bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies of Glutamate Dehydrogenase
Authors: Hucho Ferdinand , MARKAU Ursula , Sund Horst
Published: March 2005

A histidine residue has been shown to be essential for the association of glutamate dehydrogenase. After photooxidation of one histidine residue per polypeptide chain with pyridoxal 5′‐phosphate as photosensitizer the enzyme sediments with sedimentation coefficients significantly lower than the 25 S of the native enzyme. These preparations retain about 70% of their activity.

A further histidine residue has been shown to be essential for the activity of glutamate dehydrogenase. After photooxidation of a second histidine residue per polypeptide chain the enzyme is inactive. These results have been confirmed by modification experiments with diethylpyrocarbonate. The inactive enzyme has lost its ability to bind NADH.

The photooxidation of the first histidine residue is accompanied by a change in the regulatory properties of the enzyme. GTP no longer inhibits the activity while the effect of ADP is unchanged.

A novel reaction of pyridoxal‐P with the enzyme has been investigated. During irradiation of the enzyme · pyridoxal‐P complex pyridoxal‐P becomes slowly irreversibly incorporated. At the point of about 80% inactivation the glutamate dehydrogenase contains 0.43 molecules pyridoxal‐P per polypeptide chain as indicated by the incorporation of radioactivity from [4‐3H]pyridoxal‐P. The reaction is accompanied by the appearance of a new absorption maximum at 325 nm. The effect is interpreted as a photoactivated addition of an imidazole group to the double bond of a Schiff base formed between pyridoxal‐P and an ɛ‐NH2 group of a lysine residue. There is evidence that the position 97 identified by Smith et al. in 1970 (Proc. Nat. Acad. Sci. U.S.A. 67, 724) is not involved in this reaction. The reaction may be important for the determination of the relative position in the tertiary structure of the two functional groups involved.
Studies of Glutamate Dehydrogenase
Authors: Deppert Wolfgang , HUCHO Ferdinand , Sund Horst
Published: March 2005

Glyoxal inhibits the catalytic reaction of glutamate dehydrogenase competitively with respect to 2‐oxoglutarate, whereas the hydrodynamic and allosteric properties remain largely unchanged upon treatment with glyoxal. The reaction is completely reversible, but the Schiff base intermediate can be trapped by reduction with NaBH4. Determination of the free amino groups indicates that glyoxal reacts with one amino group per polypeptide chain.

From the analysis of Km and V in the range of pH 7.8 to pH 9.2 it is concluded that this amino group has a pK‐value of 8.2 and is responsible in its protonated state for the binding of the substrate 2‐oxoglutarate.

5‐Diazo‐1H‐tetrazole reacts with glutamate dehydrogenase predominantly by modifying amino groups. Depending on the reaction conditions 5‐diazo‐1H‐tetrazole first modifies that amino group which is responsible for the binding of the substrate 2‐oxoglutarate and secondly the amino group responsible for the binding of the dihydronicotinamide part of NADH. If the modification is performed in the presence of the substrate 2‐oxoglutarate the amino group which binds the substrate is protected. The modification of the enzyme with 5‐diazo‐1H‐tetrazole also conserves to a large extent the ability of the oligomer to associate and its allosteric properties.
Collagenolytic‐Cathepsin and Acid‐Proteinase Activities in the Rat Uterus during post partum Involution
Authors: Etherington David J.
Published: March 2005

The digestion rates for haemoglobin and native, insoluble collagen were determined at pH 3.5 in extracts of uteri collected from rats at 1 to 12‐days post‐partum. Both activities became concentrated during the early stages of involution, but the average total collagenolytic activity for the whole uteri was sharply reduced after the second day. Haemoglobin digestion (mainly cathepsin D) was most concentrated on days 3–5 and the total activity then slowly declined during the remainder of the 12‐day period. Collagenolytic activity was strongly affected by thiol activators and inhibitors whereas these had little effect upon haemoglobin digestion. It is concluded that cathepsin D has no action on native insoluble collagen during involution.
Effects of a Potent Catatoxic Steroid, 16α‐Cyanopregnenolone, on Microsomal Metabolism of Steroid Hormones, Sterols and Bile Acids in Rats
Authors: Einarsson Kurt , GUSTAFSSON Jan‐Åke
Published: March 2005

The effect of administration of the potent catatoxic steroid 16α‐cyanopregnenolone on the microsomal hydroxylation of 4‐[4‐14C]androstene‐3,17‐dione, 4‐[4‐14C]pregnene‐3,20‐dione, [4‐14C]‐cholesterol, 7α[6β‐3H]hydroxy‐4‐cholesten‐3‐one, and tritium‐labelled taurodeoxycholic acid was studied in rats. The 6α‐, 16α‐ and 18‐hydroxylation of 4‐androstene‐3,17‐dione increased 1.5–2 times and 6β‐hydroxylation of the same substrate increased about 5 times after administration of 16α‐cyanopregnenolone to male rats. With 4‐pregnene‐3,20‐dione as substrate 2α‐hydroxylation increased twice, 2β‐ and 16α‐hydroxylation four times, and 6β‐hydroxylation nine times in male rats; the corresponding figures for stimulation of microsomal hydroxylation of 4‐pregnene‐3,20‐dione in female rats were: 2β‐hydroxylation twice, 6α‐hydroxylation nine times, 7α‐hydroxylation four times and 6α‐hydroxylation five times. 16α‐cyanopregnenolone administration led to a significant increase (1.5 times) in male rats of the activity of 17β‐hydroxysteroid oxidoreductase active on 4‐androstene‐3,17‐dione and to a significant decrease (to about 50% of the control value) in female rats of the activity of 5α‐reductase with 4‐pregnene‐3,20‐dione as substrate. The activities of 3α‐ and 3β‐hydroxysteroid oxidoreductases were not affected. After treatment with 16α‐cyanopregnenolone the 7α‐hydroxylation of cholesterol seemed to diminish but this effect was not statistically significant; no effect was seen on the microsomal 12α‐hydroxylation of 7α‐hydroxy‐4‐cholesten‐3‐one. 7α‐Hydroxalation of taurodeoxycholic acid was stimulated about twice. It is concluded that the effects of 16α‐cyanopregnenolone administration closely resemble the effects of treatment with phenobarbital and it is suggested that these drugs have a common mechanism of enzyme induction.
Carbon‐13 Nuclear‐Magnetic‐Resonance Studies on Selected Amino Acids, Peptides, and Proteins
Authors: Freedman Murray H. , LYERLA James B. , Chaiken Irwin M. , COHEN Jack S.
Published: March 2005

Natural abundance high‐resolution carbon‐13 Fourier transform nuclear magnetic resonance (13C NMR) studies at 25 MHz have been carried out on selected amino acids, linear peptides and proteins. The pH dependence of the 13C resonances has been studied in detail in several cases. For l‐histidine, the 13C chemical shifts of all six carbon atoms are sensitive to the ionization state of the titrating carboxyl, imidazole and amino groups. The data for each carbon resonance have been computer‐fitted to a sum of three theoretical curves based on a simple proton‐association equilibrium for each transition. The extent of chemical shift change upon ionization of a nearby group has been interpreted in terms of two competing effects, inductive (through‐bond) and electric field (through‐space) effects.

The 13C NMR spectra of the amino‐terminal 1–13, 1–15, and 1–20 peptides of bovine pancreatic ribonuclease A have been analyzed. Detailed resonance assignments have been made based on comparisons with the results for the free amino acids, shift effects for amino acids in small peptides, internal peptide bond effects and differences in amino acid composition. There is considerable correspondence between the observed 13C chemical shifts of these peptides and those predicted from the shifts of the component amino acids.

Proteins of varying molecular weight have been studied to evaluate the potential of 13C NMR investigations of structure and conformation. A comparison has been made between the 13C NMR spectra of hen egg‐white lysozyme in its native and reduced states. The spectrum of the fully reduced species appears to be well represented by the sum of the spectra of the individual amino acids, both in chemical shift and line width, whereas that for the native form exhibits consistently broader resonances. Similar results were obtained for ribonuclease A. At the current level of resolution, it is impossible to discern whether this line broadening derives from shorter relaxation time effects due to restricted motion, chemical shift non‐equivalence between the same chemical groups, or both. However, it does appear that there are separately resolved peaks corresponding to Cβ‐carbons for different threonyl residues, as well as for the C‐4 ring carbons of the several tyrosyl residues in native hen egg‐white lysozyme. Spectra of carbonic anhydrases (Mr∼ 30000), which lack disulphide bonds, showed somewhat poorer resolution than the smaller proteins.
Nuclear and Cytoplasmic DNA Synthesis during the Mitotic Cycle of HeLa Cells
Authors: Volpe Pietro , EREMENKO Tamilla
Published: March 2005

In HeLa S3 cells grown in suspension and synchronized with thymidine, while nuclear DNA is replicated during the S‐phase, cytoplasmic DNA is synthesized with two peaks in the S‐ and G2‐phases: the first has its maximum 1–2 h before that of nuclear DNA; the second takes place after that of nuclear DNA, approximately at the 9th hour from the removal of synchronizing thymidine from the culture medium. Newly synthesized nuclear and G2‐phase‐cytoplasmic DNAs in CsCl gradients have buoyant density of 1.698 and 1.688 g/cm3, respectively. The buoyant density of S‐phase‐cytoplasmic DNA is 1.701 g/cm3, probably corresponding to the closed circular duplex form of mitochondrial DNA. The kinetics of synthesis of nuclear DNA is exponential, differing from that of S‐ and G2‐phase‐cytoplasmic DNAs which show an asymptotic shape of [3H]thymidine labelling. Ethidium bromide inhibits the synthesis in vivo of both S and G2 peaks of cytoplasmic DNA, but does not appreciably influence the labelling of nuclear DNA.
Size Distribution of Circular DNA from Petite‐Mutant Yeast Lacking ρDNA
Authors: Clark‐Walker George Desmond
Published: March 2005

The size distribution of circular DNA, of buoyant density 1.701 g/cm3, from a petite mutant yeast lacking DNA of buoyant density 1.684 g/cm3 (ϱDNA) has been studied by sucrose gradient analysis and electron microscopy. The DNA prepared from subcellular particles fractionated on a sucrose gradient, is found to be heterodisperse but with a major component having a length of 1.9 μm, a molecular weight of 4.02 × 106 and sedimenting as a supercoil at 22S and in the relaxed form at 15S. Minor components having lengths of 2.8, 3.9 and 5.8 μm and corresponding molecular weights of 5.71 × 106, 8.09 × 106 and 12.4 × 106 have also been observed. The significance of circular · DNA in relation to current ideas on the export of nuclear DNA to the cytoplasm is considered.
Evidence for a Common Transport System for Cytosine, Adenine and Hypoxanthine in Saccharomyces cerevisiae and Candida albicans
Authors: Polak Annemarie , GRENSON Marcelle
Published: March 2005

In Saccharomyces cerevisiae, the utilization of exogenous cytosine by pyrimidine‐requiring strains is prevented by the presence of adenine or hypoxanthine in the culture medium. Vice versa, the utilization of adenine or hypoxanthine by purine‐requiring strains is inhibited by cytosine. Neither uracil nor uridine behave as cytosine does under these conditions.

Growth behaviour of purine‐requiring mutants as well as direct uptake measurements show that adenine and hypoxanthine uptake are strongly depressed in a mutant of S. cerevisiae lacking cytosine permease. This mutant is resistant to 5‐fluorocytosine.

In Candida albicans, as well as in S. cerevisiae, the uptake of each of the substrates adenine, hypoxanthine, cytosine and 5‐fluorocytosine is competitively inhibited by the others and the apparent affinity constant (Km) for uptake of each substrate is equal to the inhibition constant (K1) of the same substance used as an inhibitor of the uptake of the others.

It is concluded that adenine, hypoxanthine, cytosine and 5‐fluorocytosine share a common recognition site for uptake and that this site is not active in the mutant lacking cytosine permease.
Studies on Energy‐Linked Reactions
Authors: Avner Philip R. , GGRIFFITHS David E.
Published: March 2005

A series of 62 mutants of Saccharomyces cerevisiae showing high levels of resistance to oligomycin and rutamycin have been isolated. These mutants can be subdivided into two classes on the basis of their cross resistance to a variety of inhibitors and uncouplers of mitochondrial energy‐conservation reactions.

Class I mutants show cross resistance to aurovertin, triethyltin, ‘1799’ (a hexafluorotriacetone derivative), uncoupling agents and inhibitors of mitochondrial protein synthesis such as chloramphenicol, mikamycin and spiramycin.

Class II mutants are specifically resistant to oligomycin and rutamycin and show no cross resistance to aurovertin, triethyltin or any of the other agents tested.

The majority of the class I mutants and one of the class II mutants exhibit cold sensitivity of the oligomycin resistance, i.e. they lose resistance at the non‐permissive temperature of 20°C, and this provides a further basis for classification of the mutants.

The growth rates of the majority of these resistant mutants from both classes were unaltered from the wild type and the growth yield of the class II mutants was also the same as that of the parental strains. The growth yield of the class I mutants, however, was 15–20% less than that of the wild type.

No differences in mitochondrial morphology or of the number of mitochondria per cell were seen on examination by the electron microscope.

The isolation and properties of specific mitochondrial oligomycin‐resistant mutants supports the biochemical evidence for different sites of action of oligomycin, triethyltin and uncoupling agents on energy‐conservation reactions.
Reciprocal Induction and Repression of Serine Dehydratase and Phosphoglycerate Dehydrogenase by Proteins and Dietary‐Essential Amino Acids in Rat Liver
Authors: Mauron Jean , MOTTU Françoise , Spohr Georges
Published: March 2005

The impact of protein nutrition upon serine biosynthesis and catabolism in rat liver was investigated by determining the activity of two key enzymes in the pathway of serine metabolism, phosphoglycerate dehydrogenase on the pathway leading from pyruvate to serine, and serine dehydratase on the catabolic way transforming serine to pyruvate.

It was found that increasing the protein content of the diet induces serine dehydratase and represses reciprocally phosphoglycerate dehydrogenase. For casein, a protein of high nutritive value, the activities of both enzymes are at equilibrium at the 20% dietary protein level, which is near the optimum casein level for rat growth. With gluten, a protein of low nutritive value, equilibrium is reached at almost the same dietary protein level (22%), although optimal growth is not attained prior to the 50% dietary protein level. In this comparison, there is no correlation between the nutritive value of the proteins (growth measurement) and their regulatory action on the two enzymes tested. Extension of the studies to other proteins, such as egg, showed however that egg, the protein of highest nutritive value, has also the greatest effect on enzyme regulation. The conclusion is that a certain relationship between nutritive value and enzyme‐regulatory action exists, but that the determining factors (amino acids) are partly different. The role of individual dietary amino acids upon serine metabolism was therefore investigated. It was first shown that only the essential amino acids as a group were responsible for the reciprocal induction and repression of the two enzymes. By feeding amino acid mixtures lacking one essential amino acid at a time to the animals, we established subsequently that four essential amino acids only, i.e. methionine, tryptophan, threonine and valine are active in regulating the two enzymes. Cystine influences only phosphoglycerate dehydrogenase. The enzyme‐regulating action of proteins can thus be expressed in terms of their content in “active” amino acids, whereas their nutritive value depends on the presence of all essential amino acids. The similar behaviour of casein and gluten in enzyme regulation in spite of the great difference in nutritive value is thus fully explained, since the deficient amino acid in gluten, i.e. lysine, is not active in the regulation of the two enzymes studied.

Finally a coherent theory for the reciprocal serine dehydratase repression and phosphoglycerate dehydrogenase induction in rat liver by lack of an “active” amino acid is proposed.

It is based on the presence of a posttranscriptional represser which both inhibits messenger translation and promotes messenger degradation, and on the fact that the translation of small proteins is less impeded by amino acid deficiency than that of bigger polypeptide chains.
Recycling of Initiation Factors IF‐1, IF‐2 and IF‐3
Authors: Benne Rob , NAAKTGEBOREN Nico , Gubbens Jos , VOORMA Harry O.
Published: March 2005

Initiation of protein synthesis in Escherichia coli is a catalytic process, provided that conditions for hydrolysis of GTP are present. This means that the initiation factors IF‐1, IF‐2 and IF‐3 are released at a certain stage of initiation complex formation, whereafter they recycle and are reutilized in another round of initiation.

Experiments presented in this paper provide evidence for the mechanism of recycling, in which the hydrolysis of GTP and IF‐1 both play a predominant role. We demonstrate that binding of fMet‐tRNA, which is dependent on IF‐2, is a stoichiometric process in a system devoid of IF‐1. Addition of IF‐1 converts it into a catalytic process, however, only if GTP can be hydrolysed. No such conversion takes place with 5′‐guanylylmethylenediphosphonate. The same observation is valid for the uncoupled IF‐2 dependent GTPase activity.

In the process of initiation IF‐1 acts as a recycling promoting factor, which displays analogous properties to elongation factor EF‐Ts. We postulate that a ribosomal‐bound IF‐2 · GDP complex is converted into an IF‐2 · GTP complex, which is released and is capable of starting a new cycle of initiation.

Furthermore, kinetic evidence is presented that IF‐3 recycles even at the level of 30‐S initiation complex formation, irrespective of whether conditions for hydrolysis of GTP are provided.
Identification of Histone Messenger RNA from HeLa Cells
Authors: Breindl Michael , GALLWTTZ Dieter
Published: March 2005

Histone mRNA from HeLa cells synchronized by a double thymidine block has been identified and characterized. Its synthesis starts immediately after the cells are released from the thymidine block.

Pulse labelling for 60 min with [5‐3H]uridine at different times of the S‐phase showed that radioactively labelled histone mRNA enters polyribosomes with a maximum during the first hour of S‐phase, i.e., before the maximum of DNA synthesis.

Histone mRNA sediments on sucrose gradients in the 8–10‐S region and separates on polyacrylamide gels into three RNA species with molecular weights of about 1.5 × 105, 1.8 × 106, and 2.1 × 105, respectively.

Polyribosomal 8–10‐S RNA from synchronized cells in S‐phase directs the synthesis of histones in a rabbit reticulocyte cell‐free system. Histones synthesized in vitro were identified by polyacrylamide gel electrophoresis and by high‐voltage paper electrophoresis of tryptic peptides. Polyribosomal 8–10‐S RNA from cells in which DNA synthesis was blocked by thymidine or hydroxyurea had only a marginal histone mRNA activity.
Studies on Soybean Trypsin Inhibitors
Authors: Koide Takehiko , IKENAKA Tokuji , Tsunasawa Susumu
Published: March 2005

For the elucidation of amino acid sequence around the reactive site of soybean trypsin inhibitor (Kunitz), fragments A and B were digested with trypsin, and the resulting peptides were separated by ion‐exchange chromatography on Dowex 50X2 or by gel filtration on Bio‐Gel P‐4. Further purification of the peptides was carried out by gel nitration on Sephadex G‐25 or by high‐voltage paper electrophoresis at pH 3.6 and 6.5.

Nine peptides were obtained in pure form from fragment A and three peptides from fragment B, and their amino acid sequences were determined by the direct Edman method and by carboxy‐peptidase digestion technique.

Overlapping peptides necessary for the alignment of the tryptic peptides from fragments A and B were obtained from a chymotryptic hydrolysate of fragment AB by gel filtration on Bio‐Gel P‐4, and their amino acid compositions and sequence analyses of some peptides made it possible to establish the amino acid sequence of an amino‐terminal region of the inhibitor consisting of 84 amino acid residues. The reactive site (Arg63‐Ile64) of the inhibitor to trypsin was involved in this region.
Studies on Soybean Trypsin Inhibitors
Authors: Koide Takehiko , IKENAKA Tokuji
Published: March 2005

For the elucidation of the amino‐acid sequence of the carboxyl‐terminal region of soybean trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the resulting peptides were separated by ion‐exchange chromatography on Dowex 50X2 or by gel nitration on Bio‐Gel P‐4.

Further fractionation and purification of the peptides were performed by ion‐exchange chromatography on Dowex 1X2, by gel filtration on Bio‐Gel P‐2 or by high‐voltage paper electrophoresis at pH 1.9 and 3.6.

Three peptides were obtained in pure form from fragment C and ten peptides from fragment D, and their amino‐acid sequences were determined by the direct Edman method and by carboxy‐peptidase digestion technique.

Overlapping peptides necessary for the alignment of the tryptic peptides from fragment D were obtained from a chymotryptic hydrolysate of fragment D.

Nine main peptides and nine minor peptides were obtained. The amino acid composition and the partial amino‐acid sequence of the 18 chymotryptic peptides of fragment D made it possible to establish the amino‐acid sequence of the carboxyl‐terminal region of the inhibitor.

The complete amino‐acid sequence of soybean trypsin inhibitor (Kunitz) deduced here was compared with that of Bowman‐Birk inhibitor, another well‐known soybean proteinase inhibitor.
Metabolic Regulation of Steroidogenesis in Adrenocortical Carcinoma Cells of Rat
Authors: Sharma Rameshwar K.
Published: March 2005

The effect of adrenocorticotropin and cyclic AMP on the transformation of pregnenolone to corticosterone has been studied in adrenocortical carcinoma and isolated normal adrenal cells of rat. This has been carried out using both chemical and double labeling techniques. Adrenocorticotropin or cyclic AMP did not shown any effect on the corticosteroidogenesis produced from pregnenolone in the isolated adrenal cells whereas the synthesis of deoxycorticosterone and corticosterone was inhibited by adrenocorticotropin in adrenal tumor cells. A similar inhibitory effect of adrenocorticotropin was observed when progesterone was used as a precursor for corticosteroidogenesis in tumor cells but the incorporation of deoxycorticosterone into corticosterone was not inhibited by this substance. These studies, therefore, suggest that the site of inhibition of adrenocorticotropin on corticosterone synthesis in the tumor lies between progesterone and deoxycorticosterone.

Cyclic AMP also inhibited the synthesis of corticosterone from pregnenolone but no inhibition of deoxycorticosterone synthesis was observed. Similar results were obtained with N6,2′‐O‐dibutyryl adenosine 3′: 5′‐monophosphate. It is suggested that the mechanism of inhibition of Corticosteroidogenesis by adrenocorticotropin in the tumor may be different from that of cyclic AMP.
Purification and Properties of a Phenol Oxidase from the Blowfly Calliphora erythrocephala
Authors: Munn Edward A. , Bufton Susan F.
Published: March 2005

Preparations of diphenol oxidase obtained from larvae of the blowfly Calliphora erythrocephala obtained by previously described methods contained large amounts of the protein calliphorin. The diphenol oxidase was obtained pure in the form of the pre‐enzyme by ammonium sulphate precipitations and by the introduction of differential and density gradient centrifugation steps to remove the calliphorin. The pre‐enzyme had a sedimentation coefficient of 9.4 S; its molecular weight determined by gel filtration was 115000. By immunoelectrophoresis, the pre‐enzyme could be resolved into three components which moved towards the cathode; all three components showed enzyme activity after activation and had some antigenic determinants in common. The activated enzyme formed a streak towards the anode from the origin on agar gel electrophoresis and was immunologically distinct from the pre‐enzyme. On density gradient centrifugation the activated enzyme yielded a wide range of sedimenting species with sedimentation values of 16–17 S and upwards, the proportion of more rapidly sedimenting (larger) forms increasing with time after activation.
Kinetics of the Interaction between Pig‐Plasma Benzylamine Oxidase and Substrate
Authors: Lindström Anders , Olsson Bengt , Pettersson Gösta
Published: March 2005

The rapid absorbance changes in the visible region observed on mixing pig plasma benzylamine oxidase with substrate under anaerobic conditions are due to reduction of a single chromophore, centered around 470 nm, in a single reaction phase reflecting a rate‐limiting combination of substrate to the enzyme. The second‐order rate constant of 1 s−1 mM−1 for this process is unaffected by the presence of oxygen.

Under aerobic conditions, addition of substrate to the enzyme leads to 470‐nm absorbance changes exhibiting a biphasic time‐dependence. A rapid phase of transient formation of the enzyme · substrate complex is followed by a slow readjustment of the steady‐state level of free enzyme, caused by substrate and oxygen consumption during the reaction.

The kinetic data obtained provide strong evidence that substrate‐binding to benzylamine oxidase is practically irreversible. They further indicate the presence of a single kinetically significant substrate‐ and oxygen‐independent rate‐limiting reaction step in the catalytic mechanism, with a velocity constant in the order of 1 s−1.

A valence change of enzyme‐bound copper could not be detected at any stage of the reaction between benzylamine oxidase and substrate, irrespective of whether or not oxygen was present in the reaction solutions.
Polyelectrolyte Behaviour of Phosvitin
Authors: Giancotti Vincenzo , Quadrifoglio Franco , Crescenzi Vittorio
Published: March 2005

The results of spectroscopic and microcalorimetric measurements on dilute aqueous solutions of phosvitin are described. Our infrared and circular dichroism spectra show that the phosphoprotein chains may assume either random‐coiled or partially helical or β‐structures depending upon the pH. The transition to the low pH β‐form occurs with a distinct cooperative character.

The strongly interdependent ionization of fixed charges densely spaced along phosvitin chains, typical of highly charged polyelectrolytes, is well illustrated by the calorimetric enthalpy of protonation data.

The interaction of phosvitin with acridine orange in dilute aqueous solution (pH ∼5) has been studied by means of spectral and equilibrium dialysis experiments. For relatively low polymer to dye concentration ratios, bound acridine orange exhibits γ‐type spectrum thus suggesting aggregation of dye molecules onto the protein. Gradual decrease in the extent of bound acridine orange aggregation is produced by increasing the concentration of the protein; the monomerically bound form is hardly obtained, however, even at relatively high phosvitin concentrations. Binding curves reveal a cooperativity character for the dye‐protein interaction.

It is suggested that about 60% only of phosphomonoester side groups of phosvitin are available for acridine orange binding, these being characterized by a high “stacking” coefficient.

The results are tentatively discussed on the basis of present information on phosvitin chemical composition and conformation.
Magnetic‐Circular‐Dichroism Studies of Cytochrome c and Cytochrome b2
Authors: Risler Jean‐Loup , Groudinsky Olga
Published: March 2005

In spite of the very different conformation of the polypeptide chain around the heme‐ligand chelate, the magnetic circular dichroism (MCD) spectra of native cytochrome c and of its heme undecapeptide dissolved in 3.3 MN‐acetyl‐dl‐methionine are identical in the Soret region. This indicates that the MCD spectrum of the heme group (in the Soret band) is not sensitive to changes in the protein conformation as long as the proper ligands are bound to the iron atom.

On the contrary, the MCD spectrum of holocytochrome b2 differs from that of its derivatives lacking the FMN group; in the Soret region a MCD band at 365 nm, observed only with the holo‐enzyme, is assigned to the δ absorption band of the heme group rather than to a flavin transition. If, as shown with cytochrome c, the MCD spectra are independent of the protein conformation, the appearance of this band upon binding of FMN can be tentatively explained by a perturbation exerted on the heme group by the flavin itself. This would imply a close proximity of the two prosthetic groups.
The Production of Enzymes Involved in Exopolysaccharide Synthesis in Klebsiella aerogenes Types 1 and 8
Authors: Norval Mary , Sutherland Ian W.
Published: March 2005

The levels of several enzymes thought to be involved in synthesis of precursors for exopolysaccharide formation in Klebsiella aerogenes have been examined in two wild‐type strains and in non‐mucoid mutants. Enzymes involved in carbohydrate anabolism and catabolism, such as hexokinase and phosphoglucose isomerase, were present at highest levels in the parent strains. Enzymes presumed to be involved in forming precursors for several polysaccharides also had high specific activities, whereas those involved solely in exopolysaccharide synthesis were present at lower levels. Loss of ability to form exopolysaccharide had little effect on the specific activities of the enzymes tested. Some non‐mucoid mutants were deficient in one of the enzymes tested or had lowered specific activity, while the specific activities of the other enzymes were unaffected. The only enzymes whose specific activities were significantly increased under conditions favouring exopolysaccharide synthesis were UDP‐glucose pyrophosphorylase and GDP‐mannose pyro‐phosphorylase.
Oxidation of Acetaldehyde by Rat‐Liver Mitochondria in Relation to Ethanol Oxidation and the Transport of Reducing Equivalents across the Mitochondrial Membrane
Authors: Grunnet Niels
Published: March 2005

The primary product of the well‐known oxidation of acetaldehyde by rat liver mitochondria is acetate. About 50% of the acetate formed from acetaldehyde is converted to ketone bodies under the experimental conditions used.

The oxidation of acetaldehyde by rat liver mitochondria is catalyzed mainly by an aldehyde: NAD oxidoreductase present in the matrix compartment of the mitochondria. About 20% of the total mitochondrial acetaldehyde dehydrogenase activity may be confined to the outer membrane or the intermembrane space.

The enzyme has a broad substrate specificity, pH‐optimum at about pH 9.0 and is inhibited by tetraethylthiuram disulfide, chloral hydrate and arsenite. The enzyme is steroid‐insensitive.

Two Km‐values for acetaldehyde, below 0.1 μM and 1.0 mM, and two Km‐values for NADM+, 2 μM and 50 μM, were obtained with a partially purified 100000 ×g supernatant. The presence of two aldehyde dehydrogenases in rat liver mitochondria could not be shown.

Oxidation of acetaldehyde by rat liver mitochondria causes an inhibition of the production of 14Co2, from [14C]palmitate. The inhibition can be accounted for by dilution of the acetyl‐coenzyme A pool of the mitochondria.

The 3‐hydroxybutyrate/acetoacetate ratio during acetaldehyde oxidation is kept at the same level as during oxidation of oleate plus coenzyme A.

The mitochondrial acetaldehyde dehydrogenase activity is 80% of the total acetaldehyde dehydrogenase activity of rat liver. The results are discussed with regard to the oxidation of acetaldehyde and to the transfer of reducing equivalents from cytosol to mitochondria during ethanol metabolism.
Multiple Effects of Rifamycin Derivatives on Animal‐Cell Metabolism of Macromolecules
Authors: Busiello Enzo , Di Girolamo Adele , Di Girolamo Mario , Fischer‐Fantuzzi Lia , Vesco Cesare
Published: March 2005

A group of rifamycin derivatives were tested against the nucleolar and nucleoplasmic RNA polymerases from three types of animal cells. It was found that several compounds effectively inhibited such enzymes without substantial discrimination. In addition, the same derivatives inhibited polyphenylalanine synthesis in an animal cell‐free system.

Experiments with intact Hela cells showed that the synthesis of RNA, the processing of rRNA, the synthesis of DNA and the uptake of nucleosides were inhibited by rifamycins.

It is concluded that the specificity of rifamycin effects obtained at the concentrations that appear to be necessary to interfere with normal (or virus‐directed) processes in animal cells can be seriously questioned.
The Molecular Weights of the Mitochondrial‐Ribosomal RNAs of Saccharomyces carlsbergensis
Authors: Reijnders Lucas , Sloof Paul , Borst Piet
Published: March 2005

We have determined the molecular weights of the mitochondrial rRNAs from the yeast Saccharomyces carlsbergensis by methods not affected by the secondary structure of the RNA. Sedimentation equilibrium centrifugation gave 1.26 and 0.68 × 106; gel electrophoresis in 8 M urea at 60 °C, 1.30 and 0.70 × 106; gel electrophoresis in 98% formamide at 30 °C, 1.34 and 0.71 × 106; electron microscopy of dimethylsulfoxide‐denatured RNA, 1.22 and 0.60 × 106. The weighted average molecular weights of 1.30 and 0.70 × 106 for these mitochondrial rRNAs are much higher than those of the rRNAs from Escherichia coli (1.10 and 0.56 × 106). This difference is not easily accounted for by the endosymbiont theory of mitochondrial origin.
Subunit Structure and Amino‐Acid Composition of Crystallized Human‐Muscle Glycogen Phosphorylase
Authors: Assaf Sáid A. , Yunis Adel A.
Published: March 2005

The subunit structure and molecular weight of the a and b forms of human muscle glycogen phosphorylase were investigated.

Electrophoresis in the presence of sodium dodecylsulfate gave a molecular weight of 94 500 for the monomer. When studied at 1 mg/ml and 20 °C by high‐speed sedimentation equilibrium, sucrose density gradient ultracentrifugation and specific activity‐concentration dependence, both a and b forms of the human enzyme were found to exist as dimers.

A slight association of human phosphorylase a was observed in the high‐speed sedimentation equilibrium values of the z‐average molecular weight (262000 for Mmacr;zvs 188000 daltons for Mmacr;w). Some association of the a enzyme was also detected by schlieren sedimentation velocity when the enzyme was studied at 5 mg/ml.

Human phosphorylase b could be fully associated to tetramer at 20 °C when Pi, Mg2+ and AMP were added but not in the presence of NaF and AMP which were found to be effective in tetramerizing rabbit phosphorylase b.

Another important difference from rabbit phosphorylase was the significantly lower proline content of the enzyme from human muscle.
The Subunit Structure of the Escherichia coli K12 Pyruvate‐Dehydrogenase Complex
Authors: Vogel Otto , Henning Ulf
Published: March 2005

It is shown that the dihydrolipoamide dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex consists of identical polypeptide chains (molecular weight: 5.6 × 104).

Amino acid and tryptic peptide analyses gave a minimum chemical molecular weight agreeing with that based on FAD content of the enzyme or that obtained by dodecylsulfate polyacrylamide gel electrophoresis. Carboxypeptidase degradation revealed one C‐terminus ‐Lys‐Lys‐CO2H per 56000 molecular weight of protein.
Thyrotropin–a Lipolytic Hormone?
Authors: Stevenson Patricia M. , Fernie Aveline , Kirkham K. E. , Ensor Janice
Published: March 2005

It is widely accepted that human thyrotropin causes lipolysis in fat cells isolated from a variety of sources. An investigation into the use of this lipolytic activity as a means of tracing thyrotropin through a fractionation procedure revealed that the biological and lipolytic activities of this hormone are separable and are probably, therefore, caused by two different entities. It is obvious that the measurement of lipolysis is an unsuitable assay for thyrotropin. It was shown that this lipotropic compound is antigenic and likely also to interfere with the accuracy of a radio‐immunoassay for thyrotropin.
Molecular Weight and Subunit Structure of the Malate‐Lactate Transhydrogenase
Authors: Allen S. H. George
Published: March 2005

The molecular weight of the malate‐lactate transhydrogenase as determined by sedimentation‐diffusion and high‐speed‐equilibrium methods is approximately 70000. Dissociation of the protein with guanidine‐HCl, sodium dodecylsulfate or citraconic acid anhydride yields a homogeneous preparation for which molecular weight values ranging from 30000 to 43000 have been found. The transhydrogenase thus appears to be a dimer, composed of two subunits and an NAD+/NADH equivalent binding weight of 35000.

Addition of a 33‐fold excess of citraconic acid anhydride based on the lysine content of the transhydrogenase was necessary for complete dissociation. This was accompanied by a complete loss of enzymatic activity and release of the bound NAD+/NADH, which, in the native enzyme, is very tightly bound. Attempts to restore enzymatic activity as well as to reform the dimer structure after removal of the citraconyl groups, even in the presence of NAD+ were unsuccessful. Partial citraconylation while destroying most of the enzymatic activity did not cause release of the NAD+/NADH prosthetic groups. The enzymatic activity of these preparations can be partially restored upon removal of the citraconyl groups. Apparently, removal of the prosthetic groups is associated with irreversible denaturation and the transhydrogenase represents a case where native configuration is extremely dependent on the binding of the prosthetic group.
Thioredoxin Reductase from Rat Liver
Authors: Larsson Agne
Published: March 2005

The thioredoxin system was first identified in microorganisms where it participates in redox reactions, such as ribonucleotide reduction, transferring electrons from NADPH to the product formed. It contains two protein components, thioredoxin and thioredoxin reductase. A similar hydrogen transport system has now been demonstrated in rat liver. Using the liver thioredoxin previously isolated as a means of assaying thioredoxin reductase this enzyme has now been purified from the same source. A 100‐fold‐purification of thioredoxin reductase was obtained by treatment at pH 5 and chromatography on Sephadex and on DEAE‐cellulose. Thioredoxin reductase was found to be specific for NADPH.

Ribonucleotide reductase, thioredoxin and thioredoxin reductase constitute a system necessary for the biosynthesis of deoxyribonucleotides. During liver regeneration, the level of ribonucleotide reductase was increased approximately 20‐fold. In contrast, the levels of thioredoxin and thioredoxin reductase remained constant.
Propriétés des formes allongées de l'acétylcholinestérase en solution
Authors: Bon Suzanne , Rieger François , Massoulié Jean
Published: March 2005

We present here several molecular properties of the asymmetric acetylcholinesterase forms, which we described in our previous publications [1–3]. From molecular sieve experiments, we determined the Stokes radius and compared its value with that calculated from micrograph measurements [3] for the A form which is a tetramer associated to an elongated element, called the “tail” (structure en grappe). A good agreement is obtained with a prolate ellipsoid of identical volume and length; this indicates that the acetylcholinesterase is, in solution, a rigid structure.

We also estimated the partial specific volume, v̄, of acetylcholinesterase, by two independent methods: first, by the difference of sedimentation velocity in gradients of different overall density (H2O, 2H2O); secondly by absolute density measurements in CsCl equilibrium gradients. Both methods are in reasonable agreement and yield a value of 0.74–0.75 for all forms of the enzyme. This enabled us to calculate, from sucrose gradient data, the sedimentation constant in water at 20 °C. Combining the values of the Stokes radius, sedimentation constant, and partial specific volume, we calculated the molecular mass of A, C, and D as 430000, 780000, and 1100000. These values justify our hypothesis that these forms possess one, two, and three tetramers, associated with the “tail”.

The methods used in this work are all based on activity measurements, which is a major advantage since only minute quantities of protein are needed. However, these are mainly comparative determinations, and it is difficult to ascertain the precision of the published data for the reference proteins.
Chirooptische Eigenschaften von schwefelhaltigen Aminosäuren
Authors: Jung Günther , Ottnad Michael , Rimpler Manfred
Published: March 2005

For the sulfur containing amino acids lanthionine, cystathionine, allocystathionine, djencolic acid, felinine, S‐alkyl cysteines and S‐alkyl homocysteines the circular dichroism spectra are recorded. In order to evaluate the effects influencing the carboxyl and in particular the sulfide chromophore a detailed investigation of the pH and temperature dependency is made. Contrary to the bands of the ultraviolet absorption and the optical rotatory dispersion, the Cotton effects of the sulfide and carboxyl chromophores can be used with advantage for comparative studies. The influence in the distance between the chiral centers and the chromophores on the ellipticities of the dissymmetrically disturbed sulfide group is described. The chiroptical behaviour of these sulfur containing amino acids in aqueous solutions is discussed with respect to their structure, dissociation equilibria, configuration and conformation.
Basic and Non‐Basic Substrates of Carboxypeptidase B
Authors: Zisapel Nava , Kurn‐Abramowitz Nurith , Sokolovsky Mordechai
Published: March 2005

The kinetics of porcine‐carboxypeptidase‐B‐catalyzed hydrolysis of several N‐blocked peptides that differ in size and in C‐terminal amino acid were examined. The data demonstrate different kinetic behaviour of carboxypeptidase‐B‐catalyzed hydrolysis of basic and non‐basic substrates. These studies are consistent with our proposed multiple loci model. The results also show a number of differences between carboxypeptidases A and B, as well as several similarities.
Modification of Carboxyl Groups in Pepsin
Authors: Matyash Ludmila F. , Ogloblina Olga G. , Stepanov Valentin M.
Published: March 2005

The chemical modification of pepsin carboxyl groups using N‐(2,4‐dinitrophenyl)‐hexamethylenediamine or compound (I) in the presence of a water‐soluble 1‐cyclohexyl‐3‐[2′‐(N‐methylmorpholino)‐ethyl]‐carbodiimide p‐toluenesulphonate or compound II at pH 5.5 resulted in the incorporation of one molecule of I per molecule of pepsin with a concomitant drop of activity to about 40% when measured against hemoglobin and 50% against N‐acetyl‐l‐phenylalanyl‐l‐tyrosine as substrates. It was established that the modification of three carboxyl groups took place: β‐carboxyl group of the aspartic acid residue, γ‐carboxyl group of the glutamic acid residue and the α‐carboxyl group of C‐terminal alanine. It is assumed that this reaction is preceded by the hydrophobic binding at some site, specific for the pepsin hydrophobic zone, with a subsequent reaction of I with the carboxyl groups localized near this zone.
Isolation of High‐Molecular‐Weight DNA from Mammalian Cells
Authors: Gross‐Bellard Maria , Oudet Pierre , Chambon Pierre
Published: March 2005

A preparative method for isolating high‐molecular‐weight DNA from animal cells is described. This method is based on the use of proteinase K, a powerful proteolytic enzyme with a broad action spectrum, which is very active in the presence of sodium dodecylsulfate and ethylene‐diamine tetraacetate. The DNA preparation is free of RNA, protein and degrading enzymes. The number‐average molecular weight of the native DNA is 190 × 106, whereas it is 90 × 106 for single‐stranded DNA, indicating that the DNA molecules do not contain single‐stranded nicks. The native DNA molecules range in molecular weight from 40 × 106 to more than 500 × 106.
Oxidative Phosphorylation in Bacteria which Contain Different Cytochrome Oxidases
Authors: Meyer David J. , Jones Colin W.
Published: March 2005

The oxidative phosphorylation efficiencies of seven species of bacteria, selected on the basis of their cytochrome oxidase composition, were determined from measurements of growth efficiencies with respect to oxygen (YO2) and →H+/O quotients of washed cell suspensions.

Organisms which contained either cytochrome oxidase aa3 (Bacillus subtilis, Microbacterium thermosphactum) or o (Xanthomonas hyacynthi, Acinetobacter lwoffi, Escherichia coli and Kurthia zopfii) as their sole terminal oxidase exhibited P/O ratios of approximately 3 whereas Acetobacter T71, which contained cytochrome oxidase a1, as its major oxidase, exhibited a P/O ratio of approximately 1.

The synthesis of cytochrome oxidases a1, and a2 by E. coli under oxygen‐limited conditions was accompanied by a sharp decrease in whole‐cell energy‐conversation efficiency. A similar decrease in efficiency was also observed as a result of the increased use by K. zopfii of a second oxidase, insensitive to cyanide and carbon monoxide, which has not hitherto been reported.

The possibility that the different cytochrome oxidases may terminate respiratory chains of different redox span or may be associated with separate phosphorylating or non‐phosphorylating chains is discussed in relation to the general properties and physiological functions of these oxidases.
Nucleoside‐Catabolizing Enzymes in Salmonella typhimurium Induction by Ribonucleosides
Authors: Nygaard Per
Published: March 2005

Addition of cytidine, uridine or adenosine to glycerol‐grown cultures of Salmonella typhimurium induces the synthesis of cytidine deaminase and uridine phosphorylase (the cyt‐enzymes) as well as the synthesis of thymidine phosphorylase, deoxyriboaldolase, phosphodeoxyribomutase and purine nucleoside phosphorylase (the deo‐enzymes).

The inducing effect of adenosine is more pronounced in mutant strains deficient in adenosine catabolism.

A regulatory mutant which synthesizes the cyt‐enzymes constitutively has been isolated. This mutant also synthesizes the deo‐enzymes in elevated amounts. However, the level of the deo‐enzymes in the regulatory mutant like in wild‐type cells can be raised further if thymidine is added to the medium.

The synthesis of the cyt‐enzymes appears to be sensitive to catabolite repression.

From induction experiments carried out in mutant strains restricted in their metabolism of cytidine and uridine it appears that both compounds may act as inducer.

Strains unable to metabolize cytidine or uridine contained increased levels of the cyt‐ and the deo‐enzymes even in the absence of external inducer. This is most likely due to endogenous induction by cytidine or uridine.