bluuebunny/crossref_metadata_2025 huggingface dataset:
RNA Polymerase from the Fungus Aspergillus nidulans
Authors: STUNNENBERG Hendrik G. , WENNEKES Lambertus M. J. , VAN DEN BROEK Hendrikus W. J.
Published: March 2005

The DNA‐dependent RNA polymerase I (or A) from the lower eukaryote Aspergillus nidulans has been purified on a large scale to apparent homogeneity by homogenizing the fungal hyphae in liquid nitrogen, extraction of the enzyme at high salt concentration, precipitation of RNA polymerase activity with polymin P (a polyethylene imine), elution of the RNA polymerase from the polymin P precipitate, ammonium sulphate precipitation, molecular sieving on Bio‐Gel A‐1.5m, binding to ion‐exchangers and DNA‐cellulose affinity chromatography. By this procedure 1.6 mg of RNA polymerase I can be purified over 2000‐fold from 500 g wet weight of starting material with a yield of 30–35%. The isolated RNA polymerase I is stable for several months at −20°C.

The subunit composition has been resolved by polyacrylamide gel electrophoresis on two‐dimensional gels, using either non‐denaturing or 8 M urea (pH 8.7) cylindrical gels in the first dimension and sodium dodecyl sulphate slab gels in the second dimension. The putative subunits have molecular weights of 190000, 135000, 63000, 62000, 43000, 29000, 29000, (28000), 16000 and probably 13000 and 12000.

Two distinct forms of RNA polymerase I (Ia and Ib) have been resolved by DEAE‐Sephadex A‐25 chromatography showing ample differences in enzymatic properties and subunit pattern.

Additional information is given on RNA polymerase II (or B) which appears to be highly insensitive to α‐amanitin at concentrations up to 400 μg/ml.
Correlation between the Induction of Mouse Interferon and the Amount of Its mRNA
Authors: RAJ N. Babu Kishan , FERNIE Bruce F. , PITHA Paula M.
Published: March 2005

Injection into Xenopus oocytes of polyadenylated cytoplasmic RNA from mouse C‐243‐3 cells induced with poly(rI) · poly(rC) and DEAE‐dextran resulted in the production of biologically active interferon. The synthesis of interferon by oocytes is linearly related to the amount of mRNA injected. Thus a titre of 160 reference units/oocyte was obtained by injecting 250 ng of total cytoplasmic poly(A)‐containing RNA.

The kinetics of interferon production in mouse C‐243‐3 cells induced with poly(rI) · poly(rC) and DEAE‐dextran are characterized by an initial lag of 3–4 h and a burst of interferon secretion between 5–8 h with a maximum accumulation at 7 h. Comparison of the relative amount of interferon mRNA at various time intervals of induction with the cellular synthesis of interferon showed a general correlation. At 9 h after induction, i.e. during the shut off phase of interferon production, the cells contained no detectable amount of interferon mRNA (< 20 units/ml).

Attempts to superinduce interferon production in mouse C‐243‐3 cells by using the cycloheximide/actinomycin D protocol as in human fibroblast cells were not successful. α‐Amanitin at concentrations similar to those required to inhibit cellular DNA‐dependent RNA polymerase form II completely inhibited the interferon production when applied during the first 3 h of induction. Addition of α‐amanitin at 3, 5 or 7 h after induction stimulated the subsequent interferon production by 2–4‐fold.
Inhibition of Protein Synthesis and Activation of Nuclease in Rabbit Reticulocyte Lysates by the Unusual Oligonucleotide pppA2′pSA2′p5′A
Authors: VAQUERO Catherine M. , CLEMENS Michael J.
Published: March 2005

The oligonucleotide 5′‐triphosphoadenylyl(2′‐5′)adenylyl(2′‐5′)adenosine (pppA2′p5′A2′p5′A) is synthesised by reticulocyte lysates and extracts from interferon‐treated cells in the presence of double‐stranded RNA. It inhibits the translation of both viral and non‐viral mRNAs and of poly‐(rU,rC) in the micrococcal‐nuclease‐treated reticulocyte lysate system. The translation of poly(rU) is insensitive in this assay, however. Inhibition develops in vitro after a lag period, the length of which depends on the nature of the mRNA present and its concentration.

The degradation of radioactive Mengo virus RNA or L cell cytoplasmic RNA is accelerated in reticulocyte lysates incubated with the oligonucleotide. This is due to activation of one or more endonucleases which degrade(s) RNA to large oligonucleotide fragments. Our results suggest that the endonuclease is responsible for most (if not all) of the inhibitory effects of the oligonucleotide on protein synthesis in the reticulocyte lysate.

In the presence of pppA2′p5′A2′p5′A, protein synthesis can be reactivated after it has ceased by addition of fresh mRNA. In contrast, initiation factor eIF‐2 is unable to prevent inhibition by the oligonucleotide under conditions where it protects the system from the effects of the haem‐controlled repressor and partially protects against inhibition by double‐stranded RNA.

These findings are discussed in relation to the regulation of protein synthesis by double‐stranded RNA and by interferon.
Distribution of Polypyrimidine · Polypurine Segments in DNA from Diverse Organisms
Authors: BIRNBOIM Hyman C. , SEDEROFF Ronald R. , PATERSON Malcolm C.
Published: March 2005

Polypyrimidine · polypurine segments are regions of duplex DNA which contain a highly asymmetric distribution of pyrimidine and purine nucleotides. A polypyrimidine in single‐stranded DNA can be detected by its ability to form a complex with poly(A, G) which will bind to hydroxy‐apatite. We tested DNA from a variety of organisms and found that most contained poly‐pyrimidines. From the shape of the curve relating DNA size to percentage bound to hydroxyapatite, we conclude that polypyrimidine · polypurine segments occur widely in DNA from higher organisms, at intervals of 6000–8000 base pairs throughout the majority of the genome. Lower levels occur in DNA from yeast and Drosophila.
Characterization of a Thermosensitive Sporulation Mutant of Bacillus subtilis Affected in the Structural Gene of an Intracellular Protease
Authors: KERJAN Pierre , KERYER Eliane , SZULMAJSTER Jekisiel
Published: March 2005

A thermosensitive sporulation mutant (ts‐15) of Bacillus subtilis has been isolated. This mutant when grown at the restrictive temperature (42°C) is unable to sporulate, shows no intracellular protease activity and no protein turnover. These three traits were recovered in two revertants (ts‐15R1 and ts‐15R2) and were also transmitted together by transformation into the wild type.

Immunological studies have shown that when ts‐15 is grown at 42°C it synthesizes a ‘cryptic’ protein with apparently the same antigenic properties as the wild type or as ts‐15 mutant grown at the permissive temperature (30°C). The intracellular proteases from the wild type and from ts‐15 grown at 30°C and 42°C were completely purified and their properties were studied with respect to their molecular weights, substrate specificity, inhibition pattern, heat inactivation and antigenicity. The molecular weight of the enzyme from the wild type or ts‐15 grown at 30°C was 64000–65000 in the absence of sodium dodecylsulfate and 31000–32000 in the presence of sodium dodecylsulfate. It was assumed therefore that the active enzyme is formed from two similar subunits. However, the intracellular protease from ts‐15 grown at 42°C showed the same molecular weight of 32000–34000 in the presence or in the absence of sodium dodecylsulfate. On the basis of this experiment and others described in the paper we concluded that the mutation in ts‐15 is most likely a point mutation in a structural gene of an intracellular protease and results in an inability to assemble the two subunits into an active form.
Studies on a Proteinase B Mutant of Yeast
Authors: WOLF Dieter H. , EHMANN Claudia
Published: March 2005

Yeast mutants lacking proteinase B activity have been isolated [Wolf, D. H. and Ehmann, C. (1978) FEBS Lett. 92, 121–124]. One of these mutants (HP232) is characterized in detail. Absence of the vacuolar localized enzyme is confirmed by checking for proteinase B activity in isolated mutant vacuoles. Defective proteinase B activity segregates 2:2 in meiotic tetrads. The mutation is shown to be recessive. Mutant proteinase B activity is not only absent against the synthetic substrate, Azocoll, but also against the physiological substrates pre‐chitin synthetase, cytoplasmic malate dehydrogenase and fructose‐1,6‐bisphosphatase. The mutant shows normal vegetative growth, a phenomenon not consistent with the idea that proteinase B might be the activating principle of chitin synthetase zymogen in vivo. Fluorescence microscopy shows normal chitin insertion. Enzymes underlying carbon‐catabolite inactivation in wild‐type cells (a mechanism proposed to be possibly triggered by proteinase B) such as cytoplasmic malate dehydrogenase, fructose‐1,6‐bisphosphatase, phosphoenolpyruvate carboxykinase and isocitrate lyase, are inactivated also in the mutant. NADP‐dependent glutamate dehydrogenase, which is found to be inactivated in glucose‐starved wild‐type cells, proceeds normally in the mutant. Mutant cells show more than 40% reduced protein degradation under starvation conditions. Sporulating diploids, homozygous for proteinase B absence, also exhibit an approximately 40% reduced protein degradation as compared to homozygous wild‐type diploids or diploids heterozygous for the mutant gene.

The time of the appearance of the first ascospores of diploid cells, homozygous for proteinase B deficiency, is delayed about 50% and sporulation frequency is reduced to about the same extent as compared to homozygous wild‐type diploids or diploids heterozygous for the mutant gene.
Purification and Some Characteristics of the Human Coagulation Factor VII
Authors: FLENGSRUD Ragnar
Published: March 2005

1. A purification procedure for factor VII (proconvertin) from human plasma is described. The procedure involves barium sulphate adsorption and elution, DEAE‐Sephadex column chromatography, barium sulphate adsorption and elution, heparin‐Sepharose column chromatography, preparative disc gel electrophoresis and finally adsorption with antiserum to prothrombin coupled to Sepharose and antiserum to albumin coupled to Sepharose. This procedure gave an approximately 8 · 105‐fold purification.

2. The factor VII obtained from the electrophoresis step was mainly a single‐chain protein with an apparent molecular weight of 53000 ± 2000.

3. After the final purification step, additional forms of factor VII, resulting from a fragmentation of the factor VII molecule were detected.

4. Amino acid composition data of the purified factor VII are given.

5. Antisera were raised in two different rabbits by injection of the purified factor VII. The antisera obtained gave a good titre against the factor VII activity and were not directed against any of the three other vitamin‐K‐dependent coagulation factors.
Site‐Specific Substituted Cobalt(II) Horse Liver Alcohol Dehydrogenases
Authors: MARET Wolfgang , ANDERSON Inger , DIETRICH Helmut , SCHNEIDER‐BERNLÖHR Helga , EINARSSON Roland , ZEPPEZAUER Michael
Published: March 2005

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1.I.1.1) has been studied with dissolved, crystalline and agaroseimmobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives.

In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier.

Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 μM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure‐stabilizing rôle of the catalytic metal ions.

The enzyme immobilised on agarose, with unchanged content of active sites [Schneider‐Bernlöhr et al. (1978) Eur. J. Biochem. 41, 475–484], was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogeneous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.
Correlation between Rates and Enzyme Levels of Increased Gluconeogenesis in Rat Liver and Kidney after Partial Hepatectomy
Authors: KATZ Norbert
Published: March 2005

The metabolic rates and key enzymes of gluconeogenesis and glycolysis were determined and correlated in perfused livers, isolated hepatocyte suspensions and kidney cortex slices of rats after two‐thirds hepatectomy. Fed unoperated, sham operated and fasted animals served as controls.

In liver the following results were obtained. Firstly, after partial hepatectomy [14C]glucose formation from [14C]lactate was increased in perfused livers to 223% after 24 h and to 173% after 48 h and in hepatocyte suspensions to 222% after 48 h. [14C]Glucose production from [14C]fructose, however, was unchanged. These metabolic rates correlated well with an increase of the level of phosphoenolpyruvate carboxykinase to 206% after 24 h and to 184% after 48 h. The levels of fructose‐1,6‐bisphosphatase and glucose‐6‐phosphatase were unaltered. Secondly, after a 24‐h fast gluconeogenesis from lactate was enhanced to 282% in perfused livers and to 300% in hepatocyte suspensions. Gluconeogenesis from fructose was slightly increased to 140%. These altered rates were accompanied by an increase of phosphoenolpyruvate carboxykinase to 213% and of glucose‐6‐phosphatase to 190%. Thirdly, after partial hepatectomy [14C]lactate formation from [14C]glucose was decreased in hepatocyte suspensions to less than 20% after 48 h. [14C]Lactate production from [14C]fructose was unchanged. These rates coincided with a decrease of the level of glucokinase to 40% and with unaltered levels of phosphofructokinase and pyruvate kinase. Fourthly, after a 24‐h fast glycolysis from glucose was lowered to below 30%. This rate was paralleled by a decrease of the level of glucokinase to 60%.

In kidney cortex the following results were obtained. Firstly, after partial hepatectomy gluconeogenesis from lactate was enhanced in tissue slices to 217% after 24 h. Gluconeogenesis from fructose was unchanged. These rates coincided with an increase of the level of phosphoenolpyruvate carboxykinase to 193% and of glucose‐6‐phosphatase to 151%. Secondly, after a 24‐h fast similar changes for the gluconeogenic rate to 200% and of phosphoenolpyruvate carboxykinase to 240% as well as of glucose‐6‐phosphatase to 136% were observed. Thirdly, neither the rates of glycolysis nor the levels of hexokinase, phosphofructokinase and pyruvate kinase were altered after partial hepatectomy or a 24‐h fast.

No significant changes neither in liver nor in kidney cortex occurred after sham operation. Thus the drastic reduction of liver tissue after partial hepatectomy was compensated by an increase of gluconeogenesis in the liver remnant and in kidney cortex, both accompanied by an enhancement of the phosphoenolpyruvate carboxykinase level, in order to maintain the glucose homeostasis.
Enrichment and Characterization of the DNA Coding for Vitellogenin in Xenopus laevis
Authors: WIDMER Heinrich J. , JAGGI Rolf B. , WEBER Rudolf , RYFFEL Gerhart U. , MUELLENER Daniel
Published: March 2005

Purified vitellogenin mRNA of Xenopus laevis was incubated with mechanically sheared DNA in high concentrations of formamide and the resulting R‐loops (i.e. RNA · DNA hybrid fragments) separated from the bulk DNA by caesium chloride buoyant density centrifugation. Hybridization with 125I‐labeled vitellogenin mRNA revealed a 15–30‐fold enrichment of the DNA coding for vitellogenin.

Restriction analysis of the R‐loop‐enriched DNA demonstrated that all known endonuclease HindIII fragments coding for vitellogenin of unfractionated Xenopus DNA were also present in the enriched material, including the specific fragments for the oligo(A)‐containing segment of the RNA.

Comparison of these restriction data with the structure found in cloned vitellogenin cDNA, indicates the presence of at least one intervening sequence in the genomic DNA coding for vitellogenin.
A DNA‐Dependent ATPase of Calf‐Thymus
Authors: ASSAIRI Liliane M. , JOHNSTON Irving R.
Published: March 2005

A DNA‐dependent ATPase has been purified from calf thymus. The enzyme hydrolyses ATP and dATP in the presence of heat‐denatured DNA. It does not hydrolyse the corresponding nucleoside triphosphates of guanine, uridine and cytosine. The Km values for ATP and dATP are both 0.62 mM. The enzyme requires magnesium or manganese ions. Its sedimentation coefficient is about 4.4 S. The catalytic activity is inhibited by N‐ethylmaleimide but is not sensitive to novobiocin and nalidixic acid which are potent inhibitors of bacterial DNA gyrase. In some cases, during purification, chromatographically distinct additional DNA‐dependent ATPase activities were detected. Limited proteolysis or covalent modification of the enzyme in the tissues, or during the first steps of its extraction, are probably responsible for the appearance of these chromatographically distinct forms.
An Electrophoretic Study of Native Myosin Isozymes and of Their Subunit Content
Authors: d'ALBIS Anne , PANTALONI Colette , BECHET Jean‐Jaques
Published: March 2005

Myosin polymorphism in muscles has been studied by a variety of electrophoretic techniques, in non‐dissociating and in dissociating conditions. The analysis of myosin isozymes in the native state was achieved in pyrophosphate buffer and required only minute amounts of protein; identical results were obtained with purified or crudely extracted myosin. The determination of the subunit content of each isozyme was done in the presence of sodium dodecyl sulphate or urea for light chain, and in a phenol, acetic acid and urea system for heavy chain screening.

Electrophoresis in non‐dissociating conditions has led to the separation of up to a dozen of myosin isozymes, differing in mobilities by as much as 30%. Muscle specificity of myosin was clearly established. Apart from a few exceptions, all the muscles tested were shown to contain more than one myosin species; fast‐twitch muscles for instance all contained the same three isozymes, but in variable ratios. Class specificity of myosin appeared related to the relative proportions of isozymes in a given muscle.

A second electrophoresis in dissociating solvents of the myosin bands first resolved in pyrophosphate buffer has then allowed a further characterization of the various isozymes. The differences in mobilities observed in the native state were shown to come either from the light chains, or from the heavy chains, or from both. The first case was illustrated by the three species present in fast muscles, which were shown to correspond to three alkali light‐chain isozymes, the heterodimer representing in some instances up to 40% of the total. Next to light‐chain muscle type specificity, electrophoresis in the phenol, acetic acid, urea system has led to the detection of differences in the heavy chains of fast, slow and cardiac myosins.

The application of these various electrophoretic techniques to the analysis of the modification of myosin isozymes during development or in pathology studies can be considered.
Studies on the Structure of the Rabbit Kidney Brush Border
Authors: KRAMERS Mary T. C. , ROBINSON Garth B.
Published: March 2005

The effects of salts and non‐ionic detergents on renal brush borders have been studied. 2 M sodium chloride, iodide or thiocyanate dissociated up to 40% of the protein from the brush borders, destroying the core filaments and resulting in the formation of membrane vesicles; EDTA had a similar effect on structure but released little protein. Triton X‐100 and Nonidet P‐40 extracted up to 60% of the protein including the major membrane glycoproteins and the enzymes trehalase, maltase and aminopeptidase (microsomal). Triton exhibited a selective effect on lipids removing phosphatidylserine, phosphatidylethanolamine and sphingomyelin but not the bulk of the phosphatidylcholine or cholesterol. The residual structures after Triton extraction comprised the core filaments associated with vesicles of lipid containing alkaline phosphatase and several other proteins. Treatment of these core‐vesicle complexes with 2 M sodium chloride dissociated the filaments, releasing the vesicles which could be recovered as a pellicle on centrifugation. It is suggested that the proteins found in the vesicles might serve to interconnect the core filaments with the lipid bilayer.
The Calcium‐Dependent Phosphatidylinositol‐Phosphodiesterase of Rat Brain
Authors: IRVINE Robin F. , HEMINGTON Norma , DAWSON Rex M. C.
Published: March 2005

1. The activity of the soluble, calcium‐dependent phosphatidylinositol‐specific phosphodiesterase (EC 3.1.4.10) against [32P] phosphatidylinositol has been investigated.

2. KCl (only at neutral pH), Mg2+, positively‐charged proteins such as histone, and phospholipids containing a choline headgroup are all inhibitory to the enzyme. Choline‐phospholipids cause a 90% inhibition at an equimolar ratio to phosphatidylinositol.

3. Other phospholipids (phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine and phosphatidic acid) are all potent stimulators of the enzyme: maximum stimulation being observed at a ratio of 1 mol activator/5–10 mol phosphatidylinositol.

4. Unsaturated amphiphiles such as oleic and oleoyl alcohol also stimulate the activity, maximum stimulation being observed at about an equimolar ratio to phosphatidylinositol. Saturated amphiphiles (such as stearic acid and stearoyl alcohol) are less effective.

5. The activation by acidic phospholipids and unsaturated amphiphiles appear to be independent as they are additive and, under certain conditions, synergistic.

6. Both types of stimulator (independently or together) can reverse the inhibition caused by histone or phosphatidylcholine.

7. Possible mechanisms of the suppression of the phosphatidylinositol phosphodiesterase in vivo, of its activation, and of the amplification of phosphatidylinositol breakdown are discussed.
Levansucrase of Bacillus subtilis
Authors: PETIT‐GLATRON Marie‐Françoise , CHAMBERT Régis , STEINMETZ Michel
Published: March 2005

A macromolecular complex including an inactive and protease‐sensitive form of levansucrase was isolated from phenol treatment of Bacillus subtilis. This complex has a weak affinity for hydroxyapatite. Its molecular weight is evaluated at 300000. This levansucrase form may be activated either by Triton X‐100, above its critical micellar concentration, or by phospholipase C. The enzyme form associated with the complex is quite different from the exocellular levansucrase, which is protease‐insensitive and detergent‐insensitive, shows high affinity for hydroxyapatite and has a molecular weight of 54000. Triton X‐100 disaggregates this complex. The active levansucrase released becomes protease‐insensitive and has the same apparent sedimentation coefficient as the exocellular enzyme. Possible relationships between the existence of this trapped complex and the secretion process of levansucrase are discussed.
Studies on the Mechanism of Transcription of Nucleosomal Complexes
Authors: WASYLYK Bohdan , CHAMBON Pierre
Published: March 2005

The mechanism of transcription by Escherichia coli RNA polymerase holoenzyme of chromatin assembled in vitro has been studied by two approaches. Using digestion with endodeoxyribonuclease EcoRI as a probe of mobility, it was found that nucleosome movement is slow compared to the time taken for RNA polymerase to transcribe through regions organised into nucleosomes. However, transcription leads to at least some displacement of nucleosomes relative to their original site on the DNA. In the second approach chromatin was reconstituted from extensively crosslinked histone octamers and simian virus 40 DNA. RNA chain elongation on this template is inhibited relative to non‐crosslinked chromatin. This can be related to a decrease in the ability of the cross‐linked histone octamers to dissociate from the DNA.
Calcium‐Sensitivity of Pig‐Carotid‐Actomyosin ATPase in Relation to Phosphorylation of the Regulatory Light Chain
Authors: MRWA Ulrike , TROSCHKA Monika , GROSS Christiane , KATZINSKI Lothar
Published: March 2005

Ca2+‐dependent phosphorylation of the 20000‐Mr regulatory light chain was found to be a necessary condition for the Ca2+ ‐sensitivity of the Mg2+‐dependent ATPase activity and super‐precipitation of pig carotid actomyosin. Actin‐myosin interaction independent of phosphorylation and Ca2+ (ATPase activity and superprecipitation) were demonstrated in aged actomyosin preparations and in preparations from which the regulatory light chains were removed by papain digestion.
The Insertion of Pig Microvillus Aminopeptidase into the Membrane as Probed by [125I]Iodonaphthylazide
Authors: NORÉN Ove , SJÖSTRÖM Hans
Published: March 2005

The insertion of pig intestinal microvillus aminopeptidase (EC 3.4.11.2) into the membrane was studied by the hydrophobic photolabel [125I]iodonaphthylazide. The aminopeptidase was either labelled in the microvillus membrane and purified, or labelled after detergent solubilization and purification in a buffer containing Triton X‐100, and then isolated from the reaction mixture.

Of the three subunits A (Mr 162000), B (Mr 123000) and C (Mr 62000) of the aminopeptidase, A and B, but not C contained radioactivity, indicating that both subunit A and B carry anchoring peptide. The radioactivity when released from the subunits by trypsin treatment was connected to low‐molecular‐weight material.
Affinity Labeling of the Escherichia coli Aspartate‐β‐Semialdehyde Dehydrogenase with an Alkylating Coenzyme Analogue
Authors: BIELLMANN Jean‐François , EID Pierre , HIRTH Christian
Published: March 2005

3‐Chloroacetylpyridine‐adenine dinucleotide phosphate (clac3PdADP+, a NADP+ alkylating analogue, irreversibly inactivates aspartate‐β‐semialdehyde dehydrogenase with pseudo‐first‐order kinetics. NADP+ and NADPH, but not the substrate, protected the enzyme from inactivation. The pH dependence of the inactivation kinetics was determined. Incorporation of 1 mol cl[14C]‐ac3PdADP+/dimer totally inactivates the enzyme.

Successive alkylation by the coenzyme analogue and by the substrate analogue, l‐2‐amino‐4‐oxo‐5‐chloropentanoic acid, was studied. After inactivation with the coenzyme analogue, no incorporation of the substrate analogue was detected. However, when the enzyme was first inactivated with the substrate analogue, the protein could subsequently be alkylated with the coenzyme analogue. The binding of NADP+ and NADPH to aspartate‐β‐semialdehyde dehydrogenase was determined by fluorescence.
Characterization of Rat‐Liver Microsomal Glutathione S‐Transferase Activity
Authors: MORGENSTERN Ralf , MEIJER Johan , DEPIERRE Joseph W. , ERNSTER Lars
Published: March 2005

Rat liver microsomes were shown to catalyze the conjugation of 1‐chloro‐2,4‐dinitrobenzene with glutathione and this activity has been characterized. It cannot be removed from the microsomes by washing or other procedures which release loosely bound material from membranes. The microsomal glutathione S‐transferase can be activated up to eight fold by treatment with N‐ethylmaleimide. This activation also affects the apparent Km of the enzyme(s) for both glutathione and 1‐chloro‐2,4‐dinitrobenzene.

Upon subcellular fractionation of the liver the N‐ethylmaleimide‐activateable glutathione S‐transferase distributes in the same manner as a marker for the endoplasmic reticulum and unlike markers for the other organelles and for the cytoplasm. Treatment of microsomes with proteases revealed that the enzyme is at least partially exposed on the cytoplasmic surface of the endoplasmic reticulum. Finally, three inducers of drug‐metabolizing systems–i.e. phenobarbital, methylcholanthrene, and trans‐stilbene oxide–all increase the activity of the cytoplasmic glutathione S‐transferases, but they do not affect the microsomal activity.

These and other considerations indicate that the microsomal glutathione S‐transferase(s) is distinct from the cytoplasmic enzymes catalyzing similar reactions. The microsomal enzyme is likely to be involved in drug metabolism and the possibility of activating it through attack on a sulfhydryl group may represent an important physiological response to certain xenobiotics.
Amino Acid Transport in Plasma‐Membrane Vesicles from Rat Liver
Authors: SIPS Herman J. , VAN AMELSVOORT Johan M. M. , VAN DAM Karel
Published: March 2005

Plasma‐membrane vesicles, isolated from rat liver, catalyze active transport of L‐alanine. The transient accumulation of L‐alanine requires the presence of a Na+ concentration gradient (outside> inside). The alanine‐Na+ symport is an electrogenic process, since it is stimulated under conditions that allow compensatory ion movements: both co‐transported anions as well as counter‐transported cations influence the rate of alanine‐Na+ symport. However, no uptake is observed in the presence of a membrane potential, when no Na+ concentration gradient is present. Sodium‐gradient‐stimulated alanine uptake is dependent on temperature and pH, stereospecific, and is affected by the presence of other amino acids. The activity of the L‐alanine transport system is influenced both by the Na+ and by the l‐alanine concentration. In the presence of 100 mM Na+, an apparent Km for alanine of 2 mM is observed; lowering the Na+ concentration results in an increase in the apparent Km, and a decrease in the apparent V. The apparent Km for Na+ is 34 mM in the presence of 0.2 mM l‐alanine. Increasing the L‐alanine concentration also results in a lower apparent Km for Na+ and a higher V.
On the Mechanism of the Cytochrome c Oxidase Reaction. Bound Fe(III) Cytochrome c as an Intermediate in the Reductive Half Reaction
Authors: PETERSEN Lars Chr. , COX Raymond P.
Published: March 2005

The reaction between cytochrome c and Cytochrome aa3 in the presence of reductant was studied using transient and steady‐state kinetic methods. To investigate the effect of complex formation the reaction was studied at both high and low ionic strength in potassium phosphate buffer (pH 7.4).

Steady‐state kinetic experiments were performed in which the oxidation level of cytochrome c was measured during the anaerobic‐aerobic transition of a system containing cytochrome aa3, cytochrome c and ascorbate. Such measurements confirm previous suggestions [Mochan and Nicholls (1972) Biochim, Biophys. Acta. 172, 37–51] that cytochrome c forms a complex with cytochrome oxidase which is not easily accessible to ascorbate reduction. The results suggest that one oxidised cytochrome c per oxidase is bound at low ionic strength.

The reduction of ‘oxygen‐pulsed’ oxidase in the presence of dithionite and various concentrations of cytochrome c was investigated by stopped flow spectrophotometry. Exposure of buffer containing cytochrome aa3, cytochrome c and dithionite to a brief pulse of oxygen caused an immediate oxidation of the oxidase and of up to one equivalent of cytochrome c per oxidase. Under conditions where complex formation is favoured the subsequent reduction of cytochrome c is many times slower than can be accounted for by the reaction between dithionite and free cytochrome c. This suggests that the bound oxidised cytochrome c is not readily accessible to dithionite reduction.

It is proposed that the complex between oxidised cytochrome c and the oxidase is an intermediate of the reductive half‐reaction and it is shown that the kinetic responses of the complex are rapid enough to be in agreement with this suggestion.
Kinetic and Thermodynamic Parameters of the Iron Spin‐State Transition in Human Aquomethemoglobin
Authors: OKONJO Kehinde O.
Published: March 2005

The rate constants for the iron spin‐state transition in human aquomethemoglobin, as well as the corresponding thermodynamic parameters, have be determined at pH 6. klh, the rate constant for the low‐to‐high spin transition, varies between 242.6 s−1, and 480.6 s−1 in the temperature range 13–27°C; khl, the rate constant for the high‐to‐low spin transition, ‐varies between 57.4 s−1 and 69.4 s−2 over the same temperature range. The activation energies are 34.59 kJ mol−1 and 9.78 kJ mol−1 respectively, while the entropy change associated with the high‐to‐low spin transition is −99.7 J mol−1 K−1.

The simple scheme postulated for formate binding to aquomethemoglobin [Okonjo, K. O. & Ilgenfritz, G. (1978) Arch. Biochem. Biophys. 189. 499–507] is shown to be inadequate. It is demonstrated that a scheme that includes a fast spin transition of the iron atoms, preceding formate binding, adequately accounts for formate binding to aquomethemoglobin in the T and R quaternary states.

Evidence is presented to show that, in a given pure solvent, the spin‐state transition appears to be insensitive to differences in solute composition. By contrast, the spin transition appears to be quite sensitive to differences in solvent composition of ethylene glycol/water mixtures.

In the presence of inositol hexakisphosphate. methemoglobin formate exhibits three relaxation times; the two with high amplitudes are affected by changes in inositol hexakisphosphate concentration. This indicates that inositol hexakisphosphate perturbs both the a and ß chains of methemoglobin and may be evidence to support the proposal that inositol hexakisphosphate, at acid pH. causes a quaternary structure transition in aquomethemoglobin.
Isolation and Immunochemical Characterization of the Euonymus europaeus Lectin Receptor from the Major Sialoglycoprotein of Human O Erythrocytes
Authors: PETRYNIAK Jerzy , PETRYNIAK Bronistawa , WASNIOWSKA Kazimiera , KROTKIEWSKI Hubert
Published: March 2005

Purified and desialized blood‐group‐M glycoprotein from human O erythrocytes precipitated with a lectin isolated from Euonymus europaeus seeds. The precipitation was inhibited completely by lacto‐N‐fucopentaose I and lactose.

The H2 N‐terminal tryptic fragment of glycoprotein M (MT), containing most of carbohydrate and all fucose of glycoprotein M [Lisowska and Jeanloz (1973) Carbohydr. Res. 29, 181 ‐ 191], did not precipitate with the lectin but did inhibit the precipitation.

Reduced alkali‐labile oligosaccharides and glycopeptide containing alkali‐stable oligosaccha‐ride were isolated from the desialized MT glycopeptide by alkaline borohydride degradation and gel nitration. Only glycopeptide with the alkali‐stable oligosaccharide inhibited the precipitation and on a molar basis it was as active as glycopeptide MT‐ Both gave 50% inhibition at about 0.76 mM.

Two desialized oligosaccharides obtained as hydrazinolysis products of glycoprotein M, were also as active as glycopeptide MT in the inhibition test. This shows that both oligosaccharides as well as alkali‐stable oligosaccharide linked to glycopeptide MT have determinants of the same structure.

The chemical composition of alkali‐stable oligosaccharides and their pattern of inhibition of precipitation strongly suggest that l‐fucose α1‐linked at the nonreducing end is an immuno‐dominant group and that grouping l‐Fucα1 → DGal →(GlcNAc, Gal, Man) is present in their molecules.

The inhibition of the precipitation by glycopeptide MT is in agreement with the presence of only one alkali‐stable oligosaccharide chain on the glycoprotein M molecule [Tomita, Furthmayr and Marchesi (1978) Biochemistry, 17, 4756 ‐ 4770]. These results as well as the restriction of lectin receptor site to alkali‐stable oligosaccharide, show that glycopeptide MT is monovalent in the reaction with the lectin.
Identification of the Residues Involved in the Oxygen‐Linked Chloride‐Ion Binding Sites in Human Deoxyhemoglobin and Oxyhemoglobin
Authors: VAN BEEK Gerard G. M. , DE BRUIN Simon H.
Published: March 2005

The number of Bohr protons released upon oxygenation of human hemoglobin was measured at 25 C and 37°C as a function of the concentration of chloride ions. From the results obtained association constants could be evaluated for the binding of chloride ions to both deoxy and oxyhemoglobin at these two temperatures. Furthermore, pK values could be determined for those protonic groups involved in chloride ion binding to deoxy and oxyhemoglobin From these data it was inferred that in oxyhemoglobin only imidazole groups are participating in chloride binding, whereas in deoxyhemoglobin the chloride binding site contained the αNH2 group of valine‐1α. The same conclusions were reached by measuring the pK values of the aminogroups of valine la and valine‐1β at different temperatures and ionic strengths. These pK values were measured by following the rate of reaction of 1 ‐fluoro‐2,4‐dinitrobenzene with the α‐NH2 group by spectrophotometric means. We further showed that binding of Cl−, Br− and I− to oxyhemoglobin follows the lyotropic or Hofmeister series, while this effect is much less for deoxyhemoglobin. This result indicates that for the binding of anions to oxyhemoglobin interactions with non‐polar groups contribute to the free‐energy change of binding
Fluorescence of Cowpea‐Chlorotic‐Mottle Virus Modified with Pyridoxal 5′‐Phosphate
Authors: KRÜS Jacob , VERDUIN Benedictus J. M. , VISSER Antonie J. W. G.
Published: March 2005

Cowpea chlorotic mottle virus (CCMV), which is stable at pH 5.0, has been modified at this pH with 0.5‐0.7 pyridoxal 5′‐phosphate molecules per protein subunit. The fluorescence properties of the labelled CCMV protein in different aggregation states of the virus provide information about the labelled part of the protein and the changes induced in its environment, when the nucleoprotein particles are swollen or dissociated. Fluorescence excitation and emission spectra indicate the presence of radiationless energy transfer from the aromatic amino acid residues to the label. Comparison of the fluorescence lifetimes of the labelled and the unlabelled protein confirms the existence of energy transfer.

The mobility of the labelled part, which can be estimated from the fluorescence polarization of pyridoxal phosphate chromophore, is higher than expected from the dimensions of the virus and the protein subunits. Polarization values and the fluorescence lifetimes depend on the presence of small amounts of NaCl or MgCl2 in the buffer solution at pH 7.5. This is due to structural changes in the vicinity of the pyridoxal phosphate label of the RNA and of the protein part.
Alterations in Enzymes Related to Adenosine 3′,5′‐Monophosphate during Compensatory Growth of Rat Lung
Authors: NIJJAR Mohinder S. , THURLBECK William M.
Published: March 2005

Left pneumonectomy in rats induced growth of the right lung which was maximum one week after the operation. This compensatory lung growth was accompanied by an increase in adenylate cyclase/cyclic AMP phosphodiesterase activity suggesting an increase in the lung adenosine 3′,5′‐monophosphate (cyclic AMP) levels after pneumonectomy. Activities of cyclic‐AMP‐dependent protein kinase, phosphorylase and glucose 6‐phosphatase were also increased suggesting the enhanced activity of the cascade system (glycogenolysis). Activities of these enzymes were maximally elevated during the first three days of operation and preceded the growth of contralateral lung, returning to the presurgery level on day 14. These data indicate that: (a)the biochemical response to pneumonectomy in rats appeared to be complete in two weeks and, (b) changes in the activities of cyclic‐AMP‐related enzymes may be important in the compensatory growth of rat lung.
Isolation and Characterization of Profilactin and Profilin from Calf Thymus and Brain
Authors: BLIKSTAD Ingrid , SUNDKVIST Ingegerd , ERIKSSON Sigbritt
Published: March 2005

Profilactin and profilin have been purified from calf thymus and calf brain. Thymus profilactin could be crystallized with a similar technique as described earlier for the spleen protein.

Preparations of profilactin from the three calf tissues spleen, thymus and brain contain a mixture of β actin and γ actin. The ratio β/γ differs between the tissues, but is constant throughout the purification steps.

The properties of profilin isolated from three sources (spleen, thymus and brain) indicate that it is a highly conserved protein.
The Nucleotide Sequences of the 5′‐Terminal T1 Oligonucleotides of Semliki‐Forest‐Virus 42‐S and 26‐S RNAs are Different
Authors: PETTERSSON Ralf F. , SÖDERLUND Hans , KÁÁRIÁINEN Leevi
Published: March 2005

To study the mechanism of the synthesis of Semliki Forest virus (SFV) 26‐S RNA, we have isolated the 5′‐terminal ‘cap’‐containing RNase‐T1‐resistant oligonucleotide (T1 cap) from the genomic 42‐S RNA and from the subgenomic 26‐S RNA and determined their nucleotide sequences. The T1 caps were purified from 32P‐labelled RNAs on two successive two‐dimensional fractionation systems: (a) electrophoresis on cellulose acetate paper followed by homochromatography and (b) two‐dimensional polyacrylamide gel electrophoresis. The T1 caps derived from the two RNAs had different mobilities in both systems. Their nucleotide sequence was found to be: m7G(5′)ppp(5′)ApUpUpGp‐for the 42‐S RNA and m7G(5′)ppp(5′)ApUpUpGp‐for the 26‐S RNA, respectively. Thus, it appears that the 26‐S RNA is not formed by initiation of the RNA polymerase at the 3′ end of the negative‐strand template followed by ‘cleavage and splicing’ or as the result of a ‘polymerase jump’. Our results, instead, favour the model of internal initiation of the polymerase on the 42‐S negative‐strand RNA template.
Yeast Mannosyl Transferases Requiring Dolichyl Phosphate and Dolichyl Phosphate Mannose as Substrate
Authors: BABCZINSKI Peter , HASELBECK Anton , TANNER Widmar
Published: March 2005

The first mannosyl unit of manno‐oligosaccharides of fungal mannoproteins is transferred in a dolichyl‐phosphate‐dependent reaction sequence to serine/threonine residues of the protein. The two membrane‐bound enzymes catalyzing this transfer in the yeast Saccharomyces cerevisiae have been solubilized by detergents. The enzyme transferring mannose from guanosine diphosphate mannose to dolichyl phosphate has been purified 18‐fold when based on membrane protein and 140‐fold when based on total cell protein. The enzyme transferring mannose from dolichyl phosphate mannose to protein has been purified 48‐fold and 380‐fold, respectively. A HCl‐treated cell‐wall mannoprotein from yeast served as acceptor protein for the second enzyme.

The solubilized enzyme catalyzing the formation of dolichyl diphosphate mannose has a Km for guanosine diphosphate mannose of 7 × 10−6 M and is saturated with about 0.15 mM yeast dolichyl phosphate. The metal requirement, pH‐optima, and the detergent concentration necessary for optimal activity have been determined for both solubilized enzymes.
Specificity of Solubilized Yeast Glycosyl Transferases for Polyprenyl Derivatives
Authors: PALAMARCZYK Grazyna , LEHLE Ludwig , MANKOWSKI Tadeusz , CHOJNACKI Tadeusz , TANNER Widmar
Published: March 2005

Four glycosyl transferases solubilized by detergents from Saccharomyces cerevisiae membranes have been tested for their specificity towards various polyprenols differing in chain length and saturation.

The formation of dolichyl diphosphate‐(N‐acetylglucosamine)1‐2 from uridine diphosphate N‐acetylglucosamine and dolichyl monophosphate showed an obligatory requirement for α‐saturated polyprenols. The apparent affinity for α‐saturated polyprenyl phosphates improves with increasing chain length; those shorter than seven isoprene units were inactive.

The rate of transfer of the chitobiosyl unit from dolichyl diphosphate chitobiose to the hexapeptide Tyr‐Asn‐Leu‐Thr‐Ser‐Val followed the sequence: α‐dihydropolyprenyl (C55) >(C80 > (C100).

The enzyme transferring the mannosyl group from guanosine diphosphate mannose to dolichyl monophosphate also showed a clear dependence on α‐saturation and chain length. In this case the chain length affects mainly the maximal velocity of the reaction. The highest value was obtained with C55α‐dihydropolyprenyl phosphate (100 nmol × h−1× mg protein−1), whereas the value with C35α‐dihydropolyprenyl phosphate was only 8 nmol × h−1× mg protein.−1.

The mannosyl transfer from dolichyl monophosphate mannose to dinitrophenylated tetrapeptide Asn‐Ala‐Thr‐Vol to form on O‐glycosidic bond strongly depended on α‐saturation and chain length of the polyprenyl phosphate. The rate of transfer correlated directly with the length of the polyprenyl residue. With C80α‐dihydropolyprenol less than half the rate of the C100 compound was observed, whereas C55 and C35α‐dihydropolyprenol are only 20 and 10% as active, respectively.
Structural Studies on the Glucose‐Heptose Region of the Proteus mirabilis R Core
Authors: RADZIEJEWSKA‐LEBRECHT Joanna , FEIGE Ulrich , JENSEN Michael , KOTELKO Krystyna , FRIEBOLIN Horst , MAYER Hubert
Published: March 2005

Methylation analysis of the core oligosaccharide of the Proteus mirabilis mutant R4 (derived from serotype 028 was carried out in order to obtain information on the internal (glucose‐heptose) region of the P. mirabilis R core.

The isolated core oligosaccharide was composed of glucose, l‐glycero‐d‐manno‐heptose, 3‐deoxy‐d‐manno‐octulosonic acid (dOclA) and phosphorus in a molar ratio of about 1:2:1:1.4. It was methylated either directly or after dephosphorylation. To localize the position of the phosphate substituents, the permethylated product was dephosphorylated with hydrogen fluoride and the oligosaccharide obtained was remethylated using C2H3I. Location of phosphate at C‐7 of the terminal heptose was shown by isolation of the sugar phosphate from partial hydrolysates and gas‐liquid chromatography/mass spectrometry of the permethylated product.

Combining the data of the methylation analysis with the data of an NMR study allows one to formulate the structure of the core oligosaccharide as follows: DGlcp(β1→¾)LZD Hepp(1 →?)dOclA. ↑(1‐ 4/3) LαD Hepp‐7‐p