bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis of a Protein‐Reactive ATP Analog and Its Application for the Affinity Labeling of Rabbit‐Muscle Actin
Authors: Faust Uwe , Fasold Hugo , Ortanderl Fhanz 
Published: March 2005

The synthesis of a protein‐reactive ATP analog, S‐dinitrophenyl‐6‐mercaptopurine riboside 5′‐triphosphate, started from isopropylidated 6‐mercaptopurine. After dinitrophenylation, the thioester riboside was phosphorylated with POCI3 and the monophosphate was converted to the triphosphate by activation with the aid of phosphoric acid diphenylester chloride, and reaction with inorganic pyrophosphate.The analog, labelled with 32P in the β and γ‐positions, was used for covalent binding of the nucleotide to rabbit muscle actin. In preparation for this affinity labelling, the one superficial—SH group of the protein had been alkylated with N‐ethylmaleimide. The nucleotide was able to depolymerize N‐ethylmaleimide‐F‐actin, and became attached to the G‐form at a ratio of 0.8 molecules per actin monomer. The product could be repolymerized to the F‐form with liberation of inorganic phosphate. The product could not be depolymerized after that by ATP solutions at low ionic strength.A single radioactively labelled peptide was discerned in fingerprints of a tryptic digest.
Studies of Glutamate Dehydrogenase: Chemical Modification and Quantitative Determination of Tryptophan Residues
Authors: Witzemann Veit , Koberstein Rudolf , Sund Horst , Rasched Ihab , Jörnvall Hans , Noack Klaus 
Published: March 2005

The effect of the modification of beef liver glutamate dehydrogenase with 2‐hydroxy‐5‐nitrobenzyl bromide was studied with respect to the association‐dissociation equilibrium as well as the enzymatic and regulatory properties. Upon incorporation of 2.1 molecules of the reagent per polypeptide chain, the association of the enzyme is strongly reduced, whereas the enzymatic activity is only slightly decreased (80% maximum velocity). The Km values and the regulation by ADP and GTP, respectively, remain unaltered. Evidence is presented that only tryptophan residues are modified. Magnetic circular dichroism measurements of the modified enzyme suggest partial disubstitntion of tryptophan residues. From the reduced incorporation of 2‐hydroxy‐5‐nitrobenzyl groups at high enzyme concentration it is concluded that tryptophan is located at the association areas of the enzyme.Quantitative determination of the tryptophan content of glutamate dehydrogenase with 2‐hydroxy‐5‐nitrobenzyl bromide, magnetic circular dichroism and peptide analysis yields four tryptophan residues per polypeptide chain contrary to three residues previously suggested from structural analysis. The fourth tryptophan, not present in the reported sequence, is recovered in a chymotryptie peptide Glu‐Trp.
The Occurrence of Multiple Activities in the High‐Molecular‐Weight DNA Polymerase Fraction of Mammalian Tissues
Authors: Holmes Andrew M. , Hesslewood Ian P. , Johnston Irving R. 
Published: March 2005

The high‐molecular‐weight DNA polymerase fraction of calf thymus, rat liver or rat spleen can be resolved into further activities differing from one another in molecular weight and charge properties. The use of Sepharose 6B columns brings about partial resolution of these activities although DEAE‐cellulose achieves a more effective separation. The high‐molecular‐weight fraction from calf thymus has so far yielded four species of activity, A, B, C and D sedimenting at 7.9 S, 5.2 S, 7.3 S and 6.0 S, respectively. In rat liver and spleen, 7.6‐S (I), 7.23 (II) and 5‐S enzymes have been detected although smaller and more variable amounts of 5‐S enzyme occur in the rat tissues compared with calf thymus where the 5.2‐5 species can represent 20–25%, of the total activity. There is no evidence for the presence of the 3.4‐S enzyme in the partially purified fractions.All enzymes exhibit anomalous gel filtration behaviour; it is suggested that this is due to asymmetry of the molecules. The molecular weights for both calf and rat enzymes are drawn from the ranges 200000–225000, 155000–170000 and 100000–110000; since these are multiples of 50000–55000, a main band on dodecylsulphate‐polyacrylamide gel electrophoresis of molecular weight 53000–54000 for one calf enzyme and one rat enzyme, is of special interest.Of the four calf thymus activities only enzyme D can utilise poly(dA) · (dT)10 to support [3H]dTMP incorporation. The pooled peaks of A, B and C from calf thymus all utilise poly(dT) · (A)10 to support [3H]dAMP incorporation. A, B and C of calf thymus have pH optima in the region of 7.8–8.1 whereas D had its pH optimum at pH 7.2; Enzymes I and II of rat liver disappear together with increasing age of the animal, and like A, B, C and D of calf thymus are especially sensitive to the level of salt in the assay.
Human Pancreatic Proteolytic Enzymes and Protein Inhibitors
Authors: Feinstein Gad , Hofstein Ronny , Koifmann Joseph , Sokolovsky Mordechai 
Published: March 2005

The purification of human proteolytic enzymes, i.e. two trypsins, two chymotrypsins and at least one elastase from activated extracts of human pancreas has been accomplished by a combination of affinity chromatography and conventional ion‐exchange chromatography on SE‐Sephadex. The enzymes thus obtained were homogeneous by several criteria: gel electrophoresis, equilibrium sedimentation and immunological assay. The molecular weight and amino‐acid composition of each preparation was determined and compared with those of other mammalian enzymes. The two trypsins showed only partial immunological identity while the two chymotrypsins were identical immunologically. The data raises the question of the number of the zymogens present in the human pancreas.In addition to the proteolytic enzymes, four protein inhibitors of proteolytic enzymes were also isolated from human pancreas. The procedure consisted of several steps that included gel filtration, affinity chromatography and isoelectric focusing. The two major components which accounted for most of the trypsin inhibitory activity were characterized and were apparently of the Kazal type. The more basic human pancreatic inhibitor had a pI of 8.7 and contained 48 amino acids with a molecular weight of 5200. The more acidic inhibitor had a pI of 6.5 and contained 39 amino acids with a molecular weight of 4300. Both inhibitors were capable of inhibiting bovine and two human trypsins. They did not inhibit two human chymotrypsins but had a weak capacity to inhibit bovine chymotrypsin.
The Specificity of the Rat‐Liver Cholesterol 7α‐Hydroxylase
Authors: Brown Maureen J. G. , Boyd George S. 
Published: March 2005

Experiments have been performed which investigate the relationship of the cholesterol 7α‐hydroxylase enzyme to the cytochrome‐P ‐450‐dependent mixed‐function oxidase system of rat‐liver microsomes. There appears to be no simple relationship between the activity of the cholesterol 7α‐hydroxylase enzyme and the cytochrome P ‐450 content of rat liver microsomes. Rat liver microsomal cholesterol 7α‐hydroxylase was sensitive to cycloheximide treatment. Its activity decayed in vivo following this treatment more rapidly than the aminopyrine demethylase enzyme or the mixed‐function oxidase components cytochrome P ‐450 and NADPH‐cytochrome c reductase. Freezing and thawing of rat liver microsomes resulted in a marked decrease in the activity of the cholesterol 7α‐hydroxylase.The specificity of the cholesterol 7α‐hydroxylase enzyme has been investigated by comparing the liver microsomal oxidation of cholesterol with that of other steroids having a similar structure to cholesterol.The substrates whose oxidation was studied were androst‐5‐en‐3β‐ol‐17‐one, pregn‐5‐en‐3β ol‐20‐one, androst‐5‐en‐3β‐ol and pregn‐5‐en‐3β‐ol. All these steroids were metabolised to one or more products when incubated with rat liver microsomes, NADPH and oxygen. Treatment of rats with cholestyramine which increased the activity of cholesterol 7α hydroxylase, had no effect on the rate of oxidation of any of the four steroids studied.The conversion of [4‐14C]cholesterol to 7α‐hydroxy[4‐14]cholesterol was significantly increased by preincubating the added tracer cholesterol with liver microsomes prior to starting the hydroxylation reaction with NADPH.It is suggested that the observed differences between the mechanism of control of the cholesterol 7α‐hydroxylase enzyme and other mixed‐function oxidase enzymes in rat liver microsomes might be due to presence of different rate‐limiting steps in the overall enzyme complex.
Reductive‐Methylation Studies on L7 and LI2 Ribosomal Proteins
Authors: Amons Reinout , Möller Wim 
Published: March 2005

L7 and L12 ribosomal proteins were reductively methylated in isolation and in situ, i.e. bound to the 50‐S ribosomal subunit. The peptide bonds involving the carboxyl groups of ε‐N‐methylated lysines are resistant to trypsin attack.
Comparison shows that most of the lysine residues of L7 and L12 proteins are more reactive in situ than in isolation. A partial unfolding or reduced association in situ is proposed. Notably when modified in solution, the reactivity of the acetylated form (L7) was greater than that of the deacetylated form (L12).
After reductive methylation the L7 and L12 proteins remain active in the GTPase reaction dependent on elongation factor G.
On the Specificity of Carbohydrate‐Lectin Recognition
Authors: Podder Sunil K. , Surolia Avadhesha , Bachhawat Bimal K. 
Published: March 2005

A galactose‐specific protein (RC1) isolated from Ricinus communis beans was found to give a precipitin reaction with concanavalin A. Its carbohydrate content amounted to 8–9% of the total protein and was found to be rich in mannose. The interaction of RC1 with galactose and lactose was measured in 0.05 M phosphate buffer containing 0.2 M NaCl (pH 6.8) by the method of conventional equilibrium dialysis. From the analysis of the binding data according to Scatchard method the association constant (Ka) at 5°C was calculated as 3.8 mM−1 and 1.2 mM−1 for lactose and galactose, respectively. In both cases the number of binding sites per molecule of RC1 with molecular weight of 120000 was found to be 2. From the temperature‐dependent Ka values for the binding of lactose, the values of –5.7 kcal/mol and –4.3 cal × mol−1× K−1 were calculated for ΔH and ΔS, respectively.The addition of concanavalin A to RC1 or vice versa led to the formation of the insoluble complex RC1· ConA4 containing one molecule of RC1 and one molecule of tetrameric concanavalin A (ConA4) which could be dissociated upon addition of concanavalin A‐specific sugars. The complex formation results in a time‐dependent appearance of turbidity in the time range from 10s to 10 min. From the measurement of the time‐dependent appearance and disappearance of the turbidity the formation (kf) and dissociation (kd) rate constants were calculated as 3 mM−1× s−1 and 0.07 ks−1 respectively. The ratio kf/kd (43μM −1), that corresponds to the association constant of complex RC1· ConA4, is higher than that of mannoside · ConA4 and thereby suggests that protein‐protein interaction contributes significantly in stabilising glycoprotein · lectin complexes. The relevance of this finding to the understanding of the chemical specificities that are involved in a model cell‐lectin interaction is discussed.
6‐O‐Methyl‐d‐glucosamine in Lipopolysaccharides of Rhodopseudomonas palustris Strains
Authors: Mayer Hubert , Framberg Karola , Weckesser Jürgen 
Published: March 2005

A hitherto unknown amino sugar was discovered in lipopolysaccharides isolated from strains of chemotype III of the photosynthetic bacterium Rhodopseudomonas palustris. The sugar (2–3% of the lipopolysaccharides dry weight) is detected as a shoulder on the descending part of the glucosamine peak obtained when acid hydrolysates of the lipopolysaccharides are passed through an amino acid analyzer. It was separated from the other amino sugars of the lipopolysaccharide (i.e. glucosamine, galactosamine, quinovosamine) by high‐voltage paper electrophoresis and subsequent paper chromatography. The mass spectrometric analysis of the isolated sugar as deuterium‐reduced alditol acetate revealed the presence of a 6‐O‐methyl‐2‐amino‐2‐deoxyhexose. Demethylation of the isolated sugar afforded glucosamine and the demethylation of its ninhydrin degradation product yielded arabinose, which showed that the parental amino sugar is glucosamine. Authentic 6‐O‐methyl‐d‐glucosamine exhibited identical properties in paper chromatography and high‐voltage electrophoresis as did its alditol acetate derivative in gas‐liquid chromatography and mass spectrometry. These data prove that the unknown amino sugar in lipopolysaccharides of Rhodopseudomonas palustris is 6‐O‐methyl‐glucosamine. The strong positive optical rotation of the isolated sugar indicates its d‐configuration.6‐O‐Methyl‐d‐glucosamine is part of the O‐specific chain and not of thelipid A moiety of the lipopolysaccharide. Periodate‐oxidation studies revealed that the sugar, like the other amino sugars of this lipopolysaccharide, does not occupy non‐reducing terminal positions to any appreciable extent.
Specific Inactivation of Fructose 1,6‐Bisphosphatase from Saccharomyces cerevisiae by a Yeast Protease
Authors: Molano Jesús , Gancedo Carlos 
Published: March 2005

A fraction has been isolated from baker's yeast that is able to inactivate fructose 1,6‐bisphos‐phatase in vitro. The inactivating fraction is heat labile, non‐dialysable and is precipitated with ammonium sulfate; thus indicating that it is a protein. Malate dehydrogenase, hexokinase, glucose phosphate isomerase, glucose‐6‐phosphate dehydrogenase, glutamate dehydrogenase and catalase from baker's yeast and fructose 1,6‐bisphosphatase from the yeast Rhodotorula glutinis were not inactivated by the preparation.A fraction which is able to inhibit the inactivating factor has also been found in baker's yeast. The inhibitor is thermoresistant, non‐dialysable and precipitable by ammonium sulfate. The possible biological significance of these findings in relation with the regulation of yeast fructose 1,6‐bisphosphatase is considered.
The Hexose‐Proton Symport System of Chlorella vulgaris
Authors: Komor Ewald , Tanner Widmar 
Published: March 2005

The specificity of the hexose transport system of Chlorella for sugars is the same as the specificity of sugar‐induced proton uptake. The Km‐values of various sugars for the rate of proton uptake agree well with the respective Km‐values for the rate of uptake of these sugars.
Under anaerobic conditions in the dark, when the rate of sugar uptake is very low, sugar‐stimulated proton uptake is also hardly observable. Under the same conditions in the light [712 nm; O2 evolution is inhibited by 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea] sugar again stimulates proton uptake. The uncoupler carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone, which inhibits sugar uptake, also prevents sugar induced proton uptake.
The Stoichiometry for 6‐deoxyglucose and for glucose under various energetic conditions is one proton taken up per sugar molecule. For other glucose analogues the Stoichiometry has been found to be larger than one. Potassium and sodium ions cannot replace protons in the hexoseproton symport system.
The Poly(adenylic acid) Sequence of Mouse Globin Messenger RNA
Authors: Mansbridge Jonathan N. , Crossley Jennifer A. , Lanyon W. George , Williamson Robert 
Published: March 2005

Mouse globin messenger RNA has been degraded by ribonuclease T1 to give poly(adenylic acid) fragments of two size classes which run electrophoretically with standard RNA molecules approximately 75 and 50 nucleotides long. These contain no internal ribonucleotides other than adenosine and occur at the 3′‐end of the mRNA molecule. The probable sequence to the 5′‐side of the poly(A) tract is ‐G‐C. Attempts to separate the α and β mRNA activities by formamide gradient elution from poly(U)‐Sepharose were unsuccessful.The evidence is more compatible with a post‐transcriptional end‐addition mechanism for the synthesis of the poly(adenylic acid) region of messenger RNA than a transcriptional mechanism.
On the Mechanism and Control of the Malonyl‐CoA‐Dependent Chain Elongation of Fatty Acids
Authors: Podack Eckhard R. , Lakomek Max , Saathoff Gisela , Seubert Werner 
Published: March 2005

The purification of the microsomal fatty acid chain elongating system from beef adrenal cortex is described.
The elongation system requires for optimal activity malonyl‐CoA, ATP, Mg2+, NADPH or NADH. Long‐chain fatty acyl‐CoA derivatives inhibit the incorporation of [1,3‐14C]malonyl‐CoA into fatty acids. Supplementation of the system with enzymes of the C‐3 oxidation sequence allows also an acetyl‐CoA‐dependent chain elongation.
Microsomes from beef adrenal cortex contain 2,3‐trans‐hexenoyl‐CoA reductase with similar properties as described for rat liver.
The ratio of the specific activities of the hexenoyl‐CoA reductase and chain elongation remains constant in the course of purification from rat liver and beef adrenal cortex. From this finding it is concluded that hexenoyl‐CoA reductase is an integrated part of the microsomal malonyl‐CoA‐dependent chain elongation system.
The physiological substrates of hexenoyl‐CoA reductase are 2,3‐unsaturated acyl‐CoA derivatives of polyenoic long‐chain fatty acids with a preference for structures present in γ‐linolenic acid.
Competitive inhibition of the microsomal enoyl‐CoA reductase by malonyl‐CoA with respect to enoyl‐CoA suggests that a multienzyme complex is involved in the microsomal chain elongation process.
Octenoyl‐CoA, octenoyl‐pantethein and octenyl‐acyl‐carrier protein are substrates of the microsomal hexenoyl‐CoA reductase from rat liver with decreasing maximal velocities and affinities. Octenoyl‐acyl‐carrier protein and malonyl‐CoA are competitive, suggesting identical mechanisms of reduction for octenoyl‐acyl‐carrier protein and octenoyl‐CoA by the multienzyme complex.
Intrinsic Luminescence Studies on the Apoenzyme and Holoenzyme of Lipoamide Dehydrogenase
Authors: Visser Antonie J. W. G. , Grande Hans J. , Müller Franz , Veeger Cees 
Published: March 2005

The following fluorescence parameters have been investigated: emission and excitation spectra, relative quantum yields, polarization values and lifetimes of the apo‐ and holoenzyme of lipoamide dehydrogenase. Furthermore, fluorescence perturbation techniques have been used to characterize qualitatively the tryptophan and flavin environment in the protein. Also emission spectra at low temperature have been recorded to examine changes occurring on strong immobilization of the enzyme. From lifetimes and quantum yields of the protein fluorescence it can be concluded that the molecular structures of the two apoenzymes, which can be prepared from this enzyme, are different. The quenched protein fluorescence of the holoenzyme is characterized by a very short fluorescence lifetime due to radiationless energy transfer from tryptophan to flavin. Excitation polarization spectra of flavin and tryptophan fluorescences show that the chromophores are rigidly bound to the protein. Luminescence studies at liquid nitrogen temperatures reveal that the triplet state of the tryptophanyl‐residues is not involved in energy transfer to the flavin.Collisional fluorescence quenching with KI shows in the dimeric holoenzyme, in contrast with the free molecules, only a small reduction in fluorescence intensity of tryptophanyl and flavin residues. Charge effects might be responsible for these observations. It can be derived from the experimental results together with the indication that only one of the two tryptophans is transferring its excitation energy to the flavin, that the distance between donor and acceptor is 1.3–1.6 nm.
Gross Conformation of Human Secretory Immunoglobulin A and Its Component Parts
Authors: Björk Ingemar , Lindh Erik 
Published: March 2005

Human secretory immunoglobulin A, several immunoglobulin A dimers and monomers and secretory component have been characterized with regard to size, shape and conformation. The molecular weights obtained were 420000 for secretory IgA, 360000 for IgA dimer, 160000 for IgA monomer and 75000 for secretory component. Secretory IgA has a markedly extended shape as evident from its frictional ratio and intrinsic viscosity. Moreover, the hydrodynamic parameters of secretory IgA are similar to those of dimeric serum IgA, which indicates similar molecular shapes of the two proteins and thus that the presence of the secretory component does not have a significant influence on the shape of the IgA part of the secretory IgA molecule. This is in contrast to results of electron‐microscopic investigations, which have suggested different shapes for secretory IgA and IgA dimer. The hydrodynamic data are compatible with an extended model for both proteins, in which the carboxy‐terminal halves of the IgA mononiers are joined end to end via a rigid or flexible link, but are in less agreement with an alternative, more compact model, in which the two IgA monomers are superimposed on each other. The IgA monomer itself has hydrodynamic characteristics suggesting a significantly more extended shape than that of IgG. Optical rotatory dispersion and circular dichroism spectra show large similarities between the conformations of IgG and both secretory and serum IgA, although minor differences do exist. As in IgG, evidence for the presence of β‐structure in the different types of IgA was obtained. The similarities of the ORD and CD spectra of secretory IgA and IgA dimer suggest that the secretory component does not exert a major effect on the internal folding of the IgA backbone.Human secretory component also has a somewhat extended shape. Its conformation, as measured by ORD and CD spectra, differs markedly from that of the immunoglobulins, although the spectra do seem to suggest the presence of some β‐structure also in secretory component. The main ORD minimum and a prominent CD band exhibited by this protein are very rarely seen in proteins and suggest that a unique type of conformation is present in secretory component.
Interactions between Membrane Functions and Protein Synthesis in Reticulocytes
Authors: Herzberg Max , Britbart Haim , Atlan Henri 
Published: March 2005

In this paper we demonstrate that two compounds which are known to enhance the permeability of membranes to potassium, valinomycin, a cyclic dodecapeptide and dicyclohexyl‐18‐crow‐6, a macrocyclic polyether, inhibit protein synthesis in reticulocytes and that this inhibition is not primarily due to changes in the intracellular potassium concentration. No effect is obtained by adding the drugs to a cell free system and the inhibition observed is likely to be membrane mediated.Dicyclohexyl‐18‐crown‐6 and low concentration of valinomycin (10 nM) act reversibly probably by interacting with the lipid phase of the membrane. This interaction in turn affects the protein synthesizing apparatus by affecting ribosomal functions. Ribosomes once isolated from treated cells are perfectly normal in structural and functional respects.High concentrations of valinomycin, which change the K+ gradient between the inside and the outside of the cell, affect the protein synthesizing apparatus of reticulocytes irreversibly by blocking incorporation of amino acids at the level of elongation or termination. Ribosomes isolated from cells treated with high valinomycin concentrations are mostly in the form of polysomes which are inactive in cell free protein synthesis.The high valinomycin block on the ribosomal fraction can be relieved either by washing ribosomes with high salt or with puromycin. It is believed that with high concentrations of valinomycin an inhibitor is released (probably from the membrane) and attached to the ribosomes. This high valinomycin effect is believed to by a supplementary phenomenon which when added to the effect of low valinomycin renders the inactivation of ribosomal functions irreversible.