bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of RNA Synthesis in Neurospora crassa Hyphae Treated with Picolinic Acid
Authors: MARTEGANI Enzo
Published: March 2005

Picolinic acid, a metal‐chelating agent, blocks the nuclei of germinating conidia of Neurosporu crassa in the G1 phase. The addition of picolinic acid to exponentially growing cultures of N. crassa in glucose medium causes an immediate inhibition of growth. Concentrations higher than 50 mM completely inhibit growth of mycelia, whereas lower concentrations (20‐ 30 mM) give only a partial inhibition. Picolinic acid, added at the concentration of 20 mM, immediately blocks the accumulation of stable RNA, while protein accumulation continues at an unchanged rate for at least 30 min and only thereafter is slightly inhibited. DNA accumul.ation rapidly slows down after the addition of the drug. The level of polyadenylated RNA decreases quickly after the addition of picolinic acid, and the rate of its synthesis also appears to be inhibited. Picolinic acid greatly affects the metabolism of stable RNA in N. crasa: both processing and transcription of rRNA are sequentially inhibited with a moderate accumulation of precursor rRNA. It is concluded that the effects of picolinic acid on the nuclear division cycle in Neurospora may be related to the requirement of specific gene products to enter the S phase, whose synthesis is inhibited following the reduction of polyadenylated RNA synthesis.
Modification of the Phosphatidylcholine‐Transfer Protein from Bovine Liver with Butanedione and Phenyiglyoxal
Authors: AKEROYD Robert , LANGE Louis G. , WESTERMAN Jan , WIRTZ Karel W. A.
Published: March 2005

Modification of arginine residues with 2,3‐butanedione and phenylglyoxal completely inhibits the transfer activity of the phosphatidylcholine transfer protein from bovine liver. Removal of borate and butanedione leads to a slow reactivation of the protein.

Both α‐dicarbonyl reagents modify three of the ten arginine residues present per protein molecule. The extent of modification is linearly related to the loss of activity.

Inactivation with butanedione is greatly diminished when the protein is bound to strongly negatively charged vesicles. Under these conditions a rapid modification of two arginine residues is observed. This suggests that the transfer protein contains one arginine residue essential for activity, probably as a binding site for the negatively charged phosphate group of the phosphatidylcholine molecule.

This study provides convincing evidence that arginine residues may play an essential role in phospholipid‐ protein interactions.
Primary Structure of the Bovine β‐Crystallin Bp Chain
Authors: DRIESSEN Huub P. C. , HERBRINK Paul , BLOEMENDAL Hans , de JONG Wilfricd W.
Published: March 2005

The major polypeptide chain of bovine β‐crystallin, βBp, was fragmented by means of cyanogen bromide treatment and by enzymatic digestions. Manual and automated Edman degradation of the resulting peptides provided the complete amino acid sequence of the 0Bp chain. The N‐terminal alanine residue was shown to be N‐α‐acetylated by mass spectrometry. The chain has a length of 204 residues and a calculated molecular weight of 23210. There is a considerable degree of homology between the N‐terminal and C‐terminal halves of the chain, presumably reflecting a tandem duplication of a shorter ancestral gene. The sequence of βBp is sufficiently related to that of γ‐crystallin II to place these proteins in the same superfamily. No sequence relationship was found with the α‐crystallin chains.
Improved Assay and Mechanism of the Reaction Catalyzed by the Chitin Synthase from Saccharomyces cerevisiae
Authors: FÄHNRICH Marianne , AHLERS Jan
Published: March 2005

An improved filtration method is introduced to perform kinetic investigations on the chitin synthase reaction. This method is especially suited for the assay of a large number of samples necessary in enzyme kinetic studies.

From initial rate data the possibility could be excluded that the two‐substrate reactions occurs by a random or a ping‐pong mechanism.

Investigations of product inhibition exclude a rapid random mechanism but favour an ordered mechanism with UDP‐N‐acetylglucosamine as the first substrate.

This result was confirmed by isotope exchange studies.
Oligosaccharides in Lysosomal Enzymes
Authors: HASILIK Andrej , VON FIGURA Kurt
Published: March 2005

The distribution of the different types of oligosaccharides in cathepsin D and in β‐hexosaminidase syn‐ thesized in cultured human fibroblasts was studied by using endo‐β‐N‐acetylglucosaminidase H as a probe for high‐mannose oligosaccharides. The enzymes were specifically labelled in the protein or the carbohydrate moiety. In both enzymes, resistant and cleavable oligosaccharides were found. The resistant oligosaccharides prevailed the secreted enzymes. Precursor molecules of cathepsin D contained two oligosaccharide side chains. Multiple forms of the precursor are synthesized with both, one or none of two oligosaccharides sensitive to the action of the endo‐β‐N‐acetylglucosaminidase H.

In fibroblasts unable to phosphorylate lysosomal enzymes (mucolipidosis 11) the excessively secreted lysosomal enzymes contained predominantly oligosaccharides resistant to endo‐β‐N‐acetylglucosaminidase H.
Evidence for a Mg2+‐Induced Conformational Change at the ATP‐Binding Site of (Na++ K+)‐ATPase Demonstrated with a Photoreactive ATP‐Analogue
Authors: REMPETERS Gerold , SCHONER Wilhelm
Published: March 2005

The 3′‐ribosyl ester of ATP with 2‐nitro‐4‐azidophenyl propionic acid has been prepared and its ability to act, as a photoaffinity label of (Na++ K+)‐ATPase has been tested.

In the dark 3′‐O‐[3‐(2‐nitro‐4‐azidophenyl)‐propionyl]adenosine triphosphate (N3‐ATP) is a substrate of (Na++ K+)‐ATPase and a competitive inhibitor of ATP hydrolysis.

Upon irradiation by ultraviolet light, N3‐ATP photolabels the high‐affinity ATP‐binding site and is covalently attached to the α‐subunit and an approximately 12000‐Mr component.

Photolabeling of the α‐subunit by N3‐ATP irreversibly inactivates (Na++ K+)‐ATPase.

Photoinactivation is strictly Mg2+‐dependent. Na+ enhances the inactivation. ATP or ADP and K+ protect the enzyme against inactivation.

Mg2+;, in concentrations required for photoinactivation, protects (Na++ K+)‐ATPase against inactivation by tryptic digestion under controlled conditions.

It is assumed that a conformational change of the ATP‐binding site of (Na++K+)‐ATPase occurs upon binding of Mg2+ to a low‐affinity site.
Adeno‐Associated Virus RNA Transcription in vivo
Authors: MARCUS Carol J. , LAUGHLIN Catherine A. , CARTER Barrie J.
Published: March 2005

We have studied RNA transcripts of the defective parvovirus, adeno‐associated virus (AAV) present in poly‐(A)rich and poly‐(A)free fractions of nuclear and cytoplasmic RNA prepared from cells infected together with a helper adenovirus. Cytoplasmic poly‐(A)rich RNA contains three overlapping spliced AAV RNAs having sizes of 3.9 × 103, 3.3 × 103 and 2.3 × 103 bases respectively. The nuclear precursors of these RNAs appear to be the coterminal unspliced poly(A)‐rich RNAs containing 4.2 × 103, 3.6 × 103 and 2.6 × 103 bases respectively. These unspliced RNAs were also found in the cytoplasm. The nuclear poly(A)‐free RNA contained a heterogenous population of AAV RNAs that were generally smaller than 2.3 × 103 bases. In addition, the Hirt pellet fraction of the nuclear RNA contained two discrete AAV poly(A)‐free RNAs having sizes of 2.5 × 103 and 2.8 × 103 bases.

The 4.2 × 103 and 3.6 × 103‐base unspliced RNAs are more abundant than the coterminal 3.9 × 103 and 3.3 × 103‐base spliced RNAs whereas the 2.3 × 103‐base spliced RNA is much more abundant than the 2.6 × 103‐ base unspliced RNA. Thus, the cytoplasmic abundance of the AAV spliced RNAs appears to be controlled in part by the post‐transcriptional events of splicing or message stability.

We also analysed the effects of AAV defective‐interfering genomes upon AAV transcription. These studies showed that when synthesis of standard AAV genomes was inhibited more than 10‐fold by defective‐interfering genomes there was no significant effect on the types or amounts of AAV RNA transcripts which accumulated. These observations indicate that interference by defective‐interfering genomes occurs mostly at the level of DNA replication rather than transcription.
Assembly of Cytochrome c. Apocytochrome c Is Bound to Specific Sites on Mitochondria before Its Conversion to Holocytochrome c
Authors: HENNIG Bernd , NEUPERT Walter
Published: March 2005

Transport of apocytochrome c across the outer mitochondrial membrane and conversion to holocytochrome c were studied in vitro. Apocytochrome c was synthesized in a cell‐free homogenate from Neurospora crassa. Transfer in vitro was accomplished in a reconstituted system consisting of the postribosomal supernatant of the cell‐free homogenate and of isolated and purified mitochondria from Neurospora. The reconstituted system has the following characteristics:

Apocytochrome c is rapidly cleared from the supernatant and holocytochrome c appears in the mitochondria with the same kinetics. More than 80% of the apocytochrome c employed is converted to holocytochrome c. No transient accumulation of apocytochrome c is found in mitochondria.

The heme group becomes covalently linked to apocytochrome c in the reconstituted system as demonstrated by analysis of tryptic peptide maps of the apoprotein and holoprotein.

Deuterohemin added to the reconstituted system but not deuteroporphyrin inhibits the formation of holocytochrome c. This inhibition is reversed by protohemin.

In the presence of deuterohemin about half of the apocytochrome c remains in the supernatant; the other half becomes associated with the mitochondria. The latter portion is tightly bound and is specifically released upon incubation of the mitochondria with excess apocytochrome c. It is converted to holocytochrome c after addition of protohemin.

We conclude from these observations that apocytochrome c is transported across the outer mitochondrial membrane via receptor sites. In the presence of the heme analogue deuterohemin, binding to the receptor sites on the cytoplasmic surface of the outer mitochondrial membrane still takes place but translocation does not. The latter step is apparently coupled to the covalent linkage of the heme group. We suggest that the formation of the thioether bonds between apoprotein and heme is catalysed by an enzyme in the intermembrane space and that deuterohemin can compete with protohemin for binding to the enzyme. Finally, the data indicate that it is the heme group and not the porphyrin group which is coupled to the apoprotein.
Rotational Diffusion of Eosin‐Labeled Pyruvate Dehydrogenase Complex of Escherichia coli
Authors: VISSER Antoniz J. W. G. , SCOUTEN William H. , LAVALETTE Daniel
Published: March 2005

The enzymatically reduced lipoyl residues of the transacetylase component of the pyruvate dehydrogenase complex from Escherichia coli were labeled with eosin maleimide. Using eosin as triplet probe, triplet‐triplet absorption dichroism measurements were performed to obtain rotational correlation times of the complex in the microsecond time domain. It was found that the hydrodynamic properties determined from the correlation times are in very good agreement with those obtained with other methods of different origin. The results can be fully explained by eosin molecules rotating with the whole complex, which consists of a mixture of heavy (60 S) and light (20 S) particles. Since no independent mobility could be detected it is suggested that thc (charged) chromophoric group is folded against the protein surface. Labeling with excess eosin maleimide tends to destabilize the complex. since the longer correlation time (60 S) decreases and the contribution of the shorter correlation time (20 S) becomes more significant upon labeling.
Messenger RNA for Human Tissue Plasminogen Activator
Authors: OPDENAKKER Ghislain , WEENING Hans , COLLEN Désiré , BILLIAU Alfons , DE SOMER Piet
Published: March 2005

A human melanoma cell line (Bowes), which secretes extrinsic (tissue‐type) plasminogen activator, was used as a source for the preparation of mRNA for extrinsic plasminogen activator. The cells were lysed and total RNA was extracted with the phenol method. A poly(A)‐rich RNA fraction was isolated by affinity chromatography on oligo(dT)‐cellulose. This preparation was translated by oocytes into (a) protein(s) that had biological activity in the assay for extrinsic plasminogen activator. On sucrose gradient centrifugation the translatable fraction of the RNA migrated with a sedimentation coefficient of approximately 19 S, a value compatible with the molecular weight of extrinsic plasminogen activator (70000). The translation product was characterized as being similar to or identical with authentic extrinsic plasminogen activator by the following criteria: (a) serological cross‐reactivity with purified extrinsic plasminogen activator in neutralization and immunoprecipitation reactions; (b) plasminogen dependency of fibrinolytic activity and (c) apparent molecular weight of 70000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis.
Alterations Induced by a Prolonged Fasting: Opposite Effects on the β‐Adrenergic Receptor‐Coupled Adenylate‐Cyclase System and on Lipolysis in Fat Cells from Rat
Authors: GIUDICELLI Yves , LACASA Daniéle , AGLI Brigitte
Published: March 2005

The maximal number of β‐adrenergic receptors in rat fat‐cell membranes from 72‐h‐fasted rats is about two‐times higher than in fed rats. However, the affinity of these receptors for β‐agonists and antagonists and the ability of guanosine 5′‐[β,γ‐imido]triphosphate (100 μM) to reduce the receptor binding affinity for β‐agonists are unaltered by fasting.

Basal and fluoride‐stimulated activities of adenylate cyclase are similar in adipocyte membranes from 72‐h‐ fasted and fed animals. However, relative to the basal activity, maximal stimulation by (—)‐isoproterenol is 33 ±5 %higher in fasted than in fed adipocytes. The sensitivity of adenylate cyclase to (—)‐isoproterenol is also increased in fat‐cell membranes from fasted animals, as the concentration of (—)‐isoproterenol required for half‐ maximal activation is three‐times lower in fasted than in fed adipocytes.

Basal adenylate cyclase activity is also about three‐times more sensitive to stimulation by guanosine 5′‐[β,γ‐imido]triphosphate in fasted than in fed adipocyte membranes. This increased sensitivity of adenylate cyclase to guanosine 5′‐[β,γ‐imido]triphosphate in fasting is much more pronounced when adenylate cyclase activity is stimulated by isoproterenol.

In contrast, although basal lipolysis is unaltered by fasting, lipolysis stimulated in vitro by isoproterenol, adrenocorticotropin or dibutyryladenosine 3′,5′‐monophosphate is severely depressed in fat cells from 72‐h‐fasted rats. However, the concentration of (—)‐isoproterenol and adrenocorticotropin required for half‐maximal activation of lipolysis are similar in fasted and fed adipocytes.

From this study, it is concluded that, contrary to the fasting‐induced decreased lipolytic responsiveness which is paradoxically observed in adipocytes in vitro, the fasting‐induced changes found in the adenylate cyclase‐ coupled‐β‐adrenergic receptor‐system of rat adipocyte are most probably physiologically relevant as these changes contribute to promote cyclic AMP synthesis and, consequently, to stimulate lipolysis, as is observed in vivo during fasting.
Ribosomal Protein S1 Associates with Escherichia coli Ribosomal 30‐S Subunit by Means of Protein‐Protein Interactions
Authors: BONI Irina V. , ZLATKIN Igor V. , BUDOWSKY Edward I.
Published: March 2005

Ribosomal proteins S1 when associated with the 30‐S subunit does not interact with 16‐S RNA but its binding is determined mostly by protein‐protein interactions. These conclusions are based on the following data.

Ultraviolet irradiation (λ=254 nm) of the 30‐S subunit does not result in the covalent cross‐linking of S1 with 16‐S RNA at irradiation doses up to 150 quanta/nucleotide, whereas the irradiation under the same conditions of S1 · polynucleotide complexes [S1 · poly(U), S1 · poly(A) and S1 · Qβphage RNA] induces effective formation of polynucleotide‐protein cross‐links.

Mild treatment of 30‐subunits lacking S‐1 with RNase A or with cobra venom endonuclease results in removal of 10–20%of the total nucleotide material but does not affect their sedimentation characteristics or their S1 binding capacity.

The association of S1 with S1‐depleted 30‐S subunits is insensitive to aurintricarboxylic acid, which is known as a strong inhibitor of complex formation between S1 and polynucleotides.

Mild trypsin treatment of S1‐depleted 30‐S subunits greatly reduces their S 1 binding capacity.
Effect of Cations on the Acetylation of Chromatin in vitro
Authors: DOD Barbara , KERVABON Aline , PARELLO Joseph
Published: March 2005

The effect of the differently charged cations Na+, Mg2+, spermidine and spermine on the acetylation of histones in vitro in soluble chromatin and in core particles was investigated. Up to a given concentration, which depends on the charge of the cation, all four cations activate the acetylation of these fractions. Above this critical concentration a gradual inhibition of the acetyltransferase activity occurs.

Spermine, spermidine and Mg2+, but not Na+, affect the relative accessibilities of the acetyltransferase to the individual histones. As the concentration of these three polyvalent cations increases there is a gradual increase in the relative acetyl incorporation in histone H3 accompanied by a corresponding decrease in H4.

Kinetic aspects of the system were also studied. A minor fraction of the soluble chromatin (mononucleosomes and dinucleosomes) which is preferentially digested by micrococcal nuclease is also acetylated preferentially. The role chromatin structure plays in determining the specificity of histone acetylation is discussed.
Primary Structure of the Glycans from Human Lactotransferrin
Authors: SPIK Gencviève , STRECKER Gérard , FOURNET Bernard , BOUQUELET Stéphane , MONTREUIL Jean , DORLAND Lambertus , VAN HALBEEK Herman , VLIEGENTHART Johdnnes F. G.
Published: March 2005

The polypeptide chain of human lactotransferrin possesses two glycosylation sites to which glycans are linked through an N‐(β2‐aspartyl)‐N‐acetylglucosaminylamine bond and which are structurally heterogenous. After chymotryptic or pronase digestions, glycopeptides with five different glycan structures could be isolated. For three of them, the structure has been determined by the application of methanolysis, methylation analysis, hydra‐zinolysis/nitrous deamination, enzymatic cleavage and 1H‐NMR spectroscopy at 360 MHz: Glycopeptides A and B : NeuAc(α2‐6)Gal(β1‐4)GlcNAc(βl‐2)Man(α‐ 3)[NeuAc(α2‐6)GaI(β‐4)GlcNAc(β1 ‐2)Man(α1‐b)]Man‐ (β1‐4)GlcNAc(β1‐4)[Fuc(α1‐6)]GlcNAc(βl‐)Asn;Glycopeptide C : NeuAc(α2‐6)(Gal(β1‐4)GlcNAc(β1‐2)Man‐ (α1 ‐3){Gal(βl‐4)[Fuc(αl‐3)]GlcNAc(β1‐2)Man(α1‐6)}Man(β1‐4)GlcNAc(β1‐4)[Fuc(α1‐6)]GlcNAc(β1 ‐)Am. Glycopeptide D : NeuAc(α2‐6)Gal(β3‐4)GlcNAc(β1‐2)Man(α1‐3)[Gal(β1‐4)GlcNAc(βl‐2)Man(α1‐6)]Man(β1‐4)Glc‐ NAc(βI‐4)[Fuc(a1‐6)]GlcNAc(βI ‐)Am.

Two other glycopeptides were obtained in very low amount and possess more complex structures.
Using 2′(3′)‐O‐Trinitrophenyl Derivatives of Adenine Nucleotides to Study the Structure and Mechanism of Functioning of Soluble Mitochondrial ATPase
Authors: KORMER Zinaida S. , KOZLOV Igor A. , MILGROM Yakov M. , NOVIKOVA Irina Yu.
Published: March 2005

The 2′(3′)‐O‐trinitrophenyl (N3ph) derivatives of the adenine nucleotides are strong competitive inhibitors of isolated mitochondrial ATPase (factor F1). Ki decreases in the order N3phAdo >N3phAMP >N3phADP and is equal to 8 nM for N3phADP. Picric acid, which activates the ATPase reaction of factor F1 without changing the Km(app.), prevents the inhibiting action of N3phADP. At pH 7.6 the inhibition of factor F1 is accompanied by the binding of one molecule of N3phADP to a molecule of the enzyme. This binding leads to changes in the absorption spectrum, but not in the intensity of the fluorescence of the N3phADP.

At pH 6.7 one or two molecules of N3phADP bind with the tight binding sites of factor F1. This binding is accompanied by the manifold enhancement of the fluorescence of N3phADP. The results obtained indicate that the sites of factor F1 that tightly bind nucleotides are immersed in the hydrophobic pocket of the protein molecule.
Purification and Characterization of Bovine Gastricsin
Authors: MARTIN Patrice , TRIEU‐CUOT Patrick , COLLIN Jean‐Claude , RIBADEAU DUMAS Bruno
Published: March 2005

The results reported in the present paper and N‐terminal sequence homologies established by other authors strongly support the assumption that the gastric protease previously called bovine pepsin I or bovine pepsin B belongs to the aspartate protease group and corresponds to gastricsin (or pepsin C) (EC 3.4.23.3) from other species.

Bovine gastricsin was prepared from commercial extracts of adult bovine vells by a procedure involving DEAE‐cellulose chromatography, gel filtration on Sephacryl S‐200 and a further DEAE‐cellulose chromatography. The preparation thus obtained was shown to be free of chymosin and bovine pepsin A by immunodiffusion, selective inactivation in urea and isoelectric focusing. Its molecular weight was estimated by gel filtration to be 32800.

Bovine gastricsin displayed microheterogeneity on isoelectric focusing with pl values of the components ranging from 3.5 to 4.0. Chromatography of bovine gastricsin on hydroxyapatite separated three fractions, none of them being homogeneous by isoelectric focusing. Concanavalin‐A‐Sepharose 4B bound bovine gastricsin to some extent, but without any significant fractionation. Proteolytic activity could be detected directly on the isoelectric focusing gel for all the components of gastricsin and its fractions from hydroxyapatite and concanavalin‐A‐ Sepharose 4B.

Bovine gastricsin and its fractions from hydroxyapatite have similar amino acid compositions, different from those of bovine chymosin and pepsin A but obviously related to those of human, simian and porcine gastricsins.

Bovine gastricsin which is inactivated by reaction with diazoacetyl‐DL‐norleucine methyl ester and with 1,2‐epoxy‐(p‐nitrophenoxy)propane in a 1:1 and 1:2 stoichiometry, respectively, is able to hydrolyse a synthetic hexapeptide, Leu‐Ser‐Phe(NO2)‐Nle‐Ala‐Leu‐OMe, used as reference substrate for aspartate proteases, and exhibits a low activity towards N‐acetyl‐l‐phenylalanyl‐l‐diiodotyrosine. Its specific clotting activity with x‐casein as substrate is only half of that of chymosin and pepsin A.
Purification and Characterization of a Chymotrypsin‐Like Enzyme from Sperm of the Sea Urchin, Hemicentrotus puleherrimus
Authors: YAMADA Yuji , MATSUI Taei , AKETA Kenji
Published: March 2005

A chymotrypsin‐like enzyme has been purified from sperm of the sea urchin, Hemicentrotus pulcherrimus, using tryptophan methyl ester (TrpOMe) linked to Sepharose 4B as an affinity column for chromatography and gel filtration. The isolated enzyme preparation is homogenous in sodium dodecylsulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 18500‐19000. This enzyme hydrolyses N‐acetyl‐l‐tyrosine ethyl ester (AcTyrOEt) and N‐benzoyl‐l‐tyrosine ethyl ester (BzTyrOEt); the optimal pH is 8.0. It does not hydrolyse N‐benzoyl‐l‐arginine ethyl ester, N‐α‐toluenesulfonyl‐l‐arginine methyl ester, N‐α‐benzoyl‐dl‐arginine‐p‐nitroanilide, hippuryl‐l‐arginine or hippuryl‐l‐phenylalanine. The Michaelis constants for AcTyrOEt and BzTyrOEt are 0.05 mM and 0.0106 mM, respectively. The enzyme activity is inhibited completely by phenyl‐ methylsulfonyl fluoride (PhMeSO2F), chymostatin and l‐I‐tosylamido‐2‐phenylethyl chloromethyl ketone (TosPheCH2Cl), and partially by soybean trypsin inhibitor and N‐7alpha;‐p‐tosyl‐l‐lysine chloromethyl ketone (TosLysCH2Cl). The enzyme is activated by CaCl2, MgC12, NaCl and KCl, and loses its activity in 5 min at 67°C. It digests the jelly coat and vitelline layer, not the fertilization membrane. The microvilli of unfertilized eggs elongate and decrease in number as the vitelline layer lyses. The vitelline layer lytic activity is inhibited completely by PhMeSO2F, TosPheCH2Cl, and chymostatin, and partially by soybean trypsin inhibitor, TosLysCH2C1, and αl‐antitrypsin. We have confirmed by transmission electron microscopy that our chymo‐trypsin‐like enzyme completely digests the vitelline layer. A result implying release of this enzyme from the acrosome vesicle is also reported
Concentration Dependence of the Order‐Disorder Transition of Carrageenans
Authors: BRYCE Thomas A. , CLARK Allan H. , REES David A. , REID David S.
Published: March 2005

The temperature‐induced changes in optical rotation which are associated with the order‐disorder transition of carrageenans have been analysed to investigate the concentration dependence and hence to attempt to derive the number of polysaccharide chains involved in the ordered species at equilibrium. The consequences of the known polydispersity have been analysed by computer‐modelling and it is shown that this need not cause misleading results. For l‐carrageenan segments in 0.1 M NaCl and 0.1 M KCl solution the results point to a two‐chain state and therefore support the proposed double helix model. For x‐carrageenan segments the apparent average number of molecules involved in the ordered state at equilibrium is 3.7, suggesting that if double helices form here they aggregate further. The method of investigation may be generally applicable to measuring the number of chains which associate in ordered states in aqueous solutions of polysaccharides.
The Nature of the Binding Site for Prothrombinase at the Platelet Surface as Revealed by Lipolytic Enzymes
Authors: BEVERS Edouard M. , COMFURIUS Paul , Robert F. A. ZWAAL
Published: March 2005

The nature of the receptor for the prothrombinase complex at the surface of non‐activated platelets was investigated by measuring the platelet prothrombin‐converting activity with a chromogenic substrate assay, after treatment of the platelets with various phospholipases or three different proteolytic enzymes. Platelet pro‐thrombin‐converting activity only decreased after treatment with those phospholipases which are able to hydrolyse phospholipids in the intact platelet and also have the ability to degrade negatively charged phospho‐lipids, phosphatidylserine and phosphatidylinositol. Those phospholipases which do hydrolyse phospholipids in the intact platelet but have no activity towards phosphatidylserine (and phosphatidylinositol) produce an in‐ crease in the platelet prothrombin‐converting activity.

Proteolytic treatment of platelets with trypsin, chymotrypsin or papain did not result in a decrease of pro‐thrombin‐converting activity. It is concluded that negatively charged phosphatidylserine and possibly phos‐phatidylinositol are involved in the prothrombin‐converting activity of non‐activated platelets. We could not demonstrate the involvement of platelet membrane proteins in a receptor for the components of the pro‐thrombinase complex.
Interaction of cAMP Derivatives with the ‘Stable’ CAMP‐Binding Site in the CAMP‐Dependent Protein Kinase Type I
Authors: de WIT Renè J. W. , HOPPE Jürgen , STEC Wojciech J. , BARANIAK Janina , JASTORFF Bernd
Published: March 2005

cAMP binding to the ‘stable’ cAMP‐binding sites in the regulatory subunit of the cAMP‐dependent protein kinase type I was investigated using a set of 18 selected derivatives. All the tested analogues were competitive with [3H]cAMP and inhibitor constants from 12 nM to 20 μM with the free regulatory subunit were determined. The cAMP molecule seemed to be bound by three specific hydrogen bonds to the 5′ and 3′ oxygen, the 2′ hydroxyl, and an ion pair interaction between the negative charge in equatorial position and a positively charged amino acid side chain. The adenine base is rather unspecifically bound with no hydrogen bonds involved. This binding specificity of the ‘stable’ site is similar to the requirement for dissociation as determined by the activation of the kinase by a respective analogue. This indicates that occupation of the ‘stable’ sites leads to activation of the protein kinase. The presence of the catalytic subunit reduced the affinity of most analogues. The binding of one derivative with the negative charge fixed in the axial position is not influenced by the addition of the catalytic subunit and ATP. A plausible model for a conformational change during the activation process in the ‘stable’ site is discussed.
Interaction of Oxidized and Reduced Uteroglobin with Progesterone
Authors: TANCREDI Teodorico , TEMUSSI Piero A. , BEATO Miguel
Published: March 2005

Binding of added progesterone to native uteroglobin requires the reduction of the disulfide bonds that hold together the two polypeptide chains of the protein. The hypothesis that in the native oxidized state of uteroglobin the steroid binding cavity is preformed and occupied by a progesterone molecule has been tested by several experimental means. The results demonstrate that progesterone does not interact with oxidized uteroglobin, and show that the majority of the oxidized uteroglobin molecules purified from pseudopregnant rabbits do not contain a progesterone molecule. Oxidation of reduced uteroglobin in the presence of saturating amounts of progesterone does not result in significant retention of the steroid inside the oxidized protein.
Impaired Phosphorylation of Lysosomal Enzymes in Fibroblasts of Patients with Mucolipidosis III
Authors: HASILIK Andrej , WAHBED Abdul , CANTZ Michael , von FIGURA Kurt
Published: March 2005

The incorporation of [3H]leucine and [32P]phosphate into three lysosomal enzymes, cathepsin D, β‐hexos‐aminidase and arylsulfatase A by fibroblasts from six patients affected with mucolipidosis III was determined. In the mutant cells the incorporation of 32P in the enzymes was reduced by 70‐97% as compared to controls. The residual phosphorylation of lysosomal enzymes is definitely higher than in fibroblasts from patients with mucolipidosis II, where apparently non‐phosphorylated enzymes are formed. In mucolipidosis III the major part of the newly formed enzymes accumulated extracellularly and the cellular enzymes were recovered mainly in their processed forms. In mucolipidosis III arylsulfatase A and the processed forms of cathepsin D exhibited a heterogeneity that was not observed in controls.

β‐Hexosaminidase and cathepsin D secreted by mucolipidosis III fibroblasts contained only a small amount of phosphorylated oligosaccharides with either one or two phosphate groups per oligosaccharide. As in controls the major fraction of phosphate was present as acid‐labile phosphodiester resistant to alkaline phosphatase.

The residual phosphorylation of lysosomal enzymes may be related to the partial intracellular retention and processing of these enzymes in fibroblasts from patients with mucolipidosis III.
The Biosynthesis and Linkage of Teichuronic Acid to Peptidoglycan in Bacillus licheniformis
Authors: J. Barrie WARD  , CURTIS Carol A. M.
Published: March 2005

Membrane and wall‐membrane preparations of Bacillus lichenformis 94 will, if supplied with the appropriate precursors, synthesize teichuronic acid and link it to peptidoglycan although teichuronic acid is absent from walls of this organism. B. lichenformis 94 lacks phosphoglucomutase activity and therefore cannot synthesize the precursor UDPglucuronic acid. The initial reaction of teichuronic acid biosynthesis is catalysed by a translocase and results in the formation of polyprenyl‐diphospho‐N‐acetylgalactosaniine and the release of UMP. This reaction is not inhibited by tunicamycin. The disaccharide repeating unit of the polymer is then formed by the transfer of glucuronic acid from UDPglucuronic acid with the release of UDP. Polymerization of the repeating units occurs by incorporation of new units at the reducing terminus of the growing teichuronic acid chain and the release of polyprenyl diphosphate. The subsequent dephosphorylation of the lipid diphosphate for reuse in the biosynthetic cycle is inhibited by bacitracin. Linkage to peptidoglycan occurs by the formation of a phosphodiester bond between the reducing N‐acetylgalactosamine terminus of the teichuronic acid chain and a 6‐hydroxyl group of a muramic acid residue in the glycan chain of peptidoglycan. Wall‐membrane preparations synthesising teichuronic acid, poly(glycero1 phosphate) teichoic acid and peptidoglycan link the teichuronic and teichoic acids to different glycan chains.
Metabolite‐Controlled Phosphorylation of Phosphofructokinase in Rat Hepatocytes
Authors: BRAND Ingeborg A. , SÖLING Hans‐Dieter
Published: March 2005

The effect of glucose on the phosphorylation of phosphofructokinase has been investigated in hepatocytes isolated froin fed and 24‐h‐starved rats by measuring the 32P radioactivity associated with the enzyme. In addition, the effects of gluconeogenic precursors and of glucagon or Bt2cAMP were followed under these conditions.

The basal incorporation of phosphate into phosphofructokinase in hepatocytes from starved and fed rats was 0.2 ± 0.1 and 0.6±0.2 mol phosphate/mol phosphofructokinase (tetramer) respectively, whereas the maximal incorporation obtained was 1.03 ± 0.2 mol phosphate/mol phosphofructokinase.

With 16 mM l‐lactate + 4 mM pyruvate or with 20 mM l‐alanine, the phosphorylation of the enzyme was lower than the basal incorporation found in hepatocytes from fed and starved rats.

Glucose (20 mM) addition to isolated hepatocytes from fed rats resulted in an increase in the incorporation phosphate into phosphofructokinase to almost maximal value.

In hepatocytes isolated from starved rats, glucose led to a 2.5‐fold increase of phosphorylation as compared to the basal incorporation.

l‐Alanine (20 mM) abolished the glucose‐induced phosphorylation in hepatocytes from starved and from fed animals.

Glucagon (0.1 μM) did not significantly enhance thc phosphorylation of phospliofructokinase in the presence of l‐lactate + pyruvate in hepatocytes from starved rats, but stimulated the phosphorylation to maximal value when cells had been preincubated in the presence of glucose.

In contrast, in liver cells isolated from fed rats. glucagon had no additional effect on phosphofructokinase pliosphorylation in the presence of glucose, but did increase the phosphorylation to maximal value when l‐lactate + pyruvate served as substrates.

l‐Alanine inhibited the glucose‐induced phosphorylation of phosphofructokinase also in the presence of glucagon.

In contrast, glucagon stimulated the phosphorylation of l‐type pyruvate kinase independently from exogenous precursors and from the nutritional state of the animals.

Bt2cAMP (0.1 mM) as glucagon, did not significantly enhance the phosphorylation of phosphofructokinase above the level obtained with glucose alone in hepatocytes from fed rats, but did increase the velocity of the phosphate incorporation in hepatocytes from starved rats, thus leading under both conditions to the maximal incorporation.

It is concluded that the degree of phosphorylation of rat liver phosphofructokinase is affected by the metabolic state, and that glucagon (or CAMP) acts indirectly, probably by changing glucose metabolism due to increased glycogenolysis rather than by an activation of a CAMP‐dependent protein kinase.
Covalent Crosslinking between Molecules of Type I and Type III Collagen
Authors: HENKEL Werner , GLANVILLE Robert W.
Published: March 2005

Several peptides containing intermolecular crosslinks were isolated from tryptic digests of the insoluble matrix of human leiomyoma and calf aorta. From leiomyoma type III collagen, two crosslinked peptides were isolated. The peptide [TN(III)]2, is a dimer which contains the 73 amino‐terminal residues of two, zero‐D staggered, crosslinked α1(III) chains (D being the distance by which molecules or chains are staggered relative to each other). After de‐blocking with pyroglutamate aminopeptidase, its amino‐terminal amino acid sequence was determined to be < Glu‐Tyr‐Asp‐Ser‐Tyr‐Asp‐Val‐Xaa‐Ser‐Gly‐Val‐Ala‐Val‐Gly‐Gly‐Leu‐Ala‐Gly‐Tyr‐Hyp‐Gly‐. The involvement of [TN(III)]2 in the formation of 4D‐staggered intermolecular bonds was demonstrated by sequence analysis of [TN(III)2]×T(III). For this three‐chained peptide, a double sequence which corresponded to TN(III) and T(III), a nine‐residue peptide from the carboxy‐terminal helical crosslinking site of type III collagen, was found.

Crosslinks between type I and type III collagens were identified by isolating a peptide TN(III)×TN(I) from leiomyoma. This peptide was characterized, first by digesting it with collagenase giving rise to ColN(III)×ColN(I) and then chymotrypsin giving C(III) and C(I). C(I) and C(III) are peptides characteristic for type I and type III collagens, respectively. Further, three‐component crosslink‐containing peptides, from both type I and type III collagen, were isolated from aorta and leiomyoma. The peptides ColN(III)× ColN(I)× T(III) from leiomyoma and Col×N(III) ColN(I)× T(I) from aorta are crosslinked amino‐terminal regions of 0D‐staggered type I and type III molecules, joined either to an adjacent 4D‐staggered type III or type I molecule. Crosslinked peptides involving only type III collagen molecules were also isolated from calf aorta [colN(III)]2 and [CoIN(III)2]×T(III)].

As intramolecular bonds between type I and III collagens can be excluded the isolation of these crosslinked peptides demonstrated that type I and type III collagen molecules are present within the same collagen fibrit with OD stagger, bound by intermolecular crosslinks. The significance of these findings with respect to the proposed models for collagen fibres and the physiological properties of the extracellular matrix, are discussed.
270‐MHz 1H Nuclear‐Magnetic‐Resonance Study of Met‐enkephalin in Solvent Mixtures
Authors: ZETTA Lucia , CABASSI Franco
Published: March 2005

1H spectra at 270 MHz of zwitterionic Met‐enkephalin pentapeptide (Tyr‐Gly‐Gly‐Phe‐Met) in (C2H3)2SO/water mixtures are reported and discussed in terms of solvent‐induced conformational transitions.

The analysis of the chemical shifts, line widths, coupling constants and rotamer populations around χ1and χ2 suggests that the conformational properties of Met‐enkephalin in the two solvents are quite different. In aqueous solution, the preferred structure, characterized by the absence of intramolecularly hydrogen‐bonded NH groups and head‐to‐tail interactions, very likely is an equilibrium of unfolded conformations with approximately equal energy. In (C2H3)2SO, the preferred structure is folded, with the Met‐5 NH intramolecularly bonded and the Gly‐3 NH protected from the solvent, while the Gly‐2 and Phe‐4 amide protons are solvent exposed.

A conformational transition of Met‐enkephalin from the intramolecularly bonded to the unbonded one takes place at about 40 mol‐% water in (C2H3)2SO, involving the Met‐5 NH proton and the Tyr‐Gly‐Gly fragment. The Phe‐4 and Met‐5θ angles do not change appreciably, which suggests that an inversion at the Gly‐3 residue of the folded form, responsible for the conformational transition, does not affect the C‐terminal moiety.

At about 70 mol‐% water in (C2H3)2SO a change in the solvent mixture properties affects the χ1 rotamer populations and the ring dynamics of the aromatic side chains. The line broadening of the Tyr‐1 δ and ɛ proton resonances indicates a specific interaction of the N‐terminal ring with the solvent.
Export of a Protein into the Outer Membrane of Escherichia coli K12
Authors: BREMER Erhard , COLE Stewart T. , HINDENNACH Ingrid , HENNING UIf , BECK Ewald , KURZ Christina , SCHALLER Heinz
Published: March 2005

The cloned ompA gene encoding the major outer membrane protein OmpA of Eschevichiu coli has been shortened in vitro by exonuclease digestion from the end corresponding to the CO2H terminus of the protein. Nine derivatives were identified which still possessed sub:tantial parts of the ompA gene and one was constructed which had suffered a small deletion early in the gene. Gene fragments encoding NH2‐terminal OmpA sequences of 45, 133, 193, and 227 residues of the 325 amino acids of OmpA were examined in detail at the DNA level and for OmpA protein fragments synthesized. The latter two fragments were incorporated into the outer membrane and all known functions of the OmpA protein were expressed whereas the fragment with 133 OmpA‐specific residues was not stably incorporated into this membrane.

In all cases where OmpA functions were observed, an OmpA‐specific polypeptide of Mr24000 was found in cell envelopes, regardless of the size of the residual ompA sequences and of the fused coding sequences in the vector DNA. Pulse‐label experiments revealed larger initial translation products, most of which were degraded to the protein of Mr 24000. The 133‐residue OmpA fragment was also detected but proved to be entirely unstable.

It is argued that the OmpA protein consists of two domains and that the NH2‐terminal moiety from residues 1 to about 180 represents the membrane domain of the polypeptide. Therefore, the loss of about 50,possibly less, CO2H‐terminal residues from this domain suffices to interfere with stable incorporation into the outer membrane.
Kinetic Investigation of the a‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide Substrates
Authors: BIZZOZERO Spartaco A. , BAUMANN Werner K. , DUTLER Hans
Published: March 2005

Peptide substrates of the general structure Ac‐Tyr‐Ly1‐Ly2…‐Lym‐NH2and Ac‐Phe‐Ly1‐NH2 have been synthesized and subjected to α‐chymotrypsin‐catalyzed hydrolysis to collect information on the interactions between the enzyme active site and the amino‐acid residues Lyl, Ly2, etc., C‐terminal to the susceptible bond of the peptide. For this purpose changes in the dissociation constants of the enzyme‐substrate complexes and in the rate constants of acylation have been related to the structural variations of the substrates. The results indicate that interactions occur with the two residues next to the scissible bond, Ly1 and Ly2, but not with residue Ly3. Structural description of individual interactions was carried out with the aid of skeletal models of the active site. From such combination of kinetic and structural data a plausible interaction scheme for the substrate side C‐terminal to the scissible bond has been deduced. This interaction scheme, which defines conformation and orientation of this part of the substrate within the active site, is characterized by the presence of a single hydrogen bond occurring between NH(Ly2) and O(Phe‐41). No donor interacting with the back‐bone carbonyl groups of residues Ly1 and Ly2 could be detected in the model of α‐chymotrypsin. The effect of modification of the side chains of residues Lyl and Ly2 on the kinetic constants was shown to be consistent with the interactions assumed to occur between the side chains of these residues and the active site. The interpretation of the results obtained from these specificity studies have led to refined concepts concerning the relative importance of different sets of enzyme‐substrate interactions in determining reactivity.
High‐Resolution 1H‐NMR Spectroscopy of Free and Glycosidically Linked O‐ Acetylated Sialic Acids
Authors: HAVERKAMP Johan , VAN HALBEEK Herman , DORLAND Lambertus , VLIEGENTHART Johannes F. G. , PFEIL Reinhard , SCHAUER Roland
Published: March 2005

A number of naturally occurring and synthetic, partially O‐acetylated derivatives of N‐acetylneuraminic and N‐glycoloylneuraminic acids have been investigated by 360‐MHz 1H‐NMR spectroscopy. O‐Acetylation causes strong downfield shifts for the resonances of neighbouring sugar‐skeleton protons. The chemical shifts of these resonances, together with their characteristic multiplet shapes, can be used for localisation of the position of O‐acetyl substituents in the molecule. The number of such substituents in the molecule can be inferred from the number of acetyl‐methyl singlets, which also have characteristic resonance positions.

This method for determination of the number and position of O‐acetyl substituents in sialic acid residues is very powerful for structural analysis of underivatized carbohydrate chains derived from glycoconjugates. This is demonstrated for the oligosaccharide N‐acetyl‐4‐O‐acetylneuraminosyl‐(α2→3)‐lactose isolated from echidna milk
1H NMR Studies of the Binding of Bacteriophage‐M13‐Encoded Gene‐5 Protein to Oligo(deoxyadeny1ic acid)s of Varying Length
Authors: ALMA Nicoletta C. M. , HARMSEN Ben J. M. , VAN BOOM Jacques H. , VAN DER MAREL Gijs , HILBERS Cornelis W.
Published: March 2005

The binding of gene‐5 protein to oligo(deoxyadeny1ic acid)s varying in length from 2 to 16 nucleotides has been studied by titrating the protein with the oligonucleotides and recording the 1H NMR spectra at 360 MHz. To obtain information about the mode of binding of the protein the aromatic parts of the spectra have been analysed by performing spectral simulations, starting from the assignments obtained from nuclear Overhauser enhancements at 500 MHz [Alma, N. C. M., Harmsen, B. J. M., Hull, W. E., Van der Marel, G., Van Boom, J. H., and Hilbers, C. W. (1981) Biochemisrry, 20, 4419‐4428]. The 1H NMR spectra of the complexes of gene‐5 protein with (dA)8, (dA)12 and (dA)16 appear to be identical except for differences in linewidth. The 1H NMR spectra of the complexes with the smaller oligonucleotides (dA)2, (dA)3 and (dA)4 differ from each other and from the spectra obtained from the complexes with longer oligonucleotides. However, binding of all oligo‐ nucleotides basically influences the same aromatic residues, namely two tyrosines and one phenylalanine. In the protein‐oligonucleotide complexes, one protein monomer covers three nucleotide residues, in contrast to the stoichiometry of 1: 4 found for protein‐polynucleotide complexes. It was found that the binding to oligo‐ nucleotides is cooperative and ionic‐strength‐dependent but far less so than found for the binding to poly‐ nucleotides.
Fibroblast Growth Factor Modulates Synthesis of Collagen in Cultured Vascular Endothelial Cells
Authors: TSENG Scheffer C. G. , SAVION Naphtali , STERN Robert , GOSPODAROWICZ Denis
Published: March 2005

Vascular endothelial cells derived from adult bovine aortic arch can be grown in two ways, either in the presence or absence of fibroblast growth factor. The types of collagen produced by cultures under these two con‐ ditions have been compared. In the presence of fibroblast growth factor, cells grow in an orderly fashion, express their normal phenotype and synthesize primarily type III collagen plus collagens types IV and V at a ratio of 10: 1: 3. Cultures grown in the absence of the factor lose their orderly pattern of growth, lose polarity and normal phenotypic expression. They devote twice the proportion of total protein‐synthesizing capacity to collagen, and now synthesize type I in addition to the other collagen types. The ratio of collagen types I: III: IV: V is approxi‐ mately 30:70:1:13. The kinds of type V collagen chains expressed are also altered. Fibroblast growth factor appears to modulate collagen synthesis, the major component of the extracellular matrix, and indirectly modulates the phenotypic expression of cultured vascular endothelial cells. In atherosclerosis, type I collagen is found in association with the intimal layer. The disorderly growth and the abnormal production of type I collagen by these vascular endothelial cells cultured in the absence of fibroblast growth factor is a model for a number of pathological situations including atherosclerotic plaque formation.