bluuebunny/crossref_metadata_2025 huggingface dataset:
Poly(A) Synthesis in T2L Phage‐Infected Escherichia coli
Authors: WUNDERLI Werner , STAEHELIN Matthys , WEHRLI Walter , HÜTTER Ralf
Published: March 2005

In crude extracts of T2L phage‐infected Escherichia coli cells an enzyme activity was found that produced poly(A) from ATP as substrate.

Purification of the extract led to the isolation of two enzymes, a polynucleotide phosphorylase and an ATPase. The polynucleotide phosphorylase possessed the same properties as the well‐known enzyme from uninfected cells and its molecular weight was about 265 000. The ATPase was purified to over 90% purity; its molecular weight was estimated to be about 165000 with three subunits of 55000. The characterization of this enzyme showed that it was different from any ATPase known so far. Mg2+ cannot be replaced by Ca2+, as it can from the membrane‐bound ATPases. The only product yielded by the enzyme was ADP; it was very specific for ATP, other ribonucleotide triphosphates being practically unaffected. The rate of ATP splitting was found to be very high, the turnover number being 2.51 × 104 min‐1 at 37°C. Even at 0°C the enzyme was still active. The optimal assay conditions for ATPase turned out to be very similar to those of polynucleotide phosphorylase. Thus the combination of the two enzymes very efficiently produced poly(A) from ATP. In this combination the polynucleotide phosphorylase was the rate‐limiting enzyme, since its turnover number was about 40 times lower than that of the ATPase. The evaluation of a variety of properties of the poly(A)‐synthesizing constituent found in the crude extracts led us to conclude that this activity arises from the combined action of ATPase and polynucleotide phosphorylase, and is not due to a poly(A) polymerase.
Highly Purified Glutamine Transaminase from Rat Brain
Authors: VAN LEUVEN Freddy
Published: March 2005

Glutamine transaminase from rat brain was purified to a high degree. The isolated enzyme appeared to be homogeneous by electrophoresis on polyacrylamide gel. The molecular weight was found to be approximately 98000; the enzyme is probably composed of two subunits. The absorbance maximum at 410 nm and the inhibition by carbonyl reagents are strong indications for the presence of pyridoxal phosphate. The enzyme showed maximal activity at pH 9.0 to 9.2. Of the amino acids tested, none could replace glutamine in the transamination reaction. Glyoxylate and phenylpyruvate were found to be the best amino acceptors. The Km values for glutamine and glyoxylate were 0.6 and 1.5 mM, respectively.
The Initial Binding of Cu(II) to Some Amino Acids and Dipeptides: A 13C Nuclear‐Magnetic‐Resonance Study
Authors: VOELTER Wolfgang , SOKOLOWSKI Gert , WEBER Ulrich , WESER Ulrich
Published: March 2005

The initial binding of Cu2+ to l‐lysine, l‐histidine, glycyl‐histidine and histidyl‐glycine in aqueous solutions was examined by 13C nuclear magnetic resonance spectroscopy. The measurements were carried out in a substantially improved way employing the pulse Fourier transform technique. Spectra of both high quality and resolution were obtained. Cu2+ complex formation with l‐lysine occurred with the α‐amino and carboxyl group attributable to the well expressed broadening effect of the 13C signals of the α‐carbon atom and the carboxyl atom. The ɛ‐amino group was not involved. Measurements of the Cu chelates using l‐histidine and glycyl‐histidine and histidyl‐glycine confirmed the ambidentate nature of the histidine residue. It was concluded that an equilibrium exists between two Cu‐complex species designated as histamine‐like and histamine‐like/glycine‐like species. In the homogeneous histamine‐like Cu complex, the Cu2+ is exclusively bound with 4 nitrogens, while in the other species one oxygen of the glycyl carboxyl group is involved in the Cu2+ binding. Blocking of this carboxyl group by peptide bonding as found in histidyl‐glycine favoured the formation of a Cu complex where the imidazole carbons of the histidyl residue were the most influenced species.
Kinetic Investigation of the α‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide‐Ester Substrates
Authors: BIZZOZERO Spartaco A. , BAUMANN Werner K. , DUTLER Hans
Published: March 2005

A number of peptide‐ester substrates of the general structure Ac‐Lxn‐.Lx2‐Lx1‐OMe have been synthesized and their α‐chymotrypsin‐catalyzed hydrolysis studied. The kinetic analysis involved varying the concentration of substrate and methanol product, and measuring rates along the entire progression curve.

For the dipeptide esters Ac‐Lx2‐Lx1‐OMe and the amino‐acid derivatives Ac‐Lx1‐OMe the following constants could be determined: the dissociation constant of the enzyme‐substrate complex, KEA, both rate constants of the acylation step, k23 and k32, and the forward rate constant of the de‐acylation step, k31. For the tripeptide ester Ac‐Ala‐Ala‐Tyr‐OMe it appears that the rate constant for the dissociation of the enzyme‐substrate complex, k21, is smaller than the rate constant for acylation, k23. Thus, for this substrate only the association and dissociation rate constants k12 and k21 could be determined and the values of k23, k32 and k31 only indirectly estimated. The influence of structural changes in the peptide moiety of the substrates on reactivity has been established by comparing the rate constants of appropriate pairs of substrates. It was found that the substrate reactivity, as measured by k23/KEA, increases with the number and strength of the secondary interactions in a manner consistent with the binding scheme which has been proposed on the basis of crystallographic studies. The effect of a particular interaction on k23 and on KEA is dependent on the nature of the other interactions. However, the effect on k23/KEA appears to be independent of the presence of the other interactions and therefore characteristic of that particular interaction. The results for these substrates are compared with those found previously for a series of peptide substrates of the structure Ac‐Lxn‐.‐Lx2‐.‐Lx1‐Gly‐NH2 which have the same acyl moiety as the peptide esters studied in this work.
Transcription of the Genome of Adenovirus Type 12
Authors: SCHEIDTMANN Karl‐Heinz , ORTIN Juan , DOERFLER Walter
Published: March 2005

In human KB cells productively infected with adenovirus type 12, viral DNA replication starts between 12 and 14 h postinfection. Virus‐specific, polysome‐associated mRNA was investigated early (6–8 h) and late (26–28 h) after infection. Most of the viral mRNA was polyadenylated and accounted for 0.46% and 24.1% of the mRNA synthesized early and late postinfection, respectively. The viral‐specific mRNA isolated both early and late after infection falls into several distinct size‐classes, ranging in molecular weights between 0.3 × 106 and 1.5 × 106 for the early RNA and between 0.6 × 106 and 2.3 × 106 for the RNA synthesized late in the infection.
Replication of Colicinogenic Factor E1 DNA in Plasmolysed Escherichia coli Cells
Authors: STAUDENBAUER Walter L.
Published: March 2005

Plasmolysed chloramphenicol‐treated Escherichia coli cells carrying the colicinogenic factor E1 utilize deoxynucleoside triphosphates for the semi‐conservative synthesis of Col E1 DNA. Col E1 DNA replication in plasmolysed cells can be dissociated into two temporally separated processes: (a) a rifampicin‐sensitive RNA synthesis, which is stimulated by adenosine 3′:5′‐monophosphate (cyclic AMP) and requires all four ribonucleoside triphosphates and (b) an ATP‐dependent DNA synthesis, which is inhibited by arabinosylnucleoside triphosphates and sulfhydryl‐blocking reagents. These two processes exhibit different sensitivities to inhibition by polyamines and actinomycin D.
Non‐histone Chromosomal Proteins
Authors: BLÜTHMANN Horst , MROZEK Stanislav , GIERER Alfred
Published: March 2005

We describe a method for fractionation of chromatin components by selective dissociation with salt in buffers containing 5 M urea in combination with chromatography on hydroxyapatite at 4 °C. This results in two histone and four non‐histone fractions which are recovered in high yield and with minimal proteolytic contamination.

Template capacity measurements of the isolated chromatins and pre‐saturation competition hybridization experiments support the idea that a group of non‐histone proteins activate the transcription of specific DNA sequences which were not transcribed from purified DNA to the same extent.

In reconstitution experiments a non‐histone protein fraction, NH4, prepared from lymphocyte chromatin by hydroxyapatite chromatography is shown to cause transcription in vitro of lymphocyte‐specific RNA sequences. A subfraction with a molecular weight of 30000 comprising 40% of the NH4 fraction protein is characteristic for this tissue and not found in liver chromatin.
Interaction between the Synthesis of α and β Globin
Authors: GARRICK Laura M. , DEMBURE Philip P. , GARRICK Michael D.
Published: March 2005

We have examined the relationship between α and β globin chain syntheses by utilizing the distribution of isoleucyl residues in rabbit hemoglobin. The α globin chain contains three isoleucyl residues while the β chain of certain rabbits contains no isoleucine. O‐Methyl‐L‐threonine, an isoleucine isostere, inhibits incorporation of radiolabeled amino acids into α chains in rabbit reticulocytes. When α chain synthesis is inhibited by 50–85%, β synthesis is stimulated by 15–50%. The excess labeled β chains are not distinguishable from authentic β chains by any of the following criteria: (a) carboxymethyl cellulose chromatography in sodium phosphate‐urea buffers, (b) electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate, and (c) electrophoresis of methionine‐containing tryptic peptides. The stimulation of β synthesis continues after the pool of excess α chains has been exhausted by preincubation with O‐methyl‐L‐threonine. The stimulation does not occur, however, when 1 mM 2‐mercaptoethanol is added to the incubation medium or when the cells are excessively diluted in the incubation mixture. The rates of β chain initiation and elongation during stimulation have been compared to the rates during normal synthesis. Although both rates are increased, the rate of elongation increases more than initiation, suggesting that initiation is the rate‐limiting step in increased β chain production. The stimulation of β synthesis when α synthesis is inhibited is interpreted as resulting from relief of competition between α and β mRNAs for limiting components of the protein synthetic apparatus.
Phage Qβ Replicase: Cell‐Free Synthesis of the Phage‐Specific Subunit and Its Assembly with Host Subunits to Form Active Enzyme
Authors: HAPPE Monika , JOCKUSCH Harald
Published: March 2005

Cell‐free translation of Qβ RNA and subsequent partial purification of the enzyme resulted in replicase activity. From 0.5 to 1.5% of all R chains synthesised were found in the 7‐S replicase complex. The presence in the 7‐S complex of the host subunits of authentic replicase, i (= S1) and EF‐Ts, was shown by the effect of antisera directed against ribosomal protein S1 and EF‐Ts, respectively. Furthermore, the presence of EF‐Ts was demonstrated by thermal denaturation of in vitro replicase made by a cell extract from an Escherichia coli mutant with a thermolabile EF‐Ts. In vitro replicase did not assemble spontaneously during protein synthesis but was formed upon subsequent purification. Assembly could be induced by ammonium sulphate precipitation (60% saturation) alone. It is concluded that the functional phage‐coded subunit synthesised in vitrorecognises i and the EF‐Tu · EF‐Ts complex among a mixture of host proteins.
The Influence of Insulin on Glucose Permeability and Metabolism of Human Granulocytes
Authors: LEROUX Jean‐Paul , MARCHAND Jean‐Claude , HONG TUAN HA Régine , CARTIER Pierre
Published: March 2005

Viable human polymorphonuclear leukocytes isolated from peripheral blood were incubated for 1 h at 37 °C with variable concentrations of insulin in a saline medium buffered at pH 7.4. The hormone increased glucose consumption by about 40% without influencing the permeability of the membranes to glucose, whose uptake followed a passive diffusion process. The measurement of intermediates localized activation of glycolysis by insulin, down to 0.36 nM, at the phosphofructokinase step. However, the spectrophotometric measurement showed no activation of phosphofructokinase after preincubation with insulin of either intact granulocytes or crude or ultracentrifuged homogenates. The level of cyclic AMP, which is known to activate phosphofructokinase, was not modified by insulin; cyclic GMP did not activate the enzyme in the granulocyte extracts: neither of the two nucleotides can therefore be considered as a direct messenger of the action of insulin on phosphofructokinase.

An important fraction of the extra glucose consumed under the influence of insulin was recovered as neither glycogen nor lactate, nor was it oxidized in the Krebs cycle. It might be assumed to have been converted into glycerolipids. However, insulin produced no detectable accumulation of tri‐glycerides and activated neither the pentose phosphate pathway nor oxidative decarboxylation of pyruvate. The fate of the extra glucose consumed under the influence of insulin therefore remains questionable.
Studies on Norepinephrine‐Induced Efflux of Free Fatty Acid from Hamster Brown‐Adipose‐Tissue Cells
Authors: BIEBER Loran L. , PETTERSSON Bertil , LINDBERG Olov
Published: March 2005

Cells were isolated from brown adipose tissue of warm‐adapted hamsters and the fate of free fatty acids released during norepinephrine‐induced lipolysis was investigated. The isolated resting cells contain between 100–400 nmoles cell‐associated free fatty acids per 106 cells; most preparations contained about 200 nmoles/106 cells. During norepinephrine‐stimulated lipolysis, the level of cell‐associated free fatty acids remains constant or decreases gradually, but does not increase, while the concentration of extracellular fatty acids increases linearly. The rate of norepinephrine‐stimulated efflux of free fatty acids was 40 ± 20 nmol x min‐1 x 106 cells‐1 (n= 11) at 37°C. The data strongly indicate that brown adipose tissue can supply free fatty acids to the circulatory system in hamster.
Requirement for Ribosome‐Releasing Factor for the Release of Ribosomes at the Termination Codon
Authors: OGAWA Koichi , KAJI Akira
Published: March 2005

With the use of 3H‐labeled R17 amB2 phage RNA having an UAG codon at the seventh triplet of the coat cistron, release of the RNA from ribosomes at the termination codon was studied. The ribosome‐releasing factor previously described was shown to stimulate the process of mRNA release at the termination codon. The stimulatory effect of the factor on this process was dependent on the termination factor (RF‐1). GTP was required for this process and guanosine 5′‐(β,γ‐methylene)triphosphate could not replace GTP. No apparent change of size of R17 RNA was observed during the release of the R17 RNA from the ribosomes. The ribosome‐releasing factor is distinct from the known termination codon‐specific factor such as RF‐1.
Control of Haemoglobin Synthesis
Authors: HUNTER Anthony R. , JACKSON Richard J.
Published: March 2005

A detailed examination of the kinetics of protein synthesis in rabbit reticulocytes in the presence of the iron chelating agent 2, 2′‐dipyridyl showed that between 30°C and 42°C there were characteristically two distinct phases of protein synthesis. An initial phase (I), in which no inhibition of protein synthesis was apparent, was followed by a gradual decline in the rate of protein synthesis leading to the second phase (II) in which protein synthesis occurred at a linear but inhibited rate for extended periods. In contrast, below 30°C, incubation in the presence of dipyridyl caused no inhibition of protein synthesis. Between 30°C and 42°C the duration and amount of protein synthesis occurring in phase I before the onset of inhibition were inversely related to temperature, as was the final rate of incorporation in phase II. During phase II, a partial reversal of the inhibition caused by dipyridyl was obtained by lowering the incubation temperature. This resulted in a burst of protein synthesis at the uninhibited rate until the amount of protein synthesis reached the same level as that in reticulocytes maintained continuously with dipyridyl at the lower incubation temperature. This burst of synthesis was observed in reticulocytes which had been held in phase II for as long as 90 min. It was also possible to reverse the inhibition by addition of haemin to cells in phase II. At any particular incubation temperature, a fixed number of rounds of protein synthesis had to occur before the onset of phase II became apparent. By the use of puromycin we showed that this was not a requirement for the synthesis of globin or of any other protein. We believe that this critical amount of protein synthesis reflects the residual ability of reticulocytes to initiate new protein chains in the absence of concurrent haem synthesis. Reticulocytes preincubated in the presence of cobaltous ions showed almost no inhibition of protein synthesis upon subsequent incubation with dipyridyl. The results are compared to those obtained in reticulocyte lysates and are discussed in terms of current theories to account for control of protein chain initiation by haemin.
Co‐fractionation of an Endonuclease Activity during the Purification of DNA Polymerase‐α from Regenerating Rat Liver
Authors: MECHALI Marcel , DE RECONDO Anne‐Marie
Published: March 2005

The presence of endonuclease activity associated with DNA polymerase was detected during the purification of high‐molecular‐weight DNA polymerase‐α from regenerating rat liver by the use of a highly sensitive test. This endonuclease activity co‐fractionated with the DNA polymerase in a great variety of purification procedures involving ion‐exchange chromatographies or molecular weight fractionation, but was further completely separated from DNA polymerase activity by using affinity chromatography on DNA‐cellulose.

The endonuclease acted on native or denatured DNA by introducing single‐strand nicks in the DNA molecules; its enzymatic properties indicate that it could act in polymerisation conditions in vitro.
Glucosylation of Galactosylhydroxylysyl Residues in Collagen in vitro by Collagen Glucosyltransferase
Authors: MYLLYLÄ Raili , RISTELI Leila , KIVIRIKKO Kari I.
Published: March 2005

Glucosylation of galactosylhydroxylysyl residues in various collagen polypeptide chains and in small peptides prepared from collagen was studied in vitro using collagen glucosyltransferase purified about 200 to 500‐fold from extract prepared from chick embryos. When various denatured polypeptide or peptide chains were compared as substrates for the enzyme, no significant differences were found between citrate‐soluble collagens from normal or lathyritic rats and isolated α1 and α2 chains. In contrast, gelatinized insoluble calf skin collagen, and peptides prepared from collagen and having an average molecular weight of about 500 were clearly less effective substrates as judged from their Km and V values.

A marked difference was found between native and heat‐denatured citrate‐soluble collagen in that no synthesis of glucosylgalactosylhydroxylysine was observed with the native collagen when the reaction was studied at 30 °C with different times, enzyme concentrations, and substrate concentrations. When the reaction was studied as a function of temperature, little glucosylation of native collagen was observed below 37 °C, but there was a sharp transition in the rate of glucosylation of native collagen at temperatures above 37 °C, similar to that observable in the melting curve of collagen. The data suggest that triple‐helical conformation of collagen prevents the glucosylation of galactosylhydroxylysyl residues.
Mitochondria and Peroxisomes from the Cellular Slime Mould Dictyostelium discoideum
Authors: PARISH Roger W.
Published: March 2005

The isolation of cell organelles from Dictyostelium discoideum was attempted using a variety of techniques. Cell homogenization (e.g. Potter‐Elvehjem, glass beads) gave poor yields of organelles which were, in addition, exceptionally fragile and unstable in density gradients.

An isolation method was developed using Triton X‐100 in buffered sorbitol/Ficoll solutions at concentrations optimal for plasma membrane rupture. Immediately following cell lysis the solutions were diluted to sub‐optimal Triton X‐100 concentrations. Sedimentabilities of malate dehydrogenase, citrate synthetase, urate oxidase and catalase of around 55%, 40%, 35% and 55% respectively could be demonstrated using this method. The organelles were more resistant to breakage during resuspension following differential centrifugation and remained largely intact during density gradient centrifugation. The distribution of adenylate kinase activity in gradients showed that at least half the mitochondria retained an intact outer membrane.

The mitochondria and peroxisomes could not be clearly separated using conventional sucrose‐Ficoll density gradients.

Separation was achieved by incubating the cell homogenate with succinate and a tetrazolium dye (2‐p‐iodophenyl‐3‐p‐nitrophenyl‐5‐phenyl monotetrazolium chloride). Succinate dehydrogenase activity of mitochondria reduced the tetrazolium dye and the product (formazan) was deposited on the mitochondrial membranes (“heavy‐labelling”). The mitochondria then sedimented to denser regions of the gradient while catalase distribution remained unchanged. The treatment left both organelles intact.

The mitochondria (1.21 g/ml) were slightly denser than the peroxisomes (1.19 g/ml). The peroxisomes contained catalase and urate oxidase; no other hydrogen‐peroxide‐producing oxidases were detected.

The slime mould urate oxidase resembled the mammalian enzyme. It had an apparent Km value of 12.5 μM, an optimum of activity at pH 8.5 in borate buffer and was competitively inhibited by trichloropurine.
Amino‐Acid Sequence of the Cyanogen‐Bromide Fragment from Human Plasminogen that Forms the Linkage between the Plasmin Chains
Authors: WIMAN Björn , WALLÉN Per
Published: March 2005

The complete amino acid sequence of a cyanogen bromide fragment (122 residues) obtained from plasminogen is described. This fragment forms the overlap between heavy (A) and light (B) chains of human plasmin. The particular arginyl‐valyl bond cleaved in the second step of the activation process is shown to be Arg98‐Val99 in this fragment. This site is not very similar to the one in the NH2‐terminal part of the molecule (Arg68‐Met69). Remarkable homologies with the ‘triple loops’ (‘kringle structures’) found in the non‐thrombin part of prothrombin are demonstrated. Homologies within the plasmin heavy (A) chain are also demonstrated, suggesting that gene duplication has occurred during evolution of this chain.
Interactions of Colipase with Bile Salt Micelles
Authors: CHARLES Maurice , ASTIER Martine , SAUVE Paul , DESNUELLE Pierre
Published: March 2005

A detailed investigation by ultracentrifugation of the colipase‐taurodeoxycholate system showed the formation of well‐defined mixed associations with a sedimentation coefficient of about 2.2 S. The fact that these associations were only detectable above the critical micelle concentration of the salt indicated that micelles rather than monomers were bound to the cofactor.

Two technical difficulties must be overcome before the weight of the associations could be measured with a reasonable accuracy. Firstly, the partial specific volume of the associations was determined using a digital microdensimeter and the interferometric system of the ultracentrifuge for concentration determinations. Secondly, due to the fact that micelle concentrations could not be equilibrated by dialysis, even after an extended period of time, an appropriate dilution of the ligand in the buffer compartment was necessary in order to compensate for its fixation by colipase in the solution. Then, the ionic strength dependence of the weight of the associations was found to vary in parallel with that of the micelles and to be in each case equal to the sum of the weights of one colipase molecule and one micelle. Therefore, colipase can be expected to contain a single high affinity site for bile salt micelle binding.
The Pyruvate‐Dehydrogenase Complex from Azotobacter vinelandii
Authors: BRESTERS Tjarda W. , DE KOK Arie , VEEGER Cees
Published: March 2005

The presence of activators (AMP and sulphate) or inhibitors (acetyl‐CoA) has no influence on the Hill coefficient of the S‐shaped [pyruvate]–velocity curve of either the pyruvate‐NAD+ overall reaction (h= 2.5) or that of the pyruvate‐K3Fe(CN)6 activity of the first enzyme (h= 1.3). pH studies indicated that the Hill coefficient is dependent on subunit ionization within the pyruvate‐containing complex and not on those in the free complex.

It is concluded that pyruvate conversion rather than pyruvate binding is responsible for the allosteric pattern. The activity is, due to absence of a protein kinase, mainly regulated at the acetyl‐CoA/CoA, and NADH/NAD+ levels and by the value of the energy charge.
Amino‐Acid Incorporation into tRNA Fragments and into Heterologous Combinations of Fragments
Authors: WÜBBELER Wolfgang , LOSSOW Christine , FITTLER Friedrich , ZACHAU Hans G.
Published: March 2005

When the CCA‐halves of tRNAAla1 (yeast) and tRNAVal1 (Escherichia coli) were incubated with the pG‐halves of tRNAVal1 (E. coli) and tRNAAla1 (yeast), respectively, heterologous complexes were detected. When a 10‐fold excess of one half was applied, up to 50% of the other half could be com‐plexed. 5–12% alanine and valine incorporation was observed into the heterologous combinations in which the pG‐halves were derived from tRNAAla1 (yeast) and tRNAVal1 (E.coli), respectively. Although the values are small they appear to be significant considering the results of a number of control experiments.

The CCA‐half of tRNASer1.2 (yeast) and another fragment of this tRNA which extends from the dihydrouridine region to the CCA‐terminus were inactive in the aminoacylation assay but they could be converted into a form which accepted serine under standard conditions even in the absence of a complementary fragment. One activation procedure involved the addition of MgCl2 to Mg2+ ‐free fragment solutions, the other consisted in a brief heating‐cooling cycle of the fragment solutions at low Mg2+ concentrations. With the two procedures up to 20% or up to 40%, respectively, of the maximal serine incorporation were achieved. At 37 °C the active conformation of the fragments persisted only for a few minutes. Analogously, the CCA‐halves of tRNAphe (yeast), tRNAAla1 (yeast), and tRNAVal1 (E. coli) could be activated although here the extent of aminoacylation varied greatly from one experiment to the other. Mischarging of the activated CCA‐halves of tRNASer1,2 (yeast) and tRNAPhe (yeast) with phenylalanine and serine, respectively, was not observed.

The results obtained with the hererologous fragment combinations and with the CCA‐halves alone, which at first sight seem to contradict each other, are discussed with respect to the confor‐mational requirements of synthetase‐tRNA recognition.
Temperature‐Sensitive Mutants of the Yeast Fatty‐Acid‐Synthetase Complex
Authors: SCHWEIZER Eckhart , KNOBLING Ansgar
Published: March 2005

By genetic complementation analysis, 88 independently isolated temperature‐sensitive fatty acid synthetase mutants have been assigned to the six different fas‐complementation groups II (fas 1), III (fas 1), Vb (fas 1), VI (fas 2), VIII (fas 2) and IX (fas 2). The complementation groups Va, Vc, Vd,IV and VII observed among nonconditional fas‐mutants have not been found among the temperature‐sensitive strains studied. From the failure to detect pantetheine‐deficient conditional fas‐mutants it is concluded that the yeast acyl‐carrier protein has an exceptionally stable tertiary structure. Furthermore, the lack of temperature‐sensitive mutants of complementation group IV possibly indicates that this group specifically represents only nonsense and frameshift mutations.

Almost half of the temperature‐sensitive fas1 and fas2 mutants studied exhibited non‐complementing characteristics. These results confirm the existence of non‐complementing fas1 and fas2 missense mutations. From this it is concluded that both fatty acid synthetase loci encode multifunctional polypeptide chains rather than several monofunctional component enzymes. The possible existence of an independent acyl‐carrier protein, as suggested by the genetic data reported in this study, is discussed.

With 10 different temperature‐sensitive fas1 and fas2 mutants the dependence of cellular growth rates on growth temperature and fatty acid supplementation was determined. With all mutants studied fatty‐acid‐independent growth was completely suppressed at non‐permissive temperatures (34–37 °C). In fatty‐acid‐supplemented media, however, these mutants exhibited the same growth characteristics as wild‐type yeast cells. In contrast to this, wild‐type yeast growth was found to be fatty‐acid‐independent at all temperatures studied. Other than in vivo, the purified fatty acid synthetase isolated from five different temperature‐sensitive fas1 and fas2 mutants exhibited in vitro no increased thermolability compared to the wild‐type enzyme. From this it is concluded that the specific conformation of fatty acid synthetase subunits either forms only at the ribosomal level during translation, or that this conformation is stabilized by the assembly of subunits into the multienzyme complex structure.
A New Method of Isolation and Some Properties of the Heavy Chain of Human Plasmin
Authors: RICKLI Egon E. , OTAVSKY Wendy I.
Published: March 2005

A method is described by which the heavy chain of human plasmin, obtained by partial reduction of urokinase‐activated plasminogen with 2‐mercaptoethanol, is adsorbed on lysine coupled to polyacrylamide. The heavy chain is recovered from the adsorbent by elution with 6‐aminohexanoic acid (yield 60–65%).

Sulfhydryl titrations of the heavy chain showed that the partial reduction involved primarily the cleavage of the sole interchain disulfide bridge of plasmin. Dodecylsulfate‐polyacrylamide electro‐phoresis gave essentially a single band corresponding to a component of about 60000 molecular weight. The NH2‐terminal amino acid was predominantly threonine.

6‐Aminohexanoic acid at different concentrations caused significant variations of the sedimentation and diffusion constants of the heavy chain indicating inhibitor‐induced conformational alterations of the protein. The present results suggest that in plasmin only the heavy chain is capable of interacting with 6‐aminohexanoic acid, and it appears that it is primarily this chain which plays an important role in the inhibition of the enzyme by 6‐aminohexanoic acid.
Further Studies on Lipid‐Peroxide Formation in Isolated Hepatocytes
Authors: HÖGBERG Johan , ORRENIUS Sten , O'BRIEN Peter J.
Published: March 2005

Lipid peroxide formation was initiated by the addition of either ADP‐complexed Fe3+or cumene hydroperoxide to a suspension of isolated hepatocytes. The reaction was monitored by malonaldehyde measurements. Upon the addition of iron, malonaldehyde production in the cells started immediately but ceased within 30–60 min, and the response was dose‐related with iron concentrations ranging from 19 to 187 μM. Malonaldehyde formation was associated with increased oxygen uptake and conjugated diene production. The additionin vitroofN,N,N′;N′‐tetramethyl‐p‐phenylenediamine, menadione orp‐benzoquinone inhibited the iron‐induced malonaldehyde production. It was also possible to demonstrate an apparent disappearance of malonaldehyde from fresh cells by addition of adequate amounts of N,N,N′,N′‐tetramethyl‐p‐phenylenediamine (100 μM).

The attenuation of the iron‐induced malonaldehyde production was found to be correlated with an increased binding of iron to an intracellular ferritin fraction. Further, malonaldehyde formation was also associated with a conversion of reduced glutathione to the oxidized form which, in turn, revealed a faster permeation out of the cells into the surrounding medium of the oxidized than of the reduced thiol. So, concomitant with the redox alterations, there was also an overall loss of glutathione from the cells.

Cumene hydroperoxide‐induced malonaldehyde production could be initiated by the addition of this peroxide in concentrations ranging from 150 μM to the liver cell incubate. With concentrations below 150 μM, a lag phase was present which seemed to be glutathione‐dependent.

It is concluded that iron enters the cell, then is probably reduced inside the cell by NADPHviathe NADPH–cytochromeP‐450 reductase, and in the reduced state initiates lipid peroxidation. The reaction is inhibited by intracellular mechanisms, the glutathione redox system being of principal importance, and possibly terminated by the iron‐apoferritin complex formation.
The Consequences of Lipid Peroxidation in Isolated Hepatocytes
Authors: HÖGBERG Johan , MOLDÉUS Peter , ORRENIUS Sten , ARBORGH Bengt , O'BRIEN Peter J.
Published: March 2005

Lipid peroxidation was initiated by the addition of either ADP‐complexed Fe3+ or cumene hydroperoxide to isolated rat hepatocytes and the resultant biochemical and morphological alterations investigated. As previously observed with microsomes, malonaldehyde formation was associated with the inactivation of glucose‐6‐phosphatase. Inhibition of microsomal oxidative drug metabolism was correlated with the release and subsequent inactivation of NADPH‐cytochrome c reductase, whereas cytochrome P‐450 destruction occurred only in the presence of high concentrations of the organic hydroperoxide which were associated with extensive malonaldehyde formation. Under these conditions there were also marked ultrastructural alterations in the hepatocytes which were not apparent after incubation in the presence of iron (≤ 187 μM Fe3+). The latter treatment was, however, associated with moderate biochemical effects such as glucose‐6‐phosphatase inactivation and increased membrane permeability. The cellular defence system against lipid peroxidation is discussed and it is concluded that the isolated liver cell system provides a valuable tool for the study of lipid peroxidation and its pathological implications.
Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin
Authors: SCHIFF Claudine , FOUGEREAU Michel
Published: March 2005

The amino acid sequence of the light chain of the mouse monoclonal MOPC 173 immunoglobulin molecule (IgG2a,x) is presented. This kappa chain contains 214 residues. Comparisons of this sequence with murine kappa chains already published by other workers bring a confirmation of the large size of the murine Vx chain pool. A complete identity was found with the constant region of the light chain of MOPC 21 from residue 97 to residue 214.
d‐Glucose‐6‐Phosphate Dehydrogenase (Entner‐Doudoroff Enzyme) from Pseudomonas fluorescens
Authors: LESSMANN Dieter , SCHIMZ Karl‐Ludwig , KURZ Gerhart
Published: March 2005

The existence of two different d‐glucose‐6‐phosphate dehydrogenases in Pseudomonas fluorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner‐Doudoroff pathway and the other to the hexose monophosphate pathway.

A procedure is described for the isolation of that d‐glucose‐6‐phosphate dehydrogenase which forms part of the Entner‐Doudoroff pathway (Entner‐Doudoroff enzyme). A 950‐fold purification was achieved with an overall yield of 44%. The final preparation, having a specific activity of about 300 μmol NADH formed per min per mg protein, was shown to be homogeneous.

The molecular weight of the Entner‐Doudoroff enzyme has been determined to be 220000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265000.

The pI of the Entner‐Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner‐Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal activity at pH 8.9.

The Entner‐Doudoroff enzyme showed specificity for NAD+ as well as for NADP+ and exhibited homotropic effects for d‐glucose 6‐phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory.

The enzyme catalyzes the transfer of the axial hydrogen at carbon‐1 of β‐d‐glucopyranose 6‐phosphate to the si face of carbon‐4 of the nicotinamide ring and must be classified as B‐side stereo‐specific dehydrogenase.
RNA‐Dependent DNA Polymerase from a Cell Line Derived from the Bone Marrow of a Patient with Polycythemia vera
Authors: WEIMANN Bernd Jürgen , SCHMIDT Jörg , KLUGE Norbert , OSTERTAG Wolfram , WOLFRUM Dirk Ingo
Published: March 2005

A RNA‐dependent DNA polymerase was isolated from a human cell line derived from the bone marrow of a patient with polycythemia vera. The purification procedure included chromatography on phosphocellulose and oligo(dT)‐cellulose, and glycerol gradient centrifugation.

The enzyme could be distinguished from polymerase A by salt elution from phosphocellulose, utilization of poly(rC) · oligo(dG) and its molecular size of about 70 000, as determined by centrifugation. Throughout the purification procedure ribonuclease H activity was co‐purified. Upon dodecyl‐sulfate‐polyacrylamide electrophoresis on microgradient gels two main bands with molecular weights of 68 000 and 66 000 and three minor bands were detected.

The enzyme preferentially used poly(rA) · oligo(dT) as template‐primer compared with poly‐(dA) · oligo(dT). It incorporated dGMP into polymer on poly(rC) · oligo(dG).
Readenylation of Polyadenylate‐Free Globin Messenger RNA Restores Its Stability in vivo
Authors: HUEZ Georges , MARBAIX Gérard , HUBERT Evelyne , CLEUTER Yvette , LECLERCQ Madeleine , CHANTRENNE Hubert , DEVOS René , SOREQ Hermona , NUDEL Uri , LITTAUER Uriel Z.
Published: March 2005

Using an ATP:RNA adenyltransferase from Escherichia coli, a polyadenylic sequence was resynthesized onto rabbit globin mRNA from which the poly(A) segment had been previously removed. Conditions for obtaining a homogenous reconstituted globin mRNA preparation containing 30 adenylic residues per message molecule were determined. The reconstituted globin mRNA was microinjected into Xenopus laevis oocytes. Its stability was very similar to that of native mRNA.
Purification and Properties of Potato 1,4‐α‐d‐Glucan: 1,4‐α‐d‐Glucan 6‐α‐(1,4‐α‐Glucano)‐Transferase
Authors: BOROVSKY Dov , SMITH Eric E. , WHELAN William J.
Published: March 2005

Q‐Enzyme, the enzyme that synthesizes the 1,6‐α‐glucosidic branch linkages of amylopectin, has been purified from potato to near homogeneity.

The molecular weight of the enzyme is 85000. The active enzyme is a monomer, with a molar activity at pH 7.0 and 24 °C of 15. The energy of activation is 25 kJ/mol below 15 °C, changing sharply to 63 kJ/mol above that temperature. Enzyme activity is not affected by Mg2+ or ATP. There are about 11 readily titratable sulfhydryl groups per molecule.

The evidence that the enzyme is a single protein entity, without hydrolytic activity towards amylose, contrasts with an earlier report that Q‐enzyme consists of two components, a hydrolase with molecular weight 70000, and a transferase with molecular weight 20000.

Q‐Enzyme acts on native and synthetic amyloses to give products resembling amylopectin in terms of average unit chain length, degree of β‐amylolysis and iodine stain. The profiles of the unit chains of these synthetic products are, however, different from that of native amylopectin. Additional branch linkages are introduced by Q‐enzyme into potato amylopectin, but the product bears no resemblance to phytoglycogen.
Structure‐Activity Relationship in the Urokinase Hydrolysis of α‐N‐Acetyl‐l‐lysine Anilides
Authors: PETKOV Dimiter , CHRISTOVA Evdokia , POJARLIEFF Ivan , STAMBOLIEVA Nikolina
Published: March 2005

The absence of both nonproductive binding and substrate activation and also the good solubility of the substrates make the urokinase‐catalysed hydrolysis of specific anilides a very suitable reaction for substrate structure–enzyme activity studies.

Derivatives of α‐N‐acetyl‐l‐lysine anilide with high σ‐value substituents in the aniline ring were synthesized. Rate constants kcat. and apparent Michaelis‐Menten constants Km(app.) are presented. From the substituent dependence of kcat. and from the fact that kcat. is 13 to 37 times smaller than the deacylation rate constant it is concluded that the rate‐limiting step proceeds prior to deacylation.

The catalytic rate constant kcat. obeys a linear free‐energy relationship of the Hammett type with ϱ=+ 0.72. Two different mechanisms implied by the results obtained from the model reaction (specific base and general acid‐base catalysed hydrolysis of N‐acetylglycine anilides under extreme conditions) are proposed in order to account for this positive and low ϱ‐value. In the first mechanism the breakdown of an enzyme tetrahedral intermediate is rate‐limiting, while in the second one its formation controls the overall rate. The discrimination between the two mechanisms, however, could not be found.
Premature Induction of Glucokinase in the Neonatal Rat by Thyroid Hormone
Authors: PARTRIDGE Nicola C. , HOH Chee Hoong , WEAVER Patricia K. , OLIVER Ivan T.
Published: March 2005

It was shown that the development of liver glucokinase in the rat coincided with a peak in the levels of circulating thyroid hormone at about the 16th postnatal day.

Administration of thyroid inhibitors blocked the development of the enzyme and administration of thyroid hormone restored activity to normal levels.

Glucokinase could be induced prematurely as early as the 2nd postnatal day by the administration of thyroid hormone followed by daily injection of glucose (10 mg/g body weight).

Glucocorticoids and corticotropin failed to induce glucokinase activity prematurely.

The postnatal increase in circulating thyroid hormone levels together with increased intake of carbohydrate at weaning may be the normal physiological stimulus for induction of this enzyme.
Protein A from Staphylococcus aureus
Authors: SJÖHOLM Ingvar
Published: March 2005

Protein A, a cell‐wall protein from Staphylococcus aureus, has been studied by spectrophotometry and spectropolarimetry. All the four tyrosines are similarily titrated with pKa= 10.25. Circular dichroism (CD) spectra show that the conformation of protein A is very stable over a large pH interval (0.99–11.8). The conformation is partly intact even in 6 M guanidine hydrochloride and at 80°C. Protein A contains about 50%α‐helical structure and 10–20%β‐structures. CD band maxima at 261 and 268 nm are ascribed to transitions in the phenylalanine residues and ellipticities between 275–285 nm to the tyrosine residues. Of the four tyrosines, 3.5 are perturbed by 20% polyethylene glycol, while all of them are perturbed by 20% dimethylsulfoxide. The role of the tyrosines in the reaction with the Fc‐region of immunoglobulins is discussed.
Ion‐Pair Formation as a Source of Enhanced Reactivity of the Essential Thiol Group of d‐Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: POLGÁR László
Published: March 2005

The reactivity and the mode of activation of the essential – SH group (Cys‐149) of d‐glyceral‐dehyde‐3‐phosphate dehydrogenase have been studied by means of a spectrophotometric method [Polgár, L., FEBS Lett. 38, 187–190 (1974)], capable of detecting the dissociated form of the thiol group in proteins.

Alkylations of Cys‐149 of NAD‐free d‐glyceraldehyde‐3‐phosphate dehydrogenase with iodoacetamide and iodoacetate were investigated. The corrected absorbance change on alkylation at 250 nm (which is a direct parameter of the dissociation of the thiol group) and the alkylation rate were determined as a function of pH. The pH profiles of both dissociation and alkylation rate of Cys‐149 conform to doubly sigmoid curves. All these curves implicate two ionizing groups (pK1= 5.5, pK2= 8.2).

It is concluded that there are two reactive forms of the –SH group in the apoenzyme between pH 5 and 10. One reactive form corresponds to the free mercaptide ion. The other can be identified with an ion‐pair composed of a mercaptide ion and some base, possibly the imidazolium group of His‐176.

The ion‐pair has lower molar absorption coefficient and nucleophilicity than the free mercaptide ion.

The two reactive forms are transformed into each other with pK2= 8.2. The ion‐pair decomposes to a nondissociated thiol group and a protonated base with pK1= 5.5.

In the presence of NAD, only the pH‐rate profile of alkylation of d‐glyceraldehyde‐3‐phosphate dehydrogenase was measured (at 370 nm). Using iodoacetamide as alkylating agent we also obtained a doubly sigmoid curve. A slight downward shift on pK1 and an upward shift in pK2 indicate that the ion‐pair exists in a somewhat wider pH‐range in the enzyme‐coenzyme complex.

An increase in the ionic strength of the reaction mixture from 0.09 to 0.45 M does not abolish the doubly sigmoid character of the curves determined either in the presence or in the absence of NAD.
Two‐Dimensional Polyacrylamide‐Gel Electrophoresis for Purification of Small RNAs Specified by Virulent Coliphages T4, T5, T7 and BF23
Authors: IKEMURA Toshimichi , OZEKI Haruo
Published: March 2005

RNAs synthesized in Escherichia coli infected with virulent phages T4, T5, T7 and BF23 were labelled with 32PO43− after phage infection. [32P]RNAs of low molecular weight were separated by two‐dimensional polyacrylamide gel electrophoresis, in which electrophoresis was carried out in two dimensions at different concentrations of acrylamide. The fractionated RNAs were characterized by RNA‐fingerprint patterns made after T1 ribonuclease digestion. The two‐dimensional gel of 10%→ 20% acrylamide was suitable for RNA of less than 200 nucleotides, while that of 5%→ 10% was preferred for RNAs of about 150–400 nucleotides.

With T4 phage, 16 RNA species were separable on a single slab gel. Among those, 11 were identified as the known RNA species, including eight T4 tRNAs, one tRNA precursor and two non‐tRNA molecules. In the case of T5 and BF23, more than 20 RNA species were separated on a slab gel; 15 or more RNAs were found in the 4‐S RNA region, and several in 5‐S and 6‐S region. The RNA‐fingerprint patterns of many BF23 RNAs were very similar to those of corresponding RNAs of T5. Pseudouridine and ribosylthymidine, minor nucleosides generally present in tRNA, were found in several BF23 4‐S RNAs tested. Possibility of those BF23 4‐S RNAs as tRNAs is discussed. With phage T7, three RNAs were detected, two of which were much smaller than tRNAs.
Properties of a DNA‐Adenylate Complex Formed in the Reaction between Mammalian DNA Ligase I and DNA Containing Single‐Strand Breaks
Authors: SÖDERHÄLL Stefan
Published: March 2005

The major DNA ligase from calf thymus (mammalian DNA ligase I) forms a covalent enzyme‐AMP complex on incubation with ATP [Söderhäll & Lindahl, J. Biol. Chem. 248, 672–675, (1973)]. The reaction of this complex with DNA has now been studied. When the ligase‐adenylate complex is incubated at 0°C for short time periods with DNA containing single‐strand breaks, a DNA‐AMP complex can be isolated from the reaction mixture by isopycnic centrifugation in CsCl. Incubation at pH 6.5 increased the amount of DNA‐AMP complex that could be isolated 10–20‐fold relative to that obtained at pH 7.4. Under the same conditions, incubation of the ligase‐AMP complex with DNA free from single‐strand breaks did not lead to detectable DNA‐AMP formation. The DNA‐AMP complex was resistant to treatment with dilute acid and alkali indicating the presence of a covalent linkage. Further, this complex was sensitive to DNase but resistant to pronase and RNase. Free AMP was released on further incubation of the isolated DNA‐AMP complex with thymus DNA ligase I and Mg2+, suggesting that the complex is a reaction intermediate. Degradation of the DNA‐AMP complex with several reagent enzymes indicated that the AMP residues were bound at the 5′ ends of the single‐strand breaks in DNA by pyrophosphate bonds.