bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Subunit Structure of the Aliphatic Amidase from Pseudomonas aeruginosa
Authors: Brown Paul R. , Smyth Maurice J. , Clarke Patricia H. , Rosemeyer Michael A. 
Published: March 2005

The molecular weight of Pseudomonas aeruginosa amidase was found to be 200000 by sedimentation equilibrium analysis; the sedimentation coefficient was 10.6 S. The subunit molecular weight determined by electrophoresis in dodecylsulphate‐acrylamide gels and gel filtration on Sephadex G‐200 in dodecylsulphate‐p‐chloromercuribenzoate solution was 33000—35000. The amino acid composition of the enzyme was determined.Only one N‐terminal amino acid, methionine, and one C‐terminal amino acid, alanine, were found indicating that the subunits were identical. Further evidence for a structure comprising 6 identical subunits came from both tryptic fingerprints and reaction of the enzyme with the cross‐linking reagent dimethyl suberimidate.
Duck‐Haemoglobin Synthesis in Frog Cells
Authors: Lane Charles‐Daniel , Gregory Caroline M. , Morel Carlos 
Published: March 2005

9‐S RNA was prepared from duck reticulocytes by dodecylsulfate treatment of the ribonucleoprotein particle derived from EDTA‐treated reticulocyte polyribosomes. The messenger content of this RNA was assayed by microinjection into oocytes of the frog Xenopus laevis. The 9‐S RNA was found to direct the synthesis of a molecular species that eluted from Sephadex G‐100 with added duck reticulocyte haemoglobin. When analysed on a cation‐exchange resin this material was found to contain the three globin‐chain species characteristic of duck reticulocyte haemoglobin. Moreover, paper electrophoresis at pH 6.5 and pH 3.5, and paper chromatography showed that these globin‐like species made in the oocyte were virtually indistinguishable, in terms of their histidine‐containing tryptic peptides, from the corresponding globin chains derived from duck reticulocyte haemoglobin.A comparison between the products made following the injection into oocytes of reticulocyte 9‐S RNA from rabbits, mice or ducks proved that it is the exogenous messenger that gives rise to the information content of the proteins whose synthesis is elicited by the injected RNA.The results also showed that the oocyte contains translational systems that are neither cell‐type nor species specific, and that duck globin messenger RNAs require no reticulocyte‐specific factors for their translation in oocytes.The construction of a saturation curve showed that there is a linear relationship between the amount of messenger injected and the amount of haemoglobin synthesized, up to the point at which haemoglobin synthesis constitutes over 35% of the total protein being made. Consequently the frog oocyte provides a convenient assay system for small quantities of duck globin mRNAs, and the messenger activity of as little as 1 ng of 9‐S RNA can be detected very simply. Thus a combination of the duck reticulocyte and frog oocyte may provide a useful system for studying post‐transcriptional control, and the formation of mRNA, in a eukaryotic cell.
Studies on the Induction and Repression of Enzymes in Rat Liver
Authors: Iwasaki Yoshifumi , Lamar Carlos , Danenberg Kathleen , Pitot Henry C. 
Published: March 2005

Tyrosine aminotransferase may be separated into four isolatable forms on hydroxyapatite columns. Form I is found in kidney, heart, brain and other tissues while forms II, III and IV appear to be unique to the liver in the rat. Forms II, III and IV were purified more than 1000‐fold from liver while form I was purified 30‐fold from kidney and heart.
The several forms have been compared kinetically and physicochemically. The Km for tyrosine of form I is significantly higher than that of the other three forms. On the other hand, in the presence of glutamate, V for all substrates studied is significantly lower for form II than for the other three forms. In addition, Kis for 2‐oxoglutarate in the presence of glutamate is somewhat lower and in the presence of pyridoxal 5′‐phosphate is significantly higher than for forms III and IV. The moledular weights and absorption spectra of forms II, III and IV could not be distinguished one from another. In contrast to forms II, III and IV the activity of form I was destroyed by brief heating at 60°C in the presence or absence of cofactors. For I also had a higher pH optimum than the other three forms.
In the fetus, glucagon, adenosine 3′:5′‐monophosphate and guanosine 3′:5′‐monophosphate induced predominantly form II. In the neonatal animal, hydrocortisone induced both forms II and III, while hydrocortisone in the presence of insulin induced form IV “prematurely”. This latter induction was insensitive to the action of actinomycin D. The rates in vivo of degradation of forms II, III and IV after hydrocortisone induction were indistinguishable. In the isolated perfused liver, hydrocortisone induced forms II, III and IV as had previously been shown in vivo, while adenosine 3′: 5′‐monophosphate induction was limited to forms II and III and insulin stimulated the level of form IV only. The significance of the differences in form I as compared to the hepatic forms, as well as the implications of selective metabolic regulation of the other three hepatic forms, are discussed.
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.
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.
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.
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.
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.
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.
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.