bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane‐Bound and Water‐Soluble Cytochrome c1 From Neurospora Mitochondria
Authors: LI Yun , LEONARD Kevin , WEISS Hanns
Published: March 2005

Cytochrome c1 is a subunit of ubiquinol‐cytochrome c reductase (EC 1.10.2.2). In Neurospora crassa wild type 74A grown in the presence of chloramphenicol, the subunit is inserted only into the bilayer of the mitochondria) inner membranes without associating with other proteins. From these modified membranes a monodisperse (cytochrome c1)‐Triton complex was isolated by subjecting the Triton‐solubilized membranes to affinity chromatography on immobilized cytochrome c. A water‐soluble pentamer of cytochrome c1 was prepared from the (cytochrome c1)‐Triton complex by removing the detergent. By limited proteolytic digestion of the cytochrome c1‐Triton complex with chymotrypsin, a water‐soluble monomeric cytochrome c1 was prepared which has a molecular weight of only 24000 as compared to 3l000 of the membrane‐bound cytochrome c1. The 24000‐Mr cytochrome c1 and the 31000‐Mr cytochrome c1 have same light absorption spectra and cytochrome‐c‐binding properties. These results are used to propose the following model. Cytochrome c1 consists of a large hydrophilic part and a small hydrophobic part. The hydrophilic part extends from the mitochondrial inner membrane into the intermembrane space. This part carries the heme and interacts with cytochrome c. The hydrophobic part anchors tile cytochrome c1 to the bilayer.
Isolation and Characterization of Dipeptidyl Peptidase IV from Human Placenta
Authors: PÜSCHEL Gerd , MENTLEIN Rolf , HEYMANN Eberhard
Published: March 2005

Human placenta is surprisingly rich in post‐proline dipeptidyl peptidase activity. Among various cell fractions, microsomes have the highest specific activity. A homogeneous enzyme preparation is obtained in a six‐step purification procedure. The final preparation appears homogeneous upon dodecyl sulfate electrophoresis, but analytical isoelectric focussing reveals various active bands with isoelectric points in the range of pH 3–4. The enzyme is a glycoprotein containing about 30% carbohydrate. Treatment with neuraminidase lowers the isoelectric points but does not reduce the heterogeneity of the band pattern. The subunit molecular weight is 120000 as estimated by dodecyl sulfate electrophoresis, whereas Mr of the native enzyme is > 200000, as can be concluded from gel filtration experiments.

The purified dipeptidyl peptidase cleaves various synthetic and natural peptides, including substance P, kentsin, casomorphin and a synthetic renin inhibitor. In general, the specificity of the placenta peptidase is similar to that of post‐proline dipeptidyl peptidase from other sources. Phenylalanylprolyl‐β‐naphthylamide (Km= 0.02 mM, V= 92 U/mg) is the best substrate among various synthetic peptide derivatives. Only peptides with a free N‐terminal amino group and proline, hydroxyproline, or alanine in position 2 of the N‐terminal sequence are cleaved. However, X‐Pro‐Pro‐… structures, e.g. as in bradykinin, are not attacked.

1 mM bis‐(4‐nitrophenyl)phosphate or 1 mM diisopropylfluorophosphate completely inactivate the peptidase within 30 min at 30°C (pH 8). The peptidase is also completely inhibited by 1 mM Zn2+ and by other heavy metals.
Intracellular Localization of Rat‐Liver Uracil‐DNA Glycosylase
Authors: COLSON Pierre , VERLY Walter G.
Published: March 2005

Most of the uracil‐DNA glycosylase of the rat liver cell is located in chromatin; there is, however, some activity in the nuclear sap and in the cytoplasm. The chromatin uracil‐DNA glycosylase has been purified; the preparation is devoid of endonuclease and exonuclease activities; the enzyme does not need divalent cations, has a broad optimum pH around 8, is strongly inhibited by increasing ionic strength and free uracil.

The apparent Km is independent of the strandeness of the DNA substrate containing uracil, but V is slightly higher with the single‐stranded substrate. The frequency of uracil substitution in the double‐stranded DNA influences the kinetic parameters: a higher frequency increases both Km and V.

The inhibitory effects of NaCl and free uracil are greater when the greater when the substrate is double‐stranded rather than single‐stranded. It is speculated that, acting either on the DNA or on the enzyme, both oppose the opening of the double helix necessary for the formation of the enzyme‐substrate complex.

The increased reaction rate with a higher frequency of uracil residues in double‐stranded DNA is interpreted as a tendency for the repair enzyme to work in a processive way. It is supposed that processivity also occurs with single‐stranded DNA and that it is opposed by both NaCl and free uracil, explaining a greater inhibition when the single‐stranded substrate has a higher uracil content.
The Ribosomal Elongation Cycle: tRNA Binding, Translocation and tRNA Release
Authors: RHEINBERGER Hans‐Jörg , SCHILLING Susanne , NIERHAUS Knud H.
Published: March 2005

Experiments concerning the ribosomal elongation cycle have been performed; the following results were obtained.

A measurements of the percentage of tightly coupled ribosomes participating in a tRNA binding reaction is obtained by determining both the stoichiometry of binding and the site location. In the case of AcPhe‐tRNA it is sufficient to determine the maximal binding since at moderate Mg2+ concentration (≤ 15 mM) one ribosome cannot bind more than one AcPhe‐tRNA molecule, which can be present at either the A or P site (exclusion principle).

tRNA binding to poly(U)‐programmed ribosomes does not follow the predictions of simple models with rigid tRNA binding sites as postulated by the classical A/P site model. For example, 2.7 molecules of tRNAPhe are found per ribosome at a ribosome concentration of 0.33 μM and a [14C]tRNAPhe excess over ribosomes of about 40‐fold, in agreement with our previous report. At a 70S ribosome concentration of 3.3 μM and a sevenfold excess of tRNAPhe a binding of 2.5 tRNA molecules/ribosome would be expected according to the binding constants reported by us, but in fact a binding of only 1.8 molecules/70S ribosome is found. These findings explain the current discrepancies concerning the number of tRNA sites on the ribosome and confirm previous reports that three tRNA molecules can be bound simultaneously to one ribosome.

The ternary complex EF‐Tu · GTP · Phe‐tRNAPhe binds to the A site at 0°C even if the P site is not occupied. Only if the P site is free does the addition of EF‐G provoke a translocation to the P site at 0°C, whereas at 37°C the Phe‐tRNAPhe‘slides’ from the A to the P site without EF‐G. This EF‐G‐independent sliding is not observed if the P site is filled by a deacylated tRNAPhe. Thus, EF‐G is directly involved in the translocation reaction, and its role is not restricted to tRNA release.

Kinetics of the puromycin reaction with Phe‐tRNA and AcPhe‐tRNA, respectively, were compared. The initial rate of AcPhe‐puromycin formation exceeds that of Phe‐puromycin formation by a factor of 3.6 (0°C, 15 mM Mg2+).

Poly(U)‐programmed ribosomes carrying only [14C]tRNA release tRNA upon addition of EF‐G. tRNA release is observed only if at least two tRNAPhe molecules are present per ribosome. The tRNA bound first is released with a higher probability than that bound second. Such a complex is not related to a classical pretrans‐location complex carrying a peptidyl‐tRNA at the A site. Therefore, when tRNA release during the course of an EF‐G‐induced translocation reaction is studied, care must be taken that no significant amounts of the complex 70S · mRNA · (deacylated tRNA) are present.
The Mechanism of Biliary Secretion of Reduced Glutathione
Authors: INOUE Masayasu , KINNE Rolf , TRAN Thao , ARIAS Irwin M.
Published: March 2005

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2‐3H]glycine–labeled GSH into an osmotically reactive intravesicular space. Althought the canalicular membrane vesicles possess γ‐glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle associated transferase by affinity labeling with L‐(αS,5,S)‐α‐amino‐3‐chloro‐4,5‐dihydro‐5‐isoxazoleacetic acid (AT‐125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle‐associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier‐mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin‐induced K+‐diffusion potential (vesicle inside positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S‐benzyl‐glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S‐transferase.
Cloning and Molecular Characterization of the ompA Gene from Salmonella typhimurium
Authors: FREUDL Roland , COLE Stewart T.
Published: March 2005

The ompA gene from Salmonella typhimurium, encoding a major heat‐modifiable protein of the outer membrane, has been cloned and extensively characterized. When expressed in Escherichia coli the gene directs the synthesis of an OmpA protein which is functionally and topologically indistinguishable from that made in S. typhimurium, thus indicating that export and membrane incorporation are very similar in the two organisms. The S. typhimurium protein effectively substitutes for the E. coli polypeptide in F‐dependent conjugation and in the uptake of certain colicins, although it cannot serve as the receptor for the OmpA‐specific phages K3 and Tull*. On examination of the primary sequence of the protein, predicted from the nucleotide sequence of its gene, it was found that those domains likely to be exposed on the cell surface were significantly different to the corresponding regions of the E. coli polypeptide. These differences in the structure of the two proteins have been used to interpret differences in their biological activities.
Methylated 7‐Deazahypoxanthines as Regiochemical Probes of Xanthine Oxidase
Authors: ROSEMEYER Helmut , SEELA Frank
Published: March 2005

7‐Deazahypoxanthine was found to be oxidised by cow's milk xanthine oxidase exclusively at carbon 2. The resulting 7‐deazaxanthine is a strong inhibitor of the enzymatic reaction. This offers a possibility for determining the structural requirements of ligand binding separately for the first step. All the monomethyl isomers of 7‐deazahypoxanthine were tested as probes by measuring their Km, Ki and V values. While the N‐3‐methyl and C‐7‐methyl isomers are still processed, the N‐9‐methyl and 6‐O‐methyl isomers are bound as inhibitors to the active site. The N‐1‐methyl compound is neither an inhibitor nor a substrate. This demonstrates that HN(1) and O = C(6) are essential for the binding. Replacement of O = C(6) by S = C(6) changes the substrate into a strong inhibitor (Ki= 9 μM), implying that the electron transfer to the enzyme is hindered. Methylation of the thioxo group (S =) reduces the inhibition significantly. In contrast to 7‐deazahypoxanthine, 2‐thioxo‐7‐deazaxanthine is an activator at concentrations below 87 μM and a partial competitive inhibitor above this concentration, which implies the presence of a second binding site.
Induction and Characterization of a Microsomal Flavonoid 3′‐Hydroxylase from Parsley Cell Cultures
Authors: HAGMANN Marie‐Luise , HELLER Werner , GRISEBACH Hans
Published: March 2005

A microsomal preparation from irradiated parsley cell cultures catalyses the NADPH and dioxygen‐dependent hydroxylation of (S)‐naringenin [(S)‐5, 7, 4′‐trihydroxyflavanone] to eriodictyol (5, 7, 3′, 4′‐tetrahydroxyflavanone). Dihydrokaempferol, kaempferol, and apigenin were also substrates for the 3′‐hydroxylase reaction. In contrast prunin (naringenin 7‐O‐β‐glucoside) was not converted by the enzyme. The microsomal preparation, which also contains cinnamate 4‐hydroxylase, did not catalyse hydroxylation of 4‐coumaric acid to caffeic acid.

3′‐Hydroxylase activity is partially inhibited by carbon monoxide in the presence of oxygen as well as by cytochrome c and NADP+. These properties suggest that the enzyme is a cytochrome P‐450‐dependent flavonoid 3′‐monooxygenase. Pronounced differences in the inhibition of flavonoid 3′‐hydroxylase and cinnamate 4‐hydroxylase were found with EDTA, potassium cyanide and N‐ethylmaleimide.

Irradiation of the cell cultures led to increase of flavonoid 3′‐hydroxylase activity with a maximum at about 23 h after onset of irradiation and subsequent decrease. This is similar to light‐induction of phenylalanine ammonialyase and cinnamate 4‐hydroxylase.

In contrast, treatment of the cell cultures with a glucan elicitor from Phytophthora megasperma f. sp. glycinea did not induce flavonoid 3′‐hydroxylase nor chalcone isomerase but caused a strong increase in the activities of phenylalanine ammonia‐lyase, cinnamate 4‐hydroxylase, and NADPH‐cytochrome reductase. The results prove that flavonoid 3′‐hydroxylase and cinnamate 4‐hydroxylase are two different microsomal monooxygenases.
Monoclonal Antibodies against Human β‐Glucocerebrosidase
Authors: BARNEVELD Ruud A. , TEGELAERS Frans P. W. , GINNS Edward I. , VISSER Pim , LAANEN Elly A. , BRADY Roscoe O. , GALJAARD Hans , BARRANGER John A. , REUSER Arnold J. J. , TAGER Joseph M.
Published: March 2005

Monoclonal antibodies were obtained against the membrane‐bound lysosomal enzyme β‐glucocerebrosidase (acid β‐glucosidase), which is deficient in Gaucher's disease.

BALB/c mice were immunized with homogeneous enzyme protein extracted from a sodium dodecyl sulphate/polyacrylamide gel. The mice were subsequently hyperimmunized with partially purified enzyme prior to fusion of spleen cells with myeloma cells. After fusion, 32 primary hybrid cell populations were obtained which continued to produce antibodies against β‐glucocerebrosidase after prolonged time of culture. All antibodies reacted with both native and denatured enzyme.

Four primary cell populations were subcloned and the antibodies produced were characterized. The antibodies were all four of the IgG1 subclass. Three of these antibodies bind to protein A whereas one does not. The results of binding assays indicated that three of the antibodies react with the same antigenic domain (epitope 1), but the fourth with a different one (epitope 2). Probably two antigenic determinants are present in epitope 1 since one of the antibodies with specificity for epitope 1 is inactivated after iodination by the chloramine‐T procedure whereas a second one is not.
Microsomal Glutathione Transferase
Authors: MORGENSTERN Ralf , DePIERRE Joseph W.
Published: March 2005

The procedure developed for purification of the N‐ethylmaleimide‐activated microsomal glutathione transferase was applied successfully to isolation of this same enzyme in unactivated form. The microsomal glutathione transferases, the unactivated and activated forms, were shown to be identical in terms of molecular weight, immunochemical properties, and amino acid composition.

In addition the microsomal glutathione transferase purified in unactivated form could be activated 15‐fold with N‐ethylmaleimide to give the same specific activity with 1‐chloro‐2,4‐dinitrobenzene as that observed for the enzyme isolated in activated form. This activation involved the binding of one molecule N‐ethylmaleimide to the single cysteine residue present in each polypeptide chain of the enzyme, as shown by amino acid analysis, determination of sulfhydryl groups by 2,2'‐dithiopyridyl and binding of radioactive N‐ethylmaleimide.

Except for the presence of only a single cysteine residue and the total absence of tryptophan, the amino acid composition of the microsomal glutathione transferase is not remarkable. The contents of aspartic acid/asparagine + glutamic acid/glutamine, of basic amino acids, and of hydrophobic amino acids are 15%, 12% and 54% respectively. The isoelectric point of the enzyme is 10.1.

Microsomal glutathione transferase conjugates a wide range of substrates with glutathione and also demonstrates glutathione peroxidase activity with cumene hydroperoxide, suggesting that it may be involved in preventing lipid peroxidation. Of the nine substrates identified here, the enzymatic activity towards only two, 1‐chloro‐2,4‐dinitrobenzene and cumene hydroperoxide, could be increased by treatment with N‐ethylmaleimide. This treatment results in increases in both the apparent Km values and V values for 1‐chloro‐2,4‐dinitrobenzene and cumene hydroperoxide.

Thus, although clearly distinct from the cytosolic glutathione transferases, the microsomal enzyme shares certain properties with these soluble enzymes, including a relative abundance, a high isoelectric point and a broad substrate specificity. The exact role of the microsomal glutathione transferase in drug metabolism, as well as other possible functions, remains to be established.
A set of non‐histone proteins isolated from the nuclei of various rat tissues
Authors: INOUE Akira , HIGASHI Youtaro , HASUMA Tadayoshi , MORISAWA Seiji , YUKIOKA Munehiko
Published: March 2005

A set of non‐histone proteins has been identified in the nuclei from liver, brain, spleen and testis tissues of the rat. Following moderate digestion of thoroughly washed nuclei with DNase I or micrococcal nuclease, EDTA was added to 5 mM to the reaction mixture and the preparation centrifuged. We found that the supernatant containeda limited amount of non‐histone proteins (fraction S1). Sodium dodecyl sulfate (SDS) gel electrophoresis revealed S1 to be composed of a remarkably simple set of proteins resolved into four groups (A – D) each possessing closely spaced doublets or a triplet. Their molecular weights were A, 76100–80000; B, 48200–49500; C, 44500–45200 and D, 39500–41500. The yield suggested that these proteins were structural constituents; however, they did not coincide with the known structural proteins of the cell nucleus. Two‐dimensional gel electrophoresis further resolved each of the SDS bands into as many as nine spots, according to various charges. Some were labelled with [32P]orthophosphate in vivo, or with [γ‐32P]ATP and purified nuclear protein kinase NII in vitro. The released proteins B – D had fairly constant relative molar ratios at various times of digestion, thereby indicating possible localizations at similar sites in the nucleus. The kinetic data together with the aggregation property at neutral pH values and the solubility in 5 mM EDTA suggest that proteins B‐D constitute a group of proteins that have several common characteristics.
Characterization of the cytochromes occurring in Methanosarcina species
Authors: KÜHN Wolfgang , GOTTSCHALK Gerhard
Published: March 2005

Several members of the genus Methanosarcina were investigated by room‐temperature and low‐temperature difference spectroscopy for the presence of cytochromes. In combination with potentiometric titrations two membrane‐bound b‐cytochromes and one membrane‐bound c‐cytochrome could be detected in cells grown on methanol or trimethylamine. Very probably acetate‐grown cells contained an additional cytochrome b. The midpoint potentials of the two b‐type cytochromes were Em1=−325 mV and Em2=−183 mV, respectively. The additional b cytochrome formed during growth on acetate exhibited a midpoint potential of Em3=−250 mV.
Uroporphyrinogen III, an intermediate in the biosynthesis of the nickel‐containing factor F430 in Methanobacterium thermoautotrophicum
Authors: GILLES Harald , THAUER Rudolf K.
Published: March 2005

Factor F430 is a nickel‐containing porphinoid present in methanogenic bacteria. The synthesis of this nickel tetrapyrrole from 5‐aminolevulinic acid was studied in Methanobacterium thermoautotrophicum. This anaerobic archaebacterium was found to accumulate [14C]uroporphyrinogen III (1.8 μmol/g) when growing on nickel‐free medium (< 50 nmol/l) supplemented with 2 mM 5‐amino‐[14C]levulinic acid. The accumulated urophorphyrinogen III was quantitatively converted to factor F430 when the cells were incubated in aminolevulinate‐free medium with 5 μM NiCl2. The newly synthesized factor had the same specific radioactivity as the precursor uroporphyrinogen III. These findings indicate that the nickelporphinoid is biosynthetically derived from uroporphyrinogen III. The presence and some properties of the enzymes catalyzing the synthesis of uroporphyrinogen III from 5‐aminolevulinic acid in M. thermoautotrophicum are also described.
Phosphorylation of purified bovine cardiac sarcolemma and potassium‐stimulated calcium uptake
Authors: FLOCKERZI Veit , MEWES Rainer , RUTH Peter , HOFMANN Franz
Published: March 2005

Sarcolemmal vesicles were prepared from bovine cardiac muscle by differential and discontinuous sucrose density gradient centrifugation. Na+/K+‐ATPase was purified 33‐fold to a specific activity of 53 ± 0.5 (12) μmol Piμmg±1×h−1, binding sites for strophantin 20‐fold to a density of 56.3 ± 5.3 (14) pmol/mg and that for the calcium antagonist nitredipine 5.5‐fold to a density of 0.72 ± 0.07 (6) pmol/mg. The specific activity of the Na+/Ca2+ exchanger was 61.1 ± 3.7 (6) nmol/mg. The vesicles had an intravesicular volume of 20 ± 4 (4) μl/mg and 56.9 ± 6 (4)% of the vesicles were right‐side‐out oriented.

Several peptides of the purified membranes were phosphorylated in the presence of Mg · ATP and EGTA. Most of the radioactive phosphate was incorporated into a peptide with an apparent molecular mass of 22 kDa. Denaturation of the membranes at 100°C changed the mobility of this peptide to 15 kDa and 11 kDa. This peptide could not be distinguished from a sarcoplasmic reticulum peptide of similar molecular mass. The phosphorylation of the sarcolemmal peptide was stimulated by Ca2+/calmodulin, cAMP and the catalytic subunit of cAMP‐dependent protein kinase. A comparison of the phosphorylation of sarcolemmal membranes with that of sarcoplasmic reticulum showed that Ca2+/calmodulin stimulated in each membrane, the phosphorylation of the 22‐kDa peptide and a 44‐kDa peptide, and in the sarcoplasmic reticulum the phosphorylation of an additional peptide of 55‐kDa. Ca2+/calmodulin‐dependent phosphorylation of a 55‐kDa peptide could not be demonstrated in sarcolemma, regardless if sarcolemmal membranes were incubated together with sarcoplasmic reticulum or if the phosphorylation was carried out in the presence of purified cardiac myosin light chain kinase or phosphorylase kinase.

‘Depolarization’ induced Ca2+ uptake which was measured according to Bartschat, D. K., Cyr, D. L. and Lindenmayer, G. E. [(1980) J. Biol. Chem. 255, 10044–10047] was 5 nmol/mg protein. This uptake was not enhanced after preincubation of the vesicles with Mg · ATP or Mg · ATP and cAMP‐dependent protein kinase. The value of 5 nmol/mg protein is in agreement with the theoretical amount of Ca2+ which can be accumulated by the bovine cardiac sarcolemma in the absence of a driving force other than the Ca2+ gradient. The potassiumstimulated Ca2+ uptake was not blocked by the organic Ca2+ channel blockers. Prolonged incubation of Mg · ATP with sarcolemmal vesicles in the presence of various ATPase inhibitors led to the hydrolysis of ATP. The liberated phosphate precipitated with Ca2+ in the presence of LaCl3. These precipitates amounted to an apparent Ca2+ uptake ranging from 50 to over 1000 nmol/mg. The results suggest that potassium‐stimulated Ca2+ uptake of bovine cardiac sarcolemmal vesicles is not enhanced in the presence of ATP or by phosphorylation of a 22‐kDa peptide.
The reaction of trout hemoglobins with isocyanides
Authors: GIARDINA Bruno , FALCIONI Giancarlo , COLETTA Massimiliano , BRUNORI Maurizio
Published: March 2005

Binding of alkylisocyanides of different bulkiness to the two major components of trout hemolysate is presented. In the case of trout hemoglobin I isocyanide binding is pH‐independent, similar to O2 and CO, and the bulkiness of the ligand is related to the endothermicity of ligand binding to the T quaternary state. On the other hand, in trout hemoglobin IV the size of the ligand seems to affect the pH dependence of affinity and cooperativity. A comparison with other ligands, like O2, allows us to hint at possible stereochemical determinants of ligand binding in these two hemoglobins.
Regulation of heat‐shock protein synthesis in chicken muscle culture during recovery from heat shock
Authors: BAG Jnanankur
Published: March 2005

Exposure of chick myotube cultures to a temperature (45°C) higher than their normal growing temperature (37°C) caused extensive synthesis of three major polypeptides of Mr= 25 000, 65 000 and 81 000 referred to as ‘heatshock polypeptides’ (hsps). When these cells were allowed to recover from heat‐shock treatment at 37°C for 6–8 h, the rate of accumulation of isotope into the 65 000‐Mr and 81 000‐Mr hsps declined to levels comparable to those in control cultures maintained at 37°C. However, incorporation of isotope in the 25 000‐Mr hsp continued at an elevated rate for a longer period than the 65 000‐Mr and 81 000‐Mr hsps. When heat‐shocked cells were allowed to recover at 37°C in the presence of actinomycin D to block new mRNA synthesis, the hsp synthesis as measured by the incorporation of radioactive isotope in these polypeptides continued at levels comparable to those in heat‐shocked cells prior to recovery. The block of recovery by actinomycin D was due to the presence of a greater amount of functional hsp mRNAs in the polysomes as compared to untreated controls. The role of competition between the mRNAs for hsps and normal cellular proteins for the translation machinery in regulating protein synthesis during the recovery from heat shock has been discussed.
Reactions in vitro of the DNA polymerase‐primase from Xenopus laevis eggs
Authors: KÖNIG Herbert , RIEDEL Hans Dieter , KNIPPERS Rolf
Published: March 2005

A form of DNA polymerase α was purified several thousandfold from a protein extract of Xenopus laevis eggs. The enzyme effectively converts, in the presence of ribonucleoside triphosphates, a circular single‐stranded phage fd DNA template into a double‐stranded DNA form and, therefore, must be associated with a DNA primase. We first show by gel electrophoresis in the presence of sodium dodecyl sulfate that both enzymatic activities, DNA polymerase and primase, most probably reside on a < 100000‐Da subunit of the DNA polymerase holoenzyme. We then assayed the polymerase‐primase at various template/enzyme ratios and found that the DNA complementary strand sections synthesized in vitro belong to defined size classes in the range of 600–2000 nucleotides, suggesting preferred start and/or stop sites on the fd DNA template strand. We show that the stop sites coincide with stable hairpin structures in fd DNA. We have used a fd DNA template, primed by a restriction fragment of known size, to show that the polymerase‐primase stops at the first stable hairpin structure upstream from the 3′‐OH primer site when the reaction was carried out at 0.1 mM ATP. However, at 2mM ATP the enzyme was able to travers this and other stop sites on the fd DNA template strand leading to the synthesis of 2–4 times longer DNA strands. Our results suggest a role for ATP in the polymerase‐primase‐catalyzed chain‐elongation reaction.
Biosynthesis of the ubiquinol‐cytochrome c reductase complex in yeast
Authors: VAN LOON Adolphus P. G. M. , KREIKE Jan , DE RONDE Antony , VAN DER HORST Gijsbertus T. J. , GASSER Susan M. , GRIVELL Leslie A.
Published: March 2005

The mitochondrial ubiquinol – cytochrome c reductase complex (complex III or cytochrome bc1 complex) is thought to consist of eight subunits, seven of which are specified by nuclear genes and synthesized in the cytoplasm. We have studied the synthesis of five of the nuclear‐encoded subunits both in vivo and in vitro and show that of these the 44‐kDa, 40‐kDa and 17‐kDa subunits are synthesized with cleavable extensions, while the 14‐kDa and 11‐kDa proteins are synthesized without detectable extra sequences. The sizes of the pre‐sequences, as determined by the relative mobility of the precursor proteins in sodium dodecyl sulphate/polyacrylamide gels, range from 0.5‐kDa for the 44‐kDa and 40‐kDa subunits to 9‐kDa for the 17‐kDa subunit. The existence in vivo of precursor forms to the 44‐kDa, 40‐kDa and 17‐kDa subunits implies that import is at least partially a post‐translational process. The precursor of the 44‐kDa subunit can be processed post‐translationally in vitro by isolated mitochondria.

The messenger RNAs for subunits of the complex have been studied. Those coding for the 44‐kDa, 40‐kDa, 14‐kDa and 11‐kDa proteins and cytochrome c1 are of different sizes, indicating that each of these subunits is synthesized as a separate protein, rather than as part of a polyprotein precursor.
Distribution of secondary structure along the fibronectin molecule
Authors: VENYAMINOV Sergei Yu. , METSIS Madis L. , CHERNOUSOV Michael A. , KOTELIANSKY Victor E.
Published: March 2005

30‐kDa, 50‐kDa and 70‐kDa gelatin‐binding, 60‐kDa central and 60–65‐kDa heparin‐binding fragments were produced by trypsin digestion of fibronectin. The secondary structure of the fragments was studied by circular dichroism and quantitative infrared spectroscopy. The structure of the 70‐kDa gelatin‐binding, 60‐kDa central and 60–65‐kDa heparin‐binding fragments in solution appeared to be very close to that in the intact fibronectin. The content of the antiparallel β‐form, the only element of the secondary structure in all the fragments studied, was shown to be 30–35%.
Studies on the mechanism of action of the histone kinase dependent on adenosine 3′,5′‐monophosphate
Authors: GABIBOV Alexander G. , KOCHETKOV Sergei N. , SASHCHENKO Lidia P. , SMIRNOV Ivan V. , SEVERIN Evgenii S.
Published: March 2005

The transient phase of histone H1 phosphorylation was studied by the quenched‐flow method. A ‘minimal’ kinetic scheme of the above process was proposed. A formal kinetic analysis was given to a four‐step mechanism of the reaction. Computer simulation of the transient‐phase kinetics of H1 phosphorylation and the steady‐state kinetics of phosphate transfer from the enzyme phosphoform to histone permitted us to estimate all kinetic constants of the proposed mechanism.
Pig heart nucleosidediphosphate kinase Phosphorylation and interaction with Cibacron blue 3GA
Authors: LASCU Ioan , POP Radu D. , PORUMB Horea , PRESECAN Elena , PROINOV Ioan
Published: March 2005

The nucleosidediphosphate kinase phosphorylation reaction led to the incorporation of 0.95 ± 0.1 phosphate groups per enzyme subunit. The equilibrium constant of the phosphorylation reaction was 0.26. The inhibition of the nucleosidediphosphate kinase activity by Cibacron blue 3GA was competitive with respect to ATP, the donor nucleotide (apparent Ki= 0.28 μM) and uncompetitive with respect to 8‐bromoinosine 5′‐diphosphate, the acceptor nucleotide (apparent Ki= 0.31 μM). By difference spectroscopy it was shown that each enzyme subunit bound one Cibacron blue 3GA molecule, whereas the phosphorylated enzyme had no affinity for the dye. ATP was an effective competitor, being able to displace the dye from its bound state. The complex behaviour noted was taken as evidence for cooperative interaction between the enzyme subunits. The data obtained using polarographic techniques agreed with these results.
Purification, characterization and redox properties of hydrogenase from Methanosarcina barkeri (DSM 800)
Authors: FAUQUE Guy , TEIXEIRA Miguel , MOURA Isabel , LESPINAT Paul A. , XAVIER António V. , DER VARTANIAN Daniel V. , PECK Harry D. , LE GALL Jean , MOURA José G.
Published: March 2005

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550‐fold purification, a 45% yield and a final specific activity of 270 μmol H2 evolved min−1 (mg protein)−1. The hydrogenase has a high molecular mass of ∼ 800 kDa and subunits with molecular masses of ∼ 60 kDa. The enzyme is stable to heating at 65°C and to exposure to air at 4°C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate‐reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non‐heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8–10 iron atoms and 0.6–0.8 nickel atom were also detected per subunit.

The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and ∼ 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g= 1.94 EPR signals, due to iron‐sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR‐active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T > 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.
Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis
Authors: BOWDEN Paul E. , QUINLAN Roy A. , BREITKREUTZ Dirk , FUSENIG Norbert E.
Published: March 2005

Keratins from the living cell layers of human and neonatal mouse epidermis (prekeratins) have been compared to those from the stratum corneum (SC keratins). Human and mouse epidermis contained four prekeratins, two of each keratin subfamily: type II basic (pI 6.5–8.5; human 68 kDa. 60.5 kDa and mouse 67kDa, 60kDa) and type I acidic (pI 4.7–5.7; human 57 kDa, 51 kDa and mouse 58 kDa, 53 kDa). While all four were present in equal amounts in adult human epidermis, two (67 kDa basic, 58 kDa acidic) were more prominent in neonatal mouse epidermis. Preliminary results with cell fractions (basal, spinous and granular) indicated that quantitative differences were a function of morphology, basal cells containing the smaller member of each subfamily and granular cells the larger. Mouse stratum corneum extracts contained four keratins (three in human): type II neutral–acidic (pI 5.7–6.7; human 65 kDa and mouse 64 kDa, 62 kDa) and type I acidic (pI 4.9–5.4; human 57.5 kDa, 55 kDa and mouse 58.5 kDa, 57.5 kDa). In both species, one‐dimensional and two‐dimensional peptide mapping (with V8 protease and trypsin respectively) indicated that while all four prekeratins were distinct gene products, similarities existed in the type II basic and the type I acidic keratin subfamilies. A strong homology also existed between type II SC keratins and the larger basic (type II) prekeratin (human 68 kDa and mouse 67 kDa) and between type I SC keratins and the larger acidic (type I) prekeratin (human 57 kDa and mouse 58 kDa). These results indicate a precursor‐product relationship within each keratin subfamily, between SC keratins and the prekeratins abundant in the adjacent granular layer. This differentiation‐related keratin processing was similar in mouse and human epidermis, and may represent a widespread phenomenon amongst keratinising epithelia.
Solubilization, purification and kinetic properties of three membrane‐bound long‐chain acyl‐coenzyme‐A thioesterases from microsomes of photosynthetic tissue
Authors: MURPHY Denis J. , MUKHERJEE Kumar D. , LATZKO Erwin , WOODROW Ian E.
Published: March 2005

Microsomal membranes of developing pea (Pisum sativum) leaves contained almost one third of the total long‐chain acyl‐CoA thioesterase activity found in the leaf cell. Three distinct forms of long‐chain acyl‐CoA thioesterase were purified by a combination of cholate‐solubilization, dialysis, ion‐exchange, and gel‐filtration chromatography. Purification factors of 4600,100 and 280 were achieved for the thioesterase forms I, II and III, respectively. Apparent molecular masses were: form I, 28 kDa; form II, 140 kDa: form III. 139 kDa. All the three thioesterases showed overlapping specificities towards palmitoyl‐CoA, stearoyl‐CoA, and oleoyl‐CoA but were inactive towards short‐chain acyl‐CoAs, such as acetyl‐CoA and malonyl‐CoA. Each thioesterase exhibited complex kinetic behavior, which was consistent with differential affinities of the enzymes for monomeric and micellar forms of their substrates. The significance of the kinetic behavior and possible regulatory role of these enzymes are discussed.
A study of the structure and dynamics of complexes between polymyxin B and phosphatidylglycerol in monolayers by fluorescence
Authors: THERETZ Alain , TEISSIE Justin , TOCANNE Jean‐François
Published: March 2005

Interactions between the antibiotic polymyxin B and monolayers of dipalmitoylglycerophosphoglycerol have been reinvestigated through a study of the structure and dynamics of the complexes by means of an interface fluorimeter of our fabrication. A fluorescence technique has been developed where the use of linearly polarized incident beams gives the simultaneous determination of the orientation and the lateral diffusion rate of a fluorescent probe inserted in the film. The present investigation was carried out with 12‐(9‐anthroyloxy)‐stearic acid, a fluorescent compound which forms non‐fluorescent photodimers upon illumination. Orientation of the probe was studied by computing the ratio of the two dimerization constants KD and the ratio of the fluorescence intensities obtained with crossed linearly polarized incident lights. The lateral diffusion rate of the probe was obtained by measuring fluorescence recovery after photobleaching (photodimerization) of the probe. Control experiments, carried out with dimyristoylglycerophosphocholine, a lipid which does not interact with polymyxin B, show that the antibiotic does not significantly modify the behaviour of the probe. Both in terms of orientation and dynamics, with respect to dipalmitoylglycerophosphoglycerol, when the antibiotic is present in the subphase (1 μM, saturating conditions), data indicate that the lipid remains in a liquid‐expanded state. This is true even at a high surface pressure (π∼37mN·m−1), above the apparent ‘transition’ which can be observed at 30–35 mN·m−1 on its compression isotherm. Computation of the contribution of polymyxin B to the film expansion leads to the conclusion that this ‘transition’ would be a structural transition between two models of interaction: one, below the ‘transition’, where the polypeptide ring penetrates between the film‐forming lipid molecules and another one, above the ‘transition’, were the antibiotic is adsorbed at the lipid‐water interface with only its hydrocarbon chain penetrating the film.
Potassium‐39 and sodium‐23 NMR studies of cation binding to phosvitin
Authors: BRAUNLIN William H. , VOGEL Hans J. , FORSÉN Sture
Published: March 2005

The binding of Na+ and K+ to hen egg‐yolk phosvitin has been studied by 23Na and 39K NMR. A transition in the binding behaviour of these cations is shown to accompany deprotonation of the phosphate groups on this highly charged protein. At lower pH the data are well described by a purely mass‐action binding model, whereas at higher pH significant polyelectrolyte effects are apparent. From single ion as well as competition experiments K+ is shown to bind more strongly to phosvitin than does Na+. The temperature and magnetic field strength dependences of the 23Na and 39K relaxation rates provide a picture of phosvitin as a highly flexible macromolecule. This work demonstrates the potential of 39K NMR as a useful tool in the study of protein–cation interactions.
Characterization of monoclonal antibodies against β‐bungarotoxin and their use as structural probes for related phospholipase A2 enzymes and presynaptic phospholipase neurotoxins
Authors: STRONG Peter N. , WOOD John N. , IVANYI Juraj
Published: March 2005

Hybridoma lines secreting monoclonal antibodies against a phospholipase‐inactive derivative of the presynaptic neurotoxin, β‐bungarotoxin, have been established. These antibodies, either of the IgG1 or IgG2b isotype with affinities in the range 1–2 × 108 l/mol, recognized a single immunodominant region of native β‐bungarotoxin, most probably located on the A (phospholipase homologue) chain of the toxin. Using plate‐adsorbed radioimmunoassay procedures, antibodies reacted with native β‐bungarotoxin and other β‐bungarotoxin isotoxins as well as with the non‐toxic phospholipase A also present in Bungarus multicinctus venom. Other phospholipase A enzymes and presynaptic phospholipase neurotoxins did not show any competition with β‐bungarotoxin in the radioimmunoassay. Globulin fractions of monoclonal antibodies partially inhibited the phospholipase activity of β‐bungarotoxin.
Expression of aldolase isozyme mRNAs in fetal rat liver
Authors: NUMAZAKI Masayoshi , TSUTSUMI Ken‐ichi , TSUTSUMI Reiko , ISHIKAWA Kiichi
Published: March 2005

The regulation of aldolase isozyme expression during development was studied by measuring the concentrations of mRNAs coding for aldolase A and B subunits in fetal and adult rat liver. Poly(A)‐containing RNAs were extracted from livers at various stages of development of fetal rats, and the aldolase A and B subunits in the in vitro translation products of these RNAs were analyzed immunologically. The content of aldolase B mRNA in 14‐day fetal liver, measured quantitatively as translational activity, was somewhat smaller than that of aldolase A mRNA; immunologically precipitable aldolase B and A amounted to 0.06% and 0.25%, respectively, of the total products. Similar experiments using RNAs from fetuses at later stages, however, showed that aldolase B mRNA increased during development, whereas aldolase A mRNA decreased. In newborn rat liver, aldolase B constituted 0.56% of the total translation products of mRNA, but there was little detectable aldolase A (0.03%).

The changes of aldolase mRNA levels were analyzed further by northern blot and dot‐blot hybridization experiments using cloned aldolase A and B cDNAs. The content of aldolase B mRNA increased in the fetal stage, and that in newborn rat liver was about 12 times that in 14‐day fetal liver. In contrast, the aldolase A mRNA content decreased during gestation and that in newborn rat liver was about one‐eighth of that in 14‐day fetal liver. These observations suggest that the switch of aldolase isozyme expression in fetal liver is controlled by the levels of the respective mRNAs.
Lipoproteins from the blood and egg yolk of the hen
Authors: BURLEY Ralph W. , SLEIGH Robert W. , SHENSTONE F. Stanley
Published: March 2005

As part of a study of the transfer of proteins and lipids from avian blood to egg yolk, some properties of lipoproteins from the blood of laying hens were compared with those of the low‐density lipoprotein (YLP) from egg yolk of the same hens. Although it is known from previous work that particles of the very‐low‐density lipoprotein (VLDL) of blood are the most likely precursors of YLP, their apoprotein patterns are different, according to electrophoresis and chromatography, with only one protein in common. YLP has the more complicated pattern which does not, however, include apoprotein B (ApoB) the main apoprotein of VLDL. It is suggested that during the transfer of VLDL to yolk, ApoB is cleaved to give smaller yolk apoproteins, especially apovitellenins IV and VI. Some evidence for this suggestion from the similarity of protein digests is presented.
A cold‐labile acetyl‐coenzyme‐A hydrolase from the supernatant fraction of rat liver
Authors: ISOHASHI Fumihide , NAKANISHI Yoko , MATSUNAGA Takaharu , SAKAMOTO Yukiya
Published: March 2005

An acelyl‐coenzyme‐A hydrolase from the supernatant fraction of rat liver is known to be rapidly inactivated at low temperature. Loss of catalytic activity is accompanied by apparent dissociation of tetrameric and dimeric forms of the enzyme into monomers. It was found that rewarming under appropriate conditions almost completely reversed the cold‐induced inactivation and dissociation of the enzyme: At a protein concentraion of 14 μg/ml, simple rewarming only partially restored the enzyme activity (less than 3% of the original activity), but at a higher concentration of the enzyme or in the presence of 1 mg/ml bovine serum albumin, the reactivation by warming was greater. Warming at 37°C appeared to be optimal for reactivation; warming at 25°C or at 43°C was less effective. Longer exposure to cold did not affect reactivation on rewarming, but on repeated inactivation and reactivation the reactivation decreased to some extent, especially at lower concentrations of enzyme protein. Among various nucleotides tested, ATP greatly enhanced the restoration of the activity, while ITP, UTP and ADP were less effective and AMP, GTP, TTP and CTP had little effect. At an enzyme‐protein concentration of 14 μg/ml, 2 mM ATP restored the enzyme activity to about 70% of that before cold treatment, while acetyl‐CoA (0.5 mM) restored the activity about 50% High concentrations of phosphate (0.92 M) and pyrophosphate (0.45 M) restored about 80% and 95%, respectively, of the original activity.

Sucrose density gradient centrifugation of the active dimer at high enzyme concentration at 4°C for 20 h produced a monomeric form without catalytic activity. Gel filtration showed that simple rewarming mostly converted the monomeric enzyme obtained in this way to the dimeric form, whereas on rewarming with ATP the monomer was mostly converted to a tetrameric form. The dimeric and tetrameric forms both had catalytic activity.
Human low‐molecular‐mass kininogen
Authors: LOTTSPEICH Friedrich , KELLERMANN Josef , HENSCHEN Agnes , RAUTH Günther , MÜLLER‐ESTERL Werner
Published: March 2005

The complete amino acid sequence of the human low‐molecular‐mass kininogen light chain has been determined. The peptide chain contains 38 amino acid residues. In position 18 the half‐cystine residue which forms the disulfide bridge to the heavy chain was found. Alignment of the present sequence with other kininogen light chain sequences indicated that the amino‐terminal part of it is species‐specific and the carboxy‐terminal part is function‐specific. Statistically significant internal homologies between various parts of the low‐molecular‐mass kininogen sequence as well as homologies between parts of the sequences of kininogen and ribonucleases were observed.