOBSON Christopher M. , HOWARTH Martin A. , POULSEN Flemming M.
Published: March 2005

Resonances of over 20 of the most slowly exchanging amide hydrogens have been identified and assigned in the 1H NMR spectrum of hen lysozyme. This was achieved by combining information about spin‐spin coupling patterns with nuclear Overhauser enhancement measurements. A computer‐based search program was used to permit the assumptions and constraints in this procedure to be closely defined and to reveal possible ambiguities. In addition, experimental values of coupling constants were compared with values calculated on the basis of the torsion angles found in the crystal structure. The close correlation between these gave further confidence in the assignment procedures and provided information concerning the nature of fluctuations about the average solution structure. The very slowly exchanging amide hydrogens are largely buried in α‐helical and β‐sheet regions of the protein structure.
Conformational changes in actin resulting from Ca2+/Mg2+ exchange as detected by proton NMR spectroscopy
Authors: BARDEN Julian A. , dos REMEDIOS Cristobal G.
Published: March 2005

Skeletal muscle actin can be maintained in a monomeric form in very low ionic strength solutions as well as in high concentrations (0.6 M) of MgCl2 or CaCl2, 400‐MHz 1H‐NMR spectra revealed characteristic changes which show that the conformation of actin alters by exchanging Ca2+ for Mg2+ in the single high‐affinity cation binding site. When all low‐affinity cation binding sites are filled (in the presence of high concentration of Ca2+ or Mg2+), the spectra show that actin conformation differs from that in low‐ionic‐strength buffer. A comparison of actin in 0.6 M CaCl2 and 0.6 M MgCl2 revealed that the environment of only a small number of protons is affected by the exchange. A new proposal for the essential steps involved in actin polymerization is presented.
N‐Bromoacetyl‐amino‐cyanopindolol: a highly potent beta‐adrenergic affinity label blocks irreversibly a non‐protein component tightly associated with the receptor
Authors: CHOREV Michael , FEIGENBAUM Allan , KEENAN Alan K. , GILON Chaim , LEVITZKI Alexander
Published: March 2005

A new chemical affinity label for the β‐adrenergic receptor, based on the structure of pindolol, has been synthesized and iodinated with 125I. The compound, N‐bromoacetylamino‐cyanopindolol (BAM‐CYP), has an apparent dissociation constant of 44 ± 7 pM towards the turkey erythrocyte membranes. This compound blocks irreversibly both the ability of β‐adrenergic receptors to bind 125I‐cyanopindolol and the ability of β‐receptors to activate adenylate cyclase in the presence of β‐agonists. Furthermore, the irreversible binding of BAM‐CYP to half of the β‐receptor sites abolishes the ligand binding activity of all the sites. These findings suggest that the β‐receptor is oligomeric in its native state. Although 125I‐BAM‐CYP blocks irreversibly and specifically the β‐adrenergic receptor, it does so by labeling a non‐protein component, most probably a water‐soluble lipid. The labeling is stereospecific since it is prevented by l‐propranolol and not by d‐propranolol. It is suggested that this lipid is tightly associated with the receptor in close proximity to the binding site. It is also suggested that this water‐soluble lipid fraction may prove crucial for the optimal interaction between the β‐adrenergic receptor and the components of adenylate cyclase.
Effects of the ribosomal subunit association on the chemical modification of the 16S and 23S RNAs from Escherichia coli
Authors: MEIER Norbert , WAGNER Rolf
Published: March 2005

70S ribosomes and 30S and 50S ribosomal subunits from Escherichia coli were modified under non‐denaturing conditions with the chemical reagent dimethylsulfate. The ribosomal 23S and 16S RNAs were isolated after the reaction and the last 200 nucleotides from the 3′ ends were analyzed for differences in the chemical modification. A number of accessibility changes could be detected for 23S and 16S RNA when 70S ribosomes as opposed to the isolated subunits were modified.

In addition to a number of sites which were protected from modification several guanosines showed enhanced reactivities, indicating conformational changes in the ribosomal RNA structures when 30S and 50S subunits associate to a 70S particle. Most of the accessibility changes can be localized in double‐helical regions within the secondary structures of the two RNAs. The results confirm the importance of the ribosomal RNAs for ribosomal functions and help to define the RNA domains which constitute the subunit interface of E. coli ribosomes.
Sheep keratins: characterization of cDNA clones for the glycine + tyrosine‐rich wool proteins using a synthetic probe
Authors: KUCZEK Elizabeth , ROGERS George E.
Published: March 2005

cDNA clones coding for the high‐glycine + tyrosine (HGT) proteins of sheep wool keratin have been isolated and sequenced. Clones were identified using a 25‐base synthetic oligonucleotide probe, deduced from the amino acid sequence of a HGT protein present at about 0.4% in the wool fibre. Southern and Northern blot analysis suggest that the gene is present as a single copy in the genome and is transcribed as an mRNA species, 600 bases in size.
Purification and characterization of the indole‐3‐glycerolphosphate synthase/anthranilate synthase complex of Saccharomyces cerevisiae
Authors: PRANTL Franziska , STRASSER Alexander , AEBI Markus , FURTER Rolf , NIEDERBERGER Peter , KIRSCHNER Kasper , HUETTER Ralf
Published: March 2005

The indole‐3‐glycerolphosphate synthase/anthranilate synthase complex from Saccharomyces cerevisiae was purified to apparent homogeneity. The native complex with Mr∼ 130000 consists of two different subunits, the TRP2 gene product with Mr= 64000 and the TRP3 gene product with Mr= 58000. The larger polypeptide was identified as anthranilate synthase and is active in vitro with ammonia as cosubstrate without need of complex formation. The smaller polypeptide carries both glutamine amidotransferase activity and indole‐3‐glycerolphosphate synthase activity. Various steady‐state kinetic parameters as well as the amino acid composition of the two polypeptides were determined.
Dual function of calmodulin (δ) in phosphorylase kinase
Authors: HESSOVÁ Zuzana , VARSÁNYI Magdolna , HEILMEYER Ludwig M. G.
Published: March 2005

The Ca2+‐independent activity of fast skeletal muscle phosphorylase kinase, A0, can be reversibly stimulated by heparin more than 20‐fold; concomitantly the Ca2+‐dependent A2 activity is abolished completely. Heparin also drastically changes the aggregation state of the enzyme; aggregated species contain significantly less δ and show an about fivefold higher A0 activity than the tetrameric form containing δ stoichiometrically. We interpret this to mean that δ has two functions in the phosphorylase kinase: an inhibitory one with respect to A0 and an activating one with respect to A2. The inhibition of A0 by Ca2+‐free δ is released, i.e. A0 increases when this subunit dissociates from the holoenzyme.

The maximally heparin‐stimulated A0 activity, A0,hep, is enriched from a crude extract to the same degree and approximately with the same yield as the major activity, A2. The phosphorylase kinase is not eluted from DEAE‐cellulose as a symmetrical bell‐shaped protein peak. The peak fraction contains the activities A2 and A0,hep, superimposed and yields a nearly homogeneous sedimentation boundary with an s20,w value of 25.5 S. The A0 yields a much broader eluation profile showing a distinct maximum from the A2 activity which contains slower sedimenting species of 12.1 S, some tetrameric enzyme of 22.7 S and higher aggregated material. Over the whole profile the activity ratio A2/A0 decreases about sevenfold whereas the ratio A2/A0,hep is constant on average. This shows that A0 is an intrinsic activity of phosphorylase kinase.

The heparin‐activated A0 activity or A0 itself in the presence of the phosphorylase phosphatase inhibitor, fluoride, can trigger a Ca2+‐independent flash activation of phosphorylase in a protein‐glycogen complex. Thus, A0 could be responsible for the conversion of phosphorylase b to a at 20 nM free Ca2+ in resting, hormone‐stimulated, muscle.
Effect of the filamentous structure of myosin on the actomyosin ATPase activity
Authors: BECHET Jean‐Jacques , d'ALBIS Anne
Published: March 2005

The ATPase activities of acto‐heavy meromyosin and of acto‐myosin minifilaments have been compared under the same conditions at low ATP (0.1 mM) and at several KCl concentrations. The activities, which are strongly salt‐dependent in both systems, have been found to be similar at high ionic strength (about 0.16 M) but different at lower ionic strength (0.06–0.07 M). Under this last condition, the catalytic constants kcat and Km are lower for acto‐myosin minifilaments than for acto‐heavy meromyosin ATPase. In addition, at low ionic strength, any decrease in the concentration of any of the ionic species (ATP, citrate, etc.) induces an increase in the interaction strength between myosin and actin filaments, as revealed by the Km changes. The presence of the troponin‐tropomyosin complex and of Ca2+ also enhances the strength of this interaction. On the other hand, the occurrence of particular interactions between F‐actin and myosin minifilaments is further substantiated by the phenomenon of superprecipitation which occurs when the ATP concentration decreases. The favourable effect of the organized structure of the myosin minifilaments on the ATPase activity of actomyosin is discussed.
Fusion of erythrocyte ghosts induced by calcium phosphate
Authors: HOEKSTRA Dick , WILSCHUT Jan , SCHERPHOF Gerrit
Published: March 2005

Using an assay which allows continouous monitoring of the mixing of aqueous contents during membrane fusion, we have investigated the kinetics of calcium‐phosphate‐induced fusion of erythrocyte ghosts. In the presence of 10 mM phosphate, the threshold concentration for Ca2+‐induced fusion was 1.25 mM, while the optimal concentration was approx. 1.75 mM Ca2+. Further enhancement of the cation concentration (≥ 2 mM) inhibited fusion of the ghosts. Initiation of fusion required the addition of phosphate prior to the addition of Ca2+, indicating that the combined interaction of Ca2+ and phosphate in or at the plane of the bilayer was a prerequisite for the induction of fusion. Furthermore, fusion was greatly facilitated upon transformation of calcium phosphate in the bulk medium from an amorphous to a solid, crystalline phase. It is suggested that membrane aggregation, and hence fusion is facilitated by the formation of crystalline calcium phosphate nucleating on the ghost membrane. La3+, Mg2+ and Mn2+ did not trigger the fusion process, although aggregation of the ghosts did occur. Under conditions where calcium phosphate precipitation was inhibited, lanthanum phosphate precipitates facilitated fusion after prior treatment of ghosts with phosphate and Ca2+. These results indicated that fusion‐prone conditions were induced prior to calcium phosphate precipitation. It is proposed that prior to calcium phosphate precipitation membrane changes are induced by separate interaction of Ca2+ and phosphate with the ghost membrane. Such an interaction could then render the ghosts susceptible to fusion and as soon as conditions are provided allowing close contact between adjacent membranes, fusion will be observed.