bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the Larval Serum Proteins of Drosophila melanogaster
Authors: BROCK Hugh W. , ROBERTS David B.
Published: July 2010

Immunological data, amino acid composition, and coordinate control during development suggest that the α, β and γ subunits of the major protein of Drosophila larval serum (LSP‐1) are coded for by genes which evolved by replications of an ancestral gene followed by mutation. In order to test this hypothesis, and to study the relationship of these genes with that coding for the second major larval serum protein (LSP‐2), we compared the one and two‐dimensional peptide maps of all four larval serum protein subunits. One‐dimensional maps generated by three different proteases showed many similarities among these proteins. Two‐dimensional peptide mapping of the methionine‐containing tryptic peptides showed that half of these peptides are common to all four larval serum protein subunits, and that about two‐thirds are common to the three LSP‐1 subunits. These observations show that the LSP‐1 subunits are more closely related to each other than any is to LSP‐2, and supported the initial suggestion that the proteins are homologous. Because the genes for the LSP‐1 subunits are each located on a different chromosome, the LSP‐1 subunits are a suitable system for investigating the evolution and dispersal of related genes, and trans control in eukaryotes.
The Catalytic Mechanism of Glutamyl‐tRNA Synthetase of Escherichia coli
Authors: KERN Daniel , LAPOINTE Jacques
Published: July 2010

The reaction pathway of tRNAGlu charging by glutamyl‐tRNA synthetase of Escherichia coli has been investigated. Comparisons of the absolute rates and of the pH dependence of the aminoacylation of tRNAGlu, the [32P]PPi– ATP isotope exchange, and the cleavage of [γ‐32P]ATP catalyzed by this synthetase show that the overall tRNAGlu charging involves at least two distinct steps, whose rate constants are very different at acidic pH but become similar at alkaline pH. Further comparisons of the rates of the AMP and PPi–dependent deacylation of Glu‐tRNAGlu and of [32P]‐PPi–ATP exchange, and the comparison of the efficiency of tRNAGlu with that of Glu‐tRNAGlu plus AMP for the [32P]PPi– ATP exchange show that the reversal of tRNAGlu charging involves at all pH values a slow step, either preceding the fast catalysis of the isotope exchange or succeeding it. Finally the study of the effects of various substrates and end‐products (PPi, tRNAGlu, AMP and hydroxylamine) on some of these reactions shows that the glutamylation process involves the participation of an intermediate complex in which glutamate is associated via a high‐energy bond, and for which PPi and tRNAGlu compete: PPi reacts faster with this intermediate at acidic, and tRNAGlu at alkaline pH. This intermediate can transfer the glutamate to hydroxylamine. These results can be the most easily interpreted in the context of a stepwise pathway of tRNAGlu charging, although some of them are compatible with a concerted pathway involving slow conformational changes.

Kinetic studies of consumption of [γ‐32P]ATP and of [3H]Glu‐tRNAGlu synthesis at the early time of the reaction show that, at acidic pH, ATP is cleaved in a stoichiometric amount to the synthetase present without concomitant synthesis of Glu‐tRNAGlu. The rate of ATP consumption during the first catalytic cycle of the enzyme largely exceeds that at the steady state, where it equals that of tRNAGlu charging during and after the first catalytic cycle. At alkaline pH there is no significant difference in the rates of ATP cleavage and tRNAGlu charging during the first turnover of the enzyme and at the steady state. In addition, no end‐product dissociation is rate‐determining for the overall tRNAGlu charging neither at acidic nor at alkaline pH. These results indicate that the aminoacylation of tRNAGlu takes place between the cleavage of ATP and the dissociation of the end‐products, and that this step is rate‐determining for the overall tRNAGlu charging at acidic pH, whereas at alkaline pH both steps, the cleavage of ATP and the synthesis of Glu‐tRNAGlu, occur at similar rates.
Flavocytochrome b2 from Baker's Yeast
Authors: POMPON Denis
Published: July 2010

It has been shown recently that the use of l‐(+)‐[2−2H]lactate as substrate instead of unlabeled l‐(+)‐lactate induces a lowering of the flavin reduction rate of cytochrome b2 by a factor of 8 [D. Pompon, M. Iwatsubo, and F. Lederer (1980) Eur. J. Biochem. 104, 479–488]. This high isotope effect enabled us to study electron transfer between prosthetic groups at a very low rate of electron entry. The kinetic scheme for electron transfer in cytochrome b2 proposed by Capeillère‐Blandin [Eur. J. Biochem. 56, 91–101 (1975)] is examined here in the light of the kinetic data reported in our preceding paper. This study indicates some disagreements, particularly at a low rate of electron entry. New kinetic schemes capable of explaining data obtained with deuterated lactate are proposed. These new schemes differ from that of Capeillère‐Blandin in that: (a) the hypothesis of simultaneous prosthetic group reduction for the two protomers of one given dimer is abandoned; (b) the limiting step of the slow phases of heme and flavin reduction is a slow interprotomer electron exchange between a heme pair, a flavin pair or heme and flavin; (c) a rather fast conformational change controlled by the redox state of heme or flavin of one protomer can modulate the rate of electron transfer in another protomer.

These new kinetic schemes allow us to determine the rate of intraprotomer and interprotomer electron transfer and to decide precisely how these steps are modified by the proteolytic cleavage of ‘intact’ enzyme to ‘cleaved’ enzyme.
Non‐Hormonal Activation of Glycogenolysis in Perfused Rat Livers
Authors: JAKOB Andrea , ALTHAUS‐SALZMANN Marianne , DIEM Susanna
Published: July 2010

Non‐hormonal activation of glycogenolysis was investigated in isolated rat livers perfused in a non‐recirculating system. Equilibration of a CO2 gradient across the cell membrane and 2,4‐dinitrophenol were used to activate glycogen breakdown.

K+, Ca2+, pH and oxygen in the effluent from the liver were continuously measured with electrodes. Perfusate glucose, lactate and pyruvate as well as the tissue adenine nucleotides and phosphorylase activities were measured to monitor the control of glycogenolysis and energy metabolism.

In livers perfused with an iso‐pH buffer system, equilibration of a CO2 gradient produced an efflux of H+ and K+ from the liver, whereas Ca2+ was taken up. Respiration and glycogenolysis were transiently enhanced. The glycogenolytic effect was diminished if Ca2+ in the perfusate was replaced with 2 mM EGTA and suppressed if a K+ medium was used for perfusion instead of the regular perfusion fluid containing Na+. No Ca2+ was taken up under these conditions.

2,4‐Dinitrophenol enhanced the release of glucose, lactate and pyruvate in the presence as well as in the absence of amytal and carboxyatractyloside. An activation of phosphorylase was correlated with a decrease of the tissue ATP content and an increase of ADP and AMP, 2 mM EGTA and K+ medium did not abolish the glycogenolytic effect of 2,4‐dinitrophenol. The phenylephrine‐induced redistributions of Ca2+ and K+ were not diminished by preperfusion with dinitrophenol and the metabolic effects of uncoupler and α‐adrenergic agonist were additive.

Our results support the view that in addition to Ca2+ redistributions, K+ movements across the plasma membrane and/or mechanisms linked to the cellular energy metabolism play a role in the control of hepatic glycogenolysis.
Purification and Properties of Assimilatory Nitrate Reductase [NAD(P)H] from Ankistrodesmus braunii
Authors: DE LA ROSA Miguel A. , DIEZ Jesús , VEGA José María , LOSADA Manuel
Published: July 2010

Assimilatory nitrate reductase [NAD(P)H] (EC 1.6.6.2) from Ankistrodesmus braunii has been purified to homogeneity by a simple procedure that utilizes as the main step affinity chromatography on Blue‐Sepharose. The best enzyme preparation has a specific activity of 61.25 units/mg protein. The enzyme has a sedimentation coefficient of 10.9 S by sucrose‐density‐gradient centrifugation, and a Stokes radius of 9.8 nm was estimated by gel filtration techniques. Its molecular weight is 460000, but only one single band of 58000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of eight subunits.

The nitrate reductase absorption spectrum shows wavelength maxima at 280 and 416 nm and a broad shoulder at 450 nm. Reduced enzyme shows maxima at 424 (Soret), 527 (β) and 557 (α) nm, and a bleaching at 450 nm. The reduced extracted heme chromophore, in pyridine and KOH, shows absorption bands at 414, 522 and 552 nm. These properties indicate the presence of a b‐type cytochrome and flavin as prosthetic groups of A. braunii nitrate reductase. A minimum of four molecules of heme has been calculated per molecule of the enzyme complex.

Redox titration of the enzyme shows a midpoint potential for the heme of –73 mV at pH 7.0.

In the presence of p‐hydroxymercuribenzoate, which inhibits the NAD(P)H‐dependent activities of the complex, the enzyme‐bound heme can be reduced with dithionite, but not with NAD(P)H.
Chicken‐Gizzard Actin
Authors: PRÓCHNIEWICZ Ewa , STRZELECKA‐GOLASZEWSKA Hanna
Published: July 2010

Interaction of actin from chicken gizzard and from rabbit skeletal muscle with rabbit skeletal muscle myosin was compared by measuring the rate of superprecipitation, the activation of the Mg‐ATPase and inhibition of K‐ATPase activity of myosin and heavy meromyosin, and determination of binding of heavy meromyosin in the absence of ATP. Both the rate of superprecipitation of the hybrid actomyosin and the activation of myosin ATPase by gizzard actin are lower than those obtained with skeletal muscle actin. The activation of myosin Mg‐ATPase by the two actin species also shows different dependence on substrate concentration: with gizzard actin the substrate inhibition starts at lower ATP concentration. The double‐reciprocal plots of the Mg‐ATPase activity of heavy meromyosin versus actin concentration yield the same value of the extrapolated ATPase activity at infinite actin concentration (V) for the two actins and nearly double the actin concentration needed to produce half‐maximal activation (Kapp) in the case of gizzard actin. A corresponding difference in the abilities of the two actin species to inhibit the K‐ATPase activity of heavy meromyosin in the absence of divalent cations was also observed.

The results are discussed in terms of the effect of substitutions in the amino acid sequence of gizzard and skeletal muscle actins on their interaction with myosin.
Identification of a Class of Lysines within the Non‐specific DNA‐Binding Site of RNA Polymerase Core Enzyme from Escherichia coli
Authors: MAKOFF Andrew J. , MALCOLM Alan D. B.
Published: July 2010

The imido ester, methyl acetimidate, which specifically amidinates lysine residues, modifies RNA polymerase core enzyme, leading to rapid loss of activity. Calf thymus DNA partially protects the enzyme against this inactivation, an effect which disappears at high salt concentration. DNA protects 17 ± 6 lysines from amidination at low salt concentration.

The dependence of amidination on methyl acetimidate concentration is examined in the presence of DNA at high and low salt concentration. Analysis of the data suggests a class of approximately 12 lysines which are protected by DNA, consistent with the above estimate. These lysines are approximately 5–10‐fold more reactive than most other available lysine residues.
Stereochemistry of the Hydrolysis of Trehalose by the Enzyme Trehalase Prepared from the Flesh Fly Sarcophaga barbata
Authors: CLIFFORD Kenneth H.
Published: July 2010

The enzyme trehalase was prepared from whole flesh flies, Sarcophaga barbata.

The rate of mutorotation of glucose released by the enzyme was compared with that of α‐d‐glucose, β‐d‐glucose and an equimolar mixture of the two.

Derivatives of the glucose released by the enzyme were prepared and analysed by gas chromatography for anomeric composition.

The enzyme was incubated with trehalose in a medium enriched with H218O and the glucose was analysed by gas chromatography/mass spectrometry.

It was found that an equimolar mixture of α‐d‐glucose and β‐d‐glucose is formed on hydrolysis of trehalose by trehalase and 18O is incorporated into β‐d‐glucose.

These results indicate that there is an inversion of configuration at C‐1 of trehalose on hydrolysis and that the hydrolysis proceeds by a bimolecular nucleophilic substitution mechanism.
Properties and Structural Role of the Subunits of Human Spectrin
Authors: CALVERT Rosalcen , BENNETT Pauline , GRATZER Walter
Published: July 2010

The subunits of spectrin from human erythrocytes were separated by ion‐exchange chromatography on hydroxyapatite in the presence of urea. When renatured from the urea solution they are found to be monomeric, although the smaller subunit (band 2) is prone to aggregation. In shape, solubility and secondary structure the subunits resemble the native spectrin dimer, indicating that subunit interaction is not essential for maintaining the native conformation. When the subunits are recombined, a dimer with the sedimentation coefficient of the native species is formed. This constitutes direct evidence that native spectrin is a heterodimer, rather than a mixture containing homologous and heterologous species. The interaction of the separated subunits with the chymotryptic fragment of the spectrin‐binding protein (protein 2.1, or ankyrin) of the erythrocyte membrane was studied. Only the smaller subunit has the ability to bind, and thus presumably contains the site by which the cytoskeleton is attached to the plasma membrane. On the other hand, the formation of a complex with F‐actin and protein 4.1 requires the presence of both subunits. A complex of these proteins with band 2 is formed, however, when traces of an additional, as yet unidentified, protein are present.
Purification and Characterization of an Aminopeptidase A from Hog Intestinal Brush‐Border Membrane
Authors: BENAJIBA Abdelouahab , MAROUX Suzanne
Published: July 2010

The aminopeptidase A of the porcine intestinal brush‐border membrane has been purified following solubilization by trypsin (p‐form) or Emulphogen (d‐form). Full purification of d‐amino‐peptidase A required the use of anti‐impurities immunoabsorbant chromatography. The d‐amino‐peptidase A constitutes about 4% of the total proteins of the membrane, compared to 8–12% for another, already characterized, brush‐border aminopeptidase N.

Both d‐form and p‐form of aminopeptidase A have been clearly shown to be dimeric. Experimental evidence is presented favoring the view that they are symmetrical dimers, with the consequence that each of the two subunits of the d‐form possesses an hydrophobic anchor holding them at the membrane surface. As already demonstrated for several other brush border hydrolases, the hydro‐phobic anchor is N‐terminal in porcine intestinal aminopeptidase A.

The molecular weight of the peptide including the anchor liberated by trypsin during the conversion of the d‐form into the p‐form has been estimated by an isotopic dilution method to be about 4500 (42 residues). This value which compares well with those recently obtained in the case of rabbit aminopeptidase N (3700–3800; 36–38 residues), indicates that the anchor is much shorter than believed earlier.

A preliminary survey of the specificity of both aminopeptidases A and N towards four synthetic amino acid p‐nitroanilides confirms that aminopeptidase A mostly cleaves acidic residues. Its activity towards neutral residues is much lower, but probably significant in certain cases.
Characterization of the Metallothioneins Induced in HeLa Cells by Dexamethasone and Zinc
Authors: KARIN Michael , HERSCHMAN Harvey R.
Published: July 2010

The metallothioneins induced in HeLa cells by dexamethasone and Zn2+ were labeled with [3H]cysteine and [35S]cysteine respectively. These labeled metallothioneins were compared to one another and to metallothioneins from human liver by (a) gel permeation chromatography on Sephadex G‐75 columns, (b) ion‐exchange chromatography on DEAE‐Sephadex A‐25 columns, (c) electrophoresis on non‐denaturing polyacrylamide gels, and (d) electrophoresis of reduced and alkylated proteins on dodecylsulfate/polyacrylamide gels. The dexamethasone‐induced metallothioneins of HeLa cells, the Zn2+‐induced metallothioneins of HeLa cells, and the metallothioneins of human liver were indistinguishable by these four criteria.
Transfer RNA Labeling of Escherichia coli Methionyl‐tRNA Transformylase
Authors: HOUNTONDJI Codjo , FAYAT Guy , BLANQUET Sylvain
Published: July 2010

Homogeneous methionyl‐tRNA transformylase from Escherichia coli can react with periodate‐treated tRNAMetf to form a Schiff s base through a free amino group (probably the ɛ‐amino group of a lysine) and the 2′,3′‐aldehyde groups created at the 3′‐terminal ribose of tRNA. This reaction is reflected by the loss of activity of the enzyme. At saturating tRNA dialdehyde and upon reduction of the Schiff's base by NaBH4, the kinetics of inactivation (37°C, pH 8.5) level off at 50% of the initial enzymic activity. In the presence of 2 mM cyanohydridoborate, a mild reducing agent which leaves intact the reacting aldehyde groups of oxidized tRNA but continuously reduces the Schiff's base in equilibrium, the activity of the enzyme can be destroyed by 100%, at a rate of 0.044min−1, with the parallel covalent incorporation of close to one tRNA molecule per enzyme molecule. Selectivity of the labeling is also supported by the demonstration that, prior to Schiff's base formation, modified tRNA binds the transformylase with equilibrium constant and stoichiometry in good agreement with those for the active binding of unmodified tRNA. Moreover intact tRNA competes for the inactivation by the dialdehyde derivative with an affinity constant identical to that for its active binding to the enzyme.
Hygromycin A, a Novel Inhibitor of Ribosomal Peptidyltransferase
Authors: GUERRERO Maria del Carmen , MODOLELL Juan
Published: July 2010

In cell‐free systems from Escherichia coli, hygromycin A inhibits polypeptide synthesis directed by either poly(U) or phage R17 RNA, and the reaction of puromycin with either natural peptidyl‐tRNA, or AcPhe‐tRNA, or the 3′‐terminal fragment of AcLeu‐tRNA (C‐A‐C‐C‐A‐LeuAc). In contrast, the antibiotic does not inhibit the enzymatic binding of Phe‐tRNA to ribosomes or the translocation of AcPhe‐tRNA. It is concluded that hygromycin A is a specific inhibitor of the peptide bond formation step of protein synthesis.

The action of hygromycin A on peptidyl transfer is similar to that of chloramphenicol, an antibiotic that shares some common structural features with hygromycin A. Both antibiotics inhibit the binding of C‐A‐C‐C‐A‐Leu to the acceptor site of peptidyl transferase and stimulate that of C‐A‐C‐C‐A‐LeuAc to the donor site of the enzyme. Moreover, hygromycin A blocks the binding of chloramphenicol to ribosomes, indicating that the binding sites of the antibiotics may be closely related. Hygromycin A is a more potent agent than chloramphenicol and binds quite strongly to ribosomes.
The Incorporation of Acetylcholinesterase from the Electric Organ into Liposomes
Authors: OCHOA Enrique L. M.
Published: July 2010

Several methods have been used to bind electric organ acetylcholinesterase to the walls of phosphatidylcholine liposomes. One of these methods [Brunner, J., Skrabal, P. & Hauser, H. (1976)Biochim. Biophys. Acta, 455, 322–331] achieved complete incorporation, although some enzyme was shown to be sequestered inside the vesicles.

The association was established either in the presence of high salt media or altered liposomal membrane fluidity.

The reconstitution process impaired the allosteric transition of acetylcholinesterase which is thought to occur when Flaxedil (gallamine triethiodide) is present in a low ionic strength medium.

It is suggested that the cholinesterase is capable of being incorporated into liposomes, possibly via hydrophobic forces.

Such a system may be an adequate one for further study of the functioning of the enzyme in a defined membrane environment.
The Indirect Mechanism of Action of the Trifluoroacetyl Peptides on Elastase
Authors: DIMICOLI Jean‐Luc , RENAUD Alain , BIETH Joseph
Published: July 2010

Trifluoroacetyl (CF3CO) dipeptide anilides are potent reversible inhibitors of elastase. Their 19F NMR spectra in the presence of the enzyme correspond therefore to slow chemical exchange. Their characteristics are very similar to those previously reported for other CF3CO‐peptides. This should correspond to a single binding mode, in which the CF3CO group lies in a specific site in close contact with protein protons.

Elastase, irreversibly inhibited by alkylation of Ser‐195 with phenylsulfonyl fluoride derivatives, binds the CF3CO‐Ala‐containing dipeptide anilides still more tightly than the native enzyme. It no longer binds the dipeptide anilides containing a bulky CF3CO‐Lys group, suggesting a location of the CF3CO site near the S1 subsite. On the other hand, the chemical shift of the CF3CO 19F resonance in the complex is the only NMR property affected by the enzyme sulfonylation, the T1 and nuclear Overhauser effect values being unmodified as compared to those in the complexes with the native enzyme. The observation of the NMR characteristics of elastases inhibited by phenylsulfonyl groups substituted with fluorine shows that complexation with CF3CO‐peptides induces a change of conformation of the catalytic site which should correspond to a displacement of His‐57 toward the ortho position of the phenylsulfonyl ring. Such a transconformation is not observed with corresponding acetylated peptides.

The phenylsulfonyl fluoride derivatives are still able to react with elastase in the presence of a large excess of CF3CO‐peptides. In such conditions the rate of inactivation is much slower but still at least 5% of that measured in the absence of the reversible inhibitors. This residual activity is hardly compatible with the presence of two exclusive modes of interaction of the CF3CO‐peptides with native elastase. On the contrary, these observations are better interpreted by a single and identical mode of binding of the peptide to the native and sulfonylated enzymes. According to this mode of binding, the reversible inhibition of elastase by CF3CO‐peptides should correspond to an indirect mechanism by which a change of conformation at the active site results in a reduced catalytic activity.
Metal Ion/Buffer Interactions
Authors: SCHELLER Kurt H. , ABEL Thomas H. J. , POLANYI Peter E. , WENK Peter K. , FISCHER Beda E. , SIGEL Helmut
Published: July 2010

The acidity constant of protonated 2‐[bis(2‐hydroxyethyl)amino]‐2(hydroxymethyl)‐1,3‐propanediol (Bistris) has been measured. The influence of hydroxo groups on the basicity of Bistris and related bases is discussed. The interaction of Bistris with the metal ions (M2+) Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I= 1.0 M, KNO3; 25°C) and the stability constants of the M(Bistris)2+ complexes were determined. Unexpectedly Ca(Bistris)2+ is the most stable among the alkaline earth ion complexes (log Kcaca(Bistris)= 2.25; the corresponding values for the Mg2+, Sr2+ and Ba2+ complexes are 0.34, 1.44 and 0.85, respectively). The ions of the 3d series follow the Irving‐Williams sequence: log KMnMn(Bistris)= 0.70, for Cu2+ 5.27 and Zn2+ 2.38. Ternary complexes containing ATP4‐ as a second ligand were also investigated: the values for Δlog KM (= log KM(ATP)M(ATP)(Bistris)–log KMM(Bistris)) are in general negative (e.g. Δlog KCa= ‐0.40 or Δllog KCu= ‐ 1.65), thus indicating that the interaction of Bistris with M(ATP)2‐ is somewhat less pronounced than with M2+. However, even in mixed‐ligand systems, complex formation may still be considerable, hence great reservations should be exercised in employing Bistris as a buffer in systems containing metal ions. Moreover, in several cases Δlog Km is relatively high [for Mg2+‐ATP4‐ Bistris even positive], indicating some cooperativity between the coordinated ligands, possibly hydrogen‐bond formation. Distributions of the complexes in dependence on pH are given, and the structures of the binary M (Bistris)2+ and the ternary M(ATP) (Bistris)2‐ complexes are discussed. The participation of Bistris hydroxo groups in complex formation is evident.
Purification and Properties of Hamamelosekinase
Authors: BECK Erwin , WIECZOREK Jürgen , REINECKE Waltraud
Published: July 2010

Hamamelosekinase (ATP: hamamelose 21‐phosphotransferase) was purified from a crude extract of Kluyvera citrophila 627 (Enterobacteriaceae) which has been grown on d‐hamamelose. Ammonium‐sulfate fractionation and twofold chromatography on DEAE‐cellulose resulted in a 51‐fold purification of the enzyme. Neither glucosekinase nor significant ATPase activity could be detected in the pure preparation. Besides d‐hamamelose only d‐hamamelitol was utilized as a substrate; however, the latter was phosphorylated at a very low rate. The molecular weight of the enzyme as estimated by gel chromatography is 21000. The Km values for hamamelose and ATP were 3 mM and 2.5 mM, respectively. The pH optimum was found at 7.5. In contrast to hexokinase, purified hamamelosekinase is very labile and could only be stabilized by addition of its substrate d‐hamamelose. The most unusual property with respect to yeast hexokinase is a pronounced substrate inhibition by hamamelose (> 5 mM) and ATP (> 7 mM), respectively, which could be interpreted as due to an economic utilization of the nutrient.

Hamamelosekinase as well as glucosekinase are inducible by growing the microorganisms on the corresponding monosaccharides.
Kinetic Studies of the Reaction of Ferric Soybean Leghemoglobins with Hydrogen Peroxide, Cyanide and Nicotinic Acid
Authors: JOB Dominique , ZEBA Boukaré , PUPPO Alain , RIGAUD Jean
Published: July 2010

A kinetic study of the reaction of two soybean leghemoglobins (components a and c) with hydrogen peroxide to form the oxidized compound (leghemoglobin IV) has been carried out over the pH range 2.5–10. Three different ionization processes of leghemoglobins with pKa values of 3, 4.7 ± 0.2 and 8.2 ± 0.1 are required to explain the rate/pH profiles. Protonation of the former group and ionization of the latter cause a decrease in the rate of reaction of the hemoproteins with H2O2. The results are compared to those obtained for the reactions of plant peroxidases and myoglobin with H2O2. The results obtained from the kinetic study of cyanide binding to soybean leghemoglobins indicate that CN− is the reactive species. Two ionization processes of leghemoglobins with pKa values of 4.7 ± 0.2 and 8.2 ± 0.1 affect the reaction rates. The association and dissociation rate constants corresponding to nicotinic acid binding to leghemoglobins a and c have been measured over the pH range 2.5–7. The dissociation rate constant is affected by ionization of a group with pKa < 2.5 for both leghemoglobin‐nicotinate complexes. In this pH range the association rate constant is only affected by ionization of a group with pKa value of 4.7 ± 0.2. The analysis of these results shows that both ionization processes corresponding to ring nitrogen atom of the ligand (pKa∼ 4.9) and to a heme‐linked group (pKa∼ 4.7 ± 0.2) influence the association rate constant. Furthermore, it appears that in the binding site of leghemoglobins the pKa value corresponding to ionization of the ring nitrogen atom of nicotinic acid is shifted from the normal value of 4.9 to a value < 2.5. This pecularity might explain the exceptional reactivity of leghemoglobins for nicotinic acid, over a large pH range. For both cyanide and nicotinic acid binding reactions, the ionizable group of leghemoglobins with pKa value of 4.7 ± 0.2 seems to act as an electrostatic gate. When this group is deprotonated, it restricts the access of anion ligands to the heme pocket. For all the three reactions studied, leghemoglobin a reacts about twice as fast as leghemoglobin c.
Analysis of Binding Interactions between Histone Core Complex and Simian Virus 40 DNA
Authors: SHEWMAKER C. Kimball , WAGNER Thomas E.
Published: July 2010

The interactions of acetylated and non‐acetylated core complex histones with simian virus 40 (SV40) DNA I have been analyzed. A modified filter‐binding assay utilizing micrococcal nuclease, which allows quantification of histone octamer binding to DNA has been developed. Using this assay it was determined that both non‐acetylated core complex histones and core complex histones acetylated with acetyl adenylate to levels existing in vivo bind cooperatively to SV40 DNA I. Although both interactions are cooperative, the magnitude of the cooperativity parameter, ω, is significantly less in the acetylated case. This difference in cooperativity is in contrast to the nearly identical intrinsic association constant, K, observed in both cases.
Chemically Induced Gene Expression
Authors: COHEN Bennett N. , BLUE William T. , WAGNER Thomas E.
Published: July 2010

We have developed a non‐enzymatic acetylating procedure, closely resembling the situation in vivo, utilizing acetyl adenylate, an acetylating agent in vivo, that mimics the enzymatic hyperacetylation of specific histone species. Analysis of the acetylated species of calf thymus histones produced from reaction with soluble chromatin yielded the same species generated in vivo and observed during active gene transcription. Four species of histone H4 and three of histone H3 occur with no alteration in histones H2A or H2B.

This procedure has been utilized to hyperacetylate simian virus 40 (SV40) minichromatin in vitro in order to study the effect of acetylated compared to non‐acetylated minichromatin in cellular transformation of cultured Balb/3T3 cells.

Transformed cell foci appeared only in the cultures infected with hyperacetylated SV40 minichromatin. To select for cellular transformation, foci were transferred to agar‐lined culture flasks and grown in a suspension of 1% methylcellulose. The selected cells were plated on slides and analyzed for the presence of T‐antigen by indirect immunofluorescence. The hyperacetylated‐minichromatin‐infected cells exhibited T‐antigen‐specific fluorescence, while non‐acetylated‐minichromatin‐treated cells and normal cells showed no specific fluorescence. These results suggest a major role for histone hyperacetylation in the mechanism of SV40 viral transformation.
Glycogen Synthase from Rabbit Skeletal Muscle
Authors: RYLATT Dennis B. , AITKEN Alastair , BILHAM Terence , CONDON Gerard D. , EMBI Noor , COHEN Philip
Published: July 2010

Glycogen synthase kinase‐3 phosphorylates three serine residues on glycogen synthase (sites 3a, 3b and 3c) which are all located in the same nine‐amino‐acid segment of the polypeptide chain. The sequence in this region is: Arg‐Tyr‐Pro‐Arg‐Pro‐Ala‐Ser(P)‐Val‐Pro‐Pro‐Ser(P)‐Pro‐Ser‐Leu‐Ser(P)‐Arg‐. These serine residues are distinct from the sites phosphorylated preferentially by cyclic‐AMP‐dependent protein kinase (sites 1a and 1b) and phosphorylase kinase (site 2).

The N‐terminal sequence of glycogen synthase containing the serine residue phosphorylated by phosphorylase kinase has been extended. The sequence in this region is: Pro‐Leu‐Ser‐Arg‐Thr‐Leu‐Ser(P)‐Val‐Ser‐Ser‐Leu‐Pro‐Gly‐Leu‐Glu‐Asp‐Trp‐Glu‐Asp‐Glu‐Phe‐Asp‐Leu‐Glu‐Asn‐Ser‐Val‐Leu‐Phe‐(Asx2,Glx2,Ala2,Val2,Lys)‐. The similarity to the N‐terminal sequence of phosphorylase is confined to the immediate vicinity of the phosphorylation site (residues 4–15).

The relationship of glycogen synthase kinase‐3 to glycogen synthase kinases that have been described by other laboratories is discussed.
Effect of N‐Acyl Substitution at Glucosamine Residues on Lysozyme‐Catalyzed Hydrolysis of Cell‐Wall Peptidoglycan and its Oligosaccharides
Authors: AMANO Ken‐ichi , ARAKI Yoshio , ITO Eiji
Published: July 2010

The effect of N‐substitution with various acyl groups at glucosamine residues on lysozyme‐catalyzed hydrolysis of cell wall peptidoglycan and its oligosaccharides was studied.

The lysozyme‐resistant cell wall peptidoglycan from Bacillus cereus, N‐unsubstituted at most of its glucosamine residues, was N‐acylated and used as substrate for lysozyme. The N‐acetylated, N‐formylated, N‐propionylated and N‐butyrylated preparations of the peptidoglycan were hydrolyzed by lysozyme with relative maximum rates of 1.00, 0.05, 0.38 and 0.10 respectively, and with apparent Km values, expressed in disaccharide unit concentration, of 0.1, 0.6, 2.4 and 10 mM respectively, whereas the N‐succinylated preparation was unsusceptible to this enzyme.

Peptidoglycan oligosaccharides, GlcN‐MurAc‐GlcNAc‐MurAc, GlcNAc‐MurAc‐GlcN‐MurAc‐GlcNAc‐MurAc and GlcN‐MurAc‐GlcN‐MurAc‐GlcNAc‐MurAc, were also N‐acylated to be examined as substrates for lysozyme. The N‐acetylated, N‐formylated, N‐propionylated and N‐butyrylated preparations from the tetrasaccharide were hydrolyzed at 0.5 mM with relative rates of 1.00, 0.04, 0.1 and 0.01 respectively, but the N‐succinylated tetrasaccharide was unaffected by lysozyme. The N‐formylated, N‐propionylated, N‐butyrylated and N‐succinylated preparations from the former hexasaccharide were hydrolyzed with relative rates of 1.5, 22, 1.6 and 0.8 respectively, as compared to the value of about 2000 for the N‐acetylated hexasaccharide. The N‐acylated preparations from the latter hexasaccharide, except the N‐succinylated one, were hydrolyzed at rates similar to those of hydrolysis of the corresponding preparations from the former hexasaccharide.

The above results seem to be accounted for by the predominant importance of the alkyl moieties of the N‐acyl substituents on the glucosamine residues located in subsite C in the enzyme‐substrate complexes.
Subcellular Distribution of Proteolytically Generated Valine in Isolated Rat Hepatocytes
Authors: SOLHEIM Anne E. , SEGLEN Per O.
Published: July 2010

A small fraction of the intracellular acid‐soluble radioactivity in [14C]valine‐labelled hepatocytes remained non‐extracted even after repeated incubations in isotope‐free medium at 37°C. This non‐extracted radioactivity was only present when the cells had been labelled under conditions allowing protein synthesis.

Approximately two‐thirds of the non‐extracted radioactivity was recovered in material with a molecular weight larger than free valine, i.e. presumably acid‐soluble peptides. The intracellular contents of these peptides were unaffected by inhibitors of hepatocytic protein degradation, and they would therefore seem to represent primary synthesis products rather than products of proteolysis.

The remaining one‐third of the radioactivity was free [14C]valine, derived from intracellular protein degradation as indicated by the reduced levels seen in the presence of inhibitors of lysosomal (ammonia, methylamine, leupeptin, chymostatin) and non‐lysosomal (chymostatin) proteolysis. The intracellular levels of twelve other amino acids were similarly reduced upon inhibition of protein degradation.

The contents of free [14C]valine in subcellular fractions were compatible with a uniform distribution of the amino acid throughout the cell. There was no evidence for any enrichment of [14C]valine in a purified lysosomal fraction, separated from mitochondria by means of isotonic metrizamide gradients.

It can be concluded that the non‐extracted valine in hepatocytes may represent a steady‐state level, maintained by intracellular protein degradation, of amino acid in transit through the cell. The non‐extractability, i.e. the maintenance of a concentration gradient towards the extracellular medium, can be calculated to be compatible with a limitation of valine efflux by the concentration‐dependent valine transport system. Since no specific subcellular compartmentation is indicated, intracellular valine can provisionally be regarded as a single, uniform pool.
A Comparison of Vesicles Derived from Terminal Cisternae and Longitudinal Tubules of Sarcoplasmic Reticulum Isolated from Rabbit Skeletal Muscle
Authors: LOUIS Charles F. , NASH‐ADLER Priscilla A. , FUDYMA Gary , SHIGEKAWA Munekazu , AKOWITZ Alfred , KATZ Arnold M.
Published: July 2010

Sarcoplasmic reticulum vesicles were separated into heavy (derived from terminal cisternae) and light (derived from longitudinal tubules) fractions, according to Meissner [Biochim. Biophys. Acta, 389, 51–68 (1975)]. The similar Ca2+ sensitivities of phosphoprotein formation, ATPase activity and calcium uptake, and the similar phosphoprotein turnover rates (ATPase/phosphoprotein formation) of both fractions indicate that the same ATPase enzyme is present in the terminal cisternae and longitudinal sarcoplasmic reticulum. The higher V for Ca2+‐activated ATPase activity and calcium uptake in the light fraction correlated with the higher concentration of ATPase enzyme per mg of membrane protein in this fraction. In both the presence and absence of calcium‐precipitating anions, the light fraction stored more calcium than the heavy. The Ca2+ dependence of calcium release after addition of EGTA appeared similat in both fractions, but the rate Of calcium release was more rapid in the light fraction.

These findings suggest that calcium release may occur more rapidly from longitudinal than terminal cisternae portions of the sarcoplasmic reticulum and that calcium release, like calcium uptake, may be mediated by the ATPase enzyme in the sarcoplasmic reticulum membrane.

Although the activation energies for Ca2+‐activated ATPase activity above and below the transition temperature were significantly different for the heavy and light fractions, their transition temperatures were similar. Partial purification of the ATPase enzyme by deoxycholate treatment modified the activation energies of the light but not the heavy fraction and caused the activation energies to become similar. The phosphoprotein levels of heavy and light vesicles did not become similar after deoxycholate treatment, although gel electrophoretograms indicated both samples contained > 90% ATPase protein. These results indicate the protein‐lipid associations in these two fractions may be different.
A Flavin‐Mononucleotide‐Binding Site in Hansenula anomala Nicked Flavocytochrome b2, Requiring the Association of Two Domains
Authors: GERVAIS Michel , LABEYRIE Françoise , RISLER Yanick , VERGNES Odile
Published: July 2010

Previous experiments in our laboratory with Saccharomyces cerevisiae flavocytochrome b2 indicated that both fragments α and β of the enzyme isolated after cleavage by yeast proteases are required to form the flavin site.

More detailed experiments have now been carried out on the nicked Hansenula anomala enzyme obtained by tryptic cleavage. A method has been devised that gives a quantitative separation in 4 M urea of β, and α with its heme still bound. The characteristics of the various species: isolated α and β and mixed α+β were studied in 4 M urea and after elimination of this reagent by dialysis in the presence of FMN and 2‐mercaptoethanol. Several methods, including heme spectroscopy, tryptophan fluorescence, sedimentation studies, and titration of bound flavin, were used. The results indicate that isolated α and β have a folded globular structure after renaturation. The flavin binding to the α+β mixture was important (50–100 %) with recovery of the flavodehydrogenase activity. In contrast, binding was not detectable (< 0.5%, Kd> 10 mM) for isolated α and β. As far as mononucleotide binding is concerned, such a cooperative requirement for two folding domains has never before been reported in other enzymes.

The present results are discussed together with others obtained in our laboratory which demonstrate that, as deduced from their sensitivity to trypsin, the structure of S. cerevisiae and H. annomala flavocytochrome b2 protomers is triglobular ‘n‐x‐β’ (n and x combined within α). The tetramer assembly, which remains intact as a nicked enzyme (αβ)4 after the first trypsin cleavage, is broken down following a second cleavage of the chain into four cytochrotne cores (n) and a functional T‐flavodelhydrogenase entity, a tetramer of the type (xβ)4.
Stepwise Cleavage of the Pro Part of Promelittin by Dipeptidylpeptidase IV
Authors: KREIL Günther , HAIML Liselotte , SUCHANEK Gerda
Published: July 2010

Melittin, the main constituent of honeybee venom, is derived from promelittin. In the amino acid sequence of the ‘pro’ region of this precursor, every second residue is either proline or alanine. The possibility has been investigated that activation of promelittin might proceed via sequential liberation of dipeptides catalyzed by a dipeptidylpeptidase IV. As substrates we used promelittin isolated from queen bees fed with radioactive proline, and enzymatic fragments of prepromelittin which contained the entire pro part and the NH2‐terminal hexapeptide of melittin.

It could first be demonstrated that pig kidney dipeptidylpeptidase IV releases dipeptides from the pro part. An enzyme of this type could then be detected in extracts from venom glands of queen bees, which also contain a dipeptidase. After inhibiting the latter enzyme with mersalyl, a stepwise cleavage of dipeptides, starting at the amino end of the pro region, could be demonstrated. This hydrolysis did not proceed into the melittin sequence. Furthermore, fragments with an extra residue at the amino end, which therefore had the wrong ‘reading frame’ for the dipeptidylpeptidase, were not hydrolyzed. With intact promelittin as substrate the rate of hydrolysis was always lower than with the fragments.

The results presented in this paper suggest a new type of precursor‐ product conversion proceeding via stepwise cleavage of dipeptide units. Our experimental evidence also ascribes a biological function to a dipeptidylpeptidase IV, it type of enzyme widely distributed in animal tissues. The evidence, that the observed reaction in vitro reflects the mechanism of promelittin activation in vivo is discussed.
Yeast Mutants Defective in Acetyl‐Coenzyme A Carboxylase and Biotin: Apocarboxylase Ligase
Authors: MISHINA Masayoshi , ROGGUENKAMP Rainer , SCHWEIZER Eckhart
Published: July 2010

Among more than 7000 mutants of Saccharomyces cerevisiae, requiring saturated fatty acids, 61 acetyl‐CoA‐carboxylase‐deficient strains have been identified. According to their mutual complementation characteristics these mutants have been assigned to two different genes, acc1 and acc2. Both acetyl‐CoA carboxylase genes tire unlinked to each other and to the fatty acid synthetase genes fas1 and fas2.

The acetyl‐CoA carboxylases of several acc1 and acc2 mutants have been purified and assayed for their overall and component enzyme activities. Besides overall acetyl‐CoA carboxylation, which was lost in all cases, both component enzymes, biotin carboxylase and transcarboxylase, were simultaneously affected in most mutants, though often to a different relative extent.

Similarly, the comparison of biochemical and genetic complementation data revealed no basis for a clear distinction between specific biotin carboxylase and transcarboxylase mutants. These results Suggest that accl is a cluster gene coding for a multifunctional protein harboring both acetyl‐CoA carboxylase component enzyme activities on the same polypeptide chain.

The acetyl‐CoA carboxylase isolated from acc2 mutants was free of biotin. Correspondingly, biotin:apoacetyl‐CoA‐carboxylase ligase activity was missing in acc2 mutants. Therefore, it is concluded that the primary defect in acc2 mutants is in the biotin:apocarboxylase ligase. In agreement with this conclusion, the acc2 acetyl‐CoA carboxylase can be activated, in the presence of biotin and ATP, by ligase preparations from wild‐type or acc1 muttant cells. By the use of these mutants, evidence was obtained that in vivo the biotinylation of both acetyl‐CoA carboxylase and pyruvate carboxylase is catalyzed by the same ligase.
Glucocorticoid‐Dependent Induction of mRNA Coding for Phosphoenolpyruvate Carboxykinase (GTP) in Rat Kidney
Authors: IYNEDJIAN Patrick B. , JACOT Michel M.
Published: July 2010

The glucocorticoids induce the synthesis of phosphorenolpyruvate carboxykinase (GTP) in rat kidney as a consequence of an increase in the level of the specific enzyme mRNA. The mRNA induction was characterized with respect to its time course after hormone administration and its sensitivity to cycloheximide. The level of rat kidney mRNA directing the synthesis of phosphoenolpyruvate carboxykinase in a wheat germ translation system nearly doubled within 2 h of a dexamethasone injection and further increased to four times the initial value at 6 h of treatment and to five times at 10 h. Cyclolieximide injected 30 min prior to dexamethasone prevented the mRNA increase. When injected 5 h after dexamethasone, the inhibitor of protein synthesis blocked the rise of phosphoenolpyruvate earboxykinase mRNA occurring normally between 5 It and 10 h after treatment with dexamethasone. Maximal inhibitions of protein synthesis on the one hand and of mRNA induction on the other were achieved at the same dose of cycloheximide, suggesting that the two effects might be related. Dexamethasone caused an increase in the functional level of several as yet‐unidentified mRNAs in addition to that coding for phosphoenolpyruvate carboxykinase. The main points emerging from this study are: (a) the virtual absence of lag between dexamethasone administration and increase in phosphoenolpyruvate carboxykinase mRNA: (b) the inhibition of phosphoenolpyruvate carboxykinase mRNA induction by cycloheximide, suggesting a possible requirement for ongoing protein synthesis; (c) the existence in the kidney of it glucocorticoidresponsive domain comprising several distinct proteins.
The Number of Functional Active Sites per Molecule of the Adenosylcobalamin‐Dependent Enzyme, Ethanolamine Ammonia‐lyase, as Determined by a Kinetic Method
Authors: HOLLAWAY Michael R. , JOHNSON Alan W. , LAPPERT Michael F. , WALLIS O. Caryl
Published: July 2010

A kinetic approach to the determination of the number of functional active sites per molecule of the adenosylcobalamin‐dependent enzyme. ethanolatnine ammonia‐lyase, is described.

Time courses for formation and breakdown of a cob(II)alamin intermediate during reaction of the enzyme, fully saturated with adensycoalaminm, with tninopropanol as substrate, were followed using a stopped‐flow spectrophotometer under two conditions: (a) enzyme concentration much greater than that of substrate, (b) substrate concentration much greater than that of enzyme.

Results were analysed in terms of a three‐step mechanism involving binding of substrate (k2+ step), cob(II)alamin formatiUn (K3+z step) and cob(II)alatnin breakdown (k3+ step). The kinetic scheme was shown to be suffitiient to account for the observed time courses and rate constants of 80 s‐1 (k+2) and 1.5 s‐1 (K+3) were determined.

The number of active sites per enzyme molecule (n) was calculated from the kinetic data in three Ways: (a) calculation from amplitude of absorbance measurements, (b) calculation from measurements of the values of rate constants and (c) analysis by computation of the kinetic data using the computer program FACSIMILE. A value for n close to 6 was calculated by each of these methods. This value is in disagreement with the literature value of about two sites‐per molecule but is consistent with the I6II6 subunit structure of the enzyme.

Kinetic analysis of data from experiments in which the adenosylcobalamin concentration was varied while substrate anti enzyme concentrations remained constant showed that all the active sites function with identical rate constants.

The principle and mathematical basis of the kinetic method for determining the value of n is given as an Appendix.