bluuebunny/crossref_metadata_2025 huggingface dataset:
The State of ADP or ATP Fixed to the Mitochondria by Bongkrekate
Authors: KLINGENBERG Martin
Published: March 2005

The reported studies are intended to clarify the binding state of ADP fixed to mitochondria under the influence of bongkrekate, and thus to discern between the affinity increase and reorientation mechanism proposed for the bongkrekate effect.

(a) The composition of the intramitochondrial adenine nucleotide pool is not changed under the influence of bongkrekate with and without added nucleotides. (b) The added ADP and ATP fixed by bongkrekate can be identified as AMP, ADP and ATP in the same proportions as in the endogenous pool. (c) The bound nucleotides respond to oxidative phosphorylation or uncoupler stimulated dephospitorylation similar as endogenous nucleotides.

It can be concluded that the ADP or ATP fixed under the influence of bongkrekate to the mitochondria are equilibrated with the intramitochondrial adenine nucleotide pool and are active in intramitochondrial phosphate transfer reactions.

The results disagree with the affinity increase mechanism but support the reorientation mechanism which postulates that ADP and ATP are trapped in the mitochondria under the influence of bongkrekate in the same amount as there are carrier sites available outside before bongkrekate addition.
Purification and Properties of an N‐Formylmethionyl‐tRNA Hydrolase
Authors: GANOZA M. Clelia , BARRACLOUGH Nada , WONG J. Tze‐Fei
Published: March 2005

The isolation and properties of a novel N‐formylmethionyl‐tRNA hydrolase (hydrolase II) from Escherichia coli are described. This enzyme is difficult to detect in crude extracts; purification, however, unmasks the activity. Sedimentation and gel filtration parameters of this enzyme differ from those of the previously described peptidyl‐tRNA hydrolase (hydrolase I), and preparations can be obtained where the two activities are free of each other. A mutant of hydrolase I has wild‐type levels of hydrolase II. These data indicate that hydrolase II is a different enzyme, or an altogether different form of hydrolase 1.

The bulk of the enzymic activity occurs in the ribosome‐free cytoplasm; the remainder is found on intact or dissociated 70‐S ribosomes.

Purified preparations of hydrolase II analyzed by two‐dimensional gel electrophoresis contain 2 protein bands. These 2 proteins do not coincide in electrophoretic mobility with any known ribosomal proteins. Analysis after mixing experiments verifies this conclusion.

The purified enzyme (hydrolase II) is inhibited by ribosomes bearing bound N‐formylmethionyl‐tRNA. The inhibition is potentiated by sparsomycin and other antibiotics that block specifically peptide‐bond synthesis. The relationship of this enzyme to other hydrolytic activities, including a newly described ribosome‐dependent hydrolase, are discussed.
On the Transport of Tripeptide Antibiotics in Bacteria
Authors: DIDDENS Heyke , ZÄHNER Hans , KRAAS Ekkehard , GÖHRING Walter , JUNG Günther
Published: March 2005

The two tripeptide antibiotics l‐2‐amino‐4‐met hylphosphinobutyryl‐alanyl‐alanine (L‐phosphinothricyl‐alanyl‐alanine) and l ‐(N5‐phosphono)methionine‐S‐sulfoximinyl‐alanyl‐alanine, both inhibitors of the glutamine svnthetase, are transported into the cell of Esclierichia coli K 12 via the oligopeptide transport system. The uptake by this system is proved first of all by cross resistance with tri‐ l ‐ornithine using oligopeptide‐transport‐deficient mutants, and secondly by antagonism tests demonstrating competitive reversal of the action of the antibiotic by several peptides which have been shown to be transported via the oligopeptide transport system, e.g. tri‐l‐alanine, tri‐l‐alanine, tri‐l‐lysine, tri‐ l ‐serine, tri‐glycine, glycyl‐glycyl‐ l‐alanine and the synthetic tripeptide l ‐azaadenyl‐aminohexanoyl‐alanyl‐alanine. On the other hand, there is no effect on the action of the antibiotic in antagonism tests with compounds which use different transport systems, such as l.‐alanyl‐alanine, l‐lysyl‐lysine, glutathione and the synthetic amino acid azaadenylaminohexanoic acid, i.e. 2‐amino‐6‐(7‐amino‐3H‐v‐triazolo‐[4,5‐d]‐pyrimidin‐3‐yl)hexanoic acid.

Another inhibitor of the glutamine synthetase, l‐methionine‐S‐dioxide (methioninesulfone) could be converted into a tripeptide form by linkage to l‐alanyl‐alanine analogously to the tripeptide antibiotics described above. Whereas the free l‐methionine‐S‐dioxide seems to be transported via the methionine transport system, the tripeptide form is transported via the oligopeptide transport system. Thus, this glutamine synthetase inhibitor can be taken up by the cell via two different transport mechanisms. Our results indicate that this could provide a synergistic effect.

The syntheses of the new tripeptides l‐azaadenylaminohexanoyl‐alanyl‐alanine and l ‐methionine‐S‐dioxidyl‐alanyl‐alanine were performed by dicyclohexylcarbodiimide couplings of the unusual N‐protected l‐α‐amino acids azaadenylaminohexanoic acid and l‐methionine‐S‐dioxide to l‐alanylalanine‐tert‐butyl ester followed by common deprotection steps. Tri‐ l‐ornithine was synthesized without carboxyl protection via two successive couplings of hydroxybenzotriazol esters of Nδ‐butoxycarbotlyl‐Nδ‐benzyloxycarbonyl‐l: ornithine to Nδ‐benzyloxycarbonyl‐l ‐ornithine
The Tentative Identification in Escherichia coli of a Multienzyme Complex with Glycolytic Activity
Authors: MOWBRAY John , MOSES Vivian
Published: March 2005

Penicillin spheroplasts of Escherichia coli were ruptured osmotically, by freezing and thawing, or mechanically. Differential centrifugation sedimented 20–30 % of the glycolytic enzymes without increasing their specific activities. There was, however, evidence of distinct groups of sedimenting enzymes; growth on different carbon sources could influence the distribution. Sucrose gradient studies gave no evidence of enzyme association but provided estimations of the molecular weight of each enzyme which were close to those subsequently observed on gel filtration. Using the determined molecular weight and a literature value for specific activity, the measured activity ratio of the enzymes was compared with that expected from an equimolar mixture. All values agreed within a factor of five, except for hexokinase. The relative roles of hexokinase and phosphotransferase in E. coli are briefly considered.

An equimolar multienzyme aggregate of all the enzymes of glycolysis would have a molecular weight of about 1.6 × 106. Chromatography on a Biogel column yielded one fraction, corresponding to a molecular weight of 1.6 × 106, which contained a proportion of all the glycolytic enzyme studied, the remaining portion of each enzyme activity was eluted from the column at the position expected from its individual molecular weight. The fraction of mol. wt 1600 000 was tested for complete glycolysis pathway activity and found not to be different from a reconcentrated mixture of the separated enzymes. Both‐the eluted and the reconstructed systems showed unexpected activity changes at different protein concentrations. The specific radioactivity of pyruvate formed by these systems from [14C]glucose 6‐phosphate was reduced by the presence of unlabelled 3‐phosphoglycerate, but by less than would have been expected had the latter been able to participate fully in glycolytic activity. This result indicates that these preparations were capable of selectivity compartmenting glycolytic intermediates.

Electron microscope investigation of both systems showed large numbers of regular 30 nm, diameter particles which, on disruption, appeared to be composed of smaller units: it is possible that these particles may have been aggregates containing glycolytic enzymes. The possible advantages of a glycolytic multienzyme complex are briefly discussed.
A Protein Factor Enhancing Activation of Inactive 30‐S Ribosomal Subunits of Escherichia coli
Authors: MISKIN Ruth
Published: March 2005

A protein factor partially purified from the S‐100 fraction (supernatant from 100000 × g spin) of Escherichia coli stimulates the conversion of 30‐S ribosomal subunits from an inactive to an active form. The subunits are normally inactivated at low concentrations of Mg2+ and reactivated when heated under appropriate ionic conditions. The factor, which differs from the known soluble factors required for protein biosynthesis as well as from proteins of the 30‐S subunit, enhances activation under conditions which by themselves allow for only a low level of activity to reappear. Fully active ribosomes and the assay reactions are not affected by the factor, indicating that its effect must be restricted to the activation process. The factor is likely to function by facilitating a conformational transition in the ribosome, and might act in vivo in a similar manner
Yeast Hexokinase: Substrate‐Induced Association‐ Dissociation Reactions in the Binding of Glucose to Hexokinase P‐II
Authors: HOGGETT James G. , KELLETT George L.
Published: March 2005

A method is described for the purification of native hexokinases P‐I and P‐II from yeast using preparative isoelectric focussing to separate the isozymes.

The binding of glucose to hexokinase P‐II, and the effect of this on the monomer‐dimer association‐dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation.

Association constants for the monomer‐dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupied in the dimer.

At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme glucose complex was 3 × 10−4 mol1 −2 and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I = 0.02 mol1−1, pH < 7.5) was also independent of enzyme concentration (in the range 10–1000.μg ml−1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non‐cooperative; values of the intrinsic dissociation constant varied from 2.5 × 10−3 mol 1−1 at pH 7.0 to 6 × 10−3 at pH 6.5.

Under intermediate conditions (pH 7.0, ionic strength = 0.15 mol l−1), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperativity (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations.

The results show that the phenomenon of half‐sites reactivity observed in the binding of glucose to crystalline hexokinase P‐II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.
Immunochemical Study of the Peptidoglycan of Gram‐Negative Bacteria
Authors: NGUYEN‐HUY Hiep , NAUCIEL Charles , WERMUTH Camille‐Georges
Published: March 2005

The specificity of antibodies directed against the peptidoglycan of gram‐negative bacteria was studied. The peptidoglycans of Proteus vulgaris, Esclierichia colt, Moraxella glucidolytica, Neisseria per/lava, give identical precipitin reactions. By means of inhibition studies with various peptidoglycan subunits and synthetic peptides, it was shown that the antibodies are essentially directed against the peptide moiety of the peptidoglycan: l‐Ala‐d‐G1u 1‐(l)‐mesoA2pm‐(l)‐d‐Ala, that the peptide reacts better with antibodies when it is not cross‐linked, and that the C‐terminal portion ‐meso‐A2pm‐d‐Ala of the peptide is immunodominant. These results explain the immunological identity of the peptidoglycans of gram‐negative bacteria, which possess the same peptide subunit. Only weak cross‐reactivity was observed with the peptidoglycans of grain‐positive bacteria (Streptococcus faecium, Micrococcus lysodeikticus, Corynebacterium poinse ttiae) where mesodiaminopimelic acid is replaced by l‐lysine pr l‐homoserine. However, the peptidoglycan of Bacillus megaterium which possesses the same peptide subunit as gram‐negative bacteria, gives only a reaction of partial identity with these bacteria. This result suggests the presence on the peptidoglycan of gram‐negative bacteria, of other undefined antigenic determinants
Reaction of N‐(3‐Pyrene)maleimide with Thiol Groups of Reticulocyte Ribosomes
Authors: LEE Terry , HEINTZ Roger L.
Published: March 2005

The reaction of N–(3–pyrene)maleimide with thiol groups of rabbit reticulocyte ribosomes offers a possible fluorescent probe for studying ribosomal structure and conformation. At relatively low concentrations of N–(3–pyrene)maleimide a group of 30–40 readily reactive sulfhydryl residues is derivatized. The major ribosomal proteins containing these thiol groups are identified as S2+S3, S5, S7, S8, S29, S31, S32, L1, L5, L6, L10+L14, L15, L18+L19, and L36. Ribosomal activity, as measured by the nonenzymic binding of phenylalanyl‐tRNA and polyphenylalanine synthesis, is inhibited by this degree of reaction with N–(3–pyrene)maleimide. This inhibition is relieved by the prior binding of polyuridylic acid to the ribosomes while the extent of derivatization by N–(3–pyrene) maleimide is diminished only slightly. The average relative polarization of the fluorescence of the ribosomal bound N–(3–pyrene)maleimide changes significantly with the degree of derivatization of ribosomal thiol groups or with the binding of polyuridylic acid, indicating the value of such a fluorescent thiol‐derivatizing agent as a probe of ribosomal structure.
The Enthalpy of Interaction of Urea with Some Globular Proteins
Authors: PAZ ANDRADE Maria I. , JONES Malcolm N. , SKINNER Henry A.
Published: March 2005

The enthalpies of interaction of urea with five globular proteins, ribonuclease A, trypsin, β‐lactoglobulin, ovalbumin and bovine serum albumin have been measured in aqueous solution at pH 7.0, I = 0.005 M and 25°C over a range of urea molality m from 0–15 mmol g−1 (where a 1 molal solution contains 1 mmol g−1). For all the proteins the interaction is exothermic, and there is an appreciable heat evolution at low urea concentrations, m < 5 mmol g−1, which increases sharply at higher urea concentrations when the proteins undergo unfolding. If account is taken of the endothermic enthalpies of unfolding of the native proteins, the enthalpies of interactions of urea per unit mass denatured protein lie in the range −45 to −75 J g−1, corresponding to an average binding enthalpy of −23 kJ mol−1 bound urea.
Investigation of the Covalent Bond Structure of Peptide‐Matrix Systems by Edman Degradation of Support‐Fixed Peptides
Authors: MECH Carla , JESCHKEIT Hans , SCHELLENBERGER Alfred
Published: March 2005

Two model peptides Ala‐Ser‐Asp‐Tyr‐Leu and Ala‐Ser‐Glu‐Tyr‐Leu have been synthesized and coupled with an without protecting groups to the amino groups of polystyrene resins. The yield in the binding operation was 93–97% by using water‐soluble carbodiimides as condensing reagents. Edman degradation of the products and quantitative estimation of the amino acid phenylthiohydantoins, as well as amino acid analysis of the partially and completely degraded resins, showed about 7–10% of the fixed peptides not to be involved in the degradation procedure.

That part of the material which was not involved in the Edman degradation during the first step also remained inactive in the following degradation operations.

The analytical data presented in the paper explain not only the course of the Edman degradation of peptides linked via the side‐chain carboxylic groups to the resin, but they also describe the chemical reactivity of the various carboxylic groups in the binding operation. 22% of the peptides are bound to the resin by side‐chain carboxylic groups in the case of the aspartate‐containing peptide, 26–27% in the case of the glutamate‐containing peptide. 75–77% and 72–75% respectively of the peptides are fixed to the resin via the C‐terminal carboxylic group. No more than 3% of the fixed material may be bound bifunctionally to the support.
Influence of Side‐Chain Structure of Aliphatic Amino Acids on Binding to Isoleucyl‐tRNA Synthetase from Escherichia coli MRE 600
Authors: FLOSSDORF Josef , PRÄTORIUS Hans‐Jörg , KULA Maria‐Regina
Published: March 2005

The binding of 10 isomeric α‐amino‐heptanoic acids, of two isomeric α‐amino‐octanoic acids, of isoleucinol and valinol, and of various α‐hydroxy acids to isoleucyl‐tRNA synthetase from Escherichic coli MRE 600 has been investigated by an ultracentrifuge method. It was found that the enzyme requires a primary amino group together with a not‐too‐small side chain as prerequisites for ligand recognition. Though the enzyme is absolutely specific for the L isomers, it is fairly tolerant against replacement of the carboxyl group of the natural substrate by more or less hydrophobic substituents. These findings can be explained in terms of Ogston's three‐point‐attachment model, if it is additionally assumed that there is no further space available in the binding region normally occupied by the α‐hydrogen atom to accept other substituents which are as bulky as the carboxyl moiety.

Similarly, the architecture of the binding region of the aliphatic side chain is discussed. Our measurements show that the free energy of binding strongly depends on the size and the structure of the remainder of the molecule. None of the isoleucine analogues employed is bound as tightly as the natural substrate itself, but there is a clear preference for side chains branched at the β‐carbon atom. The functioning of the side‐chain recognition site is best understood by imagining a two‐finger glove, of which one finger is tailored to a methyl and the other to an ethyl group. Both these fingers, together with the binding region for the glycine moiety and a steric barrier against a fourth substituent bulkier than hydrogen, are responsible for a high steric specificity towards the one side chain over its Cβ epimer.
Light Chains from Slow‐Twitch Muscle Myosin.
Authors: WEEDS Alan G.
Published: March 2005

Myosin light chains have been isolated from slow‐twitch soleus muscles of rabbit and cat. Two chemically related light chains of molecular weight about 22000 have been identified from their thiol sequences in each species, and these have been further characterized by amino acid analysis and peptide mapping studies. These light chains are related to the alkali light chains of rabbit fast‐twitch muscles and to the larger cardiac light chain from bovine heart muscle. The presence of two chemically related but phenotypically distinct light chains within single muscles suggests the presence of myosin isoenzyme or that the myosin molecule has a different light chain associated with each subfragment‐1 head. Although the stoichiometry of these two light chains had not been determined, the former of these two conclusions is favoured by analogy with experiments on fasttwitch myosins. In addition to these related light chains, soleus muscle myosin, like fast‐twitch myosins, contains a third light chain of about 19000 molecular weight. Unlike the corresponding light chain of rabbit fast‐twitch myosin, this 19000‐Mr light chain contrains no cysteine residues.

The distribution of thiol peptides together with the characteristic mobilities of these light chains on polyacrylamide gels in the presence of sodium dodecyl sulphate provides a ‘fingerprint’ of myosins from different muscle types. This has been used to look for the presence of fast‐twitch myosin in soleus muscle, the results showing both rabbit and cat soleus muscles are homogeneous in their myosin type within the sensitivity of detection of the methods. These techniques have also been used to confirm the reciprocal transformation of light chains in myosins isolated from crossreinnervated muscles. Finally the relationships between these criteria for different myosin types and histochemical procedures for muscle fibre typing are discussed.
Enhancement of Hydroxyindole‐O‐Methyltransferase and DNA‐Dependent RNA Polymerase Activities Induced by Oestradiol in Rat Pineals in Culture
Authors: MIZOBE Fumio , KUROKAWA Masanori
Published: March 2005

Hydroxyindole‐O‐methyltransferase and DNA‐dependent RNA polymerase activities were determined in the pineal gland removed from the ovariectomised rat and cultured under various experimental conditions.

The transferase activity declined very slowly during 24 h of incubation. 17β‐Oestradiol significantly increased the transferase activity within 2 h after the addition, and the extent of increase was dose‐dependent within the concentration range from 0.1 to 15 nM, being increased by 80% at 15 nM. Enhancement of the transferase activity by oestradiol was abolished not only by inhibitors of protein synthesis (cycloheximide and puromycin), but also by those of RNA synthesis (actinomycin D and α‐amanitin). It was also blocked by clomiphene citrate, an agent which is known to inhibit the binding of steroid hormones to their respective receptors.

RNA polymerase activity (forms A and B) declined rapidly during the initial period of pineal culture. Oestradiol (15 nM) increased the RNA polymerase B activity by 50% within 2 h after the addition. The increase was dose‐dependent within the concentration range from 0.1 to 15 nM, and was abolished by clomiphene citrate.

The possibility is suggested that the pineal is a target organ of oestradiol, and that the steroid‐induced reaction sequence in the pineal conforms to that is known in other target organs.
Studies on Chromatin. Free DNA in Sheared Chromatin
Authors: VARSHAVSKY Alexander J. , BAKAYEV Valery V. , ILYIN Yurii V. , BAYEV Alexey A. , GEORGIEV Georgii P.
Published: March 2005

Chromatin which has been hydrodynamically sheared in a low‐ionic‐strength buffer lacking divalent cations (I = 0.005 M) contains a heterogeneous set of deoxyribonucleoprotein particles but no molecules of free DNA. The main finding is that a transference of sheared chromatin to 1–2 mM MgCl2 or to 0.1–0.2 M NaCl results in appearance of completely free DNA molecules. A salt‐induced rearrangement of DNA‐bound histories but not a partial loss of them is responsible for the observed phenomenon. Formation of free DNA molecules is accompanied by aggregation of the majority of remaining deoxyribonucleoprotein particles.

The percentage of free DNA molecules in the chromatin which was sheared to an average DNA length of about 400 base pairs is increased from zero in the initial sample to 8–9% in 1 mM MgCl2 and further to ∼ 25% of the total DNA in 0.15 M NaCl, 2 mM MgCl2. Free DNA molecules in the sheared chromatin are observed not only upon isopycnic banding of formaldehyde‐fixed deoxyribonucleoproteins in CsCl gradients but also in non‐ionic metrizamide gradients with either fixed or unfixed deoxyribonucleoprotein samples. The process of free DNA formation is a reversible one; its direction and the equilibrium state depend in particular on the ionic conditions of the medium.
Nitrate Reductase from Chlorella vulgaris
Authors: FUNKHOUSER Edward A. , ACKERMANN Renate
Published: March 2005

Millimolar concentrations of tervalent manganese pyrophosphate can partially activate nitrate reductase which has been inactivated with NADH and HCN. The tervalent manganese complex is nevertheless not reduced by NADH in the presence of the enzyme, that is, it is not a substrate for the diaphorase moiety of the nitrate reductase. Ferric o‐phenanthroline, on the other hand, is a good diaphorase substrate, but fails to activate the inactive enzyme.
The Use of Several Energy‐Coupling Reactions in Characterizing Mutants of Escherichia coli K12 Defective in Oxidative Phosphorylation
Authors: SCHAIRER Hans U. , FRIEDL Peter , SCHMID Brigitte I. , VOGEL Günter
Published: March 2005

Oxidative phosphorylation, ATP‐32Pi exchange, ATP‐dependent quenching of acridine‐dye fluorescence, ATP‐dependent transhydrogenase and ATP‐dependent transport of thiomethyl β‐d‐galactoside are shown to be experimentally equivalent tools to study the functional state of the ATPase complex in Escherichia coli wild‐type and mutant strains defective in oxidative phosphorylation.

According to these criteria ten mutants in the ATPase complex were classified having lesions in the unc A,B region of the chromosome. The first mutant type lacks ATPase activity, but the membrane‐integrated part of the complex remains functional (class I). The second mutant type lacks a functional membrane‐integrated part, but retains ATPase activity (class II). The third mutant type is shown to be defective in both parts of the ATPase complex (class III).
NADH‐Dependent Aryl Hydrocarbon Hydroxylase in Rat Liver Mitochondrial Outer Membrane
Authors: UEMURA Tomihiko , CHIESARA Enzo
Published: March 2005

NADH‐dependent 3,4‐benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3‐methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH‐dependent 3,4‐benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH‐ to NADH‐dependent 3,4‐benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5.

Moreover, it was found that NADH‐dependent 3,4‐benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide‐insensitive, while that in microsomes was cyanide‐sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide‐insensitive cytochrome P‐450 and is inducible markedly by 3‐methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P‐450 oxidase system are discussed.
The Specificity Requirements of Bacteriophage T4 Lysozyme
Authors: JENSEN Harald B. , KLEPPE Gunnar , SCHINDLER Melvin , MIRELMAN David
Published: March 2005

A series of bacterial cell wall glycopeptides of low molecular weight and cell wall nucleotide precursors have been tested for their inhibitory action on the digestion by T4 lysozyme of a radioactively labeled linear uncrosslinked peptidoglycan. The disaccharide‐peptides GlcNAc‐MurNAc‐l‐Ala‐d‐Glu(A2pm) (C5) and GlcNAc‐MurNAc‐l‐Ala‐d‐Glu(A2pm‐d‐Ala) (C6) as well as the monosaccharide‐peptide MurNAc‐l‐Ala‐d‐Glu(A2pm) were found to be good competitive inhibitors (with similar K1 values) whereas the disaccharide‐pentapeptide GIcNAcMurNAc‐l‐Ala‐d Glu‐Gly

l‐Lys‐d‐Ala was a poor inhibitor. T4 lysozyme did not catalyse transglycosylation reactions from Escherichia coli B peptidoglycan to the disaccharide‐peptide C6. No changes were seen in the circular dichroism spectra (200–250 nm) or fluorescence emission spectra upon binding of the good inhibitors. The results obtained indicate that T4 lysozyme has a small active site capable of recognizing a unit consisting of MurNAc‐l‐Ala‐d‐Glu(A2pm).
Limited Proteolysis of Cytoplasmic and Nuclear Uterine Estradiol Receptors Yields Identical Estradiol‐Binding Fragments
Authors: VALLET‐STROUVE Claude , RAT Luce , SALA‐TREPAT José M.
Published: March 2005

Limited tryptic hydrolysis of the estradiol cytoplasmic receptor from calf uterus has been demonstrated to yield in a high‐salt buffer a stable estradiol‐binding molecule with the following characteristics: sedimentation coefficient 4,0 ± 0.1 S; Stokes radius 3.5 ± 0.05 nm; molecular weight 60000 (for an assumed v̄ value of 0.73 ml g−1) and frictional ratio 1.36.

Nuclear KCI extracts, prepared from uteri preincubated at 37°C with labeled estradiol, were analysed by Sephadex G‐200 chromatography and sucrose density gradient centrifugation. The following molecular parameters were found for the estradiol receptor complex: sedimentation coefficient 4.4 ± 0.1 S; Stokes radius 4.12 ± 0.02 nm; molecular weight 77000 and frictional ratio 1.47 (v̄= 0.73 ml g−1). Limited tryptic proteolysis of this extract gave an estradiol‐binding fragment with molecular characteristics identical to the trypsin‐modified cytoplasmic receptor. In addition, mild tryptic digestion of whole labeled nuclei allowed us to solubilize almost quantitatively the nuclear [3H]estradiol in a macromolecular bound form. The molecule thus obtained showed molecular parameters very similar to the 60000‐dalton trypsin fragments obtained from high‐salt cytoplasmic and nuclear extracts. These molecules were undistinguishable by gel electrophoresis analysis at six different acrylamide concentrations. These results in conjunction with those derived from dissociation kinetics experiments and ligand specificity studies indicate the cytosolic protein is a functional part of the nuclear receptor.

Based upon these and other studies we suggest that proteolytic cleavage of the estradiol receptor complex, which results in the removal of the estradiol‐binding sites from the nuclear recognition sites of the molecule, could play a role in the inactivation of the estradiol receptor in vivo.
The Molecular Basis of Skeletal Muscle Phosphorylase Kinase Deficiency
Authors: COHEN Patricia T. Wade , BURCHELL Ani. , COHEN Philip
Published: March 2005

The molecular basis of phosphorylase kinase deficiency was investigated in ICR/IAn mice, which show < 0.2% of normal activity in skeletal muscle (Cohen, P. T. W. and Cohen, P., 1973). The genetics of the deficiency indicate it is a single gene defect on the X‐chromosome (Lyon, J. B., 1970).

Phosphorylase kinase was purified from skeletal muscle of a control strain, C 3 H/He‐mg, by three different procedures. (a) Ammonium sulphate precipitation and gel filtration on Sepharose 4B. (b) Hydrophobic chromatography and affinity chromatography on Sepharose 4B to which antibody to rabbit muscle phosphorylase kinase has been linked covalently. (c) Precipitation from muscle extracts with anti‐phosphorylase kinase antibody.

All three procedures showed C 3 H/He‐mg phosphorylase kinases were similar to the rabbit muscle enzymes, the structures of the two isoenzyrnes being (αβ?)4 and (α'βγ)4 respectively. The proportion of the (α′βγ)4 isoenzyme relative to the (αβγ)4 isoenzyme was however about 1:1 in murine muscle compared to about 1:10 in rabbit muscle. Since the α and α′ subunits appear to be distinct gene products, the defect in ICR/IAn mice cannot be caused by a mutation in the genes coding for either the α or α′ chains, or 50% of normal activity would be observed.

All three procedures for C3H/He‐mg mice failed to detect any of the four subunits α, α′, β and γ in ICR/IAn mice, suggesting that all four chains are absent in the deficiency.

An allele for the β‐subunit was identified in rabbits, and the inheritance of the allele showed that it was determined by an autosomal gene. Assuming conservation of X‐linkage between mammals, the defect in ICR/lAn mice cannot be caused by a mutation in a β‐subunit gene.

It is proposed that ICR/IAn mice are defective in a control gene located on the X‐chromosome which is required for the expression of structural genes, at least one of which, the gene for the β‐subunit, is located on an autosome. The results imply that interchromosomal transfer of information takes place during the synthesis of phosphorylase kinase.
Glycosidases of Molluscs
Authors: REGLERO Angel , CABEZAS José A.
Published: March 2005

An α‐l‐fucosidase has been purified approximately 300‐fold from the liver (hepatopancreas) of the marine mollusc Chamelea gallina L. (=Venus gallina L.).

During the different steps of the purification procedure it was difficult to remove the contaminant N‐acetylglucosaminidase activity; but, after electrofocusing, a final preprration free of this and other glycosidades present in the crude extract was obtained. The purified enzyme has a broad specificity; it hydrolyzes p‐nitrophenyl α‐l‐fucoside and natural substrates such as oligosaccharides containing fucosidic residues with α 1–2, α 1–3 and α 1–4 linkages; also it hydrolyzes fucose‐containing glycopeptides, such as thyroglobulin glycopeptide, and glycoproteins as porcine submaxillary mucin (previously rendered free of sialic acid). The enzyme has a pH optimum of 5.2 ± 0.2, with a Km of 7 × 10−5 M using p‐nitrophenyl l‐fucoside as substrate. It is inhibited by Hg2+ and some sugars, and activated by CN−, Zn2+, Ca2+ and EDTA. It shows two peaks by isoelectric focusing (at 6.3 and 6.6). The molecular weight of the α‐l‐fucosidase by gel filtration was over 200000.
Comparative Structural and Immunochemical Properties of Leghaemoglobins
Authors: HURRELL John G. R. , NICOLA Nicos A. , BROUGHTON William J. , DILWORTH Michael J. , MINASIAN Elizabeth , LEACH Sydney J.
Published: March 2005

Circular dichroism studies on leghaemoglobins from snake bean, lupin, serradella and other plants show that, in common with soybean (reported earlier) they have a similar overall polypeptide chain conformation and haem environment and orientation. Immunochemical studies, on the other hand, suggest that the antigenic determinants on the surface of the leghaemoglobins vary considerably.

Thus, firstly the α‐helix content of the leghaemoglobins as a class is very similar (60–65%) and approaches that of the myoglobins, secondly, the sign, magnitude and shape of their circular dichroism spectra in the near ultraviolet, Soret and visible regions suggest close similarities in the environment and orientation of a structurally important tryptophan residue and of the haem moiety, and thirdly, there is comparatively weak haem‐protein interaction.

The extent of immuno cross‐reactivity was found to be best demonstrated using the Farr radioimmunoassay procedure. The results were (a) 5 leghaemoglobins from one plant (soybean) crossreacted completely but with varying affinities. (b) The extent of cross reactivity between leghaemoglobins from different plants was compared to that within a single plant; the reaction of antiserum to a soybean leghaemoglobin with a serradella leghaemoglobin was weak, with a snake bean leghaemoglobin still weaker (and incomplete) while lupin leghaemoglobins showed no cross reactivity at all. (c) The “rapid” attenuation of cross reactivity among different plant leghaemoglobins is explicable in terms of the extensive amino acid substitutions which have been demonstrated in the literature and in the present studies. (d) In view of this rapid divergence it is not surprising that sperm whale and horse heart myoglobins showed no cross reactivity with soybean leghaemoglobins.

In summary, amino acid substitutions in the leghaemoglobin family are conformationally but not immunochemically conservative.
Preferential Stimulation of the Plant mRNA Synthesis by Gibberellic Acid
Authors: WASILEWSKA Lidia D. , KLECZKOWSKI Kazimierz
Published: March 2005

Treatment of the etiolated maize seedlings with the plant hormone, gibberellic acid results in a significant enhancement of heavy polyribosome formation.

This is accompanied by highly increased incorporation of the labelled RNA precursors into RNA engaged in the polyribosotnal complex, as well as by an increased rate of protein synthesis in viva.

Determination of the specific radioactivity of particular RNA classes isolated from polyribosomes reveals that gibberellic acid stimulates mostly the synthesis of the rapidly labelled, nonribosomal RNA fraction.

A considerable amount of this rapidly labelled RNA fraction, whose synthesis is preferentially stimulated by exogenous gibberellic acid contains poly(A) sequences, as shown by affinity chromatography on oligo(dT)‐cellulose, indicating that phytohormone causes an increased transcription of mRNA in etiolated maize seedlings.

When [3H]adenosine served as the RNA precursor it was found that the ratio between the heteropolymeric and polyadenylic parts of the poly(A)‐RNA chain markedly changed under gibberellin treatment, suggesting that, in addition to an increased rate of mRNA synthesis, the plant hormone also affects the process of post‐transcriptional polyadenylation of the newly made mRNA precursors. Possible extension of the polyadenylate segment in the presence of gibberellin may account for a longer functional half‐life of the mRNA synthesized in plants treated with the phytohormone, and may explain significantly enhanced heavy polyribosome formation, as well as a higher efficiency of protein synthesis in plants treated with gibberellic acid.
Studies on the Structure and Function of Chick‐Oviduct Chromatin
Authors: STRÄTLING Wolf H. , VAN Nguyen T. , O'MALLEY Bert W.
Published: March 2005

Chick oviduct chromatin was separated into a ribonucleoprotein fraction and two chromatin fractions (early and late eluting). We utilized a gentle procedure in which moderately hydrated chromatin was subjected to chromatography on a weak ionic‐exchange resin (ECTHAM‐cellulose) eluted with a combined pH‐salt gradient. Chemical analysis of the early (fraction I) and late (fraction II) eluting fractions revealed that their histones were identical and their nonhistone proteins were markedly different. Control experiments showed that these differences were not due to protein rearrangements during chromatin preparation and/or fractionation.

The physical properties of fraction I and II differed in certain aspects. The aggregation response of fraction I to increasing concentrations of monovalent cations was five times lower than that of fraction II but the aggregation response to divalent cations was identical. Thermal denaturation assays of DNAs isolated from fractions I and II revealed identical derivative profiles of hyperchromicity vs temperature, thereby indicating similar base composition in the two fractions. Circular dichroism spectra of the purified DNAs isolated from both fractions showed identical B‐type conformations. However, DNA renaturation kinetics analyzed by computer technique indicated that fraction I DNA, contained less than half the amount of highly repetitive sequences as compared to either unfractionated chromatin or fraction II. Circular dichroism spectra of fraction I and II chromatins (at room temperature) showed significant differences in a wavelength region were only DNA is optically active (i.e: 255–320 nm). These results indicated that the DNA complexed to proteins in fraction II assumed a more C‐type conformation than the DNA in fraction I. The differences in the circular dichroism spectra could not be accounted for by differences in the RNAs or protein chromophores contained in fraction I and fraction II. When the circular dichroism spectra of fraction I and II were recorded at 55°C, the differences between the two fractions were abolished. These results were interpreted to mean that the differences in the DNA conformations found in fractions I and II were due to the differences in their nonhistone proteins. These proteins were effective in maintaining DNA conformation differences only when they were in their native form but not when heated to 55°C.

Comparison of the sedimentation coefficients of fractions I and II with their calculated molecular weights suggested a more extended structure in fraction I as compared to a more compact structure in fraction II. Only small differences were observed between fraction I and fraction II with respect to either buoyant density analysis in a metrizamide gradient or in the number of phosphate charges accessible to polylysine.
Dinitrophenol, Dicoumarol and Pentachlorophenol as Inhibitors and Parasite Substrates in the ATP Phosphoribosyltransferase Reaction
Authors: DALL‐LARSEN Torbjørn , KRYVI HåBkon , KLUNGSøYR Leiv
Published: March 2005

Adenosine‐triphosphate phosphoribosyltransferase from Escherichia coli is inhibited by dicoumarol and pentachlorophenol in competition with ATP. Ki was approximately 60 μM for dicoumarol and 50 μM for pentachlorophenol. Carbonylcyanide m‐chlorophenylhydrazone did not seem to have any kinetic effect. Dicoumarol is bound to the extent of 6 sites per enzyme hexamer with a dissociation constant Kd of 50 μM. Dicoumarol and pentachlorophenol partly prevent the binding of ATP and AMP to the transferase. The reversed reaction is inhibited by dicoumarol and pentachlorophenol without changes in [S]0.5 for phosphoribosyladenosine triphosphate. Dicumarol, dinitrophcnol and pentachlorophenol diminish the yield of phosphoribosyladenosine triphosphate in the transferase reaction apparently by acting as parasite substrates; carbonylcyanide m‐chlorophenylhydrazone had no effect.
Valyl‐tRNA, Isoleucyl‐tRNA and Tyrosyl‐tRNA Synthetase from Baker's Yeast
Authors: FREIST Wolfgang , VON DER HAAR Friedrich , FAULHAMMER Heinz , CRAMER Friedrich
Published: March 2005

Nineteen analogs of ATP have been tested in the aminoacylation reaction of valyl‐tRNA, isoleucyl‐tRNA and tyrosyl‐tRNA synthetases from baker's yeast. Four compounds are substrates for valyl‐tRNA and two for isoleucyl‐tRNA synthetase, but there is no modified substrate for the tyrosyl‐tRNA synthetase. There is one inhibitor for valyl‐tRNA synthetase, eight compounds inhibit isoleucyl‐tRNA synthetase and two compounds inhibit tyrosyl‐tRNA synthetase. Their Km and Kt and V values have been determined.

The substrate specificity shows that positions 2, 6, 7, 8, 9, 2′, and 3′ of ATP are important for catalytic action of these aminoacyl‐tRNA synthetases.
Activity and Subcellular Distribution of Protein Kinase Dependent on Adenosine 3′: 5′‐Monophosphate in Liver from Normal and Adrenalectomized Rats
Authors: ROUSSEAU Guy G. , MARTIAL Joseph , DE VISSCHER Michel
Published: March 2005

We have examined whether glucocorticoids control the activity and (or) the subcellular distribution of protein kinase dependent on cyclic'AMP (adenosine 3′: 5′‐monophosphate), since they influence, cyclic‐AMP‐dependent responses to other hormones.

Protein kinase activity was determined in rat liver homogenates and subcellular fractions, nuclear, large granular, microsomal and supernatant obtained by differential sedimentation in 0.25 M sucrose. 63% of the tissue protein kinase activity detected in absence of cyclic AMP reside in the particulate fractions. Upon addition of exogenous cyclic AMP, protein kinase activity is stimulated 1.8, 1.2, 1.2 and 4.5‐fold in nuclear, large granular, microsomal and supernatant fractions, respectively. Under these conditions, 66% of tissue activity are found in the supernatant fraction. The activity sensitive to exogenous cyclic AMP resolves into a major (84%) cytosoluble and a minor (16%) nucleomicrosomal component. The latter activity resists elution with isotonic saline and is increased in the presence of Triton X‐100.

Three groups of rats were studied: control and adrenalectomized with or without cortisol treatment. In whole liver homogenates, both protein kinase activity detected in absence of exogenous cyclic AMP andsensitivity of the enzyme to cyclic AMP were comparable in all groups. Moreover, the distribution patterns of protein kinase activity among the fractions were essentially the same in all groups of animals, whether or not particles had been treated with Triton X‐100.

Finally, in cell‐free experiments, glucocorticoids alone or in combination with their intracellular receptor did not modify protein kinase activity of rat liver.

Thus the results reported do not support the possibility that glucocorticoids influence cyclicAMP‐dependent protein kinase in rat liver. Yet, this study provides data, not available before, on subcellular distribution of this enzyme in rat liver.
Multifunctional Enzyme, Bisphosphoglyceromutase/2,3‐Bisphosphoglycerate Phosphatase/Phosphoglyceromutase, from Human Erythrocytes
Authors: IKURA Koji , SASAKI Ryuzo , NARITA Hiroshi , SUGIMOTO Etsuro , CHIBA Hideo
Published: March 2005

Bisphosphoglyceromutase and 2,3‐bisphosphoglycerate phosphatase activities responsible for 2,3‐bisphosphoglycerate metabolism in human red cells are displayed by the same enzyme protein which has phosphoglyceromutase activity [Sasaki, R., et al. (1975) Eur. J. Biochem. 50, 581–593]. This enzyme was subjected to chemical modification by trinitrobenzenesulfonate.

The three enzyme activities were inactivated by trinitrobenzenesulfonate at the same rate. The sulfhydryl content of the enzyme was unchanged during trinitrophenylation, indicating that derivatization was through the amino group. Trinitrophenylation of about one amino group per mole of the enzyme resulted in complete loss of the three activities.

Both 2,3‐bisphosphoglycerate and 1,3‐bisphosphoglycerate inhibited trinitrophenylation and effectively protected the enzyme from inactivation. Although monophosphoglycerates did not show any protective effect at concentrations which should be adequate based upon their kinetic constants, they were protective at higher concentrations.

Inactivation by trinitrophenylation was an apparent first‐order reaction. The dissociation constant of the enzyme 2,3‐bisphosphoglycerate complex was determined by analyzing the first‐order reaction on the assumption that the protective effect of 2,3‐bisphosphoglycerate was due to competition with trinitrobenzenesulfonate. The dissociation constant was in good agreement with kinetic constants of 2,3‐bisphosphoglycerate in the enzyme reactions, which indicated that 2,3‐bisphosphoglycerate did indeed exert its protective effect through competition with trinitrobenzenesulfonate for an amino group of the enzyme. The protective effect of monophosphoglycerates could be rationalized with kinetic evidence that 2‐phosphoglycerate at high concentrations interacts with the 2,3‐bisphosphoglycerate binding site.

These results indicate that the enzyme exhibits the three enzyme activities at a common active site at which one amino group essential for binding of bisphosphoglycerates is located. Based on the multifunctional properties of this enzyme, a possible mechanism was discussed for regulation of 2,3‐bisphosphoglycerate metabolism in human red.
Subunit Structure and Multifunctional Properties of Yeast Phosphoglyceromutase
Authors: SASAKI Ryuzo , UTSUMI Shigeru , SUGIMOTO Etsuro , CHIBA Hideo
Published: March 2005

A new and efficient method for preparation of pure phosphoglyceromutase from baker's yeast (Saccharomyces cerevisiae) is described. Proteolytic alterations of the enzyme during extraction can be minimized by grinding the dried yeast with aluminium oxide at low temperature.

Yeast phosphoglyceromutase contains four highly similar, probably identical subunits of molecular weight 28000, a conclusion based on the following observations. Polyacrylamide gel electrophoresis containing dodecylsulphate or urea gives a single band, indicating that the enzyme is composed of four subunits similar in their molecular weight and net charge. Cyanogen bromide cleavage and, tryptic digestion of the enzyme yield the number of peptides expected for identical subunits from the amino acid composition analysis.

The purred phosphoglyceromutase preparation has bisphosphoglyceromutase activity synthesizing‐2,3‐bisphosphoglycerate from 1,3‐bisphosphoglycerate and 3‐phosphoglycerate. It has been reported that yeast phosphoglyceromutase catalyzes the hydrolysis of 2,3‐bisphosphoglycerate at the same active site which catalyzes the phosphoglyceromutase reaction [Sasaki, R. et al. (1971) Biochim. Biophys. Acta, 227, 584–594, 595–607]. Immunological studies and chemical modification experiments indicate that bisphosphoglyceromutase activity also is due to the phosphoglyceromutase protein and involves amino groups which have been shown to be essential for the other two activities.