bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of extracellular α‐amylase and glucoamylase from the yeast Candida antarctica CBS 6678
Authors: DE MOT René , VERACHTERT Hubert
Published: March 2005

An α‐amylase and a glucoamylase were purified to homogeneity from the culture fluid of β‐cyclodextringrown Candida antarctica CBS 6678 by protamine sulfate treatment, ammonium sulfate precipitation, gel filtration (Sephadex G‐75 sf, Ultrogel AcA 54), DEAE‐Sephacel chromatography, hydroxyapatite chromatography and affinity chomatography on acarbose—AH‐Sepharose 4B.

Both enzymes were monomeric glycoproteins with fairly different amino acid compositions. Their apparent relative molecular mass, sedimentation coefficient (S°20, W), isoelectric point, absorption coefficient (280 nm), pH and temperature optima were estimated as 48 500, 4.7 S, 10.1, 1.74 cm2 mg−1, 4.2 and 57°C, respectively, for glucoamylase and as 50 000, 4.9 S, 10.3, 1.53 cm2 mg−1, 4.2 and 62°C, respectively, for α‐amylase.

Kinetic analyses indicated that both enzymes preferentially hydrolyzed high‐molecular‐mass substrates, including some raw starches. α‐Amylase was active on cyclodextrins, whereas debranching activity was demonstrated for glucoamylase. Trestatins were potent inhibitors of both α‐amylase (Ki < 1 μM) and glucoamylase (Ki < 0.1 μM), being more effective than Bay e 4609 (Ki < 10 μM). Glucoamylase was selectivity and strongly inhibited by acarbose (Ki < 0.1 μM). Activity of the latter enzyme was also affected by 1‐deoxynojirimycin (Ki < 1 mM), maltitol and amino alcohols (Ki < 10 mM).

Unlike α‐amylase, glucoamylase adsorbed strongly onto raw starch, the adsorption site being non‐identical with the active site.
A simple approach to detect active‐site‐directed enzyme‐enzyme interactions
Authors: VÉRTESSY Beáta , OVÁDI Judit
Published: March 2005

A novel approach has been elaborated to identify the mechanism of intermediate transfer in interacting enzyme systems. The aldolase/glycerol‐3‐phosphate‐dehydrogenase enzyme system was investigated since complex formation between these two enzymes had been demonstrated.

The kinetics of dihydroxyacetone phosphate conversion catalyzed by the dehydrogenase in the absence and presence of aldolase was analyzed. It was found that the second‐order rate constant (kcat/Km) of the enzymatic reaction decreases due to the formation of a heterologous complex. The decrease could be attributed to an increase of the Km value since kcat did not change in the presence of aldolase. In contrast, an apparent increase in the second‐order rate constant of dihydroxyacetone phosphate conversion by the dehydrogenase was observed if the triose phosphate was produced by aldolase from fructose 1,6‐bisphosphate (consecutive reaction). Moreover, no effect of dihydroxyacetone phosphate on the dissociation constant of the heterologous enzyme complex could be detected by physico‐chemical methods.

The results suggest that the endogenous dihydroxyacetone phosphate produced by aldolase complexed with dehydrogenase is more accessible for the dehydrogenase than the exogenous one, the binding of which is impeded due to steric hindrance by bound aldolase.
Solubilization and characterization of guinea‐pig pancreatic somatostatin receptors
Authors: ZEGGARI Mustafa , VIGUERIE Nathalie , SUSINI Christiane , GARNIER Martine , ESTEVE Jean‐Pierre , RIBET André
Published: March 2005

The solubilization of somatostatin receptors from guinea‐pig pancreas by different non‐denaturing detergents was investigated after stabilization of the receptors by prior binding of 125I‐[Tyr11]somatostatin or its analogue 125I‐[Leu8, DTrp22, Tyr25]somatostatin 28, to pancreatic plasma membranes.

The somatostatin‐receptor complexes were solubilized in a high yield by Zwittergent 3–14 (3‐[tetradecyldimethylammonio]‐1‐propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X‐100, Chaps (3‐[cholamidopropyldimethylammonio]‐1‐propanesulfonate) and octyl β‐D‐glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol.

In order to characterize solubilized somatostatin‐receptor comples, membranes receptors were covalently labelled using N‐5‐azido‐2‐nitrobenzoyloxysuccinimide as cross‐linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent Mr= 93000 which interacted with the two radioligands.

In addition, a similar component of Mr= 88000 was characterized after analysis by SDS‐PAGE of membrane receptors covalently cross‐linked with 125I‐[Leu8, DTrp22, Tyr25]somatostatin 28 by different heterobifunctional reagents: N‐5‐azido‐2‐nitrobenzoyloxysuccinimide, N‐hydroxysuccinimidyl 4‐azidobenzoate, N‐succinimidyl 6‐(4′‐azido‐2′‐nitrophenylamino)hexanoate. Optimal cross‐linking results were obtained with N‐5‐azido‐2‐nitrobenzoloxysuccinimide.

The solubilized somatostatin‐receptor complex was adsorbed to wheat‐germ agglutinin‐agarose column and eluted by specific sugars.

We concluded that the guinea‐pig pancreatic somatostatin receptor in the membrane and in the non‐denaturing detergent solution behaves as a protein monomer of apparent Mr∼ 85000–90000. The somatostatin receptor is a glycoprotein which contains complex‐type carbohydrate chains.
Apparent unmasking of [3H]GDP binding in rat brown‐fat mitochondria is due to mitochondrial swelling
Authors: NEDERGAARD Jan , CANNON Barbara
Published: March 2005

1. The presence of and biochemical background for the so‐called ‘unmasking’ phenomenon in rat brown‐fat mitochondria was investigated (i.e. the apparent increase in [3H]GDP binding to the ‘uncoupling’ protein thermogenin, without a concomitant increase in the amount of the protein).

2. It was found that an unmasking could be observed both 1 h after norepinephrine injection and after 1 h cold stress, provided that the rats were preacclimated to 28°C. The unmasking could be observed both when a filtration method and when a centrifugation method for determination of [3H]GDP‐binding capacity were used; however, the absolute values were higher with the filtration method.

3. Based on observations of slower cytochrome‐c oxidase sedimentation during centrifugation, the possibility that the matrix volume of brown‐fat mitochondria isolated from warm‐acclimated animals was smaller than that of cold‐stressed animals was investigated with 3H2O. The cold stress increased the matrix volume from being nearly non‐existent to about 1 μl/mg. A preswelling procedure in an ionic medium could similarly increase the matrix volume in mitochondria from warm‐acclimated animals but was without significant effect in the already swollen mitochondria from cold‐stressed animals or from animals adapted to a lower temperature.

4. In mitochondria from warm‐acclimated animals, the ionic preswelling procedure was fully able to increase the apparent amount of GDP binding to that observed in mitochondria from cold‐stressed animals, but it was practically without effect on GDP binding in mitochondria from cold‐stressed animals or from animals acclimated to a lower temperature.

5. It is concluded that the apparent ‘unmasking’ phenomenon, observed when the tissue is less activated than in normal control situations, is not (as hitherto anticipated) due to a specific change in thermogenin as such, but is a reflection of a general mitochondrial phenomenon.
Control of uncoupling protein in brown‐fat mitochondria by purine nucleotides
Authors: KOPECKÝ Jan , JEŽEK Petr , DRAHOTA Zdeněk , HOUŠTĚK Josef
Published: March 2005

The uncoupling protein (UP) of isolated brown adipose tissue mitochondria was studied with respect to the mechanism of control of UP function by purine nucleotides. Passive transport of H+ and Cl− was followed simultaneously in a KCl medium. With both GDP and ATP a higher sensitivity of Cl− transport (apparent Ki= 2.2 μM and 4.7 μM respectively) than of H+ transport (apparent Ki= 7.7 μM and 34 μM respectively) was observed. Chemical modification of isolated mitochondria by diazobenzenesulfonate (DA BS) up to 75 μmol/mg protein did not affect the transport, its ionic selectivity and regulation by endogenous free fatty acids. In contrast, the sensitivity to purine nucleotides of both H+ and Cl− translocation was decreased (apparent Ki increased 71 and 47 times respectively). DABS decreased the affinity of [3H]GDP for the specific nucleotide‐binding site on mitochondria (Kd increased from 2.7 μM to 13 μM) and depressed, to a smaller extent, the GDP‐binding capacity. Correlation between occupancy of the specific nucleotide‐binding site by GDP and inhibition of transport yielded a linear relationship for Cl− transport in control mitochondria, a biphasic correlation was obtained.

The results show that different structural parts of UP are involved in transport and its control by the regulatory ligands and that, in addition to binding of purine nucleotides to UP, the inhibition of ion transport by purine nucleotides depends on an intrinsic factor modulating the inhibitory effect.
Metabolism of neutral lipids in cultured fibroblasts from multisystemic (or type 3) lipid storage myopathy
Authors: RADOM Jean , SALVAYRE Robert , NEGRE Anne , MARET Arlette , DOUSTE‐BLAZY Louis
Published: March 2005

The lipid metabolism in cultured fibroblasts from multisystemic (type 3) lipid storage myopathy (MLSM) and controls has been studied through pulse‐chase experiments using radiolabelled oleic acid and acetate precursors.

1. The uptake of radiolabelled oleic acid by MLSM fibroblasts was slightly higher than in controls but did not seem to be the primary defect of the multisystemic lipid storage myopathy. The uptake of radiolabelled acetate was quite similar in MLSM and in control cells.

2. During short‐time pulse periods, using either radiolabelled oleic acid or acetate as precursors, we observed no significant difference in lipid composition between MLSM and controls. In contrast, pulse experiments using radiolabelled oleic acid as precursor showed a major accumulation of radiolabelled triacylglycerols in MLSM (around 1000% of controls); no significant increase of other neutral or polar lipids was noticed. A similar triacylglycerol storage was observed by using radiolabelled acetate as precursor, but in this case the difference between MLSM and controls was more pronounced; we also observed in MLSM cells a higher amount of polar lipids which can be due to an increased rate of fatty acid biosynthesis (from radiolabelled acetate).

3. Chase experiments, after pulse by low concentration of exogenous radiolabelled oleic acid or acetate, showed similar features: the biosynthesized triacylglycerols were not at all degraded in MLSM, but on the contrary increased, probably by accumulation of radiolabelled triacylglycerols newly synthesized from radiolabelled fatty acids liberated during the phospholipid turnover. Similarly, the triacylglycerol storage induced by high doses of fatty acids was not degraded in MLSM cells, in contrast to control cells. This suggested that the triacylglycerols synthesized in the presence of low and high levels of fatty acids were accumulated in only one subcellular cytoplasmic compartment without relationship with the lysosomal compartment, since these cells were not deficient in acid lysosomal lipase. The more probable hypothesis is a deficiency of the cytoplasmic catabolism of triacylglycerols in MLSM cells.
The reaction of human α2‐macroglobulin with α‐chymotrypsin
Authors: FAVAUDON Vincent , TOURBEZ Martine , POCHON François , MAREIX Robert , TOURBEZ Hervé
Published: March 2005

The kinetics of the conformational changes of human α2‐macroglobulin (α2M) induced by reaction with pure α‐chymotrypsin, have been analyzed using three fluorescent probes, namely protein tryptophan groups and the dye 6‐(4‐toluidino)‐2‐naphthalenesulfonate, to monitor alterations of the α2M structure, and a covalent conjugate of chymotrypsin and fluorescein isothiocyanate (Chy‐FITC). The main reaction sequence exhibits a triphasic time course with any of the labels used. Each phase is first‐order. The fixation of a single molecule of chymotrypsin to one protease‐binding site of α2M (site A) initiates the whole process and determines the access to the second site (site B). Of the three exponential phases of the reaction (20°C), phase I (k1∼ 19.6 min−1) and phase II (k2∼ 5.3 min−1) belong to site A. Phase III is related to site B transformation. It contains two steps with different responses from tryptophan (k3∼ 0.77 min−1) and Chy‐FITC (k3∼ 0.19 min−1) fluorescence measurements. The point to be stressed is that site A and site B, while presumably identical in the native form, are not equivalent with regard to their fluorescence and kinetic properties. However, the activation energy (E= 30.1 ± 2.7 kJ mol−1) is the same for the three phases of the reaction.

When present in sufficient excess, free chymotrypsin or native α2M is able to form reversible complexes with the above‐related chymotrypsin‐α2M adducts. Only the α2M site A core seems to be involved in this parallel process. In addition the conformational state of the chymotrypsin‐α2M complexes is shown to depend on the pH, with a pKa of 6.4.
Characteristics of thyroid protein kinase C
Authors: OMRI Boubaker , BRETON Marie France , PAVLOVIC‐HOURNAC Miroslava
Published: March 2005

Thyroid protein kinase C (PKc) from cytosols of porcine and rat thyroid glands has been characterized using histone H1 or endogenous proteins as substrates. As in many other tissues histone H1 is by far the preferred exogenous substrate of thyroid PKc. Kinetic studies with H1 showed that, compared to rat thyroids, porcine glands are particularly rich in PKc, the predominant kinase activity in this tissue. The cAMP‐dependent protein kinase (PKa) level, on the contrary, is very similar in both rat and porcine thyroids. Consequently, for the same type of tissue, there may be great species differences in the PKc level and the ratios between PKc and PKa kinase activities.

Chromatographic properties of thyroid PKc are similar to those described in other tissues (one major peak followed by a small shoulder) except that elution of the main peak can vary depending on the nature of the salt gradient (∼ 55 mM for NaCl and 15 mM for sodium phosphate). In the first case PKc is completely separated from the PKa activity, in the second it is coeluted with the peak of PKa type I.

The one‐dimensional PAGE pattern of proteins phosphorylated by porcine PKc is very similar to the pattern obtained by rat enzyme. Protein bands of 18 kDa, 22–25 kDa and 32–36 kDa are specific substrates of the thyroid PKc, after in vitro phosphorylation of cytosol proteins.

A great difference in Ca2+ requirement for PKc activation was noted, depending whether histone H1 or endogenous proteins were substrates. As in other tissues, calcium was absolutely necessary for phosphorylation of histone H1 by PKc. The addition of calcium was not absolutely necessary when endogenous proteins were the substrates, either for the activation of the enzyme or for phosphorylation of the PKc‐specific substrates. Almost the same rate of phosphorylation was obtained with or without calcium in the incubation medium. However the one‐dimensional PAGE pattern of phosphorylated proteins was different in the presence or absence of calcium. While addition of calcium was not absolutely necessary for the phosphorylation of a great number of proteins by the PKc, its presence was indispensable for the phosphorylation of certain endogenous substrates. However, calcium alone, in the absence of phospholipids had no effect on the phosphorylation of these proteins.

Endogenous proteins, phosphorylated by the PKc only when calcium was present, were resolved by the two‐dimensional PAGE into several distinct spots with molecular masses of 32–35 kDa and pI range of 5–7.5.

Our results suggest that only very low concentrations of Ca2+, if any, are sufficient for the phosphorylation of many endogenous substrates. Higher Ca2+ concentrations are necessary for the phosphorylation of specific proteins. It could be that modulation of Ca2+ concentration plays a role in the selection of protein substrates phosphorylated by PKc.
Ribonucleoside uptake and phosphorylation during fertilization and early development of the sea‐urchin, Strongylocentrotus purpuratus
Authors: KILLIAN Christopher E. , NISHIOKA David
Published: March 2005

Uptake of the four ribonucleosides normally present in RNA increases nearly 50‐fold shortly after fertilization in eggs of the sea‐urchin, Strongylocentrotus purpuratus. Uridine, adenosine and cytidine are phosphorylated (>95%) to their mono‐, di‐ and triphosphates immediately after transport into the fertilized egg. Although guanosine is transported to an extent equal to the other three ribonucleosides, less than 12% of it is phosphorylated after transport. In vitro nucleoside and nucleotide kinase assays of unfertilized egg homogenates indicate that the uridine, adenosine and cytidine kinases as well as the uridylate, adenylate, cytidylate and guanylate kinases are present in the egg prior to fertilization. Substrate competition measurements indicate that adenosine phosphorylation is catalyzed by a monospecific enzyme, while uridine and cytidine phosphorylations are catalyzed by a common kinase. Guanosine kinase activity was not detectable in unfertilized egg homogenates. Between 3 h and 5 h after fertilization the phosphorylation of transported guanosine begins to increase as it enters the embryo. By 7 h after fertilization, more than 95% of the guanosine entering the embryo is phosphorylated to the mono‐, di‐and triphosphates. More than 80% is phosphorylated to guanosine triphosphate. The timing of increased guanosine phosphorylation correlates with a decrease in the acid‐soluble GTP pools in the embryo, suggesting that increased guanosine kinase activity is a response to increased GTP demand. These results, in view of the importance of GTP in many cellular processes, imply a crucial role for guanosine kinase activation in GTP pool maintenance and cellular metabolism during early sea‐urchin development.
SS‐B (La) Nuclear antigen
Authors: BACH Montserrat , PALAU Jaume
Published: March 2005

Stable degradation products, obtained by digestion with endogenous and V8 proteases of calf thymus SS‐B (La) antigenic protein, have been studied. The most characteristic fragments have molecular masses of 47, 30, 23 and 17 kDa. The 47‐kDa and 30‐kDa fragments are complex and are constituted of a number of species of different isoelectric points, as has been described for the SS‐B protein molecule from other sources. Degradation products from the entire SS‐B nuclear antigen still contain the 30‐kDa SS‐B fragment, suggesting that the 30‐kDa region of the SS‐B protein molecule is firmly attached to the RNA moiety. A model is presented that implies the presence of two hinge regions sensitive to proteases and three structural domains that correspond to segments of 30 kDa, 17 kDa and 5–6 kDa.
Differential binding of mannose‐specific lectins to the carbohydrate chains of fibrinogen domains D and E
Authors: SOLIS Dolores , ESTREMERA Dolores , USOBIAGA Pilar , DÍAZ‐MAURIÑO Teresa
Published: March 2005

The interaction of fibrinogen with the mannose‐specific lectins concanavalin A (ConA), its acetyl derivative (Ac‐ConA) and Lens culinaris agglutinin (LcH) was studied. Both ConA and LcH interact specifically with individual fibrinogen Bβ and γ chains and with denatured fragments D and E. However, analysis of the binding data shows that four moles of Ac‐ConA are bound per mole of fibrinogen with two sets of binding sites (Kd1= 2.4 μM and Kd2= 16.6 μM; n1=n2= 2) while only two moles of LcH are bound per mole of fibrinogen (Kd= 2.6 μM). Ultracentrifugation studies are also in agreement with the presence in the fibrinogen molecule of two and four binding sites for LcH and Ac‐ConA, respectively. No aggregates of fibrinogen formed through LcH or Ac‐ConA linkages are observed. The use of a crosslinking reagent and ultracentrifugal analysis of the lectinfibrinogen fragments D1 and E complexes indicated that ConA, as well as Ac‐ConA, interact with both fragments D and E while LcH interacts only with fragment D. Furthermore, the binding of ConA to both D and E domains in the intact fibrinogen molecule is clearly demonstrated by using a bifunctional reagent. The bivalent character of ConA tetramers may be misinterpreted as a lack of accessibility of the lectin to two of the four carbohydrate chains of fibrinogen.

The differential binding of LcH and ConA to the carbohydrate chains of fibrinogen can be related to a different exposure of the oligosaccharide in D and E fragments and domains and to the different requirements of both lectins for their binding to glycoproteins.
Purification and characterization of d‐glyceraldehyde‐3‐phosphate dehydrogenase from the thermophilic archaebacterium Methanothermus fervidus
Authors: FABRY Stefan , HENSEL Reinhard
Published: March 2005

The d‐glyceraldehyde‐3‐phosphate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus was purified and crystallized. The enzyme is a homomeric tetramer (molecular mass of subunits 45 kDa). Partial sequence analysis shows homology to the enzymes from eubacteria and from the cytoplasm of eukaryotes. Unlike these enzymes, the d‐glyceraldehyde‐3‐phosphate dehydrogenase from Methanothermus fervidus reacts with both NAD+ and NADP+ and is not inhibited by pentalenolactone. The enzyme is intrinsically stable up to 75°C. It is stabilized by the coenzyme NADP+ and at high ionic strength up to about 90°C. Breaks in the Arrhenius and Van't Hoff plots indicate conformational changes of the enzyme at around 52°C.
Limited proteolysis reveals low‐affinity binding of N‐acetyl‐L‐glutamate to rat‐liver carbamoyl‐phosphate synthetase (ammonia)
Authors: GUADALAJARA Ana , GRISOLÍA Santiago , RUBIO Vicente
Published: March 2005

Carbamoyl‐phosphate synthetase was inactivated by elastase with first‐order kinetics, and N‐acetyl‐L‐glutamate speeded inactivation. From the dependence of the t1/2 value for inactivation on the concentration of acetylglutamate we estimate a Kd value for binding of the activator of 0.365 mM, which is approximately 600 times greater than in the presence of ATP, HCO–3, K+ and Mg2+. K+ and Mg2+ are not required for binding with low affinity, and in the absence of ATP they do not appear to increase the affinity for acetylglutamate. In the presence of acetylglutamate, mixtures of ATP, K+ and Mg2+ protect the enzyme from inactivation. ADP or AdoPP[NH]P partly replaced ATP in protecting the enzyme and thus binding of the nucleotide without further reaction is enough for protection. Two partial activities of the enzyme were inactivated by elastase to the same extent as the overall reaction, and thus elastase affects some property of the enzyme which is essential for catalysis. With other proteinases tested, inactivation was also accelerated by acetylglutamate and was slowed by mixtures of ATP, K+, Mg2+ and acetylglutamate, suggesting that changes in the accessibility of susceptible bonds are responsible for the changes in the degree of inactivation. It is concluded that elastase attacks at or close to the binding sites for ATP, and that exposure of the binding site for the ATP molecule that yields Pi (ATPA) upon binding of acetylglutamate causes the acceleration of the proteolytic inactivation.
Mode of inhibition of the DNA polymerase of Methanococcus vannielii by aphidicolin
Authors: ZABEL Hans‐Peter , HOLLER Eggehard , WINTER Josef
Published: March 2005

The mode of action of aphidicolin on DNA synthesis catalysed by the DNA polymerase of Methanococcus vannielii is competitive for dCTP, noncompetitive for dATP, dGTP and dTTP and uncompetitive for activated DNA. The kinetic data are accounted for by a mechanism in which dCTP and aphidicolin compete for the dCTP‐specific binding site on the DNA polymerase. The dissociation constant for the aphidicolin–DNA‐polymerase complex is 0.04–0.07 μM. Similar modes of inhibition of DNA synthesis exist for DNA polymerase α of higher eucaryotes but not for eubacteria or viruses and suggests a close functional relationship between the DNA polymerase of eucaryotes and of the archaebacterium M. vannielii.
Purification and immunochemical characterization of the major pertussis‐toxin‐sensitive guanine‐nucleotide‐binding protein of bovine‐neutrophil membranes
Authors: GIERSCHIK Peter , SIDIROPOULOS Dimitrios , SPIEGEL Allen , JAKOBS Karl H.
Published: March 2005

Bovine peripheral neutrophils contain high levels of a 40‐kDa pertussis toxin substrate, which was found highly enriched in a light membrane fraction upon subcellular fractionation of neutrophil homogenates. The 40‐kDa pertussis toxin substrate, referred to as αn, was purified to near homogeneity from this fraction by sequential ion‐exchange, gel‐filtration and hydrophobic chromatography. Purified αn was shown to interact with βγ subunits, undergo ADP‐ribosylation by pertussis toxin, and bind guanine nucleotides with high affinity. The mobility of purified αn on SDS/polyacrylamide gels was intermediate between those of the α subunits of Gi and Go, purified from bovine brain, and slightly lower than the mobility of the α subunit of transducin (Gt). Several polyclonal antisera against the α subunits of bovine Gt and Go did not react with αn on immunoblots. CW 6, a polyclonal antiserum reactive against the bovine αi, reacted only minimally with αn. These results suggest that the major pertussis toxin substrate of bovine neutrophils, designated Gn, is structurally different from previously identified pertussis toxin substrates and may represent a novel guanine‐nucleotide‐binding protein.
Regulation of fatty acid oxidation in isolated hepatocytes and liver mitochondria from newborn rabbits
Authors: HERBIN Catherine , PEGORIER Jean‐Paul , DUEE Pierre‐Henri , KOHL Claude , GIRARD Jean
Published: March 2005

The changes in long‐chain fatty acid oxidation during the first 24 h after birth were studied in isolated rabbit hepatocytes and liver mitochondria. The eightfold increase in this oxidation which occurs in hepatocytes between birth and 24 h was not triggered by a concomitant decrease in long‐chain fatty acid esterification. Indeed, in isolated hepatocytes from 24‐h‐old rabbits, the 75% inhibition of the oxidation by 2‐tetradecylglycidic acid, resulted in a total redirection of oleate metabolized towards triacylglycerol synthesis. Polarographic measurements of mitochondrial respiration showed that oxidative phosphorylation and respiratory chain capacity were fully functional at birth. By contrast, in liver mitochondria isolated from newborn rabbits, the rate of oxygen consumption from palmitoyl‐l‐carnitine was 60% higher than from palmitoyl‐CoA. Similarly palmitoyl‐CoA oxidation was increased 1.5‐fold in isolated mitochondria from 24‐h‐old rabbits. These results were in agreement with the twofold increase in the activity of hepatic carnitine palmitoyltransferase I between birth and 24 h. However it is unlikely that the twofold increase in this enzyme activity totally explained the eightfold increase in long‐chain fatty acid oxidation in isolated newborn rabbit hepatocytes. It was shown that the rate of the oxidation in isolated hepatocytes was inversely related to the rate of lipogenesis. Nevertheless, malonyl‐CoA concentration per se is probably not the factor involved in the regulation of the oxidation between birth and 24 h, since a 90% decrease in hepatic malonyl‐CoA concentration was not associated with a stimulation of long‐chain fatty acid oxidation. The more likely mechanism was the 30‐fold decrease in the sensitivity of carnitine palmitoyltransferase I to malonyl‐CoA inhibition.
Metabolic control and its analysis
Authors: SAURO Herbert M. , SMALL J. Rankin , FELL David A.
Published: March 2005

The matrix algebra procedure for determining the flux control coefficients of enzymes in metabolic pathways has been extended to allow determination of the concentration control coefficients. Although it is shown that the procedure is essentially unchanged in most cases, the presence of moiety‐conserved cycles in a pathway places additional limitations on the form of the equations that can be used in the matrix formulation for concentration control coefficients. In the case of branched pathways, a new coefficient has been defined, the branch distribution control coefficient, which can be obtained via the matrix procedure. Thus a single matrix equation permits calculation or algebraic evaluation of the control coefficients for flux, concentration and distribution of flux at branches, so that the complete response of a pathway to alteration of enzyme content, or to modulation by an effector, can be determined.

The relationships have been determined between flux control coefficients in isolated sections of metabolic pathways and the coefficients for the same enzymes when part of a larger metabolic system. It is shown that the control analysis of the isolated system provides useful information towards determining the control properties of the extended system.
The GTP pool in Bacillus brevis and its significance for sporulation
Authors: FEDERN Holger , RISTOW Hansjürgen
Published: March 2005

The GTP pool of Bacillus brevis as well as that of other nucleotides is highly sensitive to all kinds of environmental changes like the cell transfer procedures or nutrient depletion of the cells. In growing cultures, as well as in cells transferred from rich to nitrogen‐deficient medium, the nucleotide pool decreases significantly. This decrease is followed by the onset of sporulation only when cells are allowed to produce the peptide antibiotic tyrocidine or if tyrocidine is added to the culture. However, exogenous tyrocidine is active in triggering sporulation only when it is added within a short period of time immediately after shift down, that is when the nucleotide pool is observed to decrease.
Synthesis, accumulation and turnover of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in cultures of embryonic rat hepatocytes
Authors: van ROON Marian A. , CHARLES Robert , LAMERS Wouter H.
Published: March 2005

Glucocorticosteroid, thyroid hormones and cyclic AMP can induce the synthesis of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in cultures of hepatocytes as soon as these cells differentiate from the embryonic foregut. The low levels of both enzymes that can accumulate in such still protodifferentiated hepatocytes are due to low levels of enzyme synthesis. In cultures, the rate of synthesis of both enzymes increases continually in the presence of hormones, showing that maturation of the capacity for synthesis towards the postnatal, fully differentiated situation is occurring in these cells. The turnover rate of both enzymes in embryonic hepatocytes is lower in the presence of hormones than in the absence, but does not change during the culture period. In the presence of hormones the turnover rate is comparable to that found in adult rat liver in vivo. The development of the capacity to accumulate organ‐specific enzymes in vitro (and hence the rate of enzyme synthesis) is found to be comparable to that in utero.
[d‐Phe4]Peptide histidine‐isoleucinamide ([d‐Phe4]PHI), a highly selective vasoactive‐intestinal‐peptide (VIP) agonist, discriminates VIP‐preferring from secretin‐preferring receptors in rat pancreatic membranes
Authors: ROBBERECHT Patrick , COY David H. , De NEEF Philippe , CAMUS Jean‐Claude , CAUVIN Annick , WAELBROECK Magali , CHRISTOPHE Jean
Published: March 2005

The capacity of vasoactive intestinal peptide (VIP), peptide histidine‐isoleucinamide (PHI), secretin, and a series of analogs to discriminate between VIP‐preferring and secretin‐preferring receptors that coexist in rat pancreatic plasma membranes was evaluated by their ability to inhibit [125I]iodo‐VIP and [125I]iodo‐secretin binding and to activate adenylate cyclase. VIP, the VIP analogs [d‐His1]VIP, [d‐Ser2]VIP, [d‐Asp3]VIP and [d‐Ala4]VIP, PHI, [d‐Phe4]PHI, and secretin inhibited the binding of both ligands in a concentration range of 10−11 M to 10−5 M and with a selectivity factor varying from 18000 to 0.1. The only exception was [d‐Phe4]PHI that inhibited 125I‐VIP binding only, with an IC50 of 7 nM, and with no inhibition of 125I‐secretin binding at 10 μM.

The peptides tested stimulated adenylate cyclase in the same membranes and the slope of the dose‐effect curves indicated that all peptides, except [d‐Phe4]PHI, interacted with at least two classes of receptors: VIP‐preferring and secretin‐preferring receptors.

By contrast, the dose‐effect curve of [d‐Phe4]PHI activation of adenylate cyclase was monophasic and competitively modified by [d‐Phe2]VIP (a VIP antagonist) but not by secretin(7–27) (a secretin antagonist), indicating an interaction with VIP‐preferring receptors only. Thus, [d‐Phe4]PHI appears to be a highly selective tool to characterize these receptors.
A new agglutinin from the Tulipa gesneriana bulbs
Authors: ODA Yasuo , MINAMI Kazuo , ICHIDA Seiji , AONUMA Shigeru
Published: March 2005

Two agglutinins with different agglutinating activity exist in Tulipa gesneriana bulbs. One is the T. gesneriana lectin which agglutinates yeasts as reported previously [Oda, Y. and Minami, K. (1986) Eur. J. Biochem. 159, 239–245]. The other agglutinin is a new one which agglutinates animal erythrocytes and was purified from the tulip bulbs using affinity chromatography on thyroglobulin‐Sepharose 4B. The agglutinin agglutinated mouse and rat erythrocytes at a minimum concentration of 2 μg/ml and 30 μg/ml respectively, but did not agglutinate erythrocytes from other animals and yeasts even at a concentration of 1000 μg/ml. The agglutinin appeared homogeneous by disc gel electrophoresis at pH 4.3 and gel filtration. Its relative molecular mass was determined by gel filtration to be approximately 40000. It was suggested that the agglutinin was composed of two different subunits of 26 kDa and 14 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis. Binding of radioiodinated agglutinin to mouse erythrocytes indicated that the presence of a high‐affinity site with a dissociation constant of 2.00 × 10−9 M. In inhibition experiments thyroglobulin glycopeptides were the most potent inhibitors; thyroglobulin was also a potent inhibitor. Orosomucoid and mucin showed weak inhibition. The other glycoproteins, glycopeptides and sugars examined showed no inhibition.
Interactions of the high‐mobility‐group‐like Ceratitis capitata C1 proteins with DNA
Authors: MARQUEZ Gabriel , RODRIGUEZ Ana T. , FERNANDEZ Blanca A. , MONTERO Francisco
Published: March 2005

We have studied the interactions of the high‐mobility‐group‐like proteins (C1a1, C1a2 and C1b) from the fruit fly Ceratitis capitata with DNA. Nitrocellulose filter binding assays, thermal denaturation studies and spectrofluorimetry of the complexes revealed the existence of specific and nonspecific interactions. Thermal denaturation curves showed that the three proteins stabilized the DNA, thus suggesting a preferential binding to double‐stranded DNA. The calculation of the thermodynamic parameters of the interactions showed that the nonspecific bindings were characterized by low association constants (Ka) with values ranging from 2.7×104 M−1 to 2.0 × 106 M−1. Also, the cooperativity of these interactions was relatively high (cooperativity factor, w, values ranging over 20–35), and the number of nucleotides involved was low (1–3 base pairs). On the other hand, the existence of specific interactions between C1 proteins and DNA was suggested by two facts: (a) the retention of C. capitata [3H]DNA in nitrocellulose filters was only a low percentage of total input DNA and (b) there was a marked size dependence of the binding (25% retention of a 40‐kb DNA and only 3% retention with a DNA of 1 kb). The specific bindings had higher Ka values than the nonspecific ones, and they also were cooperative. Some differences were observed between C1b and the C1a proteins about the way they interact with C. capitata DNA.
Studies on the structure and conformation of brush border myosin using monoclonal antibodies
Authors: CITI Sandra , KENDRICK‐JONES John
Published: March 2005

We have produced and characterised five monoclonal antibodies against myosin isolated from chicken intestinal epithelial brush border cells. The binding sites of the antibodies on the rod portion of brush border myosin were localised using rotary shadowing/electron microscopy of myosin‐antibody complexes. Two antibodies were shown to bind to the tip of the myosin tail, two antibodies to sites about two thirds down the length of the rod, and one antibody about one third down the length of the rod. Brush border myosin was digested with papain, trypsin and α‐chymotrypsin, and the myosin fragments obtained were analysed by western blots with the monoclonal antibodies and polyclonal antiserum, and by gel overlay with 125I‐labelled light chains. Using this approach we were able to identify and map the protease cleavage sites and thus characterise the proteolytic substructure of brush border myosin. Solid‐phase assays, western blots and immunofluorescence were used to study the cross‐reactivity of these monoclonal antibodies against a variety of myosins from different species and cell types, to assess the immunological relatedness between brush border myosin and homologous molecules present in different tissues and species. Finally, we used a competitive solid‐phase assay to measure the ‘relative affinities’ of the antibodies towards the three possible conformational states of brush border myosin, i.e. filament, extended monomer and folded monomer.
β‐Glucosidase from Trichoderma reesei
Authors: CHIRICO William J. , BROWN Ross D.
Published: March 2005

To determine the mode of action of the β‐glucosidase from Trichoderma reesei a method was developed for synthesizing [1‐3H]cello‐oligosaccharides with specific radioactivities of approximately 3000 Ci/mol. The β‐glucosidase removed glucosyl residues from the non‐reducing end of the [1‐3H]cello‐oligosaccharides in a multiple attack mode with little tendency to attack the substrates repetitively. Values of Km were lower for longer cello‐oligosaccharides, whereas values of V remained essentially constant. A subsite map, constructed using values of V/Km for the cello‐oligosaccharides, showed that the substrate‐binding region comprises primarily three subsites.
Cobra cardiotoxins
Authors: HODGES Stephen J. , AGBAJI Abel S. , HARVEY Alan L. , HIDER Robert C.
Published: March 2005

A new preparative method for isolating homogeneous cardiotoxins from cobra venoms is described. The technique, based on reverse‐phase high‐pressure liquid chromatography, was used to isolate eight cardiotoxins of known sequence from four different venoms. In each case the method was found to be particularly efficient at removing trace quantities of contaminating phospholipase. Cardiotoxins isolated in this manner were found to retain their full biological activity.

Without exception the purified cardiotoxins lacked powerful haemolytic activity at concentrations up to 0.01 mM (about 100 μg ml−1), although some lysis of human erythrocytes was induced at higher concentrations. The cardiotoxins displayed a wide range of depolarizing activity on cultured skeletal muscle, the lowest activity being associated with the highest LD50 value.

Correlating variations in amino acid sequence and variations in depolarization potency revealed the importance of residues in the second and third loops, especially lysine‐46, serine‐48 and lysine‐52, together with a number of hydrophobic residues. Further modifications of pharmacological activity were associated with the presence of additional basic residues in the first and second loops and to minor differences in secondary structure.
The microheterogeneity of the crystallizable yeast cytoplasmic aspartyl‐tRNA synthetase
Authors: LORBER Bernard , KERN Daniel , MEJDOUB Hafedh , BOULANGER Yves , REINBOLT Joseph , GIEGÉ Richard
Published: March 2005

Yeast aspartyl‐tRNA synthetase is a dimeric enzyme (α2, Mr 125000) which can be crystallized either alone or complexed with tRNAAsp. When analyzed by electrophoretic methods, the pure enzyme presents structural heterogeneities even when recovered from crystals. Up to three enzyme populations could be identified by polyacrylamide gel electrophoresis and more than ten by isoelectric focusing. They have similar molecular masses and mainly differ in their charge. All are fully active. This microheterogeneity is also revealed by ion‐exchange chromatography and chromatofocusing. Several levels of heterogeneity have been defined. A first type, which is reversible, is linked to redox effects and/or to conformational states of the protein. A second one, revealed by immunological methods, is generated by partial and differential proteolysis occurring during enzyme purification from yeast cells harvested in growth phase. As demonstrated by end‐group analysis, the fragmentation concerns exclusively the N‐terminal end of the enzyme. The main cleavage points are Gln‐19, Val‐20 and Gly‐26. Six minor cuts are observed between positions 14 and 33. The present data are discussed in the perspective of the crystallographic studies on aspartyl‐tRNA synthetase.
The interaction of cystamine with bovine brain tubulin
Authors: BANERJEE Asok , Ann JORDAN Mary , LITTLE Melvyn , LUDUENA Richard F.
Published: March 2005

Microtubule assembly in vitro is sensitive to a variety of non‐physiological sulfhydryl‐oxidizing agents, but the physiological significance of this phenomenon is unknown, since no physiological sulfhydryl‐oxidizing agent has been shown to affect microtubule assembly in vitro. We have accordingly investigated the interaction of tubulin with cystamine. We have found that millimolar concentrations of cystamine inhibit microtubule assembly and induce an abnormal form of tubulin polymerization. Cystamine‐induced polymerization does not occur at cold temperature. Formation of the polymer requires reaction of cystamine with two sulfhydryls which become available at 37°C. In addition, cystamine reacts with about three sulfhydryls at 0°C without inducing polymerization. This latter set of sulfhydryls appear to include one or both of the previously defined βs sulfhydryls whose reaction with N, N'‐ethylene‐bis(iodoacetamide) is markedly inhibited by GTP, maytansine and vinblastine [Roach, M. C. & Luduena, R. F. (1984) J. Biol. Chem. 259, 12063–12071]. Cystamine's specific manner of interacting with tubulin suggests that it may mimic an endogenous sulfhydryl‐directed regulator of microtubule assembly.
Nitrate starvation induces homeoviscous regulation of lipids in the cell envelope of the blue‐green alga, Anacystis nidulans
Authors: GOMBOS Zoltán , KIS Mihály , PÁLI Tibor , VIGH László
Published: March 2005

Replacement of the normal culture liquid to a nitrate‐free medium resulted in an immediate drop in the ratio of protein to lipid in isolated cell envelopes of Anacystis nidulans cells. The relative fluidity of the envelope membranes or liposomes, made from the extracted lipids of the envelope, was estimated by measuring the steady‐state fluorescence polarization of 1,6‐diphenyl‐1,3,5‐hexatriene. A thermotrophic phase transition of lipids within the cytoplasmic membrane of intact cells was also revealed by detecting the temperature‐dependent absorption changes in the proportion of zeaxanthin at 390 nm. It became evident that a decrease in the proportion of protein to lipid within the cell envelope was accompanied neither by changes in the microviscosity level, nor by shifting of characteristic temperatures of the liquid‐crystalline‐to‐gel transition of lipids. In parallel with nitrate starvation, however, the proportion of saturated fatty acids of the envelope lipids increased markedly. Accumulation of saturated, longer‐chain (C18) fatty acids at the cost of C16 counterparts upon nitrate deprivation occurred in all of the complex lipids. In accordance with these findings, a pronounced decrease in the fluidity was demonstrated for the liposomes prepared from the envelope polar lipids of nitrate‐starved cells compared with the corresponding control, throughout the temperature range (45–5°C) studied.

We propose that the fluidizing effect due to a fall in the ratio of protein to lipid was compensated by a rapidly triggering regulatory process which enables the preservation of the fluidity characteristics at an optimal level within the cell envelope of A. nidulans.
Purealin, a novel activator of skeletal muscle actomyosin ATPase and myosin EDTA‐ATPase that enhanced the superprecipitation of actomyosin
Authors: NAKAMURA Yoichi , KOBAYASHI Masaki , NAKAMURA Hideshi , WU Houming , KOBAYASHI Jun'ichi , OHIZUMI Yasushi
Published: March 2005

Purealin, a novel bioactive principle of a sea sponge Psammaplysilla purea, activated the superprecipitation of myosin B (natural actomyosin) from rabbit skeletal muscle. The maximum change in the turbidity increased with increasing purealin concentrations and was three times the control value in the presence of 50 μM purealin.

The ATPase activity of myosin B was also elevated to 160% of the control value by 10 μM purealin. On the other hand, purealin inhibited the myosin ATPase in the presence of 10 mM CaCl2 and 0.5 M KCl (Ca2+‐ATPase), and the concentration for the half inhibition was 4 μM.

On the other hand, purealin activated the myosin ATPase in the presence of 5 mM EDTA and 0.5 M KCl (EDTA‐ATPase). The maximum activation by 10 μM purealin was 160% of the control value.

Furthermore, similar results concerning the modification of ATPase activities by purealin were obtained in myosin subfragment‐1 instead of myosin.

These results suggest that purealin activates the superprecipitation of myosin B by affecting the myosin heads directly. It is also an interesting observation that there is a correlation between the activities of the myosin EDTA‐ATPase and actomyosin ATPase of myosin B.
Amino acid sequence of chicken liver cathepsin L
Authors: WADA Kenji , TAKAI Toshiyuki , TANABE Tadashi
Published: March 2005

The complete amino acid sequences of the heavy and light chains of chicken liver cathepsin L have been determined by automated gas‐phase Edman degradation. The heavy and light chains contained 176 and 42 amino acid residues respectively. A glucosamine‐based oligosaccharide group was attached to Asn‐109 of the heavy chain. Chicken liver cathepsin L had high sequence homology with rat cathepsin H, but exhibited less similarity with rat cathepsin B. Comparisons of cathepsin L with plant cysteine proteinases, such as papain, actinidin and aleurain, reveal high degree of homology.
Hormonal regulation of carbamoyl‐phosphate synthetase I synthesis in primary cultured hepatocytes and Reuber hepatoma H‐35
Authors: KITAGAWA Yasuo
Published: March 2005

Regulation of carbamoyl‐phosphate synthetase I (CPS) synthesis by various hormones was compared in primary cultured hepatocytes from adult rat and in Reuber hepatoma H‐35 by pulse labeling of the cells with [35S]methionine. CPS synthesis in hepatocytes was stimulated 8‐fold and 5‐fold by dexamethasone and glucagon respectively. CPS synthesis in hepatocytes was synergically (about 50‐fold) stimulated by a combination of dexamethasone and glucagon. Less synergic stimulation was observed by combining dexamethasone with N6,O2′‐dibutyryladenosine 3′, 5′‐monophosphate (dibutyryl‐cAMP) or with isoproterenol. The basal level of CPS synthesis in hepatoma cells was higher than that in hepatocytes. CPS synthesis in hepatoma cells was stimulated by dexamethasone and dibutyryl‐cAMP but the extent was only 3‐fold and 1.8‐fold respectively. The synergic effect of combination of dexamethasone and dibutyryl‐cAMP was not observed in hepatoma cells. Neither glucagon nor isoproterenol exhibited an appreciable effect on CPS synthesis in hepatoma cells. Insulin and epinephrine suppressed CPS synthesis both in hepatocytes and hepatoma cells. The effect of epinephrine was indicated to be through α‐adrenergic receptors. The effects of insulin and epinephrine were additive on CPS synthesis both in hepatocytes and hepatoma cells.