bluuebunny/crossref_metadata_2025 huggingface dataset:
A Reinvestigation of the Phosphorylation of Rabbit Skeletal‐Muscle Glycogen Synthase by Cyclic‐AMP‐Dependent Protein Kinase
Authors: EMBI Noor , PARKER Peter J. , COHEN Philip
Published: March 2005

Glycogen synthase preparations which were free of endogenous phosphorylase kinase were phosphorylated to different extents with cyclic‐AMP‐dependent protein kinase and the phosphopeptides obtained by tryptic digestion were analysed. Limited tryptic digestion of the native enzyme quantitatively released two phosphopeptides (site 1 a and site 1 b) which were soluble in trichloroacetic acid. The trichloroacetic‐arid pellet contained a distinct phosphorylation site, which was shown to be identical to the serine residue which is phosphorylated by phosphorylase kinase (site 2).

Sites 1 a, 1 b and 2 accounted for virtually all of the phosphorylation of glycogen synthase up to 2.5 molecules phosphate incorporated per subunit. Site 1 a was initially phosphorylated 7–10‐fold faster than site 2, and 15–20‐fold faster than site 1 b. The phosphorylation of site 1 a became much slower after about 0.5 molecules of phosphate had been incorporated per subunit.

Cyclic‐GMP‐dependent protein kinase also phosphorylated glycogen synthase at sites 1 a, 1 b and 2. The order of phosphorylation was the same as that observed with cyclic‐AMP‐dependent protein kinase (site 1 a > site 2 > site 1 b).

All three sites phosphorylated by cyclic‐AMP‐dependent protein kinase could be dephosphorylated by protein phosphatase 1. Site 2 was initially dephosphorylated 5–10‐fold faster than site la and more than 100‐fold faster than site 1 b. The MgATP‐dependent protein phosphatase dephosphorylated site 2, site 1a and site 1 b to a very similar manner to protein phosphatase 1.

The different rates of phosphorylation and dephosphorylation of the three sites allowed the demonstration to be made that the activity of glycogen synthase depended on the state of phosphorylation of site 2 and site 1 a. The phosphorylation of site 1 b did not influence the activity under the conditions used.

Incubation of glycogen synthase with high levels of phosphorylase kinase did not lead to the phosphorylation of either site 1 a or 1 b, or of the three sites phosphorylated by glycogen synthase kinase 3 (sites 3a, 3b and 3c). Similarly no phosphorylation of sites 3a, 3b or 3c by cyclic‐AMP‐dependent protein kinase was detected. It is concluded that site 2 is the only site at which overlapping substrate specificity occurs between these three glycogen synthase kinases.
Evidence for New Forms of Cardiac Myosin Heavy Chains in Mechanical Heart Overloading and in Ageing
Authors: KLOTZ Catherine , SWYNGHEDAUW Bernard , MENDES Hélène , MAROTTE Franiçoise , LEGER Jean J.
Published: March 2005

Heavy chains of myosin rods and subfragment 1 were isolated from normal hearts and from mechanically overloaded hearts of young and older rats. These myosin heavy‐chain fragments were cleaved by cyanogen bromide or partially proteolysed by pronase and by chymotrypsin after denaturation with sodium dodecyl sulfate. The peptides, analyzed by electrophoresis on a one‐dimensional polyacrylamide slab gel, varied depending on the origin of the cardiac myosin heavy chains. Some bands present in the peptide patterns of the normal heart of young rats were missing from the pattern of greatly hypertrophied hearts and vice versa. We conclude that mechanical overloading of the heart stimulates the synthesis of cardiac myosin ‘isozyme’ with a heavy‐chain primary structure which is differen: from that observed in the normal heart of young rat.

The patterns from myosin heavy‐chain peptides from the hearts of older rats were different from those for peptides from young rat hearts; these results also indicate the presence of a new myosin heavy, chain specific to ageing. No difference was detected between the peptide patterns of heavy chains isolated from hypertrophied hearts of young and older rats, and those isolated from normal hearts of older rats.
Stabilisierung von Enzymen and Antiseren durch Schweres Wasser
Authors: LEMM Ursula , WENZEL Martin
Published: March 2005

Stabilisation of Enzymes and Antisera by Heavy Water

The purpose of our investigations was to study the protective effect of 2H2O on the denaturation of biochemically active proteins, e.g. the enzymes lactate dehydrogenase, hexokinase and creatine kinase and the antibodies against the steroid hormones estrone and estradiol.

The proteins were dissolved in buffer prepared either with water or heavy water. These protein solutions were exposed to heat or extremes of pH. After this the solutions were well diluted with buffer of normal water, so that the contents of heavy water was not more then 6%. The biochemical activity, i.e. enzyme‐activity or binding capacity was then measured under standard conditions.

When lactate dehydrogenase was heated in H2O its activity decreased by about 80%, whereas the activity of the enzyme incubated in 2H2O was only slightly reduced. The lesser inactivation in 2H2O is not the consequence of the generally smaller dissociation of 2H2O, because the protective effect of 2H2O was evident at pH 7.5 and pH 8.2. With the enzyme hexokinase it could be shown, that the protective effect of albumin against denaturation is even more pronounced in 2H2O. Not only proteins which are dissolved in 2H2O are protected against inactivation by heat, but also lyophilized enzymes e.g. creatine kinase are stabilized if the hydrogen bonds are exchanged by deuterium.

Besides these enzymes we tested the antibodies of the steroid hormones estrone and estradiol. A protective effect of 2H2O against heat inactivation was shown with estradiol‐antibody. The prolongation of half‐life after heating in 2H2O instead of H2O was three times. Estrone‐antibody was less sensitive against extremes of pH, if it was kept in 2H2O. 2H2O had an effect in alkaline as well as in acid ranges.

There is a sharp increase in protection at low 2H2O‐concentrations, at higher concentrations of 2H2O its protective effect increases more slowly. From a practical point of view that means: to obtain a reasonable protective effect against denaturation a 2H2O‐concentration of 15–30% is sufficient.
Inhibition of Cell Division by Interferons
Authors: GEWERT Dirk R. , SHAH Sanjay , CLEMENS Michael J.
Published: March 2005

The Daudi line of human lymphoblastoid cells shows a high sensitivity towards growth inhibition by human interferons. In cells pretreated with 70 reference units/ml of an interferon preparation for 48 h, the incorporation of exogenous [3H]thymidine into DNA is inhibited by as much as 85%. We are investigating the extent to which this effect reflects a true inhibition of the rate of DNA synthesis or whether it may be caused by changes in the metabolic utilization of exogenous thymidine by the cells. Interferon treatment results in a 30% inhibition of the rate of membrane transport and a 60% decrease in the rate of phosphorylation of [3H]thymidine in vivo. The latter effect is due to a decrease in V of thymidine kinase without any change in the value of Km for this enzyme. In addition to these changes, incorporation of [3H]uridine into DNA, which occurs as a result of the intracellular conversion of this precursor into thymidine nucleotides, is also inhibited by 75%, whereas RNA labelling by [3H]uridine is decreased by only 15% in interferon‐treated cells. Thus several different metabolic events associated with thymidine nucleotide metabolism and DNA synthesis in Daudi cells are disrupted by interferon treatment.
Cytochrome b5 as Electron Donor for Oxy‐cytochrome P‐450
Authors: NOSHIRO Mitsuhide , ULLRICH Volker , OMURA Tsuneo
Published: March 2005

The steady‐state concentration of the oxycomplex of microsomal cytochrome P‐450 in the presence of NADPH could be elevated by increasing pH and ionic strength or by adding anti‐(cytochrome b5) immunoglobulin. Low pH and low ionic strength as well as incorporation of cytochrome b5 into microsomal membranes decreased the steadystate level. Parallel to these effects, the b5 antibody inhibited monooxygenase activity at low pH and low ionic strength, but was not effective at high pH and high sonic strength. This establishes a role for cytochrome b5 in donating electrons for the reduction of oxy‐cytochrome P‐450 to the active oxygen complex of cytochrome P‐450 but also points to large variations in the importance of this role depending on the experimental conditions, the species of P‐450 involved and the substrates employed.
Chemical Structures of Three Fucogangliosides Isolated from Nervous Tissue of Mini‐Pig
Authors: FREDMAN Pam , MÅNSSON Jan‐Eric , SVENNERHOLM Lars , SAMUELSSON Bo , PASCHER Irmin , PIMLOTT Weston , KARLSSON Karl‐Anders , KLINGHARDT Georg W.
Published: March 2005

In the nervous tissue of miniature pig, type Göttingen, three fucosyl‐containing gangliosides are major gangliosides. All three were found in dorsal‐root ganglia and spinal cord, but only two of them in the forebrain. The concentrations of these gangliosides were increased in chloroquine intoxication. Their chemical structures were determined by component analysis, partial acid and enzymatic degradation and assay of the partially methylated sugars after hydrolysis, reduction and acetylation. Mass spectrometric analysis of the intact gangliosides as permethylated, permethylated‐reduced and permethylated‐reduced‐silylated derivatives confirmed the following structures of the three gangliosides.
Xenograft Rejection in Marine Sponges
Authors: MÜLLER Werner E. G. , BERND August , ZAHN Rudolf K. , KURELEC Branko , DAWES Keith , MÜLLER Isabel , UHLENBRUCK Gerd
Published: March 2005

In sponges there exists a graft rejection mechanism in which an inhibitory aggregation factor is involved. The inhibitory aggregation factor has been isolated from a culture medium containing dissociated cells of the sponge Geodia cydonium. Using ion‐exchange and gel fractionation the factor was purified and shown to be electrophoretically pure. The factor has a molecular weight of 27000 and was characterized as a glycoprotein. The activity of the inhibitory aggregation factor was not affected by heat treatment, but treatment with trichloroacetic acid resulted in the irreversible loss of activity. The inhibitory aggregation factor affects the aggregation‐factor‐mediated reaggregation of dissociated sponge cells by extension of the lag phase preceding the aggregation process; the endpoint of the reaggregation process is not changed. The inhibitory aggregation factor competes with the aggregation receptor for the binding site on the aggregation factor. The inhibitory aggregation factor is not synthesized if homologous aggregation factor is added to the dissociated cells. The results indicate that the inhibitory aggregation factor is synthesized in xenografts of the system Geodia cydonium—Geodia rovinjensis after a grafting period of at least 3 days. Following the synthesis of the factor, the concentration of the extractable aggregation factor decreases.
Interference of Nucleoside Diphosphates and Inorganic Phosphate with Nucleoside‐Triphosphate‐Dependent Calcium Fluxes and Calcium‐Dependent Nucleoside‐Triphosphate Hydrolysis in Membranes of Sarcoplasmic‐Reticulum Vesicles
Authors: WAAS Wolfgang , HASSELBACH Wilhelm
Published: March 2005

Calcium fluxes across the membranes of the sarcoplasmic reticulum vesicles supported by ATP and by GTP were analyzed with respect to the effects of the reaction products nucleoside diphosphate, Pi and intravesicular calcium. Calcium fluxes and hydrolysis of nucleoside triphosphate (NTP) were determined after cessation of net uptake of calcium and during phosphate‐supplemented net storage of calcium. After net uptake of calcium has ceased the calcium transport enzyme splits ATP and GTP with only 10% of its optimal activity, due to the inhibiting effect of high intravesicular calcium concentrations. The same maximal calcium load of 80 nmol/mg is obtained with ATP and GTP but maintained at very different rates of calcium exchange which correspond to the suppressed rates of ATP and GTP splitting. Thus, calcium influx is coupled to ATP and GTP splitting with nearly the same ratio of 0.6–0.8. The calcium‐dependent NTPase activity is less sensitive to the inhibiting effect of both nucleoside diphosphates at high concentrations than it is at low concentrations of intravesicular calcium. In contrast to the NTPase activity displayed by calcium‐filled vesicles the exchange between internal and external calcium is markedly stimulated by the addition of ADP and GDP. Evidently, at zero net uptake of calcium, calcium translocation is not coupled to phosphate liberation. In the absence of nucleoside diphosphates the addition of inorganic phosphate causes a permanently proceeding net uptake of calcium and enhances the calcium‐dependent NTPase activity. The simultaneously occurring calcium efflux, however, remains unaffected by phosphate. The resulting increment of calcium influx is matched by the increment of NTP hydrolysis, thus leaving the ratio calcium influx NTPase activity unchanged. In the presence of nucleoside diphosphates, however, the addition of phosphate accelerates calcium influx as well as calcium efflux. The rates of the resulting net uptake of calcium only reach 2% or 10%, respectively, of the rate of calcium influx supported by ATP or GTP. The rates of calcium movement together with the known rates of unidirectional calcium uptake and release are discussed in relation to the activity of the various phosphate‐transfer reactions in the transport cycle.
Purification and Properties of a Bovine Brain Thyrotropin‐Releasing‐Factor Deamidase
Authors: TATE Suresh S.
Published: March 2005

A bovine brain thyrotropin‐releasing‐factor (thyroliberin) deamidase has been purified 1100‐fold to apparent homogeneity. Molecular weight estimates by gel filtration and sodium dodecylsulfate gel electrophoresis indicate that the enzyme consists of a single polypeptide chain of molecular weight of about 62000–65000. The enzyme is inactivated by sulfhydryl blocking agents. Serine proteinase inhibitors, phenylmethanesulfonyl fluoride and benzamidine, have no effect. Besides thyroliberin, the enzyme hydrolyzes peptide bonds involving the carboxyl group of proline residues in luliberin, tuftsin, angiotensin II, melanotropin, and neurotensin. Oxytocin, vasopressin, and bradykinin are not cleaved; they are, however, strong competitive inhibitors of thyroliberin deami dation. The specificity studies indicate that the enzyme is a ‘post‐proline cleaving euzyme’ which hydrolyzes peptides of the general structure, Yaa‐Pro‐Xaa, in which Xaa = amino acid, peptide, or amide (not Pro), and Yaa = N‐blocked basic amino acid or a peptide sequence in which the C‐terminal residue (i.e. the residue prior to Pro) is a basic amino acid such as His, Lys, or Arg. The enzyme is compared to other post‐proline cleaving enzymes.
An NAD‐Linked Acetoacetyl‐CoA Reductase from Zoogloea ramigera I‐16‐M
Authors: SHUTO Hidetoshi , FUKUL Tetsuya , SAITO Terumi , SHIRAKURA Yuri , TOMITA Kenkichi
Published: March 2005

An NAD‐linked acetoacetyl–CoA reductase of Zoogloea ramigera I‐16‐M was purified to electrophoretic homogeneity. In contrast to the d(‐)‐3 ‐hydroxybutyryl‐CoA‐specific NADP‐linked acetoacetyl‐CoA reductase from the same bacterium [Saito, T. et al. (1977) Arch. Microbiol. 114, 211‐217], the purified enzyme was strictly stereospeciflc to L(+)‐3‐hydroxybutyryl‐CoA, and was active not only with NAD+ but also with NADP+ although NADP+ was less effective than NAD+ as coenzyme. The enzyme showed a pH optimum at 6.3 for the reduction of acetoacetyl‐CoA and at 8.0 for the oxidation of L(+)‐3‐hydroxybutyryl‐CoA. In the reduction reaction, Km values for acetoacetyl‐CoA and NADH were 8.8μM and 6.5μM, respectively, and in the oxidation reaction, Km values for L(+)‐3‐hydroxybutyryl‐CoA and DNA were 7.0μM and 32μM, respectively. Among various 3‐hydroxyacyl‐CoAs tested, L(+)‐3‐hydroxybutyryl‐CoA and L(+)‐3‐hydroxyvaleryl‐CoA were the most active substrates. Poly(3‐hydroxybutyrate) synthesis from acetyl‐CoA, by a system reconstituted from purified preparations of 3‐oxothiolase, acetoacetyl‐CoA reductase and poly(3‐hydroxybutyrate) synthase, was observed when the NADP‐linked but not the NAD‐linked reductase was used. These findings indicate that the NAD‐linked acetoacetyl‐CoA reductase is not directly involved in the biosynthesis of poly(3‐hydroxybutyrate).
Equilibrium Aspects of the Binding of myo‐Inositol Hexakisphosphate to Human Hemoglobin as Studied by 31P NMR and pH‐Stat Techniques
Authors: ZUIDERWEG Erik R. P. , HAMERS Laurentius F. , de BRUIN Simon H. , HILBERS Cornelis W.
Published: March 2005

The interaction of myo‐inositol hexakisphosphate (P6 ‐insitol) with human hemoglobin has been studied as a function of pH using pH‐stat techniques and 31P NMR. With the pH‐stat method the following data were obtained: the association constants for the P6 and P6 complexes at alkaline and acid pH respectively and the proton absorption curves associated with the protein/phosphate interaction for both complexes from pH 5.5 to pH 9. From these data the affinities of P6 towards deoxyhemoglobin (Hb) and carboxyhemoglobjn (HbCO) have been dalculated as a function of pH. The shape of the proton absorption curves was found to be strongly dependent on the ligation state of the hemoglobin molecule.

The pH dependence of the 31P NMR spectra of P6 bound to Hb or HbCO provides a monitor for the proton‐binding behaviour of the phosphate groups of P6‐instol when present in the central cavity of the protein. It appears that this behaviour is only slightly dependent on the ligation state of the hemoglobin molecule. The NMR spectral data were interpreted in terms of a model which takes into account the electrostatic interaction between the phosphate groups within the P6‐instol molecule as well as the electrostatic interaction between the phosphate groups and positively charged groups on the protein.

To account for the discrepancy between the pH‐stat and 31 P NMR results, i. e. a strong dependence of the proton‐absorption curves and a weak dependence of the proton‐binding behaviour of P6‐instol on the ligation state of the protein respectively, it is proposed that a conformational change takes place in HbCO upon P6‐instol binding.
31P NMR Study of the Kinetics of Binding of myo‐Inositol Hexakisphosphate to Human Hemoglobin
Authors: ZUIDERWEG Erik R. P. , HAMERS Laurentius F. , ROLLEMA Harry S. , de BRUIN Simon H. , HIBERS Corneils W.
Published: March 2005

The association and dissociation kinetics of the complexes of myo‐inositol hexakisphosphate (P6‐instol) with deoxyhemoglobin (Hb) and carboxyhemoglobin (HbCO) have been investigated by 31P NMR between pH 6.8 and pH 5.5. These complexes represent high‐affinity systems with binding constants varying between 105 M−1 and 2 × 109 M−1. 31P NMR spectra of P6‐instol were recorded in the presence of hemoglobin as a function of the P6‐inositol/hemoglobin molar ratio. It appeared that the exchange of the polyphosphate molecule between the solution and the central cavity binding site is fast on the P6‐instol NMR time scale. This observation cannot be reconciled with a single‐step binding mechanism of P6‐insitol to hemogloin.

Analysis of the spectra revealed the occurrence of additional binding of P6‐insitol to both Hb and HbCO. This binding was also observed in pH‐stat experiments performed at low ionic strength. 31P NMR exoeriments carried out with hemoglobin of which the α‐chain N termini were carbamylated, strongly suggest that these termini constitute the additional binding site for P6‐insitol

A model is proposed which accounts for the enhancement of exchange kinetics in these high‐affinity systems. In this model a rapid migration is assumed for P6‐insitol between the central cavity binding site and an entry/leaving site on the hemoglobin molecule. Based on this model 31P NMR linewidths and chemical shift patterns for this three‐site exchange problem were calculated.
Insulin Antagonism of Glucocorticoid Induction of Tyrosine Aminotransferase in Cultured Foetal Hepatocytes
Authors: HO Kevin K. W. , CAKE Max H. , YEOH George C. T. , OLIVER Ivan T.
Published: March 2005

Whereas dexamethasone is unable to induce the premature formation of hepatic tyrosine aminotransferase when administered to foetal rats in utero, the steroid can induce the enzyme in foetal rat liver if the liver is first removed from the environment in ulero and grown in culture. Dexamethasone produced a significant induction of the enzyme at a concentration of 0.1 nM in cultured foetal hepatocytes, but for optimal induction the cells were exposed to 10 nM for 15 h. Growing the hepatocytes in the presence of physiological concentrations of insulin had no effect on the enzyme activity in control cells. However, the induction of the enzyme by dexamethasone was markedly diminished in the presence of insulin. This effect of insulin is both time‐dependent and dose‐dependent with significant inhibition being obtained with 1 nM insulin. Growing foetal hepatocytes in the presence of insulin has no effect on either the cellular level of glucocorticoid receptor or on the ability of dexamethasone receptor complexes to undergo nuclear translocation suggesting that insulin inhibits some event subsequent to translocation.

The results are discussed in relation to the postnatal appearance of tyrosine aminotransferase and suggest that the marked decline in the plasma concentration of insulin, that is known to occur at birth, is a major contri butor to the postnatal induction of the enzyme.
On the Mode of Action of the Immunosuppressive Sesquiterpene Ovalicin
Authors: ZIMMERMANN Wolfgang A. , HARTMANN Guido R.
Published: March 2005

When the potent immunosuppressive sesquiterpene ovalicin is added to lymphocyte cultures one first observes a preferential inhibition of uridine incorporation into rRNA. The uptake of the nucleoside, its conversion into the triphosphate or the polymerizing activity itself are not affected A longer period of incubation with the drug results in a marked decrease in the number of ribosomes, with a concomitant reduction of the rate of leucine incorporation into all cellular proteins. After extended periods of time, the incorporation of thymidine into DNA in stimulated lymphocytes as well as in S 49.1 lymphoma cells is inhibited by 1 nM ovalicin or less, although part of the incorporation seems to be resistant to the drug even at much higher concentrations. A similar effect is observed with 3T6 mouse fibroblasts or HeLa cells. Here, however, a much longer incubation with the drug is required. This observation explains the selective effect of ovalicin on lymphocytes observed in vivo.
Purification and Properties of d(−)‐3‐Hydroxybutyrate‐Dimer Hydrolase from Zoogloea ramigera I‐16‐M
Authors: TANAKA Yoshimasa , Saito Terumi , FUKUI Tetsuya , TANIO Tomoharu , TOMITA Kenkichi
Published: March 2005

D(−)‐3‐Hydroxybutyrate‐dimer hydrolase from Zoogloea ramigera I‐16‐M was purified 7000‐fold to electrophoretic homogeneity. The molecular weight of the purified enzyme was 28000 as determined by Sephadex G‐100 gel filtration, and 30000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric point was at pH 5.7. The pH optimum for the enzyme reaction was 8.0. The dimer hydrolase was stereospecific for d(−)‐3‐[l(−)‐3‐hydroxybutyryloxy] butyric acid (dl‐dimer) but also hydrolyzed d(−)‐3‐[d(+)‐3‐hydroxybutyryloxy] butyric acid (dl‐dimer) and d(+)‐3‐[d(−)‐3‐hydroxybutyryloxy]butyric acid (ld‐dimer) at reduced rates. However, the enzyme did not attack l(+)‐3‐[l(+)‐3‐hydroxybutyryloxy]butyric acid (ll‐dimer) at all. In addition, the purified hydrolase hydrolyzed several oligomeric esters of d(−)‐3‐hydroxybutyric acid (ddd‐trimer, dddd‐tetramer and ddddd‐pentamer) faster than dd‐dimes. Time course experiments with these oligomers and analysis of hydrolytic products of dddd‐tetramer methyl ester with the hydrolase indicated that the enzyme attacked these substrates from the free hydroxyl terminus, releasing monomer units one at a time.
Occurrence of Phosphatidylcholine in Hydrogen‐Oxidizing Bacteria
Authors: THIELE Otto Wolfgang , OULEVEY Jacques
Published: March 2005

15 strains of hydrogen‐oxidizing bacteria were grown heterotrophically, harvested during the stationary phase of growth, and analyzed for their principal phospholipids. All strainsndash;with the exception of Corynebacterium autotrophicum strain SA 32 and Pseudomonas pseudoflava–contained phosphatidylcholine as a major constituent. It is concluded that the presence of phosphatidy1choline is neither characteristic of a peculiar bacterial genus or family, nor is it absolutely correlated to the ability to oxidize hydrogen. The phosphatidylcholines of all strains contain C19 cyclopropane acid which is, in some strains, predominantly located at C‐2 position of the glycerol moiety.
Size and Shape of the α Subunit of the (Ca,Mg)‐dependent Adenosinetriphosphatase from Escherichia coli in Solution in the Presence and Absence of ATP
Authors: PARADIES Hasko H.
Published: March 2005

The α subunit of the (Ca,Mg)‐dependent ATPase from Escherichia coli was studied in solution by means of X‐ray scattering experiments at variable contrast and in the presence of ATP. The experiments were carried out on an absolute scale in the range of (45.0 nm−1) <h<(1.5 nm−1) at pH 8.0. The experiments show that this system and the complex of the α subunit with ATP can be considered to be ideal and monodisperse so that the following parameters for the α subunit and its complex with ATP were determined: the radius of gyration, the volume, the degree of hydration, the maximum particle diameter, and the molecular weight. The molecular weight of the α subunit was determined as 58500 ± 3000 by means of light scattering measurements, and 57700 ± 2500 by the small‐angle X‐ray scattering experiments. The radius of gyration of the α subunit at pH 8.0 was determined to 2.64 ± 0.02 nm, the maximum chord in solution to 10.0 ± 0.5nm, the volume to 102. 4 ± 2.5 nm3, and the degree of hydration to 0.34 ± 0.02 ml. g−1. Binding of ATP to the a subunit causes structural changes. These changes are reflected in the radius of gyration, Rg= 2.45 ± 0.015 nm, in the volume, 117.6 ± 1.5 nm.3, and in the maximum chord length in solutions 7.8 ± 0.5 nm. These changes imply a decrease of the axial ratio from 2.4 to 1.4, i. e. ATP‐binding induced an increase in isometry of the α subunit.
Tertiary Structure of tRNAs in Solution Monitored by Phosphodiester Modification with Ethylnitrosourea
Authors: VLASSOV Valentin V. , GIEGÉ Richard , EBEL Jean‐Pierre
Published: March 2005

The alkylation by ethylnitrosourea of phosphodiester bonds in yeast tRNAPhe, tRNAVal and in Escherichia coli tRNAGlu, tRNAMetf, tRNAmetm and tRNAPhe was investigated under various conditions.

In unfolded tRNAs the reactivities of phosphates in various positions toward the reagent were similar. In the folded tRNAs remarkable differences in reactivities of phosphates located in various positions of the molecules were observed. In yeast and E. coli tRNAPhe, reactivities of phosphates in positions 9, 10, 11, 19, 49, 58, 59 and 60 were found to be strongly decreased. Some decrease in reactivity was observed for phosphates 23 and 24. Spermine and ethidium bromide did not influence the pattern of phosphate alkylation in the TΨC arm of yeast tRNAPhe. Our solution results fit with the crystal structure of tRNAPhe with respect to the potential availability of the phosphates in this tRNA to solvent as shown by others.

Judging from the pattern of phosphate reactivities, the structure of E. coli tRNAPhe is very similar to that of yeast tRNAPhe. Upon thermal denaturation of the yeast tRNAPhe, the reactivity of the low‐reactive phosphates increased, demonstrating a cooperative melting curve. A comparison of the pattern of phosphate alkylation in several tRNAs, essentially in their TΨC arms, revealed a striking similarity, suggesting that the folding of these tRNAs is essentially similar.
Proteolytic Digestion Studies of Chromatin Core‐Histone Structure
Authors: BÖHM Lothar , BRIAND Gilbert , SAUTIÈRE Pierre , CRANE‐ROBINSON Colyn
Published: March 2005

Trypsin digestion of chromatin results in a well‐defined set of limit peptides (P1‐P5) derived from the four core histones. Those from histones H3 and H4 have been identified. P1 is H3 residues 27‐129; P4 is H4 residues 18‐102 and P5 is H4 residues 20‐102.

The N‐terminal sequences removed correlate well with the regions that undergo post‐synthetic acetylation and which show the greatest degree of sequence conservation. Autolytic digestion of chromatin releases a peptide (P1′) from H3 representing residues 21‐135. The implications of protease digestion of the higher order structure of chromatin are discussed.
A Reinvestigation of the Purity, Isoelectric Points and Some Kinetic Properties of the Aldehyde Dehydrogenases from Sheep Liver
Authors: AGNEW Kim E. M. , BENNETT Adrian F. , CROW Kathryn E. , GREENWAY Robert M. , BLACKWELL Leonard F. , BUCKLEY Paul D.
Published: March 2005

Cytoplasmic aldehyde dehydrogenase was shown to be free of contamination by the mitochondrial enzyme by isoelectric focusing.

Both enzymes showed multiple banding in activity stains. The cytoplasmic enzyme gave two very close bands pl = 5.22 ± 0.03 whereas the mitochondrial enzyme showed seven bands, a pair at pl = 5.22 and five further bands of pl 5.48 ± 0.09, 5.56 ± 0.07, 5.56 ± 0.06, 5.70 ± 0.03 and 5.76 ± 0.02. Possible origins of the isoenzymes are discussed.

Disulfiram in a fourfold excess reduced the activity of the cytoplasmic enzyme to 9% of the initial value. The residual activity represents the activity of the disulfiram‐modified enzyme and is not due to mitochondrial contamination. This casts doubt on the role of an essential thiol group.

The mitochondrial enzyme shows a low amplitude (22%) burst in the production of 4‐nitrophenoxide ion during the hydrolysis of 4‐nitrophenyl acetate at pH 7.6. The burst rate constant was 7.3 ± 1 s−1 and the steady‐state rate constant was 0.2 s−1, values similar to those previously reported for the cytoplasmic enzyme.

The mitochondrial enzyme shows a burst in the release of protons during the oxidation of propionaldehyde at pH 7.6. The burst rate constant was 6 s−1 and the amplitude was equal to half the formal enzyme concentration. The significance of these results for the steady‐state mechanism is discussed.
Photoaffinity Labeling of Steroid Binding Proteins with Unmodified Ligands
Authors: WESTPHAL Hannes M. , FLEISCHMANN Gerhard , BEATO Miguel
Published: March 2005

Photoactivation of the α, β‐unsaturated ketones of natural and synthetic steroid molecules by light of λ≥ 330 nm allows their covalent attachment to steroid‐binding proteins. The general validity of this method is demonstrated with two steroid hormone receptors and the steroid‐binding protein uteroglobin. Progesterone can be covalently attached to the partially purified progesterone receptor and to uteroglobin, and comigrates with the binding proteins upon electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Similarly the synthetic glucocorticoid triamcinolone acetonide can be covalently bound to the partially purified glucocorticoid of rat liver. This method allows the identification of steroid hormone receptors after electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Labeling with radioactive steroids is specific since it can be prevented by the addition of an excess of non‐radioactive ligand. Digestion of the labeled binding proteins with trypsin or chymotrypsin yields a defined pattern of radioactive peptides, demonstrating that covalent attachment takes place at specific binding sites.
Antibodies to the F1‐ATPase of Rhodospirillum rubrum and Its Purified Native β‐Subunit:
Authors: PHILOSOPH Sonia , GROMET‐ELHANAN Zippora
Published: March 2005

Antibodies prepared against the Rhodospirillum rubrum F1‐ATPase (RrF1) and its purified, native β‐subunit, exhibited cross‐respectively with the following soluble preparations of R. rubrum ATPase: RrF0 . F1, RrF1 and the β‐subunit. Anti‐RrF1, but not anti‐β‐antibodies, also formed precipitin lines with soluble β‐less RrF1, indicating that antigenic determinants of both the β‐subunit and the other four RrF1‐subunits are expressed in the whole RrF1 molecule. Both antibodies agglutinated the R. rubrum chromatophores, suggesting that the β‐subunit is located on the external part of RrF1.

Both antibodies inhibited ATP synthesis and hydrolysis activities of R. rubrum chromatophores, as well as all the soluble ATPase reactions. Similar concentrations of each antibody were required for 50% inhibition of all these reactions, but anti‐RrF1 was always somewhat more effective than anti‐β. These data indicate that the β‐subunit is involved in the catalytic site of the RrF1‐enzyme.

The antibodies prepared against R. rubrum F1‐ATPase and its β‐subunit could bind the soluble chloroplast F1‐ATPase (CF1) and inhibited ATP‐linked reactions carried out by chloroplasts and by soluble CF1. In these reactions, unlike in the R. rubrum ones, anti‐β was a more potent inhibitor than the anti‐RrF1 antibody. The cross‐reaction obtained between the antibodies raised against R. rubrum F1 and its β‐subunit and the chloroplast CF1 indicates the presence of similar antigenic determinants in the photosynthetic prokaryotic and cukaryotic F1‐ATPases, which have been conserved during evolution.
Enzymic Synthesis of Lignin Precursors Comparison of Cinnamoyl‐CoA Reductase and Cinnamyl Alcohol: NADP+ Dehydrogenase from Spruce (Picea abies L.) and Soybean (Glycine max L.)
Authors: LÜDERITZ Thomas , GRISEBACH Hans
Published: March 2005

Cambial sap of Spruce (Picea abies) proved to be a good source for isolation of cinnamoyl‐CoA reductase and cinnamyl alcohol: NADP+ dehydrogenase. Apparently homogenous enzymes were obtained by a multistep procedure including dye‐ligand chromatography and for the reductase also affinity chromatography on (coenzyme A)‐agarose. An improved purification procedure for the reductase from soybean cell cultures is also reported.

Molecular weights and subunit composition of reductase and dehydrogenase from spruce are very similar to those of the corresponding enzymes from soybean.

Reduction of feruloyl‐CoA to coniferaldehyde catalysed by the reductase is a freely reversible reaction with an equilibrium constant of 5.6 × 10−4M at pH 6.25. A strong dependence of the Michaelis constants on the type of buffer was found. For reductase the Km‐value of feruloyl‐CoA in phosphate buffer (5.2 μM) is about 14‐times smaller than in citrate buffer (73 μM).

Pronounced differences in substrate specificities between the enzymes from spruce and soybean were found, which reflect the different lignin composition of gymnosperms and dicotyledenous angiosperms. From the kinetic constants of the enzymes it can be concluded that under physiological conditions feruloyl‐CoA is the preferred substrate for the reductase from both sources whereas sinapoyl‐CoA is a substrate only for the soybean reductase and sinapyldehyde a substrate only for the soybean dehydrogenase.

4‐Coumaroyl‐CoA is a poor substrate for the reductase from both spruce and soybean. This result is consistent with the low content of 4‐coumaryl alcohol units in gymnosperm and angiosperm lignin.
Effects of Detergent on Ornithine Decarboxylase from Rat Liver
Authors: KITANI Takako , FUJISAWA Hitoshi
Published: March 2005

Ornithine decarboxylase was purified approximately 37000‐fold with a 15% yield from livers of rats pretreated with thioacetamide. The specific activity of the final preparation, 1039 units/mg protein, was about four‐times higher than the highest yet reported for the rat liver enzyme. The partially purified enzyme was quite labile but the labile enzyme was dramatically stabilized by the presence of either ethylene glycol or Tween 80. The detergent appeared to serve not only stabilization of the enzyme but also renaturation of the denatured enzyme.
Identification of a Bacterial Energy‐Transducing ATPase as a Metallo (Zn2+) Protein
Authors: MOLLINEDO Faustino , LÓPEZ‐MORATALLA Natalia , PIVEL Juan P. , LARRAGA Vicente , SANTIAGO Esteban , MUÑOZ Emilio
Published: March 2005

Purified F1‐ATPase from Micrococcus/ysodeikticus contains zine in the amount of 1 mol mol of enzyme. This zine content correlates with standard values of ATPase activity (assayed with Ca 2+‐ATP as substrate) of the protein, i.e. 5‐6 μmol substrate hydrolysed. min−1 mg−1. Prolonged dialysis against EDTA results in a zinc‐free protein which concomitantly loses its ATPase activity. Chelators such as Zincon, EDTA and L‐cysteine inhibit the ATPase activity in concentration and /or time dependence related to their affinity for the metal ion involved. Reconstitution of the metallo (Zn2+) protein is demonstrated by the incorporation to the zinc‐free protein of 65Zn2+ in amount near to 1 mol/mol of enzyme. This incorporation was concomitant with the regain of ATPase activity. The inhibition by EDTA and Zincon is reversed specifically by Zn2+ while the inhibition by EDTA is prevented by Zn2+ and Mn2+ and to, a minor extent, by Cd2+.Zn2+ and Ca2+ ions are involve and are probably mandatory in the ATPase activity of M. lysodeikticus F1 but their roles appear to be different and not exchangeable. Other divalent metal ions inhibit the Ca2+‐ATPase activity of the Zn2+ protein by the following decresing order; Hg2+,Fe2+,Co2+,Cd2+,Mn2+,Mg2+. M.lysodeikticus F1‐ATPase is thus identified as a metallo (zinc) protein which requires additional divalent metal ions for ATP hydrolysis.
Studies on the Biosynthesis of Laminin by Murine Parietal Endoderm Cells
Authors: COOPER Ashley R. , KURKINEN Markku , TAYLOR Amanda , HOGAN Brigid L. M.
Published: March 2005

The biosyntnesis and processing of the polypeptides A (Mr=450×103), B1 (Mr=450×103), B2 (Mr=230×103) and C (Mr=150×103) of the extracellular matrix protein, laminin, were studied in murine parietal endoderm cells labeled with [35S]methionine. Various lines of evidence suggest that the A chains are not precursors to the smaller B chains. Firstly, in pulse‐chase experiments, radioactivity in cytoplasmic A and (B1+ B2) chains declines with the same half‐life of about 70 min. Secondly, peptide maps generated by digestion of A and (B1+ B2) chains with Staphylococcus aureus V8 protease are different. Finally, rabbit antibodies to isolated, denatured (B1+ B2) chains do not cross‐react with reduced and alkylated A chains. A, B1, B2 and C polypeptides are all glycosylated by an intracellular process involving the addition of tunicamycin and endo β‐N‐acetylglucosaminidase H. sensitive N‐linked oligosaccharide side chains. Further glycosylation probably occurs around the time of secretion. Disulphide bonding of some A and B chains can be observed in the cytoplasm within 10 min of adding [35S]methionine. However, it appears that some free A and B2 chains are present in the cytoplasm and that free A chains exist in the medium. The relationship between the 150×103‐Mr C glycoprotein and the A and B components is discussed. Although B and C chains generate different peptide maps after digestion with S, aureus V8 protease, antibodies raised against isolated, denatured C chains cross‐react with reduced and alkylated B (but not A) chains. This suggests that B and C chains may share some antigenic determinant(s).
Effects of Formamide on the Polymerization and Depolymerization of Muscle Actin
Authors: ZECHEL Kasper
Published: March 2005

Formamide was found to interfere with the polymerization of electrophoretically pure rabbit skeletal muscle G‐actin to F‐actin in vitro. It decreased the rate as well as the extent of polymerization. However, this influence was dependent on the way the polymerization reaction was intiated. If polymerization of G‐actin was induced by 2 mM MgCl2, formamide inhibited the rate and extent of the polymerization much less than if the polymerization was induced by either 2 mM CaCl2 or 0.1 M KCl. The critical protein concentration was increased when formamide was present. This effect was small in the presence of MgCl2 but an approximately tenfold increase in the critical value was observed for the CaCl2‐induced or KCl‐induced polymerization.

Depolymerization of F‐actin by molar amounts of formamide was faster and proceeded further when the polymer had been formed in the presence of KCl or CaCl2 than when it had been formed in the presence of MgCl2. It is concluded that Mg2+ stabilizes the F‐actin structure readering it more resistant than either Ca2+ or K+ against the destabilizing action of formamide.
Formation of Magnesium‐Phosphoenzyme and Magnesium‐Calcium‐Phosphoenzyme in the Phosphorylation of Adenosine Triphosphatase by Orthophosphate in Sarcoplasmic Reticulum
Authors: SUKO Josef , PLANK Brigitte , PREIS Peter , KOLASSA Norbert , HELLMANN Gertrude , CONCA Walter
Published: March 2005

The aim of the present study was to test simple reaction sequences which describe calcium‐independent plus calcium‐dependent phosphorylation of sarcoplasmic reticulum transport ATPase by orthophosphate including the function of magnesium in phosphoenzyme formation. The reaction schemes considered were based on the reaction sequence for calcium‐independent phosphorylation proposed previously; namely that the transport enzyme (E) forms a ternary complex (Mg. E. P), by random binding of free magnesium and free orthophosphate, which is in equilibrium with the magnesium‐phosphoenzyme (Mg.E‐P).

Phosphorylation, performed at pH 7.0 20°C and a constant free orthophosphate concentration using sarco‐plasmic reticulum vesicles either unloaded or loaded passively with calcium in the presence of 5 mM or 40 mM CaCl2. resulted in a gradual decrease in the apparent magnesium half‐saturation constant and an increase in maximum phosphoprotein formation with increasing calciumloads. When phosphorylation of sarcoplasmicreticulum vesicles preloaded in the presence of 5 mM CaCl2 was performed at a constant free magnesium concentration, a decrease in the apparent otthophosphate half‐saturation constant and an increase in maximum phosphoprotein formation was observed as compared with vesicles from which calcium inside has been removed by ionophore X‐537A plus EGTA treatment; however, both parameters remained unchanged by increasing free magnesium from 20 mM to 30 mM. When phosphorylation of sarcoplasmic reticulum vesicles passively loaded with calcium in the presence of 40 mM CaCl2, at which the saturation of the low‐affinity calcium binding sites of the ATPase is presumably near maximum, was performed at increasing concentrations of free orthophosphate, there was a parallel shift of phosphoprotein formation as a function of free magnesium and vice versa, with no change in the maximum phosphoenzyme formation.

Comparison of the experimental data with the pattern of phosphoprotein formation predicted from model equations for various theoretical possible reaction sequences suggests that phosphoenzyme formation from orthophosphate possesses the following features. Firstly, calcium present at the sarcoplasmic reticulum membrane binds to the free enzyme and in sequential order to E. Mg P1 or Mg E‐P or to both, but neither to E. Mg nor to E‐ P1 Secondly, calcium‐independent and calcium‐dependent phosphoproteins are magnesium‐phosphoenzymes. Calcium‐dependent phosphoenzyme is a magnesium‐calcium‐enzyme phosphate complex with 1 magnesium, 2 calciums and 1 orthophosphate (the last covalently) bound to the enzyme [Mg. E‐P. (Cal) 2], and not a ‘calcium‐phosphoprotein’ without bound magnesium.
Fragments of Ribosomal Protein S1 and Its Mutant Form m1‐S1
Authors: SUBRAMANIAN Alap R. , RIENHARDT Peter , KIMURA Makoto , SURYANARAYANA Tangirala
Published: March 2005

Escherichia coli ribosomal protein S1 and its mutant, shorter, form‐S1 were cleaved at internal methionyl residues to yield, respectively, six and five gragments of M ranging from 1000 to 24000. Methods are described to isolate the fragments in pure form. Four of the fragments (designated F2a, F2b, F3 and F4) contain between 86 and 215 amino acids and are therefore as large as other robosomal proteins. Fragments F2a, derived of S1 [S. Giorginis and A.R. Subramanian (1980) J. Mol. biol. 141, 393‐408]. Here we show that the RNA binding domain of S1 is essentially contained of S1, whose activity is modified by ligand binding, was localized in F2b, a fragment with little RNA binding capacity.

The charaterisitic RNAbinding domain and a weak riosome binding domain of S1 have previously been localized in the large trypsin‐resistent core S1‐F1 [T. Suryanarayana and A.R. Subramanian (1979) J. Mol. Biol. 127, 41–54]. Together these date indicate that two of the key functional domains S1 are located in two regions of the molecule separated by an open, exposed segment. The present study also revealed that the nonreactive ‐SH group of S1 becomes reactive m1‐S1 by the loss of remote C‐terminal region in the latter.
Cholesterol 7α‐Hydroxylase of Rat Liver
Authors: OZASA Shigeru , BOYD George S.
Published: March 2005

Cytochrome P‐450 was prepared from the liver microsomes of cholestryamine‐fed rats by solubilisation with the non‐ionic detergent Nonidet P42 followed by chromatography on a DEAE‐cellulose column and on hydroxyapatite. NADPH‐cytochrome P‐450 reductase was preapred by a technique of affinity column chromatography using 2′,5′ ADP Sepharose.

The activity of cholesterol 7α‐hydroxylase was measured in a reconstituted system of microsomal mixed function oxidase containing cytochrome P‐450 and NADPH‐cytochrome P‐450 reductase from rat liver plus cholesterol and NADPH. Endogenous cholesterol was largely depleted from the enzyme preparations by the treatment of the microsomes with cold n‐butanol/acetone.

The reconstituted system of mixed‐function oxidase catalysed a highly effective and specific 7α‐hydroxylation of cholesterol. The reconstituted system showed higher activity of cholesterol 7α‐hydroxylase than was observed in native liver microsomes. The reconstituted system had an absolute requirement for cytochrome P‐450, NADPH‐cytochrome P‐450 reductase and NADPH.

The apparent Km for cholesterol in the reconstituted system was 15 μm and the V was 1.4 nmol 7α‐hydroxycholesterol formed min−1 (nmol cytochrome P‐450)−1

The reconstituted system also catalysed the 7α‐hydroxylation of taurodeoxycholic acid, the 7α‐hydroxylation of 26‐norcholesterol and to a limited degree the 12α‐hydroxylation reactions and the 12α‐hydroxylation reaction was significantly less than the ability of the system to 7α‐hydroxylate cholesterol.
Regulation of Acetoacetyl‐CoA in Isolated Perfused Rat Hearts
Authors: MENAHAN Lawrence A , HRON W. Theodore
Published: March 2005

The role of ketone bodies and long‐chain fatty acids in regulating myocardial content of acetyl‐CoA was examined in isolated perfused hearts from fed rats. Addition of 10 mM acetoacetate to a perfusion medium containing 5.5mM glucose as exogenous sustrate resulted in a sevenfold increase of cardiac acetoacetyl‐CoA content. Inclusion of 0.75–0.94 mM palmitate in the perfusate did not augment the basal level of acetoacetyl‐CoA nor did it influence the already elevated content of the metabolic intermediate shown to exist in the presence of acetoacetate alone.

In addition to the observed increase in myocardial acetoacetyl‐CoA, a decrease in free carnitine in hearts perfused with acetoacetate was accompanied by elevated ratios of both acetylcarnitine/carnitine and long‐chain faty acylcarnitine/carnitine. Addition of palmitate in the presence of acetoacetate resulted in a further diminution of myocardial free carnitine leading to a further elevation of the long chain fatty acylcarnitine/carnitine ratio when compared to hearts perfused with the ketone body alone. The changes in myocardial CoA and their intermediates in hearts perfused with the ketone body alone. or in combination with palmitate indicate that ketone bodies can divert fatty acids away from oxidation and toward esterification as triacylglycerols. Even though the interrelationships between ketones and myocardial fatty acid metaolism have not been fully characterized, a role for ketone bodies in promoting the increase in myocardial acetoacetyl‐CoA found in diabetic ketoacidosis has been clearly defined.