bluuebunny/crossref_metadata_2025 huggingface dataset:
Ganglioside biosynthesis in rat liver
Authors: BUSAM Klaus , DECKER Karl
Published: March 2005

Three sialyltransferase activities involved in ganglioside biosynthesis were studied in Golgi‐enriched preparations of rat liver: the formation of GM3, GD3 and GD1a. The conditions for the quantitative assays of these enzymatic reactions were standardized and optimized, with Triton X‐100 being used as detergent. The apparent Km values of each sialyltransferase for N‐acetyl‐2‐(5′‐cytidylyl)neuraminic acid (1.5 mM with GM3 synthase, 0.2 mM with GD3 synthase, and 0.5 mM with GD1a synthase) and the respective glycolipid substrates (0.08 mM for lactosylceramide, 0.1 mM for GM3, and 0.5 mM for GM1) were determined. Competition experiments showed that the three sialyltransferase activities are three individual catalytic entities. Moreover, evidence was found that product inhibition may play a role in the regulation of the activity of sialyltransferases.
Isolation of a dipeptidyl aminopeptidase, a putative processing enzyme, from skin secretion of Xenopus laevis
Authors: MOLLAY Christa , VILAS Ulrike , HUTTICHER Anton , KREIL Günther
Published: March 2005

A dipeptidyl aminopeptidase has been purified to apparent homogeneity from skin secretion of Xenopus laevis. This enzyme is a glycoprotein with a molecular mass of about 98 kDa. It hydrolyzes a variety of dipeptidyl‐p‐nitroanilides and oligopeptides containing proline, alanine or glycine as the second amino acid and is inhibited by diisopropylfluorophosphate. The pH optimum was found to be around 8, while at pH 6, substrates were cleaved at about one‐third of the maximal rate. This dipeptidyl aminopeptidase has the specificity required for the cleavage of amino‐terminal extensions preceding the sequence of caerulein and xenopsin in their respective precursors.
Selective preparation and characterization of membranous and soluble forms of alkaline phosphatase from rat tissues
Authors: MIKI Akira , TANAKA Yukari , OGATA Shigenori , IKEHARA Yukio
Published: March 2005

We developed a method for selective preparation of two forms of alkaline phosphatase from rat tissues. The enzyme was extracted by n‐butanol treatment at pH 5.5 and pH 8.5 as soluble and aggregated (membranous) forms, respectively. The soluble form prepared from liver was found to be identical with the serum enzyme. Complete solubilization of the membrane‐bound enzyme without detergents had a great advantage in its purification. Rat hepatoma AH‐130 cells enriched in alkaline phosphatase were first used for purification of the livertype enzyme. The hepatoma enzyme, purified by chromatographies on concanavalin‐A–Sepharose, Sephacryl S‐300 and hydroxyapatite was used for production of antibodies specific for the liver‐type isozyme. An immunoaffinity column, prepared with anti‐(hepatoma‐enzyme) IgG was utilized for the enzyme purification from other tissues including the membranous form. Analyses of amino acid composition of the purified enzymes revealed that all the liver‐type enzymes from hepatoma, liver, kidney and serum had the same composition, whereas the intestinal type consisted of the composition distinctly different from that in the liver type. In addition, there was no significant difference in amino acid composition between the soluble and membranous forms, suggesting a possible involvement in the membranous form of a hydrophobic component other than its polypeptide domain. The present method for selective preparation of the soluble and membranous forms of alkaline phosphatase will be useful for a further investigation on the interaction of the enzyme with membranes.
Nucleotide sequence of the structural gene for dihydroorotase of Escherichia coli K12
Authors: BÄCKSTRÖM Dan , SJÖBERG Rose‐Marie , LUNDBERG Lennart G.
Published: March 2005

The nucleotide sequence of the dihydroorotase structural gene, pyrC, of Escherichia coli K12 has been determined. The DNA sequence predicts a polypeptide chain of 347 amino acid residues corresponding in size and composition to the previously purified dihydroorotase subunit. Nuclease S1 mapping indicated that transcription of pyrC is initiated around 40 base pairs upstream from the translational start. The transcriptional leader region contains a region of dyad symmetry, which allows a stable hairpin to be formed. This sequence may have regulatory functions since similar structures are found in other pyr genes. The nucleotide sequence also contains a 186‐codon open reading frame in front of pyrC. Nuclease Bal31‐deletion derivatives of pyrC plasmids indicate that this gene does not affect the expression of pyrC. The predicted polypeptide chain shows a putative signal sequence. Downstream from the structural gene a sequence similar to a rho‐independent transcriptional terminator is found. This unknown gene may thus encode a membrane protein of unknown function.
tRNA (adenine‐N1)‐methyltransferase from Dictyostelium discoideum
Authors: MUTZEL Rupert , MALCHOW Dieter , MEYER Dieter , KERSTEN Helga
Published: March 2005

An enzyme activity transferring methyl groups from S‐adenosylmethionine to endogenous tRNA was detected in the cytosol of aggregative Dictyostelium discoideum amoebae. This enzyme was purified more than 1000‐fold and was characterized as a tRNA (adenine‐N1‐)‐methyltransferase. Kinetic analysis yielded a K0.5 for S‐adenosylmethionine of 0.27 μM and competitive inhibition by S‐adenosylhomocysteine showed an I0.5 of 0.26 μM. The tRNA methyltransferase activity was stimulated by monovalent cations and the pH optimum was 7.3. tRNAs isolated from D. discoideum as well as from other eucaryotic sources could be methylated only to a minor extent. In contrast, Escherichia coli tRNA accepted up to 0.6 mol methyl group/mol tRNA, suggesting that the target nucleotide is unmethylated in procaryotic tRNA, but is commonly methylated in tRNAs from eucaryotic organisms. The activity of the methyltransferase increased 4–6‐fold during cell differentiation from the vegetative to the aggregative stage.
Inhibition of carotenoid accumulation and abscisic acid biosynthesis in fluridone‐treated dark‐grown barley
Authors: GAMBLE Patricia E. , MULLET John E.
Published: March 2005

Treatment of dark‐grown barley with 0.1 mM fluridone inhibited carotenoid accumulation but did not alter plastid biogenesis. Plastids isolated from dark‐grown control and dark‐grown fluridone‐treated plants were similar in size and protein compositions. Dehydration of dark‐grown control barley caused abscisic acid levels to increase 30–40‐fold in 4 h, while plants treated with 0.1 mM fluridone accumulated very little abscisic acid in response to dehydration. These results suggest that fluridone‐treated plants do not accumulate abscisic acid because of carotenoid deficiency rather than plastid dysfunction. Dark‐grown barley plants treated with 0.31 μM fluridone accumulated low levels of carotenoids. Dehydration of these plants resulted in a 4–8‐fold increase in abscisic acid and a decrease in antheraxanthin, violaxanthin and neoxanthin, but no change in β‐carotene or lutein plus zeaxanthin levels. This result is consistent with the suggestion that xanthophylls are precursors to abscisic acid in dehydrated plants.
Lysine‐containing DNA‐binding regions on the surface of the histone octamer in the nucleosome core particle
Authors: LAMBERT Sarah F. , THOMAS Jean O.
Published: March 2005

The DNA bound on the surface of the histone octamer in the nucleosome core particle partially protects the ɛ‐amino side‐chains of a subset of the lysine residues from reductive methylation. Most of the strongly protected lysines, which probably define the path of the DNA on the octamer surface, are in the globular (‘structured’) regions of the core histones rather than in the N‐terminal or C‐terminal ‘tails’. Analysis of the protected peptides shows that the three strongest lysine‐containing DNA‐binding sites in the core histones contain the sequence‐Lys/Arg‐Lys‐Thr/Ser‐. On the assumption that the lysine‐containing regions protected from chemical modification are also those found in lysine‐DNA cross‐links in another study [Mirzabekov et al. (1978) Proc. Natl Acad. Sci. USA 75, 4184–4188], particular DNA‐protected peptides may be tentatively assigned to particular DNA contact points. This leads to a more detailed description of the DNA‐binding regions on the octamer surface in the nucleosome core particle. Strong contacts, reflected in strongly protected lysines, may well contribute to the distortion of the DNA from smooth bending [Richmond et al. (1984) Nature (Lond.) 311, 532–537].
Site specificity of casein kinase‐2 (TS) from rat liver cytosol
Authors: MARIN Oriano , MEGGIO Flavio , MARCHIORI Fernando , BORIN Gianfranco , PINNA Lorenzo A.
Published: March 2005

The factors determining the site recognition and phosphorylation by rat liver casein kinase‐2 (CK‐2) have been explored with a set of 14 related hexapeptides each including a single phosphorylatable amino acid and five acidic plus neutral residues. Such peptides are different from each other in the following features: (a) the nature of the phosphorylatable amino acid, if any; (b) its position relative to the critically required acidic residues; (c) the extension and (d) the structure of the acidic cluster. All of them were tested as substrates and/or competitive inhibitors of CK‐2, and their kinetic and inhibition constants were determined.

The results suggest the following conclusions. (a) Under strictly comparable conditions Ser is by far preferred over Thr. Tyr not being affected at all. (b) In order to carry out its role of structural determinant the critical acidic cluster must be located on the C‐terminal side of the target residue, though not necessarily adjacent to it. (c) The affinity for the protein‐binding site, as deduced from Km and/or Ki values, is largely dependent on the number of acidic residues but it is also significantly enhanced if a hydroxylic residue is located on their N‐terminal side. (d) An acidic residue at position +3 relative to serine plays an especially important role for triggering phosphorylation, the peptide Ser‐Glu‐Glu‐Ala‐Glu‐Glu having similar Km but negligible Vmax compared to Ser‐Glu‐Ala‐Glu‐Glu‐Glu and Ser‐Glu‐Glu‐Glu‐Ala‐Glu. These data provide a rationale for the substrate specificity of CK‐2 and will give a helpful insight into the structure of the protein‐binding site of this enzyme.
Effects of the S‐adenosylhomocysteine hydrolase inhibitors 3‐deazaadenosine and 3‐deazaaristeromycin on RNA methylation and synthesis
Authors: BACKLUND Peter S. , CAROTTI Daniela , CANTONI Giulio L.
Published: March 2005

The effects of 3‐deazaaristeromycin and 3‐deazaadenosine on RNA methylation and synthesis were examined in the mouse macrophage cell line, RAW264. S‐Adenosylhomocysteine accumulated in cells incubated with 3‐deazaaristeromycin while S‐3‐deazaadenosylhomocysteine was the major product in cells incubated with 3‐deazaadenosine and homocysteine thiolactone. RNA methylation was inhibited to a similar extent by the accumulation of either S‐adenosylhomocysteine or S‐3‐deazaadenosylhomocysteine, with S‐adenosylhomocysteine being a slightly better inhibitor. In mRNA, the synthesis of N6‐methyladenosine and N6‐methyl‐2′‐O‐methyladenosine were inhibited to the greatest extent, while the synthesis of 7‐methylguanosine and 2′‐O‐methyl nucleosides were inhibited to a lesser extent. Incubation of cells with 100 μM 3‐deazaaristeromycin or with 10 μM 3‐deazaadenosine and 50 μM homocysteine thiolactone produced little inhibition of mRNA synthesis, even though mRNA methylation was inhibited. In contrast, mRNA synthesis was greatly inhibited by treatment of cells with 100 μM 3‐deazaadenosine and the inhibition of synthesis was not correlated with an inhibition of methylation.
Biosynthesis of antifreeze polypeptides in the winter flounder
Authors: HEW Choy L. , WANG Nam C. , YAN Sixu , CAI Hongyu , SCLATER Anne , FLETCHER Garth L.
Published: March 2005

The precursor proteins for winter flounder antifreeze polypeptide (AFP) were isolated from liver using gel filtration chromatography and reverse‐phase high‐performance liquid chromatography. Two major pro‐antifreezes (Mr 5000), corresponding to the precursors for AFP‐6 and AFP‐8, were characterized by amino acid analyses and automated Edman degradation. These precursors showed significant antifreeze activity. The proantifreezes were synthesized in the liver seasonally as demonstrated by immunoblotting and in vitro liver incorporation studies. No mature AFP were detected in liver, thus indicating that the processing of pro‐antifreezes, including amidation of the C‐termini, occurred mainly in the serum. The function(s) of the prosequences, if any, remain unclear.
Studies in vitro on the flavinylation of 6‐hydroxy‐d‐nicotine oxidase
Authors: BRANDSCH Roderich , BICHLER Veronika
Published: March 2005

The gene of 6‐hydroxy‐D‐nicotine oxidase (6‐HDNO), a flavoenzyme from Arthrobacter oxidans with covalently bound FAD, was expressed with the aid of an expression vector in a cell‐free coupled transcription‐translation system derived from Escherichia coli MZ9. Ultraviolet irradiation of the E. coli extract did not affect synthesis of the 6‐HDNO polypeptide nor total protein synthesis but enzymatic 6‐HDNO activity could not be detected. Addition of FAD to the irradiated cell extract restored the capability of the transcription‐translation assays to synthesize enzymatically active 6‐HDNO. However, enzymatic activity could not be restored on addition of FAD plus cell‐free extract to the ultraviolet‐inactivated assays after completion of apo‐6‐HDNO synthesis (60 min) nor to immunoprecipitates thereof. Under similar conditions, addition of [14C]FAD did not increase the protein‐bound radioactivity.

These results indicate that under conditions of limited FAD supply in the in vitro system a flavinless apo‐6‐HDNO‐polypeptide was synthesized. It was, however, not possible to bind the cofactor to the completed polypeptide chain. These findings argue for a cotranslational cofactor binding.
Simple‐kinetic descriptions of alcohol dehydrogenase after immobilization on tresyl‐chloride‐activated agarose
Authors: BILLE Vincent , REMACLE José
Published: March 2005

Yeast alcohol dehydrogenase was successfully immobilized on tresyl‐chloride‐activated agarose; the optimized conditions allowed an enzyme activity recovery of over 90%.

Comparison of free and immobilized enzyme properties showed an unchanged intrinsic activation energy of the reaction and a shift of optimum activity to a higher pH medium after immobilization. Comparison of the kinetic parameters for both substrates of the reaction showed that the Michaelis‐Menten model could not take into consideration all the constraints induced by the immobilization on the enzyme properties but that the Theorell‐Chance model was more appropriate. These results are discussed taking into consideration the factors affecting the immobilized enzyme. Finally, we discuss the possibilities of cofactor regeneration with this immobilized alcohol dehydrogenase.
Chemical and biological characterization of a truncated form of acidic fibroblast growth factor from bovine brain
Authors: GAUTSCHI Peter , FRÀTER‐SCHROEDER Marijke , MÜLLER Thomas , BÖHLEN Peter
Published: March 2005

Two forms of acidic fibroblast growth factor were isolated from bovine brain by a combination of ammonium sulfate precipitation, cation‐exchange chromatography, heparin‐Sepharose affinity chromatography, and reverse‐phase high‐performance liquid chromatography. Amino acid analysis, polyacrylamide gel electrophoresis and amino‐terminal sequence analysis showed that one form corresponds to a protein with a molecular mass of 16 kDa and the amino‐terminal sequence Phe‐Asn‐Leu‐Pro‐Leu‐Gly‐Asn‐Tyr‐Lys‐Lys‐Pro‐Lys‐Leu‐Leu‐Tyr‐and thus represents the acidic fibroblast growth factor as previously characterized [Böhlen et al. (1985) EMBO J. 5, 1951–1956]. The second mitogen form has a molecular mass of 15.5 kDa. The amino‐terminal sequence was established as Asn‐Tyr‐Lys‐Lys‐Pro‐Lys‐Leu‐Leu‐Tyr‐. This evidence indicates that the latter form represents an amino‐terminally truncated acidic fibroblast growth factor, lacking the first six amino acid residues. Both forms of the protein are biologically active and equipotent with respect to stimulation of the proliferation in vitro of mesodermal cells such as vascular endothelial and adrenal cortex cells.
Isolation of a mutant LLC‐PK1 cell line defective in hormonal responsiveness
Authors: JANS David A. , RESINK Therese J. , WILSON Lynette E. , REICH Edward , HEMMINGS Brian A.
Published: March 2005

A mutant LLC‐PK1 cell line, M18, was isolated after a single treatment of the parent culture with N‐methyl‐N′‐nitro‐N‐nitroso‐guanidine. In contrast to LLC‐PK1 cells, the mutant did not exhibit production of urokinasetype plasminogen activator (uPA) in response to the hormones calcitonin and vasopressin, but produced the expected levels of uPA upon stimulation by the receptor‐independent adenylate cyclase activators forskolin and cholera toxin, as well as by the phosphodiesterase inhibitor isobutylmethylxanthine and the 8‐bromo analogue of adenosine cyclic monophosphate, Br8cAMP. The patterns of activation of cAMP‐dependent protein kinase were identical to those of uPA induction: calcitonin and vasopressin were without effect, but the response to all other agents was normal. In similar fashion, mutant cell homogenates displayed normal activation of adenylate cyclase upon treatment with sodium fluoride, forskolin, or the non‐hydrolyzable GTP analogue guanosine 5′‐[β,γ‐imino]triphosphate, but were unresponsive to calcitonin or vasopressin.

The ability of M18 cells to bind radioactively labelled calcitonin and vasopressin was measured. The mutant possessed less than 4% of the normal levels of the receptor binding activity for both hormones. Somatic cell hybrids formed between M18 and LLC‐PK1 cells were found to retain normal hormone binding activity and responsiveness to hormones, indicating that the defect in M18 cells was recessive. M18 was concluded most probably to contain a single mutation impairing the function of two distinct polypeptide hormone receptors.
The primary structure of apolipoprotein A‐I from rabbit high‐density lipoprotein
Authors: YANG Chao‐yuh , YANG Tseming , POWNALL Henry J. , GOTTO Antonio M.
Published: March 2005

The amino acid sequence of rabbit apolipoprotein A‐I (apo A‐I) has been determined by degradation and alignment of two overlapping sets of peptides obtained from tryptic and staphylococcal digestions. All of the peptides of rabbit apo A‐I resulting from digestion by staphylococcal protease were isolated and sequenced except residues 33–37. A digestion with trypsin was employed to find overlapping and missing peptides. The N‐terminus of rabbit apo A‐I was confirmed by sequencing the intact protein up to 20 residues while the C‐terminus was identified through its homology with human apo A‐I. The protein contains 241 residues in its single chain. Its primary structure is highly homologous to the reported canine apo A‐I (80%) and human apo A‐I (78%), but exhibits less similarity with rat apo A‐I (60%). Like human apo A‐I, rabbit apo A‐I contains very little histidine (2) and methionine (1); it does however have two residues of isoleucine. Based on a comparison of the hydrophobichydrophilic character of apo A‐I residues with that of the two synthetic peptides that activated lecithin:cholesterol acyltransferase (Pownall et al. and Yokoyama et al.), we found that the five segments with the highest corresponding homologies on the protein are located within the N‐terminal half. This suggests that the N‐terminal half of apo A‐I contains the major portion of regions activating lecithin:cholesterol acyltransferase.
Nucleotide sequence of the Saccharomyces cerevisiae CTT1 gene and deduced amino‐acid sequence of yeast catalase T
Authors: HARTIG Andreas , RUIS Helmut
Published: March 2005

A 2642‐base‐pair DNA fragment containing the catalase T (CTT1) structural gene of the yeast Saccharomyces cerevisiae and its flanking regions has been sequenced. The gene codes for a protein of 562 amino acids (relative molecular mass 64,449) and appears to contain no intron. The amino acid sequence of catalase T derived from the DNA sequence shows 40.7% homology (52.2% including conservative replacements) to that of bovine liver catalase. All amino acids previously postulated to participate directly in catalysis by liver catalase and most of the amino acids of the immediate environment of hemin, the prosthetic group of catalase, are conserved in catalase T. The data obtained indicate that the folding of polypeptide chains of the two catalases compared has been conserved within a central region consisting mainly of the β‐barrel domain, which bears the prosthetic group, and a major part of the “wrapping domain”. N‐ and C‐terminal regions involved in subunit interactions are less well conserved. It is suggested that their structure is more similar to that of the corresponding regions of Penicillium vitale catalase. However, catalase T lacks the C‐terminal flavodoxin‐like domain present in this protein.
Sulphate groups are involved in the antigenicity of keratan sulphate and mask i antigen expression on their poly‐N‐acetyllactosamine backbones
Authors: TANG Ping W. , SCUDDER Peter , MEHMET Huseyin , HOUNSELL Elizabeth F. , FEIZI Ten
Published: March 2005

Conditions were established for desulphation of hexa‐, octa‐, deca‐ and larger oligosaccharides derived from corneal keratan sulphate after treatment with endo‐β‐galactosidase. The antigenicities of the desulphated oligosaccharides were compared with those of the native oligosaccharides in chromatogram binding, plastic‐plate binding or inhibition of binding assays using a novel microimmunochemical approach with oligosaccharide‐lipid conjugates (neoglycolipids). The results clearly show that sulphate residues are essential components of the antigenic determinant(s) recognised by three monoclonal antibodies to keratan sulphate, 5‐D‐4, 1‐B‐4 and MZ15, but they mask the i antigen activity of the linear poly‐(N‐acetyllactosamine) backbones of this glycosaminoglycan. Immunochemical assays, before and after β‐N‐acetylglucosaminidase treatment of desulphated linear hexa‐, octa‐ and decasaccharides derived from keratan sulphate, indicate that for reaction with one anti‐i antibody, Den, there is an absolute requirement for the non‐reducing β‐galactosyl residue of the i antigen structure to be in the terminal position, but with a second anti‐i antibody, Tho, there is in addition some reactivity with the i antigen structure having an N‐acetylglucosamine residue at the non‐reducing end. The chromatographic properties after desulphation or nitrosation of a minor keratan sulphate oligosaccharide (a dodecasaccharide), which reacts especially well with antibody 5‐D‐4, have provided the first evidence for the presence of glucosamine residues that may be N‐sulphated in corneal keratan sulphate.
Methylation in vivo of elongation factor EF‐Tu at lysine‐56 decreases the rate of tRNA‐dependent GTP hydrolysis
Authors: VAN NOORT Johannes M. , KRAAL Barend , SINJORGO Karin M. C. , PERSOON Niek L. M. , JOHANNS Earl S. D. , BOSCH Leendert
Published: March 2005

In this paper we show, that the in vivo methylation of the elongation factor Tu from Escherichia coli is correlated with the growth phase of the bacterium. Methylation occurs at one position only, i.e. Lys‐56, and initially results in monomethylation during logarithmic growth. Upon entering the stationary phase of E. coli, monomethyllysine is gradually converted into dimethyllysine.

We have undertaken an extensive comparison between the properties of the highly methylated EF‐Tu and unmodified EF‐Tu. No gross conformational differences, as measured by the rate of mild tryptic cleavage, were observed. The dissociation rates of the nucleotides GDP and GTP appear likewise to be unaffected by the methylation, just as is the stimulatory effect of the elongation factor Ts upon these rates. Whereas tRNA binding at the classical binding site of EF‐Tu (site I) also appears not to be affected by the methylation of the protein. tRNA binding at site II is. Although the apparent affinity of tRNA for site II remains unaltered upon methylation of EF‐Tu, the conformational effects of tRNA binding at this site become different. Both the GTPase activity of the protein and the reactivity of Cys‐81 are significantly less stimulated by the tRNA when EF‐Tu is methylated.

A possible physiological implication of this phenomenon is discussed.
Nucleotide sequence of a nearly full‐length cDNA coding for pepsinogen of rat gastric mucosa
Authors: ICHIHARA Yoshikazu , SOGAWA Kazuhiro , MOROHASHI Ken‐ichirou , FUJI‐KURIYAMA Yoshiaki , TAKAHASHI Kenji
Published: March 2005

A nearly full‐length rat pepsinogen cDNA was isolated from a rat gastric mucosa cDNA library and its nucleotide sequence was determined. The cDNA comprises 1370 base pairs (bp), including the 5′‐non‐coding region (60 bp), the coding nucleotide sequence (1176 bp) and the 3′‐non‐coding region (131 bp). The predicted amino acid sequence of rat prepepsinogen (392 residues) contains a 16‐residue signal sequence followed by the pepsionogen moiety of 376 residues. Rat pepsinogen has an amino acid composition characteristic of C‐type pepsinogens and is much more homologous in amino acid sequence with C‐type pepsinogens than A‐type pepsinogens. These results indicate that the major form of rat pepsinogen can be classified as a C type pepsinogen.
Light‐modulated ADP‐ribosylation, protein phosphorylation and protein binding in isolated fly photoreceptor membranes
Authors: BENTROP Joachim , PAULSEN Reinhard
Published: March 2005

Rhodopsin (P, λmax 480 nm) of blowfly photoreceptors R1–6 is converted by light into a thermally stable metarhodopsin (M, λmax 565 nm). In isolated blowfly rhabdoms photoconversion of P to M affects bacterial toxin‐catalyzed ADP‐ribosylation of a 41‐kDa protein, activates phosphorylation of opsin and induces the binding of a 48‐kDa phosphoprotein to the rhabdomeric membrane.

ADP‐ribosylation of the 41‐kDa protein is catalyzed by cholera toxin and is inhibited by P → M conversion. The 41‐kDa protein might represent the α‐subunit of the G‐protein, proposed to be part of the phototransduction mechanism [Blumenfeld, A. et al. (1985) Proc. Natl Acad. Sci. USA 82, 7116–7120].

P → M conversion leads to phosphorylation of opsin at multiple binding sites: up to 4 mol phosphate are bound/mol M formed. Dephosphorylation of the phosphate binding sites is induced by photoconversion of M to P. High levels of calcium (2 mM) inhibit phosphorylation of M and increase dephosphorylation of P.

Protein patterns obtained by sodium dodecyl sulfate gel electrophoresis of irradiated retina membranes show an increased incorporation of label from [γ‐32P]ATP also into protein bands of 48 kDa, 68 kDa and 200 kDa. Binding studies reveal that in the case of the 48‐kDa protein this effect is primarily due to a light‐induced binding of the protein to the photoreceptor membrane. The binding of the 48‐kDa phosphoprotein is reversible: after M → P conversion the protein becomes extractable by isotonic buffers. These data suggest that in rhabdomeric photoreceptors of invertebrates light‐activation of rhodopsin is coupled to an enzyme cascade in a similar way as in the ciliary photoreceptors of vertebrates, although there may be differences, e.g. in the type of G‐protein which mediates between the activated state of metarhodopsin and a signal‐amplifying enzyme reaction.
Preparation and characterization of pig plasma and platelet gelsolins
Authors: WEEDS Alan G. , GOOCH John , POPE Brian , HARRIS Harriet E.
Published: March 2005

Pig plasma gelsolin has been prepared by a revised method involving poly(ethylene glycol) precipitation, chromatography on CM‐cellulose and affinity chromatography on actin‐Sepharose. Pig platelet gelsolin has been prepared by chromatography on DEAE‐cellulose and actin‐Sepharose. Partial chemical and proteolytic cleavage shows that the two proteins are closely related in their fragmentation patterns. The amino acid sequences are identical at the N‐terminus of the platelet protein, but the plasma protein has an additional nine residues on the N‐terminal side of the common sequence. Calcium binding studies show that the plasma protein has similar calcium binding properties to both macrophage and platelet gelsolins.
Binding of pig plasma gelsolin to F‐actin and partial fractionation into calcium‐dependent and calcium‐independent forms
Authors: POPE Brian , WEEDS Alan G.
Published: March 2005

The interaction of pig plasma gelsolin with F‐actin has been studied by a sedimentation assay using 125I‐gelsolin in a Beckman Airfuge. Over 90% of the gelsolin bound to F‐actin in 0.1 mM CaCl2 in experiments using 24 μM actin and 2–10 nM 125I‐gelsolin, but only 40–50% bound in 1 mM EGTA. Addition of more F‐actin to the EGTA supernantant does not sediment this gelsolin. Demonstration of this partial calcium sensitivity depends critically on the use of F‐actin that has been prepared in the absence of calcium ions. F‐actin prepared from G‐actin in calcium or pretreated with calcium, binds 125I‐gelsolin more completely in EGTA. This suggests that gelsolin activity is influenced by transient exposure of actin to calcium.

Further evidence for partial calcium sensitivity in the interactions between gelsolin and F‐actin has been obtained by other methods, including viscometry and electron microscopy.

The gelsolin present in the EGTA supernatant is complexed to G‐actin, predominantly as binary complexes. Very low concentrations of these complexes reduce the viscosity of F‐actin in calcium but not in EGTA. Whether this effect is due to severing activity, or capping with consequent depolymerization to establish the new critical concentration, is uncertain.

The results suggest the presence of two types of gelsolin, one that requires micromolar concentrations of calcium for binding to F‐actin and one that does not. Both bind to G‐actin. Partial separation has been achieved using actin‐Sepharose.

Pig plasma gelsolin is heterogeneous on isoelectric focussing gels in urea, but the two types of gelsolin separated on actin‐Sepharose do not correspond to specific isoelectric species.
The mechanism of the protein‐synthesis elongation cycle in eukaryotes
Authors: NILSSON Lars , NYGÅRD Odd
Published: March 2005

The functional significance of the post‐translocation interaction of eukaryotic ribosomes with EF‐2 was studied using the translational inhibitor ricin. Ribosomes treated with ricin showed a decreased rate of elongation accompanied by altered proportions of the different ribosomal phases of the elongation cycle. The content of ribosome‐bound EF‐2 was diminished by approximately 65% while that of EF‐1 was unaffected. The markedly reduced content of EF‐2 was caused by an inability of the ricin‐treated ribosomes to form high‐affinity pretranslocation complexes with EF‐2. However, the ribosomes were still able to interact with EF‐2 in the form of a low‐affinity post‐translocation complex. Ricin‐treated ribosomes showed an altered ability to stimulate the GTP hydrolysis catalysed by either EF‐1 or EF‐2. The EF‐1‐catalysed hydrolysis was reduced by approximately 70%, resulting in a decreased turnover of the quaternary EF‐1 · GTP · aminoacyl‐tRNA · ribosome complex. In contrast, the EF‐2‐catalysed hydrolysis was increased by more than 400%, despite the lack of pre‐translocation complex formation. The effect was not restricted to empty reconstituted ribosomes since gently salt‐washed polysomes also showed an increased rate of GTP hydrolysis. The results indicate that the EF‐1‐ and EF‐2‐dependent hydrolysis of GTP was activated by a common center on the ribosome that was specifically adapted for promoting the GTP hydrolysis of either EF‐1 or EF‐2. Furthermore, the results suggest that the GTP hydrolysis catalysed by EF‐2 occurred in the low‐affinity post‐translocation complex.
Mucopolysaccharidases from Pseudomonas sp.
Authors: HORTON Denise S. P. Q. , MICHELACCI Yára M.
Published: March 2005

Four constitutive enzymes, capable of degrading keratan sulfate, were isolated from Pseudomonas sp.: a particulate endoglycosidase, a soluble endoglycosidase, a soluble exo‐β‐D‐galactosidase and a soluble exo‐β‐D‐N‐acetylglucosaminidase. The endoglycosidase were shown to act only upon keratan sulfate forming β‐D‐2‐acetamido‐2‐deoxy‐6‐O‐sulfoglucosyl‐(1 → 3)‐D‐galactose, as the main product. This result indicates that the enzyme catalyses the hydrolysis of β‐D‐galactose‐(1 → 4)‐N‐acetylglucosamine linkages. It was also shown that this monosulfated disaccharide inhibits the particulate keratan sulfate endoglycosidase. The bovine nucleus pulposus keratan sulfate is depolymerized at a lower rate and extent when compared to the corneal keratan sulfate. The soluble endoglycosidase is very labile, in contrast to the particulate enzyme, which has been stored at −20°C or at 4°C for at least 12 months with no loss in activity. The particulate endoglycosidase and the soluble exo‐β‐D‐galactosidase and exo‐β‐D‐N‐acetylglucosaminidase are induced when the bacteria is grown in adaptative media containing either 0.1% keratan sulfate or 0.1% chondroitin sulfate. Furthermore, particulate forms of the exoenzymes were detected. The soluble endoglycosidase specific activity, in contrast, is approximately the same in extracts of cells grown in glucose, kertan sulfate or chondroitin sulfate.

A chondroitin sulfate lyase was also identified in the soluble extracts of Pseudomonas sp. cells. This enzyme depolymerizes chondrotin 4‐sulfate, chondroitin 6‐sulfate and hyaluronic acid forming unsaturated disaccharides as main products. It is also active upon the glucuronic‐acid‐containing regions of the dermatan sulfate molecules.

The properties of the soluble enzymes, further purified by ion‐exchange chromatography, and of the particulate keratan sulfate endoglycosidase are presented.
Solution structure of a short DNA fragment studied by neutron scattering
Authors: LEDERER Hermann , MAY Roland P. , KJEMS Joergen K. , BAER Gisela , HEUMANN Hermann
Published: March 2005

The solution structure of a DNA fragment of 130 base pairs and known sequence has been investigated by neutron small‐angle scattering. In 0.1 M NaCl, the overall structure of the DNA fragment which contains the strong promoter A1 of the Escherichia coli phage T7 agrees with that expected for B‐DNA. The neutron scattering curve is well fitted by that of a rigid rod with a length of 44 nm and a diameter of 2 nm. The results were confirmed by quasi‐elastic light scattering and analytical centrifugation. The neutron measurements in H2O and D2O buffer reveal a cross‐sectional inhomogeneity not detected by X‐ray small‐angle scattering. This inhomogeneity is caused by the hydration layer around the DNA core and not by the helical structure. The primary solvent shell has a density increased by at least 4–9% compared to bulk water.
The phosphoenolpyruvate‐dependent fructose‐specific phosphotransferase system in Rhodopseudomonas sphaeroides Distribution of EIIFru over the membranes of phototrophically grown Rps. sphaeroides
Authors: LOLKEMA Juke S. , TEN HOEVE‐DUURKENS Ria H. , ROBILLARD George T.
Published: March 2005

The distribution of the fructose carrier over the membranes of Rhodopseudomonas sphaeroides was studied in cells grown under light saturation and light limitation. Three types of membranes were isolated after disruption of the cells in a French press. All three types were present in the cells grown either under the high or low light intensity, but they were present in different quantities. The cytoplasmic membrane could be separated from the photosynthetic membranes by Sephacryl S‐1000 chromatography. The cytoplasmic membrane has the highest specific density and fructose carrier content and does not contain the light‐harvesting pigments. The photosynthetic membranes could be resolved into two types by sucrose density gradient centrifugation. Type A predominates when cells are grown under light saturation, whereas type B, the chromatophores, is synthesized abundantly under light limitation. The properties of type A are in between the properties of the cytoplasmic membrane and the chromatophores. It has a slightly lower specific density and contains four times less fructose carrier than the cytoplasmic membrane, but contains half of the light‐harvesting bacteriochlorophyll of the chromatophore membrane. The fructose carrier content in the type B membranes, the chromatophores, is very low.
Purification of a functional receptor for calcium‐channel blockers from rabbit skeletal‐muscle microsomes
Authors: FLOCKERZI Veit , OEKEN Hans‐Jochem , HOFMANN Franz
Published: March 2005

The dihydropyridine receptor was purified from rabbit skeletal muscle microsomes in the presence of [3H]nitrendipine plus diltiazem or [3H](+)PN 200–110 to an apparent density of 1.5–2 nmol binding sites/mg protein. Sodium dodecyl sulfate gel electrophoresis in the absence of reducing agents yielded three peptide bands of 142, 56 and 30 kDa in a relative ratio of 11:1:1.3, whereas in the presence of 40 mM dithiothreitol bands of 142, 122, 56, 31, 26 and 22 kDa were obtained in a relative ratio of 5.5:2.2:1:0.9:14:0.09. This gel pattern was observed regardless of whether the receptor was purified as a complex with nitrendipine plus diltiazem or with (+)PN 200–110. cAMP‐dependent protein kinase phosphorylated preferentially the 142‐kDa band up to a stoichiometry of 0.82 ± 0.07 (15) mol phosphate/mol peptide. The 56‐kDa band was phosphorylated only in substoichiometric amounts.

[3H]PN 200–110 bound at 4°C to one site with apparent Kd and Bmax values of 9.3 ± 1.7 nM and 2.2 ± 0.3 (3) nmol/mg protein, respectively. The binding was stereospecific and was not observed in the presence of 1 mM EGTA. Desmethoxyverapamil interfered with the binding of [3H]PN 200–110 in an apparent allosteric manner. (–)Desmethoxyverapamil inhibited the binding of [3H]PN 200–110 at 37°C and stimulated it at 18°C. In agreement with these results, (–)desmethoxyverapamil increased the dissociation rate of [3H]PN 200–110 from 0.29 min−1 to 0.38 min−1 at 37°C and decreased it threefold from 0.046 min−1 to 0.017 min−1 at 18°C. The (+)isomer of desmethoxyverapamil inhibited PN 200–110 binding at all temperatures tested. d‐cis‐Diltiazem stimulated the binding of [3H]PN 200–110 at 37°C with an apparent EC50 of 1.4 μM and decreased the dissociation rate from 0.29 min−1 to 0.11 min−1. The stimulatory effect of d‐cis‐diltiazem was temperature‐dependent and was seen only at temperatures above 18°C.

These results suggest that the purified dihydropyridine receptor retains the basic properties of the membrane‐bound receptor and contains separate sites for at least dihydropyridines and phenylalkylamines.
Purification and properties of guanylate kinase from bovine retinas and rod outer segments
Authors: HALL Scott W. , KÜHN Hermann
Published: March 2005

The presence of three soluble nucleotide phosphotransferases in bovine rod outer segments was demonstrated: guanylate kinase (EC 2.7.4.8), nucleoside‐diphosphate kinase (EC 2.7.4.6) and adenylate kinase (EC 2.7.4.3).

The enzyme guanylate kinase, which catalyzes the reaction GMP + ATP ⇌ GDP + ADP, was purified to homogeneity from isolated bovine rod outer segments as well as from bovine retinas. The enzyme preparations obtained from both sources are identical in their chromatographic properties, molecular mass (20–23 kDa for both native enzyme and dodecylsulfate‐denatured polypeptide), Km values (13 μM for GMP and 430 μM for ATP), specific activities, and nucleotide specificities. The enzyme's turnover number was estimated to be 130 s−1. The minimum amount of enzyme found in rod outer segments is about 1 copy per 800 rhodopsin molecules. The role of the enzyme in the cyclic GMP cycle in rod outer segments is discussed.
Conformational studies of d(m5CpGpm5CpG) and d(CpGpCpG) by 1H and 31P NMR
Authors: DELEPIERRE Muriel , D'ESTAINTOT Béatrce LANGLOIS , IGOLEN Jean , ROQUES Bernard P.
Published: March 2005

Exhaustive conformational studies of d(CpG)2 and d(m5CpG)2, two convenient targets for DNA bisintercalating drugs, have been carried out by 1H and 31P NMR in low salt concentration and in the presence of 30% ethanol. Unambiguous 31P assignments of the B form are obtained by low‐power heteronuclear decoupling experiments, while 31P assignments in the Z form are obtained by two‐dimensional homonuclear chemical exchang experiments. The 31P chemical shifts and 3JH3′P coupling constants studied at various temperatures in methylated and non‐methylated tetranucleotides, are interpreted as resulting from conformational differences between the compounds. These features are corroborated by homonuclear proton nuclear Overhauser effect experiments showing the steric role of the 5‐methylcytosine in the induction of an alternating B form in d(m5CpG)2.
Binding of [1‐13C]galactose‐enriched hen ovalbumin to Erythrina cristagalli agglutinin as studied by 13C‐NMR spectroscopy
Authors: BERMAN Elisha , LIS Halina , JAMES Thomas L.
Published: March 2005

A study of the equilibrium binding of glycoproteins to lectins was undertaken using the titled compounds as a model system for such interactions. The binding of hen ovalbumin, enriched in galactose specifically 13C‐labelled at C1, to Erythrina cristagalli agglutinin was studied by 13C‐NMR spectroscopy. The lectin was shown to be bivalent for ovalbumin with an apparent estimated association constant, at infinite dilution, of about 104 M−1. The observed association is similar to that found for the corresponding disaccharide, Gal(β1–4)GlcNAc. The results strongly suggest that only the N‐acetyllactosamine moiety in the carbohydrate side chain of galactoseenriched ovalbumin participates in the binding to the lectin with little, if any, interactions from other parts of either the side chain or the polypeptide backbone of ovalbumin.
Kinetics of cadmium and terbium dissociation from calmodulin and its tryptic fragments
Authors: MARTIN Stephen R. , LINSE Sara , BAYLEY Peter M. , FORSÉN Sture
Published: March 2005

The kinetics of cadmium and terbium dissociation from bovine testis calmodulin and its tryptic fragments have been studied by stopped‐flow fluorescence methods, using the calcium indicator Quin 2.

Studies of the tryptic fragments TR1C and TR2C, comprising the N‐terminal or C‐terminal half of calmodulin, have clearly identified cadmium binding sites I and II as the low‐affinity (rapidly dissociating) sites and sites III and IV as the high‐affinity (slowly dissociating) sites. Thus the site preference of cadmium is the same as that of calcium. For terbium, however, sites I and II are the high‐affinity sites and sites III and IV are the low‐affinity sites. Thus, the site preference or terbium is not the same as that of calcium and cadmium.

In contrast to previous studies with calcium, we observe two kinetic precesses for dissociation from sites III and IV for experiments with both cadmium and terbium. Possible models for the binding of metal ions are discussed.
The transmembrane arrangement of the ADP/ATP carrier as elucidated by the lysine reagent pyridoxal 5‐phosphate
Authors: BOGNER Werner , AQUILA Heinrich , KLINGENBERG Martin
Published: March 2005

The lysine reagent pyridoxal 5‐phosphate was applied to the ADP/ATP carrier (AAC) in order to elucidate topological and functional properties of the numerous lysines within the primary structure. To establish appropriate labeling conditions, the influence of pyridoxal‐P on transport and inhibitor binding to the AAC was examined. The ADP/ATP transport is sensitive to low concentrations of pyridoxal‐P with a Ki= 0.4 mM. Binding of [3H]carboxyatractylate and [3H]bongkrekate is largely inhibited by pyridoxal‐P treatment with Ki approximately 1 mM. [3H]Carboxyatractylate is not and [3H]bongkrekate weakly removed by pyridoxal‐P, Whereas [3H]atractylate is displaced to a large extent. Under optimized conditions of pyridoxal‐P concentration, of pH and of time exposure, the AAC was exposed to [3H]pyridoxal‐P in mitochondria, in submitochondrial particles and in the detergent‐solubilized carrier. The [3H]pyridoxal‐P‐labeled AAC was isolated from mitochondria and particles. After citraconylation thermolysinolytic peptides were prepared. The pyridoxyl‐lysinecontaining peptides were purified and the pyridoxal‐P incorporation to specific lysines was determined by sequencing. The pyridoxal‐P incorporation into the AAC in various states was evaluated with regard to structural and functional aspects. First, by comparing pyridoxal‐P incorporation in mitochondria and sonic particles, the segments of the polypeptide chain exposed to the cytosolic and matrix side of the membrane are detected. Second, the additional lysine incorporation into the isolated as compared to the membrane‐bound carrier is attributed to the protein collar facing the phospholipid headgroups. Third, the difference between lysine incorporation into the carboxyatractylate‐AAC and bongkrekate‐AAC complexes reflect either conformational chages or lysines involved in the translocation channel through the protein. Fourth, the additional lysine labeled in the atractylatecarrier complex as compared to the carboxyatractylate‐carrier complex is attributed to a cationic site in the binding center. These results are incorporated into a transmembrane folding model of the carrier.
In vitro competition between adenosine(5′)tetraphospho(5′)adenosine and deoxyribonucleic acid in the reaction with diamminedichloroplatinum(II)
Authors: BACHMAIER Annelies , JUST Gerald , HOLLER Eggehard
Published: March 2005

Competition between adenosine(5′)tetraphospho(5′)adenosine (Ap4A) and DNA for the synthesis of adducts with the cis or trans isomer of diamminedichloroplatinum(II) was measured in the presence and absence of magnesium and spermidinium ions. Reaction products were analysed by circular dichroism, poly(ethyleneimine) thin‐layer chromatography and reversed‐phase chromatography. Competition was affected by the oligovalent cations that bound specifically to the dinucleotide. Platination of DNA was favoured under all conditions. Chromatin was less competitive. The mechanism was kinetic competition, DNA reacting considerably faster than Ap4A. Platinum(II) did not exchange between adducts and free DNA and Ap4A, respectively. On that basis only low amounts of Ap4A adducts were estimated to be formed under conditions of clinical chemotherapy. The cis and trans isomers of diamminedichloroplatinum(II) were equally effective.

Platinum(II) adducts of Ap4A were neither degraded by Ap4A‐specific pyrophosphohydrolases nor by phosphodiesterase nor in the presence of unfractionated extract of calf thymus. Unphysiologically high concentrations of Crotalus durissus phosphodiesterase I were required for hydrolytic splitting, the amount of which was similar for both platinum(II) isomer adducts. The results suggest that Ap4A platinum(II) adducts might accumulate during chemotherapy of cancer treatment.
Primary structure of the chromosomal protein HMb from the archaebacteria Methanosarcina barkeri
Authors: LAINE Bernard , CHARTIER François , IMBERT Marlène , LEWIS Randy , SAUTIERE Pierre
Published: March 2005

The amino acid sequence of the protein HMb, a protein of 93 residues (Mr 10757) which represents the major acid‐soluble component of the Methanosarcina barkeri nucleoprotein complex, has been established from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at aspartic acid, arginine and methionine residues.

The protein HMb is mainly characterized by a high amount of charged residues (15% of acidic residues and 26.8% of basic residues) which are distributed all along the polypeptide chain. The amino acid sequence of the protein HMb is not homologous to any eubacterial, archaebacterial or eukaryotic chromosomal proteins known up to now.