bluuebunny/crossref_metadata_2025 huggingface dataset:
Susceptibility of cartilage collagens type II, IX, X, and XI to human synovial collagenase and neutrophil elastase
Authors: GADHER Suresh J. , EYRE David R. , DUANCE Victor C. , WOTTON Sandra F. , HECK Louis W. , SCHMID Thomas M. , WOOLLEY David E.
Published: March 2005

The action of purified rheumatoid synovial collagenase and human neutrophil elastase on the cartilage collagen types II, IX, X and XI was examined. At 25°C, collagenase attacked type II and type X (45‐kDa pepsin‐solubilized) collagens to produce specific products reflecting one and at least two cleavages respectively. At 35°C, collagenase completely degraded the type II collagen molecule to small peptides whereas a large fragment of the type X molecule was resistant to further degradation. In contrast, collagen type IX (native, intact and pepsin‐solubilized type M) and collagen type XI were resistant to collagenase attack at both 25°C and 35°C even in the presence of excess enzyme. Mixtures of type II collagen with equimolar amounts of either type IX or XI did not affect the rate at which the former was degraded by collagenase at 25°C.

Purified neutrophil elastase, shown to be functionally active against soluble type III collagen, had no effect on collagen type II at 25°C or 35°C. At 25°C collagen types IX (pepsin‐solubilized type M) and XI were also resistant to elastase, but at 35°C both were susceptible to degradation with type IX being reduced to very small peptides. Collagen type X (45‐kDa pepsin‐solubilized) was susceptible to elastase attack at 25°C and 35°C as judged by the production of specific products that corresponded closely with those produced by collagenase.

Although synovial collagenase failed to degrade collagen types IX and XI, all the cartilage collagen species examined were degraded at 35°C by conditioned culture medium from IL1‐activated human articular chondrocytes. Thus chondrocytes have the potential to catabolise each cartilage collagen species, but the specificity and number of the chondrocyte‐derived collagenase(s) has yet to be resolved.
Purification and properties of (S)‐tetrahydroprotoberberine oxidase from suspension‐cultured cells of Berberis wilsoniae
Authors: AMANN Manfred , NAGAKURA Naotaka , ZENK Meinhart H.
Published: March 2005

A novel oxidase, catalyzing in the presence of oxygen the removal of four hydrogen atoms from a number of tetrahydroprotoberberines with simultaneous production of 1 mol H2O2 and H2O each, has been discovered and purified to homogeneity from Berberis wilsoniae cell cultures. This enzyme, (S)‐tetrahydroprotoberberine oxidase, exhibited strict specificity for the (S)‐enantiomer of tetrahydroprotoberberines and 1‐benzylisoquinoline alkaloids, a pH optimum at 8.9, a molecular mass of 105 kDa and consisted of two subunits each of 53 kDa and covalently bound flavin. The Km values for (S‐scoulerine and (S)‐norreticuline were 25 μM and 150 μM respectively. Concentration of the end‐products, either protoberberines or H2O2, greater than 0.5 mM caused severe enzyme inhibition. This catalyst was responsible for the conversion of (S)‐tetrahydrocolumbamine to the key intermediate, columbamine, in the metabolic pathway leading to berberine, jatrorrhizine and palmatine.
Primary sequence mapping of human apolipoprotein B‐100 epitopes
Authors: CHEN Pei‐Feng , MARCEL Yves L. , YANG Chao‐Yuh , GOTTO Antonio M. , MILNE Ross W. , SPARROW James T. , CHAN Lawrence
Published: March 2005

Differential trypsin‐accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B‐100 are both important tools for probing apoB structure and conformation on low‐density lipoproteins (LDL). In this study, we have mapped > 80% of the C‐terminal region (720 residues) of LDL apoB‐100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738–742] confirming that the C‐terminal region of about 420 residues of apoB‐100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin‐accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB‐100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB‐100 cDNAs extending from the C‐terminus of apoB‐100 cloned in the expression vector, γgt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50–150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C‐terminal 20 amino acids of apoB‐100, one (Mab 16) to residues 4154–4189, and another (Mab 20) to residues 3926–4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL‐apoB. Although epitope expression and trypsin‐accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined.
Quantitative determination of methylated CpG in satellite DNA I and in L1Rn DNA sequences extracted from rat kidney tissue and from rat kidney cell lines
Authors: DANTE Robert
Published: March 2005

The level of methylation of CpG has been determined in satellite DNA I and in an 1180‐base‐pair fragment of L1 Rn DNA sequences extracted from rat kidney tissue and from two rat kidney cell lines, NRK B77 and NRK 52E. This determination was performed by HPLC analysis of 3′‐deoxyribonucleoside monophosphates obtained after digestion of DNA labelled in vitro with [α‐32P]dGTP using DNA polymerase I. Results obtained show that L1 sequences are hypomethylated in rat cell lines (29.3% in NRK B77 and 18.6% in NRK 52E) when compared to the same fragment extracted from rat kidney tissue (47.6%). However, satellite DNA I in the cell lines is much less affected by the hypomethylation. Satellite DNA I purified from NRK B77 and NRK 52E contains 58.8% and 47.8% respectively of methylated CpG whereas it contains 62% of methylated CpG in rat kidney tissue. Therefore, the demethylation of CpG seems not to occur at random in these cell lines.
The structure of jack bean urease
Authors: TAKISHIMA Kunio , SUGA Tatsuko , MAMIYA Gunji
Published: March 2005

The amino acid sequence of jack bean urease has been determined. The protein consists of a single kind of polypeptide chain containing 840 amino acid residues. The subunit relative molecular mass calculated from the sequence is 90770, indicating that urease is composed of six subunits. Out of 25 histidine residues in urease, 13 were crowded in the region between residues 479 and 607, suggesting that this region may contain the nickel‐binding site. Limited tryptic digestion cleaved urease at two sites, Lys‐128 and Lys‐662. Proteolytic products were not dissociated and retained full enzymatic activity. Five tryptic peptides containing the reactive cysteine residues were isolated and characterized with the aid of sulfhydryl‐specific reagents, N‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphthy1)ethylenediamine and N‐(7‐dimethylamino‐4‐methyl‐3‐coumarinyl)‐maleimide. The reactive cysteine residues were located at positions 59, 207, 592, 663, and 824. The possibility that Cys‐59, Cys‐207, Cys‐663, and Cys‐824 are involved in the urease activity of the enzyme has been eliminated. Cys‐592, which is essential for enzymatic activity, is located in the above‐mentioned histidine‐rich region.
Nucleotide and thioredoxin specificity of the manganese ribonucleotide reductase from Brevibucterium ammoniagenes
Authors: WILLING Andreas , FOLLMANN Hartmut , AULING Georg
Published: March 2005

The manganese‐containing ribonucleotide reductase previously identified in gram‐positive bacteria has been purified and its nucleotide specificity and other requirements were determined. The enzyme isolated from Brevibacterium ammoniagenes is a ribonucleoside‐diphosphate reductase which, in the presence of allosteric effectors, reduces all four common substrates at comparable rates; very little activity is observed in the absence of effector nucleotides. Ribonucleoside triphosphates are reduced at 20% the rate of the diphosphates. Cytidine and uridine nucleotide reduction is specifically stimulated by ATP and dATP, adenylate reduction by dGTP, and guanosine nucleotide reduction by dTTP. Unlike the iron‐containing ribonucleotide reductase systems, high concentrations of dATP do not inhibit substrate reduction. The new bacterial enzyme tolerates high salt concentrations (up to 250 mM ionic strength) and does not require divalent metal ions for activity in vitro.

The presence of thioredoxin has been demonstrated in heat‐ and acid‐treated protein extracts of B. ammoniagenes and the protein was purified to homogeneity. It is very similar to the thioredoxins isolated from other organisms in relative molecular mass (12000), isoelectric point (4.3) and enzyme‐activating properties. In the presence of 0.3 mM dithiothreitol, the bacterial thioredoxin can serve as hydrogen donor for B. ammoniagenes ribonucleotide reductase in vitro, indicating the presence of a functional ribonucleotide reductase‐thioredoxin system in these bacteria.

The properties described in this and in our preceding paper in this journal [Eur. J. Biochem. 170, 603–611 (1988)] suggest that the B. ammoniagenes ribonucleotide reductase is intermediate in structure and specificity between the deoxyadenosylcobalamin‐dependent and the iron‐containing enzyme classes and that it is adapted to the specific requirements of deoxyribonucleotide synthesis in this organism.
Activation of inositol phosphate formation by circulating noradrenaline but not by sympathetic nerve stimulation with a similar increase of glucose release in perfused rat liver
Authors: PÜSCHEL Gerhard P. , JUNGERMANN Kurt
Published: March 2005

In the isolated rat liver perfused in situ, stimulation of the nerve bundles around the hepatic artery and portal vein caused an increase of glucose and lactate output and a reduction of perfusion flow. These changes could be inhibited completely by α‐receptor blockers. The possible involvement of inositol phosphates in the intracellular signal transmission was studied.

In cell‐suspension experiments, which were performed as a positive control, noradrenaline caused an increase in glucose output and, in the presence of 10 mM LiCl, a dose‐dependent and time‐dependent increase of inositol mono, bis and trisphosphate.

In the perfused rat liver 1 μM noradrenaline caused an increase of glucose and lactate output and in the presence of 10 mM LiCl a time‐dependent increase of inositol mono, bis and trisphosphate that was comparable to that observed in cell suspensions.

In the perfused rat liver stimulation of the nerve bundles around the portal vein and hepatic artery caused a similar increase in glucose and lactate output to that produced by noradrenaline, but in the presence of 10 mM LiCl there was a smaller increase of inositol monophosphate and no increase of inositol bis and trisphosphate.

These findings are in line with the proposal that circulating noradrenaline reaches every hepatocyte, causing a clear overall increase of inositol phosphate formation and thus calcium release from the endoplasmic reticulum, while the hepatic nerves reach only a few cells causing there a small local change of inositol phosphate metabolism and thence a propagation of the signal via gap junctions.
Nucleotide sequence and expression in Escherichia coli of the gene coding for sphingomyelinase of Bacillus cereus
Authors: YAMADA Akihiro , TSUKAGOSHI Norihiro , UDAKA Shigezo , SASAKI Takuji , MAKINO Shio , NAKAMURA Shin , LITTLE Clive , TOMITA Masahiro , IKEZAWA Hiroh
Published: March 2005

Bacillus cereus secretes phospholipases C, which hydrolyze phosphatidylcholine, sphingomyelin and phosphatidylinositol. A 7.5‐kb HindIII fragment of B. cereus DNA cloned into Escherichia coli, with pUC18 as a vector, directed the synthesis of the sphingomyelin‐hydrolyzing phospholipase C, sphingomyelinase. Nucleotide sequence analysis of the subfragment revealed that it contained two open reading frames in tandem. The upstream truncated open reading frame corresponds to the carboxy‐terminal portion of the phosphatidylcholine‐hydrolyzing phospholipase C, and the downstream open reading frame to the entire translational portion of the sphingomyelinase. The two phospholipase C genes form a gene cluster.

As inferred from the DNA sequence, the B. cereus sphingomyelinase has a signal peptide of 27 amino acid residues and the mature enzyme comprises 306 amino acid residues, with a molecular mass of 34233 Da. The signal peptide of the enzyme was found to be functional in protein transport across the membrane of E. coli.

The enzymatic properties of the sphingomyelinase synthesized in E. coli resemble those of the donor strain sphingomyelinase. The enzymatic activity toward sphingomyelin was enhanced 20–30‐fold in the presence of MgCl2, and the adsorption of the enzyme onto erythrocyte membranes was accelerated in the presence of CaCl2
Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli
Authors: FLACHMANN Ralf , KUNZ Norbert , SEIFERT Jochen , GÜTLICH Markus , WIENTJES Franz‐Josef , LÄUFER Albrecht , GASSEN Hans Günter
Published: March 2005

The two genes, nadA and nadB, responsible for quinolinate biosynthesis from aspartate and dihydroxyacetone phosphate in Escherichia coli were cloned and characterized. Quinolinate (pyridine‐2,3‐dicarboxylate) is the biosynthetic precursor of the pyridine ring of NAD.

Gene nadA was identified by complementation in three different nadA mutant strains. Sequence analysis provided an 840‐bp open reading frame coding for a 31555‐Da protein. Gene nadB was identified by complementation in a nadB mutant strain and by the l‐aspartate oxidase activity of its gene product. Sequence analysis showed a 1620‐bp open reading frame coding for a 60306‐Da protein.

For both genes, promoter regions and ribosomal binding sites were assigned by comparison to consensus sequences. The nadB gene product, l‐aspartate oxidase, was purified to homogeneity and the N‐terminal sequence of 19 amino acids was determined. The enzyme was shown to be specific for l‐aspartate.

High‐copy‐number vectors, carrying either gene nadA, nadB or nadA+nadB, increased quinolinate production 1.5‐fold, 2.0‐fold and 15‐fold respectively. Both gene products seem to be equally rate‐limiting in quinolinate synthesis.
Reductive cleavage and reformation of the interchain and intrachain disulfide bonds in the globular hexameric domain NC1 involved in network assembly of basement membrane collagen (type IV)
Authors: WEBER Sabine , DÖLZ Reinhard , TIMPL Rupert , FESSLER John H. , ENGEL Jürgen
Published: March 2005

The formation of collagen IV dimers in the extracellular space requires the association of two C‐terminal globular domains giving rise to a large hexameric structure NC1 (Mr= 170000). NC1 hexamer was purified from collagenase digests of a mouse tumor and several human tissues. It was shown by electrophoresis to consist of two kinds of cross‐linked, dimeric segments, Da and Db (Mr about 50000), and monomeric segments in a molar ratio of about 3:1. In the native hexamers free SH groups were detectable by N‐[14C]ethylmaleimide and other sulfhydryl reagents. They account for 4–11% of the total number of cysteine residues with some variations between preparations from different sources and in the distribution between monomers and dimers. Reduction with 10 mM dithioerythritol under non‐denaturing condition completely converted dimers into monomers and allowed the alkylation of all twelve cysteine residues present in each monomeric NC1 segment. A monomeric intermediate with four to six free SH groups and a higher electrophoretic mobility than the final product was observed. Generation of this intermediate from dimers Da and Db follows apparently different routes proceeding either directly or through a dimeric intermediate respectively. The time course of conversion is best described by a mechanism consisting of two (Db) or three (Da) consecutive steps with pseudo‐first‐order rate constants ranging from 0.14 ms−1 to 0.5 ms−1. Glutathione‐catalyzed reoxidation of completely reduced NC1 in the presence of 2 M urea results in a product indistinguishable from native material by ultracentrifugation and electrophoresis pattern. The data suggest that in situ formation of NC1 structures is catalyzed by a small fraction (5–10%) of intrinsic SH groups leading to the formation and stabilization of dimers by rearrangement of disulfide bonds.
Production of specific monoclonal antibodies against the active sites of human pancreatic secretory trypsin inhibitor variants by in vitro immunization with synthetic peptides
Authors: BÖLDICKE Thomas , KINDT Silke , MAYWALD Friedhelm , FITZLAFF Gabriele , BÖCHER Michael , FRANK Ronald , COLLINS John
Published: March 2005

Specific monoclonal antibodies against the active sites of two genetically engineered pancreatic secretory trypsin inhibitor (PSTI) variants (PSTI 0 and PSTI 4) were produced. The protease inhibitors PSTI 0 and PSTI 4 differ only by three amino acid substitution at their active sites. PSTI 0 inhibits trypsin, whereas PSTI 4 inhibits human granulocyte elastase and chymotrypsin. Immunization was performed in vitro with a synthetic heptapeptide that covers the mutated region of the protein. For this purpose in vitro culture conditions for the production of specific monoclonal antibodies against synthetic peptides were improved. The monoclonal antibodies obtained react specifically with the corresponding protease inhibitor variant. Competition experiments with trypsin and human elastase demonstrate that the protease displace the monoclonal antibody from the active site of PSTI 0 and PSTI 4 respectively.
Demonstration of a novel ecto‐enzyme on human erythrocytes, capable of degrading ADP and of inhibiting ADP‐induced platelet aggregation
Authors: LÜTHJE Jürgen , SCHOMBURG Axel , OGILVIE Adaling
Published: March 2005

The role of ADP as an important inducer of platelet aggregation is generally accepted. Therefore it has been postulated by many authors that the enzymatic removal of extracellular ADP from the circulation is essential to avoid platelet aggregation and thrombus formation. Here we show that erythrocytes essentially contribute to the clearance of ADP. The removal of ADP from suspensions of washed human erythrocytes was due to at least two different activities. One activity, which had already been observed by earlier workers, was identified as adenylate kinase, on the basis of the reaction products and the inhibition by adenosine(5′)pentaphospho(5′)adenosine (Ap5A). This enzyme was not associated with the cells and was always detectable in cell‐free supernatants, indicating that the enzyme had leaked from the cells into the extracellular medium.

In contrast, the second activity, which is described here for the first time, was tightly bound to the cells. The activity was not inhibited by Ap5A. The main product of the reaction was AMP, and enzyme activity depended on the presence of divalent cations. The Michaelis constant was about 28 μmol/1. This activity seemed to be an ecto‐ADPase. Studies with various inhibitors revealed that degradation of ADP was not due to a non‐specific phosphatase.

Besides the well known ADPase on the endothelium, the ecto‐activity on erythrocytes may play an important part in destroying pro‐aggregatory ADP.
Localization of a 64‐kDa phosphoprotein in the lumen between the outer and inner envelopes of pea chloroplasts
Authors: SOLL Jürgen , BENNETT John
Published: March 2005

The identification and localization of a marker protein for the intermembrane space between the outer and inner chloroplast envelopes is described. This 64‐kDa protein is very rapidly labeled by [γ‐32P]ATP at very low (30 nM) ATP concentrations and the phosphoryl group exhibits a high turnover rate. It was possible to establish the presence of the 64‐kDa protein in this plastid compartment by using different chloroplast envelope separation and isolation techniques. In addition comparison of labeling kinetics by intact and hypotonically lysed pea chloroplasts support the localization of the 64‐kDa protein in the intermembrane space. The 64‐kDa protein was present and could be labeled in mixed envelope membranes isolated from hypotonically lysed plastids. Mixed envelope membranes incorporated high amounts of 32P from [γ‐32P]ATP into the 64‐kDa protein, whereas separated outer and inner envelope membranes did not show significant phosphorylation of this protein. Water/Triton X‐114 phase partitioning demonstrated that the 64‐kDa protein is a hydrophilic polypeptide. These findings suggest that the 64‐kDa protein is a soluble protein trapped in the space between the inner and outer envelope membranes. After sonication of mixed envelope membranes, the 64‐kDa protein was no longer present in the membrane fraction, but could be found in the supernatant after a 110000 ×g centrifugation.
Phorbol 12,13‐dibutyrate and mitogens increase fructose 2,6‐bisphosphate in lymphocytes
Authors: BOSCA Lisardo , MOJENA Marina , DIAZ‐GUERRA M. José M. , MARQUEZ Carlos
Published: March 2005

The influence of tumour promoters and growth factors on glycolysis and on fructose‐2,6‐bisphosphate concentration was studied in isolated mouse spleen lymphocytes and in purified B‐cells. The intracellular concentration of fructose 2,6‐bisphosphate and the rate of lactate release were increased 2–3‐fold in spleen lymphocytes exposed to active phorbol esters, mitogenic lectins, interleukin 4 or lipopolysaccharide. The maximal effect was observed after 1 h of exposure. In these cells hexose 6‐phosphates increased 2‐fold and 6‐phosphofructo‐2‐kinase activity remained unchanged after treatment with phorbol 12,13‐dibutyrate or with lectins. Exposure of B‐cells to phorbol 12,13‐dibutyrate, interleukin 4 or lipopolysaccharide increased the glycolytic flux and the concentration of fructose 2,6‐bisphosphate without relation to their mitogenic activity.

Lymphocytes and rat liver 6‐phosphofructo‐2‐kinase were partially purified using the same procedure. The lymphocyte enzyme was not inhibited by sn‐glycerol 3‐phosphate in contrast to the potent inhibition observed in liver. Treatment of both enzymes with the catalytic subunit of the cyclic‐AMP‐dependent protein kinase failed to inactivate 6‐phosphofructo‐2‐kinase from lymphocytes. These differences suggest that lymphocytes and liver contain different forms of this enzyme.
Control of the tRNA‐tufB operon in Escherichia coli
Authors: VAN DELFT Joost H. M. , VERBEEK Hans M. , DE JONG Paul J. , SCHMIDT Denise S. , TALENS Anneke , BOSCH Leendert
Published: March 2005

‘Ribosome feedback’ effects on the expression of the genes specifying tRNA and EF‐Tu in E. coli have been studied at increased rrnB doses (rRNA gene doses). We confirm previous observations that the introduction into the cell of a multicopy plasmid carrying the rrnB operon reduces the cellular content of most tRNAs, including those encoded by the tRNA‐tufB operon, but leaves the 5S rRNA content unaffected. Increase of the dosage of intact, but not of deleted rRNA genes, causes a slight drop in total EF‐Tu that can be fully accounted for by a decrease in EF‐TuB level. The drop in EF‐TuB content (approx. 25%) is much smaller than that in tRNA content (approx. 80%). The synthesis rate of total EF‐Tu is hardly affected, indicating that the turnover of EF‐Tu has not changed. The ratio of tRNA over tuf RNA synthesis rates remains the same after elevation of rrnB dosage. Considering the large decrease in tRNA content this means that both RNA synthesis rates decrease to approximately the same extent. The relatively small drop in EF‐Tu synthesis must be due, therefore, to an enhancement of the number of EF‐Tu molecules synthesized per mRNA molecule. Apparently a post‐trans‐criptional mechanism, regulating EF‐Tu synthesis, becomes operative under these conditions.

Growth‐rate‐dependent regulation of the tRNA‐tufB operon has been studied using lysogens carrying tRNA': lacZ and tRNA‐tufB': lacZ operon fusions and a tufB': lacz' gene fusion. These experiments show that the cellular contents of tRNA, tufB RNA and EF‐TUB vary in direct proportion to the growth rate. This indicates that growth rate control of tRNA‐tufB operon transcription resembles that of stable RNA operons and not of r‐protein operons, and that the read‐through of the terminator at the end of the tRNA gene cluster remains unaltered.
Effect of histone H1, poly(ethyleneglycol) and DNA concentration on intermolecular and intramolecular ligation by T4 DNA ligase
Authors: SOBCZAK Joëlle , DUGUET Michel
Published: March 2005

The efficiency of ligation of linear DNA and the relative amounts of intramolecular versus intermolecular ligation may be triggered by a number of additive agents. The results show that it is possible to mimic the effect of poly(ethyleneglycol) 6000 by simply increasing DNA concentration about 15‐fold: both the rate and the extent of the reaction are greatly enhanced, and intermolecular ligation is largely favored. However, in this case the stimulation by salts, which occurs in poly(ethyleneglycol) solutions, is not observed; we suggest that salts enhance the hydrophobic interactions between ligase and DNA that take place in the presence of poly(ethyleneglycol). We also show that histone H1, which is involved in the formation of chromatin fibers, is able to stimulate intermolecular ligation by T4 ligase. This effect is more specific than a simple neutralisation of the phosphate groups of the DNA by positive charges of the histone; it still occurs at 125 mM NaCl and in the presence of the four core histones. The implications of the finding concerning the mode of action of histone H1 on DNA are discussed.
Porphyrin cytochrome c
Authors: KORNBLATT Jack A. , LABERGE Monique
Published: March 2005

Porphyrin cytochrome c, the iron‐free derivative of cytochrome c, has been used extensively as a fluorescent analog of cytochrome c. It appears as though its fluorescence intensity but not its relative quantum yield is affected by pH in the physiological range; an apparent pK of about 6.2 is found suggesting a histidine close to the porphyrin.

The fluorescence intensity of the porphyrin cytochrome c in the presence of cytochrome c oxidase is independent of pH; this suggests that the oxidase has the capacity to control the pK of whichever group is responsible for the pH sensitivity of the free porphyrin cytochrome c. The most likely candidate for this pH‐sensitive group is histidine‐18. The N‐3 nitrogen of this residue forms one of the axial ligands to the iron in the intact cytochrome c but it is uncoordinated in the iron‐free derivative.
Analysis of the in vivo phosphorylation state of rabbit skeletal muscle glycogen synthase by fast‐atom‐bombardment mass spectrometry
Authors: POULTER Linda , ANG Siau‐Gek , GIBSON Bradford W. , WILLIAMS Dudley H. , HOLMES Charles F. B. , CAUDWELL F. Barry , PITCHER Julie , COHEN Philip
Published: March 2005

The in vivo phosphorylation state of glycogen synthase was re‐examined by fast‐atom‐bombardment mass spectrometry and a procedure in which phosphoserine residues are first converted to S‐ethylcysteine. In animals injected with the β‐adrenergic antagonist propranolol, the phosphorylation sites in the N‐terminal (N) and C‐terminal (C) cyanogen bromide peptides were identified as the serine residues at N7, the region C28‐C39, C42, C46 and C100. In animals injected with adrenalin, the phosphorylation of N7 increased from 0.6 to 0.8 mol/mol, the region C28‐C39 from 0.7 to 1.2 mol/mol and C100 from 0.3 to 0.6 mol/mol. The phosphorylation states of C42 (0.7 mol/mol) and C46 (0.9 mol/mol) were unchanged. In addition, two further serine residues became phosphorylated at positions N10 (0.5 mol/mol) and C87 (0.5 mol/mol), which were not phosphorylated in the absence of adrenalin.

Residues N10 and C42 have not been recognized as in vivo sites of phosphorylation previously. The results suggest that N10 is phosphorylated by a novel protein kinase which may be activated by cyclic‐AMP‐dependent protein kinase. The phosphorylation of C42 is likely to be catalysed by glycogen synthase kinase 3. The protein kinases responsible for phosphorylating N7, the region C28–C39, C46, C87 and C100 in vivo and the molecular mechanisms by which adrenalin inactivates glycogen synthase in vivo are discussed.

Residue N3, a major site phosphorylated by casein kinase‐I in vitro is not phosphorylated in vivo. This and other evidence indicates that casein kinase‐I is not a glycogen synthase kinase in vivo.
A major nucleolar protein, nucleolin, induces chromatin decondensation by binding to histone Hl
Authors: ERARD Monique S. , BELENGUER P. , CAIZERGUES‐FERRER M. , PANTALONI A. , AMALRIC F.
Published: March 2005

Using circular dichroism to probe the extent of DNA condensation in chromatin, we have demonstrated that a major nucleolar protein, nucleolin, can decondense chromatin. By means of various binding assays we show that nucleolin has a strong affinity for histone H1 and that the phosphorylated N‐terminal domain, rich in lengthy stretches of acidic amino acids, is responsible for this ionic interaction. Additional experiments clearly demonstrate that nucleolin is unable to act as a nucleosome core assembly or disassembly factor and hence has little affinity for the core histone octamer.

We propose that this nucleolar protein induces chromatin decondensation by binding to histone H1, and that nucleolin can therefore be regarded as a protein of the high‐mobility‐group type.
Structural characterization of a murine myeloid leukaemia inhibitory factor
Authors: SIMPSON Richard J. , HILTON Douglas J. , NICE Edouard C. , RUBIRA Michael R. , METCALF Donald , GEARING David P. , GOUGH Nicholas M. , NICOLA Nicos A.
Published: March 2005

A leukaemia inhibitory factor (LIF) which induces macrophage differentiation in M1 murine myeloid leukaemia cells and suppresses their proliferation in vitro has been isolated in sufficient quantities (30 μg) from Krebs ascites tumour cell conditioned medium to permit its partial characterization by amino acid sequence analysis. The combination of sensitive microbore column (1.0 and 2.1 mm internal diameter) HPLC technology and microsequence analysis has enabled the positive identification of 125 of the total 179 amino acid residues (70%) in the molecule. The amino acid sequence data reported here permitted the isolation of a partial cDNA clone encoding LIF [Gearing et al. (1987) EMBO J. 6, 3995–40021. A candidate C‐terminus of the LIF molecule predicted from the amino acid sequence was confirmed by subsequent isolation of a cDNA clone corresponding to the C‐terminus of the protein.

No strong similarity was revealed when the amino acid sequence of LIF was compared with other haemopoietic growth factors, in particular granulocyte‐macrophage colony‐stimulating factor, granulocyte colony‐stimulating factor and tumour necrosis factor‐α or interleukins. The protein sequence data reported here indicate three sites of post‐translational modification (N‐linked glycosylation).
Cloning of a full‐length complementary DNA for fatty‐acid‐binding protein from bovine heart
Authors: BILLICHI Susanne , WISSEL Thomas , KRATZIN Hartmut , HAHN Ulrich , HAGENHOFF Birgit , LEZIUS Axel G. , SPENER Friedrich
Published: March 2005

A full‐length cDNA for bovine heart fatty‐acid‐binding protein (H‐FABP) was cloned from a γgt11 cDNA library established from bovine heart muscle. The cDNA sequence shows an open reading frame coding for a protein with 133 amino acids. Colinearity with the amino acid sequences of four tryptic peptides was asserted. H‐FABP isolated from bovine heart begins with an N‐acetylated valine residue, however, as derived from analysis of the tryptic, amino‐terminal‐blocked peptide and the molecular mass of the peptide obtained via secondary‐ion mass spectrometry. The molecular mass of the total protein is 14673 Da. Bovine H‐FABP is 89% homologous to rat H‐FABP and 97% homologous to the bovine mammary‐derived growth‐inhibition factor described recently by Böhmer et al. [J. Biol. Chem. 262, 15137–15143 (1987)l. Significant homologies were also found with bovine myelin protein P2 and murine adipocyte protein p422. Secondary‐structure predictions were proposed for these proteins, based on computer analysis, which reveal striking similarities.
Hyper‐regulation of pyr gene expression in Escherichia coli cells with slow ribosomes
Authors: JENSEN Kaj Frank
Published: March 2005

UTP‐modulated attenuation of transcription is involved in regulating the synthesis of pyrimidine nucleotides in Escherichia coli. Thus, expression of two genes, pyrBI and pyrE, was shown to be under this type of control. The genes encode the two subunits of aspartate transcarbamylase and orotate phosphoribosyltransferase respectively. The levels of these enzymes are inversely correlated with the intracellular concentration of UTP. Modulation of attenuation seems to be a consequence of the effect of UTP concentration on the mRNA chain growth rate. Reducing the UTP pool retards RNA polymerase movement. Mechanistically this will couple the ribosomes translating a leader peptide gene more tightly to the elongating RNA polymerase. The ribosomes will then be more prone to prevent the folding of the mRNA chains into terminating hairpin structures when RNA polymerase is at the attenuator and has to decide whether transcription should terminate or continue into the structural genes. This paper described a study of pyrBI and pyrE gene regulation in cells where the ribosomes move slowly as a result of mutation in rpsL. It appears that expression of the two genes is hyper‐regulated by the UTP pool in this type of cells.

Furthermore, the attenuator model can only account for the results if it is assumed that UTP‐concentration‐dependent pausing of transcription occurs in vivo in the two pyr gene leaders such that RNA polymerase waits for the coupled ribosomes before transcribing into the attenuator regions.
Isolation, molecular properties and kinetic studies of a strict β‐fucosidase from Lactuca sativa latex
Authors: GIORDANI Roger , NOAT Georges
Published: March 2005

A β‐fucosidase located in the latex serum of Lactuca sativa has been isolated and purified to homogeneity. This enzyme greatly differs from the other fucosidases already known in that it is strictly specific for the fucosyl residue and for the anomeric β carbon. It is the first time that such an enzyme is shown to exist.

The enzyme is a monomer and its molecular mass is close to 37 kDa. Its sedimentation constant is equal to 2.8 S. It is very stable at pH 5.5 in citrate/phosphate buffer but extensive denaturation occurs up to pH 7.5. Kinetic studies have shown that two ionization steps probably control the rate of the enzymatic hydrolysis.

No precise information could be obtained about the possible in vivo role of this β‐fucosidase. However, the pure enzyme can release fucose from the cell walls obtained from hypocotyls of L. sativa. This result may be taken as evidence for the presence of β‐fucosidic links in these walls so that the enzyme could be involved in the differentiation of articulated laticifers.
Ornithine‐containing lipids of some Pseudomonas species
Authors: KaAWAI Yohko , YANO Ikauya , KANEDA Kenji , YABUUCHI Eiko
Published: March 2005

Ornithine‐containing lipids purified by thin‐layer chromatography were found to represent 2–15% of the total extractable cellular lipids in two or three strains each of four Pseudomonas species: P. aeruginosa, P. fluorescens, P. stutzeri and P. cepacia. The structures of the ornithine‐containing lipids were elucidated by chemical analysis, thin‐layer chromatography, gas‐liquid chromatography, gas‐liquid chromatography/mass spectrometry (electron impact or secondary ion) and infrared absorption spectroscopy. At least six molecular species of ornithine‐containing lipids were present in common in all of the preparations of the four Pseudomonas species. The structure which was the most abundantly in P. fluorescens (about 60% of the total amount of the ornithine‐containing lipid) was 3‐hydroxyhexadecanoic acid amide‐linked to ornithine and esterified to hexadecanoic acid. In addition to this structure, 3‐hydroxyoctadecenoic acid amide‐linked to ornithine and esterified to hexadecanoic acid was a dominant structure in the ornithine‐containing lipids of P. aeruginosa, P. stutzeri or P. repacia. In P. cepacia, another ornithine‐containing lipids with a terminal polar fatty acid, 3‐hydroxyhexadecanoic acid amide‐linked to ornithine and esterified to 2‐hydroxynonadecacyclopropanoic acid or 2‐hydroxyoctadecenoic acid, was found; its content, which represented 8–11% of the total extractable cellular lipids, was higher than that of the ornithine‐containing lipids with a terminal nonpolar fatty acid. These ornithine‐containing lipids exhibited hemagglutinating activity. Additionally, it was very interesting that hydroxy fatty acids included in the ornithine‐containing lipids were not found in the phospholipids which represented more than 80% of the total extractable cellular lipids.
Purification and properties of the cellular and scrapie hamster prion proteins
Authors: TURK Eric , TEPLOW David. B. , HOOD Leroy E. , PRUSINER Stanley B.
Published: March 2005

During scrapie infection an abnormal isoform of the prion protein (PrP), designated PrPsc, accumulates and is found to copurify with infectivity; to date, no nucleic acid has been found which is scrapie‐sepecific. Both uninfected and scrapie‐infected cells synthesize a Prp isoform, denoted PrPc, which exhibits physical properties that differntiate it from PrPsc was purified by immunoaffinity chromatography using a PrP‐ sepecific monoclonal antibody cross‐linked to protein‐A‐Avidgel. PrPsc was purified by detergent extraction, poly(ethylene glycol) precipation and repeated differentila centrifugation of PrPsc polymers. Both PrP isoforms were found to have the same N‐terminal amino acid sequence which begins at a predicted signal peptide cleavage site. The first 8 residues of PrPc found to be KKXPKPGG and the first 29 residues of PrPc were found to be KKXPKPGGWNTGGSXYPGQGSPGGNRYPP. Arg residue 3 and 15 in PrPsc and 3 PrPc appear to be modified since no contain an intramolecular disulfide bond, liking Cys 179 and 214, which creates a loop of 36 amino acid containing the two N‐ linked glycosylation sites. Developement of purification of how PrPsc is synthesized either form PrPc or a precursor.
Opposite regulation of the mRNAs for parvalbumin and p19/6.8 in myotonic mouse muscle
Authors: KLUXEN Franz‐Werner , SCHÖFFL Fritz , BERCHTOLD Martin W. , JOCKSUCH Harald
Published: March 2005

The gene mutation in the mouse, ‘arrested development of rightingresponse’, adr, causes a defect of chloride conductance of the muscle fibre membrane leading to the symptoms of myotonia [Mehrke, G., Brinkmeier, H. and Jockusch, H. (1988) Muscle & Nerve 11, 440‐446]. In fast muscle, the myotonic phenotype is accompanied by a drastic reduction of the Ca2+‐binding protein, parvalbumin. Messenger RNA levels in organs of myotonic (ADR) mice were analysed. In fast muscles of the mutant, in‐vitro‐translatable parvalbumin mRNA was strongly reduced, whereas the mRNA for the slow‐muscle‐specific protein, p19/6.8, was increased. In contrast, the parvalbumin mRNA in thecerebellum was not affected by the adr mutation. A reduction of the two parvalbumin mRNA species (700 and 1100 nucleotides) in ADR fast muscle and unaltered prvalbumin cDNA as a probe. The mRNA level for another Ca2+‐binding protein, calmodulin, was low in muscle and high in the central nervous system but was unaffected by the mutation. When adr/adrmice were fed a diet containing the membrane‐stabilising drug, tocainide, the levels inmuscle of the mRNAs for parvalbumin and p19/6.8 were partially normalised.
Comparison of the amino acid sequences of the transacylase components of branched chain oxoacid dehydrogenase of Pseudomonas putida, and the pyruvate and 2‐oxoglutarate dehydrogenases of Escherichia coli
Authors: BURNS Gayle , BROWN Tracy , HATTER Kenneth , SOKATCH John R.
Published: March 2005

The nucleotide sequence of bkdB, the structural gene for E2b, the transacylase component of branched‐chain‐oxoacid dehydrogenase of Pseudomonas putida has been determined and translated into its amino acid sequence. The start of bkdB was identified from the N‐terminal sequence of E2b isolated from branched‐chain‐oxoacid dehydrogenase of the closely related species, P. aeruginosa. The reading frame was composed of 65.5% G + C with 82.3% of the codons ending in G or C. There was no intergenic space between bkdA2 and bkdB. No codons requiring minor tRNAs were utilized and the codon bias index indicated a preferential codon usage. The bkdB gene encoded 423 amino acids although the N‐terminal methionine was absent from E2b prepared from P. aeruginosa. The relative molecular mass of the encoded protein was 45134 (45003 minus methionine) vs 47000 obtained by SDS/polyacrylamide gel electrophoresis. There was a single lipoyl domain in E2b compared to three lipoyl domains in E2p, and one domain in E2o, the transacylases of pyruvate and 2‐oxoglutarate dehydrogenases of Escherichia coli respectively. There was significant similarity between the lipoyl domain of E2b and of E2p and E2o as well as between the E1‐E2 binding domains of E2b, E2p and E2o. There was no similarity between the E3 binding domain of E2b to E2p and E2o which may reflect the uniqueness of the E3 component of branched‐chain‐oxoacid dehydrogenase of P. putida. The conclusions drawn from these comparisons are that the trans‐acylases of prokaryotic pyruvate, 2‐oxoglutarate and branched‐chain‐oxoacid dehydrogenases descended from a common ancestral protein probably at about the same time.
Purification and caracterization of DNA polymerase α from plasmodia of Physarum polycephalum
Authors: WEBER Cornelia , FISCHER Heinrich , HOLLER Eggehard
Published: March 2005

DNA polymerase α from Physarum polycephalum has been purified fromfreshly harvested microplasmodia. An inhibitory activity was removed by precippitation with poly(ethyleneimine) and interfering type‐β‐like DNA polymerase by chromatography on phosphocellulose. The preparation was free of endonucleases and exonucleases.

The DNA‐polymerizing polypeptide had a molecular mass of 140kDa by polyacrylamide gel electrophoresis under denaturing conditions. It was contained in purified samples and in crude cell extracts. Peptides of smaller size that reacted with antibodies against this protein were generated during purification and prolonged standing. Molecular sizing under non‐denaturing conditions resulted inhigh‐molecular‐mass forms.

The isoelectric point (pI) was 6.7 ± 0.2, the pH optimum at pH 6.8, and the temperature optimum at 40°C. Monovalent salts, Li+, Na+, NH4+, K+, were inhibitory except for small activation maxima at 10mM, 75 mM and 100 mM in the case of Na+, NH4+ and K+ respectively. The bivalent cations Mg2+ and Mn2+ had broad activity maxima at 3‐20 mM concentrations, which were shifted to 0.05‐0.1 mM in the case of Mn2+ and synthetic DNA homopolymers.

The numbers of molecules of DNA polymerases in Physarum nuclei were calculated and compared with the established number of molecules of DNA polymerasesinhigher eucaryotes
Lectin‐carbohydrate interactions
Authors: BHATTACHARYYA Lokesh , BREWER C. Fred
Published: March 2005

The binding of galactose‐specific lectins from Erythrina indica (EIL), Erythrina arborescens (EAL), Ricinus communis (agglutinin; RCA‐I), Abrus precatorius (agglutinin; APA), and Bandeiraea simplicifolia (lectin I; BSL‐I) to fluoro‐, deoxy‐, and thiogalactoses were studied in order to determine the strength of hydrogen bonds between the hydroxyl groups of galactose and the binding sites of the proteins. The results have allowed insight into the nature of the donor/acceptor groups in the lectins that are involved in hydrogen bonding with the sugar. The data indicate that the C‐2 hydroxyl group of galactose is involved in weak interactions as a hydrogen‐bond acceptor with uncharged groups of EIL and EAL. With RCA‐I, the C‐2 hydroxyl group forms two weak hydrogen bonds in the capacity of a hydrogen‐bond acceptor and a donor. On the other hand, there is a strong hydrogen bond between the C‐2 hydroxyl group of galactose, which acts as a donor, and a charged group on BSL‐I. The C‐2 hydroxyl group of the sugar is also a hydrogen‐bond donor to APA. The lectins are involved in strong hydrogen bonds through charged groups with the C‐3 and C‐4 hydroxyl groups of galactose, with the latter serving as hydrogen‐bond donors. The C‐6 hydroxyl group of the sugar is weakly hydrogen bonded with neutral groups of EIL, EAL, and APA. With BSL‐I, however, a strong hydrogen bond is formed at this position with a charged group of the lectin. The C‐6 hydroxyl groups is a hydrogen‐bond acceptor for EIL and EAL, a hydrogen‐bond donor for APA and BSL‐I, and appears not to be involved in binding to RCA‐I. The data with the thiosugars indicate the involvement of the C‐l hydroxyl group of galactose in binding to EIL, EAL, and BSL‐I, but not to RCA‐I and APA.
Analysis of the thermal unfolding of porcine procarboxypeptidase A and its functional pieces by differential scnning calorimetry
Authors: SANCHEZ‐RUIZ José M. , LOPEZ‐LACOMBA José L. , MATEO Pedro L. , VILANOVA María , SERRA Miguel A. , AVILES Francisco X.
Published: March 2005

Porcine pancreatic procarboxypeptidase A and its tryptic peptides, carboxypeptidase A and the activation segment, have been studied by high‐sensitivity differential scanning calorimetry (DSC). The thermal denaturation of the zymogen and the active enzyme has been carried out at two pH values, 7.5 and 9.0, at different ionic strengths and at different scan rates. The endothermic transitions for these two proteins were always irreversible under all conditions investigated. The denaturation behaviour of both proteins seems to fit very well with the kinetic model for the DSC study of irreversible unfolding of proteins recently proposed by one of our groups. Fromm this model, the activation energies obtained for the denaturation of the pro‐and carboxypeptidase were 300 ± 20 kJ mol−1 and 250 %% 14 kJ mol −1 respectively. On the other hand, the isolated activation segment appears as athermostable piece with a highly reversible thermal unfolding which follows a two‐state process. The denaturation temperature observed for the isolated segment was always at least 15 K higher than those of the zymogen and the avtive enzyme.