bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of Lipids in Functions of Photosynthetic Membranes Revealed by Treatment with Lipolytic Acyl Hydrolase
Authors: RAWYLER André , SIEGENTHALER Paul‐André 
Published: March 2005

1. Thylakoid membranes and subchloroplast particles I and II enriched in photosystem I and photosystem II, respectively, were treated with a potato lipolytic acyl hydrolase.2. In the thylakoid membrane fraction, this treatment inhibited electron flows involving both photosystems and the associated photophosphorylations. However, electron flows involving either photosystem I of photosystem II were still preserved. The treatment of thylakoid membranes by lipolytic acyl hydrolase brought about a temporal convergence of different events such as maximal activity of reduced dichloroindophenol‐supported electron flows, complete inhibition of photophosphorylations and electron transport activities through photosystem II + I, onset of the decay N,N,N′,N′‐tetramethyl‐p‐phenylenediamine‐supported activity of photosystem activity and of the stimulation of photosystem activity (from reduced dichloroindophenol to NADP+ by exogenous plastocyanin.3. Lipolytic acyl hydrolase catalyzed a limited hydrolysis of each lipid but in a stepwise manner, the galactolipids being attacked before the ionic lipids. The extent of the hydrolysis was not more than 50% for each lipid. Most of the hydrolytic process occurred before any significant change in photochemical activities could be observed.4. In subchloroplast particles I, a treatment by lipolytic acyl hydrolase did not greatly affect the electron transport whilst lipid hydrolysis was almost complete.5. In subchloroplast particles II, neither the electron flow activities nor lipid content were significantly altered by lipolytic acyl hydrolase.6. The sites of lipolytic acyl hydrolase action appeared to be localized between plastoquinones and P700. It is suggested that it is not possible to establish a quantitative and/or temporal correlation between the extent of lipid hydrolysis and the inhibition of photochemical activities.7. The profile of the hydrolysis of lipids in thylakoid membranes suggests that ionic lipids are less accessible to lipolytic acyl hydrolase than galactolipids.
Origin of the Ammonia Found in Protein‐Free Extracts of Rat‐Liver Mitochondria and Rat Hepatocytes
Authors: WANDERS Ron J. A. , HOEK Jan B. , TAGER Joseph M. 
Published: March 2005

1. Protein‐free extracts of isolated rat‐liver mitochondria contain 5.17 ± 0.19 nmol ammonia/mg protein [cf. Harris, E. J. and Bassett, D. J. (1971) FEBS Lett. 19, 214–2171.2. The ammonia found in the protein‐free extracts does not originate from lysosomes contaminating the mitochondrial preparation.3. When isolated mitochondria are incubated with ornithine, 14CO2 and a source of ATP a small amount of citrulline is formed. This amount is stoichiometrically equivalent to the ammonia that disappears from the extramitochondrial space, whereas the amount of ammonia found in the protein‐free extracts of the mitochondria remains unchanged. Similar results were obtained when the reductive amination of 2‐oxoglutarate was used as an ammonia‐consuming reaction.4. When isolated mitochondria are incubated under conditions such that the glutamate dehydrogenase and 3‐hydroxybutyrate dehydrogenase reactions reach equilibrium, the thermodynamically active concentration of ammonia is not equal to the concentration measured in the protein‐free extracts.5. About 80% of the ammonia found in protein‐free extracts of rat‐liver mitochondria is derived from a component or components with a molecular weight of ≧ 50000.6. Protein‐free extracts of isolated rat‐liver cells contain considerable amounts of ammonia. After digitonin fractionation this ammonia is found in the protein‐free extract of the particulate fraction.7. It is concluded that the ammonia found in protein‐free extracts of rat‐liver tissue is derived from a component or components in the mitochondria and is released during deproteinization.
The Energy‐State of Mitochondria during the Transport of Ca2+
Authors: LÖTSCHER Hans‐Ruedi , WINTERHALTER Kaspar H. , CARAFOLI Ernesto , RICHTER Christoph 
Published: March 2005

The fluctuations of the membrane potential during mitochondrial Ca2+ transport have been monitored with an electrode sensitive to tetraphenylphosphonium. The following conclusions have been reached.1. The membrane becomes depolarized during the influx of Ca2+. When the bulk of the Ca2+ pulse has been taken up, it repolarizes, but not completely.2. If all of the accumulated Ca2+ is released from mitochondria and cycling is inhibited, the membrane repolarizes completely.3. The accumulation of Ca2+ alone does not induce mitochondrial damage. In the presence of inorganic phosphate, the uptake of Ca2+ may lead to complete and irreversible depolarization, depending on the amount of Ca2+ and phosphate accumulated. The irreversible damage observed in the presence of phosphate is prevented by Mg2+.
Reconstitution of Mitochondrial Oligomycin and Dicyclohexylcarbodiimide‐Sensitive ATPase
Authors: GLASER Elzbieta , NORLING Brigitta , ERNSTER Lars 
Published: March 2005

1. Oligomycin and dicyclohexylcarbodiimide‐sensitive ATPase was isolated from beef‐heart mitochondria and treated with 3.5 M NaBr in order to remove F1. The residue, called F0, was found to consist of seven components. Five of these are stained by Coomassie blue after dodecyl‐sulfate‐polyacrylamide‐gel electrophoresis. Two of them correspond to the oligomycin‐sensitivity‐conferring protein and coupling factor F6, with apparent molecular weights of 21000 and 9400, respectively. Three additional polypeptides of molecular weights 23000, 10500 and 8600 were not identified with known proteins. Two components not stained with Coomassie blue were detected by autoradiography of the gels of F0 preincubated with [14C]dicyclohexylcarbodiimide. These two components probably represent monomeric and oligomeric forms of the dicyclohexylcarbodiimide‐binding protein.2. F0 induced an oligomycin and dicyclohexylcarbodiimide‐sensitive enhancement of K++ valinomycin‐driven proton translocation across the membrane of artificial phospholipid vesicles.3. The interaction of F0 with purified, soluble beef heart F1 was investigated. F0 was capable of binding F1 and conferring oligomycin and dicyclohexylcarbodiimide sensitivity and cold stability on its ATPase activity. Furthermore F0 was found to diminish the specific activity of F1‐ATPase. A comparison of these effects at varying F0/F1 ratios shows that F0 binds F1 in both an oligomycin‐sensitive and an oligomycin‐insensitive manner, and that both types of binding involve a conferral of cold stability and a decrease in specific activity. High F0/F1 ratios favoured the oligomycin‐sensitive type of binding, indicating that F1 binds preferentially to oligomycin‐sensitivity‐conferring sites. Treatment of ATPase complex with trypsin resulted in an F0 with a decreased proportion of oligomycin‐sensitivity‐conferring binding sites and a diminished ability to lower the specific activity and cold lability of F1.4. Reconstitution of F0 treated with trypsin and F1, oligomycin‐sensitivity‐conferring protein and F6 showed that at a constant amount of F1 bound, both oligomycin‐sensitivity‐conferring protein and F6 increased the oligomycin sensitivity of ATPase activity. It was therefore concluded that both of these coupling factors are involved in the conferral of oligomycin sensitivity.5. The effect of the order of addition of F1, oligomycin‐sensitivity‐conferring protein and F6 to F0 on the reconstitution of oligomycin‐sensitive ATPase activity, and of F1 and oligomycin‐sensitivity‐conferring protein to submitochondrial particles on the reconstitution of respiratory control, was investigated. The highest values of oligomycin sensitivity and respiratory control were obtained when F1 was added as the first component, indicating that F1 plays a directing role in the organisation of the components.
Pyruvate Carboxylase and Propionyl‐CoA Carboxylase as Anaplerotic Enzymes in Skeletal Muscle Mitochondria
Authors: DAVIS E. J. , SPYDEVOLD Ø. , BREMER Jon 
Published: March 2005

Oxygen uptake in skeletal muscle mitochondria respiring on pyruvate or on acetylcarnitine plus propionylcarnitine is stimulated 3–4‐fold by bicarbonate. The stimulation is highly dependent on ATP. The respiration rate obtained amounts to 1/4–1/3 of the rate obtained with pyruvate‐malate in the presence of ADP. With decreasing ATP/ADP ratios in the medium, a decreasing stimulation by bicarbonate is obtained. Similar results were obtained with heart mitochondria.With ATP added, a pyruvate‐dependent build up of citric acid cycle intermediates takes place in incubations with skeletal muscle mitochondria amounting to about 0.5 nmol × min−1× mg protein−1.In 14CO2‐fixation experiments, the activity of pyruvate carboxylase (EC 6.4.1.l) amounts to about 3 nmol × min−1× mg protein−1 under similar conditions. With propionylcarnitine plus acetylcarnitine a similar stimulation of respiration and fixation of bicarbonate is observed. In this case the respiration and the propionyl‐CoA carboxylase (EC 6.4.1.3) is less inhibited by ADP.The results are discussed in relation to the regulation of the level of citric acid cycle intermediates in muscle tissues.It is concluded that pyruvate carboxylase is an important anaplerotic enzyme in skeletal muscle mitochondria.
Activities of Enzymes Involved in Amino‐Acid Metabolism in Developing Rat Placenta
Authors: REMESAR Xavier , AROLA Lluís , PALOU Andreu , ALEMANY Marià 
Published: March 2005

Aspartate transaminase, alanine transaminase, glutamate dehydrogenase, arginase, serine dehydratase, tyrosine transaminase, glutamine synthetase, glutaminase and adenylate deaminase activities were measured in crude homogenates of 12, 19 and 21‐day rat placentae. There is a considerable quantitative importance in enzymes able to produce free ammonia, such as adenylate deaminase and glutamate dehydrogenase, activity that progressively decrease with the age of placenta. The glutamine synthetase and tyrosine transaminase activities increase with age, while serine dehydratase decreases considerably and aspartate and alanine transaminase do not change practically. Arginase shows a maximum at 19, with lower 12 and 21‐day activities. No measurable glutaminase activity has been found.The possible implications of the enzymes studied upon the ammonia‐producing activity of rat placenta are discussed together with the relative decreasing role of placenta for the overall metabolic activity of the foetus, especially during the last phases of its development.
A Flow‐Quench Apparatus for Cryoenzymic Studies
Authors: BARMAN Thomas E. , BRUN André , TRAVERS Franck 
Published: March 2005

The flow‐quench method was adapted to sub‐zero conditions. Two apparatus were constructed: a rapid flow‐quench apparatus (which takes samples in the 5–300‐ms time range) and a time delay flow‐quench apparatus (0.5 s and longer). The apparatus were constructed so that the reagents are only in contact with chemically inert materials: glass, poly(tetrafluoroethylene) and poly(trifluoro‐chloroethylene).The modified flow‐quench apparatus were used to study the initial formation of creatine phosphate by creatine kinase at –15°C in the time range 5 ms to 10 s. As at +4°C [Travers, F., Barman, T. E. and Bertrand, R. (1979) Eur. J. Biochem. 100, 149–1551, the time course of product formation was complex and consisted of three phases: a lag phase, a burst phase and the steady‐state phase.The apparatus were also used to test chemically for reaction intermediates involving labile phosphate and phospho‐enzyme complexes on the creatine kinase reaction pathway at –15°C. Since neither type of intermediate could be detected down to 5 ms, this enzyme probably proceeds via a direct in‐line type of mechanism.
Determination of the Stability Constants of Mn2+ and Mg2+ Complexes of the Components of the NADP‐Linked Isocitrate Dehydrogenase Reaction by Electron Spin Resonance
Authors: KUCHEL Philip W. , REYNOLDS C. Hugh , DALZIEL Keith 
Published: March 2005

1. The stability constants (Ks) of Mn2+ and Mg2+ complexes of isocitrate, 2‐oxoglutarate, NADP and NADPH have been estimated by using electron spin resonance to measure free Mn2+ in ligand–metal‐ion solutions.2. The values of Ks for the Mn2+ complexes at 25°C, in triethanolamine buffer containing NaCl, pH 7.0 and ionic strength 0.15 M, are 497 M−1 for isocitrate, 39 M−1 for 2‐oxoglutarate, 467 M−1 for NADP and 943 M−1 for NADPH.3. For the Mg2+ complexes under the same conditions, the Ks values are 357 M−1, 25 M−1, 133 M−1 and 179 M−1 respectively. The large difference between the stabilities of the isocitrate and 2‐oxoglutarate complexes is thus largely responsible for the observed variation of the apparent equilibrium constant of the NADP‐linked isocitrate dehydrogenase reaction with magnesium ion concentration.4. NADP‐linked isocitrate dehydrogenase from bovine heart mitochondria binds Mn2+, and the stability constant of the complex is about 2.2 × 104 M−1. The formation of this complex may explain the inhibition of the enzyme‐catalysed reaction observed with Mn2+ concentrations greater than 0.2 mM in initial rate measurements.
Binding of Cibacron Blue F3GA to the Flavin and NADH Sites in Cytochrome b5 Reductase
Authors: POMPON Denis , GUIARD Bernard , LEDERER Florence 
Published: March 2005

The behaviour of cytochrome b5 reductase holoenzyme and apoenzyme toward blue‐dextran–Sepharose has been studied. Holoenzyme was adsorbed at low ionic strength and could be eluted with 100 μM NADH or NAD+. Flavin‐free enzyme was even more strongly bound and could be eluted with 1 M NaCl, or 100 μM NADH + 10 μM FAD. Separately the cofactors were without effect. FMN was less effective than FAD. ADP and AMP eluted nothing.Cibacron blue F3GA was found to exert a mixed inhibition on NADH oxidation. Dye binding to holoenzyme elicited a characteristic red shift in its spectrum. Comparison of the difference spectrum amplitudes at 680 and 585 nm showed the presence of a second binding mode at higher dye concentrations.These results point to the existence for cytochrome b5 reductase of two binding sites with high affinity for blue‐dextran‐Sepharose: the NADH binding site and the flavin binding site. For the latter it is clear that the isoalloxazine pocket must play a role in dye binding. Cytochrome b5 reductase is the second flavoenzyme which has been shown to have affinity for immobilized dye at the flavin site, the first one being flavocytochrome b2, an FMN‐dependent enzyme [D. Pompon and F. Lederer (1978) Eur, J. Biochem. 90, 563–569].
A Photochemical Crosslinkinig Study of the Subunit Structure of Membrane‐Associated Spectrin
Authors: MIDDAUGH C. Russell , JI Tae H. 
Published: March 2005

The subunit structure of membrane‐associated spectrin was investigated by crosslinking human erythrocyte membranes with a variety of cleavable photosensitive heterobifunctional reagents by flash photolysis millisecond crosslinking. Crosslinked complexes were analyzed on agarose‐polyacrylamide gels whose high exclusion limit permits the resolution of molecules with Mr larger than 106.Crosslinking of membrane ghosts produced a series of new bands with apparent molecular weights of approximately 420000, 710000 and 910000, as well as a band at the top of the gels. No significant bands were detectable between the 910000‐Mr band and the material at the gel top. The molecular weights of the three bands correspond to spectrin dimer, trimer, and tetramer, suggesting a limiting tetrameric stoichiometry for membrane‐associated spectrin.The rate of formation of spectrin oligomers was examined by increasing crosslinking time. Dimers accumulate before trimer and tetramer, and as tetramer increased, dimer diminished. A simple kinetic model is employed which is also consistent with spectrin tetramer existing on the cytoplasmic surface of the erythrocyte membrane as the basic structural unit.
Comparative Immunochemical Studies of Cytochrome P‐450CAM of Pseudomonas putida and of Cytochrome P‐450SCC, of Bovine Adrenocortical Mitochondria
Authors: DUS Karl M. , LITCHFIELD William J. , HIPPENMEYER Paul J. , BUMPUS John A. , OBIDOA Onyechi , SPITSBERG Vitaly , JEFCOATE Colin R. 
Published: March 2005

Antibodies raised in rabbits against highly purified preparations of two cytochromes (cytochrome P‐450CAM from Pseudomonas putida and cytochrome P‐450scc from bovine adrenocortical mitochondria) were found to be specific for their respective antigens in Ouchterlony doublediffusion assays. In each case a single sharp curve was observed and no response was seen with other P‐450 hemeproteins.
In enzyme inhibition studies with reconstituted monooxygenase systems both the precipitating antibodies and soluble, monovalent Fab−1 fragments obtained from them by peptic digestion were effective inhibitors. In contrast to the Ouchterlony assay, anti(P‐450CAM) not only stopped camphor hydroxylation completely but also caused measurable inhibition of the cholesterol side‐chain cleavage (desmolase) reaction while anti(P‐450scc) inhibited camphor hydrolxylation significantly in addition to abolishing desmolase activity.
Iodination of cytochromes P‐450CAM and P‐450scc with the help of chloramine‐T produced soluble, radiolabeled preparations with specific activities ranging from 3 × 108 to 1 × 109 counts × min−1×μmol−1 which showed undiminished enzymatic activities and gave a single precipitin curve each in the Ouchterlony assay with anti(P‐450CAM) and anti(P‐450scc), respectively. In the iodinated preparations 60–70% of the tyrosine residues were found to be present as monoiodotyrosines while no other amino acid was labeled.
A sensitive radioimmunoassay was developed, measuring inhibition of binding between 125I‐labeled cytochrome P‐450CAM and its antibodies by cross‐reacting material, which revealed substantial cross‐reactivity of cytochromes P‐45011β (88%), P‐450scc (64%) and P‐450LM‐2 (60%). Conversely, competitive binding o.125I‐labeled cytochrome P‐450sccand unlabeled cross‐reacting antigens to anti(P‐450scc demonstrated cross‐reactivities for cytochromes P‐450sccβ (60%), P‐450CAM (75%) and P‐450LM2 (90%). Incubation of cytochrome P‐450scc with adrenodoxin, and of cytochrome P‐450CAM with putidaredoxin, prior to interaction with anti(P‐450scc) and anti(P‐450CAM), respectively, caused marked decreases in the ability of the corresponding P‐450 hemeproteins to bind to their antibodies.
Even the BrCN‐derived hemepeptides of cytochromes P‐450CAM and P‐450ssc were still able to recognize the antibodies elicited by their parent proteins as evidenced by significant crossreactivities in the radioimmunoassay. It is concluded that the four P‐450 hemeproteins tested in this investigation, cytochromes P‐450CAM, P‐450ssc, P‐45011β, and P‐450LM‐2, have one or more antigenic determinants in common and that a major antibody binding site is associated with the hemepeptide.
Comparison of the Mg2+ and Ca2+ Binding Properties of Troponin Complexes P1‐TI2C and TI2C
Authors: JAHNKE Ulrike , HEILMEYER Ludwig M. G. 
Published: March 2005

The phosphoserine present in troponin T of freshly isolated skeletal muscle troponin P1‐TI2C was dephosphorous by alkaline phosphatase and the resulting troponin TI2C characterized by phosphorous content and gel electrophoresis in presence of sodium dodecylsulfateBoth complexes bind Ca2+ in an identical manner with a K0.5 of 5.3 × 10−9 M for the Ca2+/ Mg2+ binding sites and of 1.1 × 10−6 M for the Ca2+‐specific sites. 3.5 mM Mg2+ lower the K0.5 value at the Ca2+/Mg2+ binding sites to 1.3 × 10−7 M in the phospho‐troponin P1‐TI2C and leaves nearly unchanged the value of the dephosphorylated troponin TI2C at 1.2 × 10×8 M. At 10 mM Mg2+ only one dissociation constant of about 1.0 −6 M is determined with both complexes.In analogy dephosphorylation of troponin P1‐TI2C reduces the affinity for Mg2+ at the Ca2+/Mg2+ binding sites from 6.7 × 10−5 M to 2.0 × 10−3 M. Again the Mg2+‐specific sites are uninfluenced. The possibility is discussed that removal of the phosphate group from troponin T allows the interaction of the N‐terminal domin of troponin T with other amino acid side chains of troponin.
Isolation and Partial Characterization of the Plasma Membrane of Purified Bovine Neutrophils
Authors: MOTTOLA Cristina , GENNARO Renato , MARZULLO Antonio , ROMEO Domenico 
Published: March 2005

Large amounts of granulocytes can be isolated from bovine blood by differential centrifugation and hypotonic lysis of erythrocytes, followed by separation of neutrophils and eosinophils by centrifugation through a gradient of colloidal silica.
Careful homogenization of the purified neutrophils and subfractionation of the postnuclear supernatant by centrifugation through a discontinuous sucrose gradient provides a membrane fraction (at the 20/32%, w/w, sucrose interface), which collects about 20–35% of the activity of plasma membrane marker enzymes.
Treatment of the plasma membrane fraction with 0.5 M KCl removes some protein and activity of granule enzymes, leading to an about 20–35‐fold enrichment in specific activity of plasma membrane marker enzymes. In particular, there is a 25‐fold enrichment in a Ca2+‐dependent ATPase, whose half‐maximal reaction velocity is reached at 2.2 × 10‐7 M Ca2+.
High‐resolution sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals a complex composition of the neutrophil plasma membrane, with about 40 polypeptides stained by Coomassie blue. Ten of these peptides are more intensely stained by the dye; their apparent molecular weight ranges from 81000 to 34000. All these ten polypeptides but one (which is likely to be actin) are markedly enriched in the plasma membrane fraction over the original homogenate.
Since bovine blood can be obtained in unlimited amounts, the procedures here described can be applied to a large‐scale purification of the neutrophil plasma membrane, suitable for biochemical studies.
Spontaneous Dissociation of a Cytochrome Core and a Biglobular Flavoprotein after Mild Trypsinolysis of the Bifunctional Saccharomyces cerevisiae Flavocytochrome b2
Authors: Gervais M. , TEGONI Marietta 
Published: March 2005

Saccharomyces cerevisiae flavocytochrome b2 is known as a bifunctional enzyme which behaves as the association of an FMN flavodehydrogenase with its specific acceptor, a b5‐like cytochrome. Mild trypsinolysis gives rise to three complementary fragments (n, X, β′), both prosthetic groups being still bound. After such proteolysis the separation of a biglobular flavoprotein domain (carrying FMN) from a cytochrome domain (with the home) is obtained by molecular sieving under non‐denaturing conditions.The marked lack of affinity between the tetrameric flavoprotein (X, β′)4 and the monomeric cytochrome core (n) leads to the hypothesis that the two domains are not tightly associated in the native molecule and might move relative to each other. Their respective mobility is possibly required for the catalytic mechanism. The comparison with previous trypsinolysis studies on the flavocytochrome b2 from Hansenula anomala suggests the presence of two common zones of hypersensitivity to proteases, along the protomeric polypeptide chain, and strongly supports the validity of the triglobular model for both flavocytochromes.