bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitrogen Fixation by Extracts of Mycobacterium flavum 301
Authors: Biggins David R. , Postgate John R.
Published: March 2005

Crude extracts of lactate‐grown Mycobacterium flavum 301 reduced acetylene to ethylene with NADH as electron donor provided benzyl viologen was present; ATP was required. Lactate, pyruvate, H2 or glucose‐6‐phosphate were also active donors via appropriate dehydrogenases.

Crude extracts also reduced cyanide to methane with NADH, ATP and benzyl viologen.

Debris (composed of wall and membrane fragments) had lactic, pyruvic and NADH dehydrogenase activities. They reduced acetylene with sodium dithionite or NADH. Benzyl viologen was not required with NADH; ATP and O2 inhibited the reaction.

Crude extracts were as sensitive to inactivation by oxygen as extracts from anaerobic bacteria. Glutathione decreased and ATP increased oxygen‐sensitivity.

Debris preparations were not inactivated by oxygen.

These results suggest that the nitrogen‐fixing system of M. flavum 301 resembles those of other aerobes, but that its mechanisms for protecting nitrogenase from oxygen are more primitive.
Partial Enzymic Digestion of 50 S Ribosomal Subunits from Escherichia coli
Authors: Spencer Margaret E. , Walker Ian O.
Published: March 2005

50 S ribosomal subunits from Escherichia coli were incubated with an endonuclease specific for single‐stranded nucleic acid. After incubation the sedimentation coefficient remained unaltered but gel filtration showed that 10% of the nucleic acid in the ribosome had been degraded to small fragments. When the enzyme‐treated native subunits were subsequently unfolded in EDTA their sedimentation properties were similar to controls. Thermal denaturation in the presence of formaldehyde produced small fragments of RNA with a sedimentation coefficient of 2 S.

Subunits which had first been unfolded in EDTA and then incubated with enzyme were rapidly degraded to small fragments with a sedimentation coefficient of 2 S. The degraded fragments had 20% hyperchromism on thermal denaturation which showed that the secondary structure of the RNA had remained intact. These experiments were interpreted in terms of the hairpin‐loop model of RNA. They show that on the surface of the native subunit there is no interhelical single‐stranded RNA which is accessible to enzyme whereas there is a significant amount of intrahelical single‐stranded RNA. In the unfolded subunit the interhelical regions become exposed to attack by the enzyme.

A digest of unfolded subunits was separated by gel filtration on Sephadex G‐100 into three fractions. Fraction I, which eluted at the exclusion volume of the column, was mainly aggregated protein. Fraction II, which contained 65% of the ultraviolet absorbing material and no protein, was hyperchromic and the circular dichroic spectrum was consistent with this fraction having double‐helical secondary structure. Fraction II moved as a single, sharp boundary on sedimenta‐tion with s20= 1.7 S and had a molecular weight of 15700. Fraction III, which contained 35% of the ultraviolet absorbing material and no protein, was low molecular weight nucleotide with no secondary structure.
The Immunochemistry of Peptidoglycan
Authors: Schleifer Karl H. , Krause Richard M.
Published: March 2005

Peptidoglycans or cell walls of Staphylococcus epidermidis, Lactobacillus acidophilus, Micrococcus lysodeikticus, and Corynebacterium poinsettiae, solubilized by ultrasonic treatment, gave precipitin reactions with streptococcus Group A‐variant antisera. By means of inhibition studies with the pentapeptide L‐Ala‐D‐Glu‐γ‐L‐Lys‐D‐Ala‐D‐Ala and N‐acetyl glucosamine, it was demonstrated that the peptidoglycan of S. epidermidis and cell walls of L. acidophilus reacted with antibodies to the pentapeptide, whereas trichloroacetic acid extracted cell walls of M. lysodeikticus and C. poinsettiae precipitated predominantly antibodies directed against the glycan part of the peptidoglycan. This is explained by the occurrence of peptide subunits ending with C‐terminal D‐Ala‐D‐Ala in the peptidoglycans of S. epidermidis and L. acidophilus and the lack of this determinant in the peptidoglycan of M. lysodeikticus and C. poinsettiae. Antibodies to the pentapeptide were isolated by means of an immunoabsorbent column. The gammaglobulin of these antibodies were determined by microzone electrophoresis and immunoelectrophoresis. The antibodies were able to agglutinate whole cells of streptococcus Group A‐variant but not the cells of Groups A or C. The light chains of antibodies reacting with A‐variant carbohydrate, C. poinsettiae, and S. epidermidis peptidoglycans were resolved by gel disc electrophoresis. The patterns were quite different. Antibodies reacting with S. epidermidis yielded the same light chain pattern as antibodies to the pentapeptide prepared by immunoabsorbent chromatography. The antibodies directed against the peptidoglycan showed a limited heterogeneity, especially those reacting with the glycan.
Effect of Phytohemagglutinin on Lymphocyte Membrane Transport
Authors: Peters J. Hinrich , Hausen Peter
Published: March 2005

Experiments with the non‐metabolizable glucose analogue 3‐O‐methyl‐[U‐14C]glucose revealed that this substance is transported through lymphocyte membranes by “facilitated diffusion”. The uptake process is stimulated immediately upon addition of phytohemagglutinin. Counter‐transport and saturation experiments indicate that the stimulation provoked by phytohemagglutinin is due to an activation of carrier sites in the membrane.
Structure of Phycocyanobilin
Authors: Schram Bernard L. , Kroes Hanno H.
Published: March 2005

The structure of phycocyanobilin, the prosthetic group of phycocyanin, has been investigated by means of mass spectral, infrared, and nuclear magnetic resonance studies. The linear tetrapyrrolic compound has a molecular weight of 588 and contains an intramolecular hydrogen bond, the rupture of which forms the basis for several of the chemical transformations. Esterification, for example, is accompanied by dehydrogenation, so the molecular weight of the dimethyl ester is 614.

The structure of the phycobilin P 655 has been deduced from an analysis of the properties of phycocyanobilin; this structure is nearly identical to the one proposed by Cole, Chapman, and Siegelman. For the phycobilin P 608 only a tentative structure can be presented.
Metabolism of the Reserve Polysaccharide of Streptococcus mitis
Authors: Walker Gwen J. , Builder Janet E.
Published: March 2005

The properties of a purified branching enzyme (α‐1,4‐glucan : α‐1,4‐glucan 6‐glycosyltransferase) isolated from intracellular extracts of several strains of Streptoccus mitis were studied with maltodextrins, amylose, amylopectin and glycogen as substrates. Maltodextrins with an average degree of polymerization above 20 were good substrates, and the rate of branching enzyme action increased with chain‐length. The enzyme had a higher affinity towards branched substrates than linear polysaccharides, and evidence of its ability to convert glycogen into a more highly branched product is presented. The rate of synthesis of glycogen by glycogen synthetase was increased when branching enzyme was present. The activating and protective effect of citrate ions on branching enzyme is described, and the way in which citrate could regulate the degree of branching that occurs during glycogen synthesis is discussed.
The Respiratory System of Azotobacter vinelandii
Authors: Ackrell Brian A. C. , Jones Colin W.
Published: March 2005

A detailed procedure is described for the isolation of stable respiratory membranes of relatively high phosphorylation efficiency from nitrogen‐fixing cultures of Azotobacter vinelandii (P/O, NADH = 0.90–1.10).

Three phosphorylation sites are detectable. These are located at the level of NADH dehydrogenase (Site I; P/2e 0.45), between the primary dehydrogenases and the cytochromes (Site II; P/2e 0.53) and on the minor terminal pathway, to the oxygen side of the c‐type cytochromes (Site III; P/2e 0.22).

Switching of electron flow between the terminal oxidation pathways with low concentrations of KCN and 2‐n‐alkyl‐4‐hydroxyquinoline‐N‐oxide causes changes in oxygen uptake and phosphate esterification which are compatible with the postulated locations of the three phosphorylation sites and which demonstrate, indirectly, the non‐phosphorylating nature of the b1→a2 major terminal pathway.

Phosphorylation efficiences with ascorbate plus purified A. vinelandii cytochromes c4 and c5, horse heart cytochrome c and the artificial electron electron mediators 2,6‐dichlorophenol indophenol and N,N,N′,N′‐tetramethyl‐p‐phenylene diamine indicate that the minor terminal pathway is further split into a slow, phosphorylating pathway (c5→oxygen) and a faster, non‐phosphorylating pathway (c4→oxygen).
Turnover of the Deoxyribonucleoside Triphosphates in Escherichia coli 15 T during Thymine Starvation
Authors: Neuhard Jan , Thomassen Elisabeth
Published: March 2005

Since hydroxyurea specifically inhibits the biosynthesis of deoxyribonucleotides in Escherichia coli, analysis of the deoxyribonucleoside triphosphate pools following hydroxyurea addition makes possible a determination of the turnover of these compounds in vivo. This method has been used in the present work to study the effect of thymine starvation of E. coli 15 T on the turnover of dGTP, dATP, and dCTP. From the results obtained the following conclusions may be made.

In exponentially growing cultures DNA synthesis can account for the entire turnover of the deoxyribonucleoside triphosphates.

Removal of thymine from the growth medium results in the appearance of catabolic reactions that degrade dATP and dCTP. During the first 20 min of thymine starvation the rates of degradation of dATP and dCTP are of the same magnitude as those accounted for by DNA polymerization during exponential growth. After 75 min, however, the decay rate of dCTP has increased 5‐fold while the rate of dATP decay remains constant. During the entire period of thymine starvation the rate of decay of dGTP is very low, i.e. one sixth of that observed during exponential growth.

The end products of the catabolic reactions involved are to a large extent excreted into the growth medium as diphenylamine‐reacting material.

By determining the rate of accumulation of the individual deoxyribonucleoside triphosphates following thymine deprivation, and adding these values to the decay rates, determined in the presence of hydroxyurea, it is possible to estimate the endogenous rate of synthesis of dGTP, dATP and dCTP under these conditions. The results show that removal of thymine from E. coli 15 T results in a significant reduction in the rate of dGTP synthesis, essentially no effect on the rate of dATP synthesis, and an increasing rate of dCTP synthesis reaching 6‐fold values after 75 min of starvation.
The Sensitivity of Exchange Reactions of Tricarboxylate, 2‐Oxoglutarate and Dicarboxylate Transporting Systems of Rat Liver Mitochondria to Inhibition by 2‐Pentylmalonate, p‐Iodobenzylmalonate, and Benzene 1,2,3‐tricarboxylate
Authors: Robinson Brian H. , Williams G. Ronald , Halperin Mitchell L. , Leznoff Clifford C.
Published: March 2005

A new specific inhibitor, benzene 1,2,3‐tricarboxylate is described for the tricarboxylate transporting system in rat liver mitochondria. This compound, together with the ibhibitors 2‐pentylmalonate and 2‐p‐iodobenzylmalonate, has been used to elucidate the exchange reactions of the citrate, 2‐oxoglutarate and malate transporting system in rat liver mitochondria. It is proposed that both the 2‐oxoglutarate and citrate transporting systems are able to catalyse l‐malate/l‐malate exchange in addition to the l‐malate/l‐malate exchange catalysed by the dicarboxylate transporting system. p‐Iodobenzylmalonate was found to inhibit all three transporting systems investigated. The implications of these findings are discussed.
Isolation and Characterization of Seryl‐tRNA Synthetase from Yeast
Authors: Heider Henning , Gottschalk Ellen , Cramer Friedrich
Published: March 2005

A novel method for the isolation in 29% overall yield of seryl‐tRNA synthetase from baker's yeast is described. The method includes opening of the cells by autolysis with toluene, two ammonium sulfate fractionations, a combined RNAase and DNAase digestion of the nucleic acid components and successive chromatographic fractionations on phosphocellulose and DEAE‐cellulose. The enzyme shows an unusual affinity for phosphocellulose.

The purified enzyme is homogeneous by equilibrium ultracentrifugation, by disc electrophoresis and by dodecylsulfate gel electrophoresis. The enzyme consists of two subunits, the molecular weight of which in the denatured and native states is 60 000 and 61 000, respectively, as determined by ultracentrifugation and dodecylsulfate gel electrophoresis. The total enzyme dissociates easily into the subunits, even in dilute buffer solution. The molecular weight of the total enzyme was determined by ultracentrifuge measurements to be 115000. Even under these conditions at lower concentration the enzyme tends to dissociate; the data point to an active dimer of molecular weight 120 000.

No free N‐terminal amino acid could be found. The enzyme was further characterized by determination of its ultraviolet spectrum and content of tryptophan and tyrosine.
X‐Ray and Neutron Small‐Angle Scattering from Hemoglobin in Aqueous Solution and in Crystal
Authors: Schneider Rudolf , Mayer Adalbert , Schmatz Werner , Schelten Jakob , Franzel Rüdiger , Eicher Hermann
Published: March 2005

The small‐angle scattering function of hemoglobin for X‐rays and neutrons has been calculated from the atomic coordinates of hemoglobin, established by X‐ray diffraction analysis. These theoretical scattering functions were compared with the experimental scattering data on aqueous hemoglobin solutions reported previously. The good agreement of the two sets of data in and beyond the region of the Guinier approximation gives confidence to the statement that the three‐dimensional structures of hemoglobin in single crystal and in aqueous solution are the same.
Diacetyl (Acetoin) Reductase from Aerobacter aerogenes
Authors: Hetland Øyvind , Olsen Bjørn R. , Christensen Terje B. , Størmer Fredrik C.
Published: March 2005

The native and subunit molecular weights of diacetyl (acetoin) reductase from Aerobacter aerogenes have been investigated by sedimentation equilibrium analysis and gel electrophoresis. The values obtained are 100000 for the native enzyme and 25000 for the subunit. The native enzyme is dissociated into four equal‐sized subunits by exposure to guanidine hydrochloride, urea, or dodecylsulfate. The subunits can reassociate in the presence of bovine serum albumin to yield the native enzyme.

Electron micrographs of enzyme molecules, negatively stained, show that they appear primarily as tetramers with four subunits at the corners of squares. The molecular weight as calculated from the diameter of the subunits is consistent with the values obtained by sedimentation equilibrium and gel electrophoresis.
Diacetyl (Acetoin) Reductase from Aerobacter aerogenes Evidence for Multiple Forms of the Enzyme
Authors: Hetland Øyvind , Bryn Klaus , Størmer Fredrik C.
Published: March 2005

A homogeneous preparation of diacetyl (acetoin) reductase from Aerobacter aerogenes has been shown to consist of several isoelectric species. The native enzyme consists of at least 12 species, all possessing enzymatic activity. No differences in the band patterns were observed when curd extract, purified enzyme, purified enzyme in the presence of NAD, and reactivated enzyme from urea treatment, were subjected to isoelectric focusing in polyacrylamide gels. Electrofocusing of the monomers in urea gels gave at least 5 bands. The isoelectric points for the bands were in the range of 5.8 to 6.8.
Deoxyribonucleotide Phosphatase from Rat Liver and Cultured Mouse Fibroblasts
Authors: Magnusson Göran
Published: March 2005

An enzyme which catalyzes the preferential dephosphorylation of the 5′‐phosphates of deoxythymidine, deoxyuridine and deoxyguanosine was purified 100‐fold from rat liver. The preparation appeared to be free from contaminating phosphatase activities. The dephosphorylation of deoxyribonucleotides was dependent on the presence of Mg2+ ions and had a pH optimum around 6.0. The Km value for the dephosphorylation of deoxythymidine 5′‐phosphate was 5.7 mM.

High activities of the enzyme were found in cultured mouse fibroblasts. In parasynchronously growing cells no changes of enzyme activity were found during the cell cycle.
Metabolism of Corticosterone in the Isolated Perfused Rat Liver
Authors: Eriksson Håkan , Gustafsson Jan‐Åke
Published: March 2005

[4‐14C]Corticosterone was perfused through isolated livers from male and female rats. Bile, perfusate and liver tissue were extracted and analysed separately. No specific differences were observed in the distribution of various isomers of corticosterone metabolites isolated from these three sources. However, the radioactive metabolites with the chromatographic property of steroid glucuronides were mainly present in the bile. Male rat livers metabolized corticosterone into the following 20‐hydroxy compounds: 5α‐pregnane‐3α, 11β, 20β(and 20α), 21‐tetrol and 5α‐pregnane‐3β, 11β, 20β,21‐tetrol. In female rat livers, on the other hand, only small amounts of 5α‐pregnane‐3β,11β,20β,21‐tetrol were formed. The predominant corticosterone metabolites isolated after perfusion of female rat livers were identified as 3α(and 3β), 11β,15α,21‐tetrahydroxy‐5α‐pregnan‐20‐one and 3α(and 3β),11β,21‐trihydroxy‐5α‐pregnan‐20‐one. Furthermore, female rat livers produced a larger amount of mono‐and disulphurylated metabolites than male livers. All these findings agree well with what has previously been found concerning the excretion of steroids in faeces from germfree rats. The present results therefore show that an important reason for the large sexual differences observed in rats in the faecal and urinary steroid patterns is a different hepatic metabolism of steroids in male and female rats.
Immunochemical Studies on Citrobacter O Antigens (Lipopolysaccharides)
Authors: Keleti Juraj , Lüderitz Otto , Mlynaršék Dušan , Sedlák Jiři
Published: March 2005

An investigation on the sugar compositions of lipopolysaccharides (O antigens) derived from 45 well–defined Citrobacter serotypes was carried out. As a result, a chemical classification into 20 chemotypes was achieved, of which 11 were identical with chemotypes occuring in Salmonella and Escherichia coli. All Citrobacter lipopolysaccharides contained glucosamine, glucose, heptose, 2‐keto‐3‐deoxyoctonate and galactose, the latter, in some cases, in very small amounts. These sugars probably represent building blocks of the basal core polysaccharides, whose structures however may not be identical for all Citrobacter lipopolysaccharides. In addition to the basal sugars, the following monosaccharides were detected as constituents of the various Citrobacter lipopolysaccharides: d‐xylose, d‐mannose, fucose, rhamnose, 6‐deoxytalose, 4‐deoxy‐d‐idose, abequose, galactosamine, d‐fucosamine, 3‐amino‐3, 6‐dideoxy‐glucose and ‐galactose. The sugar compositions of serologically related Citrobacter and Salmonella O antigens were compared. While some cross‐reacting pairs belonged to the same chemotype, others proved to be distinct with respect to their sugar constituents.
The Thiol Groups of Rat Liver Cystathionase
Authors: Dème Danielle , Durieu‐trautmann Odile , Chatagner Fernande
Published: March 2005

Rat liver cystathionase (homoserine hydrolyase) catalyzes the deamination of homoserine and the desulfhydration of cysteine. A study has been made of the SH groups of the highly purified enzyme. The titration of the SH groups by p‐chloromercuribenzoate and 5,5′‐dithiobis‐(2‐nitrobenzoic acid) indicated that the enzyme contains approximatively 12 SH per mole (mol. wt. 170000) of native enzyme and 20 SH per mole of urea‐denaturated enzyme.

On the basis of their reactivity towards SH reagents the SH groups of cystathionase have been classified into 4 classes.

Addition of pyridoxal phosphate, the coenzyme of cystathionase, prior to treatment with SH reagents did not modify the availability of SH groups for SH reagents; however, addition of homoserine, a substrate for cystathionase, or of l‐alanine, a competitive inhibitor for both homoserine deamination and cysteine desulfhydration, led to a striking protection of several SH groups, 4 and 8, respectively.

The over‐all results are discussed in relation to the hypothesis of a “double” active center composed of one site necessary to the binding of homoserine and one other involved in the binding of cysteine. In addition the hypothesis that cystathionase may consist of subunits is considered, taking into account the symmetry of the SH groups in each class and the number of pyridoxal phosphate binding sites on the protein.
Imidazole Group‐Catalyzed Reactions in Formaldehyde
Authors: Martin Charles J. , Oza Narenda B. , Marini Mario A.
Published: March 2005

The catalysis of p‐nitrophenyl acetate hydrolysis by imidazole compounds in formaldehyde solution occurs at a rate lower than in water.

Kinetically determined pK' values of imidazole and its derivatives agree with titrimetric values and the catalytically active species is the free base form of the N‐hydroxymethyl adduct of the imidazole ring.

With imidazole as catalyst in formaldehyde, N‐acetyl‐N′‐hydroxymethylimidazolium cation is apparently a short‐lived intermediate in the reaction. The hydroxymethyl adduct, although removable by dilution, is not displaced by substrate during catalysis.

These results provide additional support for the occurrence of a reaction between for‐aldehyde and the imidazole ring and its kinetic consequences parallel those seen in the effect of formaldehyde on chymotrypsin‐catalyzed reactions.
Modification of Methionyl‐tRNA Synthetase by Proteolytic Cleavage and Properties of the Trypsin‐Modified Enzyme
Authors: Cassio Doris , Waller Jean‐Pierre
Published: March 2005

Earlier studies have shown that native methionyl‐tRNA synthetase from Escherichia coli K12 is composed of four, probably identical, subunits having a molecular weight of 43 000 each.

Incubation of the purified enzyme with trypsin results in irreversible conversion into an enzymatically active modified form which, by a number of criteria, including molecular size, catalytic and antigenic properties, electrophoretic and chromatographic behaviour, appears to be undistinguishable from the altered methionyl‐tRNA synthetase previously observed after incubation at 37°C of a crude extract. Furthermore, a similar modification of methionyl‐tRNA synthetase resulted upon incubation of the purified enzyme with several other proteolytic enzymes, including papain, subtilisin and pronase.

The structural and catalytic properties of the homogenous trypsin‐modified methionyl‐tRNA synthetase were examined in detail. It has a molecular weight of 64000 established by equilibrium ultracentrifugation, and dissociates in the presence of 8 M urea to yield two, apparently identical subunits of molecular weight 32000. Furthermore, it is shown that proteolysis of the native enzyme by trypsin is accompanied by release of enzymatically inactive fragments corresponding to approximately 20% of the original protein. The simplest interpretation of these results is that proteolysis selectively removes a portion of each original subunit, corresponding to a quarter of its size, with consequent dissociation of the original tetramer into dimers, each now composed of modified subunits.

No significant differences in the Michaelis constants for any of the substrates or inhibitors tested were found between native and modified enzyme. Nor was conversion accompanied by any detectable change in the specificity towards the amino acid. The molecular activity (expressed as moles of substrate converted/mole subunit) of the homogenous trypsin‐modified enzyme was found to be 20% lower than that of native methionyl‐tRNA synthetase, for both reactions catalyzed by the enzyme. An interesting difference between the two forms of the enzyme appeared in their magnesium ion requirement for the aminoacylation of tRN As. Thus, under otherwise identical assay conditions, the trypsin‐modified enzyme required significantly less magnesium ions for the aminoacylation of both tRNAMetf and tRNAMetm at optimal rates. Another remarkable feature of the trypsin‐modified enzyme is its enhanced stability against heat inactivation, compared to the native enzyme.

A structural model is proposed for methionyl‐tRNA synthetase which attempts to account for its great susceptibility to limited proteolysis, and for the maintenance of its catalytic properties despite the resulting radical changes in its molecular structure.
The Synthesis and Circular Dichroism of a Series of Peptides Possessing the Structure (l‐Tyrosyl‐l‐alanyl‐l‐glutamyl)n
Authors: Schechter Bilha , Schechter Israel , Ramachandran Janakiraman , Conway‐jacobs Abigail , Sela Michael
Published: March 2005

A series of peptides possessing the structure (l‐Tyr‐l‐Ala‐l‐Glu)n, with n = 1,2,3,4,7,9, and 13 was synthesized. The oligomers up to n = 4 were prepared by stepwise synthesis using the N‐hydroxysuccinimide ester of the tripeptide derivative for the elongation of the chain. The oligomers with n = 7,9, 13 were prepared by a polycondensation technique, followed by gel filtration.

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

Antibodies were provoked against a random copolymer of l‐tyrosine, l‐alanine, and l‐glutamic acid, denoted as(Tyr,Ala,Glu)n, and against the α‐helical high molecular weight polymer containing the repeating sequence l‐tyrosyl‐l‐alanyl‐l‐glutamyl, denoted as (Tyr‐Ala‐Glu)n. Significant crossreactions were found between anti‐(Tyr‐Ala‐Glu)n antibodies and (Tyr,Ala,Glu)n, as well as between anti‐(Tyr,Ala,Glu)n antibodies and (Tyr‐Ala‐Glu)n.

Small peptides, obtained from a 45‐min pronase digestion of (Try,Ala,Glu)n efficiently inhibited precipitin reactions between anti‐(Tyr,Ala,Glu)n and (Tyr,Ala,Glu)n, anti‐(Tyr,Ala,Glu)n and (Tyr‐Ala‐Glu)n, and anti‐(Tyr‐Ala‐Glu)n and (Tyr,Ala,Glu)n. The peptides did not affect at all the reaction between anti‐(Tyr‐Ala‐Glu)n and (Try‐Ala‐Glu)n. It was concluded that the cross‐reaction between the (Tyr‐Ala‐Glu)n and (Tyr,Ala,Glu)n systems were due to sequential determinants in (Tyr‐Ala‐Glu)n rather than helix‐dependent determinants in (Tyr,Ala,Glu)n.

The cross‐reactive fraction from anti‐(Tyr‐Ala‐Glu)n sera was isolated on a (Tyr,Ala,Glu)n immunoadsorbent and the purified antibodies were found to precipitate to a much larger extent with (Tyr,Ala,Glu)n than with (Tyr‐Ala‐Glu)n. These antibodies were probably produced against some degradation products of (Tyr‐Ala‐Glu)n produced in vivo.
Properties of the DNA Synthesizing Activity in DNA‐Membrane Complexes from Bacterial Cell Extracts
Authors: Strätling Wolf , Knippers Rolf
Published: March 2005

A fast sedimenting complex of gently lysed pol A− cells (an Escherichia coli strain lacking DNA polymerase I) included fragmented cell wall structures, lipid components, about 20% of the cellular protein and most of the bacterial chromosome. A major fraction of the DNA synthesizing activity in these strains cosedimented with this complex. Several biochemical properties of this complex are described. The DNA synthesizing activity can be released from the complex by ultrasonication and by various detergents. The results are interpreted to mean that the DNA synthesizing activity is attached to the bacterial chromosome.
Microbial Formation of 17α‐C21 Steroids
Authors: Björkhem Ingemar , Eriksson Håkan , Gustafsson Jan‐Åke
Published: March 2005

After anaerobic incubation in deuterated water of 3β‐hydroxy‐5,16‐pregnadien‐20‐one with caecal contents from rats, 3β‐hydroxy‐17α‐pregn‐5‐en‐20‐one with deuterium label in ring D was isolated. Enolization of this compound with alkali yielded 3β‐hydroxy‐17β‐pregn‐5‐en‐20‐one with about 0.4 atoms of deuterim in ring D. Incubation of this compound with rat liver microsomes fortified with NADPH yielded 3β,16α‐dihydroxy‐17β‐pregn‐5‐en‐20‐one devoid of deuterium in ring D. The results show that the saturation of the Δ16‐double bond, catalyzed by intestinal microorganisms, involves a trans addition of hydrogen to the 16α‐ and 17β‐positions.
Limited Proteolysis of Horse Heart Cytochrome c
Authors: Schejter Abel , Goldkorn Tzipora , Sokolovsky Mordechai
Published: March 2005

Proteolysis of cytochrome c with pepsin results in the rapid preferential cleavage of the peptide bond between residues 66 and 67. The resulting 1–66 heme peptide is biologically inactive, binds readily cyanide and carbon monoxide, is oxidized in air, but is not reduced by ascorbate. The spectrum of this heme peptide has the same maxima observed for shorter heme peptides, but the absorption coefficients of their maxima are significantly lower.
Molecular Weight, Single‐Chain Structure and Amino Acid Composition of Human Lactoferrin
Authors: Querinjean Pierre , Masson Pierre L. , Heremans Joseph F.
Published: March 2005

A molecular weight of 75000 was computed for human lactoferrin on the basis of a sedimentation coefficient s°20, W= 4.93, a partial specific volume v̄20= 0.715 and a diffusion coefficient D°20, W= 5.6×10−7 cm2xsec−1. A molecular weight of 76800 ± 1600 was estimated from electrophoreses in agarose acrylamide gel containing sodium dodecylsulphate. No decrease of the molecular weight was observed when the reduced and alkylated or aminoethylated protein was submitted to 6 M urea, indicating that the molecule of lactoferrin consists of a single polypeptide chain. From the analysis of the amino acid composition of human lactoferrin a theoretical molecular weight of 76400 was computed by means of a statistical test. A comparison of the amino acid compositions of human lactoferrin and transferrin suggests that the two proteins are more closely related to one another than was suspected before.
Acidic Structural Proteins of Connective Tissue
Authors: Wolff Ivar , Fuchswans Werner , Weiser Max , Furthmayr Heinz , Timpl Rupert
Published: March 2005

From the insoluble structural matrix of skin and other connective tissues the collagen was removed by collagenase digestion. The remaining acidic structural proteins were then separated from elastin by solubilization with 8 M urea followed by treatment with mercaptoethanol in 8 M urea. Sodium dodecylsulfate‐polyacrylamide electrophoresis demonstrated up to 15 bands in some samples. The molecular weights were in the range of 15000 to 88000. Partial resolution of this mixture was achieved by molecular sieve chromatography. Treatment with NaBH4 converted most of the components into polypeptide chains with a molecular weight of 15000. The observed heterogeneity may thus reflect to some extent polymerization of the same polypeptide chain. The presence of different protein species is, however, suggested, since in gel diffusion two to four antigens were discernible. Most of them were destroyed by reduction. An antigen stable towards reduction could be demonstrated by passive hemagglutination. This antigen was, at least in the rat skin, common to the proteins of the urea as well as the urea‐mercaptoethanol extract.
Cell Walls of a Teichoic Acid Deficient Mutant of Bacillus subtilis
Authors: van Heijenoort Jean , Menjon Danièle , Flouret Bernard , Szulmajster Jekisiel , Laporte Jean , Batelier Gérard
Published: March 2005

The Cbl‐1 mutant of Bacillus subtilis 168 WT is the result of a spontaneous mutation pressenting a pleiotropic character. In this paper, the results of the morphological and chemical analyses of the cell walls isolated from bothe strains are compared. Whenm either trhe whole cells or the isolated cell walls of both strains were fixed with osmium tetroxide, the examination by electron microscopy showed extensive morphological differences in structure due to the partial destruction of the mutant cell walls. The chemical investigaions performed on the isolated cell walls revealed surprising differences in chemical structure. In the cells walls of the mutant, teichoic acid was completely missing and was replaced by protein. A major protein‐containing fraction accounting for about 50% of the material soulubilozed by autolysis of the mutant cell walls was isolated. Polyacrylamide gel electrophoresis showed that this fraction was composed mainly of a single protein of high molecular weight. When denatured by soduim dodecylsufate treactment in the pressence of β‐mercaptoethanol, the protein yielded by gel electrophoresis two major bands with molecular weights of approximately 105000 and 155000, repectively. The molecular weight of the Cbl‐1‐cell wall protein determined by high speex sedimentation equilibrium was 255600.
Metabolism of Prostaglandin F2α in Guinea Pig Lung
Authors: Granström Elisabeth
Published: March 2005

[9β‐3H]Prostaglandin F2α was incubated with the particle free fraction of a homogenate of guinea pig lung. One less polar metabolite appeared, which was assigned thje strcuture 9α.11α‐dihydroxy‐15‐ketoprost‐5‐enoic acid. The identification was based on gas chromatographic and mass spectrometric analyses of the trimethylsilyl ether, acetate, methoxime‐trimethlsily ether and methoxime‐acetate derivatives of the methyl ester. The position of the double bond was established by oxidative ozonolysis of the methlester and acetate followed by gas chromatogralphymass spectrometry.
Phospholipase A from Bee Venom
Authors: Shipolini Rudolf A. , Callewaert George L. , Cottrell Richard C. , Doonan Shawn , Vernon Charles A. , Banks Barbara E. C.
Published: March 2005

The phospholipase A from the venom of the common European honey bee (Apis mellifica) has been completely purified. The final product (13 g from 700 g of crude venom) readily crystallizes and is homogeneous with respect to starch gel electrophoresis at pH 8.0, isoelectric focussing in polyacrylamide gel in the pH range 3–10, and sedimentation and diffusion analysis in the ultracentrifuge.

Only one N‐terminal residue, isoleucine, can be detected either by the Edman or dansyl methods. Quantitative N‐terminal analysis and gel filtration on Sephadex G‐100 give values for the molecular weight of about 19000. Ultracentrifugation studies lead to a value of about 40000: in concentrated solution the molecule exists, therefore, as a dimer.

The identity of the enzyme as a phospholipase of the A2 type has been confirmed since, with 1‐oleyl‐2‐isolauroyl phosphatidyl ethanolamine as substrate, isolauric acid is liberated in 100% yield whereas no oleic acid is released. A method for the assay of the enzyme based on continuous titrimetric estimation of hexanoic acid liberated from 1,2‐dihexanoyl lecithin has been used to study various aspects of the activity of the enzyme.
Uncoupling of Monooxygenation and Electron Transport by Flurocarbons in Liver Microsomes
Authors: Ullrich Volker , Diehl Horst
Published: March 2005

Perfluoro‐n‐hexane in contrast to n‐hexane is not hydroxylated by the microsomal monooxygenation system in liver microsomes of phenobarbital treated rats. However, the fluorocarbon forms an enzyme‐substrate complex and, therefore, stimulates NADPH oxidation. Fluorocarbons can be regarded as dead‐end inhibitors which uncouple electron transport from monooxygenation. This effect must be taken into consideration when fluorocarbons are used as oxygen carriers in liver perfusion.
Isolation of the Ribosomal RNA Genes of Salmonella typhimurium
Authors: Udvardy Andor , Venetianer Pál
Published: March 2005

A procedure is described for the purification of the ribosomal RNA genes of Salmonella typhimurium LT‐2. Its steps are: ultrasonic fragmentation of the denatured DNA, separation of the complementary strands, hybridization of the rRNA transcribing strand with rRNA, and separation of the hybrid from bulk DNA by means of repeated chromatography on deoxycholate‐treated benzoylated‐DEAE‐cellulose. The purification was 190‐fold with approximately 6% yield. The purity of the final product was 64%.
Identification of Lysines Reactive with Pyridoxal 5′‐Phosphate in Glyceraldehyde‐3‐phosphate Dehydrogenase
Authors: Forcina Bruno G. , Ferri Giuseppina , Zapponi Maria Carla , Ronchi Severino
Published: March 2005

Rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase is almost completely inhibited by reaction of 4 moles of pyridoxal 5′‐phosphate per mole of subunit chain of holoenzyme.

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

Various sub‐cellular fractions of rat liver exert catalytic effects on the oxidation of [4‐14C]‐cholesterol to 7α‐hydroxy‐[4‐14C]cholesterol and to other [4‐14C]cholesterol oxidation products. Some of these oxidation products created in these studies in vitro appear to be aberrant oxidation products in that there is no evidence that they are intermediates in the physiological oxidation of cholesterol. These aberrant oxidation products have been classified as autoxidation products despite the fact that the autoxidation reactions in these cell fractions appear to require the participation of NADPH, native proteins and perhaps certain nucleotides for the production of these compounds.

The enzyme system catalysing the oxidation of [4‐14C]cholesterol to 7α‐hydroxy‐[4‐14C]cholesterol could be assayed in a 18000 ×g supernatant fraction of rat liver. This enzymic reaction was adversely affected by extensive homogenisation of liver tissue and by prolonged incubation periods, both of which tended to increase the autoxidation reactions. The cholesterol‐7α‐hydroxylase enzyme showed optimal activity in the presence of NADPH and oxygen.

In the washed liver microsomal fraction, [4‐14C]cholesterol was extensively oxidised in the presence of NADPH, ADP and ferrous ions to 7β‐hydroxycholesterol, cholestan‐3β, 5α, 6β‐triol and 7‐ketocholesterol. Further investigation showed that another compound was also formed, having the same mobility as 7‐ketocholesterol in the thin layer solvent systems used. Reduction and further metabolism of this compound compared with reference standards suggested that it may be a cholesterol oxide, formed under conditions where peroxidation of microsomal lipids could occur. A comparison of the effects of Tris and phosphate buffers, and various nucleotides suggests that cholesterol autoxidation in the microsomal fraction may be related to the general lipid peroxidation systems.

The boiled 100000 ×g liver supernatant preparation or 2‐mercaptoethylamine both effectively quenched cholesterol peroxidation, at the same time stimulating the cholesterol‐7α‐hydroxylase system. A scheme has been suggested to explain the liver microsomal cholesterol autoxidation or peroxidation and to contrast this process with the mixed function oxidase involved in the enzymic cholesterol‐7α‐hydroxylase reaction.
Acquisition and Loss of Rotenone Sensitivity in Torulopsis utilis
Authors: Katz Richard , Kilpatrick Laurie , Chance Britton
Published: March 2005

Torulopsis utilis yeast cells growing exponentially on synthetic medium with ethanol as carbon and energy source are insensitive to rotenone; the cells acquire the rotenone‐sensitive component of the mitochondrial electron transport chain upon entering an ethanol‐depleted stationary phase. The rotenone‐sensitive site is lost when growth is restimulated with ethanol, but not when growth is restimulated with glycerol or acetic acid. Growth limitation by iron or by glucose or glycerol is also characterized by acquisition of the rotenone‐sensitive site, upon entering stationary phase. The transition from rotenone‐sensitive to ‐insensitive can also be studied by the use of semi‐continuous culture in a chemostat. Studies with the protein synthesis inhibitors cycloheximide and chloramphenicol indicate that protein synthesis on cytoplasmic ribosomes is required both for the acquisition and the loss of the rotenone‐sensitive site. The acquisition of rotenone sensitivity is apparently associated with an increased phosphorylation efficiency, as evidenced by measurements of P/O ratios.
6‐Methylsalicylic Acid Synthesis by Isolated Barley Chloroplasts
Authors: Kannangara C. Gamini , Henningsen Knud W. , Stumpf Paul K. , von Wettstein Diter
Published: March 2005

Chloroplasts isolated from dark grown barley leaves greened for 6 h rapidly incorporate [1‐14C]acetate into 6‐methylsalicylic acid. Etioplasts synthesize little 6‐methylsalicylic acid and mature chloroplasts lack this activity.

Carbon dioxide and CoA are essential cofactors for the synthesis. While light increases the synthesis of 6‐methylsalicylic acid, a considerable amount of the acid is formed in the dark. Externally supplied NADPH has no effect on the incorporation of [1‐14C]acetate into 6‐methylsalicylic acid by isolated chloroplasts.

The biosynthesized 6‐methylsalicylic acid has been purified by thin layer chromatography. Characterization of the labeled compound is done by radio gas liquid chromatography, by repeated crystallization to constant specific radioactivity and by identification of its nitration product as 3‐methyl‐2,4,6‐trinitrophenol.

Since 75% of the radioactivity of 6‐methylsalicylic acid synthesized by isolated chloroplasts is confined to the aromatic nucleus, a mechanism of biosynthesis involving head to tail condensation of acetyl CoA with malonyl CoA is indicated.
The Linkage of Pyrophosphorylethanolamine to Heptose in the Core of Salmonella minnesota Lipopolysaccharides
Authors: Lehmann Volker , LÜDERITZ Otto , Westphal Otto
Published: March 2005

Mild acid hydrolysis of the P+ lipopolysaccharide of Salmonella minnesota mRz, a UDP‐glucose synthetase‐less mutant, leads to the formation of 5 degradation products whose structures were studied. Free 2‐keto‐3‐deoxyoctonate (KDO), KDO‐7‐phosphorylethanolamine, and fraction P− (Hep‐Hep‐KDO) have been identified previously as degradation products of a P− lipopolysaccharide (glycolipid). Fractions P+ (Hep‐(Hep‐4‐phosphate)‐KDO) and PPN (Hep‐(Hep‐4‐pyrophosphorylethanolamine)‐KDO) occur only in the P+ mutant. It was shown that fraction P+ is not an artifact which was formed from fraction PPN during partial hydrolysis. The simultaneous occurrence of fractions P+ and PPN reflects microheterogenicity of the lipopolysaccharide. Possible reasons for this are discussed.
Hydroxymethylhydroperoxide as Inhibitor and Peroxide Substrate of Horseradish Peroxidase
Authors: Marklund Stefan
Published: March 2005

Bis(hydroxymethyl)peroxide, formerly reported to inhibit horseradish peroxidase, was found not to influence the enzyme.

Hydroxymethylhydroperoxide, which may be formed from bis(hydroxymethyl)peroxide by hydrolysis, irreversibly inhibits horseradish peroxidase in a second‐order reaction, k= 3.0X102/sec−1XM−1 at pH 4.27 in 6.7 mM guaiacol.

Hydroxymethylhydroperoxide is a peroxide substrate of horseradish peroxidase.

The rate constant (k1) for the formation of the enzyme‐substrate complex (Cpd I) between hydroxymethylhydroperoxide and horseradish peroxidase was indirectly determined to be 2X105 sec−1XM−1 at pH 4.27.
DNA Synthesis in Nucleotide Permeable Escherichia coli Cells
Authors: Geider Klaus , Hoffmann‐Berling Hartmut
Published: March 2005

Cells made permeable to nucleotides by treatment with ether synthesize DNA from exogenous deoxynucleoside triphosphates in a semi‐conservative fashion. The reaction proceeds from sites of previous synthesis on the chromosome, and not detectably from new sites. Short chains appear as precursors of long units and have a mean sedimentation coefficient of about 10 S in alkali, like Okazaki‐pieces when synthesized in the presence of 10 to 20 μM deoxynucleoside triphosphates, and of only 5 S when synthesized in the presence of 1 μM dNTP. Under the former conditions short chains contain not more than half of the newly synthesized DNA; under the latter conditions they can contain more than half, the remainder of the newly synthesized DNA being in long units. Nascent short chains labelled after several minutes of synthesis in the presence of 5‐bromodeoxyuridine 5′‐triphosphate at high deoxynucleoside triphosphate concentrations show bimodal density distribution in an alkaline Cs2SO4 equilibrium gradient with peaks of material corresponding to the density of heavy single strands and almost light single strands, respectively. The implications of these findings are discussed. Judged from controls, repair synthesis was not a source of error in these experiments.

In contrast with results reported for intact cells, ether‐treated polA1− (Kornberg‐polymerase deficient) cells join nascent short chains as efficiently as do polA+ cells.
Release of Ethanolamine Pyrophosphate during Mild Acid Hydrolysis of the Lipopolysaccharide of Pseudomonas aeruginosa
Authors: Drewry David T. , Gray George W. , Wilkinson Stephen G.
Published: March 2005

Low molecular weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudomonas aeruginosa were isolated from the fraction containing the partially degraded polysaccharide, by successive chromatography on columns of Sephadex G‐75 and G‐10, followed (as necessary) by preparative high voltage paper electrophoresis. The major components identified were 2‐keto‐3‐deoxyoctonic acid, basic amino acids (free and bound), inorganic orthophosphate, ethanolamine phosphate and ethanolamine pyrophosphate. Ethanolamine pyrophosphate has not previously been found in acid hydrolysates of lipopolysaccharides. Ethanolamine phosphate and some of the orthophosphate were apparently produced by breakdown of ethanolamine pyrophosphate.