bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunochemical Studies on Human Fibrinogen and Its Fragments
Authors: KUDRYK Bohdan , REUTERBY Jan , BLOMBÄCK Birger 
Published: March 2005

A radioimmunoassay has been developed for the NH2‐terminal portion of human fibrinogen (N‐DSK) as well as for the Bβ chain fragment (residues 1–115) of N‐DSK. This assay has proved valuable in the estimation of the amount of cross‐reaction between N‐DSK and related fragments. The effective range for each assay described was found to be between 1 nmol and 1 pmol.The first system studied was 125I‐labeled N‐DSK–anti‐N‐DSK (System 1). Reduced and alkylated (S‐carboxymethylated) N‐DSK was found to be a strong competitor in this system. The inhibition plateau with the latter was significantly lower than that obtained with unlabeled N‐DSK at equivalent concentrations. This suggests that intact antisera contain a population of antibodies which are elicited to conformational determinants of N‐DSK. However, this antisera must also contain antibodies to altered N‐DSK since a displacement at identical inhibition levels was observed at lower concentrations by S‐carboxymethylated N‐DSK as poposed to intact N‐DSK. These results indicate that 125I‐labeled N‐DSK is in fact “immunologically damaged” by the labeling procedure and consequently reacts with antibodies elicited to structurally altered N‐DSK. Fibrinogen was found to inhibit system 1 to only a very small degree at the concentration levels investigated.Using another N‐DSK antisera and 125I‐labeled N‐DSK (System 1 A), both unlabeled N‐DSK and the NH2‐terminal portion of plasmic fragment E (Aα 1–51, Bβ 54–115, γ 1–58)2, obtained by cleavage with CNBr, were shown to be strong competitors. However, the parent molecule, fragment E itself, (Aα 1–86, Bβ 54–119, γ1–58)2, was shown to be a poor competitor. The significance of these findings is discussed.The 125I‐labeled Bβ fragment–anti‐Bβ fragment assay System (system 2) demonstrated that the latter is a highly immunogenic structure of intact N‐DSK. This was established by a comparison of the inhibition curves obtained in this system using N‐DSK and unlabeled Bβ fragment. In addition, low levels of inhibition were also shown by two fragments of the Bβ chain of N‐DSK. It would appear that the latter fragments, obtained by digestion of Bβ fragment with reptilase and arvin respectively, have few, if any, antigenic determinants of intact Bβ fragment. Fragment E was shown to be a poor competitor in System 2.
Stable and Labile Products of Mitochondrial Protein Synthesis in vitro
Authors: WHEELDON Leslie W. , DIANOUX Anne‐Christine , BOF Mireille , VIGNAIS Pierre V. 
Published: March 2005

1. This paper describes some features of the protein synthesis in isolated rat liver mitochondria and reports on the stability of the product of mitochondrial protein synthesis in vitro.2. Chase experiments performed after a 30‐min pulse of [14C]leucine indicated that a large part of the product is degraded to an acid‐soluble form (labile product) almost as rapidly as it is terminated and released from the mitoribosome. Only a small proportion of the completed product (between 20 and 40 %) is conversed in acid‐insoluble form (stable product).2. Both labile and stable products have an exceptional degree of hydrophobicity, shown by their marked resistance to acid and alkaline hydrolysis and by their solubility in organic solvents.4. A 14C‐labelled peptide with an apparent molecular weight of 12000–13000, as assessed by gel electrophoresis in the presence of dodecylsulfate, is released after heat treatment of mitoribosomes isolated from mitochondria pulsed with [14C]leucine.
Activation of Cholesteryl Esterase in Bovine Adrenal Cortex
Authors: TRZECIAK Wieslaw H. , BOYD George S. 
Published: March 2005

The influence of adenosine 3′:5′‐monophosphate (cyclic AMP) and ATP on the activity of cholesteryl esterase has been studied in bovine adrenal cortex.It has been shown that cyclic AMP and ATP stimulated the activity of cholesteryl esterase in vitro. The increase in cholesteryl esterase activity was dependent upon the presence of cyclic‐AMP‐dependent protein kinase. After removal of protein kinase from cholesteryl esterase preparation no stimulation upon the addition of cyclic AMP and ATP was observed.The addition of purified protein kinase, which was virtually free from cholesteryl esterase activity resulted in approx. 3‐fold stimulation of cholesteryl esterase activity in the presence of cyclic AMP and ATP. Without cyclic AMP and ATP no stimulation of partially purified cholesterol esterase by protein kinase was observed.Preincubation of crude cholesteryl esterase in the presence of 5 mM MgCl2 caused a marked decrease in cholesteryl esterase activity. Under these conditions cyclic AMP and ATP protected the enzyme against inactivationPreincubation of partially purified cholesteryl esterase with [γ‐32P]ATP, cyclic AMP and cyclic AMP‐dependent protein kinase resulted in an increase in cholesteryl esterase activity with a concomitant transfer of 32P‐labelled phosphate into the protein fraction. Incubation of the phosphorylated protein fraction in the presence of 5 mM MgCl2 revealed a decrease in cholesteryl esterase activity with a concomitant release of 32P‐labelled phosphate from phosphorylated protein. The decrease in cholesteryl esterase activity and the release of 32P‐labelled phosphate from phosphorylated protein exhibited similar kinetics.It is postulated that the bovine adrenal cortex cholesteryl esterase is activated by cyclic AMP‐dependent protein kinase and that the activation involves a transfer of a terminal phosphate group from ATP onto the cholesteryl esterase molecule.
Horse Pancreatic Ribonuclease
Authors: Scheffer Albert Jan , Beintema Jaap J. 
Published: March 2005

Horse ribonuclease was purified after acid extraction of pancreas by ammonium sulfate fractionation and chromatography on CM‐cellulose. It is a glycoprotein with an average molecular weight of about 18000. The pure enzyme was chromatographically and electrophoretically heterogeneous due to the heterogeneity of the carbohydrate moiety.The amino‐acid sequence was determined from four series of peptides obtained by different cleavage methods. All but four peptide bonds were overlapped by one or more peptides. The polypeptide chain contains 125 amino‐acid residues and carries oligosaccharide side‐chains at positions 21, 34, and 62; part of Asn‐21 occurs in the carbohydrate‐free form. There are two additional amino acids at the C‐terminus and a deletion at position 39. Including these, horse ribonuclease differs in 35 positions from the bovine enzyme. Horse ribonuclease is more closely related to the artiodactyl ribonucleases than is the rat enzyme.Except for the deletion, all differences can be accommodated in the three‐dimensional model of bovine ribonuclease‐S without altering the folding of the backbone. Model building of the loop containing the deletion offered an explanation for the deviating titration behaviour of one of the tyrosine residues in horse ribonuclease.
On the Biosynthesis of Bacitracin by a Soluble Enzyme Complex from Bacillus licheniformis
Authors: FRØYSHOV Øystein , LALAND Søren G. 
Published: March 2005

1. An enzyme (bacitracin synthetase) which catalyzes synthesis de novo of bacitrain A from the L‐isomers of the constituent amino acids has been purified 11 × from Bacillus licheniformis. The enzyme reaction is dependent on ATP and Mg2+ (or Mn2+). When the l‐isomer of glutamic acid, ornithine, phenylalanine or asparagine was replaced by the corresponding d‐isomer, bacitracin synthesis occurred except in the case of d‐ornithine.2. The partially purified enzyme was fractionated on a Sephadex G‐200 column into two fractions (peak I and peak II) which were both required for bacitracin synthesis. Peak I catalyzed the 32PPi‐ATP exchange reaction in the presence of the l‐isomers of eight of the ten amino acids in bacibracin. These were isoleuoine, cysteine, leucine, glutamic acid, phenylalanine, histidine, aspartic acid and asparagine. Peak II catalyzed the exchange reaction in the presence of the l‐isomers of the remaining two amino acids lysine and ornithine. Aminoacyl‐tRNA synthetases were absent in both peaks.3. Peak I and peak II had the ability to bind covalently amino acids when incubated with ATP, Mg2+ and one of the l‐isomers found to catalyze the 32PPi‐ATP exchange reaction. Peak I predominantly bound isoleucine, cysteine, leucine, glutamic acid, phenylalanine, histidine, aspartic acid, asparagine. Peak II predominantly bound lysine and ornithine. The binding of one amino acid did not seem to affect the binding of a second amino acid. The experiments suggest that the covalently bound amino acids are thioester linked. In contrast to what was found with gramicidin S and tyrocidine synthetases no [14C]AMP‐ATP exchange reaction was detected in the presence of the amino acids.4. The labelled amino acids covalently bound to respectively peak I and peak II were intermediates in bacitracin synthesis.5. After treatment with alkali and alkaline phosphatase, peak I liberated pantothenic acid which was determined using Lactobacillus plantarum. The results suggest that bacitracin synthetase contains covalent bound phosphopantetheine.
The Mechanism of Enzymatic Cellulose Degradation
Authors: Berghem Lars E. R. , Pettersson L. Göran 
Published: March 2005

1. A β‐glucosidase (cellobiase) has been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride.2. The crude material was pre‐fractionated by chromatography on Bio‐Gel P‐10 and by DEAE‐Sephadex chromatography as previously described [1]. Further fractionation methods utilized in the present paper included ammonium sulfate precipitation, SE‐Sephadex chromatography, isoelectiic focusing, and chromatography on Bio‐Gel P‐60.3. A yield of 53 mg enzyme was obtained per 100 g commercial cellulase. The specific activity of the purified enzyme represented an approximately 60‐fold increase from that of the commercial product.4. The isolated enzyme was homogeneous in polyacrylamide gel electrophoresis at pH 8.0 by isoelectric focusing in a polyacrylamide gel and also in free zone electrophoresis.5. No enzyme activity towards Avicel (microcrystalline cellulose) or towards carboxymethyl‐cellulose could be detected in the purified material under the assay conditions used.6. A molecular weight of 47000 was determined for the enzyme by dodecylsulfate‐polyacrylamide gel electrophoresis. The same molecular weight value was obtained by chromatography of the native enzyme on Bio‐Gel P‐100 and also by chromatography of the reduced and alkylated enzyme on Sepharose 6B in 6 M guanidine‐HCl.7. The amino‐acid analysis showed that the enzyme was rich in aromatic (9.5%) and acidic (19.0%) amino acids. The half‐cystine content was 6 residues per molecule indicating 3 disulfide bridges, since no free sulfhydryl group was detectable. The purified enzyme contained no carbohydrate (as hexose).8. The enzyme was isoelectric at pH 5.74 (10 °C) and the electrophoretic mobility was determined to be 1.49 × 10−5 cm2 s−l V− at pH 8.0.9. The Km‐values for the enzyme towards the substrates p‐ nitrophenyl‐β‐d‐glucoside, cellobiose, and cellotetraose were found to be 0.28, 1.5, and 0.35 mM, respectively.
The Mechanism of Reaction of Cys‐149 of d‐Glyceraldehyde‐3‐phosphate Dehydrogenase with p‐Hydroxy‐mercuribenzoate
Authors: BATKE József , KELETI Tamá , FISCHER Éva 
Published: March 2005

1. During the reaction of the Cys‐149 residue of pig muscle d‐glyceraldehyde‐3‐phosphate dehydrogenase holoenzyme with p‐hydroxymercuribenzoate a ternary complex is first formed. The process follows second‐order kinetics as measured by the disappearance of the absorption band at 360 nm characteristic of the enzyme‐coenzyme charge‐transfer interaction. Mercaptide bond formation takes place in this ternary complex as a first‐order intramolecular transformation. The firmly bound coenzyme molecules are also released in a first‐order reaction, as detected by the disappearance of the absorption band at 272 nm characteristic of the enzyme‐coenzyme complex, simultaneously with the formation of mercaptide bond.2. Both the formation of mercaptide bond and the release of NAD show biphasic first‐order kinetics, which indicates that there are differences in the reactivities of Cys‐149 residues and between the coenzyme binding sites within the tetramer. The biphasic first‐order kinetics of mercaptide bond formation was also observed in the reaction of the apoenzyme with p‐hydroxy‐mercuribenzoate.
Relationship between Fatty‐Acid and Phenol Synthesis in Aspergillus fumigatus
Authors: WARD Alan C. , PACKTER Neville M. 
Published: March 2005

Investigations have been conducted into the relationship between fatty acid and phenol synthesis in submerged cultures of Aspergillus fumigatus. Both groups of metabolites are derived from acetate but phenol synthesis only occurs at a late stage in the incubation period and is dependent on a change in the internal environment of the mycelium. Secretion of acetate‐derived phenols commenced many hours after exhaustion of ammonium ions from the culture medium, or transfer of mycelium to glucose solution. Lipid synthesis, however, was maintained at this stage and continued into the phenol‐secreting phase when sufficient glucose was present to provide the necessary substrates. The additional lipid formed was identified as triglyceride.The composition of fatty acids in the total lipids was very similar to that found in other Aspergillus sp. and related Ascomycetes and consisted principally of C18:2 with c16:0, c16:1, C18:0 and C18:1.Addition of 4‐fluorophenylalanine, but not cycloheximide, caused a rapid reduction in fatty acid synthetase activity and lipid synthesis, and abolished formation of orsellinic acid and trihydroxytoluene, the normal secreted products.Certain enzymes examined, including the dehydrogenases of the pentose phosphate pathway, maintained their specific activity during the “stationary” phase whereas fatty acid synthetase was present at greatly reduced activity. The activity of ATP citrate lyase was severely reduced to an undetectable level after approximately 40 h incubation; acetyl‐CoA would therefore not be made available for fatty acid, phenol or ergosterol synthesis after this time.Initiation of phenol synthesis cannot be ascribed simply to diversion of substrates from fatty acid synthesis or to reduction in fatty acid synthetase activity.
Preparation of a Soluble, Bifunctional Enzyme Aggregate and Studies on Its IGnetic Behaviour in Polymer Media
Authors: MATTLASSON Bo , JOHANSSON Ann‐Christin , MOSBACH Klaus 
Published: March 2005

Soluble, bifunctional enzyme aggregates have been prepared by crosslinking sequentially acting enzymes with glutaraldehyde. Aggregates of β‐glucosidase and glucose oxidase with molecular weights in the range 200000–300000 have been characterized from such a preparation. The sequentially working enzymes malate dehydrogenase and citrate synthase have also been aggregated using the same method. The kinetic behaviour of a system comprising two sequentiaily acting enzymes, either separately or as an aggregate, was studied in media containing increasing concentrations of poly(ethyleneglycol). A plot of transition time against polymer concentrations was bell‐shaped in the case of β‐glucosidase and glucose oxidase whereas in the system malate dehydrogenase–citrate synthase almost no lagphase was observed and comequently no transition time was registered. In both enzyme systems the steady‐state rate in the overall reaction was stimulated up to 30% at lower polymer concentrations, whereas reduced rates were obtained in more concentrated polymer solutions. These effects are interpreted in terms of (a) an exclusion effect leading to a relative enrichment of the rate‐limiting intermediate and an apparently higher concentration of the enzymes in the sequence and (b) a decreased diffusion rate which lowers the apparent V.In a similar polymer medium glucose oxidase alone shows decreased apparent Km for glucose on increasing the polymer content.
Chitinase in Goat Serum
Authors: LUNDBLAD Gunnar , LIND Jan , STEBY Maria , HEDERSTEDT Bengt 
Published: March 2005

1. A glycol‐chitin‐splitting enzyme without lysozyme (muramidase) activity was found in serum from various animals. Goat, cow, hen, sheep and pig possessed high activity. No activity was found in serum from man, monkey, horse, dog, cat, rabbit, guinea‐pig or hamster.2. Glycol chitin has been used as a substrate in purification and characterization. A viscosimetric assay with the use of this substrate was found to be a convenient method.3. By means of ammonium sulphate fractionation of goat serum and subsequent gel chromatography a 100‐fold purification of the enzyme was obtained.4. The purified enzyme has its optimal activity around pH 1.65 with glycol chitin as a substrate and, when incubated at 50 °C for 60 min, the optimal stability is in the pH interval 3.5–6.5. The pI determined by isoelectric focusing is 4.85 and the molecular weight assessed by gel chromatography is around 60000.5. The purified goat serum enzyme degrades colloidal chitin with an optimum at pH 5.5 and is thus defined as a chitinase. Goat anti‐human lysozyme serum does not inhibit the purified chitinase.6. To elucidate the origin of the serum chitinase, the concentration of the enzyme was determined in various organ tissues and body fluids. The highest activity with the exception of serum was found in the wall of the fourth stomach of goat and cow.
The Interaction of Vasoactive Intestinal Polypeptide and Secretin with Liver‐Cell Membranes
Authors: DESBUQUOIS Bernard 
Published: March 2005

125I‐labelled derivatives of vasoactive intestinal polypeptide (VIP) and secretin have been used to examine the nature of the interaction of these hormones with isolated liver‐cell membranes of the rat.Incorporation of one atom of iodine per molecule of hormone does not appreciably affect the ability of VIP to stimulate the activity of adenylate cyclase in liver membranes, but causes a two to three‐fold decrease in the apparent affinity of secretin towards this enzyme.Specific binding of 125I‐labelled VIP to membranes is a saturatable process with respect to VIP. A maximum of 0.8 and 3.1 picomoles of hormone per milligram of protein, respectively, binds to microsomal and to plasma membranes. Native VIP and secretin compete with 125I‐labelled VIP for binding in a way expected from the respective affinities of these hormones towards adenylate cyclase activity; about 100 times greater concentrations of secretin are necessary to achieve effects similar to those observed with VIP. Synthetic secretin displaces the VIP‐membrane complex identically to natural secretin, indicating that the biological effects of the latter probably do not result from a contamination by VIP. Glucagon, 2–27‐gastrin, cholecystokinin‐pancreozymin, angiotensin and bradykinin do not compete for binding.The labelled material dissociated from the VIP‐membrane complex retains the ability of unused VIP to bind to membranes, but VIP in the incubation medium is rapidly inactivated upon exposure to membranes. Inactivation of the medium VIP is inhibited by certain polypeptides, such as bacitracin, and by the metal‐complexing agent 1,10‐phenantroline; these compounds increase the binding of 125I‐labelled VIP to membranes when subsaturating concentrations of hormone are used.The VIP‐membrane interaction has been characterized kinetically under conditions where inactivation of the medium VIP is inhibited. At 23°C, the rate of VIP‐membrane association is 1.3 × 107 M−1 s−1 and the rate of dissociation is 2.3 × 10−3 s−1. The dissociation constant based on these rate constantsis 0.17 nM; a value of 0.16 nM is obtained separately from equilibrium data. The rates of association and of dissociation are equally sensitive to temperature, resulting in a dissociation constant that is not temperature‐dependent.Binding of VIP to membranes is markedly reduced by increasing the ionic strength of the medium. Maximum binding occurs at pH 7.4. Nucleoside di‐ and triphosphates decrease the binding of VIP to membranes at equilibrium and increase the rate of dissociation of the VIP‐membrane complex; guanyl nucleotides are most effective. Digestion of the membranes with trypsin and phospholipase A results in a loss of the ability of these membranes to bind VIP. 125I‐labelled secretin binds to liver membranes in a way expected from the ability of native secretin to displace the 125I‐labelled VIP‐membrane complex; binding of iodinated secretin to membranes is inhibited by native secretin and VIP similarly to that of iodinated VIP.
Métabolisme phospholipidique dans les macrophages alvéolaires de poumon
Authors: SOULA Georges , CHAMPANET Catherine 
Published: March 2005

Phospholipid Metabolism in Alveolar Macrophages of Lungs [32P]Orthophosphate Incorporation in Pulmonary Surfactant Phospholipids in Rat in vivo and in vitroPulmonary surfactant phospholipids play an important role in the surface tension activity of the lung.Incorporation of [32P]orthophosphate into rat‐lung pulmonary‐surfactant phospholipids was studied in vitro and in vivo after separation on silicic‐acid‐column chromatography and by one and two‐dimensional thin‐layer chromatography. While they represent a small part of the phospholipids of the whole lung, pulmonary‐surfactant phospholipids show a rapid turnover, particularly in vitro.After in vitro incubation, phosphatidylcholine and lysophosphatidylcholine show very important specific activities; by contrast, phosphatidylethanolamine and sphingomyelin have a very low turnover. Thus phosphatidyldimethylmethanolamine‐methylation pathway, demonstrated in the lung, is not very important.Phosphatidylglycerol is labeled more markedly in vivo than in vitro indicating, for this lipid, a possible transfer from lung tissue to pulmonary‐surfactant cells. In diphosphatidylglycerol specific activities are always very low.The incorporation of radioactive phosphate into phosphatidylinositol, more important in vitro than in vivo, is probably related to the phagocytosis activity of these cells.Our work demonstrates the existence of a phospholipid biosynthesis into pulmonary surfactant but cannot explain the metabolic relations between lung tissue and macrophagic alveolar cells investigated as a possibility in our results.
Modification of Cysteine Residues with Sodium 2‐Bromoethanesulfonate
Authors: NIKETIC Vesna , THOMSEN Johannes , KRISTIANSEN Karsten 
Published: March 2005

A procedure for alkylation of cysteine residues with sodium 2‐bromoethanesulfonate is described. The reaction is performed under mild conditions and complete modification is obtained without side reactions. S‐Sulfoethylated proteins are comparable to performic acid oxidized or S‐sulfonated proteins with respect to solubility properties and behaviour in ion exchange chromatography. S‐Sulfoethylcysteine was found to be stable during acid hydrolysis. S‐Sulfoethylated peptides were suitable for automatic Edman degradation due to their polarity, since losses during extraction are reduced, especially after the coupling stage, as shown by degradation of modified peptides.