bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions entre pigments et acides nucléiques
Authors: Courtois Y. , Guschlbauer W. , Fromageot P.
Published: March 2005

In this study, several properties of actinomycin D have been examined:

The effect of different solvents upon absorption, optical rotary dispersion and circular dichroism show that the optical activity does not coincide with the absorbance maximum around 440 mμ. Further an association between the polypeptide rings of actinomycin in aqueous solutions is quite possible.

The increase in optical rotatory dispersion when the actinomycin‐DNA complex is formed shows that the molecular asymmetry of the actinomycin molecule is enhanced. This is in contrast to the observations made upon acridine‐DNA complexes where the optical activity decreases with increasing DNA concentration. This clearly shows that the optical activity of the actinomycin‐DNA complex does not result from interactions between dye molecules specifically oriented by the DNA, but by interactions between DNA and the dye. The fixation of actinomycin on helical DNA imposes a highly specific orientation of both the phenoxazine and the two lactone rings, the result of which is the increase of the optical activity of the dye.

The optical rotatory dispersion of the weak actinomycin‐deoxyguanosine complex can clearly be distinguished from the much firmer actinomycin‐DNA complex. The optical rotatory dispersion curve of this complex is different both in amplitude and shape compared with that of the actinomycin‐DNA complex. This is due to the greater sterical freedom which imposes much less constraint to the conformation of the bound actinomycin molecule.

Acid denaturation of the actinomycin‐DNA complex below pH 3 shows the appearance of an association of the type actinomycin‐deoxyguanosine. While it is difficult to separate these changes by absorbance measurements it can unequivocally be distinguished by optical rotatory dispersion.

No complex formation could be detected between actinomycin and polydeoxyguanylic acid.
Formation of Malonaldehyde from Phospholipid Arachidonate during Microsomal Lipid Peroxidation
Authors: Niehaus W. G. , Samuelsson B.
Published: March 2005

The thiobarbituric acid reacting material produced during enzymatic microsomal lipid peroxidation has been identified as malonaldehyde. The malonaldehyde was condensed with urea to form 2‐hydroxy‐pyrimidine, which was identified by its ultraviolet spectrum, chromato‐graphic properties, and mass spectrum. Incubations with phosphatidyl choline labelled with tritiated arachidonate yielded 2‐hydroxy‐pyrimidine with a specific activity nearly equal to that of the phospholipid arachidonate. Incubations with tritiated arachidonic acid yielded 2‐hydroxy‐pyrimidine with a specific activity nearly 2 orders of magnitude less than that of free arachidonic acid. Therefore, phospholipid arachidonate has been established as the major source of the malonaldehyde produced during microsomal lipid peroxidation.
Malate Dehydrogenases of Ox Kidney
Authors: Dupourque D. , Kun E.
Published: March 2005

Isolation of malate dehydrogenase from ox kidney mitochondria consists of repeated salt and ethanol fractionation and DEAE + CM cellulose chromatography of an extract of mitochondrial acetone powder, resulting in 150‐fold purification and 60% recovery. Molecular and catalytic properties of the isoenzymes of both mitochondrial and cytoplasmic origin are compared in terms of modifiability by phosphate and 1 mM oxalacetate.
Structure of Porcine Cholecystokinin‐Pancreozymin
Authors: Mutt V. , Jorpes J. E.
Published: March 2005

Cholecystokinin‐pancreozymin has been found to have the partial structure Lys (Ala1, Gly1, Pro1, Ser1) · Arg · Val (Ile1, Met1, Ser1) Lys · Asn (Asx1, Glx1, His1, Leu2, Pro1, Ser2) Arg. Ile (Asp1, Ser1) Arg · Asp [Gly1, Met2, Trp1, Tyr (SO3H)] Asp · Phe · NH2.

Of the four peptide bonds in this polypeptide, three arginyl and one lysyl, that are cleaved by trypsin only the arginyl‐valine bond is cleaved by thrombin, with retention of the cholecystokinin and pancreozymin activities of the original molecule in the C‐terminal heptacosapeptide. The C‐terminal tryptic octapeptide too has strong cholecystokinin and pancreozymin activity. Except for the replacement of a threonine with methionine the eight amino acid units of this peptide are the same as those that comprise the corresponding part of the molecule of caerulein. Also like that in caerulein, the phenolic group of the only tyrosine of cholecystokinin‐pancreozymin is esterified with sulphuric acid.
Binding of Glycolytic Enzymes to Structure Proteins of the Muscle
Authors: Arnold H. , Pette D.
Published: March 2005

Only 60–70% of the total activity of aldolase can be extracted from rat or rabbit muscle homogenates with aqueous solutions of relatively low ionic strength. The extraction of aldolase from muscle tissue is only complete in aqueous solutions with an ionic strength greater than 0.2. The fraction of aldolase which is set free at high ionic strength is not located within a special cellular compartment but is present in a bound form and is desorbed in dependence of the ionic strength of the extraction medium.

F‐actin, myosin, acto‐myosin and stroma‐protein were prepared from rabbit muscle and the binding of aldolase to each of these structure proteins was studied in vitro. A completely reversible binding of aldolase to F‐actin, actomyosin, myosin and stroma protein was found. F‐actin possesses by far the highest binding capacity. 100% of the enzyme is bound when 1 mg of aldolase is added to 1 mg of highly purified F‐actin. Under identical experimental conditions acto‐myosin binds 40%, myosin 25%, and stroma protein only 15% of the aldolase activity.

A modified Langmuir isotherm is derived, and the analytical evaluation supports the assumption that the binding of aldolase to F‐actin occurs at two different binding sites.

The binding of aldolase to F‐actin and other structure proteins depends on the ionic strength. At 150 mM KCl, a complete desorption occurs, and 50% of the actin‐bound aldolase is set free at a concentration of 80 mM KCl. The sigmoid shape of the desorption curve indicates a cooperative mechanism of the binding phenomenon.

Within the physiological range, the pH does not influence the binding of aldolase to F‐actin.

Similar to aldolase, glyceraldehydephosphate dehydrogenase is bound to F‐actin, and under the experimental conditions, 1 mg of F‐actin binds up to 1.2 mg of glyceraldehydephosphate dehydrogenase.

Studies with a purified preparation of myogen reveal that also fructose‐6‐phosphate kinase, and in a lower degree phosphoglycerate kinase, pyruvate kinase and lactate dehydrogenase can be adsorbed to F‐actin. No binding occurs in the case of creatine kinase.

The possible significance of the binding phenomenon is discussed with respect to the location of the Embden‐Meyerhof system at the site of the actin filaments within the isotropic zones of the cross‐striated muscle fiber.
Carboxyl‐Terminal Region of Human and Bovine Erythrocyte Carbonic Anhydrases
Authors: Nyman P. O. , Strid L. , Westermark G.
Published: March 2005

Carboxyl‐terminal sequences have been determined for forms B and C of the human erythrocyte carbonic anhydrase and form B of the bovine enzyme. The proteins were degraded with CNBr, and peptide fragments of 20 residues were isolated from human enzyme C and bovine enzyme B while the human B form gave a fragment consisting of only 19 residues. The sequences of these fragments were determined and by carboxypeptidase digestion of the whole proteins it was shown that they represent the carboxyl‐terminal part of the proteins.

Comparison of the sequences of the enzymes gives strong support for evolutionary homology. Greater similarities in sequence are found between bovine carbonic anhydrase and human carbonic anhydrase C than between the two forms of the human enzyme.

All sequences contain an Arg‐Pro peptide bond. In two of the sequences this bond was found to be hydrolyzable with trypsin.
Reinigung und Eigenschaften der Carnitindehydrogenase aus Pseudomonas aeruginosa
Authors: Aurich H. , Kleber H.‐P. , Sorger H. , Tauchert H.
Published: March 2005

Carnitine dehydrogenase from Pseudomonas aeruginosa A7244 catalyzes the oxidation of carnitine to 3‐dehydrocarnitine and is specific for l‐carnitine and NAD+. The enzyme is formed by induction. l‐carnitine and 3‐dehydrocarnitine are inducers. Both substances can be utilized by the bacteria under aerobic conditions as sole source of carbon and nitrogen. After treatment of bacteria with lysozyme carnitine dehydrogenase is found in the supernatant of 90000 ×g and has been purified about 200‐fold by means of ammonium sulfate fractionation (between 40 and 55% saturation), gel filtration (Sephadex G‐200) and hydroxyl apatite chromatography. The purified enzyme did not react with d,l‐3‐hydroxybutyrate, l‐lactate, ethanol, l‐malate and d,l‐isocitrate (final concentration 25 mM). The optimal temperature is 35° for the forward reaction (Q10= 1.71; energy of activation 8.5 kcal/mole) as well as the reverse reaction (Q10= 1.99; energy of activation 8.2 kcal/mole). The oxidation reaction with carnitine as substrate exhibited optimum activity between pH 9.0 and 9.5. The reduction reaction with 3‐dehydrocarnitine and NADH as substrates showed optimum activity at pH 7.0. The apparent Michaelis constants, Km, for the substrates carnitine and NAD+ and for the products of the reaction 3‐dehydrocarnitine and NADH were determined. The pK/pH‐ and – log [V/Km]/pH‐profiles of the forward reaction allow the assumption of thiol groups in the active center of the enzyme. Analogues of l‐carnitine (d‐carnitine, glycine betaine and choline) are competitive inhibitors of l‐carnitine oxidation. The equilibrium constant of the reaction of carnitine dehydrogenase was determined and found to be 1.3 ×10−11 (standard error: ± 0.5 ± 10−11) at 30°. Possibilities of an optical test to determine l‐carnitine and 3‐dehydrocarnitine using carnitine dehydrogenase are discussed.
Steroids in Germfree and Conventional Rats
Authors: Eriksson H. , Gustafsson J.‐Å. , Sjövall J.
Published: March 2005

Gas chromatography‐mass spectrometry was used to identify 3α‐hydroxy‐5α,17α‐pregnan‐20‐one, its 17β epimer and, tentatively, 3α‐hydroxy‐5α‐pregn‐16‐en‐20‐one in faeces from conventional rats. These steroids were present as conjugates having the chromatographic properties of sulphates. They could not be found in faeces from germfree rats. Incubations of 3β‐hydroxy‐pregna‐5,16‐dien‐20‐one and 3β,16α‐dihydroxy‐5α‐pregnan‐20‐one with intestinal microorganisms yielded as major products 3β‐hydroxy‐17α‐pregn‐5‐en‐20‐one and 3β‐hydroxy‐5α,17α‐pregnan‐20‐one, respectively. Smaller amounts of different isomers of these compounds were also obtained. It is suggested that the major pathway for the formation of 17α‐pregnane derivatives in the rat is a bacterial conversion of 16α‐hydroxylated precusors via aΔ16 intermediate.
Steroids in Germfree and Conventional Rats
Authors: Gustafsson J.‐Å. , Sjövall J.
Published: March 2005

The C19 steroids in faeces from germfree rats have been studied by gas chromatography‐mass spectrometry. 3α,11β‐Dihydroxy‐5α‐androstan‐17‐one and 3α,7α‐dihydroxy‐5α‐androstan‐17‐one were the predominant steroids in this group and 5α‐androstane‐3α,7α,17β‐triol, 3α,19‐dihydroxy‐5α‐androstan‐17‐one were also identified. Evidence was obtained for the presence of 3α,15α‐dihydroxy‐5α‐androstan‐17‐one, 5α‐androstane‐3α,15α,17β‐triol and 5α‐androstane‐3α,17β‐diol. The latter compounds were present in small amounts and their stereochemistry could not be definitely established. The steroids were present in faeces as monosulphates as evidenced by their chromatographic behaviour on Sephadex LH‐20 and carboxymethyl Sephadex LH‐20. The possible significance of the predominance of 3α,5α steroid isomers in faeces from germfree rats is discussed.
The Energy‐Dependent Incorporation of Serine into Phospholipids of Isolated Rat Liver Mitochondria
Authors: Bygrave F. L. , Bücher Th.
Published: March 2005

A Mg++‐requiring, energy‐dependent incorporation of labelled serine by isolated rat liver mitochondria has been studied. Possible contributions to the incorporation by bacteria and microsomes are negligible. The serine is incorporated mostly into phosphatidylserine (85%) with small amounts into phosphatidylethanolamine (7%). The energy requirement is satisfied by ATP which can be generated via oxidative phosphorylation or provided exogenously. Hypothetical high energy intermediates of oxidative phosphorylation are not obligatory. Subfractionation of the mitochondria following the incorporation of serine shows that the radioactivity is associated mostly with the outer membrane. The isolated inner and outer membranes also incorporate serine but not as efficiently as intact mitochondria. The data is discussed in relation to the biosynthesis of the mitochondrial membranes.
Formation and Metabolism of Cholest‐5‐ene‐3β,12α‐diol
Authors: Einarsson K.
Published: March 2005

When labeled cholesterol was incubated with the 20,000 ×g supernatant fluid of rat liver homogenate, cholest‐5‐ene‐3β,12α‐diol could be isolated only in very small amounts indicating that cholest‐5‐ene‐3β,7α‐diol is the predominant intermediate in the conversion of cholesterol into cholest‐5‐ene‐3β,7α,12α‐triol. In the presence of 20,000 ×g supernatant fluid tritium‐labeled cholest‐5‐ene‐3β,12α‐diol was converted into 7α,12α‐dihydroxycholest‐4‐en‐3‐one mainly by means of the intermediary formation of cholest‐5‐ene‐3β,7α,12α‐triol. In the presence of microsomal fraction fortified with NADP, cholest‐5‐ene‐3β,12α‐diol was converted into 12α‐hydroxycholest‐4‐en‐3‐one. Tritium‐labeled 12α‐hydroxycholest‐4‐en‐3‐one was converted in the presence of 100,000 ×g supernatant fluid into 5β‐cholestane‐3α,12α‐diol by means of the intermediary formation of 12α‐hydroxy‐5β‐cholestan‐3‐one. When 12α‐hydroxycholest‐4‐en‐3‐one was incubated with the 20,000 ×g supernatant fluid, small amounts of 5β‐cholestane‐3α,7α,12α‐triol were formed. No 7α‐hydroxylated metabolites could be identified when tritium‐labeled 12α‐hydroxy‐5β‐cholestan‐3‐one and 5β‐cholestane‐3α,12α‐diol were incubated with the 20,000 ×g supernatant fluid.

After administration of tritium‐labeled 12α‐hydroxycholest‐4‐en‐3‐one to bile fistula rabbits 2–3% of the bile acids formed consisted of cholic acid. After administration of tritium‐labeled 12α‐hydroxy‐5β‐cholestan‐3‐one and 5β‐cholestane‐3α,12α‐diol no significant amounts of cholic acid could be identified.

It is concluded that the initial reaction in the conversion of cholesterol into cholic acid is predominantly a 7α‐hydroxylation.
Ribosome‐Catalyzed Peptidyl Transfer
Authors: Maden B. E. H. , Monro R. E.
Published: March 2005

Effects of cations and pH value on ribosome‐catalyzed peptidyl transfer were studied, using as assays the reaction of puromycin with (a) washed, polyphenylalanine‐charged Escherichia coli ribosomes and (b) terminal fragments from formlyl‐methionyl‐tRNA (CAACCA‐Met‐F and CCA‐Met‐F) in the presence of purified 50 S ribosomal subunits. In both systems the reaction required the presence of suitable divalent and monovalent cations, and was progressively inhibited below pH 8.5.

In the polyphenylalanine system Mg2+ was active over the range 0.3 mM to 1 M, with a broad optimum from approximately 3 to 100 mM. Mg2+ was replaceable by Ca2+ or Mn2+. Other divalent metal ions were inert or inhibitory. Spermidine was also inert. Monovalent cations were active in the order, NH4+ > K+= Rb+ > Cs+. NH4+ and K+ were progressively more effective from 10 mM to at least 1 M. Na+ and Li+ were inert.

The reaction of formyl‐methionyl oligonucleotides with puromycin showed similar responses to the polyphenylalanine system except that (a) Mg2+ was active over the range 5 mM to 1 M with an optimum at approximately 100 mM, (b) Ca2+ was inert, (c) K+ was inactive below 100 mM, and (d) NH4+ was only weakly active.

The bearing of the results on the mechanism of ribosome‐catalyzed peptidyl transfer is discussed, and a possible application of the results is noted.
Studies on the Metabolism of C19‐Steroids in Rat Liver
Authors: Gustafsson J.‐Å. , Lisboa B. P. , Sjöpvall J.
Published: March 2005

The formation of androstanetriols after incubation of testosterone with 105,000 ×g microsomes of adult male rat liver was investigated. The steroids were isolated by thin‐layer chromatography and were identified by gas chromatography‐mass spectrometry as 5α‐androstane‐2β,3α,17β‐triol, 5α‐androstane‐3α,7α,17β‐triol and 5α‐androstane‐3α(and 3β),16α,17β‐triol. Incubation of 2β‐ and 7α‐hydroxytestosterone only gave androstanetriols with a 3α‐configuration while incubation with 6β‐ and 16α‐hydroxytestosterone resulted in the formation of both the 3α‐ and 3β‐epimers of the respective androstanetriols. After incubation with 5α‐dihydrotestosterone the following androstanetriols were isolated: 5α‐androstane‐2β,3α(and 3β),17β‐triol, 5α‐androstane‐3α(and 3β),7α,17β‐triol and 5α‐androstane‐3α(and 3β),16α,17β‐triol. No steroid with a 6β‐hydroxy group could be isolated after this incubation. When 5α‐androstane‐3α,17β‐diol was incubated only 5α‐androstane‐2β,3α,17β‐triol was formed, and 5α‐androstane‐3β,17β‐diol was not hydroxylated in the microsomes. These findings indicate a substrate specificity for the 2β‐, 6β‐, 7α‐ and 16α‐hydroxylases.

After incubation of testosterone with 105,000 ×g liver microsomes from male germfree rats the same metabolites were isolated as after incubation with microsomes from conventional rats.
1. Preparation and Characterisation of Highly Purified 5 S RNA from Escherichia coli
Authors: Geroch M. E. , Richards E. G. , Davies G. A.
Published: March 2005

A simple yet rapid method of preparing pure 5 S RNA from Escherichia coli cells is described. Starting from 100 g of wet frozen cells, the yield is of the order of 10 mg of RNA judged to be better than 98% pure. The efficiency of each stage in the preparative procedure was assayed by gel electrophoresis. Values of the mean residue absorptivity and sedimentation coefficient under standard conditions are given and some evidence is presented that the molecule can exist in several structure forms.
Hexopyranoside: Cytochrome c Oxidoreductase from Agrobacterium tumefaciens
Authors: van Beeumen J. , de Ley J.
Published: March 2005

An inducible enzyme, catalyzing the formation of 3‐ketosugars, has been isolated in a pure state from Agrobacterium tumefaciens, grown on lactose. It occurs in the cytoplasm. It is a FAD‐protein with a sedimentation coefficient of 5.1 S and a molecular weight of 85,000 × 7,700. The pH optimum varies with the nature of the electron acceptor: with cytochrome c it is 7.0, with 2,6‐dichlorophenolindophenol 6.0 and with 5‐methylphenazinium methyl sulphate 7.9.

p‐Chloromercuribenzoate, and to some extent atabrine, have an inhibitory effect. In view of its lability, the enzyme is best preserved in a nitrogen atmosphere at pH 7 and 0°. The substrate specificity as studied with a variety of sugars, justifies the name of hexopyranoside: cytochrome c oxidoreductase for the enzyme. As a rule, disaccharides are more easily oxidized than monosaccharides. The highest affinity is observed with cellobiose and lactobionic acid (Km=2.0 × 10‐4M). Most of the 3‐ulose endproducts were detected by thin layer and/or polarography. The minimal structural requirements of a substrate sugar allow the proposal of a molecular model for the active centre of the enzyme.

The oxidation‐reduction potential at pH 7 and 25° of the system lactose/3‐ketolactose is calculated to be Eo=+0.16 V.
Proteine aus Neurospora crassa
Authors: Friemel H. , Peseke H. , Kleist H. , Brock J. , Bienengräber V. , Mücke D.
Published: March 2005

The mycelium of Neurospora crassa was cultivated in a minimal liquid medium in surface culture. The moist mycelium was lyophilized, powdered, and eluted using 0,14 M sodium chloride solution for extracting the proteins. The enrichment of these proteins was carried out by means of ultrafiltration (vacuum and pressure filtration). The extracted proteins of N. crassa were characterized by paper‐, agar gel‐ and immuno‐electrophoresis. The antisera were obtained by immunising rabbits for 21 weeks.

By means of agar gel electrophoresis 5 zones of proteins were separated in N. crassa 3a6A; by means of immunoelectrophoresis 17 bands resulted from the same protein mixture. The electrophoretic mobilities of the precipitation arcs were calculated. The immunoelectrophoretic patterns of N. crassa E 5256 obtained by N. crassa 3a6A antiserum nearly agreed with the bands of N. crassa 3a6A antigen. However there were no corresponding results in the patterns obtained from N. crassa 46001–1–1a and N. crassa 6994a with the same antiserum. It may be assumed that these differences depend on the different genetic enzyme patterns of the wild and mutant strains; they are also responsible for the differences in the rates of synthesis depending on the duration of cultivation.
Proteine aus Neurospora crassa.
Authors: Kleist H. , Friemel H. , Brock J. , Bienengräber V. , Mücke D.
Published: March 2005

The cytoplasmic proteins of Neurospora crassa 3a6A (wild strain) depending on the time of mycelial growth–are characterized by means of agar gel electrophoresis, acetate‐foil electrophoresis, immunelectrophoresis, agar gel diffusion technique, labelling with 59Fe and 58Co and by detection of the lactate dehydrogenase‐isoenzymes. The protein composition of the mycelium changes with age. During mycelial growth the fraction of watersoluble proteins increases. Simultaneously, quantitative differences are evident in the water‐soluble protein fraction. The patterns of immunelectrophoretical precipitation depend on mycelial age. 2 Fe‐labelled precipitation arcs are detectable. Also the stability of the Co‐proteides depends on the age of the mould. The distribution of the lactate dehydrogenase‐isoenzymes varies during mycelial growth.
Proteine aus Neurospora crassa.
Authors: Bienengräber V. , Friemel H. , Brock J. , Kleist H. , Mücke D.
Published: March 2005

In additional experiments on characterizing cytoplasmic proteins in Neurospora crassa (N. crassa) the enzyme and isoenzyme patterns of this fungus were analyzed. By agar gel electrophoresis the following isoenzymes were found in several N. crassa strains: 5 isoenzymes of Lactate dehydrogenase, 5 isoenzymes of Glycerolphosphate dehydrogenase, 5 isoenzymes of Glutamate dehydrogenase and 3 isoenzymes of Malate dehydrogenase. Carboxylesterase was separated into 4 fractions by means of agar gel electrophoresis.

By immunoelectrophoresis some precipitation arcs proved to be carrying enzymatic activities. Depending on the tested N. crassa strain the activity of Carboxylesterase could be localized in 2–4 precipitation arcs. ρ‐Diphenoloxidase was found as an elongated precipitation line by means of immunoelectrophoresis. Ribonuclease was seen in 2 precipitation arcs on the anodic and cathodic side of the starting point. 2 isoenzymes of Peroxidase were demonstrated in N. crassa 3a6A. One precipitation arc near the starting point indicated the activity of Catalase.
Enzymic Flux Rates in vivo through the Embden‐Meyerhof Pathway and the Nucleotides of the Mouse Liver
Authors: Reich J. G. , Till U. , Günther J. , Zahn D. , Tschisgale M. , Frunder H.
Published: March 2005

The enzymic flux rates through the Embden‐Meyerhof pathway and the nucleotides of the liver have been measured in vivo with the [32P]Pi‐tracer technique. The specific radioactivities of 15 positions in 11 metabolites have been measured within the first minute after injection of carrier‐free [32P]Pi. The results have been treated with the analogue computer technique. The main conclusions are:

The turnover of all liver compounds examined is in the range of from seconds to minutes. The velocity of oxidative phosphorylation is so high that within 30 sec after the tracer injection prestationary kinetics is observed, indicating that the turnover of the γ‐P position of ATP amounts to more than 25 μmoles per g fresh liver per min.

The labelling of ATP‐γ‐P is rapidly redistributed within the other nucleotide β‐ and γ‐P‐positions, the reactions of the nucleoside diphosphokinase type being more rapid than with the monophosphokinase type.

The rate of net glycolysis or gluconeogenesis is less than 0.1 μmoles per g per min in the postabsorptive state.

Synthesis of glycogen or UDP‐glucuronic acid via UDPG is less than 0.3 μmoles per g per min.

The absolute turnover rate of glucose 6‐phosphate is about 3 μmoles per g per min.

The pathway of glucose 6‐phosphate formation and the fate of glucose 6‐phosphate cannot be measured on the basis of 32P‐experiments alone.

Phosphofructokinase as well as aldolase and fructose 1,6‐biphosphate phosphohydrolase are inhibited in vivo. In the latter two cases, adequate kinetic explanation of this phenomenon is not available from in vitro results of the literature.

The isotope flux via glucosephosphate isomerase amounts to 0.4 μmoles per g per min. This is about one order of magnitude smaller than expected from the maximal activity and from the kinetic behaviour of the enzyme in vitro.

Synthesis of about 0.7 μmoles of glycerol 3‐phosphate from ATP and glycerol takes place per g per min. At least 0.7 μmoles of phosphoenolpyruvate are synthesized per g per min without being converted to fructose 1,6‐biphosphate. The fate of phosphoenolpyruvate is unknown. Reconversion to pyruvate or conversion to lipid glycerol is possible.
Analogue Computer Analysis of Tracer Flow Patterns through the Glycolytic and Related Pathway in Erythrocytes and other Intact Metabolic Systems
Authors: Reich J. G.
Published: March 2005

The technique and theoretical implications of an analogue computer processing of tracer data in complex metabolic systems are outlined. The procedure may be used for the prediction and analysis of experimental results. The analysis is carried out in three steps: model construction and mathematical formulation; construction of one particular set of fluxes compatible with the data; generalization to give all compatible flux combinations and to fix restrictions on permissible flux values.

Usually, metabolic systems are characterized by a slow input of radioactivity. The resulting numerically ill‐conditioned matrices either restrict the extractibility of certain parameters or lead to ambiguous results. Uses and limitations of the procedure are discussed in terms of glycolytic net flow in erythrocytes. In contrast to intuitive conclusions in previous papers, a detailed treatment of the literature data reveals that some 50% of the net flow goes via the biphospho‐glycerate shunt. Usually, tracer experiments yield the isotopic shuttle rather than the net flux rate. However, an expression of the shuttle in terms of the free enthalpy or the equilibrium of the reaction may be derived. Thus, absolute flow and net flow are of similar information content for regulation problems. The applicability of free‐energy considerations in vivo may be tested directly with the tracer method.
Zur Biosynthese der Apiose
Authors: Sandermann H. , Grisebach H.
Published: March 2005

The ethanolic extract of 8‐week‐old parsley plants was chromatographed on Dowex‐1×2 with a concave gradient using the buffer system of Hurlbert. After further purification on carboncelite columns, paper chromatography and high‐voltage electrophoresis, 120 μmoles of a homogeneous UDP‐sugar fraction were obtained. The sugars present in this fraction were identified after acid or enzymatic hydrolysis (Crotalus adamanteus venom, acid phosphatase) by various chemical and enzymatic tests. A modified Dische test which is specific for apiose and a quantitative determination of d‐xylose with d‐glucose–oxidase were developed.

According to the analytical investigations the UDP‐sugar fraction had the following composition (μmol): UDP‐d‐glucose (84), UDP‐d‐galactose (27), UDP‐d‐xylose (4), UDP‐l‐arabinose (2), UDP‐fructose (0.42), UDP‐l‐rhamnose (2.4), and UDP‐apiose (0.18). The presence of UDP‐apiose in parsley strongly corroborates the proposed biosynthetic pathway for the formation of d‐apiose via UDP‐d‐glucuronic acid.
Purification of an Extracellular Proteinase from Penicillium notatum
Authors: Makonnen B. , Porath J.
Published: March 2005

An extracellular proteolytic enzyme from the culture medium of Penicillium notatum, obtained as a dry, tannin‐precipitated powder from a commercial source, has been isolated. The purification procedures used include extraction of the crude proteinase with buffer from the commercial product, precipitation with ammonium sulphate, chromatography on DEAE‐Sephadex A‐25, gel filtration on Sephadex G‐75, followed by ion‐exchange chromatography on SE‐Sephadex C‐50, and finally zone electrophoresis on a cellulose column. Sufficient evidence is presented to show the purity of the isolated proteinase. Arguments for and against the presence of two distinct proteolytic enzymes from the same source are also presented.
On the Catabolism of Deoxyribonucleosides in Cells and Cell Extracts of Escherichia coli
Authors: Munch‐Petersen A.
Published: March 2005

Extracts of Escherichia coli cells differ considerably from whole cells in their deoxyribosidecatabolizing activities.

Whole cells catalyze an efficient transfer of the deoxyribosyl group from thymine to adenine, whereas phosphorolysis of the deoxyribonucleosides proceeds more slowly. The nucleoside purine pyrimidine deoxyribosyltransferase activity is shown to depend on the presence in the cells of thymidine phosphorylase as well as purine nucleoside phosphorylase.

Cell extracts show a considerable loss of the nucleoside purine pyrimidine deoxyribosyltransferase activity, but catalyze the phosphorolysis of the deoxyribonucleosides much more efficiently than whole cells.

Whole cells readily catabolize the deoxyribose moiety of thymidine while cell extracts have partially lost this capacity. The loss seems to be due to a destruction of deoxyribomutase activity during sonic treatment of the cells, while thymidine phosphorylase and deoxyriboaldolase activities are resistant to this treatment.

All the above mentioned enzymes, involved in catabolism of deoxyribonucleosides, are released in the medium when the cells are subjected to osmotic shock‐treatment. They are all induced by growth of the culture in the presence of deoxyribonucleosides.

These observations have led to the assumption that the enzymes concerned with breakdown of deoxyribonucleosides in E. coli structurally and functionally constitute a complex, located at or near the cell membrane, and depend on the intact structure of the cells for proper function. Uptake of exogenous thymine into cellular DNA is probably controlled by this complex of enzymes, since the cellular pool of deoxyribosyl groups, necessary for the formation of thymidine from thymine, may be regulated through an interplay between these enzymes.
The Reaction of Fatty Acyl Thiolesters with Proteins
Authors: Marsh J. B.
Published: March 2005

The enzyme fraction precipitated from rat liver cell sap at pH 5 catalyzes the formation of fatty acyl coenzyme A thiolesters. During the incubation, about 3% of the labeled fatty acid appears covalently bound to protein. The fatty acyl CoA esters were the intermediates in the reaction. The covalent linkage of the fatty acid to the protein was not enzymatic, since labeled palmityl CoA reacted directly with protein. Proof for the covalent nature of the binding was obtained in three ways: (a) the label was not removed by a series of solvent extractions which removed free fatty acid or fatty acyl CoA, (b) labeled palmityl peptides were isolated from peptic digests of labeled protein, (c) peptides isolated from protein labeled with [1‐14C]palmitate were not decarboxylated by azide in the Schmidt reaction.

Covalent labeling of the pH 5 enzyme protein was extensive only with fatty acids above 10 carbon atoms. Acetyl CoA gave less than 10% of the reaction given by palmityl CoA. These results suggested a preliminary binding of the hydrocarbon tail of the CoA esters with hydrophobic regions of the protein.

Evidence for the involvement of protein hydroxyl and amino groups in the reaction was obtained. Reduction of pronase peptides of protein labeled with palmitic acid with LiAlH4 gave rise to labeled cetyl alcohol. SH Groups were not involved as judged by the failure of neutral hydroxylamine to remove the label and by the fact that gamma globulin, which has no free SH groups, was capable of reacting. Acid or alkaline hydrolysis at 100° removed less than half of the label, suggesting the presence of amide bonds. Blockage of amino groups by forming the picryl derivative of the protein greatly reduced the extent of the reaction.
Structural and Functional Aspects of Ribosome Interaction with Endoplasmic Reticulum Membranes
Authors: Comorosan S. , Lugojan P.
Published: March 2005

The different biochemical components of endoplasmic reticulum membranes (phospholipids, phosphoproteins and RNA) were investigated as to their ability to interact with the ribosomal protein and RNA.

Light‐scattering determinations, viscosity measurements and gel‐filtration techniques were employed.

A complex between a fraction of ribosomal protein (dialyzed against urea) and a phosphoprotein fraction from endoplasmic reticulum membranes, and a complex between a fraction of ribosomal protein (dialyzed against water) and two different phosphoprotein fractions from endoplasmic reticulum membranes, were obtained. No complexes could be detected between the other components under investigation. Likewise no complexes could be detected in the control experiments with lecithin, pepsin and cytochrome c, tested with ribosomal protein fractions and endoplasmic reticulum membranes phosphoproteins.

These results suggest an additional new mechanism concerning the interaction of ribosomes with the endoplasmic reticulum membranes.
Thioredoxin. 5. Amino Acid Sequences of the Tryptic Peptides of Peptide A
Authors: Holmgren A.
Published: March 2005

Tryptic digestion of peptide A, the C‐terminal fragment of thioredoxin, obtained by cyanogen bromide cleavage, resulted in the isolation of nine peptides. The complete amino acid sequence of each was determined, and the N‐terminal and C‐terminal fragments of peptide A were identified. Together the nine tryptic peptides accounted for all 71 residues of peptide A. Hydrazinolysis and studies with carboxypeptidase A established ‐Asn‐Leu‐Ala to be the C‐terminal sequence of peptide A and of thioredoxin.
Synthesis of N‐Acetylated Deoxyribonucleoside 5′‐Triphosphates and their Utilization in Enzymatic Formation of Single‐Stranded Polydeoxyribonucleotides
Authors: Hayes F. N. , Hansbury E. , Mitchell V. E. , Ratliff R. L. , Williams D. L.
Published: March 2005

The N‐acetylated deoxyadenosine and deoxyguanosine 5′‐triphosphates (dAAcTP and dGAcTP) have been synthesized and found competent as substrates for enzymatic chain lengthening of polydeoxyribonucleotides without concomitant loss of the acetyl group. The enzyme used was terminal deoxyribonucleotidyltransferase. The addition of dGAc units proceeds without aggregation that self‐limits addition of nonacetylated dG units. The resulting polymer 3′‐ended with dGAc units is an efficient initiator for subsequent chain lengthening. Hydrogen bonding is not destroyed by N‐acetylation of the dA‐dT Watson‐Crick pair, although it is considerably weakened. As a practical result of this, a polymer 3′‐ended with dAAc units is a much more efficient initiator for enzymatic chain lengthening by dT units than is a corresponding polymer with dA units. Venom phosphodiesterase does not hydrolyze a polymer 3′‐ended with dAAc units.
Protonated Polynucleotide Structures
Authors: Courtois Y. , Fromageot P. , Guschlbauer W.
Published: March 2005

The change of the optical rotatory dispersion spectra as a function of pH of DNAs of different base composition and the synthetic polynucleotides poly d(A–T) and poly (dG) · poly (dC) has been studied.

It is shown that the acid titration of DNA when followed by optical rotatory dispersion is considerably more complex than it appears from ultraviolet absorption data. The gradual appearance of a peak at 260–270 mμ between pH 3 and 4 before acid denaturation, indicates a change of conformation of the guanosine residues in this pH region.

DNA methylated in N‐7 of guanine does not show this inversion of Cotton effects. It is concluded that this position plays an important role in the acid titration of DNA.

Protonation on N‐7 of guanine, rotation of guanine out of the helix, reversion into the syn‐position, pairing in Hoogsteen manner and thus sharing the proton with N‐1 on cytosine is suggested as a possible interpretation of these results.

From the data presented it is concluded that poly (dG) · poly (dC) has a conformation and/or structure different from that of DNA (B‐form). In analogy with published data on nucleo‐sides, it is suggested that guanosine is in the syn‐conformation. Three possible structures seem to be feasible: (a) Watson‐Crick pairing with parallel strands; (b) Hoogsteen pairing with anti‐parallel strands; (c) Watson‐Crick pairing with antiparallel strands in a left handed helix.
The Dissociation into Subunits of Aspartate Aminotransferase from Pig Heart Muscle
Authors: Banks B. E. C. , Doonan S. , Gauldie J. , Lawrence A. J. , Vernon C. A.
Published: March 2005

Ultracentrifugation studies show that, at concentrations of about 0.1 mM the aspartate aminotransferase of pig heart muscle does not dissociate into subunits either in 6 M guanidine hydrochloride, pH 7.5, or 8 M urea solution, pH 7.5. On prolonged treatment (8 weeks, 0°) with urea, however, complete dissociation occurs and the measured value of the molecular weight drops to approx. 40,000. The protein so obtained is enzymically inactive and migrates on electrophoresis in starch gel at pH 8.0 as a single, fast‐moving band.

Gel filtration experiments show that the holoenzyme (pyridoxal form) partly dissociates into two subunits at a concentration of about 3 nM. The specific activity of the enzyme does not change in this region; the monomer and dimer have, therefore, the same catalytic activity per mole of cofactor.

The apoenzyme is unstable at high dilution and its dissociation has not been directly demonstrated by gel filtration. However, indirect evidence obtained from a study of the kinetics of its recombination with cofactor suggests that dissociation occurs at a concentration of about 50 nM.
The Interconversion of Liver Glycogen Synthetase a and b in vitro
Authors: de Wulf H. , Hers H. G.
Published: March 2005

The activation of liver glycogen synthetase b into a in vitro is greatly accelerated by caffeine and theophylline, and is inhibited by fluoride, EDTA, glycogen, Pi and sulfate. The inhibition by fluoride and EDTA is partially released by an excess of magnesium but not by the methyl‐xanthines. This activation is also suppressed when ATP is added to the homogenate or when the concentration of endogenous ATP is maintained at a high level by shaking the preparation in the air.

The inactivation of synthetase a into b in vitro can be studied after its previous activation performed either in vitro at 20° or in vivo by treatment of the animals with glucose or with glucocorticoids. The inactivation occurs only in the presence of exogenous or endogenous ATP and is accelerated by 3′,5′‐cyclic AMP. It is inhibited by glucose 6‐phosphate, even in the presence of the cyclic AMP, and by glycogen only in the absence of the nucleotide. A half maximal inactivation is obtained in the presence of 0.2 μM 3′,5′‐AMP.

All these findings are in agreement with the hypothesis that the activation of liver glycogen synthetase is catalyzed by a specific phosphatase whereas its inactivation is a phosphorylation at the expense of ATP under the action of a specific kinase.
The Preparation and Some Properties of Bromelain Covalently Attached to O‐(Carboxymethyl)‐Cellulose
Authors: Wharton C. W. , Crook E. M. , Brocklehurst K.
Published: March 2005

Purified bromelain has been attached to CM‐cellulose by reaction with its acid azide derivative to yield insoluble preparations which contain 6.5–11.0 mg of bromelain/100 mg of product.

The result of an extensive washing procedure strongly suggests that this protein is attached chemically to the CM‐cellulose.

The apparent catalytic constant for the catalysis of the hydrolysis of α‐N‐benzoyl‐l‐arginine ethyl ester (BzArgOEt) at pH 7.0, I= 0.2 and 25.0° by CM‐cellulose‐bromelain is 52% of the corresponding value for the bromelain‐catalysed hydrolysis when the enzyme concentration is estimated by protein analysis and 77% of the value for the bromelain‐catalysed hydrolysis when the enzyme concentration is estimated by analysis of the thiol content of CM‐cellulose‐bromelain.

The Michaelis constant for the CM‐cellulose‐bromelain‐catalysed hydrolysis of BzArgOEt is about an order of magnitude lower than the corresponding value for the bromelain‐catalysed hydrolysis at low ionic strength and approaches the value for the bromelain‐catalysed hydrolysis at high ionic strength.

The attachment of bromelain to CM‐cellulose protects the enzyme from oxidation at 4° but offers little or no protection from heat denaturation.
The Nature of the Perturbation of the Michaelis Constant of the Bromelain‐Catalysed Hydrolysis of α‐N‐Benzoyl‐l‐Arginine Ethyl Ester Consequent upon Attachment of Bromelain to O‐(Carboxymethyl)‐Cellulose
Authors: Wharton C. W. , Crook E. M. , Brocklehurst K.
Published: March 2005

The apparent Michaelis parameters for the catalysis of the hydrolysis of α‐N‐benzoyl‐l‐arginine ethyl ester (BzArgOEt) at pH 7.0 and 25.0° by bromelain and by CM‐cellulose‐bromelain have been measured over a wide range of ionic strength.

An equation has been derived which relates the apparent Km to the ionic strength of the assay medium for the catalysis of a reaction involving a charged substrate by an enzyme attached to a solid matrix which carries a charge opposite in sign to that of the substrate.

This equation has been shown to describe the change observed in the apparent Km for the catalysis of the hydrolysis of BzArgOEt by CM‐cellulose‐bromelain consequent upon changing the ionic strength of the assay medium. This finding supports the interpretation of the perturbation of the Km of the free enzyme catalysis by attachment of the enzyme to CM‐cellulose as mainly the result of charge‐charge interactions.

The effect of changing the ionic strength on Vmax for the catalysis by bromelain and by CM‐cellulose‐bromelain is discussed briefly and compared with the ionic strength effect on the rate of non‐enzymic hydrolysis of BzArgOEt.
Mechanism of Methionine Biosynthesis in Rat Liver
Authors: Burton E. , Sakami W.
Published: March 2005

Synthesis of methionine in rat liver by the B12‐independent N5‐methyltetrahydropterolyltri‐glutamate: homocysteine methyltransferase reported by Wang, Koch and Stokstad has been investigated. In agreement with their results it was found that methionine was formed by crude rat liver supernatant fraction and dialyzed mitochondrial extract on aerobic incubation with N5‐methyltetrahydropteroyltriglutamate, homocysteine and magnesium chloride. However, the folate derivative could not have been involved in this process since N5‐[14C]methyl‐tetrahydropteroyltriglutamate did not incorporate 14C into the methionine. In the supernatant fraction, methionine was formed by the transmethylation of endogenous betaine with added homocysteine. Rat liver supernatant fraction was found to contain 250 nmoles/ml betaine. In the mitochondrial extract, methionine formation occurred by proteolysis. It is concluded that a B12‐independent N5‐methyltetrahydropteroyltriglutamate: homocysteine methyltransferase does not occur in rat liver.
Activation, Inhibition, and pH‐Dependence of the Hydrolysis of α‐N‐Benzoyl‐l‐Arginine Ethyl Ester Catalyzed by Kallikrein from Porcine Pancreas
Authors: Fiedler F. , Werle E.
Published: March 2005

Kallikrein from porcine pancreas is inhibited by heavy metal ions and activated by sulfhydryl compounds or chelating agents. This activation is interpreted as a reversal of heavy metal inhibition. From the experiments the conclusion is drawn that kallikrein is neither a “sulfhydryl protease” nor a metal‐dependent protease.

The pH dependency of the hydrolysis of α‐N‐benzoyl‐l‐arginine ethyl ester by kallikrein at low substrate concentrations indicates three basic groups with pK 7.9 and 5.7 in the free enzyme and pK 6.9 in the enzyme‐substrate complex which participate in catalysis. This pH‐dependence resembles trypsin‐ and especially cholinesterase‐catalyzed hydrolyses. From the most alkaline pK value the participation of an amino group and from the other pK values the participation of a histidine residue in kallikrein‐catalyzed hydrolysis is inferred.
Oxidative Metabolism in Cells Isolated from Brown Adipose Tissue
Authors: Prusiner S. B. , Cannon B. , Ching Te May , Lindberg O.
Published: March 2005

Non‐stimulated brown fat cells exhibit a respiration controlled by availability of phosphate acceptor systems.

Cells stimulated by nor‐epinephrine gradually lose the respiratory control as demonstrated by a diminishing response to carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP).

The initial phase of increased respiration upon norepinephrine addition is characterized by an increased availability of activated fatty acids.

A mechanism cannot be envisaged which could utilize all the ATP which would be produced by a tightly coupled oxidative phosphorylation system.

A fine metabolic control must exist which regulates a balance between ATP production for fatty acid activation and a loosening of coupling between electron transport and phosphorylation.
3α‐Hydroxysteroid: NAD Oxidoreductase Activity in Crystalline Preparations of 20β‐Hydroxysteroid: NAD Oxidoreductase
Authors: Pocklington T. , Jeffery J.
Published: March 2005

Crystalline 20β‐hydroxysteroid: NAD oxidoreductase preparations from Streptomyces hydrogenans were shown to have 3α‐hydroxysteroid: NAD oxidoreductase activity. Compounds of both the 5α‐androstane and 5α‐pregnane series served as substrates.

Among the compounds tested, no evidence was found of 3β‐hydroxysteroid: NAD oxidoreductase activity. The 3α‐hydroxysteroid: NAD oxidoreductase activity did not extend to compounds having 5β‐H, a 4,5 double bond, a 5,10 double bond, or 1,2 and 4,5 double bonds together. In the 5α‐H series, a 1,2 double bond prevented the compound from serving as a substrate.

Substituents at C‐11, ‐16 and ‐17 altered the kinetic constants for the 3α‐hydroxysteroid: NAD oxidoreductase reaction, and in the case of an 11β‐hydroxy or 11‐oxo group the effects were similar to the effects reported for these groups for 20β‐hydroxysteroid: NAD oxidoreductase activity.

Kinetic data were consistent with the view that two types of 3α‐hydroxysteroid: NAD oxidoreductase were present which involved different active centres, neither of which was the active centre for the 20β‐hydroxysteroid: NAD oxidoreductase activity. It is not yet established whether these enzymic activities are associated with the same or different molecules. The 3α‐hydroxysteroid: NAD oxidoreductase activities do not correspond to those of previously characterised enzymes (3α‐hydroxysteroid dehydrogenase and α‐hydroxycholanate dehydrogenase.
Kinetic Evidence for the Existence of α‐Globin Pool in β‐Thalassemic Reticulocytes
Authors: Bargellest A. , Pontremoli S. , Menini C. , Conconi F. , Melloni E.
Published: March 2005

The kinetics of incorporation of 3H‐labeled amino acids into α, β, and γ globin has been determined, following incubation of intact reticulocytes, in stroma‐free hemolysates and in purified Hb F and Hb A from homozygous β‐thalassemic subjects.

The labeled globins present in the cytoplasm as well as in purified Hb F and Hb A at various incubation times, were separated by CM‐cellulose chromatography, and the ratios of [3H]α to [3H]counterpart globins were determined.

The results obtained show that in the initial stages of incubation, the ratios of [3H]α to [3H]‐counterpart globins are higher than 1 in the total lysate and in purified hemoglobins; these ratios, after long periods of incubation, decrease to values below 1.

These findings suggest that the excess of newly synthesized α molecules flows very rapidly from the polyribosomes to the hemoglobins, and then, more slowly, to a “stromal”α pool.

A model is presented, showing the most likely mechanism for the overall process.