bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on 1‐β‐D‐Arabinofuranosyl‐cytosine‐Resistant Mutants of Chinese‐Hamster Fibroblasts
Authors: Robert de Saint‐Vincent Bruno , Buttin Gérard
Published: March 2005

The biochemical basis of resistance to 1‐β‐D‐arabinofuranosyl‐cytosine (Ara‐Cyt), an analog of cytidine, was explored in two lines of Chinese‐hamster fibroblasts, one of which exhibits a temperature‐dependent resistance to the drug. In both cases, an absolute defect in the capacity to phosphorylate Ara‐Cyt was demonstrated under growth conditions which confer complete resistance.

Both lines are unable to utilize appreciably exogenous deoxycytidine (dCyd) for nDNA synthesis, thus suggesting that in hamster fibroblasts, as in other previously studied mammalian cells Ara‐Cyt and dCyd are phosphorylated by the same pathway.

However, the level of dCyd kinase activity in crude extracts of the Ara‐Cyt‐kinase‐deficient cells is not greatly reduced. This is explained by the existence in wild‐type cells of two dCyt kinases which can be separated by DEAE‐cellulose chromatography: dCyd kinase 1, which is devoid of activity on Ara‐Cyt, is present in both wild‐type and mutant cells, while dCyd kinase 2, which phosphorylates Ara‐Cyt, is detectable in wild‐type Ara‐Cyt‐sensitive cells only.

The inefficiency of dCyd kinase 1 alone to ensure conversion of exogenous dCyd for its incorporation into nDNA is best accounted for by the mitochondrial location of this enzyme: this is established both by subcellular fractionation experiments and by the demonstration that incorporation of exogenous dCyd into mtDNA is unchanged in the Ara‐Cyt‐resistant mutants.
Substrate Studies for Insulin‐Specific Protease
Authors: Baskin Frankie K. , Kitabchi Abbas E.
Published: March 2005

Insulin‐specific protease, a soluble cellular enzyme from rat skeletal muscle which has been purified recently as a single enzyme, has been studied in regard to its substrate specificity, using various immunoreactive and biologically active insulin and proinsulin intermediates. The rate of degradation of pork insulin taken as 100% was compared to other insulin and proinsulin derivatives. Porcine proinsulin intermediates consisting of cleaved proinsulin, desdipeptide, desnonapeptide and destridecapeptide‐proinsulin, as well as desalanine, monoarginine and diarginine‐insulin, were degraded at 19.8, 25.6, 63.5, 73.7, 101.5, 98 and 98% of the activity of insulin, respectively. Rates of degradation of beef proinsulin, and intermediates I and II were 6, 20.8 and 5.9% of that of insulin with insulin‐specific protease, respectively. Studies of Km and V determinations of pork insulin and proinsulin and their intermediates revealed that all the substrates had similar V values (1.0 pmol/min); whereas the Km values (nM) were as follows: insulin, 22.2; desalanine‐insulin, 15.8; monoarginine‐insulin, 24.4; diarginine‐insulin, 24.4; proinsulin, 857.2; cleaved pro‐insulin, 234.2; desdipeptide‐proinsulin, 176.0; desnonapeptide‐proinsulin, 55; and destridecapep‐tideproinsulin, 44. Reduced proinsulin, labeled with iodo[14C]acetamide, did not show increased degradability by insulin‐specific protease as compared to native proinsulin. These studies suggest that one requirement for optimal substrate activity may be the deblocking of the amino end of the A chain of insulin. The blocking of the amino end, which is present in proinsulin or proinsulin intermediate, will reduce degradability of these substrates by insulin‐specific protease. The fact that the cleavage of disulfide bonds of proinsulin resulted in no further activation of proinsulin supports the above and indicates that steric hindrance may have a lesser role in the proinsulin molecule's inability to be degraded by insulin‐specific protease.
Membrane Adenosine Triphosphatase of Micrococcus lysodeikticus
Authors: Andreu Jose Manuel , Muñoz Emilio , Albendea Jose Antonio
Published: March 2005

The adenosine triphosphatase of Micrococcus lysodeikticus has been purified by Sephadex G‐200 filtration in 1 mM EDTA‐50 mM NH4HCO3 buffer pH 9.0, after its selective release (“shock wash”) from the membranes. The use of the EDTA‐ammonium bicarbonate buffer, instead of the Tris‐CI buffers previously used (Muñoz et al., 1969), shortened the process of purification and improved its yield. The purified ATPase did not show trypsin‐stimulated activity, unlike the crude soluble enzyme which presented an approximately 50% ATPase stimulation by trypsin. The purified protein gave a single band in 7% or 10% polyacrylamide‐gel electrophoresis. Sedimentation‐velocity experiments provided new criteria of protein purity, although they indicated a possible dissociation of the protein at low concentrations. We calculated a so20, w value of 13.6 S by linear extrapolation. Sedimentation‐equilibrium experiments confirmed both the homogeneity and partial dissociation of the native enzyme. Hence, we determined molecular weights of 330000–350000. The amino acid composition of M. lysodeikticus ATPase resembles those of other ATPases, but contains lower percentages of proline and lysine. It reveals a medium hydrophobicity, which agrees with its properties as membrane‐associated enzyme. A partial specific volume of 0.74 ml/mg was calculated from the amino acid composition. Sodium dodecylsulphate—7% polyacrylamide electrophoresis revealed five components (relative mobilities were: 0.71, 0.75, 0.79, 0.92 and 1.0). The componente(s) of faster mobility appeared to be contaminant(s). The other four components were considered as the ATPase subunits (α, β, γ and δ). Similar subunit patterns were obtained by dissociation with phenol‐acetic acid and urea‐acetic acid. The α and β‐subunits were present in greater amounts than the γ and δ‐subunits. The γ‐subunits showed variations depending on the enzyme preparation. The other three subunits (“intrinsic” subunits) were found in all preparations with approximately constant molar ratios. We therefore conclude that the 33000—350000 molecular weight must correspond to a complex of the “intrinsic” subunits. The molecular weight of each subunit calculated from its relative mobility in dodecylsulphate‐polyacrylamide electrophoresis were: α= 52500 ± 5000, β= 47000 ± 4500, γ= 41500 ± 4000 and δ= 28500 ± 2000. We propose a formula α3β3δ(+γ) for the native ATPase non‐stimulated by trypsin.

M. lysodeikticus APTase shows marked structural similarities with beef‐heart mitochondrial ATPase. The presence of minor subunits (γ, δ) does not seem to account for the trypsin stimulation of the enzyme.
Induction Capacity of Enzyme Synthesis during the Cell Cycle of Saccharomyces cerevisiae
Authors: Sebastian Jesus , Carterr Bruce L. A. , Halvorson Harlyn O.
Published: March 2005

The activities of the enzymes ornithine transaminase in Saccharomyces cerevisiae and β‐galactosidase in Saccharomyces lactis increase periodically once per cell cycle. This step synthesis of the enzymes is observed under either basal or induced conditions; however, when the cells are growing under non‐induced conditions, addition of the inducer at any stage of the cell cycle results in an enhancement of the enzyme activity for both enzymes. The amount of enzyme activity induced is constant for much of the cell cycle but doubles over a portion of the cycle, probably as a result of replication of the structural gene of these enzymes during the S phase. These results suggest that whereas there is periodic expression of structural genes during the cell cycle of yeasts grown in a constant environment, these genes can, upon induction, be expressed at any time during the cell cycle.
Brown‐Adipose‐Tissue Mitochondria
Authors: Nicholls David G. , Lindberg Olov
Published: March 2005

Endogenously uncoupled mitochondria from the brown adipose tissue of cold‐adapted hamsters, rats and guinea‐pigs swell in KC1, KBr, KNO3 and KCNS in the presence of valinomycin. Identical rates of swelling are obtained employing the ammonium salts or the potassium salts in the presence of nigericin.

Albumin has no effect on the swelling rates in these potassium salts plus valinomycin, but is strongly inhibitory with both potassium salts plus nigericin and with ammonium salts. Albumin inhibits swelling in potassium phosphate plus valinomycin but is without effect in potassium phosphate plus nigericin or in ammonium phosphate. All these inhibitions may be reversed by uncoupler.

These results are interpreted in terms of electrogenic entry of Cl‐, Br‐, NO3‐ and CNS‐, and of a high native proton permeability which is abolished by albumin.

GDP inhibits swelling in KCl and KBr in the presence of either valinomycin or nigericin. The inhibition is not reversed by uncoupler. Exchange of 36Cl‐ both into and out of the sucroseimpermeable space is strongly inhibited by GDP. The effectiveness of different nucleotides in inhibiting 36Cl‐ efflux is identical to their effectiveness previously observed for uncoupler‐releasable respiratory inhibition in the presence of albumin.

GDP only partially inhibits NO3‐ permeation and is without demonstrable effect on thiocyanate, phosphate or acetate entry, but only if no proton movements are involved. Otherwise, in these systems, some limitation of proton permeation is observed which may be reversed by carbonyl cyanide p‐trifluoromethoxyphenylhydrazone.

The entry of sodium appears to be by electroneutral exchange and not to be affected by albumin or nucleotide.

The use of the ion permeability properties as a tool to investigate the relationship between membrane structure and energy conservation is discussed.
2‐Amino‐2,6‐Dideoxy‐D‐Glucose (D‐Quinovosamine): a Constituent of the Lipopolysaccharides of Vibrio cholerae
Authors: Jann Barbara , Jann Klaus , Beyaert Guido O.
Published: March 2005

From the lipopolysaccharides of Vibrio cholerae strains Inaba and Ogawa, an unusual amino sugar was isolated.

By sequential oxidation with sodium metaperiodate and hypoiodide and by oxidation with periodate followed by detection of the acetaldehyde formed, the amino sugar was characterized as a 2‐amino‐dideoxyaldose.

The hydrochloride of the amino sugar was compared with those of 2‐amino‐2,6‐dideoxy‐d‐glucose, 2‐amino‐2,6‐dideoxy‐l‐galactose, 2‐amino‐2,6‐dideoxy‐d‐mannose, 2‐amino‐2,6‐dideoxy‐d‐gulose, and 2‐amino‐2,6‐dideoxy‐d‐allose in an amino acid analyzer. The amino sugar alcohols were studied by paper electrophoresis in molybdate buffer.

From the results obtained the amino sugar was identified as 2‐amino‐2,6‐dideoxyglucose (quinovosamine). Its optical rotation indicated it to be of the d‐configuration.
Purification and Properties of an Iodide Peroxidase from Submaxillary Gland of Goat
Authors: Mahajani Usha , Haldar Indira , Datta Asoke G.
Published: March 2005

An iodinating enzyme has been identified and partially purified from the homogenate of goat submaxillary gland. The iodinating enzyme was purified 130‐fold and found to be closely associated with iodide peroxidase activity in every step of purification. The finally purified preparation, although not homogeneous, exhibited both iodinase and peroxidase activities. The iodinase and the iodide peroxidase activities have comparable pH curves, hydrogen peroxide requirement and similar reactions towards some common reagents. Partial inactivation by pronase digestion also affects the two activities in a similar manner and fails to dissociate them. These observations led us to suggest that a single enzyme is responsible for the oxidation of I− and incorporation of active iodine to the tyrosine moiety. The enzyme catalyses a production of 31.0 μmol triiodide × min−1× mg−1 and can also catalyse effectively the oxidation of o‐dianisidine which is stimulated 2000% in presence of KI. The enzyme also catalyses the iodination of thyroglobulin and albumin, other than free tyrosine, with the formation of iodotyrosines.
Membranes of Glyoxysomes from Castor‐Bean Endosperm
Authors: Bieglmayer Christian , Ruis Helmut , Graf Jürg
Published: March 2005

A procedure was developed for the isolation of membranes of glyoxysomes from the endosperm of germinating castor beans (Ricinus communis L. var. zanzibariensis). These membranes were prepared by osmotic‐shock treatment of isolated glyoxysomes. In some cases this was followed by mild sonication. They were isolated by centrifugation on to a 57% (w/w) sucrose layer and purified by isopycnic sucrose gradient centrifugation.

The membrane preparations were characterized by electronmicroscopy and assayed for enzyme activities. They consisted mainly of ghost‐type vesicles and were found to contain a major part of fatty acid β‐oxidation, malate synthase, citrate synthase and antimycin‐A‐insensitive NADH‐cytochrome c reductase activities of the whole glyoxysomes. Malate dehydrogenase may also be bound to the membrane to a significant extent. A tentative scheme is postulated describing the structural organization of fatty acid β‐oxidation and of the glyoxylate cycle in glyoxysomes.
A Nuclear‐Magnetic‐Resonance Study of the Manganese · Thiamine‐Pyrophosphate Complex in Solution
Authors: Grande Hans J. , Veeger Cees , Houghton Ray L.
Published: March 2005

Manganese causes a broadening of the resonance lines in the nuclear magnetic resonance (NMR) of thiamine pyrophosphate (TPP). This broadening was used to calculate values for the transverse relaxation times T2. The longitudinal relaxation time T1 was determined using progressive power saturation and also by Fourier‐Transform NMR. Since the paramagnetic contribution to T2−1 is always larger than to T1−1, T1 was free of scalar quantities and therefore it was used to evaluate internuclear distances according to the Solomon‐Bloembergen relationships. From these calculated distances a folded structure was derived. The effect of temperature in the proton absorptions in NMR indicate a τM of 10−6 s similar to that of ADP and ATP. Combinations of 1H and 31P NMR showed that coupling occurred between the 5′‐β‐CH2 and the adjacent phosphorus and that at higher temperatures the pyrophosphate moiety was rotating more freely as indicated by a narrowing of the phosphate resonances. EPR measurements showed that a 1:1 complex of TPP and Mn2+ was formed. The association constant and free energy (ΔG298) of the complex are given.
Studies on the Induction of Nitrate Reductase by Nitrite in Bean‐Seed Cotyledons
Authors: Lips S. Herman , Kaplan Drora , Roth‐Bejerano Nurit
Published: March 2005

Nitrate reductase of sterilized bean seed cotyledons is more readily induced by nitrite (10 mM) then by nitrate (100 mM) when incubated in these solutions for 24 h. Inhibition of induction by tungstate, dependence on NADH and presence in extracts of endogenous nitrate at effective substrate concentrations were observed with both inducers. Nitrite was oxidized to nitrate after absorption but the concentration produced thereby was less than 5% of that produced by direct absorption of nitrate from the incubation medium. Nitrite may be a direct inducer of nitrate reductase in bean seed cotyledons.
The Reaction of Active‐Site Inhibitors with Elastase Using a New Assay Substrate
Authors: Thomson Alan , Denniss Iain S.
Published: March 2005

1. Some active‐site directed chloroketone derivatives of elastase have been synthesised and characterised. Their inhibition constants and rate constants for inhibition of elastase have been determined.

2. A new spectrophotometric substrate of elastase has been developed.

3. Inhibition of elastase increases in efficiency with length of polypeptide chain except that in N‐acetylated derivatives the dipeptide is significantly less reactive when bound to the enzyme than is a simple alanine derivative.
Immunochemical Studies on Rat‐Liver Microsomal NAD Glycohydrolase
Authors: Bock Karl Walter , Matern Siegfried
Published: March 2005

Rabbit antiserum was prepared against rat liver microsomal NAD glycohydrolase solubilized by lipase treatment and subsequently purified by successive DEAE‐cellulose, Sephadex G‐100 chromatography and polyacrylamide gel electrophoresis. The antibody inhibited the purified enzyme 60–80%, but the membrane‐bound and Triton X‐100 solubilized enzyme up to 20–30% only. A single precipitation line was produced when the antibody reacted with the purified enzyme, and with crude lipase and Triton X‐100 solubilized enzyme preparations. NAD glycohydrolase from liver rough‐microsomal and plasma‐membrane fractions reacted identically.

The antibody to rat liver NAD glycohydrolase similarly inhibited the lipase or Triton X‐100 solubilized and membrane‐bound NAD glycohydrolase from rat spleen and brain as well as from mouse liver but not from bovine liver.
Purification and Some Properties of 2‐Decylhomocitrate Synthase from Penicillium spiculisporum
Authors: Måhlén Anders
Published: March 2005

2‐Decylhomocitrate synthase, which catalyses the condensation between lauroyl‐CoA and 2‐oxoglutarate has been purified from Penicillium spiculisporum to a specific activity of 1.2μmol×min−1×mg−1. It also accepts decanoyl‐CoA as substrate. The peptide chain weight is about 43000, and s020,W for the native enzyme is 8.3 S. The apparent pH optimum is 8.3 and apparent Km values are for lauroyl‐CoA ≤ 1 μM at 3.3 mM oxoglutarate and for oxoglutarate, 0.75 mM at 28 μM lauroyl‐CoA. Oxaloacetate is an inhibitor competitive with oxoglutarate with Ki of 0.4–0.8mM. Palmitoyl‐CoA is partially competitive with lauroyl‐CoA. 5,5′‐Dithiobis(2‐nitrobenzoic acid) inactivates the enzyme but this is partially prevented by lauroyl‐CoA or palmitoyl‐CoA, and strongly prevented by both substrates or combinations of substrate and inhibitor. Great similarities between this enzyme and 2‐decylcitrate synthase from another strain of the mold are found in kinetic behaviour, molecular size and SH sensitivity, but a preliminary peptide mapping experiment reveals considerable differences in their primary structure.
Evidence that Fusidic Acid Inhibits the Binding of Aminoacyl‐tRNA to the Donor as well as the Acceptor Site of the Ribosomes
Authors: Otaka Tadahiko , Kaji Akira
Published: March 2005

A relatively high concentration of fusidic acid (3 mM) inhibits the binding of aminoacyl‐tRNA to the donor as well as the acceptor site of ribosomes in the absence of elongation factor G (EF‐G). In the presence of EF‐G a low concentration of fusidic acid (1 mM) inhibited the nonenzymatic binding of aminoacyl‐tRNA to the donor and the acceptor site approximately 30% and 80%, respectively. On the other hand, binding of aminoacyl‐tRNA to the acceptor site which is dependent on elongation factor T, was specifically inhibited by, EF‐G, GTP and fusidic acid. From these observations, it is suggested that the binding site of fusidic acid, GDP and EF‐G may not be strictly located at the acceptor site.
Paradoxical Effects of Theophylline and Its Interaction with Insulin on Glucose Metabolism in Adipose Tissue
Authors: Trueheart Pamela A. , Herrera M. Guillermo , Jungas Robert L.
Published: March 2005

The effects of theophylline on the metabolism of glucose by rat epididymal adipose tissue from fed and fasted rats were studied. The addition of 0.5 mg theophylline per ml to the incubation medium caused a large decrease in the conversion of medium glucose to fatty acids, glyceride glycerol, and CO2 and a large increase in lipolysis in tissue from both fed and fasted rats. This increase in lipolysis was not blocked by large concentrations of insulin even though insulin was able to overcome the inhibition of glucose metabolism. When tissue from fed rats was incubated 1 h with theophylline, rinsed, and then incubated 2 h without theophylline, the glucose uptake during the latter period was increased. Glucose oxidation to CO2, conversion to fatty acids and to glyceride glycerol were all enhanced. This paradoxical “preincubation effect” did not appear when tissue from fasted rats was studied. The possible role of cyclic AMP in regulating the utilization of glucose is discussed.
Further Studies on the Mechanism of Action of UDP‐Apiose/UDP‐Xylose Synthase from Cell Cultures of Parsley
Authors: Baron Diethard , Grisebach Hans
Published: March 2005

1. When uridine diphopho‐d‐[U‐14C]xylose was incubated with the UDP‐apiose/UDP‐xylose synthase from cell suspension cultures of parsley no radioactivity was detected in UDP‐apiose (UDP‐Api).

2. UDP‐d‐glucuronic acid (UDP‐GlcUA) labeled with tritum at either C‐4 or C‐5 was converted to UDP‐Api/UDP‐Xyl synthase. Free apiose was reduced to apiitol which was oxidized with periodic acid to glycolic acid. In both cases oxidation of glycolic acid with glycollate oxidase from spinach to glyoxalic acid released all the tritium into the water. This result proves that during the hydride shift from UDP‐[4‐3H]GlcUA tritium is transfered only to the pro‐R position at C‐3′ of apiose and that inversion of configuration occurs at C‐4 of apiose in the synthase reaction.

3. When the UDP‐Api/UDP‐Xyl synthase reaction was carried out in a 3H1HO/2H2O mixture, a total of 1.5 mol 3H per mol apiose was incorporated with correction made for the product isotope effect. By degradation of apiose it was proved that 1 mol 3H was located at C‐4 and 0.5 3H per mol sugar at C‐3′ of apiose.

4. After addition of NaB3H4 to the enzyme incubation with UDP‐d‐[U‐14C]GlcUA and hydrolysis of the nucleiotide sugars, arabinose, xylose and glucuronic acid with a constant 3H/14C ratio were isolated after extensive purification. it was shown by degradation of xylose that over 90% of the tritium was located at C‐4 of this sugar. These results prove the existence of a 4‐keto intermediate and the formation of UDP‐4‐keto‐glucuronic acid as an intermediate in the decarboxylation step.

5. The results presented can best be explained by an aldolase type mechanism for the UDP‐Api/UDP‐Xyl synthase reaction.
Bacteriophage T7‐DNA Replication in vitro: an Experimental System
Authors: Ferdinand Franz Josef , Krause Elke , Knippers Rolf , Strätling Wolf
Published: March 2005

We describe an experimental system which consists of intact double‐stranded T7 DNA and an unfractionated protein extract from bacteriophage‐infected cells. Under proper conditions, DNA replication is initiate. Evidence will be presented that, at least the viral DNA polymerase and the function of the viral gene‐4‐protein are required for initiation. Circumstantial evidence suggests that the viral RNA polymerasee is also necessary for initiation.

After successful initiation a part of the viral genome is semiconservatively replicated.
Studies on the Metabolic Effect Induced in the Rat by a High‐Fat Diet
Authors: Zaragoza‐Hermans Nelly M.
Published: March 2005

Adipose tissue of rats fed from weaning a fat diet or a control carbohydrate diet, was incubated with [U‐14C]glucose or [6‐14C]glucose (20 mM) or with unlabelled glucose (20 mM) and trace amount of [U‐14C]pyruvate, in the presence of insulin (1000 μU/ml). Fluxes of carbon through various metabolic pathways were calculated.

In adipose tissue of fat‐fed rats, the rate of glucose carbon flow at the level of pyruvate decarboxylation and in fatty acid synthesis from acetyl‐CoA, was reduced to 5% or less of the control. The rate of glycolysis was only decreased to 20% while the greatest part of intermediary pyruvate was diverted into lactate.

Recycling of pyruvate through a partial reversal of glycolysis was also demonstrated and was concomitant with an exchange of 14C with CO2, accounting for up to 35% of the total 14CO2 production from [U‐14C]glucose.

A balance of production and utilization of reduced nucleotide coenzymes in the cytoplasm during glucose metabolism was established. In contrast with the control tissue, a net excess was obtained in adipose tissue of fat‐fed rats. Alteration of the cytoplasmic redox state was also indicated by an abnormally high lactate/pyruvate ratio.

Several aspects of the glucose metabolism show the existence of a high energetic level or ATP:ADP ratio.

The significance of the findings is discussed with regard to the control of lipogenesis in adipose tissue.
Structural Characterization of a Soluble Fraction from Lens‐Capsule Basement Membrane
Authors: Olsen Bjorn R. , Alper Robert , Kefalides Nicholas A.
Published: March 2005

A soluble fraction was isolated from anterior lens capsule basement membrane by treatment with a dilute, acidic solution of sodium citrate. This fraction had an amino acid and carbohydrate composition similar to that of the intact basement membrane. Upon electron microscopy, this fraction was found to contain two morphologically distinct components: thin strands measuring 2–3 nm in diameter and globular units measuring 5–20 nm diameter. Treatment with pepsin destroyed the globular component leaving the filaments intact. The amino acid composition of the pepsin‐treated extract was similar to that of basement membrane collagen. Treatment with bacterial collagenase resulted in the destruction of the filamentous components leaving the globular components intact. These data indicate that the filamentous structure was collagenous whereas the globular component was not. Reduction of disulfide bonds resulted in an apparent dissociation of the globular unit from the filamentous component. The data indicate that the protein extracted with sodium citrate represents a structural subunit of the lens capsule basement membrane.
The Threonine‐Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K 12
Authors: Véron Michel , Saari John C. , Villar‐Palasi Carlos , Cohen Georges N.
Published: March 2005

The interactions between the catalytic regions of the subunit of the bifunctional enzyme aspartokinase I‐homoserine dehydrogenase I, although not necessary for the catalytic functions, are shown to be of utmost importance for the regulation of the activities of the enzyme.

The study of the effect of a number of –SH reagents on the protein shows that the inhibition of the homoserine dehydrogenase activity by the allosteric effector l‐threonine is closely correlated with the existence of an active aspartokinase region.

The kinetic study of the proteolytic reaction leading to the previously described homoserine dehydrogenase fragment indicates that a dissociation is a necessary requirement for the digestion of the aspartokinase region.

Cross‐linking with dimethylsuberimidate and proteolytic degradation of the fully cross‐linked tetramer have been used to show that the oligomeric interactions, necessary to build the tetrameric structure, involve the kinase region only.

A tentative model is proposed showing aspartokinase I‐homoserine dehydrogenase I as an isologous tetramer in which the aspartokinase regions, organized as a “core”, are surrounded by the homoserine dehydrogenase regions.
On the Binding of Rifampicin to the DNA‐Directed RNA Polymerase from Escherichia coli
Authors: Lill Ute I. , Hartmann Guido R.
Published: March 2005

The capacity of RNA polymerase to bind the antibiotic rifampicin is rather sensitive to conditions which lead to conformational changes in the enzyme. Repeated freezing and thawing in 1 M LiCl as well as treatment with guanidinium chloride destroys the binding affinity of the enzyme for the inhibitor. Similarly rifampicin does not associate with any of the isolated subunits α, β or β′. Surprisingly, the reaction of an excess of mercurials with the enzyme leads only to a partial loss of binding capacity suggesting that –SH groups are not directly involved in the formation of the complex with rifampicin. During chain elongation very little rifampicin is bound to the enzyme. Simultaneously enzymatic activity is not affected by the inhibitor. RNA cannot protect the enzyme against the attachment of rifampicin although the binding capacity is reduced. Treatment of the complex of enzyme and antibiotic with RNA does not release rifampicin from the complex. These results support the hypothesis that the tight binding of rifampicin to RNA polymerase is the principal cause of the inhibitory action of the antibiotic.
On the Mechanism of Bongkrekate Effect on the Mitochondrial Adenine‐Nucleotide Carrier as Studied through the Binding of ADP
Authors: Klingenberg Martin , Buchholz Marlies
Published: March 2005

Further unique effects of bongkrekate on the binding of ADP to the inner mitochondrial membrane are described in addition to a preceding report by us and explained so as to demonstrate the reorientation of a mobile carrier across the membrane.

1. The dependence of the bongkrekate‐induced increase of ADP binding is nonlinear with respect to the bongkrekate concentration. Conscquently the apparent Kd for ADP decreases nonlinearly with bongkrekate concentration. The nonlinearity is kinetic in nature and is not explained by regulatory (allosteric) effects. The nonlinearity is diminished at lower pH.

2. The binding of bongkrekate appears to be dependent on ADP indicating a reciprocal enhancement effect between ADP and bongkrekate binding. As a consequence the binding of high‐affinity nucleotide (such as ADP) is relatively more increased by bongkrekate the binding of nucleotides with less affinity such as dADP. This specificity preference for ADP is also increased by bongkrekate as compared to other analogues such as formycin diphosphate, and the imidodiphosphate and methylene diphosphate derivations of adenylate. The counteraction of bongkrekate and atractylate on ADP binding also depends on the cooperation between ADP and bongkrekate.

3. An apparent irreversibility of the bongkrekate‐induced ADP binding is demonstrated by several effects. In the course of time bongkrekate + ADP supersede the counteraction of atractylate, even at higher atractylate concentration. Also carboxy‐atractylate, with still higher affinity than atractylate, cannot abolish the bongkrekate. [14C]ADP once fixed with bongkrekate at low pH cannot be removed by exchange with excess ADP or atractylate even at pH 8, although at this pH bongkrekate is unable to induce a binding increase.

4. The results can be explained by the “reorientation mechanism” of the bongkrekate effect by which the ADP · carrier complex is translocated inside and all carriers become trapped inside by bongkrekate as the immobile carrier · bongkrekate complex. The reciprocal enhancement of bongkrekate and ADP binding is explained by the fact, that ADP is required to move the carrier inside where bongkrekate can bind to the carrier. The irreversibility of the bongkrekate effect results from the shift of the carrier inside and the resulting removal of the carrier out of the equilibrium with external ligands such as atractylate or ADP. The results thus produce the most direct evidence so far available on the molecular level, for the existence of a mobile metabolite carrier.
Isolation and Characterization of Ribosome‐Free Cytoplasmic Messenger‐Ribonucleoprotein Complexes from Avian Erythroblasts
Authors: Gander Eugen S. , Stewart Andrew G. , Morel Carlos M. , Scherrer Klaus
Published: March 2005

Two RNA · protein complexes were isolated from duck erythroblast postribosomal supernatants. Their nominal sedimentation values on sucrose gradients are 12 S and 20 S, respectively. The 12‐S particle contains a 4–6 S RNA, in the 20‐S particle a 9‐S RNA is found. This 9‐S RNA is shown to direct the synthesis of all duck globin chains in a cell‐free, messenger RNA‐dependent protein‐synthesizing system.

The protein moiety of these ribosome‐free particles is described and compared with the proteins found in the mRNA · protein complex liberated by EDTA from polyribosomes. We show that in the free‐cytoplasmic particles no protein can be found which is identical to any of the polypeptides associated with polyribosomal mRNA. Some of these protein are phosphorylated and contain phosphoserine. The electrophoretic patterns of phosphorylated proteins from the two globin mRNA‐containing complexes differ, as do those of the unlabelled polypeptides. We conclude that the mRNA‐associated protein population is exchanged when the mRNA enters the translation machinery.

The possible role of the RNA‐associated proteins in the post‐transcriptional and translational control of eukaryotic protein synthesis is discussed.
A New Purification Procedure for Yeast Phosphofructokinase Minimizing Proteolytic Degradation
Authors: Diezel Wolfgang , Böhme Hans‐Joachim , Nissler Karl , Freyer Renate , Heilmann Werner , Kopperschläger Gerhard , Hofmann Eberhard
Published: March 2005

A new and efficient method for preparation of pure yeast phosphofructokinase is described, leading rapidly to an enzyme with minimum proteolytic alterations. Essential is an affinity chromatography step on the reactive dye Cibacron blue F3G‐A® which has been covalently bound to Sephadex G‐100. The enzyme prepared by this method has a significantly higher sedimentation constant (20.5 S) than those forms of yeast phosphofructokinase purified by procedures previously described (17.8 S) and is composed of two different kinds of subunits having molecular weights of 130000 and 96000, respectively. By gel filtration various enzyme fractions have been obtained, containing either mainly subunits of the 130000 or of the 96000 type, respectively. Samples of phosphofructokinase which have been prepared by the original and by the new method were compared by means of analytical gel chromatography and dodecyl‐sulphate electrophoresis.

In the course of the preparation procedure described previously, the enzyme is evidently proteolytically converted to the 570000 molecule by degradation of the subunits having 130000 molecular weight to those of 96000.
Transfer of Valine into Rabbit Haemoglobin from Various Isoaccepting Species of Valyl‐tRNA Differing in Codon Recognition
Authors: Takemoto Toshiyuki , Takeishi Keiichi , Nishimura Susumu , Ukita Tyunosin
Published: March 2005

The transfer of valine into haemoglobin from various isoaccepting species of Val‐tRNA obtained from Escherichia coli and yeast, which differ in the specificity for codon recognition has been investigated using a rabbit reticulocyte cell‐free system. It was demonstrated that the Val‐tRNA species which bind to ribosomes in response to the codon GUG preferentially transferred valine into both α and β‐globin chains and that the other species of Val‐tRNA which cannot repond to GUG were scarcely utilized in the haemoglobin synthesis. Several possibilities to explain this phenomenon were examined and consequently it has been strongly suggested that valine codons in mRNA for rabbit globin, at least for α‐globin chain, are very rich in GUG and possibly GUA.
Enzymatic Iodination of Protein
Authors: Pommier Jacques , Sokoloff Louis , Nunez Jacques
Published: March 2005

1. The initial rate of I2 formation catalyzed by horse‐radish peroxidase exhibited a clear sigmoid relationship with respect to the concentration of I−. These data were found to fit an equation based on a model which predicts a second‐order dependence on iodide concentration and are consistent with a bimolecular reaction between two I−ions on the surface of the enzyme. Such a model presumes two sites for the substrate on the enzyme.

2. The initial rate of lysozyme iodination also exhibited a clear sigmoid relationship with respect to concentration of I−. The sigmoidicity increased with the lysozyme concentration. These experimental data fit a random‐ordered sequence of substrate fixation but with one of the two possible sequences kinetically preferred. These results also suggest that lysozyme interacts with the enzyme and that there are two sites for substrate addition on the surface of the enzyme.

3. When the ratio of the concentrations of both substrates, iodide and lysozyme, is varied, the rates of formation of each product, I2 or iodinated protein, also vary; the lower the iodide/protein ratio, the lower the I2 yield and the higher the rate of protein iodination. Studies on the influence of pH on the nature of the product showed that I2 formation is favored at acidic pH and protein iodination at more alkaline pH. These results are also consistent with a two‐site model for the enzyme, both sites being able to fix either two iodide ions or one iodide ion and one lysozyme molecule. The affinity of the sites for each one of the two substrates differs according to the pH. This conclusion was confirmed by the observation that iodination of free tyrosine could be obtained with very good yields provided that the pH of the reaction was adjusted to avoid either complete dimerization of free tyrosine, which is favored at alkaline pH, or I2 formation which is favored at low pH.

4. These results and other quantitative data on the stoichiometry of H2O2 consumption do not establish unequivocally which is the oxidized iodide‐reacting species, I+ or I°. However, they indicate that I2 is not the iodinating species in the protein iodination reaction catalyzed by horse‐radish peroxidase.
Ribonuclease Activity in Escherichia coli Polyribosomes
Authors: Mangiarotti Giorgio , Turco Emilia
Published: March 2005

Polyribosomes containing mRNA differentially labelled with [3H]uracil and [14C] uracil in the 5′‐ and the 3′‐proximal protions were isolated from Escherichia coli cells. mRNA degradation to alcohol‐soluble products occurred when polyribosomes were incubated in conditions of protein synthesis and proceeded in the 5′ to 3′‐direction. 15% of the mRNA was degraded at the end of a 10‐min period of incubation.

The mRNA‐degrading enzyme (or enzymic system) was found to be bound to polyribosmes; it could be removed by washing in low salt. The content in RNAase activity per ribosomes in polyribosomes increased three to five‐fold when polyribosomes were isolated from cells in which extensive run‐off of ribosomes from mRNA had been induced. On the contrary the RNAase activity in free ribosomes was not affected. This is taken as an indication that the enzyme(s) responsible for the degradation of endogenous mRNA in vitro is not aspecifically adsorbed to ribosomes, but specifically located towards the 5′‐end of the mRNA chains in polyribosomes.
The β‐Oxidative Cleavage of Long‐Chain Fatty Acids in Rat‐Liver Cytoplasm
Authors: Fiecchi Alberto , Galli‐Kienle Marzia , Scala Antonio , Galli Giovanni , Paoletti Rodolfo
Published: March 2005

A β‐oxidative cleavage of oleyl‐ and stearyl‐CoA has been obtained with soluble enzymes of rat‐liver homogenate. The transformation requires NAD+ as an oxido‐reductive cofactor and is enhanced by ATP and CoA. Under the reported conditions 2,9‐octadecadienoic, 3,9‐octadecadienoic, 3‐hydroxy‐9‐octadecenoic, 7‐hexadecenoic, 3‐hydroxy‐7‐hexadecenoic and 5‐tetradecenoic acids are present after incubation of oleyl‐CoA, while no detectable amounts of shorter fatty acids are formed. A similar oxidation pattern is obtained with stearyl‐CoA when Mg2+ ions are also added to the system. The cytoplasmic origin of these enzymes is established by comparing their activity with that of the mitochondrial β‐oxidation enzymes.
The Mechanism of the α‐Chymotrypsin and Trypsin‐Catalyzed Hydrolysis of Amides
Authors: Berezin Ilya V. , Kazanskaya Novella F. , Klyosov Anatole A. , švedas Vytautas K.
Published: March 2005

The applicability of the acylenzyme mechanism to the tryptic hydrolysis of the amide bond has been studied. It was found that trypsin acetylated at the hydroxyl group of the “active” serine or acetyl (Ser‐183)trypsin displays no activity with respect to both esters (N‐benzoyl‐l‐arginine ethyl ester and p‐nitrophenyl‐p′‐guanidinobenzoate) and anilides (N‐carbobenzoxy‐l‐arginine m‐nitroanilide and N‐benzoyl‐dl‐arginine p‐nitroanilide). It was also found, that deacetylation of acetyl(Ser‐183)trypsin is effectively promoted by N‐carbobenzoxy‐dl‐arginine m‐nitroanilide (Z‐Arg‐Nan) taken at a high concentration. As was found by means of a detailed kinetic analysis of experimental results, the binding of this anilide with acetyl(Ser‐183)trypsin increases the rate constant of deacylation of the latter by as many as 23 times. This effect which results in a rapid increase in the concentration of native trypsin in the reaction system, causes a marked increase in both kcat and Km(app) of tryptic hydrolysis of Z‐Arg‐Nan). Apparently, the phenomena is the main reason for the erroneous conclusions of Bresler et al. (1969, 1971), who stated: trypsin with an acylated active serine remains a highly efficient catalyst of amide hydrolysis. Finally, the data of Bresler et al. on tryptic hydrolysis and hydroxylaminolysis which contradict the acylenzyme mechanism of hydrolysis of the amide bond can be readily accounted for by chemistry of hydroxylamine, as was done by Bender et al. (1964) and Epand and Wilson (1963).

Thus, the data obtained so far strongly support the acyl‐enzyme mechanism of the hydrolysis of the amide bond catalyzed by trypsin and α‐chymotrypsin.
Reconstruction of Double‐Stranded Bacterial rDNA from the Partially Purified Complementary Strands
Authors: Udvardy Andor , Droppa Magdolna , Venetianer Pál
Published: March 2005

Earlier we described a method for the purification of the transcribing strand of Salmonella typhimurium rDNA. Here we report the purification of the complementary (minus) strand of Escherichia coli and Salmonella typhimurium rDNA, by means of partial reassociation of total bacterial DNA, chromatography on hydroxyapatite, on methylalbumin‐kieselguhr, and again on hydroxyapatite. A method is presented for the measurement of rDNA(minus). From the purified complementary strands of rDNA a stable duplex has been reconstructed with nearly 50% rDNA content.
Specific Binding of Phosphate by a Chloroform‐Soluble Protein from Rat‐Liver Mitochondria
Authors: Kadenbach Bernhard , Hadváry Paul
Published: March 2005

If an aqueous solution of [32P]phosphate is shaken with a chloroform‐extract of rat liver mitochondria, the label is bound specifically by a number of chloroform‐soluble proteins.

Several chloroform‐soluble proteins contain free SH‐groups, as indicated by the binding of [14C]ethacrynate, a specific reagent for SH‐groups.

One fraction of chloroform‐soluble proteins binds phosphate in high amounts, and this binding of phosphate is specifically inhibited by SH‐reagents.

Among various labeled anions (phosphate, malate, citrate, glutamate and ADP) only phosphate was shown to be bound at a significant amount by a chloroform‐extract of rat liver mitochondria.
Large‐Scale Preparation of Soluble Adenovirus Hexon, Penton and Fiber Antigens in Highly Purified Form
Authors: Boulanger Pierre A. , Puvion Francine
Published: March 2005

A three‐step mild procedure is described, that permits isolation of an adenovirus type‐2 soluble hexon, penton and fiber antigens in an extensively purified form. This procedure comprises ammonium sulfate precipitation, Sephadex ion‐exchange chromatography and adsorption chromatography on an hydroxyapatite column. The final antigen preparations were homogeneous with regard to immunological, electrophoretic and electron microscopic criteria.
A Highly Sensitive Method for Amino‐Acid Analysis by a Double‐Isotope‐Labelling Technique Using Dansyl Chloride
Authors: Brown Joseph P. , Perham Richard N.
Published: March 2005

A method for amino acid analysis by double isotope labelling has been developed using 3H‐labelled dansyl chloride and 14C‐labelled amino acids as internal standards.

As little as 20 pmol of an amino acid can be accurately measured by this method.

The amino acid compositions of several peptides, determined using about 50 pmol peptide, are reported and compared with the results obtained using conventional high‐sensitivity ion‐exchange chromatography. The correspondence is very good.

The amino acid composition of a tryptic peptide containing the active site disulphide bridge of the lipoamide dehydrogenase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli has been shown to be identical to that of the corresponding peptide from the 2‐oxoglutarate dehydrogenase complex of the same organism. This result is consistent with the view that the lipoamide dehydrogenase components of both complexes have a common amino acid sequence.
Changes of the Fluorescence of l‐Anilino‐8‐naphtalenesulfonate, Associated with the Membranes of the Sarcoplasmic Reticulum, Induced by General Anesthetics
Authors: Augustin Jan , Hasselbach Wilhelm
Published: March 2005

The fluorescence of 1‐anilino‐8‐naphthalene sulfonate (ANS), dissolved in buffer, is enhanced by the narcotics ether and halothane.

Ether and halothane considerably reduce the quantum yield of ANS, bound to native and lipid‐depleted sarcoplasmic vesicles. Obviously the effect of the anesthetics takes place at the same sites before and after delipidation.

The excitation spectrum of ANS, associated with the sarcoplasmic membranes, is resolved by halothane into at least two maxima with a new separate maximum at 394 nm. The emission spectrum is not influenced.

The narcotics are only able to influence the fluorescence of those ANS molecules which can be displaced by oleic acid, a noncompetitive inhibitor of the interaction of the dye with the membranes of native and lipid‐depleted vesicles.

Higher homologues of diethylether did not influence the fluorescence. Fluorescence quenching by ether and halothane is enhanced with rising temperatures. The polarisation of the fluorescence of membrane‐bound ANS is enhanced by halothane.

The intrinsic fluorescence of native and lipid‐depleted vesicles is considerably decreased by halothane, hardly influenced by ether. Concentrations of 2.2 M sucrose completely abolish the fluorescence quenching by the narcotics.

All effects produced by ether and halothane are reversible.
The Dynamics of Nucleic‐Acid Single‐Strand Conformation Changes
Authors: Pörschke Dietmar
Published: March 2005

The dynamics of the single‐strand conformation changes of oligo‐ and polyriboadenylic acids has been investigated by a fast temperature‐jump technique. Chemical rexalation processes have been observed in the range of 0.1 to 1 μs. The relaxation times are independent of the nucleotide concentration and also do not depend upon the pH within the range 6.9 to 9.2. The amplitudes correspond to those expected from thermal melting curves. According to these results the relaxation is assigned to the “single‐strand helix‐to‐coil” transition.

Measurements af different wavelengths show that the relaxation times observed in the spectral range 240 nm to 270 nm are different from those observed around 285 nm by a factor of 2 to 3. This result demonstrates that the “helix‐coil” transition of adenylic acid chains cannot be described by a simple all‐or‐none model with two reaction states.

As expected for a single‐strand helix‐coil transition there is a relatively small ionic strength dependence: a decrease of the salt concentration leads to an increase of the relaxation time. At high ionic strengths of monovalent ions (1 M and more) the appearance of relaxation times at 5 to 10 μs and in the ms range seem to indicate aggregation.

The presence of Mg2+ leads to a strong decrease of the amplitude around 285 nm, whereas the amplitude around 245 nm remains almost unchanged; the time constant of the 245‐nm relaxation increases with increasing Mg2+ concentration. Apparently Mg2+ ions induce a specific shift of the riboadenylic acid chain conformation.

Measurements of the temperature dependence in the range of 0 to 50°C revealed an increasing rate of the conformation change with increasing temperature. The relaxation process measured around 245 nm is characterised by a higher apparent activation enthalpy than that measured around 285 nm.

The relaxation behaviour found for the polymer is also shown by oligoriboadenylic acids. The chain length dependence of the relaxation times is relatively small. The difference of the relaxation behaviour observed around 245 nm and around 285 nm extends to the dimer. Thus the observation of two different relaxation times cannot be explained by a cooperative effect.

The relaxation time constants observed for oligo‐ and polyriboadenylic acids indicate the existence of energy barriers in the conformational transition. Similar time constants have been observed previously for the elementary process of base pairing in adenylic‐uridylic acid double helices.

The observation of two relaxation time constants for the conformation change of riboadenylic acid chains demonstrates the existence of more than two different conformation states.