bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biochemical comparison of the Neurospora crassa wild‐type and the temperature‐sensitive leucine‐auxotroph mutant leu‐5
Authors: AIRAS R. Kalervo , SCHISCHKOFF Jörg , CRAMER Friedrich 
Published: March 2005

The cytoplasmic leucyl‐tRNA synthetases were purified from a wild‐type Neurospora crassa and from a temperature‐sensitive leucine‐auxotroph (leu‐5) mutant. A detailed steady‐state kinetic study of the aminoacylation of the tRNALeu from N. crassa by the purified synthetases was carried out.These enzymes need preincubation with dithioerythritol and spermine before the assay in order to become fully active. The Kappm value for leucine was lowered by high ATP concentrations and correspondingly the Kappm, ATP was lowered by high leucine concentrations. The Kappm, Leu was lowered by high pH, a pK value of 6.7 (at 30°C) was calculated for the ionizable group affecting the Km. At the concentrations of 2 mM ATP, 20 μM leucine, 0.3 μM tRNALeu, and pH 7 the apparent Km values were Kappm, ATP= 1,3 mM, Kappm, Leu= 49 μM and Kappm, tRNA= 0.15 μM. No essentially altered cytoplasmic leucyl‐tRNA synthetase was produced by the temperature‐sensitive mutant strain when kept at 37°C.In none of these experiments could we find any difference between the wild‐type enzyme and the enzyme from the mutant strain (wheter grown at permissive temperature, 28°C, or grown at permissive temperture for 24 h followed by growth at 37°C). We therefore think that the small difference in the Km value for leucine of the wild‐type and mutant enzyme, established in some earlier investigations, is not due to a difference in the kinetic properties of the enzyme molecules but to an external influence. The almost total lack of the mitochondrial leucyl‐tRNA synthetase in the mutant strain besides the leucine autotrophy remains the only difference between the wild‐type and mutant strains.
The isolation, purification and amino‐acid sequence of insulin from the teleost fish Cottus scorpius (daddy sculpin)
Authors: CUTFIELD John F. , CUTFIELD Susan M. , CARNE Alan , EMDIN Stefan O. , FALKMER Sture 
Published: March 2005

Insulin from the principal islets of the teleost fish, Cottus scorpius (daddy sculpin), has been isolated and sequenced. Purification involved acid/alcohol extraction, gel filtration, and reverse‐phase high‐performance liquid chromatography to yield nearly 1 mg pure insulin/g wet weight islet tissue. Biological potency was estimated as 40% compared to porcine insulin. The sculpin insulin crystallised in the absence of zinc ions although zinc is known to be present in the islets in significant amounts. Two other hormones, glucagon and pancreatic polypeptide, were copurified with the insulin, and an N‐terminal sequence for pancreatic polypeptide was determined.The primary structure of sculpin insulin shows a number of sequence changes unique so far amongst teleost fish. These changes occur at A14 (Arg), A15 (Val), and B2 (Asp). The B chain contains 29 amino acids and there is no N‐terminal extension as seen with several other fish. Presumably as a result of the amino acid substitutions, sculpin insulin does not redily form crystals containing zinc‐insulin hexamers, despite the presence of the coordinating B10 His.
Synaptosomal bioenergetics
Authors: KAUPPINEN Risto A. , NICHOLLS David G. 
Published: March 2005

The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea‐pig cerebral cortex is characterized.

Essentially all synaptosomes contain functioning mitochondria.
There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration.
Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose.
In glucose‐free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios.
In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide.
Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes.
The in‐situ mitochondrial membrane potential (ΔΨm) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20‐mV depolarization, while added pyruvate causes a 6‐mV hyperpolarization even in the presence of glucose.
Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by α‐cyano‐4‐hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted.
It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.
Demonstration of cGMP‐dependent protein kinase and cGMP‐dependent phosphorylation in cell‐free extracts of platelets
Authors: WALDMANN Rainer , BAUER Silvia , GÖBEL Claus , HOFMANN Franz , JAKOBS Karl H. , WALTER Ulrich 
Published: March 2005

Homogenates, membranes and cytosol of rat and human platelets were found to contain cGMP‐dependent protein kinase immunoreactivity. Specific cGMP‐dependent protein kinase immunoreactivity was about 1.7 pmol protein kinase/mg protein for homogenates of human platelets and 0.7 pmol/mg for homogenates of rat platelets; the majority appeared to be associated with the membrane fraction.In membranes of platelets low concentrations of cAMP (0.5–2 μM) stimulated the phosphorylation of five major proteins with apparent relative molecular masses, Mr, of 240000, 130000,50000,42000 and 22000 while low concentrations of cGMP (0.5–2 μM) stimulated the phosphorylation of three major proteins with apparent Mr of 130000, 50000 and 46000. An affinity‐purified antibody against the cGMP‐dependent protein kinase was prepared which specifically inhibited the activity of cGMP‐dependent protein kinase. In membranes of human platelets this affinity‐purified antibody inhibited the cGMP‐stimulated phosphorlation of the three proteins with Mr of 130000, 50000 and 46000 while it had no effect on the cAMP‐dependent and cyclic‐nucleotide‐independent protein phosphorylation.The results demonstrate that platelets contain a cGMP‐dependent protein kinase and at least three specific substrates for this enzyme. Two of these substrates, the proteins with apparent molecular Mr of 130000 and 50000, are substrates for both cAMP‐ and cGMP‐dependent protein kinase. The protein with apparent Mr of 130000 appears to be closely related to an intrinsic plasma membrane protein of vascular smooth muscle cells which is a substrate for a membrane‐associated cGMP‐dependent protein kinase. Therefore, cGMP‐dependent protein kinase and cGMP‐regulated phosphoproteins may mediate in platelets the intracellular effects of those hormones, vasodilators and drugs which elevate the level of cGMP and inhibit platelet aggregation.
Purification and characterization of N,N‐dimethylformamidase from Pseudomonas DMF 3/3
Authors: SCHÄR Hans‐Peter , HOLZMANN Werner , RAMOS TOMBO Gerardo M. , GHISALBA Oreste 
Published: March 2005

The N,N‐dimethylformamide‐hydrolyzing enzyme (DMFase) from Pseudomonas DMF 3/3 has been purified to apparent electrophoretic homogeneity with an overall 49‐fold purification, a 24% yield and a final specific activity of 1.98 μmol N,N‐dimethylformamide (DMF) hydrolyzed min−1 (mg protein)−1. The native DMFase has a relative molecular mass of 250000 and is composed of two light‐chain (Mr= 15000) and two heavy‐chain (Mr= 105000) subunits. The stability of DMFase is optimal at pH values above 7.5 and at temperatures below 20°C. The activity of the enzyme is inhibited by metal‐chelating agents such as EDTA and 2,2′‐dipyridyl. Emission and atomic absorption spectroscopy measurements showed that iron is present in significant amounts in DMFase, indicating that it is an iron‐containing amidohydrolase. In the ultraviolet/visible spectrum prominent bands were observed at 224 nm, 280 nm and 396 nm and shoulders are present at 418 nm and 467 nm.DMFase from Ps. DMF 3/3 has an isoelectric point of 7.7. The enzyme exhibits optimal activity between pH 5 and 6 and at 40°C. The substrate spectrum is rather narrow. The enzyme hydrolyzes preferentially substituted short‐chain aliphatic amides such as DMF, N‐ethylformamide and N‐methylformamide. N,N‐diethylformamide, N,N‐dimethylacetamide and unsubstituted amides, e.g. formamide, prolinamide, acetamide, acrylamide and butyramide are substrates as well, but are hydrolysed at significantly lower rates. DMFase obeys Michaelis‐Menten kinetics and its Km and Vmax values for DMF are 13.8 mM and 1.89 U/mg, respectively, as determined from a Lineweaver‐Burk plot.
Isolation and characterization of the 10‐kDa and 22‐kDa polypeptides of higher plant photosystem 2
Authors: LJUNGBERG Ulf , ÅKERLUND Hans‐Erik , ANDERSSON Bertil 
Published: March 2005

Two polypeptides of 10 kDa and 22 kDa, shown to be components of the higher plant photosystem 2, were purified and examined. A NaCl/Triton X‐100 treatment was designed, which released these two polypeptides from the thylakoid membrane, in concert with the extrinsic 16‐kDa and 23‐kDa proteins, concomitant with a loss in oxygen‐evolution activity. After this treatment the oxygen‐evolving activity of the photosystem 2 membranes devoid of the 10‐kDa and the 22‐kDa polypeptides could be restored with CaCl2, but not by readdition of the purified 23‐kDa protein. This defiency was caused by an inability of the 23‐kDa protein to rebind to the photosystem 2 membranes. In analogy, the oxygen‐evolution activity of a highly purified photosystem 2 core preparation, devoid of the 10‐kDa and 22‐kDa polypeptides, was stimulated by CaCl2, but not by the 23‐kDa protein. We, therefore, suggest that the 10‐kDa or the 22‐kDa polypeptides provide a binding‐site for the extrinsic 23‐kDa protein to the thylakoid membrane.The 10‐kDa and 22‐kDa polypeptides were isolated through ion‐exchange chromatography in the presence of detergents. They both displayed hydrophobic properties, verified by their low proportion of polar amino acid residues and their partition to the hydrophobic phase during Triton X‐114 fractionation. The purified polypeptides did not contain metallic cofactors or substances with absorption in the visible region of the spectrum.
Dimeric ubiquinol:cytochrome c reductase of Neurospora mitochondria contains one cooperative ubiquinone‐reduction centre
Authors: LINKE Petra , BECHMANN Georg , GOTHE Alexander , WEISS Hanns 
Published: March 2005

Dimeric ubiquinol:cytochrome c reductase of Neurospora mitochondria was isolated as a protein‐Triton complex and free of ubiquinol (Q). The enzyme was incorporated into phosphatidylcholine membranes together with Q. The effects of varying the molar ratio of Q to enzyme on the electron transfer from duroquinol (DHQ2) to the cytochromes c, c1 and b were studied.The rate of electron flow from DQH2 to cytochrome c was 15 times increased by Q and was maximal when one molecule of Q was bound to one enzyme dimer. The apparent Km value for DQH2 of the Q‐free enzyme was 5 μM and of the Q‐supplemented enzyme 25 μM. The pre‐steady‐state rate of electron transfer from DQH2 to cytochrome c1 was also 15 times increased by Q and was maximal with one Q molecule bound to one enzyme dimer. This effect of Q was inhibited by antimycin. The pre‐steady‐state rate of electron transfer from DQH2 to cytochrome b was 5 times decreased when Q was bound to the enzyme and this effect of Q was insensitive to myxothiazol. The H+/2 e− stoichiometry with DQH2 as substrate of the Q‐supplemented enzyme was 3.6.These results are interpreted in accordance with a Q‐cycle mechanism operating in a dimeric cytochrome reductase. Each enzyme monomer catalyses a single electron transfer from the QH2‐oxidation centre to the Q‐reduction centre and the two monomers cooperate in the reduction of Q to QH2 at one Q‐reduction centre. This centre contains two different binding sites for Q. DQH2 does not properly react at the QH2‐oxidation centre. DQH2, however, binds to the loose Q‐binding site of the Q‐reduction centre and reduces the Q bound to the tight Q‐binding site of the centre. The QH2 thus formed at the Q‐reduction centre serves as electron donor for the QH2‐oxidation centre.
The effects of pressure on yeast cytochrome c peroxidase
Authors: KORNBLATT Jack A. , ENGLISH Ann M. , HUI BON HOA Gaston 
Published: March 2005

The effects of pressure on cytochrome c peroxidase [CcP(FeIII)], its cyano derivative (CcP · CN) and its enzyme‐substrate complex (ES) have been studied. The effects of pressure on the binding of the substrate analog porphyrin cytochrome c (porphyrin c) to CcP · CN and ES have also been studied.High pressure causes CcP(FeIII) to undergo a high‐spin to low‐spin transition but has no detectable effect on either CcP · CN, which is already low spin, or on ES. The low‐spin CcP(FeIII) structure at pressure is similar to the low‐spin form at low temperature and the low‐spin form of horseadish peroxidase at high pressure. ΔV° associated with the spin equilibrium is about 30 ml/mol and is independent of temperature. ΔG° is small, 4.7 kj/mol at 0 °C, while ΔH° is 14.2 kJ/mol at 1 bar (100 kPa).Pressure has no detectable effect on the binding equilibria of mixtures of CcP · CN plus porphyrin c or ES plus porphyrin c. This indicates that the interaction of CcP and porphyrin c results in little or no volume change; the same is true in the case of cytochrome c oxidase and porphyrin c.
Structural analysis of poly(ADP‐ribose) polymerase in higher and lower eukaryotes
Authors: SCOVASSI A. Ivana , IZZO Raffaella , FRANCHI Elisabetta , BERTAZZONI Umberto 
Published: March 2005

A phylogenetic survey for the poly(ADP‐ribose)polymerase has been conducted by analyzing enzyme activity in various organisms and determining the structure of the catalytic peptides by renaturation of functional activities of the enzyme in situ after electrophoresis in denaturating conditions (activity gel).The enzyme is widely distributed in cells from all different classes of vertebrates, from arthropods, mollusks and plant cells but could not be detected in echinoderms, nematodes, platyhelminthes, thallophytes (including yeast) and bacteria. The presence on activity gels of a catalytic peptide with Mr= 115000–120000 was demonstrated in vertebrates, arthropods and mollusks but no activity bands were recovered in many lower eukaryotes, in plant cells and bacteria. By using an immunological procedure that used an antiserum against homogeneous calf thymus poly(ADP‐ribose) polymerase, common immunoreactive peptides were visualized in mammals, avians, reptiles, amphibians and fishes, while lacking in non‐vertebrate organisms.Our results indicate that the structure of poly(ADP‐ribose) polymerase is conserved down to the mollusks suggesting its important role for DNA metabolism of multicellular organisms.
Interaction of enzymes involved in triosephosphate metabolism
Authors: TOMPA Péter , BÄR Jörg , BATKE József 
Published: March 2005

The affinity of baker's yeast (Saccharomyces cerevisiae) fructose‐1, 6‐bisphosphate aldolase towards the metabolically related enzymes phosphofructokinase and glyceraldehyde‐3‐phosphate dehydrogenase was tested by using a fluorescence‐probe technique with fluorescein isothiocyanate attached covalently to the enzymes. The dissociation constants of the enzyme‐enzyme complexes, as well as the rate constants of association and dissociation, were determined. Data were compared with the parameters derived from a mammalian (rabbit muscle) system, known from the literature and determined under the same conditions (pH 7.5 or 8.5 in 0.05 M Tris/HCl buffer at 20°C). The comparison reveals similarities in the supramolecular organization of these cytoplasmic enzymes in phylogenetically distant species. Moreover, the fact that in vitro hybrid complexes are formed of stability comparable to that of non‐hybrid complexes indicates that this ancient characteristic is probably conserved during evolution.A possible regulatory mechanism is presented, based on the dynamic competition, with each other, of the enzymes involved in triosephosphate metablism.
Synthesis of ATP by soluble mitochondrial F1 ATPase and F1–inhibitor‐protein complex in the presence of organic solvents
Authors: GÓMEZ PUYOU Armando , TUENA DE GÓMEZ PUYOU Marietta , de MEIS Leopoldo 
Published: March 2005

The F1 and F1–inhibitor‐protein complex synthesized tightly bound ATP from ADP and Pi when the organic solvents dimethylsulfoxide (20–50% v/v), ethyleneglycol (20–60% v/v) or poly(ethyleneglycol) 4000 and 8000 (30–50% w/v) were included in the assay media. There was no synthesis of tightly bound ATP in the absence of organic solvents.In the presence of 50% dimethylsulfoxide, maximal synthesis of ATP was obtained at pH values between 6.5 and 7.7. In both F1 and F1 ‐inhibitor‐protein there was no synthesis of ATP in the absence of MgCl2. The rate of ATP synthesis became faster as the MgCl2 concentration in the medium was raised from 0.1–10 mM. The Km for Pi of F1 was in the range of 0.8–1.5 mM. The Km for Pi of the F1 ‐inhibitor‐protein was much higher than that of F1 and could not be measured.In the presence of 10 mM MgCl2 and 2 mM Pi, the rate constants of ATP synthesis by F1 and F1–inhibitor‐protein were 5.2–10.4 h−1 and 3.5–5.9 h−1 respectively. For both enzymes the rate constant of ATP hydrolysis was 0.69 h−1. The tightly bound ATP of F1 and F1‐inhibitor‐protein were hydrolyzed at a much slower rate when either the Pi concentration or the MgCl2 concentration was suddenly decreased.Both in presence and absence of Mg2+, 40–60% of the radioactive tightly bound ATP synthesized by F1 was hydrolyzed when non‐radioactive ATP was added to the assay medium. This was not observed when F1 ‐inhibitor‐protein was used.
Calcium transport in membrane vesicles ofStreptococcus cremoris
Authors: DRIESSEN Arnold J. M. , KONINGS Wil N. 
Published: March 2005

Rightside‐out membrane vesicles ofStreptococcus cremoriswere fused with proteoliposomes containing the light‐driven proton pump bacteriorhodopsin by a low‐pH fusion procedure reported earlier [Driessen, A. J. M., Hellingwerf, K. J. & Konings, W. N. (1985)Biochim. Biophys. Acta 808, 1–12]. In these fused membranes a proton motive force, interior positive and acid, can be generated in the light and this proton motive force can drive the uptake of Ca2+. Collapsing ΔΨ with a concomitant increase in ΔpH stimulates Ca2+uptake while dissipation of the ΔpH results in a reduced rate of Ca2+uptake. Also an artificially generated ΔpH, interior acid, can drive Ca2+uptake inS. cremorismembrane vesicles.Ca2+uptake depends strongly on the presence of external phosphate while Ca2+‐efflux‐induced proton flux is independent of the presence of external phosphate. Ca2+accumulation is abolished by the divalent cation ionophore A23187.Calcium extrusion from intact cells is accelerated by lactose. Collapse of the proton motive force by the uncoupler carbonylcyanidep‐trifluoromethoxyphenylhydrazone or inhibition of the membrane‐bound ATPase byN,N′‐dicyclohexylcarbodiimide strongly inhibits Ca2+release. Further studies on Ca2+efflux at different external pH values in the presence of either valinomycin or nigericin suggested that Ca2+exit from intact cells is an electrogenic process. It is concluded that Ca2+efflux inS. cremorisis mediated by a secondary transport system catalyzing exchange of calcium ions and protons.
13C NMR evidence for bacteriochlorophyll c formation by the C5 pathway in green sulfur bacterium, Prosthecochloris
Authors: OH‐HAMA Tamiko , SETO Haruo , MIYACHI Shigetoh 
Published: March 2005

The 13C NMR spectra of the pheophorbide of bacteriochlorophyll c, formed in the presence of l‐[1‐13C]glutamate and [2‐13C]glycine and [13C]bicarbonate in Prosthecochloris aestaurii, were analysed. The isotope in the glutamate was specifically incorporated into the eight carbon atoms in the tetrapyrrole macrocycle derived from the C‐5 of 5‐aminolevulinic acid, while no specific enrichment of these eight carbon atoms was observed in the spectrum of the pigment formed in the presence of [2‐13C]glycine. These labelling patterns provide evidence for the operation of the C5 pathway of 5‐aminolevulinic acid synthesis for bacteriochlorophyll c formation in the bacterium.The labelling of bacteriochlorophyll c by [13C]bicarbonate is consistent with its formation from 5‐[1,4,5‐13C]aminolevulinic acid formed by the C5 pathway from [1,2,5‐13C]glutamic acid. It is proposed that this glutamate is the transamination product of 2‐[1,2,5‐13C]oxoglutaric acid, arising by carboxylation of [1,4‐13C]succinyl‐CoA with 13CO2 catalysed by 2‐oxoglutaric acid synthase activity, and that the labelled succinyl‐CoA is, in turn, derived by the fixation of 13CO2 by the reductive tricarboxylic acid cycle.The 13C chemical shifts of two sp2 quaternary carbons of bacteriopheophorbide c methyl ester (C‐2 and C‐4) were reassigned.
Further characterization of an early expressed glycoprotein of the rabbit small intestinal brush border
Authors: GORVEL Jean‐Pierre , RIGAL Alain , BERNADAC Alain , MAROUX Suzanne 
Published: March 2005

Mouse 23 B 921 monoclonal antibody recognized a rabbit brush‐border antigen with an apparent molecular mass 140 kDa (140‐kDa Ag) which, unlike most hydrolases, is expressed in the poorly differentiated crypt cells of the small intestine. Immunoelectron microscopy of brush‐border vesicles showed that the 23 B 921 bound to an epitope localized on the outside of the membrane. As is the case with hydrolases the external domain of the 140‐kDa Ag constitutes the main part of the molecule, which can be released by papain treatment of brush‐border vesicles. The presence of a small hydrophobic domain, anchoring the molecule into the membrane and responsible for its amphipatic character, was shown by its affinity for Triton‐X114 micelles. The topological organization in the membrane of 140‐kDa Ag seemed to be identical to that of hydrolases. Unlike hydrolases, however, the native structure of the antigen was found from its sensitivity to proteolysis to be very dependent on its integration into the lipid bilayer. Nevertheless, detergent‐extracted 140‐kDa Ag can be purified by immunoaffinity chromatography although it cannot be stocked for more than 48 h even in the presence of protease inhibitors. The carbohydrate moiety of 140‐kDa Ag, bearing the human blood group A determinant, amounts to 20% of the total molecular mass.The existence of some privileged relationship was established between 140‐kDa Ag and hydrolases: (a) in the membrane, hydrolases protect the 140‐kDa Ag from papain action; (b) after detergent extraction, 140‐kDa Ag is strongly associated with several hydrolases particularly disaccharidases.