bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between protein/solvent proton exchange and progressive conformation and fluctuation changes in hemoglobin
Authors: ANTRI Saïd El , SIRE Olivier , ALPERT Bernard
Published: March 2005

The pH dependence of the tertiary structural changes and the quaternary reorganization of the αβ interface of oxyhemoglobin in solution has been previously observed in our laboratory. The present work aims to establish whether or not these progressive structural changes with pH result from proton exchange between the protein and the solvent. We therefore have used infrared spectroscopy, acid/base titration and 1H/2H exchange to assess the effect of external proton concentration on the structural and dynamic properties of the hemoglobin molecule in solution. This study is also performed on the carbonmonoxy form since the affinity of hemoglobin for CO is much higher than for oxygen. The present findings demonstrate that affinity changes are closely related to protein fluctuations, as well as structural modifications. An increased affinity, induced either by ligand replacement or pH variation, is associated with the constrained chains exhibiting a lower degree of fluctuation, together with a looser şβ interface association.
Acceptor specificity and tissue distribution of three human α‐3‐fucosyltransferases
Authors: MOLLICONE Rosella , GIBAUD Anne , FRANÇOIS Anne , RATCLIFFE Murray , ORIOL Rafael
Published: March 2005

Based on the capacity to transfer α‐L‐fucose onto type‐1 and type‐2 synthetic blood group H and sialylated acceptors, a comparison of the α‐3‐fucosyltransferase activities of different human tissues is shown. Three distinct acceptor specificity patterns are described: (I) myeloid α‐3‐fucosyltransferase pattern, in which leukocytes and brain enzymes transfer fucose actively onto H type‐2 acceptor and poorly onto sialylated N‐acetyllactosamine; (II) plasma α‐3‐fucosyltransferase (EC 2.4.1.152), in which plasma and hepatocyte enzymes transfer, in addition, onto the sialylated N‐acetyllactosamine; (III) Lewis α‐3/4‐fucosyltransferase (EC 2.4.1.65), in which gall‐bladder, kidney and milk enzymes transfer, in addition, onto type‐1 acceptors. The small amount (<10%) of α‐3‐fucosyltransferase activity found in the plasma of an α‐3‐fucosyltransferase‐deficient individual had a myeloid‐type acceptor pattern, suggesting that this small proportion of the plasma enzyme is derived from leukocytes. In addition to the three acceptor specificity patterns, these enzyme activities can be differentiated by their optimum pH: 8.0–8.7 for the enzymes from myeloid cells and brain, 7.2–8.0 for liver enzymes and 6.0–7.2 for gallbladder enzymes. Milk samples had two α‐3‐fucosyltransferase activities, the Lewis or α‐3/4‐fucosyltransferase under control of the Lewis gene and an α‐3‐fucosyltransferase with plasma acceptor pattern which was independent of the control of the Lewis gene. The apparent affinity for GDP‐fucose of the myeloid‐like enzyme was weaker than those of the plasma and Lewis‐like enzymes. The apparent affinities for H type 2 and sialylated N‐acetyllactosamine were stronger for exocrine secretions as compared to the plasma and myeloid enzymes. The plasma type of α‐3‐fucosyltransferase activity was more sensitive to N‐ethylmaleimide and heat inactivation than the samples with myeloid‐like α‐3‐fucosyltransferase activity.
Purification and properties of N5, N10‐methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum (strain Marburg)
Authors: MA Kesen , THAUER Rudolf K.
Published: March 2005

The reduction of N5,N10‐methylenetetrahydromethanopterin (CH2= H4MPT) to N5‐methyltetrahydromethanopterin (CH3‐H4MPT) is an intermediate step in methanogenesis from CO2 and H2. The reaction is catalyzed by CH2= H4MPT reductase. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) was found to be specific for reduced coenzyme F420 as electron donor; neither NADH or NADPH nor reduced viologen dyes could substitute for the reduced 5‐deazaflavin. The reductase was purified over 100‐fold to apparent homogeneity. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed only one protein band at the 36‐kDa position. The apparent molecular mass of the native enzyme was determined by gel filtration to be in the order of 150 kDa. The purified enzyme was colourless. It did not contain flavin or iron. The ultraviolet/ visible spectrum was almost identical to that of albumin, suggesting the absence of a chromophoric prosthetic group. Reciprocal plots of the enzyme activity versus the substrate concentration at different constant concentrations of the second substrate yielded straight lines intersecting at one point on the abscissa to the left of the vertical axis. This intersecting pattern is characteristic of a ternary complex catalytic mechanism. The Km for CH2= H4MPT and for the reduced coenzyme F420 were determined to be 0.3 mM and 3 μM, respectively. Vmax was 6000 μmol · min−1· mg protein−1 (Kcat= 3600 s−1). The CH2= H4MPT reductase was stable in the presence of air; at 4°C less than 10% activity was lost within 24 h.
Isolation of two high‐molecular‐mass proteinases from human erythrocytes
Authors: SACCHETTA Paolo , SANTARONE Stella , BATTISTA Pasquale , Di COLA Domenico
Published: March 2005

Two forms of a neutral – alkaline high‐molecular‐mass proteinase (termed A1 and A2) have been purified from human erythrocytes by a procedure including a DEAE‐cellulose batchwise treatment of erythrocyte cytosol, gel filtration and DEAE‐cellulose chromatography. Both enzymes show distinctive properties of multicatalytic proteinases. They have an apparent molecular mass of 700 kDa and are composed by eight major subunits (23 – 32 kDa). Both enzymes show a proteinase activity towards casein and hydrolyze synthetic peptides with tyrosine, arginine or lysine at the P1 position. Among the synthetic peptides tested, the tetrapeptide succinyl‐leucyl‐leucylvalyl‐tyrosyl‐7‐amino‐4‐methylcoumarin and tripeptides with arginine in the P1 position (benzyloxycarbonyl‐valyl‐leucyl‐arginyl‐4‐methoxy‐2‐naphthylamide and benzyloxycarbonyl‐alanyl‐arginyl‐arginyl‐4‐methoxy‐2‐naphthylamide) are the most effective substrates. The proteinases are devoid of amino and diaminopeptidase activity. Both enzymes are completely inhibited by hemin, chymostatin and thiol‐group reagents. However, the enzymes can be distinguished by the isoelectric point, the different effect of nucleotides, glutathione disulphide, sodium dodecyl sulfate and cations on the catalytic activity.
The influence of glycosylation on the fate of renin expressed in Xenopus oocytes
Authors: NAKAYAMA Kazuhisa , HATSUZAWA Kiyotaka , KIM Won‐Sin , HASHIBA Kiyomi , YOSHINO Teruhiko , HORI Hitoshi , MURAKAMI Kazuo
Published: March 2005

It has been recently reported that, in Xenopus oocytes injected with the mRNA for human renin, this secretory renal glycoprotein acquires phosphomannosyl residues on its asparagine‐linked oligosaccharide chains, remains intracellular and undergoes a proteolytic cleavage which removes the prosegment. To understand the influence of glycosylation on the fate of renin in Xenopus oocytes and whether it is specific for human renin, we have expressed human renin and mouse Ren1 renin, which are glycosylated at two and three selected asparagine residues, respectively, and mouse Ren2 renin, which is not glycosylated, in Xenopus oocytes. The majority of human and Ren1 renins remained intracellular and underwent proteolytic cleavage, whereas mouse Ren2 renin was secreted efficiently. When human and Ren1 renins were expressed in oocytes treated with tunicamycin, both were secreted efficiently. A mutant of human renin, which had amino‐acid substitutions at both glycosylation sites, was also secreted efficiently, whereas that mutated at one of the two sites was not. These results indicate that the majority of all of the glycosylated renin molecules remain intracellular and undergo proteolytic cleavage, probably due to the acquisition of phosphomannosyl residues, and the human renins remains intracellular if it is only glycosylated at one of the two sites.
Structure and biosynthesis of the xylose‐containing carbohydrate moiety of rice α‐amylase
Authors: HAYASHI Makoto , TSURU Akiko , MITSUI Toshiaki , TAKAHASHI Noriko , HANZAWA Hiroyuki , ARATA Yoji , AKAZAWA Takashi
Published: March 2005

Suspension‐cultured cells of rice secrete α‐amylase into the culture medium. It has been shown that the mature form of the α‐amylase contains xylose‐bearing N‐linked oligosaccharide:

image

and

image

We demonstrate that suspension‐cultured cells of rice secrete α‐amylase containing oligomannose‐type oligosaccharides in the presence of 1‐deoxymannojirimycin or tris(hydroxymethyl)aminomethane. On the other hand, α‐amylase purified from germinated rice seedlings contains several kinds of oligomannose‐type and N‐acetyllactosamine‐type oligosaccharides. The processing pathway of oligosaccharide moieties in rice cells is discussed on the basis of a comparison of these oligosaccharides structures.
Transcriptional control of the cysG gene of Escherichia coli K‐12 during aerobic and anaerobic growth
Authors: PEAKMAN Timothy , BUSBY Stephen , COLE Jeffrey
Published: March 2005

The 74‐min region of the Escherichiu coli chromosome includes five open reading frames of known sequence. The first and last of these genes, nirB and cysG, are transcribed in the same direction and both are essential for NADH‐dependent nitrite reductase activity. The functions of the other genes, nirD, nirE and nirC, which are located between nirB and cysG, are unknown. The nirB gene is transcribed from a promoter which is anaerobically induced, expression being dependent on the transcription activator protein, Fnr. Here we show that the nirD, nirE, nirC and cysG genes are also expressed from the nirB promoter. After subcloning cysG, a second promoter was located less than 100 bases upstream of cysG. Two groups of transcription start points separated by 40 bases were detected in this region by S1 mapping. Rates of transcription from the isolated cysG promoter were the same during aerobic growth and anaerobic growth in the presence or absence of nitrite. However, when the nirB gene and its promoter were cloned back upstream from the cysG promoter, the rate of transcription was higher during anaerobic growth than during aerobic growth and was further induced by nitrite. These increases were totally dependent on a functional fnr gene and were shown by S1 mapping experiments to be due to transcriptional read‐ through from the Fnr‐dependent nirB promoter.

No promoter activity was associated with DNA fragments between the BamHI site located within the N‐terminal coding region of the nirB gene and the cysG promoter located at the C‐terminus of nirC.
Recognition of different nucleotide‐binding sites in primary structures using a property‐pattern approach
Authors: Bork Peer , Grunwald Christian
Published: March 2005

Consensus sequence patterns for β‐α‐β folds binding FAD, NAD and GTP were constructed on the basis of 11 steric and physicochemical properties. These property patterns permit detection and distinction of the respective nucleotide‐binding sites on the basis of amino acid sequence analysis alone. The SWISS‐PROT database (release 9) was screened with the three calculated patterns, and nucleotide‐binding sites identified are presented. They correspond to existing structure data (if known). For the detected sequence segments we are able to predict the β‐α‐β motif as well as the respective binding sites. For some of the proteins so detected a nucleotide‐binding capacity has not previously been reported.
2‐Aminobenzoyl‐CoA monooxygenase/reductase, a novel type of flavoenzyme
Authors: Langkau Bernd , Ghisla Sandro , Buder Reiner , Ziegler Klaus , Fuchs Georg
Published: March 2005

In a previous report we have described some properties of a novel flavoenzyme from a denitrifying Pseudomonas species which catalyzes the oxygen‐ and NAD(P)H‐dependent conversion of 2‐aminobenzoyl‐CoA [Buder, R., Ziegler, K., Fuchs, G., Langkau, B. & Ghisla, S. (1989) Eur. J. Biochem. 185, 637–634]. In this paper, we report on the identification of the three products formed from 2‐aminobenzoyl‐CoA in this reaction. The spectroscopic data and the chemical properties of these compounds and those of their degradation products are compatible with the structures of 2‐amino‐5‐hydroxybenzoyl‐CoA, 2‐amino‐5‐hydroxycyclohex‐1‐enecarboxyl‐CoA and of 2‐amino‐5‐oxocyclohex‐1‐enecarboxyl‐CoA. The latter is the main product and was found to be rather unstable since it hydrolyzes and decarboxylates readily at pH < 5. Ammonia is released from the decarboxylation product in the neutral pH range to yield 1,4‐cyclohexanedione. Conditions were optimized such that the CoA thioester of 2‐amino‐5‐hydroxybenzoate is the product obtained at > 98% yield. 2‐amino‐5‐hydroxycyclohex‐1‐enecarboxyl‐CoA is the product which is formed when the mixture of the reaction products is treated with sodium borohydride before separation.
Localization of membrane‐associated (NiFe) and (NiFeSe) hydrogenases ofDesulfovibrio vulgaris using immunoelectron microscopic procedures
Authors: ROHDE Manfred , FÜRSTENAU Ursel , MAYER Frank , PRZYBYLA Alan E. , PECK Harry D. , Le GALL Jean , CHOI Eui S. , MENON Nanda K.
Published: March 2005

The intracellular location of membrane‐associated (NiFe) and (NiFeSe) hydrogenases of Desulfovibrio vulgaris was determined using pre‐embedding and post‐embedding immunoelectron microscopic procedures. Polyclonal antisera directed against the purified (NiFe) and (NiFeSe) hydrogenases were raised in rabbits. One‐day‐old cultures of D. vulgaris, grown on a lactate/sulfate medium, were used for all experiments in these studies. For post‐embedding labeling studies cells were fixed with 0.2% glutaraldehyde and 0.3% formaldehyde, dehydrated with methanol, and embedded in the low‐temperature resin Lowicryl K4M.

Our post‐embedding studies using antibody‐gold or protein‐A‐gold as electron‐dense markers revealed the location of the two hydrogenases exclusively at the cell periphery; the precise membrane location was then demonstrated by pre‐embedding labeling. Spheroplasts were incubated with the polyclonal antisera against (NiFe) and (NiFeSe) hydrogenase followed by ferritin‐linked secondary antibodies prior to embedding and sectioning. The observed labeling pattern unequivocally revealed that the antigenic reactive sites of the (NiFe) hydrogenase are located in the near vicinity of the cytoplasmic membrane facing into the periplasmic space, whereas the (NiFeSe) hydrogenase is associated with the cytoplasmic side of the cytoplasmic membrane.
How do MgATP analogues differentially modify high‐affinity and low‐affinity ATP binding sites of Na+/K+ ‐ATPase?
Authors: SERPERSU Engin Halit , BUNK Sabine , SCHONER Wilhelm
Published: March 2005

The exchange‐inert tetra‐ammino‐chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na +/K+ ‐ATPase slowly by interacting with the high‐affinity ATP binding site. The inactivation proceeded at 37°C with an inactivation rate constant of 1.34 × 10−3 min−1 and with a dissociation constant of 0.62 μM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K+ ‐ATPase by Cr(NH3)4ATP, and its H2O‐substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4ATP was 29‐fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active‐site residues does not seem to play a significant role in the inactivation of Na+/K+ ‐ATPase by Cr(III)‐ATP complexes.

Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high‐affinity ATP site with an apparent dissociation constant of 1.8 μM. The observed inactivation rate constant of 2.11 × 10‐3 min−1 became higher when Na + or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP‐inactivated enzyme.

Co(NH3)4ATP inactivates Na+/K+ ‐ATPase by binding to the low‐affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)‐ATP or Rh(III)‐ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low‐affinity ATP site. Inactivation of Na+/K+ ‐ATPase was faster by the Δ isomer of Co(NH3)4ATP than by the Δ isomer. Co(NH3)4ATP, but not Cr(H2O)4ATP or adenosine 5′ ‐[β,γ‐methylene]triphosphate competitively inhibited K+ ‐activated p‐nitrophenylphosphatase activity of Na+ K+ ‐ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low‐affinity ATP binding site.
Metabolic control analysis
Authors: SMALL J. Rankin , FELL David A.
Published: March 2005

This paper illustrates a method to calculate the sensitivities of control coefficients to the elasticities which determine their values and it is shown that these sensitivities are systemic properties. We show, both theoretically and with a practical example, how they can be used to investigate: (a) the relative importance of a particular elasticity in the determination of the value of a control coefficient; (b) the effect of experimental error on the values of the control coefficients and (c) the construction of confidence limits around the values of the control coefficients.
Protein kinase C inhibitor proteins
Authors: TOKER Alex , ELLIS Christine A. , SELLERS Lynda A. , AITKEN Alastair
Published: March 2005

Potent inhibitors of protein kinase C have been isolated from sheep brain by DEAE‐cellulose, phenyl‐Sepharose CL‐4B and Mono Q anion‐exchange chromatography. Analysis by one‐ and two‐dimensional SDS/polyacrylamide gel electrophoresis showed the purified preparation to contain three bands ranging over 29–33 kDa in molecular mass, each consisting of several charge isomers with similar pI values (5.4–5.7). Peptide mapping, amino acid analysis and sequencing suggested that the proteins are related, with the possibility that some species are distinct gene products. The concentration of inhibitor proteins required for half‐maximal inhibition of protein kinase C activity is 1.7 μM. Inhibitory activity could not be affected by increasing the substrate, cofactor or ATP concentration in the standard protein kinase C assay, but was abolished by heat treatment. The inhibitor preparation did not affect the binding of phorbol dibutyrate to protein kinase C and could inhibit phosphorylation over a wide range of calcium concentrations. Inhibitory activity could be removed by immunoprecipitation of the purified inhibitor proteins with polyclonal antibodies raised against synthetic peptides, the sequences corresponding to those of peptide fragments obtained from protein digests. Amino acid sequence analysis of the inhibitors confirms they are novel proteins although similarities exist with a neuronal specific protein termed 14‐3‐3 and the carboxy terminus of the calcium‐lipid binding series (endonexin/calpactin/lipocortin).
Proteolytic activation of protein kinase C‐ɛ
Authors: SCHAAP Dick , HSUAN Justin , TOTTY Nick , PARKER Peter J.
Published: March 2005

Proteolysis of native protein kinase C‐ɛ (PKC‐ɛ) is shown to occur through tryptic attack at multiple sites within the PKC‐ɛ V2/V3 domain. Following initial cleavage of PKC‐ɛ with trypsin, the kinase activity using a synthetic peptide substrate was found to be lipid/phorbol‐ester independent, as observed for other members of this kinase family. Interestingly, there is also an increase in the histone kinase activity, indicating that there is an influence of the regulatory domain of the enzyme on substrate specificity. This is discussed in the context of alternatively spliced PKC‐ɛ mRNAs that are shown to be present in brain and lung tissues.
Expression of cytochrome P‐450 enzymes in cultured human hepatocytes
Authors: MOREL Fabrice , BEAUNE Philippe H. , RATANASAVANH Damrong , FLINOIS Jean‐Pierre , YANG Chung S. , GUENGERICH F. Peter , GUILLOUZO André
Published: March 2005

Hepatocytes from adult and newborn humans were put into primary culture and exposed to phenobarbital, 3‐methylcholanthrene, or rifampicin, three well‐known inducers of cytochrome P‐450 in animals. The expression of four cytochrome P‐450 enzymes (or groups of enzymes, namely P‐450 IIIA, P‐450 IIC8/9/10, P‐450 IIE1, and P‐450 IA2) was investigated. These enzymes were found to remain expressed during the period of culture studied. Treatment with the inducers for three days resulted in different responses, depending upon the inducer and the enzyme. Phenobarbital and rifampicin increased P‐450 IIC8/9/10 mRNA transcripts and the corresponding protein, while 3‐methylcholanthrene was ineffective. Both P‐450 IIIA mRNA and protein were strongly induced by rifampicin. All of the hepatocytes were found to synthesize P‐450 IIIA in response to rifampicin, as shown by immunoperoxidase staining. P‐450 IIIA expression was not affected by phenobarbital and was decreased by 3‐methylcholanthrene. P‐450s IA2 and IIE1 decreased to 25–50% of the initial level during these cultures. P‐450 IA2 and ethoxyresorufin O‐deethylase activity (which is a monooxygenase activity related to P‐450 IA family) were increased only by 3‐methylcholanthrene and did not respond to the other inducers. P‐450 IIE1 was not induced by any of these compounds. P‐450 IIC8/9/10 and P‐450 IIIA mRNA levels were also measured in human hepatocytes from one newborn. P‐450 IIC8/9/10 was barely expressed in freshly isolated cells but increased dramatically with time in culture. P‐450 IIIA transcripts were abundant in both freshly isolated and cultured cells derived from a newborn.

These results clearly demonstrate that human hepatocytes continue to express cytochrome P‐450 enzymes and respond to inducers in culture. This model system provides a useful approach for investigating the effects of drugs on maturation and expression of drug‐metabolizing enzymes in human liver.
Isolation of a specific granulopoiesis inhibitor from calf spleen and identification as glutathione
Authors: FETSCH Jörg , ECKART Klaus , MAURER H. Rainer
Published: March 2005

A small peptide was isolated from calf spleen, specifically inhibiting murine granulopoiesis in vitro. Purification involved ultrafiltration, anion‐exchange chromatography with a FPLC system and reversed‐phase chromatography on HPLC. Determination of the amino acid composition, following acid hydrolysis and phenyl isothiocyanate and dabsyl chloride derivatization, revealed the amino acids glutamic acid, cysteine and glycine. Although the N‐terminal amino group was not blocked, peptide sequencing with common techniques was not possible. Comparison of the isolated peptide with the well‐known tripeptide glutathione by HPLC and fast atom bombardment (FAB)/tandem mass spectrometry showed the identify of both substances. Moreover, glutathione was found to be a specific granulopoiesis inhibitor in vitro at 10–100 nM, a so far unknown property of this well‐known peptide.
Comparison of the carbohydrate‐binding specificities of seven N‐acetyl‐D‐galactosamine‐recognizing lectins
Authors: PILLER Véronique , PILLER Friedrich , CARTRON Jean‐Pierre
Published: March 2005

Seven plant lectins, Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia agglutinin (GSA, isolectin A4), Helix pomatia agglutinin (HPA), soybean (Glycine max) agglutinin (SBA), Salvia sclarea agglutinin (SSA), Vicia villosa agglutinin (VVA, isolectin B4) and Wistaria floribunda agglutinin (WFA), known to be specific for N‐acetyl‐D‐galactosamine‐(GalNAc) bearing glycoconjugates, have been compared by the binding of their radiolabelled derivatives, to eight well‐characterized synthetic oligosaccharides immobilized via a spacer on an inert silica matrix (Synsorb). The eight oligosaccharides included the Forssman, the blood group A and the T antigens, as well as αGalNAc coupled directly to the support (Tn antigen) and also structures with GalNAc linked α or β to positions 3 or 4 of an unsubstituted Gal. The binding studies clearly distinguished the lectins into αGalNAc‐specific agglutinins like DBA, GSA and SSA, and lectins which recognize α‐ as well as β‐linked GalNAc residues like HPA, VVA, WFA and SBA. HPA was the only lectin which bound to the βGall → 3αGalNAc‐Synsorb adsorbent (T antigen) indicating that it also recognizes internal GalNAc residues. Among the αGalNAc‐specific lectins, DBA strongly recognized blood group A structures while GSA displayed weaker recognition, and SSA bound only slightly to this affinity matrix. In addition, DBA and SSA were able to distinguish between GalNAc linked α1 → 3 and GalNAc linked α1 → 4, to the support, the latter being a much weaker ligand.

These results were corroborated by the binding of the lectins to biological substrates as determined by their hemagglutination titers with native and enzyme‐treated red blood cells carrying known GalNAc determinants, e.g. blood group A, and the Cad and Tn antigens. For SSA, the binding to the αGalNAc matrix was inhibited by a number of glycopeptides and glycoproteins confirming the strong preference of this lectin for αGalNAc‐Ser/ Thr‐bearing glycoproteins.
Isolation and physical properties of the DNA‐directed RNA polymerase from Thermus thermophilus HB8
Authors: WNENDT Stephan , HARTMANN Roland K. , ULBRICH Norbert , ERDMANN Volker A.
Published: March 2005

The DNA‐directed RNA polymerase from the extremely thermophilic eubacterium Thermus thermophilus HB8 was purified employing a new and rapid method. The subunit pattern of the enzyme, analyzed by SDS gel electrophoresis, was interpreted as: 140 kDa and 170 kDa for β and β′, 40 kDa for α and 92 kDa for ó. The RNA polymerase is active at elevated temperatures (65°C). Kinetic data provide evidence for the existence of two NTP binding sites with very strong cooperativity. The promoter site specificity of the isolated enzyme has been proven by in vitro transcription employing two T. thermophilus templates whose in vivo starts of transcription were characterized by nuclease S1 mapping.
Structural requirements for signal transduction of the insulin receptor
Authors: FLÖRKE Ralf‐Rüdiger , Klein Helmut W. , Reinauer Hans
Published: March 2005

Structural requirements for signal processing by human placental insulin receptors have been examined. Insulin binding has been found to change the physico‐chemical properties of (αβ)2 receptors solubilized with Triton X‐100, indicating a marked alteration of the form, i.e. size and shape, of the molecular complex. (a) The Stokes radius decreases from about 9.5 nm to 7.9 nm, as determined by PAGE with Triton X‐100 in the buffer (Triton X‐100/PAGE), and from 9.1 nm to 8.7 nm, as assessed by gel filtration. (b) The sedimentation coefficient s2o,w rises from 10.1 S to 11.4 S. Upon dissociation of the receptor‐hormone complex, the alterations are reversed. After autophosphorylation of hormone‐bound (αβ)2‐insulin receptors, phosphate incorporation was found for 7.9‐nm receptor forms when receptor‐insulin complexes were crosslinked with disuccinimide suberate prior to Triton X‐100/PAGE. However, phosphate incorporation was demonstrated for the 9.5‐nm receptor forms when receptor‐insulin complexes were not prevented from dissociation. This strongly indicates that the (αβ)2) receptor is autophosphorylated after assuming its 7.9‐nm form upon insulin binding. Moreover, the insulin‐dependent structural alterations are not affected by autophosphorylation.

In contrast to (αβ)2 receptors, the diffusion and the sedimentation behaviour of αβ receptors, which carry a dormant tyrosine kinase even in the hormone‐laden state, has been found to be insensitive to insulin binding.

Different molecular properties of αβ and (αβ)2 receptors have also been detected by hormone binding studies. Insulin binding to (αβ)2 and αβ receptors differs markedly with respect to pH, ionic strength, and temperature. This might indicate that the structure of the hormone binding domain of αβ receptor changes on association into the (αβ)2 species. Alternatively, distinct hormone‐induced conformational alterations at the molecular level of αβ and (αβ)2 receptor species may lead to the different binding properties.

Our data demonstrate that the (αβ)2‐insulin receptor undergoes extended conformational alterations upon insulin binding. This capacity for structural changes coincides with the hormone‐inducable enhancement of tyrosine autophosphorylation of the 7.9‐nm insulin‐bound receptor form. In contrast, αβ receptors appear to be looked in an inactive nonconvertable state. Thus, interaction between two αβ receptor units is required to allow extended conformational alterations. which are assumed to be the triggering event for augmented autophosphorylation.
Purification and characterization of five different α subunits of guanine‐nucleotide‐binding proteins in bovine brain membranes
Authors: KOBAYASHI Ichiro , SHIBASAKI Hisayuki , TAKAHASHI Katsunobu , TOHYAMA Keita , KURACHI Yoshihisa , ITO Hiroyuki , UI Michio , KATADA Toshiaki
Published: March 2005

We have purified five different α subunits of guanine‐nucleotide‐binding proteins (G proteins) from bovine brain membranes as active forms bound to guanosine 5′‐[γ‐thio]triphosphate (GTP[γS]). All the purified α subunits were interacted with βγ subunits and served as a substrate for pertussin‐catalyzed ADP‐ribosylation. Based on the findings of immunoblot analyses using specific antibodies raised against various α subunits of G proteins, three of them were identified as αi‐1.αi‐2 and αi‐33, and the other two were classified into α0 type. One of the α0‐type proteins was the most abundant in the brain membranes (termed α0), and the other (α02) appeared to differ from α0 in its proteolytic digestion data. The physiological properties of these purified GTP[γS]‐bound α subunits towards adenylate cyclase and atrial muscarinic K+ channels were studied. The nucleotide‐bound forms of αi‐1, αi‐2. αi‐3 and α02 inhibited the adenylate cyclase activity of S49 cyc− membranes which had been reconstituted with GTP[γS]‐treated Gs; this inhibition appeared to be mainly competitive with the activated Gs, αi‐1 having the most potent inhibitory activity among them. GTP[γS]‐bound α0, however, could not inhibit the Gs‐stimulated activity at all. On the other hand, all the GTP[γS]‐bound α subunits activated atrial muscarinic K+ channels, accompanied by a lag time, at picomolar concentrations. The βγ subunits resolved from G proteins also activated the K+ channels without a lag time at nanomolar concentration. The maximum activation by the βγ subunits appeared to be more potent than that by any of the a subunits. These results suggest that α and βγ subunits might activate the K+ channels by mechanisms different from each other.
Expression in Escherichia coli of ferredoxin: NADP+ reductase from spinach
Authors: ALIVERTI Alessandro , JANSEN Thomas , ZANETTI Giuliana , RONCHI Severino , HERRMANN Reinhold G. , CURTI Bruno
Published: March 2005

A cDNA clone for the preprotein of spinach ferredoxin: NADP+ reductase has been modified to allow the expression in Escherichia coli of the mature flavoprotein form that lacks the transit peptide. An expression vector, pFNR1, was constructed by subcloning the fragment into the plasmid pDS12/RBSII, SphI. In the crude extracts of transformed cells after induction, two active holoproteins of 35 kDa and 32 kDa, respectively, were found. The 32‐kDa protein, purified by immunoaffinity chromatography, was found to lack the first 28 residues of the spinach protein sequence and to have a methionine as the N‐terminal residue instead of Val29. A new expression plasmid, pFNR2, was obtained by in vitro mutagenesis of the codon GTG for Val29 to the synonymous GTT; in this case, only the 35‐kDa protein was expressed by transformed cells.

Both the 35‐kDa and 32‐kDa enzymes were purified and characterized. All the properties analyzed of the cloned 35‐kDa enzyme were very similar to those of the spinach flavoprotein. The 32‐kDa form showed the same catalytic efficiency of the spinach enzyme as a diaphorase but its interaction with oxidized ferredoxin was partially impaired.
Effect of polyamines on the activity of malarial α‐like DNA polymerase
Authors: BACHRACH Uriel , ABU‐ELHEIGA Lutfi
Published: March 2005

DNA polymerase from the malarial parasite Plasmodium falciparum required Mg2+ for activity, Putrescine (1 mM) caused a twofold increase in enzyme activity in the presence of a suboptimal concentration of MgCl2 (2 mM). Spermidine (1.5–2.0 mM) or spermine (0.1–0.3 mM) increased the activity of malarial DNA polymerase, in the presence of 2 mM MgCl2, by factors of 6 and 3–5, respectively. The activity of DNA polymerase from calf thymus or from NIH 3T3 cells transformed by the ras oncogene were not stimulated by these polyamines to the same extent. These findings suggest that in malaria‐infected erythrocytes, polyamines, at physiological concentrations, serve as a cofactor for the parasitic α‐like DNA polymerase. Malarial parasites grown in cultured human erythrocytes did not synthesize DNA after treatment with α‐difluoromethylornithine, which caused polyamine depletion in the infected cells. DNA synthesis was resumed after adding putrescine to the polyamine‐depleted cultures. DNA synthesis was also initiated when actinomycin D was added along with putrescine to polyamine‐depleted cells. It thus appears that polyamines are essential for the translation of the DNA polymerase mRNA and that polyamines play an important role in regulating the cell cycle of the malarial parasite.
Inhibition of Clostridium histolyticum collagenases by phosphonamide peptide inhibitors
Authors: DIVE Vincent , YIOTAKIS Athanasios , NICOLAOU Anna , TOMA Flavio
Published: March 2005

Several phosphonamide peptides having the general structure R‐PO(OH)‐Xaa‐Yaa‐Zaa were synthesized and tested for inhibition of Clostridium histolyticum collagenase. Inhibition was found to depend on the nature of R, Xaa, Yaa and Zaa such that the maximal affinity (Ki= 5 nM) was observed when R =p‐nitrophenylethyl, Xaa = Gly, Yaa = Pro and Zaa = 2‐aminohexanoic acid; this represents the tightest binding of inhibitor reported to date for any bacterial collagenase. Substitution of the p‐nitrophenylethyl by a methyl group led to a 500‐fold decrease of the potency, highlighting the existence of optimal interaction between the nitrophenylethyl side chain and one subsite of the enzyme. Replacement of the NH group in glycine residue (Xaa position) by ‐O‐ or ‐N‐CH3 produces significantly less potent inhibitors, presumably due in part to the loss of a hydrogen bond between the inhibitor and collagenase active site. These phosphonamidates are thought to be acting as transition‐state analogues of the peptide substrate.
Comparative structural and enzymatic properties of skeletal muscle myosin from neonatal and adult rabbits
Authors: HOUADJETO Maurice , BECHET Jean‐Jacques , d'ALBIS Anne
Published: March 2005

Several structural and enzymatic properties of myosin from skeletal muscles of neonatal and adult rabbits were compared.

Electrophoretic analyses and proteolysis experiments indicated that differences between the two myosin types could be attributed to their heavy subunits. Circular dichroism measurements of subfragment‐1 species, and trypsin‐digested derivatives showed that the neonatal protein contained less α‐helices than the adult form.

The Mg2+ ‐ATPase activity of neonatal myosin was lower than that of adult myosin, especially in the presence of actin. In comparison with adult subfragment‐1, it was found that the binding of ATP analogues such as adenosine 5′‐[β,γ‐imino]triphosphate and PPi, or that of ATP (as deduced from the apparent KATPm) to neonatal subfragment‐1 in the presence of actin was enhanced, while that of ADP was decreased. On the other hand, the association of actin with the ADP ‐ neonatal‐subfragment‐1 complex was weaker. These features must be expressed in the cyclical actin‐myosin association/dissociation steps occurring in ATP hydrolysis, and more particularly in the reassociation of actin with the ATP‐hydrolysis‐products – myosin complex.
Escherichia coli DNA polymerase I: inherent exonuclease activities differentiate between monofunctional and bifunctional adducts of DNA and cis‐ or trans‐diamminedichloroplatinum(II)
Authors: BERNGES Frithjof , DÖRNER Gertrud , HOLLER Eggehard
Published: March 2005

[3H]dGMP‐3′‐labelled, activated salmon testis DNA and [32P]dGMP‐5′‐labelled open circular M13 DNA were reacted with cis‐diamminedichloroplatinum(II), cis‐diamminechloroaquaplatinum(II), cis‐diamminediaquaplatinum(II) or trans‐diamminechloroaquaplatinum(II). The reaction was arrested after arbitrary times by adjustment to slightly alkaline solution conditions. The platinum‐containing DNA was digested with Escherichia coli DNA polymerase I. The progress of nucleotide release was measured by acid precipitation of undigested DNA. Solubilized nucleotides and adducts were analysed by HPLC.

The 3′‐5′‐exonuclease activity liberated single‐coordinated dGMP‐platinum(II) adducts from both cis‐ and trans‐platinum(II) treated salmon testis DNA and a small fraction of adducts of cis‐platinum(II) that coordinated two molecules of dGMP. The bisadduct was derived from non‐neighboring guanine residues probably located at or close to 3′‐termini. This nuclease activity neither cut between nor after neighboring guanine residues crosslinked by cis‐platinum(II). No bisadduct was liberated for trans‐platinum(II).

The 5′‐3′‐exonuclease activity did not liberate any nucleotide adducts from cis‐platinum(II)‐treated DNA. However, it removed single‐coordinated guanine adducts of trans‐diamminedichloroplatinum(II).

From the kinetics of the appearance of dGMP monoadducts and the inhibition of digestion, a reaction scheme is formulated for the reaction of platinum(II) complexes with DNA that confirms and extends the previously published one [W. Schaller, H. Reisner & E. Holler (1987) Biochemistry 26, 943–950]. The longevity of the dGMP monoadduct intermediate is discussed in the context of the efficiency of cis‐diamminedichloroplatinum(II) as an antitumor drug.
Nonribosomal biosynthesis of peptide antibiotics
Authors: KLEINKAUF Horst , von DÖHREN Hans
Published: March 2005

Peptide antibiotics are known to contain non‐protein amino acids, D‐amino acids, hydroxy acids, and other unusual constituents. In addition they may be modified by N‐methylation and cyclization reactions. Their biosynthetic origin has been connected in many cases to an enzymatic system referred to as the ‘thiotemplate multienzymic mechanism’. This mechanism includes the activation of the constituent residues as adenylates on the enzymic template, the acylation of specific template thiol groups, epimerization or N‐methylation at this thioester stage, and polymerization in the sequence directed by the multienzymic structure with the aid of 4′‐phosphopantetheine as a cofactor, including possible cyclization or terminal modification reactions. The reaction sequences leading to gramicidin S, tyrocidine, cyclosporine, bacitracin, polymyxin, actinomycin, enniatin, beauvericin, δ‐(L‐α‐aminoadipyl)‐L‐cysteinyl‐D‐valine and linear gramicidin are discussed. The structures of the multienzymes, their genetic organization, the biological functions of these peptides and results on related systems are discussed.
Chondroitin proteoglycans from squid skin
Authors: KARAMANOS Nikos K. , ALETRAS Alexis J. , ANTONOPOULOS Christos A. , HJERPE Anders , TSIGANOS Constantine P.
Published: March 2005

Two populations of proteochondroitins were isolated from 4 M guanidine hydrochloride extracts of squid skin by a combination of ion exchange, gel chromatography and density gradient centrifugation. The proteoglycans, Mr 4.8 × 105 and 2.8 × 105, contained four and two chondroitine chains respectively and unusual oligosaccharides with uronic acid and sulphate groups, and had different amino acid and neutral sugar composition. The chondroitin chains isolated after alkaline borohydride treatment contained varying amounts of glucose, galactose, mannose, fucose and xylose, most likely as branches. Both proteoglycans were antigenic to the rabbit and showed considerable cross‐reactivity as assessed by competition experiments using the ELISA technique. The proteoglycans reacted neither with exogenous hyaluronic acid nor with each other to form aggregates.
Interaction of a new photosensitive derivative of vinblastine, NAPAVIN, with tubulin and microtubules in vitro
Authors: NASIOULAS Georgios , GRAMMBITTER Klaus , HIMES Richard H. , PONSTINGL Herwig
Published: March 2005

We have synthesized a new photoreactive vinblastine derivative, 3‐({[2‐amino(4‐azido‐2‐nitrophenyl)ethyl]‐amino}‐carbonyl)‐O4‐decetyl‐3‐de(methoxycarbonyl)‐vincaleukoblastine (NAPAVIN), which can be photo‐activated with light in the 455‐nm region as well as with ultraviolet irradiation. Previous studies had shown that photoactivated NAPAVIN is much more effective than vinblastine in inhibiting cell proliferation of multidrug resistant cell lines. The experiments reported here demonstrate that the unirradiated derivative is very similar to vinblastine in its interactions with brain tubulin and microtubules, regarding inhibition of in vitro assembly, binding, aggregation, and production of protofilament spirals. Irradiation of [3H]NAPAVIN in the presence of tubulin led to covalent binding of the drug to both subunits of the protein. Labeling also occurred when NAPAVIN was first irradiated, then incubated with tubulin in the dark, indicating the production of a fairly stable reactive species with a half‐life of about 400 min. We conclude that labeling by this compound, under some conditions, occurs not by a nitrene but by an electrophilic photoproduct.
Structure of the genes of two homologous intracellularly heterotopic isoenzymes
Authors: JURETIĆ Nenad , MATTES Urs , ZIAK Martin , CHRISTEN Philipp , JAUSSI Rolf
Published: March 2005

The genes of mitochondrial and cytosolic aspartate aminotransferase of chicken were cloned and sequenced. In both genes nine exons encode the mature enzyme. The additional exon for the N‐terminal presequence that directs mitochondrial aspartate aminotransferase into the mitochondria is separated by the largest intron from the rest of the gene. A comparison of the two genes of chicken with the aspartate aminotransferase genes of mouse [Tsuzuki, T., Obaru, K., Setoyama, C. & Shimada, K. (1987) J. Mol. Biol. 198, 21–31; Obaru, K., Tsuzuki, T., Setoyama, C. & Shimada, K. (1988) J. Mol. Biol. 200, 13–22] reveals closely similar structures: in the gene of both the mitochondrial and the cytosolic isoenzyme all but one intron positions are conserved in the two species and five introns out of nine are placed at the same positions in all four genes indicating that the introns were in place before the genes of the two isoenzymes diverged.

The variant consensus sequence (T/C)11T(C/T)AG at the 3′ splice site of the introns of the genes for nuclearencoded mitochondrial proteins, which had been deduced from a total of 34 introns [Juretić, N., Jaussi, R., Mattes, U. & Christen, P. (1987) Nucleic Acids Res. 15, 10083–10086], was confirmed by including an additional 22 introns into the comparison. The position −4 at the 3′ splice site is occupied by base T in 43% of the total 56 introns and appears to be subject to a special evolutionary constraint in this particular group of genes.

The following course of evolution of the aspartate aminotransferase genes is proposed. Originating from a common ancestor, the genes of the two isoenzymes intermediarily evolved in separate lineages, i.e. the ancestor eukaryotic and ancestor endosymbiontic cells. When endosymbiosis was established, part of the endosymbiontic genome, including the aspartate aminotransferase gene, was transferred to the nucleus. This process probably led to the conservation of certain splicing factors specific for nuclear‐encoded mitochondrial proteins. The presequence for the mitochondrial isoenzyme was acquired by DNA rearrangement. In the eukaryotic lineage, the mitochondrial isoenzyme evolved more slowly than its cytosolic counterpart.
Kinetics and specificities of two closely related β‐glucosidases secreted by Schizophyllum commune
Authors: LO Amy C. , BARBIER Jean‐René , WILLICK Gordon E.
Published: March 2005

Two β‐glucosidases (I and II) were isolated from Schizophyllum commune, and their physical and chemical properties studied. The two enzymes have very similar sequences, as shown by HPLC analysis of tryptic digests and partial amino acid sequencing. As judged by their circular dichroism spectra, they have almost identical secondary structure. The estimates for α‐helix, β‐sheet, and other structures were 21%, 40% and 39%, respectively, for β‐glucosidase I and 27%, 32% and 41% for β‐glucosidase II. Their near‐ultraviolet spectra were identical. β‐Glucosidase I was more highly glycosylated than β‐glucosidase II, having 2 mol N‐acetylglucosamine/mol enzyme 36, mol mannose/mol enzyme and 1.2 mol glucose/mol enzyme vs 1.2, 17 and 3 mol/mol, respectively, in β‐glucosidase II. The native glycosylated form of β‐glucosidase I had a molecular mass of 102 kDa, and that of β‐glucosidase II, 96 kDa. As estimated from sensitivity to N‐glycanase, β‐glucosidase II sugars were mainly asparagine linked, but much of the sugar in β‐glucosidase I was not removed by this treatment and was apparently serine or threonine linked.

Kinetic analysis showed that both forms had similar Km values (0.3–2.1 mM) for oligosaccharides of 2–6 residues, but the kcat values of β‐glucosidase II were lower by 30–75% than those of β‐glucosidase I. The substrate dependence of kcat/Km indicated that both enzymes had binding sites for three glucose residues. The pH optimum of β‐glucosidase I was higher than that of β‐glucosidase II (5.8 vs 5.1). Both had similar specificities for several (R)‐β‐D‐glucosides tested. Both enzymes were competitively inhibited by their glucose product, but β‐glucosidase II was consistently less inhibited than β‐glucosidase I. Cellobiase activity was much more markedly inhibited than the activity with higher oligosaccharides, and the result of this, plus the lower hydrolytic rate with cellobiose, resulted in an accumulation of cellobiose as higher oligosaccharides were digested. Glucono‐δ‐lactone inhibited both enzymes and the hydrolysis of all oligosaccharide substrates similarly (Ki= 4 μM). We conclude that the catalytic site is identical in both enzymes, but subtle structural differences are reflected in a differential activity on the higher oligosaccharides and in the differential effects of the glucose product as an inhibitor. Furthermore, ethanol had a stimulatory effect on β‐glucosidase I but inhibited β‐glucosidase II, which presumably reflects differential effects of ethanol on the conformations of the two species.