bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of Ubiquinone‐9 Biosynthesis in Rat‐Liver Slices by Exogenous Mevalonate
Authors: Aiyar Anant S. , Olson Robert E.
Published: March 2005

Mevalonate, added in the incubation medium, increases by nearly two‐fold the biosynthesis of ubiquinone in liver slices as evidenced by enhanced incorporation of radioactivity from labeled precursors of the benzoquinone moiety such as [1‐14C]benzoate and [Me‐14C]methionine. Other intermediates of isoprenoid biosynthesis such as mevalonic acid 5‐pyrophosphate and isopentenyl pyrophosphate show a qualitatively similar effect on labeling of ubiquinone. The stimulation due to mevalonate is even more marked under conditions in which its endogenous synthesis and/or utilisation is known to be impaired such as in liver slices from fasted or cholesterol‐fed rats and in liver slices from normal animals incubated in the presence of vanadium ions. These results strongly suggest that the rate of biosynthesis of mevalonate, the precursor of the poly‐isoprenoid side chain, is the probable limiting factor in the biogenesis of ubiquinone in the rat.
The Complete Amino‐Acid Sequence of Human α‐Lactalbumin
Authors: Findlay John B. C. , Brew Keith
Published: March 2005

α‐Lactalbumin was isolated from human milk in a yield of 1.8 mg/ml of milk. The purification procedure involved ammonium sulphate fractionation (30% to 80% saturation) and pH‐4.0 precipitation, followed by gel filtration with Sephadex G‐100. A final purification stage using DEAE‐cellulose was necessary in some preparations.

Peptides derived from the reduced, S‐aminoethylated protein by treatment with cyanogen bromide and digestion of these CNBr fragments with trypsin, chymotrypsin or thermolysin, were purified by gel filtration, ion exchange chromatography and high‐voltage paper electrophoresis.

From the sequences of these peptides it has proved possible to deduce unambiguously the complete primary structure of the protein. Comparison with bovine α‐lactalbumin shows an identity in 72% of the residues with a further 6% being chemically similar amino acids. The corresponding figures for the human α‐lactalbumin/human lysozyme comparison, are 39% and 12%, respectively. The significance of some of the amino acid replacements is discussed.
A New Polynucleotide Complex Poly(s2C) · poly(I)
Authors: Faerber Peter , Scheit Karl‐Heinz , Sommer Hans
Published: March 2005

Poly(s2C) · poly(I) complex formation (where s2C = 2‐thiocytidylate) was shown by spectrophotometric titration, absorption‐difference spectroscopy and hydroxyl‐ion titration. The absorption temperature profile of the complex does not display a transition between 10 and 100°C. Cooperative melting with a transition midpoint at 77°C was observed only after destabilisation by 30% ethyleneglycol. A stabilizing effect of s2CMP residues in hellces is further shown by the observation that 25% s2CMP in poly(C3,s2C) · poly(I) significantly increased the thermal stability compared to poly(C) · poly(I). The ultraviolet absorption spectrum, first derivative absorption spectrum and circular dichroism spectrum suggest a weak n—π* transition at 320 nm in poly(s2C) · poly(I). Unlike other nucleoside diphosphates the polymerisation of s2CDP by polynucleotide phosphorylase from Escherichia coli occurred with a pronounced lag phase which disappeared after the addition of oligonucleotide primers.
Degradation of the Murein · Lipoprotein Complex of Escherichia coli Cell Walls by Dictyostelium Amoebae
Authors: Braun Volkmar , Hantke Klaus , Wolff Helga , Gerisch Günther
Published: March 2005

Amoebae of the cellular slime mold Dictyostelium discoideum can be grown in submerged culture on Escherichia coli W7. This strain is deficient in the synthesis and decarboxylation of diaminopimelate, the amino acid specific for murein. With [3H]diaminopimelate‐labeled E. coli W7, it was found that the murein · lipoprotein complex (rigid layer) of the bacterial cell wall is degraded completely by the amoebae, and diaminopimelate is converted to lysine which is then incorporated into protein. Cleavage of the bond between N‐acetylmuramic acid and l‐alanine and release of alanine from the murein peptide side chain is shown in vitro. Several glycosidases which degrade the polysaccharide chains of the murein were separated and both muramidase and N‐acetyl‐β‐d‐glucosaminidase activity could be demonstrated. The amoebae also contain proteolytic and lipolytic enzymes which degrade the lipoprotein in vitro. From the lipid which is covalently bound to the protein, palmitic acid was released. All these hydrolases have an optimum of pH 3 which suggests a lysosomal origin.

Amoebae of Dictyostelium discoideum strain ax‐2, grown in a bacteria‐free medium take up isolated murein · lipoprotein complex. These amoebae contain, without prior growth on E. coli cells, the hydrolases which are responsible for the degradation of the murein · lipoprotein complex and for the conversion of diaminopimelate into lysine.
Localization of Antigenic Determinants on Bovine Encephalitogenic Protein
Authors: Bergstrand Håkan
Published: March 2005

By peptic or tryptic proteolysis of different fragments of bovine encephalitogenic protein, a set of small fragments covering the following regions of the native protein have been obtained: 44–68, 44–89, 75–91, 93–116, 114–130, 134–150, and 154–170 (the amino acid residues are numbered according to [1]). These fragments were used in the macrophage migration inhibition technique in guinea pigs (an assay which is vividly considered as a correlate in vitro to delayed type hypersensitivity) to study the localization of antigenic determinants in the protein. The results indicate the presence of at least one determinant in each of regions 44–68, 134–140, and 154–170. Furthermore, the rather strong determinant, which has previously been localized around the single tryptophan residue in the protein does not seem to be contained within peptide 114–130, as this peptide is considered unreactive in the assay. On the other hand, this peptide shows full encephalitogenic activity.
The Function of Amino Groups in the Binding of Iron by Transferrin
Authors: Zschocke Rainer H. , Chiao Marie T. , Bezkorovainy Anatoly
Published: March 2005

Succinylation and a combination of succinylation with reductive alkylation and trinitrophenylation procedures were used to investigate the function of amino groups of transferrin. It was shown that apo‐transferrin had two types of amino groups: those that were easily accessible to chemical modification reagents (60–61 in number) and those that were more difficult to modify (6–7 in number), which were also concerned with the maintenance of the conformation of the protein. The latter type of amino groups became even less accessible to the environment upon the binding of the iron by transferrin. However, the unreactive amino groups were not directly involved in the binding of iron by transferrin, since iron‐binding properties could be partially recovered in the extensively succinylated protein. It was concluded that the function of the amino groups in question was probably to stabilize the three‐dimensional structure of transferrin.
Medium Hypertonicity and Polyribosome Structure in Hela Cells
Authors: Wengler Gerd , Wengler Gisela
Published: March 2005

The influence of hypertonicity of the growth medium on polyribosome structure in Hela cells was measured after double labelling of the cells under conditions where the ribosomal RNA and the polyribosomal messenger RNA were labelled with [14C]uridine and [3H]uridine, respectively.

Increase of the tonicity of the growth medium by addition of NaCl or sucrose leads to a total disaggregation of the cellular polyribosomes within less than 10 min. The process is fully reversible, even if RNA synthesis is blocked by high doses of actinomycin D, and can be repeated at least twice under these conditions.

This reaction is probably caused by a specific block of initiation of protein synthesis under these conditions.

The ribosomes are set free as 80‐S monoribosomes free of messenger RNA, which is found in the lysate in the form of ribonucleoprotein complexes of widely varying sedimentation velocities.

Some possible implications of these findings are discussed.
Sequence of the Murein · Lipoprotein and the Attachment Site of the Lipid
Authors: Braun Volkmar , Bosch Valerie
Published: March 2005

The primary structure of a covalent lipoglycoprotein (murein · lipoprotein) from the cell wall of Escherichia coli is presented. The amino acid sequence consists of 57 residues. The lipid is attached to the N‐terminal serine and the murein is bound to the 6‐amino group of the C‐terminal lysine of the lipoprotein. The repetitive design of the sequence suggests a very conservative evolution in which a structural gene coding for 15 amino acids was duplicated once and then only half of this gene, thus coding for 7 amino acids was fused four times with the first 29 amino acids. Some deletions but only few exchanges of amino acids apparently occurred.

This structural membrane lipoprotein aggregates strongly when released from the murein by lysozyme digestion. The molecular weight was therefore determined in 2%, 0.5% and 0.1% dodecylsulfate by polyacrylamide gel electrophoresis and gel chromatography. The molecular weight of 13000 thus found deviated from that of 10000 calculated from the sum of the amino acids, the lipid as known so far and the part of the murein remaining bound to the lipoprotein. This was not due to a different amount of dodecylsulfate binding by the lipoprotein compared with proteins containing no lipid but may be due to a more spherical shape of the small molecule in contrast to the rod‐like shape of standard proteins in dodecylsulfate.
Metabolism of Prostaglandin F2α in the Rabbit
Authors: Svanborg Kerstin , Bygdeman Marc
Published: March 2005

[17,18‐3H2]Prostaglandin F2α was injected intravenously into female rabbits and the structures of six urinary metabolites were determined. The compounds were 5α, 7α,11‐trihydroxytetranorprost‐9‐en‐1,16‐dioic acid, 5α, 7α,11‐trihydroxytetranorprosta‐1,11‐dioic acid, 5α,7α‐dihydroxy‐11‐oxotetranorprosta‐1,16‐dioic acid, 5α,7α,11‐trihydroxytetranorprost‐9‐enoic acid, 5α,7α,11‐trihydroxytetranorprostanoic acid and 5α,7α‐dihydroxy‐11‐oxotetranorprostanoic acid. Two of these have not been found previously in any species.
The Structural Subunit of Amyloid
Authors: Zuckerberg Avraham , Gazith Joseph , Rimon Abraham , Reshef Tamar , Gafni Joseph
Published: March 2005

“Water‐soluble” amyloid, prepared from three organs obtained from two patients (one with genetic and one with secondary amyloidosis), was purified by sequential treatment with 10% NaCl and 90% acetone. After dissociation in 5 M guanidine‐HCl, ultrafiltration isolated a low molecular weight component which we designated F‐protein. On polyacrylamide‐dodecylsulfate electrophoresis, F‐protein proved to be essentially homogeneous and had a mobility of 1.07 relative to bromophenol blue marker. In all three preparations, the amino acid composition, the 16.4–17.1% nitrogen content, the < 0.25% neutral sugars and the glycine amino terminal indicate a single polypeptide in pure state. This polypeptide, in contrast to the parent material, showed neither green birefringence after Congo red staining nor fibrillar structure on electron microscopy.

Reorganization into fibrils and recovery of staining properties identifying amyloid was obtained by solution of the isolated polypeptide in 8M urea and sequential exchange of the solvent by 0.05 M NaHCO3 and distilled water.

These observations suggest that F‐protein is the structural subunit of the amyloid fibril.
Galactose Transport in Escherichia coli and the Galactose‐Binding Protein
Authors: Vorisek Josef , Kepes Adam
Published: March 2005

Galactose uptake and galactose‐binding protein have been examined in a galactokinase‐deficient mutant of Escherichia coli K108.

[14C]Galactose was found to be accumulated up to one‐hundred‐thousand‐fold in the cytoplasm and to be subject to permanent turnover with unchanged velocity.

The concentration‐dependence curve exhibits sigmoïdicity and reveals a very high affinity of the system for galactose. The spectrum of specificity was explored by competition.

Metabolic inhibitors such as fluoride and uncouplers have weak or moderate effects, only a combination of several inhibitors can cause an efflux at a rate comparable to the rate of uptake.

Among inactivating agents N‐ethylmaleimide, parachloromercuribenzoate and dinitrofluorobenzene have no short term effects. Mercuric chloride and formaldehyde are strong inactivators. The effect of HgCl2 is reversible by excess thiol.

Only small amounts of barium‐precipitable derivatives of galactose (e.g. galactose 6‐phosphate) are observed and these do not exhibit turnover.

The concentrated shock fluid binds [14C]galactose in equilibrium dialysis experiments. The binding curve is sigmoïdal with respect to galactose.

The binding is not abolished by HgCl2. The implications of these findings on possible transport mechanisms are discussed.
Resolution of the Hemolytic and the Hydrolytic Activities of Phospholipase‐C Preparation from Clostridium perfringens
Authors: Sabban Esther , Laster Yehudith , Loyter Abraham
Published: March 2005

Phospholipase‐C preparations from Clostridium perfringens can induce hemolysis of chicken erythrocytes in the presence of EDTA. Under these conditions hydrolysis of membrane phospholipids does not take place. When Ca2+ is added to the incubation media both hemolysis and hydrolysis of phospholipids occur. However, when Mn2+ or UO22+ is added, both the hemolysis and hydrolysis of membrane phospholipids are strongly inhibited.

When phospholipase‐C (Cl. perfringens) preparations are heated at 56°C in the presence of Ca2+, the hemolytic activity is inhibited while the hydrolytic activity of the enzyme is retained. Such non‐hemolytic phospholipase‐C barely hydrolyse phospholipids from intact chicken red‐blood cells. However, it readily hydrolyses phospholipids from erythrocyte ghosts.

Thus it appears that membrane phospholipids are unavailable to the enzyme unless they are exposed by hemolysis.
An Alkaline Protease from Acremonium kiliense
Authors: VAN Heyningen Simon
Published: March 2005

A protease, purified from the growth medium of Acremonium kiliense Grütz, has a pH optimum of 10.5 for the hydrolysis of casein.

It hydrolyzes the reduced, carboxymethylated B‐chain of bovine insulin at 13 points, principally those next to the carboxyl group of aromatic and hydrophobic amino acids, but not at the basic amino acid residues.

It does not hydrolyze dipeptides, benzoyl arginine ethyl ester or glutaryl‐l‐phenylalanine‐p‐nitroanilide.

The enzyme can be titrated with N‐trans‐cinnamoyl imidazole and assayed with the p‐nitrophenyl esters of benzoyloxycarbonyl amino acids.

The enzymic hydrolysis of N‐acetyl phenylalanine ethyl ester can be followed by an absorbance change at 221 nm or by means of a pH‐stat. Analysis of the variation of Km and V with pH for this substrate suggests that there is a group involved in the activity which has a pK of 6.6 in the free enzyme and in the active enzyme · substrate complex.

It is inhibited by diisopropylphosphofluoridate and phenylmethylsulphonyl fluoride, suggesting that it is a serine protease. It is unaffected by metals, metal‐chelating agents, iodoacetate or 2‐mercaptoethanol.
Thermochemistry of the Avidin‐Biotin Reaction
Authors: Suurkuusk Jaak , WadsÖ Ingemar
Published: March 2005

The enthalpy for the binding of biotin to avidin has been determined at 25, 34 and 43°C (pH 9, ammonium acetate buffer): ΔH(25°C) =–22.5 ± 0.1 kcal/mol biotin; ΔCp=–237 ± 12 cal × mol biotin−1× K−1.

Solubilities and enthalpies of solution of biotin in water and in ethanol were determined at 25°C. The transfer process for biotin between water and ethanol was considered as a simple model for the protein binding process.
Influence of Spironolactone and Dexamethasone on Phosphoprotein Phosphatase in the Rat Liver, Kidney and Spleen
Authors: JapundŽIĆ Ivanka , MimiĆ‐OKA Jasmina , JapundŽIĆ Milorad , SzabÓ Sándor
Published: March 2005

Phosphoprotein phosphatase activity is markedly increased in the rat liver, barely stimulated in the kidney, but not influenced in the spleen, by spironolactone treatment. Dexamethasone is ineffective in these respects, whereas cycloheximide, dactinomycin and SKF 525‐A significantly interfered with the action of spironolactone. Our findings demonstrate that spironolactone can induce both microsomal and extramicrosomal enzymes.
Synthesis of ATP‐ and dATP‐Substituted Sepharoses and Their Application in the Purification of Phage‐T4–Induced Ribonucleotide Reductase
Authors: Berglund Ove , Eckstein Fritz
Published: March 2005

p‐Aminophenylesters of ATP and dATP were synthesized and coupled to Sepharose 4B activated by cyanogen bromide. On chromatography of a crude extract of T4‐infected Escherichia coli cells on ATP‐ or dATP‐Sepharose columns a limited number of proteins was adsorbed. They could subsequently be eluted from such columns with ATP or dATP as revealed by dodecylsulfate‐polyacrylamide gel electrophoresis. T4 ribonucleotide reductase was purified on dATP‐Sepharose to about 70% purity from a crude extract, and essentially to homogeneity from a crude ammonium sulfate fraction.
The Threonine‐Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K12
Authors: Véron Michel , Falcoz‐Kelly Françoise , Cohen Georges N.
Published: March 2005

Aspartokinase I‐homoserine dehydrogenase I from Escherichia coli K 12 was subjected to mild proteolysis. A fragment carrying only the desensitized homoserine dehydrogenase activity was purified. It is a dimer having a subunit molecular weight of 55000 as opposed to 86000 for the subunit of the native tetrameric enzyme.

On the other hand, a threonine sensitive aspartokinase devoid of homoserine dehydrogenase activity was extracted from the mutant Gif 108. The purified protein is shown to have subunits shorter than those of the wild‐type enzyme (molecular weight 47000).

It was shown that the two catalytic activities of aspartokinase I‐homoserine dehydrogenase I are sequentially distributed on the single polypeptide chain: the aspartokinase activity is located in the amino‐terminal section and the homoserine dehydrogenase in the carboxyl‐terminal section.

The results are discussed in relation to the configuration of the native enzyme and with the possible origin of the bifunctional protein.
Characterization of Chicken Red‐Cell RNAase
Authors: Sánchez De Jiménez Estela , Léon De Miles Ma. de la Paz , Ruth Román P.
Published: March 2005

RNAase from 13‐day‐old chicken‐embryo red cells was purified. Nuclei and cytoplasmic fractions were used as source of the enzyme. The nuclear enzyme was purified 1440‐fold and the one from cytoplasm, 1683‐fold.

Both enzyme preparations showed the same optimum pH and ion specificity, but different ionic strength dependence and temperature stability.

Kinetic studies with these RNAase fractions indicated that both of them are endonucleases with specificity for pyrimidine nucleotide bases in the substrate, hydrolyzing poly(U) or poly‐(A,U,G) faster than poly(C) or poly(A,C,G). Both enzyme fractions showed strong dependence on the sedondary structure of RNA, and did not hydrolyze single stranded DNA. The implications of these characteristics on the enzyme role are discussed.
Protein Kinase from Rainbow‐Trout‐Testis Ribosomes
Authors: Jergil Bengt
Published: March 2005

A protein kinase specifically associated with the ribosomal fraction has been isolated from rainbow trout testis. The enzyme was extracted from isolated trout‐testis ribosomes in 0.6 M KCl, and was separated from trout testis protamine kinase by hydroxylapatite chromatography. The ribosomal protein kinase is a Mg2+‐dependent enzyme that will transfer the terminal phosphoryl group from ATP into O‐phosphoseryl linkages of the substrate. The enzyme will catalyze the phosphorylation of several basic proteins, and the slightly lysine‐rich histone IIb2 is most readily phosphorylated followed by protamine and ribosomal proteins. Histones I, IIb1 and III are phosphorylated at an intermediate rate, while the arginine‐rich histone IV and an acidic protein such as phosvitin are poor substrates. The enzyme is stimulated by the addition of dithiothreitol, and cyclic AMP enhances enzyme activity between 10 and 25%.
Purification and Characterisation of Crystalline β‐Amylase from Barley
Authors: Visuri K. , Nummi M.
Published: March 2005

Pure crystalline β‐amylase was prepared from ungerminated barley in the presence of mercaptoethanol or dithiothreitol. The preparation consists of four isoenzymes with different isoelectric points. The preparation is monodisperse and has a molecular weight of 57200 in sedimentation equilibrium analysis. One mole of the enzyme binds 2.6 moles of p‐chloromercuribenzoate, indicating a minimum of 3 sulfhydryl groups per molecule. The enzyme polymerizes very rapidly through the sulfhydryl groups in the absence of reducing agents. p‐Chloromercuribenzoate inhibits the polymerization and the enzymic activity. The activity can be restored completely with the reducing agents mercaptoethanol or dithiothreitol. The amino acid composition was found to be relatively similar to that of wheat β‐amylases.
Oxidation‐Reduction Potentials and Ionization States of Two Turnip Peroxidases
Authors: Ricard Jacques , Mazza Gilbert , Williams Robert J. P.
Published: March 2005

In the present paper, differences in the environment of the iron atom of two turnip isoperoxidases, called P1, and P7, have been established. This study is performed by way of spectrophotometric titrations, and by the analysis of oxidation‐reduction potential variations with regard to hydrogen ion concentration.

Turnip peroxidases P1 and P7 exhibit near infrared bands with intensities and energies almost identical to those of metmyoglobin and horseradish peroxidase.

In the case of turnip hemoprotein P1, an ionization, with an apparent pK close to 10, has been shown only in the oxidized form of the enzyme. Two other ionizations, in the ferric and the ferrous forms, respectively, occur also. They possess the same apparent pK, equal to 4.3, and seem to correspond to the same group. Another ionization is seen, with an apparent pK equal to 6.3, which specifically occurs in the reduced peroxidase. These results are very close to those obtained by various workers on horseradish peroxidase.

In the case of turnip hemoprotein P7, the situation is very different. An ionization, with a pK close to 6.5, occurs in the ferric form of the enzyme. Two other ionizations, one in the oxidized form, the other in the reduced form of the enzyme, occur also. They both possess the same apparent pK equal to 8.4. A further ionization pertaining to the ferrous form only, is also seen, with an apparent pK close to 5.

The above results are in agreement with the existence of the same axial ligands, namely histidine and water, in both hemoproteins. A likely interpretation of the ionizations detected in the acidic pH range is to assume that the distal groups of the proteins, for both peroxidases P1 and P7, are different. The occurrence, respectively, of a carboxyl group and a histidine residue would agree with the spectrophotometric and potentiometric titration data.

The high value of the oxidation‐reduction potentials of peroxidase P7, and the unexpected stability of the oxygenated compound of its ferrous form, are also compatible with an oxygenase function of this hemoprotein.
Structure of the Sugar Components of the Quinocycline Complex
Authors: Matern Ulrich , Grisebach Hans , Karl Wolfgang , Achenbach Hans
Published: March 2005

The sugar components of quinocycline A and isoquinocycline A and of quinocycline B and isoquinocycline B could be obtained as methylglycosides by methanolysis of the antibiotics with methanol‐trifluoroacetic acid. The NMR spectrum of the α‐ and β‐methylglycosides of the sugar from quinocycline A and isoquinocycline A is fully consistent with the structure 2,6‐dideoxy‐4‐C‐[1′‐hydroxyethyl]‐l‐xylo‐hexopyranose (Fig, 1, compound III, R = H, α‐anomer) deduced from previous chemical studies and from an X‐ray analysis of isoquinocycline A. The absolute configuration at C‐5 of compound III was confirmed as l by isolation of l‐(+)‐lactic acid after periodate oxidation. The α‐ and β‐methylglycosides of the sugar from quinocycline B and isoquinocycline B show in the mass spectrum a molecular ion (C9H16O5) possessing 2 hydrogen atoms less than that of the methylglycosides of compound III (C9H18O5). For one of the two anomeric methylglycosides from quinocycline B and isoquinocycline B the structure α‐O‐methyl–2,6‐dideoxy‐4‐C‐[1′‐oxoethyl]‐l‐xylo‐ or l‐ribo‐hexopyranoside (Fig.3, compound IVa or IVb) was deduced mainly from the NMR spectrum and by isolation of l‐(+)‐lactic acid after periodate oxidation. The relative configuration of the hydroxyl groups at C‐3 and C‐4 as trans (compound IVa) was established by the fact that the methylglycoside does not form a borate complex. The carbonyl function of compound IVa gives a sharp band at 1740 cm−1 in the infrared.

The structure of compound IVa was confirmed by the formation of the α‐methylglycoside of compound III (R = CH3) as one product of sodium borohydride reduction of the α‐methylglycoside, compound IVa. The sugar of quinocycline B and isoquinocycline B is therefore 2,6‐dideoxy‐4‐C‐[1′‐oxoethyl]‐l‐xylo‐hexopyranose.
Plant‐Leaf Microbodies as the Intracellular Site of Nitrate Reductase and Nitrite Reductase
Authors: Lips S. Herman , Avissar Yael
Published: March 2005

The subcellular location of nitrate reductase and nitrite reductase has been studied. This was done in order to test the feasibility of the functional coupling of the pathways of nitrate reduction and glycolate oxidation. Nitrate reductase and nitrite reductase sediment together with catalase‐containing microbodies in tobacco leaves. Nitrate reductase seems to be located on the membrane of the microbodies and can be easily separated from it without apparently affecting the integrity of the organelle. Nitrite reductase and catalase are located within the microbodies.
Enzyme Localization in Beef‐Heart Mitochondria
Authors: Addink Albert D. F. , Boer Pieter , Wakabayashi Takashi , Green David E.
Published: March 2005

Treatment of heavy beef heart mitochondria with different concentrations of digitonin, followed by centrifugation of the treated suspension resulted in a release of mitochondrial enzymes in a certain order. Monoamine oxidase and rotenone‐insensitive NADH cytochrome c reductase were found primarily in the outer membrane fraction. At low concentrations of digitonin adenylate kinase and creatine kinase were in part released pointing to an intracristal localization; the remainder of these enzymes was found to be associated with the inner membrane‐matrix fraction. The soluble citric cycle enzymes were found in the inner membrane‐matrix preparation. They were released from the matrix in two groups: (a) isocitrate dehydrogenase, malate dehydrogenase, fumarase and aconitase and (b) 2‐oxoglutarate dehydrogenase, pyruvate dehydrogenase and lipoate dehydrogenase. Enzymes intrinsic to the inner membrane were released at concentrations of digitonin higher than 0.4 mg/mg protein (cytochrome oxidase, succinate dehydrogenase, ATPase and 3‐hydroxybutyrate dehydrogenase). The absence of citric cycle enzyme activity, except succinate dehydrogenase in submitochondrial particles prepared by sonication, also points to a localization of these enzymes in the matrix.

From the experiments with the digitonin method it is concluded that the majority of the shared mitochondrial enzymes has a similar localization both in beef heart and rat liver mitochondria.

Detergents such as digitonin, Lubrol WX and Titron X‐100 induce the “twisted” configuration of the inner membrane of beef heart mitochondria, suspended in a sucrose medium, as visualized electron microscopically.
The Preparation and Properties of Triosephosphate Isomerase from Chicken Muscle and a Comparison with that from Rabbit Muscle
Authors: Mcvittie John D. , Esnouf Michael P. , Peacocke Arthur R.
Published: March 2005

A method is described for the purification of the enzyme triosephosphate isomerase from chicken breast muscle. The physical properties are compared with those of triosephosphate isomerase from rabbit muscle.

The enzyme prepared from chicken muscle was homogeneous as judged by electrophoresis in polyacrylamide gels and equilibrium sedimentation studies.

The molecular weight of the native chicken muscle enzyme was 48400 ± 700 (S.D.) and the subunits had a molecular weight of 25000 ± 1000 (S.D.). The values obtained for the rabbit muscle enzyme were 50500 ± 1500 (S.D.) and 26000 ± 1000 (S.D.), respectively.

On the basis of sedimentation velocity runs with various molarities of guanidinium chloride as solvent, the separation of subunits and the unfolding of these subunits was shown to occur simultaneously upon denaturation of the chicken muscle enzyme.

Some preparations of guanidinium chloride, which was used as a denaturant, were shown to possess lytic activity.
The Structure of Erythrocyte Membranes
Authors: Capaldi Roderick A.
Published: March 2005

The membrane proteins of beef erythrocyte ghosts have been fractionated on the basis of their different interactions with lipid. The individual proteins in each fraction have then been characterized by polyacrylamide gel electrophoresis. Fractions I and II, which were solubilized from the membrane by distilled water and 1.2 M NaCl, respectively, amounted to 46% of the total membrane protein and included virtually all the polypeptide‐chains with molecular weights greater than 200000. These proteins must be linked to the membrane by predominantly ionic interactions, indicating that they are extrinsic proteins of the erythrocyte membrane.

A considerable proportion of membrane protein (fraction III) was not liberated by distilled water or 1.2 M NaCl but could be separated from lipids by sodium dodecylsulphate, a reagent which disrupts hydrophobic interactions. 10 different polypeptides were identified in this fraction, including four glycoproteins. One polypeptide of molecular weight 87000 accounted for about half of the protein‐staining components. The proteins in this fraction are likely to be intrinsic membrane proteins which penetrate deeply into the lipid bilayer of the erythrocyte membrane.
The Effect of α‐Amanitin and (NH4)2SO4 on RNA Synthesis in Nuclei and Nucleoli Isolated from Physarum polycephalum at Different Times during the Cell Cycle
Authors: Grant William D.
Published: March 2005

Nuclei isolated from various points in the mitotic cycle were examined for RNA synthesis in the presence of Mg2+ or Mn2++ (NH4)2SO4. A peak of activity 2.5—3 h after mitosis was only slightly increased by Mn2++ (NH4)2SO4 over that in the presence of Mg2+, and inhibited some 50% by low levels of α‐amanitin in the presence of either Mg2+ or Mn2++ (NH4)2SO4. A G2 peak of activity 2.5—3 h before mitosis was 100% increased by Mn2++ (NH4)2SO4 over that observed in the presence of Mg2+, the increase being completely sensitive to low levels of α‐amanitin, whereas the Mg2+ activity remained insensitive. Nucleoli isolated some 2.5—3 h before mitosis exhibited RNA synthesis which was unstimulated by Mn2++ (NH4)2SO4 and insensitive to α‐amanitin. Similarities in RNA synthesis in isolated nucleoli and isolated G2 nuclei in the presence of Mg2+ with respect to pH optima and actinomycin‐D sensitivity were observed. It is concluded that there is a high basal level of α‐amanitin‐insensitive RNA synthesis activity which is nucleolar and therefore probably ribosomal, rising to a peak some 2.5—3 h before mitosis, and a major peak of α‐amanitin‐sensitive nucleoplasmic RNA synthesis activity some 2.5—3 h after mitosis, thereafter dropping to a low level.
Effects of a Glucocorticoid on the Concentrations of CoA and Carnitine Esters and on Redox State in Bovine Liver
Authors: Baird G. David , Heitzman Raymond J. , Snoswell Alan M.
Published: March 2005

In this study, the hepatic concentrations of free and acetylated CoA and carnitine, and also hepatic redox state, have been determined in normal lactating cows and in lactating cows to which voren (dexamethasone 21‐pyridine‐4‐carboxylate) was administered 48 h previously.

The values for the hepatic [CoA]/[acetyl‐CoA] and [carnitine]/[acetylcarnitine] ratios in the normal cow were found to be lower than the corresponding values previously reported for other species. Voren administration brought about an increase in both these ratios. Only the increase for the [CoA]/[acetyl‐CoA] ratio was statistically significant, however.

Using the lactate dehydrogenase and glutamate dehydrogenase systems, respectively, values were obtained for the cytosolic and mitochondrial [NAD+]free/[NADH]free ratios in normal bovine liver that were similar to the corresponding values previously obtained for rat liver. Following voren administration there was a fall in the cytosolic value for this ratio and a rise in the mitochondrial value, so that the equilibrium between the cytosolic and mitochondrial redox states appeared to be disrupted.

Following voren administration there was an increase in the degree of reduction of total NAD.

The results are discussed in relation to the antiketogenic action of voren in the cow.
Proton‐Relaxation‐Enhancement Studies on the Binding to Yeast Pyruvate Kinase of a Substrate and Effectors
Authors: Fell David A. , Peacocke Arthur R. , Dwek Raymond A.
Published: March 2005

The binding to yeast pyruvate base of Mn2+ has been determined from the enhancement of the proton relaxation rate which it causes. There were further changes in this enhancement in the presence of a substrate (phosphoenolpyruvate) and the effector (fructose diphosphate) and, from these changes, information was derived about their binding.

Both strong and weak binding sites for Mn2+ on the enzyme were detected.

The affinity of the enzyme for phosphoenolpyruvate was increased when the fructose diphosphate · Mn complex was bound to the enzyme.

The proton relaxation enhancement results indicated that there were not more than two sites for fructose diphosphate · Mn, and two sites for phosphoenolpyruvate in the presence of the fructose diphosphate · Mn complex, but that there may have been more than two sites for phosphoenolpyruvate in the presence of Mn2+ only.
Structural Investigations on Cell Walls of Nocardia sp.
Authors: Vacheron Marie‐Jeanne , Guinand Micheline , Michel Georges , Ghuysen Jean‐Marie
Published: March 2005

The walls of Nocardia kirovani are composed of three main constituents: the peptidoglycan matrix, a polysaccharide polymer and a variety of free and bound lipids. The free lipids represent 17.5% (dry weight) of the walls and consist for the major part of C16–18 fatty acids and nocardic acids, and for the minor part, of nocardones, triglycerides and carotenoid pigments. The nocardic acids were identified as tri‐ and tetra‐unsaturated, α‐branched, β‐hydroxylated compounds C58H110O3—C66H124O3 the nocardones as tri‐and tetra‐unsaturated ketones C57H106O—C63H1180O, and the main carotenoid pigment as phlei‐xantophylle palmitate. Esters of glycerol with C14, C16, C18 fatty acids and, for some of them, with odd numbered poly‐unsaturated acids containing 35 to 45 carbon atoms, were also identified. Bound lipids represent about 20% (dry weight) of the walls and consist mainly of nocardic acids probably ester‐linked to an arabinogalactan polymer. The peptidoglycan (about 40% dry weight) is composed of β‐1,4‐N‐acetylglucosaminyl‐N‐glycolylmuramic acid disaccharide units that are substituted by diamidated l‐Ala‐d‐αGln‐(l)‐A2pm‐(d)‐NH2 tripeptides and diamidated l‐Ala‐d‐αGln‐(l)‐A2pm‐(d)‐NH2‐(l)‐d‐Ala tetrapeptides, where A2pm is meso‐diaminopimelic acid. Crosslinking between some of the peptide units is mediated through d‐Ala‐(d)‐A2pm linkages (peptidoglycan of chemotype I).
Structural Studies of Alcohol Dehydrogenase from Rat Liver
Authors: Jörnvall Hans , Markovič Oskar
Published: March 2005

The amino acid composition of alcohol dehydrogenase from rat liver was determined. Tryptic peptide maps of the carboxymethylated protein were compared with those of the corresponding derivative of the horse enzyme. Large peptides, not ordinarily resolved by this method, were redigested with thermolysin or chymotrypsin to permit a comparison of the entire proteins. The amino acid sequences of peptides of special interest were determined.

A subunit molecular weight close to 40000 is established for rat liver alcohol dehydrogenase. The protein is homologous to the corresponding equine and human enzymes; sequence analysis of about one‐fifth of all residues in the protein chain of the rat enzyme shows a structural identity of about 80% among the subunits of the enzymes from man, horse and rat. The rate of evolution in alcohol dehydrogenase is estimated. Species differences of special structural or functional interest include exchanges of cysteine residues and of amino acids adjacent to the “active‐site” cysteine residue.

No evidence was detected for subunits of different primary structures in rat liver alcohol dehydrogenase; neither the subunit differences characteristic for the human nor for the equine isoenzymes were found. It thus seems as if isoenzymes of liver alcohol dehydrogenase have evolved independently in these three species.
Are Serum Lipoproteins Esterases?
Authors: Kaminski Marie , Dubois Philippe
Published: March 2005

The immune precipitates of high‐density lipoprotein of duck serum with rabbit antisera are active on an esterase substrate, while the high‐density lipoprotein itself is not active.

The immune precipitates formed by high‐density lipoprotein with isolated immunoglobulins, either in agar gel or in liquid medium, were also shown to be inactive.

Such inactive immune precipitates can become active through interaction with a component of rabbit serum.

Investigations on the conditions of the reaction and first observations concerning the nature of the activating component are reported.
Über die Wechselwirkung des Actinomycin C3 mit Mono‐, Di‐ und Oligonucleotiden
Authors: Schara Reinhard , Muller Werner
Published: March 2005

Sequence specificity in the binding of the actinomycins to DNA has been studied by measuring the dissociation constants of several deoxyribodinucleotide‐actinomycin complexes by spectral‐titration. Additional data for some mononucleotides and for a deoxyribotetranucleotide have been collected for comparison. The results show that:

G‐Containing dinucleotides form complexes of roughly 10 times higher stability than G‐free dinucleotides. These data parallel the corresponding results for mononucleotides [16].

The G‐containing dinucleotides may be subdivided into three classes of the following sequential types:

a) d(pX‐G), which bind the actinomycins with similar affinity as the dGMP,

b) d(pG‐X), which bind the actinomycins less strongly than dGMP, and

c) d(pG‐C), which binds the actinomycins in a 2:1 complex (two dinucleotides per dye molecule) of higher stability than all the other dinucleotides.

The tetranucleotide d(pC‐T‐A‐G) behaves as the dinucleotides of type a.

Molecular weights of the complexes as determined by sedimentation equilibrium measurements confirmed the stoichiometry of the actinomycin‐d(pG‐C) complex obtained by titration. Corresponding measurements for the actinomycin complexes of d(pC‐G) and d(pC‐T‐A‐G) revealed that the antibiotic strongly favours double helix formation of these self complementary oligonucleotides, yielding 2:2 complexes with two equivalent binding sites at the ends of the helices.

All data are consistent with fixation of the actinomycin chromophore on the 3′‐side of the dG‐residue as in the actinomycin‐dG crystal [8].
The “Free” Lipids of Two Different Strains of Hydrogen‐Oxidizing Bacteria in Relation to Their Growth Phases
Authors: Thiele Otto Wolfgang , Dreysel Jörg , Hermann Dieter
Published: March 2005

Two strains of hydrogen‐oxidizing bacteria were used: Hydrogenomonas eutropha strain H16 and strain 11/x. The former is a gram‐negative flagellated rod and has been relatively well known with respect to its morphological, physiological, and biochemical properties. The latter is a gram‐positive, non spore‐forming, motile small rod the definite classification of which is unknown as yet.

Both strains were grown chemolithotrophically in submerged culture with a limited nitrogen‐source and supplied with a gas mixture of H2, O2, and CO2. The lipid composition of the bacteria was determined at the end of the exponential phase (I), at the storage phase (II), and after reutilization of storage material (III).

Along with the accumulation of poly(3‐hydroxybutyric acid) as the major storage material in strain H16 the amount of other lipids was shown to increase 1.8‐fold at phase II and to decrease at phase III. There is little or no change in the lipid pattern in various growth phases. It is suggested that the 1.8‐fold increase of the latter lipids during phase II is due to the synthesis of membrane lipids that are required for the formation of membranes surrounding the intra‐cellular granules of accumulating poly(3‐hydroxybutyric acid).

In strain 11/x the amount of lipids increases 7 times along with an equal increase of carbohydrates at phase II. The majority of accumulated lipids consists of triglycerides free of significant amounts of unusual acyl group. It is suggested that there is a true storage of triglycerides that are reutilized during phase III.

The lipid pattern of strain H16 is characterized by a high proportion of “strongly bound lipids” that consist predominantly of phospholipids. Characteristic lipid components are phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol, while coenzyme Q8 (0.41 μmol/g bacterial dry weight) has been found among the neutral lipids. A C17 cyclopropane acid (probably cis‐9,10‐methylene hexadecanoic acid) is present in all saponifiable lipids.

The lipid pattern of strain 11/x is characterized by a high proportion of “loosely bound lipids” that consist predominantly of neutral lipids, particularly of triglycerides. Among the polar lipids, diphosphatidylglycerol and phosphoinositides are most abundant, and several unknown phospholipids are also present. No cyclopropane fatty acid has been found. The possible use of strain 11/x as a source of nutritional fat is discussed.
The Conversion of [4‐3H]Fructose and of [4‐3H]Glucose to Liver Glycogen in the Mouse
Authors: Hue Louis , Hers Henri‐Géry
Published: March 2005

[4‐3H,6‐14C]Fructose or [4‐3H,6‐14C]glucose were injected into mice and the recovery of the two isotopes in liver glycogen was measured. As a mean, the 3H/14C ratio in glycogen was 36.6 and 51.8% of that in the injected fructose and glucose, respectively. From the comparison of these two ratios, the loss of tritium proper to fructose metabolism was equal to 29.2%. In the same experiments, the C‐6 to C‐1 randomization proper to fructose metabolism was, as a mean, equal to 27.4%. These findings are in agreement with the hypothesis that the glyceraldehyde formed in the liver upon aldolic splitting of fructose 1‐phosphate is metabolized by phosphorylation into glyceraldehyde 3‐phosphate under the action of triokinase. The oxidation of d‐glyceraldehyde into d‐glycerate would have resulted in a complete loss of tritium in glycogen. Furthermore, the 3H/14C ratio in d‐glyceraldehyde formed from fructose was the same as in the precursor despite the fact that the aldehyde dehydrogenase discriminates against [1‐3H]glyceraldehyde. Our results therefore preclude any important participation of the glycerate pathway in the metabolism of fructose.

In the isolated rat diaphragm, the loss of tritium relative to 14C was of the order of 20%, for both glucose and fructose, confirming that, in muscle these two sugars are converted to glycogen by the same pathway.

When incubated in vitro with a liver preparation deprived of glucose 6‐phosphatase, doubly labelled UDPG lost no 3H relative to 14C. Doubly labelled hexose 6‐phosphates underwent a rapid loss of tritium in the same conditions, and this loss was cancelled by fructose diphosphate. It appears probable that the tritium loss is due to an interconversion of hexosephosphates with pentosephosphates, the hydrogen exchange occurring at the level of the pentosephaosphate 3‐epimerase.

In agreement with an exchange of hydrogen at the level of hexose 6‐phosphates in the liver, it was observed that the decay of [4‐3H]glucose in the blood is faster than that of [U14C]glucose.
Deoxyribonucleoside Triphosphate Pools of Normal and Transformed Baby‐Hamster‐Kidney Cells
Authors: Bjursell K. Gunnar , Reichard Peter A. , Skoog K. Lambert
Published: March 2005

Pools of the four deoxyribonucleoside triphosphates were determined during growth in tissue culture of baby hamster kidney normal cells and cells transformed either by wild‐type polyoma virus or a temperature‐sensitive mutant of polyoma (ts‐3). The cells are transformed by ts‐3 at 31°C but lose most of their transformed character at 39°C. The three cell lines did not show any major differences with respect to pool sizes at 39°C: during rapid growth, dCTP formed the largest and dGTP the smallest pool, while the dATP and dTTP pools were of intermediate size. On cessation of growth all pools decrease in size; the decrease was very pronounced for dCTP but slight for dATP.

The results at 31°C were similar to those at 39°C with one exception: the two transformed lines contained about twice as much dTTP as the normal cell line under comparable growth conditions. Our results suggest that after transformation a change occurs in the control of one or several of the enzymes leading to the synthesis of dTTP. This change is manifested only at the lower temperature.
Transcription in vitro of T3 DNA by Escherichia coli and T3 RNA Polymerases
Authors: Dunn John J. , Mcallister William T. , Bautz Ekkehard K. F.
Published: March 2005

RNA isolated from Escherichia coli infected by bacteriophage T3 was sedimented through sucrose gradients. The size distributions of the mRNA species coding for three T3‐specific enzymes, the S‐adenosyl‐l‐methionine cleaving enzyme (adenosylmethioninase), phage RNA polymerase and lysozyme, were then investigated by using the fractionated RNA to program a cell‐free protein‐synthesizing system followed by enzymatic analysis of the translation products. Identical analyses of transcripts produced in vitro by E. coli and T3 RNA polymerases were performed and it was found that E. coli RNA polymerase, in conjuncton with termination factor (ϱ), was capable of synthesizing early mRNA species (mRNA for adenosylmethioninase and phage polymerase) apparently identical in size and coding capacity to those isolated from infected cells. Transcripts synthesized by the T3 RNA polymerase, while consisting predominantly of late RNA species, were found to contain functional mRNAs for adenosylmethioninase, phage RNA polymerase and lysozyme.

The transcription patterns obtained are consistent with the assignment of one promotor for the region situated near the left terminus of the genome.