bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Octanoate and Palmitate on the Metabolism of Valine in Perfused Hindquarter of Rat
Authors: SPYDEVOLD Øystein
Published: March 2005

The effect of octanoate and palmitate on the oxidation of 14C‐labelled valine has been studied in perfused hindquarter of rat.

The oxidation rate of valine increases 30 times when the concentration of valine is increased from 0.1 mM to 5 mM.

Octanoate at a 5 mM concentration effected a 10‐fold increase in the flux through the α‐keto‐isovalerate dehydrogenase step and a 5‐fold increase at 0.5 mM concentration.

Palmitate (1 mM) effects only a moderate increase in the valine oxidation.

With no octanoate there was a great accumulation of α‐ketoisovalerate in both the muscle and the perfusion medium.

With octanoate little α‐ketoisovalerate accumulated whereas 3‐hydroxyisobutyrate was found in high concentration both in the muscle and in the medium.

Octanoate stimulated the production of citric‐acid‐cycle intermediates and lactate.

The results are discussed in relation to valine metabolism in the body.
3′ Esters of ADP as Energy‐Transfer Inhibitors and Probes of the Catalytic Site of Oxidative Phosphorylation
Authors: SCHÄFER Günter , ONUR Günes
Published: March 2005

A large series of 3′ esters of ADP has been synthesized. Several of these can serve as photoaffinity labels; others exhibit fluorescent properties. The corresponding AMP and ATP derivatives have also been synthesized in some cases.

The influence of the 3′‐O‐acyl nucleotides on energy‐linked functions of beef‐heart submitochondrial particles has been investigated. The following results were obtained.

a) 3′ Esters of ADP are powerful and highly specific inhibitors of oxidative phosphorylation. The inhibition is competitive to ADP and Ki values as low as 0.05 μM, for the 3′‐O‐(1)naphthoyl ester of ADP, could be observed.

b) The inhibition of oxidative phosphorylation by 3′ esters of ADP appears to be non‐competitive versus inorganic phosphate.

c) The nucleotide analogs are not phosphorylated themselves. The corresponding ATP analogs can not drive energy‐linked processes.

d) The 3′ esters of AMP are ineffective as inhibitors, whereas the ATP derivatives are only comparatively weak inhibitors.

e) Uncoupled or solubilized ATPase is almost two orders of magnitude less sensitive against inhibition by 3′ esters than coupled systems. The analogs exert maximal inhibition specifically in systems involving an ‘energized’ state of the coupling device.

f) Azido‐group‐bearing analogs can be used for irreversible photoinactivation of the coupling ATPase. Photoinactivation also is most efficient when carried out with ‘energized’ particles.

g) The inhibitory properties are similar also in ATP‐driven NAD+ reduction by succinate, and in the uncoupler‐sensitive ATP ⇌ Pi exchange. The required concentrations for half‐maximal inhibition are somewhat higher than in oxidative phosphorylation, but lower than with uncoupled ATPase.

From molecular models, from substituent properties, and from the conditions required for inhibition it is concluded that these highly effective analogs of ADP may act as conformation‐specific probes at the catalytic site of oxidative phosphorylation. The results are interpreted in terms of a model suggesting that, in the process of ATP synthesis, a hydrophobic cavity on the enzyme is exposed only in the energized state, accepting the large 3′ substituent. The substituent is assumed to inhibit phosphoryl transfer and/or conformational transitions inherent in the process of ADP phosphorylation by steric hinderance.
The Excision of N‐Methyl‐N‐nitrosourea‐Induced Lesions from the DNA of Chinese Hamster Cells as Measured by the Loss of Sites Sensitive to an Enzyme Extract that Excises 3‐Methylpurines but not O6‐Methylguanine
Authors: SHACKLETON Jason , WARREN William , ROBERTS John J.
Published: March 2005

An enzyme extract from Micrococcus luteus excises 3‐methyladenine and 3‐methylguanine but not O6‐methylguanine, 7‐methylguanine, 1‐methyladenine or 7‐methyladenine from DNA reacted with N‐methyl‐N‐nitrosourea. The extract was used to detect lesions in the DNA of Chinese hamster cells treated in culture with N‐methyl‐N‐nitrosourea. It was concluded that 3‐methyladenine is excised from these cells with a half‐life of about 2.3 h.
The Structure of a Transcriptional Unit on Colicin E1 Plasmid
Authors: MORITA Masayuki , OKA Atsuhiro
Published: March 2005

In an RNA‐synthesizing system in vitro, a low‐molecular‐weight RNA consisting of about 110 residues (RNA‐I) was efficiently synthesized on DNA of colicin E1 plasmid (ColE1) and its deletion derivatives. The promoter site for RNA‐I was analysed by testing the RNA polymerase‐binding ability and template activity of restriction fragments; it was mapped in the region between the replication initiation site and the colicin immunity gene of ColE1. The direction of transcription was determined by hybridization tests to the separated strands of the template. The DNA region directing RNA‐I was sequenced, and RNA‐I was assigned on the sequence based on the nearest‐neighbour data of RNA. The sequences of its promoter and terminator regions were also deduced. Although the function of this small RNA species is unknown, a unique secondary structure could be constructed from its sequence and sensitivity to RNase.
The Association of Actin with a Thyroid Lysosomal Fraction
Authors: DICKSON John G. , MALAN P. Gordon , EKINS Roger P.
Published: March 2005

A lysosome‐enriched fraction was prepared from bovine thyroid tissue using sucrose gradient centrifugation. An inhibitor of DNAase I was found to co‐sediment and co‐purify with the lysosomal fraction. This inhibitory activity is blocked by heavy meromyosin in the absence of ATP, and a component of 42000 molecular weight can be isolated by affinity chromatography on DNAase I linked to Sepharose. These results are consistent with the presence of an actin‐like protein in a lysosome‐enriched preparation from bovine thyroid tissue. Also, an increase in the level of membrane‐associated actin is observed in response to thyrotropin stimulation of the thyroid tissue.
Yeast Phenylalanyl‐tRNA Synthetase
Authors: BALTZINGER Mireille , FASIOLO Franco , REMY Pierre
Published: March 2005

The localization of the binding sites of the different ligands on the constitutive subunits of yeast phenylalanyl‐tRNA synthetase was undertaken using a large variety of affinity and photoaffinity labelling techniques. The tRNAPhe was cross‐linked to the enzyme by non‐specific ultraviolet irradiation at 248 nm, specific irradiation in the wye base absorption band (315 nm), irradiation at 335 nm, in the absorption band of 4‐thiouridine (s4U) residues introduced in the tRNA molecule, or by Schiff's base formation between periodate‐oxidized tRNAPhe (tRNAPheox) and the protein. ATP was specifically incorporated in its binding site upon photosensitized irradiation.

The amino acid could be linked to the enzyme upon ultraviolet irradiation, either in the free state, engaged in the adenylate or bound to the tRNA.

The tRNA, the ATP molecule and the amino acid linked to the tRNA were found to interact exclusively with the β subunit (Mr 63000). The Phenylalanine residue, either free or joined to the adenylate, could be cross‐linked with equal efficiency to either type of subunit, suggesting that the amino acid binding site is located in a contact area between the two subunits.

The Schiff's base formation between tRNAPheox and the enzyme shows the existence of a lysyl group close to the binding site for the 3′‐terminal adenosine of tRNA. This result was confirmed by the study of the inhibition of yeast Phenylalanyl‐tRNA synthetase with pyridoxal phosphate and the 2′,3′‐dialdehyde derivative of ATP, oATP.
Immunochemical Studies of Beef Pancreas Tryptophanyl‐tRNA Synthetase and Its Fragments
Authors: SCHEINKER Vladimir Sh. , BERESTEN Sergey F. , MAZO Alexander M. , AMBARTSUMYAN Noona S. , ROKHLIN Oskar V. , FAVOROVA Ol'ga O. , KISSELEV Lev L.
Published: March 2005

The immunoglobulin G (IgG) fraction of the antiserum from rabbits immunized with homogeneous beef pancreas tryptophanyl‐tRNA synthetase inhibits the enzyme activity in the reactions of both tRNATrp aminoacylation and tryptophan activation. Fab fragments of IgG act in a similar way. Common antigenic determinants have been detected in tryptophanyl‐tRNA synthetases from beef, pig, chicken and rat livers using pure antibodies against beef pancreas tryptophanyl‐tRNA synthetase. This observation indicates the evolutional stability of certain structural features of tryptophanyl‐tRNA synthetases.

The interaction of antibodies with the fragments of beef tryptophanyl‐tRNA synthetase produced by endogenous and tryptic proteolysis of the enzyme has been studied. One third of the antiserum antibodies interacting with the C‐terminal fragment of the enzyme (Mr∼ 40000) inhibits its activity whereas the antibodies to the N‐terminal fragment (Mr∼ 20000) have no effect on the enzyme activity.

The immunochemical identity of the two synthetase fragments differing in their enzymatic activity supports the assumption that the loss of enzymatic activity of the tryptic fragment is caused by lack of a small peptide which is retained in case of endogenous proteolysis; probably the amino acid residues of this peptide participate in formation of active centre of tryptophanyl‐tRNA synthetase.

A radioimmunochemical method is described for determining the number of antigenic determinants. One molecule of tryptophanyl‐tRNA synthetase was found to bind 9(± 1) molecules of Fab fragments.

Antibodies against tryptophanyl‐tRNA synthetase from beef pancreas do not inhibit noticeably the activity of reverse transcriptase from avian myeloblastosis virus. No antigenic determinants in common have been detected in reverse transcriptase and tryptophanyl‐tRNA synthetase by radio‐immunochemical assays.
Interaction of ADP with Skeletal and Cardiac Myosin and Their Active Fragments Observed by Proton Release
Authors: KARDAMI Elizavetta , DE BRUIN Simon , GRATZER Walter
Published: March 2005

The technique of proton release measurement has been used to explore the binding of ADP to skeletal and cardiac myosins and their active fragments in a variety of conditions. It has proved possible to obtain binding profiles on intact myosin in the filamentous, undissolved form in physiological solvent conditions. Binding constants are given. At higher ionic strength (0.5 M potassium chloride) the binding profile of magnesium‐ADP is compatible with the presence of two types of site, differing from one another both in respect of affinity and the number of protons released per site. Studies with cardiac myosin reveal no such indications of heterogeneity, and are consistent with the presence of a single population of thermodynamically indistinguishable sites. In the absence of divalent cations, in solutions containing potassium ions and EDTA, ADP binds with absorption rather than liberation of protons. The pH profile of proton absorption at saturation can be fitted in terms of an ionising group with an unperturbed pK of 9.4, and at least one of lower pK (5.9). The dissociation constant (pH 8 at 5°C) is about 8 μM, and the affinity for uncomplexed ADP is thus only slightly weaker than that for magnesium‐ADP.
Mitochondrial ATPase Complex of Aspergillus nidulans and the Dicyclohexylcarbodiimide‐Binding Protein
Authors: TURNER Geoffrey , IMAM Gamal , KÜNTZEL Hans
Published: March 2005

The dicyclohexylcarbodiimide‐binding protein of Aspergillus nidulans has been identified as the smallest subunit of the mitochondrial ATPase complex, and has a molecular weight of approximately 8000. It is extractable from whole mitochondria and from the purified enzyme in neutral chloroform/methanol, contains 30% polar amino acids, and the N‐terminal amino acid has been identified as tyrosine. Using a double‐labelling technique in the absence and presence of cycloheximide, followed by immunoprecipitation of the enzyme complex with antiserum against Neurospora crassa F1 ATPase, it has been shown that this subunit is synthesized on cytoplasmic ribosomes.
Aggregation of Sponge Cells
Authors: MÜLLER Werner E. G. , ZAHN Rudolf K. , KURELEC Branko , MÜLLER Isabel , VAITH Peter , UHLENBRUCK Gerd
Published: March 2005

From the cell membranes of the sponge Geodia cydonium a component was isolated and purified which inhibits the aggregation factor isolated from the same source; the component was termed anti‐aggregation receptor. This molecule was characterized as a glycoprotein (54% neutral carbohydrate) and its molecular weight is in the range of 18000. One biological site of the anti‐aggregation receptor was determined to be d‐galactose. Indirect evidence presented seems to indicate that this molecule is present in an active form in aggregation‐deficient cells and absent in aggregation‐susceptible cells.
A New Purification Scheme for Elongation Factor 1 from Rabbit Reticulocytes and Investigation of the Homology of the Subunits with Those of Initiation Factor 2
Authors: MORETTI Simonetta , STAEHELIN Theophil , TRACHSEL Hans , GORDON Julian
Published: March 2005

The aim of this work was to compare the subunits of the elongation factor EF‐1 and the initiation factor eIF‐2 from rabbit reticulocytes. We devised a simple procedure for the purification of EF‐1: stepwise Chromatography on heparin‐Sepharose, separation of the heavy form by sucrose gradient centrifugation, and a final step of stepwise chromatography on RNA‐Sepharose. The heparin‐Sepharose column also clearly separated EF‐1 and EF‐2 within one chromatographic step. The EF‐1 was 350‐fold purified and the yield was 10%. This preparation showed after electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate three bands corresponding to those described by others as the subunits, with Mr of 54000, 49000 and 29200. An additional band of Mr 34000 was present but no others. The 49000‐Mr and 34000‐Mr bands corresponded exactly in molecular weight to two of three subunits of eIF 2. A more detailed comparison was therefore made of all subunits of EF‐1 and eIF‐2. This was done by examination of chymotryptic fingerprints on polyacrylamide gel electrophoresis. No evidence for homology between EF‐1 and eIF‐2 was found. However, the two larger subunits of eIF‐2 had a majority of chymotryptic fragments in common, thus indicating some homology between these polypeptides.
Chemical Evidence for a Codon‐Induced Allosteric Change in tRNALys Involving the 7‐Methylguanosine Residue 46
Authors: WAGNER Rolf , GARRETT Roger A.
Published: March 2005

[32P]tRNALyS, from Escherichia coli, was modified with kethoxal, in the presence and absence of the oligonucleotide codon (A)4. The presence of the codon resulted in a faster modification rate of the tRNA at three guanine sites which were identified by a diagonal fingerprint method. A large increase in the modification rate occurred at the 7‐methylguanosine residue 46 (m7G‐46) in the presence of the codon; weakly enhanced modification was observed at G‐15 and G‐57.

It is concluded that the formation of a codon‐anticodon complex induces, primarily, a confor‐mational change involving disruption of the m7G‐46 from the m7G‐46 · G‐22 · C‐13 base triple. Subsequently, the guanines of G‐15 and G‐57, in the D and T loops, respectively, become slightly more reactive, suggesting a weak tendency for these two interacting arms to unfold.

The results are interpreted in terms of an equilibrium between two main conformers and a third minor one; the possible significance of these conformers, in protein biosynthesis, is considered.
Identification of the Translation Products of Human Fibroblast Interferon mRNA in Reticulocyte Lysates
Authors: WEISSENBACH Jean , ZEEVI Menachem , LANDAU Tamar , REVEL Michel
Published: March 2005

Messenger RNA was purified from human foreskin fibroblasts FS11, a high interferon‐producer line, after induction with synthetic double‐stranded RNA. The mRNA was translated in a cell‐free protein‐synthesis system from rabbit reticulocytes. The translation products, containing biologically active human interferon, were immunoprecipitated by a serum from rabbits immunized against partially purified interferon. Analysis of the immunoprecipitate by polyacrylamide gel electrophoresis in dodecylsulfate shows that the product of human fibroblast interferon mRNA is a 23000‐Mr polypeptide. Methods are described for the synthesis and rapid identification of this polypeptide, which should be useful for structural analysis of interferon and isolation of its mRNA.
A Lipopolysaccharide‐Binding Cell‐Surface Protein from Salmonella minnesota
Authors: GEYER Rudolf , GALANOS Chris , WESTPHAL Otto , GOLECKI Jochen R.
Published: March 2005

Protein extracts obtained from Salmonella minnesota Re mutant cells by treatment with EDTA/NaCl solution contain a protein which exhibits high affinity to bacterial lipopolysaccharides. The isolation and partial characterization of this lipopolysaccharide‐binding protein is described.

The protein was purified from EDTA extracts by a two‐step procedure consisting of ion‐exchange chromatography on CM‐Sephadex and preparative polyacrylamide gel electrophoresis at pH 9.5. The yield of the total purification procedure was around 16%.

The resulting protein preparation was homogeneous on the basis of disc gel electrophoresis, dodecylsulfate gel electrophoresis, isoelectric focusing in polyacrylamide gel and immunoelectrophoresis.

The isoelectric point of the protein was found to be 10.3 at 4°C. Its molecular weight determined by dodecylsulfate gel electrophoresis is 15000. Its amino acid composition is characterized by the absence of histidine and proline, a low content in tyrosine and high amounts of alanine, lysine, aspartic and glutamic acid residues, or their respective amides.

The lipopolysaccharide‐protein association was shown to be mainly due to ionic interactions of the basic protein with negatively charged groups (probably phosphate and pyrophosphate groups) of the lipid A moiety.

Purified lipopolysaccharide‐binding protein is immunogenic in rabbits, thus enabling the preparation of specific antiserum.

The protein is located at the surface of Salmonella minnesota Re mutant cells as revealed by antiserum absorption with total bacteria. Ferritin‐labelling studies further demonstrated that it is evenly spread over the entire cell surface.

Comparative antiserum absorption studies using smooth and rough strains of Salmonella minnesota, Salmonella typhimurium, Escherichia coli, Klebsiella and Shigella revealed the presence of lipopolysaccharide‐binding protein (or a serologically cross‐reacting antigen) in most of the strains tested. From these results the protein can be considered as a common antigen of Enterobacteriaceae.
Characterization of Long Guanosine‐Free RNA Sequences from the Dahlemense and U2 Strains of Tobacco Mosaic Virus
Authors: KUKLA Brice A. , GUILLEY Hubert A. , JONARD Gerard X. , RICHARDS Kenneth E. , MUNDRY Kurt W.
Published: March 2005

Four naturally occurring strains of tobacco mosaic virus, U2, Dahlemense, CV4, and the bean form of tobacco mosaic virus, were tested for the existence of long T1 RNAase oligonucleotides analogous to the oligonucleotide Ω found in the common or U1 strain of tobacco mosaic virus and which makes up the 5′ non‐coding region of the RNA molecule. U2 and Dahlemense RNA were each found to contain this type of long T1 RNAase oligonucleotide with chain lengths of 54 and 74–77 residues, respectively. The sequence of the two oligonucleotides was determined mostly by using 5′‐32P‐labelled material in vitro and rapid polyacrylamide gel sequencing techniques.
Binding of Coenzyme and Substrate and Coenzyme Analogues to 6‐Phosphogluconate Dehydrogenase from Sheep Liver
Authors: ABDALLAH Mohamed A. , ADAMS Margaret J. , ARCHIBALD Ian G. , BIELLMANN Jean‐François , HELLIWELL John R. , JENKINS Susan E.
Published: March 2005

The analogues of the coenzyme NADP+, nicotinamide–8‐bromo‐adenine dinucleotide phosphate (Nbr8ADP+) and 3‐iodopyridine–adenine dinucleotide phosphate (io3PdADP+), were prepared. Nbr8ADP+ was found to be active in the hydrogen transfer and io3PdADP+ is a coenzyme competitive inhibitor for 6‐phosphogluconate dehydrogenase. The binding of NADP+, NADPH and NADPH together with 6‐phosphogluconate as well as that of both analogues to crystals of the enzyme 6‐phosphogluconate dehydrogenase has been investigated at 0.6‐nm resolution using difference electron density maps. The molecules bind in a similar position in a cleft in the enzyme subunit distant from the dimer interface. The orientation of the coenzyme in the site has been determined from the io3PdADP+–NADP+ difference density. The ternary complex difference density extends beyond that of the nicotinamide moiety of the coenzyme and tentatively indicates substrate binding. No clear identification of the bromine atom of Nbr8ADP+ can be made. However, the analogue is bound more deeply in the cleft than is NADP+. The NADPH density is the most clearly defined and has thus been used to fit a molecular model using an interactive graphics system, checking for preferred geometry. A possible conformation is presented which is significantly different from that of NAD+ in the lactate dehydrogenase ternary complex.
Formylmethionyl‐tRNA Binding Properties of Escherichia coli Ribosomal Protein S1
Authors: LI Percy T. , SHEA Timothy , ELLIS Steven , CONWAY Thomas W.
Published: March 2005

Formylmethionyl‐tRNA has been found to bind to Escherichia coli ribosomal protein S1. Complexes of S1 and methionine‐labeled fMet‐tRNAMetf were detected by binding to cellulose nitrate membranes. Complex formation was stimulated by incubation at low ionic strength, at 37°C and in the absence of Mg2+. Protein S1 also bound [3H]poly(rC) under the same conditions or in buffers of higher ionic strength, 10 mM in Mg2+, and during incubation at 0°C. Unlabeled poly(rC) was an effective inhibitor of the binding of labeled fMet‐tRNA when both nucleotides were added simultaneously to the reaction mixtures; however, approximately 80% of previously bound f[35S]Met‐tRNA remained bound after the subsequent addition of unlabeled poly(rC). Binding of f[35S]Met‐tRNA, but not the binding of [3H]poly(rC), was inhibited by polyamines or aminoglycoside antibiotics. Protein S1 that had been reacted with N‐ethylmaleimide failed to bind f[35S]Met‐tRNA but was still capable of binding [3H]poly(rC).

The data support the concept of two distinct RNA‐binding sites on the S1 molecule. Initiator tRNA appears to occupy a site concerned with the RNA unwinding property of S1, whereas poly(rC) binds primarily at a separate site. During initiation on the ribosome, the poly(rC) site may interact with the 3′ end of 16‐S ribosomal RNA, while the other site may interact with either mRNA or fMet‐tRNA.
Mitochondrial Protein‐Synthesizing Machinery in Saccharomyces cerevisiae Grown in Different Metabolic Conditions
Authors: BALDACCI Giuseppe , FALCONE Claudio , FRANCISCI Silvia , FRONTALI Laura , PALLESCHI Claudio
Published: March 2005

The isoacceptor patterns of mitochondrial seryl and alanyl‐tRNAs from Saccharomyces cerevisiae grown under different conditions and committed to fermentative or respiratory metabolism have been studied by reversed‐phase chromatography.

An extensive variability of the chromatographic patterns of the four isoacceptors for serine and of the three isoacceptors for alanine has been observed as a function of carbon source and stage of growth, but the main differences were observed in the different stages of growth on glucose.

In order to distinguish the effects due to stage of growth from those due to relief from glucose repression, the isoacceptor patterns of mitochondrial tRNA were compared in repressed and derepressed resting cells; results show that some of the mitochondrial species are almost undetectable in resting repressed cells, but are the major ones after recovery of respiration.

Two of the serine isoacceptors (species 1 and 2), one of which is absent or not acylable in repressed resting cells, are different gene products.
Properties of Herpes Simplex Virus DNA Polymerase and Characterization of Its Associated Exonuclease Activity
Authors: KNOPF Karl‐Werner
Published: March 2005

Herpes simplex virus (HSV) DNA polymerase was isolated on a large‐scale from African green monkey kidney cells infected with HSV type 1 (HSV‐1) strain Angelotti. After DNA‐cellulose chromatography the enzyme showed a specific activity of 48000 units/mg protein. Three major single polypeptides with molecular weights of 144000, 74000 and 29000 were copurified with the enzyme activity at the DNA‐cellulose step. By its chromatographic behavior and by template studies, the HSV DNA polymerase activity was clearly distinguishable from cellular α, β and γ DNA polymerase activities. Two exonucleolytic activities were found in the DNA‐cellulose enzyme preparation. The main exonucleolytic activity, which degraded both single‐stranded and double‐stranded DNA to deoxynucleoside 5′‐monophosphates, was separated by subsequent velocity sedimentation. The remaining exonucleolytic activity was not separable from the HSV DNA polymerase by several chromatographic steps and by velocity sedimentation at high ionic strength. This novel exonuclease and HSV DNA polymerase were equally sensitive both to phosphonoacetic acid and Zn2+ ions, inhibitors of the viral polymerase. Similar to the 3′‐to‐5′‐exonuclease of procaryotic DNA polymerases and mammalian DNA polymerase δ, the HSV‐polymerase‐associated exonuclease catalyzed the removal of 3′‐terminal nucleotides from the primer/template as well as the template‐dependent conversion of deoxynucleoside triphosphates to monophosphates.
Ontogeny of the Adenosine‐3′:5′‐Phosphate‐Dependent Protein‐Kinase System during Early Uterine Development
Authors: EPPENBERGER Urs , ROOS Werner , FABBRO Doriano , SURY Adrian , WEBER Judith , BECHTEL Elke , HUBER Peter , JUNGMANN RICHARD A.
Published: March 2005

The development of protein kinase and estrogen‐binding proteins was studied in the nuclear and cytosol fractions of rat uteri as a function of postnatal age.

Analysis of cyclic‐AMP‐dependent protein kinase activity, with protamine sulfate as substrate in the presence of 3 μM cyclic AMP, revealed that the cytosol as well as the nuclear cAMP‐dependent protein kinase activity increased markedly between the third and fifth postnatal days. The total cytosol protein kinase activity remained constant after the age of 5 days (850 pmol [32P]phosphate incorporated × min−1× mg cytosol protein−1), but nuclear cAMP‐dependent protein kinase activity decreased sharply (60%), coinciding with a time period during which nuclei acquire considerable estrogen‐binding activity.

Analysis of the multiplicity of cyclic‐AMP‐dependent protein kinases by chromatography of uterine cytosol on DEAE‐cellulose revealed the presence of type I and type II cyclic‐AMP‐dependent protein kinase. Type I isoenzyme levels decreased relative to the type II isozyme from the time of birth to the age of 20 days, type II isozyme did not undergo any major developmental changes.

The specific estrogen binding activity in utertine cytosol reached a maximum in 12‐day‐old rats (1.7 pmol of 17β‐estradiol bound/mg protein). It declined thereafter and reached constant levels in 17‐day‐old and older rats. Concomitantly nuclear levels of estrogen binding protein increased progressively from very low levels (0.1 pmol) in 5‐day‐old rats to 1.2 pmol of 17β‐estradiol bound/mg of nuclear protein in 12‐day‐old and 20‐day‐old rats.

The results indicate the early existence of a functional uterine cyclic‐AMP/protein kinase system in the neonatal rat and suggest that the protein kinase system and specific estrogen‐binding activity develop in a reciprocal manner.
Purification and Properties of Nitrogenase from the Cyanobacterium, Anabaena cylindrica
Authors: HALLENBECK Patrick C. , KOSTEL Paul J. , BENEMANN John R.
Published: March 2005

The nitrogenase complex was isolated from nitrogen‐starved cultures of Anabaena cylindrica. Sodium dithionite, photochemically reduced ferredoxin, and NADPH were found to be effective electron donors to nitrogenase in crude extracts whereas hydrogen and pyruvate were not. The Km for acetylene in vivo is ten‐fold higher than the Kmin vitro, whereas this pattern does not hold for the non‐heterocystous cyanobacterium, Plectonema boryanum. This indicates that at least one mechanism of oxygen protection in vivo involves a gas diffusion barrier presented by the heterocyst cell wall. The Mo‐Fe component was purified to homogeneity. Its molecular weight (220000), subunit composition, isoelectric point (4.8), Mo, Fe, and S2− content (2, 20 and 20 mol/mol component), and amino acid composition indicate that this component has similar properties to Mo‐Fe‐containing components isolated from other bacterial sources. The isolated components from A. cylindrica were found to cross‐react, to varying degrees, with components isolated from Azotobacter vinelandii, Rhodospirillum rubrum, and P. boryanum.
Cryoenzymological Studies on Myosin Subfragment 1
Authors: TRAVERS Franck , HILLAIRE Dominique
Published: March 2005

The ATPase activities of myosin subfragment 1 were studied under subzero conditions with ethylene glycol as the antifreeze.

The pH profile of the Ca ATPase activity was affected by both ethylene glycol and the temperature but the pH profile of the Mg ATPase activity was only slightly affected.

At 20°C the Mg ATPase activity was not affected by the solvent. The Ca and acto‐Mg ATPase activities decreased to similar extents as the solvent concentration was increased. These results are discussed in terms of the accessibility of the active site.

The Mg, Ca and acto‐Mg ATPase activities were studied down to −20°C, in 40% ethylene glycol. All three systems showed an increase in activation energy at low temperatures. These results were compared to previous work and they were analysed in terms of a change in the rate‐limiting step.

When myosin S1 is mixed with ATP, difference spectra are obtained whose amplitudes decrease with time and which are a manifestation of the intermediate M**· ADP · Pi (the product complex). The results obtained show that the disappearance of this complex occurs in the post‐steady‐state phase at 20°C but in the pre‐steady‐state phase at –20°C. These studies provide the necessary data for planning work on the stabilization of the reaction intermediates on, in particular, the actomyosin pathway. By working at –20°C in 40% ethylene glycol a 1000‐fold decrease in this activity is obtained.
A Centrifugation Study of Rat‐Liver Mitochondria, Lysosomes and Peroxisomes during the Perinatal Period
Authors: MERTENS‐STRIJTHAGEN Jeanine , DE SCHRIJVER Christian , WATTIAUX‐DE CONINCK Simone , WATTIAUX Robert
Published: March 2005

We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de Duve et al. (1955). In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose‐6‐phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5′‐Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme we used, does not appreciably differ for the late foetal, early postnatal and adult rat livers.

An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distributions of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal: foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H2O gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.
The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by 1H Nuclear Magnetic Resonance
Authors: LENSTRA Johannes A. , BOLSCHER Ben G. J. M. , STOB Sijtze , BEINTEMA Jaap J. , KAPTEIN Robert
Published: March 2005

1. The aromatic proton resonances in the 360‐MHz 1H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments.

2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His‐1 19) and two tyrosines are accessible to photo‐excited flavin. This permitted the identification of the C‐4 proton resonance of His‐119.

3. The resonances of the ring protons of Tyr‐25, Tyr‐76 and Tyr‐1 15 and the C‐4 proton of His‐12 were identified by comparison with subtilisin‐modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr‐73, Tyr‐92 and Phe‐46.

4. On addition of active‐site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe‐120.

5. Four of the six tyrosines of bovine RNase, identified as Tyr‐76, Tyr‐115 and, tentatively, Tyr‐73 and Tyr‐92, are titratable above pH 9. The rings of Tyr‐73 and Tyr‐115 are rapidly rotating or flipping by 180° about their Cβ‐Cγ bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr‐25 is involved in a pH‐dependent conformational transition, together with Asp‐14 and His‐48. A scheme for this transition is proposed.

6. Binding of active‐site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH‐dependent transition in the region of Asp‐14, Tyr–25 and His‐48.

7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.
Properties of Bacillus subtilis ATP‐Dependent Deoxyribonuclease
Authors: SHEMYAKIN Michael F. , GREPACHEVSKY Alexander A. , CHESTUKHIN Arsenii V.
Published: March 2005

A purification procedure described previously resulting in electrophoretically pure Bacillus subtilis ATP‐dependent DNAase has now been modified by adding a fractionation stage with Polymin P to permit large‐scale isolation of the enzyme. It has been found that the enzyme molecule (Mr= 300000) consists of two large subunits with Mr 155000 and 140000. The purified enzyme has three activities: (1) DNAase on linear single‐stranded and double‐stranded DNAs, (2) DNA unwinding and (3) ATPase. Circular duplex DNAs were not affected by the enzyme. Study of the dependence of these activities on temperature, pH, and ATP and Mg2+ concentrations has revealed two different states of the enzyme. At low ATP concentrations and alkaline pH, it showed chiefly nuclease action, degrading considerable amounts of DNA to small fragments five residues long on average. At higher ATP concentrations and neutral pH (more physiological conditions) it predominantly unwound DNA. Simultaneously it cut preferentially one of the duplex strands to fragments more than 1000 residues in length. The results obtained suggest that the energy of the enzymecleaved ATP is mainly expended on unwinding rather than on degrading DNA molecules.
Investigation of Soluble Mitochondrial ATPase by the Reacting Enzyme Sedimentation Method
Authors: CHERNYAK Victor Ya. , KOZHANOVA Zinayda E. , CHERNYAK Boris V. , KOZLOV Igor A.
Published: March 2005

Soluble mitochondrial ATPase from bovine heart (factor F1) loses its activity during ATP hydrolyses. The inactivation is accelerated by moderate pressure, which is generated in an ultracentrifuge cell. The rate of inactivation slows down if the concentration of the substrate (MgATP) is diminished. ATP hydrolysis proceeds at an almost constant rate if the substrate concentration is as low as 0.05 mM.

One intersubunit cross‐link formed by dimethylsuberimidate per molecule of factor F1, prevents its inactivation during the ATPase reaction both without pressure and in an ultracentrifuge.

Sedimentation coefficients measured by the reacting enzyme centrifugation method of both unmodified factor F1 at a low (about 0.05 mM MgATP) substrate concentration and of its dimethylsuberimidate cross‐linked form in the presence of 10 mM MgATP, were determined to be s20,w= 12.4 ± 0.4 S. The value is the same as that obtained by the conventional boundary sedimentation method in the absence of the substrate.

This result testifies to the fact that the conformation of reacting factor F1 in solution is similar to that of the enzyme in the absence of the substrate.
Sodium and Calcium Binding to Panulirus interruptus Hemocyanin as Studied by 23Na Nuclear Magnetic Resonance
Authors: NORNE Jan‐Erik , GUSTAVSSON Hans , FORSÉN Sture , CHIANCONE Emilia , KUIPER Harm A. , ANTONINI Eraldo
Published: March 2005

Addition of Panulirus hemocyanin to NaCl solutions produces marked changes in the 23Na relaxation parameters; they show that sodium ions interact with binding sites on the protein and exchange rapidly with the bulk. The observed non‐lorentzian lineshapes and the non‐exponential decay of the transverse magnetization indicate that non‐extreme narrowing conditions apply and give information on the dynamics of the interaction.

Panulirus hemocyanin has at least two classes of Na+ binding sites; the binding constant of the more strongly bound sodium ions is in the order of 1 × 102 M−1. Competition between Na+ and Ca2+ for protein binding sites is demonstrated by the effect of Ca2+ on the 23Na relaxation parameters. However, only the more strongly bound Na+ are displaced by Ca2+. The number of Ca2+ needed to displace these sodium ions is 3–5 per oxygen binding site.

The 23Na relaxation parameters are influenced also by the state of oxygenation of the protein, indicating a linkage between Na+ and oxygen binding. The simplest interpretation of the data is that sodium ions bind more strongly to oxyhemocyanin in agreement with oxygen equilibrium experiments.
The Proton‐Translocating Adenosine Triphosphatase of the Obligately Anaerobic Bacterium Clostridium pusteuriunum
Authors: CLARKE David J. , FULLER Frances M. , MORRIS J. Gareth
Published: March 2005

1. The cell‐membrane ATP phosphohydrolase of vegetatively grown Clostridium pasteurianum was specifically Mg2+‐dependent, but demonstrated significant activity with GTP, CTP and UTP. It displayed approximate Michaelis‐Menten kinetics only in the presence of certain effectors (e.g. phosphoenolpyruvate, fructose 1,6‐bis‐phosphate) which decreased the Km, for ATP (to below 2 mM) but also V, whilst extending to pH 5.8 the effective pH range of activity of the enzyme.

2. ATP phosphohydrolase activity of the membrane ATPase (BF0Fl) was inhibited by N,N′‐dicyclohexylcarbodiimide, butyricin 7423, Dio‐9, 4‐chloro‐7‐nitrobenzofurazan, efrapeptin, leucinostatin and quercetin, and to a lesser degree by aurovertin and citreoviridin. The enzyme was not inhibited by oligomycin, spegazzinine, tributyl tin, triethyl tin or venturicidin. The soluble ATPase (BF1) component differed in not being inhibited by N,N′‐dicyclohexylcarbodiimide, butyricin 7423 or leucinostatin.

3. The ATPase (BFoF1) complex and its soluble (BF1) component were separately purified.

4. Dodecylsulphate/polyacrylamide gel electrophoresis separated only four polypeptide components in the purified ATPase (BF0F1), with approximate molecular weights (± 10%) as follows: subunita, 65500; subunit c, 57500; subunit d,a, 43000; subunit fa, 15000. The soluble (BF1, component contained only the three polypeptide subunits a, c and d2. These were present in the BF0F1 preparation in the ratio 2:1:2; the contribution of subunit fa could not satisfactorily be quantified.

5. Subunit a was identified as the component binding 4‐chloro‐7‐nitrobenzofurazan and sub‐unit fa as the component binding N,N′‐dicyclohexylcarbodiimide. The ATP phosphohydrolase activity of the membrane ATPase was not activated by trypsin treatment and the ATPase (BFoF1) contained no trypsin‐sensitive inhibitor protein subunit.

6. Purified ATPase (BF0F1) was incorporated into artificial proteoliposomes which demonstrated ATP‐dependent enhancement of 8‐anilinonaphthalene‐1‐sulphonate fluorescence and ATP‐dependent proton influx. These reactions were abolished by proton conductors (e.g. carbonylcyanide m‐chlorophenylhydrazone), by valinomycin in the presence of a high external concentration of K+, or by N,N′‐dicyclohexylcarbodiimide, butyricin 7423, Dio‐9, 4‐chloro‐7‐nitrobenzofurazan or leucinostatin. Oligomycin, tributyl tin, triethyl tin and venturicidin were not inhibitory.

7. When stripped of the soluble BF1 component, such ATPase‐proteoliposomes demonstrated nil ATP phosphohydrolase activity and did not display ATP‐dependent enhancement of 8‐anilino‐naphthalene‐1‐sulphonate fluorescence or ATP‐dependent protein influx. All of these activities were restored by incubation of the BF1‐depleted proteoliposomes with a purified preparation of the soluble BF1 component.
The Proton‐Translocating Adenosine Triphosphatase of the Obligately Anaerobic Bacterium Clostridium pasteurianum
Authors: CLARKE David J. , MORRIS J. Gareth
Published: March 2005

1. Vesicles which demonstrated ATP‐dependent proton influx were produced from cell membranes of Clostridium pasteurianum by a cholate‐dialysis procedure which was also employed to introduce bacteriorhodopsin into such membrane vesicles and into artificial proteoliposomes.

2. ATP synthetase activity was assayed using illuminated bacteriorhodopsin‐containing crude membrane vesicles plus a glucose and hexokinase ‘ATP trap’. The membrane‐bound ATPase of vegetatively grown cells of Cl. pasteurianum displayed measurable ATP synthetase activity in this assay.

3. ATPase‐proteoliposomes constructed of purified ATPase (BF0F1) of Cl. pasteurianumw with bacteriorhodopsin and a mixture of phospholipids accomplished light‐dependent synthesis of ATP from ADP plus Pi. The reaction was inhibited by N,N′‐ dicyclohexylcarbodiimide and by proton conductors such as tetrachlorosalicylanilide. The specific ATP synthetase activity of the purified Cl. pasteurianum ATPase was significantly less than that of similarly purified ATPases (BF0F1) from Escherichia coli, Streptococcus faecalis and Streptococcus pleomorphus. The specific ATP synthetase activity of the ATPase of Clostridium formicoaceticum was greater when the enzyme complex was derived from fumarate‐grown cells then when it was purified from organisms grown on fructose.

4. The apparent Km value (for Mg2+‐ADP‐Pi) displayed by the ATPase of Cl. pasteurianum when acting as an ATP synthetase was much higher than the apparent Km, value (for ATP) in ATP phosphohydrolysis. A similar disposition to serve as an ATP phosphohydrolase was displayed by the ATPase of fructose‐grown Cl. formicoaceticum, but the ATPase from fumarate‐grown cells of this organism was substantially more effective in ATP synthesis.

5. The ATP synthetase activity of Cl. pasteurianum ATPase (BF0F1) was as susceptible as was its ATP phosphohydrolase activity to inhibition by dicyclohexylcarbodiimide, butyricin 7423, Dio‐9, 4‐chloro‐7‐nitrobenzofurazan, quercetin and citreoviridin and was similarly insensitive to inhibition by triethyl tin and tributyl tin.
Characterization of Human Platelet Proteins Solubilized with Triton X‐100 and Examined by Crossed Immunoelectrophoresis
Authors: HAGEN Inger , BJERRUM Ole Jannik , SOLUM Nils Olav
Published: March 2005

Whole human platelets and platelet membranes have been solubilized in 1% Triton X‐100, and the solubilized proteins examined by crossed immunoelectrophoresis using rabbit antibodies raised against either whole platelets or isolated membranes. 90% of the platelet proteins were solubilized by this extraction. About twenty immunoprecipitates were observed using the extracts obtained from whole platelets, whereas normally eight immunoprecipitates were seen with extracts from isolated membranes. Albumin, factor VIII and fibrinogen were identified with monospecific antibodies.

Correlation of the patterns obtained for platelets or membranes was obtained by addition experiments, by crossed‐line immunoelectrophoresis and by crossed immunoelectrophoresis of a mixture of extracts from unlabeled whole platelets and membranes isolated from platelets labeled by lactoperoxidase‐catalyzed 125I iodination.

Four sialoglycoproteins were identified by their reduced electrophoretic migration after neuraminidase treatment, and six proteins interacted with various lectins, indicating them to be glycosylated.

Seven amphiphilic proteins were identified by charge‐shift crossed immunoelectrophoresis, and nine by crossed hydrophobic interaction immunoelectrophoresis with phenyl‐Sepharose.

The topographical arrangement of the membrane proteins was examined with lactoperoxidase‐catalyzed 125I‐labeled platelets as antigens, and by antibodies absorbed with a suspension of whole platelets. Four and six radioactively labeled precipitates could be identified using the platelet and membrane extracts, respectively, indicating them to be exposed at the outer platelet surface. This was confirmed by the use of antibodies absorbed with intact platelets.
Biological Role of Gramicidin S in Spore Functions
Authors: MARAHIEL Mohamed A. , DANDERS Wolf , KRAUSE Michael , KLEINKAUF Horst
Published: March 2005

Gramicidin‐S‐negative mutants of Bacillus brevis ATCC9999 have been isolated with a remarkly higher yield after ethidium bromide or acridine orange treatment, than after N‐methyl‐N'‐nitro‐N‐nitrosoguanidine treatment. Four (MIV, Smr170, R5 and EB 16) of 38 isolated mutants were characterized with respect to the lesion in gramicidin‐S‐synthesizing activity. The mutants sporulate to the same extent as the parental strain except mutant Smr 170 which sporulates less. However, mutant spores were more heat‐sensitive and possessed a reduced level of dipicolinic acid content. No significant difference was observed in the germination time of wild‐type and mutant spores. All spores germinated after 80–110 min, but the outgrowth time was different: all gramicidin‐S‐negative mutants grew out immediatly after germination whereas wild‐type spores required a lag period of 9–10 h. When the mutants were allowed to sporulate in the presence of gramicidin S, the spores were found to be heat‐resistant and their outgrowth postponed to the same period as the parent spores. The addition of gramicidin also eliminated the deficiency of dipicolinic acid.

A new class of gramicidin‐S‐negative mutant, R5, which only activates L‐valine and L‐leucine, is described. A possible biological function of gramicidin S in the heat‐resistance and in the timing of spore outgrowth is discussed.