bluuebunny/crossref_metadata_2025 huggingface dataset:
Acquisition of an Insulin‐Sensitive Activity of Adenosine‐3′,5′‐Monophosphate Phosphodiesterase during Adipose Conversion of 3T3‐L2 Cells
Authors: MURRAY Thomas , RUSSELL Thomas R.
Published: March 2005

Mouse 3T3‐L2 fibroblasts differentiate when cultured and express a new phenotype which is characteristic of adipose cells. One aspect of this differentiation is the acquisition of hormone sensitivities typical of adipocytes. The sensitivity of adenosine‐3′,5′‐monophosphate (cAMP) phosphodiesterase to brief insulin treatment in 3T3‐L2 cells at various stages of adipose conversion was examined. The capacity to increase the activity of a low‐Km form of the enzyme in response to acute insulin exposure is a property which is acquired as the cells begin to express the adipose phenotype. Enzyme activity in preadipocytes is not affected by insulin, nor is there an effect in the non‐differentiating 3T3‐C2 line under any of the conditions tested. Since insulin receptors and cAMP phosphodiesterase are both present in the preadipocytes, one result of this cytodifferentiation is to effectively couple receptors to the enzyme activity.

Insulin‐sensitive cAMP phosphodiesterase in 3T3‐L2 adipocytes is characterized by a 40–50% increase in enzyme activity during insulin treatment when determined at 0.1 μM cAMP. This activation results from an increase in the apparent V of the enzyme and does not involve a change in enzyme‐substrate affinity. Maximal stimulation is seen within 2–5 min and is sustained for at least 45 min when high levels of insulin (850 nM) are used. Lower concentrations of insulin (1.7 nM) also bring about rapid activation, although the activation is not completely sustained during longer incubations. Stimulated activity falls off to about 60% of peak values by 30 min. Re‐addition of insulin after 30 min raises enzyme activity back to the maximal level. Further, the insulin response is completely reversible in that the insulin‐sensitive cAMP phosphodiesterase activity disappears within 10 min after removal of insulin from the cultures.
Pancreatic Ribonucleases of Mammals with Ruminant‐Like Digestion
Authors: HAVINGA Johan , BEINTEMA Jaap J.
Published: March 2005

High levels of pancreatic ribonucleases are found in ruminants, species that have a ruminant‐like digestion and several species with coecal digestion. Pancreatic ribonucleases from several independently evolved species with ruminant‐like digestion were investigated to test a hypothesis that glycosylation of ribonucleases may have some function in species with coecal digestion and that glycosylation of the enzyme may not be advantageous for ruminants. Ribonucleases from the hippopotamus, two‐toed sloth and three‐toed sloth were isolated by extraction with sulfuric acid and affinity chromatography. Complete amino acid sequences were determined for the ribonucleases from the hippopotamus and two‐toed sloth and a partial sequence for the enzyme from the three‐toed sloth. The amino acids 75–78 of hippopotamus ribonuclease were positioned by homology with other artiodactyl ribonucleases.

In hippopotamus ribonuclease a heterogeneity was found at position 37, half of the molecules containing glutamine and the other half lysine. Hippopotamus ribonuclease differs less from pig and bovine ribonuclease than these differ from each other, because more ancestral characteristics have been retained. Although hippopotamus ribonuclease contains all four Asn‐X‐Ser/Thr sequences previously found to be glycosylation sites in one or more pancreatic ribonucleases, only the sequence Asn‐Met‐Thr (34–36) is glycosylated in the variant with glutamine at position 37, while the variant with lysine at this position is carbohydrate‐free.

Both sloth ribonucleases are completely glycosylated at the sequence Asn‐Met‐Thr (34–36) with a simple type of carbohydrate chain. The amino acid sequence of two‐toed sloth ribonuclease shows some interesting coupled replacements.
Cation Binding by the Rat‐Incisor‐Dentine Phosphoprotein
Authors: COOKSON David J. , LEVINE Barry A. , WILLIAMS Robert J. P. , JONTELL Mats , LINDE Anders , de BERNARD Benedetto
Published: March 2005

Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy have been used to quantify metal binding to the phosphoprotein extractable from demineralized rat incisor dentine. Paramagnetic cation probes enable identification of the metal binding sites. Cations are able to diffuse across the protein surface while forming a relatively long‐lived metal‐phosphoprotein complex. The ability of the protein to sequester surface‐mobile Ca(II) is discussed in terms of its ability to act as a possible nucleation site for the initial localization of Ca(II) within the dentine matrix.
Complete Amino‐Acid Sequences of DNA‐Binding Proteins HU‐1 and HU‐2 from Escherichia coli
Authors: LAINE Bernard , KMIECIK Daniel , SAUTIERE Pierre , BISERTE Gérard , COHEN‐SOLAL Michel
Published: March 2005

The DNA‐binding protein HU from Escherichia coli is a heterodimer constituted of two poly‐peptide chains termed HU‐1 and HU‐2, of 90 residues each. Their primary structures were established from structural data obtained from tryptic peptides of each monomer in addition to the structural data provided by the automated Edman degradation of the dimer and by peptides derived from cleavage of the dimer with trypsin, chymotrypsin, V8 staphylococcal protease and dilute acid. The results presented in this paper confirm the amino‐terminal and carboxy‐terminal sequences of the dimer HU reported previously [Laine et al. (1978) FEBS Lett. 89, 116–120]. The amino acid sequences of proteins HU‐1 and HU‐2 are identical to those of proteins NS‐1 and NS‐2 respectively, determined independently by Mende et al. [FEBS Lett. (1978) 96, 395–398].

The amino acid sequences of proteins HU‐1 and HU‐2 are closely related but differ by 28 residues. These proteins are characterized by their high content of hydrophobic residues represented mostly by alanine. In both proteins, half of the basic residues are scattered along the polypeptide chain and the remainder is found within two short sequences located in the carboxy‐terminal part of the molecule.

No sequence homology could be established between the proteins HU‐1 and HU‐2 and any one of the five histones from different eukaryotes.
Unified Mechanism for Proton‐Transfer Reactions Affecting the Catalytic Activity of Liver Alcohol Dehydrogenase
Authors: KVASSMAN Jan , PETTERSSON Gösta
Published: March 2005

The effect of pH on substrate binding to liver alcohol dehydrogenase has been examined over the pH range 6–10 by transient‐state and steady‐state kinetic methods. The results provide evidence that there is no significant effect of pH on benzaldehyde binding to the enzyme. Benzyl alcohol association to the binary enzyme · NAD+ complex requires protonation of an ionizing group with a pKa of 7.6 in the binary complex. Substrate dissociation from the enzyme · NAD+· alcohol complex is regulated by an ionizing group with a pKa of 6.6 (6.4) in the complex formed with naphthyl alcohol (benzyl alcohol). Alcohol desorption from the ternary complex occurs exclusively when the ionizing group is in the protonated form.

A reaction mechanism is proposed which accounts for all major effects of pH on liver alcohol dehydrogenase catalysis over the investigated pH range. The reactivity of the enzyme · NAD+ (enzyme · NAD+· alcohol) complex is suggested to be regulated by the ionization state of a water (alcohol) molecule bound at the catalytic zinc atom of the enzyme. Zinc‐bound water does not function as a binding site for substrates or as a mediator of catalytic proton transfer from substrate to solution at the binary‐complex level. Catalytic proton transfer takes place at the ternary‐complex level, probably through an alcohol/alcoholate ion interconversion of the enzyme‐bound substrate. This proton transfer step can be envisaged to serve the purpose of facilitating hydride transfer during alcohol oxidation and alcohol desorption during aldehyde reduction. The kinetics of proton uptake/release during naphthaldehyde reduction at pH 6 are shown to be consistent with the proposed mechanism of enzyme action.
The Presence of a New 3‐Oxoacyl‐CoA Thiolase in Rat Liver Peroxisomes
Authors: MIYAZAWA Shoko , OSUMI Takashi , HASHIMOTO Takashi
Published: March 2005

A novel 3‐oxoacyl‐CoA thiolase was found in rat liver. This thiolase, mitochondrial general 3‐oxoacyl‐CoA thiolase and acetoacetyl‐CoA thiolase were purified from the rat liver after the induction of these activities by the administration to rats of di(2‐ethylhexyl)phthalate, which enhanced the peroxisomal β‐oxidation activity. The new 3‐oxoacyl‐CoA thiolase was distinguished from mitochondrial and cytoplasmic thiolases by the following: DEAE‐cellulose chromatography, phosphocellulose chromatography, immunochemical titration, and substrate specificity. Subcellular fractionation of liver and sucrose‐density‐gradient centrifugation of the light mitochondrial fraction revealed that the new thiolase was mainly in peroxisomes.
Acetylation of Nucleosomal Histones in vitro
Authors: BÖHM Joachim , SCHLAEGER Ernst‐Jürgen , KNIPPERS Rolf
Published: March 2005

A new histone‐specific acetyltransferase, which is closely associated with nucleosomes prepared from lymphocyte nuclei by treatment with micrococcal nuclease, is described. The acetylating enzyme transfers [3H]acetyl groups from [3H]acetyl‐coenzyme A to the endogenous histones H2A, H2B, H3 and H4 in nucleosomes as well as to free histones added to the reaction mixture. Histone H1 is not acetylated by this enzyme. The acetyltransferase was partially purified by DEAE‐Sephadex and DNA‐cellulose chromatography. The nucleosome‐associated enzyme binds to DNA cellulose at low salt concentrations (DNA‐binding acetyltransferase), while the previously described histonespecific acetyltransferases have no affinity to DNA under these conditions. This high affinity for DNA may explain the association of DNA‐binding acetyltransferase with nucleosomes.
Isolation and Characterization of Chicken Secretin
Authors: NILSSON Ann , CARLQUIST Mats , JÖRNVALL Hans , MUTT Viktor
Published: March 2005

Chicken secretin has been isolated and its structure determined. It is composed of 27 amino acid residues, and has an amidated C terminus. The armino acid Sequence is His‐Ser‐Asp‐Gly‐Leu‐Phe‐ThrSer‐Glu‐Tyr‐Ser‐Lys‐Met‐Arg‐Gly‐Asn‐Ala‐Gln‐Val‐G ln‐Lys‐Phe‐Ile‐Gln‐Asn‐Leu‐Met‐NH2. The structure shows distinct similarities to that of porcine secretin, amino acid identities occur at 14 positions (residues 1–4, 6–9,11,14,17, 20, 24 and 26) but considerable differences are also present among the remaining 13 positions. Chicken secretin further shows a clear structural similarity to the vasoactive intestinal peptide (37% identical positions), the gastric inhibitory peptide (30% identical positions) and to glucagon (52% identical positions), suggesting wide evolutionary and functional relationships.
The Sequestration, Processing and Retention of Honey‐Bee Promelittin Made in Amphibian Oocytes
Authors: LANE Charles D. , CHAMPION Janet , HAIML Liselotte , KREIL Gunther
Published: March 2005

Messenger RNAs from one kind of secretory cell can be introduced into the cytoplasm of another: the heterologous proteins formed by the recipient cell are usually processed and topologically segregated in the manner characteristic of the donor cell. Xenopus oocytes injected with honey‐bee venom gland RNA provide some support for this generalization, but also reveal important exceptions to it. Thus, the frog cell makes a small polypeptide whose partial sequence matches perfectly that of insect promelittin, except that the product formed in oocytes ends at the C terminus with a glycine as opposed to a glutamine amide residue. N‐terminal heterogeneity is seen in protoxin made in oocytes and venom gland cells, and species shorter by two residues are seen in both tissues. We suggest that the oocyte contains a dipeptidylpeptidase. Promelittin made in oocytes is barely detectable in the cytosol but is found associated with a vesicle fraction which also contains some newly synthesized endogenous oocyte proteins. The association with vesicles is long‐lasting; thus promelittin is retained slightly more efficiently than sequestered oocyte proteins, and an incubation period of about two weeks is required to reduce by half the amount of these endogenous vesicle proteins. Thus neither promelittin nor any products derived from it are secreted rapidly. Gel analysis fails to reveal promelittin in the medium surrounding the oocyte, although traces can be detected by assaying for a characteristic heptapeptide. Such small amounts could result from slow secretion or leakage. Melittin could not be detected by gel analysis or peptide assay. The retention of the honey‐bee protein within the frog cell is discussed in terms of the specificity of the processing systems and secretory pathways of venom gland cells and oocytes. We suggest that whilst some export mechanisms function efficiently in a wide variety of cells, others do not, and may even be restricted to specific cell types.
Interactions between Phage λ Replication Proteins, λ DNA and Minicell Membrane
Authors: ŻYLICZ Maciej , TAYLOR Karol
Published: March 2005

Gentle methods for minicell lysis and lysate fractionation have been elaborated: lysis by T4 lysozyme without detergents, and fractionation by equilibrium sedimentation in a metrizamide density gradient, both at low ionic strength. In the lysates of phage‐λ‐infected minicells the λ DNA, trapped at a prereplicative step [Witkiewicz, H. and Taylor, K. (1979) Biochim. Biophys. Acta 564, 31–36], appeard in two peaks of different buoyant densities: as a membrane‐bound and a free λ DNA. The covalently‐closed‐circular form of λ DNA appeared exclusively in the membrane fraction.

The λ‐coded proteins, synthesized in λ‐infected minicells, appeared in two major fractions: as membrane‐bound and as free proteins, and in one minor fraction, bound with free λ DNA. Neither λ protein engaged in the initiation of DNA replication was present in the fraction of free proteins: the P‐gene product was membrane‐associated, and the O‐gene product formed a complex with free λ DNA.

The efect of high ionic strength (KCl) and of detergents (Triton X‐100 and sarcosyl) on the binding of replication proteins with λ DNA and with the membrane was studied. The non‐ionic detergent, Triton X‐100 caused displacement of a part of λ DNA from the membrane to the free λ DNA peak; both γ replication proteins were bound with free γ DNA. The binding of the O protein with λ DNA was relatively stable, but was destroyed by the ionic detergent, sarcosyl.
The Amino‐Acid Sequence of the α Subunit of the Mitogenic Lectin from Vicia sativa
Authors: GEBAUER Günter , SCHILTZ Emil , RÜDIGER Harold
Published: March 2005

The complete amino acid sequence of the α chain of the mitogenic lectin from Vicia sativa has been determined. The polypeptide was digested by trypsin and chymotrypsin. The fragments were isolated and most of them were sequenced by automatic solid‐phase Edman degradation. A total sequence of 52 amino acid residues was obtained which is homologous to the α subunits of the lectins from Pisum sativum, Lens culinaris and Vicia faba. The central part (residues 13–32) in particular is completely conserved in all four proteins. In contrast to the three other known sequences, the V. sativaα subunit has an additional serine at the N terminus.

The differences of the related amino acid sequences of these lectins and concanavalin A, the lectin from Canavalia ensiformis, have been compared using the relative substitution frequency found in homologous proteins by McLachlan. The degree of homology decreases in the order: V. sativa > P. sativum > V. faba > L. culinaris > C. ensiformis.

Prediction of the secondary structure according to Chou and Fasman reveals that the lectin α chain is similar to concanavalin A in the first half of the molecule whereas the C‐terminal part apparently tends to form an α helix.
The Amino Acid Sequence of the δ Subunit (Calmodulin) of Rabbit Skeletal Muscle Phosphorylase Kinase
Authors: GRAND Roger J. A. , SHENOLIKAR Shirish , COHEN Philip
Published: March 2005

The amino acid sequence of the phosphorylase kinase δ subunit has been determined using cyanogen bromide peptides. These peptides were ordered by isolation of the tryptic peptides containing carboxy[14C]methylmethionine. The protein is identical to bovine uterus calmodulin and differs from bovine brain calmodulin only in amide assignments. The δ subunit contains 148 amino acids, including one residue of trimethyllysine, and has a molecular weight of 16680. The N terminus is blocked, probably with an acetyl group, and the protein has a net overall charge at pH 7 of –24. The asparagine residues 60 and 97 have been shown to be partially deamidated in the protein. The sequence of the N‐terminal tripeptide and the amide assignments on residues 58 and 60 were not determined unequivocally.
Comparisons of Proteins Associated with Duck‐Globin mRNA and Its Polyadenylated Segment in Polyribosomal and Repressed Free Messenger Ribonucleoprotein Complexes
Authors: VINCENT Alain , GOLDENBERG Samuel , SCHERRER Klaus
Published: March 2005

EDTA dissociation of polyribosomes from duck erythroblasts allowed us to isolate the 15‐S globin messenger ribonucleoproteins (mRNP) by sucrose gradient centrifugation or affinity chromatography on poly(U)‐Sepharose or oligo(dT)‐ce1lu1ose columns. Their protein composition was compared by one and two‐dimensional electrophoresis in sodium dodecyl sulfate to the free 20‐S mRNP containing the repressed fraction of globin mRNA [Vincent, A., Civelli, O., Maundrell, K., and Scherrer, K. (1980) Eur. J. Biochem. 112, 617–633]. The protein composition of the 15‐S mRNP isolated by these methods in different ionic strength conditions, was characterized by a major 73000‐Mr polypeptide and seven minor polypeptides with Mr ranging from 45000 to 68000, all of which are slightly basic, and about five acidic ones in the 80000–130000‐Mr range. All these are retained in the 15‐S mRNP core particle isolated at 0.5 M KCl. At low ionic strength, in addition, a specific group of acidic polypeptides in the Mr range 35000–105000 was also found associated with globin mRNA. Oligo(dT)‐cellulose chromatography of mRNP digested with ribonucleases A and T1 indicated that the 73000‐Mr major protein is bound to the poly(A) segment; some other proteins resolved as minor components interact with both the poly(A) and non‐poly(A) regions of globin mRNA.

Characterization of proteins interacting with the poly(A) segment of non‐polyribosomal globin mRNA in 20‐S free mRNP demonstrated the absence of the polyribosomal 73000‐Mr poly(A)‐binding protein. Furthermore, it confirmed that the protein compositions of translatable polyribosomal and repressed free globin mRNP are very different. Indeed, the respective core (0.5 M KCl) particles contain only two possibly common polypeptides. The specificity of proteins associated with globin mRNA in two different functional states shown here supports the hypothesis of a role of mRNP proteins in translational control of mRNA.
Preparative Isolation of Apo(Ca2+‐ATPase) from Sarcoplasmic Reticulum and the Reactivation by Lysophosphatidylcholine of Ca2‐Dependent ATP Hydrolysis and Partial‐Reaction Steps of the Enzyme
Authors: NESTRUCK‐GOYKE A. Christine , HASSELBACH Wilhelm
Published: March 2005

A preparative method for the isolation of the lipid free apoprotein, the Ca2+‐ATPase of sarcoplasmic reticulum, from the partially purified lipoprotein. Ca2‐ATPase vesicles, is presented.

By enzymatic hydrolysis of the phospholipids and removal of the splitting products and endogenous neutral lipids, the apoprotem was consistently delipidated to 0.02 μmol Pi/mg protein.

Reactivation of the splitting of ATP and the pseudo substrate. dinitrophenyl phosphate, was demonstrated with a variety of lipids and detergents.

A total reactivation of ATP splitting was achieved after a mild ultrasonication of the apoprotein with myristoylglycerophosphocholine which resulted in solubilization of the enzyme as an optically clear solution.

The stable resolubilized enzyme could be stored for several weeks maintaining full enzymatic activity. Gel chromatography suggested that under the assay conditions, the monomeric form of the enzyme predominated.

In comparison with the native enzyme, the resolubilized enzyme showed differences in the temperature dependence of the activation of ATP hydrolysis and a reduced apparent affinity for MgATP.

The phosphate‐transferring activities of the resolubilized enzyme were only partially reactivated in the forward direction, and none of the reverse partial‐reaction steps of the enzyme could be demonstrated.
Cation Dependence of Restriction Endonuclease EcoRI Activity
Authors: WOODHEAD J. Lesley , BHAVE Neetin , MALCOLM Alan D. B.
Published: March 2005

Restriction endonuclease EcoRI cleaves the DNA sequence under optimum digestion conditions. A variation in pH and ionic strength can result in EcoRI* activity when is cut.

A divalent cation, usually Mg2+, is required for enzyme activity, though Mn2+ can also be used. Eight different cations with ionic radius/charge ratios similar to Mg2+ were tested and CO2+ and Zn2+ were also found to act as cofactors for EcoRI.

A comprehensive study has been made of the effect of NaCl and pH on the EcoRI/EcoRI* transition in the presence of the above four cations. Generally, a decrease in NaCl and/or an increase in pH caused a decrease in enzyme specificity. The changeover depended on the cation. They may be placed in order of their ability to increase EcoRI specificity thus: Co2+ > Zn2− > Mg2+ > Mn2+.

The Km of EcoRI for ColE1 DNA, in the presence of CO2+, was found to be 0.4 nM, compared to 3 nM with Mg2+, whereas the turnover was only one double‐stranded scission/min with Co2+ compared to eight/min with Mg2+.

The implications of all these findings on the enzyme's mechanism are discussed.
Mechanism of Action of a 16‐Membered Macrolide
Authors: SIEGRIST Sylvie , LAGOUARDAT Jacques , MOREAU Nicole , LE GOFFIC François
Published: March 2005

Rosaramicin is a new macrolide antibiotic with activity similar to that of erythromycin. In this paper, we describe the synthesis of [3H]dihydrorosaramicin and show by sucrose gradient centrifugation, gel exclusion chromatography and equilibrium dialysis that rosaramicin and its dihydroderivative bind specifically to the 70‐S ribosome and 50‐S ribosomal subunit of Escherichia coli. Binding to the 30‐S subunit is not detectable. The parameters of the binding interaction were evaluated by equilibrium dialysis. The affinities of E. coli ribosomes for rosaramicin and dihydrorosaramicin are in good agreement with the mininal inhibitory concentration of these drugs for microorganisms.
Reactivation of the Phospho Form of the NAD‐Dependent Glutamate Dehydrogenase by a Yeast Protein Phosphatase
Authors: HEMMINGS Brian A.
Published: March 2005

A protein phosphatase was isolated from the yeast, Candida utilis, which could reactivate (dephosphorylate) the phosphorylated form of the NAD‐dependent glutamate dehydrogenase. The protein could also dephosphorylate casein, histone and kemptide (a heptapeptide corresponding to the phosphorylation site of liver pyruvate kinase).

Reactivation of the phosphorylated glutamate dehydrogenase was stimulated by the simultaneous addition of NAD and l‐glutamate; 2‐oxoglutarate, NH+4 and NADH had no effect. The reactivation of phosphorylated glutamate dehydrogenase could be inhibited by phosphate, pyrophosphate and fluoride.
Occurrence of Heterogenous Forms of the Subunits of Creatine Kinase in Various Muscle and Nonmuscle Tissues and Their Behaviour during Myogenesis
Authors: ROSENBERG Urs B. , EPPENBERGER Hans M. , PERRIARD Jean‐Claude
Published: March 2005

Purified, homodimeric creatine kinases from chicken were subjected to two‐dimensional gel analysis under dissociating conditions. Each of the subunits M‐creatine kinase and B‐creatine kinase was resolved into a basic and an acidic subspecies with very similar mobilities in the sodium dodecylsulfate dimension. The M‐creatine kinase subspecies were found in myogenic cells, fast muscle, slow muscle and the B‐creatine kinase subspecies were present in heart, gizzard and brain. The creatine kinase subunits were identified in these tissues by a variety of methods like immunoreplicas of two‐dimensional gels, immunoprecipitations, or coelectrophoresis with purified creatine kinase and all gave the same results. In the course of myogenic development in vitro the subspecies were synthesized coordinately and no indication was found for a differential regulation of any of the subspecies of the creatine kinase subunits. No radioactive phosphorus was incorporated into either one of the subspecies, hence phosphorylation could be ruled out as the source of heterogeneity. Furthermore, peptide mapping analysis of partial proteolytic digests did not reveal differences among the subspecies of the same subunit. Not only chicken but also rat creatine kinase displayed this type of heterogeneity. All subspecies were observed after translation of chicken RNA in a cell‐free protein‐synthesizing system. The heterogeneity probably might best be explained by the existence of multiple, but closely related genes for the creatine kinase subunits.
Induction by Hormones of Phenylalanine Ammonia‐Lyase in Bean‐Cell Suspension Cultures
Authors: JONES D. Hugh , NORTHCOTE Don H.
Published: March 2005

Transfer of dark‐grown suspension cultures of Phaseolus vulgaris cells from a maintenance medium to medium with reduced auxin and increased cytokinin content resulted in an increase in phenylalanine ammonialyase activity 3–5 days later. This increase in phenylalanine ammonia‐lyase activity may be used to examine changes in control of gene expression following plant hormone action.

An inhibitor of transcription (aetinomycin D) and an inhibitor of translation [d‐2‐(4‐methyl‐2,6‐dinitroanilino)‐N‐methylpropionamide) were used to investigate this phenylalanine ammonia‐lyase response under conditions of controlled cell density and optimised inhibitor dose.

The rising phase of the enzyme activity was inhibited by either actinomycin D or the propionamide derivative while cytoplasmic streaming was unaffected by these inhibitors. This indicates that both transcription and translation are required for the response.

The falling phase of phenylalanine ammonia‐lyase specific activity is due to inactivation or degradation of the enzyme since the decrease cannot be explained by dilution out of the enzyme during cell growth. This falling phase was insensitive to the propionamide inhibitor, but application of actinomycin D resulted in an increase in phenylalanine ammonia‐lyase activity (superinduction) after 12 h.

Superinduction by actinomycin D suggests that availability of phenylalanine ammonia‐lyase mRNA for translation may be controlled to produce the change in enzyme activity. Lack of superinduction by the propionamide inhibitor indicates that translation of a protein which degrades either phenylalanine ammonia‐lyase or its mRNA is not required to cause the fall in phenylalanine ammonia‐lyase activity. The superinduction is, therefore, probably caused by an alteration in competition between mRNAs and the increased enzyme synthesis results from preferential translation of relatively stable phenylalanine ammonia‐lyase mRNA already transcribed during the rising phase of activity. Actinomycin D is postulated to suppress competition from other less stable mRNAs for the translation machinery, by inhibiting their transcription.

A model based on de novo synthesis of both phenylalanine ammonia‐lyase mRNA and its encoded enzyme has been shown to apply to other phenylalanine ammonia‐lyase responses. This model assumes a constant rate of degradation of the enzyme throughout the response, and is only compatible with the mRNA‐competition hypothesis for superinduction.
Nucleotide Sequence Coding for the Respiratory NADH Dehydrogenase of Escherichia coli
Authors: YOUNG Ian G. , ROGERS Bruce L. , CAMPBELL Hugh D. , JAWOROWSKI Anthony , SHAW Denis C.
Published: March 2005

The nucleotide sequence of the structural gene coding for the respiratory NADH dehydrogenase of Escherichia coli has been determined by the chain‐termination method. The reading frame for the protein starts with the unusual initiation codon UUG and predicts an amino acid sequence of 434 residues (Mr= 47304). The reading frame was confirmed by protein chemical studies including determination of the N‐terminal sequence of the protein. The product made in vivo was found to have threonine as its N‐terminal residue, indicating that the initiating N‐formylmethionine had been removed by post‐translational processing.
Effect of Threonylcarbamoyl Modification (t6A) in Yeast tRNAArgIII on Codon‐Anticodon and Anticodon‐Anticodon Interactions
Authors: WEISSENBACH Jean , GROSJEAN Henri
Published: March 2005

The effect of N‐[9‐(β‐d‐ribofuranosyl) purin‐6‐ylcarbamoyl]threonine (t6A) adjacent to anticodon U*‐C‐U of yeast tRNAArgIII (where U* is a modified U), compared to its unmodified adenosine counterpart, has been evaluated by three independent methods: (a) the polynucleotide‐directed binding of tRNA on ribosomes, (b) the ribosome‐free trinucleotide binding to the anticodon. (c) the anticodon‐anticodon binding test.

The results obtained by these three methods indicate a small but significant stabilization effect of t6A on the binding of yeast tRNAArgIII with (a) poly(A,G) in the presence of Escherichia coli ribosomes, (b) free A‐G‐A triplet, and (c) E.coli tRNAServ (anticodon G‐G‐A).

We therefore conclude that the stabilization effect of t6A occurs on U · A and U · G base pairs adjacent to the 5′ side of the modified nucleoside, most probably by stacking.
Stability of Non‐polyadenylated Viral mRNAs Injected into Frog Qucytes
Authors: McCRAE Malcolm A. , WOODLAND Hugh R.
Published: March 2005

The naturally non‐polyadenylated mRNAs of reovirus were shown to have a half‐life in excess of 3 days when injected into Xenopus laevis oocytes. Hybridization analysis and gel electrophoresis showed that all 10 mRNA species had a similar high stability, despite being translated at widely differing rates. We have confirmed previous findings indicating a role for the 5′‐terminal cap structure in determining reovirus mRNA stability [Furuichi et al. (1977) Nature (Lond.) 266, 235–239]. The significance of these results in relation to a general role for poly(A) in messenger RNA function is discussed.
Immunological Evidence for a Proteolytic Cleavage at the Active Site of Antithrombin in the Mechanism of Inhibition of Coagulation Serine Proteases
Authors: WALLGREN Per , NORDLING Kerstin , BJÖRK Ingemar
Published: March 2005

Previous studies have shown that a modified form of antithrombin, cleaved at a single‐Arg‐Ser bond near the carboxy‐terminal end of the chain, is formed during the reaction with thrombin concurrent with the formation of the inactive enzyme‐inhibitor complex. A variety of evidence suggests that this cleavage site is the active site of antithrombin. In this work, antisera against intact antithrombin, the modified form of antithrombin and the anti thrombin‐thrombin complex were used in immunodiffusion analyses to probe the state of the inhibitor in its complexes with coagulation serine proteases. The resuiis show that new antigenic determinants not present in intact antithrombin are created in modified antithrombin by the single peptide‐bond cleavage. The same antigenic determinants are found also in complexes between antithrombin and thrombin or factor Xa. No evidence for the exposure of other new determinants in the complexes was obtained. The most likely conclusion from these results is that antithrombin exists in its complexes with the serine proteases as the modified, two‐chain form of the inhibitor. This suggests that the mechanism of inhibition involves proteolytic cleavage of the active site of antithrombin by the protease.
Arylsulfatases of Human‐Lung Tumors Transplanted into Athymic Mice
Authors: GASA Shinsei , MAKITA Akira , KAMEYA Toru , KODAMA Tetsuro , KOIDE Tsutomu , TSUMURAYA Masaru , KOMAI Tomoyoshi
Published: March 2005

The activities and properties of arylsulfatases A and B from human lung carcinoma transplanted into athymic mice were demonstrated. The activities of arylsulfatases A and B from transplanted carcinomas with four histological types were more than twofold higher as compared to those from surgical tumors, except for arylsulfatase A activity in blastoma. Arylsulfatase B in transplanted tumors was almost completely replaced, except for blastoma, by an anionic B variant (B1) which was a minor component of arylsulfatase B in surgical lung tumor and absent in normal human lung. The properties of arylsulfatases A and B from transplanted tumors were essentially identical, respectively, with those from normal lung or surgical tumors in respect of molecular weight, heat stability, pH optimum, isoelectric point (pI), Km time course profile and substrate specificity. Arylsulfatase B1 showed the properties similar to B enzyme except for net charge.

The cause of the negative charge of tumor B1 enzyme was investigated. By the action of phosphatase, which was added exogenously or had been persistently included in the partially purified enzyme preparation, B1 enzyme (pI 7.5) shifted to about pI 8.2. Treatment of B1 enzyme with neuraminidase, concomitant with the endogenous phosphatase, resulted in marked increase (pI 9.5) of the isoelectric point, identical to that of arylsulfatase B. Thus, it is most probable that tumor B1 enzyme is modified by additional sialic acid and phosphate bound to arylsulfate B.
Evidence for the Presence of Anion‐Recognition Sites in Pig‐Liver Aldehyde Reductase
Authors: BRANLANT Guy , TRITSCH Denis , BIELLMANN Jean‐François
Published: March 2005

Aldehyde reductase from pig liver is completely inactivated by phenyl glyoxal, a substrate of this enzyme. Inactivation, exhibiting pseudo‐first‐order kinetics and a reaction order of nearly one, is observed in the presence or absence of the coenzyme analogue 1,4,5,6‐tetrahydronicotinamide‐adenine dinucleotide phosphate (h5.62NADPH). These results suggest that one single residue, not located in the coenzyme‐binding site, is modified. Loss of activity is directly proportional to incorporation of [14C]phenyl glyoxal. Amino acid analysis demonstrates the modification of one arginine residue, on average. Nevertheless, the ternary complex enzyme · h5.62NADPH ·p‐carboxybenzaldehyde does not afford complete protection against phenyl glyoxal inactivation. In contrast, (±)2,3‐dimethylsuccinic acid, a strong inhibitor of the reductase, completely prevents inactivation of reductase by phenyl glyoxal. A pseudo‐affinity labeling is observed with p‐carboxyphenyl glyoxal, used as a specific arginine‐modifying agent. Such a mechanism differs from an affinity labeling in that the formation of the binary complex enzyme ·p‐carboxyphenyl glyoxal does not precede the alkylating step. At high concentration, p‐carboxybenzaldehyde protects aldehyde reductase from inactivation by p‐carboxyphenyl glyoxal.

All these results suggest the presence of two anion‐binding sites on this reductase, one being responsible for the better binding of substrates bearing a carboxyl group to the enzyme‐coenzyme complex, and the second one being modified by specific arginine reagents and implicated in the binding of uncompetitive inhibitors like (±)2,3‐dimethylsuccinic acid.

Results obtained by W. S. Davidson and T. G. Flynn [J. Biol. Chem. 254, 3724–3729 (1979)] on the modification of pig kidney reductase by 2,3‐butanedione conflicted somewhat with our results. This has led us to compare these two reductases. No differences between the enzymes from either source could be detected, thus it is likely that they are both the same protein.
pH‐Dependence of the ATP‐Driven Uptake of Noradrenaline by Bovine Chromaffin‐Granule Ghosts
Authors: SCHERMAN Daniel , HENRY Jean‐Pierre
Published: March 2005

The kinetic parameters of noradrenaline uptake by chromaffin granule ghosts have been measured at external pH values between 6.5 and 8.5. The log of the Km for noradrenaline decreased linearly with pH with a slope of –1.0, indicating that the observed affinity increase originated in deprotonation of a single chemical group. This result is interpreted as showing that the neutral form of monoamines is the true substrate for the amine carrier. The Km of the carrier for the neutral form of noradrenaline was calculated as 0.1 μM. The maximal velocity, V, of the uptake reaction was constant from pH 6.5 to 8.0 and decreased at more alkaline pH values. Since the proton electrochemical gradient ΔμH+ generated by the membrane H+‐pump was independent of the pH in the range 6.5–9.5, the pH dependence of the maximal velocity of uptake reflects the pH profile of the monoamine transporter.
Chloroquine‐Induced Accumulation of Gangliosides and Phospholipids in Skeletal Muscles
Authors: NILSSON Olle , FREDMAN Pam , KLINGHARDT Georg W. , DREYFUS Henri , SVENNERHOLM Lars
Published: March 2005

High doses of the lysosomotropic drug chloroquine result in lipid storage in many organs in animals. We used miniature pigs, type Gottingen, to study the lipid accumulation in skeletal muscle after chloroquine intoxication for more than 200 days. The lipids of the quadriceps muscle in intoxicated and in age‐matched control pigs were characterized and determined. The lipid storage was larger in skeletal muscle than in any other organ of the intoxicated pigs. The concentration of phospholipids was increased threefold, acidic phospholipids relatively more than neutral ones. The lysosome‐specific acidic phospholipid bis(monoacylglyceryl)phosphate content was almost 50‐fold larger in the intoxicated pigs than in the controls. Cholesterol was increased slightly more than the phospholipids, but there was no particular accumulation of cholesteryl esters, which has been shown to occur in the liver. For the first time a storage of gangliosides, relatively more pronounced than of other lipids, was demonstrated in skeletal muscle in drug‐induced lipidosis. The concentration of total gangliosides was increased 10–15‐fold, and the pattern of gangliosides showed some distinct changes resulting in at least a 100‐fold increase in the concentration of ganglioside GM2 (II3NeuAc‐GgOse4Cer).
Purification and Properties of Two 2‐Oxoacid:Ferredoxin Oxidoreductases from Halobacterium halobium
Authors: KERSCHER Lorenz , OESTERHELT Dieter
Published: March 2005

Pyruvate: ferredoxin oxidoreductase and 2‐oxoglutarate: ferredoxin oxidoreductase were obtained from cell‐free extracts of Halobacterium halobium as homogeneous proteins after ammonium sulfate precipitation, salting‐out chromatography with ammonium sulfate on unsubstituted agarose, gel filtration and chromatography on hydroxyapatite. The respective molecular weights are 256000 and 248000. Both enzymes consist of two sets of nonidentical subunits of Mr 86000 and 42000 in the case of the pyruvate‐degrading enzyme and of 88000 and 36000 in the case of the 2‐oxoglutarate‐degrading enzyme. Analyses indicate that an intact enzyme molecule contains two [4 Fe‐4S]2+(2+.1+) clusters and two molecules of thiamin diphosphate.

Flavin nucleotides, lipoic acid and pantetheine are absent. Thus the enzymes are very similar to the 2‐oxoacid: ferredoxin oxidoreductases from fermentative and photosynthetic anaerobes described previously, but are clearly different from the 2‐oxoacid dehydrogenase multienzyme complexes which commonly occur in aerobic organisms.
The Biosynthesis of Wall Teichoic Acid by Toluenised Cells of Bhacillus subtilis W23
Authors: HANCOCK Ian C.
Published: March 2005

Toluenised cells of Bacillus subtilis W23 synthesized the teichoic acid, poly(ribitol phosphate), from exogenous precursors. The synthesis was dependent on concomitant synthesis of the linkage unit that joins teichoic acid to peptidoglycan. Under conditions that reduced cell autolytic activity, a large proportion of the teichoic acid became linked to the cell wall, independently of peptidoglycan synthesis. The specific activity of the system was more than 30 times that of isolated membranes, so that activity could be measured readily in the cells from 2 ml of an exponential culture of bacteria.
Drastic Changes in Accumulation and Synthesis of Plasma‐Membrane Proteins during Aggregation of Dictyostelium discoideum
Authors: ONO Ken‐ichi , TODA Katsumi , OCHIAI Hiroshi
Published: March 2005

Protein compositions of plasma membranes of Dictyostelium discoideum and their developmental changes during aggregation were analyzed by two‐dimensional polyacrylamide gel electrophoresis. More than 150 polypeptide species could be detected with the growth‐phase cells by staining. Most of them (about 120 spots) were conserved during aggregation, although their relative intensities of staining changes. On the initiation of cell differentiation, new polypeptides were accumulated on the plasma membrane and at least 100 new polypeptides were detected at the late‐aggregation stage. This accumulation of new components was completely inhibited when the growth‐phase cells were incubated in a liquid medium containing 5 mMEDTA. Studies on protein synthesis suggested that the accumulation and appearance of the new protein components was due to de novo synthesis.

Pulse‐label experiments with [35S]methionine showed that out of 200 proteins detected during aggregation, about 90 proteins showed various stage‐specific patterns of synthesis and the rest were synthesized consistently.

Actin and discoidin present in the plasma membrane were identified by reference to purified actin and discoidin. Actin synthesized de novo accumulated progressively on the plasma membrane during aggregation. Discoidin also accumulated on the plasma membrane, but a large amount was also recovered in a cytoplasmic soluble fraction. Discoidin I was separated into two spots with different pI values on a two‐dimensional gel.
Isolation and Properties of Chloroplast Coupling Factor from Wheat
Authors: MOASE Elaine H. , GREEN Beverley R.
Published: March 2005

Wheat chloroplast coupling factor (CF1) was estracted with a modification of the chloroform extraction method of Younis et al. (J. Biol. Chem. 252, 1814‐1818, 1977). A one‐step purification on an 8‐25% sucrose gradient yielded a CF1 which was at least 98% pure as judged by sodium dodecyl sulfate/polyacrylamide gel eletrophoresis.

Inclusion of proteolysis inhibitors during extraction and purification consistently gave a CF1containing all five subunits. Selective loss of the δ and ɛ subunits was observed when proteolysis inhibitors were omitted.

Proteolysis inhibitors prevented the release of wheat CF1 from the thylakoid by the low‐ionic‐strength wash method of Strotmann et al. (Biochim. Biophys. Acta, 314, 202‐210, 1973). The enzyme extracted with chloroform and low ionic strength were compared by electrophoresis and no evidence of a difference in molecular weight of any subunit was observed. This suggests that a proteolytic event is not required for release of wheat CF1 by the low‐ionic‐strength method, even though release is inhibited by proteolysis inhibitors.

The γ subunit of wheat CF1 probably contains at least one internal disulfide bridge, as the electrophoretic mobility of this subunit is lower in the presence of reducing agent than in its absence.

Wheat CF1 was viewed by the electron microscope after negative staining. Discrete particles, many appearing hexagonal, were observed at high magnifications. Markham rotational analysis confirmed that the enzyme has sixfold symmetry in at least one of its orientations.
Correlation between Protein Phosphorylation and Progesterone Synthesis in Bovine Luteal Cells Stimulated by Lutropin
Authors: DARBON Jean‐Marie , URSELY Jsocelyne , LEYMARIE Pierre
Published: March 2005

The implication of cytosolic protein phosphorylation in the regulation by lutropin of steroidogenesis in luteal tissue was investigated, in vitro by incubating selected small bovien luteal cells in the presence of 32P‐labelled phosphate. The incorporation of 32P into cytosolic proteins was measured after fractionation by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and autoradiography of the gel. Progeserone synthesis, 32P incorporation into cytosolic proteins, cyclic AMP content and protein kinase activity were measured simultaneously aafter incubation for various times and with increasing doses of lutropin.

As previously reporte, the gonadotropin as well as dibutyryladenosine 3′, 5′ monophosphate enchanced by 60–150% the degree of phosphorylation of six cytosolic proteins of molecular weight ranging from 50000–115000.

Time‐course studies in the presence of a saturating dose of lutropin demonstrated a close correlation between progesterone synthesis, protein phosphorylation and cyclic AMP production. The three parameters were increased within 5 min, the level of cylci AMP and the extent of protein phosphorylation reaching a maximum value after 10‐20 min.

Dose‐response curves for progesterone synthesis and protein phosphorylation were nearly identical with a half‐maximal stimulation occurring for both parameters with a physiological concentration of lutropin (3ng/ml). Conversely dose‐response curves for cyclic AMP and protein kinase activity were clearly shifted, as expected, toward the higher concentration of lutropin (half‐maximal stimulation for 90 ng/ml of lutropin).

Further characterization of the phosphorylated proteins using a highly resolutive two‐dimensional eletrophoretic technique demonstrated that the number of proteins phosphrylated under lutropin and the degree of stimulation was distinctly higher than suspeted from monodimensional fractionation technique. The 32P incorporation fro two of the nine proteins phosphorylated by lutropin appeared at least ten‐times higher than in the control.

The present result are compatible with the implication of at least some of the observed unduced protein phosphorylations in the steroidogenic effect of lutropin in luteal tissue. They sustain the hypothesis of a cyclic‐AMP‐dependent protein kinase activation as a part of the mechanism of lutropin action.
Interrelationships between 3‐Hydroxy‐3‐Methylglutaryl‐CoA Synthase, Acetoacetyl‐CoA and Ketogenesis
Authors: MENAHAN Lawrence A. , HRON W. Theodore , HINKELMAN Doloris G. , MIZIORKO Henry M.
Published: March 2005

Kinetic and physical approaches have been employed to investigate the binding of acetoacetyl‐CoA to hydroxymethylgluaryl‐CoA synthase. The enzyme has an apparent Km for acetoacetyl‐CoA (0.35μM) which is more than an order of magnitude lower than the K(6‐10μM) measured for substrate inhibition by this metabolite. Hepatic acetoacetyl‐CoA concentration, as measured by a sensitive and highly specific radioactive assay appears to be in the 1‐ 10 μM range; the concentration decreases during diabetic ketoacidosis. Total hepatic activity of hydroxymethylglutaryl‐CoA synthase and levels of mitochondrial enzyme protein, determined by radioimmunoassay, are not appreciably different in livers from control or ketoacidotic animals.

In contrast to the decrease in hepatic acetoacetyl‐CoA concentration observed during ketoacidosis, myocardial acetoaetyl‐CoA levels are increased by aaat least tenfold when compared to controls. Elevated acetoacetyl‐CoA levels may serve to inhibit fatty acid utilization y the heart. Thus, a consideration of the multiple interactions of acetoacetyl‐CoA with the enzymes involved in ketone ody production and utilization may be useful in evaluating the metabolic significance of this intermediate.