bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Comparison of Vesicles Derived from Terminal Cisternae and Longitudinal Tubules of Sarcoplasmic Reticulum Isolated from Rabbit Skeletal Muscle
Authors: LOUIS Charles F. , NASH‐ADLER Priscilla A. , FUDYMA Gary , SHIGEKAWA Munekazu , AKOWITZ Alfred , KATZ Arnold M. 
Published: July 2010

Sarcoplasmic reticulum vesicles were separated into heavy (derived from terminal cisternae) and light (derived from longitudinal tubules) fractions, according to Meissner [Biochim. Biophys. Acta, 389, 51–68 (1975)]. The similar Ca2+ sensitivities of phosphoprotein formation, ATPase activity and calcium uptake, and the similar phosphoprotein turnover rates (ATPase/phosphoprotein formation) of both fractions indicate that the same ATPase enzyme is present in the terminal cisternae and longitudinal sarcoplasmic reticulum. The higher V for Ca2+‐activated ATPase activity and calcium uptake in the light fraction correlated with the higher concentration of ATPase enzyme per mg of membrane protein in this fraction. In both the presence and absence of calcium‐precipitating anions, the light fraction stored more calcium than the heavy. The Ca2+ dependence of calcium release after addition of EGTA appeared similat in both fractions, but the rate Of calcium release was more rapid in the light fraction.These findings suggest that calcium release may occur more rapidly from longitudinal than terminal cisternae portions of the sarcoplasmic reticulum and that calcium release, like calcium uptake, may be mediated by the ATPase enzyme in the sarcoplasmic reticulum membrane.Although the activation energies for Ca2+‐activated ATPase activity above and below the transition temperature were significantly different for the heavy and light fractions, their transition temperatures were similar. Partial purification of the ATPase enzyme by deoxycholate treatment modified the activation energies of the light but not the heavy fraction and caused the activation energies to become similar. The phosphoprotein levels of heavy and light vesicles did not become similar after deoxycholate treatment, although gel electrophoretograms indicated both samples contained > 90% ATPase protein. These results indicate the protein‐lipid associations in these two fractions may be different.
A Flavin‐Mononucleotide‐Binding Site in Hansenula anomala Nicked Flavocytochrome b2, Requiring the Association of Two Domains
Authors: GERVAIS Michel , LABEYRIE Françoise , RISLER Yanick , VERGNES Odile 
Published: July 2010

Previous experiments in our laboratory with Saccharomyces cerevisiae flavocytochrome b2 indicated that both fragments α and β of the enzyme isolated after cleavage by yeast proteases are required to form the flavin site.More detailed experiments have now been carried out on the nicked Hansenula anomala enzyme obtained by tryptic cleavage. A method has been devised that gives a quantitative separation in 4 M urea of β, and α with its heme still bound. The characteristics of the various species: isolated α and β and mixed α+β were studied in 4 M urea and after elimination of this reagent by dialysis in the presence of FMN and 2‐mercaptoethanol. Several methods, including heme spectroscopy, tryptophan fluorescence, sedimentation studies, and titration of bound flavin, were used. The results indicate that isolated α and β have a folded globular structure after renaturation. The flavin binding to the α+β mixture was important (50–100 %) with recovery of the flavodehydrogenase activity. In contrast, binding was not detectable (< 0.5%, Kd> 10 mM) for isolated α and β. As far as mononucleotide binding is concerned, such a cooperative requirement for two folding domains has never before been reported in other enzymes.The present results are discussed together with others obtained in our laboratory which demonstrate that, as deduced from their sensitivity to trypsin, the structure of S. cerevisiae and H. annomala flavocytochrome b2 protomers is triglobular ‘n‐x‐β’ (n and x combined within α). The tetramer assembly, which remains intact as a nicked enzyme (αβ)4 after the first trypsin cleavage, is broken down following a second cleavage of the chain into four cytochrotne cores (n) and a functional T‐flavodelhydrogenase entity, a tetramer of the type (xβ)4.
Stepwise Cleavage of the Pro Part of Promelittin by Dipeptidylpeptidase IV
Authors: KREIL Günther , HAIML Liselotte , SUCHANEK Gerda 
Published: July 2010

Melittin, the main constituent of honeybee venom, is derived from promelittin. In the amino acid sequence of the ‘pro’ region of this precursor, every second residue is either proline or alanine. The possibility has been investigated that activation of promelittin might proceed via sequential liberation of dipeptides catalyzed by a dipeptidylpeptidase IV. As substrates we used promelittin isolated from queen bees fed with radioactive proline, and enzymatic fragments of prepromelittin which contained the entire pro part and the NH2‐terminal hexapeptide of melittin.It could first be demonstrated that pig kidney dipeptidylpeptidase IV releases dipeptides from the pro part. An enzyme of this type could then be detected in extracts from venom glands of queen bees, which also contain a dipeptidase. After inhibiting the latter enzyme with mersalyl, a stepwise cleavage of dipeptides, starting at the amino end of the pro region, could be demonstrated. This hydrolysis did not proceed into the melittin sequence. Furthermore, fragments with an extra residue at the amino end, which therefore had the wrong ‘reading frame’ for the dipeptidylpeptidase, were not hydrolyzed. With intact promelittin as substrate the rate of hydrolysis was always lower than with the fragments.The results presented in this paper suggest a new type of precursor‐ product conversion proceeding via stepwise cleavage of dipeptide units. Our experimental evidence also ascribes a biological function to a dipeptidylpeptidase IV, it type of enzyme widely distributed in animal tissues. The evidence, that the observed reaction in vitro reflects the mechanism of promelittin activation in vivo is discussed.
Yeast Mutants Defective in Acetyl‐Coenzyme A Carboxylase and Biotin: Apocarboxylase Ligase
Authors: MISHINA Masayoshi , ROGGUENKAMP Rainer , SCHWEIZER Eckhart 
Published: July 2010

Among more than 7000 mutants of Saccharomyces cerevisiae, requiring saturated fatty acids, 61 acetyl‐CoA‐carboxylase‐deficient strains have been identified. According to their mutual complementation characteristics these mutants have been assigned to two different genes, acc1 and acc2. Both acetyl‐CoA carboxylase genes tire unlinked to each other and to the fatty acid synthetase genes fas1 and fas2.The acetyl‐CoA carboxylases of several acc1 and acc2 mutants have been purified and assayed for their overall and component enzyme activities. Besides overall acetyl‐CoA carboxylation, which was lost in all cases, both component enzymes, biotin carboxylase and transcarboxylase, were simultaneously affected in most mutants, though often to a different relative extent.Similarly, the comparison of biochemical and genetic complementation data revealed no basis for a clear distinction between specific biotin carboxylase and transcarboxylase mutants. These results Suggest that accl is a cluster gene coding for a multifunctional protein harboring both acetyl‐CoA carboxylase component enzyme activities on the same polypeptide chain.The acetyl‐CoA carboxylase isolated from acc2 mutants was free of biotin. Correspondingly, biotin:apoacetyl‐CoA‐carboxylase ligase activity was missing in acc2 mutants. Therefore, it is concluded that the primary defect in acc2 mutants is in the biotin:apocarboxylase ligase. In agreement with this conclusion, the acc2 acetyl‐CoA carboxylase can be activated, in the presence of biotin and ATP, by ligase preparations from wild‐type or acc1 muttant cells. By the use of these mutants, evidence was obtained that in vivo the biotinylation of both acetyl‐CoA carboxylase and pyruvate carboxylase is catalyzed by the same ligase.
The Number of Functional Active Sites per Molecule of the Adenosylcobalamin‐Dependent Enzyme, Ethanolamine Ammonia‐lyase, as Determined by a Kinetic Method
Authors: HOLLAWAY Michael R. , JOHNSON Alan W. , LAPPERT Michael F. , WALLIS O. Caryl 
Published: July 2010

A kinetic approach to the determination of the number of functional active sites per molecule of the adenosylcobalamin‐dependent enzyme. ethanolatnine ammonia‐lyase, is described.
Time courses for formation and breakdown of a cob(II)alamin intermediate during reaction of the enzyme, fully saturated with adensycoalaminm, with tninopropanol as substrate, were followed using a stopped‐flow spectrophotometer under two conditions: (a) enzyme concentration much greater than that of substrate, (b) substrate concentration much greater than that of enzyme.
Results were analysed in terms of a three‐step mechanism involving binding of substrate (k2+ step), cob(II)alamin formatiUn (K3+z step) and cob(II)alatnin breakdown (k3+ step). The kinetic scheme was shown to be suffitiient to account for the observed time courses and rate constants of 80 s‐1 (k+2) and 1.5 s‐1 (K+3) were determined.
The number of active sites per enzyme molecule (n) was calculated from the kinetic data in three Ways: (a) calculation from amplitude of absorbance measurements, (b) calculation from measurements of the values of rate constants and (c) analysis by computation of the kinetic data using the computer program FACSIMILE. A value for n close to 6 was calculated by each of these methods. This value is in disagreement with the literature value of about two sites‐per molecule but is consistent with the I6II6 subunit structure of the enzyme.
Kinetic analysis of data from experiments in which the adenosylcobalamin concentration was varied while substrate anti enzyme concentrations remained constant showed that all the active sites function with identical rate constants.
The principle and mathematical basis of the kinetic method for determining the value of n is given as an Appendix.
Sulfhydryl Reagents Prevent Dephosphorylation and Proteolysis of Histones in Isolated HeLa Metaphase Chromosomes
Authors: PAULSON James R. 
Published: July 2010

It has been shown by D'Anna et al. [Nucleic Acids Res. 5, 3195–3207 (1978)] that histones H1 and H3, which are highly phosphorylated during mitosis in mammalian cells, become rapidly dephosphorylated during conventional metaphase chromosome isolation procedures.We show here that this dephosphorylation can be completely prevented by including sulfhydryl reagents, such as p‐chloromercuriphenyl sulfonate or 5,5′‐dithiobis(2‐nitrobenzoate), in the chromosome isolation buffers. These reagents also efficiently inhibit the endogenous proteases present in isolated HeLa chromosomes and nuclei.
The Metabolic Fate of Mitochondrial Hydrogen Peroxide
Authors: NOHL Hans , JORDAN Werner 
Published: July 2010

Mitochondrial H2O2formation is not in equilibrium with defence mechanisms that counteract an accumulation of H2, in rat‐heart cell.A model for the accumulation kinetics is proposed which is consistent with the data presented.Four different pathways of H2O2metabolism are described in rat‐heart mitochondria. The major site for metabolic branching of H2O2Via different routes was found to be the mitchondrial catalase.Glutathione (GSH) peroxidase accounts for only 15% of intramitochondrial H2O2metabolism, while catalase‐mediated destruction is four times more rapid.Catalase activity is limited by its structural compartmentation in the matrix, while GSH peroxidase activity was found to be dependent on the availability of free GSH.Catalase was shown to protect rat‐heart mitochondria from upsetting redox states of GSH and pyridine nucleotides following H2O2, decomposition by GSH peroxidase.Computer simulations of experimental data Suggest the existence of a third sink for mitochondrial H2O2, possibly due to mitochondrial formation of OH′ radicals; another fraction of the H2O2matrix pool may cross the mitochondrial membrane and accumulate in the cytosol.
Localization of Phosphoproteins and of Protein Kinases in Chromatin from Hepatoma Tissue‐Cultured Cells
Authors: KITZIS Alain , TICHONICKY Lydie , DEFER Nicole , KRUH Jacques 
Published: July 2010

An important role in the control of gene expression has been attributed to phosphoproteins present among chromatin, non‐histone proteins. In a previous work we have shown that at least part of these phosphoproteins are associated with nucleosomes. In this work we wanted to establish whether this association occurs with all nucleosomes or with the nucleosomes present in fragments preferentially released by a mild micrococcal nuclease digestion, which originated essentially from active parts of chromatin.Phosphoproteins were labelled in vivo by incubating hepatoma tissue‐cultured cells with [32P]phosphate and chromatin was submitted to a limited micrococcal nuclease digestion. The released fragments were fractionated by preparative gel electrophoresis. [32P]Phosphoproteins were essentially found in the smallest released fragments; monomers and dimers of nucleosomes. The same result was obtained when the phosphoproteins were labelled in vitro by incubating each fragment obtained by the preparative electrophoresis in the presence of [γ‐32P]ATP. It indicates that part of the protein kinase activity was strongly bound to the particles. The bound phosphoproteins were analysed by sodium dodecylsulfate/polyacrylamide gel electrophoresis. Two main polypeptides were characterized: phosphopeptide a, Mr 41000, present in all small fragments; phosphopeptide b, Mr 31000, present in all small fragments, except in the fastest moving nucleosomes.Phosvitin kinase was found associated with the small released fragments, its specific activity was by far the highest in the fraction which includes the dieters of nucleosomes.It is concluded that phosphoproteins and protein kinases are associated with the nucleosomes of the active parts of chromatin, which suggests a role of these proteins in the control of gene expression.
The Neutral and Acidic Glyceroglucolipids from Gastric Mucous Barrier of Rat‐Stomach Antrum, Body and Forestomach
Authors: SLOMIANY Bronislaw L. , GALICKI Nina I. , KOJIMA Koichi , SLOMIANY Amalia 
Published: July 2010

Distribution of glyceroglucolipids in the mucous barrier of rat‐stomach antrum, body and forestomach was investigated. Surface mucus lining and preformed intracellular mucus were obtained by perfusion in vivo of the ligated stomach compartments with saline and 2 M NaCl. Lipids were extracted from dialyzed and lyophilized samples, and separated on DEAE‐Sephadex into neutral and acidic fractions. The glyceroglucolipids contained in each fraction were quantified and then separated into individual components by thin‐layer chromatography. Significant differences in the concentration of glyceroglucolipids in the separated areas of the stomach were found. The content of glyceroglucolipids in the antral portion of the stomach was five‐times greater than that of the forestomach. The level of sulfated glyceroglucolipids was eight‐times greater in the antrum as compared to the body and four‐times greater as compared to the forestomach. One sulfated glyceroglucolipid was present in antral perfusates, whereas the body and forestomach contained two additional acidic glyceroglucolipids. Based on data of partial acid and alkaline methanolyses, oxidation with periodate and chromium trioxide, and methylation of the native and desulfated compound, the structure of antral glyceroglucolipid is proposed to be: SO3H(→6)Glc(α1→6)Glc(α1→6)Glc(α1→6)Glc(α1→3)‐1(3)‐O‐alkyl‐2‐O‐acylglycerol. The neutral glyceroglucolipid fractions from each area of the stomach were similar in composition and only variations in their concentration were observed.The data obtained suggest that the sulfated glyceroglucolipids are involved in the protection of the gastric mucosa of the antrum, since their concentration is greatest in this most acidic portion of the stomach which is also most often exposed to duodenal reflux.
The Binding of Oxidised Coenzyme to Bovine‐Liver Glutamate Dehydrogenase Studied by Circular‐Difference Spectroscopy
Authors: BAYLEY Peter M. , O'NEILL Kevin T. J. 
Published: July 2010

The binding of NAD+ to glutamate dehydrogenase may be followed quantitatively by titration, using high‐sensitivity circular dichroism (CD) difference spectroscopy.
The CD of the bound coenzyme in the binary complex E · NAD closely resembles that of bound ADP, although the affinity is much lower, being 350‐fold less for NAD+ at 20°C in 0.1 M phosphate, pH 7.
A family of CD spectra may be analysed by unconstrained linear regression assuming only three components: free enzyme, free coenzyme, and a single binary complex, E · NAD.
Taking the molar CD of bound ADP as representing the molar CD of the adenine chromophore of bound NAD+, the linear regression shows the formation of a simple 1:1 complex E · NAD with Kd= 0.72 mM in a simple binding process without positive or negative cooperativity.
NADP+ binding is more than 10‐fold weaker than NAD+ binding.
From the similarity of the CD of bound ADP and bound NAD+ it is probable that NAD+, in forming a simple binary complex, binds preferentially at the regulatory (adenine nucleotide) binding site (site II).
Direct evidence has been obtained for the binding of a second molecule of NAD+ to the ternary complex E · NAD‐glatarate. This process occurs with low affinity and is probably also located at the adenine regulatory site.
This second‐site binding of NAD+ may contribute to the phenomena of non‐Michaelis‐Menten kinetics and apparent negative homotropic interactions in the binding of NAD+, previously attributed to subunit‐subunit cooperative interactions.
Non‐specific Biosynthesis of Hopane Triterpenes by a Cell‐Free System from Acetobacter pasteurianum
Authors: ROHMER Michel , ANDING Claude , OURISSON Guy 
Published: July 2010

A cell‐free syqtem from the bacterium Acetobacter pasteurianum was incubated with [12‐3H]‐squalene; diploptene and diplopterol, hopanoids normally present in the bacterium, were labelled. Their radioactivity was confirmed by purification using thin‐layer chromatography, synthesis of derivatives and recrystallization to constant specific activity. This demonstrates the direct cyclization of squalene into diploptene and diplopterol, catalysed by a squalene cyclase activity in A. pasteurianum.
The same cell‐free system transformed (RS)‐2,3‐epoxy‐2,3‐dihydro‐[12,13‐3H]squalene into labelled 3α‐hydroxyhop‐22(29)‐ene, 3β‐hydroxyhop‐22(29)‐ene, hopane‐3α,22‐diol and hopane3β,22‐diol. Their radioactivity was similarly confirmed. This bacterial homogenate is thus capable of cyclizing an unnatural substrate, 2,3‐epoxy‐squalene, into 3‐hydroxyhopanoids normally absent in the bacterium.
The 3α‐hydroxy and 3β‐hydroxyhopanoids could have been enzymatically interconverted via the 3‐oxo compound. Synthetic racemic (RS)‐2,3‐epoxy‐2,3‐dihydro‐[3‐3H]squalene was incubated and gave rise to 3‐3H‐labelled 3α and 3β‐hydroxyhopanoids. This excludes an isomerization via a 3‐oxo compound which would give unlabelled 3‐hydroxyhoparoids.
In conclusion, the cyclase of A. pasteurianum accepts the replacement of the normal substrate, saualene, by the corresponding cpoxide. Furthermore it is not selective in the stereochemistry of the epoxide and cyclizes both enantiomers, contrary to the epoxysqualene cyclase of eukaryotes.
Metabolism of NAD and N1‐Methylnicotinamide in Growing and Growth‐Arrested Cells
Authors: JOHNSON George S. 
Published: July 2010

Nicotinamide is metabolized primarily into NAD and N1‐methylnicotinamide in cultured cells of normal rat kidney. Tne metabolic pathways for the nicotinamide metabolites are independently regulated and are influenced by the growth stage of the cells. N1‐Methylnicotinamide levels are 1.5‐2‐fold elevated to cells growth‐arrested by treatment with histidinol, thymidine, or picolinic acid, or by serum starvation. This increase is due to a more rapid rate of synthesis rather than a decrease in excretion. The rates of both synthesis and degradation of NAD are increased in serumstarved cells so that the NAD concentration is the same as it is in growing cells. NAD and N1methylnicotinamide levels are not significantly increased when the intracellular nicotinamide concentration is increased 20‐fold by addition of excess nicotinamide to the culture medium, demonstrating that the size of the nicotinamide pool does not limit synthesis of these compounds.In medium containing normal amounts of nicotinamide, the apparent first‐order rate constant for the decay of NAD, radioactively labeled in the nicotinamide moiety, is about 4 h‐1. Labeled N1‐methylnicotinamide is not metabolized, but rather is excreted into the medium with a first‐order rate constant of 3.9 h−1. The rate of loss of label from NAD, but not from N1‐methylnicotinamide, is increased about twofold by addition of excess nicotinamide to the culture medium. This could be explained by a dilution of a labeled nicotinamide pool which is formed during NAD degradation and which is recycled into NAD but not into N1‐methylnicotinamide.The results demonstrate a rapid turnover of NAD at the bond joining nicotinamide and ADPribose, in agreement with previous studies. In addition, the results show that nicotinamide is metabolized into N1‐methylnicotinamide with what appears to be a carefully regulated synthetic mechanism. The existence of significant amounts of N1‐methylnicotinamide in cultured cells raises the question of the physiological importance of this compound.
Benzil, a Potent Activator of Microsomal Epoxide Hydrolasein vitro
Authors: SEIDEGÅRD Janeric , DEPIERRE Joseph W. 
Published: July 2010

Benzil was found to be a very potent activator of microsomal epoxide hydrolase activity (measured with styrene oxide as substrate)in vitro. The activating effect was uncompetitive and benzil causes approximately ninefold increases in both the apparentVand the apparentKmof the enzyme(s). The half‐maximal effect on activity was obtained as a 0.3 mM concentration of benzil.The activating effect obtained with benzil was found to be very specific, since a variety of structurally related compounds had little or no effect on microsomal epoxide hydrolase activity. In order to obtain indications for the existence of more than one microsomal epoxide hydrolase the effect of benzil on this activity from rats induced with phenobarbital, 3‐methylcholanthrene, 2‐acetylaminofluorene,trans‐stilbene oxide, and benzil was tested. The differences observed were minor.