bluuebunny/crossref_metadata_2025 huggingface dataset:
Amino acid sequence of PR‐39
Authors: AGERBERTH Birgitta , LEE Jong‐Youn , BERGMAN Tomas , CARLQUIST Mats , BOMAN Hans G. , MUTT Viktor , JÖRNVALL Hans
Published: March 2005

We recently isolated from pig intestine and characterized a 31‐residue antibacterial peptide named cecropin P1 with activity against Escherichia coli and several other Gram‐negative bacteria. The isolation involved a number of batch‐wise steps followed by several chromatography steps. The continued investigation of these antibacterial peptides has now yielded another antibacterial peptide with high activity against both E. coli and Bacillus megaterium. Amino acid analysis showed a very high content of proline (49 mol%) and arginine (26 mol%), an intermediate level of phenylalanine and low levels of leucine, tyrosine, isoleucine, and glycine. The primary structure was determined by a combination of Edman degradation, plasma desorption mass spectrometry and C‐terminal sequence analysis by carboxypeptidase Y degradation using capillary zone electrophoresis for detection of liberated residues. The calculated molecular mass was 4719.7 Da, which is in excellent agreement with 4719 Da obtained by plasma desorption mass spectrometry. The peptide was named PR‐39 (proline‐arginine‐rich with a size of 39 residues). The lethal concentration of the peptide was determined against six Gram‐negative and four Gram‐positive strains of bacteria.
Purification and properties of l‐alanine aminotransferase from Chlamydomonas reinhardtii
Authors: LAIN‐GUELBENZU Blanca , CÁRDENAS Jacobo , MUÑOZ‐BLANCO Juan
Published: March 2005

An enzyme which catalyzes the transamination of l‐alanine with 2‐oxoglutarate has been purified 157‐fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. The enzyme showed maximal activity at pH 7.3 and 50°C, has an apparent molecular mass of 105 kDa as estimated by gel filtration, and consists of two identical subunits of 45 kDa each as deduced from PAGE/SDS studies. A stoichiometry of two moles pyridoxal 5‐phosphate/mole enzyme was calculated. The enzyme has an isoelectric point of 8.3 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of l‐alanine. Pyridoxal 5‐phosphate protected activity against heat inactivation and, to a minor extent, l‐alanine and 2‐oxoglutarate, but not l‐glutamate. Spectral data and activity inhibition and protection studies strongly support the involvement of pyridoxal 5‐phosphate in enzyme catalysis through a Schiff's base formation. The purified enzyme was able to transaminate only l‐alanine and l‐glutamate with glyoxylate out of ten amino acids tested. l‐Alanine aminotransferase exhibited hyperbolic kinetic for 2‐oxoglutarate, pyruvate, and l‐glutamate, and nonhyperbolic behaviour for l‐alanine. Apparent Km values were 0.054 mM for 2‐oxoglutarate, 0.52 for l‐glutamate, 0.24 mM for pyruvate, and 2.7 mM for l‐alanine. Transamination of l‐alanine in C. reinhardtii is a bisubstrate reaction with a bi‐bi ping‐pong mechanism, and is not inhibited by substrates.
Functional expression of the cDNAs encoding rat 11β‐hydroxylase [cytochrome P450(11β)] and aldosterone synthase [cytochrome P450(11β, aldo)]
Authors: NONAKA Yasuki , OKAMOTO Mitsuhiro
Published: March 2005

Expression plasmids containing two cDNAs of a rat cytochrome P450(11β) family, pcP450(11β)‐62 [Nonaka, Y., Matsukawa, N., Morohashi, K., Omura, T., Ogihara, T., Teraoka, H. & Okamoto, M. (1989) FEBS Lett. 255, 21–26] and pcP450(11β, aldo)‐46 [Matsukawa, N., Nonaka, Y., Ying, Z., Higaki, J., Ogihara, T. & Okamoto, M. (1990) Biochem. Biophys. Res. Commun. 169, 245–252], were constructed and introduced into COS‐7 cells by electroporation. Enzymatic activities of the expressed cytochromes P450(11β) and P450(11β, aldo) were determined by using 11‐deoxycorticosterone, corticosterone, 18‐hydroxy‐11‐deoxycorticosterone, 18‐hydroxycorticosterone, or 19‐hydroxy‐11‐deoxycorticosterone as a substrate. Cytochrome P450(11β) catalyzed 11β‐, 18‐and 19‐hydroxylations of 11‐deoxycorticosterone and 19‐oxidation or 19‐hydroxy‐11‐deoxycorticosterone at substantial rates, 18‐hydroxylation of corticosterone at a very low rate, but no aldosterone production. Cytochrome P450(11β, aldo) catalyzed 11β‐ and 18‐hydroxylations of 11‐deoxycorticosterone, 18‐hydroxylation of corticosterone and aldosterone production from 11‐deoxycorticosterone or corticosterone. But neither 19‐hydroxylation of 11‐deoxycorticosterone nor 19‐oxidation of 19‐hydroxy‐11‐deoxycorticosterone was catalyzed by cytochrome P450(11β, aldo).
Structure and properties of a ubiquitously expressed protein kinase C, nPKCδ
Authors: MIZUNO Keiko , KUBO Kyoko , SAIDO Takaomi C. , AKITA Yoshiko , OSADA Shin‐ichi , KUROKI Toshio , OHNO Shigeo , SUZUKI Koichi
Published: March 2005

cDNA clones coding for novel protein kinase C δ (nPKCδ) were isolated from a mouse brain cDNA library. Mouse nPKCδ consists of 674 amino acid residues and has sequence identity of 95% with rat nPKCδ. Antiserum raised against a C‐terminal peptide of rat nPKCδ identified a 79‐kDa protein in COS cells transfected with a mouse nPKCδ cDNA expression plasmid. nPKCδ expressed in COS1 cells had phorbol‐ester‐binding activity and protein kinase activity in a phorbol‐ester‐or diacylglycerol‐dependent manner, like conventional protein kinase C (cPKC) isozymes and nPKCδ. However, nPKCδ, like nPKCδ, is not activated by Ca2+, a known activator of cPKCs, and requires lower concentrations of Mg2+ for full activation than cPKCs. Moreover, apparent kinetic constants for synthetic oligopeptides (MBP4–14, EGFR peptide and ɛ‐peptide) were quite different between nPKCδ and cPKC in two different conditions. Among various phospholipids tested, phosphatidylinositol is the most potent activator of nPKCδ, in clear contrast to cPKCs and nPKCδ. Limited proteolysis of nPKCδ generated a C‐terminal active fragment with a cofactor‐independent kinase activity. Northern blot analysis indicated that nPKCδ, like cPKCα, is widely distributed in almost all the tissues and cells examined and, in some cases such as fibroblast cells, exists as a major PKC type. These results suggest that nPKCδ is involved in fundamental cellular functions regulated by diacylglycerols and mimicked by phorobol esters.
Effect of the amine non‐leaving group on the structure and stability of DNA complexes with cis‐[Pt(R‐NH2)2(NO3)2]
Authors: BUTOUR Jean‐Luc , ALVINERIE Paul , SOUCHARD Jean‐Pierre , COLSON Pierre , HOUSSIER Claude , JOHNSON Neil P.
Published: March 2005

The antitumor compound cis‐[Pt(NH3)2Cl2] (cisplatin), conserves two ammine ligands during gthe reaction with its cellular garget DNA. Modifications of these non‐ileaving groups change the antineoplastic properties of this ompound and its enotoxic effects. It is therefore of interest to determine the influence of non‐leaving groups on the structure and stability of DNA in vitro. We have investigated platinum‐DNA adducts formed by cis‐[Pt(RNH2)2(NO3)2] (where R‐NH2= NH3, metylamine, cyclobutylamine, cyclopentylamine and cyclohexylamine) as a function of DNA binding. All compounds quantitatively reacted with DNA in less than 1 h at 37°C. They formed bifunctional adducts with adjacent nucleotides judging from the displacemetn of teh intercalating molecule ethidium bromide, ultraviolet absorption spectroscopy and circular dichroism. Substitution of a H on the NH3 ligand by alkyl groups dramatically destablized the platinum‐DNA complex. Thermal stability dereased progressively with an increasing number of carbon atoms, Δtm=–4.4°C for 3 cclohexylamine‐platinum‐DNA adducts/1000 nucleotides, conditions where cisplatin had no effect. DNA adducts with cycloutylamine and cyclothexylasmine ligands inhibited the hydrolysis of platinum‐DNA complexes by S1 nuclease. Km for the digestion of DNA containing these lesions was 2.3 times reater than for cisplatin, indicating steric inhibitionof enzyme‐substrate complex formation. These results showthat the non‐leaving groups of substituted cis‐Pt(II) compounds may destabilize DNA and interfere with protein‐DNA interactions. These perturbations may have consequences for the genotoxic and antitumor activigties of platinum compounds.
Molecular cloning of soluble aminopeptidases from Saccharomyces cerevisiae
Authors: GARCIA‐ALVAREZ Nieves , CUEVA Rosario , SUÁREZ‐RENDUELES Paz
Published: March 2005

Plasmids capable of complementing lap1, lap2 and lap3 mutations [R. J. Trumbly and G. Bradley (1983) J. Bacteriol. 156, 36–48] were isolated from a yeast YEp13 library by screening for activity against the chromogenic aminopeptidase substrate L‐leucine β‐naphthylamide in intact yeast colonies. The genomic inserts were shown to contain the structural genes for aminopeptidases yscII, yscIII and ysclIV. Plasmids containing the gene encoding aminopeptidase yscII of Saccharomyces cerevisiae, APE2 (LAP1) were analyzed in detail. APE2 was determined by DNA blot analysis to be a single‐copy gene located on chromosome XI. The cloned fragment was used to idenify a 2.7‐kb mRNA. The cloned APE2 gene was sequenced and found to consist of an open reading frame of 2583 bp encoding a protein of 861 amino acids. The protein sequence contains two putative N‐glycosylation sites. A significant amino acid similarity was detected between the APE2 gene product and members of the zinc‐dependent metallopeptidase gene family. Chromosomal disruption of the APE2 gene completely abolished the distinct activity band previously identified as aminopeptidase yscII [H. H. Hirsch, P. Suárez‐Rendueles. T. Achstetter and D. ri. Wolf (1988) Eur. J. Biochem 173, 589–598] in crude extrats subjected to non‐denaturing polyacrylamide gel electrophoresis and subsequent aminopeptidase activity staining. No vital consequence of aminopeptidase yscII absence on cell growth could be detected.
The effect of enhanced α‐helicity on the activity of a winter flounder antifreeze polypeptide
Authors: CHAKRABARTTY Avijit , HEW Choy L.
Published: March 2005

The antifreeze polypeptide (AFP) from the winter flounder displays partial α‐helix formation at lower temperatures. To investigate the relationship between antifreeze activity and α‐helical structure, we designed and then chemically synthesized an AFP analog with enhanced α‐helicity, and compared its conformation and antifreeze properties with those of the native AFP. The synthetic analog was more helical than the native AFP; however the antifreeze activity of both peptides were identical. The antifreeze activity of the peptids displayed a strong pH dependence, which paralleled pH‐induced changes in helix content. At pH 8.5, the antifreeze activity of both peptides displayed identical concentration dependences. In addition to antifreeze activity measurements, the effects of the peptides on the rate of ice crystal growth were also measured. While both peptides affected the a‐ and c‐ axis growth rates of ice crystals, the highly helical analog was able to exert its effects on ice crystal growth rates at 7–8‐fold lower concentrations than the native AFP. These data indicate that there is a direct but complex relationship between α‐helicity and antifreeze activity.
Surface topography of histidine residues in lysozymes
Authors: ZHAO Yun‐juan , SULKOWSKI Eugene , PORATH Jerker
Published: March 2005

Several avian and mammalian c‐type lysozymes were chromatographed on chelated (to iminodiacetate) and immobilized transition metal ions (Co2+, Ni2+, Cu2+ and Zn2+) under a variety of experimental conditions. The varied affinity of evolutionary variants of the lysozyme family for chelated metal ions, IDA‐M(II), can be rationalized primarily in terms of the presence, multiplicity and microenvironments of histidine residues. The chromatographic resolution of some of these closely related proteins attests to the analytical power of immobilized metal‐ion affinity chromatography.
The primary structure of ammodytin L, a myotoxic phospholipase A2 homologue from Vipera ammodytes venom
Authors: KRIŽAJ Igor , BIEBER Allan L. , RITONJA Anka , GUBENšEK Franc
Published: March 2005

A new myotoxic phospholipase A2 homologue, having a serine residue in position 49 instead of highly conserved aspartic acid, was found in the venom of Vipera ammodytes. The primary structure revealed additional mutations in the positions important for enzymatic activity. Tyr28 is exchanged for a histidine and Gly33 for asparagine. These changes render earlier‐reported weak enzymatic activity unlikely. The role of this rather abundant venom fraction is apparently in myotoxicity, which was confirmed in the muscle‐cell culture from neonatal rats. The muscle‐cell culture proved to be a good tool to investigate the effects of various myotoxins on muscle cells.
Mechanism of action of the calpain activator protein in rat skeletal muscle
Authors: MICHETTI Mauro , VIOTTI Pier Luigi , MELLONI Edon , PONTREMOLI Sandro
Published: March 2005

Rat skeletal muscle contains a calpain activator protein characterized by a high specificity for calpain II, the high Ca2+‐requiring isoform of this class of proteinases. The activator protein increases the rate of intramolecular conversion of the native 80‐kDa catalytic subunit of calpain into the autolysed 75‐kDa forms with maximal rate at concentrations of calcium approximately 25 times lower than those required by the native proteinase. The activator protein interacts with native calpain II forming a 1:1 complex; interaction does not occur with the fully activated form, produced by autoproteolysis. Even after immobilization to membranes, the activator binds to calpain, which then undergoes sequential activation and release from its bound form. The activator is itself resistant to digestion by calpain II, whereas it increases the rate at which homologous calpastatin is degraded by the proteinase. Taken together, these results are indicative of the existence in rat skeletal muscle of an activating system specific for calpain II which is potentially involved in the regulation of the inhibitory efficiency of calpastatin, through modulation of its intracellular level.
NMR studies of lantibiotics
Authors: VAN DE VEN Frank J. M. , VAN DEN HOOVEN Henno W. , KONINGS Ruud N. H. , HILBERS Cornelis W.
Published: March 2005

Nisin is a posttranslationally modified protein of 34 amino acids, and is a member of the class of bacteriocidal polypeptides known as lantibiotics, that contain the unusual amino acid lanthionine. Its structure in aqueous solution has been determined on the basis of NMR data, i.e. interproton distance constraints derived from nuclear Overhauser enhancement spectroscopy and torsion angle constraints derived from double‐quantum‐filtered correlated spectroscopy. Translation of the NMR constraints into a three‐dimensional structure was carried out with the distance‐geometry program DISMAN, followed by restrained energy minimization using CHARMm. The internal mobility of the peptide chain prohibited the determination of a precise overall folding of the molecule, but parts of the structure could be obtained, albeit sometimes with low resolution. The structure of nisin can best be defined as follows. The outermost N‐terminal and C‐terminal regions of nisin appear quite flexible, the remainder of the molecule consists of an amphiphilic N‐terminal fragment (residues 3–19), joined by a flexible ‘hinge’ region to a rigid double‐ring fragment formed by residues 23–28. The latter fragment has the appearance of a somewhat overwound α‐helix. It is suggested, by assuming the presence of a (transient) α‐helical structure in this part of prenisin, that the coupling between residues 23 and 26, as well as between 25 and 28, by thioether bridges, and the inversion of the Cα chiralities at positions 23 and 25, can be rationalized.
Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus
Authors: CONSALVI Valerio , CHIARALUCE Roberta , POLITI Laura , VACCARO Rosaria , DE ROSA Mario , SCANDURRA Roberto
Published: March 2005

The hyperthermophilic archaebacterium Pyrococcus furiosus contains high levels of NAD(P)‐dependent glutamate dehydrogenase activity. The enzyme could be involved in the first step of nitrogen metabolism, catalyzing the conversion of 2‐oxoglutarate and ammonia to glutamate. The enzyme, purified to homogeneity, is a hexamer of 290 kDa (subunit mass 48 kDa). Isoelectric‐focusing analysis of the purified enzyme showed a pI of 4.5. The enzyme shows strict specificity for 2‐oxoglutarate and l‐glutamate but utilizes both NADH and NADPH as cofactors. The purified enzyme reveals an outstanding thermal stability (the half‐life for thermal inactivation at 100°C was 12 h), totally independent of enzyme concentration.

P. furiosus glutamate dehydrogenase represents 20% of the total protein; this elevated concentration raises questions about the roles of this enzyme in the metabolism of P. furiosus.
Sequence and properties of β‐xylosidase from Bacillus pumilus IPO
Authors: XU Wei‐Zhong , SHIMA Yasufumi , NEGORO Seiji , URABE Itaru
Published: March 2005

The nucleotide sequence of the β‐xylosidase (xynB) gene from Bacillus pamilus has been reported previously [Moriyama, H., Fukusaki, E., Crespo, J. C., Shinmyo, A. & Okada, H. (1987) Eur. J. Biochem. 166, 539–545]. However, the sequence indentified in the present study is quite different from the previously reported one. The total lenght of the PstI‐EcoRI fragment of a plasmid pOXN295 containing the xnyB gene is 2201 bp from our sequencing, while the length of the fragment in the previous data was 2466 bp. The sequences are similar in the N‐terminal (500 bp) and C‐terminal (260 bp) regions, but those in the central region are completely different. From the following observations, the previous sequence seems to have no reliable experimental basis. First, the restriction sites observed for pOXN295 are quite different from the sites deduced from the sequence. Second, the amino acid composition deduced from the sequence and the composition identified by amino acid analysis of the purified β‐xylosidase are very different. It is confirmed, on the other hand, that our new sequence agrees well with these experimental data. The enzyme was purified to homogeneity from Bacillus pumilus and Escherichia coli harboring a hybrid plasmid which highly expresses the xynB gene. The molecular mass of the enzyme was estimated to be 190 kDa by high performance gel filtration chromatography using TSK‐G3000SW and 56 kDa by SDS/polyacrylamide gel electrophoresis. The pH optimum was 7.0, and the optimum temperature was 40°C. The Vm value was estimated to be 1.23±0.14 μkat/mg (for p‐nitrophenyl β‐d‐xylosied) and 0.14±0.011 μkat/mg (for xylobiose), while Km was estimated to be 3.9±0.59 mM (for p‐nitrophenyl β‐d‐xylosied) and 8.9±1.19 mM (for xylobiose).
Hydrogen‐forming and coenzyme‐F420‐reducing methylene tetrahydromethanopterin dehydrogenase are genetically distinct enzymes in Methanobacterium thermoautotrophicum (Marburg)
Authors: von BÜNAU Rudolf , ZIRNGIBL Carmen , THAUER Rudolf K. , KLEIN Albrecht
Published: March 2005

A coenzyme‐F420‐reducing and an H2‐forming methylenetetrahydromethanopterin dehydrogenase have been isolated from Methanobacterium thermoautotrophicum (Marburg). Indirect evidence suggested that the former enzyme (32 kDa) might be derived from the latter enzyme (42 kDa) by proteolysis. To test this hypothesis the gene sequence of the H2‐forming dehydrogenase was determined and compared with the N‐terminal amino acid sequence of the F420‐reducing dehydrogenase. No corresponding sequences were found indicating that the two dehydrogenases are genetically distinct enzymes. With purified enzyme preparations it is shown that the activity of the F420‐reducing dehydrogenase is inhibited in the presence of the H2‐forming enzyme. This finding is discussed in terms of substrate competition.
Cloning and molecular characterization of the HgiCI restriction/modification system from Herpetosiphon giganteus Hpg9 reveals high similarity to BanI
Authors: ERDMANN Dirk , DÜSTERHÖFT Andreas , KRÖGER Manfred
Published: March 2005

The genes coding for the GGYRCC specific restriction/modification system HgiCI from Herpetosiphon giganteus Hpg9 have been cloned in Escherichia coli in three steps. As an initial step, the methyltransferase gene could be obtained after heterologous in vitro selection of a plasmid gene bank by cleavage with the isoschizomeric restriction endonuclease BanI. The adjacent endonuclease gene was cloned following Southern blot analysis of flanking genomic regions. The two genes code for polypeptides of 420 amino acids (M.HgiCI) and 345 amino acids (R.HgiCI). Establishing a functional endonuclease gene could only be achieved using a tightly regulated expression system or by methylation of the genomic DNA prior to transformation of the endonuclease gene. The methyltransferase M.HgiCI shows significant similarities to the family of 5‐methylcytidine methyltransferases. Striking similarities could be found with both the isoschizomeric endonuclease and methyltransferase of the BanI restriction/modification system from Bacillus aneurinolyticus.
Hypochlorous acid and myeloperoxidase‐catalyzed oxidation of iron‐slfur clusters in bacterial respiratory dehydrogenases
Authors: HURST James K. , BARRETTE William C. , MICHEL Bryce R. , ROSEN Henry
Published: March 2005

Hypochlorous acid and related oxidants derived from myeloperoxidase‐catalyzed reactions contribute to the microbicidal activities of phagocytosing nuetrophils and monocytes. Microbial ironsulfur (Fe/S) clusters have been suggested as general targets of myeloperoxidase‐derived oxiations, but no susceptible Fe/S site has yet been identified. In this study, the effects of HOCl and myeloperoxidase‐catalyzed peroxidation of chloride ion upon EPR‐detectable Fe/S clusters in Escherichia coli and Pseudomonas aeruginosa were examined. Increasing amounts of oxidant produced progressive loss of signal amplitudes from the S‐1 and S‐3 Fe/S clusters of succinate: ubiquinone oxidoreductase in respiring membrane fragments. These changes were compared to loss of microbial viability, succinate uptake rates, succinate dehydrogenase activity and succinate‐dependent respiration. The amounts of oxidant required to destroy Fe/S clusters exceeded the amounts required to kill organisms or inhibit respiratory function by fators of four or five. Power saturation characteristics of the S‐1 signal indicated tht the S‐2 signal was also resistant to modification, even in highly oxidized membranes. Loss of succinate‐dependent respiration was closely associated with HOCl and myeloperoxidase‐mediated microbicidal activity against P. aeruginosa and was also an early event in the oxidant‐mediated metabolic dysfunctiosn fo E. coli. However, these effects were not caused by the destruction of the Fe/S clusters within the succinat: ubiquinone oxidoreductase. Rather, the major respiration‐inhibiting lesion(s) appeared to reside at points in the respiratory chain between the Fe/S clusters and the ubiquinone reductase site.
Substitution of the cysteinyl residue (Cys21) of subunit b of the ATP synthase from Escherichia coli
Authors: KAUFFER Sabine , DECKERS‐HEBESTREIT Gabriele , ALTENDORF Karlheinz
Published: March 2005

The Fo complex of the ATP synthase (F1Fo) of Escherichia coli contains only two cysteinly residues, Cys21, of the two copies of subunit b. Modification of Cys21 with the hydrophobic maleimide N‐(7‐dimethylamino‐4‐methyl‐coumarinyl)maleimide resulted in impairment of Fo functions [Schneider, E. & Altendorf, K. (1985) Eur. J. Biochem. 153, 105–109]. We replaced this resdue (via cassette mutagenesis) by Ser, Gly, Ala, Thr, Asp and Pro. None of the replacements resulted in detectable alterations of the function of the ATP synthase, making a functional role for these sulfhydryl residues unlikely. Due to its high tolerance towards amino acid substitutions, the region around Cys21 seems not to be a protein‐protein contact area.
Purification and characterization of the F1 portion of the ATP synthase (F1Fo) of Streptomyces lividans
Authors: HENSEL Michael , DECKERS‐HEBESTREIT Gabriele , ALTENDORF Karlheinz
Published: March 2005

The F1 complex of the ATP synthase of Streptomyces lividans was isolated and purified. The procedure involved the solubilization of F1 from membranes with buffer of low ionic strength in the presence of EDTA, ionexchange chromatography and gel filtration. The purified F1 complex from S. lividans (SLF1) consists of five subunits α,β,γ,δ and ɛ with molecular masses of 58000, 50000, 36000, 28000 and 13000, respectively and exhibits immunological cross‐reactivity with the F1 portion purified from Escherichia coli (ECF1). The enzymatic properties of SLF1 were determined by the use of microtiter‐plate‐based assay and compared with data obtained for ECF1. ATPase activity of SLF1 (specific activity: 20–30 U/mg) was only observed in the presence of high concentrations of Ca2+ (10 mM). Stimulation of the ATPase activity by Mg2+ was not detectable; quite to the contrary, Mg2+ inhibited the Ca2+‐stimulated activity of SLF1. SLF1 was re‐bound to F1‐stripped membranes of S. lividans, but not to F1‐stripped membrane vesicles of E. coli. In contrast, ECF1 could be cross‐reconstituted with F1‐stripped membranes of S. lividans; however, a structural but not a functional reconstitution of the hybrid F1Fo complex was observed.
The activation of expressed cGMP‐dependent protein kinase isozymes Iα and Iβ is determined by the different amino‐termini
Authors: RUTH Peter , LANDGRAF Wolfgang , KEILBACH Alexandra , MAY Boris , EGLEME Cecile , HOFMANN Franz
Published: March 2005

cDNA of bovine cGMP‐dependent protein kinase (cGMP kinase) isozymes Iα and Iβ differ only in their amino‐terminal domains (amino acids 1–89 and 1–104, respectively). Each recombinant isozyme (rIα and rIβ) was transiently expressed in COS‐7 cells and its properties were compared with the cGMP kinase isozymes P‐I and P‐II purified from bovine trachea. The subunit of P‐I, P‐II, rIα and rIβ had a molecular mass of about 75 kDa. rIα and rIβ had S20,w values of 7.6 and 7.2, respectively, indicating that they were present as dimeric holoenzymes. Immunostaining with specific antibodies showed that P‐I and rIα, and P‐II and rIβ, were immunologically indistinguishable. P‐I, P‐II, rIα and rIβ had the same catalytic activity. However, rIα and rIβ were half‐maximally activated at 0.1 μM and 1.3 μM cGMP, and 0.3 μM and 12 μM 8‐bromoguanosine 3′,5′‐(cyclic)phosphate (Br8‐cGMP), respectively. P‐I and P‐II had a similar shift in their apparent KA values. P‐I and rIα bound 2 mol cGMP/mol subunit to high‐affinity (site 1) and low‐affinity (site 2) cGMP‐binding sites. The exchange rates were 0.005–0.009 min‐1 for site 1 and 3.7 min‐1 for site 2. In contrast, P‐II and rIβ bound 2 mol cGMP/mol enzyme subunit at only two low‐affinity binding sites (site 2) with k‐1 values of 0.92 min‐1 and 4.8 min‐1. These results suggest that a change from the Iα amino‐terminal domain to that of Iβ increases the apparent KA value for cGMP 10‐fold by altering the binding properties of binding site 1. The differential expression of the cGMP kinase isozymes could be an important mechanism in vivo to dampen the effect of long‐term elevation of cGMP level.
Molecular cloning and analysis of two tandemly linked genes for pyruvate kinase of Trypanosoma brucei
Authors: ALLERT Sylvie , ERNEST Isabelle , POLISZCZAK Annick , OPPERDOES Fred R. , MICHELS Paul A. M.
Published: March 2005

In Trypanosoma brucei (stock 427) genes encoding the glycolytic enzyme pyruvate kinase are present on two homologous chromosomes. We have cloned and characterized one of the alleles. Two large, tandemly arranged open reading frames were found, each coding for a pyruvate kinase polypeptide of 498 amino acids. The gene sequences differ at 15 positions, resulting in five amino acid substitutions. The calculated molecular masses of the polypeptides are 54 378 Da and 54 363 Da. These values are somewhat smaller than those reported for the subunit molecular mass of the purified protein, which is 57–59 kDa. However, in vitro translation of the DNA region corresponding to the open reading frame, and translation of the RNA in a wheat‐germ lysate, yielded a product that comigrated exactly with the native polypeptide in SDS/PAGE.

The overall identity between the sequences of the trypanosomal enzyme and the enzymes from other sources is 41–51%. The conserved residues are not equally distributed over the polypeptide. The primary structure of domains A and, to a lesser extent, B, which constitute the active site, are rather well conserved. In contrast, the sequence of domain C, which supposedly is involved in the regulation of the enzyme activity, is much more variable.

The cytosolically located pyruvate kinase of T. brucei lacks the specific features found in the majority of the glycolytic enzymes of this organism that are sequestered in a microbody‐like organelle, the glycosome. It has neither a relatively high subunit molecular mass, due to unique insertions or terminal extensions, nor a high excess of positively charged amino acids. The polypeptide is shorter than that of most other pyruvate kinases and the calculated net charge is only + 3.
β subunit of mitochondrial F1‐ATPase from the fission yeast
Authors: FALSON Pierre , LETERME Serge , CAPIAU Carine , BOUTRY Marc
Published: March 2005

The Schizosaccharomyces pombe nuclear gene, atp2, encoding the β subunit of the mitochondrial ATP synthase, was sequenced and found to contain a 1575‐bp open reading frame. Two adjacent transcription‐initiation sites were found at positions 34 and 44 nucleotides upstream of the translation‐initiation codon. The deduced polypeptide sequence was composed of 525 amino acid residues (molecular mass = 56875 Da). The mature polypeptide starts at residue 45 (molecular mass = 51685 Da), indicating the presence of a presequence of 44 residues, presumably involved in mitochondrial targeting.

The atp2 mutant B59‐1 [Boutry, M. & Goffeau, A. (1982) Eur. J. Biochem. 125, 471–477] and its related revertant allele R4‐3 [Jault, J. M., Di Pietro, A., Falson, P., Gautheron, D. C., Boutry, M. & Goffeau, A. (1989) Biochem. Biophys. Res. Commun. 158, 392–399] were also cloned and sequenced. A single nonsense mutation, CAG (Gln170) → TAG (stop) in mutant B59‐1, became a missense mutation, TAG (stop) → TAC (Tyr) in revertant R4‐3. Gln170 is located between the first and second elements belonging to the nucleotide‐binding site. Its substitution by a tyrosine residue increases the enzyme affinity towards ADP, the amount of endogenous nucleotides and the apparent negative cooperativity for ATPase activity.
Tissue‐specific expression of high‐voltage‐activated dihydropyridine‐sensitive L‐type calcium channels
Authors: BIEL Martin , HULLIN Roger , FREUNDNER Stefanie , SINGER Dafna , DASCAL Nathan , FLOCKERZI Veit , HOFMANN Franz
Published: March 2005

The cloning of the cDNA for the α1 subunit of L‐type calcium channels revealed that at least two genes (CaCh1 and CaCh2) exist which give rise to several splice variants. The expression of mRNA for these α1 subunits and the skeletal muscle α2/δ, β and γ subunits was studied in rabbit tissues and BC3H1 cells. Nucleic‐acid‐hybridization studies showed that the mRNA of all subunits are expressed in skeletal muscle, brain, heart and aorta. However, the α1‐, β‐ and γ‐specific transcripts had different sizes in these tissues. Smooth muscle and heart contain different splice variants of the CaCh2 gene. The α1, β and γ mRNA are expressed together in differentiated but not in proliferating BC3H1 cells. A probe specific for the skeletal muscle α2/δ subunit did not hybridize to poly(A)‐rich RNA from BC3H1 cells. These results suggest that different splice variants of the genes for the α1, β and γ subunits exist in tissues containing L‐type calcium channels, and that their expression is regulated in a coordinate manner.
The mouse S‐antigen gene
Authors: TSUDA Masahiko , KIKUCHI Takanobu , YAMAKI Kunihiko , SHINOHARA Toshimichi
Published: March 2005

We have characterized a gene for mouse S‐antigen and compared its sequence with that of corresponding human and two recently published Drosophila S‐antigen genes. The mouse S‐antigen gene was approximately 50 kbp in length and consisted of 16 exons and 15 introns. The length of most exons was less than 100 bp and the smallest one was only 10 bp. In contrast, the length of most introns was larger than 2 kbp and the gene consisted of 97% intron and 3% exon. Both splice sites for donor and accepter were in good agreement with the GT/AG rule. S‐antigen genes in human and mouse were highly conserved. In contrast, genes for the Drosophila 49‐kDa arrestin homolog and arrestin consist of three introns and four exons and two introns and three exons, respectively. The 5′‐flanking region of the mouse S‐antigen gene, approximately 1.0 kbp long, had no regulatory elements for transcription such as the TATA, CAAT and GC boxes, while a Drosophila arrestin gene has TATA and CAAT boxes. Interestingly, the 5′‐flanking region of the mouse gene had promoter activity in an in vitro transcription assay using a nuclear extract of rat brain. A major transcription start site was found at 387 bp upstream from the translation start codon ATG in mouse. From our results, and those of others, we suggest that the S‐antigen gene has evolved from a comman ancestor gene by either insertion or deletion of introns. Such an alteration of gene structure may have played a role in the evolution of the S‐antigen.
Cloning and sequence analysis of cDNA encoding two crustacean hyperglycemic hormones from the lobster Homarus americanus
Authors: TENSEN Cornelis P. , DE KLEUN Dominique P. V. , VAN HERP François
Published: March 2005

Using the polymerase chain reaction with degenerated oligonucleotides, we have isolated cDNA clones that encode two structurally different (92% identity) crustacean hyperglycemic hormones (CHH) from the lobster Homarus americanus. The deduced amino acid sequences fully agree with previously published data on partial amino acid sequences, amino acid compositions and molecular masses of hyperglycemic peptides in the lobster. A comparative analysis between the deduced primary structure of two lobster CHH and the crab CHH sequence reveals a phylogenetic relationship and allows the prediction of biologically important regions within the structures of these novel neuropeptides.
Binding of proteins of HeLa S3 cell extract to oligonucleotides containing the consensus interferon‐response sequence (IRS) and to the IRS‐containing fragment of the human c‐myc gene
Authors: TURPAEV Kirill T. , ITKES Alexander V. , ALEXANDROVA Nadja M. , POKROVSKAYA Olga V. , IMAMOVA Laele R. , CHERNOV Boris K. , KISSELEV Lev L.
Published: March 2005

The human c‐myc proto‐oncogene was recently found to contain a regulatory sequence similar to the consensus interferon‐response sequence (IRS) of interferon‐activating genes. Binding of regulatory protein(s) to this sequence of cloned fragment of c‐myc, lacking the main part of 5′‐nontranscribing region, regulates in vitro transcription from I1/I2 initiation sites located in the first intron of the gene.

Here, we have shown that HeLa S3 nuclear extract contains different protein factors, at least two, that bind preferentially to the IRS sequence of either the c‐myc gene or the interferon‐dependent 6–16 gene. Moreover, each of these factors ‘cross‐binds’ to the region of the other gene, although affinity of this interaction is lower. Binding constants of these proteins to oligonucleotide fragments of c‐myc and 6–16 genes were determined.

In vitro transcription of the human full‐length c‐myc gene (i.e. the gene containing the complete 5′ ‐noncoding region) initiated from I1/I2 sites, that is controlled by the IRS region, was demonstrated to be blocked. A possible physiological role for the mechanisms described is discussed.
The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex
Authors: JEKEL Peter A. , HARTMANN J. Bernard H. , BEINTEMA Jaap J.
Published: March 2005

The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex, has been determined predominantly with conventional non‐automatic methods. The positions of three disulfide bridges have been determined. The sequence has about 60% identity with that of a chitinase from cucumber and 95% with the N‐terminal sequence of the lysozyme/chitinase of Parthenocissus quinquefolia. The half‐cystine residues in hevein and cucumber chitinase are located at identical positions. Hevamine is a basic protein from the lutoids (vacuoles) of rubber latex and may have a role in plugging the latex vessels and cessation of latex flow. The differences in cellular location, charge properties and sequence between hevamine and cucumber chitinase are similar to those between class I and class II chitinases from tobacco and other plant species.
Complementation of soluble phosphofructokinase activity in yeast mutants
Authors: GAYATRI Archana G. , MAITRA Pabitra K.
Published: March 2005

We describe here the genetic and biochemical analyses of two classes of mutations in the soluble phosphofructokinase (PFK I) of Saccharomyces cerevisiae: those leading to the loss of activity and those giving rise to a kinetically altered enzyme. Complementation and allele‐testing between these two classes of mutants show that loss of enzyme activity in vitro can come about not only by mutations in the catalytic subunit but also in the regulatory subunit. Also, a mutation in the catalytic subunit can give rise to an enzyme altered in its kinetic properties in a manner phenomenologically similar to that caused by a mutation in the regulatory subunit.

The results of the complementation studies in diploids suggest that, in spite of their distinct functions, both the subunits are essential for activity to be detected in vitro. This is confirmed by the reconstitution of an active PFK I enzyme by mixing cell‐free extracts of two complementing parents, each of which lacks the enzyme activity. PFK activity appears in the mixture, reaching a maximum value of 60–100% of that of the diploid in 15–30 min at 24°C. Unlike the catalytic subunit which exists in various multimeric states in cell‐free extracts of the mutant bearing only this subunit, the regulatory subunit exists largely as a monomer in a mutant devoid of the catalytic subunit. The reconstituted enzyme, however, is indistinguishable from that of the wild type, as analysed by sedimentation studies and Western blot analysis, demonstrating that only the heteromeric complex of the two subunits is active, while neither of the individual subunits displays activity in vitro.
Amino acid alterations essential for increasing the catalytic activity of the nylon‐oligomer‐degradation enzyme of Flavobacterium sp.
Authors: KATO Ko , FUJIYAMA Kazuhito , HATANAKA Haruyo Sawai , PRIYAMBADA Irfan Dwidya , NEGORO Seiji , URABE Itaru , OKADA Hirosuke
Published: March 2005

The structural genes of two homologous enzymes, 6‐aminohexanoate‐dimer hydrolase (EII; nylB) and its evolutionally related protein EII′ (nylB′) of Flavobacterium sp. KI72 have an open reading frame encoding a peptide of 392 amino acids, of which 47 are different, and conserved restriction sites. The specific activity of EII towards 6‐aminohexanoate dimer is about 1000‐fold that of EII'. Construction of various hybrid genes obtained by exchanging fragments flanked by conserved restriction sites of the two genes demonstrated that two amino acid replacements in the EII′ enzyme, i.e. Gly181 → Asp (EII type) and His266 → Asn (EII type), enhanced the activity toward 6‐aminohexanoate dimer 1000‐fold.
Evidence for a peroxisomal fatty acid β‐oxidation involving d‐3‐hydroxyacyl‐CoAs
Authors: ENGELAND Kurt , KINDL Helmut
Published: March 2005

A novel d‐(–)‐3‐hydroxyacyl‐CoA hydro‐lyase, forming 2‐trans‐enoyl‐CoA and formerly designated as epimerase (EC 5.1.2.3), was extracted from fat‐degarding cotyledons of cucumber seedling. The enzyme, called d‐3‐hydroxyacyl‐CoA hydro‐lyase or d‐specific 2‐trans‐enoyl‐CoA hydratase, is shown to be required for the degradation of unsaturated fatty acids that contain double bonds extending from even‐numbered C atoms. The d‐3‐hydroxyacyl‐CoA hydro‐lyase was exlusively localized within peroxisomes. A 10000‐fold purification by chromatography on a hydrophobic matrix, a cation exchanger, on hydroxyapatite and Mono S led to two proteins of apparent homogeneity, both exhibiting Mr of 65000. The d‐3‐hydroxyacyl‐CoA hydro‐lyases are homodimers with slightly differing isoelectric points around pH = 9.0. They catalyze the conversion of 2‐trans‐enoyl‐CoA into d‐3‐hydroxyacyl‐CoA. The reverse reaction was observed but no reaction with 2‐cis‐enoyl‐CoAs or l‐3‐hydroxyacyl‐CoAs. 2‐trans‐Decenoyl‐CoA was converted 10‐times faster than 2‐trans‐butenoyl‐CoA. The conversion of 4‐cis‐decenoyl‐CoA into octenoyl‐CoA was demonstrated in vitro with purified proteins with an assay mixture containing acyl‐CoA oxidase, multifunctional protein, thiolase and the d‐3‐hydroxyacyl‐CoA hydrolyase. Comparisons of enzyme activities present in the cotyledons or isolated peroxisomes clearly show that the pathway via dienoyl‐CoA reductase in much less effective than the sequence involving d‐3‐hydroxyacyl‐CoA hydro‐lyase.
Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase
Authors: DE MEIS Leopoldo , SUZANO Valdecir A. , CALDEIRA Teresa , MINTZ Elisabeth , GUILLAIN Florent
Published: March 2005

The first step towards ATP synthesis by the Ca2‐ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35°C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10‐fold, whereas it had no effect on the apparent affinity for Mg2+.

In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations.

Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+.

The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.
Characterization of a quinole‐oxidase activity in crude extracts of Thermoplasma acidophilum and isolation of an 18‐kDa cytochrome
Authors: GÄRTNER Peter
Published: March 2005

A quinol‐oxidase activity was detected in crude extracts of the thermoacidophilic archaebacterium Thermoplasma acidophilum. The activity was optimal at pH 5.4 and 50 °C. The Km for ubiquinol‐10 was 18 μM. The enzyme was inhibited by 2n‐heptyl‐4‐hydroxyquinoline N‐oxide with a Ki of 150 nM. Ubiquinols with different side‐chain lengths were oxidized at similar rates, whereas menaquinols were converted at 6–12‐fold higher rates compared to ubiquinols. Membranes from T. acidophilum contain cytochromes of b, d and a1 types, as shown by optical spectroscopy. CO difference spectroscopy suggests the existence of a cytochrome o. A b‐type cytochrome with an apparent molecular mass of 18 kDa was purified in the presence of high detergent concentrations. It readily forms dimers on SDS/PAGE. This cytochrome also contains Cu, as shown by atomic‐absorption spectroscopy. Redox titration suggests that the 18‐kDa cytochrome may contain 2 heme groups; b558 with a midpoint potential of 75 mV and b562/553 with a midpoint potential of – 150 mV.
Quantitative assessment of regulation in metabolic systems
Authors: HOFMEYR Jan‐Hendrik S. , CORNISH‐BOWDEN Athel
Published: March 2005

We show how metabolic regulation as commonly understood in biochemistry can be described in terms of metabolic control analysis. The steady‐state values of the variables of metabolic systems (fluxes and concentrations) are determined by a set of parameters. Some of these parameters are concentrations that are set by the environment of the system; they can act as external regulators by communicating changes in the environment to the metabolic system. How effectively a system is regulated depends both on the degree to which the activity of the regulatory enzyme with which a regulator interacts directly can be altered by the regulator (its regulability) and on the ability of the regulatory enzyme to transmit the changes to the rest of the system (its regulatory capacity). The regulatory response of a system also depends on its internal organisation around key variable metabolites that act as internal regulators. The regulatory performance of the system can be judged in terms of how sensitively the fluxes respond to the external stimulus and to what degree homeostasis in the concentrations of the internal regulators is maintained. We show how, on the level of both external and internal regulation, regulability can be quantified in terms of an elasticity coefficient and regulatory capacity in terms of a control coefficient. Metabolic regulation can therefore be described in terms of metabolic control analysis. The combined response relationship of control analysis relates regulability and regulatory capacity and allows quantification of the regulatory importance of the various interactions of regulators with enzymes in the system. On this basis we propose a quantitative terminology and analysis of metabolic regulation that shows what we should measure experimentally and how we should interpret the results. Analysis and numerical simulation of a simple model system serves to demonstrate our treatment.