bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ontogeny of the Adenosine‐3′:5′‐Phosphate‐Dependent Protein‐Kinase System during Early Uterine Development
Authors: EPPENBERGER Urs , ROOS Werner , FABBRO Doriano , SURY Adrian , WEBER Judith , BECHTEL Elke , HUBER Peter , JUNGMANN RICHARD A. 
Published: March 2005

The development of protein kinase and estrogen‐binding proteins was studied in the nuclear and cytosol fractions of rat uteri as a function of postnatal age.Analysis of cyclic‐AMP‐dependent protein kinase activity, with protamine sulfate as substrate in the presence of 3 μM cyclic AMP, revealed that the cytosol as well as the nuclear cAMP‐dependent protein kinase activity increased markedly between the third and fifth postnatal days. The total cytosol protein kinase activity remained constant after the age of 5 days (850 pmol [32P]phosphate incorporated × min−1× mg cytosol protein−1), but nuclear cAMP‐dependent protein kinase activity decreased sharply (60%), coinciding with a time period during which nuclei acquire considerable estrogen‐binding activity.Analysis of the multiplicity of cyclic‐AMP‐dependent protein kinases by chromatography of uterine cytosol on DEAE‐cellulose revealed the presence of type I and type II cyclic‐AMP‐dependent protein kinase. Type I isoenzyme levels decreased relative to the type II isozyme from the time of birth to the age of 20 days, type II isozyme did not undergo any major developmental changes.The specific estrogen binding activity in utertine cytosol reached a maximum in 12‐day‐old rats (1.7 pmol of 17β‐estradiol bound/mg protein). It declined thereafter and reached constant levels in 17‐day‐old and older rats. Concomitantly nuclear levels of estrogen binding protein increased progressively from very low levels (0.1 pmol) in 5‐day‐old rats to 1.2 pmol of 17β‐estradiol bound/mg of nuclear protein in 12‐day‐old and 20‐day‐old rats.The results indicate the early existence of a functional uterine cyclic‐AMP/protein kinase system in the neonatal rat and suggest that the protein kinase system and specific estrogen‐binding activity develop in a reciprocal manner.
Cryoenzymological Studies on Myosin Subfragment 1
Authors: TRAVERS Franck , HILLAIRE Dominique 
Published: March 2005

The ATPase activities of myosin subfragment 1 were studied under subzero conditions with ethylene glycol as the antifreeze.The pH profile of the Ca ATPase activity was affected by both ethylene glycol and the temperature but the pH profile of the Mg ATPase activity was only slightly affected.At 20°C the Mg ATPase activity was not affected by the solvent. The Ca and acto‐Mg ATPase activities decreased to similar extents as the solvent concentration was increased. These results are discussed in terms of the accessibility of the active site.The Mg, Ca and acto‐Mg ATPase activities were studied down to −20°C, in 40% ethylene glycol. All three systems showed an increase in activation energy at low temperatures. These results were compared to previous work and they were analysed in terms of a change in the rate‐limiting step.When myosin S1 is mixed with ATP, difference spectra are obtained whose amplitudes decrease with time and which are a manifestation of the intermediate M**· ADP · Pi (the product complex). The results obtained show that the disappearance of this complex occurs in the post‐steady‐state phase at 20°C but in the pre‐steady‐state phase at –20°C. These studies provide the necessary data for planning work on the stabilization of the reaction intermediates on, in particular, the actomyosin pathway. By working at –20°C in 40% ethylene glycol a 1000‐fold decrease in this activity is obtained.
A Centrifugation Study of Rat‐Liver Mitochondria, Lysosomes and Peroxisomes during the Perinatal Period
Authors: MERTENS‐STRIJTHAGEN Jeanine , DE SCHRIJVER Christian , WATTIAUX‐DE CONINCK Simone , WATTIAUX Robert 
Published: March 2005

We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de Duve et al. (1955). In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose‐6‐phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5′‐Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme we used, does not appreciably differ for the late foetal, early postnatal and adult rat livers.An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distributions of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal: foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H2O gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.
The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by 1H Nuclear Magnetic Resonance
Authors: LENSTRA Johannes A. , BOLSCHER Ben G. J. M. , STOB Sijtze , BEINTEMA Jaap J. , KAPTEIN Robert 
Published: March 2005

1. The aromatic proton resonances in the 360‐MHz 1H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments.2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His‐1 19) and two tyrosines are accessible to photo‐excited flavin. This permitted the identification of the C‐4 proton resonance of His‐119.3. The resonances of the ring protons of Tyr‐25, Tyr‐76 and Tyr‐1 15 and the C‐4 proton of His‐12 were identified by comparison with subtilisin‐modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr‐73, Tyr‐92 and Phe‐46.4. On addition of active‐site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe‐120.5. Four of the six tyrosines of bovine RNase, identified as Tyr‐76, Tyr‐115 and, tentatively, Tyr‐73 and Tyr‐92, are titratable above pH 9. The rings of Tyr‐73 and Tyr‐115 are rapidly rotating or flipping by 180° about their Cβ‐Cγ bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr‐25 is involved in a pH‐dependent conformational transition, together with Asp‐14 and His‐48. A scheme for this transition is proposed.6. Binding of active‐site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH‐dependent transition in the region of Asp‐14, Tyr–25 and His‐48.7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.
Investigation of Soluble Mitochondrial ATPase by the Reacting Enzyme Sedimentation Method
Authors: CHERNYAK Victor Ya. , KOZHANOVA Zinayda E. , CHERNYAK Boris V. , KOZLOV Igor A. 
Published: March 2005

Soluble mitochondrial ATPase from bovine heart (factor F1) loses its activity during ATP hydrolyses. The inactivation is accelerated by moderate pressure, which is generated in an ultracentrifuge cell. The rate of inactivation slows down if the concentration of the substrate (MgATP) is diminished. ATP hydrolysis proceeds at an almost constant rate if the substrate concentration is as low as 0.05 mM.One intersubunit cross‐link formed by dimethylsuberimidate per molecule of factor F1, prevents its inactivation during the ATPase reaction both without pressure and in an ultracentrifuge.Sedimentation coefficients measured by the reacting enzyme centrifugation method of both unmodified factor F1 at a low (about 0.05 mM MgATP) substrate concentration and of its dimethylsuberimidate cross‐linked form in the presence of 10 mM MgATP, were determined to be s20,w= 12.4 ± 0.4 S. The value is the same as that obtained by the conventional boundary sedimentation method in the absence of the substrate.This result testifies to the fact that the conformation of reacting factor F1 in solution is similar to that of the enzyme in the absence of the substrate.
Sodium and Calcium Binding to Panulirus interruptus Hemocyanin as Studied by 23Na Nuclear Magnetic Resonance
Authors: NORNE Jan‐Erik , GUSTAVSSON Hans , FORSÉN Sture , CHIANCONE Emilia , KUIPER Harm A. , ANTONINI Eraldo 
Published: March 2005

Addition of Panulirus hemocyanin to NaCl solutions produces marked changes in the 23Na relaxation parameters; they show that sodium ions interact with binding sites on the protein and exchange rapidly with the bulk. The observed non‐lorentzian lineshapes and the non‐exponential decay of the transverse magnetization indicate that non‐extreme narrowing conditions apply and give information on the dynamics of the interaction. Panulirus hemocyanin has at least two classes of Na+ binding sites; the binding constant of the more strongly bound sodium ions is in the order of 1 × 102 M−1. Competition between Na+ and Ca2+ for protein binding sites is demonstrated by the effect of Ca2+ on the 23Na relaxation parameters. However, only the more strongly bound Na+ are displaced by Ca2+. The number of Ca2+ needed to displace these sodium ions is 3–5 per oxygen binding site.The 23Na relaxation parameters are influenced also by the state of oxygenation of the protein, indicating a linkage between Na+ and oxygen binding. The simplest interpretation of the data is that sodium ions bind more strongly to oxyhemocyanin in agreement with oxygen equilibrium experiments.
The Proton‐Translocating Adenosine Triphosphatase of the Obligately Anaerobic Bacterium Clostridium pusteuriunum
Authors: CLARKE David J. , FULLER Frances M. , MORRIS J. Gareth 
Published: March 2005

1. The cell‐membrane ATP phosphohydrolase of vegetatively grown Clostridium pasteurianum was specifically Mg2+‐dependent, but demonstrated significant activity with GTP, CTP and UTP. It displayed approximate Michaelis‐Menten kinetics only in the presence of certain effectors (e.g. phosphoenolpyruvate, fructose 1,6‐bis‐phosphate) which decreased the Km, for ATP (to below 2 mM) but also V, whilst extending to pH 5.8 the effective pH range of activity of the enzyme.2. ATP phosphohydrolase activity of the membrane ATPase (BF0Fl) was inhibited by N,N′‐dicyclohexylcarbodiimide, butyricin 7423, Dio‐9, 4‐chloro‐7‐nitrobenzofurazan, efrapeptin, leucinostatin and quercetin, and to a lesser degree by aurovertin and citreoviridin. The enzyme was not inhibited by oligomycin, spegazzinine, tributyl tin, triethyl tin or venturicidin. The soluble ATPase (BF1) component differed in not being inhibited by N,N′‐dicyclohexylcarbodiimide, butyricin 7423 or leucinostatin.3. The ATPase (BFoF1) complex and its soluble (BF1) component were separately purified.4. Dodecylsulphate/polyacrylamide gel electrophoresis separated only four polypeptide components in the purified ATPase (BF0F1), with approximate molecular weights (± 10%) as follows: subunita, 65500; subunit c, 57500; subunit d,a, 43000; subunit fa, 15000. The soluble (BF1, component contained only the three polypeptide subunits a, c and d2. These were present in the BF0F1 preparation in the ratio 2:1:2; the contribution of subunit fa could not satisfactorily be quantified.5. Subunit a was identified as the component binding 4‐chloro‐7‐nitrobenzofurazan and sub‐unit fa as the component binding N,N′‐dicyclohexylcarbodiimide. The ATP phosphohydrolase activity of the membrane ATPase was not activated by trypsin treatment and the ATPase (BFoF1) contained no trypsin‐sensitive inhibitor protein subunit.6. Purified ATPase (BF0F1) was incorporated into artificial proteoliposomes which demonstrated ATP‐dependent enhancement of 8‐anilinonaphthalene‐1‐sulphonate fluorescence and ATP‐dependent proton influx. These reactions were abolished by proton conductors (e.g. carbonylcyanide m‐chlorophenylhydrazone), by valinomycin in the presence of a high external concentration of K+, or by N,N′‐dicyclohexylcarbodiimide, butyricin 7423, Dio‐9, 4‐chloro‐7‐nitrobenzofurazan or leucinostatin. Oligomycin, tributyl tin, triethyl tin and venturicidin were not inhibitory.7. When stripped of the soluble BF1 component, such ATPase‐proteoliposomes demonstrated nil ATP phosphohydrolase activity and did not display ATP‐dependent enhancement of 8‐anilino‐naphthalene‐1‐sulphonate fluorescence or ATP‐dependent protein influx. All of these activities were restored by incubation of the BF1‐depleted proteoliposomes with a purified preparation of the soluble BF1 component.
The Proton‐Translocating Adenosine Triphosphatase of the Obligately Anaerobic Bacterium Clostridium pasteurianum
Authors: CLARKE David J. , MORRIS J. Gareth 
Published: March 2005

1. Vesicles which demonstrated ATP‐dependent proton influx were produced from cell membranes of Clostridium pasteurianum by a cholate‐dialysis procedure which was also employed to introduce bacteriorhodopsin into such membrane vesicles and into artificial proteoliposomes.2. ATP synthetase activity was assayed using illuminated bacteriorhodopsin‐containing crude membrane vesicles plus a glucose and hexokinase ‘ATP trap’. The membrane‐bound ATPase of vegetatively grown cells of Cl. pasteurianum displayed measurable ATP synthetase activity in this assay.3. ATPase‐proteoliposomes constructed of purified ATPase (BF0F1) of Cl. pasteurianumw with bacteriorhodopsin and a mixture of phospholipids accomplished light‐dependent synthesis of ATP from ADP plus Pi. The reaction was inhibited by N,N′‐ dicyclohexylcarbodiimide and by proton conductors such as tetrachlorosalicylanilide. The specific ATP synthetase activity of the purified Cl. pasteurianum ATPase was significantly less than that of similarly purified ATPases (BF0F1) from Escherichia coli, Streptococcus faecalis and Streptococcus pleomorphus. The specific ATP synthetase activity of the ATPase of Clostridium formicoaceticum was greater when the enzyme complex was derived from fumarate‐grown cells then when it was purified from organisms grown on fructose.4. The apparent Km value (for Mg2+‐ADP‐Pi) displayed by the ATPase of Cl. pasteurianum when acting as an ATP synthetase was much higher than the apparent Km, value (for ATP) in ATP phosphohydrolysis. A similar disposition to serve as an ATP phosphohydrolase was displayed by the ATPase of fructose‐grown Cl. formicoaceticum, but the ATPase from fumarate‐grown cells of this organism was substantially more effective in ATP synthesis.5. The ATP synthetase activity of Cl. pasteurianum ATPase (BF0F1) was as susceptible as was its ATP phosphohydrolase activity to inhibition by dicyclohexylcarbodiimide, butyricin 7423, Dio‐9, 4‐chloro‐7‐nitrobenzofurazan, quercetin and citreoviridin and was similarly insensitive to inhibition by triethyl tin and tributyl tin.
Characterization of Human Platelet Proteins Solubilized with Triton X‐100 and Examined by Crossed Immunoelectrophoresis
Authors: HAGEN Inger , BJERRUM Ole Jannik , SOLUM Nils Olav 
Published: March 2005

Whole human platelets and platelet membranes have been solubilized in 1% Triton X‐100, and the solubilized proteins examined by crossed immunoelectrophoresis using rabbit antibodies raised against either whole platelets or isolated membranes. 90% of the platelet proteins were solubilized by this extraction. About twenty immunoprecipitates were observed using the extracts obtained from whole platelets, whereas normally eight immunoprecipitates were seen with extracts from isolated membranes. Albumin, factor VIII and fibrinogen were identified with monospecific antibodies.Correlation of the patterns obtained for platelets or membranes was obtained by addition experiments, by crossed‐line immunoelectrophoresis and by crossed immunoelectrophoresis of a mixture of extracts from unlabeled whole platelets and membranes isolated from platelets labeled by lactoperoxidase‐catalyzed 125I iodination.Four sialoglycoproteins were identified by their reduced electrophoretic migration after neuraminidase treatment, and six proteins interacted with various lectins, indicating them to be glycosylated.Seven amphiphilic proteins were identified by charge‐shift crossed immunoelectrophoresis, and nine by crossed hydrophobic interaction immunoelectrophoresis with phenyl‐Sepharose.The topographical arrangement of the membrane proteins was examined with lactoperoxidase‐catalyzed 125I‐labeled platelets as antigens, and by antibodies absorbed with a suspension of whole platelets. Four and six radioactively labeled precipitates could be identified using the platelet and membrane extracts, respectively, indicating them to be exposed at the outer platelet surface. This was confirmed by the use of antibodies absorbed with intact platelets.
Biological Role of Gramicidin S in Spore Functions
Authors: MARAHIEL Mohamed A. , DANDERS Wolf , KRAUSE Michael , KLEINKAUF Horst 
Published: March 2005

Gramicidin‐S‐negative mutants of Bacillus brevis ATCC9999 have been isolated with a remarkly higher yield after ethidium bromide or acridine orange treatment, than after N‐methyl‐N'‐nitro‐N‐nitrosoguanidine treatment. Four (MIV, Smr170, R5 and EB 16) of 38 isolated mutants were characterized with respect to the lesion in gramicidin‐S‐synthesizing activity. The mutants sporulate to the same extent as the parental strain except mutant Smr 170 which sporulates less. However, mutant spores were more heat‐sensitive and possessed a reduced level of dipicolinic acid content. No significant difference was observed in the germination time of wild‐type and mutant spores. All spores germinated after 80–110 min, but the outgrowth time was different: all gramicidin‐S‐negative mutants grew out immediatly after germination whereas wild‐type spores required a lag period of 9–10 h. When the mutants were allowed to sporulate in the presence of gramicidin S, the spores were found to be heat‐resistant and their outgrowth postponed to the same period as the parent spores. The addition of gramicidin also eliminated the deficiency of dipicolinic acid.A new class of gramicidin‐S‐negative mutant, R5, which only activates L‐valine and L‐leucine, is described. A possible biological function of gramicidin S in the heat‐resistance and in the timing of spore outgrowth is discussed.
Identification of the C‐1‐Phosphate‐Binding Arginine Residue of Rabbit‐Muscle Aldolase
Authors: PATTHY László , VÁRADI András , THÉSZ János , KOVÁCS Katalin 
Published: March 2005

The arginine‐specific reagent 1,2‐cyclohexanedione reacts selectively with the arginine residue of the C‐1‐phosphate‐binding site of aldolase and inactivates the enzyme. The labeled peptide isolated from tryptic digests of inactivated aldolase was found to correspond to the sequence Leu‐43 to Arg‐56, the residue modified by cyclohexanedione being Arg‐55. This peptide was absent from digests of aldolase treated in the same way but protected from inactivation by the presence of substrate, thus correlating modification of Arg‐55 with loss of activity.Selective isolation of the peptide containing the modified arginine residue was effected by chemisorption chromatography on boric acid gel, a procedure exploiting the specific interaction of matrix‐bound boric acid groups with vicinal m‐hxdroxyl groups of cyclohexanedione‐modified arginine side chains.
The Two Major Isozymes of Yeast Alcohol Dehydrogenase
Authors: WILLS Christopher , JÖRNVALL Hans 
Published: March 2005

The structures of the two cytoplasmic isozymes of alcohol dehydrogenase in a haploid strain of the yeast Saccharomyces cerevisiae have been compared and related to a commercial preparation of the protein that was previously analyzed. The constitutive isozyme I (purified from a petite cell incapable of mitochondrial respiration) is found to correspond closely to the commercially available but taxonomically uncharacterized enzyme. After analysis of 86 % of all positions, two amino acid differences were found among the 298 residues compared. The enzymes from these sources are thus about 99 % identical.The inducible isozyme II (purified from aerobically grown grande cells) differs substantially from isozyme I. After analysis of 82% of all positions, there are 15 amino acid exchanges in relation to isozyme I, and 17 in relation to the commercial preparation among the 284 residues compared. Some of the differences established between isozymes I and II explain heterogeneities previously observed in yeast alcohol dehydrogenase.The amino acid sequences can be matched without insertions or deletions, and 15 of the 17 differences are interpretable as single base changes. The close homology of the two isozymes shows that they were produced from a gene duplication which is unexpectedly recent in view of the functional differences of the isozymes.Differences in patterns of subunit cooperativity and in Km values have been found between the isozymes. Reflecting this, four of the differences between forms I and II appear to correspond to positions close to the interface between subunits in the tertiary structure of the related liver alcohol dehydrogenase, while two apparently correspond to residues near the inter‐domain cleft of that subunit. No residues with known catalytic functions are, however, exchanged.Normally, the amino terminus of the yeast enzyme is blocked by an acyl group, but a significant amount of the isozyme II preparation was found to have a free amino terminus. The presence of unblocked molecules is very rare among acetylated proteins, but may lead to further insight into possible roles of α‐amino‐acetylation.