bluuebunny/crossref_metadata_2025 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 inactivation

Preincubation 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 vitro

Pulmonary 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.
A Comparison on the Coordination Tendency towards Cu2+ of the Base Moieties in Guanosine, Inosine and Adenosine 5′‐Triphosphates
Authors: SIGEL Helmut , NAUMANN Christoph F. , PRUS Bernhard
Published: March 2005

The chemical shift of the proton H(8) in the proton magnetic resonance spectrum of GTP was measured in aqueous solution in dependence on pH (2 to 12). Based hereon the acidity constants for protonation at N(7) and deprotonation at N(1) were determined. From the line broadening effects in the presence of Cu2+ it is concluded that this metal ion interacts with N(7) of the guanine moiety in Cu(GTP)2−. Hence, the structure of this latter complex corresponds to the one of Cu(ATP)2−, while in Cu(ITP)2− only a very minor interaction with N(7) occurs (cf. Eur. J. Biochem. 41, [1974] 209–216). There is also evidence that in the complex Cu(GTP‐1H)3− a 5‐membered chelate involving N(7) and O(6) is formed, while in Cu(ITP‐1H)3− the interaction with Cu2+ occurs only with the N(1), O(6)‐site of the base moiety. The decrease in line broadening observed at higher pH indicates that the Cu2+‐nucleic‐base interaction is depressed in CU(ATP)(OH)3−, Cu(GTP‐1H)(OH)4− and Cu(ITP‐1H)(OH)4−, as well. In all the mixed‐ligand systems which consist of Cu2+, 2,2′‐bipyridyl (Bipy) and one of the three nucleotides (NTP) the ternary complexes, Cu(Bipy) (NTP)2−, are formed. Within these ternary complexes a charge‐transfer interaction occurs between the purine part of the nucleotides and 2,2′‐bipyridyl.
Interaction of Biguanides with Mitochondrial and Synthetic Membranes
Authors: SCHÄFER Günter , RIEGER Ekkehard
Published: March 2005

Primary reactions of the energy‐conserving pathway in oxidative phosphorylation are inhibited by biguanides and alkylguanidines. The inhibitors bind to phospholipids of mitochondria and synthetic membranes with identical affinities and are assumed to modify the physical properties of membranes, as for example cation conductance.

The study compares effects of biguanides on respiratory‐linked ion fluxes across mitochondrial membranes with those on the conductance and the electrical capacitance of phospholipid bilayers. The following observations have been made:

1. The rate of energy‐linked mitochondrial swelling and the rate of proton uptake during phosphorylation of ADP is inhibited in presence of biguanides. The activity of different compounds is proportional to their binding affinity to phospholipids.

2. Oscillatory volume changes of mitochondria are largely abolished by biguanides.

3. Biguanides cause a small increase of electrical conductance of phospholipid bilayers. This is only in part due to charge transfer by the lypophilic biguanide cations. It is suggested that also structural changes are induced by biguanides altering the surface area per molecule of phospholipid. At high biguanide concentrations structural reorganization is also expressed by changes in electrical capacitance. Both effects are saturable and proportional to binding affinity.

4. Biguanides affect the electrical conductance of bilayers in the opposite way but to the same extent with positively charged species (K+/ionophore) and with negatively charged species (uncouplers of oxidative phosphorylation). This is explained by generation of a positive surface potential at phospholipid containing membranes.

5. The data fit excellently the application of diffuse double‐layer theory to lipid bilayers. The relative effectiveness of different biguanides on model membranes is identical to that on ion fluxes with mitochondrial membranes. From this it is concluded that a similar mechanism of action applies to synthetic and natural membranes as well, suggesting that screening of functional negative sites interferes with the translocation of cations linked to electron transport and the primary reactions in conservation of respiratory energy.
Acidic Phosphoproteins of the 60‐S Ribosomal Subunits from HeLa Cells
Authors: HORAK Ivan , SCHIFFMANN Dietmar
Published: March 2005

Two‐dimensional analysis of the ribosomal proteins from 60‐S subunits of HeLa cells revealed a triplet of acidic proteins, L40a, L40b and L40c, of identical molecular weight (13700). which can be separated only on the basis of their charge differences. Two of the spots, L40b and L40c, become labeled after incubation of the cells with inorganic [32P]phosphate. The electrophoretic behavior and molecular weights of these proteins support the notion that the proteins L40b and L40c. are phosphorylated forms of the protein L40a. The same proteins can be phosphorylated also in vitro by a HeLa protein kinase on 60‐S subunits but not on 80‐S ribosomes. The inaccessibility of L40 proteins to the phosphorylation in vitro on 80‐S ribosomes suggests that they are located in the interface between the 40‐S and 60‐S subunits.
Circular Dichroism Studies of the Binding of o‐Nitrophenyl‐β‐d‐fucoside and o‐Nitrophenyl‐β‐d‐galactoside to Lac Repressor
Authors: MAURIZOT Jean‐Claude , CHARLIER Michel
Published: March 2005

The binding of o‐nitrophenyl‐β‐D‐fucoside and o‐nitrophenyl‐β‐D‐galactoside to Escherichia coli lac repressor was investigated by circular dichroism in the wavelength range 300–400 nm corresponding to the o‐nitrophenyl chromophores. The CD signal of both ligands drastically changed when they bound to lac repressor due to the asymmetric interaction of the o‐nitrophenyl ring with chemical groups of protein. The CD spectra of bound ligands indicate close similarity between the environment of o‐nitrophenyl‐β‐D‐fucoside and o‐nitrophenyl‐β‐D‐galactoside on lac repressor. The CD signal is used to calculate the binding parameters (K and n) to lac repressor. It is demonstrated that the limited proteolytic digestion of lac repressor which gives a ‘core protein’ does not affect the environment of both ligands on the protein.
Different Pools of Free Myoinositol in Chick‐Embryo Cells as Indicated by Infection with Newcastle‐Disease Virus
Authors: DIRINGER Heino , ROTT Rudolf
Published: March 2005

Infection of chicken fibroblasts with Newcastle‐disease virus indicates that cellular inositol is compartmented in at least two pools. Only the smaller pool is directly connected with the biosynthesis of phosphatidylinositol. Entrance of exogenous inositol into this pool is inhibited by phlorizin but not by the virus. Three hours after infection Newcastle‐disease virus blocks the entrance of inositol from the small pool into one (or more) subsequent larger pool(s). About five hours after infection the virus enhances the catabolism of phosphatidylinositol in chicken cells and about seven hours after infection the permeability of the plasma membrane increases.
Interferon, Double‐Stranded RNA and RNA Degradation
Authors: RATNER Lee , SEN Ganes C. , BROWN Glenn E. , LEBLEU Bernard , KAWAKITA Masao , CABRER Bartolome , SLATTERY Elizabeth , LENGYEL Peter
Published: March 2005

Extracts from interferon‐treated Ehrlich ascites tumor cells differ in various biochemical characteristics from extracts from control cells. Thus, as reported earlier, double‐stranded RNA (dsRNA) promotes the phosphorylation by ATP of at least two proteins in extracts from interferon‐treated cells, but not, or to only a lesser extent, in extracts from control cells. Moreover reovirus mRNAs are degraded faster in reaction mixtures containing extracts from interferon‐treated cells than in those containing extracts from control cells but only if the reaction mixtures are supplemented with dsRNA and ATP. The faster RNA degradation in extracts from interferon‐treated cells is due to enhanced endonuclease action. We designated the agent(s) catalyzing this as endonucleaseINT. There is some enhancement of nuclease activity by dsRNA and ATP also in extracts from control cells. The extent of this enhancement is, however, much smaller than in extracts from interferon‐treated cells. We report now that the promotion of mRNA cleavage in extracts from interferon‐treated cells by dsRNA and ATP is apparently not a consequence of a possible impairment in the attachment of the mRNA to ribosomes since (a) the protein synthesis inhibitors (sparsomycin and edeine) do not affect the degradation and (b) dsRNA and ATP enhance RNA degradation also in the 200000 × g supernatant fraction from extracts of interferon‐treated cells and this fraction is essentially free of ribosomes. (The ribosome concentration in our 200000 × g supernatant fraction is less than 1% of that in the 30000 × g extract.) GTP or the ATP analogs AMP(CH2)PP or AMP‐P(CH2)P do not substitute for ATP and dsDNA or DNA · RNA hybrids do not substitute for dsRNA in activating endonucleaseINT. The optimal concentration of ATP in activating endonucleaseINT is 1 mM or higher. The addition of dsRNA and ATP to extracts from interferon‐treated cells affects the degradation of some RNAs much more than that of others. Thus it promotes the degradation of reovirus mRNA from the large size class strongly, the degradation of those from the medium size class to an intermediate extent and that of those from the small size class the least. Furthermore it enhances the degradation of mRNA from either control or interferon‐treated Ehrlich ascites cells more than that of Ehrlich ascites cell ribosomal RNA or mouse globin mRNA. Reovirus dsRNA is not cleaved by endonucleaseINT. The partially purified mouse interferon preparation (specific activity 8 × 108 NIH reference standard units/mg protein) used in several experiments was free of nuclease activity under our experimental conditions. Moreover the addition to extracts from control cells (or extracts from interferon‐treated cells) of this interferon preparation together with dsRNA and ATP did not enhance nuclease activity.
The Modification of Nuclear Proteins by ADP‐ribosylation
Authors: RICKWOOD David , MacGILLIVRAY Alexander J. , WHISH William J. D.
Published: March 2005

When incubated in vitro purified mouse nuclei incorporate NAD into poly(ADP‐Rib). Analysis of the product on CsCl/urea gradients showed that a large proportion of the poly(ADP‐Rib) was not attached to protein. The free poly(ADP‐Rib) did not appear to arise through degradation and its chain length was significantly longer than the poly(ADP‐Rib) attached to proteins. Fractionation of the proteins on hydroxyapatite revealed that tissue‐specific modification of both the histones and non‐histone proteins, had occurred. In the case of one protein species there was evidence for the existence of several forms with different numbers of ADP‐Rib residues.
Deficiency of Subunit Two of Cytochrome Oxidase in the mi‐3 Cytoplasmic Mutant of Neurospora crassa
Authors: BERTRAND Helmut , WERNER Sigurd
Published: March 2005

The formation of cytochrome oxidase was examined in the cytochrome‐aa3‐deficient mi‐3 cyto‐plasmic mutant ofNeurospora crassa. The treatment of mitochondria of mutant mi‐3 with antibody to cytochrome oxidase yields a precipitate of cross‐reacting material consisting predominantly of cytochrome oxidase subunits 5 (mol. wt 14000) and 6 (mol. wt 12000) and small amounts of subunits 4 (mol. wt 16000) and 7 (mol. wt 10000), but devoid of the mitochondrially synthesized, high molecular weight polypeptides, namely subunits 1 (mol. wt 41000), 2 (mol. wt 28000) and 3 (mol. wt 21000).

On the other hand, a qualitative examination of the products of mitochondrial translation in mutant mi‐3 revealed that large molecular weight polypeptides are synthesized. However, a comparison of the dodecyl sulfate acrylamide gel electrophoresis patterns of the mitochondrial translation products of mutant mi‐3 and wild‐type indicated that the cytoplasmic mutant is deficient in polypeptides in the molecular weight ranges of 28000 and 21000, respectively.

By means of subunit specific antibodies it could be shown that the mitochondria of mutant mi‐3 contain virtually undetectable amounts of subunit 2 of cytochrome oxidase, but synthesize easily detectable quantities of subunits 1 and 3. Those subunits that are present in the mitochondria of mutant mi‐3 do not appear to form a definitive complex with each other.

The above results are discussed in view of earlier observations indicating that mutant mi‐3 may be defective in a regulatory circuit that controls the formation of cytochrome aa3. The synthesis of subunit 2 of cytochrome oxidase could be the affected process, and the relative deficiency of this polypeptide may limit the assembly of cytochrome aa3 in the mutant.
Topography of Escherichia coli Ribosomal Protein L12 in situ
Authors: HASNAIN Sadiq , VISENTIN Louis Peter , KAPLAN Harvey
Published: March 2005

The ionization constants of each individual free amino group of Escherichia coli ribosomal protein L12 as it exists on the native intact 50‐S subunit has been determined. All these amino groups are exposed and have pK values varying from 9.7 to 11.0. The amino terminus, on the other hand, is unreactive and therefore appears to be buried. In addition, the reactivities indicate that the aminoterminal region of residues 1–59 undergoes a pH‐dependent conformational change on the 50‐S subunit.
Synthesis and Transport of Myosin in Physarum polycephalum
Authors: SCHWÄRZLER Markus , JOCKUSCH Brigitte M. , HALL Len , BRAUN Richard
Published: March 2005

Immunological techniques have been used to study the rate of synthesis and intracellular transport of myosin in the slime mould Physarum polycephalum. Quantitative precipitation of myosin in homogenates of Physarum was achieved using an antimyosin antibody produced in rabbit in response to purified Physarum myosin. Dodecylsulphate‐gel electrophoresis revealed that about 50% of the precipitated material is myosin.

The rates of synthesis of total cellular protein and myosin were measured over the mitotic cycle. Both were found to increase exponentially or linearly between two successive nuclear divisions. Similarly, no difference in the proportion of myosin‐synthesising polysomes, assayed by precipitation with antimyosin serum, could be detected between the S phase and G2 phase of the mitotic cycle.

Myosin makes up nearly 2% of total plasmodial proteins. Its transport into the nucleus occurs predominantly during the G2 phase.
Stability of the Disulfide Bonds of Fibrinogen and Identification of Specific Subsets of Surface‐Oriented Histidine Residues Highly Susceptible to Alkylation
Authors: PLOW Edward F.
Published: March 2005

The alkylating agents, iodoacetate and iodoacetamide, were spontaneously incorporated into fibrinogen at pH above 5.5 and 37°C. These alkylating agents were covalently bound by fibrinogen, and the association was not dependent upon the presence of factor XIII or plasminogen. Approximately 18 mol of either alkylating agent were bound per mol of fibrinogen. By amino acid analysis, no S‐carboxymethylcysteine was detected; but the histidine derivatives, 3‐carboxymethylhistidine and 1,3‐dicarboxymethylhistidine, were identified as the only modified residues, and these were present at an approximate 2: 1 ratio. The alkylation reaction was not altered by systematic cleavage of fibrinogen by plasmin, and the alkylated residues in the D · E complex were again restricted to the 3‐carboxymethylhistidine and 1,3‐dicarboxymethylhistidine derivatives in an approximate 2: 1 ratio. Approximately 72% of the incorporated alkylating agent associated with the D region of fibrinogen and 28% with the E region. Alkylation of fibrinogen prolonged its thrombin time, and alkylated fibrinogen inhibited the coagulation of unmodified fibrinogen. This effect was associated with interference with the polymerization of fibrin monomers. The results indicate that the disulfide bonds of native fibrinogen are stable, and plasmin‐mediated catabolism of fibrinogen does not induce disulfide interchange. Specific subsets of surface‐oriented histidine residues located within the relatively plasmin‐resistant D · E core of fibrinogen are highly susceptible to alkylation, and some of these specific histidine residues may play an important role in the intermolecular alignment and polymerization of fibrin monomers.
Synthesis and Kinetic Properties of a New NAD+ Derivative Carrying a Vinyl Group
Authors: MURAMATSU Minoru , URABE Itaru , YAMADA Yasuhiro , OKADA Hirosuke
Published: March 2005

A method for the synthesis of NAD+‐N6‐[N‐(N‐acryloyl‐1‐methoxycarbonyl‐5‐aminopentyl)‐propioamide] (monomeric NAD+ derivative), a new NAD+ derivative carrying a vinyl group, was described. By copolymerization with acrylamide 90% of the NAD+ derivative was converted to a water‐soluble macromolecular NAD+ derivative (polymeric NAD+ derivative). Both NAD+ derivatives were reduced completely with yeast alcohol dehydrogenase.

High cofactor activities relative to free NAD+ and NADH were obtained for the monomeric derivatives of NAD+ (71–86%) and NADH (66–87%) with yeast alcohol, horse liver alcohol, lactate, and malate dehydrogenases. Lower but substantial relative cofactor activities were obtained for the polymeric derivatives of NAD+ (18–33%) and NADH (28–60%) with yeast alcohol, horse liver alcohol, and malate dehydrogenases. But lactate dehydrogenase had negligible activity both for the polymeric NAD+ and NADH derivatives.

Kinetic studies were carried out with yeast alcohol dehydrogenase and lactate dehydrogenase, and the following kinetic constants were determined: the maximum velocity (V), the limiting Michaelis constants for coenzyme (Ka) and for substrate (Kb), and the dissociation constant of the enzyme‐coenzyme complex (Kia). In the reaction system with the monomeric NAD+ derivative and yeast alcohol dehydrogenase, the Kia value was similar to that for NAD+ and the values of V, Ka, and Kb were 38, 30, 21% of those for NAD+. In the reaction system with the polymeric NAD+ derivative and yeast alcohol dehydrogenase, the values of V, Ka, Kb, and Kia were 0.5, 4, 3, 3 times larger than those for the monomer. In the reaction system with the monomeric NAD+ derivative and lactate dehydrogenase, the values of V, Ka, Kb and Kia were 39, 16, 36, 57% of those for NAD+. From these results the following conclusions were obtained: (a) the modification of NAD+ by alkylating at position 6 in the adenine moiety of NAD+ caused a decrease in the V value, possibly due to configurational alterations of the binary or ternary coenzyme complexes of the enzymes resulting in decreased cofactor activity of NAD+, but the decrease in the activity was somewhat compensated by the decrease in the values of Ka, Kb, and Kia also caused by the modification; (b) the incorporation of the NAD+ derivative into the side chain of polyacrylamide caused decrease in the affinity for the enzyme and further decrease in the V value, and thus decreased the cofactor activity to below that of the monomeric NAD+ derivative.
The Effect of Electron Donors and Acceptors on Light‐Induced Absorbance Changes and Photophosphorylation in Rhodospirillum rubrum Chromatophores
Authors: SILBERSTEIN Binah R. , EPEL Bernard L. , MALKIN Shmuel , GROMET‐ELHANAN Zippora
Published: March 2005

Light‐induced difference spectra between 400 and 640 nm of Rhodospirillum rubrum chromatophores were performed in the presence and absence of exogenous electron donor acceptor systems and compared with the chemical oxidation spectrum. The results indicate that the component previously defined as P430 is not a unique entity but rather represents different species, or a mixture of species, under various conditions. Under all conditions in which the reaction center bacteriochlorophyll is reversibly photooxidized, as indicated by the bleaching around 600 nm. it is also contributing to the absorbance increase around 430 nm. In one case, in presence of reduced dichloroindophenol and in the absence of oxygen, the photooxidation of reaction center bacteriochlorophyll is fully supressed. Under these conditions an irreversible change around 430 nm is still observed and seems to be due to the Soret band of a b‐type cytochrome. In the presence of reduced dichloroindophenol and absence of oxygen there is a marked inhibition of photophosphorylation. This inhibition is apparently due to the complete reduction of the cyclic electron carriers. Addition of the low potential dye benzyl viologen facilitates an almost complete recovery of the reversible photooxidation of reaction center bacteriochlorophyll as well as of photophosphorylation. These results indicate that the apparent mid‐point potential of the primary electron acceptor in Rhodospirillum rubrum chromatophores is probably in the range of that of benzyl viologen (E′o=−340 mV).
The Regulation of Glutamate Metabolism in Candida utilis
Authors: HEMMINGS Brian A. , SIMS Anthony P.
Published: March 2005

An earlier observation from this laboratory (J. Gen. Microbiol. 64, 423–427) that NAD‐dependent glutamate dehydrogenase activity is modulated by rapid inactivation has been extended to show that this mechanism is completely reversible. Changes in properties of the enzyme accompany inactivation and two different forms, active (a) and inactive (b) of the enzyme with distinctive properties have been isolated. Incubation of the inactive enzyme with magnesium in vitro produced a rapid increase of activity; this was accompanied by a change in the properties of the enzyme to those of the a form. This control mechanism of enzyme interconversion appears widespread among yeasts. Its probable role in modulating glutamete synthesis and degradation is discussed.
Analysis of Calf‐Thymus Satellite DNA: Evidence for Specific Methylation of Cytosine in C‐G Sequences
Authors: GAUTIER Fritz , BÜNEMANN Hans , GROTJAHN Lutz
Published: March 2005

Digestion of purified calf tymus satellite I (θ= 1.714 g/cm3) with a series of restriction enzymes shows that modification in this satellite occurs preferentially in the sequence C‐G. This was also shown to be the case in the other satellites and in bulk chromosomal calf thymus DNA.

Cloning of purified satellite I DNA in Escherichia coli makes sites, previously modified, available for cutting with certain restriction enzymes. All these ‘new’ sites contain the sequence C‐G.

High‐resolution mass spectroscopy establishes that the satellites contain a low concentration of 5‐methylcytosine. This iners that methylation which inhibits restriction enzyme cutting must occur preferentially in the sequence C‐G. Hybridization of cRNA of cloned satellite I DNA with the satellites III (θ= 1.706 g/cm3) and IV (θ= 1.710 g/cm3) shows that there is no or little sequence homology between these satellites. Digestion of calf thymus satellite I DNA with endoR ·EcoRI and subsequent hybridization studies with the fragments shows two EcoRI fragments in addition to the usual 1400‐base‐pair EcoRI repeat unit.
Accumulation of Hypothiocyanite Ion during Peroxidase‐Catalyzed Oxidation of Thiocyanate Ion
Authors: AUNE Thomas M. , THOMAS Edwin L.
Published: March 2005

An oxidized form of thiocyanate accumulated during incubation of lactoperoxidase or myeloperoxidase with thiocyanate ion and low concentrations of peroxide. Addition of one mole of peroxide resulted in loss of one mole of thiocyanate and formation of one mole of the oxidation product. The oxidation product reacted with two moles of sulfhydryl to yield one mole of disulfide and one mole of thiocyanate. The same product was obtained by alkaline hydrolysis of thiocyanogen. The product is proposed to be hypothiocyanite ion. The peroxidases may catalyze the oxidation of thiocyanate to hypothiocyanite, or to thiocyanogen. Hypothiocyanite may be the antimicrobial agent formed during peroxidase‐catalyzed oxidation of thiocyanate.

Hypothiocyanite decomposed slowly with a second‐order rate constant of 0.1 M−1 s−1 at 25°C. When the amount of peroxide added approached the amount of thiocyanate, the yield of hypothiocyanite reached a maximum and then declined. This result suggested that hypothiocyanite was oxidized by excess peroxide. Efforts to produce high concentrations of hypothiocyanite were unsuccessful, even when thiocyanate was well in excess of peroxide. As the hypothiocyanite concentration approached 0.5 mM, decomposition was accelerated and small amounts of cyanide were produced. Hypothiocyanite reacted stoichiometrically with cyanide to give products that did not oxidize sulfhydryls. Hydrolysis of thiocyanogen at neutral pH also resulted in rapid decomposition and formation of cyanide.