bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of a cDNA and characterization of the 5′ flanking region of the gene encoding the type I regulatory subunit of the cAMP‐dependent protein kinase
Authors: NOWAK Ines , SEIPEL Katja , SCHWARZ Manuela , JANS David A. , HEMMINGS Brian A.
Published: March 2005

A bovine cDNA probe for the type I regulatory subunit of the cAMP‐dependent protein kinase [Lee et al. (1983) Proc. Natl Acad. Sci. USA 80, 3608–3612] was used to screen two λgt11 libraries constructed from poly(A)‐rich RNA from the porcine kidney cell line, LLC‐PK1. A series of overlapping clones were isolated and characterized. The largest clone, λR115, of 1426 bp was found to code for the entire R1 protein but was apparently missing the 3′ end of the mRNA. The porcine cDNA codes for a protein of 389 amino acids that shows 99% homology to bovine R1 and hybridizes to two major mRNA transcripts of approximately 2.0 kb and 4.5 kb from LLC‐PK1 cells.

The porcine cDNA for R1 was used to screen a genomic library of LLC‐PK1 DNA constructed in the EMBL‐3 vector and several clones were isolated and characterized. By using a probe from the 5′ end of the R1 cDNA we isolated the 5′ end of the gene and 700 bp of the promoter region of the gene were sequenced. The promoter region lacks a characteristic TATA box but contains two inverted CAAT boxes and is rich in G + C residues. Several sequence motifs were identified in the 5′ promoter region which could be responsible for the regulation of synthesis of this gene. Multiple transcription initiation sites were identified by S1 nuclease mapping.
Actions of extracellular UTP and ATP in perfused rat liver
Authors: HÄSSINGER Dieter , STEHLE Thomas , GEROK Wolfgang
Published: March 2005

1. In perfused rat liver infusion of UTP and ATP in micromolar concentrations increased the portal pressure, with UTP being three times more effective than ATP at concentrations below 50μM. Whereas ATP (up to 100 μM) increased oxygen consumption, there was a dose‐dependent inhibition of oxygen uptake by UTP.

2. Both nucleotides stimulated hepatic glucose output; however, the time‐courses were different. Withdrawal of UTP, but not of ATP (up to 100 μM) caused a further transient, but substantial stimulation of glucose output.

3. ATP led to a transient net K+ uptake by the liver being followed by a K+‐release phase. Similar changes were observed with UTP; however, the initial K+ uptake was prolonged compared to ATP (1.9 min versus 3.5 min) and withdrawal of UTP, but not of ATP, stimulated hepatic K+ release markedly.

4. Metabolic and hemodynamic effects comparable to those induced by ATP were obtained with β‐and γ‐thio substituted ATP, whereas β, γ‐methylene‐substituted ATP was much less effective. The characteristic effects of UTP on glucose output, portal pressure and K+ fluxes were preserved during constant infusion of ATP or its β,γ‐methylene derivative, pointing to additive effects.

5. ATP (20 μM) led to a net Ca2+ release (50–60 nmol/g liver) within 2–3 min. When the extracellular Ca2+ concentration was lowered from 1.25 mM to 0.3 mM, this Ca2+ release was increased to about 110 nmol/g liver whereby its time course remained largely unchanged. With 1.25 mM Ca2+, UTP induced Ca2+ movements only near the detection level (i.e. below 10–20 nmol/g liver); however, with 0.3 mM Ca2+ in influent perfusate, there was a slow Ca2+ release (not completed within 5–6 min). The maximal rates of Ca2+ efflux following ATP and UTP (20 μM each) were 70 nmol and 30 nmol g−1 min−1. Withdrawal of UTP led to a short Ca2+ release supcrimposing a phase of net Ca2+ uptake.

6. The data show that extracellular UTP is a potential and effective regulator of hepatic metabolism, ion fluxes across the hepatocyte membrane and hemodynamics. Compared to ATP, UTP seems to be more effective and the responses to both nucleotides are different. The data suggest that the action of UTP could involve a receptor distinct from the purinergic P2 receptor, whereas the ATP action involves predominantly the P2Y purinoceptor subtype.
The Noc region of Ti plasmid C58 codes for arginase and ornithine cyclodeaminase
Authors: SANS Notker , SCHRÖDER Gudrun , SCHRÖDER Joachim
Published: March 2005

Plant tumors induced by Agrobacterium tumefaciens synthesize a group of substances (opines) which can serve as sole source of carbon and nitrogen for the bacteria. We investigate Ti‐plasmid‐coded genes and enzymes involved in catabolism of the opine N2‐(1,3‐dicarboxypropyl)‐L‐arginine (nopaline) with a novel approach: expression and mapping of protein‐coding regions in Escherichia coli minicells, followed by identification of enzyme functions in the heterologous E. coli background. The results show that a specific part of the nopaline catabolism (Noc) region of Ti plasmid C58 is packed with closely spaced protein‐coding regions which can be expressed into polypeptides of distinct sizes in E. coli. We identify and map three enzyme activities: nopaline oxidase, arginase and ornithine cyclodeaminase, an unusual protein converting ornithine directly into proline. Nopaline oxidase requires two different Noc‐gene‐encoded proteins for function and the latter two enzymes are new discoveries in the Noc region. These three enzyme activities together constitute a catabolic pathway leading from nopaline through arginine and ornithine to proline.
EcoR124 and EcoR124/3: the first members of a new family of type I restriction and modification systems
Authors: PRICE Clive , PRIPFL Theres , BICKLE Thomas A.
Published: March 2005

We have purified the EcoR124 and EcoR124/3 restriction enzymes and shown that they are type I enzymes by several criteria: subunit composition, DNA and S‐adenosylmethionine‐dependent ATPase activity, and site‐specific DNA methylase activity. By immunochemical criteria these enzymes are related to each other but are unrelated to the two previously investigated families of type I restriction enzymes. They form therefore a new family which we call type IC. The arrangement of the structural genes coding for these enzymes and their transcriptional organisation have been determined. These are different from the common arrangement found for the other two families of type I enzymes.
Identification of protein phosphatases‐1 and 2A and inhibitor‐2 in oocytes of the starfish Asterias rubens and Marthasterias glacialis
Authors: PONDAVEN Pierre , COHEN Philip
Published: March 2005

Protein phosphatases present in the particulate and soluble fractions of oocytes of the starfish Asterias rubens and Marthasterias glacialis have been classified according to the criteria used for these enzymes from mammalian cells.

The major protein phosphatase activity in the particulate fraction had very similar properties to protein phosphatase‐1 from mammalian tissues, including preferential dephosphorylation of the β subunit of phosphorylase kinase, sensitivity to inhibitor‐1 and inhibitor‐2, inhibition of phosphorylase phosphatase activity by protamine and heparin, and retention by heparin‐Sepharose. The major protein phosphatase in the soluble fraction had very similar properties to mammalian protein phosphatase‐2A, including preferential dephosphorylation of the α subunit of phosphorylase kinase, insensitivity to inhibitors‐1 and 2, activation by protamine and heparin, and exclusion from heparin‐Sepharose.

An acid‐stable and heat‐stable protein was detected in the soluble fraction of starfish oocytes, whose properties were indistinguishable from those of inhibitor‐2 from mammalian tissues. It inhibited protein phosphatase‐1 specifically, and its apparent molecular mass on SDS polyacrylamide gels was 31 kDa. Furthermore, an inactive hybrid formed between the starfish oocyte inhibitor and the catalytic subunit of mammalian protein phosphatase‐1 could be reactivated by preincubation with MgATP and mammalian glycogen synthase kinase‐3.

The remarkable similarities between starfish oocyte protein phosphatases and their mammalian counterparts are indicative of strict phylogenetic conservation of these enzymes. The results will facilitate further analysis of the role of protein phosphorylation in the control of starfish oocyte maturation by the hormone 1‐methyladenine.
Improved purification of pyruvate decarboxylase from wheat germ
Authors: ZEHENDER Hartmut , TRESCHER Dorothea , ULLRICH Johannes
Published: March 2005

An improved procedure was developed for the isolation of pyruvate decarboxylase from wheat germ. Its final step, an electrophoresis of the native apoenzyme in concave pore gradient polyacrylamide gels, followed by superficial activity‐staining, produced two bands of different molecular masses and chain compositions. The high‐molecular‐mass band occurred in low quantity and consisted of, probably eight, apparently identical chains of Mr= 33000, as judged from sodium dodecyl sulfate electrophoreses. The low‐molecular‐mass band contained two types of chains with Mrα= 63000–65000 and Mrβ= 61000–62000. The N termini of both chains were threonine, whereas their C‐terminal sequences were different: α, ‐(Val)‐(Ser)‐(Ala)‐Leu; β, ‐(His)‐(Asp)‐(Ala)‐Ser. Their amino acid composition was too different to be compatible with our original concept of one chain being produced from the other by proteolytic shortening. Limited proteolysis by Staphylococcus aureus V8 proteinase yielded peptides partly identical size and partly quite different.

In all properties investigated, the low‐molecular‐mass enzyme largely resembled yeast pyruvate decarboxylase; the holoenzyme appeared to possess (αβ)2 structure, the apoenzyme αβ. SH reagents inactivated the enzyme. Binding and fluorescence of 2‐p‐toluidinonaphthalene‐6‐sulfonate indicated a similar lipophilicity of the active site as found earlier for the yeast enzyme. 2‐Hydroxy‐5‐nitrobenzyl modification of exposed tryptophan residues left the holoenzyme intact, but in the apoenzyme it destroyed most of the cofactor‐binding ability and hence the activity. The strength of cofactor binding and the maximal specific activity were found somewhat lower than in yeast pyruvate decarboxylase.
Sequential processing reactions in the formation of hormone amides
Authors: BLEAKMAN Alexandra , SMYTH Derek G.
Published: March 2005

The substrate specificity of an enzyme with amidating activity, present in porcine pituitary, was investigated by examining its ability to convert the synthetic peptides D‐Tyr‐Val‐Gly and D‐Tyr‐Val‐Gly‐Lys‐Arg to the dipeptide amide D‐Tyr‐Val‐CONH2. The purified enzyme catalysed the amidation reaction with the tripeptide but did not accept the pentapeptide as a substrate. With the mixture of enzymes present in a membrane fraction from porcine pituitary or the enzymes in a secretory granule fraction, both the tripeptide and pentapeptide substrates gave rise to D‐Tyr‐Val amide; the formation of dipeptide amide from the pentapeptide, however, involved a latency period after which amidation occurred at a similar rate with the two substrates. Evidence was obtained that arginine and lysine were released from the C terminus of the pentapeptide before amidation took place since the rate of formation of dipeptide amide was reduced at pH values that were compatible with amidation but unfavourable to the action of carboxypeptidase H. In addition formation of the dipeptide amide from the pentapeptide was blocked by guanidinoethylmercaptosuccinic acid and glycylarginine, which are inhibitors of carboxypeptidase enzymes.

The experiments demonstrate that removal of basic residues from the C terminus of a peptide and amidation at C‐terminal glycine are reactions that take place consecutively. These prohormone‐processing reactions, which are intrinsic to the formation of hormone amides, did not synergise.
Computer‐graphics interpretations of residue exchanges between the α, β and γ subunits of human‐liver alcohol dehydrogenase class I isozymes
Authors: EKLUND Hans , HORJALES Eduardo , VALLEE Bert L. , JÖRNVALL Hans
Published: March 2005

Three‐dimensional models of human alcohol dehydrogenase subunits have been constructed, based on the homologous horse enzyme, with computer graphics. All types of class I subunits (α, β, and γ) and the major allelic variants (β1/β2 and γ1/γ2) have been studied. Residue differences between the E‐type subunit of the horse enzyme and any of the subunits of the human isozymes occur at 64 positions, about half of which are isozyme‐specific. About two thirds of the substitutions are at the surface and all differences can be accommodated in highly conserved three‐dimensional structures.

The model of the γ isozyme is most similar to the crystallographically analyzed horse liver E‐type alcohol dehydrogenase, and has all the funtional residues identical to those of the E subunit except for one which is slightly smaller: Val‐141 in the substrate pocket. The residues involved in coenzyme binding are generally conserved between the horse enzyme and the α, β, and γ types of the human enzyme. In contrast, single exchanges of these residues are the ones involved in the major allelic differences (β1 versus β2 and γ1 versus γ2), which affects the overall rate of alcohol oxidation since NADH dissociation is the rate‐determining step. Residue 47 is His in β2 and Arg in the β1, γ1, and γ2 subunits, and in horse liver alcohol dehydrogenase. Both His and Arg can make a hydrogen bond to a phosphate oxygen atom of NAD; hence the lower turnover rate of β1 apparently derives from a charge effect. The substitution to Gly in the α subunit results in one less hydrogen bond in NAD binding, and consequently in rapid dissociation. This may explain why the overall rate is an order of magnitude faster than that of β1. The important difference between γ1 and γ2 is an exchange at position 271 from Arg to Gln which can give a hydrogen bond from Gln in γ2 to the adenine of NAD. The tighter binding to γ2 can account for the slower overall catalytic rate in this isozyme.

The kinetics and interactions of cyclohexanol and benzyl alcohol with the isozymes were judged by docking experiments using an interactive fitting program. Most of the differences in Km values for alcohols of the β and γ subunits could be related to one amino acid difference, i.e. Thr‐48 in the β subunit, compared with Ser in the γ and the horse liver enzyme subunits. The substrate pocket of our model of the α subunit differs most markedly both from those of the other subunits of the human isozymes and from that of the horse enzyme. Substitution of Ala‐93 in α for the Phe in the others makes the substrate cleft much wider close to the active‐site zinc atom and its ligand Cys‐174. Our model of the α subunit suggests that the substrate specificity of isozymes with this subunit differs significantly from that of other isozyme subunits.
Characterization of a polysaccharide component of lipopolysaccharide from Pseudomonas aeruginosa IID 1008 (ATCC 27584) as D‐rhamnan
Authors: YOKOTA Shin‐ichi , KAYA Shunji , SAWADA Shuzo , KAWAMURA Takashi , ARAKI Yoshio , ITO Eiji
Published: March 2005

Structural studies were carried out on a rhamnose‐rich polysaccharide isolated from the O‐polysaccharide fraction of lipopolysaccharide in Pseudomonas aeruginosa IID 1008 (ATCC 27584) after destruction of the major O‐specific chain by alkaline treatment. The isolated polysaccharide contained rhamnose, 3‐O‐methyl‐6‐deoxyhexose, glucose, xylose, alanine, galactosamine and phosphorus in a molar ratio of 67:6.9:4.3:2.1:1.1:1.0:4.1. Data from analysis involving Smith degradation, methylation, 1H‐NMR spectroscopy and optical rotation measurement showed that the polysaccharide was built up of three moieties, a rhamnan chain composed of about 70 D‐rhamnose residues, the core chain and an oligosaccharide chain comprising 3‐O‐methyl‐6‐deoxyhexose, xylose, rhamnose and probably glucose. The repeating unit of the rhamnan chain was indicated to have the following structure: →3)D‐Rha(α1 → 3)D‐Rha(α1 → 2)D‐Rha(α1 →. This structure is identical with that proposed previously for the repeating unit of the side chain of lipopolysaccharide from plant pathogenic bacteria Pseudomonas syringae pv. morsprunorum C28 [Smith, A. R. W., Zamze, S. E., Munro, S. M., Carter, K. J. and Hignett, R. C. (1985) Eur. J. Biochem. 149, 73–78].
cDNA cloning and in vitro synthesis of the Dolichos biflorus seed lectin
Authors: SCHNELL Danny J. , ALEXANDER Danny C. , WILLIAMS Bill G. , ETZLER Marilynn E.
Published: March 2005

The Dolichos biflorus seed lectin contains two structurally related subunits. A cDNA library was constructed using RNA isolated from D. biflorus seeds actively synthesizing the seed lectin. The library was expressed in Escherichia coli using a lambda Charon 16 vector, and lectin‐specific antiserum was used to isolate a seed lectin cDNA. Hybridization of the D. biflorus seed lectin cDNA to RNA isolated from seeds actively producing both lectin subunits identifies a single‐size RNA of 1100 bases. An oligodeoxyribonucleotide probe, constructed from an amino acid sequence common to both lectin subunits, detects the same size RNA. Translation of seed mRNA in vitro and immunoprecipitation of translation products using a lectin‐specific antiserum yields a single polypeptide of slightly higher molecular mass than the largest seed lectin subunit. This seed lectin precursor is indistinguishable from a polypeptide synthesized from mRNA hybrid selected by the seed lectin cDNA. These data support the existence of a single polypeptide precursor for both subunit types of the D. biflorus seed lectin and suggest that differences between the subunit types arise by posttranslational processing.
Purification and characterization of the SpoOA protein of Bacillus subtilis from an overproducing strain of Escherichia coli
Authors: IKEUCHI Toshihiko , TSUNASAWA Susumu , SAKIYAMA Fumio
Published: March 2005

The spoOA gene of Bacillus subtilis is essential for the earliest stage of sporulation. To purify and characterize the product of the spoOA gene, we constructed a fusion plasmid in which the spoOA coding region was placed under the control of the Ptac promoter. When expression of the spoOA gene was induced in Escherichia coli cells by derepression of Ptac, the SpoOA protein constituted 15% of total cellular protein. The SpoOA protein was purified to homogeneity from these cells. We found that the NH2‐terminal amino acid sequence of the purified protein was essentially the same as that of the SpoOA protein (spoOA‐cat protein) from B. subtilis, and that the NH2‐terminal methicnine of the SpoOA protein from E. coli was formylated presumably because of insufficient amounts of the deformylating enzyme. The T signal [Ganoza, M. C., Marliere, P., Kofoid, E. C. and Louis, B. G. (1985) Proc. Natl Acad. Sci. USA 82, 4587–4591], in addition to the Shine‐Dalgarno signal to determine the initiation codon of the spoOA gene, is considered to function in E. coli as well as in B. subtilis. We also found that the purified SpoOA protein had a DNA‐binding activity. It was preferentially bound to the 175‐bp BclI fragment of φ105 DNA, and was released in the presence of 0.3 M KCl.
Comparison of the sex and subcellular distributions, catalytic and immunochemical reactivities of hepatic epoxide hydrolases in seven mammalian species
Authors: MEIJER Johan , LUNDQVIST Gerd , De PIERRE Joseph W.
Published: March 2005

Sex and species differences in hepatic epoxide hydrolase activities towards cis‐ and trans‐stilbene oxide were examined in common laboratory animals, as well as in monkey and man.

In general trans‐stilbene oxide was found to be a good substrate for epoxide hydrolase activity in cytosolic fractions, whereas the cis isomer was selectively hydrated by the microsomal fraction (with the exception of man, where the cytosol also hydrated this isomer efficiently). The specific cytosolic epoxide hydrolase activity was highest in mouse, followed by hamster and rabbit. Epoxide hydrolase activity in the crude ‘mitochondrial’ fraction towards trans‐stilbene oxide was also highest in mouse and low in all other species examined. Microsomal epoxide hydrolase activity was highest in monkey, followed by guinea pig, human and rabbit, which all had similar activities. Sex differences were generally small, but where significant, male animals had higher catalytic activities than females of the same species in most cases.

Antibodies raised against microsomal epoxide hydrolase purified from rat liver reacted with microsomes from all species investigated, indicating structural conservation of this protein. Antibodies directed towards cytosolic epoxide hydrolase purified from mouse liver reacted only with liver cytosol from mouse and hamster and with the ‘mitochondrial’ fraction from mouse in immunodiffusion experiments. Immunoblotting also revealed reaction with rat liver cytosol. The cytosolic and ‘mitochondrial’ epoxide hydrolases in all three mouse strains and in both sexes for each strain were immunochemically identical.

The anomalies in human liver epoxide hydrolase activities observed here indicate that no single common laboratory animal is a good model for man with regard to these activities.
Amino acid sequence of endothiapepsin
Authors: BARKHOLT Vibeke
Published: March 2005

The amino acid sequence of endothiapepsin, the aspartic protease from Endothia parasitica has been determined. The enzyme consists of 330 residues. The sequence determination was performed exclusively at the protein level. The homology of this fungal milk‐clotting enzyme with aspartic proteases is demonstrated by alignment with pepsin, chymosin, gastricsin, renin, and cathepsin D from various vertebrates and proteinase A from Saccharomyces cerevisiae showing 25–30% identity. The identity with mucor rennin from Mucor pucillus was 21% and with penicillopepsin from Penicillium janthinellum 53%, the fungal enzymes thus representing the lowest as well as the highest degree of homology.
The gangliosides of adult human aorta: intima, media and plaque
Authors: PROKAZOVA Nina V. , OREKHOV Alexander N. , MUKHIN Dmitry N. , MIKHAILENKO Irina A. , KOGTEV Leonid S. , SADOVSKAYA Valentina L. , GOLOVANOVA Natalia K. , BERGELSON Lev D.
Published: March 2005

The composition, structure and localization of gangliosides of aorta taken from subjects who had died after myocardial infarction were studied. Individual gangliosides were purified by high‐performance liquid chromatography and high‐performance thin‐layer chromatography and were characterized on the basis of their chromatographic mobility, carbohydrate composition, neuraminidase hydrolysis and methylation analysis. The main aortic gangliosides were identified as GM3, GM1, GD3, GD1a and GT1b. Significant differences in the ganglioside composition of intima and media were detected and the ganglioside profile of atherosclerotic plaques was found to differ markedly from that of unaffected intima. The latter was characterized by high content of GD3, a ganglioside thought to be associated with membrane permeability, cell interaction, adhesiveness and growth and to supress unspecific immune responses. Possible implications of the results in low‐density lipoprotein binding to the arterial wall and in immunological changes induced by atherosclerotic lesions are discussed.
Some pecularities of functioning of H+‐ATPase from the membranes of the anaerobic bacterium Lactobacillus casei
Authors: MILEYKOVSKAYA Evgenia I. , ABULADZE Anna N. , KORMER Svetlana S. , OSTROVSKY Dmitrii N.
Published: March 2005

Radiation inactivation analysis gave the target sizes of 176 + 5 kDa and 275 + 33 kDa for ATPase from anaerobic Lactobacillus casei and aerobic Micrococcus luteus bacteria respectively. The values are close to the known molecular masses of the enzymes. Thus, to function the L. casei ATPase, like the F1‐ATPases, requires a complete structure composed of all the enzyme subunits. L. casei ATPase is inhibited by 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole owing to modification of an amino acid residue(s) with pK > 8.5. L. casei ATPase consists of six identical subunits and differs from α3β3γδɛ‐type F1‐ATPases in a number of catalytic properties. Namely, ATP hydrolysis under the ‘unisite’ conditions proceeds at a relatively high rate suggesting the absence of cooperative interactions between the catalytic sites. Contrary to mitochondrial F1‐ATPase. L. casei ATPase does not form an inactive complex with ADP. These findings imply essential differences in the operating mechanism for L. casei ATPase and F1 ATPase.
Are calcium‐dependent protein kinases involved in the regulation of glycolytic/gluconeogenetic enzymes?
Authors: MIESKES Gottfried , KUDUZ Josko , SÖLING Hans‐Dieter
Published: March 2005

Changes in glycolytic flux have been observed in liver under conditions where effects of cAMP seem unlikely. We have, therefore, studied the phosphorylation of four enzymes involved in the regulation of glycolysis and gluconeogenesis (6‐phosphofructo‐1‐kinase from rat liver and rabbit muscle; pyruvate kinase, 6‐phosphofructo‐2‐kinase and fructose‐1,6‐bisphosphatase from rat liver) by defined concentrations of two cAMP‐independent protein kinases: Ca2+/calmodulin‐dependent protein kinase and Ca2+/phospholipid‐dependent protein kinase (protein kinase C). The results were compared with those obtained with the catalytic subunit of cAMP‐dependent protein kinase. The following results were obtained.

Ca2+/calmodulin‐dependent protein kinase phosphorylates 6‐phosphofructo‐1‐kinase and L‐type pyruvate kinase at a slightly lower rate as compared to cAMP‐dependent protein kinase.

6‐Phosphofructo‐1‐kinase is phosphorylated by the two kinases at a single identical position. There is no additive phosphorylation. The final stoichiometry is 2 mol phosphate/mol tetramer. The same holds for L‐type pyruvate kinase except that the stoichiometry with either kinase or both kinases together is 4 mol phosphate/mol tetramer.

Rabbit muscle 6‐phosphofructo‐1‐kinase is phosphorylated by cAMP‐dependent protein kinase but not by Ca2+/calmodulin‐dependent protein kinase.

Fructose‐1,6‐bisphosphatase from rat but not from rabbit liver is phosphorylated at the same position but at a markedly lower rate by Ca2+/calmodulin‐dependent protein kinase when compared to the phosphorylation by cAMP‐dependent protein kinase.

6‐Phosphofructo‐2‐kinase is phosphorylated by Ca2+/calmodulin‐dependent protein kinase only at a negligible rate.

Protein kinase C does not seem to be involved in the regulation of the enzymes examined: only 6‐phosphofructo‐2‐kinase became phosphorylated to a significant degree. In contrast to the phosphorylation by cAMP‐dependent protein kinase, this phosphorylation is not associated with a change of enzyme activity. This agrees with our observation that the sites of phosphorylation by the two kinases are different.

The results indicate that Ca2+/calmodulin‐dependent protein kinase but not protein kinase C could be involved in the regulation of hepatic glycolytic flux under conditions where changes in the activity of cAMP‐dependent protein kinase seem unlikely.
Subunit II of cytochrome c oxidase from Paracoccus denitrificans
Authors: STEINRÜCKE Peter , STEFFENS Guy C. M. , PANSKUS Gunnar , BUSE Gerhard , LUDWIG Bernd
Published: March 2005

Cytochrome c oxidase from the bacterium Paracoccus denitrificans, while being related to the mitochondrial enzyme in many ways, consists of only two to three different subunits. For the identification of its genes, a Paracoccus DNA library was constructed and screened with specific antibodies for expression of cloned inserts in E. coli. A positive clone expressing immunoreactive products in the molecular mass region of authentic subunit II revealed a high homology of its DNA‐deduced amino acid sequence with subunit II sequences of the mitochondrial oxidases; several typical features, such as the transmembrane folding pattern and the presumed copper‐binding site, are highly conserved between prokaryotic and mitochondrial polypeptides. A comparison with peptide sequencing data of the purified subunit established the presence of a characteristic N‐terminal extension as well as a longer C terminus in the initial translation product of the Paracoccus subunit; by mass spectroscopy, the first N‐terminally blocked residue of the mature polypeptide was identified as a pyroglutamate. No code abnormalities, but a highly specific codon usage were observed; no evidence for a localization of the subunit I gene directly adjacent to this gene has been obtained.
Monoclonal antibodies directed against the human A431 β‐adrenergic receptor recognize two major polypeptide chains
Authors: KAVERI Srinivas V. , CERVANTES‐OLIVIER Paulina , DELAVIER‐KLUTCHKO Colette , STROSBERG A. Donny
Published: March 2005

A serum‐albumin‐alprenolol conjugate was used to isolate β‐adrenergic receptors from the human A431 cell lysates. Three monoclonal antibodies were obtained from BALB/c mice immunized with these receptors. These antibodies: BRK‐1, BRK‐2, BRK‐3, were respectively of the IgM, IgG2a and IgG3 classes. All three antibodies recognized photoaffinity‐labelled receptors, immunoprecipitated ligand‐binding activity and identified the 65‐kDa and 55‐kDa polypeptides corresponding to the β2‐adrenergic receptors of A431 cells. BRK‐2 and BRK‐3 recognized both β1 and β2‐adrenergic receptors of several mammalian cells. All three antibodies visualized, by immunofluorescence, the β2‐adrenergic receptors at the surface of A431 cells. The monoclonal antibodies are directed against the protein portion of the β‐adrenergic receptors since partial or complete removal of the carbohydrate moieties by treatment with endoglycosidase such as endo‐F (endo‐β‐N‐acetylglucosaminidase F) and periodate oxidation did not affect the immunoreactivity. These antibodies will be of value to immunopurify the β‐adrenergic receptors.
The double‐stranded RNA‐dependent protein kinase is also activated by heparin
Authors: HOVANESSIAN Ara G. , GALABRU Julien
Published: March 2005

The double‐stranded(ds)‐RNA dependent protein kinase from human cells is a Mr 68000 protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by dsRNA, the p68 kinase becomes autophosphorylated. The phosphorylated p68 kinase then can catalyze the phosphorylation of exogenous substrates, such as eIF2 and histone. The second phosphorylation step can take place in the absence of dsRNA. Here we show that, besides dsRNA other polyanions, especially heparin, can also activate the p68 kinase for the autophosphorylation reaction. Heparin activation of the p68 kinase is reversible since it can be prevented by addition of antithrombin III, heparin‐binding protein. However, when antithrombin III is added after autophosphorylation of the p68 kinase then phosphorylation of histone is not affected. The p68 kinase binds to heparin‐Sepharose. Further evidence that the p68 kinase can be activated by heparin was provided by photoaffinity labeling with 8‐azido‐[α‐32P]ATP. This ATP analog can bind to the p68 kinase only in the presence of heparin or dsRNA. Thus suggesting that the activation of the p68 kinase triggers a conformational modification allowing the binding of ATP. Basic proteins, histone and protamine, prevent the activation process induced by heparin. This is probably due to binding of these basic proteins to heparin and thus sequestering the activator of the protein kinase.
Characterization of epitopes on a variant surface glycoprotein from Trypanosoma congolense by six monoclonal antibodies
Authors: REINWALD Erwin , GREISER‐WILKE Irene , ARTAMA Wayan , RISSE Hans‐Jörg , MÖLLING Karin
Published: March 2005

Monoclonal antibodies were isolated from mice immunized with variant surface glycoprotein of Trypanosoma congolense. Five out of the six monoclonals were able to detect epitopes at the cell surface in an indirect immunofluorescence analysis. One antibody did not react. Using protein‐A‐containing bacterial adsorbent all monoclonal antibodies precipitate glycosylated as well as non‐glycosylated variant surface glycoprotein. Carbohydrate chains therefore do not appear to be part of the immunodeterminant structure recognized by the various monoclonal antibodies.

Interaction of the monoclonal antibodies with protein fragments obtained by partial proteolysis with V8 protease from Staphylococcus aureus or papain allows the classification of the antibodies into three groups with different epitope specificity.
1 H‐NMR studies on the gene‐5‐encoded single‐stranded DNA binding protein of the filamentous bacteriophage IKe
Authors: de JONG Evert A. M. , KONINGS Ruud N. H. , HARMSEN Ben J. M. , PRINSE Carla W. J. M. , HILBERS Cornelis W.
Published: March 2005

Under physiological conditions and at concentrations needed for NMR studies, severe aggregation of the gene‐5 protein of the filamentous phage IKe occurs. Conditions are described for which well‐resolved 1H‐NMR spectra of the protein can be obtained. The aromatic part of the spectrum is analyzed by means of two‐dimensional NMR techniques; a complete interpretation is presented. Oligonucleotide binding studies reveal that just one phenylalanyl residue and one tyrosyl residue are influenced by the binding of rAMP, (dA)2, (dA)3, (dA)4, (dA)6, d(pT)3 or (dT)4. Upon binding, the aromatic resonances of these amino acid residues are shifted upfield by about 0.4–0.5 ppm. NMR measurements at different pH values demonstrate that only one of the two histidyl residues is freely titratable. From CIDNP experiments it is concluded that three out of five tyrosyl residues are located at the surface of the protein. Measurements carried out as a function of protein concentration indicate the occurrence of specific protein‐protein interactions between dimeric gene‐5‐protein molecules. The data obtained are compared with those available for the gene‐5 protein of M13. It follows from the comparison that these proteins mimic each other in almost every respect.
Effect of ethylene treatment on the concentration of fructose‐2,6‐bisphosphate and on the activity of phosphofructokinase 2/fructose‐2,6‐bisphosphatase in banana
Authors: MERTENS Emmanuel , MARCELLIN Pierre , VAN SCHAFTINGEN Emile , HERS Henri‐Géry
Published: March 2005

Preclimacteric bananas fruits were treated for 12 h with ethylene to induce the climacteric rise in respiration. One day after the end of the hormonal treatment, the two activities of the bifunctional enzyme, phosphofructokinase 2/fructose‐2,6‐bisphosphatase started to increase to reach fourfold their initial value 6 days later. By contrast, the activities of the pyrophosphate‐dependent and of the ATP‐dependent 6‐phosphofructo‐1‐kinases remained constant during the whole experimental period, the first one being fourfold greater than the second. The concentrations of fructose 2,6‐bisphosphate and of fructose 1,6‐bisphosphate increased in parallel during 4 days and then slowly decreased, the second one being always about 100‐fold greater than the first. The change in fructose 2,6‐bisphosphate concentration can be partly explained by the rise of the bifunctional enzyme, but also by an early increase in the concentration of fructose 6‐phosphate, the substrate of all phosphofructokinases, and also by the decrease in the concentration of glycerate 3‐phosphate, a potent inhibitor of phosphofructokinase 2. The burst in fructose 2,6‐bisphosphate and the activity of the pyrophosphate‐dependent phosphofructokinase, which is in banana the only enzyme known to be sensitive to fructose 2,6‐bisphosphate, can explain the well‐known increase in fructose 1,6‐bisphosphate which occurs during ripening.
Two hydrophobic low‐molecular‐mass protein fractions of pulmonary surfactant
Authors: CURSTEDT Tore , JÖRNVALL Hans , ROBERTSON Bengt , BERGMAN Tomas , BERGGREN Per
Published: March 2005

Hydrophobic low‐molecular‐mass proteins were isolated from minced pig lungs and separated into two fractions. Electrophoresis of protein fraction 1 showed two major bands. Calculations of molecular masses from the electrophoretic mobilities are unreliable because of the extreme hydrophobicity of the peptides. However, the two bands were at positions corresponding to apparent molecular masses of about 3 kDa and 14 kDa, while sequence degradation disclosed only one major structure. Electrophoretic separation of protein fraction 2 revealed one band, at an apparent molecular mass of about 6 kDa. Microheterogeneities at the N terminus of both fractions were observed. However, the two fractions had different N‐terminal structures and amino acid compositions. Consequently they are concluded to represent different polypeptides without common segments. Bronchoalveolar lavage from humans also contains surfactant polypeptides and at least the fraction 2 peptide is highly similar in human and porcine surfactants.

Artificial surfactant preparations, obtained by recombination of protein fraction 1 or 2 with a mixture of synthetic phospholipids, were evaluated with the pulsating bubble method and in experiments on artificially ventilated premature newborn rabbits. The addition of protein fraction 1 to the phospholipid mixture improved surface adsorption from more than 300 s to about 2 s and reduced minimum surface tension from more than 20 mN/m to nearly 0 as measured with a pulsating bubble. When this surfactant preparation was instilled into the airways of newborn rabbits, the tidal volumes at insufflation pressure 25 cm H2O was increased about twentyfold compared to the volumes obtained in non‐treated controls. Preparations based on protein fraction 1 had better in vitro and in vivo properties than those based on protein fraction 2. Both these protein‐based preparations were decidedly more effective than phospholipids alone.
Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells
Authors: SARKAR Nilima , LIST James F. , BANFALVI Gaspar
Published: March 2005

Simian virus 40 (SV40) DNA replication was studied in monolayers of infected monkey CV‐1 cells, permeabilized with lysolecithin, by incubation with [α‐32P]dTTP, the other dNTPs and rNTPs and an ATP‐regenerating system. Analysis of the labeled SV40 DNA by sedimentation in alkaline sucrose gradients showed that about 30% of the material synthesized by the permeable cells in the course of 60 min consisted of covalently closed circular SV40 DNA (form I), with the remainder sedimenting as relaxed circles (form II) and replicative intermediates between 18 S and 4 S. The synthesis of SV40 DNA in the permeabilized cell system required the presence of all four dNTPs and was completely inhibited by aphidicolin, consistent, with the involvement of DNA polymerase α. A detailed analysis of the distribution of radioactivity in the DNA synthesized involved cleavage with BstNI restriction endonuclease, followed by polyacrylamide gel electrophoresis and radioautography. The extent of labeling of all restriction fragments was nearly proportional to their length, suggesting that the entire SV40 chromosome was being replicated. This was confirmed by the careful comparison of the rate of labeling of a DNA fragment which includes the replication origin, and a fragment which includes the replication terminus. Their labeling was proportional to their size, regardless of the time for which the labeling was carried out. This demonstrated that the replication of the entire SV40 chromosome occurred in a steady state and that the start and termination of replication continuously occurred throughout the labeling period. The availability of an in vitro system in which replication of SV40 DNA undergoes multiple replication cycles should be of considerable value in the analysis of the mechanism of replication of this viral genome.
Modification of flow‐force relationships by external ATP in yeast mitochondria
Authors: RIGOULET Michel , GUERIN Bernard , DENIS Michel
Published: March 2005

The purpose of this work is to measure protonmotive force and cytochrome reduction level under different respiratory steady states in isolated yeast mitochondria. The rate of respiration was varied by using three sets of conditions: (a) different external phosphate concentrations with a fixed concentration of ADP (ATP synthesis) and (b) different concentrations of carbonylcyanide m‐chlorophenylhydrazone in the presence of oligomycin and carboxyatractylate (uncoupling) either in the absence or (c) in the presence of external ATP.

ADP plus phosphate stimulates respiration more than uncoupler at the same protonmotive force value. However, the relationships between respiratory rate and protonmotive force were similar when stimulation was induced either by ADP + Pi or by carbonylcyanide m‐chlorophenylhydrazone in the presence of ATP.

At the same respiratory rate, cytochrome a+a3 is more reduced by uncoupler than by ADP + Pi additions. However, the relationships between respiratory rate and reduction level of cytochrome‐c oxidase are similar both under ATP synthesis and with uncoupling conditions in the presence of external ATP.

Control of respiration exerted by cytochrome‐c oxidase was measured under the three conditins mentioned above. This control was low when the respiratory rate was varied by the ATP synthesis rate; it increased as a function of the respiratory rate with uncoupler in the absence of ATP. ATP decreased this control under uncoupling conditions.

These results suggest a regulatory effect of external ATP on cytochrome‐c oxidase, and support the view that the relationships between respiratory rate and protonmotive force, on the one hand, and respiratory rate and the reduction level of cytochrome‐c oxidase, on the other, depend respectively on the kinetic regulations of the system.
Characterization of a mannosidase acting on α1 → 3‐ and α1 → 6‐linked mannose residues of oligomannosidic intermediates of glycoprotein processing
Authors: MONIS Elizabeth , BONAY Pedro , HUGHES R. Colin
Published: March 2005

Baby hamster kidney (BHK) cell extracts catalyze the conversion of [3H]mannose‐labelled (Man)5GlcNAc and (Man)6GlcNAc oligosaccharides to a (Man)3GlcNAc species that retains affinity for concanavalin‐A‐Sepharose and appears to be Manα1 → 3[Manα1 → 6]Manβ1 → 4GlcNAc. The properties of the (Man)5GlcNAc‐hydrolase activity differ from lysososomal α‐mannosidases as well as previously described processing mannosidases acting on oligosaccharide intermediates of N‐glycan assembly. Mosquito cell extracts catalyze hydrolysis of (Man)6GlcNAc but lack the (Man)5GlcNAc hydrolase activity detected in BHK cell extracts.

Glycopeptide analysis has been carried out on a ricin‐resistant BHK mutant RicR14 that lacks N‐acetylglucosaminyl transferase I and fails to convert oligoigomannosidic N‐glycans to complex‐type chains, and mosquito cells that constitutively lack N‐acetylglucosaminyl transferase I. In both cells lines, the cellular glycoproteins contain (Man)5GlcNAc oligosaccharide as the major stable component equivalent to a 15–20‐fold increase compared with normal BHK cells. Although containing very high amounts of asparagine‐linked (Man)5(GlcNAc)2, RicR14 cells exhibit (Man)5GlcNAc hydrolase activity at levels similar to wild‐type BHK cells. This result, together with previous work [Foddy, L., Feeney, J. & Hughes, R. C. (1986) Biochem. J. 233, 697–706] showing the complete inhibition of conversion of oligomannosidic intermediates to complex‐type N‐glycans in BHK cells treated with swainsonine, an inhibitor of mannosidase II but not the (Man)5GlcNAc hydrolase activity, argues against a major role for the (Man)5GlcNAc hydrolase activity in N‐glycan asembly and suggesting other functions for the mannosidase activity in BHK cells.
Type 1, blue copper proteins constitute a respiratory nitrite‐reducing system in Pseudomonas aureofaciens
Authors: ZUMFT Walter G. , GOTZMANN Dany J. , KRONECK Peter M. H.
Published: March 2005

Pseudomonas aureofaciens truncates the respiratory reduction of nitrate (denitrification) at the level of N2O. The nitrite reductase from this organism was purified to apparent electrophoretic homogeneity and found to be a blue copper protein. The enzyme contained 2 atoms of copper/85 kDa, both detectable by electron paramagnetic resonance (EPR) spectroscopy. The protein was dimeric, with subunits of identical size (40 ± 3 kDa). Its pl was 6.05.

The EPR spectrum showed an axial signal with g‖ at 2.21(8) and g‖ at 2.04(5) The magnitude of the hyperfine splitting (A‖= 6.36 mT) indicated the presence of type 1 copper only. The electronic spectrum had maxima at 280 nm, 474 nm and 595 nm (ɛ= 7.0 mM−1 cm−1), and a broad shoulder around 780 nm.

A copper protein of low molecular mass (15 kDa), with properties similar to azurin, was also isolated from P. aureofaciens. The electronic spectrum of this protein showed a maximum at 624 nm in the visible range (ɛ= 2.5 mM−1 cm−1) and pronounced structures in the ultraviolet region. The EPR parameters were g‖= 2.26(6) and g‖= 2.05(6), with A‖= 5.8 mT. The reduced azurin transferred electrons efficiently to nitrite reductase; the product of nitrite reduction was nitric oxide.

The specific nitrite‐reducing activity with ascorbate‐reduced phenazine methosulfate as electron donor was 1 μmol substrate min−1 mg protein−1. The reaction product again was nitric oxide. Nitrous oxide was the reaction product from hydroxylamine and nitrite and from dithionite‐reduced methyl viologen and nitrite. No ‘oxidase’ activity could be demonstrated for the enzyme.

Our data disprove the presumed exclusiveness of cytochrome cd1 as nitrite reductase within the genus Pseudomones.
The degree of branching in (α1,4)‐(α1,6)‐linked glucopolysaccharides is dependent on intrinsic properties of the branching enzymes
Authors: TOLMASKY Diana Silvia , KRISMAN Clara Rebeca
Published: March 2005

Branching enzymes from rat and rabbit liver, as well as from potato and maize were prepared. They were almost free from contaminating glucan‐degrading enzymes.

In ‘sweet corn’ maize, two separate fractions with (α1,4)glucan: (α1,4)glucan α6‐glycosyltransferase activities were obtained. One of them synthesized amylopectin, the branched component of starch, in the presence of phosphorylase and Glc1P, while the other fraction synthesized phytoglycogen. Furthermore, in a maize variety which does not accumulate phytoglycogen, only one fraction of branching activity was found, that formed amylopectin under the above‐mentioned conditions.

Comparative analyses performed with native (α1,4)‐(α1,6)glucopolysaccharides, and those synthesized in vitro with the branching enzyme from the same tissue, demonstrated a close similarity between both glucans.

It may be concluded that the branching enzyme is responsible for the specific degree of (α1,6) branch linkages found in the native polysaccharide.