bluuebunny/crossref_metadata_2025 huggingface dataset:
Amino acid sequence of a region on the glycogen‐binding subunit of protein phosphatase‐1 phosphorylated by cyclic AMP‐dependent protein kinase
Authors: Caudwell F.Barry , Hiraga Akira , Cohen Philip
Published: July 2002

The amino acid sequence of a region on the glycogen‒binding (G)‒subunit of protein phosphatase‒1G that is phosphorylated by cyclic AMP‒dependent protein kinase has been determined. The sequence is: This finding will facilitate studies of the effects of hormones on the phosphorylation state of the G‒subunit in vivo.
Veratryl alcohol as a mediator and the role of radical cations in lignin biodegradation by Phanerochaete chrysosporium
Authors: Harvey P.J. , Schoemaker H.E. , Palmer J.M.
Published: July 2002

The oxidative capacity of the ligninase from Phanerochaete chrysosporium toward monomethoxylated and dimethoxylated aromatic compounds was investigated. Phenylacetic acid derivatives were shown to be decarboxylated by the ligninase, via C‐C bond cleavage. Dimethoxylated substrates were much more readily oxidised by the ligninase than were monomethoxylated derivatives, but oxidation of the latter could be stimulated by catalytic amounts of 1,4‐dimethoxybenzene or veratryl alcohol. A mechanism based upon the ability of radical cations to function as one‐electron oxidants is described. The role of redox mediators in lignin degradation, and the biological significance of veratryl alcohol as a secondary metabolite of P. chrysosporium are discussed.
The protein kinase C activator phorbol‐12‐myristate‐13‐acetate enhances cyclic AMP accumulation in pheochromocytoma cells
Authors: Hollingsworth Elizabeth B. , Ukena Dieter , Daly John W.
Published: July 2002

The protein kinase C activator, phorbol‐12‐myristate‐13‐acetate (PMA), augments the cyclic AMP accumulation induced by forskolin in pheochromocytoma (PC 12) cells with an EC50 value of 14 nM, while having no effect on basal values. At a concentration of 100 nM PMA markedly augmented the magnitude of the forskolin response and, in addition, caused a slight increase in the potency of forskolin. PMA also enhanced the maximal cyclic AMP accumulation produced by 2‐chloroadenosine, and caused a slight increase in potency of the adenosine analog. Since PMA mimics the effect of diacylglycerols that form during the turnover of the membrane lipid, phosphatidylinositol, the results suggest an interrelationship between the systems involved in phosphatidylinositol turnover and cyclic AMP generation in PC 12 cells.
Active‐site geometry of proteinase K
Authors: Betzel Christian , Pal Gour P. , Struck Mark , Jany Klaus-Dieter , Saenger Wolfram
Published: July 2002

Proteinase K (EC 3.4.21.14) from the fungus Tritirachium album Limber is the most active known serine endopeptidase. The sequence of its 275‐residue long polypeptide chain and its three‐dimensional folding show a high degree of homology with the bacterial subtilisin proteases. Using difference Fourier methods, the binding mode of the synthetic carbobenzoxy‐Ala‐Ala‐chloromethyl ketone inhibitor to the active site of proteinase K was determined. In several cycles of restrained least‐squares, the enzyme‐inhibitor complex was refined to a current R = 12% for 9400 X‐ray diffraction data between 2.2 and 5.0 Å resolution. The inhibitor is attached to proteinase K by two covalent bonds; one between the methylene carbon of the inhibitor and Nϵ2 of the catalytic His 68, the other between the ketone carbon atom of the inhibitor and Oγ of the catalytic Ser 221. In addition, two hydrogen bonds donated by the peptide NH of Ser 221 and by the side chain NH2 of Asn 160 hold the hemiketal O− in the oxyanion hole. The peptide inhibitor is further hydrogen bonded to the proteinase polypeptide chain in a three‐stranded antiparallel pleated sheet.
Cobamide‐containing membrane protein complex in Methanobacterium
Authors: Schulz Harald , Fuchs Georg
Published: July 2002

In Methanobacterium thermoautotrophicum, most of the sole cellular cobamide, 5‐hydroxybenzimidazolylcobamide (factor III [(1986) Anal. Biochem., submitted]) is bound to a membrane protein. This corrinoid protein is solubilized as a protein complex of Mr
~500 000 by 5% nonpolar detergents. The complex is stable in 7 M urea. In SDS, two smaller fractions of Mr
~ 30 000 and 60 000 carrying the cobamide are found. The unusual properties of the corrinoid enzyme complex suggest a novel function in the metabolism of CO2 and Hz in methanogens.
The structure of 5 S ribosomal RNA in the methanogenic archaebacteria Methanolobus tindarius and Methanococcus thermolithotrophicus
Authors: Willekens Peter , Stetter Karl O. , Vandenberghe Antoon , Huysmans Erik , De Wachter Rupert
Published: July 2002

The nucleotide sequences of 5 S rRNA from archaebacteria Methanolobus tindarius and Methanococcus thermolithotrophicus were determined. They can be fitted into a secondary structure model that shows features specific for archaebacterial 5 S RNAs, as documented in a previous study. Reconstruction of a phylogenetic tree on the basis of sequences from 15 archaebacterial species presently investigated allows one to discern five branches diverging at deep evolutionary levels within this primary kingdom.
LLC‐PK1 cell mutants in cAMP metabolism respond normally to phorbol esters
Authors: Jans David A. , Hemmings Brian A.
Published: July 2002

Mutants of the pig kidney cell line, LLC‐PK1, affected in cAMP metabolism, were examined for cAMP‐dependent protein kinase (cAMP‐PK) activity and for cAMP‐mediated induction of urokinase‐type plasminogen activator (uPA). The FIB4 and FIB6 mutant cell lines possessed about 10% parental levels of cAMP‐PK activity and concomitantly reduced uPA production (10–20% parental) in response to calcitonin, forskolin and 8‐bromo cAMP. The FIB1, FIB2 and FIB5 mutant cell lines had about 70% parental levels of cAMP‐PK and the synthesis of uPA was 40–60% parental. Thus, cAMP‐mediated induction of uPA showed a dependence on the absolute levels of cAMP‐PK. However, uPA synthesis in response to phorbol‐12‐myristate‐13‐acetate by all of the mutants was similar to parental, which indicates that enzyme induction mediated by phorbol esters does not involve cAMP or cAMP‐PK.
The growth of ordered two‐dimensional sheets of 70 S ribosomes from Bacillus stearothermophilus
Authors: Piefke J. , Arad T. , Gewitz H.S. , Yonath A. , Wittmann H.G.
Published: July 2002

Well ordered two‐dimensional sheets of intact 70 S ribosomes from Bacillus stearothermophilus have been obtained in vitro using salt‐alcohol mixtures. These sheets consist of relatively small unit cells with dimensions of 200 ± 20 Å and 400 ± 30 Å. Diffraction patterns of electron micrographs of these sheets stained with uranyl acetate contain features to 42 Å resolution.
D‐Mannose‐specific interaction between xanthan and D‐galacto‐D‐mannan
Authors: Tako Masakuni , Nakamura Sanehisa
Published: July 2002

A gelation occurred in a mixed solution of xanthan and locust‐bean gum at room temperature; in contrast, gelation did not occur in a solution of xanthan and guar gum. The maximum dynamic modulus was obtained when the mixing ratio of xanthan and locust‐bean gum was 1:2 at 0.2% total gums. A mixture of deacetylated xanthan and locust‐bean gum showed the highest dynamic modulus, about twice that of the mixture of native xanthan. We concluded that the intermolecular interaction between xanthan and locustbean gum might occur between the side chains of the former and back‐bone of the latter molecules in a lock‐and‐key arrangement.
The NH2‐terminal cleavage of Escherichia coli translational initiation factor IF3
Authors: Lammi Matilde , Pon Cynthia L. , Gualerzi Claudio O.
Published: July 2002

A short form of Escherichia coli translational initiation factor IF3, repeatedly found both in vivo and in vitro, lacking the positively charged N‐terminal hexapeptide has been produced by mild trypsinization. The properties of this short form of IF3 have been studied. Compared to the long native form of the factor, the shortened IF3 displays a markedly decreased thermal stability and affinity for the 30 S ribosomal sub‐unit, as well as a reduced biological activity in protein synthesis. Following the loss of the N‐terminal hexapeptide, a second peptide bond (Lys‐90Val‐91) becomes easily accessible to proteolytic attack suggesting that formation of the short IF3 may be the first step in the physiological degradation of the factor.
Nucleotide sequence of the α‐amylase gene (ALP1) in the yeast Saccharomycopsis fibuligera
Authors: Itoh Tetsuya , Yamashita Ichiro , Fukui Sakuzo
Published: July 2002

The complete nucleotide sequence of the secretable α‐amylase gene ALP1 from the yeast Saccharomycopsis fibuligera has been determined. The ALP1 DNA hybridized to a polyadenylated RNA of 2.0 kilobases. A single open reading frame encodes a 494‐amino acid protein which is highly homologous with α‐amylase (Taka‐amylase) of a fungus Aspergillus oryzae.
Eukaryotic elongation factor Tu is present in mRNA‐protein complexes
Authors: Greenberg Jay R. , Slobin Lawrence I.
Published: July 2002

By two‐dimensional gel electrophoresis, partial peptide mapping, and antibody binding we have shown that eukaryotic elongation factor Tu is in close contact with mRNA in rabbit reticulocytes. It can be crosslinked to mRNA by irradiating both polysomes and 40–80 S mRNA‐protein complexes with short‐wave UV light. To our knowledge this is the first case in which a known translation factor has been shown to be associated with mRNA in native ribonucleoproteins.
Enzyme and organic solvents: Horse liver alcohol dehydrogenase in non‐ionic microemulsion: Stability and activity
Authors: Lee Kang Min , Biellmann Jean-François
Published: July 2002

In a microemulsion made with Triton X‐100, the stability of the enzymatic activity was higher than in ionic microemulsions. The stability increased with water content. The kinetic constants (Michaelis constant of NAD+ and maximum velocity) were close to those found in the previously studied microemulsions. The Michaelis constant of NAD+ expressed with respect to the buffer volume was higher than in water. The pH dependence of the kinetic constants in this microemulsion was determined. The activity determined by NAD+ reduction decreased with water content, whereas the redox activity determined via butanol oxidation coupled to retinal reduction was only slightly reduced.
Conversion of ATP‐actin to ADP‐actin reverses the affinity of monomeric actin for Ca2+ vs Mg2+
Authors: Selden L.A. , Gershman L.C. , Kinosian H.J. , Estes J.E.
Published: July 2002

Monomeric ATP‐actin binds Ca2+ 3–4‐times more strongly than Mg2+ at pH 8. On conversion of G‐ATP‐actin to G‐ADP‐actin, the relative affinity of actin for the divalent cations is reversed, so that Mg2+ is bound 6‐times more strongly than Ca2+. The dissociation rate constant of Ca2+ from Ca‐ADP‐actin is 50‐fold higher than that for Ca2+ from Ca‐ATP‐actin, suggesting that this reversal of divalent cation affinities is due primarily to a higher equilibrium dissociation constant for Ca‐ADP‐actin. These results demonstrate an interaction between the actin‐bound nucleotide and divalent cation or their binding sites.
Degradation by the 2',5'‐phosphodiesterase activity of mouse cells requires the presence of a ribo hydroxyl group in the penultimate position of the oligonucleotide substrate
Authors: Torrence Paul F. , Alster David , Huss Sophie , Gosselin Gilles , Imbach Jean-Louis
Published: July 2002

A series of 9‐β‐D‐xylofuranosyladenine (xyloA or xyloadenosine) substituted analogs of 2‐5A core trimer and tetramer were examined for their ability to be degraded by the 2',5'‐phosphodiesterase activity of cytoplasmic extracts of mouse L cells. Two distinct groups of xyloA‐substituted analogs could be readily discriminated. The first group contained xyloadenosine at the 2'‐termini and included A2'p5'A2'p5'(xyloA) and A2'p5'A2/p5'A2'p5'(xyloA). These oligomers behaved as did their parent oligoadenylates in that they were equally sensitive to degradation by the 2',5'‐phosphodiesterase activity. The second group of oligonucleotides bore a xyloadenosine residue in the penultimate nucleotide residues of the oligomers and included A2'p5'(xyloA)2'p5'(xyloA), (xyloA)2'p5'(xyloA)2'p5'(xyloA), A2'p5'A2'p5'(xyloA)2'p5'(xyloA) and (xyloA)2'p5' (xyloA)2'p5'(xyloA)2'p5'(xyloA). This group was quite resistant to 2',5'‐phosphodiesterase activity. In all, the findings demonstrate that the ribo configuration 3'‐hydroxyl group in the penultimate nucleotide of the oligonucleotide substrate is a prerequisite for the 2',5'‐phosphodiesterase activity.
Possible involvement of prostaglandins in vasoconstriction induced by zymosan and arachidonic acid in the perfused rat liver
Authors: Dieter Peter , Altin Joseph.G. , Bygrave Fyfe L.
Published: July 2002

Exposure of perfused livers to zymosan, arachidonic acid or phenylephrine but not to latex particles, stimulates hepatic constriction. The effects of arachidonic acid are rapid, reach a maximum after 2–3 min and then decline. They are blocked by the cyclooxygenase inhibitor indomethacin but not by the lipoxygenase inhibitor nordihydroguaiaretic acid. This suggests a role for prostaglandins in this action. Zymosan progressively increases hepatic pressure after a lag time of about 1 min. Perfusion of bromophenacyl bromide, indomethacin and nordihydroguaiaretic acid only partially inhibits the zymosan‐induced vasoconstriction. None of these inhibitors affect the phenylephrine‐induced response. Repeated infusion of arachidonic acid leads to homologous desensitization of the response whereas the response of the liver to phenylephrine is unaffected. The present data indicate that prostaglandins, produced and released within the liver, affect vasoconstriction in this organ.
Sites of arrestin action during the quench phenomenon in retinal rods
Authors: Zuckerman Ralph , Cheasty James E.
Published: July 2002

The target proteins for arrestin (48 kDa protein) action during the quench of cGMP phosphodiesterase (PDE) activation in retinal rod disk membranes were identified by the use of a cross‐linking reagent. A heterobifunctional, cleavable, photoactivatable cross‐linker (sulfo‐SADP) was coupled to purified arrestin. Under precise weak visible light bleach and nucleotide conditions of quench, the cross‐linker was UV flash‐activated at a time when quench was well established. The target proteins covalently linked to arrestin by cross‐linker activation were identified by immunoblotting. In the presence of ATP arrestin cross‐linked to both PDE and rhodopsin during the quench phenomenon. Removal of ATP from the reaction mixture essentially abolished the cross‐link with PDE, just as ATP omission abolishes quench, but significantly increased the cross‐link to rhodopsin. The absence of a cross‐link to the plentiful β‐subunit of transductin, as well as the results of competition studies employing arrestin without attached cross‐linker, suggest that the observed cross‐links are specific and reflect true binding interactions of arrestin during quench. The data are consistent with a model of quench in which photolyzed rhodopsin (R*) catalyses the formation of an activated form of arrestin, which dissociates from R* in the presence of ATP, and binds to PDEs, thereby deactivating them.
Developmentally regulated alternative splicing of brain myelin‐associated glycoprotein mRNA is lacking in the quaking mouse
Authors: Fujita Nobuya , Sato Shuzo , Kurihara Tadashi , Inuzuka Takashi , Takahashi Yasuo , Miyatake Tadashi
Published: July 2002

Evidence is presented that expression of the two myelin‐associated glycoprotein mRNAs is developmentally regulated in mouse brain. In quaking mouse, the mRNA without a 45‐nucleotide exon portion was scarcely expressed throughout development. We conclude that the mechanism of splicing out the 45‐nucleotide exon portion is lacking in quaking mouse.
Psoralen photofootprinting of protein‐binding sites on DNA
Authors: Zhen Wei-ping , Jeppesen Claus , Nielsen Peter E.
Published: July 2002

Using a BAL31 exonuclease assay to determine the sites of 4,5′,8‐trimethylpsoralen photocrosslinking in DNA we have shown that 5′‐TA sites which are accessible to psoralen DNA interstrand photocrosslinking in naked DNA become inaccessible when protein, in casu, λ‐repressor E. coli or RNA polymerase are bound at their recognition DNA sequences (OR1 operator or deol promoter, respectively). These results show that psoralens can be used as photofootprinting reagents to study specific protein‐DNA interactions.
DNA‐binding protein(s) interacts with a conserved nonameric sequence in the upstream regions of wheat histone genes
Authors: Kawata Takefumi , Nakayama Takuya , Mikami Koji , Tabata Tetsuya , Takase Hisabumi , Iwabuchi Masaki
Published: July 2002

A nuclear protein(s), HBP‐2, that binds to the upstream region of the wheat histone H4 gene was identified from a fractionated nuclear extract of wheat germ by DNase I footprinting. The DNase I‐protected region contained the conserved nonameric motif, CATCCAACG. Cross‐competition experiments that used the mobility shift assay showed that this nuclear protein(s) binds specifically to the upstream sequence that has been postulated to be a cis element of the wheat H3 gene. Our findings suggest that this DNA‐binding protein(s) may be a trans‐acting factor in the regulation of the transcription of wheat histone genes.
Receptor‐regulated formation of GTP[γS] with subsequent persistent Gs‐protein activation in membranes of human platelets
Authors: Wieland Thomas , Jakobs Karl H.
Published: July 2002

Preincubation of human platelet membranes with the ATP analog ATP[γS] led to persistent adenylate cyclase activation. This stimulation was increased by copreincubation with PGE1 and obliterated by removing endogenous GDP by the NTP‐regenerating system, creatine phosphate plus creatine kinase. PGE1 partially reversed the action of the regenerating system. Control formation of GTP[γS] from ATP[γS] and GDP in platelet membranes was apparently not stimulated by PGE1. In contrast, in the presence of creatine phosphate plus creatine kinase, which prevented formation of GTP[γS], PGE1 stimulated formation of this GTP analog, by partially reversing the action of the NTP‐regenerating system. The data indicate that GTP[γS] can be formed by a membrane‐associated nucleoside diphosphokinase from ATP[γS] and GDP, resulting in persistent Gs‐protein activation, and that this process can be stimulated by an agonist‐activated receptor.
Characterization of sites of serine phosphorylation in human placental insulin receptor copurified with insulin‐stimulated serine kinase activity by two‐dimensional thin‐layer peptide mapping
Authors: Smith David M. , Sale Graham J.
Published: July 2002

Insulin receptor was copurified from human placenta together with insulin‐stimulated kinase activity that phosphorylates the insulin receptor on serine residues. Analysis of phosphorylated insulin receptor by two‐dimensional tryptic peptide mapping showed that sites of insulin stimulated serine phosphorylation in the insulin receptor were recovered in the same peptides as those known to be phosphorylated on serine in vivo in response to insulin. This indicates that the serine kinase copurified with the insulin receptor represents a physiologically important enzyme involved in the insulin triggered serine phosphorylation of the insulin receptor in vivo.
Gene transfer in plant protoplasts Inhibition of gene activity by cytosine methylation and expression of single‐stranded DNA constructs
Authors: Hershkovitz Maty , Gruenbaum Yosef , Zakai Nehama , Loyter Abraham
Published: July 2002

Transfection of petunia protoplasts with the plasmid pUC118CaMVCAT, bearing the bacterial chloramphenicol acetyltransferase (CAT) gene, resulted in the appearance of CAT activity. No CAT activity was detected in protoplasts transfected with the hemimethylated form of pUC118CaMVCAT. Transfection of petunia protoplasts with ss DNA constructs bearing the coding and noncoding sequences of the CAT gene also resulted in the appearance of CAT activity, indicating expression of ss DNA constructs in transfected protoplasts. The relevance of our observations to control of gene expression in plant cells is discussed.
Secondary structure prediction for RNA binding domain in RNP proteins identifies βαβ as the main structural motif
Authors: Ghetti A. , Padovani C. , Di Cesare G. , Morandi C.
Published: July 2002

In eukaryotic cells transcript processing is strictly dependent upon binding of specific proteins. Nuclear RNA binding proteins share a common domain, which is involved in RNA binding. In order to characterize RNP‐RNA interactions we have performed a secondary structure prediction based both on statistical algorithms and comparative analysis of different proteins. A high conservation for secondary structure propensity between different RNPs was observed.
Colicin N and its thermolytic fragment induce phospholipid vesicle fusion
Authors: Massotte D. , Pattus F.
Published: July 2002

Colicin N, a bacteriocin encoded on a plasmid belonging to the pore‐forming class of colicins, induces phospholipid vesicle fusion at acidic pH as demonstrated by fluorescence resonance energy transfer. Its C‐terminal thermolytic fragment has properties very similar to the native molecule. The fusion is protein concentration‐dependent and is regulated by (a) group(s) with a pK of approximately 4.6. The physiological relevance of this characteristic common to all colicins tested so far is discussed.
RNA editing at a splicing site of NADH dehydrogenase subunit IV gene transcript in wheat mitochondria
Authors: Lamattina Lorenzo , Weil Jacques-Henry , Grienenberger Jean-Michel
Published: July 2002

Comparison between the sequence of the gene coding for the wheat mitochondrial NADH dehydrogenase subunit IV (nad4) and the cDNA sequence obtained by reverse transcription, using total wheat mtRNA as template, has shown the presence of a uridine residue, not encoded by the genomic sequence, at the exon2‐exon3 junction of the spliced transcript. This U creates a non‐encoded CUG leucine codon which is essential for maintaining the reading frame, as shown by the conservation of the amino acid sequence of the NAD4 protein in various species. The addition of a U or the specific post‐transcriptional conversion of a C to a U could explain this phenomenon.
Acyl coenzyme A: 6‐aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans
Authors: Whiteman P.A. , Abraham E.P. , Baldwin J.E. , Fleming M.D. , Schofield C.J. , Sutherland J.D. , Willis A.C.
Published: July 2002

A study of the final stages of the biosynthesis of the penicillins in Penicillium chrysogenum has revealed two types of enzyme. One hydrolyses phenoxymethyl penicillin to 6‐aminopenicillanic acid (6‐APA). The other, also obtained from Aspergillus nidulans, transfers a phenylacetyl group from phenylacetyl CoA to 6‐APA. The acyltransferase, purified to apparent homogeneity, had a molecular mass of 40 kDa. It also catalyses the conversion of isopenicillin N (IPN) to benzylpenicillin (Pen G) and hydrolyses IPN to 6‐APA. In the presence of SDS it dissociates, with loss of activity, into fragments of ca 30 and 10.5 kDa, but activity is regained when these fragments recombine in the absence of SDS.
The carboxyl terminus heptapeptide of the R2 subunit of mammalian ribonucleotide reductase inhibits enzyme activity and can be used to purify the R1 subunit
Authors: Yang Fu-De , Spanevello Rolando A. , Celiker Incila , Hirschmann Ralph , Rubin Harvey , Cooperman Barry S.
Published: July 2002

The heptapeptide, FTLDADF, identical in sequence to the last seven amino acid residues of the carboxyl terminus of the R2 subunit of mouse ribonucleotide reductase (RR), and its Nα‐acetyl derivative both inhibit calf thymus RR. The Nα‐acetyl derivative is considerably more potent, displaying a K
i of 20 μM. The same K
i was found for N‐AcFTLDADF inhibition of a reconstituted ribonucleotide reductase from calf thymus R1 and mouse R2, indicating that the C‐termini of calf R2 and mouse R2 might be identical. Our results, taken together with previous results of others on inhibition of viral RR, suggest that inhibition of RRs by peptides mimicking the C‐terminus of R2 may be a general phenomenon. In addition, we have shown that an affinity column, FTLDADF‐Sepharose 4B, can be used to prepare ~95% pure calf thymus R1, devoid of contamination with R2, in a very simple procedure that should be generally applicable to Rl purification from many sources.
Allicin, a naturally occurring antibiotic from garlic, specifically inhibits acetyl‐CoA synthetase
Authors: Focke Manfred , Feld Andrea , Lichtenthaler Hartmut K.
Published: July 2002

Allicin is shown to be a specific inhibitor of the acetyl‐CoA synthetases from plants, yeast and mammals. The bacterial acetyl‐CoA‐forming system, consisting of acetate kinase and phosphotransacetylase, was inhibited too. Non‐specific interaction with sulfhydryl‐groups could be excluded in experiments with dithioerythritol and P‐hydroxymercuribenzoate. Binding of allicin to the enzyme is non‐covalent and reversible. [14C]‐Acetate incorporation into fatty acids of isolated plastids was inhibited by allicin with an I50
‐value lower than 10 μM. Other enzymes of the fatty acid synthesis sequence were not affected, as was shown using precursors other than acetate.
Rhodopsin reconstitution in bleached rod outer segment membranes in the presence of a retinal‐binding protein from the honeybee
Authors: Pepe I.M. , Cugnoli C. , Schwemer J.
Published: July 2002

The physiological role of a retinal‐binding protein from honeybee is investigated. This protein, upon previous loading with all‐trans retinal and subsequent irradiation with monochromatic light of wavelength 490 nm, is able to promote the reconstitution of rhodopsin when added to a suspension of opsin membranes from bleached bovine rod outer segments. In this respect this retinal‐binding protein could have a role very similar to that postulated for the well‐known cephalopod retinochrome, that serves to catalyze the formation in the presence of light of 11‐cis retinal in photoreceptor cells and to provide it for the reconstitution of rhodopsin during the visual cycle.
Inhibition of thrombin by synthetic hirudin peptides
Authors: Binnie Cameron G. , Erickson Bruce W. , Hermans Jan
Published: July 2002

To investigate the role of different regions of hirudin in the interaction with the proteinase thrombin, segments of hirudin containing 15‐51 residues were synthesized. The C‐terminal segment 40‐65 inhibited the fibrinogen clotting activity of thrombin but not amidolysis of tosyl‐Gly‐Pro‐Arg‐p‐nitroanilide. Central peptide 15–42 was insoluble at pH 7, but peptide 15‐65 inhibited fibrinogen clotting and amidolysis to an equal extent. The N‐terminal loop peptide 1‐15 had no inhibitory activity and did not affect the potency of peptide 15‐65. These data suggest that the central region inhibits catalysis.
Guanosine and formycin triphosphates bind at non‐catalytic nucleotide binding sites of CF1 ATPase and inhibit ATP hydrolysis
Authors: Guerrero Karen J. , Ehler Linda L. , Boyer Paul D.
Published: July 2002

Guanosine triphosphate and formycin triphosphate (FTP) in the presence of excess Mg2+ can bind to empty non‐catalytic sites of spinach chloroplast ATPase (CF1). This results in a greatly reduced capacity for ATP hydrolysis compared to the enzyme with non‐catalytic sites filled with ATP. With two GTP bound at non‐catalytic sites the inhibition is about 90%; with two FTP bound about 80% inhibition is obtained. Binding and release of the nucleotides from the non‐catalytic sites are relatively slow processes. Exposure of CF1 with one or two empty non‐catalytic sites to 5–10 μM FTP or GTP for 15 min suffices for about 50% of the maximum inhibition. Reactivation of CF1 after exposure to higher FTP or GTP concentrations requires long exposure to 2 μM EDTA. The findings show that, contrary to previous assumptions, GTP can bind tightly to non‐catalytic sites of CF1. They suggest that the presence of adenine nucleotides at non‐catalytic sites might be essential for high catalytic capacity of the F1 ATPases.
Direct identification and quantification of the cofactor in glutamate semialdehyde aminotransferase from pea leaves
Authors: Nair Sean P. , Harwood John L. , John Robert A.
Published: July 2002

Glutamate asmialdehyde aminotransferase, a key enzyme in the synthetic pathway leading to chlorophyll was purified from pea, (Pixum sativum) leaves. Although the preparation contained a single contaminant the enzyme could be unambiguously identified as a dimer of subunit molar mass 45 kDa having an absorption spectrum consistent with the presence of pyridoxamine phosphate as cofactor. The cofactor was released by treatment with strong phosphate at low pH and was identified and quantified fluorimetrically. The specific activity of the enzyme (1.4 μmol·min−1·mg−1; 23 nkatal·mg−1) is very much higher than previously reported.
Three‐dimensional structure of ribonuclease Ms*3′‐guanylic acid complex at 2.5 Å resolution
Authors: Nonaka Takamasa , Mitsui Yukio , Irie Masachika , Nakamura Kazuo T.
Published: July 2002

The crystal structure of ribonuclease Ms*3+‐guanylic acid complex has been determined by molecular replacement methods based on the known structure of ribonuclease T1. The pattern of hydrogen‐bonds between the enzyme and the guanine base is similar to that discovered by Arni et al. [(1988) J. Biol. Chem. 263, 15358–15368] in the crystal structure of ribonuclease T1*2′‐guanylic acid complex. As for the possible general base in the trans‐phosphorylation step of the catalysis, 0 c 1 of Glu17 is within the hydrogen‐bond distance (2.7 Å) of the 2′‐0 of the nucleotide while N c 2 of His14 is significantly more distant (3.4 Å) from the 2′‐0.
Yeast α‐matching factor receptor‐linked G‐protein signal transduction suppresses Ras‐dependent activity
Authors: Arkinstall Stephen J. , Papasavvas Savvas G. , Payton Mark A.
Published: July 2002

Homologues of mammalian Ras conserved in Saccharomyces cerevisiae mediate glucose‐stimulated cyclic AMP formation and we used this response to test for regulation of yeast Ras activity by the α‐mating factor signal transduction pathway. α‐Mating factor suppresses glucose‐stimulated cyclic AMP formation by up to 57 ± 12.6% (n = 5) and similar inhibition was observed in four different yeast strains (MATa cells). Moreover, this response is potent (IC50 = 0.14 ± 0.19 μM (n = 4)), rapid (maximal within 1–2 min), and displays an absolute requirement for both the α‐mating factor receptor (STE2) and associated G‐protein β‐subunit (STE4). Inhibition appears independent of both phosphodiesterase activation and α‐mating factor‐stimulated cytoplasmic alkalinization. Also, basal cyclic AMP levels are unaffected by pheromone. This is the first demonstration that a cell‐surface receptor linked to a heterotrimeric G‐protein can suppress Ras‐dependent activity and could provide important insight into mechanisms controlling p21
ras
in man. Inhibition of Ras‐dependent cyclic AMP formation could also be a key event facilitating responses characteristic of yeast mating.
The cystatins: Protein inhibitors of cysteine proteinases
Authors: Turk Vito , Bode Wolfram
Published: July 2002

The last decade has witnessed enormous progress of protein inhibitors of cysteine proteinases concerning their structures, functions and evolutionary relationships. Although they differ in their molecular properties and biological distribution, they are structurally related proteins. All three inhibitory families, the stefins, the cystatins and the kininogens, are members of the same superfamily. Recently determined crystal structures of chicken cystatin and human stefin B established a new mechanism of interaction between cysteine proteinases and their inhibitors which is fundamentally different from the standard mechanism for serine proteinases and their inhibitors.
Primary structure of bovine cathepsin S Comparison to cathepsins L, H, B and papain
Authors: Wiederanders Bernd , Broemme Dieter , Kirschke Heidrun , Kalkkinen Nisse , Rinne Ari , Paquette Thomas , Toothman Penelope
Published: July 2002

The primary structure of bovine cathepsin S was determined by combining results of protein and peptide sequencing with the sequence deduced from nucleic acid sequencing. Using polymerase chain reaction (PCR) technology, cDNA clones commencing at amino acid 22 of the mature enzyme and continuing through the 3′ untranslated region of bovine cathepsin S mRNA were isolated and sequenced. The open reading frame in these overlapping clones correctly predicts the determined amino acid sequence of 13 tryptic peptides derived from purified bovine spleen cathepsin S. The deduced amino acid sequence shows that mature bovine cathepsin S consists of 217 amino acids corresponding to a molecular weight of 23.7 kDa. Cathepsin S belongs to the papain superfamily of lysosomal cysteine proteinases and shares 41% identity with papain. Amino acid sequence identities of bovine cathepsin S to human cathepsins L, H, and B are 56%, 47% and 31% respectively.
Reptilian alcohol dehydrogenase Heterogeneity relevant to class multiplicity of the mammalian enzyme
Authors: Hjelmqvist Lars , Ericsson Monica , Shafqat Jawed , Carlquist Mats , Siddiqi Abdur Rehman , Höög Jan-Olov , Jörnvall Hans
Published: July 2002

Liver alcohol dehydrogenase of the ethanol‐active type (‘class I enzyme’) from the lizard, Uromastix hardwickii, was purified and screened for relationships with other vertebrate forms of the enzyme. Two differernt acetylated N‐termini (acetyl‐Gly and acetyl‐Ser) and further positional differences already in the N‐terminal segments establish the presence of two types of protein chain. The multiplicity is different from that hitherto detected within vertebrate class I alcohol dehydrogenase isozymes but typical of that which would be expected for subunits of different classes. In particular, relationships to class II or to class II‐related forms appear likely. This may indicate yet further vertebrate alcohol dehydrogenase multiplicity or discovery of a class II non‐mammalian enzyme. The results give prospects of defining gene duplications corresponding to more than one alcohol dehydrogenase class split to at an early vertebrate stage.
Differential regulation of metalloprotease steady‐state mRNA levels by IL‐1 and FGF in rabbit articular chondrocytes
Authors: Chandrasekhar Srinivasan , Harvey Anita K.
Published: July 2002

The expression of collagenase and stromelysin is believed to be coordinately regulated. In this report however, we provide evidence that suggests subtle differences may exist in the early events of the induction of these enzymes. Rabbit articular chondrocytes treated with interleukin‐1, either alone or in combination with fibroblast growth factor, accumulated steady‐state mRNA levels for both the enzymes, with the latter treatment more effective in inducing greater levels and within a shorter time. Further, the induction of the enzymes by either protocol was blocked by cycloheximide co‐treatment. Cycloheximide added 1 h post‐stimulation with interleukin‐1 + fibroblast growth factor failed to block stromelysin mRNA expression, but was able to block collagenase steady‐state mRNA levels. Transforming growth factor‐β, another inhibitor of metalloprotease induction, showed no such differential activity. The results suggest that collagenase and stromelysin may have subtle variations in their induction pathways. Our studies further show that the enzyme induction by interleukin‐1 alone or in combination with fibroblast growth factor occurs through different, but related mechanisms.
Appearance of the Na+‐motive terminal oxidase in Bacillus FTU grown under three different conditions lowering the Δ\̄gmH+ level
Authors: Semeykina Anna L. , Skulachev Vladimir P.
Published: July 2002

The terminal oxidases and coupled Na+ transport have been studied in intact cells and inside‐out subcellucar vesicles of alkalo‐ and halotolerant Bacillus FTU grown under different conditions. Cells grown at pH 7.5 are shown to possess a system or respiration‐dependent Na+ transport which is (i) inhibited by protonophorous uncoupler and (ii) activated by Δψ‐discharging agent valinomycin, suggesting that the Na+ transport is due to cooperation of the H+‐motive oxidase and Na+/H+ antiporter. On the other hand, growth under conditions lowering the Δ\̄gmH+ level, namely (i) pH 8.6, (ii) pH 7.5 in the presence of protonophore, and (iii) pH 7.5 in the presence of low cyanide concentrations results in appearance of terminal oxidase‐supported Na+ transport which is stimulated by protonophores (the Na+‐motive oxidase). In all three cases, the appearance of ascorbate (+ TMPD) oxidation resistant to low and sensitive to high cyanide concentrations was found to occur. It is concluded that not only alkaline pH but also other conditions which lower Δ\̄gmH+ can cause substitution of Na+ for H+ as a coupling ion.
Differential inhibition of abortive transcription initiation at different promoters catalysed by E. coli RNA polymerase Effect of rifampicin on purine or pyramidine‐initiated phosphodiester synthesis
Authors: Kumar K.Prasanna , Chatterji Dipankar
Published: July 2002

The action of rifampicin on the RNA chain initiation catalysed by E. coli RNA polymerase over different templates has been studied. The steady‐state formation of dinucleoside tetraphosphate under the condition of abortive initiation reaction was assayed. It was observed that rifampicin shows a spectrum of inhibitory effects on transcription initiation at different promoters. At two different promoters with a pyrimidine nucleotide at the 5′‐initiation site, e.g. rrnB P2 having CTP and lac P2 having UTP. the effect of rifampicin on the abortive synthesis of the First phosphodiester bond was found to be total, even at low concentrations of the antibiotic. On the other hand, in most cases the effect of rifampicin on the abortive synthesis with a purine nucleotide at the 5′‐initiation site was found to be only partial, with the exception of the T7A2 promoter, where rifampicin stimulates the abortive synthesis of pppGpC, It was also noticed that if there was a purine nucleotide at the second position of a dinucleotide which had already been synthesised by the enzyme, then further addition of the third nucleotide was not blocked in the presence of rifampicin. It appeared that a purine nucleotide at the initiation site or at the product terminus site of a translocated dinucleotide behaved similary towards rifampicin. In the same way, if this position was occupied by a pyrimidine, rifampicin would inhibit further phosphodiester synthesis. even at a very low concentration. The stimulatory effect of rifampicin at the T7A2 promoter was presumably because here a ternary complex containing the promoter, enzyme and the abortive transcript pppGpC was initially stable, but dissociated upon addition of rifampicin, resulting in the rapid turn‐over of the product.
Accumulation of the preassembled membrane arm of NADH:ubiquinone oxidoreductase in mitochondria of manganese‐limited grown Neurospora crassa
Authors: Schmidt Michael , Friedrich Thorsten , Wallrath Jürgen , Ohnishi Tomoko , Weiss Hanns
Published: July 2002

The NADH:ubiquinone oxidoreductase (complex I) of mitochondria is constructed from two arms arranged perpendicular to each other. The peripheral arm producing into the matrix contains the proximal section of the electron pathway, and the membrane arm with all mitochondrially encoded subunits contains the distal section of the electron pathway. When Neurospora crassa is grown under manganese limitation the formation of the peripheral arm is disturbed, but the membrane arm containing the iron—sulfur cluster N‐2, is accumulated. An extra‐polypeptide, assumed to be a chaperon, is found to be associated with this pre‐assembled membrane arm.
Complementary DNA sequences of two 14.5 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria Completion of the primary structure of the complex?
Authors: Arizmendi Jesús M. , Skehel J.Mark , Runswick Michael J. , Fearnley Ian M. , Walker John E.
Published: July 2002

The amino acid sequences of two nuclear‐encoded subunits of complex I from bovine heart mitochondria have been determined. Both proteins have an apparent molecular weight of 14.5 kDa and their N‐α‐amino groups are acetylated. They are known as subunits B14.5a and B14.5b. Neither protein is evidently related to any known protein and their functions are obscure. A total of 34 nuclear‐encoded subunits of bovine complex 1 have now been sequenced and it is thought that the primary structure of the complex is now complete, although with such a complicated structure it is difficult to be certain that there are no other subunits remaining to be sequenced. Seven additional hydrophobic subunits of the enzyme are encoded in mitochondrial DNA, and therefore bovine heart complex I is an assembly of about 41 different proteins. If it is assumed that there is one copy of each protein in the assembly, these polypeptides contain 7,955 amino acids in their sequences, more than are found in the Escherichia coli ribosome, which contains 7,336 amino acids in its 32 polypeptides.
Progesterone action through aggregation of a receptor on the sperm plasma membrane
Authors: Tesarik J. , Mendoza C. , Moos J. , Fénichel P. , Fehlmann M.
Published: July 2002

Rapid steroid effects, reported in several cell types, have pointed out the possibility of non‐genomic mechanisms of action, presumably on cell surface receptors. Here we analyzed the effects of antibody‐mediated aggregation of a novel type of progesterone receptor on the plasma membrane of human sperm cells. We report that aggregation of hormone‐receptor complexes induces Ca2+ influx and a Ca2+‐dependent exocytotic event in this system. These data suggest a possible mechanism for rapid steroid‐induced events. Further research is warranted to examine if a similar mechanism is involved in rapid steroid effects in other cell types.
Quantitative study of the binding of cysteine proteinases to basement membranes
Authors: Guinec N. , Dalet-Fumeron V. , Pagano M.
Published: July 2002

Binding of cysteine proteinases of the papain superfamily (papain and cathepsins B, B‐like and L) to basement membranes was studied by using the enzymatic activity of these proteinases against their specific fluorogenic substrates. Papain inactived by E64 was used for K
d determination by competition experiments. The binding was characterized using the following parameters, the equilibrium constant, K
d, and the number of substract sites, n, values of which were in the range of 10−7 and 1012, respectively. Such results would be of significant interest for the understanding of the biological role of cysteine proteinases in tumour invasion and other types of tissue remodeling.
Molecular cloning of a novel protein‐tyrosine phosphatase SH‐PTP3 with sequence similarity to the src‐homology region 2
Authors: Adachi Masaaki , Sekiya Masuo , Miyachi Toshiki , Matsuno Keiki , Hinoda Yuji , Imai Kohzoh , Yachi Akira
Published: July 2002

Protein‐tyrosine phosphorylation and dephosphorylation are directly associated with cellular growth, signal transduction, and neoplastic transformation. Here we report the isolation of a complementary DNA (cDNA) clone encoding a novel protein‐tyrosine phosphatase (PTP) from a human T cell PEER cDNA library. The predicted open reading frame encodes a ∼68‐kDa protein composed of 593 amino acids which contains two src‐homology region 2's (SH2 domains) at the N terminus; this PTP is designated as SH‐PTP3. Northern blot analysis revealed that SH‐PTP3 mRNA was expressed throughout many tissues and the transcriptional size was consistent at about 6.0 kb. As with other SH2 domains in src‐family kinases, the SH2 domains of SH‐PTP3 may play a crucial role in interactions with tyrosine phosphorylated signaling proteins, including itself and protein tyrosine kinases (PTKs), to regulate tarpets' enzyme activity.
Staurosporine suppresses replicon initiation in mammalian cells
Authors: Gekeler Volker , Wilisch Annette , Probst Gudrun , Kugel Andrea , Brischwein Klaus , Engelcke Michael , Probst Hans
Published: July 2002

Replication in cellular replicons of mouse Ehrlich ascites, human CCRF‐CEM and hamster BHK‐21 cells was analyzed, after exposition of the cells to Staurosporine, by measuring the overall DNA synthesis rate, by alkaline sedimentation analysis of length distributions of growing daughter strand DNA and by DNA fibre autoradiography. The results consistently indicated that micromolar concentrations of Staurosporine caused, in all three cell lines, a fast suppression of replicon initiation which was reversible if the drug treatment did not exceed about 2 h. The inhibition of initiation was accompanied by a slight reduction of rates of propagation of replication forks. The data are interpreted in terms of the existence of a so far unknown factor which seems to be involved relatively directly in the initiation process of cellular replicons and has to be activated, like the large T antigen of SV 40 for the replication initiation in the viral genome, by a specific phosphorylation event. Unlike several other protein phosphorylations of cellular regulation, the kinase concerned here seems to be inhibited only by relatively high Staurosporine concentrations.
Structural relationship of streptavidin to the calycin protein superfamily
Authors: Flower Darren R.
Published: July 2002

Streptavidin is a binding protein, from the bacteria Streptomyces avidinii, with remarkable affinity for the vitamin biotin. The lipocalins and the fatty acid‐binding proteins (FABPs), are two other protein families which also act by binding small hydrophobic molecules. Within a similar overall folding pattern (a β‐barrel with a repeated +1 topology), large parts of the lipocalin, FABP, and streptavidin molecules can be structurally equivalenced. The first structurally conserved region within the three‐dimensional alignment, or common core, characteristic of the three groups corresponds to an unusual structural feature (a short 310 helix leading into a β‐strand, the first of the barrel), conserved in both its conformation and its location within their folds, which also displays characteristic sequence conservation. These similarities of structure and sequence suggest that all three families form part of a larger group: the calycin structural superfamily.
Isolation and characterization of autophagy‐defective mutants of Saccharomyces cerevisiae
Authors: Tsukada Miki , Ohsumi Yoshinori
Published: July 2002

Autophagy in the yeast is similar to that in mammalian cells. A mutant designated as apg1 (autophagy) defective in accumulation of autophagic bodies in the vacuoles was isolated by selection using a light microscope from a mutagenized proteinase‐deficient strain. In the apg1 strain, which has normal vacuolar proteinases, nitrogen starvation did not induce protein degradation. The apg1 mutant lost its viability faster than wild‐type cells during nitrogen starvation. By using the loss of viability as a first screening test, 75 other apg mutants were selected. These apg mutants including apg1 fell into 15 complementation groups. Genetic analyses of representative apg mutants revealed that they all had single recessive chromosomal mutations. Strains with each apg mutation were defective in protein degradation in the vacuoles induced by nitrogen starvation and homozygous diploids for each apg mutation did not sporulate. These results on the apg mutants suggest that autophagy via autophagic bodies is indispensable for protein degradation in the vacuoles under starvation conditions, and that at least 15 APG genes are involved in autophagy in yeast.