bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of GM1 Ganglioside with Bovine Serum Albumin Formation and Isolation of Multiple Complexes
Authors: TOMASI Maurizio , RODA L. Giorgio , AUSIELLO Clara , D'AGNOLO Giuliano , VENERANDO Bruno , GHIDONI Riccardo , SONNINO Sandro , TETTAMANTI Guido
Published: March 2005

The binding of ganglioside GM1 to bovine Serum albumin has been studied by using absorption and fluorescence properties of the protein chromophores. Differences to the ultraviolet absorption spectrum and in fluorescence quenching, as well as a marked shift of the wavelength at the fluorescences maximum provide information about the binding of this ganglioside to albumin. Ultracentrifugal studies showed that there are two forms of the GM1‐protein complexes which differ markedly in their molecular weight. These two forms have been separated on this basis, by a chromatographic sieving procedure, and designated as complexes I and II. Both complexes are characterized by it GM1: protein ratio of one ganglioside micelle per albumin polypeptide chain. Complex II polymerizes slowly and irreversibly to a dimer, complex I. These results have been correlated with the optical studies in order to draw limited inferences its to the environment of the binding sites on the native protein. The interaction between GM1 micelles and albumin is mostly hydrophobic and the two complexes are actually mixed ganglioside‐protein micelles. At submicellar concentrations of ganglioside a binding of ganglioside GM1 to albumin also occurs. This process is due, however, to an aspecific, reversible adhesion of GM1 molceules on the albumin surface with no apparent perturbation of the albumin structure.
Localisation and Importance of the Sugar Part of Human Casein
Authors: FIAT Anne‐Marie , JOLLÈS Jacqueline , AUBERT Jean‐Pierre , LOUCHEUX‐LEFEBVRE Marie‐Henriette , JOLLÈS Pierre
Published: March 2005

The study of glycopeptides obtained after enzymic digestions of human caseinoglycopeptide demonstrated the presence of several (up to 10) prosthetic sugar groups distributed throughout the peptide chain instead of only one in the case of cow and sheep χ‐caseinoglycopeptides. One of the glycopeptide, allowed us, to complete the C‐terminal sequence of human caseinoglycopeptide previously published in part by others. The predicted secondary structure was in accordance with the high number of sugar groups situated all but one in β‐turns. A significant microheterogeneity seems to occur at the sugar level of human caseinoglycopeptide, which might thus present differences from one milk to another.
Surface Labeling of Membrane Glycoproteins and Their Drastic Changes during Development of Dictyostelium discoideum
Authors: TODA Katsumi , ONO Ken‐ichi , OCHIAI Hiroshi
Published: March 2005

Surface glycoproteins on plasma membranes from Dictyostelium discoideum were labeled by sodium inctaperiodate oxidation and sodium bore[3H]hydride reduction. The amount of incorporation of tritium from NaB3H4 reached a platcau after 10 min at a periodate concentration of 20 mM. The density analysis carried out by sucrose density gradient centrifugation showed that the plasma membranes were selectively labeled by this technicue. About 84% of the radioactivity incorporated was released by hydrolysis with 0.25 M H2SO4 for 3 h at 100° C. The released materials were eluted at it bed volume after chromatography using a Sephadex G‐50 column. From the paper chromatographic analysis of the eluate, four radioactive spots were detected. Two of them were glycerol and glyceraldehyde and the other two spots seemed to be oligosaccharides.

Using the above method, the plasma membranes from aggregation‐phase cells were labeled four times more than those front growth‐phase cells. The labeled plasma membrane fraction during the aggregation phase was separated into at least 39 distinct glycoprotein bands on a one‐dimensional dodecylsulfate/polyacrylamide gel. Six of the 39 bands increased markedly in density and two new bands appeared. In contrast, four bands decreased in density in the aggregation phase.

Using this method, the surface glycoproteins can be analyzed directly by gel electrophoresis of the lystate of labeled whole cells without preparing the plasma membranes. Changes during development of glycoproteins on outer cell surfaces were also confirmed by O'Farrell's method of twodimensional polyacrylamide gel electrophoresis. Utilizing this technique, glycoproteins on plasma membranes of D. discoideum were separated into 63 individual spots; 45 of these 63 spots changed during the early developmental course of D. discoideum. This is in contrast to the slight change in the soluble and membrane proteins during this phase. This fact also suggests that the glycoproteins have important roles in the process of aggregation.
RNA Polymerase III from Drosophila hydei Pupae
Authors: GUNDFLFINGER Eckart , SAUMWEBER Harry , DALLENDÖRFER Angelika , STEIN Hans
Published: March 2005

DNA‐dependent RNA polymerase III, which is usually highly resistant to α‐amanitin, has been purified from Drosophila hydei pupae. The enzyme is insensitive to α‐amanitin concentrations up to 0.1 μM; at 100 μM α‐amanitin the enzyme activity is inhibited by approximately 10%. The enzyme was extracted at low ionic strength and purified to homogeneity by six purification steps; 0.1–0.2 mg enzyme/kg pupae could be obtained. The subunit composition of the enzyme was determined after sucrose‐gradient centrifugation by sodium dodecyl sulphate electrophoresis in polyacrylamide gels. The enzyme was resolved into putative subunits of molecular weight 154000, 135000, 62000, 58000, 38000, 32000, 31000, 27200, 26500, 21500 and 17500. This subunit composition is in general accord with that of eucaryotic class III enzymes. The catalytic properties (salt‐activation profile, ratio of activity with denatured DNA to that with native DNA) and the order of elution of the enzyme from DEAE‐cellulose with respect to RNA polymerase II agree with the classification of the isolated enzyme as an RNA polymerase III.
Preparation and Characterization of Inverted Cell Envelopes of Halobacterium halobium
Authors: GARTY Haim , DANON Arlette , CAPLAN S. Roy
Published: March 2005

Inside‐out cell envelopes from Halobacteriun hulobium R1M1 have been isolated by sonication and subsequent sucrose‐density‐gradient fractionation. The inverted cell envelopes transport pr otons from the exterior of the vesicles by a light‐dependent, uncoupler‐inhibited process. Enzymes usually facing the inside of the cell are exposed to the external medium in this preparation.
Molecular Cloning of Bovine Thyroglobulin Complementary DNA
Authors: CHRISTOPHE Daniel , BROCAS Huguette , GANNON Frank , DE MARTYNOFF Guy , PAYS Etienne , VASSART Gilbert
Published: March 2005

Double‐stranded thyroglobulin complementary DNA (cDNA) was synthesized from purified 33‐S bovine thyroglobulin mRNA. This synthetic structural gene has previously been shown to contain three sites for the restriction endonuclease HindIII, yielding two internal fragments of 1900 and 2500 base pairs respectively. Recombinant molecules were prepared by ligating the HindIII‐restricted cDNA to the plasmid pBR322 which had been linearized by the same enzyme. When Escherichia coli was transformed with this mixture, it yielded two kinds of colonies each harboring recombinant plasmids containing one of the two cDNA fragments. Both recombinant molecules hybridized specifically to translatable thyroglobulin mRNA. Sequence homology between the two cloned DNAs could not be detected by cross‐hybridization experiments; this argues against the existance of internal structural repetition in thyroglobulin Subunits. Together, the two cloned DNA fragments represent 55% of the 8000‐base‐pair double‐stranded thyroglobulin DNA.
The Quaternary Structure of Bovine α‐Crystallin
Authors: SIEZEN Roland J. , BINDELS Jaques G. , HOENDERS Herman J.
Published: March 2005

The stability of the native quaternary structure of bovine α‐crystallin was studied, by sedimentation analysis and electron microscopy, as a function of pH (7–11), ionic strength (0.01–0.5), temperature (6–60° C) and calcium ion concentration (0 and 10 mM).

Three successive transitions are distinguished at 20° C. Firstly, a slow transconformation step, which is independent of pH, ionic strength or calcium ions. Secondly, an irreversible primary dissociation step, favoured by increasing pH above 8 and/or a lower ionic strength, with formation of ‘alkali‐modified α‐crystallin′, which is spherically shaped like the native protein but has a smaller average diameter, sedimentation coefficient and molecular weight. Thirdly, with further increase of pH above 9, a rapidly reversible dissociation of alkali‐modified α‐crystallin characterized by a single reaction boundary in sedimentation velocity analysis. In the presence of calcium ions the quaternary structure is stabilized to the extent that no dissociation is observed up to at least pH 10.3.

Upon increase of temperature, at pH 7.3, a slow irreversible dissociation and swelling run parallel until a limit is reached around 37° C with formation of ‘temperature‐modified α‐crystallin′, which is indistinguishable from the native protein by electron microscopy, but has a higher relative viscosity and lower sedimentation coefficient and molecular weight. Calcium ions have little or no effect on this transition. Above 37° C a reversal of this transition or aggregation is indicated.

These findings, together with previous structural data of microheterogeneity, reassociation from urea, and aging of α‐crystallin in vivo, are incorporated into a hypothetical scheme of transitions, based on a three‐layer model for the quaternary structure.
Inhibition of Cholesterol Synthesis in Rat Liver by Physiological Doses of Taurocholate
Authors: WEIS Elmar Eugen , BARTH Christian Arthur
Published: March 2005

This report deals with the controversial problem whether bile acids exert a direct inhibitory effect on the rate of hepatic cholesterol synthesis. For this purpose rats have been provided with an ‘extracorporeal bile duct′, an experimental model which makes it possible to initiate a bile fistula two weeks after last surgery. In the animals that had recovered completely from operative trauma, a 6‐h infusion of 33.4 μmol sodium taurocholate × (100 g body weight)‐1× h−1, prevented the threefold rise of hepatic cholesterol synthesis following bile diversion.

The rate of cholesterol synthesis was monitored by incorporation of [14C] acetate in vitro.

There was no difference whether the animals were infused from 4–10 h (dark/light transition) or from 16–22 h (light/dark transition) besides the fact that taurocholate caused a more extensive inhibition during the light‐dark transition.

A parallel kind of response was observed for the activity of hepatic 3‐hydroxy‐3‐methylglutaryl‐CoA rcductase (EC 1.1.1.34), the regulatory enzyme of cholesterol synthesis.

A twofold rise of hepatic fatty acid synthesis was observed following bile diversion which was not seen in case of simultaneous bile salt infusion.

It is concluded that bile salts exert direct feedback inhibition of hepatic cholesterol synthesis and that opposite results reported by other investigators are probably due to the infusion of too low doses of taurocholate.
The Efects of Protein Extraction on the Structure and Filtration Properties of Renal Basement Membranes
Authors: ROBINSON Garth B. , LIGLER Frances S.
Published: March 2005

Basement membranes were isolated from rabbit kidney using the detergent N‐dodecyl sarcosine. Treatment of the membranes with 6 M urea or 5% sodium dodecyl sulphate in the presence of reducing agent at 37° C extracted approximately 50% of the protein. Amino acid analysis and polyacrylamide gel electrophoresis demonstrated that the residual proteins were collagenous and of molecular weight. Extracted membranes retained their continuity of structure and exhibited a matrix composed of fibrous and globular elements when examined by electron microscopy. When the filtration properties of the membranes were studied in vitro they showed an enhanced capacity to retain proteins. The results suggest that basement membranes contain a matrix of fibrous protein, which is responsible for the sieving characteristics of the native membranes.
Membrane Mutants: A Yeast Mutant with a Lesion in Phosphatidylserine Biosynthesis
Authors: KOVÁČ Ladistav , GBELSKÁ Iveta , POLIACHOVÁ Vlasta , ŠUBÍK Július , KOVÁČvÁ Vlasta
Published: March 2005

A single‐gene nuclear choline‐requiring mutant of Saccharomyces cerevisiae was studied. Choline as a growth supplement to synthetic media could be substituted by low concentrations of d imethylethanolamine, monomethylethanolamine or ethanol amine. DL‐Serine also supported growth, but only at high concentrations: on a molar basis it was approximately one hundred times less effective than choline. When cultured in unsupplemented medium the mutant cells soon ceased to grow. The growth‐arrested cells contained less than one fifth of the phosphatidylethanolamine present in wild‐type cells and only traces of phosphatidylserine. The relative content of the two phospholipid species was raised by growing the mutant cells in the presence of choline of the other supplements but still remained lower than in wild‐type cells.

The mutant cells depleted of phosphatidylethanolamine and phosphatidylserine had greatly diminished ability to fuse with other cells in mating and their protoplasts showed increased resistance to hypotonic lysis. Respiration was not substantially affected by the deficit of the two phospholipid species in the mutant.

In cell‐free preparations, the affinity of the phosphatidylserine synthesizing system for serine was found to be almost two orders of magnitude lower in the mutant than in the wild‐type. The impairment of phosphatidylserine synthesis accounts for growth requirement and the abnormal phospholipid composition of the mutant cells.
The Identity of the Serotonin‐Sensitive Aryl Acylamidase with Acetylcholinesterase from Human Erythrocytes, Sheep Basal Ganglia and Electric Eel
Authors: GEORGE Shaji T. , BALASUBRAMANIAN Aiylam S.
Published: March 2005

The identity of the serotonin‐sensitive aryl acylamidase with acetylcholinesterase from three diverse sources, namely sheep basal ganglia, human erythrocyte membrane: and electric eel, was examined. Both the enzymes co‐purified with constant ratios of specific activity from all the three sources by different affinity chromatographic techniques. The ratio of acetylcholinesterase to aryl acylarnidase activity was highest for basal ganglia, less for erythrocyte and lowest for eel enzymes. Both the purified enzymes co‐migrated on polyacrylamide gel electrophoresis either as a single species or multiple species under different conditions. Gel density gradient electrophoresis indicated identical migration rates of both the enzymes. Extraction of the enzymes from the three sources by different techniques of membrane disruption and subsequent gel filtration on Sepharose 6B showed multiple peaks of enzyme activity. Both the enzymes had identical elution profiles on Sepharose 6B gel filtration. All the enzyme peaks from Sepharose 6B on gel electrophoresis showed co‐migration of the enzyme activities. Apart from inhibition by serotonin and acetylcholine the purified aryl acylamidases from all the three sources were potently inhibited by neostygmine, eserine and BW284C51, all strong inhibitors of acetylcholinesterase. It is suggested that the serotonin‐sensitive aryl acylamidase is identical with acetylcholinesterase.
Aldrin Epoxidation Catalyzed by Purified Rat‐Liver Cytochromes P‐450 and P‐448 High Selectivity for Cytochrome P‐450
Authors: WOLFF Thomas , GREIM Helmut , HUANG Mou‐Tuan , MIWA Gerald T. , LU Anthony Y. H.
Published: March 2005

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat livermicrosotnal cytochrome P‐450 or P‐448, NADPH‐cytochrome c reductase, dilauroylphosphatidylcholine and sodium Cholate. Cytochrome P‐450, purified from hepatic microsomes of phenobarbital‐treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observedin the absence of the lipid and sodium cholate was increased threefold by addition of dilauroyl‐phosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparentKm for aldrin in the complete system was 7 ± 2 μM. SKF 525‐A, at a concentration of 250 μM, inhibited aldrin epoxidation by 65% whereas 7,8‐benzoflavone had no inhibitory effect at concentrations up to 250 μM. Addition of ethanol markedly increased epoxidase activity. The increase was three fold in the presence of 5% ethanol.

When cytochrome P‐448 purified from hepatic microsomes of 3‐methylcholanthrene‐treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P‐450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 ± 7 μM. The modifiers of monooxygenase reactions, 7,8‐benzoflavone, SKF 525‐A and ethanol, inhibited the activity mediated by cytochrome P‐448. The I50 was 0.05, 0.2 and 800 mM, respectively.

These results indicate that aldrin is a highly selective substrate for cytochrome P‐450 species present in microsomes of Phenobarbital‐treated animals and is a poor substrate for cytochrome P‐448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent K, and their sensitivity to modifiers, like 7,8‐benzoflavone and ethanol.
Phosphorylase Kinase from Rabbit Skeletal Muscle Identification of the Calmodulin‐Binding Subunits
Authors: PICTON Colin , KLEE Claude B. , COHEN Philip
Published: March 2005

Phosphorylase kinase has the structure (αβγδ)4 where the δ‐subunit is identical to the calciumbinding protein termed calmodulin [Shenolikar et al. 1979) Eur. J. Biochem. 100, 329–337].

The δ‐subunit was tightly bound to phosphorylase kinase in the absence of calcium ions, and its rate of exchange with [14C]calmodulin was only 15% per week. The δ‐subunit remained associated with phophorylase kinase in the presence of 8 M urea provided that calcium ions were present and this property enabled electrophoretic techniques to be used which demonstrated that the δ‐subunit was associated with the γ‐subunit. This finding was confirmed by cross‐linking experiments with dimethylsuberimidate which resulted in the formation of a γδ complex.

Phosphorylase kinase was shown to bind one additional molecule of calmodulin per αβγδ unit, termed the δ‐subunit. Glycerol gradient centrifugation in the presence of [14C]calmodulin indicated that the interaction of the δ‐subunit with phosphorylase kinase only occurred in the presence of calcium ions, and that the Kd value was near 0.01 μM. This was similar to the concentration of δ‐subunit which produced half‐maximal activation. The δ‐subunit did not remain associated with phosphorylase kinase in the presence of 8 M urea, either in the presence or absence of calcium ions. The very slow exchange between the δ‐subunit and [14C]calmodulin, and the calcium‐dependcnt binding of the δ‐subunit allowed cross‐linking experiments to be used which demonstrated that the δ‐subunit was bound to both the α and β subunits. This result was supported by the finding that selective proteolysis of either the δ‐subunit, or the α and β subunits, decreased or abolished the ability of phosphorylasc kinase to bind to calmodulin‐Sepharose.

The roles of the different subunits in the regulation of phosphorylase kinase activity are discussed.
Enzymatic Hydrolysis of Disaccharide Unit of Collagen
Authors: HAMAZAKI Hideaki , HOTTA Kyoko
Published: March 2005

The hydroxylysine‐linked disaccharide unit, 2‐O‐α‐d‐glucopyranosyl‐galactopyranosylhydroxylysine (Glc‐Gal‐Hyl), prepared from collagens, was hydrolyzed by a glucohydrolase present in rat spleen. by homogenates of intestines, livers, and kidneys, which had a high a‐D‐glucosidase activity for neutral glucosides. The Glc‐Gal‐Hyl glucohydrolase was purified from rat spleens by affinity chromatography and gel filtration to the extent that sodium dodecyl sulfate/polyacrylamide gel electrophoresis gave a single band stained by Coomassie blue G‐250. This purified glucohydrolase had a pH optimum around 5.8, and the Michaelis constant was 5.9 mM when Glc‐Gal‐Hyl was used as a substrate. This enzyme did not hydrolyze neutral glucosides. It is concluded that this Glc‐Gal‐Hyl glucohydrolase is responsible for catabolism of the hydroxylysine‐linked disaccharide unit derived from collagens in mammals.
Messenger RNA for Cataractous Lens Proteins Are also Present on Normal Lens Polyribosomes
Authors: WEILL Jean‐Claude , LECA Gérald , VINCENT Alain , CIVELLI Olivier , POULIQUEN Yves
Published: March 2005

We have previously described an experimental model in vivo where cataract is induced by injection of the antimitotic bleomycin in the newborn rat. The first opacity of the lens appears 12–15 days after the injection of the drug concomitantly with a group of precise biochemical modifications among the soluble crystallins. These modifications are mainly the accumulation of two additional low‐molecular‐weight βLcrystallin subunits and of several smaller α‐crystallin polypeptides. Messenger RNA isolated from normal and cataractous lenses was assayed for translation in it cell‐free wheat germ extract. Analysis of the translation products by one‐dimensional and twodimensional gel electrophoresis indicated that the messenger RNAs coding for the two βL subunits are also present on normal lens polyribosomes. Purification and subsequent analysis by peptide mapping of the cataractous βL subunits suggest that they are precursor polypeptides of the normal low‐molecular‐weight β‐crystalline, which are no longer processed after translation and therefore accumulate in the pathological lens cell. On the other hand, in the translation of the cataractous mRNA in vitro, only the normal a chains are detected. These cataractous α‐crystalline are therefore post‐translational degradation products of the normal α polypeptides. This phenomenon seems similar to the one observed in the senescent lens. The possible involvement of these modifications in the etiology of this experimental cataract is discussed further.
Characterization and Quantification of the Androgen and Glucocorticoid Receptors in Cytosol from Rat Skeletal Muscle
Authors: SNOCHOWSKI Marek , DAHLBERG Erik , GUSTAFSSON Jan‐Åke
Published: March 2005

The binding of the radioactive synthetic hormonal steroids [3H]dexamethasone (9α‐fluoro‐11β, 17α, 21‐trihydroxy‐16α‐methyl‐1, 4‐pregnadiene‐3,20‐dione) and [3H]methyltrienolone (17β‐hydroxy17‐α‐methyl‐4,9,11‐estratrien‐3‐one) to cytosol from rat skeletal muscle was studied using dextrancoated charcoal to separate unbound and receptor‐bound steroid. The rates of association, dissociation, and degradation of the complexes of dexamethasone and methyltrienolone with receptor were highly dependent on temperature. The temperature dependence of association was greater for dexamethasone, and that of degradation was greater for methyltrienolone. Dissociation rates were insignificant for both steroid‐receptor complexes compared to association and degradation rates. The apparent equilibrium dissociation constants for the binding of dexamethasone and methyltrienolone to their receptor binding sites were about 7 and 0.3 nM, respectively, regardless of temperature (0, 15 or 23° C). The lack of influence of temperature on the equilibrium constants indicate that the binding was of hydrophobic character, and the corresponding free energy changes upon binding of dexamethasone and methyltrienolone to their respective binding sites were –41 and –49 kJ mol‐1 under equilibrium conditions at 0° C. The apparent maximum number of binding sites determined from Scatchard plots under these conditions was about 1900 fmol/g of tissue, 3500 fmol/mg of DNA or 30 fmol/mg of protein in the case of the dexamethasone receptor, and the corresponding figures for the methyltrienolone were about 100 fmol/g of tissue, 200 fmol/mg of DNA or 2 fmol/mg of protein. The ligand specificities of the binding sites for dexamethasone and methyltrienolone were typical of a glucocorticoid and an androgen receptor, respectively.

Both steroid‐receptor complexes were retained on DNA‐cellulose columns, and were eluted by NaCl at an ionic strength of 0.1. The DNA‐cellulose step purified about 20 times, and was used to allow gel exclusion chromatography and electrofocusing. Both steroid‐receptor complexes were excluded from a column of Sephadex G‐150. Electrofocusing in preparative columns gave reproducible patterns consisting of three peaks for each receptor. The apparent isoelectric points were 5.4, 5.6 and 0.2 for the glucocorticoid receptor, and 5.9, 6.2 and 8.5 for the androgen receptor.
Opposite Effects of Thiocyanate on Tyrosine Iodination and Thyroid Hormone Synthesis
Authors: VIRION Alain , DEME Danièle , POMMIER Jacques , NUNEZ Jacques
Published: March 2005

The effect of a pseudohalide, SCN−, an anion with the same molecular size as iodide, was studied on two reactions: thyroglobulin iodination and thyroid hormone synthesis (coupling reaction) catalyzed by peroxidases. The coupling reaction was studied separately from the iodination reaction by using labelled thyroglobulin samples previously iodinated but containing little or no hormones.

SCN− inhibits iodide oxidation (I−→ I2) whatever the enzyme, thyroid, lactoperoxidase or horseradish peroxidase. The amount of SCN− required to completely inhibit this reaction varies depending on the enzyme. Similarly tyrosine iodination is inhibited by SCN− with large variations, depending on the peroxidase, in the concentration of this anion required for inhibition.

In contrast SCN− stimulates the coupling reaction: (a) this effect is seen with the thyroid and lactoperoxidases but not with horseradish peroxidase; (b) the concentration of SCN− required for half‐maximal stimulation of the coupling reaction is much lower (0.5–1 μM) than that required for the inhibition of iodide oxidation (60–80 μM); (c) CIO−4, an anion with the same molecular size as SCN− and I−, has no effect on the coupling reaction; (d) this stimulatory effect of SCN− does not depend on a modification of the thyroglobulin molecule since it is not seen with horseradish peroxidase or in purely chemical coupling conditions.

The stimulatory effect of SCN− is therefore seen as resulting from the binding of this anion to a limited number of high‐affinity sites present at the surface of both thyroid and lactoperoxidases. The inhibitory effect depends, in contrast, on the binding of SCN− to the substrate site with lower affinities. Since iodide also behaves both as a substrate for the iodination reaction and as a stimulatory ligand for the coupling reaction, these data provide further support in favour of the existence of an enzyme‐iodide (or SCN−) complex with catalytic properties different from those of the native peroxidase.
Conformational Characteristics of the Trinucleoside Diphosphate dApdApdA and Its Constituents from Nuclear Magnetic Resonance and Circular Dichroism Studies
Authors: OLSTHOORN Cornelis S. M. , BOSTELAAR Lein J. , VAN BOOM Jacques H. , ALTONA Cornelis
Published: March 2005

Proton NMR studies at 360 MHz were carried out on the trinucleoside diphosphate dApdApdA, the dinucleoside monophosphate dApdA and the methylphosphate esters of the monomers pdA, pdAp and dAp. The compounds were also investigated by means of circular dichroism at varying temperatures. Complete NMR spectral assignments are given. The thermodynamics of stacking derived from the circular dichroic spectra was used to extrapolate the observed coupling constants, measured at a range of temperatures, to values appropriate to the pure stacked states of the dimer and the trimer.

The data were interpreted in terms of the N and S deoxyribose pseudorotational ranges [Altona, C. and Sundaralingam, M. (1972) J. Am. Chem. Soc. 94, 8205–8212] and it is shown that the right‐handed single helix of the trimer occurs in two distinct forms: the major one corresponds to a B‐DNA‐like structure, S‐S‐S; the minor one (30%) has the sugar rings in a hitherto unsuspected S‐S‐N conformational sequence. The dimer behaves quite similarly, a mixture of 70% S‐S and 30% S‐N stacks being indicated. The detailed geometry of the S‐type sugar rings is not invariable but appears to undergo a slight shift when another base stacks on top of a given nucleotide unit. The backbone angles of the fully stacked B‐DNA‐like single helix in solution are (starting the notation at P) β= 187°, γ= 50°, δ= 138°, ɛ= 186°.
Changes in Polygalacturonase Isoenzymes during the ‘Ripening’ of Normal and Mutant Tomato Fruit
Authors: TUCKER Gregory A. , ROBERTSON Neil G. , GRIERSON Donald
Published: March 2005

Polygalacturonase activity is not detectable in mature green tomato fruits but appears as fruits begin to change colour and continues to increase during the ripening period. There is a sequential appearance of two isoenzymes, polygalacturonase 1 and 2, during ripening. These isoenzymes have been purified and their properties compared. Polygalacturonase 1 has a Mr of 100000, is 50% inactivated at 78° C and has a density of 1.343 g cm−3 in caesium chloride. Polygalacturonase 2 has a Mr of 42000, is 50% inactivated at 57° C and has a density of 1. 300 g cm−3 in caesium chloride.

Fruits from isogenic lines homozygous for the ‘Neverripe’ (Nr) mutation do not ripen normally and contain reduced amounts of polygalacturonase. Only polygalacturonase 1 is produced in Nr fruit. Tomatoes from isogenic lines homozygous for the ‘ripening inhibitor’ (rin) mutation do not ripen normally and produce very little detectable polygalacturonase.

Although polygalacturonases 1 and 2 have different properties they both give rise to a single polypeptide on electrophoresis in polyacrylamide gels in the presence of sodium dodecylsulphate (Mr= 46000). A comparison of the major fragments produced by limited proteolysis of polygalacturonase 1 and 2 with chymotrypsin suggests that the polypeptides from the two isoenzymes are similar. The same conclusion was reached from a comparison of polygalacturonase 1 and 2 by radioimmunoassay, using antibody prepared against polygalacturonase 2 and 125I‐labelled polygalacturonase 2.

The results from radioimmunoassay of extracts from green and ripening fruits suggest that the increase in polygalacturonase activity during ripening is due to net synthesis of protein.
Cathepsin‐D‐Dependent Initiation of the Hydrolysis by Lysosomal Enzymes of Apoprotein B from Low‐Density Lipoproteins
Authors: VAN DER WESTHUYZEN Deneys R. , GEVERS Wieland , COETZEE Gerhard A.
Published: March 2005

The degradation of 135I‐apoprotein B of human low‐density lipoprotein by cell extracts of cultured bovine aortic smooth muscle cells was determined by measuring the formation of acidsoluble products and by analyzing the electrophoretic patterns of digested apoprotein in gels containing sodium dodecyl sulfate. Degradation resulted in an initial rapid accumulation of a limited number of distinct smaller fragments. Two products with apparent molecular weights of 220000 and 200000 predominated. Pepstatin inhibited proteolysis almost completely, as measured by either assay.

Leupeptin decreased hydrolysis to acid‐soluble products by approximately 50%, but had no effect on the initial cleavage of intact apoprotein B. Similar results were found in the case of extracts from cultured human skin fibroblasts and from adult bovine arterial smooth muscle. Leupeptin inhibited intracellular degradation of 125I‐apoprotein B in cultured cells by approximately 50%. It is concluded that the intralysosomal degradation of apoprotein B involves an initial limited endoproteolytic attack at susceptible sites by cathepsin D. This and other enzymes, including cathepsin B, then act synergistically to bring degradation to completion.
Sequencing of Peptide Mixtures by Edman Degradation and Field‐Desorption Mass Spectrometry
Authors: SHIMONISHI Yasutsugu , HONG Yeong‐Man , KITAGISHI Toyoko , MATSUO Takekiyo , MATSUDA Hisashi , KATAKUSE Itsuo
Published: March 2005

A new procedure is described for sequencing of peptide mixtures by a combination of Edman degradation and field‐desorption mass spectrometry. The procedure involves measurement of the mass values of quasi‐molecular ions ([M + H]+) of constituent peptides in the mixture and their fragments degraded by the Edman method. Calculation of all the possible mass differences of the mass values before and after degradation and identification of the phenylthiohydantoins released reveal the N‐terminal amino acids of individual peptides in the mixture. Repetition of these operations gives the amino acid sequences of individual peptides in the mixture. As typical examples, the sequencing of peptide mixtures prepared by proteolytic digestion of glucagon are described. In addition, a computer program was designed for determining the possible sequences of proteins from the partial sequences and mass values of their proteolytic peptides. Output data showed that the entire amino acid sequence of glucagon can be determined by only four and two cycles of degradation of chymotryptic and tryptic peptides, respectively.
Expression of Bacteriophage M13 DNA in vivo
Authors: SMITS Mari A. , SCHOENMAKERS John G. G. , KONINGS Ruud N. H.
Published: March 2005

Hybridization studies have indicated that on the M13 genome there are located regions which are transcribed in vivo with a high as well as with a low frequency. The region with the highest transcriptional activity coincides with the region coding for the polypeptides which during the infection cycle are needed most abundantly (gene V and gene VIII protein). The hybridization studies furthermore have indicated that at least a number of the promoters previously detected in vitro are operative in the infected cell. In addition the experimental data are consistent with the presence of three transcription termination signals on the M13 genome.

With the aid of analytical and preparative hybridization studies we have been able to establish that during the infection cycle at least eleven M13‐specific RNA species are made which range in size from 280 to 2000 nucleotides. The six major RNA species (i.e. an 8‐S RNA with 370 nucleotides, a 9‐S with 420, three 11‐S species with 760, 810 and 860 nucleotides and a 14‐S species with 1140 nucleotides) are encoded by the region with the highest transcriptional activity (located between the N‐terminal end of gene X and the C‐terminal end of gene VIII). Characterization of these RNA species with respect to their genetic origin, codogenic properties and nucleotide sequences has indicated that their synthesis is terminated at the previously detected rho‐independent transcription termination signal T0.25, which is located immediately after gene VIII. From the data obtained it furthermore could be deduced that the 5′ termini of these RNA species are located as follows: for the 8‐S RNA immediately in front of gene IX (promoter G0.18), the 9‐S RNA within or immediately distal to the 5′ end of gene VII, the three 11‐S RNAs within the C‐terminal end of gene X and the 14‐S RNA immediately in front of gene X. Analyses of the 5′ ends of the major RNA chains synthesized in vivo have indicated that four (i.e. the 9‐S and three 11‐S species) out of the six RNA species are the result of processing of precursor molecules. For this processing the RNA processing enzymes RNase III, RNase E, and RNase P are not required. The meaning of these results, in terms of the mechanisms by which the expression of the M13 genome is regulated, is discussed.
Specific Protein Phosphorylation during Stimulation of Amylase Secretion by β‐Agonists or Dibutyryl Adenosine 3′,5′‐Monophosphate in the Rat Parotid Gland
Authors: JAHN Reinhard , UNGER Clemens , SÖLING Hans‐Dieter
Published: March 2005

The present study was undertaken in order to examine the possible involvement of protein phosphorylation during β‐adrenergic stimulation in the rat parotid gland. Isolated parotid gland slices were stimulated by either isoproterenol or dibutyryl adenosine 3′,5′‐monophosphate (Bt2cAMP) in the presence or absence of propranolol. Amylase output was measured as a parameter for the degree of stimulation of secretion.

Stimulation of secretion by either isoproterenol or Bt2AMP was associated with phosphorylation of three protein bands as revealed by sodium dodecylsulfate/polyacryla:nide gel electrophoresis and autoradiography. The apparent molecular weights of the three proteins were 35100 (protein I), 25 700 (protein II) and 20400 (protein II).

After cell fractionation by differential and gradient centrifugation, protein I was enriched in a light membrane fraction most likely corresponding to the plasma membrane as revealed by marker enzyme analysis. Proteins II and III were recovered in a denser fraction containing mainly mitochondria and rough micrcsomes. The effect of isoproterenol but not that of Bt2cAMP on phosphorylation of all three protein bands was completely abolished by propranolol.

The different time course in the stimulation of amylase secretion by isoproterenol and Bt2cAMP respectively was reflected by corresponding differences in the time course of protein phosphorylation.
Inducible β‐Xyloside Permease as a Constituent of the Xylan‐Degrading Enzyme System of the Yeast Cryptococcus albidus
Authors: KRÁTKÝ Zdeněk , BIELY Peter
Published: March 2005

The yeast, Cryptococcus albidus, depending on whether it is grown on xylan or glucose, differs remarkably in the ability to take up inducers of extracellular endo‐1,4‐β‐xylanase synthesis. In washed, glucose‐grown cells the initially low ability to take up xylobiose or methyl β‐d‐xylopyranoside, increases during incubation with these compounds after a lag‐phase shorter than the induction time of the extracellular β‐xylanase. Using of methyl β‐d‐[U‐14C]xylopyranoside as a very slowly metabolizable inducer of β‐xylanase it has been established that the increase of the rate of xylobiose or methyl xyloside uptake is due to induction of an active transport system for methyl β‐d‐xyloside and β‐1,4‐xylooligosaccharides. The system is called β‐xyloside permease. The permease activity of induced cells decreases in the absence of β‐xylanase inducers. The induction of permease as well as its inactivation (degradation) can be prevented with cycloheximide, thus both events appear to be dependent on de novo protein synthesis. In analogy with other active transport systems, β‐xyloside permease function can be effectively blocked by inhibitors of energy metabolism in the cells.

The demonstrated example of induction of a permease, for inducers and products of hydrolysis of an extracellular polysaccharide hydrolase, points to a new feature of induction of extracellular enzymes in eucaryotic microorganisms.
Cockroach Collagen: Isolation, Biochemical and Biophysical Characterization
Authors: FRANCOIS Jean , HERBAGE Daniel , JUNQUA Simone
Published: March 2005

Collagen fibrils from the mesenteric connective sheath of the adult cockroach Periplaneta americana were extracted by enzymatic digestion with pepsin and were purified. Chromatographic studies and sodium dodecylsulfate electrophoresis revealed the presence of a single chain. It was demonstrated that the structure of this collagen could be represented by the formula (α)3. The amino acid composition is typical of collagens (one‐third glycine, and a high imino acid content) and similar to that of type II. The carbohydrate content was high (8.8%), and the cyanogen bromide pattern was different from that of known collagens. The chains were linked by the stable intermolecular bond dihydroxylysinonorleucine. The banding patterns of the segment‐long‐spacing crystallites and of the reconstituted fibrils were similar to type I collagen. The molecular weight (Mr 280000) and length (285 nm) were typical, but the denaturation temperature was high (38.5°C). It was concluded that cockroach mesenteric collagen showed the characteristic features of invertebrate mesodermal collagens, except that of the thermal stability of the triple‐helical structure.
Functional Studies on Ribosomes Lacking Protein L1 from Mutant Escherichia coli
Authors: SUBRAMANIAN Alap R. , DABBS Eric R.
Published: March 2005

The function of protein L1 in protein biosynthesis has been examined using ribosomes from two independently derived mutants of Escherichia coli, both of which lacked this protein. In systems in vitro with phage MS2 RNA or poly(U) as message, the mutant ribosomes showed from 40% to 60% of the activity of wild‐type ribosomes. The reduction in activity was apparent in the kinetics of [14C]phenylalanine incorporation throughout the incubation period. The activities were restored fully to the wild‐type level by the addition of purified protein L1. These results show on the one hand that protein L1 is not essential for protein biosynthesis but, on the other, its presence can significantly increase the overall rate of this process. The data further indicate it as likely that protein L1 exerts its effect at the elongation step.
Modification of Carboxylate Groups in Bovine Pancreatic Phospholipase A2
Authors: FLEER Eduard A. M. , VERHEIJ Hubertus M. , de HAAS Gerard H.
Published: March 2005

Carboxylase groups in bovine pancreatic phospholipase A2 were modified using a water‐soluble carbodiimide and semicarbazide. At pH 5.5 rapid modification of 13 out of the total of 15 carboxylates occurred, leaving Asp‐39 and Asp‐99 unmodified. Subsequent modification with radioactive semicarbazide at pH 3.5 resulted in the labelling of Asp‐39. In the modified proteins both Ca2+ binding and enzymatic activity are completely lost.

Modification of the enzyme at pH 5.5 in the presence of Ca2+ ions yielded a protein with three unmodified carboxylates which is capable of binding Ca2+ ions, while 15% residual activity was found. A second reaction, using [14C]semicarbazide at pH 5.5 in the absence of Ca2+, leads to the incorporation of one mole of [14C]semicarbazide/mole of protein with loss of enzymatic activity and Ca2+ binding. The label was found to be attached to Asp‐49, demonstrating the essential role of Asp‐49 in Ca2+ binding.

Studies on the pH dependence of the Ca2+ binding to the protein suggested that Asp‐49 has an apparent pK of 5.2.
Mass Spectrometric Sequence Studies of a Superoxide Dismutase from Bacillus stearothermophilus
Authors: AUFFRET Anthony D. , Blake Timothy J. , WILLIAMS Dudley H.
Published: March 2005

A partial sequence of the manganese‐containing superoxide dismutase from Bacillus stearothermophilus has been determined by mass spectrometry. A new fragmentation reaction at N‐terminal proline is described and an ‘in‐chain’ fragmentation, also occurring at proline, has been observed. There is some evidence that the latter cleavage may also occur at other residues. Some differences from the classically determined sequence are reported, and the validity of these observations is discussed.
The Electrochemical Proton Gradient Generated by the Fumarate‐Reductase System inEscherichia coli and Its Bioenergetic Implications
Authors: HELLINGWERF Klaas J. , BOLSCHER Jan G. M. , KONINGS Wil N.
Published: March 2005

Proton translocation, coupled to electron transfer in the fumarate reductase system, generates and electrochemical potential gradient for protons (). The magnitude of this free energy gradient has been determined in theEscherichia coli strains ML 208–225 and AN 283. The measurements were performed in (inverted) membrane particles, right‐side out membrane vesicles and EDTA‐treated intact cells in external media of various ionic compositions and pH.

The maximal values of in these three systems were +103, −101 and −105 mV, respectively. This implicates that in E. coli, upon transition from oxygen to fumarate as electron acceptor, the magnitude of the decreases considerably. This change of has substantial consequences for the cellular metabolism.
Frequency and Diversity of RNA Sequences in Different Cell Types of the Fungus Schizophyllum commune
Authors: ZANTINGE Bert , HOGE J. Harry C. , WESSELS Joseph G. H.
Published: March 2005

In the basidiomycete Schizophyllum commune an RNA complexity of 13500 average‐sized (1100 nucleotides) RNA sequences was found by means of RNA excess hybridization to complementary DNA. 600 abundant poly(A)‐containing RNA sequences were present in about 100 copies per cell, whereas the rare 13000 poly(A)‐containing RNA sequences were present in about 5 copies per cell on average.

Homologous and heterologous cDNA hybridizations to poly(A)‐rich RNAs revealed some quantitative, but no significant qualitative differences between the monokaryotic and the dikaryotic cell type. Heterologous hybridization and subsequent hybridizations of remaining single‐stranded cDNA to poly(A)‐rich RNAs from both cell types showed qualitative differences amounting to 2–3% of the rare sequences in the two cell types. However, from the results of similar hybridizations with total RNAs, i.e. including non‐polyadenylated RNA, it could be calculated that the RNA sequences in monokaryons and dikaryons are at least 99% the same.

Identity of RNA sequences in monokaryons and dikaryons was also inferred by comparison of the patterns of polypeptides synthesized by translation of total RNA in cell‐free systems.
Identification in Xenopus laevis of a Class of Oocyte‐Specific Proteins Bound to Messenger RNA
Authors: DARNBROUGH Christopher H. , FORD Peter J.
Published: March 2005

Proteins associated with poly(A)‐rich RNA in Xenopus laevis oocytes have been identified in immature ovary homogenates using sucrose gradient centrifugation, oligo(dT)‐cellulose fractionation and analysis by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and two‐dimensional acid/urea/sodium dodecyl sulphate gel electrophoresis. At least eight proteins are associated with ribonucleoprotein complexes and have the following properties.

Four major proteins, mRNP1–4, cosediment with poly(A)‐rich RNA at 40–200 S, bind to oligo(dT)‐cellulose and are oocyte‐specific. These are very basic proteins with molecular weights of about 50 × 103, 52 × 103, 56 × 103 59 × 103.

Two further proteins, mRNP5,6, molecular weights 75 × 103 and about 100 × 103, also co‐isolate with poly(A)‐rich RNA. mRNP7(Mr, 16 × 103) cosediments with poly(A)‐rich RNA but fails to bind to oligo(dT)‐cellulose. mRNP8 (Mr, 22 × 103) binds to oligo(dT)‐cellulose but sediments more slowly than the bulk of poly(A)‐rich ribunucleoprotein, at 30–70 S.

mRNP1–4 are present in immature oocytes but only mRNP3–4 are found in full‐grown oocytes, while none of them could be detected in Xenopus liver or reticulocytes. These proteins are largely cytoplasmic, associated with free mRNP particles, and differ in molecular weight from proteins isolated from polysomal mRNPs in somatic tissues.

mRNP1–4 are very abundant in immature oocytes; sedimentation properties are consistent with a protein: RNA ratio of 4:1 (w/w). The sedimentation constant of mRNP particles is resistant to concentrations of ribonuclease A which degrade ribosomes, although mRNA isolated from these ribonuclease‐treated particles appears to be partially degraded.

The role of ribonucleoprotein complexes in the stability and storage of mRNA during oogenesis is discussed.
Purification and Some Properties of NAD‐Glycohydrolase from Conidia of Neurospora crassa
Authors: MENEGUS Faustino , PACE Mario
Published: March 2005

NAD‐glycohydrolase from conidia of Neurospora crassa was purified by affinity chromatography, using 4‐methylnicotinamide adenine dinucleotide as ligand immobilized onto Sepharose through a hydrophilic spacer arm.

The pure enzyme is a glycoprotein with an isoelectric point of 5.5 and a molecular weight of 33000 as determined by sodium dodecylsulphate gel electrophoresis.

The specific activity is the highest so far found for NAD‐glycohydrolases and in various aspects the enzyme is different from that isolated from mycelia ofN. crussa grown in a ‘zinc‐deficient’ medium.