bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biospecific Affinity Chromatography in Aqueous‐Organic Cosolvent Mixtures
Authors: LOWE Christopher R. , MOSBACH Klaus 
Published: March 2005

The effect of the weak polarity‐reducing agent, ethylene glycol, on the binding of pig heart lactate dehydrogenase to N6‐(6‐aminohexyl)‐AMP‐Sepharose has been investigated. In the absence of the reagent and under the conditions used, a non‐specific interaction of the enzyme with the adsorbent led to recoveries of enzyme activity as low as 60% when the enzyme was eluted from the columns with a linear NADH gradient. The inclusion of low concentrations of ethylene glycol in column irrigants considerably improved the recovery of enzyme activity with quantitative recoveries being obtained in the presence of 20–30%.Concentrations of ethylene glycol above 35% altered the native conformation of lactate dehydrogenase and led to a decreased affinity for the immobilised ligand. Under these conditions, the altered protein fluorescence and decreased ability to bind NADH could be correlated with the chromatographic behaviour of the enzyme on columns of N6‐(6‐aminohexyl)‐AMP‐Sepharose. These effects were exploited to elute the enzyme from a column of immobilised‐AMP with a linear gradient of ethylene glycol.
Diaminobenzidine an Electron Donor to Photosystem 1 and to Photosystem 2 in Chloroplasts
Authors: BEN‐HAYYIM Gozal , DRECHSLER Ziwia , GOFFER Judith , NEUMANN Joseph 
Published: March 2005

3,3′‐Diaminobenzidine was shown to serve as an electron donor to photosystem 1 [in the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea. In Tris‐treated chloroplasts diaminobenzidine serves as an electron donor to photosystem 1 and to photosystem 2; the latter is sensitive to 3‐(3,4‐dichloro‐pheny 1)‐1,1‐dimethylurea.
Addition of diaminobenzidine to Tris‐treated chloroplasts causes an increase in fluorescence yield.
Diaminobenzidine‐dependent electron transport mediated by photosystem 2 is coupled to synthesis of ATP even in the absence of an electron acceptor. This phosphorylation which is presumably supported by cyclic electron flow, is sensitive to 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea.
Diaminobenzidine‐dependent ATP formation, in Tris‐treated chloroplasts exhibits the red‐drop phenomenon.
The diaminobenzidine‐induced cyclic photophosphorylation (mediated by photosystem 2) is resistant to a large extent to KCN‐treatment which is known to inhibit reactions catalyzed by photosystem 1.On the other hand ATP formation supported by electron transport from diaminobenzidine to methyl viologen in the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dirnethylurea] is largely inhibited by KCN‐treatment. This observation suggests that there are two coupling sites of ATP formation, one catalyzed by diaminobenzidine as a donor to photosystem 1 (in the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea), and the other supported by diaminobenzidine which acts both as a donor to photosystem 2 (in Tris‐treated chloroplasts) and as an acceptor (in its oxidized form) from a carrier located between the two photosystems.
Synthesis and Hydrolysis by Pepsin and Trypsin of a Cyclic Hexapeptide Containing Lysine and Phenylalanine
Authors: TERADA Shigeyuki , KATO Tetsuo , IZUMIYA Nobuo 
Published: March 2005

A cyclic hexapeptide, cyclo(‐Gly2‐Phe2‐Gly‐Lys‐), and the corresponding open‐chain hexa‐peptides, Gly2‐Phe2‐Gly‐Lys and Phe‐Gly‐Lys‐Gly2‐Phe2 have been synthesized and their susceptibilities to the hydrolytic action of pepsin and trypsin were determined.
The cyclic peptide was hydrolyzed slowly by trypsin to a hexapeptide Gly2‐Phe2‐Gly‐Lys2 the value of the Michaelis constant for this reaction being Km= 0.010 M, whereas Phe‐Gly‐Lys‐Gly2‐Phe was hydrolyzed rapidly to yield Phe‐Gly‐Lys and Gly2‐Phe; Km= 0.00022 M.
The cyclic peptide was not cleaved by pepsin at all, but Gly2‐Phe2‐Gly‐Lys was hydrolyzed rapidly at a Phe‐Phe bond; Km= 0.0091 M.
The cyclic peptide inhibits the hydrolysis of Gly2‐Phe2‐Gly‐Lys by pepsin in a linear non‐competitive manner, the value of the inhibition constant being Ki= 0.004 M.
The Binding of Atractylate and Carboxy‐atractylate to Mitochondria
Authors: KLINGENBERG Martin , GREBE Karin , SCHERER Burkhard 
Published: March 2005

35S‐labelled atractylate and carboxy‐atractylate are produced biosynthetically and used for studying the binding of these specific ligands to the ADP, ATP carrier in beef heart mitochondria. The following results are obtained.

Inhibition of translocation activity goes parallel to the increase of binding by [35S]atractylate. No additional binding is observed after full inhibition of translocation is reached giving evidence that atractylate binds exclusively to the carrier.
The maximum number of binding sites of both atractylates is about 1.6 μmol/g protein in beef heart mitochondria and decreases on treatment of the membrane by Pi, freezing, ageing, etc. The dissociation constants of the binding are approximately for atractylate Kd= 5.10−8M and for carboxy‐atractylate Kd= 10−8M. The mass action plots of the concentration dependence for the binding are nonlinear‐convex in particular with carboxy‐atractylate and more linear with atractylate. Nonlinearity appears to be caused by some retardation of equilibration in the case of very high affinity binding.
The binding of atractylate and carboxy‐atractylate is relatively fast in intact mitochondria and slower in aged membranes. There is a slower and a faster binding portion.
The atractylates remove ADP in a nearly 1:1 stoichiometry from untreated mitochondria. In aged and Pi‐treated membranes the ratio Δ; ADP/Δatractylate approaches 0. Obviously binding of carrier sites to ADP is more sensitive to alterations then that of the atractylates. The assumption is maintained that the binding site for atractylate is identical with that for ADP and ATP.
Bongkrekate prevents binding of both atractylates. However, when added after, it only removes atractylate but not the carboxy compound because of its different tight binding. The removal of atractylate depends on the synergistic effect of bongkrekate with ADP.
The binding studies with [35S]atractylate and in particular the interaction with bongkrekate support the reorienting carrier model in which atractylate as an impermeable ligand fixes the binding site of the carrier outside while with bongkrekate the carrier site is turned to the inside.
Kinetics of Binding of Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) in which the Reactive‐Site Peptide Bond Lys‐15‐Ala‐16 is Cleaved, to α‐Chymotrypsin and β‐Trypsin
Authors: QUAST Ulrich , ENGEL Jürgen , STEFFEN Erna , MAIR Gerhard , TSCHESCHE Harald , JERING Helmut 
Published: March 2005

Equilibrium measurements of the binding of reactive‐site‐cleaved (modified) bovine trypsin‐kallikrein inhibitor (Kunitz) to α‐chymotrypsin and β‐trypsin show a stoichiometric 1:1 association with high binding constants. At least in the case of chymotrypsin much evidence is presented that the reaction with modified inhibitor leads to the same complex as the reaction with virgin inhibitor does. The association rate constant of modified inhibitor with chymotrypsin at pH 7, 22.5° C is 15.8 M−1 s−1. This is about 2×104 times slower than the binding of virgin inhibitor to that enzyme. In the analogous reaction of modified inhibitor with β‐trypsin, however, the association rate constant (1.2 × 104 M−1 s−1 at pH 6.9, 22.5° C) is of about the same order of magnitude as it is in the reaction of virgin inhibitor and trypsin. These and analogous phenomena observed in the reactions of virgin and modified soybean trypsin inhibitor (Kunitz) with α‐chymotrypsin and β‐trypsin suggest that the specificity of both inhibitors to trypsin is strongly reflected in the association rate constants of the modified forms.The dissociation rate constants of the complexes of trypsin‐kallikrein inhibitor with chymotrypsin or with trypsin towards the modified inhibitor are estimated to be unmeasurably slow (half‐life times of 45 or 1.5 × 104 years, respectively).
Complexes of Metal Phthalocyanines with Globin as the Models of Heme Proteins
Authors: PRZYWARSKA‐BONIECKA Helena , TRYNDA Liliana , ANTONINI Eraldo 
Published: March 2005

The reaction between iron and cobalt tetrasulfonated phthalocyanines and globin results in the formation of the green complexes, as has been proved by difference spectroscopy. Spectrophotometric titration data indicate the formation of those complexes at the molar ratio 1:1. The complexes of ferrous, ferric and cobaltous tetrasulfonated phthalocyanines with globin have been isolated from the reaction mixtures by separation on Sephadex G‐50 and precipitation of the protein fractions with ammonium sulfate. The visible spectra of these complexes are characterised by the main intensive peak at 641 nm, 678 nm, and 675 nm for ferric, ferrous and cobaltous derivatives, respectively. The new globin complexes have the property of reversible combination with oxygen and coordination with cyanide ions.It is evident from the results of the spectrophotometric titrations of hemoglobin and methemoglobin with cobaltous tetrasulfonated phthalocyanine that iron protoporphyrins are displaced by this cobalt derivative; this suggests that phthalocyanine and porphyrin are bonded in a similar manner.
The Mitochondrial ATPase
Authors: FERGUSON Stuart J. , LLOYD William J. , LYONS Michael H. , RADDA George K. 
Published: March 2005

Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4‐chloro‐7‐nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents.
In sodium dodecyl sulphate, the nitrobenzofurazan group on tyrosine is transferred to newly exposed sulphydryl groups on the enzyme.
The rate of transfer of the nitrobenzofurazan moiety from the enzyme to sulphydryl compounds is compared with that for transfer from the model compound N‐acetyl‐tyrosine‐O(7‐nitrobenzofurazan) ethyl ester, the synthesis and properties of which are also described.
The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4‐chloro‐7‐nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue.
The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondrial ATPases.
When the ATPase activity of submitochondrial particles is inhibited by 4‐chloro‐7‐nitrobenzofurazan, there is a parallel decrease in the extent of the energy‐linked fluorescence enhancement of 1‐anilino‐naphthalene‐8‐sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sulphydryl reagents.
Two Enzymically Active Forms of Glycyl‐tRNA Synthetase from Bacillus brevis
Authors: SURGUCHOV Andrei P. , SURGUCHOVA Irina G. 
Published: March 2005

Using sucrose density centrifugation and gel filtration of a 105000 ×g supernatant of Bacillus brevis two enzymic activities of glycyl‐tRNA synthetase were separated. Enzyme catalyzing the aminoacylation of tRNA (E1) elutes in a high‐molecular‐weight region. Enzyme active in glycylhydroxamate formation (E2) elutes from a Sephadex gel column and sediments in sucrose density gradient in a region of relatively low molecular weight. The presence of two enzymic activities does not depend on the method of cell disruption; their proportion does not change when protease inhibitor (diisopropylphosphorofluoridate) is added to the extraction buffer.Both E1 and E2 were purified to a nearly homogeneous state. Sedimentation coefficients (Sw.20) were found to be 8.6 S and 3.6 S and molecular weights 226000 and 66000 for E1 and E2, respectively. During storage, E1 dissociates into two components, one of which has electrophoretic mobility identical to E2. The molecular weight of the other component is about 160000.Electrophoresis of E1 in the presence of sodium dodecylsulfate reveals two bands corresponding to molecular weights of 81000 and 30000. Under these conditions, E2 dissociates into a polypeptide with a molecular weight of 30000.Valine was found to be the N‐terminal amino acid for E2 and both valine and glutamic acid were N‐terminal amino acids for E1.It is concluded that E1 is a tetrameric protein consisting of two large and two small subunits (α2β2). E2 is a component of E1 with a structural formula α2.
Proton‐Magnetic‐Resonance Studies of the Lysine Residues of Ribonuclease A
Authors: BROWN Larry R. , BRADBURY J. Howard 
Published: March 2005

The amino groups of ribonuclease A (RNase‐A) have been methylated with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance (PMR) studies. Although enzymatic activity is lost, PMR difference spectroscopy and PMR studies of thermal denaturation show native conformation is largely preserved in methylated RNase‐A. Resonances corresponding to the NH2‐terminal α‐amino and 10 ɛ‐amino N‐methyl groups are titrated at 220 MHz to obtain pK values. After correction for the effects of methylation, using values previously derived from model compound studies, a pK of 6.6 is found for the α‐amino group, a pK of 8.6 for the ɛ‐amino group of lysine‐41 and pK values ranging from 10.6 to 11.2 for the other lysine ɛ‐amino groups. Interactions between lysine‐7 and lysine‐41 or between the α‐amino and ɛ‐amino groups of lysine‐1 have been proposed to account for deviations from simple titration behaviour.The correct continuities for the titration curves of the histidine H‐2 proton resonances have been confirmed by selective deuteration of the H‐2 protons. Titration curves for the H‐2 proton resonances of histidine‐12 and histidine‐119 of methylated RNase‐A show deviations from the titration curves for the native enzyme, indicating some alteration of the active‐site conformation. In the presence of phosphate, titration curves for the H‐2 proton resonances of histidine‐12 and histidine‐119 of methylated RNase‐A indicate binding of phosphate at the active site, but these curves continue to show deviations from the titration behaviour of native RNase‐A. The titration curve for the N‐methyl resonance of lysine‐41 is perturbed considerably by the presence of phosphate, which indicates a possible catalytic role for lysine‐41.
The Isolation of Nuclei and Basic Nucleoproteins from the Cellular Slime Mold Dictyostelium discoideum
Authors: CHARLESWORTH Marilyn Chapleo , PARISH Roger W. 
Published: March 2005

A method is described for the isolation of nuclei from an axenic strain of Dictyostelium discoideum using a sorbitol/Ficoll solution and low concentration of Triton X‐100.Basic proteins have been extracted from the nuclei and on polyacrylamide gel electrophoresis yield a consistent pattern in which five major groups or bands predominate. Four of these five fractions comigrate with calf thymus histones and one fraction seems to be unique to D. discoideum.The slowest moving of the five fractions is soluble in 0.5 M perchloric acid and comigrates with calf thymus histone F1. After recovery from the perchloric acid solution by precipitation with acetone this fraction yielded one major band on electrophoresis.
Translational Step Inhibited in vivo by Aflatoxin B1 in Rat‐Liver Polysomes
Authors: SARASIN Alain , MOULÉ Yvonne 
Published: March 2005

Aflatoxin B1 strongly inhibits protein synthesis in rat liver cells. We previously demonstrated that this inhibition could be divided into two steps: up to 5h aflatoxin blocks protein synthesis directly and specifically at the polysome level; beyond 7h protein synthesis inhibition appears chiefly as a consequence of transcription impairment due to drug action.This paper confirms the foregoing results and represents an attempt to localize the translational step inhibited in vivo by aflatoxin B1. We used the simulation study developed by Li, Kisilevsky, Wasan and Hammond, 1972 (Biochim. Biophys. Acta, 272, 451–462) to determine precisely the site inhibited in vivo after drug intoxication. This analysis is based on two parameters: the kinetics of polysome labeling to follow the nascent peptide synthesis, and the kinetics of supernatant labeling to follow the completed protein synthesis.Up to 5h after dosing, aflatoxin specifically inhibits the elongation and/or termination steps during protein synthesis; after longer periods of time inhibition occurs essentially at the initiation step. When the intracellular concentration of aflatoxin is too high, particularly 2h after dosing, each step of protein synthesis is blocked.Polypeptide synthesis by the postmitochondrial supernatants isolated from aflatoxin‐treated animals is impaired in the same proportion as protein synthesis in vivo. The damage caused by aflatoxin is mostly observed on microsomes. However, purified polysomes isolated from aflatoxin‐treated rats synthesize proteins in vitro to the same extent as those from controls. These results suggest that aflatoxin metabolite(s) are bound to polysomes with noncovalent bonds. These active metabolites are probably lost during polysome isolation procedures.Finally, relationships between protein metabolism and aflatoxin carcinogenesis are discussed.
Interaction of a Non‐Histone Chromatin Protein (High‐Mobility Group Protein 2) with DNA
Authors: GOODWIN Graham H. , SHOOTER Kenneth V. , JOHNS Ernest W. 
Published: March 2005

The interaction with DNA of the calf thymus chromatin non‐histone protein termed the high‐mobility group protein 2 has been studied by sedimentation analysis in the ultracentrifuge and by measuring the binding of the 125I‐labelled protein to DNA. The results have been compared with those obtained previously by us [Eur. J. Biochem. (1974) 47, 263–270] for the interaction of high‐mobility group protein 1 with DNA. Although the binding parameters are similar for these two proteins, high‐mobility group protein 2 differs from high‐mobility group protein 1 in that the former appears to change the shape of the DNA to a more compact form.
The molecular weight of high‐mobility group protein 2 has been determined by equilibrium sedimentation and a mean value of 26000 was obtained.
A low level of nuclease activity detected in one preparation of high‐mobility group protein 2 has been investigated.
Spin‐Labeling Studies of Urea‐Treated Leucine Aminopeptidase
Authors: LASSMANN Günter , DAMERAU Werner , SKLENAR Gisela , SCHWARZ Dieter , FROHNE Marlies , LUDEWIG Manfred , HANSON Horst 
Published: March 2005

Leucine aminopeptidase (EC 3.4.11.1) from bovine eye lens was spin‐labeled at the most reactive thiol groups with 2,2,6,6‐tetramethyl‐4‐[2‐iodoacetamido]‐piperidine‐1‐oxyl.
Electron spin resonance spectra show two spectral parts corresponding to two local conformational states in the environment of bound label. One state (A) exhibits a strong immobilizing effect on the mobility of the bound label whereas the other one (B) immobilizes weakly. Independently on the degree of labeling a ratio of A:B ∼ 4:1 was estimated. In B a hydrophobic environment of label was observed.
Treatment of leucine aminopeptidase by 6.2 M urea leads to the following structural changes.a) An additional weakly immobilizing conformational state (B′) with reduced hydrophobic interactions and increased mobility representing an unfolded conformational state appears. B′ shows a time‐dependent increase of its extent at the expense of B and A′ (half conversion time about 0.5 h). The extent of this conformational change is larger, if the enzyme is additionally complexed with Mn2+.b) Mn2+ complexed with the protein is partly released producing hydrated Mn2+.c)After withdrawal of urea the observed conformational changes in leucine aminopeptidase are fully reversible, giving the initial ratio of A:B ∼ 4:1 even after long incubation.
6.2 M urea is not able to destroy the strongly immobilizing conformational state A completely.
Mechanism of 2‐Deoxy‐d‐glucose Inhibition of Cell‐Wall Polysaccharide and Glycoprotein Biosyntheses in Saccharomyces cerevisiae
Authors: KRÁTKÝ Zdeněk , BIELY Peter , BAUER Štefan 
Published: March 2005

The mechanism of inhibition by 2‐deoxy‐d‐glucose of the synthesis of yeast wall polysaccharides and glycoproteins was investigated in Saccharomyces cerevisiae cells and protoplasts. The extent of the inhibition of mannan and glucan synthesis was found to be dependent on whether glucose or mannose was used as the carbon source in the medium. During growth on glucose, 2‐deoxy‐d‐glucose inhibited more intensively mannan than glucan formation. Biosynthesis of wall glucan was strongly suppressed in mannose medium. Selective incorporation of 2‐deoxy‐d‐glucose occurred into that polysaccharide, synthesis of which was more inhibited under given conditions. Suggestive evidence has been obtained that the decisive factor for the proportion of glucan and mannan in the walls is the direction of glucose 6‐phosphate/mannose 6‐phosphate interconversion dependent on the exogeneous hexose.No close correlation was found between the inhibition of mannan synthesis and the appearance of the mannan‐protein enzymes invertase and acid phosphatase. Effect of 2‐deoxy‐d‐glucose was therefore investigated on the parallel synthesis of protein, mannan and several extracellular and intracellular enzymes in protoplasts grown on glucose and mannose. The results obtained pointed out that the hindrance of the secretion of mannan‐protein enzymes is of a complex nature and related more to the inhibition of synthesis of the protein moiety than to the inhibition of glycosylation. Synthesis of several enzymes was found to be a subject of a metabolic control by 2‐deoxy‐d‐glucose or its metabolites.
Modification of Amino Groups of Human‐Erythrocyte Glycoproteins and the New Concept on the Structural Basis of M and N Blood‐Group Specificity
Authors: LISOWSKA Elwira , DUK Maria 
Published: March 2005

Various kinds of modification of amino groups of M and N blood group glycoproteins abolished their capacity to inhibit rabbit and human anti‐M and anti‐N sera.
The reversible modification of amino groups revealed that M and N blood group activity was restored after the removal of amino‐group‐blocking residues.
Modification of amino groups had an entirely different effect on the reactivity of red cell glycoproteins with Vicia graminea agglutinin. The serological activity of N glycoprotein towards Vicia graminea anti‐N agglutinin was unchanged, whereas the weak activity of M glycoprotein towards this anti‐N agglutinin was increased to the level of that of N glycoprotein.
These results indicate that there is a structural difference between M and N glycoproteins, which resides beyond the oligosaccharide chains. It suggests in turn that M and N blood group specificity is determined by amino acid sequence in the peptide chains of red cell glycoproteins.
Effects of Ricin on the Ribosomal Sites Involved in the Interaction of the Elongation Factors
Authors: CARRASCO Luis , FERNANDEZ‐PUENTES Carmen , VAZQUEZ David 
Published: March 2005

The effects of ricin on the different steps of the elongation cycle of protein synthesis in a rabbit reticulocyte cell‐free system are studied in this paper. The toxin most probably acts by catalytically inactivating the ribosomes, since a single molecule of the toxin can inactivate 300 ribosomes for poly(U)‐directed phenylalanine incorporation. The effect of the toxin on the ribosome is irreversible.Ricin specifically inhibits elongation‐factor‐1‐dependent aminoacyl‐tRNA binding to ribosomes but has no effect on the non‐enzymic binding of aminoacyl‐tRNA. Ricin also inhibits formation of the complex elongation‐factor‐2 · ribosome · nucleotide with GTP, GDP or GMP‐P(CH2)P. However, the toxin has no effect on translocation. These apparently conflicting results are discussed in this study.