bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of Messenger RNA Coding for Eggshell Protein of the DNA‐Eliminating Nematode Ascaris lumbricoides
Authors: ZULAUF Erich , GUT Charlotte
Published: March 2005

Poly(A)‐containing RNA from polyploid uterine epithelial cells of Ascaris lumbricoides has been isolated by poly(U)‐Sepharose chromatography. The bulk of poly(A)‐containing RNA migrates as 18‐S RNA in formamide/polyacrylamide gels. In a cell‐free wheat germ system, this RNA directs the synthesis of a polypeptide with identical migration behavior in dodecylsulphate/urea/polyacrylamide gels as the polypeptide isolated from the proteinaceous eggshell. The two proteins reveal almost identical peptide patterns in fingerprint analysis. The authentic eggshell protein has been identified as a glycoprotein with a molecular weight of about 10 000, as determined by dodecylsulphate/polyacrylamide gel electrophoresis. The apparent discrepancy between mRNA length and the required coding length for the protein is discussed.
Fluorescence Energy‐Transfer Studies on the Pyruvate Dehydrogenase Complex Isolated from Azotobacter vinelandii
Authors: SCOUTEN William H. , DE GRAAF‐HESS Adriana C. , DE KOK Arie , GRANDE Hans J. , VISSER Antonie J. W. G. , VEEGER Cees
Published: March 2005

Fluorescence energy transfer has been employed to estimate the minimum distance between each of the active sites of the 4 component enzymes of the pyruvate dehydrogenase multienzyme complex from Azotobacter vinelandii. No energy transfer was seen between thiochrome diphosphate, bound to the pyruvate decarboxylase active site, and the FAD of the lipoamide dehydrogenase active site. Likewise, several fluorescent sulfhydryl labels, which were specifically bound to the lipoyl moiety of lipoyl transacetylase, showed no energy transfer to either the flavin or thiochrome diphosphate. These observations suggest that all the active centers of the complex are quite far apart (≧40 nm), at least during some stages of catalysis. These results do not preclude the possibility that the distances change during catalysis.

Several of the fluorescent probes used possessed multiple fluorescent lifetimes, as shown by determination of lifetime averages by both phase and modulation measurements on a phase fluorimeter. These lifetimes are shown to result from multiple factors, not necessarily related to multiple protein conformations.
Reactions of Antibodies against Ferredoxin, Ferredoxin‐NADP+ Reductase and Plastocyanin with Spinach Chloroplasts
Authors: BÖHME Herbert
Published: March 2005

Purified antisera against ferredoxin, ferredoxin‐NADP+ reductase and plastocyanin agglutinated osmotically shocked and washed spinach chloroplasts, prepared according to standard procedures. The monomeric antibody (immunoglobulin G fraction) of the reductase antiserum agglutinated chloroplasts specifically and directly, indicating that protruding structures (for example, the coupling factor) do not act as steric hindrances as has been suggested.

With ferredoxin antiserum, the presence of a pentameric antibody (immunoglobulin M fraction) was obligatory to observe a positive agglutination reaction. Immunoglobulin G only inhibited ferredoxin‐dependent reactions, like NADP+ ‐photoreduction, but did not cause agglutination. Ferredoxin seems to be located in depressions of the membrane, possibly caused by a partial release of this protein in shocked chloroplasts.

Similar results were obtained with purified immunoglobulins from a plastocyanin antiserum. Again the immunoglobulin G fraction inhibited electron transport reactions catalyzed by plastocyanin, whereas immunoglobulin M showed a positive agglutination, but had no influence on electron transport. It is concluded that ferredoxin, ferredoxin‐NADP+ reductase and plastocyanin are peripheral electron transport components, located at the outer thylakoid membrane.
Association of Poly(A) and Poly(U) Polymerases with Cytoplasmic Ribosomes
Authors: MILCHEV Georgui I. , HADJIOLOV Asen A.
Published: March 2005

Cytoplasmic ribosomes of Ehrlich ascites tumour cells were isolated under conditions where ribonuclease contamination is minimized. These ribosomes displayed poly(A) and poly(U) polymerase activity with [3H]ATP or [3H]UTP as substrates, while incorporation of [3H]GTP or [3H]CTP was not observed. Poly(A) polymerase is Mn2+ ‐dependent with an optimum at 1 mM, while poly(U) polymerase is activated by Mg2+ with a broad optimum at 5–20 mM. Both enzyme activities remained associated with the two ribosomal subparticles upon EDTA treatment and sucrose density gradient fractionation. The product of poly(U) polymerase was bound to endogenous 28‐S and 18‐S rRNA. About 40% of the poly(A) polymerase product was retained by oligo(dT)‐cellulose and the radioactivity distributed in 28‐S and 18‐S rRNA as well as in 4–16‐S RNA material. Addition of poly(A) stimulates markedly the activity of poly(A) polymerase, but addition of poly(U) is without effect on either enzyme. The poly(U) polymerase of the small and the large subparticle did not display a preferential use of endogenous versus exogenous RNA as primer. The same was true for poly(A) polymerase of the small subparticle. However, the poly(A) polymerase of the large subparticle showed a marked preference for exogenous cytoplasmic 18‐S RNA before or after purification through oligo(dT)‐cellulose. This effect was not observed with 18‐S rRNA obtained from previously isolated small ribosomal subparticles. It is suggested that ribosome‐associated poly(A) polymerase may display a preference for mRNA lacking poly(A) as primer.
Studies on the Purification and Properties of a 6.8‐S DNA Polymerase Activity Found in Calf‐Thymus DNA Polymerase‐α Fraction
Authors: HESSLEWOOD Ian P. , HOLMES Andrew M. , WAKELING William F. , JOHNSTON Irving R.
Published: March 2005

The heterogeneity of calf thymus DNA polymerase‐α has been further investigated. In particular, an enzyme (enzyme D) which exhibits higher activity on poly(dA) · (dT)10 (A:T = 20:1) compared with that on activated DNA, has been further purified and its properties compared with two other activities of the DNA polymerase‐α fraction (enzymes A1 and C) which do not show a preference for poly(dA) · (dT)10 over activated DNA.

As with A1 and C, enzyme D was shown to have many of the characteristic properties of DNA polymerase‐α in that it is an acidic protein as judged by its binding to DEAE‐cellulose, has a molecular weight of about 140000, does not use a poly(A) · (dT)10 template‐initiator complex and is inhibited by N‐ethylmaleimide. It exhibits anomalous gel filtration behaviour on Sepharose 6B and it binds relatively weakly to DNA‐cellulose compared with DNA polymerase‐β. The extreme sensitivity of enzyme D to inhibition by N‐ethylmaleimide distinguishes it from A1 and C, as does its elution position from a DEAE‐cellulose column. On the other hand enzymes C and D are readily inactivated by heating at 45 °C unlike enzyme A1.

The possible interrelationships of the multiple activities of calf thymus DNA polymerase‐α are discussed.
Purification and Properties of Phosphorprotein Phosphatases with Different Substrate and Divalent Cation Specificities from Canine Heart
Authors: LI Heng‐Chun , HSIAO Kwang‐Jen , CHAN Wanda W. S.
Published: March 2005

Three phosphoprotein phosphatases have been isolated from canine heart by a procedure involving treatment of the enzyme preparation with 80% ethanol at room temperature. They are a divalent‐cation‐independent, nonspecific enzyme (Mr= 35000) referred to as phosphatase S, an Mg2+ ‐activated enzyme (Mr= 49300) with activity predominately toward phosphocasein and an Mn2+ ‐activated enzyme (Mr= 61000) with activity predominately toward phosphohistone. Phosphatase S has been purified over 10000‐fold and the two divalent‐cation‐dependent enzymes have been purified over 1000‐fold from the crude extract. The purified preparation of phosphatase S showed a single broad protein stain band which coincided with the enzymic activities when examined with polyacrylamide gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis of the enzyme preparation also showed a single protein band corresponding to a molecular weight of 35000. Phosphatase S exhibited a broad specificity and was about equally active toward phosphorylase a, phosphorylated histone, casein and cyclic‐AMP‐dependent protein kinase. Phosphatase S showed an optimum between pH 6.5–8 and was inhibited by ATP, other nucleoside phosphates, Pi, PPi and NaF. The Km values of phosphatase S for phosphorylase a, phosphorylated histone, and casein are 4, 27, and 16 μM, respectively. Evidence indicates that phosphatase S is a catalytic subunit of several nonspecific, divalent‐cation‐independent enzymes reported previously [Biochim. Biophys. Acta, 483, 337–347 (1977)].

The present findings indicate that at least three basic catalytic entities of phosphorprotein phosphatase can be isolated from canine heart extract. These phosphatases which differ from one another in substrate specificity and divalent cation dependence may play different roles in the regulations of protein dephosphorylation in the cell.
Replication of M‐13 DNA in Plasmolysed Escherichia coli Cells
Authors: KESSLER‐LIEBSCHER Barbara E. , STAUDENBAUER Walter L. , HOFSCHNEIDER Peter Hans
Published: March 2005

DNA molecules with restricted binding of intercalating dyes are observed as replicative intermediates during the replication of bacteriophage M‐13 duplex DNA in a cellular system in vitro prepared by plasmolysis of M‐13‐am5‐infected Escherichia coli cells. Restriction of dye binding is abolished by heating the DNA to 80 °C, but can be recovered by slow cooling of the heat‐treated DNA. Radioactive pulse‐label incorporated by these molecules is found exclusively in elongated viral strands of more than one genome length. In the electron microscope this DNA fraction is seen to contain a significant number of duplex DNA rings with two single‐stranded tails protruding from the same region of the ring. It is proposed that these structures arise by branch migration during the isolation of replicating molecules containing only one single‐stranded tail. The topological constraint in these molecules is most likely caused by base‐pairing between partially complementary regions of the two single‐stranded tails.
Purification and Characterization of DNA Polymerases from the Plasmacytoma MOPC 104E and Abelson Murine Leukemia Viruses
Authors: WEIMANN Bernd J. , SCHMIDT Jörg , TAKACS Bela
Published: March 2005

RNA‐dependent DNA polymerases of intracisternal A particles from the mouse plasma cell tumor MOPC 104E and of Abelson murine leukemia virus (A‐MuLV) were isolated from particle preparations by Nonidet P40 and ultrasonic treatment and purified by column chromatography on DEAE‐cellulose and phosphocellulose, followed by centrifugation in linear sucrose gradients. Both DNA polymerases were very similar in their elution patterns from phospho and DEAE‐cellulose, template specificities, requirements for optimum activity and inactivation by anti‐(reverse transcriptase) antiserum. They are associated with ribonuclease H activity. For molecular weight determinations, antibody‐precipitated enzymes were bound to staphylococcal‐protein‐A‐Sepharose, solubilized and run on dodecylsulfate/polyacrylamide gels. Their apparent molecular weight was estimated to be 80000.
Porcine Follitropin
Authors: CLOSSET Jean , MAGHUIN‐ROGISTER Guy , HENNEN Georges , STROSBERG A. Donny
Published: March 2005

The amino acid séquence of the porcine β subunit has been established by studies of peptides isolated after tryptic, thermolytic and staphylococcal protease treatments of the reduced and carboxymethylated chain. The primary structure of the amino‐terminal region of the molecule has been solved by automatic sequencing of the reduced and tritium‐labeled carboxymethylated subunit.

The amino acid sequence of porcine follitropin β subunit differs from that of its human counterpart by several amino acid replacements, deletion or addition of one or several residues. The porcine chain appears shorter at both its amino and carboxy‐terminal ends. The chemical evolution of follitropin is briefly considered and compared to these of thyrotropin and lutropin.
Conformation of Erabutoxins a and b in Aqueous Solution as Studied by Nuclear Magnetic Resonance and Circular Dichroism
Authors: INAGAKI Fuyuhiko , MIYAZAWA Tatsuo , HORI Hideaki , TAMIYA Nobuo
Published: March 2005

The 270‐MHz nuclear magnetic resonance (NMR) spectra of erabutoxins a and b have been observed in 2H2O solution. By the use of convolution difference and double resonance techniques, proton signals in the aromatic and methyl regions have been assigned. From the pH dependence of NMR chemical shifts, the pKa value of His‐26 of erabutoxin b is found to be 5.8, whereas His‐7 of erabutoxins a and b is not protonated at pH above 3. The imidazole ring of His‐7 is protonated upon the denaturation at pH 2.85. The acid denaturation process has been followed by the His‐26 and methyl proton signals and is found to be reversible but is slow as compared with NMR chemical shift time scale. The circular dichroism (CD) of erabutoxin b has also been observed. The denaturation is found to involve a major change from the β‐rich conformation to a disordered one. The NMR and CD changes upon acid denaturation are satisfactorily explained by the two‐state process. The deuterium exchange rates of the C‐2 protons of His‐26 and His‐7 of erabutoxin b indicate that His‐26 is exposed to the solvent whereas His‐7 is tightly buried in the interior of the protein globule. The pKa value of Tyr‐25 is as high as about 12.0, possibly due to the hydrogen bond formation between the hydroxyl group of Tyr‐25 and a carboxylate group. The hydroxyl group of Tyr‐25 is reversibly titrated so that this group is not buried tightly in the interior of the protein globule. The line width of the aromatic proton signals of Tyr‐25 is significantly broad at room temperature, suggesting a restricted rotation of the aromatic ring. The aromatic proton signals of Trp‐29 are fairly sharp; this aromatic ring is exposed and mobile. Except for His‐7, the micro‐environments of Tyr‐25, His‐26, and Trp‐29 residues and methyl proton signals of valine and isoleucine are consistent with the locations of α carbon atoms as elucidated by X‐ray crystal analyses.
Structure and Replication of Echovirus Type 12
Authors: ROSENWIRTH Brigitte , EGGERS Hans J.
Published: March 2005

A technique as been devised which allows us to label predominantly echovirus‐12‐specific proteins: upon infection in the presence of d‐(−)‐2‐(α‐hydroxybenzyl)benzimidazole plus actinomycin D the shut‐off of host‐cell protein synthesis takes place as in infected, untreated cells, but the bulk of viral protein synthesis is inhibited. Upon removal of 2‐(α‐hydroxybenzyl)benzimidazole a peak of viral protein synthesis is visible 3 h later.

The time course of appearance of viral proteins is followed by dodecyl sulfate/polyacrylamide gel electrophoresis of the proteins which had been pulse‐labeled at different times post‐infection.

The protein patterns induced in the infected cells by echovirus 12 and poliovirus 2 are compared; though they are different, some analogies are observed. The molecular weights of the polypeptides are determined.

In the presence of amino acid analogs cleavage of viral proteins is impaired. A group of large proteins up to Mr 220 000 is detectable in the infected cell which ordinarily is not observed. The 220 000‐Mr protein may represent the translation product of the total viral RNA. The hierarchy of the virus‐specific proteins originating from post‐translational cleavage is discussed.
Two Different Types of Bonds Linking Single ADP‐Ribose Residues Covalently to Proteins
Authors: BREDEHORST Reinhard , WIELCKENS Klaus , GARTEMANN Axel , LENGYEL Helgard , KLAPPROTH Karin , HILZ Helmuth
Published: March 2005

Single ADP‐ribose residues covalently bound to protein of rat liver and Ehrlich ascites tumor cells were rendered acid soluble by treatment with neutral hydroxylamine. Incubation of the acid‐soluble extract with 1 M NaOH at 56°C converted the released ADP‐ribose selectively to 5′‐AMP, which could then be quantified by a highly specific radioimmunoassay for 5′‐AMP.

Direct treatment of the acid‐insoluble fractions with alkali yielded a higher amount of ADP‐ribose equivalents than NH2OH treatment, indicating the release of additional ADP‐ribose residues linked to the acceptors by an NH2OH‐resistant, alkali‐labile linkage, as already observed in vitro. In adult rat liver 5300 pmol mono(ADP‐ribose) residues/mg DNA linked by NH2OH‐labile bonds, and 12600 pmol mono(ADP‐ribose) residues released by NaOH were found. The corresponding values for Ehrlich ascites tumor cells (stationary growth phase) were 480 pmol and 1660 pmol respectively. Opposite to the ratios found in vitro, and in spite of the nearly tenfold difference in total releasable mono(ADP‐ribose) residues, both tissues exhibited higher levels of ADP‐ribose residues bound to the acceptors by the NH2OH‐resistant linkage than residues linked by NH2OH‐susceptible bonds. These data also show that the bulk of the ADP‐ribosylated proteins in eukaryotic cells is modified by single ADP‐ribose rather than by poly(ADP‐ribose) chains.

Extraction of histone H1 with perchloric acid prior to the determination of ADP‐ribose residues indicated that histone H1 in vivo carried only a very small fraction of the total protein‐bound mono(ADP‐ribose) residues.
Properties ofHuman Hemoglobin Immobilized on Sepharose 4B
Authors: ROSSI FANELLI Maria Rosaria , AMICONI Gino , ANTONINI Eraldo
Published: March 2005

This paper reports the properties of human hemoglobin covalently bound to Sepharose 4B both in ‘high‐affinity’ and ‘low‐affinity’ conformations.

The results suggest that the coupling reaction is strongly affected by the conformational changes linked to oxygenation of the protein. The rate and the extent of the reaction are different for the oxy and deoxyderivatives, probably due to the change in reactivity of the amino groups in the liganded and unliganded tetramer.

The data on the equilibrium which is established between matrix‐bound and soluble subunits, measured by the ‘subunit‐exchange chromatography’, indicate that the system displays a minimal heterogeneity when hemoglobin is coupled to the gel in the deoxy state at intermediate protein concentration and pH 8.

Matrix‐bound hemoglobin is characterized by a higher oxygen affinity and by decreased homotropic and heterotropic interactions with respect to hemoglobin in solution, but the changes depend strongly on the conditions used in the coupling procedure.
Studies on Polypeptide‐Chain‐Elongation Factors from an Extreme Thermophile, Thermus thermophilus HB8
Authors: ARAI Ken‐Ichi , OTA Yoshimi , ARAI Naoko , NAKAMURA Shun , HENNEKE Christina , OSHIMA Tairo , KAZIRO Yoshito
Published: March 2005

Polypeptide chain elongation factors have been purified from an extreme thermophile, Thermus thermophilus HB8. By chromatography on a DEAE‐Sephadex column, the factors were separated into two peaks; peak I contained a complex of EF‐Tu and EF‐Ts, while peak II was composed of EF‐Tu · GDP and EF‐G. These factors were subsequently purified to homogeneous states and crystallized.

The EF‐Tu · EF‐Ts complex could be resolved into EF‐Tu and EF‐Ts by chromatography on a Sephadex G‐200 column in the presence of 8 M guanidine‐HCI. The complex could be reconstituted from EF‐Tu and the renatured EF‐Ts.

No immunological cross‐reaction was detected between EF‐Tu, EF‐Ts, and EF‐G from T. thermophilus and the antibodies to their corresponding Escherichia coli factors.

The molecular weight of EF‐Tu · GDP determined by sedimentation equilibrium and sodium dodecylsulfate/polyacrylamide gel electrophoresis was 49000 and 51000 respectively. On the other hand, the molecular weight of EF‐Ts was estimated as 27000 and 64000, respectively, by sodium dodecylsulfate/polyacrylamide gel electrophoresis and Sephadex gel filtration, suggesting that the protein existed probably as a dimer. The molecular weight of the EF‐Tu · EF‐Ts complex determined by sedimentation equilibrium and by gel filtration, was 142000 and 220000, respectively. Since the molar ratio of EF‐Tu to EF‐Ts in the EF‐Tu · EF‐Ts complex was one to one, it was suggested that the complex was composed of 2 mol each of EF‐Tu and EF‐Ts. The molecular weight of EF‐G was estimated as 85000, 80000 and 78000 by equilibrium centrifugation, gel filtration, and sodium dodecylsulfate/polyacrylamide gel electrophoresis respectively.
Effects of Gibberellic Acid and of Tunicamycin on Glycosyl‐Transferase Activities and on α‐Amylase Secretion in Barley
Authors: SCHWAIGER Hubert , TANNER Widmar
Published: March 2005

A crude membrane fraction was prepared from isolated aleurone layers, the secretory tissue of barley grains. The layers were pre‐incubated in the presence or absence of the phytohormone gibberellic acid. The membranes catalyzed the transfer of [14C]mannose from GDP‐[14C]mannose and of N‐[14C]acetylglucosamine from UDP‐N‐[14C]acetylglucosamine to endogenous and exogenous dolichyl monophosphate. When gibberellic acid was present during the pretreatment the activity of the transferases was increased by a factor of two to three. A significantly increased activity was observable within four hours after the addition of gibberellic acid, whereas the gibberellic‐acid‐induced secretion of the glycoprotein α‐amylase started only after 12 h.

Tunicamycin inhibited the secretion of α‐amylase by 60 to 80%. Intracellularly, however, no α‐amylase was found to accumulate. On the other hand, tunicamycin did not inhibit the rate of total protein synthesis by more than 10%. The possibility is discussed that the synthesis of the protein portion of glycoproteins is specifically inhibited, when glycosylation is prevented.
Isolation and Characterisation of Peptide Hydrolases from the Maize Root
Authors: SHANNON John D. , WALLACE William
Published: March 2005

The maize root has two main proteinase and carboxypeptidase components. Proteinase I and carboxypeptidase I, which predominate in older plants, appear to have a serine group at their active sites and have been estimated to have molecular weights of approximately 54000 and 77000 respectively. Proteinase I, which has been purified up to 500‐fold, degrades haemoglobin and azocasein with maximum activity at pH 4 and 9–10 respectively, while on maize root protein it gives most hydrolysis in the neutral pH range. The main portion of the nitrate‐reductase‐inactivating activity in the maize root extract is due to proteinase I. Carboxypeptidase I, like several other plant carboxypeptidases such as carboxypeptidase C which have now (IUB Recommendations 1978) been classified as serine carboxypeptidases (EC 3.4.16.1), has maximum activity around pH 5 and has esterase activity.

A second group of proteases, proteinase II and carboxypeptidase II, separated from the above on carboxymethyl‐cellulose, were shown to have different molecular weight properties and be equally sensitive to serine and thiol group inhibitors. Proteinase I1 degrades haemoglobin, but not azocasein and does not mediate nitrate reductase inactivation. Associated with this second group of proteases was a macromolecular component which inactivated nitrate reductase but, unlike the action of proteinase I, was not inhibited by phenylmethylsulphonyl fluoride or casein. It was inhibited by metal chelating agents which were without effect on nitrate reductase inactivation due to proteinase I.
Properties of Microsomal Subfractions Isolated from Developing Rabbit Skeletal Muscle
Authors: ZUBRZYCKA Elzdot;bieta , MICHALAK Marek , KOSK‐KOSICKA Danuta , SARZALA M. Gabriela
Published: March 2005

A heterogeneous muscle microsomal fraction isolated from 3–4‐day‐old rabbits was separated into two subfractions by continuous sucrose‐density‐gradient centrifugation. The two subfractions differ from each other morphologically and biochemically.

Negative staining reveals that the heavier subfraction (U2) consists of ‘thin‐contour’ vesicles resembling those of sarcoplasmic reticulum membrane from mature muscle. The surface of many of them (about 60%) is covered by 4‐nm particles.

The lighter subfraction (U1) contains smooth vesicles with a contour thicker than that of the vesicles of subfraction U2. This difference is probably due to the high lipid content in the vesicles of subfraction U1. The extraction of loosely bound lipids (60% of total lipid content) with anhydrous diethyl ether changes the ultrastructure of these vesicles; their contour becomes thin and the vesicles resemble those in subfraction U2.

The activity of Mg2+ ‐dependent ATPase is very high in subfraction U1, but this subfraction lacks calcium uptake and Ca2+‐dependent ATPase activity. Contrary to that, subfraction U2 shows the activity of Ca2+‐dependent ATPase and calcium‐loading capacity in the presence of oxalate.

Sodium dodecyl sulphate/polyacrylamide gel electrophoresis shows that the protein pattern of both subfractions is more complicated than that of sarcoplasmic reticulum vesicles from adult muscle. Both subfractions contain proteins of molecular weight between 12000 and 105000. The antibody precipitation technique indicates the presence of Ca2+‐dependent ATPase in subfraction U2 and the presence of calsequestrin in both microsomal subfractions.
The Charge Heterogeneity of Soluble Human Galactosyltransferases Isolated from Milk, Amniotic Fluid and Malignant Ascites
Authors: GERBER Andreas Ch. , KOZDROWSKI Irene , WYSS Sonja R. , BERGER Eric G.
Published: March 2005

UDP‐galactose: N‐acetylglucosamine galactosyltransferase was isolated from pooled human milk, pooled amniotic fluid and from two different individual samples of malignant ascites. The purification procedure involving two successive affinity chromatography steps on N‐acetylglucosamine–agarose and α‐lactalbumin–agarose yielded an enzyme preparation homogeneous by size. Under non‐denaturing conditions the ascites and amniotic fluid enzymes had identical electrophoretic mobility, but they moved faster than the milk enzyme. Isoelectric analysis in the presence and absence of urea resolved the milk enzyme into at least 13 different forms, nine of which had the same isoelectric points after refocusing. All enzyme forms showed similar activity when free N‐acetyl‐glucosamine, ovalbumin, sialic‐acid‐free ovine submaxillary mucin and glucose, in the presence of α‐lactalbumin, were used as acceptor substrates. Comparative isoelectric focusing of the three galactosyltransferases revealed identical patterns of the amniotic and ascites enzymes, but only partial overlap with the milk enzyme, which was less negatively charged. Neuraminidase treatment of ascites and milk galactosyltransferases produced very similar focusing patterns. The possible structural basis for this charge heterogeneity is briefly discussed.
Kinetic Estimation of the Base Sequence Complexity of Poly(A)‐Containing mRNA in Ehrlich‐Ascites‐Tumor Cells and Its Abundance in Nuclear RNA
Authors: SAMAL Babrubahan , BEKHOR Isaac
Published: March 2005

Poly(A)‐containing messenger RNA was isolated from polysomes of Ehrlich ascites tumor cells, and analyzed for sequence complexity by hybridization to its complementary DNA. The results indicate the presence of about 27000 diverse mRNA species in mouse Ehrlich ascites tumor cells. Total nuclear RNA was also hybridized to cDNA transcribed from polysomal poly(A)‐containing mRNA up to an r0t of 3000 M · s. It was found that all classes of the polysomal poly(A)‐containing mRNA sequences were also present in the nucleus, although the distribution varied. About 2% of the total nuclear RNA sequence were expressed as total polysomal poly(A)‐containing mRNA. We also report that the total percentage of the haploid mouse genome transcribed in Ehrlich cells is significantly higher than that found in other mouse cells previously examined for poly(A)‐containing mRNA sequence complexity.
Translation in vitro of Tetrahymena pyriformis Polyadenylated mRNA
Authors: PORTIER Marie‐Madeleine , MILET Michele , HAYES Donal H.
Published: March 2005

The capacity of poly(A)‐containing RNA of the protozoan ciliate Tetrahymena piriformis to direct the synthesis of proteins in vitro has been tested using two cell‐free systems: a wheat germ extract and a rabbit reticulocyte lysate. The results obtained with these two systems are compared and the identification of α and β tubulins among the products of protein synthesis in vitro, after separation by one‐dimensional and two‐dimensional electrophoresis, is described. By isoelectric focusing in polyacrylamide gels, each species of tubulin is resolved into several bands, suggesting that the main subunits are more heterogeneous than has been generally described. Poly(A)‐containing RNA has also been fractionated on a 70% formamide/sucrose gradient and it is shown that α and β tubulins are coded by separate mRNAs.
Relationship between Single‐Stranded DNA Isolated from Cultured Muscular Cells during Differentiation and the Transcription of Messenger RNA
Authors: LEIBOVITCH Serge Alexandre , LEIBOVITCH Marie‐Pierre , KRUH Jacques , HAREL Jacques
Published: March 2005

Single‐stranded DNA (ssDNA), equivalent to about 2% of the total nuclear DNA, was isolated by an improved method of hydroxyapatite chromatography from native nuclear DNA of rat myoblast cells and myotubes, of the L6 line. Small quantities of 125I‐labelled ssDNA were annealed with a large excess of unlabelled DNA, cytoplasmic RNA and mRNA from myoblasts or myotubes. The results indicated that ssDNA belongs to the non‐repetitious portion of the cell genome and is formed of two distinct molecular fractions. The major ssDNA fractions (75%) consist of non‐self‐reassociating DNA sequences and the minor fraction (25%) consists of self‐reassociating DNA sequences. About 30–32% and 25–26%, of ssDNA from myoblast represent DNA sequences complementary to total cytplasmic RNAs and polyadenylated RNAs respectively. Hybridizations of ssDNA with an excess of RNA from myoblasts and/or myotubes show differences in the abundance and the diversity of mRNA during muscular differentiation. These differences were confirmed by DNA‐driven reactions between 125I‐labelled polyadenylated RNA and ssDNA in great excess.
Purification and Properties of Long‐Chain Acyl‐Coenzyme‐A Synthetase from Rat Liver
Authors: TANAKA Takao , HOSAKA Kohei , HOSHIMARU Minoru , NUMA Shosaku
Published: March 2005

Long‐chain acyl‐coenzyme‐A synthetase from the microsomes as well as from the mitochondrial fraction of rat liver has been purified to homogeneity as evidenced by dodecylsulfate/polyacrylamide gel electrophoresis, amino‐terminal analysis and the elution profile at the final chromatography step. The purification procedure involves resolution of the cellular particles with Triton X‐100 and chromatography on Blue‐Sepharose, hydroxyapatite and phosphocellulose. The purified enzymes from both sources have a specific activity of 26–29 units/mg protein at 35°C, which is more than 100‐fold higher than those of long‐chain acyl‐CoA synthetases of animal and bacterial origin hitherto reported. The purified enzymes exhibit a molecular weight of approximately 76000 as estimated by dodecylsulfate/polyacrylamide gel electrophoresis and catalyze the activation of saturated fatty acids with 10–18 carbon atoms and unsaturated fatty acids with 16–20 carbon atoms most efficiently. The purified enzyme from the microsomes and that from the mitochondrial fraction, which are obtained by essentially identical procedures, are indistinguishable from each other with respect to all molecular and catalytic properties examined, including molecular weight, amino acid composition, amino‐terminal residue, heat stability, specific activity, pH optimum and substrate specificity regarding fatty acid, acyl acceptor and nucleoside 5′‐triphosphate.
The Effect of Urea on the Structure of Nuclear Ribonucleoprotein Particles from Rat Liver
Authors: NORTHEMANN Wolfgang , KLUMP Horst , HEINRICH Peter C.
Published: March 2005

Ribonucleoprotein particles were isolated in the presence of cytosolic RNase inhibitor from rat liver nuclei and exposed to increasing concentrations of urea. The average sedimentation coefficient of 90 S in a sucrose gradient decreased slightly with urea concentrations of up to 2 M. At 5 M urea the particles sedimented at about 20 S. The protein‐to‐RNA ratio decreased from 4 to 1.4 after treatment with 5 M urea. The proteins which are preferentially detached from the ribonucleoprotein complex are in the molecular weight range of 30000–45000. In addition, small nuclear RNAs (snRNA) were dissociated from the ribonucleoprotein particles by high urea concentrations (> 2M). From the fact that snRNAs are dissociated from the ribonucleoprotein complex by high urea concentrations and not dissociated by high NaCl concentrations even after extensive proteolytic digestion, it is concluded that hydrogen bonds are involved in their binding to heterogeneous nuclear RNA (hnRNA).

The change in the RNA conformation after addition of increasing concentrations of urea was studied by circular dichroism measurements of ribonucleoprotein particles and protein‐free particle RNA. In the presence of 5 M urea the molar ellipticities at 264 nm decreased from 15800 to 9400, 21000 to 19700, 19500 to 16600 and from 20900 to 16800 deg × cm2× dmol−1 in the case of ribonucleoprotein particles, protein‐free particle RNA, hnRNA and snRNA respectively. From the results of the circular dichroism measurements it is concluded that the high‐molecular‐weight proteins, which are not dissociated from the particles, are responsible for a conformational restraint on hnRNA.

snRNA, hnRNA, as well as the physiological mixture of both, exhibit the same gradual increase in absorbance with temperature. In the presence of 0.1 M NaCl melting temperatures of about 43°C were obtained in all three cases. The gradual nature of the thermal transition is due to the shortness and heterogeneity of the helical regions. The average helix length is about 5 base pairs long.
Use of Protein‐Mediated Lipid Exchange in the Study of Membrane‐Bound Enzymes
Authors: DYATLOVITSKAYA Ella V. , LEMENOVSKAYA Alla F. , BERGELSON Lev D.
Published: March 2005

The ability of liver lipid‐exchange proteins to introduce foreign phospholipids into microsomes was used in a study of the lipid dependence of glucose‐6‐phosphatase. Supplementation of intact rat liver and hepatoma microsomes with exogeneous aminophospholipids prevents the decline of glucose‐6‐phosphatase activity during incubation, whereas the introduction of exogeneous phosphatidylcholine has no protective effect. On the contrary with deoxycholate‐disrupted hepatoma microsomes, introduction of additional phosphatidylcholine causes activation while phosphatidyl‐ethanolamine has only little effect. The results are explained by assuming that the transport unit and the catalytic moiety of the glucose‐6‐phosphatase system have different lipid requirements, the activity of the former protein depending mainly on phosphatidylethanolamine and phosphatidylserine and that of the catalytic protein depending on phosphatidylcholine.

In deoxycholate‐disrupted liver microsomes (in which both the glucose‐6‐phosphatase activity and the phosphatidylcholine content are much higher than in hepatoma microsomes) incubation with phosphatidylcholine and lipid‐exchange proteins alters neither the phospholipid composition nor the enzyme activity. This suggests that the diminished activity of glucose‐6‐phosphatase in hepatomas may be partly due to a low level of phosphatidylcholine.
Fluorimetric Study of the Complex between Yeast Phenylalanyl‐tRNA Synthetase and tRNAPhe
Authors: EHRLICH Ricardo , LEFEVRE Jean‐François , REMY Pierre
Published: March 2005

The interaction between yeast tRNAPhe and phenylalanyl‐tRNA synthetase was studied by monitoring different emission properties of the wybutine residue. The following results were established: (a) the isolated tRNAPhe exists in solution under at least two forms in a magnesium‐dependent equilibrium; (b) in the complex with the cognate synthetase, the tRNA still has two different conformations, slightly different from the above conformers. Mg2+ ions appear to play a crucial role in the adaptation of both macromolecules one to another: for concentrations of the order of 1 mM, magnesium ions trigger a conformational change of the anticodon loop of tRNA, resulting in the expulsion of the wybutine from a stacked region. Furthermore, experimental evidence suggests that the anticodon region lies in a groove of the protein.

This local conformation change, occurring in the anticodon loop, can be correlated to structural modifications of the enzyme as shown by the modification of circular dichroism and tryptophan fluorescence.

During these different conformation changes, an energy transfer from tryptophan residues to wybutine is triggered in the critical magnesium concentration range.
Synthesis and Properties of N‐Acetyl‐N′‐(p‐glyoxylylbenzoyl)cystamine, a New Reagent for RNA‐RNA and RNA‐Protein Cross‐Linking
Authors: EXPERT‐BEZANÇON Alain , HAYES Donal
Published: March 2005

The synthesis of N‐acetyl‐N′‐(p‐glyoxylylbenzoy)cystamine (Gbz‐Cyn2‐Ac) is described. Like other glyoxal‐type reagents it reacts with guanine and arginine. The kinetics and pH dependence of these reactions are studied. (Gbz‐Cyn2‐Ac) reacts with non‐base‐paired guanines at about 20 sites in 16‐S rRNA. After reduction of disulfide bonds each derivatized guanine residue carries a free –SH group which can be used to create RNA‐RNA bridges or, after introducing an additional photoactivatable derivative, RNA‐protein bridges.
Isolation and Characterization of Membrane‐Bound Arylamidases from Human Placenta and Kidney
Authors: HIWADA Kunio , ITO Taketoshi , YOKOYAMA Masako , KOKUBU Tatsuo
Published: March 2005

Membrane‐bound arylamidases (EC 3.4.11.2) from human placenta and kidney were purified. The enzymes were solubilized from membrane fractions using trypsin. Purification procedures included DEAE‐cellulose column chromatography, concanavalin‐A–Sepharose 4B column chromatography, hydroxyapatite column chromatography and Sephadex G‐200 gel filtration. The final recoveries of placental and kidney enzymes were 22.0% and 20.8%, respectively.

Polyacrylamide gel electrophoresis, analytical isoelectric focusing, immunoelectrophoresis and ultracentrifugation indicated the homogeneity of both purified enzymes. Equilibrium ultra‐centrifugation showed molecular weights of 193000 for placental enzyme and 211000 for kidney enzyme. Electrophoresis of the enzymes under denaturing conditions on dodecylsulfate/polyacrylamide gel indicated that each enzyme was a dimer consisting of two identical subunits. Isoelectric points of placental and kidney enzymes were 4.20 and 4.32 at 4°C, respectively. The amino acid compositions of the two membrane‐bound arylamidases were similar. The carbohydrate accounted for 18.2% of placental enzyme and 17.4% of kidney enzyme.

The purified placental enzyme had a specific activity of 60.1 μmol min−1 (mg protein)−1 and the kidney enzyme had a specific activity of 96.4 μmol min−1 (mg protein)−1. Kcat values of placental and kidney enzymes were 187.8 s−1 and 328.6 s−1, respectively. The hydrolytic coefficient (Kcat/Km) of placental enzyme for l‐alanyl‐β‐naphthylamide was 2159 mM−1 s−1 and that of kidney enzyme was 3778 mM−1 s−1.

Rabbit antibodies against placental and kidney membrane‐bound arylamidases inhibited the activities of the corresponding antigens, but the inhibitions were never complete. Three membrane‐bound arylamidases from placenta, kidney and renal cell carcinoma were immunochemically indistinguishable.
Isomeric Incorporation of the Haem into Monomeric Haemoglobins of Chironomus thummi thummi
Authors: RIBBING Wilfried , RÜTERJANS Heinrich
Published: March 2005

With high‐resolution NMR spectroscopy it has been possible to detect various types of isomerisms in the monomeric component III of the haemoglobins of Chironomus thummi.

In the component III prepared from commercially available larvae of Ch. thummi a chemical heterogeneity has been found to occur at the position E6 (Ile or Thr). The imidazole C‐2 proton resonance of the adjacent His‐E7 is split because of this alternative occupancy.

From larvae of an inbreeding of the single subspecies Chironomus thummi thummi a component III material has been isolated in which the position E6 is occupied by one single amino acid. An additional isomerism of this haemoglobin III is produced by an isomeric incorporation of the haem group into the haem pocket. This isomerism has been established from the complex signal pattern of the mesoproton resonances of the diamagnetic ligated haemoglobin III. Other evidences of this isomerism have been obtained from an analysis of the high‐field proton resonances.
Turnover of the Poly(A) Moiety of mRNA in Wheat‐Germ Extract
Authors: ABRAHAM Abraham K. , PIHL Alexander , JACOB Samson T.
Published: March 2005

The synthesis and degradation of poly(A) were examined in wheat germ lysates under conditions used for protein synthesis. The lysate contained both poly(A)‐polymerizing and poly(A)‐hydrolytic activities. The synthetic activity was dependent on the presence of either poly(A) or polyadenylated mRNA as primer. Concurrent with the synthesis of poly(A), radioactivity was released from labeled poly(A) and from the poly(A) region of mRNA. Both poly(A) synthesis and hydrolysis were independent of Mn2+ and could proceed in the presence of Mg2+, the major divalent metal ion required for protein synthesis.

The synthetic activity was favoured at high (1 mM) ATP concentration, whereas the hydrolytic activity was maximal in the absence of ATP or at low (0.1 mM) ATP concentration. These data indicate that the steady‐state length of the poly(A) moiety of mRNAs in the cytoplasm may be regulated by the levels of ATP. Translation products of polyadenylated mRNAs isolated from myeloma cells before and after partial deadenylation were separated by dodecylsulfate/polyacrylamide gel electrophoresis. Shortening of the poly(A) moiety did not alter the relative amounts of the peptides synthesized, indicating that this process does not involve a preferential breakdown of certain species of mRNA.
Modulation of the Nucleosome Structure by Histone Acetylation
Authors: BODE Jürgen , HENCO Karsten , WINGENDER Edgar
Published: March 2005

A rapid procedure for the isolation of core particles from Chinese hamster ovary cells is described which permits measurements, usually at the day of their preparation. Particles of 145 ± 5 base pairs, derived from interphase cells, will be compared with the analogue specimens from butyrate‐treated cells, metaphase cells and a standard preparation from chicken erythrocytes. Butyrate cause an increase in the acetylation of histones H3 and H4, which induces alterations of the interhistone and histone‐DNA interactions. Changes in the interhistone contacts, correlated to an extension of a‐helical segments, lead to an altered accessibility of the H3 cysteine side‐chains and to a different histone displacement by protamines. On the other hand, histone‐DNA contacts are loosened in parts and this is particularly evident from the changes in the premelting region of a thermal‐denaturation profile.
Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii
Authors: VOORDOUW Gerrit , VAN DER VIES Saskia M. , EWEG Jan Kees , VEEGFR Cees , VAN BREEMEN Jan F. L. , VAN BRUGGEN Ernst F. J.
Published: March 2005

Pyridine nucleotide transhydrogenase from Azotobacter vinelandii was purified with a scaled‐up procedure. In a typical purification 500 ml cell‐free extract from 200 g cells is loaded on an Ado‐2′, 5′‐P2‐Sepharose 4B affinity column (20 ml bed volume). After washing, the enzyme is desorbed with 2′AMP at neutral pH and further purified by Sephadex G‐200 gel chromatography. The enzyme (10–12 mg) is obtained in 40–60% yield and is homogeneous its judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.

The homogeneity of the purified enzyme is also apparent from electron microscopy studies, where the enzyme appears as a polydisperse set of polymers without contaminating, structures and from fluorescence lifetime studies by the method of single‐photon counting. The flavin fluorescence appears to decay with a single lifetime τ= 2.5 ns. The polymeric nature of transhydrogenase can be aptly demonstrated by density gradient centrifugation in the presence of KBr. After centrifuging for 50 h at 160000 × g and 10° C the enzyme is concentrated in a narrow fluorescent band with buoyant density Qb= 1.305 g cm−3.

The arrangement of subunits in the transhydrogenase polymer has been derived from optical diffraction studies of electron micrographs. The polymers are built tip from a linear assembly of tetramers. Four subunits are placed in a rhomb with sides of 13.5 mm and an angle of 45° (135°) between the sides. A second tetramer is located staggered on top of the first one. Since a variety of other studies have indicated that the polymers dissociate into octamers under alkaline condition [Voordouw, G. et al. (1979) Eur. J. Biochem. 98, 447–454] we conclude that this smallest functional unit is built up from two tetramers.
Fluorescence Polarization and Energy‐Transfer Studies on the Pyruvate Dehydrogenase Complex of Escherichia coli
Authors: SCOUTEN William H. , VISSER Antonie J. W. G. , GRANDE Hans J. , DE KOK Arie , DE GRAAF‐HESS Adriana C. , VEEGER Cees
Published: March 2005

We have attached eosin maleimide specifically to the lipoyl group of the pyruvate dehydrcgenase complex isolated from Escherichia coli. Using this as the fluorescence acceptor and the intrinsic FAD of the lipoamide dehydrogenase subunit as the fluorescence donor, we confirmed previous measurements with other probes, in which it was suggested that the flavin moiety is at a substantial distance (over 4.5 nm) from the labeled lipoyl group. Since the lipoyl group must supply electrons to the FAD during the catalytic decarboxylation of pyruvate, we have investigated several potential mechanisms whereby this could happen. Movement within the camplex, possibly triggered by the presence of substrate, seemed to be a strong possibility. Complex labeled with fluorophores on the accessible sulfhydryls, or on the lipoyl functions, did not give evidence of such triggering upon addition of substrate as judged by both static and dynamic fluorescence depolarization.

The mobility of the subunits of labeled lipoamide dehydrogenase exceeded that expected for the total complex. Pyrene maleimide bound to the lipoyl functions also exhibited considerably faster rotations than the predicted one of the whole complex (τc > 3 μs). This suggests that a constant movement within the complex, coupled with the rotation of the lipoyl group, may bring the active sites of the complex transiently close enough together to interact on a time scale much faster than enzyme turnover. At the same time, the lipoyl group and the active sites of the complex can spend most of their time at points which are rather distant from each other.
Purification and Chemical Characterization of the Vitamin‐B12‐Dependent 5‐Methyltetrahydrofolate: Homocysteine Methyltransferase from Escherichia coli B
Authors: PAESSENS Arnold , RÜDIGER Harold
Published: March 2005

The transferase was isolated by means of hydrophobic chromatography and combination of ionexchange and gel filtration at different pH values and ionic strengths. As judged by disc electrophoresis, the enzyme is homogeneous. Electrophoresis in the presence of sodium dodecylsulfate reveals only one band with Mr= 49500 ± 10%. In gel filtration the native enzyme has a Mr of 200000. The subunits can be crosslinked by iminothiolane followed by hydrogen peroxide oxidation. In sodium dodecylsulfate electrophoresis this results in a band pattern of integer multiples of 50000 up to 200000 but not higher. The high‐Mr aggregates disappear on splitting the crosslinks by reduction. Thus the enzyme appears to be composed of four subunits identical or nearly identical in Mr.

By the dansyl method, only phenylalanine and methionine were found as the amino‐terminal residues, suggesting the existence of two different types of subunits.