bluuebunny/crossref_metadata_2025 huggingface dataset:
Yeast Hexokinase A
Authors: ROSSI Antonio , MENEZES Luiz C. , PUDLES Julio
Published: March 2005

Yeast hexokinase A (ATP: d‐hexose 6‐phosphotransferase, EC 2.7.1.1) dissociates into its sub‐units upon reaction with succinic anhydride. The chemically modified subunits could be isolated in a catalytically active form. The Km values found for ATP and for glucose were of the same order as those found for the native enzyme.

Of the 37 amino groups present per enzyme subunit, 2–3 of these groups might be located in the proximity of the region of subunit interactions. The 50% loss of the initial activity, which follows the succinylation of these more reactive amino groups, does not seem to be due to the modification of a residue on the enzyme active site or to a change of the tertiary structure of the protein. This 50 % loss of the enzyme activity may be related to the dissociation of the dimer into monomers.

Both native enzyme and the succinylated subunits have the same pH‐dependent denaturation rate profiles in response to 2 M urea. Moreover, the apparent pK of the group involved in the transition from a more stable conformation of the protein in the acid range to a less stable one at alkaline pH seems to be similar to the pK of the group implicated in the transition between the protonated inactive form of the enzyme and an active deprotonated form.

The succinylated subunit presents ‘negative co‐operativity’ with respect to ATP at slightly acid pH; however, the burst‐type slow transient in the reaction progress curve and the activation effect induced by physiological polyanions, effects observed for the native enzyme, were not detected in the standard experimental conditions with the succinylated subunit.
Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin
Authors: ROCCA‐SERRA José , MILILI Michéle , FOUGEREAU Michel
Published: March 2005

The complete amino acid sequence of CNBr fragment H4 of the murine immunoglobulin MOPC 173 (IgG2a,x) has been determined, thus completing the sequence determination of the entire heavy chain.

The H4 fragment contains 150 residues, and extends from residue 105 to residue 254 of the heavy chain, which appears thus to be composed of 447 amino acids residues. This fragment contains the end of the V region, the switch peptide, the CH1 domain, the hinge region and the beginning of CH2. Sequence comparisons suggest that the CH1 domain is highly conserved in evolution, and allows the definition of two additional isotypic‐specific regions.
Properties of Immobilized Flavodoxin from Peptostreptococcus elsdenii
Authors: MAYHEW Stephen G. , STRATING Marijke J. J.
Published: March 2005

The small flavoprotein, flavodoxin, isolated from Peptostreptococcus elsdenii, has been covalently coupled to CNBr‐activated Sepharose 4B. The immobilized protein replaces ferredoxin as an electron carrier in hydrogen production from dithionite or pyruvate in the presence of ferredoxin‐free extracts of P. elsdenii; compared with soluble flavodoxin, its activities in these systems are 13% and 3.5% respectively.

Acid treatment reversibly dissociates FMN from the immobilized protein. The dissociation constant of the complex with FMN, determined by fluorimetric titration, is 1.5 (± 0.4) nM, and is therefore very little different from that of soluble flavodoxin. Like soluble apoflavodoxin, the immobilized apoprotein is highly specific for flavins with an N‐10 side‐chain of 5 carbon atoms and a C‐5′ phosphate group. Approximately half of the flavin impurity in commercial preparations of FMN (12–15% of the total flavin), and similar impurity in synthetic analogues of FMN, is not separated by conventional purification procedures, but it is readily and conveniently removed by affinity chromatography with apoflavodoxin as the immobilized ligand. The immobilized protein is stable for long periods; its capacity for FMN decreases by only 20% after 15 cycles of flavin dissociation and reassociation during several months.
Microbody of Methanol‐Grown Yeasts
Authors: FUKUI Saburo , KAWAMOTO Susumu , YASUHARA Shigeki , TANAKA Atsuo , OSUMI Masako , IMAIZUMI Fusako
Published: March 2005

Profuse appearance of microbodies was observed in the cells of methanol‐utilizing yeasts in connection with the enhanced catalase activity. These microbodies were isolated successfully by means of sucrose gradient centrifugation from the methanol‐grown cells of Kloeckera sp. no. 2201. Localization of a flavin‐dependent alcohol oxidase as well as characteristic microbody enzymes (catalase and d‐amino acid oxidase) were ascertained in the isolated microbodies, whereas formaldehyde and formate dehydrogenases were detected in the cytoplasmic region. Localization of catalase in the isolated microbody was also demonstrated by the cytochemical technique with 3,3′‐diaminobenzidine.
Reversible, Covalent Immobilization of Enzymes by Thiol‐Disulphide Interchange
Authors: CARLSSON Jan , AXÉN Rolf , UNGE Torsten
Published: March 2005

α‐Amylase and α‐chymotrypsin have been immobilized by covalent attachment to mercaptohydroxypropyl ether agarose gel. The technique involves two steps: (a) thiolation of the enzymes by methyl 3‐mercaptopropioimidate, (b) coupling of the thiolated enzymes to a mixed disulphide derivative of agarose obtained by reacting mercaptohydroxypropyl ether agarose with 2,2′‐dipyridyl disulphide.

The immobilization technique can be performed so that most of the inherent activity of the enzymes is conserved. However, diffusion limitations and steric factors prevent full manifestation of the immobilized activities.

Immobilized α‐amylase was used in a packed‐bed reactor for the continuous hydrolysis of starch. When the enzymically active gel had lost its activity it could be regenerated in situ by reductive uncoupling of the inactive protein and attachment of a new portion of thiolated α‐amylase.
On the Mechanism of Protein‐Synthesis Inhibition by Abrin and Ricin
Authors: BENSON Stephen , OLSNES Sjur , PIHL Alexander , SKORVE Jon , ABRAHAM Abraham K.
Published: March 2005

The mechanism of protein synthesis inhibition by the toxic lectins, abrin and ricin, has been studied in crude and in purified cell‐free systems from rabbit reticulocytes and Krebs II ascites cells.

In crude systems abrin and ricin strongly inhibited protein synthesis from added aminoacyl‐tRNA, demonstrating that the toxins act at some point after the charging of tRNA.

Supernatant factors and polysomes washed free of elongation factors were treated separately with the toxins and then neutralizing amounts of anti‐toxins were added. Recombination experiments between toxin‐treated ribosomes and untreated supernatant factors and vice versa showed that the toxin‐treated ribosomes had lost most of their ability to support polyphenylalanine synthesis, whereas treatment of the supernatant factors with the toxins did not inhibit polypeptide synthesis.

Recombination experiments between toxin‐treated isolated 40‐S subunits and untreated 60‐S subunits and vice versa showed that only when the 60‐S subunits had been treated with the toxins was protein synthesis inhibited in the reconstituted system. The incorporation of [3H]puromycin into nascent peptide chains was unaffected by the toxins, indicating that the peptidyl transferase is not inhibited.

Both the EF‐1‐catalyzed and the EF‐2‐catalyzed ability of the ribosomes to hydrolyze [γ‐32P]GTP was inhibited by abrin and ricin.

An 8‐S complex released from the 60‐S subunit by EDTA treatment possessed both GTPase and ATPase activity, while the particle remaining after the EDTA treatment had lost most of its GTPase activity. Both enzyme activities of the 8‐S complex were inhibited by abrin and ricin.

The present data indicate that there is a common site on the 60‐S subunits for EF‐1‐ and EF‐2‐stimulated GTPase activity and they suggest that abrin and ricin inhibit protein synthesis by modifying this site.
Adenylate Cyclase from Rat‐Liver Plasma Membrane: Inhibition by Mersalyl and Other Mercurial Derivatives
Authors: MAVIER Philippe , HANOUNE Jacques
Published: March 2005

The adenylate cyclase activity from a rat liver plasma membrane preparation was inhibited by low concentrations (1–10 μM) of the mercurial diuretic mersalyl. Complete inhibition was obtained with 0.1 mM mersalyl. Similar effects were observed whether the adenylate cyclase preparation was assayed in the presence of 10 μM GTP, 0.1 μM glucagon, 10 mM NaF or without any addition. The effect of mersalyl was not due to inhibition of the regenerating system present in the incubation medium, since the effect of mersalyl was preserved and even enhanced in its absence. The inhibition brought about by mersalyl was due to both a decrease of the maximal velocity of the reaction and of the affinity of the enzyme for the substrate. It was immediate, and irreversible spontaneously, but it was reversed by the simultaneous additions of 2‐mercaptoethanol, in a dose‐dependent fashion. Other ‐SH reagents were found to have an effect equal to, or lower than, that of mersalyl. Mersalyl had no effect upon Mg2+ ‐ATPase, although it inhibited the (Na+ ‐K+) activated ATPase. Since mersalyl is known to be a ‘non‐penetrant’ reagent, it is postulated that a catalytically important, mercurial‐sensitive, part of adenylate cyclase is at the surface of the plasma membrane. This view is supported by the following facts: (a) mersalyl acted with a similar dose‐response curve upon an intact as well as a detergentdispersed cyclase preparation while no effect was observed upon a solubilized Mg2+ ‐ATPase preparation; (b) a covalent p‐chloromercuribenzoate‐Sephadex preparation (but not its supernatant) inhibited the cyclase from intact membranes. It is proposed that mercurial derivatives, by their relative specificity of action (no effect on Mg2+ ‐ATPase), can serve as useful probes in the elucidation of the multicomponent structure of the cyclase system.
Extracellular Enzyme System Utilized by the Fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the Breakdown of Cellulose
Authors: STREAMER Margaret , ERIKSSON Karl‐Erik , PETTERSSON Bert
Published: March 2005

A strong synergistic response was observed between the five endo‐1,4‐β‐glucanases and the exo‐1,4‐β‐glucanase obtained from culture solutions of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum), when these enzymes were allowed to degrade de‐waxed cotton and Avicel. No synergism was observed if Walseth cellulose, an acid‐swollen cellulose, was used.

If de‐waxed cotton was pretreated with endo‐1,4‐β‐glucanases, the exo‐1,4‐β‐glucanase enzyme released much more degradation products than from an untreated cotton. This strongly supports the theory that the endo‐1,4‐β‐glucanases, acting randomly over the cellulose chain, go in first and open up chain ends where the exo‐enzyme can act.

In an earlier paper [Eriksson, K.‐E. & Pettersson, B. (1975) Eur. J. Biochem. 51, 193–206] physico‐chemical differences between the endo‐1,4‐β‐glucanases were demonstrated. Functional differences have now been found. The five endo‐enzymes give rise to different degradation products from Avicel, show differences in preferred mode of attack on cellodextrins, and exhibit pronounced differences in synthetic activity.

The exo‐1,4‐β‐glucanase released its products in the α‐configuration.

A method for the separation of cellodextrins on a Biol‐Sil A column is also described.
Factors Affecting the Molecular Structure and the Agglutinating Ability of Concanavalin A and Other Lectins
Authors: HUET Marianne
Published: March 2005

Ultracentrifugation analyses were performed on lectins under varying conditions of pH, ionic strength and temperature.

It has been demonstrated that the phytohemagglutinin from Phaseolus vulgaris, the wheat germ agglutinin and the soybean agglutinin are stable when these parameters are varied, whereas the concanavalin A molecule exhibits a striking reversible dimer‐tetramer transition with variation in pH (from 6.0 to 7.2) and temperature (from 4° up to 37°C).

It has also been demonstrated that, in agglutination experiments undertaken at different temperatures, cells do eventually aggregate with the first three lectins provided that incubation time is sufficient, whereas the concanavalin‐A‐induced agglutination was previously found to be temperature‐sensitive.

These results strongly suggest that the effect of temperature on agglutination by lectins may essentially be due to a structural transition of the lectin itself and not only to modification of cell surface properties.
The Interaction of Actinomycin C3 and Actinomine with DNA
Authors: ZIPPER Peter , BÜNEMANN Hans
Published: March 2005

Small‐angle X‐ray scattering was applied to solutions of calf thymus DNA and calf thymus DNA complexed with various amounts of actinomycin C3 or actinomine in phosphate‐saline buffer at pH 6.9 and I= 0.2. From the measurements of DNA in the absence of dye, two cross‐section radii of gyration of Rc, = 0.875 ± 0.015 nm and Rc2, = 0.81 ± 0.02 nm, and a mass per unit length of M/l= 1906 ± 43 daltons/nm resulted. The investigation of DNA complexed with dye revealed a decrease of the cross‐section radii of gyration as compared to those for the DNA in the absence of dye and a relatively low increase of the mass per unit length on binding of actinomycin and a slight decrease of M/l on binding of actinomine.

The latter results are interpreted on the basis of a length increase of the DNA double helix by 0.47 ± 0.03 nm per actinomycin molecule and by 0.355 ± 0.03 nm per actinomine molecule bound. The results for Rc, and Rc2 obtained for the various samples of complexed DNA were extra polated to the limiting binding ratio where each dye molecule is associated with a minimum of six nucleotide pairs. According to this extrapolation, the cross‐section radii of gyration of such a complex would amount to (Rc)b= 0.805 ± 0.015 nm and (Rc2)b= 0.76 ± 0.015 nm for the complex with actinomycin, and to (Rc)b= 0.77 ± 0.015 nm and (Rc2)b= 0.75 ± 0.01 nm for the actinomine complex. On the basis of a core and shell model for solvated DNA, these results can be understood as to indicate a decrease of the radial dimensions of both the core and the shell when the dye is bound.

The experimental results are compared with theoretical data calculated from the atomic coordinates of the detailed intercalation model for the actinomycin · DNA complex as recently proposed by Sobell and Jain. The model proves to be consistent fairly well with our data on the length increase of the double helix, but it appears to be unable to explain the experimentally observed decrease of Rc2, on binding of dye.
Conformational Changes in Fibrous Elastin Due to Calcium Ions
Authors: HORNEBECK William , PARTRIDGE S. Miles
Published: March 2005

A column packed with calcium‐free bovine aorta elastin provided good separations of mixtures of bile salts when water was the moving phase. Tritium‐labelled cholesterol was applied to the column using dilute solutions of taurodeoxycholate in Tris‐NaCl buffers as solvent. The cholesterol was quantitatively eluted as a narrow peak in a rising gradient of taurodeoxycholate. When Na+ in the buffer was replaced by Ca2+ elution of the labelled cholesterol was delayed. Control experiments in which the elastin fibres were replaced as the column packing by an inert stationary phase consisting of n‐butanol immobilized by silane‐treated Celite showed that the effect of the change from Na+ to Ca2+ on the solvent properties of taurodeoxycholate was small and in the opposite direction. The experiments indicated that the replacement of sodium by calcium as the ionic environment of fibrous elastin produced a configurational change towards increasing hydrophobic character.
The Hormonal Control of Activity of Skeletal Muscle Phosphorylase Kinase
Authors: COHEN Philip , WATSON David C. , DIXON Gordon H.
Published: March 2005

Two tryptic phosphopeptides containing the sites on the α and β subunits of phosphorylase kinase which are phosphorylated by protein kinase, dependent on adenosine 3′: 5′‐monophosphate (cyclic AMP), have been isolated and their amino acid sequences have been determined.

32P‐labelled phosphorylase kinase, containing 1.9 mol phosphate per mol enzyme, was digested with an equimolar quantity of trypsin for 2.5 min at pH 7.0, 20°C. This treatment released nearly all the 32P radioactivity associated with the β subunit as trichloroacetic‐acid‐soluble material. Only a small proportion of the 32P radioactivity associated with the α subunit was solubilised, the remainder being removed in the trichloroacetic acid pellet.

The β‐subunit tryptic phosphopeptide was completely resolved from traces of the α‐subunit phosphopeptide by gel filtration on Sephadex G‐25. Further purification by peptide mapping separated the phosphopeptide into four components, each derived from the same nine‐amino‐acid segment of the βchain, which was found to possess the sequence:Gln‐Ser‐Gly‐Ser(P)‐Val−IleTyr‐Pro‐Leu‐Lys. The four components were produced by the partial cyclisation of the N‐terminal glutaminyl residue, and by the presence of two alleles for the β subunit in the rabbit population, which led to a valine‐isoleucine ambiguity.

The α‐subunit phosphopeptide was liberated from the trichloroacetic acid pellet by redigestion with trypsin. It was the largest component in the digest which remained soluble in 5% trichloroacetic acid, and obtained in a highly purified form by a single filtration on Sephadex G‐50. The peptide comprised 39 amino acids of which nine were serine and three were threonine residues. Only one residue, the serine at position three from the amino terminus, was phosphorylated. The amino‐terminal sequence of the peptide was shown to be: Arg‐Leu‐Ser(P)‐Ile‐Ser‐Thr‐Glu‐Ser‐Glx‐Pro‐Asx‐Gly.

The sequences confirm the stoichiometry of the reaction and the absolute specificity of cyclic‐AMP‐dependent protein kinase for just two of the 200 serine residues in the enzyme. These results and an inspection of the rate of phosphorylation of a number of skeletal muscle proteins, including each enzyme of the glycolytic pathway, lead to the conclusion that cyclic‐AMP‐dependent protein kinase is an extremely specific enzyme. The molecular basis of this specificity is discussed.
Soluble and Membrane‐Bound Enzyme‐Active Antigens of Rat‐Liver Lysosomes
Authors: BERZINS Klavs , BLOMBERG Fred , PERLMANN Peter
Published: March 2005

Secondary lysosomes were isolated from rat liver and separated into a soluble and a membrane fraction. Plasma membranes and microsomes were also isolated and antisera against the various fractions were prepared in rabbits. Lysosomal content and detergent‐solubilized membrane fractions were analysed in two‐dimensional immunoelectrophoresis (crossed immunoelectrophoresis). The immunoprecipitates were stained by histochemical procedures for different enzyme activities such as phosphatases, non‐specific esterase, arylsulphatase, glycosidases and l‐leucyl‐β‐naphthylamidase.

When lysosomal content was tested against its corresponding antiserum, 17 different precipitates could be seen. Most of the enzyme activities tested were shown to reside separately in one or a few precipitates each. In contrast, when the membrane extracts were investigated, a more polymorphic pattern of enzyme‐active precipitates appeared. Thus, when lysosomal membrane extracts were reacted with homologous antiserum 11 precipitates with acid phosphatase activity were obtained. Several of the antigens were electrophoretically different and immunologically non‐identical. As expected from the biology of secondary lysosomes, many of their antigens were also found in microsomes and/or plasma membranes, but several antigens unique for lysosomes were detected concomitantly.

Closer analysis of these results indicated that several seemingly identical enzyme‐active proteins occurred both in soluble and membrane‐associated forms. However, while many of the membrane antigens expressed 2–4 different enzyme activities, only one activity was detected in individual precipitates of the lysosomal content. Thus, acid phosphatase activity was found together with esterase activity in three membrane‐associated antigens. The precipitates formed by two of these also stained for arylsulphatase and nucleoside tri‐, di‐ and monophosphatase activities. l‐Leucyl‐β‐naphthylamidase activity was found in one additional acid‐phosphatase‐active precipitate.
Extracellular Enzyme System Utilized by the Fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the Breakdown of Cellulose
Authors: ERIKSSON Karl‐Erik , PETTERSSON Bert
Published: March 2005

An exo‐1,4‐β‐glucanase from culture solution of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum) grown on powder cellulose as the sole carbon source has been extensively purified and characterized with respect to some physico‐chemical properties. The purification has been carried out in a five‐step procedure comprising chromatography on DEAE‐Sephadex, gel filtration on polyacrylamide P‐150, activation on a Dowex 2‐X8 anion exchanger, chromatography on Concanavalin A‐Sepharose and chromatography on SP‐Sephadex.

The purified enzyme was found to be pure and homogeneous by analytical polyacrylamide electrophoresis, by electrophoresis on dodecylsulphate gels and by analytical isoelectric focusing. A single symmetrical peak was obtained with the free zone electrophoresis method. The purification factor is about 15 and the yield of exo‐1,4‐β‐glucanase activity 7%.

After purification, the enzyme showed no viscosity‐decreasing activity towards carboxymethyl‐cellulose solutions.

The exo‐1,4‐β‐glucanase was isoelectric at pH 4.3 (4°C).

A molecular weight of 48600 was calculated on the basis of a knowledge of the partial specific volume, ultracentrifugation data and the amino acid composition. The enzyme contained no carbohydrate.
Crystalline Adenylate Kinase from Carp Muscle
Authors: NODA Lafayette , SCHULZ Georg E. , Von ZABERN Inge
Published: March 2005

The concentration of adenylate kinase in carp muscle is about 0.3 mg/g. An improved isolation procedure makes use of a dilute solution of the substrates, ATP and AMP, to elute the enzyme from a phosphocellulose column in overall yields of 60% before crystallization. By the hexokinase–pH‐stat assay the specific activity is 3550 units/mg. The preparation has been found to be essentially homogeneous by dodecylsulfate gel electrophoresis, isoelectrofocusing and gel filtration.

The molecular weight has been determined to be 22000 by several methods. The absorbance of a 1% solution at 280 nm is 6.9 and the isoelectric point by electrofocusing is pH 5.9.

The crystals of carp adenylate kinase have the space group P4122 or P4322.

The amino acid composition has been determined. There is no tryptophan, no cystine. There is one amino acid residue each of cysteine and histidine which are at or close to the catalytic center.

Several peptides derived by tryptic hydrolysis have been isolated and identified with corresponding peptides of porcine adenylate kinase. Consideration is given to histidine and cysteine being a part of the active site.
Multiple Forms of Tryptophanyl‐tRNA Synthetase from Beef Pancreas
Authors: LEMAIRE Geneviève , GROS Claude , EPELY Sylvie , KAMINSKI Marie , LABOUESSE Bernard
Published: March 2005

Three different forms of tryptophanyl‐tRNA synthetase can be isolated from pancreatic extracts. Structural, immunological and catalytic properties of these various forms have been compared. The native enzyme is a dimeric molecule of molecular weight 108000. Two other forms, of molecular weight 85000 and 82000, are composed of two polypeptide chains identical with the carboxyl terminal region of the native subunits. These molecules are supposed to derive from the original protein by removal, from the amino‐terminal part of each subunit, of a fragment of 11000 to 13000 molecular weight. Such removal modifies the shape and the stability of the molecule and decreases its specific activity.

The origin of the derived forms is attributed to proteolysis. In fact, limited proteolysis of purified tryptophanyl‐tRNA synthetase, in its native form, by elastase, results in the formation of an active compound, similar to one of the tryptophanyl‐tRNA synthetase derived forms. Furthermore, incubation with “elastolytic fractions” prepared from pancreatic extracts presenting a particularly high level of proteolytic activity produces the same cleavage in tryptophanyl‐tRNA synthetase polypeptide chain.
On the Mechanism of Action of Isocitrate Lyase
Authors: DIMROTH Peter , MAYER Klaus , EGGERER Hermann
Published: March 2005

The enzymes citrate lyase and isocitrate lyase catalyse similar reactions in the cleavage of citrate to acetate plus oxaloacetate and of isocitrate to succinate plus glyoxylate, respectively.

Nevertheless, the mechanism of action of each enzyme appears to be different from each other. Citrate lyase is an acyl carrier protein‐containing enzyme complex whereas isocitrate lyase is not. The active form of citrate lyase is an acetyl‐S‐enzyme but that of isocitrate lyase is not a corresponding succinyl‐S‐enzyme.

In contrast to citrate lyase, the isocitrate enzyme is not inhibited by hydroxylamine nor does it acquire label if treated with appropriately labelled radioactive substrate.

Isotopic exchange experiments performed in H218 with isocitrate as a substrate produced no labelling in the product succinate. This was shown by mass‐spectrometric analysis.

The conclusion drawn from these results is that no activation of succinate takes place on the enzyme through transient formation of succinic anhydride or a covalently‐linked succinyl‐enzyme, derived from this anhydride
The Primary Structure of the Major Cytoplasmic Valine tRNA of Mouse Myeloma Cells
Authors: PIPER Peter W.
Published: March 2005

This paper describes the derivation of the primary structure of the major valine tRNA in the cytoplasm of mouse myeloma cells. Approximately 75% of the nucleotide sequence of this tRNA is also shared by the tRNA1val of yeast, this homology serving as a further indication of the extreme conservation of the structures of the tRNAs of different eukaryotic organisms. A novel feature of mouse myeloma tRNA1val is its loop IV sequence: ‐U‐ψ‐C‐G‐m1 A‐A‐A‐. This particular loop IV sequence has not previously been found in a tRNA structure. In addition, tRNA1val possesses some unusual nucleoside modifications. 5‐Methyluridine (T) was not found to occur within loop IV of this tRNA, although this minor nucleoside is also absent from certain other mammalian tRNAs. Only one other tRNA, mammalian tRNAFfMet, has been found to possess 2‐methylguanosine (m2G) in the position between the (b) and (c) stems of the cloverleaf. Numerous tRNAs have m22G in this location, and it would appear that the second methylation of this guanosine is characteristically absent from certain mammalian tRNA species.
An Affinity‐Column Procedure Using Poly(l‐proline) for the Purification of Prolyl Hydroxylase
Authors: TUDERMAN Leena , KUUTTI Eeva‐Riitta , KIVIRIKKO Kari I.
Published: March 2005

An affinity column procedure is reported for purifying prolyl hydroxylase. The procedure is based on the affinity of the enzyme for its competitive polypeptide inhibitor, and involves affinity chromatography in a column containing poly(l‐proline) of molecular weight 30000 linked to agarose, and the elution of the enzyme from the column with poly(l‐proline) of molecular weight 5700. The enzyme is finally separated from this polyproline by gel filtration.

The procedure was employed for purifying prolyl hydroxylase from an ammonium sulphate fraction of chick embryo extract. The recovery of enzyme activity varied in ten enzyme preparations from 50 to 82%, and the purified preparations synthesized from 59.3 to 91.5 pmol hydroxyproline per mg enzyme per h at 37°C with a saturating concentration of (Pro‐Pro‐Gly)5 as substrate. The enzyme was pure when examined by polyacrylamide gel electrophoresis as a native protein or in the presence of sodium dodecyl sulphate, and the amino acid composition of the enzyme agreed with that reported previously with only minor exceptions. The molecular weight of the enzyme measured by equilibrium in an analytical ultracentrifuge was 240000, indicating that the enzyme had been isolated in the tetramer form.
Studies on New Intracellular Proteases in Various Organs of Rat
Authors: KATUNUMA Nobuhiko , KOMINAMI Eiki , KOBAYASHI Keiko , BANNO Yoshiko , SUZUKI Kunio , CHICHIBU Kenji , HAMAGUCHI Yoshitaka , KATSUNUMA Tsunehiko
Published: March 2005

Specific proteases which inactivate the apo‐proteins of many pyridoxal enzymes were found in skeletal muscle, liver and small intestine of rats. The protease from these three organs were purified and their properties were compared.

The purified proteases from liver and skeletal muscle appeared homogeneous on acrylamide gel electrophoresis. Two different proteases were separated from small intestine. A homogeneous, crystalline enzyme was obtained from the muscle layer while enzyme from the mucosa was partially purified.

They showed substrate specificity for pyridoxal enzymes. Their pH optima were in an alkaline region. They showed activity with the substrate of chymotrypsin, N‐acetyl‐l‐tyrosine ethyl ester, but not with that of trypsin, p‐toluenesulfonyl‐l‐arginine ethyl ester. They were inhibited by pyridoxal phosphate or pyridoxamine phosphate and seryl residues were involved in their active center.

The four enzymes differed in the following characters: (a) molecular weights; (b) patterns of elution from a CM‐Sephadex column; (c) rates of inactivation of substrate enzymes; (d) rates of cleavage of N‐acetyl‐l‐tyrosine ethyl ester; (e) reactivities with antiserum against the enzyme from the muscle layer of small intestine; (f) specific activities.

The amino acid composition and effect of chemical modifications of the crystalline enzyme from the muscle layer of small intestine were examined to elucidate its active sites and mode of action. Serine and histidine residues were found to be essential for protease activity. A tyrosine residue was also necessary for activity. Modifications of its sulfhydryl group, amino residues and carboxyl group had no effect on its activity.
Studies on New Intracellular Proteases in Various Organs of Rat
Authors: BANNO Yoshiko , SHIOTANI Taiichi , TOWATARI Takae , YOSHIKAWA Daisuke , KATUNUMA Nobuhiko , KATSUNUMA Tsunehiko , AFTING Ernst‐Günter
Published: March 2005

To study the role of group‐specific protease in enzyme degradation, alternation of its activity under various physiological conditions was examined.

Studies on the distribution of group‐specific protease in various organs of rats showed high activity in skeletal muscle and the muscle layer of small intestine, and rather low activity in liver. The activity varied in different muscles, but red muscle tended to have higher activity than white muscle. Activity was much lower in the muscles of the stomach and colon than in those of the small intestine.

Group‐specific protease in skeletal muscle increased under various dietary conditions (starvation, protein‐free diet or high protein diet), but the activities in the muscle layer of the small intestine and liver were not greatly influenced by dietary conditions. None of the hormones tested (i.e. hydrocortisone, glucagon, insulin, growth hormone and estrogen) influenced the activity of group‐specific protease in liver.

The level of group‐specific protease in skeletal muscle was increased markedly fifteen days after denervation, with a reciprocal decrease in the level of muscle phosphorylase, which is a good substrate of the protease.

Liver protease activity appeared in the late suckling period. The activity in skeletal muscle was high at the time of birth and attained the adult level 3 weeks after birth. The activity in the muscle layer of the small intestine did not change after birth. Thus the mechanisms for evoking these three specific proteases during development are apparently different. The activity of liver protease began to decrease approximately 12 h after partial hepatectomy and reached a minimum after about 72 h. Recovery of the protease activity was very slow and activity had not returned to the normal value 7 days after the operation. This observation seems to be consistent with the fact that there is little or no protease activity in liver in the neonatal period.
Relation between Structure and Function in Some Partially Synthetic Ribonucleases S′
Authors: FILIPPI Bruno , MORODER Luis , BORIN Gianfranco , SAMARTSEV Michele , MARCHIORI Fernando
Published: March 2005

Some analogues have been prepared of S‐peptide, the peptide obtained together with S‐protein from subtilisin‐modified beef pancreatic RNase A. The syntheses are described of [Orn10, Asn14]‐S‐peptide and 1ɛ, 7ɛ,10δ‐triguanidino‐[Orn10, Asn14]‐S‐peptide. The S‐peptide analogues are able to activate S‐protein at the level of the parent [Orn10]‐S‐peptide and 1ɛ,7ɛ‐diguanidino‐S‐peptide respectively, although at high peptide‐to‐protein molar ratios. After their recombination with S‐protein the buried character of Tyr‐25 was restored, as judged from difference absorption and circular dichroism spectra in the near‐ultraviolet region. These findings indicate that the asparaginyl residue is a possible naturally occurring substituent in the RNase A sequences whose state of amidation in position 14 has not yet been defined.
Dynamic Control on the Rate of the Reduction of the b Type Cytochromes in Submitochondrial Particles
Authors: EISENBACH Michael , GUTMAN Menachem
Published: March 2005

In the presence of antimycin and KCN the reduction of cytochrome b in phosphorylating submitochondrial particles followed a biphasic first‐order kinetics. The transition from the first, rapid phase to the second, slow phase occurred while the reduction of cytochromes c + c1 and a through or around the antimycin block was still linear with time. Thus, the phase transition was due to a fall‐off in the rate of cytochrome b reduction.

The biphasic reduction of cytochrome b was observed over a wide temperature range (0 – 30°C), with succinate of NADH as electron donors and with phosphorylating particles or coupled rat‐heart mitochondria. With rat‐heart mitochondria the same biphasic reduction was observed in the presence of either carbonyl cyanide p‐trifluoromethoxyphenylhydrazone or oligomycin.

In both the rapid and the slow phases, the rate of reduction of cytochrome b‐561 was equal to that of b‐565. Thus both cytochromes b‐561 and b‐565 were affected by the mechanism which determined the reduction‐rate. Furthermore, each of these cytochromes could be reduced individually with rate constants typical of the slow phase.

The proportion of rapidly reduced to slowly reduced cytochrome b was independent of the degree of its reducibility and could be controlled by the experimental conditions. When antimycin was used as the only inhibitor, 96% of the b‐type cytochromes were reduced in the rapid phase. If the c and a‐type cytochromes were first reduced by ascorbate and tetramethyl‐p‐phenylenediamine in the presence of KCN and antimycin, all the b‐type cytochromes were fully reduced at the slow‐rate.

With succinate, the rate of the rapid phase depended on the activation level of the succinic‐dehydrogenase. The rate constant of the second phase was unaffected by the succinic dehydrogenase activity, if the preparation was more than 20% active. Furthermore, the rate constant of the slow reduction was the same with succinate, NADH, or even with durohydroquinone (which reacted directly with cytochromes b).

It is suggested that cytochrome b can exist in two forms: kinetically active or sluggish. The active form is rapidly reduced by the endogenous quinone (QH2) or durohydroquinone. The rate of the reduction of the active form by succinate or NADH is probably determined by the rate of the reduction of Q by the dehydrogenases. The second form of cytochrome b is characterized by its sluggish reduction by QH2 or durohydroquinone.

It is proposed that the transformation from the active to the sluggish form is induced by the reduction of a controlling group, named Y, located on the oxygen side of the antimycin inhibition site. When Y is oxidized, cytochrome b is in its active form, and when Y is reduced, cytochrome b is in its sluggish form. The nature of this kinetic control and a comparison with the mechanism controlling the reducibility of cytochrome b are discussed.
Mouse Immunoglobulin Genes: Studies on the Reiteration Frequency of Light‐Chain Genes by Hybridisation Procedures
Authors: RABBITTS Terence Howard , MILSTEIN Cesar
Published: March 2005

The partially purified immunoglobulin light chain messenger RNA fraction from P3K (MOPC 21) mouse myeloma tissue‐culture cells has been employed in hybridisation studies. Fragments of the messenger RNA were generated by alkali hydrolysis. 6‐S fragments not containing poly(A) showed the characteristic biphasic hybridisation profile seen with the intact RNA fraction. 12‐S and 6‐S poly(A)‐containing fragments, however, showed single transitions lacking the rapidly hybridising component. Complementary DNA copies of the intact messenger RNA fraction were prepared with RNA‐dependent DNA polymerase and the DNA populations fractionated on acrylamide gels. Hybridisation experiments with complementary DNA fractions up to 800 bases in length showed annealing to single (or a few) genes. A rapidly hybridising component (about 200 copies) appears in the cDNA fraction containing the largest transcripts. We conclude that the kappa constant region gene and the MOPC 21 variable region gene are present as one or a few copies in the haploid genome and that the rapidly hybridising component is not due to variable region genes.
Lens Glycoproteins: Biosynthesis in Cultured Epithelial Cells of Bovine Lens
Authors: HUGHES R. Colin , LAURENT Maryvonne , LONCHAMPT Marie‐Odile , COURTOIS Yves
Published: March 2005

Radioactivity from [3H]glucosamine is rapidly incorporated into cellular fractions of lens epithelial cells cultured in vitro. The incorporated isotope appears largely in glycoproteins of the cell surface that are exposed to trypsin and are released into a soluble form by proteolysis of intact cells. Glycoproteins are also secreted by cultured cells and can be recovered in the culture fluids. Sodium dodecylsulphate – polyacrylamide gel electrophoresis shows that a range of glycoproteins with apparent molecular weights from approximately 14000 to 120000 are present. The relationships of these glycoproteins to collagen and the non‐collagenous glycoproteins of lens basement membranes are discussed.

A plasma membrane fraction obtained from non‐trypsinised lens epithelial cells contains one major glycoprotein of apparent molecular weight 120000. A major non‐glycosylated polypeptide of molecular weight about 38000 detectable by Bloemendal et al.(1972) in plasma membranes of differentiated lens fibre cells was not prominent in lens epithelial cell membranes.

Examination of lens basement membranes extracted in various ways failed to reveal major glycoproteins of low molecular weight. Higher molecular weight glycoproteins, some of them related to collagen, were present.
Purification and Studies on Some Properties of the 4‐Aminobutyrate: 2‐Oxoglutarate Transaminase from Rat Brain
Authors: MAÎTRE Michel , CIESIELSKI Lucien , CASH Christopher , MANDEL Paul
Published: March 2005

Using various chromatographic procedures, 4‐aminobutyrate: 2‐oxoglutarate transaminase from rat brain has been purified 2400 times with respect to the initial brain homogenate. The purified protein, which has a specific activity of 10 μmol × min−1× mg−1, gave a single band by acrylamide gel electrophoresis and isoelectric focusing. It has a molecular weight of 105000 ± 5000 and an isoelectric point of 6.8 In the presence of 0.1% sodium dodecylsulphate, a single protein band is seen on polyacrylamide gel, corresponding to a molecular weight of 57000 ± 5000. N‐terminal analysis reveals two chains with the same N‐terminal amino acid, thus the enzyme may be considered as a dimer consisting of two identical subunits. The pH optimum for enzyme activity is 8.5

Studies of the enzymic reaction show that the general mechanism is of the ping‐pong bi‐bi model. The Km for 2‐oxoglutarate at saturating 4‐aminobutyrate concentration is 5.5 mM and V is 22 μmol × min−1× mg−1 whereas the km for 4‐aminobutyrate extrapolated to saturating 2‐oxoglutarate concentration is 4 mM. 2‐Oxoglutarate competitively inhibits the enzyme with respect to 4‐aminobutyrate, with a Ki of 1.8 × 10−4 M. The same phenomenon is seen for the reverse reaction where the Ki is 6.6 × 10−4 M for succinic semi‐aldehyde.
Distinct Ribonuclease H Activities in Calf Thymus
Authors: BÜSEN Werner , HAUSEN Peter
Published: March 2005

Three enzymes with ribonuclease H activity are present in calf thymus. They have been separated on the basis of chromatographic behaviour and molecular weight. They are further distinguished from one another by their ionic requirements and sensitivity to the −SH reagent N‐ethylmaleimide. Two of these enzymes are classified as ribonuclease H, the third is obtained in a fraction which possesses ribonuclease H activity but also degrades double‐stranded RNA and poly(rA). No association between any of the enzymes and cellular DNA polymerases could be demonstrated.
Polysome Translational State during the Cell Cycle
Authors: EREMENKO Tamilla , VOLPE Pietro
Published: March 2005

HeLa cells were synchronized with a double thymidine block. Ribosomal subunits, monomers and polyribosomes have been quantitatively analysed at hourly intervals, during interphase, and every 15 min, during mitosis. This analysis was performed on linear 7–47% sucrose gradients.

From the beginning of G1 up to the end of S phase, a certain equilibrium among ribosomal sub‐units, monomers and polyribosomes is maintained, while from the time of entering G2 to M the translation machinery appears to be mobilized in the sense of polysome formation. Under these conditions, the amount of polysomes per cell during the mitotic cycle is expressed by a bi‐phasic pattern showing pre‐ and post‐mitotic peaks with a falling‐off during S. The G1 peak, meanwhile, is much lower than the G2 peak.

The incorporation of [3H]leucine into nascent polypeptide chains on polysomes, as well as into bulk cell proteins and into nuclear and cytoplasmic proteins considered separately, is also represented by a bi‐phasic curve which shows, however, a higher peak in G1 and a lower peak in G2, with two fallings‐off during S and M, respectively. Since between the G1 and the G2 amino acid pools there are not strong differences of leucine concentration, the discrepancy between the amount of polysomes and the rate of labelling is discussed on the basis of the differences of polysome shape found at the different stages of the cycle. In young cells, in fact, there is an abundance of small polysomes, while in the old cell large polysomes predominate.

It is suggested that, in the old cell, the rate of translation on large polysomes could be relatively lower or that among these heavy aggregates a given number of “frozen” polysomes could be present. The ribosome state is considered as a probable limiting‐factor of translation, particularly in mitosis.
Studies of Glutamate Dehydrogenase: Analysis of Functional Areas and Functional Groups
Authors: HUCHO Ferdinand , RASCHED Ihab , SUND Horst
Published: March 2005

It is shown by limited tryptic digestion of beef liver glutamate dehydrogenase under native conditions that the amino terminus of the polypeptide chain is located at the surface of the molecule. End‐group analysis after trypsin treatment yields aspartic acid as the new N‐terminal amino acid while the C‐terminal threonine remains unchanged.

NADH, especially in the presence of 2‐oxoglutarate, protects the enzyme against tryptic degradation. In the absence of the coenzyme, glutamate dehydrogenase is rapidly inactivated.

The regulatory effects of ADP and GTP are only slightly altered by trypsin. A small shift of the pH dependence of the activation by ADP is observed.

The quaternary structure of the unimer of the enzyme is not affected by limited tryptic digestion indicating that the N‐terminal part of the polypeptide chain is not located in the contact domains between the polypeptide chains. The association of the hexamer to large associated particles is reduced but not abolished.

It is shown by treatment of the enzyme with iodo[2‐14C]acetic acid as well as with Ellman's reagent that the six –SH groups of the polypeptide chain are buried and not accessible to these reagents in phosphate buffer. In Tris buffer they become exposed and react in the order 89, 55, 197, 115, 270, 319. This together with the result that in Tris buffer the rate of inactivation caused by trypsin is higher than in phosphate buffer indicates that Tris buffer changes drastically the properties of the enzyme.

Cross‐linking of the enzyme molecule with bifunctional reagents and subsequent dodecyl‐sulfate‐polyacrylamide electrophoresis shows that the six identical polypeptide chains are arranged in two groups of three.

The implications of these results for the tertiary and quaternary structure of beef liver glutamate dehydrogenase are discussed.
Regulation of Muscle Contraction: Binding of Troponin and Its Components to Actin and Tropomyosin
Authors: HITCHCOCK Sarah E.
Published: March 2005

The binding of troponin components to actin and tropomyosin has been studied by cosedimentation with actin and affinity chromatography. It is shown that troponin binds to actin and tropomyosin in the presence and absence of calcium but the binding to actin is sensitive to ionic strength. Troponin‐I + C binds to actin‐tropomyosin in the absence of calcium but not to actin or tropomyosin alone. Troponin‐I binds to actin and the binding is improved in the presence of tropomyosin even though troponin‐I does not bind to tropomyosin alone. Troponin‐C does not bind to actin or tropomyosin. The results suggest that the binding of troponin by actin is influenced by tropomyosin. A model of regulation by troponin is proposed.
Bacterial Respiration‐Linked Proton Translocation and Its Relationship to Respiratory‐Chain Composition
Authors: JONES Colin W. , BRICE Joan M. , DOWNS Andrew J. , DROZD Jan W.
Published: March 2005

The relationship between respiratory chain composition and the efficiency of respiration‐linked proton translocation was studied in nine bacterial species of widely differing taxonomic and ecological status.

All the bacteria investigated contained respiratory chain dehydrogenases, ubiquinone and/or menaquinone, cytochrome b and cytochrome oxidase aa3 and/or o. In addition, some of these organisms also contained pyridine nucleotide transhydrogenase and/or cytochrome c.

→H+/O ratios of whole cell suspensions oxidising endogenous substrates were in the approximate range 4–8 mol H+ translocated per g‐atom oxygen consumed. It was concluded from the observed →+H+/O ratios of cells loaded with specific substrates that proton‐translocating loops 1 and 2 were present in all of the organisms investigated, but that loops 0 and 3 were dependent upon the presence of pyridine nucleotide transhydrogenase and cytochrome c respectively.

The wide range in energy conservation efficiency which was observed in these organisms is discussed in relation to their respiratory chain composition and natural habitat.
The Mechanism of Template Activation by Exonuclease V
Authors: FERDINAND Franz‐Josef , KNIPPERS Rolf
Published: March 2005

In crude extracts from Escherichia coli cells the ATP‐dependent exonuclease V was found to be most active in converting double‐stranded DNA into a suitable template for DNA polymerases. This phenomenon was studied in some detail with isolated exonuclease V and T7 DNA polymerase. We found that, at ATP concentrations around 1 mM, the exonuclease produces a broad spectrum of DNA fragments. One class of fragments is largely single stranded with hydrogen‐bonded small primer sequences. These structures allow the synthesis of remarkably homogeneous polynucleotide strands by T7 DNA polymerase.