bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Cations on High‐Affinity and Low‐ Affinity ATP‐Binding Sites of (Na+, K+)‐ATPase as S+tudied by Disulfides of Thioinosine Triphosphate and Its Analogue
Authors: PATZELT‐WENCZLER Rosemaric , MERTENS Wolfgang 
Published: March 2005

Binding of the disulfides of thioinosine triphosphate and thioinosine 5′‐[β,γ‐methylene]triphosphate, (SnoPPP), and (SnoPP[CH2]P)2, to the (Na+,K+)‐ATPase in the presence of 10mM EDTA results in an inactivation of the enzyme.
K+, Rb+ and Cs+ protect the high‐affinity and low‐affinity binding sites of (Na+,K+)‐ATPase against inactivation by (SnoPPP), and (SnoPP[CH2]P)2. The protection constants increase with the atomic radii of the monovalent cations.
The protective effects of low concentrations of K +, Rb+ and Cs+ on the inactivation of the (Na+,K+)‐ATPase by (SnoPP[CH2]P)2 are smaller than the effects on the inactivation by (SnoPPP)2. The electronegativity of the bridge atom between β and θ phosphate groups obviously has a function on the mode of action of monovalent cations.
High concentrations of K+, Rb+ and Cs+ accelerate the inactivation of the high‐affinity and low‐affinity binding sites of the (Na+,K+)‐ATPase by (SnoPP[CH2]P)2.
Na+ concentrations below 10 mM advance the inactivation of the high‐affinity and low‐affinity binding sites of the (Na+, Kplus;)‐ATPase by (SnoPPP)2 and (SnoPP[CH2]P)2. Na+ concentrations above 10 mM protect against inactivation.
K+ raises the activatingeffect in the presence of low concentrations of Na+ by inactivation through (SnoPPP)2 and (SnoPP[CH2]P)2. The protective effect of higher concentrations of Na+ is amplified by K+, when using (SnoPPP)2, whereas it is reduced in the case of (SnoPP[CH2]P)2.
Mg2+ concentrations up to 5 mM advance the inactivation of the high‐affinity and low‐affinity binding sites of the (Na+, K+)‐ATPase by (SnoPPP)2 and (SnoPP[CH2]P)2. Higher concentrations of Mg2+ have a protective effect.
The monovalent cations (Na+, K+, Rb+, Cs+) and Mg2+ act in a noncompetitive manner with respect to nucleotide binding. It is therefore concluded, that they are effective by inducing conformational changes of the enzyme.
Control of Ketogenesis and Fatty‐Acid Synthesis at the Mitochondrial Branch‐Point for Acetyl‐CoA in the Chick Liver Cell: Effect of Adenosine 3′,5′‐Monophosphate
Authors: MOONEY Robert A. , LANE M. Daniel 
Published: March 2005

Previous work has shown that dibutyryl adenosine 3′,5′‐monophosphate (Bt2cAMP) markedly activates ketogenesis from fatty acids released during the mobilization of cytoplasmic triacylglycerol‐rich vesicles in the chick liver cell. In the present investigation, activation of the formation of [14C]acetoacetate and 3‐[14C]hydroxybutyrate from [1‐14C]oleate by the cyclic nucleotide was shown to occur at two mitochondrial sites: (a) the l‐(–)‐carnitine‐dependent translocation of the fatty acid across the inner mitochondrial membrane and (b) the intramitochondrial metabolism of acetyl‐CoA.The hypothesis was tested that cAMP regulates ketogenesis and fatty acid synthesis by controlling acetyl‐CoA flux at the mitochondrial branch‐point leading into the 3‐hydroxy‐3‐methylglutaryl‐CoA cycle or to citrate formation. Bt2cAMP caused an immediate activation of acetoacetate formation and a concomitant inhibition of fatty acid synthesis from acetate. Evidence is presented that these effects are due to a cyclic‐nucleotide‐induced fall in the mitochondrial concentration of oxaloacetate. An assay for oxaloacetate, sufficiently sensitive to analyze extracts of cells in monolayer culture, was employed. With this assay, Bt, cAMP was shown to cause a > 90% decrease in cellular oxaloacetate concentration. The calculated level to which intramitochondrial oxaloacetate fell was below the Km for oxaloacetate in the citrate‐synthase‐catalyzed reaction and, therefore, was sufficient to curtail citrate formation.Lactate, a precursor of oxaloacetate and an antiketogenic agent, reversed the effects of Bt2cAMP. Under conditions where glucose was sole precursor of oxaloacetate and citrate, the activation of ketogenesis and the inhibition of fatty acid synthesis by Bt2cAMP were reversed by lactate. Lactate also reversed the Bt2cAMP‐induced decrease in oxaloacetate and citrate concentration. The fact that lactate enters the pathway between glucose and oxaloacetate indicated that the site of inhibition by Bt, cAMP lies above pyruvate in the glycolytic pathway. Based on these and previous results it appears that, by inhibiting glycolysis, cAMP exerts long‐range effects on two major pathways in the chick hepatocyte, ketogenesis and fatty acid synthesis.
The Effect of pH and d‐Glycerate 2,3‐Bisphosphate on the O2 Equilibrium of Norma1 and SH(β93)‐Modified Human Hemoglobin
Authors: ANTONINI Eraldo , CONDÒ Saverio G. , GIARDINA Bruno , IOPPOLO Carmela , BERTOLLINI Albcrto 
Published: March 2005

The present paper reports data for the effect of pH and d‐glycerate 2,3‐bisphosphate (d‐glycerate‐2,3‐P2) on the oxygen equilibrium of norma1 and SH(β93)‐modified human hemoglobin. At sufficiently high d‐glycerate‐2,3‐P2 concentrations, both oxy and deoxy forms of HbA are saturated with the organic phosphate at a1l pH va1ues between 6 and 9. Furthermore the difference in the affinity for d‐glycerate‐2,3‐P2 between deoxy and oxy hemoglobin remains constant with pH, implying that the pK va1ues of the Bohr effect groups in deoxy and oxyhemoglobin are not affected by the presence of d‐glycerate‐2,3‐P2 on the hemoglobin molecule.In the hemoglobins modified in position β93, the difference in affinity between deoxy and oxy hemoglobin for d‐glycerate‐2,3‐P2 decreases with decrease in pH due mainly to a decrease in the affinity of the deoxy form for d‐glycerate‐2,3‐P2. The effect of the chemica1 modification appears to be primarily a change in pK of a Bohr group in deoxy hemoglobin which is especia1ly pronounced in the presence of phosphates.
Isolation and Some Properties of the Restriction Endonuclease BcnI from Bacillus centrosporus
Authors: PETRUŠYTĖ Marytė , JANULAITIS Arvydas 
Published: March 2005

A specific type–II restriction endonuclease BcnI from Bacillus cenfrosporus has been purified to electro–phoretic homogeneity in three chromatographic steps. Around 15 μg of such a preparation can be isolated from 1g of the cell paste. The yield of the enzyme is higher than that of any type–II restriction endonuclease so far reported.The molecular weight of the enzyme determined by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate equals 27 500 and 28000 respectively.The activity of the restriction endonuclease is maximal at pH 9.2 and 40–45 °C. The optimal magnesium concentration was estimated to be 7.5 mM. The activity of BcnI may also be observed in the presence of Co2+, Mn2+, Ni2+ and Zn2+ but it is markedly less than in the presence of Mg2+.
Purification and Some Properties of Tyrosine 3‐Monooxygenase from Rat Adrenal
Authors: OKUNO Sachiko , FUJISAWA Hitoshi 
Published: March 2005

Tyrosine 3‐monooxygenase was purified to homogeneity, as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, from rat adrenal. The specific activity of the final preparation was approximately 1600 nmol min−1 mg protein−1, which was much higher than the highest yet reported. The enzyme was markedly stabilized in the presence of glycerol, Tween 80 and EDTA. As judged by gel filtration on Ultrogel AcA 34, sodium dodecyl sulfate/polyacrylamide gel electrophoresis and cross‐linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 59000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 6.6 in its absence. Amino acid analysis of the enzyme revealed a fairly high content of serine residues in this protein.Purification of the enzyme caused changes in the kinetic properties of the enzyme. The Km for 2‐amino‐4‐ hydroxy‐6‐methyl‐5,6,7,8‐tetrahydropteridine decreased from 220 μM to 58 μM. The pH profile for the enzyme activity became more broad and the pH optimum was changed from an acid pH to a neutral pH. Although polyanions, such as heparin and dextran sulfate, markedly stimulated the activity of crude enzyme by increasing the V, they were much less effective in the activation of purified enzyme. A marked stimulation of the enzyme activity by phospholipids, such as phosphatidylserine, phosphatidylinositol, phosphatidylcholine and phos‐ phatidylethanolamine, were not observed in both pure and crude preparations even at low concentrations of the pterin cofactor.
Effect of Secondary Structure of Messenger Ribonucleic Acid on the Formation of lnitiation Complexes with Prokaryotic and Eukaryotic Ribosomes
Authors: ZAGÓRSKA Ludwika , CHROBOCZEK Jadwiga , KLITA Stefan , SZAFRAŃSKI Przemystaw 
Published: March 2005

The effect of modification of the secondary structure of phage f2 RNA and brome mosaic virus (BMV) RNA 3 on the formation of initiation complexes in Escherichia coli and wheat germ protein‐synthesizing systems was studied. Modification of the RNAs was achieved by using O‐methylhydroxylamine, which specifically reacts with cytosines; this leaves the initiation codons unchanged and, under denaturing conditions, leads to irreversible unfolding of the RNA. E. coli ribosomes interact with newly exposed AUG/GUG codons in the modified templates forming polysomes, whereas they form monosomes with native f2 RNA or BMV RNA 3. With wheat germ ribosomes, disomes are formed in the presence of BMV RNA 3, either native or modified. With f2 RNA, eukaryotic ribosomes form monosomes, independent of the secondary structure of the template.The results indicate that, in contrast to prokaryotic ribosomes, binding of eukaryotic ribosomes to f2 RNA or BMV RNA 3 is not affected by modification of the secondary structure of these messengers.
Demonstration of a Stable Occluded Form of Ca2+ by the Use of the Chromium Complex of ATP in the Ca2+‐ATPase of Sarcoplasmic Reticulum
Authors: SERPERSU Engin Halit , KIRCH Ulrïke , SCHONER Wilhelm 
Published: March 2005

Sarcoplasmic reticulum Ca2+‐ATPase is slowly inactivated by the β, γ‐bidentate of the chromium(III) complex of ATP (CrATP). The rate of inactivation depends on the presence of calcium ions in the incubation medium. Half‐maximal inactivation rate is reached at 80 nm Ca2+. The dissociation constant of enzyme‐CrATP complex at 26°C is 31 μM. Inactivation of the enzyme by CrATP is protected competitively by ATP. From this a dissociation constant of the enzyme ATP complex of 6.2 μM is calculated.The inactivation of Ca2+‐ATPase is proportional to the incorporation of 32P from [γ‐32P]CrATP into the 100000‐Mr protein. Concomitant with the inactivation there is also an incorporation of 45Ca2+ into the purified Ca2+‐ATPase. A ratio of 45Ca2+ binding to phosphorylation of 1.7‐1.9 is calculated from the capacities of both processes as well as from their kinetics.When Ca2+ uptake into sarcoplasmic reticulum is fuelled by ATP, treatment of the vesicles with the Ca2+ ionophore X‐537‐A or the production of leaks by adding deoxycholate leads to a loss of already accumulated 45Ca2+. Such treatment, however, leaves the 45Ca2+ in the vesicles of Ca2+‐ATPase unaffected, if ATP had been replaced by CrATP during the Ca2+ uptake. It is possible to localize 45Ca2+ together with the phosphointermediate in the dodecylsulfate electrophoresis on the 100000‐Mr protein and to a minor degree on the 200000‐Mr dimer. This apparently occluded 45Ca2+ is rapidly lost by treatment of the enzyme with trichloroacetic acid.It is concluded, that CrATP acts as an ATP analogue on Ca2+‐ATPase of sarcoplasmic reticulum. Hydrolysis of CrATP, as in (Na++ K+)‐ATPase [Pauls, H., Bresenbröcker, B., and Schoner, W. (1980) Eur. J. Biochem. 109, 523‐533] leads to a stable phosphointermediate, which appears to bind 45Ca2+ in a stable occluded form. Both processes apparently lead to the suicide of the Ca2+‐ATPase.
Similarities between Mouse and Rat‐Liver Microsomal Cytochromes P‐450 Induced by 3‐Methylcholanthrene
Authors: CHEN Yuan‐Tsong , LANG Matti A. , JENSEN Nancy M. , NEGISHI Masahiko , TUKEY Robert H. , SIDRANSKY Ellen , GUENTHER Thomas M. , NEBERT Daniel W. 
Published: March 2005

Anti‐(P1‐450) and anti‐(P‐448) are two antibodies developed against distinctly different forms of 3‐methyl‐ cholanthrene‐induced cytochrome P‐450 in C57BL/6N mouse liver microsomes [Negishi and Nebert, J. Biol. Chem. 254, 11015‐11023 (1979)]. The effects of these antibodies on Long‐Evans and Sprague‐Dawley rat liver microsomes and rat ‘minimal deviation’ hepatoma 7777 microsomes were studied.Rat microsomal aryl hydrocarbon hydroxylase activity, induced by polycyclic aromatic compounds, is prefer‐ entially inhibited by anti‐(P1‐450) more so than anti‐(P‐448). Polycyclic‐aromatic‐induced rat liver microsomal acetanilide 4‐hydroxylase activity is preferentially inhibited by anti‐(P‐448) more so than anti‐(P1‐450). Anti (P1‐450) precipitates a 56‐kDa moiety in polycyclic‐aromatic‐induced rat liver microsomes. Anti‐(P‐448) precipitates a 55‐kDa moiety in control, phenobarbital‐treated, or polycyclic‐aromatic‐induced rat liver microsomes. A heterogeneity was found amongst individual Morris ‘minimal deviation’ hepatomas 7777, indicating the 3‐methylcholanthrene‐induced aryl hydrocarbon hydroxylase and acetanilide 4‐hydroxylase'activities must represent different forms of rat P‐450.Clone 46 DNA, a portion of the mouse cloned P1‐450 structural gene, hybridizes to one gene (or a small number of genes) in rat liver and to 3‐methylcholanthrene‐induced rat 23‐S P1‐450 mRNA. As has already been shown in the mouse [Tukey, Nebert and Negishi, J. Biol. Chem. 256, 6969‐6974 (1981)], rat liver P1‐450 induction is under transcriptional control, and there is no evidence for gene amplification or some gross form of DNA rearrangement.These data demonstrate striking similarities between rat and mouse for the P1‐450 structural gene, the P1‐450 mRNA, the P1‐450 protein and the P‐448 protein. Polycyclic‐aromatic‐induced P1‐450, and its association with polycyclic‐aromatic‐induced aryl hydrocarbon hydroxylase activity, therefore does not appear to be closely correlated with polycyclic‐aromatic‐induced P‐448 in either rat or mouse.
Identity Problems Concerning Subunits of the Membrane Factor of the Mitochondria1 ATPase of Saccharomyces cerevisiae
Authors: SOMLO Marie , COSSON Jacky , CLAVILIER Lea , KRUPA Mireille , LAPORTE Iréne 
Published: March 2005

As confirmed by fingerprint analysis, high‐resolution acrylamide gel electrophoresis identifies subunits of the mitochondrial ATPase complex directly at the level of Coomassie‐blue‐stained yeast mitochondria. Using this technique, the following results were obtained.

Three classes of subunits have been found in the ATPase complex. (a) The classical F1 peptides of cytoribosomal origin (57, 53 and 30.5 kDa). (b) Two peptides of mitoribosomal origin with high cycloheximide‐ resistant label: one of 7.8 kDa (the OLI1 gene product), the second of 20 kDa (the OLI2 gene product). Neither of these peptides is readily stained by Coomassie blue in the purified ATPase complex. (c) Four Coomassie‐blue‐stained peptides (27.5, 25, 23.5 and 22.5 kDa), the last one being subunit 6, which (like class b) functionally qualify as subunits of the membrane factor of the ATPase complex. As shown by labeling with 35SO2−4 and 3H‐labeled amino acids, their biosynthesis is only very slightly cycloheximide‐resistant, although it is submitted to coordinate regulation distinct from that of the class F1 peptides.
Consequently it was shown that the OLI2 gene product, a 20‐kDa peptide with high cycloheximide‐ resistant label, which was generally taken to be ‘subunit 6’ of the ATPase, is not in fact identical to this peptide.
Studies on the Biosynthesis of Microsomal Membrane Proteins
Authors: OKADA Yoshiie , FREY Alan B. , GUENTHNER Thomas M. , OESCH Franz , SABATINI David D. , KREIBICH Gert 
Published: March 2005

The site of synthesis and mechanism of insertion into membranes of several microsomal polypeptides was studied using translation system programmed in vitro with polysomes or with mRNA extracted from free and membrane‐bound rat liver polysomes. Primary translation products of cytochrome b5, NADH: cytochrome b5 oxidoreductase, NADPH: cytochrome P‐450 oxidoreductase and epoxide hydrolase were isolated by specific immunoprecipitation and compared with the mature proteins. The following observations were made:

While cytochrome b5 and NADH: cytochrome b5 oxidoreductase are synthesized in free polysomes, NADPH: cytochrome P‐450 oxidoreductase and epoxide hydrolase are made in membrane‐bound polysomes.
In all cases the radioactively labelled products synthesized in vitro comigrated with the purified native polypeptides. Since these proteins are not glycoproteins, it can be inferred that in vivo they do not undergo proteolytic cleavage during or after translation.
Levels of translatable mRNA coding for NADPH: cytochrome P‐450 oxidoreductase or epoxide hydrolase were increased by phenobarbital or by various carcinogens respectively.
A comparison of amino‐terminal segments of the native epoxide hydrolase and that synthesized in vitro showed that this protein retains an amino‐terminal polypeptide sequence which resembles the transient insertion signal found in presecretory proteins.
The amino‐terminal region of NADPH: cytochrome P‐450 oxidoreductase contains 7 leucine residues in its first 15 amino acids and can, therefore, be presumed to be the hydrophobic portion which anchors this poly‐ peptide to the membrane.

Mechanisms which may account for the spatial disposition of the membrane proteins and for their selective accumulation in endoplasmic reticulum membranes are discussed.
Investigation of the Iron‐Sulfur Clusters in Some Mitochondria1 Mutants of Saccharomyces cerevisiae
Authors: CAPEILLERE‐BLANDIN Chantal , OHNISHI Tomoko 
Published: March 2005

We have undertaken a systematic analysis of the iron‐sulfur clusters present in strains of Saccharomyces cerevisiae carrying mutations in the COB‐BOX region of the mitochondrial DNA coding for cytochrome b, and in OXI loci of the mitochondrial DNA coding for subunits of cytochrome oxidase. The iron‐sulfur clusters and cytochromes in complexes II and III of the yeast mitochondrial respiratory chain have been largely identified. Mutant phenotypes are defined in terms of succinate dehydrogenase and cytochrome c reductase activities, low‐ temperature absorption spectra and electron paramagnetic resonance spectra.From quantitative comparison of iron‐sulfur clusters in mitochondrial preparations of BOX, OXI and rho° mutants, we have made the following two sets of observations. (a) Among the components of complexIII, the appearance of Rieske's iron‐sulfur cluster seems to be correlated only to the presence or absence of the cytochrome b; moreover Rieske's cluster is absent even when polypeptide fragments of apocytochrome b are known to be synthesized. This observation constitutes a very interesting example illustrating perturbation of the incorporation of a cytoplasmically made protein (Rieske's protein) by structural alterations of a mitochondrially made protein (cytochrome b), both being assembled within the same multiprotein complex. The situation differs with another cytoplasmically‐made protein (cytochrome c1). (b) Clusters S‐1, S‐2 and S‐3 associated with complex II are present, irrespective of the type of mutation, at about ∼ 10‐20 % of the level of that in the wildtype species; this partial repression could be a result of the respiratory deficiency of these mutants.
Limited Proteolysis and Proton NMR Spectroscopy of Bacillus stearothermophilusPyruvate Dehydrogenase Multienzyme Complex
Authors: DUCKWORTH Harry W. , JAENICKE Rainer , PERHAM Richard N. , WILKIE Andrew O. M. , FINCH John T. , ROBERTS Gordon C. K. 
Published: March 2005

The pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was treated with chymotrypsin at pH 7 and 0°C. Loss of the overall catalytic activity lagged behind the rapid cleavage of the lipoate acetyltransferase polypeptide chains, whose apparent M, fell from 57000 to 45000 as judged by sodium dodecylsuphate/poly‐ acrylamide gel electrophoresis. The inactive chymotrypsin‐treated enzyme had lost the lipoic‐acid‐con‐ taining regions of the lipoate acetyltransferase chains, yet remained a highly assembled structure. Treatment of this chymotryptic core complex with trypsin at pH 7.0 and 0°C caused a further shortening of the lipoate acetyltransferase polypeptide chains to an apparent Mr of 28000 and was accompanied by disassembly of the complex. The lipoic‐acid‐containing regions are therefore likely to be physically exposed in the intact complex, protruding from the structural core formed by the lipoate acetyltransferase component between the subunits of the other component enzymes.Proton nuclear magnetic resonance spectroscopy demonstrated that the enzyme complex contains large regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after excision of the lipoic‐acid‐containing regions with chymotrypsin. It is likely that the highly mobile regions are in the lipoate acetyltransferase component and facilitate movement of the lipoic acid residues. Such polypeptide chain mobility provides the molecular basis of a novel system of active‐site coupling in the 2‐oxo acid dehydrvgenase multienzymc complexes.
Activity of Carbamoyl‐Phosphate Synthetase (Ammonia) in Isolated Rat‐Liver Mitochondria: Cycling of Carbamoyl Phosphate in the Absence of Ornithine
Authors: LOF Cor , WANDERS Ron J. A. , MEIJER Alfred J. 
Published: March 2005

When NH3was added to isolated rat‐liver mitochondria incubated with succinate and bicarbonate, oxidation of succinate was stimulated to a greater extent than could be accounted for by the net formation of carbamoyl phosphate.Measurement of the rate of incorporation of [14C]bicarbonate into carbamoyl phosphate, after the mito‐ chondria had been preincubated with NH3and unlabelled bicarbonate, revealed that flux through carbamoyl‐phosphate synthetase (ammonia) was much greater than the net formation of carbamoyl phosphate indicated.It is concluded that part of the carbamoyl phosphate produced in the absence of ornithine is degraded. About 20% of the degradation can be accounted for by non‐enzymatic reactions of carbamoyl phosphate outside the mitochondria. It is proposed that the remainder of the degradation of carbamoyl phosphate occurs by partial reversal of the reaction of carbamoyl‐phosphate synthetase.
Structural Studies on Rat Prostatic Binding Protein
Authors: PEETERS Ben , MOUS Jan , ROMBAUTS Wilfried , HEYNS Walter 
Published: March 2005

The amino acid sequence of component C1, the polypeptide specific for subunit F of prostatic binding protein, the major secretory glycoprotein of the rat ventral prostate, has been determined. Its structure was established using the manual Edman degradation on the intact protein and on the most relevant fragments isolated from trypsin, chymotrypsin, thermolysin and Staphylococcus aureus protease digests of the 14C‐labelled S‐carboxamidornethylated component C1.Component C1 contains 88 amino acids corresponding to a molecular weight of 10246. It is an acidic polypeptide due to the presence of 17 acidic residues; its thrce cysteine residues are almost symmetrically distributed over the peptide chain. Highly polar regions are found in positions 17‐27 and 37‐47, while the C‐terminal part of the molecule contains two hydrophobic segments