bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
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.
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.
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.