bluuebunny/crossref_metadata_2025 huggingface dataset:
The Mechanism of the Halothane‐Dependent Efflux of Calcium from Rat‐Liver Mitochondria
Authors: GRIST Elizabeth M. , BAUM Harold
Published: March 2005

The halothane‐dependent, calcium‐induced loss of respiratory control in rat liver mitochondria [1,2] is Mg2+‐dependent and is accompanied by an enhanced mitochondrial swelling. It is suggested that this swelling reflects an increase in calcium activity in the matrix space, due to a decrease in binding of the accumulated cation. This change in the partition of intramitochondrial calcium is correlated with an inhibition by halothane of energy‐independent, calcium‐induced swelling.

The enhanced swelling associated with the active accumulation of calcium in the presence of halothane does not lead to a marked increase in permeability to other ions. Nevertheless, under conditions of energised calcium uptake, and in the presence of Mg2+, a halothane‐dependent, ruthenium red‐insensitive efflux of calcium is observed. This is consistent with the proposed halothane‐dependent increase in the matrix activity of accumulated Ca2+. It is suggested that this mechanism accounts for the previously postulated [2] futile cycle of calcium uptake and release induced by halothane in rat liver mitochondria.
Character of the Stimulatory Response of Uridine Kinase in Rat Livers to Cycloheximide Treatment
Authors: ČIHAK Alois
Published: March 2005

Administration of cycloheximide to adult rats resulted in the enhancement of uridine kinase activity in the liver. The increase of enzyme activity was not affected by adrenal secretion or uptake of food. Actinomycin D, hydroxyurea and puromycin injected simultaneously with cycloheximide depressed the increase of enzyme activity. On the contrary, administration of 5‐azacytidine following cycloheximide resulted in further enhancement of uridine kinase. While the half‐time of template RNA for uridine kinase in control rat livers was 24 h, for cycloheximide‐stimulated enzyme it was about 11 h. At higher dose levels cycloheximide leads to an increased incorporation of both uridine and orotic acid into liver RNA; however, this effect was not observed in the starved animals. The mechanism of action of cycloheximide is briefly mentioned.
Lysosomal Enzymes as Agents of Turnover of Soluble Cytoplasmic Proteins
Authors: DEAN Roger T.
Published: March 2005

The degradation of cytosol proteins in vitro by purified cathepsin D and cathepsin B1 and by mixtures of lysosomal enzymes, was studied. By means of a double‐labelling method, it was shown that the relative rates of degradation of cytosol proteins by the purified enzymes and by mixtures of enzymes under a wide range of conditions in vitro correlated well with their relative rates of turnover in vivo.

The complex mixture of cytosol proteins was degraded less rapidly after denaturation than in the native state, both by the purified proteases and by the mixture of lysosomal enzymes. This contrasts with previous results on proteolysis of single purified proteins. The possible role of lysosomal enzymes in turnover in vivo was discussed.
Studies on Plasma Free‐Fatty‐Acid Metabolism and Triglyceride Synthesis of Brown Adipose Tissue in vivo during Cold‐Induced Thermogenesis of the Newborn Rabbit
Authors: SCHENK Herwart , MENDE Thomas , GOETZE Eberhard , HEIM Tibor , VARGA Ferenc
Published: March 2005

Parameters of plasma free fatty acid metabolism (pool size, half time, disappearance rate, turnover time and absolute turnover rate), the influx of plasma free fatty acids into the glycerides of brown adipose tissue and the pathway of triglyceride synthesis in brown adipose tissue (glycerol‐1‐phosphate versus monoglyceride pathway) were examined after intravenous injection of [1‐14C]palmitate in newborn rabbits. In the thermoneutral environment of 35°C the turnover rate of plasma free fatty acids was 10.20 μmol/min per 100 g body weight and its flux into the glycerides of brown adipose tissue 0.367 μmol/min per 100 g body weight. Cold exposure at an ambient temperature of 20°C caused a decrease to 5.84 μmol/min and 0.207 μmol/min per 100 g body weight, respectively.

Both under basal conditions at an ambient temperature of 35°C and under cold‐induced thermogenesis at an ambient temperature of 20°C triglyceride synthesis in brown adipose tissue ran through the glycerol 1‐phosphate pathway.
A Fluorescence Study of the Temperature‐Dependent Polymerization of Bovine β‐Casein A1
Authors: PEARCE Kevin N.
Published: March 2005

The intrinsic fluorescence properties of bovine β‐casein A1 solutions have been studied under a variety of conditions. The protein shows both tyrosine and tryptophan fluorescence emission, the former being more pronounced at low temperature (5°C) and in the presence of urea. Approximate relative quantum yields for the tyrosine and tryptophan residues were determined using free tyrosine and free tryptophan respectively as standards. The tryptophan emission intensity of β‐casein shows an increase with temperature indicating a temperature‐dependent transition of the protein. Two‐state analysis of the emission‐intensity‐temperature data yielded positive enthalpy and entropy values for the transition over a range of protein concentrations (0.018–0.18%). A marked protein concentration effect was apparent which indicated that the transition was a consequence of polymerization rather than a prerequisite for polymerization. The size of the polymers appeared to be concentration‐dependent. Decreased ionic strength and increased pH both caused a reduction in the increase in emission intensity when the temperature was increased. At higher pH both the enthalpy and the entropy for the transition were reduced. Experimental precision was insufficient to allow the effect of decreased ionic strength on these parameters to be determined. Many of the properties of the temperature‐dependent transition can be explained if the transition is considered to be one of micelle formation.
Biochemical Research on Oogenesis
Authors: DENIS Herman , MAZABRAUD André , WEGNEZ Maurice
Published: March 2005

The properties of tRNA from oocytes of Xenopus laevis were compared with those of tRNA from somatic cells of the same species. Both types of tRNAs were found to have the same average length and to contain an equal variety of modified nucleotides. However, tRNA from small oocytes differed from somatic tRNA by its chromatographic behavior on methylated‐albumin kieselguhr columns.

The elution profiles from reversed‐phase chromatography 5 columns of several aminoacyl‐tRNAs were compared after charging somatic and oocyte tRNAs with 3H or 14C‐labelled amino acids. Striking differences in peak position were observed when tRNA from small oocytes was mixed and co‐chromatographed with somatic tRNA. The differences were less important when tRNAs from large oocytes and from somatic cells were compared. Mixtures of egg and somatic tRNAs gave completely or almost completely coincident elution profiles. Only one isoacceptor, tRNA1Met (initiator tRNA) had the same position in all reversed‐phase chromatography 5 chromatograms.

The results are discussed in terms of possible post‐transcriptional modifications of tRNA in the course of oogenesis. An alternative explanation resorting to changes in the tRNA population of the growing oocyte is also envisaged.
Characterization of Yeast Ribosomal DNA
Authors: RETÈL Jan , VAN KEULEN Harry
Published: March 2005

High‐molecular‐weight yeast ribosomal DNA, purified by preparative Hg2/Cs2SO4 gradient centrifugation, was characterized using RNA‐DNA hybridization, CsCl gradient analysis and denaturation‐renaturation experiments. The following conclusions are drawn from the results of these studies.

55–60% of the total yeast rDNA is made up of sequences coding for 42‐S ribosomal precursor RNA.

The mean length of the 42‐S precursor cistrons corresponds to a molecular weight of 6.2 × 106.

The overall G + C content of yeast rDNA is 42%. On the other hand, both the segments coding for the nonribosomal excess RNA of the 42‐S precursor and those specifying 26‐S and 17‐S rRNA have a higher G + C content of 46.9–47.5%.

The 42‐S precursor cistrons are closely bound up with sequences rather low in G + C content (34–35%), which account for 40–45% of the total yeast rDNA. In contrast to the multiple 42‐S precursor cistrons which show very little or no base sequence divergence these intervening (A + T)‐rich sequences display a considerable heterogeneity in base sequence.
The Location and Function of Cytochrome c2 in Rhodopseudomonas capsulata Membranes
Authors: HOCHMAN A. , FRIDBERG I. , CARMELI C.
Published: March 2005

Two fractions of membrane preparations, a heavy and a light one were isolated from mildly broken Rhodopseudomonas capsulata cells. The light fraction which contained vesicles similar to the regular chromatophores obtained by sonication and a heavy fraction which appeared in electron micrographs to consist of cell fragments which were designated as heavy chromatophores and were composed of broken cell envelopes containing closely packed vesicles enclosed within the cytoplasmic membrane. Both types of chromatophores catalyzed photophosphorylation. However, cytochrome c2 could be washed out only from the heavy chromatophores. Photophosphorylation activity which was lost by the removal of the cytochrome could be restored by addition of either cytochrome c2 or phenazine methosulphate.

Light induced proton efflux in heavy chromatophores in contrast to proton influx in regular chromatophores. The washed heavy chromatophores did not lose the light induced proton movement. Light induced quenching of 9‐aminoacridine and atebrin fluorescence in chromatophores, while the fluorescence was enhanced in the heavy chromatophores. The washing did not affect the fluorescence changes of the heavy chromatophores but caused a reduction of the steady state of the carotenoid absorbance shift.

It is suggested that the membrane in the heavy chromatophores is oriented inside out with respect to the membrane in regular chromatophores. Cytochrome c2 which is attached to that side of the membrane facing the outside medium could be removed from the heavy chromatophores and reconstituted to them. The role of cytochrome c2 in photophosphorylation is discussed.
Structure and Synthesis of a Lipid‐Containing Bacteriophage
Authors: SCHÄFER Rolf , FRANKLIN Richard M.
Published: March 2005

A polymerase activity is associated with protein IV, a protein which is associated with the DNA in bacteriophage PM2. The native enzyme unit is probably a dimer. Manganese ions are required for the polymerisation reaction and there is a well‐defined Mn2+ optimum at 2.5 mM. The pH optimum is at 8.1, the temperature optimum at 28°C. The activity is a polynucleotide‐pyrophosphorylating reaction in the presence of ribo‐ or deoxyribonucleoside triphosphates. The polymerisation reaction is stimulated in the presence of nucleic acids or polynucleotides as effectors. The product is not covalently linked to the effector.
Carboxymethylation of Horse‐Liver Alcohol Dehydrogenase in the Crystalline State
Authors: ZEPPEZAUER Eila , OHLSSON Ingrid , JÖRNVALL Hans
Published: March 2005

Horse liver alcohol dehydrogenase (isozyme EE) in the crystalline state was alkylated with iodoacetate under conditions resulting in the single substitution of Cys‐46, which is a ligand to the active‐site zinc atom. Alkylation was facilitated by the prior formation of a complex with imidazole bound to the zinc atom. Extent and specificity of the reaction were determined by use of 14C‐labelled iodoacetate and by analyses of radioactive peptides after cleavage with trypsin. Ternary complexes of the enzyme with coenzymes and inhibitors effectively protected the protein against alkylation. ADP‐ribose, Pt(CN)2‐4, 1,10‐phenanthroline, Au(CN)‐2 and AMP also prevented alkylation with decreasing effectiveness.

Crystallographic studies of the alkylated enzyme show that the carboxymethylated sulfur atom of Cys‐46 is still liganded to the active‐site zinc atom and that the iodide ion liberated during alkylation is bound as the fourth ligand to zinc, displacing imidazole. Crystallographic analyses were also performed of the binding of AMP and Pt(CN)2‐4 to the enzyme. It was found that Arg‐47 interacts with the phosphate moiety of the nucleotide. Lys‐228 and Arg‐47 interact in the platinate complex with the bulky anion, the center of which coincides with the position of the nucleotide phosphate. Some of the cyano‐ligands to platinum occupy a crevice between the coenzyme phosphate binding site and the active‐site zinc atom.

The results of the combined studies on primary and tertiary structures confirm previous suggestions that iodoacetate enters the active site via reversible binding to an anion‐binding site. This site interacts with the negatively charged groups of the coenzyme as well as with ADP‐ribose, Pt(CN)2‐4 and to a lesser extent Au(CN)‐2 and AMP, which therefore prevent the reversible binding of iodoacetate. 1,10‐Phenanthroline does not block the binding site but interferes with alkylation presumably by changing the coordination of zinc.

Identification of this labelled residue in both chemical and crystallographic studies correlates the primary and tertiary structures. Characterizations of the active‐site zinc region and the general anion‐binding site are also presented.
Adenosine‐3′: 5′‐Monophosphate‐Dependent and Plasma‐Membrane‐Associated Protein Kinase from Bovine Corpus Luteum
Authors: AZHAR Salman , MENON K. M. Jayaram
Published: March 2005

The solubilization of plasma membrane fractions FI and FII associated protein kinases has been attempted using monovalent salts of high ionic strength and various detergent treatments. Extraction of FI and FII plasma membranes with high ionic strength salt solutions did not release more than 20% of the protein kinase activity. Similarly, monovalent salts released little adenosine 3′: 5′‐monophosphate (cyclic AMP) binding activity, but after extraction binding capacity of cyclic [3H]AMP to plasma membranes was increased about 150–200%. Triton X‐100 was a better solubilizing agent than Lubrol WX or deoxycholate. In addition to solubilization, 0.1% Triton X‐100 also stimulated the protein kinase activity 150‐200%. The properties of Triton X‐100 solubilized FI and FII and purified cytosol KII were characterized with respect to protein substrate specificity, effect of cyclic AMP, cyclic nucleotide specificity, effects of divalent metal ion and gonadotropins. Upon sucrose density gradient centrifugation, FI solubilized protein kinase and cyclic AMP binding activities co‐sedimented with a sedimentation coefficient of 6.3 S. The FII solubilized protein kinase sedimented as two components with sedimentation coefficients of 7.7 S and 5.5 S. The cyclic AMP binding activity also sedimented as two components with sedimentation coefficient 6.7 S and 5.5 S. Cyclic AMP caused dissociation of solubilized protein kinase from FI into a single catalytic (4.8 S) and two cyclic AMP binding subunits (8.1 S and 6.7 S). FII solubilized enzyme was dissociated into one catalytic (4.8 S) and one cyclic AMP binding subunit (6.3 S). Fractionation of FI and FII solubilized enzymes on DEAE‐cellulose column chromatography resolved them each into two peaks Ia, Ib and IIa, IIb, respectively. Peaks Ib and IIb were more sensitive to cyclic AMP stimulation than Ia and IIa peaks. From these studies it is concluded that the plasma‐membrane‐associated and cytosol protein kinases have similar catalytic properties but differ in some of their physical properties.
Modulation of the Enzymic Activity of Chicken Pepsin by the Covalent Modification of Its Single ‐ SH Group
Authors: SHECHTER Yoram , RUBINSTEIN Menachem , BECKER Rosette , BOHAK Zvi
Published: March 2005

The single cysteinyl residue of chicken pepsin was modified with a wide spectrum of reagents to produce mixed disulfides or alkylated derivatives. All these derivatives showed enhanced catalytic activity towards the synthetic peptide Z‐Ala‐Ala‐Phe‐Phe‐OPrP, where OPrP is the 3‐(4‐pyridyl)‐propyl‐1‐oxy group. The overall catalytic constant kcat/Km for these derivatives was 4‐25‐fold larger than that of the native enzyme. The activity of the enzyme towards denatured hemoglobin was slightly decreased (10–45%) by these modifications. When the mixed disulfide derivatives were treated with excess mercaptan, the sulfhydryl group was regenerated and activity reverted to that of the native enzyme.

The ‐SH group of chicken pepsin reacted preferentially with reagents containing an aromatic group. The reaction was found to depend on the ionization of a single group, presumably the ‐SH itself, with a pKa= 7.5. The rate of reaction of the fully deprotonated species with various disulfides was 100–1000‐fold smaller than that of the ‐ SH group of glutathione.

It is suggested that the groups attached covalently to the sulfhydryl also interact with other amino acid side chains in the protein thereby affecting the active center of chicken pepsin.
Ion‐Binding to Phospholipids
Authors: HAUSER Helmut , PHILLIPS Michael C. , LEVINE Barry A. , WILLIAMS Robert J. P.
Published: March 2005

Surface chemical and nuclear magnetic resonance (NMR) techniques have been used to study the interaction of Ca2+ and lanthanides with lecithins. With both methods positive reactions were detected at metal concentrations > 0.1 mM.

1H and 31P high‐resolution NMR spectra obtained with single bilayer vesicles of lecithin were invariant up to Ca2+ concentrations of 0.1 M indicating that there is only a loose association between Ca2+ and the phospholipid.

The weak interaction between Ca2+ and lecithin is confirmed by both surface chemical and NMR techniques showing that the packing of egg lecithin molecules present in bilayers does not change up to Ca2+ concentrations of about 0.1 M. The packing was also independent of pH between 1–10.

Contradictory results have been reported in the literature concerning the question of Ca2+ binding to lecithins. The conflicting results are shown to have arisen from differences in the experimental conditions and differences in the sensitivity of the physical methods used by various authors to study Ca2+‐lecithin interactions.

An estimate of the strength of binding and molecular details of the interaction were derived using paramagnetic lanthanides as isomorphous replacements for Ca2+. From the changes in chemical shifts induced in the presence of lanthanides an apparent binding constant KA∼ 30 l/mol was calculated at lanthanide concentrations > 10 mM. Using surface chemical methods it was shown that this KA is up to 10 times larger than that for Ca2+ binding.

The complete assignment of the 1H NMR spectrum of lecithin, including the resonances from the relatively immobilized glycerol group, was determined to derive molecular details of the cationlecithin interaction.

From spin‐lattice relaxation‐time measurements and line broadening in the presence of GdCl3 it is concluded that the cations are bound to the phosphate group and that this is the only binding site.

The absolute proton shifts induced by paramagnetic lanthanides depended on the nature of the ion, but the shift ratios standardised to the shift of the O3POCH2 (choline) signal were invariant throughout the lanthanide series indicating that the shifts are purely pseudocontact. In contrast the 31P shifts were found to contain significant contact contributions. These findings are consistent with a weak interaction and with the phosphate group being the binding site.

The absolute shifts but not the shift ratios depended on the anion present indicating that the cation binding may be accompanied by binding of anions.

Contrary to negatively charged phospholipids the interaction of lanthanides with lecithins was enhanced as the ionic strength was increased by adding NaCl. This was explained in terms of steric hindrance due to the extended conformation of the lecithin polar group.
Identity and Activities of Lysosomal Enzymes in Parenchymal and Non‐Parenchymal Cells from Rat Liver
Authors: VAN BERKEL Theodorus J. C. , KRUIJT Johan K. , KOSTER Johan F.
Published: March 2005

Intact parenchymal and non‐parenchymal cells were isolated from rat liver. The parenchymal cells were purified by differential centrifugation, while non‐parenchymal cells were obtained free of parenchymal cell contamination by preferentially destroying the parenchymal cells with the aid of pronase (0.25%).

The ability to isolate pure intact parenchymal and non‐parenchymal cells permitted the characterization and measurement of specific activities of various lysosomal enzymes, representing the main functional hydrolytic activities of the lysosomes in these distinct cell types.

Lysosomal enzymes catalysing the hydrolysis of the terminal carbohydrate moiety of glycoproteins and glycolipids were not particularly enriched in the non‐parenchymal cells as compared to parenchymal cells. The ratio of the specific activities of non‐parenchymal cells over parenchymal cells varied between 0.7 for N‐acetyl‐β‐d‐hexoseaminidase to 2.1 for α‐glucosidase. This suggests no specific role of the non‐parenchymal cells in the hydrolysis of terminal carbohydrate moieties of glycoproteins and glycolipids.

The enzymes acid phosphatase and aryl sulphatase, representing the phosphate and sulphate hydrolyzing activities, were enriched in the non‐parenchymal cells as compared to the parenchymal cells by a factor of 2.5.

The most important peptidase cathepsin D, representing protein breakdown capacity, is enriched in the non‐parenchymal cells as compared to parenchymal cells by a factor 6.0, suggesting a possible specific function of non‐parenchymal cells in protein breakdown.

The most enriched lysosomal enzyme, representing lipid hydrolysis, is acid lipase, which is enriched in the non‐parenchymal cells with a factor of 10.

The distribution of lysosomal enzymes between parenchymal and non‐parenchymal cells suggests different functional roles of the lysosomes in these cell types. It can be concluded that the non‐parenchymal cells possess a set of lysosomal enzymes which makes them extremely suitable for a phagocytic and antimicrobial function in the liver.
Control of Fatty‐Acid‐Synthetase Biosynthesis in Saccharomyces cerevisiae
Authors: DIETLEIN Gerhard , SCHWEIZER Eckhart
Published: March 2005

143 out of 308 fasl mutants (47%) and 139 out of the 443 fas2‐mutants (32%) genetically studied in this laboratory fail to complement with any other fas‐mutant (deficient in fatty acid synthetase) of the same gene locus. From these noncomplementing fas‐mutants no mutant fatty acid synthetase can be isolated using the wild‐type enzyme purification procedure. Furthermore the noncomplementing fas‐mutants generally contain no material immunologically crossreacting with a specific fatty acid synthetase antiserum. However, subunits obtained after dissociation of the complex with sodium dodecylsulfate still cross react with this antiserum. Therefore, it is concluded that noncomplementing fas‐mutants contain no fatty acid synthetase component proteins, though one of the two fas‐loci is mutationally unaffected. This conclusion was further confirmed by 14C‐labeled amino acid incorporation studies which indicated that in noncomplementing fas‐mutants, other than in wild type and complementing fas‐mutant cells, no label was incorporated into fatty acid synthetase subunits or precursor proteins. At nonpermissive temperature, the same biochemical and immunological characteristics were observed with temperature‐sensitive non‐complementing fas‐mutants.

These results suggest that noncomplementing fas‐mutants either represent regulatory mutants unable to induce the mutationally unaffected other fas‐gene locus or that they are association‐defective mutants. In both cases the resulting individual subunits of the complex may be rapidly degraded by intracellular proteases.
Immunochemical Studies of Factor V
Authors: GREENQUIST Alfred C. , WEINBERG Robert M. , KUO Alice L. , COLMAN Robert W.
Published: March 2005

Native bovine factor V exhibits a molecular weight of 300000 as determined by gel filtration of untreated plasma. Highly purified factor V exhibits multiple molecular weight forms which range from small active fragments to aggregates of several million which are generated during the purification on cellulose phosphate. Isoelectric focusing of a single high‐molecular‐weight species produced a single protein and activity peak at pH 4.65. Factor V activity is associated with each protein band observed following polyacrylamide gel electrophoresis. Antisera to factor V prepared in rabbits produces a time‐dependent and concentration‐dependent inhibition of factor V activity in plasma and purified factor V. The multiple molecular weight forms of factor V appear equivalent upon immunodiffusion and on immunoelectrophoresis migrate as an alpha globulin between albumin and fibrinogen. Immunoprecipitation arcs are equivalent in plasma and serum.

Factor V consists of two major types of subunits, a light chain (73000), aggregates of which form the higher‐molecular‐weight species, and a heavy chain (125000). Using preparations containing one or both chains isolated by disc gel electrophoresis, antiserum was shown to contain two families of antibodies, one against each subunit. Cross reactivity with both light and heavy chain antigens is observed in sheep and goat but not monkey or human plasma. The antisera also neutralized goat and sheep factor V activity.
Animal DNA‐Dependent RNA Polymerases
Authors: HOSSENLOPP Paul , CHAMBON Pierre , WELLS David
Published: March 2005

DNA‐dependent RNA polymerase was solubilized from normal and adenovirus‐2 infected HeLa cells. Multiple peaks of enzyme activity were separated by DEAE‐Sephadex chromatography. In addition to class A and B enzyme activities (respectively insensitive and sensitive to inhibition by 10 nM α‐amanitin), three peaks of class C enzyme activity were found which are sensitive to inhibition by α‐amanitin only at much higher concentrations (0.1 mM). Rechromatography of these class C peaks indicates that they are not chromatographic artifacts. Class C enzymes differ from class A and B enzymes by several criteria including inhibition by α‐amanitin, immunological properties, and the ability to transcribe native calf thymus DNA at high ionic strength. However, the ionic strength optimum and the divalent cation requirements of class C enzymes are not invariant characteristics of the enzymes and are markedly dependent on the nature and the amount of template in the reaction. No differences, either qualitative or quantitative, were found between the multiple enzymes isolated from normal or adenovirus‐2 infected cells.

All of the partially purified HeLa cell RNA polymerases were able to transcribe an intact double‐stranded adenovirus‐2 DNA under conditions where no transcription occurred with purified calf thymus AI and B RNA polymerases. Since the multiple enzymes were devoid of endonuclease and exonuclease activities, the ability of the partially purified enzymes to transcribe adenovirus‐2 DNA cannot be ascribed to initiation of RNA synthesis at nicks or single‐stranded regions of the DNA. No differences in transcriptional ability were found between corresponding enzyme classes from normal or infected cells, but a comparison of the ability of the various enzyme classes to transcribe intact viral DNA revealed large differences. Although partially purified HeLa class A and B enzymes were able to initiate on the intact viral DNA to a limited extent only, it appears that the class C enzymes transcribe intact duplex DNA much more efficiently than any other class of eukaryotic polymerase yet reported.
Fractionation of Rat‐Liver‐Chromatin Nonhistone Proteins into Two Groups with Different Metabolic Rates
Authors: RUSSEV George , ANACHKOVA Boika , TSANEV Roumen
Published: March 2005

In the pH interval 10.5–11.8, 70% of the nonhistone proteins normally present in rat liver chromatin were dissociated. The rest remained complexed with DNA even at pH 13.

Dodecylsulfate‐polyacrylamide gel electrophoresis revealed that the majority of the high‐molecular‐weight nonhistone proteins together with a few characteristic fractions with molecular weights of 40000–60000 remained in the alkali‐resistant group.

l‐[14C]Leucine pulse‐labelling experiments showed that the specific radioactivity of the alkalilabile nonhistone proteins was 2–3 times higher than that of the alkali‐resistant nonhistone proteins, which, in turn, had the same specific radioactivity as that of the histones. The same held true for chromatin from regenerating rat liver.

In the course of a 21‐day chase the specific radioactivity of the alkali‐labile nonhistone proteins gradually decreased and finally became 3 times lower than that of the alkali‐resistant nonhistone proteins. On the contrary, the ratio of the specific radioactivities of the alkali‐resistant nonhistone proteins and of the histones to the specific radioactivity of DNA remained constant during the chase.

A conclusion can be drawn that a fraction of liver nonhistone proteins exists which is alkali‐resistant and is conserved in chromatin like histones.
On the Dissociation and Association of Escherichia coli Ribosomes
Authors: WALTERS Hans , SMITS Willem , VAN KESSEL Jan , VAN OS Gerard
Published: March 2005

The dissociation and association behaviour of 70‐S ribosomes of Escherichia coli has been studied. It has been shown that the dissociation‐association reaction can be both a real dynamic equilibrium and a non‐equilibrium reaction, dependent upon the ionic conditions of the solvent. At relatively high ionic strength (I= 0.15 M or more) the dissociation‐association reaction is an equilibrium reaction, whereas at lower ionic strength (I= 0.1 M or less) there is no dynamic equilibrium between 70‐S ribosomes and its subunits. In the latter case a hysteresis in the dissociation‐association reaction is observed.

Whether there is a dynamic equilibrium or not can be demonstrated by a single centrifugation experiment, using the analytical ultracentrifuge.
Heterogeneity of the Lipopolysaccharide from Pseudomonas aeruginosa
Authors: CHESTER Ian R. , MEADOW Pauline M.
Published: March 2005

Lipopolysaccharide isolated from Pseudomonas aeruginosa PAC1 and its phage‐resistant mutant was degraded by mild acid hydrolysis into lipid A and three major polysaccharide‐containing fractions which were separated on Sephadex G‐75. The low‐molecular‐weight fraction contained glucose, rhamnose, heptose, galactosamine, alanine and phosphate. The higher‐molecular‐weight fractions consisted mainly of glucose, rhamnose and glucosamine together with amino compounds. Alkaline degradation of the lipopolysaccharide produced at least four different species each of which contained a low‐molecular‐weight polysaccharide similar if not identical to that produced by acid hydrolysis. Under certain growth conditions an abnormal lipopolysaccharide was produced which was defective in the low‐molecular‐weight polysaccharide and contained mainly high‐molecular‐weight material. Strains of different serotype yielded lipopolysaccharides which also exhibited heterogeneity but contained a low‐molecular‐weight polysaccharide similar to that obtained from strain PAC1 and PAC1R. It is suggested that each strain of P. aeruginosa may produce several lipopolysaccharides each containing a polysaccharide common to all. The relative proportions of the various lipopolysaccharides may be changed by growth conditions.
Polynucleotide Kinase from Rat‐Liver Nuclei
Authors: TERAOKA Hirobumi , MIZUTA Kazutaka , SATO Fumiyasu , SHIMOYACHI Mari , TSUKADA Kinji
Published: March 2005

A polynucleotide kinase, which catalyzes the phosphorylation of 5′‐hydroxyl ends of deoxyribo‐nucleic acid in the presence of adenosine triphosphate, has been purified 260‐fold with a yield of 14% from 0.15 M NaCl extracts of rat liver nuclei.

The purified enzyme has a pH optimum of 5.5. The enzyme is reversibly inhibited by p‐chloro‐mercuribenzoate. The S0.5 value (ligand concentration required for a half‐maximal activity) for ATP is 2.5 μM. A bivalent cation is essential for the reaction and S0.5 values for Mg2+, Ca2+ and Mn2+ are 3.3 mM, 4 mM and 0.05 mM respectively. Pyrophosphate remarkably inhibits the activity with I0.5 value (ligand concentration required for a half‐maximal inhibition) of 0.2 mM, and sulfate, with I0.5 of 0.5 mM, whereas phosphate weakly inhibits the activity with I0.5 of about 20 mM.

An apparent molecular weight of the purified enzyme is estimated to be 8 × 104 by gel filtration on a column of Sephadex G‐150, and the Stokes radius of the enzyme molecule is shown to be about 0.36 nm. Sucrose density gradient centrifugation reveals that the enzyme has a sedimentation coefficient of about 4.4 S.
Ultrastructural Organization and Biochemical Characterization of Chromatin · RNA · Protein Complexes Isolated from Mammalian Cell Nuclei
Authors: BACHELLERIE Jean‐Pierre , PUVION Edmond , ZALTA Jean‐Pierre
Published: March 2005

Extranucleolar elements were isolated from mammalian cell nuclei and characterized by correlative biochemical‐ultrastructural studies. These complex structures were shown to retain a supra‐particle arrangement closely resembling the organizaton in situ of definite nuclear areas. In particular the morphological RNA · protein species, namely perichromatin fibrils and granules, were detected in association with characteristic regions of chromatin. By autoradiographic experiments, we were able to show that perichromatin fibrils represent the morphological state of newly formed heterogeneous nuclear RNA (hnRNA).

Chromatin · RNA · protein complexes were further fractionated by means of a treatment dissociating the chromatin component. A minor part of DNA, possibly involved in the biosynthesis and the processing of hnRNA, remains linked to the resulting RNA · protein network.
Phenol Hydroxylase from Yeast
Authors: NEUJAHR Halina Y. , GAAL Andràs
Published: March 2005

Thiol groups in phenol hydroxylase were measured using two different –SH reagents and amino acid analysis. Stepwise blocking of the –SH groups was correlated with enzyme activity and FAD content.

The results indicate that the enzyme contains 16 –SH groups per molecule of Mr 1.48 × 105. At least four –SH groups are not accessible without the use of a denaturing agent. There is seemingly no disulphide bridge.

On the whole, the reactivity towards p‐hydroxymercuribenzoate is much greater than towards 5,5′‐dithio‐bis(2‐nitrobenzoic acid). The two reagents seem to have a different specificity with respect to which –SH groups they attack. Either reagent dislocates FAD from the holoenzyme, leaving a characteristic mercaptide derivative of the apoenzyme. Such derivatives were used to prepare the apoenzyme. The –SH groups in the apoenzyme are much more reactive towards 5,5′‐dithiobis(2‐nitrobenzoic acid) than the –SH groups in the holoenzyme.

The stoichiometry of the reaction with 5,5′‐dithio‐bis(2‐nitrobenzoic acid) indicates that at least 8 –SH groups are located in spatially close pairs. The most reactive pair of all does not appear to be of importance for enzyme activity. The two subsequent –SH pairs are essential for enzyme activity and are involved in FAD attachment.

The reactivity of the –SH groups decreases dramatically in the presence of substrate, even at substrate concentrations equivalent to the level of the catalytic sites.

The isolated apoenzyme has a tendency to aggregate. A large proportion of –SH groups in such aggregate(s) is buried, especially when EDTA is not used throughout the preparation of the apoenzyme. The aggregates are enzymically inactive.
Precursor mRNA from Erythroid‐Enriched Bone‐Marrow Cells of the Rabbit
Authors: LADHOFF Axel‐M. , THIELE Bernd J. , COUTELLE Charles
Published: March 2005

Precursor mRNA (pre‐mRNA) molecules, sedimenting at > 45 S, from erythroid‐enriched bone marrow cells of the rabbit and hemoglobin mRNA molecules from rabbit reticulocytes were investigated by electron microscopy. Four of 98 measured pre‐mRNA molecules had a length between 15 and 17.1 μm. In some of the pre‐mRNA molecules a characteristic condensed structure was observed at one end, strikingly resembling the structure of the mRNA molecules.
The Amino‐Acid Sequence of Isoinhibitor K from Snails (Helix pomatia)
Authors: TSCHESCHE Harald , DIETL Thomas
Published: March 2005

Performic‐acid‐oxidized isoinhibitor K of snails (Helix pomatia) was subjected to argininedirected tryptic proteolysis. Six peptide fragments including one overlap peptide from limited cleavage of the Arg‐3‐Pro‐4 bond were purified to homogeneity. Four arginine peptides and the C‐terminal peptide were sequenced by automatic Edman degradation using a special peptide program. The phenylthiohydantoins were all identified by chemical ionization mass spectrometry, except four cysteic acid residues that were identified on an amino acid analyzer after acid hydrolysis. Quantitative evaluation of the phenylthiohydantoins by chemical ionization mass spectrometry using total molecular‐ion beam integration greatly facilitated sequencing. The mass spectrum of the dipeptide <Glu‐Gly revealed that the N‐terminus was blocked by pyroglutamic acid.

The complete amino acid sequence of isoinhibitor K was determined. An almost 50% homology between the sequences of the snail inhibitor and the bovine trypsin‐kallikrein® inhibitor (Kunitz) became obvious. A comparison of all homologous sequences of this particular class of proteins known to date is presented.
Patulin Biosynthesis: The Metabolism of m‐Hydroxybenzyl Alcohol and m‐Hydroxybenzaldehyde by Particulate Preparations from Penicillium patulum
Authors: MURPHY Gillian , LYNEN Feodor
Published: March 2005

The ring hydroxylation of m‐hydroxybenzyl alcohol to gentisyl alcohol by a particulate preparation from Penicillium patulum has been characterised. The activity was shown to be closely associated with, but not necessarily identical to, m‐cresol 2‐hydroxylase activity of the 105 000 x g microsomal fraction. As with both the m‐cresol hydroxylases of this system, m‐hydroxybenzyl alcohol hydroxylase requires oxygen and NADPH for activity. A Km value for m‐hydroxybenzyl alcohol of 15 μM was measured. Inhibition of the hydroxylase activity and its reversal by light, as well as the action of cytochrome c, KCN and other effectors suggested a mixed‐function oxidase reaction of the cytochrome P‐450, NADPH‐cytochrome reductase type.

m‐Hydroxybenzaldehyde was not ring hydroxylated by any preparation from P. patulum. Apart from the previously described conversion to m‐hydroxybenzyl alcohol by a predominantly soluble dehydrogenase, m‐hydroxybenzaldehyde was metabolized to m‐hydroxybenzoic acid by a particulate fraction. This activity required NADPH.

It was concluded that the main biosynthetic pathway to patulin must be through m‐hydroxybenzyl alcohol, gentisyl alcohol and gentisaldehyde.
Studies Concerning the Mechanism by which Translational‐Control RNA Regulates Protein Synthesis in Embryonic Muscle
Authors: HEYWOOD Stuart M. , KENNEDY Doris S. , BESTER André J.
Published: March 2005

Muscle translational‐control RNA (tcRNA) has been separated into two classes, polysomal and messenger ribonuclear protein (mRNA · protein), which have different sizes as determined by acrylamide gel electrophoresis. While normally translation of mRNA · protein mRNA is inhibited by tcRNA derived from the same mRNA · proteins, this inhibition does not occur if the messenger is previously de‐adenylated. This suggests that the poly(A) segment of mRNA is required for the tcRNA activity. Utilizing different mRNA · protein fractions from muscle, myosin mRNA · protein and small mRNA · proteins (<30 S), we have been able to demonstrate that a degree of specificity exists in the interaction of tcRNA and mRNA derived from the same mRNA · proteins. This is illustrated by the facts that (a) each tcRNA only inhibits the translation of its respective mRNA and (b) the highest percentage of structural change occurs when each tcRNA is hybridized to its respective mRNA as measured by its resistance to T1 and T2 RNase.
Tightness and Orientation of Vesicles from Guinea‐Pig Kidney Estimated from Reactions of Adenosine Triphosphatase Dependent on Sodium and Potassium Ions
Authors: WALTER Horst
Published: March 2005

In order to study the “sidedness” of the ligands of the Na+, K+‐ATPase in the phosphorylation from [32P] ATP, tight vesicles were prepared from guinea pig kidney and partially purified by a two‐stage sucrose and Ficoll gradient centrifugation procedure. These vesicles were derived presumably from plasma membrane fragments resealed after the initial disruption of the cells during homogenization. Tightness of the vesicles was estimated according to activation by the nonionic detergent, Triton X‐100. Treatment with Triton X‐100 increased both the activity of the Na+, K+‐ATPase and its Na+‐dependent phosphorylation from [32P] ATP at least three‐fold. Activation of both functions also appeared when the vesicles were shocked osmotically. These results suggest that the preparation contains a major population of tight normal vesicles (∼ 75%) in which the phosphorylation site faces faces the intravesicular solution.

In the response to ouabain breakdown of the phosphoenzyme was inhibited in vesicles treated with Triton X‐100 but not in intact ones as if ouabain could not get to its binding site. Correspondingly in phosphorylation from ATP pretreatment with ouabain in the presence of inorganic phosphate produced less inhibition in intact vesicles than in those disrupted with Triton X‐100 beforehand.

These data suggest the presence of an everted vesicle fraction in the preparation (∼ 20%). Apparently only a small fraction of the vesicles was leaky.

In the everted vesicles the action of K+ on the phosphoenzyme was slow. In order to accelerate the dephosphorylation in intact vesicles as effectively as in disrupted ones, K+ had to be added before the start of phosphorylation. This supports the view that K+ was acting from the side of the membrane opposite to that where the γ‐phosphoryl group was accepted from ATP.
Identification of Ribitol Phosphate as a Constituent of the Lipopolysaccharide from Proteus mirabilis, Strain D52
Authors: GMEINER Jobst
Published: March 2005

A polyol was released from the lipopolysaccharide of Proteus mirabilis, strain D52, during alkaline hydrolysis and its phosphate ester was isolated after acid hydrolysis. This polyol has been identified as ribitol by comparison of the free polyol, its phosphate ester and its anhydro derivative formed after acid treatment with authentic xylitol, D‐and L‐arabitol, ribitol and their corresponding derivatives on paper and gas‐liquid chromatography.
Gram‐Scale Purification of Methionyl‐tRNA and Tyrosyl‐tRNA Synthetases from Escherichia coli
Authors: BRUTON Chris , JAKES Ross , ATKINSON Tony
Published: March 2005

A procedure is presented for the purification of 3 g of homogeneous methionyl‐tRNA synthetase and 1 g of homogeneous tyrosyl‐tRNA synthetase from 50 kg batches of Escherichia coli. The procedure permits the isolation of many enzymes simultaneously and the elution positions of seven other aminoacyl‐tRNA synthetases, catalase, rhodanese, phosphofructokinase, elongation factor Tu and cytochrome b‐562 are indicated. The problems of extraction work on this scale are discussed.
The Pyruvate‐Dehydrogenase Complex from Azotobacter vinelandii
Authors: BRESTERS Tjarda W. , DE ABREU Ronney A. , DE KOK Arie , VEEGER Cees , VISSER Jaap
Published: March 2005

The pyruvate dehydrogenase complex from Azotobacter vinelandii was isolated in a five‐step procedure. The minimum molecular weight of the pure complex is 600000, as based on an FAD content of 1.6 nmol · mg protein−1. The molecular weight is 1.0–1.2 × 106, indicating 1 mole of lipoamide dehydrogenase dimer per complex molecule. Sodium dodecylsulphate gel electrophoretical patterns show that apart from pyruvate dehydrogenase (Mr 89000) and lipoamide dehydrogenase (Mr monomer 56000) two active transacetylase isoenzymes are present with molecular weight on the gel 82000 and 59000 but probably actually lower.

The pure complex has a specific activity of the pyruvate‐NAD+ reductase (overall) reaction of 10 units · mg protein−1 at 25°C. The partial reactions have the following specific activities in units · mg protein−1 at 25°C under standard conditions: pyruvate‐K3Fe(CN)6 reductase 0.14, transacetylase 3.6 and lipoamide dehydrogenase 2.9. The properties of this complex are compared with those from other sources. NADPH reduced the FAD of lipoamide dehydrogenase as well in the complex as in the free form. NADP+ cannot be used as electron acceptor.

Under aerobic conditions pyruvate oxidase reaction, dependent on Mg2+ and thiamine pyrophosphate, converts pyruvate into CO2 and acetate; V is 0.2 μmol O2· min−1· mg−1, Km (pyruvate) 0.3 mM. The kinetics of this reaction shows a linear 1/velocity – 1/[pyruvate] plot. K3Fe(CN)6 competes with the oxidase reaction. The oxidase activity ist stimulated by AMP and sulphate and is inhibited by acetyl‐CoA.

The partially purified enzyme contains considerable phosphotransacetylase activity. The pure complex does not contain this activity. The physiological significance of this activity is discussed.
Isolation and Properties of an Arginine‐Binding Protein from Saccharomyces cerevisiae
Authors: OPEKAROVÁ Miroslava , KOTYK Arnošt , HORÁK Jaroslav , KHOLODENKO Vasiliy P.
Published: March 2005

Transfer of exponentially growing cells of Saccharomyces cerevisiaeΣ 1278 b to a fresh medium (or simply to distilled water) resulted in the loss of ability to transport arginine (and lysine), accompanied by the release of several proteins from the membrane surface or periplasmic space. Fractionation by ultrafiltration, Sephadex G‐50 chromatography and freeze‐drying yielded a homogeneous protein (55 mg per 100 g dry weight of cells) with specific binding ability for l‐arginine (Kd= 3.8 × 10−4 M) and l‐lysine (Ki= 4.2 × 10−4 M). The protein contains over 40 amino acid residues and has a molecular weight of about 5000. In solution, it appears to aggregate as its concentration is raised, thereby decreasing the overall binding capacity for arginine. Addition of the protein to a depleted culture does not restore the transport of arginine. It is apparently the recognition protein for the specific arginine‐transporting system of Saccharomyces cerevisiae but it occurs in almost identical amounts in the MG 168 mutant with impaired arginine transport.
Arginine Decarboxylase from Lathyrus sativus Seedlings
Authors: RAMAKRISHNA Seethala , ADIGA P. Radhakantha
Published: March 2005

Arginine decarboxylase which makes its appearance in Lathyrus sativus seedlings after 24 h of seed germination reaches its highest level around 5–7 days, the cotyledons containing about 60% of the total activity in the seedlings at day 5. The cytosol enzyme was purified 977‐fold from whole seedlings by steps involving manganese chloride treatment, ammonium sulphate and acetone fractionations, positive adsorption on alumina C‐γ gel, DEAE‐Sephadex chromatography followed by preparative disc gel electrophoresis. The enzyme was shown to be homogeneous by electrophoretic and immunological criteria, had a molecular weight of 220000 and appears to be a hexamer with identical subunits. The optimal pH and temperature for the enzyme activity were 8.5 and 45 °C respectively. The enzyme follows typical Michaelis‐Menten kinetics with a Km value of 1.73 mM for arginine. Though Mn2+ at lower concentrations stimulated the enzyme activity, there was no dependence of the enzyme on any metal for the activity. The arginine decarboxylase of L. sativus is a sulfhydryl enzyme. The data on co‐factor requirement, inhibition by carbonyl reagents, reducing agents and pyridoxal phosphate inhibitors, and a partial reversal by pyridoxal phosphate of inhibition by pyridoxal · HCl suggests that pyridoxal 5′‐phosphate is involved as a co‐factor for the enzyme. The enzyme activity was inhibited competitively by various amines including the product agmatine. Highest inhibition was obtained with spermine and arcain. The substrate analogue, l‐canavanine, homologue l‐homoarginine and other basic amino acids like l‐lysine and l‐ornithine inhibited the enzyme activity competitively, homoarginine being the most effective in this respect.