bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modification of Methionyl‐tRNA Synthetase by Proteolytic Cleavage and Properties of the Trypsin‐Modified Enzyme
Authors: Cassio Doris , Waller Jean‐Pierre 
Published: March 2005

Earlier studies have shown that native methionyl‐tRNA synthetase from Escherichia coli K12 is composed of four, probably identical, subunits having a molecular weight of 43 000 each.Incubation of the purified enzyme with trypsin results in irreversible conversion into an enzymatically active modified form which, by a number of criteria, including molecular size, catalytic and antigenic properties, electrophoretic and chromatographic behaviour, appears to be undistinguishable from the altered methionyl‐tRNA synthetase previously observed after incubation at 37°C of a crude extract. Furthermore, a similar modification of methionyl‐tRNA synthetase resulted upon incubation of the purified enzyme with several other proteolytic enzymes, including papain, subtilisin and pronase.The structural and catalytic properties of the homogenous trypsin‐modified methionyl‐tRNA synthetase were examined in detail. It has a molecular weight of 64000 established by equilibrium ultracentrifugation, and dissociates in the presence of 8 M urea to yield two, apparently identical subunits of molecular weight 32000. Furthermore, it is shown that proteolysis of the native enzyme by trypsin is accompanied by release of enzymatically inactive fragments corresponding to approximately 20% of the original protein. The simplest interpretation of these results is that proteolysis selectively removes a portion of each original subunit, corresponding to a quarter of its size, with consequent dissociation of the original tetramer into dimers, each now composed of modified subunits.No significant differences in the Michaelis constants for any of the substrates or inhibitors tested were found between native and modified enzyme. Nor was conversion accompanied by any detectable change in the specificity towards the amino acid. The molecular activity (expressed as moles of substrate converted/mole subunit) of the homogenous trypsin‐modified enzyme was found to be 20% lower than that of native methionyl‐tRNA synthetase, for both reactions catalyzed by the enzyme. An interesting difference between the two forms of the enzyme appeared in their magnesium ion requirement for the aminoacylation of tRN As. Thus, under otherwise identical assay conditions, the trypsin‐modified enzyme required significantly less magnesium ions for the aminoacylation of both tRNAMetf and tRNAMetm at optimal rates. Another remarkable feature of the trypsin‐modified enzyme is its enhanced stability against heat inactivation, compared to the native enzyme.A structural model is proposed for methionyl‐tRNA synthetase which attempts to account for its great susceptibility to limited proteolysis, and for the maintenance of its catalytic properties despite the resulting radical changes in its molecular structure.
The Synthesis and Circular Dichroism of a Series of Peptides Possessing the Structure (l‐Tyrosyl‐l‐alanyl‐l‐glutamyl)n
Authors: Schechter Bilha , Schechter Israel , Ramachandran Janakiraman , Conway‐jacobs Abigail , Sela Michael 
Published: March 2005

A series of peptides possessing the structure (l‐Tyr‐l‐Ala‐l‐Glu)n, with n = 1,2,3,4,7,9, and 13 was synthesized. The oligomers up to n = 4 were prepared by stepwise synthesis using the N‐hydroxysuccinimide ester of the tripeptide derivative for the elongation of the chain. The oligomers with n = 7,9, 13 were prepared by a polycondensation technique, followed by gel filtration.The circular dichroism spectra of the above oligopeptides were measured in the wavelength range 200–330 nm in a solution of 0.15 M sodium chloride –0.02 M sodium phosphate, pH 7.4, conditions at which the high molecular weight polytripeptide (Tyr‐Ala‐Glu)n exists in a helical conformation. The circular dichroic spectrum of the high molecular weight polymer exhibits two negative ellipticity bands: an intense band at 220 nm ([Θ]max= 8700) and a weak band at 273 nm ([Θ]max= 360). The tripeptide (n = 1) exhibits a positive ellipticity band at 227 nm ([Θ]max= 4000) and a broad, positive, weak band at 270 nm ([Θ]max= 115). The other oligopeptides (n = 2 to 13) exhibit positive bands in the 227 nm region, with ellipticity values that decrease as the degree of polymerization increases. Except for (Tyr‐Ala‐Glu)13, which has a negative peak centered at 216 nm, circular dichroic spectra of the other oligomers (n = 1 to 9) in the 200–210 nm region resemble that of random coils. In the 260–290 nm region, the circular dichroic curves of the oligopeptides gradually approach the 272 nm band of the polypeptide (Tyr‐Ala‐Glu)n, as the degree of polymerization increases. Under physiological conditions, only the (Tyr‐Ala‐Glu)13 shows an indication of helical content.
A Comparison of the Antigenic Specificity of Random and Ordered Linear Polypeptides Composed of l‐Tyrosine, l‐Alanine and l‐Glutamic Acid
Authors: Conway‐jacobs Abigail , Schechter Bilha , Sela Michael 
Published: March 2005

Antibodies were provoked against a random copolymer of l‐tyrosine, l‐alanine, and l‐glutamic acid, denoted as(Tyr,Ala,Glu)n, and against the α‐helical high molecular weight polymer containing the repeating sequence l‐tyrosyl‐l‐alanyl‐l‐glutamyl, denoted as (Tyr‐Ala‐Glu)n. Significant crossreactions were found between anti‐(Tyr‐Ala‐Glu)n antibodies and (Tyr,Ala,Glu)n, as well as between anti‐(Tyr,Ala,Glu)n antibodies and (Tyr‐Ala‐Glu)n.Small peptides, obtained from a 45‐min pronase digestion of (Try,Ala,Glu)n efficiently inhibited precipitin reactions between anti‐(Tyr,Ala,Glu)n and (Tyr,Ala,Glu)n, anti‐(Tyr,Ala,Glu)n and (Tyr‐Ala‐Glu)n, and anti‐(Tyr‐Ala‐Glu)n and (Tyr,Ala,Glu)n. The peptides did not affect at all the reaction between anti‐(Tyr‐Ala‐Glu)n and (Try‐Ala‐Glu)n. It was concluded that the cross‐reaction between the (Tyr‐Ala‐Glu)n and (Tyr,Ala,Glu)n systems were due to sequential determinants in (Tyr‐Ala‐Glu)n rather than helix‐dependent determinants in (Tyr,Ala,Glu)n.The cross‐reactive fraction from anti‐(Tyr‐Ala‐Glu)n sera was isolated on a (Tyr,Ala,Glu)n immunoadsorbent and the purified antibodies were found to precipitate to a much larger extent with (Tyr,Ala,Glu)n than with (Tyr‐Ala‐Glu)n. These antibodies were probably produced against some degradation products of (Tyr‐Ala‐Glu)n produced in vivo.
Phospholipase A from Bee Venom
Authors: Shipolini Rudolf A. , Callewaert George L. , Cottrell Richard C. , Doonan Shawn , Vernon Charles A. , Banks Barbara E. C. 
Published: March 2005

The phospholipase A from the venom of the common European honey bee (Apis mellifica) has been completely purified. The final product (13 g from 700 g of crude venom) readily crystallizes and is homogeneous with respect to starch gel electrophoresis at pH 8.0, isoelectric focussing in polyacrylamide gel in the pH range 3–10, and sedimentation and diffusion analysis in the ultracentrifuge.Only one N‐terminal residue, isoleucine, can be detected either by the Edman or dansyl methods. Quantitative N‐terminal analysis and gel filtration on Sephadex G‐100 give values for the molecular weight of about 19000. Ultracentrifugation studies lead to a value of about 40000: in concentrated solution the molecule exists, therefore, as a dimer.The identity of the enzyme as a phospholipase of the A2 type has been confirmed since, with 1‐oleyl‐2‐isolauroyl phosphatidyl ethanolamine as substrate, isolauric acid is liberated in 100% yield whereas no oleic acid is released. A method for the assay of the enzyme based on continuous titrimetric estimation of hexanoic acid liberated from 1,2‐dihexanoyl lecithin has been used to study various aspects of the activity of the enzyme.
Identification of Lysines Reactive with Pyridoxal 5′‐Phosphate in Glyceraldehyde‐3‐phosphate Dehydrogenase
Authors: Forcina Bruno G. , Ferri Giuseppina , Zapponi Maria Carla , Ronchi Severino 
Published: March 2005

Rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase is almost completely inhibited by reaction of 4 moles of pyridoxal 5′‐phosphate per mole of subunit chain of holoenzyme.Reduction of the pyridoxal 5′‐phosphate inactivated holoenzyme with NaBH4 followed by carboxymethylation of thiol groups, and subsequent tryptic digestion, allowed the separation of two strongly fluorescent peptides. These have been identified as peptides T19+T20 (residues 184 to 192) and peptides T22+T23 (residues 198 to 216) of the sequence published by Harris and Perham for porcine enzyme, in which lysines 191 and 212 are blocked by pyridoxal 5′‐phosphate. Evidence for other lysyl residues reacting to a lesser extent with the reagent is reported: among such residues, only lysine‐2(contained in the N‐terminal peptide) has been unambigously identified. No reaction takes place at lysine‐183. The homology between the sequence around lysine‐212 and the sequence around the essential lysine residue in glutamate dehydrogenase is stressed. The role of pyridoxal 5′‐phosphate reactive lysines in the mechanism of glyceraldehyde‐3‐phosphate dehydrogenase is discussed.
The Oxidation of Cholesterol in Rat Liver Sub‐Cellular Particles
Authors: Mitton John R. , Scholan Norma A. , Boyd George S. 
Published: March 2005

Various sub‐cellular fractions of rat liver exert catalytic effects on the oxidation of [4‐14C]‐cholesterol to 7α‐hydroxy‐[4‐14C]cholesterol and to other [4‐14C]cholesterol oxidation products. Some of these oxidation products created in these studies in vitro appear to be aberrant oxidation products in that there is no evidence that they are intermediates in the physiological oxidation of cholesterol. These aberrant oxidation products have been classified as autoxidation products despite the fact that the autoxidation reactions in these cell fractions appear to require the participation of NADPH, native proteins and perhaps certain nucleotides for the production of these compounds.The enzyme system catalysing the oxidation of [4‐14C]cholesterol to 7α‐hydroxy‐[4‐14C]cholesterol could be assayed in a 18000 ×g supernatant fraction of rat liver. This enzymic reaction was adversely affected by extensive homogenisation of liver tissue and by prolonged incubation periods, both of which tended to increase the autoxidation reactions. The cholesterol‐7α‐hydroxylase enzyme showed optimal activity in the presence of NADPH and oxygen.In the washed liver microsomal fraction, [4‐14C]cholesterol was extensively oxidised in the presence of NADPH, ADP and ferrous ions to 7β‐hydroxycholesterol, cholestan‐3β, 5α, 6β‐triol and 7‐ketocholesterol. Further investigation showed that another compound was also formed, having the same mobility as 7‐ketocholesterol in the thin layer solvent systems used. Reduction and further metabolism of this compound compared with reference standards suggested that it may be a cholesterol oxide, formed under conditions where peroxidation of microsomal lipids could occur. A comparison of the effects of Tris and phosphate buffers, and various nucleotides suggests that cholesterol autoxidation in the microsomal fraction may be related to the general lipid peroxidation systems.The boiled 100000 ×g liver supernatant preparation or 2‐mercaptoethylamine both effectively quenched cholesterol peroxidation, at the same time stimulating the cholesterol‐7α‐hydroxylase system. A scheme has been suggested to explain the liver microsomal cholesterol autoxidation or peroxidation and to contrast this process with the mixed function oxidase involved in the enzymic cholesterol‐7α‐hydroxylase reaction.
6‐Methylsalicylic Acid Synthesis by Isolated Barley Chloroplasts
Authors: Kannangara C. Gamini , Henningsen Knud W. , Stumpf Paul K. , von Wettstein Diter 
Published: March 2005

Chloroplasts isolated from dark grown barley leaves greened for 6 h rapidly incorporate [1‐14C]acetate into 6‐methylsalicylic acid. Etioplasts synthesize little 6‐methylsalicylic acid and mature chloroplasts lack this activity.
Carbon dioxide and CoA are essential cofactors for the synthesis. While light increases the synthesis of 6‐methylsalicylic acid, a considerable amount of the acid is formed in the dark. Externally supplied NADPH has no effect on the incorporation of [1‐14C]acetate into 6‐methylsalicylic acid by isolated chloroplasts.
The biosynthesized 6‐methylsalicylic acid has been purified by thin layer chromatography. Characterization of the labeled compound is done by radio gas liquid chromatography, by repeated crystallization to constant specific radioactivity and by identification of its nitration product as 3‐methyl‐2,4,6‐trinitrophenol.
Since 75% of the radioactivity of 6‐methylsalicylic acid synthesized by isolated chloroplasts is confined to the aromatic nucleus, a mechanism of biosynthesis involving head to tail condensation of acetyl CoA with malonyl CoA is indicated.
Hydroxymethylhydroperoxide as Inhibitor and Peroxide Substrate of Horseradish Peroxidase
Authors: Marklund Stefan 
Published: March 2005

Bis(hydroxymethyl)peroxide, formerly reported to inhibit horseradish peroxidase, was found not to influence the enzyme.
Hydroxymethylhydroperoxide, which may be formed from bis(hydroxymethyl)peroxide by hydrolysis, irreversibly inhibits horseradish peroxidase in a second‐order reaction, k= 3.0X102/sec−1XM−1 at pH 4.27 in 6.7 mM guaiacol.
Hydroxymethylhydroperoxide is a peroxide substrate of horseradish peroxidase.
The rate constant (k1) for the formation of the enzyme‐substrate complex (Cpd I) between hydroxymethylhydroperoxide and horseradish peroxidase was indirectly determined to be 2X105 sec−1XM−1 at pH 4.27.
DNA Synthesis in Nucleotide Permeable Escherichia coli Cells
Authors: Geider Klaus , Hoffmann‐Berling Hartmut 
Published: March 2005

Cells made permeable to nucleotides by treatment with ether synthesize DNA from exogenous deoxynucleoside triphosphates in a semi‐conservative fashion. The reaction proceeds from sites of previous synthesis on the chromosome, and not detectably from new sites. Short chains appear as precursors of long units and have a mean sedimentation coefficient of about 10 S in alkali, like Okazaki‐pieces when synthesized in the presence of 10 to 20 μM deoxynucleoside triphosphates, and of only 5 S when synthesized in the presence of 1 μM dNTP. Under the former conditions short chains contain not more than half of the newly synthesized DNA; under the latter conditions they can contain more than half, the remainder of the newly synthesized DNA being in long units. Nascent short chains labelled after several minutes of synthesis in the presence of 5‐bromodeoxyuridine 5′‐triphosphate at high deoxynucleoside triphosphate concentrations show bimodal density distribution in an alkaline Cs2SO4 equilibrium gradient with peaks of material corresponding to the density of heavy single strands and almost light single strands, respectively. The implications of these findings are discussed. Judged from controls, repair synthesis was not a source of error in these experiments.In contrast with results reported for intact cells, ether‐treated polA1− (Kornberg‐polymerase deficient) cells join nascent short chains as efficiently as do polA+ cells.
The Effects of Thyroxine on Hepatic Gluconeogenesis and Ketogenesis in Dairy Cows
Authors: Heitzman Raymond J. , Hibbitt Kenneth G. , Mather Ian 
Published: March 2005

Four daily injections of l‐thyroxine were given to healthy cows in early lactation. Blood and liver samples were taken during the course of the experiment and assayed for the following metabolites and enzymes: glucose, glycogen, free fatty acids, lactate, pyruvate, 3‐hydroxybutyrate, acetoacetate, citrate, 2‐oxoglutarate, phosphoenolpyruvate, 2‐phosphoglycerate, 3‐phosphoglycerate, malate, NAD+, NADH, glucose‐6‐phosphatase, pyruvate kinase, phosphopyruvate carboxylase and α‐glycerolphosphate dehydrogenase.The changes in the liver enzyme activities and metabolite levels following the thyroxine injections indicate an increase in the rate of gluconeogenesis and ketogenesis.Out of a group of 12 cows only 2 animals showed the early clinical signs of ketosis, otherwise no other unusual clinical signs were observed. Thyroxine elevated the milk yields of the cows, although this increase was not maintained in the animal showing the more severe signs of ketosis.
Influence of Ring Substituents on the Action of Phenols on Some Dehydrogenases, Phosphokinases and the Soluble ATPase from Mitochondria
Authors: Stockdale Martin , Selwyn Michael J. 
Published: March 2005

The effects of phenols on the isolated mitochondrial ATP phosphohydrolase (ATPase) have been compared with their effects on several dehydrogenases, phosphokinases, catalase and acid phosphatase. 2,4‐Dinitrophenol and pentachlorophenol inhibit several of these enzymes at phenol concentrations similar to those which inhibit the ATPase. Stimulation of enzyme activity, by certain phenols which have a para‐substituent, is observed with the ATPase but not with any of the other enzymes.
The concentrations of phenols required to inhibit lactate dehydrogenase, hexokinase and the ATPase, and to produce maximal stimulation of the ATPase have been measured. These biological effects have been correlated with constants which describe the effect of ring substituents on the hydrophobic bonding and electronic properties of the phenols.
The kinetics of the inhibition of lactate dehydrogenase and hexokinase by phenols have been investigated. These inhibitions are competitive with the adenine‐containing substrate and are second order in phenol concentration. When the adenine in NADH is replaced by hypoxanthine the inhibition becomes first order in phenol concentration. When ATP is replaced by ITP as the substrate for the ATPase the inhibition by phenols changes from second to first order.
It is concluded that the effects of phenols on the coupling factor ATPase are not related to their ability to uncouple oxidative phosphorylation. The observations suggest that phenols bind to sites in enzymes which normally bind an adenine residue in a substrate.
Calcium and the Exportable Protein in Rat Parotid Gland
Authors: Wallach David , Schramm Michael 
Published: March 2005

Most of the calcium (>80%) present in rat parotid gland slices was secreted concomitantly with the exportable protein. Detailed kinetics showed that secretion induced by catecholamines and by N6 monobutyryl adenosine 3′:5′‐monophosphate proceeded at an approximately constant ratio of calcium to amylase (60 nmoles calcium/mg exported protein). It was calculated that the calcium which is tightly bound to amylase could account for only 11% of the exported calcium.Among the subcellular fractions of the gland, the secretory granules showed the highest calcium/protein ratio which was identical with that found for the material secreted by the slices. Both amylase and calcium were sedimented in a crude granule fraction to the extent of 50% of the total present in the homogenate of the gland.Various treatment that caused release of amylase from the crude secretory granule fraction also caused release of calcium to a similar extent. The calcium in secretory granules was not readily exchangeable when the granules were suspended in a medium containing 45Ca. The possible role of calcium in the aggregation of the exportable proteins in the secretory granules is discussed in view of the information on the presence of relatively high concentrations of bivalent cations in some other gland systems.
l‐Threonine Deaminase of Rhodospirillum rubrum
Authors: Feldberg Ross S. , Datta Prasanta 
Published: March 2005

A procedure for the purification of threonine deaminase from Rhodospirillum rubrum is described. The 2200‐fold purified enzyme was judged to be pure by disc‐gel electrophoresis and ultracentrifugation.Sedimentation velocity centrifugation yielded an s°20,w value of 8.1 S and a D20,w of 4.65 × 10−7 cm2× sec−1. Assuming a partial specific volume of 0.74 g/ml the molecular weight of 164000 was calculated from the Svedberg equation. However, from calibrated Sephadex G‐200 columns, a D20,w of 4.15 × 10−7 cm2× sec−1 was obtained and using this equation, the molecular weight was calculated to be 180000.Electrophoresis of the enzyme on polyacrylamide gels in the presence of sodium dodecyl sulfate revealed that the native enzyme was composed of four subunits, each of about 46000 molecular weight. In addition to the native tetrameric molecule, an active octameric species was detected in polyacrylamide gel electrophoresis.The spectrum of the pure enzyme showed absorption maxima at 279 nm and 412 nm, characteristic of pyridoxal 5′‐phosphate‐containing enzymes. Analysis of the cofactor content revealed the presence of 4.2 moles of pyridoxal phosphate per 180000 g of protein.The enzyme displayed a normal Michaelis‐Menten substrate saturation curve in the absence of isoleucine. High concentrations of isoleucine inhibited the enzyme activity somewhat only at low levels of the substrate; the inhibition was weak as compared to all other biosynthetic threonine deaminases. No other amino acid or any nucleotide tested produced any significant activation or inhibition.In assays at pH values greater than 8.4, a transient build up of a 245 nm absorbing species was observed. It is suggested that this species corresponds to one of the two intermediates of the deamination reaction, α‐aminocrotonate or α‐iminobutyrate, and that at high pH the rate‐limiting step in the reaction is the hydrolysis of the intermediate to the final products, 2‐oxobutyrate and ammonia.
Transcription of Mitochondrial DNA in vitro from Neurospora crassa
Authors: Schäfer Klaus P. , Bugge Gisela , Grandi Maria , KÜNTZEL Hans 
Published: March 2005

Intact mitochondrial DNA from Neurospora crassa (linear molecules of contour length 25–26 μm) has been transcribed in vitro with RNA polymerase from Escherichia coli.At low concentrations (6 μg/ml) mitochondrial DNA is transcribed as efficiently as DNA from E. coli or calf thymus. The transcription product has the low G + C content characteristic of mitochondrial RNA.After formation of an initial complex between enzyme and mitochondrial DNA only a limited number of bound enzymes can start RNA synthesis in the presence of rifampicin. The sigma factor is required to confer rifampicin resistance.By comparing the initial rate of rifampicin resistant transcription of bacteriophage and mitochondrial DNA under saturating enzyme concentrations, the number of specific binding sites per mitochondrial genome (molecular weight 50 to 60 × 106) has been estimated to be 8 to 9. The transcription product synthesized in the presence of rifampicin has been characterized by competition‐hybridization and by RNAase digestion in solution. About 50% of the sequences hybridizing with mitochondrial DNA are homologous to RNA isolated from intact mitochondria; 35% of the product consists of rapidly renaturing double‐stranded regions characteristic of ribosomal RNA, and 5% of slowly renaturing double strands.25% of the sequences synthesized in vitro form double strands only after annealing for 16 h at 62 °C but do not contain sequences complementary to stable mitochondrial RNA synthesized in vivo.From these results it is concluded that bacterial RNA polymerase recognizes mitochondrial DNA sequences resembling bacteriophage initiation sites, and that enzymes bound to binding sites conferring rifampicin resistance transcribe asymmetrically cistrons coding for mitochondrial RNA.