bluuebunny/crossref_metadata_2025 huggingface dataset:
Further Studies on the Rotation of Globular Proteins in Polymer Solutions
Authors: ÖBrink Björn , Laurent Torvard C.
Published: March 2005

The rotational diffusion of pyrene‐butyric‐labelled human serum albumin and dansyl‐labelled bovine fetuin, β‐lactoglobulin, α‐lactalbumin and pancreas ribonuclease has been studied by fluorescence polarization in concentrated dextran solutions (up to 0.4 g/ml). The relaxation times of the proteins increased only moderately even at very high polysaccharide concentrations. The results confirmed the conclusions drawn from studies on dansyl‐labelled serum albumin that the friction exerted by the polysaccharide on the rotation of a protein is low. The very strong inhibition of translational diffusion in polymer media can therefore be ascribed to steric hindrance.

The effect of dextran on the rotational diffusion of different proteins varied and the reason for this is discussed. The measurements indicate that conformational changes may occur in some proteins at high polymer concentrations.
Composition of the Photosynthetic Apparatus of Normal Barley Leaves and a Mutant Lacking Chlorophyll b
Authors: Thornber J. Philip , Highkin Harry R.
Published: March 2005

Comparison of the composition of the energy‐trapping apparatus of normal barley photo‐synthetic membranes and those lacking chlorophyll b shows that not only is chlorophyll b absent from the mutant, but all constituents (chlorophyll a, chlorophyll b, carotenoids and the protein moiety) of the major chlorophyll‐protein complex of normal higher plant chloroplast membranes are also missing; other chlorophyll‐containing components, as far as can be ascertained, are essentially unaffected by the mutation. The nuclear gene which codes for the protein moiety of this complex is suggested as a possible site of the mutation in this barley mutant. This chlorophyll α/β‐protein complex which accounts for such a large proportion of the chlorophyll and protein of the photosynthetic apparatus of higher plants is thus not essential for a plant to grow photosynthetically, and therefore a new name, light‐harvesting chlorophyll‐protein, is proposed for the complex, which was formerly termed the photosystem II chlorophyll‐protein. The biosynthetic machinery in the mutant is unable to compensate for the loss of the light‐harvesting chlorophyll‐protein by adding extra chlorophylls into alternative sites in the membrane, thus the chlorophyll/P700 ratio in the mutant is two‐thirds that of the normal plant. The light‐harvesting chlorophyll‐protein is the major location for chlorophyll b in higher plant membranes. The function of the complex is thought to be analogous to that of the algal biliproteins, i.e. light energy absorbed by the complex is fed preferentially, but not exclusively, to the photosystem II reaction‐center or trap. It is reasoned that the complex exerts a strong influence on the organization of photosynthetic lamellae in higher plant chloroplasts by maintaining lamellae in contact with each other.
The Heterogeneity of Mouse‐Chromatin Nonhistone Proteins as Evidenced by Two‐Dimensional Polyacrylamide‐Gel Electrophoresis and Ion‐Exchange Chromatography
Authors: Macgillivray Alexander J. , Wood David RICK
Published: March 2005

A two‐dimensional gel electrophoresis procedure employing a combination of isoelectric focusing and dodecylsulfate electrophoresis has been used to analyse the components of 32P‐labelled nonhistone protein fractions obtained by the chromatography of salt‐urea dissociated chromatins on hydroxyapatite.

In this way the nonhistone proteins eluted from hydroxyapatite by 0.05 M phosphate (fraction H2) have been found to consist of a heterogeneous mixture of components of a molecular weight range of 15000 to 200000 and to have isoelectric points between pH 4.5 and 9. The components of the fraction eluted from hydroxyapatite by 0.2 M phosphate (fraction H3) appeared to consist of a smaller group of proteins with a molecular weight range similar to that of the H2 proteins but whose isoelectric points lay between pH 2 and 6. Both fractions consisted of a mixture of phoshorylated and nonphosphorylated protein species.

Comparison of the components of H2 and H3 protein fractions by this electrophoretic procedure showed that many of the phosphorylated and nonphosphorylated proteins were common to mouse liver, kidney and brain chromatins. Only a few species were found to be tissue specific.

It was found that chromatography on QAE‐Sephadex did not follow the same separation pattern as that obtained by isoelectric focusing, suggesting that the fractionation on this ion‐exchange material is not entirely dependent on the overall charge of the polypeptide.

Preparation of individual nonhistone protein species has been attempted using this chromatographic procedure in conjunction with gel filtration in guanidine hydrochloride.
Three Forms of the 5.8‐S Ribosomal RNA Species in Saccharomyces cerevisiae
Authors: Rubin Gerald M.
Published: March 2005

The existence of three stable forms of the 5.8‐S ribosomal RNA species in Saccharomyces cerevisiae has been demonstrated. The major form constitutes approximately 90% of the total 5.8‐S RNA and the two minor forms each constitute approximately 5%. The nucleotide sequence of the major form begins pA‐A‐A‐Cp. The sequences of the minor forms begin pU‐A‐U‐U‐A‐A‐A‐A‐A Cp and pA‐U‐A‐U‐U‐A‐A‐A‐A‐A‐Cp. The remainders of the sequence of all three forms appear to be identical.
Kinetic Studies of Levansucrase of Bacillus subtilis
Authors: Chambert Régis , Treboul Geneviève , Dedonder Raymond
Published: March 2005

The mechanism of action of levansucrase of Bacillus subtilis was investigated by initial velocity measurements of reactions catalyzed by this enzyme and initial velocity of isotopic exchange at equilibrium. Comparison of experimental results with theoretical results derived from various postulated mechanisms, supports a “ping‐pong” mechanism, involving the intermediate participation of a fructosyl‐enzyme. Rate constants for each step of this sequential mechanism were estimated.
Sterols in Porphyridium Species
Authors: Beastall Graham H. , Tyndall Alison M. , Rees Huw H. , Goodwin Trevor W.
Published: March 2005

The sterol components of the Rhodophyceae, Porphyridium cruentum and Porphyridium aerugineum have been investigated. The 4α‐methyl sterol fraction of P. cruentum contained two novel sterols, 4α‐methyl‐5α‐cholesta‐8,22‐dien‐3β‐ol and 4α,24ζ‐dimethyl‐5α‐cholesta‐8,22‐dien‐3β‐ol, together with 4α‐methyl‐5α‐cholest‐8‐en‐3β‐ol, 4α,24ζ‐dimethyl‐5α‐cholest‐8‐en‐3β‐ol and obtusifoliol. It is particularly significant that the two new sterols are the first reported 4‐methyl sterols to contain δ22 unsaturation. Broken cell preparations of both organisms cyclized squalene‐2,3‐oxide exclusively to cycloartenol. A possible biosynthetic pathway based on the structures of all the sterols isolated from P. cruentum is proposed, which allows for the introduction of δ22 unsaturation at the 4α‐methyl level.
Affinity Chromatography on Immobilised Adenosine 5′‐monophosphate
Authors: Lowe Christopher R. , Harvey Michael J. , Dean Peter D. G.
Published: March 2005

The effect of pH on the binding of lactate dehydrogenase to 6‐aminohexanoyl‐NAD+‐Sepharose and N6‐(6‐aminohexyl)‐5′‐AMP‐Sepharose has been examined and related to its behaviour in free solution. The response to pH was dependent upon the nature of the inert matrix and the coupling procedure used. The difference in interaction of several enzymes with N6‐(6‐aminohexyl)‐5′‐AMP‐Sepharose in response to pH has been investigated. Complex enzyme mixtures have been resolved on N6‐(6‐aminohexyl)‐5′‐AMP‐Sepharose with pH gradients.
A Sequence of 83 Nucleotides Containing the Replicase Cistron Ribosome Binding Site of Phage Qβ RNA
Authors: Porter Alan G. , Hindley John , Billeter Martin A.
Published: March 2005

70‐S initiation complexes of Escherichia coli ribosomes and 32P‐labelled Qβ RNA were digested with T1 ribonuclease at 0°C, and the ribosome‐protected Qβ initiator fragments resolved by poly‐acrylamide gel electrophoresis. The sequence of one of the fragments (83 nucleotides long) has been determined. It begins with the sequence of 32 nucleotides already determined for the ribosome binding site of the replicase cistron. The sequence of the remaining 51 nucleotides establishes the order of a further 17 amino acids in the phage‐coded replicase subunit. Seven out of the first 11 amino acids in the replicase subunits of phages MS2 and Qβ are identical.
Sensitivity to Ampicillin and Cephalothin of Enzymes Involved in Wall Peptide Crosslinking in Escherichia coli K12, Strain 44
Authors: Nguyen‐Distèche Martine , Pollock Jerry J. , Ghuysen Jean‐Marie , Puig Juan , Reynolds Peter , Perkins Harold R. , Coyette Jacques , Salton Milton R. J.
Published: March 2005

After extraction of the membranes of Escherichia coli K12 strain 44 by Brij‐36T, each of the four enzyme activities (natural transpeptidase, unnatural transpeptidase, carboxypeptidase and endopeptidase) of the wall peptide crosslinking system, occurs in two forms characterized by large differences in their sensitivity to ampicillin (but much smaller differences in their sensitivity to cephalothin). The fractionation of the enzyme activities into two groups of low and high sensitivity to ampicillin is achieved essentially by chromatography of the membrane extract on DEAE‐cellulose.
Studies of Glutamate Dehydrogenase
Authors: Krause Jobst , Bühner Manfred , Sund Horst
Published: March 2005

The binding of NADH and NADPH to beef liver glutamate dehydrogenase has been investigated using fluorescence titration and the preparative as well as the analytical ultracentrifuge over a wide range of protein and coenzyme concentrations. The glutamate dehydrogenase oligomer (molecular weight 336000, six polypeptide chains) binds independent of enzyme concentration a total of twelve molecules of each reduced coenzyme, NADH and NADPH. The first six binding sites, the “active sites”, bind NADH and NADPH in the same manner, whereas the weaker binding to the other six sites, the “nonactive sites”, is remarkably different for both coenzymes: probably due to the steric hindrance or electrostatic repulsion the affinity of the enzyme for NADPH is about 10 times lower than that for NADH. This result explains the different kinetic behaviour of the two coenzymes. From the results obtained at different protein concentrations it is concluded that the coenzyme binding sites and the association sites are located at different parts of the enzyme surface.

A procedure is described for three‐parameter fitting of titration curves using a programmable desk calculator with on‐line x‐y recorder.
The Bacterial Nitrate Reductases
Authors: Forget Pierre
Published: March 2005

Nitrate reductase of Escherichia coli has been solubilized from particle fractions by a double treatment: first an akali‐acetone precipitation and then a solubilization in a buffered sodium deoxycholate. The enzyme has been purified 50‐fold with a yield of 1 to 2%. Polyacrylamide‐gel electrophoresis and ultracentrifugation show the preparation to be nearly homogeneous. The protein has a molecular weight of 320000 and an iso‐electric point at pH 4.2. The absorbance which increases continuously from 600 to 280 nm does not reveal the presence of a heme or a flavin group but the spectrum resembles that of some bacterial ferroproteins. The estimation of metals indicates 1.5 atoms Mo and 20 atoms Fe per mole. Approximately one labile sulfide is found per iron atom. It is likely that nitrate reductase A is an iron‐sulfur protein containing molybdenum. The purified protein uses as substrates NO3‐ and CIO3‐ and as electron donors reduced benzyl‐ and methyl‐viologens, FMNH2 and FADH2 but not NADH or NADPH. It should be pointed out that the solubilization does not modify the enzymatic properties of nitrate reductase. CN‐ and N3‐ are strong inhibitors. Azide is a competitive inhibitor and the nitrate reductase affinity for this inhibitor is 1000 times greater than for nitrate. The type of inhibition observed and the metal chelating nature of the inhibitor suggest that a metal, Fe or Mo, or both, play a role in the formation of enzyme‐substrate complex.
Phenol Extraction of Heavy Rapidly‐Labeled Nuclear RNA from Chick Embryos
Authors: Olszanska Bozenna , Grabczewska Ewa , Lassota Zofia
Published: March 2005

Heavy rapidly‐labeled RNA from chick embryo nuclei separates on electrophoresis in 2.4% polyacrylamide gel into at least four fractions with the mobilities of rRNA precursors, and a fraction of “giant” RNA remaining at the start.

The heavy fractions with mobilities of rRNA precursors pass into solution without heating during phenol treatment at pH 8.3 or during phenol‐1.6% dodecylsulphate treatment at pH 5.

The “giant” RNA can be partially solubilized without heating by phenol‐1.6% dodecylsulphate treatment at pH 5 but the extraction becomes efficient only on heating to 65°C or on raising the pH to 8.3.

Precautions are discussed to prevent the degradation of heavy RNA at elevated pH and the aggregation of rRNA at elevated temperature during phenol extraction.
Transient Kinetics of Benzaldehyde Formation during the Catalytic Action of Pig‐Plasma Benzylamine Oxidase
Authors: LindstrÖM Anders , Olsson Bengt , Pettersson Gösta
Published: March 2005

The kinetics of benzaldehyde formation during the oxidative deamination of benzylamine by pig plasma benzylamine oxidase have been studied by stopped‐flow techniques. In the presence of saturating amounts of benzylamine and 0.01–0.25 mM oxygen, the reaction exhibits a transient first‐order burst of benzaldehyde formation approximating to one mole of product released per mole of enzyme. These observations confirm that benzylamine oxidase contains only one active site, and provide clear evidence that benzaldehyde is formed prior to the rate‐limiting interaction between oxygen and the substrate‐reduced from of the enzyme.

Apparent first‐order rate constants for the transient burst are linearly dependent on the benzylamine concentration, and agree quantitatively with those determined previously for reduction of the enzyme by substrate. Hence it may be concluded that benzaldehyde formation and reduction of the enzyme are kinetically indistinguishable by the stopped‐flow technique, both processes being rapid and governed by the same rate‐limiting second‐order interaction between enzyme and substrate.
Separation and Characterisation of Subunits of Histidine Decarboxylase from Micrococcus sp.n.
Authors: Prozorovski Vladimir , Jörnvall Hans
Published: March 2005

Histidine decarboxylase from Micrococcus sp.n. was carboxymethylated and two types of subunits, I and II, were separated in excellent yield by exclusion chromatography on Sephadex G‐100 in 8 M urea. Total compositions, number of tryptic peptides and N‐terminal groups of the subunits were analyzed. The larger subunit I is composed of all amino acids commonly found in proteins and has a blocked N‐terminal residue, whereas the smaller subunit II does not contain proline, histidine or cysteine and has N‐terminal methionine. The number of tryptic peptides detected in peptide mapping experiments of subunit I is about 27 and that of subunit II is about 15. These figures are in good agreement with the total number of lysine and arginine residues determined by amino acid analysis, if subunit I contains about 270 residues, corresponding to a molecular weight of 29000, and subunit II about 70 residues, corresponding to a molecular weight of 7000. It is concluded that histidine decarboxylase contains two apparently homogeneous but completely different polypeptide chains and that the native enzyme of molecular weight 110000 is a hexamer with three subunits of each type.
Participation of Soluble Phophatidylserine Synthetase in Phosphatidylethanolamine Biosynthesis in Escherichia coli Membrane
Authors: Ishinaga Masataka , Kito Makoto
Published: March 2005

Phosphatidylserine synthetase occurs in a soluble and a ribosome‐bound form in Escherichia coli. The solble enzyme of E. coli B was partially purified by DEAE‐Sephadex chromatography. The ribosomal enzyme was obtained by differential centrifugation. Two types of the enzyme had the same Km (150 μM) for l‐serine. The soluble enzyme was required for the biosynthesis of phosphatidylethanolamine from phosphatidic acid via CDP‐diglyceride in the membrane. The ribosomal enzyme was less effective to the phosphatidylethanolamine formation than the soluble enzyme. By the sucrose‐density‐gradient centrifugation method the soluble enzyme was proved to be loosely associated with the membrane.
Thermodynamics of the Helix‐Coil Transition of (dG‐dC) Oligomers
Authors: Pohl Fritz M.
Published: March 2005

The dissociation of short double‐helical DNA with the alternating sequence (dG‐dC)n. (dG‐dC)n into the corresponding single strands was studied as a function of chain length, oligomer concentration and temperature in 1.0M NaCl, pH 7.2 by hypochromicity and equilibrium centrifugation. The melting curves are analyzed according to an “all‐or‐none” model and the following thermodynamic parameters were deduced.

The change in free energy for formation of a dG · dC base pair adjacent to another one, ΔG0s, =−2.2 kcal/mol at 37°C; the corresponding change in enthaply, ΔH0s, =−11.1 ± 0.5kcal/mol and in entropy, ΔS0s, =−27.9 ± 1 cal · K−1· mol base pair−1. The stability constant, s, is 104 ± 5 at 25°C. The formation of the first base pair from two single strands involves a nucleation parameter, β, = 1.6 10−4 M−1 at 25°C, which is similar to the corresponding values determined for different RNA oligomers. The enthaply change associated with the formation of the first base pair, −ΔH0βs, =+3±5 kcal/mol which is very close to zero.

These thermodynamic parameters are compared with those for the dA · dT base pair. At physiological conditions, it is estimated that the stability of one dG · dC pair is very close to that of two dA.dT pairs. The data imply that the opening of a unique sequence of DNA in a cell is a very improbable event.
Guanidination of Lysine‐15 in the Active Site of the Basic Pancreatic Trypsin Inhibitor
Authors: Vincent Jean‐Pierre , Schweitz Hugues , Lazdunski Michel
Published: March 2005

Selective modification of lysine‐15 at the active site of the basic pancreatic trypsin inhibitor strongly alters its association with trypsin. Ka, the equilibrium constant for the association, and kd, the first‐order rate constant for the dissociation are 20nM−1 and 1.0×10−4s−1 for the trypsin. · 15‐guanidinated‐inhibitor complex as compared to 16 pM−1 and 6.6×10−8s−1 for the trypsin · native‐inhibitor complex. Guanidination of lysine residues (Lys26, Lys41 and Lys46) outside the active site of the inhibitor does not affect the stability of the binding with trypsin.

The selective guanidination of lysine‐15 in the pancreatic inhibitor does not alters its association with chymotrypsin.

Reduction and carboxamidomethylation of the disulfide bridge Cys14‐Cys38 in the selectively guanidinated inhibitor decreases Ka by a factor of 4.6 and increases kd by a factor of 55. The same chemical modification of the Cys14‐Cys38 bridge in the non‐guanidinated inhibitor decreases Ka by a factor of 2700 and increases kd by a factor of 330.
A Kinetic Study of the Isozymes Determined by the Three Human Phosphoglucomutase Loci PGM1, PGM2 and PGM3
Authors: Quick Christine B. , Fisher Rachel A. , Harris Harry
Published: March 2005

The isozyme products (PGM1, PGM2 and PGM3) of the three human phosphoglucomutase genes (PGM1, PGM2 and PGM3) were separated and compared as phosphoglucomutases, phosphoribomutases and glucose 1,6‐bisphosphatases.

The Km values for glucose 1‐phosphate (Glc‐1‐P) were estimated at various glucose 1,6‐bisphosphate concentrations. When the glucose 1,6‐bisphosphate concentration was less than 10 μM, PGM1 was seen to be much more effective at catalysing the phosphoglucomutase reaction than either PGM2 or PGM3. The effect of the coenzyme (glucose 1,6‐bisphosphate) concentration was studied and was found to affect the Km for Glc‐1‐P of PGM2. As the glucose 1,6‐bisphosphate concentration was increased the Km decreased; this effect was particularly noticeable over the concentration range 100 μM to 400 μM where a small alteration in glucose 1,6‐bisphosphate concentration produced a marked fall in the value of the Km for Glc‐1‐P of PGM2. The concentration of glucose 1,6‐bisphosphate in a range of human tissues was estimated and was found to be within the range 10 μM to 600 μM in most tissues.

PGM2 was found to be more effective at catalysing the phosphoribomutase reaction than PGM1. The Km for ribose 1‐phosphate for neither the PGM1, nor the PGM2 isoenzymes was affected by the concentration of glucose 1,6‐bisphosphate. The possibility of glucose 1,6‐bisphosphate acting on PGM2 and thereby directing whether it functions as a phosphoribomutase or a phosphoglucomutase has been discussed. Ribose 1‐phosphate inhibited the phosphoglucomutase activity of the products of all three loci. The ribose 1‐phosphate appeared, from Dixon plots, to be competing with glucose 1‐phosphate.

The effect of 2,3‐bisphosphoglycerate on the phosphoglucomutase activity of the PGM1 and PGM2 isozymes was examined; this compound was found to inhibit by competing with glucose 1,6‐bisphosphate in both cases.

The ability of the isoenzymes of all three genes to catalyse a glucose 1,6‐bisphosphatase reaction has been reported. The Km values for glucose 1,6‐bisphosphate for this reaction were estimated as being of the order of 1000 μM.
Spectral Properties of Rat Adrenal‐Mitochondrial Cytochrome P‐450
Authors: Jefcoate Colin R. , Simpson Evan R. , Boyd George S.
Published: March 2005

Difference spectra produced by the binding of steroids to rat adrenal‐mitochondrial cytochrome P‐450 indicate distinct interactions with, respectively, pregnenolone, 11‐deoxycorti costerone and 25‐hydroxycholesterol. These so‐called type I (deoxycorticosterone and hydroxy‐cholesterol) or type II (pregnenolone) spectral changes have been compared in bovine adrenalcortex mitochondria and in adrenal mitochondria from rats. In the latter species the animals were cortex mitochondria and in adrenal mitochondria from rats. In the latter species the animals were subjected to one of three pretreatments; rats were either given an ether stress to increase the plasma concentration of adrenocorticotrophic hormone or were injected with the protein‐synthesis inhibitor cycloheximide, or kept in a quiescent state. These experiments substantiate the theory that distinct P‐450 cytochromes are involved in cholesterol side‐chain cleavage (P‐450SCC) and steroid 11β‐hydroxylation (P‐45011β).

A pH‐sensitive cholesterol complex of cytochrome P‐450SCC was quantitated by means of the pregnenolone difference spectrum. Adrenal mitochondria from “stressed” rats had about twice as much of this cholesterol complex of cytochrome P‐450SCC as adrenal mitochondria from rats kept quiescent or subjected to cycloheximide treatment. The pH‐dependence of the cytochrome P‐450SCC‐cholesterol complexs was unaffected by the pretreatment of the animals.

The type‐I spectral change produced by 25‐hydroxycholesterol was unaffected by stress or by changes in pH but was almost removed by sonication. The binding of 25‐hydroxycholesterol to adrenal mitochondrial cytochrome P‐450 was competitively inhibited by pregnenolone but was unaffected by deoxycorticosterone. The 25‐hydroxycholesterol difference spectrum may detect a distinct form of cytochrome P‐450SCC.

Sonication of adrenal mitochondria from quiescent and cycloheximide‐treated rats increased both the absorbance change produced by pregnenolone and the cholesterol side‐chain cleavage activity to values approaching those found in mitochondria from stressed rats. The rate of side‐chain cleavage of added 25‐hydroxycholesterol was greater than that observed with endogenous cholesterol and this rate was unaffected by prior stress to the rats. It is suggested that the low cholesterol side‐chain cleavage activity in adrenal mitochondria from quiescent rats and rats given cycloheximide may be due to the existence within the mitochondrial membrane of specific “restraints” upon the interaction between a certain fraction or pool of cholesterol and cytochrome P‐450SCC. The availability (or restraint) of this choleterol pool may be altered by the action of adrenocorticotrophic hormone, and mediated by a labile protein factor.
Substrate Stereochemistry of 3‐Hydroxy‐3‐Methylglutaryl‐Coenzyme A Synthase
Authors: Cornforth John Warcup , Phillips Gareth T. , Messner Burkhard , Eggerer Hermann
Published: March 2005

Synthetic specimens ofR‐[2H1,3H1]acetate and ofS‐[2H1,3H1]acetate were converted into coenzyme A thiolesters.

Each of these thiolesters was condensed with acetoacetyl‐CoA on 3‐hydroxy‐3‐methyl‐glutaryl‐CoA synthase.

The products, which were found to be hydroxymethylglutaryl‐phosphopantetheines owing to a secondary cleavage of the coenzyme A moieties, were reduced chemically to 3Rmevalonates.

Each mevalonate specimen, after mixing with [2‐14C]mevalonate, was converted into squalene and cholesterol by a rat liver preparation.

Each cholesterol specimen was converted into androst‐1,4‐diene‐3,17‐dione by incubation with a strain ofMycobacterium phleiin the presence of an inhibitor.

When the procedures 4 and 5 were carried out with authentic specimens of 2R‐ and 2S‐[2‐3H1]mevalonate, it was found that the androstadienedione had lost nearly all tritium derived from the 2S‐mevalonate but had retained nearly all tritium (relative to14C) derived from the 2R‐mevalonate.

The androstadienedione derived, by procedures 1–5, fromR‐acetate showed a loss of more than half of its tritium (relative to14C). The androstadienedione derived by the same procedure fromS‐acetate lost less than half of its tritium.

It is concluded that the reaction on hydroxymethylglutaryl‐CoA synthase is stereospecific and is associated with an intramolecular hydrogen isotope effect. If this effect is normal (kH/kD>1) then the condensation proceeds with inversion of configuration at the methyl group.
Secondary Isotope Effect in the Phosphorylase Reaction
Authors: Firsov Leonid M. , Bogacheva Tatjana I. , Bresler Simeon E.
Published: March 2005

The mechanism of action of phosphorylase B was studied by means of both α deuterium and α‐tritium secondary kinetic isotope effects of the enzymatic reaction. Two pairs of labelled substrates were synthesized : [1‐3H]glucose 1‐phosphate, [U‐14C]glucose 1‐phosphate and [1‐14C, 1‐2H]glucose‐1‐phosphate with [6‐3H]glucose 1‐phosphate. The measurements showed that the isotope effects of the enzymatic synthesis of glycogen as well as of the reverse reaction of glycogen phosphorolysis were equal to zero.

The secondary α‐tritium isotope effect of the acid hydrolysis of glucose‐1‐phosphate was estimated. The ratio KH/K3H in this case was found to be 1.21.

These data are consistent with the mechanism of direct displacement at the C‐1 atom of glucose and the formation of an intermediate enzyme · glucose‐1‐phosphate complex depending on the presence of glycogen.
Sub‐Ribosomal Particles of Cultured Chinese‐Hamster Cells
Authors: Enger M. Duane , Walters Ronald A. , Hampel Arnold E. , Campbell Evelyn W. , Hanners John L.
Published: March 2005

Low‐molecular‐weight methylated RNAs exist in the cytoplasm of mechanically or chemically disrupted cultured Chinese hamster ovary cells as nucleoproteins sedimenting in the region of 10—30 S. A differential population of tRNA, representing about 4% tRNA of the cells, is also recovered from this region as is a differential population of aminoacyl synthetases The relationship of these RNA populations to each other and protein moieties of the 10—30‐S sub‐ribosomal region is the subject of this investigation. As regards preparation of the sub‐ribosomal particles, it is found that maximal yield of 10—30‐S material with retention of enzyme activity is achieved when cytoplasm is prepared using the non‐ionic detergent nonidet P‐40. The ratio of protein‐to‐RNA in this region, which contains 2% protein of the cells, is about 17:1. Concentration and resedimentation of fractions within the 10—30‐S region resolve two peaks of approximetaly 18 S and 21 S comprised mainly of protein and a primarily nucleic acid peak of about 20 S which contains most of the tRNA. Low‐molecular‐weight methylated RNAs are recovered from the entire 18—20‐S region with the exception of the largest species, which is preferentially recovered from the 21‐S region. Isopycnic cesium chloride analysis of the 18—20‐S region after formaldehyde fixation resolves most of the RNA from the majority of the protein. The particulate RNA bands at 1.32—1.38g/ml before and at >1.7g/ml after pronase treatment. Sedimentation values of the protein particles are unaffected by ribonuclease or by prolonged storage at‐15 °C or 4 °C. However, tRNA is released as free tRNA as a function of time at 4 °C without concurrent degradation of the 18‐S and 21‐S protein particles and without release of low‐molecular‐weight methylated RNA. Thus, it appears that most of the particle tRNA is unassociated with the major sub‐ribosomal protein particles or with the particles containing low‐molecular‐weight methylated RNA.
On the Peptidoglycan of the Cell Walls of Pseudomonas aeruginosa
Authors: Heilmann Hans‐Dietrich
Published: March 2005

The peptide side chains of the peptidoglycan of the Pseudomonas aeruginosa cell envelope have the sequence H2N‐l‐Ala‐d‐Glu‐A2pm‐d‐Ala‐COOH. The d‐glutamate is linked to the meso‐2, 5‐diaminopimelate (A2pm) by a peptide bond formed by the C‐5 carboxyl group of d‐glutamate. The C‐1 carboxyl group of the glutamate does not occur in the amidated state in the cell wall. The configurations of alanine, glutamate, and 2, 5‐diaminopimelate have been confirmed.
5′‐Ends of Ribosomal and Ribosomal Precursor RNAs from Xenopus laevis
Authors: Slack Jonathan M. W. , Loening Ulrich E.
Published: March 2005

The 5′‐ends of 18‐S, 28‐S and 40‐S RNA from Xenopus laevis have been studied by two techniques. The first involves fractionation of alkaline hydrolysates on columns of DEAE‐Sephadex at pH 7.8 and 2.7, followed by thin‐layer chromatography on polyethyleneimine cellulose. The second involves labelling in vitro of the RNA species at the 5′‐end using polynucleotide kinase.

The 40‐S molecule is shown not to bear a terminal 5′‐triphosphate. It has the 5′‐terminus guanosine 5′‐phosphate. Both the 18 and 28‐S RNAs have the 5′‐terminus uridine 5′‐phosphate. The 28‐S RNA has the 5′‐terminal sequence pU‐N‐N‐N‐Gp.

These results are discussed in relation to the transcription of the ribosomal genes, the possible heterogeneity of the ribosomal precursor and the position of the 28‐S sequence within the precursor molecule.
28‐S RNA from Xenopus laevis Contains a Sequence of Three Adjacent 2′‐O‐Methylations
Authors: Slack Jonathan M. W. , Loening Ulrich E.
Published: March 2005

Evidence is presented to show that an alkaline hydrolysis product from 40‐S and 28‐S RNA of Xenopus laevis is a tetranucleotide containing three adjacent 2′‐O‐methylations.

This is the longest such sequence yet reported and is believed to be Gm‐Am‐Am‐Ap.
Transfer of Heme from Ferrihemoglobin and Ferrihemoglobin Isolated Chains to Hemopexin
Authors: Hrkal Zbyněk , Vodrážka Zdeněk , Kalousek Ivan
Published: March 2005

Incubation of ferrihemoglobin as well as of hemoglobin isolated chains in ferri‐form with hemopexin at pH 7.0 leads to the gradual transfer of heme to the hemopexin molecules until the equilibrium is attained.

The reaction of ferrihemoglobin with hemopexin can be described as a four‐stage process involving successive transfer of the four heme groups from hemoglobin tetramer to the four molecules of hemopexin. The heme binding sites of the hemoglobin molecule are nonequivalent and cooperative.

The transfer of heme from the isolated hemoglobin chains in ferri‐form occurs as the simple competitive reaction which can be characterized by a single equilibrium constant.

The affinity constant value for the binding of heme to the single binding center of hemopexin molecule was estimated as 1.9 × 1014× M−1.

The direct transfer of heme from hemoglobin to hemopexin may occur in vivo as a part of hemoglobin degradation process.
Induction of Ethylmorphine Demethylation and Aniline p‐Hydroxylation by Various Catatoxic Steroids in Regenerating Rat Liver
Authors: Japundžić Ivanka , Japundžić Milorad , Szabo Sandor
Published: March 2005

In rats, the administration of a single dose of 3β‐hydroxy‐20‐oxopregn‐5‐ene‐16α‐carbonitrile and 9α‐fluoro‐11β,‐17 ‐dihydroxy‐3‐oxo‐4‐androstene‐17α‐propionic acid, SC‐11927 at the time of partial hepatectomy or 20 h later, resulted in a 3 to 5‐fold increase in ethylmorphine demethylase and aniline hydroxylase activity in the regenerating liver. Using the inhibitors of protein synthesis dactinomycin and cycloheximide it was shown that the induction process in regenerating liver, as well as in the liver of sham‐operated controls, could be blocked at transcriptional and translational levels. The extent of induction of the same enzymes by 17‐hydroxy‐7α‐thioacetyl‐3‐oxo‐4‐androstene‐17α‐propionic acid γ‐lactone, spironolactone in regenerating liver was only one third of that in sham‐operated controls. Treatment with spironolactone for three days before operation did not increase the degree of enzyme induction in regenerating liver.

Evidence is presented against a mutual exclusion of simultaneous cell proliferation and induction of mixed function oxidase. Both, liver growth rate and hydrolxyase activity can be simultaneously stimulated by catatoxic steroids. The possible occurrence of these two processes in different cell areas of the regenerating liver is discussed.
Fluorescent Labelling of Polynucleotides by 9‐Bromomethylanthracene
Authors: Pochon François , Perrin Martine
Published: March 2005

The action of 9‐bromomethylanthracene on poly(A) either single stranded or complexed with poly(U) has been investigated. Reaction of the polycyclic hydrocarbon occurs specifically on the amino group of adenylic residues in the polymer whereas the N1 position is also conjugated in the monomer using the same experimental conditions. Poly(G) reacts to a lower extent than poly(A) with the formation of one conjugate instead of two for the monomer. Cross linking congujation occurs between the two strands of DNA. The different factors governing the specificity and the extent of the reaction of this reagent with polynucleotides are discussed, the different geometry of double‐stranded structure inducing various reactions of conjugation. The extent of reaction was tested for several polynucleotides and their complexes. The B form of DNA was found to be the most sensitive one. The conjugates provide convenient labels for conformational studies of polynucleotides using fluorescence spectra and polarisation data.
The Amino‐Acid Sequence of Porcine Adenylate Kinase from Skeletal Muscle
Authors: Heil Albert , Müller Gudrun , Noda Lafayette , Pinder Thomas , Schirmer Heiner , Schirmer Ilse , von Zabern Inge
Published: March 2005

Adenylate kinase (ATP:AMP phosphotransferase) has been purified 490‐fold from porcine muscle with a final yield of 60 mg/kg muscle.

The amino‐acid composition is Asp11, Asn2, Thr14, Ser11, Glu19, Gln6, Pro6, Gly19, Ala8, Cys2, Val17, Met6, Ile9, Leu18. Tyr7, Phe5, Lys21, His2, Arg11.

The protein molecule is a single polypeptide chain of 194 amino‐acid residues with an acetyl‐methionine at the N‐terminus and a lysine residue at the C‐terminus.

Cyanogen bromide cleavage of carboxymethylated adenylate kinase yielded six fragments which were further degraded by using trypsin, chymotrypsin, thermolysin, subtilisin or α‐protease. Sequence data on the resulting peptides are summarized in the present report, full details are given in a supplementary paper which has been deposited at CNRS from where copies can be obtained.

The primary structure of porcine adenylate kinase is: Ac‐Met‐Glu‐Glu‐Lys‐Leu‐Lys‐Lys‐Ser‐Lys‐Ile10‐Ile‐Phe‐Val‐Val‐Gly‐Gly‐Pro‐Gly ‐ Ser‐Gly20‐Lys‐Gly‐Thr‐Gln‐Cys‐Glu‐Lys‐Ile‐Val‐Gln30‐Lys‐Tyr‐Gly‐Tyr‐Thr‐His‐Leu‐Ser‐Thr‐Gly40‐Asp‐Leu‐Leu‐Arg‐Ala‐Glu‐Val‐Ser‐Ser‐Gly50‐Ser‐Ala‐Arg‐Gly‐Lys‐Met‐Leu‐Ser‐Glu‐Ile60‐Met‐Glu‐Lys‐Gly‐Gln‐Leu‐Val‐Pro‐Leu‐Glu70‐Thr‐Val‐Leu‐Asp‐Met‐Leu‐Arg‐Asp‐Ala‐Met80‐Val‐Ala‐Lys‐Val‐Asp‐Thr‐Ser‐Lys‐Gly‐Phe90‐Leu‐Ile‐Asp‐Gly‐Tyr‐Pro‐Arg‐Glu‐Val‐Lys100‐Gln‐Gly‐Glu‐Glu‐Phe‐Glu‐Arg‐Lys‐Ile‐Gly110‐Gln‐Pro‐Thr‐Leu‐Leu‐Leu‐Tyr‐Val‐Asp120‐Ala‐Gly‐Pro‐Glu‐Thr‐Met‐Thr‐Lys‐Arg‐Leu‐Leu130‐Lys‐Arg‐Gly‐Glu‐Thr‐Ser‐Gly‐Arg‐Val‐Asp140‐Asp‐Asn‐Glu‐Glu‐Thr‐Ile‐Lys‐Lys‐Arg‐Leu150‐Glu‐Thr‐Tyr‐Tyr‐Lys‐Ala‐Thr‐Glu‐Pro‐Val160‐Ile‐Ala‐Phe‐Tyr‐Glu‐Lys‐Arg‐Gly‐Ile‐Val170‐Arg‐Lys‐Val‐Asn‐Ala‐Glu‐Gly‐Ser‐Val‐Asp180‐Asp‐Val‐Phe‐Ser‐Gln‐Val‐Cys‐Thr‐His‐Leu190‐Asp‐Thr‐Leu‐Lys.
Characterization of Blood‐Group‐H‐Active Ceramide Tetrasaccharide from Hog‐Stomach Mucosa
Authors: Slomiany Bronislaw L. , Slomiany Amalia , Horowtz Martin I.
Published: March 2005

A fucose‐containing ceramide tetrasaccharide exhibiting blood‐group‐H activity was isolated from a lipid extract of hog stomach mucosa by column fractionations on DEAE‐cellulose and Florisil followed by purification on thin‐layer plates.

By partial acid hydrolysis, sequential degradation with specific glycosidases and methylation analysis, the structure of this glycolipid was identified as: Fucα1 →, 2Galα1 → 3Galβ1 → 4Glc1 → 1Cer; where Fuc = fucose; Gal = glucose; Gal = glucose and Cer = ceramide.
Stepwise Dissociation of Histones from Rat‐Liver Chromatin in Alkaline Solutions
Authors: Russev Georgi , Venkov Christo , Tsanev Roumen
Published: March 2005

Rat liver chromatin was sedimented in a sucrose density gradient under alkaline conditions. It is shown that in the pH interval 12.2—12.8 four histone species are dissociated stepwise in the sequence: F2b, F1, F2al and F3. Histone F2a2 remains bound to DNA even at pH 13.

About 70% of the non‐histonc proteins dissociate from the chromatin at pH values lower than 12.2 and 30% remain bound to DNA in the range of pH studied.
ATPase and GTPase Activities Isolated from Rat Liver Ribosomes
Authors: Grummt Friedrich , Grummt Ingrid , Erdmann Volker A.
Published: March 2005

An RNA · protein complex containing ribosomal 5‐S RNA has been isolated by treatment of rat liver 60‐S ribosomal subunits with EDTA. The protein moiety of this complex shows one band in dodecylsulfate gel electrophoresis whose mobility suggests a molecular weight of about 32000. The complex exhibits both GTPase and ATPase activities. The optimal conditions for both enzymic activities have been studied. Kinetic experiments revealed that GTPase and ATPase both had a Km of 3.7 μM, whereas V values for GTPase and ATPase were 5.6 and 2.2 pmol/min, respectively. Inhibition studies have shown that two different enzymatic sites are involved in both reactions.
Further Investigations on the Subunit Structure of Microsomal Carboxylesterases from Pig and Ox Livers
Authors: Junge Wolfgang , Krisch Klaus , Hollandt Heinrich
Published: March 2005

Using sedimentation equilibrium studies, the molecular weight of pig liver esterase was found to be 163000. Sedimentation velocity experiments yielded a sedimentation coefficient s020,w of 7.9 S; the diffusion coefficient was found to be 40.7 μm2· s–1 by ultracentrifugation and 40.3 μm2· s–1 by analytical gel filtration. From these data a molecular weight of 181 000 was calculated. By analytical disc electrophoresis a molecular weight of 183000 was estimated.

Disc electrophoresis of ox liver esterase revealed three enzymatically active bands. The molecular weight of two of them was 54500 and that of the third 187000. Extrapolation of the sedimentation coefficients measured at very low enzyme concentrations to infinite dilution yielded a minimum value of S020,w= 4.0 S. Based on gel filtration results a diffusion coefficient of 66.0 μm2· s–1 was calculated. From these data a molecular weight of 57300 was obtained. Sedimentation equilibrium experiments of the ox enzyme showed a non‐linear plot of In absorbance versus r2 indicating heterogeneity with respect to molecular weight. The smallest species present had a molecular weight of 59000.

By sedimentation equilibrium experiments in the presence of 6 M guanidine‐HCl and 0.1 M 2‐mercaptoethanol, the subunit weight of pig liver esterase was found to be 58000. For the maleylated enzyme, values of 52500 (sedimentation equilibrium) and 56500 (from S20,w and D20,w) were obtained.

By covalent cross linking of the subunits with dimethyl suberimidate and subsequent polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, three bands of molecular weights 60000, 120000 and 180000 were found.

All these results give strong evidence for a three subunit structure of pig liver carboxylesterase.
Purification and Characteristics of Hybridase (Ribonuclease H) from Rat‐Liver Cytosol
Authors: Roewekamp Walter , Sekeris Constantin E.
Published: March 2005

Ribonuclease H or hybridase, the enzyme cleaving the RNA moiety of DNA · RNA hybrids has been extensively purified from rat‐liver cytosol by ammonium sulphate precipitation, DEAE‐cellulose chromatography, Sephadex G‐200 and G‐150 superfine gel filtration, isoelectric focusing, phosphocellulose and hydroxyapatite chromatography. Neither native or denatured DNA, nor single or double‐stranded natural or synthetic ribopolynucleotides are degraded by the enzyme. The pH optimum of the reaction is 7.9. Enzyme activity is totally dependent on the divalent cations Mg2+ and Mn2+, Mg2+ being optimal for the reaction. The enzyme acts on the hybrid as an endonuclease resulting in end products with a chain length less than 15, having a 5′‐phosphate and a free 3′‐hydroxyl end group. Synthetic homopolymer hybrids are digested only if they are composed of purine bases. The enzyme has a molecular weight of 120000–125000 and seems to be composed of two subunits with molecular weights of 85000 and 43000.
Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin: Amino‐Acid Sequence of the Fc Fragment
Authors: Bourgois Alain , Fougereau Michel , Rocca‐Serra José
Published: March 2005

The amino‐acid sequence of the Fc fragment of a mouse monoclonal IgG2a molecule is presented. With the exception of one deletion, this region possesses the same length as that of the human γ1 chain or that of the rabbit γ chain. Identities between the Fc fragments of 3 animal species (human, mouse and rabbit) average 50% and are markedly more pronounced for the CH2 domain than for the CH3 domain, observation which is in agreement with an independent evolution of each homology region. Strikingly, the degree of conservation of the vH region is of the same order of magnitude as that observed for the Fc fragment. This parallelism in evolution is discussed.
Kinetic Properties of the Phosphofructokinase from Erythrocytes of Rats and Rabbits
Authors: Kühn Brigitte , Jacobasch Gisela , Gerth Christa , Rapoport Samuel M.
Published: March 2005

The activity of the phosphofructokinase from erythrocytes of rats and rabbits, and presumably of the enzyme from other sources depend on the presence of either K+ or NH4+ ions. Their effects are not due to ionic strength. The apparent activation constant, KD, is about 1 mM for NH4+ and 10 mM for K+. While K+ ions appear to act only as activators of the catalytic site, NH4+ ions have a double action. They act both as activators and as positive effectors which abolish the cooperativity, presumably by affecting the interaction between the subunits. They increase the affinity of the enzyme for fructose 6‐phosphate and ATP as substrates, while the affinity for ATP as inhibitor is weakened. These effects are probably secondary to the changed interaction between the subunits. It is concluded that only data on the phosphofructokinase in K+ system are representative of normal intracellular conditions. In certain physiological and pathological states the effect of NH4+ ions on the allosteric properties of the enzyme is probably important.

Pi is an activator of both the K+‐ and the NH4+‐activated enzyme, with an approximate KD of 1.6 mM. Its effect is therefore of importance under physiological and pathological conditions.

The data on the purified K+‐activated enzyme are in good agreement with changes of the metabolic pattern of the intact erythrocyte.
Kinetic Properties of the Phosphofructokinase from Erythrocytes of Rats and Rabbits
Authors: Kühn Brigitte , Jacobasch Gisela , Gerth Christa , Rapoport Samuel M.
Published: March 2005

The phosphofructokinase of the red cells of rats is exquisitely sensitive to inhibition by ATP. The apparent k1 for ATP is decreased with lower pH values and fructose‐6‐phosphate concentrations. 2,3‐Bisphophoglycerate, which is present in the red cells of man and many mammals, inhibits the phosphofructokinase in a synergistic manner with ATP. The inhibitory effect of phosphoenolpyruvate is biologically insignificant. It may be estimated that the enzyme would be practically completely inactive under cellular conditions without the action of positive effectors.

There exist a multitude of positive effectors. Significant activations under cellular conditions are exercised by AMP, glucose 1,6‐bisphosphate and inorganic phosphate. Fructose 1,6‐bisphosphate activates to a limited extent and ADP only at alkaline pH values. All positive effectors produce a decreased inhibition by ATP and increased affinity of the enzyme for fructose 6‐phosphate. AMP is the activator with the highest affinity, while glucose 1,6‐bisphosphate may be considered as a baseline activator owing to its sluggish turnover.

The data may be fitted in a qualitative manner to the model of Monod, Wyman and Changeux. It is possible that the negative anionic effectors act on the same site.
Enzymic Modification of Cyclopropane Sterols in Yeast Cell‐Free System
Authors: Anding Claude , Parks Leo W. , Ourisson Guy
Published: March 2005

Cycloartenol, cycloeucalenol and 24‐methylene pollinastanol have been incubated in a yeast cell‐free system. Esters of the substrates are formed but there is no evidence of enzymatic cyclopropane cleavage.
Comparative Studies of Mouse (H‐2) and Human (HL‐A) Histocompatibility Antigens
Authors: Hess Maxime , Smith William
Published: March 2005

A purification procedure applied to H‐2 antigens (controlled by the H‐2 locus) was used for the characterization of HL‐A substances (HL‐A2 and HL‐A7) (controlled by the HL‐A locus) solubilized from lymphoid cell membranes by delipidation. Upon Sephadex chromatography the serologically active material had an apparent Mr of 32000, and displayed, in relation to H‐2 antigens, a faster electrophoretic mobility and higher pI values. One sulfhydryl group and one (intrachain) disulfide bond were present in partially purified HL‐A preparations. Reduction and aminoethylation in 4 M urea did not influence the electrophoretic migration of HL‐A active substances, suggesting that, as already demonstrated for H‐2 alloantigens, in HL‐A molecules the genetic determinants are present on single polypeptide chains. Highly purified HL‐A alloantigens, like H‐2 substances, revealed a high degree of microheterogeneity. These findings further support the concept of the genetic and structural homology of the main histocompatibility systems in mouse and man.