bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Polypeptide Components from Light‐Harvesting Pigment‐Protein Complex II (B800–850) of Rhodopseudomonas capsulata
Authors: TADROS Monier H. , ZUBER Herbert , DREWS Gerhart 
Published: March 2005

A new procedure for isolation, purification and identification of the three polypeptides of the membrane‐bound light‐harvesting complex II (B800–850) of Rhodopseudomonas capsulata has been developed.The polypeptides were extracted from crude intracytoplasmic membranes with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60. The peak fractions were transferred to solvents of different polarity and separated by gel filtration or ion‐exchange chromatography. The three major polypeptides isolated by this two‐step chromatography were found to be homogenous and identical with the three polypeptides of the light‐harvesting complex II, as judged by amino acid analysis and N‐terminal sequence determination.Contaminating minor polypeptides, of which the functions are unknown, were different from the polypeptides of the B800–850 complex studied by the same criteria.
Bile‐Salt‐Binding Polypeptides in Plasma Membranes of Hepatocytes Revealed by Photoaffinity Labelling
Authors: KRAMER Werner , BICKEL Ulrich , BUSCHER Hans‐Peter , GEROK Wolfgang , KURZ Gerhart 
Published: March 2005

Photoaffinity labelling of a subfraction of plasma membranes of rat liver, enriched with sinusoidal surfaces, with the sodium salts of (3β‐azido‐7α, 12α‐dihydroxy‐5β‐cholan‐24‐oyl)‐2‐amino[2‐3H(N)]ethanesuifonic acid, (7, 7‐azo‐3α, 12α‐dihydroxy‐5β‐cholan‐24‐oyl)‐2‐amino[2‐3H(N)]ethanesulfonic acid and (11ξ‐azido‐12‐oxo‐3α, 7α‐dihydroxy‐5β‐cholan‐24‐oyl)‐2‐amino[2‐3H(N)]ethanesulfonic acid resulted with each derivative in a clear covalent incorporation of radioactivity into polypeptides with the apparent molecular weights of 67000, 52000, 48000, 43000 and about 20000.
Photoaffinity labelling of a membrane subfraction predominantly composed of bile canalicular membranes by the photolabile derivatives of the conjugated bile salts also showed covalent incorporation of radioactivity into polypeptides of the same apparent molecular weights as with the subfraction enriched with the sinusoidal membranes.
The extent of photoaffinity labelling of the different membrane polypeptides is dependent upon the photolabile bile‐salt derivative used. However, with each of the photolabile derivatives the relative ratio of the labelling of the different membrane polypeptides was similar for both membrane subfractions. Provided that the uptake as well as the secretion of bile salts by hepatocytes are carrier‐mediated processes, this suggests the participation of the same polypeptides in both processes.
The Role of Amino‐Terminal Alanine in the Control of Conformation and Activity of α‐Chymotrypsin
Authors: SHARMA Satish K. , HOPKINS Thomas R. 
Published: March 2005

Novel acetylated derivatives of three different three‐chained chymotrypsins were prepared from bovine chymotrypsinogen A and their catalytic properties and kinetics of denaturation in urea were compared with those of the corresponding non‐acetylated enzymes.Measurements of the Km (apparent) as a function of pH confirmed earlier findings of Valenzuela and Bender [J. Biol. Chem. 248, 4909–4914 (1973)] that the α‐species of chymotrypsin is much more sensitive to reversible inactivation at high pH compared to its sister three‐chained chymotrypsins. α1 and K‐chymotrypsin.Similarly, the denaturation rate constants in 8 M urea of α‐chymotrypsin were much more sensitive to high pH than α1 and K‐chymotrypsin. The urea denaturation study showed a transition at about pH 8 to a more urea‐sensitive from of α‐chymotrypsin, whereas α1 and K‐chymotrypsin were relatively insensitive to a change in pH from 6.5 to 10.When the N‐terminal Ala149 of α‐chymotrypsin was acetylated, thus preventing protonation of the N terminus, the active enzyme derivative displayed the same Km (app) vs pH profile as α1 and K‐chymotrypsin. Urea denaturation studies with this masked derivative also showed that the pH‐dependent transition of native α‐chymotrypsin at pH 8 was eliminated.These results demonstrate that it is the presence of the protonated Ala149 residue in α‐chymotrypsin that accounts for much of the hypersensitivity of this enzyme species to inactivation and urea denaturation in the pH region 7.5–10.
Lipoxygenase mRNA in Rabbit Reticulocytes
Authors: THIELE Bernd J. , ANDREE Hartwig , Höhne Marina , RAPOPORT Samuel M. 
Published: March 2005

The synthesis of the erythroid lipoxygenase, an enzyme which is of importance for the degradation of mitochondria during the maturation of reticulocytes to erythrocytes, was studied in reticulocytes from bone marrow and in density‐separated fractions from peripheral blood of anemic rabbits.Lipoxygenase mRNA was enriched to about 75% by digestion of polysomes with protease K, poly(U)‐Sepharose chromatography and repeated sucrose gradient centrifugation. From sucrose gradient centrifugation, electrophoresis and electron microscopy a molecular weight of about 106 was calculated.Synthesis of lipoxygenase is absent in erythroblasts, in very young reticulocytes obtained from bone marrow, of in the lightest fractions of reticulocytes from the peripheral blood. More mature blood reticulocytes show a considerable synthesis of the enzyme. The induction of the synthesis of the lipoxygenase seems to be initiated when reticulocytes have reached the peripheral blood.It is shown that lipoxygenase mRNA is present in reticulocytes as a translationally inactive free cytoplasmic messenger ribonucleoprotein (mRNP) particle. After deproteinization isolated mRNA obtained from masked mRNP codes for authentic lipoxygenase in a cell‐free protein‐synthesizing system of reticulocytes.
A Residue Critical for Flavin Binding in Flavocytochrome b2 from Baker's Yeast
Authors: POMPON Denis , LEDERER Florence 
Published: March 2005

The reagent 2‐keto‐3‐butynoic acid is the product formed in the reaction between the suicide reagent 2‐hydroxy‐3‐butynoate and a number of flavoproteins. We describe in this paper the inactivation of flavin‐free flavocytochrome b2 by 2‐keto‐3‐butynoate, in a rapid reaction which introduces 0.9 mol reagent for total inactivation. The modification results in loss of affinity for flavin and affects a cysteine residue.We also describe in this paper a simple enzymatic method for preparing 2‐keto‐3‐butynoate, as well as some properties of the reagent, in particular its stability and susceptibility to nucleophilic attack. We show that at neutral pH it is highly specific for thiol compounds. Some properties of the adduct formed with glutathione are described. These experiments should pave the way for the use of 2‐keto‐3‐butynoate with other proteins.
The Role in CaCO3 Crystallization of an Acid Ca2+‐Binding Polysaccharide Associated with Coccoliths of Emiliania huxleyi
Authors: BORMAN Anton H. , de JONG Elisabeth W. , HUIZINGA Marlies , KOK Dik J. , WESTBROEK Peter , BOSCH Leendert 
Published: March 2005

Coccoliths of the unicellular marine alga Emiliania huxleyi are formed intracellularly in a specialized vesicle. Closely associated with the CaCO3 crystals of the coccoliths is an acid Ca2+‐binding polysaccharide. The latter is considered to fulfil a regulatory function in CaCO3 crystallization. In this study it is demonstrated that the coccolith polysaccharide is able to inhibit CaCO3 precipitation in vitro. The degree of inhibition is dependent on the nature of the cations bound to the acid groups of the polysaccharide. After substitution of Na+ by Ca2+ ions the polysaccharide is far less effective in inhibiting CaCO3 precipitation.The coccolith polysaccharide contains two types of acid groups: uronic acids and sulphate esters. Only the uronic acids are responsible for the inhibition of CaCO3 precipitation. Desulphated polysaccharide inhibits precipitation to the same extent as the native molecule whereas the carboxyl‐reduced polysaccharide is unable to block CaCO3 crystallization.Inhibition of CaCO3 precipitation can be suppressed by the presence of ethanol. Presumably the conformation of the molecule is altered under these conditions.Alginic acid [poly(mannuronic‐iduronic acid)] and poly(galacturonic acid) also inhibit CaCO3 precipitation. Inhibition of precipitation is effected by the coccolith polysaccharide and alginic acid at comparable concentrations. The concentration of poly(galacturonic acid) needed to obtain the same result is about 20‐times higher. Contrary to the coccolith polysaccharide, both poly(uronic acid)s inhibit CaCO3 precipitation in the absence as well as in the presence of ethanol. These results suggest that conformational changes of the coccolith polysaccharide may play a role in CaCO3 crystallization in vivo.
On the Enigma of Old Yellow Enzyme's Spectral Properties
Authors: EWEG Jan Kees , MÜLLER Franz , VAN BERKEL Willem J. H. 
Published: March 2005

Old yellow enzyme (NADPH oxidoreductase) in the free and complexed state was thoroughly investigated by the following techniques: absorption, circular dichroism, fluorescence/phosphorescence and electron paramagnetic resonance spectroscopy and fluorescence/phosphorescence decay measurements, applied over a wide range of temperature(7–293 K). The data obtained were interpreted by comparison with results from similar measurements on free FMN, existing spectral data on isoalloxazine model systems and theoretical data.The results clearly demonstrate the inadequacy of a simple phenolate‐FMN donor‐acceptor charge‐transfer complex to explain the phenomena occurring upon the addition of phenols to old yellow enzyme. Instead it was found that the phenolate anion interferes strongly with an existing tight complex between FMN and the apoprotein, probably an H‐bonded structure in which FMN is tautomerized and interacts with an L‐chiral center. This is concluded from a separate electronic transition with an origin at 496 nm, thus far not recognized as such, and the circular dichroism observed.The emission is dominated by that of free FMN, although protein‐bound FMN seems also to become luminescent in glassy solution at 143 K. A second fluorescence/phosphorescence emission appears upon excitation of both native and complexed old yellow enzyme in the ultraviolet. This emission is quenched by the addition of phenol to the enzyme, shows a large (3000‐cm–1) blue shift on going to a low‐temperature glass and is tentatively assigned to excimers of nucleic acids. Long‐wavelength excitation with a synchronously pumped, mode‐locked Rhodamine 6‐G dye laser revealed a third, extremely weak emission in both native old yellow enzyme and its complexes. It decays with a lifetime of about 3 ns at 143 K.Electron paramagnetic resonance spectra revealed the presence of a low amount of an unpaired spin in old yellow enzyme. Owing to an unusual relaxational behaviour it could only be observed below 15 K and, again, the signal was measured in both the native enzyme and its complexes. Possible assignment and consequences of this observation are discussed.In frozen aqueous solutions of the enzyme‐phenolate complex, a phase transition was discovered at which the colour of the complex reverted to that of the native enzyme. Subsequent melting restored the original colour.The observed phenomena and existing literature data lead to the conclusion that the only model from which no apparent inconsistencies emerge is that of a very complicated network of hydrogen‐bonded structures in the protein. These involve several, partly unknown, chromophores. Phenols interfere with this network, leading to the formation of the long‐wavelength absorption band in old yellow enzyme.
cDNA Cloning and Analysis of Chick‐Embryo‐Liver Cytochrome P‐450 mRNA Induced by Porphyrinogenic Drugs
Authors: BROOKER John D. , O'CONNOR Reina 
Published: March 2005

Hepatic microsomal cytochrome P‐450 was elevated in 17‐day chick embryos by combined administration of the porphyrinogenic drugs 2‐allyl‐2‐isopropylacetamide and 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine. Increased apoprotein levels were the result of de novo protein synthesis; in vitro obtained translation data suggested that cytochrome P‐450 mRNA levels were elevated.A 1000‐base cDNA sequence for the drug‐induced cytochrome P‐450 mRNA was isolated from a chick embryo cDNA ‘library’ and this was used as a specific probe to investigate drug‐mediated induction of cytochrome P‐450 mRNA.RNA‐DNA ‘dot’ hybridisation studies demonstrated that drug treatment led to a 3–5‐fold increase in the level of this mRNA and that the mRNA was predominantly associated with membrane‐bound polyribosomes. Treatment of embryos with the drugs individually demonstrated that both of them induced synthesis of the same mRNA.These studies show directly that treatment of chick embryos with 2‐allyl‐2‐isopropylacetamide or 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine caused increased levels of cytochrome P‐450 mRNA and suggest that this involved increased transcription of the gene.
Structure of Fibronectin and Its Fragments in Electron Microscopy
Authors: PRICE Todd M. , RUDEE M. L. , PIERSCHBACHER Michael , RUOSLAHTI Erkki 
Published: March 2005

Human plasma fibronectin and a series of its large proteolytic fragments were analyzed by electron microscopy using tungsten shadowing on carbon and polystyrene films. On carbon, intact fibronectin appeared as a randomly coiled strand, while on polystyrene it appeared as an elongated structure. Two fragments of fibronectin, Mr=205000 and 190000, which lack the NH2‐terminal domain of fibronectin and retain the collagen‐binding, cell‐attachment and heparin‐binding functions, and a Mr= 170000 fragment, which retains the collagen‐binding and cell‐attachment functions, were seen as rods with varying degrees of nodularity while a Mr= 100000 fragment, which only binds to collagen, had two clear‐cut domains.These results support the existing biochemical evidence that the segregation of the functional activities in the fibronectin molecule is based on distinct structural domains and provides evidence for the existence of an additional structural domain not revealed by biochemical and functional studies.
Purification and Chemical Characterisation of Membrane Glycoproteins from Rat Thymocytes and Brain, Recognised by Monoclonal Antibody MRC OX 2
Authors: BARCLAY A. Neil , WARD Harry A. 
Published: March 2005

The MRC OX 2 monoclonal antibody recognises antigens present on rat thymocytes, brain, follicular dendritic cells in lymphoid organs, vascular endothelium, some smooth muscle and B‐lymphocytes. The OX 2 antigens recognised by this antibody were purified from brain and thymus, by solubilisation with sodium deoxycholate, affinity chromatography with MRC OX 2 antibody and gel filtration. The purified brain and thymoeyte OX 2 antigens were glycoproteins with apparent M, 41000 and 47000 respectively as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. Rabbit antisera raised against the purified antigens were analysed by radioimmunoassay and immunoperoxidase‐staining of tissue sections. The brain and thymocyte OX 2 antigens were antigenically very similar to those on the other tissues. This indicates that the unusual pattern of distribution was not the result of fortuitous cross‐reaction of the MRC OX 2 antibody, as the rabbit sera would be expected to recognise more determinants on the antigen than that recognised by the monoclonal antibody.The amino acid compositions of brain and thymus OX 2 antigens were very similar but with no distinguishing features. Carbohydrate compositions showed that the OX 2 antigens were highly glycosylated, with brain OX 2 antigen containing 24% and thymocyte OX 2antigen 33% by weight of carbohydrate. Both OX 2 antigens contained carbohydrate residues typical of structures N‐linked to asparagine but lacked galactosamine, indicating the absence of O‐linked structures. Thymocyte OX 2 contained higher levels of galactose and sialic acid but less fucose than brain OX 2. Similar differences had been observed for brain and thymocyte Thy‐1 antigens and were also observed in pooled glycoproteins purified by lentil lectin affinity chromatography from these tissues, reflecting overall differences in the patterns of glycosylation in the two tissues. The OX 2 antigens showed many similarities to Thy‐1 antigens in their odd patterns of distribution, characteristic migration on polyacrylamide gels in sodium dodecyl sulphate, and carbohydrate compositions. It is possible that OX 2 antigens, like Thy‐1 antigens, have homologies with immunoglobulin domains. A possible role for OX 2 antigens in cell interactions necessary for tissue organisation is discussed.
Limited Proteolysis of the 94000‐Dalton Subunit of Panulirus interruptus Hemocyanin the Carbohydrate Attachment Site
Authors: VEREIJKEN Johan M. , SCHWANDER Edwin H. , SOETER Nell M. , BEINTEMA Jaap J. 
Published: March 2005

Limited proteolysis of the native monomeric 94‐kDa subunit of Panulirus interruptus hemocyanin by trypsin, plasmin and subtilisin produces an 18‐kDa fragment, a 71‐kDa fragment and a small glycopeptide. In the plasmin digest a 23‐kDa precursor of the 18‐kDa fragment has been observed. Automatic Edman degradations demonstrated that the 18‐kDa fragments have their origin at the N terminus of the 94‐kDa subunit and incubations with carboxypeptidase A showed that the 71‐kDa fragments originate from the C terminus. The glycopeptide is situated in between. The amino acid sequence of the glycopeptide has been determined. Its carbohydrate content accounts for the total carbohydrate of the 94‐kDa subunit.All three proteases cleave the subunit at two positions within a very restricted area of the polypeptide chain, which indicates the presence of an exposed loop, carrying the carbohydrate chain, in the corresponding part of the native molecule.
Amino‐Acid Sequence of an α‐Parvalbumin, PI = 4.88, from Frog Skeletal Muscle
Authors: JAUREGUI‐ADELL Juan , PECHERE Jean‐Franqois , BRIAND Gilbert , RICHET Colette , DEMAILLE Jaques G. 
Published: March 2005

The primary structure of the most basic (PI = 4.88) of the two major parvalbumins from frog skeletal muscle (Rana esculenta) has been determined by partial automatic sequencing of the protein which exhibits a free N terminus, and a study of overlapping peptides obtained by cyanogen bromide cleavage and digestion with trypsin, thermolysin and Armillaria mellea protease.This protein shows the typical structure of an α‐parvalbumin. Comparison of the primary structure of ionbinding loops of α anld β‐parvalbumins does not provide a clear‐cut explanation of their differences in ionbinding properties [Haiech, J., Derancourt, J., Pechere, J. F., and Demaille, J. G. (1979) Biochemistry, 18, 2752‐2758].
Effects of Streptozotocin‐Induced Diabetes Mellitus on the Binding and Uptake of Asialoorosomucoïd by Isolated Hepatocytes from Rats
Authors: DODEUR Michèle , DURAND Dominique , DUMONT Jean , DURAND Genévieve , FEGER Jeanne , AGNERAY Jean 
Published: March 2005

The binding and the total uptake of [3H]asialoorosomucoíd was studied in normal rats, streptozotocïn‐diabetic rats and insulin‐treated diabetic rats.

The binding of [3H]asialoorosomucoi'd to normal rat hepatocytes gave a curvilinear plot when analysed by the Scatchard method, suggesting either two classes of independent receptor sites with different apparent association constants or a negative cooperativity in binding. The data, plotted according to the method of De Meyts and Roth showed a decrease of the affinity constant with respect to site occupancy.
The Scatchard plot obtained for the [3H]asialoorosomucoid binding to streptozotocin‐diabetic rat hepatocytes was curvilinear. According to the independent site model, the receptor number of each class was decreased, without any change of the apparent association constants. According to the model of De Meyts and Roth, no modification of the interaction factor and apparent affinity constants was observed, whilst the total number of receptor sites was decreased by half in streptozotocin‐diabetic rats as compared with normal rats.
Study of the total uptake of [3H]asialoorosomucoId by hepatocytes from each group of rats showed a decrease by half of the endocytosis in streptozotocin‐diabetic rats as compared with normal rats.
Insulin treatment of the streptozotocin‐diabetic rats restored both parameters studied to normal level, whilst the withdrawal of insulin treatment decreased their level to that of streptozotocin‐diabetic rats.These results are related to possible modifications of the hepatic binding protein, receptor for the plasma asialoglycoproteins, in the diabetic state.
The Glucose/Glucose‐6‐Phosphate Cycle in the Periportal and Perivenous Zone of Rat Liver
Authors: JUNGERMANN Kurt , HEILBRONN Regine , KATZ Norbert , SASSE Dieter 
Published: March 2005

Periportal and perivenous hepatocytes contain different activities (V) of antagonistic key enzymes such as glucokinase and glucose‐6‐phosphatase. In order to get an insight into the metabolism of the periportal and perivenous area the flux rates (v) of the glucose/glucose‐6‐phosphate cycle were calculated on the basis of the Michaelis‐Menten equation using the measured zonal concentrations of glucose and glucose 6‐phosphate, the zonal activities of glucokinase and glucose‐6‐phosphatase previously reported and the half‐saturating substrate con‐ centrations (Km) of the two enzymes found in the literature. The concentrations of glucose were obtained as a first approximation by measuring the concentrations in portal (= periportal) and hepatovenous (= perivenous) blood; those of glucose 6‐phosphate were calculated from the levels determined in microdissected periportal and perivenous liver tissue.The calculations showed (a) that the overall cycling rates agreed remarkably well with those reported for intact animals and (b) that during a normal feeding rhythm the periportal zone should catalyze net glucose output and the perivenous zone should mediate net glucose uptake, as proposed by the model of ‘metabolic zonation’.
Complexing of Glycolipids and Their Transfer between Membranes by the Activator Protein for Degradation of Lysosomal Ganglioside GM2
Authors: CONZELMANN Ernst , BURG Josef , STEPHAN Günther , SANDHOFF Konrad 
Published: March 2005

The lysosomal degradation of ganglioside GMZ by hexosaminidase A depends on the presence of the specific activator protein which mediates the interaction between micellar or membrane‐bound ganglioside and water‐soluble hydrolase. The mechanism and the glycolipid specificity of this activator were studied in more detail.

It could be shown with three different techniques (isoelectric focusing, centrifugation and electrophoresis) that the activator protein extracts glycolipid monomers from micelles or liposomes to give water‐soluble complexes with a stoichiometry of 1 mol of glycolipid/mol of activator protein. Liposome‐bound ganglioside Gm2 is considerably more stable against extraction and degradation than micellar ganglioside.
In the absence of enzyme the activator acts in vitro as glycolipid transfer protein, transporting glycolipids from donor to acceptor membranes.
The activator protein is rathcr specific for ganglioside GM2. Other glycolipids (GM3, GM1, GDla and GA2) form less stable complexes with the activator and are transferred at a slower rate (except for ganglioside G, than ganglioside GM2.