bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analytical Subcellular Fractionation of Rat Liver with Special Reference to the Localisation of Putative Plasma Membrane Marker Enzymes
Authors: SMITH Geoffrey D. , PETERS Timothy J. 
Published: March 2005

A method for the subcellular fractionation of rat liver using whole homogenates of rat liver and analytical sucrose density gradient centrifugation is presented. The distributions in the sucrose gradients of marker enzymes for all organelles have been determined for control homogenates and for homogenates prepared in the presence of selective membrane perturbants. This technique is not subject to potential loss of information inherent in the use of postnuclear supernatants as starting material for fractionation experiments.Particular attention has been paid to the distributions of putative plasma membrane marker enzymes, up to 50% of which may be found in the nuclear pellet. γ‐Glutamyltransferase has been found to be entirely plasma membrane in location but has a different distribution pattern when compared with other plasma membrane markers. Particulate alkaline phosphatase and alkaline phosphodiesterase are shown to have bimodal distribution, one peak of which is coincident with 5′‐nucleotidase. The other peak is coincident with that of the golgi marker, galactosyltransferase, but the membrane structure containing these activities shows characteristics of plasma membrane rather than golgi apparatus.
The Activated Glucocorticoid Receptor of Rat Liver
Authors: WESTPHAL Hannes M. , BEATO Miguel 
Published: March 2005

The activated form of the hepatic glucocorticoid receptor has been purified 60000‐fold taking advantage of the differential affinity for phosphocellulose of the activated and the nonactivated forms of the receptor. Rat liver cytosol was incubated with [3H]triamcinolone acetonide at low temperature and low ionic strength and adsorbed to a large excess of phosphocellulose. The unbound fraction was heat‐activated, passed through a column of DEAE‐cellulose and precipitated with ammonium sulfate. The activated receptor was then bound to a small phosphocellulose column and eluted with a gradient of NaCl. Additional steps included chromatography on DNA‐cellulose, precipitation with ammonium sulfate and centrifugation through a sucrose density gradient.The final preparation was identified as the steroid‐receptor complex by electrophoresis on polyacrylamide gels in the presence of heparin; it exhibits a single band of molecular weight 40000 ± 4000 in gels containing sodium dodecyl sulfate. An independent calculation of the molecular weight under nondenaturing conditions based on the Stokes' radius (2.7 nm) and the sedimentation coefficient (s20,w= 3.0 S) yields similar results, indicating that the activated form of the receptor we have isolated is composed of a single polypeptide chain and contains a single steroid‐binding site per molecule. Since this homogenous preparation of receptor has retained its affinity for DNA it may be useful for cell‐free studies.
Quantitative Study of the Interaction of Formylmethionyl‐tRNAfMet with Ribosomes of Escherichia coli
Authors: IVANOV Yuri V. , GRAJEVSKAJA Rosa A. , SAMINSKY Eugen M. 
Published: March 2005

fMet‐tRNAfMet binds reversibly to the donor site (P‐site) of Escherichia coli ribosomes both in the absence of messenger and in the presence of ApUpGp and some other oligonucleotides or poly(U). Kinetics of interaction conforms the second‐order law. The equilibrium constants and the rate constants of binding are estimated at 0°C.Not only the cognate trinucleotide ApUpGp but also some other oligonucleotides and even poly(U) stimulate the binding. The presence of total deacylated tRNA considerably increases the selectivity of association.
Investigations on the Possible Involvement of Phospholipids in the Glucose‐6‐Phosphate Transport System of Rat‐Liver Microsomal Glucose‐6‐Phosphatase
Authors: SCHULZE Hans‐Ulrich , SPETH Maria 
Published: March 2005

Interrelationships between the catalytic behavior of glucose‐6‐phosphatase and the structure of rat‐liver microsomal membranes were investigated.

Rabbit anti‐microsomal serum completely inhibited glucose‐6‐phosphate hydrolysis in detergent‐modified microsomes but showed no inhibitory effect on the enzyme activity of intact or mechanically disrupted vesicles.
Controlled proteolysis of intact microsomes using carboxypeptidase A and/or aminopeptidase M largely denatured enzymes situated on the outer surface of the microsomal vesicles such as monodehydroascorbate reductase and cytochrome c reductase. However, it did not affect the glucose‐6‐phosphatase activity at all, which remained in a latent state within the membrane.
Temperature studies on glucose‐6‐phosphatase have revealed that only the enzyme activity of intact microsomes exhibited a nonlinear Arrhenius plot, whereas detergent‐modified microsomes showed a linear temperature response.
Treatment of microsomes with phospholipase C and toluene‐2,4‐diisocyanate resulted in an apparent loss of about 65% and 85% of the original glucose‐6‐phosphatase activity and was closely correlated with hydrolysis and chemical modification of phosphatidylethanolamine, respectively. These apparent inactivations could be reversed by addition of Triton X‐114 alone without any phospholipid supplementation.

These observations indicate that glucose‐6‐phosphatase is buried within the microsomal membrane, not exposed on either side. They also suggest that phospholipids are involved in the glucose‐6‐phosphate transport mechanism.
Lipoprotein Lipase
Authors: BENGTSSON Gunilla , OLIVECRONA Thomas 
Published: March 2005

The rate at which lipoprotein lipase hydrolyzes triglycerides in lipQproteins and in synthetic emulsions decreases sharply with the amount of products formed unless albumin is present. Three factors which contribute to this inhibition as follows. (a) The fatty acids and the monoglycerides formed on hydrolysis locate at the lipid‐water interface of the emulsion and, since they are substrates for the enzyme, act as competitive inhibitors of triglyceride hydrolysis. (b) The enzyme forms complexes with fatty acids. Therefore, as fatty acids accumulate in the system some of the enzyme will be sequestered into enzyme‐fatty acid complexes. Albumin can prevent formation of such complexes since it has a higher affinity for fatty acids than the enzyme has. (c) Activator proteins do not enhance triglyceride hydrolysis unless a fatty acid acceptor is present.The strong inhibition of the enzyme itself and the loss of lipolysis‐stimulating effects of activator proteins provide a feed‐back regulation of the action of the lipase on lipoproteins at the capillary endothelium, ensuring that products are not generated more rapidly than they are utilized by the tissue.
Metabolism of Extracellular Glutathione in Rat Small‐Intestinal Mucosa
Authors: GRAFSTRÖM Roland , STEAD A. Howard , ORRENIUS Sten 
Published: March 2005

Metabolism of exogenous glutathione was investigated in suspensions of freshly isolated rat small‐intestinal mucosal cells. The cells catalyzed the oxidation of reduced glutathione (GSH) to glutathione disulfide (GSSG). Neither serine · borate nor methionine significantly influenced this reaction. Formed GSSG was further metabolized as indicated by its disappearance from the medium. Degradation of GSSG was stimulated by methionine and inhibited by serine · borate. Separation and identification of GSSG metabolites were achieved by high‐performance liquid chromatography. The results indicate that the preferred route for GSSG metabolism to the constituent amino acids in small intestine, is by hydrolytic removal of the two γ‐glutamyl groups of GSSG to yield cystinyl‐bisglycine which is subsequently hydrolyzed to cystine.γ‐Glutamyltransferase activity was compared in isolated intestinal, kidney and liver cells using γ‐glutamyl‐p‐nitrocarboxyanilide as substrate. Kiedney cells were approximately 5‐fold and 150‐fold more active than intestinal and liver cells, respectively. Serine · borate markedly inhibited, and glycyl‐glycine stimulated, hydrolysis of γ‐glutamyl‐p‐nitrocarboxyanilide in all cell types confirming the involvement of γ‐glutamyltransferase in the reaction. The hydrolysis of γ‐glutamyl‐p‐nitrocarboxyanilide was inhibited to approximately the same extent by either GSH or GSSG suggesting that both compounds interact at the donor site of γ‐glutamyltransferase.Comparison of the rates of glutathione metabolism by isolated intestinal and kidney cells suggests that the intestinal contribution to the degradation of extracellular glutathione may be physiologically more important than has previously been assumed.
Localization of Individual Lysine‐Binding Regions in Human Plasminogen and Investigations on Their Complex‐Forming Properties
Authors: LERCH Peter G. , RICKLI Egon E. , LERGIER William , GILLESSEN Dieter 
Published: March 2005

After partial digestion of human plasminogen with elastase, followed by chymotryptic cleavage of one of the fragments produced, two polypeptides with molecular weights of approximately 10000 and with lysine‐binding sites still intact were isolated by means of affinity chromatography and gel filtration.One fragment, which was completely sequenced (88 residues), was identified as the fourth kringle, whereas the other, according to partial sequence analysis represented the first kringle. Equilibrium, dialysis against 6‐aminohexanoic acid yielded for the first kringle one high‐affinity binding site (Ka= 60 mM−1) and for the fourth kringle one single low‐affinity binding site (Ka= 28 mM−1). Moreover, interactions were detected between the first kringle and the N‐terminal CNBr fragment of plasminogen and also fibrin. In these cases an additional lysine‐ binding site, though of low affinity, appears to be involved. Thus, the first kringle seems to play important roles, structurally by contributing to the maintenance of a compact structure of plasminogen through an intramolecular interaction with its N‐terminal polypeptide region, and functionally by increasing the fibrin affinity of Lys‐plasminogen (plasminogen lacking the first 76 residues) and plasmin.
Defective Assembly of the Small Ribosomal Subunit in a Temperature‐Sensitive Mutant of Escherichia coli
Authors: HAYES Françoise , SCHMITT Sophie 
Published: March 2005

Comparison of the properties in vitro of total 30‐S ribosomal subunit proteins and purified protein S4 of Escherichia coli D10 (wild type) and E. coli 219tS2 (temperature‐sensitive) has given the following results.

Reconstitution of functional 30‐S subunits in vitro occurs when total 30‐S subunit proteins of either strain are used even at temperatures which are not permissive for 30‐S subunit assembly in vivo in E. coli 219tS2. The yield of reconstitution is, however, twofold lower with 30‐S subunit proteins of E. coli 219tS2 than with wild‐type proteins.
The yield of complex formation between 16‐S rRNA and protein S4 of E. coli 219ts2 is temperature‐ sensitive and lower at all temperatures tested (33–42°C) than that observed when wild‐type S4 is used.
The conformational stability of complexes between 16‐S rRNA and S4 from 219ts2 is more temperature‐sensitive than that of analogous complexes containing wild‐type S4.

These observations provide an explanation for the temperature sensitivity of 30‐S subunit assembly in E. Coli 219ts2.
A Trichodermin‐Resistant Mutant of Saccharomyces cerevisiae with an Abnormal Distribution of Native Ribosomal Subunits
Authors: CARTER Christopher J. , CANNON Michael , JIMÉNEZ Antonio 
Published: March 2005

A yeast mutant (CLP‐8), resistant at the ribosome level to the trichothecene antibiotic trichodermin, differs from its parent in having an unusual distribution of native ribosomal subunits. Sucrose gradient analysis of cytoplasmic extracts from this mutant revealed a large excess of material sedimenting at 60 S with little or no material sedimenting at 40 S.
The excess 60‐S material consists predominantly of functionally active 60‐S ribosomal subunits, as indicated by both analysis of ribosomal RNA and studies in vitro using a poly(U)‐directed protein‐synthesizing system.
Using the poly(U) system it was found that high‐salt‐washed particles derived from either the excess 60‐S peak or 80‐S ribosomes of CLP‐8 exhibited very similar levels of resistance to the antibiotic fusarenon‐X, a drug closely related chemically to trichodermin. The same level of resistance to fusarenon‐X was also shown by high‐salt‐washed 60‐S ribosomal particles obtained from a further trichodermin‐resistant yeast strain (TR‐1), although this strain has a normal distribution of native ribosomal subunits. In addition, both CLP‐8 and TR‐1 are equally resistant to inhibition of protein synthesis by trichothecene antibiotics, as assayed in vivo.
Genetic analysis of CLP‐8 indicates that the trichodermin‐resistant trait can be segregated from the lesion responsible for the inbalance of native ribosomal subunits. However, the latter defect is only expressed phenotypically in cells that retain the trichodermin‐resistant character.
CLP‐8 has a further defect in that both in vivo and in vitro it fails to generate native 40‐S ribosomal subunits from 80‐S particles. There may be a lesion in the protein factor normally required for this process.
The Relationship between Intramitochondrial N‐Acetylglutamate and Activity of Carbamoyl‐Phosphate Synthetase (Ammonia)
Authors: HENSGENS Hubertus E. S. J. , VERHOEVEN Arthur J. , MEIJER Alfred J. 
Published: March 2005

The relationship between urea synthesis, intracellular N‐acetylglutamate and the capacity of rat‐liver mitochondria to synthesize citrulline was investigated.
Treatment of rats with glucagon prior to killing results not only in an increased intra‐mitochondrial ATP concentration and an increased capacity of the mitochondria to synthesize citrulline, but also in an increased concentration of intramitochondrial N‐acetylglutamate.
Comparison of the rate of citrulline synthesis in mitochondria from glucagon‐treated and from control rats, incubated under different conditions, shows that the increased N‐acetylglutamate concentration after glucagon treatment is at least in part responsible for the observed increased capacity of the mitochondria to synthesize citrulline.
Ureogenic flux in isolated hepatocytes under different incubation conditions correlated with the intracellular concentration of N‐acetylglutamate and with the capacity of the mitochondria to synthesize citrulline.
When isolated hepatocytes were incubated with NH3, ornithine, lactate and oleate, intracellular N‐acetylglutamate increased about eightfold in the first 10 min; during this period the rate of urea synthesis increased considerably.
It is concluded that the concentration of intramitochondrial N‐acetylglutamate plays an important role in the short‐term control of flux through the urea cycle under different nutritional and hormonal conditions.
The Quaternary Structure of Bovine α‐Crystallin
Authors: SIEZEN Roland J. , BINDELS Jacques G. , HOENDERS Herman J. 
Published: March 2005

Reaction of calf α‐crystallin, which consists of about 30 A and 10 B subunits, with various bisimido esters at pH 8.0–8.5 and room temperature leads to inter‐subunit crosslinking. Little difference is observed in the efficiency of crosslinking by reagents of a homologous series with different lengths (from C6 to C12). At any stage of the reaction an exponential decrease is found in the amounts of crosslinked dimer, trimer, tetramer etc., with no preference for the formation of any particular oligomer, suggesting that all subunits on the surface of the spherical α‐crystallin molecule are in quasiequivalent positions.Molecular weight analysis by dodecylsulfate gel electrophoresis and reversible crosslinking show that both A and B subunits occur in an apparently constant ratio in the crosslinked oligomers, from which we infer that both types of subunits are randomly arranged on the surface of the α‐crystallin molecule.Not all of the subunits can form inter‐chain crosslinks, as a limiting value of about 60% of the subunits is found in oligomeric form. A simple explanation could be a core of subunits which is inaccessible to certain chemical reagents, although other alternatives are also discussed.
Complex‐Forming Properties of Spinach NADP+ Reductase with Ferredoxin, Ferrocyanide and NADP+
Authors: RICARD Jacques , NARI Joannès , DIAMANTIDIS Grigorios 
Published: March 2005

The flavoprotein NADP+ reductase from spinach chloroplasts may form a ternary complex with one molecule of NADP+ and one molecule of ferredoxin. Spectroscopic titration studies show that the NADP+ binding site and the ferredoxin binding site are totally independent, that is previous binding of ferredoxin does not modify binding of NADP+, and conversely.Since NADP+ reductase conditions the diaphorase reaction, that is an electron transfer between NADPH and various acceptors such as ferricyanide, the binding of ferrocyanide and its possible interaction with NADP+ and ferredoxin has been studied. Ferrocyanide behaves as a competitive inhibitor with respect to both NADP+ and ferredoxin. This seems paradoxical since NADP+ and ferredoxin are independently bound at two different non‐overlapping sites of the flavoprotein. This apparent paradox may be resolved by a theoretical analysis of the interactions between either ferrocyanide and NADP+, or ferrocyanide and ferredoxin. Theory shows that if ferrocyanide is non‐specifically bound at two independent sites, namely the NADP+ and the ferredoxin binding sites, it appears competitive with respect to both NADP+ and ferredoxin, although ternary flavoprotein‐ferredoxin‐ferrocyanide and flavoprotein‐NADP+‐ferrocyanide complexes are formed. The binding constants of NADP+, ferredoxin and ferrocyanide for the enzyme have been determined. These results are discussed in connection with the possible mechanism of the diaphorase reaction.
A Two‐step Synthesis of New Water‐Soluble Polymers of NAD+ and ADP
Authors: LE GOFFIC François , SICSIC Sames , VINCENT Christian 
Published: March 2005

Alkylation at the N‐1 position of the adenine moiety of NAD+ ADP or ATP with 2,3‐epoxypropylacrylatc, followed by polymerization with or without acrylamide at pH 8, gave water‐soluble polymers of NAD+ and ADP where the alkyl chain was located at the exocyclic adenine C‐6 amino group. Cofactor incorporations were good to high: 145–447 μmol NAD+/g polymer and 667 μmol ADP/g polymer.About 30%, of the bound NAD+ could be reduced with rabbit muscle lactate dehydrogenase, yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase; 84%, of the bound ADP was phosphorylated with rabbit muscle creatine kinase.High cofactor activities were obtained with polymerized NAD+ with alcohol dehydrogenase as enzyme: the initial rate of NAD+ polymer reduction was 35–81% that of free NAD+. These values remained substantially high with agarose‐immobilized alcohol dehydrogenase (15–36%) and should eventually allow their use in continuous enzymatic reactors.Enzymatic phosphorylation of ADP polymer by creatine kinase gave an ATP polymer with high biological activity: 480 μmol ATP/g polymer were transformed with yeast hexokinase.
Distribution of Radioactive Lipids between Envelopes and Thylakoids from Chloroplasts Labelled in vivo
Authors: JOYARD Jacques , DOUCE Roland , SIEBERTZ Heinz Peter , HEINZ Ernst 
Published: March 2005

Spinach leaves were labelled with 14CO2 for subsequent isolation of radioactive chloroplasts, which were separated into envelopes and thylakoids. The analyses carried out with the lipid extracts from these membranes were concerned with the following questions: are chloroplast envelopes a functional interface between endoplasmic reticulum and thylakoids, do they also play a predominant role in galactolipid biosynthesis in vivo, and is it possible to demonstrate galactolipid export from envelopes into thylakoids?Taken together the results show that lipid export is apparently too fast in vivo to be followed by the labelling and separation technique used, since thylakoid lipids contained always far more total label than envelope counterparts, whereas the specific activity of envelope lipids was higher. Phosphatidylcholine, which has been suggested to function as acyl carrier between endoplasmic reticulum and chloroplasts, was never found labelled to any extraordinary extent in envelopes. Envelopes may be regarded as small, but rapidly turned over lipid pools.
Structure and Activity of the Prosthetic Group of Methanol Dehydrogenase
Authors: DUINE Johannis A. , FRANK Jzn Johannes , VERWIEL P. Eugène J. 
Published: March 2005

The reconstitutive ability of the isolated prosthetic group of methanol dehydrogenase with the apoenzyme of glucose dehydrogenase and the results of electron spin resonance measurements suggest that the prosthetic group has not been modified during the isolation. This result, and the properties of the directly isolated prosthetic group and derivatives, confirm the suggestion that its structure is 2,7,9‐tricarboxy‐1H‐pyrrolo[2,3‐f]quinoline‐4,5‐dione.From the activity shown by derivatives of the prosthetic group and of structural analogues in the apoenzyme test it is concluded that the o‐quinone structure is essential for activity. Hence the trivial name pyrrolo‐quinoline quinone would be appropriate. The testing of the analogues also shows that the pyrrolo ring and the 9‐carboxylic acid group are not essential for activity as they can be replaced by a pyridinol ring and a 9‐hydroxy group respectively.The determination of the molar absorption coefficient of the prosthetic group (18400 M−1 cm−1 at 249 nm) enables its quantitative analysis. Thus it could be established that methanol dehydrogenase contains one prosthetic group per enzyme molecule. The consequences of this result in relation to already known properties of this ‘quinoprotein’ dehydrogenase are discussed.
Lactose Carrier Protein of Escherichia coli Structure and Expression of Plasmids Carrying the Y Gene of the lac Operon
Authors: TEATHER Ron M. , BRAMHALL John , RIEDE Isolde , WRIGHT J. Keith , FÜRST Monika , AICHELE Gabriele , WILHELM Ursula , OVERATH Peter 
Published: March 2005

The previously described hybrid plasmid pC7 which carries lacI+ O+Δ(Z)Y+ A+ on a 12.3 × 106‐Mr DNA fragment [Teather et al. (1978) Mol. Gen. Genet. 159, 239–248] was partially digested with the restriction endonuclease EcoRI under conditions reducing the recognition sequence to ↓ d(A‐A‐T‐T) and ligated to the vector pBR322. lacY‐carrying inserts of various sizes (Mr, 1.5–4.7 × 106) were obtained.Hybrid plasmid pTE18 (2300‐base‐pair insert) carries part of the I (repressor) gene, the promotor‐operator region, part of the Z (β‐galactosidase) gene, the Y (lactose carrier) gene and part of the A (transacetylase) gene. Upon induction of pTE18‐harbouring strains the Y‐gene product is expressed at a nearly constant rate for several generations and accumulates to a level of 12–16% of the total cytoplasmic membrane protein. Integration into the membrane leads to active carrier as judged by binding and transport measurements.