bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of Hydrogen Translocating Shuttles during Ethanol Oxidation in Hepatocytes from Euthyroid and Hyperthyroid Rats
Authors: HENSGENS Lambert A. M. , NIEUWENHUIS Benedictus J. W. M. , VAN DER MEER Roelof , MEIJER Alfred J.
Published: March 2005

A quantitative study of the contribution of the malate‐aspartate and glycerol‐3‐phosphate cycles to the translocation of reducing equivalents from cytosol to mitochondria during ethanol oxidation has been made in hepatocytes from euthyroid and hyperthyroid rats.

1. In hepatocytes from euthyroid rats both cycles have an almost equal capacity and their relative contribution to total hydrogen transport to the mitochondria depends on the conditions chosen.

2. In hepatocytes from hyperthyroid rats maximal rates of ethanol oxidation were significantly lower than in hepatocytes from euthyroid rats, even though the capacity of the glycerol‐3‐phosphate cycle was increased. This was due to a decreased activity of alcohol dehydrogenase.
Three‐Step Purification of Glucocorticoid Receptors from Rat Liver
Authors: GOVINDAN Manjapra V. , MANZ Bernhard
Published: March 2005

The 90000‐Mr glucocorticoid receptor was purified to homogeneity according to sodium dodecyl‐sulfate/polyacrylamide gel electrophoresis. An affinity column containing either dexamethasone‐17β‐carboxylic acid or dexamethasone‐21‐methanesulfonate bound to the matrix with the help of a disulfide bond is used in this study. Using this affinity matrix, in a single step, 8700‐fold purification of glucocorticoid receptor from rat liver cytosol could be achieved. Following the method of activation and DNA‐cellulose chromatography the 90000‐Mr receptor subunit was purified to homogeneity.
Neutral Deoxyribonucleases of HeLa S3 Cells
Authors: SLOR Hanoch , LEV‐SOBE Tarns , ZÖLLNER Jürgen
Published: March 2005

Extracts of HeLa S3 cells were electrophoresed on polyacrylamide gels; gel slices were eluted and the eluates were assayed for DNase activities against native and denatured DNA substrates in the presence of MgCl2 or Na2EDTA. Aliquots of each eluate were also assayed for their ability to nick the circular supercoiled PM2 phage DNA to distinguish endonucleases from exonucleases. Peaks of endonuclease activities were characterized as forming 3′‐phospho‐oligonucleotides or 5′‐phospho‐oligonucleotides by the use of oligonucleotides produced by these enzymes as substrates for the 5′‐phosphate‐specific snake venom exonuclease. The total activity of DNases in gel eluates was much higher than that in cell extract applied to the gel, indicating the presence of inhibitors in the cell extract.
Isomeric Incorporation of the Haem into Monomeric Haemoglobins of Chironomus thummi thummi
Authors: KRÜMPELMANN Dieter , RIBBING Wilfried , RÜTERJANS Heinrich
Published: March 2005

13C‐NMR studies on 13CO complexes of the components III and IV of the monomeric haemoglobins from the subspecies Chironomus thummi thummi have been carried out in order to confirm the existence of two conformational isomers differing by the isomeric incorporation of the haem group and the extent of the Bohr effect. In addition, the allosteric linkage between the ligand binding site and the Bohr proton binding site in the component IV is described from investigations of various pH‐dependent proton resonances including the C‐2 proton resonances of the titratable histidines. In comparison to the data obtained for the component III it is assumed that in both conformational isomers of the component IV the allosteric linkage between the distal site of the haem and the main Bohr proton donating group is present.

From corresponding NMR investigations of the component I an isomeric incorporation of the haem group into this monomeric haemoglobin seems unlikely. Also, any correlation between the ligand binding site and a titratable group of the protein has not been found in this haemoglobin, in agreement with previous results of other laboratories that a Bohr effect in the component I cannot be detected.
Lack of Gross Protein Structure Changes in the Working Cycle of (Na+, K+)‐Dependent Adenosinetriphosphatase
Authors: CHETVERIN Alexander B. , VENYAMINOV Sergei Yu. , EMELYANENKO Viktor I. , BURSTEIN Edward A.
Published: March 2005

Infrared and tryptophan fluorescence spectra of practically all sufficiently stable functional complexes of a highly purified preparation of membrane‐bound (Na+, K+)‐dependent ATPase have been measured. The formation of any functional complex was not accompanied by any considerable change of either shape or position of the tryptophan fluorescence spectrum. Only in the presence of adenine nucleotides was there a small decrease of fluorescence intensity (by 5–8%), which apparently results from a change of the sample light scattering. Analysis of the results obtained leads to the conclusion that the environment of no more than one or a few tryptophan residues may differ in all the (Na+,K+)‐ATPase complexes studies. A comparison of infrared protein spectra in the region of amide I band showed that at any wavenumber the differences between them did not exceed 3% of the maximum absorption. This means that no more than 3% of protein peptide groups can change their conformation upon transition between the enzyme functional states. These results, obtained by two independent techniques, allow us to conclude that even if changes of the internal protein structure occur during the working cycle of this transport system, they have an extremely local character.
Structure and Synthesis of a Lipid‐Containing Bacteriophage Studies on the Structure of the Bacteriophage PM2 Nucleocapsid
Authors: SATAKE Haruhiko , AKUTSU Hideo , KANIA Malgosia , FRANKLIN Richard M.
Published: March 2005

The nucleocapsid of bacteriophage PM2, prepared according to Schäfer et al. [Eur. J. Biochem. 92, 579–588 (1978)], was studied by biochemical and biophysical methods. It was not possible to isolate the lipid‐free nucleocapsid. More than 95% of the lipids were associated with the nucleocapsid. The asymmetric distribution of phospholipids across the viral membrane was retained in the nucleocapsid since less than 10% of the phosphatidylethanolamine was accessible to the non‐penetrable membrane probe, 2,4,6‐trinitrobenzenesulfonate.

Micro‐dissection of the nucleocapsid with thermolysin demonstrated the asymmetric orientation of core proteins across the nucleocapsid membrane. Protein III was embedded deeply in the lipid bilayer and about 20% of the molecule extended to the exterior. Protein IV interacts with PM2 DNA and is partially in the inner leaflet of the bilayer.

Small‐angle X‐ray scattering studies on the nucleocapsid enabled us to localize the lipid bilayer structure at radii from 20.5 nm to 24.0 nm.
Lipid A Induces cells, Uniquely Present in Bone Marrow, to Secrete Proteins other than Immunoglobulins
Authors: GOLLAPUDI Sastry V. S. , KERN Milton
Published: March 2005

The lipid‐A moiety of lipopolysaccharide induced freshly isolated bone marrow cells incubated with radioactive leucine to exhibit enhanced release of proteins other than immunoglobulins. Such stimulation by lipopolysaccharide was essentially not observed with cell suspensions from spleen, thymus, appendix, peritoneal exudate, whole blood, lymph node, liver, testis, buffy coat of blood and reticulocyte‐enriched blood. The extracellular appearance of proteins was shown to be due to secretion by excluding other alternatives. Thus, the rate of cell death and/or leakage of cellular contents, as well as the rate of shedding of surface membrane protein, was unaffected measurably by lipopolysaccharide. Secretion of non‐immunoglobulin proteins was selective as judged by the finding that the rate of immunoglobulin released by bone marrow cells during the usual four‐hour pulse‐label period was not stimulated by lipopolysaccharide. Enhancement of mitogenesis and enhancement of secretion of non‐immunoglobulin protein by lipopolysaccharide appeared to occur at independent sites or even in different cells because splenocytes which readily exhibited mitogenic response to lipopolysaccharide essentially did not exhibit stimulation of secretion of non‐immunoglobulin protein.
Isolation and Characterization of Two Molecular Forms of Octopine Dehydrogenase from Pecten maximus L.
Authors: MONNEUSE‐DOUBLET Marie‐Odile , LEFEBURE Francine , OLOMUCKI Anna
Published: March 2005

Two forms, A and B, of octopine dehydrogenase from Pecten maximus L. striated adductor muscles were separated by ion‐exchange chromatography and purified to homogeneity.

Their kinetic properties were similar and, among others, the mnemonical behaviour, previously found for octopine dehydrogenase, was confirmed. Structural features, determined by polyacrylamide gel electrophoresis with and without sodium dodecylsulfate, amino acid composition, immunological tests and heat stability were compared. The only differences between the two forms are their charge and their sensibility to various chemical treatments. Assays of reciprocal conversion of the two forms by oxidation, reduction or deamidation failed. No tissue specificity and no relation to any physiological conditions could be observed. However, a constant ratio A:B = 1:4 was statistically found in crude extracts as well as in the purified enzyme. It therefore seems possible to assume that the two forms of octopine dehydrogenase pre‐exist in living P. maximus, although their genetic origin has to be established.
Structure de la chrysantelline B, nouvelle saponine isolée de Chrysanthellum procumbens Rich
Authors: BECCHI Michel , BRUNETEAU Maud , MICHEL Georges , TROUILLOUD Michel , COMBIER Henri , PONTANIER Henri
Published: March 2005

Structure of Chrysantellin B, a New Saponin Isolated from Chrysanthellum procumbens Rich

A new saponin, chrysantellin B, has been found in a tropical plant Chrysanthellum procumbens Rich as a minor companion of chrysantellin A which was previously studied. The structure of chrysantellin B was determined by mass spectrometry, proton and 13C nuclear‐magnetic resonance. The aglycone part is a triterpene: 3β,16α,23‐trihydroxy‐olean‐12‐en‐28‐oic acid or caulophyllogenin. Carbohydrate components are D‐glucose, D‐xylose and L‐rhamnose in a molar ratio 1:2:2, The structure of chrysantellin B was established as 3‐O‐β‐D‐glucopyranosyl[L‐rhamnopyranosyl‐(α1 → 3)‐D‐xylopyranosyl‐(β1 → 4)‐D‐rhamnopyranosyl‐(α1 → 2)‐D‐xylopyranosyl‐(β1 → 28)]caulophyllogenin.
Evidence that Ionization of Zinc‐Bound Water Regulates the Anion‐Binding Capacity of the Coenzyme‐Binding Site in Liver Alcohol Dehydrogenase
Authors: ANDERSSON Pia , KVASSMAN Jan , LINDSTRÖM Anders , OLDÉN Bertil , PETTERSSON Gösta
Published: March 2005

1. The mechanistic and structural origin of the pKa 9.2 dependence of coenzyme association and anion binding to liver alcohol dehydrogenase has been investigated by titrimetric and spectrophotometric binding studies involving ligands (imidazole, 1,10‐phenanthroline and 2,2′‐bipyridine) which combine to the catalytic zinc ion of the enzyme subunit with displacement of zinc‐bound water.

2. Imidazole abolishes the pKa 9.2 dependence of NADH binding to the enzyme. The pH dependence of ADP‐ribose and Pt(CN)2‐4 binding is similarly abolished by imidazole, as well as by 1,10‐phenanthroline and 2,2′‐bipyridine. It is concluded from these results that the pKa 9.2 dependence of coenzyme and anion binding most likely derives from ionization of zinc‐bound water.

3. Evidence is presented showing that the protonation state of the pKa 9.2 group also regulates ligand binding to the catalytic zinc ion, which lends strong support to the conclusion that this ionizing group can be identified as zinc‐bound water. The pKa, 9.2 dependence of bipyridine binding is abolished by ADP‐ribose and Pt(CN)2‐4, indicating that complex formation at the anion‐binding subsite of the coenzyme‐binding site perturbs the pKa of zinc‐bound water to a value above 10.

4. The cooperative interrelationship between ionization of zinc‐bound water and complex formation at the anion‐binding subsite is proposed to be attributable to a competition between the zinc‐bound hydroxyl ion and external anionic ligands for salt bridge formation with the guanidinium group of Arg‐47. This explains why ionization of zinc‐bound water affects the anion‐binding capacity of the enzyme and why complex formation at the anion‐binding subsite affects the pKa of zinc‐bound water.

5. The kinetics of complex formation with bipyridine are consistent with a two‐step binding mechanism in which a pKa 9.2 dependent rapid pre‐equilibration between enzyme and ligand is followed by a pH independent rate‐limiting formation of the chromophoric binary complex. Desorption of zinc‐bound water, therefore, is likely to take place in the primary association step rather than in the subsequent rate‐limiting step. This renders the possibility less likely that productive ternary complexes formed during catalysis may contain a zinc‐bound penta‐coordinate water molecule.
Relation of the Antiproliferative Action of Methylglyoxal‐bis(guanylhydrazone) to the Natural Polyamines
Authors: SEPPÄNEN Pauli , ALHONEN‐HONGISTO Leena , JÄNNE Juhani
Published: March 2005

Enzymic determinations of intracellular concentrations of methylglyoxal‐bis(guanylhydrazone), an anticancer drug which inhibits the synthesis of the polyamines spermidine and spermine, in cultured tumor cells revealed that the drug was remarkably effectively concentrated inside the cell. A concentration gradient across the cell membrane as great as 500–1000‐fold was formed in cells exposed to the drug for 1–2 days. An exposure of cultured Ehrlich ascites carcinoma cells to increasing concentrations of the drug indicated that the cells could tolerate intracellular concentrations up to 1 mM with only slight changes in their proliferation rate. Micromolar concentrations of spermidine or spermine, but not putrescine, effectively blocked the uptake of methylglyoxal‐bis(guanylhydrazone) and reduced the intracellular concentration of the drug below the levels required for growth inhibition.

Analysis of cellular polyamine contents in Ehrlich ascites cells exposed to rising concentrations of methylglyoxal‐bis(guanylhydrazone) gave little support to the view that the drug‐induced growth inhibition was solely produced by an intracellular polyamine deprivation.

Not only was the uptake of the drug inhibited in the presence of spermidine and spermine, but it was likewise washed out by polyamines from the cells that had been previously exposed to the drug and then transferred into drug‐free medium in the presence of polyamines.

For the inhibition of methylglyoxal‐bis(guanylhydrazone) uptake by amines, three or more amino/imino groups were apparently required, since low concentrations of aliphatic diamines were either without any effect (short‐chain diamines) or only marginally prevented (long‐chain diamines) the uptake of the drug. High concentrations of Mg2+ ions, however, markedly inhibited the transport of the drug into Ehrlich ascites tumor cells.
Induction of Plasma Proangiotensin by Steroid Hormones in Nephrectomized Rats
Authors: VOIGT Jürgen , KÖSTER Hubert
Published: March 2005

The response of plasma proangiotensin to various steroids was studied in bilaterally nephrectomized rats. In male animals, estradiol and testosterone increased the proangiotensin level up to 250% and 180% respectively. In female animals, both hormones lead to an increase of proangiotensin up to 220% of the controls, when given at optimal dose. The dose dependence on estradiol and testosterone was about the same in male rats, whereas in female animals the formation of proangiotensin was stimulated by much lower doses of estradiol as compared to testosterone. Adrenalectomy plus nephrectomy reduced the proangiotensin level to 20–30% of the value measured in animals nephrectomized only. Cortisol caused a rapid increase of plasma proangiotensin up to 1000% in adrenalectomized animals. Independent from the adrenal state, the amounts of cortisol necessary to induce proangiotensin were very low as compared to the dose response of other proteins, biosynthesis of which is regulated by cortisol. 21‐Dehydrocortisol and aldosterone induced proangiotensin with the same efficiency as cortisol, whereas several other chemically related steroids were less active or inactive. Comparing the biological activity of the various steroids tested, it has to be concluded that the Δ4‐ene‐3‐one structure of ring A, the 11β‐OH group of ring C and the carbonyl group at C‐20 of the ketol side‐chain of cortisol are very important with respect to proangiotensin induction. The response of proangiotensin to cortisol, 21‐dehydrocortisol and aldosterone could be inhibited by actinomycin D and cycloheximide, whereas the effects of estradiol and testosterone could be reversed by cycloheximide only.
Structure of the 5‐S Ribosomal RNA Gene and Its Adjacent Regions in Torulopsis utilis
Authors: TABATA Satoshi
Published: March 2005

A DNA segment spanning one repeating unit of ribosomal RNA(rRNA) genes in a yeast strain, Torulopsis utilis, has been cloned on a bacterial plasmid pBR322. The size of the cloned segment was about Mr, = 8.0 × 106. All of the genes for the four species of rRNA were linked on it, and there was a long spacer between the 5‐S and 18‐5 rRNA coding regions.

The nucleotide sequences of the regions flanking the 5‐S rRNA gene were determined and compared with those in the corresponding regions of Saccharomyces cerevisiae [Valenzuela, P., Bell, G. I., Masiarz, F. R., DeGennaro, L. J., and Rutter, W. J. (1977) Nature, 267, 641–643; Maxam, A. M., Tizard, R., Skryabin, K. G., and Gilbert, W. (1977) Nature, 267, 643–645]. The sequence of the T. utilis 5‐S rRNA is identical with that of S. cerevisiae except for two residues at positions 18 and 61. However, its upstream region contained a quite different sequence, and a sequence which showed some homology was only found at positions – 21 to – 28. The sequences were d(T‐G‐T‐A‐A‐C‐C‐T) in T. utilis and d(T‐A‐T‐C‐A‐C‐C‐T) in S. cerevisiae. Although the presence of various repeat sequences having the same or opposite directions was noted, these repeats occurred at different positions in the two yeast species.

In the downstream region, the common sequence was only seven dT deoxynucleotides, which occurred immediately after the 5‐S rRNA coding sequence. Significant direct and inverted repeat sequences were found in T. utilis, but such repeats are not seen in S. cerevisiae.
Hemolysis of Rat Erythrocytes by Replacement of the Natural Phosphatidylcholine by Various Phosphatidylcholines
Authors: LANGE Louis G. , VAN MEER Gerrit , OP DEN KAMP Jos A. F. , VAN DEENEN Laurens L. M.
Published: March 2005

Vesicles of a variety of types of phosphatidylcholine have been shown to be suitable donors of phosphatidylcholine to intact rat erythrocytes in the presence of a specific phosphatidylcholine exchange protein. Coincident with the progression of phosphatidylcholine exchange is the onset of hemolysis, occurring at degrees of exchange characteristic for the type of phosphatidylcholine employed. During exchange with the dimyristoyl, dipalmitoyl and distearoyl species, hemolysis starts when 27%, 25% and 22% of the native phosphatidylcholine is replaced by these disatured species, respectively. In the case of dielaidoylglycerophosphocholine and 1‐stearoyl‐2‐oleoylglycero‐phosphocholine hemolysis starts after the introduction of 30% and 28%, respectively. In contrast, the replacement of native erythrocyte phosphatidylcholine with egg phosphatidylcholine, and the dioleoyl species up to levels of 60% does not result in rapid hemolysis. Despite such functional consequences, overall contents of the individual phospholipids and cholesterol are normal. Accompanying the biochemical events are morphologic changes, including echinocyte and spherocyte formation. The structure‐function implications and possible mechanisms of hemolysis are discussed.
The Modification of the Peptidyltransferase Activity of 50‐S Ribosomal Subunits, LiCl‐Split Proteins and L16 Ribosomal Protein by Pyridoxal Phosphate
Authors: BAXTER Ross M. , WHITE Vivienne T. , ZAHID Nasir D.
Published: March 2005

Pyridoxal phosphate photoinactivates the peptidyltransferase activity of 504 ribosomal subunits, LiCl split proteins and protein L16. Ethyromycin exhibits significant protection.

These results, taken together with earlier reports, indicate the involvement of the single histidine of L16 in peptidyltransferase activity. The adjacent association in L16 of histidine and lysine indicates that pyridoxal phosphate should represent a selective inhibitor of peptidyltransferase activity.
Reversible Phosphorylation and Inactivation of Acetyl‐CoA Carboxylase from Lactating Rat Mammary Gland by Cyclic AMP‐Dependent Protein Kinase
Authors: HARDIE D. Grahame , GUY Paul S.
Published: March 2005

Acetyl‐CoA carboxylase has been purified from lactating rat mammary gland using a combination of ammonium sulphate and poly(ethyleneglycol) precipitations. The enzyme was purified from 35–70‐fold with a yield of over 50%, the exact figures being difficult to estimate because of activation of the enzyme that occurs during the preparation. The preparation was homogeneous by the criterion of polyacrylamide gel electrophoresis in sodium dodecyl sulphate and had a single subunit of molecular weight 240000, containing 1.02 ± 0.04 molecules of biotin and 3.1 ± 1.7 molecules of alkali‐labile phosphate per subunit. The purified enzyme was phosphorylated and inactivated rapidly when incubated in the presence of [γ‐32P]ATP and magnesium ions with the purified catalytic subunit of cyclic‐AMP‐dependent protein kinase from rabbit skeletal muscle. Both phosphorylation and inactivation are blocked by the heat‐stable protein inhibitor of cyclic‐AMP‐dependent protein kinase, and can be reversed by incubation with purified protein phosphatase‐1 from rabbit skeletal muscle. The inactivation by the protein kinase and reactivation by the protein phosphatase correlate with the near‐stoichiometric phosphorylation and dephosphorylation of site(s) located in a single tryptic peptide. Phosphorylation does not affect the Km for substrates, but brings about a twofold decrease in V and a twofold increase in the apparent dissociation constant for the allosteric activator, citrate. We also present evidence that the activation of rabbit mammary acetyl‐CoA carboxylase by protein phosphatase‐1 described previously [Hardie and Cohen (1979) FEBS Lett. 103, 333–338] is due to dephosphorylation at site(s) which are not phosphorylated by either cyclic‐AMP‐dependent protein kinase or acetyl‐CoA carboxylase kinase‐2.

These results suggest that the rapid inactivation of acetyl‐CoA carboxylase, and hence fatty acid synthesis, by adrenaline in adipose tissue, or glucagon in the liver, is due to phosphorylation of the enzyme by cyclic‐AMP‐dependent protein kinase.
Cytochrome c Interaction with Yeast Cytochrome b2
Authors: VANDERKOOI Jane M. , GLATZ Peter , CASADEI Jan , WOODROW George V.
Published: March 2005

Fluorescent derivatives of cytochrome c were prepared by replacing the heme iron with closed‐shell metals such as zinc or tin. The iron‐free derivatives of cytochrome c bind to yeast lactate dehydrogenase (cytochrome b2) stoichiometrically and with high affinity. Spectral overlap exists between the fluorescence of porphyrin, Zn(II) or Sn(IV) cytochrome c and the absorption of the heme of cytochrome b; therefore dipole‐dipole interaction is possible as predicted by Forster's theory of energy transfer. Changes in the fluorescence yield and the fluorescent decay profile of the cytochrome c derivatives are consistent with the view that the heme distance is sufficiently close for dipolar interactions. The distance calculated from the data depends upon assumptions in the theory for energy transfer and uncertainties in the experiment. It can be argued that due to the symmetry of the metalloporphyrins the relative orientations of the two hemes do not introduce a significant uncertainty in the calculation. However the decay profiles of the iron‐free cytochromes are complex, possibly reflecting structural rearrangement of the polypeptide chain during the fluorescent lifetime. The steady‐state fluorescent yields would indicate that the mean distance is around 1.8 nm
Taurocholate Inhibits the Glucocorticoid‐Induced Rise of 3‐Hydroxy‐3‐methylglutaryl‐CoA Reductase in Primary Culture of Hepatocytes
Authors: BARTH Christian A. , HILLMAR Ingrid
Published: March 2005

The influence of taurocholate, the major bile acid of the rat, on 3‐hydroxy‐3‐methylglutaryl‐CoA reductase [mevalonate: NADP+ oxidoreductase (acylating CoA); EC 1.1.1.34], the regulatory enzyme of cholesterol synthesis, was studied in primary cultures of rat hepatocytes. The basal activity of the enzyme was not altered by adding up to 10 μM taurocholate to the culture medium.

On the contrary, 1 nM to 10 μM taurocholate caused a dose‐dependent inhibition of enzyme activity within 6 h if added simultaneously with 10 μM dexamethasone. Because this glucocorticoid causes a cycloheximide‐sensitive rise of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase activity in this system the results are taken as evidence that bile salts inhibit the synthesis of the enzyme. The induction of tyrosine transaminase (L‐tyrosine: 2‐oxoglutarate aminotransferase; EC 2.6.1.5) by dexamethasone was not influenced by taurocholate, which demonstrates that the glucocorticoid sensitivity of the cells was not impaired by the bile salt.

It is concluded that there is a direct control of hepatic cholesterol synthesis by bile salts.
Antigenic Relationships between Pore Proteins of Escherichia coli K12
Authors: OVERBEEKE Nico , VAN SCHARRENBURG Guus , LUGTENBERG Ben
Published: March 2005

Antisera were raised against the purified Escherichia coli K12 outer membrane proteins ompA‐, ompC‐ and ompF proteins and protein e. Several immunological methods were used to investigate the specificity of the antisera and the immunological relationship between the major outer membrane proteins. Although the antisera had been raised against highly purified proteins, several of them contained activity against lipopolysaccharide and lipoprotein due to minor impurities in the immunogens. The three general porins ompF protein, ompC protein and protein e were shown to be cross‐reactive. Anti‐(ompA protein) serum only reacted with the homologous protein. None of these antisera reacted with the phage lambda receptor protein or with protein III. Pore protein preparations isolated from Salmonella typhimurium, Klebsiella aerogenes, Enterobacter cloaceae and Proteus mirabilis were found to be structurally related to the E. coli K12 porins as they reacted with the antisera raised against E. coli K12 porins.
Role of Prolactin in the Transcription of β‐Casein and 28‐S Ribosomal Genes in the Rabbit Mammary Gland
Authors: TEYSSOT Bertrand , HOUDEBINE Louis‐Marie
Published: March 2005

Isolated mammary nuclei were incubated in the presence of HgCTP and the neosynthesized RNA was isolated with a SH‐Sepharose column. The concentration of β‐casein mRNA and 28‐S ribosomal RNA in the neosynthesized RNA fractions was evaluated using [3H]DNA probes complementary to β‐casein mRNA and 28‐S rRNA respectively. In the unstimulated pseudopregant rabbit, the transcription of both genes was easily detectable. Injections of prolactin progressively enhanced the transcription rate of both genes and preferentially the β‐casein gene. A comparison between the transcription rates and the accumulation of the corresponding gene products in the cell revealed that there is a good correlation between these two parameters for the 28‐S rRNA gene. By contrast, the acceleration of β‐casein gene transcription by prolactin is unable to account for the simultaneous accumulation of β‐casein mRNA, indicating that prolactin is a potent stabilizer of casein mRNA. Injections of CB154 into lactating rabbits (a drug which suppresses the secretion of prolactin by hypophysis), induced a rapid drop of β‐casein mRNA concentration and a slow decline of β‐casein gene transcription. Simultaneously the drug was responsible for a marked and rapid decrease of 28‐S rRNA gene transcription, while the concentration of the rRNA remained elevated. During weaning the transcription of the β‐casein gene and, to a lower degree, the transcription of the 28‐S rRNA gene proceeded more slowly and this phenomenon was accompanied by a progressive decline of the RNA concentrations.
Isolation of Polymerization‐Competent Cytoplasmic Actin by Affinity Chromatography on Immobilized DNAse I Using Formamide as Eluant
Authors: ZECHEL Kasper
Published: March 2005

Formamide dissociates the G‐actin · DNAse‐I complex and is therefore suitable as an alternative to the eluant containing 3 M guanidinium hydrochloride, suggested originally by Lazarides and Lindberg [Proc. Natl Acad. Sci. USA, 71, 4742–4746 (1974)], to elute actin from immobilized DNAse‐I–agarose. Formamide provides the advantage of being a much weaker denaturant than guanidinium hydrochloride and being a nonionic substance. In the concentration necessary for the dissociation of the G‐actin · DNAse‐I complex (approximately 10 M) formamide denatures actin only slowly (half‐time approximately 150 min at 2°C) and thus allows the recovery of a large fraction of actin in a polymerization‐competent form from the affinity column.

Based on these findings a rapid two‐step procedure for the isolation of non‐muscle G‐actin from cultured cells is described. The actin is obtained in high yield and purity (greater than 90%) and can readily polymerize to F‐actin.
Respiratory Properties of Cytochrome‐c‐Deficient Mutants of Azotobacter vinelandii
Authors: HOFFMAN Paul S. , MORGAN T. Vance , DERVARTANIAN Daniel V.
Published: March 2005

Cytochrome‐c‐deficient mutants of Azotobacter vinelandii have been isolated following mutagenesis with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine. These mutants grow well under nitrogen‐fixing conditions and studies of the physiology and energy conservation efficiency show no apparent differences from those of the parent strain. Under oxygen‐limited growth conditions, the growth rate of the cytochrome‐c‐deficient mutant was slightly slower (approx. 15%) than that of the parent strain. Cytochromes of the c‐type are required for the oxidation of artificial electron donors such as reduced N,N,N,′,N′‐tetramethyl‐p‐phenylenediamine [Ph(NMe2)2]. This study could not demonstrate a physiological role for the c‐type cytochromes which further supports the idea that the minor Ph(NMe2)2‐oxidizing pathway of the electron transport chain may be independent of the major pathway terminated by cytochrome d.
Perchlorate Binding to Cytochrome c
Authors: ANDERSSON Thomas , ÅNGSTRÖM Jonas , FALK Karl‐Erik , FORSÉN Sture
Published: March 2005

1. The effects of perchlorate on cytochrome c have been investigated by 1H and 35Cl NMR, electron paramagnetic resonance and optical spectroscopy.

2. The pK values for the formation and disappearance of the major alkaline conformation were found to be displaced from 9.3 to 8.3 and from 10.4 to 10.9, respectively. The displacement was dependent on the ClO4− concentration below 0.1 M.

3. Competition experiments between perchlorate and chloride show that ClO4− binds both to the neutral and alkaline forms but with a higher affinity for the latter. The appearance of a new binding site in the alkaline form accounts for the markedly enhanced relaxation rate of 35ClO4− in this pH range. Complex formation between cyanide and the alkaline species results in the loss of this binding site, which probably is located close to or within the heme crevice.

4. The neutral form of ferricytochrome c also binds perchlorate strongly as evidence by the unique appearance of a high‐spin signal dependent on pH and perchlorate concentration. This signal disappears with the same pK value as the neutral form.

The effects of perchlorate on cytochrome c are due to specific binding of this ion.
Purification and Properties of the Major Apurinic/Apyrimidinic Endodeoxyribonuclease of Rat‐Liver Chromatin
Authors: THIBODEAU Lise , BRICTEUX Suzanne , VERLY Walter G.
Published: March 2005

Two nucleases active on alkylated‐depurinated DNA have been extracted from rat liver chromatin with 1 M KCl. The major enzyme was purified to near homogeneity; it has a molecular weight of 12500 (although some dimerization might occur), needs Mg2+ or Mn2+ for activity. The endonuclease activity is specific for apurinic/apyrimidinic sites in DNA; the enzyme has no associated exonuclease activity.
Cryoenzymic Studies on the Transition‐State Analog Complex Creatine Kinase ‐ ADPMg ‐ Nitrate ‐ Creatine
Authors: TRAVERS Franck , BARMAN Thomas E.
Published: March 2005

Concomitant with the formation of the transition state analog complex creatine kinase ‐ ADPMg. nitrate ‐ creatine [Milner‐White, E. J. and Watts, D. C.1971 Biochem. J. 122, 727–7401 there results a large difference spectrum. The shape of the spectrum was characteristic of tryptophan exposure.

The kinetics of formation and final amplitude of the spectrum were studied at – 15°C using a stopped‐flow apparatus. The results obtained allowed for a plausible reaction pathway for nitrate fixation: the ordered addition of nitrate and creatine to the binary enzyme‐ADPMg complex, a slow protein isomerization and the final addition of a molecule each of nitrate and creatine.

The kinetic parameters for the formation of the transition state analog complex were compared with those already known for the overall reaction obtained under the same conditions [Barman, T. E., Brun, A. and Travers, F.1980 Eur. J. Biochem. 110, 397–403], This comparison revealed a striking analogy between the two processes. This suggests that the conformation of creatine kinase in the analog complex is very similar to that in the transition state complex on the catalytic pathway.
Identification of Proteolytic Activities in the Cytosolic Compartment of Mature Human Erythrocytes
Authors: PONTREMOLI Sandro , MELLONI Edon , SALAMINO Franca , SPARATORE Bianca , MICHETTI Mauro , BENATTI Umberto , MORELLI Alessandro , DE FLORA Antonio
Published: March 2005

Individual lysates from human erythrocyte suspensions, completely deprived of leucocytes and platelets, were assayed for a number of proteolytic activities using both naturally occurring and synthetic substrates. Removal of hemoglobin by batchwise DEAE‐cellulose chromatography did not modify the complement of the various proteolytic activities which were then fractionated by means of chromatography on a column of DEAE‐cellulose, followed by conventional techniques such as gel chromatography and preparative electrophoresis. This procedure allowed a number of proteinases to be identified in the erythrocyte cytosol while providing a tool for their selective though partial separation. The following peptidases were found to be present in the soluble fraction of mature human erythrocytes: (a) a neutral endopeptidase having an approximate molecular weight of 110000; (b) three acidic endopeptidases, with pH optima between 2.5 and 3.5, showing molecular and functional properties almost identical with those of the three proteinases previously purified from solubilized erythrocyte membranes [Pontremoli et al. (1979) Biochem. J. 181, 559–568]; (c) two dipeptidylaminopeptidases whose molecular weights are around 80000 and tentatively identified as dipeptidyl aminopeptidases II and III, respectively, on the basis of their substrate specificities and pH optima; (d) presumably two aminopeptidases, having an approximate molecular weight of 80000 and classified as an aminopeptidase with broad substrate specificity and an aminopeptidase B, respectively. No evidence for any carboxypeptidase activity was found in the cytosolic compartment of mature human erythrocytes.
An Endonuclease from Yeast Mitochondrial Fractions
Authors: MOROSOLI Rolf , LUSENA Charles V.
Published: March 2005

A membrane‐bound endonuclease has been isolated from mitochondrial fractions of Saccharomyces cerevisiae. The enzyme is present in a stable complex and has an approximate molecular weight of 14000. It requires Mg2+ or Mn2+ for activity, and has an optimum pH of 7.0. Its activity with native DNA is five times less than with denatured DNA in 0.05 M KC1 and is very low in 0.2 M KCl. The activity with RNA is 40% of that with denatured DNA; the two substrates are competitive. Its mode of action is endonucleolitic, it cuts both strands of native λ DNA at the same or nearby sites. After mild digestion of DNA, analysis of 5′‐end groups of the digestion products indicated a marked preference for deoxythymidylic and deoxyguanilic acid residues as the site of enzymatic cleavage. After exhaustive digestion of DNA, mononucleotides (2.4%), dinucleotides (70.5%) and trinucleotides (27%) ending in 5′‐phosphate are produced.
Stereochemistry of Valine Biosynthesis
Authors: CROUT David H. G. , HEDGECOCK Charles J. R. , LIPSCOMB Elizabeth L. , ARMSTRONG Frank B.
Published: March 2005

When α‐aceto[1,3,5‐13C3]lactate(2‐hydroxy‐2‐methyl‐3‐oxo[1,3,5‐13C3]butanoate) was incubated with a cell‐free system prepared from Salmonella typhimurium, the valine produced was labelled in the C‐4 pro‐S position. This result proves that during the tertiary ketol rearrangement catalysed by the reductoisomerase of the isoleucine‐valine pathway, the methyl group transfer is to the re face of the trigonal centre at C‐3 of α‐acetolactate.
The Penicillin‐Binding Proteins in Streptococcus faecalis ATCC 9790
Authors: COYETTE Jacques , GHUYSEN Jean‐Marie , FONTANA Roberta
Published: March 2005

Streptococcus faecalis ATCC 9790 possesses seven membrane‐bound penicillin‐binding proteins. They have been characterized with respect to their apparent molecular weights, relative abundance, specificity profiles for 15 different β‐lactam antibiotics and stability under various conditions. In water and at 37°C, all the native penicillin‐binding proteins have half‐lives longer than 20 h except protein 3b (half‐life of about 600 min) and protein 4 (half‐life of about 175 min). The short‐lived 80000‐Mr protein 4 is spontaneously converted into a 73000‐Mr water‐soluble, penicillin‐binding protein 4*. Similarly, the short‐lived 82000‐Mr protein 3b seems to be the protein from which the 72000‐Mr water‐soluble protein X spontaneously originates during incubation of the membranes. Release of both proteins 4* and X from the membrane is maximal under alkaline conditions; it is not inhibited by various protease inhibitors. After exposure to trypsin, the 43000‐Mr membrane‐bound penicillin binding protein 6 (a DD‐carboxypeptidase) gives rise to a 30000‐Mr water‐soluble protein 6*. Like the parent protein, protein 6* exhibits both DD‐carboxypeptidase activity and penicillin‐binding ability. With proteins 6 and 6*, low dose levels of p‐chloromercuribenzoate prevent both enzyme activity and combination with penicillin, thus strongly suggesting that a thiol group is involved in the enzyme active center. We have shown previously [Coyette et al. in Eur. J. Biochem. 88, 297–305 (1978) and 75, 231–239 (1977)] that the DD‐carboxypeptidase protein 6 fragments the benzylpenicillin molecule with formation of phenylacetylglycine. Breakdown of the complex formed between [14C]benzylpenicillin and the 140000‐Mr membrane‐bound protein 1 is also ‘enzyme‐catalysed’. Most likely, however, the released product is penicilloate. With all the other penicillin‐binding proteins whose molecular weights are intermediate between those of proteins 1 and 6, breakdown of the complexes formed with [14C]benzylpenicillin results from proteolysis and is not due to the release of the bound metabolite. None of the penicillin‐binding proteins behaves, by itself, as a lethal target for β‐lactam antibiotic action on the living cells.
Transcriptional and Translational Control of Glucose‐Stimulated (Pro)insulin Biosynthesis
Authors: JAHR Henning , SCHRÖDER Dieter , ZIEGLER Brigitte , ZIEGLER Manfred , ZÜHLKE Hartmut
Published: March 2005

Islets of Langerhans were isolated from the pancreata of fed or 48‐h‐fasted Wistar rats. The islets were incubated with either [3H]leucine of [3H]uridine. Inhibition of RNA synthesis by actinomycin D or by α‐amanitin for 4 h had no influence on the (pro)insulin biosynthesis of isolated islets of fed rats. The (pro)insulin biosynthesis was not inhibited after two days incubation of islets of fed rats with α‐amanitin either. Incorporation of labelled uridine into total RNA for 3 h was stimulated by glucose in islets of fasted, but not of fed rats. Therefore, it was concluded that transcriptional control does not participate, even for longer periods than believed previously, in acute regulation of (pro)insulin biosynthesis of islets isolated from fed rats. Despite the strong and preferential stimulation of (pro)insulin biosynthesis of islets of fed rats by glucose the radioactivity of the [3H]uridine‐labelled polysomes active in proinsulin synthesis remained unchanged.

To interprete these experimental data we suggest that glucose triggers the transformation of a translationally inactive form of pre‐proinsulin mRNA to a translationally active form.
A Switch from Translational Control to Transcriptional Control of Protein Synthesis in Mid‐exponential Growth Phase of Bacterial Cultures
Authors: BAHRAMIAN Mohammad B. , HARTLEY Brian S.
Published: March 2005

1. We present evidence suggesting a sudden switch from translational control to transcriptional control of protein synthesis in mid‐exponential growth of bacterial batch cultures. At a critical cell density a switch from large to small polysomes occurs during a short period of exponential growth. The profile of specific polysomes engaged in synthesis of a constitutive enzyme, ribitol dehydrogenase, changes at the same point but in an opposite way: a linear profile peaking at monosomes changes to a dome‐shaped profile peaking at about 15 ribosomes/mRNA, which persists into late exponential phase despite a gradual reduction in the total polysome population. The switch in the pattern of protein synthesis is exhibited dramatically by changes in the specific activity of ribitol dehydrogenase in cell extracts at different stages of batch culture. In early exponential phase the specific activity of the enzyme is constant, but it begins to rise suddenly, at the same point at which the polysome profiles change, and continues to increase up to the end of exponential phase. This effect is exhibited by the strains of Klebsiella aerogenes taht are inducible for (in the presence of the inducer), consitutive for, or superproducers of ribitol dehydrogenase, and it appears to be unrelated to catabolite repression.

2. The above results depend on improved techniques for production of large amounts of bacterial polysomes and the ability to label nascent peptides attached to polysomes very specifically with radioactive antibody to ribitol dehydrogenase. Our success was due to the observation that sodium heparin completely abolishes non‐specific interactions of the antibody with the polysomes.
Factors Affecting the Quaternary Structure of the Allosteric L‐Lactate Dehydrogenase from Lactobacillus casei and Lactobacillus curvatus as Investigated by Hybridization and Ultracentrifugation
Authors: MAYR Ulrich , HENSEL Reinhard , KANDLER Otto
Published: March 2005

The allosteric L‐lactate dehydrogenases of Lactobacillus curvatus and Lactobacillus casei exist in the tetrameric form (molecular weight about 145000) at pH 5.0–5.5 even in the absence of the effectors Mn2+ and Fru(1,6)P2 (fructose 1,6‐bisphosphate), but undergo reversible dissociation to monomers (molecular weight about 35000) at higher pH values or in the presence of urea.

In the range between pH 5.5 (tetrameric state) and pH 7.4 (monomeric state) the L. curvatusL‐lactate dehydrogenase exists in a dissociation‐association equilibrium comprising tetramers, dimers and monomers as indicated by the sc20,w values and the results of hybridization experiments. The simultaneous addition of both effectors [Mn2+ and Fru(1,6)P2] at pH 7.4, however, resulted in the stabilization of the tetrameric form. The addition of Fru(1,6)P2 alone at pH 7.4 had almost no influence on the quarternary structure, whereas the addition of Mn2+, as well as that of NADH, largely prevented dissociation.

The L‐lactate dehydrogenase of L. casei showed similar properties, although the enzyme dissociates only at about pH ≧ 7.8. As in the case of the L. curvatus enzyme, Fru(1,6)P2 has no influence on the pH‐dependent dissociation of the L. casei enzyme, whereas Mn2+ stabilizes the tetrameric structure. Reconstitution of a mixture of the two dissociated enzymes results in the formation of all statistically possible, enzymatically active hybrids. No hybridization between the allosteric enzymes from L. casei and L. curvatus and the non‐allosteric ones of Lactobacillus plantarum and Lactobacillus acidophilus was observed.
A High Production Rate of Translatable IgG mRNA Accounts for the Amplified Synthesis of IgG in Myeloma Cells
Authors: WALLACH Michael , LASKOV Reuven
Published: March 2005

The aim of the present work was to determine whether the accumulation of Ig mRNA in myeloma cells is due to a high rate of production or to a high stability of these molecules. Specific mRNAs for the light and heavy polypeptide chains of IgG were isolated from the murine MPC‐11 myeloma tumor cells by immune precipitation of polysomes which synthesize these chains. It was found that the immune‐precipitated polysomes were enriched 10 –30‐fold in the γ and χ mRNA sequences respectively. In the wheal germ cell‐free system the χ mRNA preparation was translated mainly into three polypeptides of Mr 25000, 18000, and 15000. The method of immune precipitation of polysomes was also used to characterize three variant clones of MPC‐11 myeloma. It was found that little if any γ‐chain polysomes are present in the L‐chain producer and non‐producer clones, while a substantial amount of χ‐chain polysomes was present in the non‐producer clone. This may be due to the presence in the non‐producer cells of the constant region χ‐chain fragment.

In order to determine the relative synthesis rate of χ and γ mRNAs, pulse‐labeled polysomes were immune precipitated using antibodies to χ and γ chains. It was found that χ and γ mRNA molecules are produced at a very high relative rate each accounting for 10–15% of the total labeled mRNA after 1 h of labeling. These values are higher than the steady‐state pool size of χ and γ mRNA, which was 5–6%, and indicates that the half‐life of these molecules is not unusually high. It is concluded that the amplified synthesis of immunoglobulin chains in myeloma cells is mainly due to a high rate of production of translatable χ and γ mRNAs.
Stoichiometry of Interaction of Chicken Ovoinhibitor with Pancreatic Trypsin, Chymotrypsin and Elastase I
Authors: GERTLER Arieh , BEN‐VALID Isaac
Published: March 2005

1. The interaction of chicken ovoinhibitor with pancreatic trypsin, chymotrypsin and elastase I was studied in the absence of substrate by affinity chromatography, electrophoresis and gel filtration.

2. It was found that ovoinhibitor has, at least, five separate and non‐overlapping binding sites: two for trypsin, two for chymotrypsin and one for elastase. Most likely, all five sites may be occupied simultaneously.

3. Electrophoretic studies indicated that two binding sites for trypsin and probably also for chymotrypsin differ in their affinity toward the respective enzyme.
Studies of Liposome Interactions with Rat Thymocytes
Authors: KRAMERS Mary T. C. , PATRICK John , BOTTOMLEY Juliana M. , QUINN Peter J. , CHAPMAN Dennis
Published: March 2005

1. The consequences of incubating liposomes with rat thymocytes have been studied using liposomes of dipalmitoylphosphatidylcholine and cholesterol or dipalmitoylphosphatidylcholine only.

2. Dipalmitoylphosphatidylcholine‐cholesterol liposomes do not bind to the cells and can be removed by washing. An increase in cellular cholesterol is observed. However dipalmitoylphosphatidylcholine liposomes bind rapidly to the cells and cannot be removed by repeated washing. Cholesterol is removed from the cells.

3. There are small changes in intracellular cations in the cholesterol‐enriched cells, but no transport studies have been made. Cells depleted of cholesterol lose K+ with little change in intracellular Na+. Na+ influx is increased. The majority of this increase appears to be ouabain‐sensitive, indicating an increase in pump‐mediated Na+ influx, K+ influx is reduced.

4. The significance of these results is discussed.