bluuebunny/crossref_metadata_2025 huggingface dataset:
18O‐Studies with Citrate Synthase and Malate Synthase
Authors: Wunderwald P. , Eggerer H.
Published: March 2005

The labelling pattern of a β‐hydroxyacid, synthesized on nucleophilic enzymatic reaction of acetyl‐CoA with an α‐oxoacid in H218O, yields information about the mechanism of this type of enzyme action. Two related reactions catalyzed by citrate synthase and malate synthase were investigated in H218O. Malate was formed as the product in both cases.

A micro‐scale procedure is described for the stepwise and specific removal of the oxygen groups present in malate, by enzymatic degradation.

Mass spectrometric analyses of the enzymatically obtained 18O‐labelled malates and of their degradation products revealed the labelling of one oxygen atom of one carboxyl group of malate. The significance of this result is discussed.
Ribonucleotide Reductase from Regenerating Rat Liver
Authors: Larsson A.
Published: March 2005

Ribonucleotide reductase was purified 150‐fold from crude extracts of 48 hour regnerating rat liver using centrifugation at 100 000 ×g, pH 5 precipitation, ammonium sulfate fractionation, and Cγ‐alumina adsorption. The enzyme activity was assayed by the conversion of [3H]cytidine nucleotides to deoxycytidine nucleotides. For optimal activity the enzyme required addition of ATP, Mg ions, Fe ions, and a dithiol reducing agent.

Ribonucleotide reductase could be demonstrated in crude extracts of regenerating livers but not in livers of normal or sham operated rats. After purification, however, all three types of livers were found to contain the enzyme, regenerating livers having the highest levels. After partial hepatectomy ribonucleotide reductase activity increased approximately 20‐fold with a maximum at 50 hours after operation. Within the next 50 hours the enzyme levels decreased to those of normal liver tissue.
Relative Proportions of Mitochondrial and Nuclear DNA in Yeast under Various Conditions of Growth
Authors: Fukuhara H.
Published: March 2005

The relative proportions of mitochondrial and nuclear DNA were compared between various types of cells including anaerobically grown cells, aerobically grown glucose‐repressed and highly derepressed cells. The comparison was made by the use of a labelled reference yeast, which was mixed with various types of cells before extraction of DNA, so that the results could not be influenced by any unequal losses of DNA components during extraction.

Relative proportions of mitochondrial DNA and nuclear DNA were estimated from the specific radioactivities of the two DNA components which were separated by column chromatography on hydroxyapatite.

Contrary to the previous observation by other authors, no major differences were found between completely derepressed cells grown aerobically on galactose up to the stationary phase and cells grown on high glucose under strict anaerobiosis or cells growing under strong aerobic glucose repression.

By the same technique, the mitochondrial DNA content of some other strains including a δ– mutant was examined.
Isolation of a Disulfide Bridge Containing Fragment of the Variable Part of Pig Immunoglobulin λ‐Chains
Authors: Franěk F. , Novotny J.
Published: March 2005

The pig immunoglobulin λ‐chains with intact disulfide bonds were subjected to hydrolysis by trypsin. Gel filtration of the hydrolyzate and icon‐exchange chromatography on SE‐Sephadex yielded a fragment containing 70—71 amino acid residues. It is composed of two sections which can be separated after reduction of a disulfide bond. The amino acid composition of resulting peptides shows that they contain variable sequences. One of the peptides has a blocked N‐terminal amino group. All data indicate that the isolated fragment originates in the variable part of the chain and that one of its sections includes the N‐terminus of the chain.
Effect of Photooxidation on Human Caeruloplasmin
Authors: Bannister W. H. , Wood E. J.
Published: March 2005

Human caeruloplasmin showed a rapid loss of p‐phenylene‐diamine oxidase activity when subjected to photooxidation in the presence of methylene blue. Major conformational changes did not occur. The kinetic characteristics of the loss of phenylene‐diamine oxidase activity were comparable to those of the destruction of histidine residues, and the relative extent of the two processes indicated a one‐to‐one correspondence between them. Copper was lost at a slower rate than that of destruction of histidine residues. It is suggested that the site responsible for the oxidase activity of caeruloplasmin may contain an essential histidine residue and that, although histidine residues may also be involved in copper binding, they are not exclusive copper ligands.
Oxidative Phosphorylation and Compartmentation of Fatty Acid Metabolism in Brown Fat Mitochondria
Authors: Hittelman K. J. , Lindberg O. , Cannon B.
Published: March 2005

Mitochondria freshly isolated from brown adipose tissue by a standard procedure consistently fail to display normal oxidative phosphorylation. Incubation of these particles with ATP and carnitine in the absence of exogenous substrate results in a rapid burst of respiration which eventually slows to a low resting rate. Oxidation of the endogenous substrate is absolutely dependent upon the presence of both ATP and carnitine and is completely inhibited by rotenone. In the absence of added CoASH, respiration is also inhibited by atractyloside, and this inhibition in completely released by addition of CoASH.

After the ATP + carnitine‐induced respiration has slowed to a low resting rate, oxidative phosphorylation is observed during the oxidation of added α‐ketoglutarate, succinate, or α‐glycerophosphate.

If the mitochondria are incubated with ATP and carnitine in the presence of rotenone, coupled behavior is also observed upon subsequent oxidation of succinate or α‐glycerophosphate.

Under appropriate conditions, the time‐course of appearance of oxidative phosphorylation corresponds closely to the time‐course of appearance of significant levels of acylcarnitine.

It is concluded that the endogenous substrate of brown fat mitochondria is free fatty acids which lie initially outside the acyl‐CoA barrier, and that these are responsible for the uncoupled behavior of the freshly isolated particles. By providing the mitochondria with the appropriate components for activation and transport of free fatty acids, the redistribution which occurs relieves the uncoupling and hence both oxidative phosphorylation and classical sensitivities to inhibitors become evident.

The significance of the fatty acid transport mechanism in these mitochondria is discussed with respect to a possible role in regulating the degree of uncoupling which may occur during active thermogenesis by brown adipose tissue.
Brain Lipid Analysis of a Myelin Deficient Mutant, the “Quaking” Mouse
Authors: Jacque C. M. , Harpin M. L. , Baumann N. A.
Published: March 2005

The “Quaking” mouse is a mutant which has an inherited disease involving the process of myelination in the central nervous system.

Fractionation of brain lipids and fatty acid methyl ester analysis of each lipid isolated have been performed. No qualitative abnormalities have been detected, either in ethanolamine, serine and choline phosphoglycerides, or in cholesterol. Proof has now been obtained that the considerable reduction in long chain fatty acids (C24), occurs in cerebrosides, sulphatides (where they are normally major constituents) and in sphingomyelin. Long chain hydroxylated fatty acids are comparatively less diminished than non‐hydroxylated one. This metabolic defect, detected in all the sphingolipids that contain this type of fatty acids could be in favour of a mutation related to fatty acid elongation which is very important at the time of myelin formation.
Biosynthesis of Salmonella Lipopolysaccharide. The in vitro Transfer of Phosphate to the Heptose Moiety of the Core
Authors: Mühlradt P.
Published: March 2005

An enzyme involved in the biosynthesis of the core part of Salmonella lipopolysaccharide, an ATP‐dependant lipopolysaccharide phosphorylating enzyme, has been studied in an in vitro system. The system requires as acceptor the heated 20 000 ×g cell wall fraction from Salmonella minnesota mR5, a mutant which lacks the phosphotransferase (P–), and as enzyme source 1000 00 ×g supernatant of protein extracts from rough P+ mutants of either S. minnesota or S. typhimurium. Phosphate donor is ATP labelled with 32P in the γ‐position. The enzyme system requires a high concentration of Mg++ ions (0.1 M) for optimal functioning.

The phosphotransferase is solubilised by repeated washings of cell debris obtained after lysis of the cells by EDTA and lysozyme with dilute Tris buffer.

Isolation and characterisation of two fractions consisting of heptose phosphate from the lipopolysaccharide of S. minnesota mRz, a UDP‐synthetase‐less P+ mutant, is described. Products of partial hydrolysis of acceptor after 32P‐incorporation were compared with these fractions and found to be identical. In this way the incorporation product was identified to be core‐lipopolysaccharide (glycolipid) which is phosphorylated at the heptose moiety.
Structure covalente de la myoglobine de cheval
Authors: Dautrevaux M. , Boulanger Y. , Han K. , Biserte G.
Published: March 2005

The complete sequence (153 amino acids) of horse heart myoglobin has been established.

The sequence of a peptide isolated from chymotrypsin hydrolyzates of globin allowed to fill a gap in the tentative sequence previously proposed; this peptide was studied by means of pepsin hydrolysis, action of N‐bromosuccinimide on histidyl bonds, and Edman degradation method associated with dansylation.

After hydrolysis of globin by chymotrypsin treated by TLCK, the same major peptides were found as after hydrolysis by non‐treated chymotrypsin. These peptides were identified by ion‐exchange resins and bidimensional paper chromatography, and also by the determination of their amino‐acid composition.

The suppression of secondary cuts explains the presence of new peptides, whose the composition and N‐terminal sequences were determined. It was then possible to confirm several sequences which could be previously deduced from the study of split products after cyanogen bromide treatment. The specificity of chymotrypsin towards certain types of peptide bonds is discussed.

Horse myoglobin, as compared with sperm whale myoglobin, contains 18 differences: 17 substitutions and one inversion. The major part of the substitutions are located in N‐ and C‐terminal sequences; they are all punctiform, resulting from the replacement of only one base in each codon.
Pyruvate Formate‐Lyase Reaction in Escherichia coli
Authors: Knappe J. , Schacht J. , Möckel W. , Höpner Th. , Vetter H. , Edenharder R.
Published: March 2005

Fractionation of the cell extract led to partial isolation of four protein fractions that are involved in the dissimilation of pyruvate into acetyl‐CoA and formate: Enzyme I, molecular weight about 140000; enzyme II, molecular weight about 30000, which requires an activation by ferrous ion and dithiols; enzyme III, molecular weight 25000, which is a flavorprotein with flavin‐monoucleotide as coenzyme; and a hitherto less characterized fraction IV. The system in addition comprises the cofactors S‐adenosylmethionine and thiamine diphosphate.

Enzyme III, fraction IV, and thiamine diphosphate could be replaced by the reagents cobaltous ion and thiols which form complexes of high reducing power. This part of the enzyme system obviously plays an auxiliary role, most likely by providing an appropriate redox potential via pyruvate as electron donor.

The ability to dissimilate pyruvate was found to arise only and specifically from an interaction between enzyme I, ferrous ion‐activated enzyme II and adenosylmethionine, in presence of either of the above auxiliary systems. Enzyme I is thereby converted into an active form, possibly by reduction, which alone is responsible for the catalysis of the pyruvate dissimilation reaction. The identification of this pyruvate formate‐lyase was accomplished by sedimentation experiments in anaerobic sucrose gradients.
Structural Investigations on T1 Lipopolysaccharides
Authors: Berst M. , Hellerqvist C. G. , Lindberg B. , Lüderitz O. , Svensson S. , Westphal O.
Published: March 2005

The structure of the lipopolysaccharide from Salmonella friedenau T1 form has been investigated. The T1‐specific chains are composed of almost equal amounts of β‐d‐ribofuranose and β‐d‐galactofuranose residues. The former are linked to the 2‐position, the latter to the 3‐position or, to a smaller extent, to the 6‐position. A low percentage of branching, through β‐d‐galactofuranose residues, substituted in the 3‐ and 6‐position, is demonstrated. The core region of the T1 lipopolysaccharide seems to have the same general structure as that of other Salmonella lipopolysaccharides.
Biosynthesis of 19‐Hydroxy‐Prostaglandin A1
Authors: Israelsson U. , Hamberg M. , Samuelsson B.
Published: March 2005

Prostaglandin A1, prostaglandin E1 and 11α‐hydroxy‐9,15‐diketoprostanoic acid have been incubated with guinea pig liver microsomes plus boiled high speed supernatant supplemented with NADPH. Prostaglandin A1 was converted in 10–18% yield into 19‐hydroxy‐prostaglandin A1 (35%) and 20‐hydroxy‐prostaglandin A1 (65%). Prostaglandin E1 gave a very small yield (less than 3%) of a hydroxylated derivative, and 11α‐hydroxy‐9,15‐diketoprostanoic acid was not hydroxylated at all. In the latter case, however, a reduction of the side chain keto group took place, affording 11α,15‐dihydroxy‐9‐ketoprostanoic acid.
Chromatography of 32P‐Labelled Oligonucleotides on Thin Layers of DEAE‐Cellulose
Authors: Brownlee G. G. , Sanger F.
Published: March 2005

A two‐dimensional fractionation procedure has been developed for separating radioactively‐labelled oligonucleotides of up to 50 residues long, using uniformly 32P‐labelled 5S RNA of Escherichia coli as a model compound. The method uses ionophoresis on cellulose acetate at pH 3.5 in the first dimension; and ascending chromatography with a concentrated mixture of oligonucleotides on thin layers of mixed DEAE‐cellulose and cellulose in the second dimension.
γ‐Glutamyl Phosphate Attached to Glutamine‐Specific tRNA
Authors: Wilcox Michael
Published: March 2005

B. subtilis Gln‐tRNA formation, in vitro, involves the initial acceptance of glutamic acid by tRNAGln to form a missense Glu‐tRNAGln intermediate which is converted to Gln‐tRNA by a subsequent amidation step, catalyzed by a specific amido‐transferase, and requiring divalent cations, ATP, and l‐glutamine or l‐asparagine as amide donor. This reaction is associated with stoichiometric cleavage of glutamine and ATP to yield glutamic acid and Pi, respectively. Amidation proceeds via the formation of an activated intermediate which is shown to be γ‐phospho‐Glu‐tRNAGln (P‐γ‐Glu‐tRNAGln). P‐γ‐Glu‐tRNAGln, bound to amido‐transferase, is detected following incubation in the absence of amide donor. Subsequent addition of l‐glutamine to the system leads to the rapid loss of the phosphate moiety from the intermediate concomitantly with the formation of Gln‐tRNA, which is released from the enzyme. Consequences of the pathway, if it is duplicated in vivo, are the separation of the synthesis of glutamine destined for protein from that of free glutamine and, further, the coupling of the synthesis of the amino acid with that of protein. These implications are discussed with regard to their bearing on possible functions of the pathway.
On the Duplication of Ribosomal RNA Cistrons in Chinese Hamster Cells
Authors: Amaldi F. , Giacomoni D. , Zito‐Bignami R.
Published: March 2005

Because of the redundancy of ribosomal RNA cistrons and in analogy with a few known special cases, one may postulate that a few “master genes” for rRNA direct the synthesis of multiple copies of themselves per cell cycle. We have shown that in Chinese hamster cells rRNA cistrons duplicate like the bulk of DNA, once in a cell cycle. The time during the S‐phase at which rRNA genes duplicate has also been determined by means of RNA‐DNA hybridization studies.

The time of duplication of rRNA genes occurs between t= 1.5 and t= 3.0 hours from the beginning of the S‐phase.
The dAMP‐Ag+ Complex and the Solubilization of Benzo[a]pyrene
Authors: Green B. , Homer R. B. , Mcdonough G. R.
Published: March 2005

The solubilization of the polycyclic hydrocarbon benzo[a]pyrene by aqueous solutions of dAMP‐Ag+ complexes has been investigated. At high pH solubilization is efficient at Ag+/dAMP ratios not more than 1 but is markedly diminished when the ratio of total silver ions added per base residue is greater than 2. The components separately show little solubilizing activity.

The ultraviolet absorption and circular dichroism spectra of silver complexes of dAMP and poly A under various conditions have been compared.
Functional Properties of Hemoglobin Zürich
Authors: Winterhalter K. H. , Anderson N. M. , Amiconi G. , Antonini E. , Brunori M.
Published: March 2005

Hemoglobin Zürich (β63 His → Arg) has a higher affinity for oxygen and for ethyl isocyanide than hemoglobin A. It shows a decreased value of n in the Hill equation (∼ 1.8 for O2 and 1.0 for ethyl isocyanide). However, the Bohr effect is normal, thus excluding the distal histidine as the donor of the Bohr effect protons. The kinetics of combination with CO differs largely from that of hemoglobin A.
Steroids in Newborns and Infants
Authors: Eneroth Peter , Gustafsson Jan‐Åke , Nyström Ernst
Published: March 2005

Lipid extracts of human meconium were separated by chromatography on silicic acid and lipophilic Sephadex. Trimethylsilyl ethers of sterols in the free and ester fractions were analyzed by gas chromatography‐mass spectrometry. Cholesterol and lathosterol made up 93% of the total sterols and were the predominating sterols in the unesterified fraction (relative abundance: 96%). The remaining 4% in this fraction consisted of lanosterol, 24,25‐dihydro‐Δ8‐lanosterol, 24,25‐dihydro‐Δ9‐lanosterol, 4α,14α‐dimethyl‐5α‐cholest‐7 (and 8)‐en‐3β‐ol, and Δ7‐ and Δ8‐methostenol. The sterol ester fraction contained 4,4‐dimethyl‐5α‐cholest‐8‐en‐3β‐ol, 4,4‐dimethyl‐5α‐cholest‐8,14‐dien‐3β‐ol, β‐sitostanol, cholesterol, lathoesterol, lanosterol, 24,25‐dihydro‐Δ8‐lanosterol, 4α,14α‐dimethyl‐5α‐cholest‐7(and 8)‐en‐3β, and Δ7‐ and Δ8‐methostenol. However, this fraction contained a smaller proportion of Δ7‐sterols than the free fraction. The percentage of cholesterol and lathosterol was also relatively smaller (75%) than in the free sterol fraction.
Intracellular Localization of Phosphodiesterases I and II in Rat Liver
Authors: Erecińska Maria , Sierakowska Halina , Shugar David
Published: March 2005

Differential and density gradient centrifugation techniques have been employed to study the intracellular distribution of phosphodiesterases I and II in rat liver, using as specific substrates the p‐nitrophenyl esters of thymidine‐5′‐phosphate and thymidine‐3′‐phosphate. Some of the results for phosphodiesterase I were also examined with the use of a 5′‐terminated DNA core as substrate.

Phosphodiesterase I was found to be largely (>80%) localized in the plasma membrane, whereas purified nuclei, rough microsomes, and the inner mitochondrial membrane are essentially free of this enzyme. The possible sources of apparent phosphodiesterase I activity of the outer mitochondrial membrane, the lysosomes and the smooth microsomal membrane are discussed.

Phosphodiesterase II is localized largely in the soluble fraction of the lysosomes, and exhibits the latency typical of lysosomal hydrolases.

The overall findings are examined in relation to those of other investigators. Possible reservations regarding the absolute specificities of the substrates employed, and their bearing on the localization results, are discussed.
Affinity Chromatography of Bovine Pancratic Ribonuclease A
Authors: Wilchek Meir , Gorecki M.
Published: March 2005

5′‐(4‐Aminophenyl‐phosphoryl)‐uridine‐2′(3′)‐phosphate was synthesized and coupled to Sepharose by activation with cyanogen bromide. This conjugate was used as a specific adsorbent for purification of ribonuclease A by affinity chromatography. Totally reduced and oxidized RNase preparations which are enzymatically inactive were not adsorbed or retarded by the RNase‐specific column, while S‐protein is strongly retarded.
Enzymic Flux Rates within the Mononucleotides of the Mouse Liver
Authors: Zahn D. , Klinger R. , Frunder H.
Published: March 2005

Within the first 30 sec after intravenous injection of [32P]Pi the P‐flux was measured within the mononucleotide pool of the normal mouse liver. It is only in this short time interval that the dynamic phase of tracer kinetics is met, permitting proper calculations of flux rates of the mononucleotides with high turnover.

The simplified model applied for the calculation of the stated flux rates describes fairly adequately the main pathways within the complex network of possible mononucleotide interconversions.

The P‐flux from the β‐P of ADP to the β‐P of ATP, catalyzed preferentially by oxidative and substrate phosphorylation amounts to 33 μmoles per g fresh liver per min.

12, 0.7, 0.3 and 0.01 μmoles γ‐P of ATP are transfered per g per min to the β‐P of ADP, UDP, GDP and CDP, respectively, via the group nucleoside monophosphate kinases. These flux rates are 1–2 orders of magnitude less than expected from the kinetics behaviour of the involved enzymes in vitro. The reverse reaction of the nucleoside monophosphate kinases is probably small.

9, 4 and 0.5 μmoles of the γ‐P of ATP are transferred per g per min to the γ‐P of GTP, UTP and CTP via nucleoside diphosphate kinase.
Bactoprenol, ATPase and Acetate Activating Enzymes of a Vesicular Fraction from Lactobacillus casei
Authors: Thorne K. J. I. , Barker D. C.
Published: March 2005

The lipids formed by L. casei from [14C]mevalonic acid, principally bactoprenol, were released into the medium when the bacteria were treated with trypsin, followed by lysozyme and EDTA. This released lipid was found to be attached to protein in a particle of size about 35 million. Electron microscopy showed that the bactoprenol‐protein complex had a vesicular structure and may have originated from the mesosomes of the cell. The enzymes ATPase, acetokinase and phosphotransacetylase were released from the bacterial cell together with the bactoprenol‐protein complex. ATPase and acetokinase could be separated from the vesicular fraction by gel filtration. The significance of these findings in relation to the role of bactoprenol and of mesosomes in cell wall biosynthesis is discussed.
Studies on the Bacteriophage MS2
Authors: Vandenberghe A. , van Styvendaele B. , Fiers W.
Published: March 2005

Undegraded, uniformly 32P‐labelled replicative form and replicative intermediate were prepared and fractionated according to sedimentation rate.

For each fraction the amount of 5′‐terminal residues, which after alkaline hydrolysis are released as 5′‐triphosphoryl, 3′(2′)‐phosphoryl nucleosides (nucleoside tetraphosphates), was quantitatively determined. The replicative form contained O, and the replicative intermediate 2.5 nascent chains, if the assumption is made that the latter are on the average half completed. The number of nascent chains apparently increases with increasing sedimentation constant of the replicative intermediate molecules.

The nucleoside tetraphosphate, released from each fraction, was identified as exclusively pppGp. This is interpreted as evidence for a 5′ to 3′ polarity in the synthesis of viral RNA, i.e. a growing 3′OH end.

The replicative form and replicative intermediate were also prepared from cells, in which the 32P‐label was chased for 5 min before harvest. The amount of nucleoside tetraphosphate released agreed with a single end group per complete chain. Presumably 50 to 100% of the residual labelled terminal was derived from the complementary strand, and again only pppGp was found. It follows that the 3′‐terminal adenosine residue of the viral RNA is presumably not part of the genetic information transferred during replication.
Relationship between Age and Properties of Human and Rabbit Erythrocyte Glucose‐6‐Phosphate Dehydrogenase
Authors: Fornaini G. , Leoncini G. , Segni P. , Calabria G. A. , Dacha M.
Published: March 2005

The properties of human and rabbit erythrocyte glucose‐6‐phosphate dehydrogenase have been investigated during aging in vivo. The erythrocytes of different age have been prepared by 2 procedures: (a) by inducing a reticulocytosis by phenylhydrazine administration to rabbits and (b) by separating the young and old red cells of man and rabbit according to the different resistence to osmotic lysis.

During aging, human and rabbit glucose‐6‐phosphate dehydrogenase shows a decrease of the catalytic activity and some marked modifications of its properties. Thermolability and NADPase‐dependent inactivation increase; the protective effect of NADP and the affinity for both glucose‐6‐phosphate and NADP substrates are decreased; no modifications have been demonstrated as regards pH optimum and electrophoretic patterns in starch gel. It is possible that these modifications begin after the expulsion of ribosome material.

According to the literature concerning the behaviour of other enzymes in the physiological state and of glucose‐6‐phosphate dehydrogenase of some pathological conditions, it is possible that the modified properties of the enzyme during aging in vivo can be related to some structural modifications of the protein.
Polysaccharides in Vegetative and Aggregation‐Competent Amoebae of Dictyostelium discoideum
Authors: Malchow D. , Lüderitz O. , Kickhöfen B. , Westphal O. , Gerisch G.
Published: March 2005

Amoebae of Dictyostelium discoideum, cultivated on bacteria as the sole nutrient, secrete into the medium a degraded, water‐soluble form of the corresponding bacterial lipopolysaccharide. Amoeba‐degraded polysaccharide of Salmonella london isolated from the culture medium of amoebae by the phenol‐water method, was purified by fractional precipitation with ethanol. Its chemical analysis showed that it contained all the sugar constituents of the parent lipopolysaccharide including the glucosamine backbone of lipid A, as well as the O‐acetyl groups linked to galactose units of the O‐specific chains, and the N‐acetyl groups linked to glucosamine units of the core. The degraded polysaccharide, however, was completely devoid of the ester‐ and amide‐linked long chain fatty acids present in the lipid A component of the parent lipopolysaccharide. The molecular weight was determined as 15,400. Serological investigations showed that, as expected, the degraded polysaccharide of S. london retained its serological O specificity, but failed to sensitize erythrocytes for passive hemagglutination. When tested in mice the degraded polysaccharide was significantly less toxic as compared with the parent lipopolysaccharide. Chemical analysis of amoeba‐degraded polysaccharide derived from Escherichia coli B/r led to analogous results, and it is concluded that amoebae of Dictyostelium discoideum contain esterases and amidases which specifically cleave the long chain fatty acids from the lipid A component of bacterial lipopolysaccharides.
The Reversible Removal of one Specific Copper(II) from Fungal Laccase
Authors: Malkin R. , Malmström B. G. , Vänngård T.
Published: March 2005

One of the four copper atoms of fungal laccase forms a Cu1+‐chelate with bathocuproine disulfonate at pH 4.0 in 1 M guanidine hydrochloride and in the presence of excess ascorbate.

Electron paramagnetic resonance (EPR) analysis of the native and copper‐depleted proteins has shown that one of the two Cu2+ in the protein has been removed under these conditions. The Cu2+ removed has been identified as the Type 2 or “non‐blue” Cu2+. The spectral characteristics of the other Cu2+ present in the molecule (the Type 1 Cu2+) appear unchanged after removal of the second Cu2+.

The copper‐depleted protein is almost completely devoid of enzymic activity after removal of the Type 2 Cu2+ but both the activity and original copper content can be restored after incubation of the copper‐depleted protein with Cu2+ and ascorbate under anaerobic conditions. No activity is restored by the addition of Cu2+ alone.

The rate of removal of the Type 2 Cu2+ closely parallels the rate of decrease of enzymic activity of the protein during the reaction with the chelating agent. These results show that the Type 2 Cu2+ is an integral part of the laccase molecule and has a functional role in the catalytic reaction.
Partial Purification and some Properties of the (R)‐Citrate Synthase from Clostridium acidi‐urici
Authors: Gottschalk G.
Published: March 2005

The enzyme (R)‐citrate synthase which catalyzes essentially the same biochemical reaction as the citrate synthase but differs in its stereospecificity, has been studied. From cell‐free extracts of Clostridium acidi‐urici the enzyme has been purified 80‐fold by isoelectric precipitation and by chromatography on DEAE‐cellulose and DEAE‐Sephadex. The purification is made difficult by the instability of the enzyme. With the aid of the stereospecific cleavage of citrate by citrate lyase it was demonstrated that the purified (R)‐citrate synthase is not contaminated by the usual citrate synthase.

The (R)‐citrate synthase differs in some properties from the usual citrate synthase: it is inactivated by exposure to oxygen, anaerobic conditions and a sulfhydryl compound being required for maximum activity. (R)‐citrate synthase has a specific requirement for manganese ions, the enzyme is inhibited by EDTA, and this inhibition can be reversed by manganese ions but not by magnesium ions.
Characterization of a New Type of Arginase from Chicken Liver
Authors: Rossi N. , Grazi E.
Published: March 2005

This paper reports the partial purification and characterization of a new type of arginase from chicken liver.

The enzyme can be demonstrated only in less than 10% of a fasting chicken population, and has never been found in fed animals.

The new arginase differs with respect to several properties (chromatographic behaviour, sedimentation coefficient, Km for arginine) from the arginase normally found in chicken liver and is more like the ureotelic arginase isolated from rat liver.
Rat Liver Phenylalanine Hydroxylase
Authors: Nielsen K. H.
Published: March 2005

The activity of rat liver phenylalanine hydroxylase has been measured in systems which, in addition to the components necessary for hydroxylation, contained ascorbic acid and catalase. 6‐Methyl‐tetrahydropterine or the corresponding 6,7‐dimethyl compound was used as tetrahydropterine cofactor. Simple kinetic experiments lead to the determination of rate constants for the following reactions: the aerobic oxidation of the tetrahydropterine cofactor, the reduction of the dihydropterine cofactor by ascorbic acid and other reductants and the conversion of the dihydropterine cofactor from an active to an inactive form. These rate constants are used for the calculation of the actual concentration of tetrahydropterine cofactor in the hydroxylase system and for correction of the measured rates of hydroxylation for the inactivation of the dihydropterine cofactor.

Ascorbic acid, as well as other reductants that may be present, increase the instability of phenylalanine hydroxylase, but this effect is negligible at sufficiently high catalase concentrations. Certain non‐reducing inhibitors can also stabilize the enzyme.

The initial rate of reaction at 30° is linear during the first 5 or 6 min. In contrast at temperatures of 25° and below there is a lag period, whose duration may be reduced by preincubation of the enzyme with tetrahydropterine cofactor. A similar preincubation of the enzyme with l‐phenylalanine seems to activate the hydroxylase briefly.
The Threonine‐Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K 12
Authors: Truffa‐Bachi P. , van Rapenbusch R. , Janin J. , Gros C. , Cohen G. N.
Published: March 2005

The complex protein carrying the two threonine sensitive activities aspartokinase I and homoserine dehydrogenase I is composed of six subunits of a molecular weight 60,000. Furthermore, quantitative determination of the N‐terminal amino acids indicates the presence of six methionine residues per mole of native enzyme (mol. wt. 360,000).

Equilibrium sedimentation studies of N‐ethylmaleimide treated protein shows that its six disulfide bridges, revealed by chemical analysis, are intra‐chain.

Fingerprints of tryptic digests of the protein fail to reveal any difference between the subunits.
Ion Transport in Liver Mitochondria
Authors: Rossi E. , Azzone G. F.
Published: March 2005

The relationship between translocation of K+ and respiration has been studied under conditions where large variations of the transmembrane K+ concentration gradient were obtained. It is found that the energy expenditure is a function of the number of ions translocated and not of the transmembrane concentration gradient. The K+/∼ ratio is slightly below 3 when measured on the total oxygen uptake and between 3 and 4 when measured on the Δ oxygen uptake.

The maximal [K+]i/[K+]o ratio maintained by respiring liver mitochondria is about 1,600 corresponding to an equilibrium potential of 192 mV. When the [K+]i/[K+]o ratio exceeds 2,000 times addition of valinomycin under aerobic conditions causes an efflux instead of an uptake of K+.

Mitochondria behave as perfect osmometers in a wide range of osmolarities. It can be calculated that the concentration of osmotically active internal K+ corresponds to 45% of the total osmolarity of the medium.
Glycoprotein Biosynthesis: Purification and Properties of Glycoprotein
Authors: Bosmann H. Bruce
Published: March 2005

A glycoprotein :N‐acetylglucosamine transferase utilizing endogenous acceptors and a fetuin: N‐acetylglucosamine transferase utilizing a defined exogenous fetuin acceptor from which sialic acid, galactose and N‐acetylglucosamine had been removed were characterized from guinea pig liver. Both glycoprotein :N‐acetylglucosamine systems used UDP‐N‐acetylglucosamine as substrate, required Mn++ and had optimum pH of 5.4. Both enzyme systems were solubilized by extraction of the liver with 0.1% Triton X‐100. The fetuin :N‐acetylglucosamine transferase was separated from endogenous acceptors and purified more than 24‐fold on Sephadex G‐200. The endogenous system had a Km of 0.6 μM and the exogenous fetuin :N‐acetylglucosamine transferase had a Km of 3.8 μM with respect to N‐[14C]acetylglucosamine. The fetuin :N‐acetylglucosamine transferase was highly specific; 11 glycoproteins and 20 mono‐ and disaccharides and amino acids were negative when tested for acceptor activity. The fetuin :N‐acetylglucosamine transferase did not transfer any other nucleotide diphosphate sugar onto the acceptor; even UDP‐N‐acetylgalactosamine was not transferred. It appeared that the exogenous and endogenous systems may well be identical except that the endogenous acceptor was removed during purification of the enzyme.
The pH 6 Acetolactate‐Forming Enzyme from Serratia marcescens
Authors: Malthe‐Sørenssen Didrik , Størmer Fredrik C.
Published: March 2005

The pH 6 acetolactate‐forming enzyme from Serratia marcescens has been purified to homogeneity, and some of its characteristics have been described.

Cocarboxylase and manganese have been completely removed from the enzyme. The addition of FAD to the enzyme did not stimulate the formation of acetolactate, and no FAD was released from the enzyme after trichloro‐acetic acid treatment.

The enzyme did not crystallize as the same enzyme from Aerobacter aerogenes does, but the kinetic properties of the enzyme are similar. The pH optimum in acetate is 6.0, and the enzyme activity is inhibited by phosphate and sulfate. These compounds cause sigmoidal kinetics, and apparent Michaelis kinetics are observed in acetate.
Studies on the Interaction of the Inhibitor O‐Mercuriphenyl β‐d‐Galactoside Chloride with β‐Galactosidase from Escherichia coli K12
Authors: Loontiens Frank G. , Wallenfels Kurt , Weil Rudolf
Published: March 2005

The effect of the sulfhydryl reagent O‐mercuriphenyl β‐d‐galactoside chloride (synthesis given) on β‐galactosidase from Escherichia coli K 12 was investigated in view of the suggestion by Wallenfels and Malhotra in 1960 that cysteine participates in the catalytic mechanism of the enzyme. This reagent inactivated β‐galactosidase in amounts smaller than 8 sulhydryl equivalents, corresponding to one tenth of the total amount of protein SH groups. No change in the ratio of hydrolase and transferase activities was found in the partially inactivated enzyme. Competitive inhibitors were partially protective and inactivation could be reversed with 2‐mercaptoethanol.

It was established that O‐mercuriphenyl β‐d‐galactoside chloride could not be regarded as an active site directed reagent for β‐galactosidase. The compound is enzymatically hydrolyzed and its reaction product O‐mercuriphenyl chloride is a more effective inhibitor than the galactoside itself.

The sedimentation behaviour of the inactivated enzyme was comparable to the native enzyme. Yet upon standing, the modified protein tended to dissociate into the monomer of molecular weight 130 000 and to aggregate.

The modified and inactivated enzyme still exhibited a strong binding capacity: 4 independent binding sites and a dissociation constant equal to 21 μM for the competitive inhibitor 3‐nitro‐4‐aminophenyl β‐d‐thiogalactoside were found by ultracentrifugation studies with absorption optics.

Although some SH groups seem to be indirectly involved in enzyme function, it is evident that their modification, resulting in inactivation, does not significantly reduce substrate binding in the tetramer and that no SH group is involved in the catalytic mechanism itself.
Immunochemical Studies on Hen's Egg‐White Lysozyme
Authors: Strosberg Arthur D. , Kanarek Louis
Published: March 2005

By specific chemical modification of amino acid residues of egg white lysozyme it is demonstrated that the carbamylation of the α amino group and carboxymethylation of the unique histidine residue do not affect the reaction of the enzyme with the anti‐lysozyme antibodies.

In contrast, it appears from this work that carbamylation or acetylation of the ɛ amino groups, carboxymethylation of the methionine residues as well as CNBr cleavage of the polypeptide chain do alterate the antigenic structure of lysozyme.
The Chemical and Electronic Structure of the Neutral Flavin Radical as Revealed by Electron Spin Resonance Spectroscopy of Chemically and Isotopically Substituted Derivatives
Authors: Müller Franz , Hemmerich Peter , Ehrenberg Anders , Palmer Graham , Massey Vincent
Published: March 2005

The neutral flavosemiquinone has been studied in detail by electron spectroscopy. Isotopic (15N, 2H) and chemical substitutions have been carried out. A hyperfine coupling scheme of the neutral flavosemiquinone is described where N(5), N(10), CH3(10), CH3(8) and H(6) are involved. The highest spin density is located at N(5) as has also been found for the anionic and chelated form of the flavosemiquinone. The exchangeable proton in the neutral flavosemiquinone is attached to N(5) having a coupling constant of 7.6 G which is about the same magnitude as the coupling constant to N(5). The hyperfine couplings are interpreted in terms of spin densities and comparison is made with available quantum chemical calculations. Evidence is presented that two tautomeric forms of the neutral flavosemiquinone have to be considered which can be obtained in a pure form by suitable substitution of flavin derivatives. The two types of neutral flavosemiquinones are easily distinguishable by their electron spin resonance and light absorptions.
Protonated Polynucleotide Structures
Authors: Sarocchi Marie‐Thérèse , Courtois Yves , Guschlbauer Wilhelm
Published: March 2005

The interaction between poly(C) and guanylic monomers (guanosine, Guo‐5′‐P, Guo‐3′‐P and Guo‐2′/3′‐P) at millimolar concentrations was investigated by optical methods.

All monomers formed complexes of stoichiometry of 1 guanosine for 2 cytidines although under sensibly different conditions: while guanosine, Guo‐3′‐P and Guo‐2′/3′‐P formed complexes stable over several pH units, the interaction between poly(C) and Guo‐5′‐P is only stable within 0.1 pH units. The reasons for this surprising behaviour must be searched for in a conformation of Guo‐5′‐P which differs from the other guanyl monomers.

All complexes observed follow cooperative binding. The interactions are only stable in the presence of a large excess of monomer. Nearest neighbour interaction energies computed are found to be between 2 and 3 kcal/mole, sensibly lower than found in polymer interactions.

From the results presented it appears that the poly(C)‐guanyl monomer complexes have similar or identical geometry as poly(C) + poly(G) at acid pH.
Pools and Turnover Rates of Nuclear Ribosomal RNA in Tetrahymena pyriformis
Authors: Leick Vagn , Andersen Stig Bryde
Published: March 2005

The labeling kinetics of rRNA in exponentially growing Tetrahymena pyriformis were analyzed quantitatively in order to determine pool‐sizes and turnover rates of nuclear 17 and 25 S rRNA.

The mathematical analysis of the system indicates that, during the steady state of growth, there is an inverse proportionality between pool‐size and turnover rate of nuclear rRNA. Thus, from the labeling kinetics of rRNA and the growth rate constant (4.6 × 10−3 min−1), the pool‐size of macronuclear 17 S rRNA was estimated at 0.5% of that of cytoplasmic 17 S rRNA corresponding to a turnover rate constant of 0.92 min−1. The analogues figures for 25 S rRNA were estimated at 1.6% and 0.29 min−1.

Since the RNA content of macronuclei is about 2% of the cellular RNA content, it is concluded that during growth, most nuclear RNA is found in precursors of ribosomes and that macronuclei contain few, if any, finished ribosomes.

Apart from its use on rRNA, the method described here may be generally useful in determining pool‐sizes and turnover rates of other precursors, such as free ribosomal proteins.
The Compartmentation of CoA and Fatty Acid Activating Enzymes in Rat Liver Mitochondria
Authors: Skrede Sverre , Bremer Jon
Published: March 2005

The effect of disulphides on acyl‐carnitine formation and fatty acid oxidation in rat liver mitochondria has been studied.

Tetrathionate, which does not penetrate the inner membrane, inhibited the carnitine dependent oxidation of long chain fatty acid oxidation almost completely. This oxidation was reactivated by CoA but not by other thiols.

Carnitine increased the inhibition by tetrathionate, indicating that a small pool of preformed acyl‐CoA is present at the outer membrane.

By means of a new, sensitive method for the determination of CoA, a leakage of CoA from the mitochondria during the incubations was demonstrated. The leakage was increased by Pi and 2,4‐dinitrophenol.

The observations in paragraphs 3 and 4 explain why carnitine stimulates the oxidation of fatty acids in the absence of added CoA.

It was confirmed that an ATP‐dependent fatty acid‐CoA synthetase with great capacity is localized in the outer mitochondrial compartment.

A small residual capacity for ATP‐dependent long chain fatty acid activation was demonstrated even in the presence of tetrathionate, indicating an “internal” localization. This activity may depend on the medium chain synthetase or on a separate enzyme in the matrix compartment. The activation of short chain fatty acids was not inhibited by tetrathionate.

Atractyloside inhibited both long chain acyl‐CoA synthetase and carnitine palmityltransferase. The inhibition of both enzymes was counteracted by CoA.

It is concluded that atractyloside is unsuitable for the study of the compartmentation of fatty acid activating enzymes in mitochondria.

From the studies with disulphides it is concluded that fatty acid oxidation in mitochondria can be explained by a simple two compartment model where the inner membrane is the permeability barrier. Such a functional model is in agreement with previous enzyme localization studies.
The Conformation of Small Proteins
Authors: Chicheportiche Robert , Lazdunski Michel
Published: March 2005

The conformation of neurotoxin II of Androctonus Australis Hector has been studied by optical rotatory dispersion and ultraviolet difference spectrophotometry. Depending upon pH and temperature, the protein exists under, at least, four different molecular forms, I, II, III and D which are in rapid equilibrium.

A state‐diagram has been obtained which describes the different zones of temperature and pH in which forms I, II, III and D are stable. A physico‐chemical analysis of all transconformations has been carried out. The acidic as well as the alkaline transition (I ⇌ II, and I ⇌ III respectively) are controlled by the unmasking of structurally important ionisable groups, one of them being probably the carboxylic side‐chain of an aspartic or a glutamic acid. Form I, which is predominant between pH 4 and 9 and below 30°, contains a high degree of ordered structure. It is very stable toward heat denaturation and remains folded in 9.5 M urea, while form II, its conformational isomer at acidic pH, is completely unfolded at 50° or in high concentrations of urea. A very low cooperativeness has been observed for the unfolding of form II in urea or at high temperatures.
Influence of pH and Metal Ions on the Fluorescence of Polycyclic Hydrocarbons in Aqueous DNA Solution
Authors: Green Brian
Published: March 2005

The fluorescence of benzo[α]pyrene or perylene solubilized in DNA solutions of low ionic strength is strongly quenched either by addition of silver ions at a ratio Ag+:DNA phosphate < 0.15 or by lowering the pH from 7.5 to 5. In poly d(A‐T) solutions the effects are much less marked than in DNA. Cu2+ ions are also effective quenchers in this system and Co2+ and Ni2+ have some activity but little effect is seen with Mn2+, Zn2+, Cd2−, Mg2+ or Na+ ions.

The results establish that GC pairs play the dominant role in these quenching reactions and show that the DNA bases have a specific role in the DNA/hydrocarbon interaction. They also suggest that GC‐containing sites are the major sites of physicochemical fixation of hydrocarbons to DNA.

No gross difference in the general nature of the DNA binding sites was found for the carcinogenic benzo[α]pyrene and non carcinogenic perylene.
Substrate Specificity of Escherichia coli Peptidyl‐Transferase
Authors: Panet Amos , de Groot Nathan , Lapidot Yehuda
Published: March 2005

The kinetics of the puromycin interaction with different ribosomal bound peptidyl‐tRNAs were studied. The rates of the puromycin reaction with AA‐Phe‐tRNA and AA2‐Phe‐tRNA using three different amino acids (AA) were compared. At 4° all the three dipeptidyl‐tRNAsPhe reacted considerably slower than the three tripeptidyl‐tRNAsPhe. Acetyl‐Phe‐tRNA reacted faster than the dipeptidyl‐tRNA, but slower than the tripeptidyl‐tRNA. Glyn‐Phe‐tRNA (n= 3, 6 and 9) reacted with puromyein much faster than Gly‐Phe‐tRNA and at a rate similar to that of Gly2‐Phe‐tRNA.

Lauroyl‐Phe‐tRNA reacted with puromyein much slower than acetyl‐and octanoyl‐Phe‐tRNA both at 4° and 30°