bluuebunny/crossref_metadata_2025 huggingface dataset:
Diamine Oxidase in Rabbit Liver
Authors: Argento‐Cerüg Maria Paola , Sartori Claudia , Autuori Francesco
Published: March 2005

Preliminary studies carried out in order to find the optimal conditions for the assay of diamine oxidase activity in rabbit liver homogenate, showed that the oxidation of the substrates histamine and cadaverine at alkaline pH (∼ 8.0) was not inhibited by aminoguanidine and hydroxylamine (specific inhibitors of diamine oxidase), but was blocked by octanol (specific inhibitor of monoamine oxidase) which was uneffective at lower pH values. Borate inhibited diamine oxidase but not monoamine oxidase activity, since its inhibitory effect, at different pH values, was very similar to that of aminoguanidine and hydroxylamine. Diamine oxidase from rabbit liver seemed to be active only at almost neutral pH values. At alkaline pH values, the oxidation of histamine and cadaverine was operated by monoamine oxidase. Furthermore cadaverine‐oxidizing activity, when assayed at pH 8.8, exhibited the same subcellular distribution as monoamine oxidase. When assayed at a pH near neutrality the bulk of diamine‐oxidizing activity (both with cadaverine and histamine as substrates) was present in the microsomal fraction.
Diamine Oxidase in Rabbit Liver
Authors: Argento‐Cerù Maria Paola , Sartori Claudia , Autuori Francesco
Published: March 2005

Subfractionation of the microsomes according to Dallner [4] showed that diamine oxidase is concentrated in the smooth microsomes.

Centrifugation of the post‐mitochondrial supernatant treated with CsCl on a discontinuous three‐step gradient consisting of 0.8 M sucrose (density 1.12 g/ml) layered on 1.3 M sucrose (density 1.20 g/ml) resulted in three fractions: fraction 1 at the interface 1.034/1.12 g/ml; fraction 2 at the interface 1.12/1.20 g/ml; fraction 3 a pellet at the bottom of the tube. No diamine oxidase activity is present in fraction 1, but it is significantly more concentrated in fraction 2 than in fraction 3.

Biochemical and morphological analysis of the subfractions revealed that fraction 1 mainly consists of plasma membrane fragments, fraction 2 of smooth vesicles and fraction 3 of rough vesicles and free ribosomes.
DNA Synthesis in Isolated HeLa Nuclei
Authors: Hershey Howard V. , Stieber Joseph F. , Mueller Gerald C.
Published: March 2005

A nuclear system from synchronized HeLa cells is described which continues DNA replication at sites which were active in vivo. Under the defined optimal conditions the initial rates of DNA replication in isolated nuclei range from 50 to 100% of that in the cells of origin; 1—2% of the HeLa DNA can be replicated in vitro. Proteins of the cytosol are essential to the stabilization of the replication system. Experiments in which the parental strands were fractionally labelled with bromodeoxyuridine provide evidence that the ordered nature of DNA replication in vivo is preserved in the system in vitro. Repair replication of homopolymer synthesis was insignificant under the conditions of study.
Isolation and Amino‐Acid Sequence of the C‐Peptide of Duck Proinsulin
Authors: Markussen Jan , Sundby Finn
Published: March 2005

C‐peptide from the duck was isolated and its amino acid sequence determined. The duck C‐peptide contains 26 amino acids and differs substantially from the known mammalian C‐peptides. It is the first C‐peptide shown to contain the amino acids histidine, tyrosine and phenylalanine. The sequence was found to be as follows: Asp‐Val‐Glu‐Gln‐Pro‐Leu‐Val‐Asn‐Gly‐Pro‐Leu‐His‐Gly‐Glu‐Val‐Gly‐Glu‐Leu‐Pro‐Phe‐Gln‐His‐Glu‐Glu‐Tyr‐Gln.

A comparison of duck C‐peptide with the known mammalian C‐peptides shows that the terminals are homologous and that the five invariable positions of the nine known mammalian C‐peptides can be retained by means of four deletions in the duck C‐peptide. It is likely that their common ancestor, the reptile C‐peptide, contained at least 31 amino acids and that deletions at various positions were involved in the evolution of ox, pig, dog and duck C‐peptides
Purification and Properties of DNA Polymerase III
Authors: Otto Bernd , Bonhoeffer Friedrich , Schaller Heinz
Published: March 2005

DNA polymerase III was purified 15000 to 20000‐fold from crude extracts of Escherichia coli cells. Purification was followed by two assays, a standard polymerase assay and a complementation test in which DNA polymerase III stimulates the DNA replication in vitro of a dnaE mutant. The enzyme seems to be a single polypeptide chain with a molecular weight of about 140000. There are about 10 molecules per cell.

For maximal activity the enzyme requires all four deoxynucleoside triphosphates, Mg2+, and a high concentration of primer sites on a single‐stranded DNA template. The rates of nucleotide incorporation were determined to be as high as 5000 nucleotides per min and enzyme molecule. The high sensitivity of the enzyme to salt (0.1 M KCl) is eliminated if enzyme and template are used in high concentration. In adition to its polymerizing activity the enzyme has an exonucleolytic activity on single‐stranded DNA.
Specificity of Aeromonas Aminopeptidase toward Oligopeptides and Polypeptides
Authors: Wilkes Stella H. , Bayliss Mary E. , Prescott John M.
Published: March 2005

A variety of oligopeptides and polypeptides was subjected to the action of Aeromonas amino‐peptidase and the sequential release of amino acid residues was determined quantitatively. The residues that proved susceptible to hydrolysis included all hydrophobic, aromatic and basic amino acids plus proline. Aspartyl, glutamyl and cysteic acid residues were not removed from the amino‐terminus of any substrate tested, even at high enzyme concentrations but in contrast, asparagine, glutamine and aminoethylated cysteine were released. Glycine was generally resistant to hydrolysis, but was slowly released from some substrates dependent upon the adjacent residues. The specificity and ease of purification of this aminopeptidase should make it a useful tool for sequence determination.
Termination of Polypeptide Synthesis in Bacteria and the Exchange of Ribosomal Subunits
Authors: Perazzolo Clovis A. , Azzam Mansur E. , Algranati Israel D.
Published: March 2005

A cell‐free system able to elongate and terminate nascent peptide chains has been prepared with polyribosomes and supernatant fraction from Escherichia coli D10. When labeled 30‐S subunits were added in several conditions and at different moments, the exchange of radioactivity between 70‐S and 30‐S particles could be measured during and after termination of protein synthesis. The subunit exchange taking place during completion of polypeptide synthesis was not modified by the presence of spermidine, used to stabilize 70‐S ribosomes. This result led to the conclusion that upon termination and release of polypeptide chains, ribosomes dissociate and detach from polysomes as 30‐S and 50‐S subparticles. The “hybrid” 70‐S monomer formed after the exchange with added 30‐S subunits, behaves as a run‐off ribosome.
Kinetics of Transcription by the Bacteriophage‐T3 RNA Polymerase in vitro
Authors: Mcallister William T. , Küpper Hans , Bautz Ekkehard K. F.
Published: March 2005

Analysis of the kinetics of RNA synthesis by the bacteriophage T3 RNA polymerase revealed a critical dependence of enzyme activity on the concentration of GTP. The nature of the Lineweaver‐Burk plot for this substrate suggested that on a T3 DNA template the enzyme initiates RNA chains with the sequence pppGpG. The observation that a high level of pyrophosphate exchange is obtained in the presence of GTP alone as a substrate confirmed this interpretation. Similar experiments show that on a T7 DNA template the T3 RNA polymerase initiates RNA chains with the sequence pppGpA. As a result of the kinetic analysis, it was proposed that the enzyme has only two substrate binding sites, and that the Km for the initiation site is 5 to 7‐times greater than that for the polymerization site.

Analysis of the products made during incubation with GTP alone, and with T3 DNA as a template, demonstrate that only a dinucleotide is formed under these conditions. This is not sufficient to establish a stable transcription complex. Incubation with GTP and ATP together results in the production mainly of tetranucleotides and in the formation of a stable transcription complex that is also resistant to heparin.

At low ionic strength (0.05 M KCl) synthesis of RNA begins with no detectable lag phase at 18°C. At high ionic strength (0.15M KCl) a lag phase in the initiation of RNA synthesis is apparent. Preincubation with GTP alone does not remove the lag phase, but preincubation with GTP, ATP and UTP abolishes the lag phase and under these conditions the initial rate of synthesis is identical to that observed at low ionic strength.
Immunochemical Studies on Shigella boydii Lipopolysaccharides
Authors: Dmitriev Boris A. , Backinowsky Leon V. , Kochetkov Nikolay K. , Khomenko Natalja A.
Published: March 2005

An investigation of the sugar composition of lipopolysaccharides derived from fifteen well‐defined Shigella boydii serotypes was carried out. All Sh. boydii lipopolysaccharides contained glucosamine, glucose, galactose, heptose and 3‐deoxyoctulosonic acid. These sugars were considered to represent the core polysaccharides and those except heptose and 3‐deoxyoctulosonic acid could represent the constituents of the O‐specific polysaccharide chains. In addition to the basal sugars, the following monosaceharides were detected as constituents of the various Sh. boydii lipopolysaccharides: mannose, rhamnose, l‐quinovosamine, galactosamine and 3,6‐di‐deoxyhexose. As a result, a chemical classification of Sh. boydii lipopolysaccharides into eight chemotypes was achieved and this differs from the accepted Sh. boydii classification.
The Modification of Deoxyribonucleohistone by Trypsin and Chymotrypsin
Authors: Chatterjee Sanjukta , Walker Ian O.
Published: March 2005

Trypsin and chymotrypsin completely degraded whole histone in less than 15 min as judged by changes in the peptide dichroic band at 220 nm and gel electrophoresis patterns. By contrast, the action of trypsin in deoxyribonucleohistone under conditions of low ionic strength and relatively low concentrations of substrate (200—600 μg/ml) produced partial degradation of f1 and f3 only after 3‐h incubation with enzyme. Finally, all fractions were degraded.

Chymotrypsin caused rapid and almost complete degradation of f1 in nucleohistone in less than 1 h and f3 in less than 3 h. F2b was degraded after a lag of about 30 min. All molecules of f1, f3 and f2b were attacked inside 6 h. The degradation of f2b gave rise to a large peptide fragment, not observed in digests of whole histone, which remained bound to the DNA in 0.7 M NaCl. F2al and f2a2 were not significantly degraded.

Degradation of f3 was accompanied by an increase in viscosity and the nucleohistone lost its rigid, rod‐like properties, becoming more flexible. Thus f3 is involved in maintaining the supercoil in its rigid conformation.

The sites of trypsin attack are not initially accessible in bound histone fractions f2a1, f2a2 and f2b, whereas these sites appear to be partially accessible in f1 and f3. On the other hand the chymotryptic cleavage points are accessible in bound f1, f3 and f2b but not in f2a1 and f2a2. The all‐or‐nothing character of the degradation suggests that f3 and f2b are regularly arranged along the axis of the supercoiled nucleohistone.
Spectral Studies of Interactions of Detergents with Tryptophyl Compounds
Authors: Bello Jake , Bello Helene R.
Published: March 2005

The interactions of sodium dodecylsulfate and hexadecyltrimethylammonium chloride with typtophyl model compounds were investigated by fluorescence, classical difference spectra and thermal perturbation spectra. Oppositely charged models and detergents interact to produce marked spectral changes. Apolar interactions also contribute. The ease of interaction with sodium dodecylsulfate decreases in the order: tryptamine, Lys‐Trp‐Lys, Leu‐Trp‐Leu, Gly‐Trp‐Gly, Glu‐Trp‐Glu shows little or no interaction. Lys‐Trp‐Lys forms both soluble and insoluble complexes with sodium dodecylsulfate.
Peptidyl‐Transferase Activity of Escherichia coli Ribosomes Digested by Ribonuclease T1
Authors: ČErná Jiřina , RychlÍK Ivan , JonÁK Jiří
Published: March 2005

Digestion of ribosomes by ribonuclease T1 results in the inactivation of peptidyl transferase and of the binding ability of the donor site. However, the binding activity of the acceptor site is not affected by ribonuclease digestion. The binding sites of ribonuclease‐treated ribosomes retain their original sensitivity to chloramphenicol, spiramycin, erythromycin and lincomycin.

Digestion of 50‐S ribosomal subunits by ribonuclease T1 results in the loss of peptidyl transferase activity and of the binding ability of the donor site in the same way as digestion of 70‐S ribosomes either intact or recombined from 30‐S and 50‐S subunits. The acceptor activity of the acceptor binding site of 50‐S ribosomal subunits does not change after T1ribonuclease digestion.

Erythromycin, bound to the ribosomes before digestion by ribonuclease T1, decreases the rate of inactivation of peptidyl transferase, whereas spiramycin does not change the effect of ribonuclease on transfer activity.
Active‐Site Titration of Pig‐Plasma Benzylamine Oxidase with Hydrazine Derivatives
Authors: LindstrÖM Anders , Pettersson Gösta
Published: March 2005

The interaction between pig plasma benzylamine oxidase and hydrazine derivatives has been investigated kinetically and spectrophotometrically. The hydrazines tested combine irreversibly to the enzyme and there is a one‐to‐one correspondence between inactivation of the enzyme and hydrazone formation with enzyme‐bound pyridoxal phosphate. Spectrophotometric titration with hydrazines established that benzylamine oxidase contains one mole of reactive pyridoxal phosphate per mole of protein, whereas the copper content is at least two atoms per protein molecule.
Replication of Bacteriophage M‐13
Authors: Staudenbauer Walter L. , Hofschneider Peter Hans
Published: March 2005

A mutant of bacteriophage M‐13 temperature‐sensitive in gene‐5 was isolated. The effect of this mutation on phage DNA synthesis was studied at the restrictive temperature in the presence and absence of the dnaB function which is required for early double‐strand replication. From the results obtained it is concluded that gene‐5 protein has two functions: (a) as already known it controls negatively double‐strand replication and (b) it is directly involved in progeny single‐strand synthesis.
The Effect of Changes in Functional Activity on Ubiquinone Redox Status in Isolated Atria
Authors: Liu Mai‐Shian , Hoffmann Philip C. , Siess Manfred
Published: March 2005

Changes in the mechanical activity of isolated guinea pig atria were induced by a variety of procedures. The atria were freeze‐stopped in liquid nitrogen and the redox status of ubiquinone (Q) determined as a measure of the redox behavior of the mitochondrial compartment in intact cardiac tissue.

40% of the total Q was oxidized in spontaneously contracting atria under aerobic conditions at 30°C. 75% of Q was reduced when the atria were exposed to anoxic conditions (N2 and KCN). Maximal oxidation of Q (87%) was observed when atria were treated with 2μM rotenone followed by stimulation with 6 μM epinephrine.

At the height of the inotropic and chronotropic response to epinephrine alone (1.5 min) Q redox status was shifted slightly towards the reduced side (31% oxidized). At later time points (10 and 30 min), when the activity of the atria was still increased but to a lesser extent, the Q redox status had shifted slightly to the oxidized side (49% oxidized).

Larger shifts of Q redox status to the oxidized side 30 min after epinephrine were observed in atria concomitantly exposed to the respiratory inhibitors, malonate, amytal and rotenone. Under these conditions, there was a direct correlation between the contractile activity at the time of freeze‐stop and the proportion of Q in the oxidized form. Ouabain in doses producing positive inotropic responses did not lead to any increase in the amount of oxidized Q.

Lowering the calcium level in the bath medium below 10% of its normal value essentially abolished atrial contractility, and Q was found to be slightly reduced (33% oxidized). Moreover, epinephrine produced only a minimal inotropic response under these conditions and Q showed no redox change. On the other hand, atria in a 10% calcium medium exhibited only 12% of the contractile amplitude shown in the normal calcium medium and the Q redox status was not detectably different. In addition, no difference in Q redox status could be demonstrated in left atria which showed no spontaneous activity and similar atria electrically stimulated to contract at 180/min.

The small shifts in atrial Q redox status (except in the presence of respiratory inhibitors) in response to marked changes in mechanical activity of the tissue suggests that changes in the rates of Q oxidation and reduction are balanced in such a way as to maintain redox status relatively constant.
Structural and Immunogenic Properties of a Major Antigenic Determinant in Neutral Salt‐Extracted Rat‐Skin Collagen
Authors: Stoltz Michael , Timpl Rupert , Kühn Klaus , Furthmayr Heinz
Published: March 2005

The major antigenic determinant of neutral salt‐extracted rat‐skin collagen is located in the C‐terminal CNBr peptide α1‐CB6b of the α1‐chain. This peptide is shortened by 20 amino acid residues from its C‐terminal nonhelical end if compared with α1‐CB6 obtained from collagen α1‐chains extracted under denaturating conditions. As reported earlier, degradation by tissue proteases during extraction was assumed as the main reason for the observed reduction in size.

The C‐terminal sequence of α1‐CB6b was determined as Tyr‐Asp and was found to be essential for antigenic activity. Proportional loss of binding to antibodies with decrease in size was observed for appropriate proteolytic fragments derived from α1‐CB6b. Since this peptide extends only five amino acid residues beyond the helical collagen sequence, an important contribution of the conformational structure for this antigenic determinant is suggested. Decrease in ability to induce high antibody titers to α1‐CB6b was already observed at a higher structural level, e.g. after denaturation of triple‐helical collagen to random‐coiled α‐chains. The same result could be obtained if adjuvant was omitted in the priming injection.
On the Reconstitution of Photophosphorylation in Chloroplast Membranes
Authors: Shoshan Varda , Shavit Noun
Published: March 2005

A high degree of reconstitution of photophosphorylation was achieved using resolved chloroplast particles obtained by incubation with 1 mM EDTA at relatively high chlorophyll concentrations (about 400 μg/ml). The resolved particles lost about 95% and 75% of their phosphorylative and proton transport activity respectively. Upon reconstitution with either crude or purified chloroplast coupling factor preparations, almost full activity was recovered. Control of electron transport by phosphorylating agents was also observed in these reconstituted preparations.

Highly reconstituted partieles were also obtained by incubation of chloroplasts, at low chlorophyll concentrations, with EDTA up to 10 mM. Above this concentration of EDTA, poor uncoupling was observed.

Gel electrophoresis of supernatant fractions obtained from incubations with EDTA under conditions which produced resolved particles with a poor degree of reconstitution revealed a protein fraction not appearing in supernatants from highly reconstituted particles. The degree of reconstitution was inversely correlated with the appearance of this protein band.
Effect of Iodide Concentration on Thyroxine Synthesis Catalysed by Thyroid Peroxidase
Authors: Pommier Jacques , Deme Danièle , Nunez Jacques
Published: March 2005

Thyroglobulin with a low content of iodine atoms per molecule of protein was incubated with thyroid peroxidase and increasing iodine concentrations (0.01 to 4 mM).

When iodide was in great excess (4 mM), I2 was the only product, protein iodination being markedly or completely inhibited. At low iodide concentrations (0.2 mM and below) no I2 was formed. A clear competition between these two possible reactions catalysed by thyroid peroxidase was therefore demonstrated.

When iodide concentration was less than 0.5 mM, thyroid peroxidase catalysed only the iodination of thyroglobulin. Thyroid hormones began to accumulate when the iodination reaction leveled off. Different observations suggest that thyroxine was synthesised mainly by the coupling of two diiodotyrosine residues rather than by the iodination of 3,5,3′‐triiodothyronine residues.

With very low iodide concentration, the synthesis of hormones began soon after the formation of hormonogenic residues. However the iodination reaction being faster than the coupling reaction, highly iodinated thyroglobulin could be obtained which did not contain thyroid hormones.

From these results it appears that thyroid peroxidase is able to enter into at least three reactions: iodide peroxidation, tyrosine residues iodination and coupling of iodotyrosine residues to thyroid hormones. Surprisingly, this relative unspecificity of thyroid peroxidase may have some regulatory physiological significance.
Enzymatically Active Half‐Monomers of Acetylcholinesterase
Authors: Millar David B. , Grafius Melba A. , Palmer David A. , Millar Geraldine
Published: March 2005

An enzymatically active 7.4‐S species of acetylcholinesterase has been isolated which has a molecular weight of 134000 ± 6%. It apparently has its origin in a Triton‐X‐100‐mediated cleavage of higher polymers of acetylcholinesterase but not from cleavage of purified 10.8‐S enzyme. The half‐monomer has a Km for acetylthiocholine almost twice that of 10.8‐S enzyme but the Ki for eserine is the same. The subunit molecular weight of the 7.4‐S enzyme, as determined by sodium dodecylsulfate gel electrophoresis, is about 65000 indicating the enzyme to be a dimer. The enzyme is maximally heat stable in 0.1 M ammonium sulfate. Under certain conditions we observe a speed dependency of the sedimentation coefficient. These properties may make the half‐monomer a useful model for studying the kinetics and subunit interactions in acetylcholinesterase.
A Preliminary Three‐Dimensional Arrangement of the Proteins in the Escherichia coli 30‐S Ribosomal Sub‐Particle
Authors: Morgan Joan , Brimacombe Richard
Published: March 2005

30‐S ribosomal subparticles from Escherichia coli were hydrolysed with ribonuclease T1, pancreatic ribonuclease or micrococcal nuclease in the presence of 2 M urea, and various concentrations of magnesium and ethanol. The RNA · protein fragments produced were separated on 5% polyacrylamide/agarose composite gels, and fractions from these gels were subjected to protein analysis on 17.5% periodate‐soluble polyacrylamide gels run in the detergent sarkosyl, using the technique already published.

A wide range of RNA · protein fragments was obtained by this procedure, each containing a few specific ribosomal proteins. The strict criteria already published for determining the specificity of the proteins in each fragment were applied. The RNA · protein fragments divide into two distinct groups, those containing some or all of proteins S7, S9, S10, S13, S14 and S19, and those containing some or all of proteins S4, S5, S6, S8, S11, S15, S16(17), S18 and S20. Proteins S1, S2, S3, S12 and S21 were not found in specific fragments.

The individual proteins found together in specific RNA · protein fragments are interpreted as being close neighbours in the 30‐S particle. The range of fragments observed is sufficient to enable the data to be combined with Nomura's “assembly map” and data from protein crosslinking experiments, into a preliminary three‐dimensional arrangement of the proteins.
Effects of 5′‐Amido Analogues of Adenosine 3′: 5′‐Monophosphate and Adenosine 3′: 5′‐Monophosphothioate on Protein Kinase, Binding Protein and Phosphodiesterases
Authors: Jastorff Bernd , Bär Hans‐Peter
Published: March 2005

The biological effects of 5′‐amido analogues of adenosine 3′:5′‐monophosphate (cAMP) and monophosphothioate, with and without N‐substituents, were investigated.

The unsubstituted amino analogues stimulated the cAMP‐dependent protein kinase from bovine muscle while N‐substitution prevented this effect. Some of the N‐alkyl derivatives, however, inhibited the protein kinase activity. Only adenosine 5′‐amido 3′:5′‐monophosphate was found to compete for the binding of [3H]cAMP to cAMP binding protein from bovine muscle.

Phosphodiesterases from bovine heart and rabbit brain hydrolyzed 5′‐amido‐adenosine 3′: 5′‐monophosphate, but only the former hydrolyzed the corresponding phosphothioate. The heart diesterase was inhibited competitively by N‐substituted derivatives of 5′‐amido‐adenosine 3′: 5′‐monophosphate and 5′‐amido‐adenosine 3′: 5′‐monophosphothioate, but 5′‐N‐n‐octyl‐amino‐adenosine 3′:5′‐monophosphate did not inhibit any of the brain diesterases tested.

Results suggest that steric and electronic requirements of the cyclophosphate ring determine the catalytic effects of the cAMP on protein kinase, while alteration in other regions of the molecule exercise an appreciably smaller effect and largely affect binding parameters. Phosphodiesterases show similar requirements for binding and reactivity.
Thiol Function and Tertiary Structure of RNA Polymerase of Escherichia coli
Authors: Nicholson Bruce H. , King Andrew M. Q.
Published: March 2005

RNA polymerase requires thiol compounds to maintain optimum activity during isolation, although it is not a thiol enzyme.

Total thiols have been determined for RNA core polymerase by reaction with p‐chloro‐mercuribenzoate, 2‐chloromercuri‐4‐nitrophenol and 5,5′‐dithio‐bis(2‐nitrobenzoic acid). Their distribution between the subunits has been determined as ααββ' = 4.4.8.8.

An arbitrary division of the reaction rate profile shows that of the 18 thiols reacting with 5,5′‐dithio‐bis(2‐nitrobenzoec acid) in 18 h, over 10 react in 1% of this time. Most of these are buried thiols since only 2 or 3 react with N‐[p‐(‐methyl‐2‐benzthiazole)phenyl]maleimide.

Enzyme activity is lost gradually on titration with 5,5′‐dithio‐bis(2‐nitrobenzoic acid), but rapidly with p‐chloromercuribenzoate until 10–12 thiols have reacted per mole; polymerase then undergoes a large reversible change in activity, although with 5,5′‐dithiobis(2‐nitrobenzoic acid) reversibility is slow and depends on the groups/mole that have reacted.

This inactivation by 5,5′‐dithio‐bis(2‐nitrobenzoic acid) may be prevented by pre‐initiating the enzyme which causes the reaction to stop at 11 thiols/mole, leaving 60–70% of the original activity. DNA on its own does not produce this resistance, which is proportional to the amount of initiated complex present.

Measurements with fluorescent probes show that considerable destabilisation of the tertiary structure occurs on reaction with p‐chloromercuribenzoate. Consideration of this together with the known subunit distribution of thiols strongly suggests that DNA acts by stabilising the structure rather than by direct steric hindrance.
A Yeast Mutant with Temperature‐Sensitive Mitochondrial Ribosomes
Authors: Thomas David Y. , Scragg Alan H.
Published: March 2005

A mutant of Saccharomyces cerevisiae has been selected which has temperature‐sensitive mitochondrial protein synthesis. This has been demonstrated by incorporation into cycloheximide‐inhibited whole cells, isolated mitochondria and hybrid cell‐free systems. The temperature sensitivity is associated with the mitochondrial ribosomes and the mutation is recessive and nuclear.
Macromolecular Structure of Lipopolysaccharides from Gram‐Negative Bacteria
Authors: Hannecart‐Pokorni Eleonora , Dekegel Daniel , Depuydt Freddy
Published: March 2005

The cell wall lipopolysaccharide constituent of Shigella flexneri, Salmonella typhimurium and Salmonella minnesota appears in the electron microscope as long slender filaments having a molecular weight of several millions. Such lipopolysaccharides are constituted of subunits held together by low‐energy bonds of chelating and hydrophobic nature due to the lipid part of the molecule. Physicochemical studies gave molecular weights in the range of 18000 to 25000 for the subunits of S strains depending upon the bacterial species, and 10000 in the case of the Salmonella minnesota R mutants. The subunits seem to be long ellipsoids of revolution made up of a lipid part and two polysaccharide side chains.

Studies of the dissociation and the reassociation of the lipopolysaccharide subunits showed the existence of a cylindrical intermediate “unit structure” having a molecular weight of about 5·105. The arrangement of the subunits in that unit structure seems to be of a micellar type, the lipids being located in the central part of this structure. This latter model is compatible with a skew arrangement of unit structures into the larger polymer molecules.
Pyrimidine‐Ribonucleotide Pools and Their Turnover in Phage T4‐Infected Escherichia coli Cells
Authors: Fast Robert , Sköld Ola
Published: March 2005

Exogenous uracil was shown to be taken up efficiently into ribonucleotide pools at all times during the cycle of T4 phage infection, in which stable host RNA synthesis is known to be turned off. This uptake was shown to be effected by ribonucleotide pool expansion and a net increase in mRNA content after phage infection. By comparing different phage mutants, the draining of ribonucleotide pools into DNA synthesis by ribotide reduction, was also found to be instrumental for pool labeling from exogenous uracil. The UDPG pool expanded as much as 3‐fold during DNA negative mutant infection, presumably as a result of absent DNA glucosylation.

From measurements of specific activities of nucleotides by double labeling and thin‐layer chromatography, it was concluded that the incorporation into RNA of a radioactive, exogenous base can be reliably used for following T4 phage transcription.
A Study of the Interaction of Manganese Ions with ATP by 31P Fourier‐Transform Nuclear‐Magnetic Resonance
Authors: Brown Frank F. , Campbell Iain D. , Hirst Christopher W. J. , Richards Rex E. , Henson Raymond
Published: March 2005

The spin‐lattice relaxation rates of the phosphorous nucelei in ATP have been measured as a function of temperature in the presence of manganese perchlorate using pulsed nuclear magnetic resonance methods. Detailed interpretation of the results in terms of the dipolar interaction between the electron and nuclear spins leads to the conclusion that the α‐phosphorous atom is more distant from the manganese ion than the β‐ or γ‐phosphorous atoms, which are equidistant from the metal ion within experimental error. The rate constant for the manganese ion dissociating from the complex is found to be 150 ms−1 at 25°C.
Properties of Arginine Kinase from Drosophila melanogaster
Authors: Wallimann Theo , Eppenberger Hans M.
Published: March 2005

1. Starch‐gel electrophoresis of extracts of several strains of the genus Drosophila reveals neither allelic variants nor isoenzymes of arginine kinase. Arginine kinase of the drosophilid Zaprionus vittiger migrates differently.

2. Arginine kinase is non‐uniformly distributed in tissues of Drosophila melanogaster. The activity in muscle tissue represents about 70% of the total activity. A characteristic fluctuation of the enzyme activity during the development from the egg to the adult stage can be observed.

3. Purification of arginine kinase of Drosophila melanogaster has been achieved. Several physico‐chemical properties of the enzyme have been determined. The molecular weight in the range of 40 000 is comparable to other arthropod arginine kinases and suggests that the active enzyme is a monomer.
Purification and Properties of Pig‐Heart Hexokinase
Authors: Easterby John S. , O'Brien Michael J.
Published: March 2005

Hexokinase has been purified from pig heart to a specific activity of 80 units/mg.

The enzyme has an s20 of 5.11 ± 0.15 S and a molecular weight of 97000. Dodecylsulphate‐polyacrylamide electrophoresis and maleylation of the enzyme each suggest that it contains a single polypeptide chain. Amino‐acid analysis reveals similarities between the compositions of heart and brain hexokinases. Steady‐state kinetic investigations at sub‐optimal substrate concentrations are consistent with a mechanism in which at least one of the substrates is in equilibrium with its enzyme · substrate complex.
Crystalline 6‐Phosphogluconate Dehydrogenase from Sheep Liver
Authors: Silverberg Michael , Dalziel Keith
Published: March 2005

6‐Phosphogluconate dehydrogenase has been obtained in the pure, crystalline form from sheep liver by an extensive modification of an earlier purification procedure. The absorption coefficient, A1%280, estimated from dry weight measurements, is 11.4. The amino acid composition is reported. The molecular weight of the enzyme from measurement by several methods is 94000 ± 2000, and there are two subunits in the molecule. From studies of the reaction of the native enzyme with 5,5′‐dithiobis(2‐nitrobenzoic acid) and p‐hydroxymercuribenzoate, there appear to be two reactive thiol groups per subunit which are essential for activity and are protected by 6‐phosphogluconate or NADPH.
Carbon‐Dioxide Concentration and the Distribution of Monocarboxylate and H+ Ions between Intracellular and Extracellular Spaces of Hemoglobin‐Free Perfused Rat Liver
Authors: Sies Helmut , Noack Gerhild , Halder Karl‐Heinz
Published: March 2005

H+ movement and monocarboxylate‐monocarboxylate exchange accompanying monocarboxylate gradient equilibration in hemoglobin‐free perfused rat liver have been studied in an isopH system using different buffer systems.

1. At 5–10 mM concentrations of added monocarboxylate, there is an approximately linear dependence of net H+ uptake by the liver on added monocarboxylate. Net H+ uptake at 10 mM additions is 1.5–1.6 μmol per gram liver from aromatic weak acids like benzoate or salicylate, and 0.6–0.8 μmol per gram liver for aliphatic weak acids like acetate, lactate, pyruvate, and also 5,5‐dimethyloxazolidine‐2,4‐dione.

2. Practically no net H+ movement is observed with dicarboxylates like phthalate or succinate and also not with choline chloride.

3. Monocarboxylate‐monocarboxylate exchange is demonstrated by preloading the hepatocytes with benzoate, lactate and pyruvate, and observation of their release upon addition of other monocarboxylates.

4. Net H+ uptake increases as total CO2 in perfusate is decreased at constant extracellular pH, in accordance with an intracellular acidification by the CO2 system. This is supported by measurement of distribution of 5,5‐dimethyloxazolidine‐2,4‐dione, and by monocarboxylate release from the cells during increases of CO2 concentration.

5. Intracellular pH poise is low when bicarbonate buffer is replaced by zwitterionic buffers like N‐2‐hydroxyethylpiperazine‐N′‐ethane sulfonic acid or morpholinopropane sulfonic acid, resulting in calculated intracellular alkalinization of up to 0.5 pH unit. 5,5‐Dimethyloxazolidine‐2,4‐dione as a permeant buffer system is suggested as a more suitable replacement of bicarbonate buffer than the zwitterionic buffers with respect to intracellular pH poise.
On the Mechanism of Formation of a Partially Active Aldolase by Tryptic Digestion
Authors: Biszku Etelka , Sajgó Mihály , Solti Magda , Szabolcsi Gertrud
Published: March 2005

The limited proteolysis of a fully active mercury derivative of rabbit muscle aldolase affects all four subunits of the enzyme, but not in a uniform way. At least four out of the nine peptides liberated are cleaved off from only two subunits. The specific activity of the product (aldolase‐T) is 50% of that of the native enzyme. The combined kinetic and peptide analytical study of aldolase‐T formation has shown that the diminished activity is the consequence of the splitting off of a particular tyrosine‐containing peptide from all four subunits of aldolase, and of the cleavage of a peptide bond in the vicinity of residue Cys‐341 in only two subunits. The data presented, together with the results of the polyacrylamide gel electrophoresis of aldolase‐T in the absence and presence of sodium dodecylsulfate, support the idea that the structure of oligomeric aldolase is of type AABB. Large segments of one kind of the subunits can be split off without affecting the tetrameric structure. A tentative sequence of the C‐terminal 60 amino acids of aldolase is also presented.
Action of Protease I of Escherichia coli on RNA Polymerase of Same Origin
Authors: Hermier Brigitte , Pacaud Michèle , Dubert Jean‐Marie
Published: March 2005

The recently isolated protease I of Escherichia coli leads to the partial degradation of RNA polymerase of E. coli and causes, in particular, the splitting of subunits ββ′ into fragments of approximately the same size as that of factor σ. This action is similar to that of papaïn or α‐chymotrypsin employed for short periods at much lower concentrations. The immunochemical study presented leads to the identification of the origin of the fragments obtained by limited proteolysis and shown the lack of cross reaction between subunits ββ′ and σ factor.
Synthesis and Translation of Ribopolymers in Escherichia coli Made Permeable by Treatment in High Sucrose Concentration
Authors: Ben‐Hamida Fakher , Gros François
Published: March 2005

An Escherichia coli“permeabilized” cell system obtained by treatment in high sucrose concentration, because of its depletion from endogenous metabolites, its low nuclease, phosphatase and phosphokinase content, catalyzes a very efficient polymerisation of the common ribonucleoside diphosphates (UDP, CDP, ADP and GDP) presumably through the polynucleotide phosphorylase retained within the cells after osmotic shock. Accordingly incorporation of nucleotides into polymers is insensitive to actinomycin D and does not take place in the presence of Mg2+ions with E. coli Q13 a strain in which the polynucleotide phosphorylase is a Mn‐dependent enzyme. Polymers containing U, C, G, or A as the only constituting nucleosides have been found to accumulate for several hours at a linear rate, in the supernatant phase from osmotically shocked E. coli D10. If, following the arrest of polymer formation the amino acid coded for by the corresponding triplet is added to the incubation medium together with an energy‐yielding system, a very efficient incorporation of this amino acid (and of this amino acid only) into an acid‐precipitable material takes place. The resulting product (presumably an homopolypeptide) is associated with the cell bodies. Translation of the preformed polyribonucleotide is concomitant with its nucleolytic cleavage by a K‐dependent nuclease.
The Interaction between α‐Chymotrypsin and Pancreatic Trypsin Inhibitor (Kunitz Inhibitor)
Authors: Vincent Jean‐Pierre , Lazdunski Michel
Published: March 2005

1. The association constant, Ka, of the 1:1 complex formed between α‐chymotrypsin and the pancreatic trypsin inhibitor is 110 μM−1 at pH 8.0 and 25 °C. The second‐order rate constant for the association, ka, is 110 mM−1 s−1 and the first‐order rate constant for the dissociation, kd, is 10−3 s−1 under the same conditions. Thermodynamic parameters for complex formation at 25 °C, pH 8.0 are ΔGa0= ‐11.0 kcal×mol−1, ΔHa0= 3 kcal×mol−1 and ΔSa0= 48 cal. · mol−1· K−1.

2. Temperature and pH‐dependences of the rate constants ka and kd have been studied.

3. The difference in stability between the α‐chymotrypsin · inhibitor complex and the trypsin · inhibitor complex (Ka= 16 pM−1, ΔGa0=–18.1 kcal×mol−1) is due essentially to differences of kd values. The dissociation of the α‐chymotrypsin · inhibitor complex is 1.5×104 times faster than that of the trypsin · inhibitor complex. Differences in Ka and kd values are discussed in terms of differences in stabilizing interactions.

4. The Cys14‐Cys38 disulfide bridge of the inhibitor, which is highly succeptible to reduction when the inhibitor is free, is masked in the α‐chymotrypsin · inhibitor complex. Reduction of the Cys14‐Cys38 bridge in the free inhibitor does not prevent association with α‐chymotrypsin. Values of ka and kd for the formation of the 1:1 α‐chymotrypsin · reduced‐inhibitor complex and for the complex formed with the native inhibitor are very similar. This situation differs from that observed with the trypsin · pancreatic‐inhibitor complex. In that case, reduction of Cys14‐Cys38 bridge considerably decreases the stability of the complex formed with trypsin; Ka is decreased by a factor of 3×104 and kd is increased by a factor of 8.6×103.
Sites of Synthesis of Chloroplast‐Membrane Proteins
Authors: Apel Klaus , Schweiger Hans‐Georg
Published: March 2005

The protein synthesis of isolated chloroplasts of Acetabularia has been measured by the in‐corporation of labeled amino acids into the trichloroacetic‐acid‐insoluble fraction as well as into individual membrane fractions.

Chloramphenicol inhibited the incorporation into the trichloroacetic‐acid‐insoluble material only up to 60%, as did cycloheximide. The effect of the two antibiotics on the incorporation into the trichloroacetic‐acid‐insoluble material of isolated chloroplasts is additive.

By the treatment of chloroplasts with EDTA most of the originally membrane‐bound proteins were dissolved and could be separated quantitatively from EDTA‐insoluble material. Only three protein components could be detected in the membrane fragments. Two of these membrane proteins were synthesized in isolated chloroplasts. The synthesis of one of these two proteins is inhibited completely by cycloheximide, while the labeling of the other protein has scarcely been affected. The synthesis of the cycloheximide‐insensitive protein is almost completely blocked by chloramphenicol, but the cycloheximide‐sensitive protein still has been labeled distinctly in the presence of chloramphenicol. It is concluded that these chloroplast proteins are synthesized on two different groups of ribosomes, both of which are present in the isolated chloroplasts. The presence of 80‐S ribosomes in this chloroplast preparation has been indicated by finding 26‐S rRNA. Thus it is probable that the cycloheximide‐sensitive particles in the chloroplast preparation are identical with 80‐S ribosomes.

While in isolated‐chloroplasts only two membrane proteins have been synthesized, all three EDTA‐insoluble membrane proteins have been labeled in whole cells. It is concluded that the synthesis of the third protein takes place on a ribosome group in the cytosol which is active only in the intact cell.
The Degradation of CoA: Subcellular Localization and Kinetic Properties of CoA‐and Dephospho‐CoA Pyrophosphatase
Authors: Skrede Sverre
Published: March 2005

1. An enzyme degrading dephospho‐CoA with 5′‐AMP as the reaction product is present in the plasma membranes of rat liver. CoA is degraded at less than 10% of the rate of dephospho‐CoA.

2. The presence of plasma membranes in nuclear and microsomal fractions, which also contain significant amounts of the plasma‐membrane marker enzymes, probably explains the presence of dephospho‐CoA pyrophosphatase in these fractions. The enzyme is different from inorganic pyrophosphatase present in nuclei and microsomes with respect to pH optimum and sensitivity to inhibitors.

3. Dephospho‐CoA pyrophosphatase is inactivated by dialysis against EDTA, and is partially reactivated by Mn2+ and a number of other divalent cations.

4. The pyrophosphatase degrading dephospho‐CoA and CoA is probably identical with nucleotide pyrophosphatase from plasma membranes. The degradation of dephospho‐CoA is inhibited competitively by nucleotides with a pyrophosphate bond; most strongly by NADH, which has the highest affinity for nucleotide pyrophosphatase.

5. Competitive inhibitors of dephospho‐CoA degradation are less efficient when a ribose 3′‐phosphate is present in the nucleotide. Km for dephospho‐CoA is 0.02 mM, for CoA about 0.3 mM. These results indicate that the 3′‐phosphate group is a sterical hindrance for the access of the nucleotides to the active site. V for dephospho‐CoA in plasma membranes was about 200 nmol×mg protein−1×min−1, but for CoA one order of magnitude less.

6. Also nucleotide pyrophosphatase from Crotalus adamanteus degraded CoA at a much lower rate than dephospho‐CoA (below 1%).
Kinetic Analysis of a Rat‐Liver Nucleotidase Activated by Deoxyribonucleic‐Acid Constituents
Authors: Fritzson Per
Published: March 2005

A nucleotidase present in rat liver cytosol which hydrolyzes certain 3′‐ and 5′‐nucleotides, is subject to allosteric activation by dIno, dGuo, dThd, dUrd, and the corresponding 5′‐deoxy‐ribonucleotides. A systematic study of enzyme activation as a function of effector concentration was carried out with Urd‐3′‐P or [14C]dThd‐3′‐P as substrate. It was found that optimal effector concentration was 6–8 mM for the nucleosides, 4 mM for dThd‐5′‐P, and 1–2 mM for the other 5′‐deoxyribonucleotides. Deoxyinosine was a stronger activator than any of the other effectors and afforded a 3.3‐fold increase in the rate of Urd‐3′‐P hydrolysis at optimal concentration. At 1 mM concentration the enzyme was stimulated 2.3 times by dIno and, furthermore, by dGuo dGuo‐5′‐P, dThd‐5′‐P, dUrd‐5′‐P, and dIno‐5′‐P in decreasing order of effectiveness. The di‐ and triphosphates of dThd were more stimulatory than the monophosphate at concentrations above 3 mM. The p‐nitrophenyl ester of dThd‐5′‐P showed stimulation similar to that of the unesterified compound.

A kinetic enzyme model was developed which explains all the observed relations between enzyme activity and effector concentration. The parameters of the model were calculated by means of a programmed electronic computer and showed that the apparent dissociation constants for binding of dIno and the 5′‐monophosphates of dIno, dGuo, and dUrd to the regulatory site were about 1.7 mM. The other effectors had higher dissociation constants. The apparent Km values of the 5′‐deoxyribonucleotides, calculated by means of the computer, were about 0.2 mM. This value is similar to the Km values of the 3′‐nucleotides.

The stimulation of enzyme activity is due to an increase in V without a detectable change in the apparent Km, a mechanism which is less common for regulatory enzymes.
Structural Studies on the Heptose Region of Salmonella Lipopolysaccharides
Authors: Hämmerling Günter , Lehmann Volker , Lüderitz Otto
Published: March 2005

Degradation studies performed on the original or dephosphorylated core oligosaccharides derived from lipopolysaccharides of Salmonella Ra, Rb, and RcP+ mutants revealed the presence of a branched heptose trisaccharide, −3(HepIII‐1,7‐)‐HepII‐1,3‐HepI–. The branching heptose III is linked to position 7 of heptose II. Heptose II is also substituted (at either position 2, 4, or 6) by a phosphate group. Other previously identified substituents of the heptose units are a pyrophosphoryl‐ethanolamine group linked to position 4 of heptose I and the hexose oligosaccharide of the core linked to position 3 of heptose II. The degree of substitution of the heptose main chain with heptose III varies in different lipopolysaccharides from 20 to 90%. A lipopolysaccharide of chemotype RcP− was analyzed and found not to contain the third heptose unit.
The Reactivity of Escherichia coli Ribosomal Sulphydryl Groups with 5,5′‐Dithiobis(2‐nitrobenzoic Acid)
Authors: Johnson Peter M. , Walker Ian O.
Published: March 2005

A study has been made of the reactivity of the sulphydryl groups of Escherichia coli ribosomes with 5,5′‐dithiobis(2‐nitrobenzoic acid). Our results show that all the sulphydryl groups of the ribosomal proteins will react with this sulphydryl‐specific reagent.

Measurements of the rate of reaction provide evidence for conformational changes, independent of the association –dissociation reaction. The presence of 100 mM K+ or Na+ will markedly reduce the reactivity of the sulphydryl groups of 70‐S ribosomes or the ribosomal subunits. This conformational change has been interpreted in terms of a “tightening” of the ribosomal structure by the binding of monovalent cations.

A similar conformational change was observed on decreasing the temperature of the reaction mixture. The difference in the reaction rate was more than can be explained by the temperature dependence of the kinetics of the simple reaction, investigated using a model sulphydryl compound, 2‐mercaptoethanol.