bluuebunny/crossref_metadata_2025 huggingface dataset:
ADP‐Ribosylation of DNA‐Dependent RNA Polymerase of Escherichia coli by an NAD+: Protein ADP‐ribosyltransferase from Bacteriophage T4
Authors: ROHRER Hermann , ZILLIG Wolfram , MAILHAMMER Reinhard
Published: March 2005

A protein from bacteriophage T4 responsible for the alteration of host DNA‐dependent RNA polymerase and absent in T4 alt− phage was purified from T4 phage and enriched from T4‐infected cells. It is injected during infection together with the known internal proteins. It has a molecular weight of about 70000 and catalyses the release of nicotinamide and the transfer of the ADP‐ribosyl moiety from NAD+ to arginyl residues of various proteins including itself.

RNA polymerase from Escherichia coli accepts ADP‐ribosyl residues in all four subunits; the α subunit reacts with very high specificity. Only half of the α subunits are labelled, 45 with one, 5 % with two residues. The main product shows the same electrophoretic mobility as α subunits altered or modified in vivo. The a subunit in modified RNA polymerase is no acceptor.
The Trypsin and Chymotrypsin Inhibitors in Chick Peas (Cicer arietinum L.)
Authors: BELEW Makonnen , PORATH Jerker , SUNDBERG Lars
Published: March 2005

From a crude extract of chick peas (Cicer arietinum L.) inhibitors of trypsin and chymotrypsin were isolated by affinity chromatography on a column of trypsin‐Sepharose 6B. The content of inhibitors was found to be 1.5 g/kg. They were further separated into six isoinhibitors by ion‐exchange chromatography on DEAE‐Sephadex A‐25. Two of the isoinhibitors accounted for about 50 % of the isolated inhibitors and were further purified to a homogeneous state.

The isoinhibitors had a molecular weight of about 10000 as determined by molecular‐sieve chromatography on Sephadex G‐75. They were stable towards extremes of pH and temperatures up to 75 C or towards digestion by pepsin. They were also stable in 6 M urea but not in 6 M guanidineHCI. The intact inhibitors were destroyed when the peas were cooked at 100°C or when they were toasted at 130°C.

The four major inhibitors had similar amino acid compositions and did not contain detectable amounts of free sulfhydryl groups, tryptophan or carbohydrate. Cysteine is the dominant amino acid residue in all of them and accounted for about 20 % of their amino acid content. The isoelectric point of the isoinhibitors lies in the range of pH 4.9‐8.6 and two of the major inhibitors had isoelectric points of pH 4.75 and pH 4.96. They inhibited chymotrypsin to the same extent but differed in their inhibitory activities towards trypsin, indicating that they are mixtures of native and trypsinmodified forms and that they probably have separate sites for the two enzymes. They did not inhibit other proteolytic enzymes belonging to two groups (i.e., serine or cysteine enzymes) or originating from different sources (i.e., animals, plants or bacteria).
The Major Proteins of the Escherichia coli Outer Cell‐Envelope Membrane
Authors: GARTEN Wolfgang , HINDENNACH Ingrid , HENNING Ulf
Published: March 2005

The cyanogen bromide fragments of protein I, a major protein of the Escherichia coli outer cell envelope membrane, have been isolated and characterized. There appear to be two methionineserine or methionine‐threonine sequences causing incomplete cleavage but complete conversion of methionine to homoserine. Largely due to the existence of these overlapping fragments the order of 5 of the 6 fragments present could be deduced.

None of the fragments exhibits any remarkable low degree of polarity, and the tryptic fingerprint of the largest fragment (comprising about 60% of protein I) also does not show any conspicuous large fraction of lipophilic peptides. It is concluded that the domain of protein I that may be buried in the lipid phase of the outer membrane in all likelihood is not very large, and there is, in fact, no definite proof yet that protein I is a membrane protein sensu stricto.
Time‐Dependent Inhibition of Diamine Oxidase by Carbonyl‐Group Reagents and Urea
Authors: CRABBE M. James C. , CHILDS Robert E. , BARDSLEY William G.
Published: March 2005

The behaviour of several carbonyl group reagents and urea as time‐dependent inhibitors of both pig kidney and human placental diamine oxidase is described.

Plots of log (vt/v0) against time were not linear with these reagents as the usual theories predict.

This was particularly the case with aminoguanidine and phenylhydrazine and a thorough study of the effects of these compounds on the human placental diamine oxidase is described.

By applying a new theory for time‐dependent inhibition, the inhibition of diamine oxidase by aminoguanidine and phenylhydrazine is adequately accounted for.

The time‐dependent recovery of activity on addition of sodium pyruvate suggested that the compounds used are acting solely as carbonyl group reagents, inhibiting by Schiff‐base formation at the active‐site carbonyl group.
The Interaction of Organic Phosphates with Human and Chicken Hemoglobin
Authors: BRYGIER Jeanne , DE BRUIN Simon H. , VAN HOOF Peter M. K. B. , ROLLEMA Harry S.
Published: March 2005

In this study the binding properties of inositol hexaphosphate and 2,3‐bisphosphoglycerate to chicken and human deoxyhemoglobin and carboxyhemoglobin were compared. It appeared that in all cases the binding to chicken hemoglobin is much stronger than to human hemoglobin. This is very probably due to the fact that 4 out of the 12 residues, responsible for the binding of phosphates in chicken hemoglobin, are arginines. These are absent in human hemoglobin, where the binding site is made up by only 8 residues.

For chicken hemoglobin one strong binding site could be observed in both unliganded and liganded hemoglobin. From these observations we conclude that the same binding site is involved in both the oxy‐ and deoxy structure showing a different affinity to phosphates in the two conformational states. For human hemoglobin we reached the same conclusion.
Hepatic Nucleases
Authors: BARTHOLEYNS Jacques , PEETERS‐JORIS Chantal , BAUDHUIN Pierre
Published: March 2005

In the large granule fraction of rat liver, the density distribution of inhibitor‐sensitive neutral ribonuclease is similar to that for acid hydrolases and its density distribution is similarly modified by Triton WR‐1339 accumulation in lysosomes. Particulate neutral ribonuclease is latent; the enzyme is unmasked by very low digitonin concentrations or hypoosmotic shock. These observations demonstrate that the bulk of liver neutral ribonuclease is associated with the lysosomal system.

In view of the neutral pH optimum of the enzyme and of some particularities of its distribution in fractionation experiments, the possibility of an extrahepatic origin of neutral ribonuclease has been investigated. After partial pancreatectomy, a significant decrease is observed in both plasma and liver neutral ribonuclease. The effect is specific, for it does not occur for other lysosomal enzymes. Also, labelled bovine pancreatic ribonuclease, when injected intravenously, is taken up by the liver. The sedimentable labelled enzyme has a density distribution similar to the distribution of other foreign proteins, horseradish peroxidase or yeast invertase. These results are explained by the uptake of plasmatic neutral ribonuclease from pancreatic origin by the liver.
DNA Synthesis in Circulating Erythroblasts of Anemic Duck
Authors: MODAK Sohan P. , COMMELIN Dominique , GROSSET Lise , MONNAT Marianne , IMAIZUMI Marie‐Thérése , SCHERRER Klaus
Published: March 2005

In the circulating blood of anemic ducks, 5 % of all erythroid cells synthesize DNA. Immature erythroblasts, at all stages of differentiation, synthesize DNA although to a varying degree, while reticulocytes and erythrocytes do not. In the erythroid cell population labeled in vitro 2 h with 32Pi, half of the labeled DNA sediments as small‐molecular‐weight molecules, suggesting that these molecules fail to integrate into the high‐molecular‐weight components.

Labeled DNA is found in the cytoplasmic postmitochondrial fractions and it is in a form of deoxyribonucleoproteins which cosediment with ribosomes as well as subribosomal particles in sucrose gradients. However, fixation with HCHO and centrifugation to equilibrium in CsC1 gradient of these particles shows that the deoxyribonucleoprotein bands at the density different than the ribosomes and, thus, not physically linked to them. In EDTA‐dissociated ribosomes, the deoxyribonucleoprotein particles cosediment with ribosomal subunits in such a way that the larger the particle, the larger the molecular weight of the DNA cosedimenting with it.

The specific radioactivity of the cytoplasmic ribosome‐derived and postribosomal‐particle‐derived DNAs and the small molecular‐weight nuclear DNA is similar and 10‐20‐fold higher than that of the bulk nuclear DNA. The former three DNA species sediment between 4‐14 S. It is concluded that the cytoplasmic nonmitochondrial DNA species are of the nuclear origin. Less than 0.5% of the total cellular nonmitochondrial DNA can be purified from the nucleus and the cytoplasm as fast‐labeled small‐molecular‐weight components. All of the cellular nonmitochondrial DNA species band at the same mean buoyant density in Cs2SO4/urea gradients. All behave as native structures in hydroxyapatite and contain less than 5% of their length as single‐stranded regions.
Inhibition of Collagenase from Achromobacter iophagus by Synthetic Peptides
Authors: SVENSSON Birte , SIFFERT Odile , KEIL Borivoj
Published: March 2005

Peptides of the general structure Pro‐Leu‐X‐Pro have been synthesized, with proline in l or d‐form and with either glycine or sarcosine in the position of X. Their inhibitory action on the cleavage of the peptide Cbz‐Gly‐Pro‐Leu‐Gly‐Pro by pure collagenase from Achromobacter iophagus was studied.

Competitive inhibition was found in all cases, the most efficient by Pro‐Leu‐MeGly‐Pro. One product of cleavage of the synthetic substrate, Cbz‐Gly‐Pro‐Leu, also inhibits the enzyme.
Turnover of Polyadenylated Messenger RNA in Fission Yeast
Authors: FRASER Ronald S. S.
Published: March 2005

Polyadenylated RNA was isolated from fission yeast (Schizosaccharomyces pombe) total RNA using oligo(dT)‐cellulose, and was studied as a model for messenger RNA. The half‐life of polyadenylated RNA was measured by two independent methods. (a) The rate of labelling of polyadenylated RNA during incubation of cells with [5 3H]uridine was measured. A half‐life of 40‐45 min was found by comparing the experimental data with theoretical curves calculated for labelling of RNAs with various half‐lives. The influence of precursor‐pool specific activity on RNA labelling kinetics is considered. (b) Cells were labelled with [5‐3H]uridine then further RNA synthesis was inhibited by addition of 8‐hydroxyquinoline. The rate of loss of radioactivity from polyadenylated RNA indicated a half‐life of 50 min. The half‐life found by these two methods is about one‐third of the cell doubling time, and is much longer than previous estimates by indirect methods of yeast messenger RNA half‐life. Both experimental methods provided evidence for the existence of two populations of polyadenylated RNA. The larger population has a half‐life of 40‐50 min; a much smaller population is probably turning over more rapidly.

After inhibition of RNA synthesis by 8‐hydroxyquinoline, the rate of total protein synthesis declined much more rapidly than the polyadenylated RNA content of the cells. However, 60 min after inhibition of RNA synthesis there was a small rise in the rate of protein synthesis. These data are interpreted as evidence for mechanisms controlling protein synthesis which operate at the level of messenger RNA translation.
The Tryptophan Synthase from Escherichia coli
Authors: KIRSCHNER Kasper , WISKOCIL Robert L. , FOEHN Martha , REZEAU Laurel
Published: March 2005

An improved method is described for the purification of the α‐subunit of tryptophan synthase from Escherichia coli. The standard manganese chloride and acid‐precipitation steps have been replaced by rapid and efficient chromatographic procedures.

Indoleethanol phosphate, indolepropanol phosphate and indolebutanol phosphate have been synthesized. They are not cleaved by tryptophan synthase and are strictly competitivee inhibitors versus indoleglycerol phosphate. The inhibition constant decreases as the number of methylene groups in the side chain increases. This may reflect an improved accommodation of the indole and phosphate moieties to their respective subsites. The difference spectra generated by binding indole, indoleglycerol phosphate and indolepropanol phosphate to the α‐subunit are very similar. This reflects the transfer of the indole moiety to an hydrophobic environment within the active center. The binding of indolepropanol phosphate to the α2β2‐complex perturbs the spectrum of pyridoxal 5′‐phosphate located in the β2‐subunit. This demonstrates direct or indirect interactions between the component active sites.

Binding studies by spectrophotometric titration and equilibrium dialysis with indolepropanol [32P]phosphate show that there is only one binding site per equivalent of α‐subunit. Complex formation with the β2‐subunit increases the affinity of the α‐subunit for indolepropanol phosphate. It is a general consequence of protein‐protein interaction in this system.
Heterogeneity of Human Milk β(1 – 4)‐d‐Galactosyltransferase
Authors: PRIEELS Jean‐Paul , MAES Eddy , DOLMANS Marcel , LEONIS José
Published: March 2005

β(1 – 4)‐Galactosyltransferase from human milk (the A protein of lactose synthase) has been found to be heterogeneous when fractionated by affinity chromatography against insolubilized a‐lactalbumin, using a linear gradient of decreasing N‐acetylglucosamine concentration. Three forms were isolated. Molecular weights of the different species, as determined by sodium dodecylsulphate gel electrophoresis, were found to be 38000, 43000 and 50000. The 38000 and 50000 species were studied for their catalytic ability to synthesize either lactose in the presence of α‐lactalbumin, or N‐acetyllactosamine in the presence and absence of the ‘specifier’ protein.

Appreciable difference was observed between the two enzyme forms with respect to their catalysis of lactose synthesis with a‐lactalbumins from various sources. Differences in the rate of production of N‐acetyllactosamine in the presence of α‐lactalbumin were also observed. For the lowest‐molecular‐weight species it was found that the inhibitory effect of a‐lactalbumin upon N‐acetyllactosamine syn‐ thesis becomes an activating effect at higher α‐lactalbumin concentrations, while no such inversion was observed for the other species.

The results suggest that the conformation at the site of association of the enzyme with the acceptor saccharide or α‐lactalbumin has been changed, presumably by a partial enzymic hydrolysis.
Nitration of Tyrosyl Residues in Human α‐Lactalbumin
Authors: PRIEELS Jean‐Paul , DOLMANS Marcel , LEONIS José , BREW Keith
Published: March 2005

α‐Lactalbumin isolated from human milk was reacted with tetranitromethane in molar excess of 8–32 mol/mol of tyrosine. After gel filtration on Sephadex G‐75, followed by chromatographic fractionation using DEAE‐Sephadex A‐25, three main components were separated, which differed from one another in the extent of nitration.

These protein fractions were found to contain, respectively, one and two nitrotyrosine residues, or two nitrotyrosine residues together with one nitrotryptophan. The lactose synthase specifier activity of each of these components was measured and compared with that of unsubstituted cr‐lactalbumin.

Comparison of kinetic parameters showed the chemically modified proteins to be only slightly less active when tyrosines were the sole residues modified. In sharp contrast the additional nitration of a single tryptophan residue totally abolished the specifying activity of α‐lactalbumin.

Circular dichroism spectra of the tryptophan derivative revealed some structural alteration when compared with the other two and with the native protein. The conclusion could also be confirmed by using a double‐immunodiffusion technique. After hydrolysis of the derivatives with thermolysin, it was possible to localize the substituted residues in the known sequence of human a‐lactalbumin. Tyrosine‐103 was found to be more easily nitrated than tyrosine‐18. These two residues seem, there‐fore, to be on the outer surface of the molecule and more exposed than tyrosine‐36 and tyrosine‐50.

Some precautions are indicated in the use of tetranitromethane as a nitrating agent on the basis of complex products observed in the nitration of the free amino acids tyrosine and tryptophan and their derivatives.
Some Actions of Purified Toxins from Bungarus fasciatus Venom on Isolated Pig‐Heart Mitochondria
Authors: LEBLANC Pierre , LIEVREMONT Maurice
Published: March 2005

In the presence of succinate as an oxidation substrate, neurotoxins α, β and γ induce the following. Firstly, an increasing stimulation of oxygen uptake, which is potentiated by 25 μM Ca2+. Mg2+ 1.3 mM completely inhibits the effect of toxin α but not of toxins β and γ. Secondly, a depletion of the Mg2+, Ca2+ and K+ content of the water‐soluble and membrane‐bound mitochondrial compartments, following complex kinetics, which suggest a multistep interaction mechanism of the toxins with the mitochondria. 25 μM Ca2+ also potentiates the effect of the toxins on these ionic flows. Thirdly, no decrease of turbidity with toxin α, and a limited decrease with toxins β and γ.

In the absence of respiration, the neurotoxins induce a cationic depletion, the kinetics of which are different than with succinate, suggesting an instantaneous maximal effect on the inner mem‐brane. Toxins β and γ (but not α) induce, under these conditions, a turbidity decrease of large amplitude, which is proportional to the amount of toxin added and tends to reach a maximum. With γ toxin this turbidity decrease is faster than the rate of water uptake (which never exceeds 18%) indicating that it is due rather to structural modifications than to swelling. The same is observed with β toxin, provided the mitochondria1 protein concentration to be lower than 0.7 mg/ml. For higher concentrations, a continuous decrease of turbidity together with a considerable uptake of water probably reflects the onset of phospholipasic activities.

It is postulated that structural modifications of the mitochondrial membranes are initiated which lead to the loss of their selective impermeability. The simultaneous loss of respiratory control with succinate may be due to the direct (though Ca2+‐potentiated) displacement of the fraction of the membrane‐bound Mg2+ ions which controls its energy‐transducing properties.

In addition, correlations between the effects of the toxins on mitochondria and their neuro‐toxicity in vivo are discussed.
Calcium‐Related Changes of Enzyme Activities in Energy Metabolism of Cultured Embryonic Chick Myoblasts and Myotubes
Authors: SCHUDT Christian , GAERTNER Udo , DÖLKEN Gottfried , PETTE Dirk
Published: March 2005

Changes in activity of enzymes involved in energy metabolism have been determined in unfused, fused as well as in fusion‐inhibited chick embryo muscle cells in vitro. Functionally related enzymes which supposedly are coded by “gene clusters” show a similar degree and rate of enzyme activity increase. Hexokinase and glucose‐6‐phosphate dehydrogenase reveal only slight activity changes during muscle cell development under the conditions studied. The elevation of phosphofructokinase can be distinguished from that of the other glycolytic enzymes by its higher rate of increase and from that of phosphorylase by its time‐course of activity change. The Ca2+ dependence of the phos‐phorylase activity increase runs parallel to myoblast fusion rate. Experiments in which calcium was removed from cultures which had reached the final morphological state of mature myotubes 24 h after onset of fusion show that increases of enzyme activities are irreversible and that these increases proceed at unchanged rates. Experimental evidence suggests that although fusion and enzyme syntheses may be uncoupled, both are similarly triggered by being dependent on Ca2+ concentration.
Ligand Binding of Organic Sulfides to Microsomal Cytochrome P‐450
Authors: NASTAINZCYK Wolfgang , RUF Hans Heinrich , ULLRICH Volker
Published: March 2005

Octyl methyl‐, butyl methyl‐ and pentamethylene sulfide react with about 50 % of oxidized cyto‐chrome P‐450 in liver microsomes from phenobarbital‐pretreated rats by formation of optical dif‐ference spectra with maxima at 435 and 552 nm and concomitant shifts in the electron paramagnetic resonance spectrum. Reduction by NADPH or sodium dithionite yielded a Soret absorption band at 449 nm and α and β bands at 573 and 545 nm, respectively. The ligand metyrapone and the substrate n‐octane competitively inhibited the formation of these difference spectra and pentamethylene sulfide was a strong competitive inhibitor of the O‐dealkylation of 7‐ethoxycoumarin. These results indicate a direct ligand binding of the sulfides to cytochrome P‐450 with concomitant blocking of the hydrophobic substrate binding site. Some sulfides did not interact as ligands but as substrates, in variation, however, with the source of microsomes.
An ATPase Depending an the Presence of Single‐Stranded DNA from Mouse Myeloma
Authors: HACHMANN Henning J. , LEZIUS Axel G.
Published: March 2005

An ATPase was purified from mouse myeloma MOPC 70E the activity of which depends an the presence of single‐stranded DNA and divalent cations such as Mg2+, Mn 2+, Ca 2+, Ni2+ or Fe2+. The enzyme splits both ribonucleoside and deoxyribonucleoside triphosphates but preferentially ATP and dATP yielding nucleoside diphosphates and inorganic phosphate. The enzyme has an absolute requirement for single‐stranded DNA. Alternating double‐stranded polydeoxynucleotides are only slightly effective, and native double‐stranded DNA, single‐stranded and double‐stranded RNAs as well as DNA. RNA hybrids are ineffective in stimulating the ATPase. The enzyme was further characterized by sedimentation in a sucrose density gradient (S20, w= 5.5 S) and by isoelectric focussing in an ampholine pH gradient (pI = 6.5).
Purification and Properties of an Esterase B4 from Human Liver
Authors: COATES Paul M. , EDWARDS Yvonne H. , HOPKINSON David A.
Published: March 2005

A butyryl esterase, designated B4, has been purified from human liver and some of its properties described. The activity of this enzyme comprises 0.48 of the total butyryl esterase activity found in human liver.

Esterase B4 has been distinguished from other butyryl esterases by its preference for the esters of the fluorogenic compounds 4‐methyl umbelliferone and fiuorescein over naphthyl esters as substrates. Other distinguishing features of this esterase include a relatively high pI (pH 8.7) a monomeric structure of low molecular weight (20000) and high solubility in solutions of ammonium sulphate.
Preparation and Characterization of the Active Derivative of Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) with the Reactive Site Lysine‐15 – Alanine‐16 Hydrolyzed
Authors: JERING Helmut , TSCHESCHE Harald
Published: March 2005

The derivative of the trypsin‐kallikrein® inhibitor (Kunitz), TKI+, was prepared with the reactivesite peptide bond Lys‐15‐Ala‐16 hydrolyzed. This was achieved by selective borohydride reduction of the Cys‐14‐Cys‐38 disulfide bond, followed by tryptic cleavage of the reactive‐site peptide bond, air reoxidation of the half‐cystine residues, and purification by ion‐exchange chromatography. The derivative corresponds to the hypothetical ‘modified’ inhibitor TKI+, which so far could not be obtained from virgin inhibitor by a direct modification reaction (partial proteolysis).

The derivative isolated was homogeneous as revealed by amino acid analysis, disc electrophoresis, inactivation by carboxypeptidase B, and inactivation by sodium borohydride reduction. The inhibitory activity of the sodium‐borohydride‐reduced inhibitor was fully recovered after air reoxidation. The site of cleavage in the inhibitor was confirmed by performic acid oxidation and subsequent isolation of the two corresponding peptides containing residues 1–15 and 16–58 of the entire polypeptide chain. From several aminopeptidases tested only aminopeptidase K rapidly cleaved Ala‐16 and Arg‐17 from the modified inhibitor and at a reduced rate Ile‐18. Des‐(Ala16,Arg17)‐inhibitor and des‐Ala16‐inhibitor are both lacking a strong inhibitory activity against bovine trypsin. This indicates a decrease in the association constant by factor of at least 108 ‐1010. The reactive‐site‐modified inhibitor is not subject to further enzymic breakdown and therefore is a permanent inhibitor of trypsin. However, the modified inhibitor forms the inactive complex much slower than virgin inhibitor.

In the modified inhibitor the hydrolyzed peptide bond was resynthesized to yield virgin inhibitor by forming the complex with trypsin and subjecting the complex to kinetic control dissociation. This proves that the bond Lys‐15‐Ala‐16 is at the reactive site of this inhibitor. Preparation of a modified and still active inhibitor (Kunitz) is in agreement with the general model proposed for the interaction of proteinase‐inhibitor‐proteinase interactions. This presents new evidence that this model is generally applicable.
A Mutant Strain of Chlamydomonas reinhardi Exhibiting Altered Ribulosebisphosphate Carboxylase
Authors: NELSON Patricia E. , SURZYCKI Stefan J.
Published: March 2005

A mutant, ac i72, of Chlamydomonas reinhardi possessing an altered ribulosebisphosphate carboxylase and unable to grow on minimal medium has been isolated and characterized. Comparison of ribulosebisphosphate carboxylase purified from both wild type and ac i72 strains is given. The enzyme from ac i72 shows alterations in several characteristics : (a) the specific activity is reduced to 35% that of wild type, (b) the V for both substrates is reduced 3–6‐fold, (c) the Mg2+ requirement for maximal activity is 3 times greater, (d) the inhibitory effect of Cl− is greater, and (e) the isoelectric point is changed (6.0 for wild type and 5.8 for ac i72). However, the ribulosebisphosphate carboxylase from ac i72 is identical to that from wild type with respect to pH requirement, temperature sensitivity, subunit structure, and sedimentation characteristic.

Other photosynthetic properties of wild type and ac i72 cells were also compared. CO2 fixation in ac i72 in vivo is reduced proportionally to the reduction in activity of the enzyme, but the level of O2 evolution is the. same as in wild‐type cells. Photosynthetic electron transport, 70‐S ribosome content, and chlorophyll content are unaltered in ac i72. The chloroplast ultrastructure of ac i72 cells is distinctly different from that of wild‐type cells. The inheritance of the mutation is Mendelian.
Purification and Characterization of Dopamine β ‐Hydroxylase from Bovine Adrenal Medulla
Authors: LJONES Torbjørn , SKOTLAND Tore , FLATMARK Torgeir
Published: March 2005

A new purification procedure that permits large‐scale purification of dopamine β‐hydroxylase from bovine adrenal medulla was developed. Whole adrenal medullas were extracted with 0.1 Triton X‐ 100, and the enzyme was purified by precipitation with polyethylene glycol, chromatography on DEAE‐cellulose, and adsorption to concanavalin A linked to agarose. The yield of protein and the specific activity were high compared with previously published methods. The enzyme appeared essentially homogenous by the criteria of polyacrylamide gel electrophoresis in the presence or absence of dodecylsulfate, and sedimentation velocity analysis.

The purified protein was subjected to amino acid and carbohydrate analyses, and the results were compared with previously published data. We found about 3 mol of copper per mol of protein (tetramer of 290000 daltons). No free sulfhydryl groups could be found. Analysis for NH2‐terminal amino acids with [14C]dansyl chloride revealed 2 residues of alanine and 2 residues of serine per tetramer. We found the NH2‐terminal amino acid of chromogranin A to be leucine. The results of our analysis for amino acid composition and NH2‐terminal amino acids do not support the suggestion that dopamine β‐hydroxylase and chromogranin A contain identical peptide chains.
Influence of Unsaturated Fatty Acids in Chloroplasts
Authors: SIEGENTHALER Paul‐André , DEPÉRY Françoise
Published: March 2005

Linolenic acid (C18:3) is the main endogenous unsaturated fatty acid of thylakoid membrane lipids, and seems in its free form to exert significant effects on the structure and function of photosynthetic membranes. In this investigation the effect of linolenic acid was studied at various pH values on the electron flow rate in isolated spinach chloroplasts and related to ΔpH, the proton pump and the pH of the inner thylakoid space (pHi). The ΔpH and pHi were estimated from the extent of the fluorescence quenching of 9‐aminoacridine.

Linolenic acid caused a shift (approximately one unit) of the pH optimum for electron flow toward acidity in the following systems: (a) photosystems II + I (from H2O to NADP+ or to 2,6‐dichlorophenolindophenol) coupled or non‐coupled; (b) photosystem II (from H20 to 2,6‐dichloro‐phenolindophenol in the presence of dibromothymoquinone).

In photosystem I conditions (phenazine methosulphate), the ΔpH of the control increased as a function of external pHo with a maximum around pH 8.8. When linolenic acid was added, the ΔpH dropped, but its optimum was shifted toward more acidic pHo. The same phenomena were also observed in photosystems II + I (from H2O to ferricyanide) and in photosystem II conditions (from H2O to ferricyanide in the presence of dibromothymoquinone). However, the ΔpH was smaller and the sensitivity of the proton gradient toward linolenic acid was eventually higher than for photosystem I electron flow activity. The proton pump which might be considered as a measure of the internal buffering capacity of thylakoids was optimum at pHo 6.7 in the controls. An addition of linolenic acid diminished the proton pump and shifted its optimum toward higher pHo.

As a consequence, pHi increased when pHo was raised. At the optimal pHo 8.6 to 9, pHi were 5 to 5.5. Additions of increasing concentrations of linolenic acid displaced the curves toward higher pHi. A decrease of pHo was therefore required to maintain the pHi in the range of 5 ‐ 5.5 for maximum electron flow.

In conclusion, the electron flow activity seems to be delicately controlled by the proton pump (buffer capacity), ΔpH, pHi and pHo. Fatty acids damage the membrane integrity in such a way that the subtle equilibrium between the factors is disturbed.
Protein Degradation and Proteinases during Yeast Sporulation
Authors: BETZ Heinrich , WEISER Ulrike
Published: March 2005

During ascospore formation in Saccharomyces cerevisiae, at least 60–70% of the pre‐existing vegetative protein was broken down at a rather constant rate until the time mature asci appeared. Under the same conditions in a non‐sporulating haploid derived from the same strain the rate of protein degradation, although initially comparable to that of sporulating cells, decreased much more rapidly. Proteins synthesized at different times during sporulation had approximately the same degradation rates as the vegetative proteins. Similar rates of degradation were observed for the vegetative proteins in all fractions obtained from cell homogenates by differential centrifugation.

Protein breakdown after transfer to sporulation medium was blocked by uncouplers and inhibitors of energy metabolism, and was partially inhibited by cycloheximide.

Polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate, of the proteins extracted from vegetative cells and from isolated asci and ascospores revealed that ascus formation was accompanied by a shift of the cellular proteins to a lower molecular weight.

From several proteinase inhibitors tested, only tosyl‐p‐lysine chloromethylketone slightly reduced the rate of ascus formation. During sporulation the total activity of proteinase A increased more than twofold with a maximum at 18 h after transfer to sporulation medium. Total proteinase B activity showed a striking increase in the first hours after transfer to sporulation medium and after that remained constant throughout sporulation. The levels of carboxypeptidase Y and of the proteinase B inhibitor were not significantly altered during sporulation. The proteinases and the proteinase B inhibitor were present within the mature ascospore. The proteinases, from both vegetative and sporulating cells were eluted with the same ionic strength from DEAÉ‐Sephadex, and they were undistinguishable in their sensitivity to different proteinase inhibitors. No additional proteolytic activities could be detected in sporulating cells using 3H‐labelled denatured yeast protein as a substrate.
Formation of Uridine Diphosphate 2‐Deoxy‐d‐glucose and Guanosine Diphosphate 2‐Deoxy‐d‐glucose in vitro Using Animal Enzymes
Authors: SCHWARZ Ralph T. , SCHMIDT Michael F. G.
Published: March 2005

The enzymic formation of guanosine diphosphate 2‐deoxy‐d‐glucose and uridine diphosphate 2‐deoxy‐d‐glucose from synthetically prepared 2‐deoxy‐d‐glucose 1‐phosphate is described: Intubation of 2‐deoxydglucose 1‐phosphate with an enzyme preparation from bovine mammary glands and either GTP or UTP gives rise to the corresponding nucleoside‐diphosphate derivative of 2‐deoxydglucose. Uridine diphosphate 2‐deoxydglucose could also be obtained by incubation of 2‐deoxydglucose 1‐phosphate with UTP and UDP glucose pyrophosphorylase from beef liver
Regulation of Cyanide‐Insensitive Respiration in Neurospora
Authors: EDWARDS David L. , ROSENBERG Emiko
Published: March 2005

Inhibition of either mitochondrial transcription or translation in Neurospora crassa results in the rapid production of the cyanide‐insensitive pathway of mitochondrial respiration. Protein synthesis an cytoplasmic ribosomes is required for the appearance of cyanide‐insensitive respiration in the culture.

Removal of the inhibition of transcription or translation results in a rapid return to cyanide‐sensitive respiration. Additional protein synthesis in the cytoplasm is required for the loss of cyanide‐insensitive respiratory activity while additional mitochondrial protein synthesis has no effect.

These studies indicate that a mitochondrial gene product is involved in a negative manner in the regulation of cyanide‐insensitive respiration. When the mitochondrial product is present, the pathway is not expressed, when the mitochondrial product is absent, the pathway is expressed.

Studies with forced heterokaryons formed from respiratory‐deficient mutants having cyanideinsensitive respiration and respiratory‐competent auxotrophs indicate that the site of action of the mitochondrial gene product is external to the mitochondrion.
Phase Transitions in the Membrane of a Marine Bacterium, Pseudomonas BAL‐31
Authors: TSUKAGOSHI Norihiro , PETERSEN Marianne H. , HUBER Ursula , FRANKLIN Richard M. , SEELIG Joachim
Published: March 2005

An unsaturated fatty acid auxotroph, strain UFA, isolated from the marine pseudomonad Pseudomonas BAL‐31, host cell of the lipid‐containing bacteriophage PM2, was grown in media supplemented with different unsaturated fatty acids. Under these conditions the fatty acid composition of the cell could be altered drastically. The phase transition in the native membrane and in the extracted lipids was analyzed by electron spin resonance using a nitroxide spin probe. Membranes prepared from strain UFA grown in cisl6: 1 or trans16:1 showed one transition at 9.4 °C and 12.4 °C respectively. Extracted lipids in both cases had almost the same transition temperature as that of the intact membrane. Membranes prepared from Pseudomonas BAL‐31 had one transition at ∼ 12 °C, on the other hand there was no clear cut phase transition using extracted lipids.

Replication of bacteriophage PM 2 took place below the transition temperature of the membrane lipids in the case where strain UFA was grown in trans 16: 1. Other cases were not studied.
Binding of Secretory Component to Dimers of Immunoglobulin A in vitro
Authors: LINDH Erik , BJÖRK Ingemar
Published: March 2005

A complex between secretory component and an immunoglobulin A (IgA) myeloma dimer has been studied in vitro as a model to elucidate the mechanism of the formation of disulfide bonds during assembly in vivo of secretory immunoglobin A. A small amount of free thiol groups, totally about 0.4 groups per mole of protein, were shown to be present on both the heavy and light chains of the IgA dimer, but not on its J‐chain, while no such groups could be demonstrated on free secretory component. The SH‐groups on IgA most likely exist as a result of incomplete oxidation of some intraor interchain disulfide bonds of the molecule, analogous to what has been suggested for IgG. Several types of evidence indicated that the disulfide bonds between secretory component and IgA are formed after the noncovalent association of the two proteins by a sulfhydryl group – disulfide bond exchange reaction, in which the small amount of free sulfhydryl groups on the IgA dimer initiate the reaction by reducing a reactive disulfide bond on secretory component. This exchange reaction, which thus proceeds by the mechanism of so‐called disulfide interchange reactions, requires certain conformational features of one or both of the proteins and leads to the formation of presumably two new interchain disulfide bonds between secretory component and IgA. The reaction does not progress to completion, however, but ends in an equilibrium so that a small proportion of the secretory component molecules always are unattached by disulfide bonds.
On The Mechanism of Amylose Branching by Potato Q‐Enzyme
Authors: BOROVSKY Dov , SMITH Eric E. , WHELAN William J.
Published: March 2005

When potato Q‐enzyme converts amylose into an amylopectin‐like molecule, the action is by a random, endo‐type transglycosylation of the substrate chains.

Inter‐chain transfer takes place during the formation of the amylopectin branch linkage. This is seen in experiments in which radioactive label was transferred between substrates of disparate molecular weight. Intra‐chain transfer, leading to the formation of a branch linkage, is not excluded by these experiments.

The minimum length of amylose chain that can act as an acceptor in the transglycosylation reaction, under the experimental conditions described, is greater than 40 glucose units.

The requirement of Q‐enzyme for substrate chains at least 40 glucose units in length is interpreted as meaning that a stabilized secondary and tertiary structure must be established in the substrate before it can be utilized by Q‐enzyme, and that the forces that provide such conformation are sufficiently strong only when the chains are longer than the minimum. Inter‐chain transfer is seen as taking place by one of two mechanisms. The first involves the reaction of the enzyme with a chain that has a stabilized (helical?) conformation. An enzyme‐donor chain intermediate is formed, that then reacts with an acceptor chain to complete the transglycosylation. The second mechanism envisages the substrate for the enzyme as being a complex formed between two chains (a double helix?). The enzyme encounters the complex and carries out an inter‐chain transglycosylation reaction.
Molecular‐Weight Determination of Animal‐Cell RNA by Electrophoresis in Formamide under Fully Denaturing Conditions on Exponential Polyacrylamide Gels
Authors: SPOHR Georges , MIRAULT Marc‐Edouard , IMAIZUMI Tereza , SCHERRER Klaus
Published: March 2005

A method for electrophoretic analysis of RNA under fully denaturing conditions on exponential gradient polyacrylamide gels is described. Full denaturation, and strand separation of DNA · RNA hybrids and double‐stranded RNA is obtained in dry formamide only if electrophoresis is carried out at 45° and 55 °C, respectively.

In such conditions, the effects of secondary structure of RNA, important in aqueous medium, are suppressed and a linear correlation is obtained between the logarithm of the molecular weight of an RNA and its final position in the gel over the entire molecular weight range of 104–107.

Based on absolute molecular weight standards, obtained from sequenced rRNA of Escherichia coli and tRNA and extrapolating to higher molecular weights, the size of animal cell RNA was reexamined. Precursor rRNA (45 S) from HeLa cells migrates according to a molecular weight of 4.1 × 106. Nascent precursor mRNA has molecular weights of up to 5 × 106 in the case of duck erythroblasts and of up to 107 in HeLa cells. This seems to represent the largest size of non‐viral animal‐cell RNA molecules.
Deoxyribosyl Transfer Catalysis with trans‐N‐Deoxyribosylase
Authors: DANZIN Charles , CARDINAUD Robert
Published: March 2005

Kinetic studies were carried out in order to investigate the enzymic mechanism of a 215‐fold‐purified purine(pyrimidine) nucleoside: purine(pyrimidine) deoxyribosyl transferase fraction from Lactobacillus helveticus. A variety of natural deoxyribonucleosides and bases were used as substrates. Initial velocity, product inhibition and isotopic exchange studies are consistent with a ping‐pong bi‐bi mechanism. The kinetic parameters are used to show that this fraction is free from any contamination by a specific purine nucleoside: purine deoxyribosyl transferase also found in the same strain of L. helveticus.
The Linkage of Lysine in the O‐Specific Chains of Proteus mirabilis 1959
Authors: GROMSKA Wieslawa , MAYER Hubert
Published: March 2005

Lipopolysaccharides of qualitatively identical but quantitatively different sugar composition were extracted from Proteus mirabilis strain 1959. The lipopolysaccharide with the higher percentage of typical O‐specific constituents was subjected to partial acid hydrolysis. An oligosaccharide B22 was separated by paper chromatography and electrophoresis. It was found to be composed of equimolar amounts of d‐galacturonic acid, d‐galactosamine and l‐lysine. Dinitrophenylation of the oligosaccharide as well as of the genuine lipopolysaccharide afforded e‐dinitrophenyl‐l‐lysine after acid hydrolysis, showing that lysine was linked to the disaccharide via its α‐amino group. Further studies including the Morgan‐Elson and Elson‐Morgan reactions, NaBH4‐reduction, hydrazinolysis and periodate oxidation revealed the structure of oligosaccharide B22 as d‐galacturonyl‐(1 → 4)‐d‐galactosamine with lysine attached to the carboxylic group of galacturonic acid via its α‐amino group. Judged from its high inhibition capacity this oligosaccharide has to be considered as an essential part of the serological determinant of Proteus mirabilis 1959. The frequent occurrence of lysine and galacturonic acid in Proteus mirabilis O‐serogroups and their possible significance for the respective serological specificities are discussed.
Studies on New Intracellular Proteases in Various Organs of Rats
Authors: KOMINAMI Eiki , KATUNUMA Nobuhiko
Published: March 2005

Homogenates of the muscle layer of rat small intestine irreversibly inactivated endogenous ornithine aminotransferase at 37°C. Addition to the homogenate of coenzymes and the various keto‐acids which act as substrate inhibited conversion of the holoenzyme to the apoenzyme and its subsequent degradation. Addition of protease inhibitors including soybean trypsin inhibitor, chymostatin and phenylmethylsulfonyl fluoride almost completely prevented inactivation of the enzyme.

Immunological activity decreased during inactivation of the enzyme, but its rate of decrease was much slower than that of loss of enzyme activity. Antigen‐antibody precipitates from homogenates containing inactivated enzyme. were separated by electrophoresis on sodium dodecylsulfatepolyacrylamide gels. In this way breakdown products of the enzyme were found, indicating that the enzyme in homogenates was inactivated by limited proteolysis. These results obtained in vitro support our previous suggestion (1975) of a stepwise mechanism for degradation of pyridoxal enzymes.

However, in vivo there was no difference between the rates of decrease of enzyme activity and of immunological activity during the decrease of ornithine aminotransferase following inhibition of protein synthesis. Moreover, no degradation products of the enzyme were found under either control, or pyridoxine‐deficient conditions. But the rate of decay of ornithine aminotransferase in the muscle layer of the small intestine was faster in pyridoxine deficiency. The apparent half‐life in vitamin‐B6‐depleted rats was estimated as 5 h, which was shorter than that in control rats (8 h). These results suggest that a step in the conversion of the holoenzyme to the apoenzyte is ratelimiting in degradation of the enzyme in vivo.
The Binding of Lipid to the Lipid‐Free Adenosine Triphosphatase Protein of Sarcoplasmic Reticulum
Authors: HARDWICKE Peter M. D.
Published: March 2005

Dinitrophenylated dipalmitoyl phosphatidylethanolamine and its lyso derivative have been shown to bind to the lipid‐free ATPase protein derived from the sarcoplasmic reticulum. The binding of these lipids is accompanied by the quenching of up to 95% of the tryptophyl fluorescence of the protein. This effect is reversed by 9–10 mM deoxycholate.

The solubility of the lipid‐free ATPase protein in the absence of deoxycholate and the solubility of submillimolar concentrations of the dinitrophenylated monopalmitoyl phosphatidylethanolamine anion in aqueous media allowed binding experiments using this lipid ligand to be carried out in a simple buffer system. It is shown that in the case of this lipid the initial phase of the binding process displays an apparent positive co‐operativity. Data from the second phase in the saturation of the protein with this lipid is consistent with binding to independent, equivalent, non‐interacting sites with a microscopic (intrinsic) association constant of 1.63 ×106 M−1, the fluorescence being quenched in a geometric fashion. Altogether a total of about 15 molecules of this lipid may be bound by the protein.
The Trypsin and Chymotrypsin Inhibitors in Chick Peas (Cicer arietinum L.)
Authors: BELEW Makonnen , EAKER David
Published: March 2005

The two principal isoinhibitors P‐5 and P‐6 isolated earlier from the seeds of chick peas (Cicer arietinum L.) by a procedure involving biospecific affinity chromatography on active, matrix‐bound trypsin are shown to be the virgin and trypsin‐modified forms of the same inhibitor. The virgin inhibitor P‐5 consists of a single peptide chain of 66 amino acid residues cross‐linked by seven disulfide bridges. Upon interaction with the matrix‐bound trypsin under the conditions used the virgin inhibitor P‐5 is about 50% converted to P‐6 by cleavage of a single Lys‐Ser linkage at position 14– 15 in the molecule. The resulting tetradecapeptide remains bound to the rest of the molecule by two or four disulfide bridges, but the two fragments representing residues 1–14 and 15–66 separate readily by molecular‐sieve chromatography following reductive or oxidative cleavage of the disulfide linkages. The sequence of residues 1‐25 was established by Edman degradation.

Cleavage of the Lys14–Ser15 linkage at the trypsin‐reactive site of the virgin inhibitor significantly reduces its trypsin inhibitory activity without affecting its anti‐chymotryptic activity. Upon treatment with carboxypeptidase B the trypsin‐modified inhibitor loses its anti‐tryptic activity but remains fully active against chymotrypsin. A similar treatment has no effect on the virgin inhibitor. Furthermore, the complex of the inhibitor with trypsin is inactive towards trypsin but remains fully active against chymotrypsin and vice versa. It is thus concluded that the principal inhibitor in chick peas inhibits both enzymes simultaneously and is clearly of the ‘double‐headed’ type with separate and independent reactive sites for the two enzymes. The molecule appears to be highly homologous to the Bowman‐Birk soybean inhibitor and to the lima bean inhibitor.