bluuebunny/crossref_metadata_2025 huggingface dataset:
The Isolation of an α‐Lactalbumin with Three Disulphide Bonds
Authors: Barman Thomas E.
Published: March 2005

Three forms of α‐lactalbumin have been isolated from pooled cows' milk: α‐lactalbumin B, glyco‐α‐lactalbumin B and a new form of α‐lactalbumin, α‐lactalbumin III. α‐Lactalbumin III accounts for about 5% of the total α‐lactalbumin of the milk. This protein has very similar physical and kinetic properties to α‐lactalbumin B but its amino acid composition is different; in particular it has only three disulphide bonds rather than the four usually found in α‐lactalbumin.
Reduced Pyridine‐Nucleotides Balance in Glucose‐Growing Saccharomyces cerevisiae
Authors: Lagunas Rosario , Gancedo Juana M.
Published: March 2005

The quantitative importance of transhydrogenation from NADH to NADP in Saccharomyces cerevisiae growing on glucose has been investigated. To this aim, the contribution of the different metabolic pathways to glucose metabolism has been measured and a balance between the requirements and the production of reduced pyridine nucleotides has been established. The results suggest that the oxidative hexose monophosphate pathway accounts for all, or almost all, the cellular requirements for NADPH and that the reoxidation of NADH is effected for a part by the respiratory chain but mainly by enzymatic reactions coupled to the reduction of organic compounds like acetaldehyde or dihydroxyacetone phosphate. A significant transhydrogenation between pyridine nucleotides is therefore not likely to occur in S. cerevisiae growing on glucose.
Replication of Colicinogenic Factor El in Escherichia coli
Authors: Sherratt David J. , Helinski Donald R.
Published: March 2005

Replication of the colicinogenic factor E1 (Col E1) has been studied in exponentially growing cells and in cells treated with chloramphenicol. Whereas about 80% of non‐replicating Co1E1 DNA is isolated from the cytoplasm, the membrane fraction is enriched for newly replicated DNA. Equal amounts of pulse label are incorporated into each of the strands of Col E1 supercoils isolated after a short pulse, indicating a symmetrical replication mechanism. The replication time of the Co1E1 plasmid is about 2 min at 33°C. The synthesis of Co1E1 supercoils was reduced to less than 10% of the control level 4 min after shift to 42 °C in a temperature‐sensitive mutant for DNA polymerase I.
Nature and Linkages of the Fatty Acids Present in Lipopolysaccharides from Vibrio metchnikovii and Vibrio parahemolyticus
Authors: Rietschel Ernst Th. , Palin William J. , Watson Dennis W.
Published: March 2005

The lipopolysaccharides of Vibrio metchnikovii and Vibrio parahemolyticus have been analyzed for their fatty acid composition. These fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues within the lipid A component of the lipopolysaccharides. With regard to nature and to type of linkage of the fatty acids, both lipopolysaccharides are almost identical. Approximately equal amounts of myristic, palmitic and 3‐lauroxylauric acid (3‐O‐lauroyl‐3‐hydroxylauric acid) are involved in ester linkages. In addition, varying amounts of an unsaturated fatty acid are ester‐bound. 3‐Hydroxymyristic acid, whose hydroxyl group is free, is exclusively amide‐bound. The similarities and differences regarding the nature and distribution of fatty acids in Vibrio lipopolysaccharides as compared to Salmonella lipopolysaccharides are discussed.
Electrophoretic Separation of Escherichia coli Ribosomal Particles on Polyacrylamide Gels
Authors: Talens Anneke , van Diggelen Otto P. , Brongers Menno , Bosch Leendert , Popa Liviu M.
Published: March 2005

Escherichia coli ribosomes display a remarkable heterogeneity when submitted to polyacrylamide‐gel electrophoresis (4% acrylamide). Both the 70‐S ribosomes and 50‐S ribosomal subunits are resolved into at least four and the 30‐S ribosomal subunits into three particle subclasses.

After reversal of the current, all particles refocus into one single band at the origin. Ribosomes recovered from the gel after electrophoresis have retained some 40–100% of their capacity to synthesize peptide bonds. Their sedimentation behaviour has remained unaltered. Upon reelectrophoresis, no new peaks are detectable although the relative proportions of the various subclasses are frequently changed. The ratio in the occurrence of these subclasses is readily affected by a variety of factors, however, some of which are not directly related to the electrophoretic technique itself.

The three classes of ribosomes and subunits (70‐S, 50‐S and 30‐S) display approximately equal electrophoretic mobilities when the sieving action is reduced to a minimum. This conclusion follows from experiments in which the ribosomes are submitted to electrophoresis in a sucrose gradient or in 0.4% agarose gels. In contrast, electrophoresis in polyacrylamide gels with a gradient in pore size reveals the same ribosomal diversity as noted in gels with a fixed acrylamide concentration of 4%. In these pore‐gradient gels (3‐8% acrylamide), the migration rate of each ribosomal subclass decreases continuosly when the time of electrophoresis is prolonged. After about 30 h, further penetration seems to be blocked.

All three classes consist of particles with low electrophoretic mobility (designated I particles) and those which move faster (designated II particles). The I particles are partially converted to II particles by incubation at 37 °C and/or pelleting prior to electrophoresis. The II particles are converted to I particles upon raising the ribosome concentration. The electrophoretic profiles displayed by the various 50‐S subclasses are not strongly affected by variations in Mg2+ concentration in the range 0.5‐10.0 mM. The electropherograms of 30‐S subunits become more diffuse when the Mg2+ concentration is raised. The possible occurrence of ribosomal dimers and/or conformation changes is discussed.
Stimulation of Ribonucleic‐Acid Synthesis in Chick‐Embryo Fibroblasts by Exogenous Adenosine 3′:5′‐Monophosphate
Authors: Koblet Hans , Kohler Ursula , Wyler Robert
Published: March 2005

In the chick embryo fibroblast cell culture adenosine cyclic 3′:5′‐monophosphate (cyclic AMP) stimulates ribonucleic acid biosynthesis (+50%), rRNA and tRNA as well as mRNA. The RNA pool size is enlarged and the specific activity elevated. Optimal concentration of cyclic AMP is 3—4 mM; it has to be present for 3 h in order to exert its full action. During this time the effect can be prevented by inhibitors of protein biosynthesis.

The stimulation is not due to altered transport of the precursor [3H]uridine. Adenosine and adenosine 5′‐monophosphate exhibit similar effects which, however, seem to result from another mechanism. It is felt that the action of cyclic AMP and similar compounds can only be explained through a pleiotropic effect (phosphorylation of histones, of RNA polymerase, induction of initiation and/or regulatory factors).
Carbohydrate and Amino‐Acid Composition of Human Glomerular‐Basement‐Membrane Fractions Purified by Affinity Chromatography
Authors: Mahieu Philippe M. , Winand Roger J.
Published: March 2005

Human glomerular basement membranes can be substantially solubilized by autoclaving in 0.1 M Tris‐acetate buffer pH 7.4, at 110°C, during 3 h or by treatment with 8 M urea containing mercaptoethanol. The best solubilization (80% of soluble material) was obtained by autoclaving. Therefore it was this material which has been purified from tritiated glycoproteins, by affinity chromatography, using Sepharose cyanogen‐bromide coupled with rabbit anti‐human glomerular‐basement‐membrane γ‐G globulin; at least 80% of solubilized basement membranes are adsorbed onto this γ‐globulin.

Unadsorbed material contains the same carbohydrate units as the whole membranes but are richer in heteropolysaccharide units. Its molecular weight is lower than 69000. Adsorbed glyco‐protein components, eluted from the Sepharose by acidic medium at 4°C, are further purified by preparative polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate. Four major fractions exhibiting molecular weights from 80000 to 300000 are obtained; they have the same carbohydrate units as the whole membranes, but have more disaccharide units. Similarly, amino acid composition of these purified fractions exhibits more 3‐hydroxyproline and hydroxy‐lysine residues than the whole membranes. This procedure appears therefore to be a valuable technique for preparing large quantities of labelled glomerular‐basement‐membrane antigens containing both types of oligosaccharidic chains.
Isolation and Characterization of Rat‐Liver Mitochondrial Ribosomes Highly Active in Poly(U)‐Directed Polyphenylalanine Synthesis
Authors: Greco Margherita , Cantatore Palmiro , Pepe Gabriella , Saccone Cecilia
Published: March 2005

The procedure used to obtain mitochondrial ribosomes from rat liver highly active in catalyzing a poly(U)‐dependent polyphenylalanine synthesis is described.

Two kinds of particles with sedimentation coefficients of 80‐S and 55‐S, present in the mitochondrial lysate, are able to catalyze protein synthesis in vitro.

Sensitivity to specific inhibitors of protein synthesis demonstrates that the activity of 55‐S particles is only of mitochondrial origin whereas that of 80‐S particles is partly due to mitochondrial ribosomes and partly to contaminating cytoplasmic ribosomes.

Characterization of the two kinds of mitochondrial particles has been carried out by studying their sedimentation pattern in different experimental conditions and by analyzing the RNA extracted by polyacrylamide gel electrophoresis. 16‐S and 12‐S RNA seem to be specific mitochondrial ribosomal species.

The results suggest that the 55‐S particle represents the basic unit for protein synthesis in rat liver mitochondria. Particles with higher sedimentation value probably represent aggregates of mitochondrial monosomes.
Studies on Plant Aspartate Transcarbamylase
Authors: ACHAR Balebail S. , SAVITHRI Handanahal S. , VAIDYANATHAN Chelakara S. , APPAJI RAO Naropantul
Published: March 2005

Aspartate transcarbamylase is purified from mung bean seedlings by a series of steps involving manganous sulphate treatment, ammonium sulphate fractionation, DEAE‐cellulose chromatography, followed by a second ammonium sulphate fractionation and finally gel filtration on Sephadex‐G 100. The enzyme is homogeneous on ultracentrifugation and on polyacrylamide gel electrophoresis. It functions optimally at 55°C. It has two pH optima, one at 8.0 and the other at 10.2. The enzyme follows Michaelis‐Menten kinetics with l‐aspartate as the variable substrate. However, it exhibits sigmoid saturation curves at both the pH optima when the concentration of carbamyl phosphate is varied. The enzyme is allosterically inhibited by UMP at both the pH optima. Increasing phosphorylation of the uridine nucleotide decreases the inhibitory effect. The enzyme is desensitized to inhibition by UMP on treatment with p‐hydroxymercuribenzoate, gel electrophoresis indicating that the enzyme is dissociated by this treatment; the dissociated enzyme can be reassociated by treatment with 2‐mercaptoethanol. The properties of the mung bean enzyme are compared with the enzyme from other sources.
Role of Manganous Ion in the Kinetics of Pig‐Heart NAD‐Specific Isocitrate Dehydrogenase
Authors: COHEN Phyllis F. , COLMAN Roberta F.
Published: March 2005

The NAD‐dependent isocitrate dehydrogenases require the presence of divalent metal cation for activity. In order to elucidate the role of metal in this reaction, Michaelis constants for manganese were determined as a function of pH and isocitrate concentration. At constant pH, the Km values for total manganese as well as free manganous ion decreased as the concentration of isocitrate in the reaction mixture was increased; in contrast, the Michaelis constant in terms of the metal · isocitrate complexes was invariant, suggesting that manganese binds as a metal chelate of isocitrate. A previous analysis of the Michaelis constants for isocitrate [Cohen, P. F., and Colman, R. F., Biochemistry (1972) 11, 1501] indicated that in the presence of 1–10 mM manganese, the substrate binds as free dibasic isocitrate. A model is proposed which allows for the formation of the active enzyme · manganese · isocitrate complex from either of two pathways: in the presence of low concentrations of manganese, the enzyme may combine directly with the metal chelate of dibasic isocitrate; alternatively, in the presence of high concentrations of manganese, the enzyme first binds metal ion to yield an enzyme · manganese complex which subsequently reacts with free dibasic isocitrate.

The affinity of the enzyme for the manganese · dibasic‐isocitrate complex varies with pH, exhibiting a lower Michaelis constant at pH 8 than 6. It is shown that the catalytic function of the free enzyme is dependent on the ionization of an amino acid residue with pKe= 7.26. The measurable PK of this ionizable group is decreased to 6.39 in the enzyme · manganese and enzyme · manganese isocitrate complexes.

The Km for manganese was determined in the presence of various chelating agents. Although citrate and GDP increase the Michaelis constant in terms of total manganese, the Km for the actual substrate (manganese · dibasic‐isocitrate) is not changed. This result can be attributed to the competition between these chelators and isocitrate for manganous ion. In contrast, ADP lowers the Km values for the metal · isocitrate complex, as well as that for total manganese, suggesting that a direct interaction with the enzyme is involved.
Yeast Inorganic Pyrophosphatase: Studies on Metal Binding
Authors: BAYKOV Alexander A. , AVAEVA Svetlana M.
Published: March 2005

A study has been made of the interaction of yeast inorganic pyrophosphatase with two activating cations, Mg2+ and Zn2+, and one inhibitory cation, Ca2+ at pH 6.5 and ionic strength of unity. These cations were found to partially protect the enzyme against inactivation by iodine. Dissociation constants of enzyme metal complexes estimated from protection studies were 11, 500 and 4000 μM for Zn2+, Mg2+ and Ca2+, respectively. An additional, lower affinity site, was found for Zn2+ (KD= 3.4 mM). Competition studies showed that magnesium and calcium ions compete for the same site as the tighter bound zinc ion and do not compete for the lower affinity site. Binding of Zn2+ to both sites is pH‐dependent; the interaction with the high‐affinity site is controlled by a group with pK= 6.6. The binding constant for Mg2+ is nearly the same as the lower activation constant for this cation in enzymatic hydrolysis of pyrophosphate. Two activating and one inhibitory site were found for Zn2+ in ATP hydrolysis. The inhibitory site was identified as the lower affinity site deduced from protection studies. Both activation constants for Zn2+ are higher than the dissociation constant for the tightly bound zinc deduced from protection studies.
The Synthesis of Three AMP‐Analogues: N6‐(6‐Aminohexyl)‐adenosine 5′‐Monophosphate, N6‐(6‐Aminohexyl)‐adenosine 2′,5′‐Bisphosphate, and N6‐(6‐Aminohexyl)‐adenosine 3′,5′‐Bisphosphate and Their Application as General Ligands in Biospecific Affinity Chromatography
Authors: BRODELIUS Peter , LARSSON Per‐Olof , MOSBACH Klaus
Published: March 2005

Phosphorylation of 6‐chloropurine riboside with phosphorus oxychloride and phosphorus trichloride gave a mixture of the two isomers, 6‐chloropurine‐riboside 2′,5′‐bisphosphate and 6‐chloropurine‐riboside 3′,5′‐bisphosphate. Reaction with 1,6‐diaminohexane followed by resolution of the isomeric mixture on a Dowex 1‐X2 column yielded N6‐(6‐aminohexyl)‐adenosine 2′,5′‐bisphosphate and N6‐(6‐aminohexyl)‐adenosine 3′,5′‐bisphosphate.

The inhibition of several NADP+‐dependent and NAD+‐dependent dehydrogenases by N6‐(6‐aminohexyl)‐adenosine 2′,5′‐bisphosphate, N6‐(6‐aminohexyl)‐adenosine 3′,5′‐bisphosphate and N6‐(6‐aminohexyl)‐adenosine 5′‐monophosphate was examined.

These three AMP‐analogues were attached to Sepharose 4B by the cyanogen bromide method and the binding of several NAD(P)+‐dependent enzymes were investigated. NADP+‐dependent enzymes were bound to Sepharose substituted with N6‐(6‐aminohexyl)‐adenosine 2′,5′‐bisphosphate, whereas NAD+‐dependent enzymes were not bound under the same conditions. Conversely, when N6‐(6‐aminohexyl)‐adenosine 5′‐monophosphate was used as the immobilised ligand only the NAD+‐dependent enzymes were bound, as well as glucose‐6‐phosphate dehydrogenase showing weak affinity. These observations strongly suggest that these two immobilised analogues represent true biospecific and group‐specific adsorbents. The enzymes were eluted with their complementary nucleotides, NAD(H) and NADP(H). These techniques were utilised to purify several NADP+‐dependent enzymes from a crude Candida utilis extract by chromatography on the new biospecific adsorbent.
Evidence in Favour of the Modification in vivo of RNA Polymerase Subunits during Sporogenesis in Bacillus thuringiensis
Authors: KLIER Andreé , LECADET Marguerite‐M.
Published: March 2005

The subunit composition of RNA polymerase from Bacillus thuringiensis during Sporogenesis has been examined in crude extracts, through immunoprecipitation experiments. Modifications of the different subunits, especially of the β' and sigma subunits were demonstrated and the timing of these modifications have been specified. As regards the origin of these modifications, arguments in favour of the proteolysis in vivo were obtained from such experiments: analysis of labelled RNA polymerase extracted from sporulating cells, mature spores and from mixtures of vegetative and sporulating cells allowed us to exclude neosynthesis or any artefactual modification resulting from a proteolysis in vitro. On the other hand the same experiments revealed the appearence of new components characteristic for sporulating cells: a polypeptide chain of molecular weight 75000 and two new subunits named Y and Z, previously found in the sporal enzyme. In the same way, new components of low molecular weight, associated with RNA polymerase were also detected in vegetative cells infected with phage B 1715 V1.
Translation Products in vitro of Mitochondrial Messenger RNA from Neurospora crassa
Authors: KÜNTZEL Hans , BLOSSEY Hans‐Christian
Published: March 2005

A submitochondrial polypeptide‐synthesizing system prepared from Neurospora crassa actively incorporates radioactive amino acids in vitro in the absence of added messenger RNA. Translation products were fractionated into what are most probably single polypeptide chains by filtration through Sephadex G‐100 in the presence of dodecylsulfate. The majority of incorporated amino acids is present in two partially resolved fractions of apparent molecular weights 12000 (P1) and 10000 (P2). P2 (but not P1) forms large‐molecular‐weight aggregates during concentration in the presence of 0.5% dodecylsulfate. Both P1 and P2 aggregate in the presence of 0.01% dodecylsulfate. P1 can be separated from P2 by selective extraction with 90% methanol.

Purified mitochondrial DNA from Neurospora crassa was transcribed in vitro by Escherichia coli RNA polymerase. The resulting transcript was translated in a cell‐free system from E. coli into two polypeptide fractions having apparent molecular weights identical to P1 and P2.

These data suggest that in Neurospora crassa the major proteins coded by mitochondrial cistrons and synthesized by endogenous mitochondrial polyribosomes are polypeptides of apparent molecular weights 12000 and 10000.
Control of the Rate of Hepatic Uptake and Catabolism of Liposome‐Entrapped Proteins Injected into Rats. Possible Therapeutic Applications
Authors: GREGORIADIS Gregory , NEERUNJUN Diane E.
Published: March 2005

131I‐ or 125I‐labelled albumin and β‐fructofuranosidase were entrapped in liposomes composed of phosphatidylcholine, cholesterol and a charged lipid (molar ratio 7:2:1). Investigations on the fate of such liposomes injected into rats revealed the following.

1. Initially there is an increased rate of removal from plasma (rapid phase) followed by a decreased one (slow phase). The rate of removal during the rapid phase was highest for negatively charged liposomes and lowest for positively charged liposomes with neutral liposomes in between. Uniform rates of removal, regardless of charge, were obtained during the slow phase.

2. The rate of hepatic uptake of such liposomes paralleled that observed for their removal from plasma during the rapid phase.

3. Concurrent administration of a large quantity of positively charged liposomes and of a smaller quantity of negatively charged liposomes led to a diminution of the rapid phase of the latter suggesting a common site of uptake for liposomes of either charge.

4. Blockade of the reticuloendothelial system with carbon led to an increased hepatic localization of liposomes during the rapid phase suggesting a bimodal uptake of liposomes with both the Kupffer and the liver parenchymal cells involved.

5. Cross‐linking of liposome‐entrapped albumin and β‐fructofuranosidase increased the stability of the latter and following injection into rats rendered both proteins less vulnerable to degradation by the liver lysosomal enzymes.

6. The possibility of controlling both the rate of removal from plasma and catabolism of liposome‐entrapped proteins adds more scope to the use of liposomes in therapy.
d‐Galactose Dehydrogenase from Pseudomonas fluorescens
Authors: BLACHNITZKY Ernst‐Otto , WENGENMAYER Friedrich , KURZ Gerhart
Published: March 2005

1. A procedure is described for the isolation of d‐galactose dehydrogenase from Pseudomonas fluorescens, grown on d‐galactose as carbon source. A 750‐fold purification was achieved with an overall yield of about 40%. The final preparation had a specific activity of about 850 μmol NADH formed per min per mg protein.

2. The enzyme has been shown to be homogeneous by ultracentrifugation, by disc gel electrophoresis, by gel isoelectric focusing and by dodecylsulfate gel electrophoresis.

3. The molecular weight of the native enzyme has been determined to be 64000 by sedimentation equilibrium studies and by gel permeation chromatography.

4. The enzyme dissociates in the presence of 0.1% sodium dodecylsulfate to polypeptide chains, whose molecular weight was determined as 32000 by electrophoresis, indicating that the native enzyme is composed of two subunits.

5. The amino acid composition of d‐galactose dehydrogenase has been determined.

6. The pI of the enzyme has been shown to be 4.28. The pH optimum is between 9.1 and 9.5. At pH 9.1 and 30 °C the limiting Michaelis constant for NAD+ is Ka= 0.24 mM, for NADP+Ka= 2.3 mM and for d‐galactose with NAD+ and with NADP+ as cosubstrate Kb= 0.7 mM. The dissociat on constant for NAD+ is Kia= 0.54 mM and for NADP+Kia= 6.2 mM.

7. d‐Galactose dehydrogenase from Ps. fluorescens catalyzes the dehydrogenation of d‐galactose by either NAD+ or NADP+. The enzyme oxidizes the following sugars arranged according to relative rates in decreasing order: d‐fucose > d‐galactose > l‐arabinose > 2‐deoxy‐d‐galactose ≫ 4‐deoxy‐d‐galactose > 2‐deoxy‐2‐amino‐d‐galactose.

8. The correspondence between amino acid composition, subunit size and structure and between specificity of d‐galactose dehydrogenase from Ps. fluorescens and from Ps. saccharophila is discussed.
Comparative Sequence Studies on α2‐CB2 from Calf, Human, Rabbit and Pig‐Skin Collagen
Authors: FIETZEK Peter P. , KÜHN Klaus , FURTHMAYR Heinz
Published: March 2005

The entire sequence of α2‐CB2 from calf skin collagen, a cyanogen‐bromide‐derived peptide from the central portion of the α2 chain containing 30 amino‐acid residues was established. The sequence of 22 N‐terminal residues of α2‐CB2 from human and rabbit skin collagen and of 26 residues of the corresponding peptide from pig skin collagen were also determined. Comparison of the sequences revealed six definite positions within the first 21 amino‐acid residues of the peptide in which substitutions occurred, suggesting a portion of high sequence variability within the α2 chain.
Adenosine 3′ :5′‐Monophosphate and Hormone Interrelationships in the Mammary Gland of the Rat during Pregnancy and Lactation
Authors: SAPAG‐HAGAR Mario , GREENBAUM A. Leslie
Published: March 2005

The activities of adenyl cyclase, the phosphodiesterases of low and high Km and the tissue content of adenosine 3′:5′‐monophosphate (3′: 5′‐AMP) in the mammary glands of rats at different stages of the lactation cycle have been measured. The effect of in vitro F−, Ca2+ and of the hormones progesterone, β‐estradiol, hydrocortisone, insulin and prolactin on the adenyl cyclase activity of membrane fractions isolated from the mammary glands of pregnant and lactating rats has been investigated. The results show that there is a coordinated change of the activities of the enzymes adenyl cyclase and 3′:5′‐AMP phosphodiesterase and the tissue content of 3′:5′‐AMP at different stages of the lactation cycle with the activity of adenyl cyclase and the level of the cyclic nucleotide rising to a peak at the end of pregnancy and then falling sharply after parturition. The reverse pattern was observed for the 3′:5′‐AMP phosphodiesterase activity. F−(1–10 mM) consistently produced an inhibition (40%) of adenyl cyclase activity of mammary glands from pregnant rats while having no effect on the enzyme from the lactating gland. Low concentrations of Ca2+ (1 μM) activated the enzyme from pregnant glands and from those of early lactation by about 40% but had little effect on the glands taken from rats of mid‐lactation. Increasing the Ca2+ concentration led to a decline of this effect and eventual inhibition at 5 mM, the effect being most marked on the fully lactating gland. Progesterone and β‐estradiol consistently caused a considerable stimulation (80 and 50%, respectively) of the adenyl cyclase activity of the pregnant gland; hydrocortisone, insulin and prolactin did not. None of the hormones tested activated the adenyl cyclase activity of the lactating gland and insulin appeared to inhibit it severely. The results, which suggest that the growth and development of the mammary gland is related to the tissue content of 3′: 5‐AMP while the initiation and scale of lactation may be related to its removal, are discussed in relation to development, lactogenesis and milk ejection and the complex endocrine control of these processes.
Metabolism of Palmotoxins B0 and G0in vitro
Authors: BASSIR Olumbe , EMEROLE Godwin O.
Published: March 2005

The metabolism of the palmotoxins is yet unknown. Our preliminary report (1973) shows that they are probably metabolized in vitro in a similar manner to the aflatoxins. This involves demethylation and a possible hydroxylation.

The rate of demethylation of 50 nmol toxin attains a maximum in about 75 min. Optimum pH was between 7.5 and 7.6 using 0.3 M phosphate buffer, In comparison with aflatoxin B1, palmotoxins B0 and G0 were found to be demethylated and hydroxylated at a slower rate. Two fluorescent metabolites were identified for palmotoxin B0 on thin‐layer chromatography and only one for palmotoxin G0. Fluorescence spectra of the isolates from the toxins showed excitation and fluorescence maxima similar to those of the parent compounds. Ultraviolet spectra of the isolates showed a shift to higher wavelength when compared with the parent compound. Glucuronide conjugates were identified with both the palmotoxins and aflatoxin B1. Phenobarbitone was found to induce the palmotoxins B0 and G0 as well as the aflatoxin‐B1‐metabolizing enzymes. The activity of these enzymes were inhibited by carbon monoxide aeration.
Replication of the Double‐Stranded Replicative‐Form DNA of Bacteriophage M‐13 in Plasmolysed Escherichia coli Cells
Authors: STAUDENBAUER Walter L.
Published: March 2005

Escherichia coli cells, infected with bacteriophage M‐13 am‐5, when plasmolysed by treatment with 2 M sucrose incorporate radioactivity from deoxyribonucleoside triphosphates into phage‐specific DNA. After suppressing host DNA synthesis by treatment with mitomycin C prior to infection, more than 80% of the incorporated label is found in superhelical replicative form I molecules. If the incorporation is carried out in the presence of bromodeoxyuridine triphosphate, the radioactive label is found in equal amounts in two classes of hybrid replicative form molecules in which either the viral or the complementary strand is labeled.

DNA synthesis depends on a functional gene‐2 product and shows a specific requirement for ATP and magnesium as divalent cation. Incorporation is inhibited by mitomycin C, nalidixic acid, phenyl ethanol, and sulfhydryl‐blocking agents. It is concluded that the observed DNA synthesis occurs by a semi‐conservative replication process resembling replicative form replication in vivo.
The Stimulus‐Secretion Coupling of Glucose‐Induced Insulin Release
Authors: MALAISSE Willy J. , SENER Abdullah , MAHY Marianne
Published: March 2005

1. The possible significance of the insular sorbitol pathway in glucose‐induced insulin release was investigated.

2. Glucose, but neither fructose nor galactose, stimulated sorbitol synthesis by isolated islets. Aldose reductase inhibitors such as 2,4‐dimethylglutaric and 3,3‐tetramethylene glutaric acid abolished the glucose‐induced sorbitol formation, but failed to affect glucose‐induced insulin release. About 75% of the sorbitol formed by the islets was recovered in the incubation medium, the efflux of sorbitol occurring independently of insulin release. Exogenous fructose and sorbitol exerted a modest insulinotropic action characterized by a shift to the left of the sigmoidal curve relating insulin release to glucose concentration. The relative insulinotropic potency of glucose (100%), fructose (16%) and sorbitol (7%) paralleled their rate of oxidation by the isolated islets.

3. These data suggest that metabolism through the sorbitol pathway plays little role in the process of glucose‐induced insulin release, although exogenous sorbitol exerts a modest insulinotropic action.
The Peptide Moiety of Blood‐Group‐Specific Glycoproteins
Authors: GOODWIN Shirley D. , WATKINS Winifred M.
Published: March 2005

A purified glycoprotein with blood‐group‐A specificity, was digested with insoluble pronase to give a serologically active product enriched in peptide‐bonded serine and threonine residues; this was degraded with 0.25 M sulphuric in glacial acetic acid for 24 h. The resultant N‐acetylgalactosaminyl‐peptides were hydrolysed with 0.2 M hydrochloric acid for 24 h at 110°C. Fractionation of the products on Sephadex G‐10, followed by preparative separation on an amino‐acid analyser and paper electrophoresis at pH 1.9 yielded the following homogeneous oligopeptides; one tripeptide with the N‐terminal sequence Thr‐Thr‐Ser, three tetrapeptides with the sequences, Thr‐Ser‐Thr‐Ser, Thr‐Pro‐Thr‐Ser and Pro‐Thr‐Thr‐Ser, one pentapeptide with the sequence Thr‐Pro‐Thr‐Ser‐Ser, one hexapeptide with the sequence Pro‐Thr‐Thr‐Thr‐Pro‐Ser and one heptapeptide with the sequence Ala‐Pro‐Thr‐Thr‐Ser‐Gly‐Ser. Galactosamine was present in several of these fragments and the hexapeptide and heptapeptide both contained sufficient of this sugar for all the serine and threonine residues to be glycosylated.

The following five tetrapeptides sequences were deduced from mixtures of two peptides which differed by the amino‐acid substituent at only one position in the chain; Pro‐Thr‐Ala‐Ser, Thr‐Thr‐Pro‐Ser, Thr‐Thr‐Ala‐Ser, Ser‐Pro‐Thr‐Ser, and Ser‐Ala‐Thr‐Ser. A tripeptide mixture which differed only in the nature of the third amino acid yielded the following three tripeptide sequences: Pro‐Thr‐Ser, Pro‐Thr‐Gly and Pro‐Thr‐Ala.

These peptides do not indicate a clearly defined amino‐acid sequence surrounding the glycosylated hydroxy amino acids but establish that in the human blood‐group‐specific glycoproteins there is close packing of the serine and threonine residues in the region of the peptide moiety carrying the carbohydrate chains.
Glucose Synthesis in Tritiated Water
Authors: ROGNSTAD Robert , KATZ Joseph , CLARK Dallas G.
Published: March 2005

Isolated rat or hamster liver parenchymal cells or kidney cortex segments were incubated in a medium containing 3HHO and the incorporation and partial distribution of tritium in the glucose formed was determined. Gluconeogenesis from lactate or pyruvate caused about 80 to 90% of the possible amount (7 μatoms per glucose molecule) of fixation of tritium from 3HHO in glucose, with a high percentage labelling on C‐6. Glucose formation from glycogen breakdown under anaerobiosis caused less than 20% of the possible fixation of tritium, with most of the labelling on C‐2. This technique may be of eventual use in determining rates and sources of glucose formation.
Polyribosome Aggregation in Isolated Rat‐Liver Cells
Authors: GRANT Anne G. , BLACK Elizabeth G.
Published: March 2005

Polyribosomes were prepared from isolated rat liver cells by homogenisation or Triton X‐100 lysis. Triton X‐100 lysis proved the more effective method of preparation of ribosomes and was therefore used to study polyribosome profiles obtained from isolated liver cells incubated in Krebs‐Ringer phosphate and Eagle's basal essential medium for various times. Polyribosome aggregation decreased throughout the three‐hour incubation period but cells in the basal essential medium maintained a more aggregated profile and higher rate of albumin synthesis than those in Krebs‐Ringer phosphate.
Reduction of Adenosine 5′‐phosphosulfate to Cysteine in Extracts from Chlorella and Mutants Blocked for Sulfate Reduction
Authors: SCHMIDT Ahlert , ABRAMS William R. , SCHIFF Jerome A.
Published: March 2005

Techniques for preparation of enzyme extracts from Chlorella pyrenoidosa chick are described which yield a stable system which converts adenosine 5′‐phosphosulfate to sulfide and/or cysteine in the absence of added active thiols. Since these thiols (such as dithiothreitol) have been shown to yield sulfite, among other products, from the adenosine‐5′‐phosphosulfate sulfotransferase reaction, we have studied the adenosine 5′‐phosphusulfate reducing system in the presence and absence of dithiothreitol. In all cases dithiothreitol increases the activity of the reducing system forming cysteine from adenosine 5′‐phosphosulfate. Mutant Sat−2, which cannot grow on sulfate, is shown to lack thiosulfonate reductase activity but to retain sulfite reductase activity. Extracts from this mutant will form sulfide and cysteine only in the presence of dithiothreitol, while wild‐type extracts will form these products in presence or absence of dithiothreitol, the activity with dithiothreitol being higher. Mutants Sat−1,3–6 which are blocked in the adenosine‐5′‐phosphosulfate sulfotransferase step are blocked in the formation of sulfide and cysteine from adenosine 5′‐phosphosulfate. Binding studies with adenosine 5′‐phosphosulfate indicate that the sulfonyl group is transferred to a carrier to form carrier‐S‐SO−3 and the available evidence suggests that this carrier is a prosthetic group of the thiosulfonate reductase. On the basis of this evidence, two pathways in the cell‐free extracts are thought to be operating. One, in the absence of active thiols, is a pathway consisting of bound intermediates in which the sulfonyl group of adenosine 5′‐phosphosulfate is transferred via adenosine‐5′‐phosphosulfate sulfotransferase to carrier on the thiosulfonate reductase where it is reduced to the thiol level by ferredoxin; the thiol is then transferred via O‐acetyl‐l‐serine sulfhydrylase to form cysteine. The other, or free intermediate pathway operates in the presence of dithiothreitol which releases free sulfite from the adenosine‐5′‐phosphosulfate sulfotransferase reaction. This sulfite is then reduced via sulfite reductase to sulfide which forms cysteine via O‐acetyl‐l‐serine sulfhydrylase. Since mutant Sat−2, which cannot grow on sulfate nor reduce it in vivo, lacks thiosulfonate reductase activity but yields sulfite reductase activity in extracts, we conclude that the bound intermediate pathway is the preferred pathway of sulfate reduction in vivo; the free intermediate pathway is probably operative only when free sulfite is available either from occasional side reactions in vivo or when sulfite is fed to cells from outside.
On the Nature of DNA Promoter Conformations
Authors: TRAVERS Andrew
Published: March 2005

Low concentrations (up to 20% v/v) of glycerol and dimethylsulphoxide stimulate RNA synthesis in vitro, the extent of stimulation being dependent on the nature of the DNA template. This stimulation results mainly from an increase in initiation. The target of these compounds is the DNA template rather than RNA polymerase. Ribosomal RNA synthesis is affected in a temperature‐dependent manner, 20% glycerol lowering the transition temperature between the open and closed forms of the promoter by 4–5°C. Thus in a manner analogous to its effect on gal mRNA synthesis in vitro glycerol partially releases rRNA synthesis from the constraints essential for control.
A Ribonuclease H from Ustilago maydis
Authors: BANKS Geoffrey R.
Published: March 2005

A ribonuclease, which can degrade about 90% of the RNA strand of an RNA · DNA hybrid, has been extensively purified from Ustilago maydis. It is highly specific for a calf thymus RNA · DNA hybrid, little or no degradation of the RNA strands occurring after heat denaturation of the hybrid, or of single and double‐stranded DNA. With the exception of poly(rU) · poly(dA), all the ribonucleotide strands in hybrid homopolymers were susceptible to degradation, the double‐stranded homo‐polyribonucleotides being resistant. Both poly(rU) and poly(rI), however, were degraded.

The enzyme possesses a molecular weight of 100000–130000 and requires magnesium ions, ammonium sulphate and a sulphydryl reagent for maximum activity. It is an endoribonuclease, releasing oligoribonucleotides terminated by 5′‐phosphate and 3′‐hydroxyl groups, but is unable to remove a ribonucleotide covalently joined to the 3′‐terminus of DNA. Two mutant strains of U. maydis, which are temperature‐sensitive for DNA synthesis, possess elevated levels of the enzyme. This organism contains at least two other enzymes which can degrade the hybrid substrates.
The Mode of Action of Pleuromutilin Derivatives
Authors: HODGIN Louise A. , HÖGENAUER Gregor
Published: March 2005

The pleuromutilin derivative 14‐deoxy‐14‐[(2‐diethylaminoethyl)‐mercaptoacetoxy]‐mutilin HCl interferes strongly with cell‐free poly(U)‐directed polyphenylalanine synthesis in an extract from Escherichia coli (supernatant obtained by centrifugation at 30000 ×g). The compound is unable to inhibit the assembly of initiation complexes and does not significantly influence Phe‐tRNA attachment to the ribosomal A‐site catalyzed by elongation factor T. It does, however, reduce the puromycin release of formylmethionine from initiator tRNA bound to the P‐site, indicating that either the peptidyl transferase is affected or the binding of puromycin is abolished by this antibiotic. The influence of this agent on protein synthesis performed by E. coli polysomes is less pronounced than the inhibition of polyphenylalanine synthesis. The opposite effect has been reported for chloramphenicol. This mirror‐image‐like behaviour seems to suggest that both antibotics are related in their mode of action. The formation of the dipeptide fMet‐Ala coded for by R‐17 phage RNA is also impeded by the pleuromutilin derivative. The binding of Ala‐tRNA to the A‐site is affected in this reaction. Since attachment of Phe‐tRNA to ribosomes with an empty P‐site (dependent on elongation factor T) is not hindered, the inhibition of the tRNA binding to initiation complexes seems to indicate that the antibotic requires an occupied P‐site in order to prevent binding of aminoacyl‐tRNA to the A‐site.
Le centre actif de la 3‐oxostéroide Δ1‐déshydrogénase d'Arthrobacter simplex
Authors: PÉNASSE Lucien , NOMINÉ Gérard
Published: March 2005

The Active Center of the 3‐Oxosteroid Δ1‐dehydrogenase from Arthrobacter simplex

The kinetic parameters of the semi‐purified, hydrocortisone induced 3‐oxosteroid Δ1‐dehydrogenase from Arthrobacter simplex have been determined with twenty different steroids. The values thus obtained with nortestosterone and six of its stereoisomers and with derivatives bearing substituents at positions 10, 13 and 17, show that for the reaction to take place, first the presence of a 3‐oxo group, a Δ4‐double bond and axial hydrogens at 1α and 2β positions, are necessary, secondly no bulky substituents must be present in the middle part of the α‐face and at position 4; thirdly at positions 10β and 13β no substituents with more than two carbon atoms must be present. With steroids bearing a methyl group in position 10, the chirality of this center has to be natural, and with 19‐norsteroids, the conformation of the whole molecule has to be almost flat.

The active center could be represented as a cleft in the protein molecule one side of which is in contact with the α‐face of the steroid, the other side being located a few tenths of nanometer above the β‐face, The Δ4‐double bond is required for the reaction but not for binding. There is no contact point in the area of carbon atom 17.
Purification and Properties of α‐d‐Mannosidase from Medicago sativa L.
Authors: DE PRIJCKER Jozef , VERVOORT Antoine , DE BRUYNE Clement
Published: March 2005

A purification method for α‐mannosidase from the seeds of Medicago sativa L. is described. It includes ammonium sulphate precipitation, phosphocellulose chromatography, DEAE‐Sephadex ion‐exchange chromatography, and Sephadex G‐200 gel filtration. The enzyme has a molecular weight of about 220000. At pH values below neutrality, α‐mannosidase undergoes inactivation. This process can be prevented by Zn2+, whereas EDTA accelerates inactivation. The influence of pH on Km and V have been determined with p‐nitrophenyl α‐d‐mannoside as substrate. From the pH dependence it could be deduced that a dissociable group with pK∼ 6 and functioning as a proton donor is necessary for catalytic activity. p‐Chloromercuribenzoate and other sulfhydryl reagents were not inhibitory. Glycon specificity studies reveal strict requirements at C‐3, C‐4 and C‐5 of the mannopyranosyl moiety. The trans‐diaxial position of the C‐2 hydroxyl and the aglycon group seems necessary for hydrolysis, but not for binding. The strong inhibitory power of mannono‐(1 → 5)‐lactone suggests that the glycon part of the substrates is bound to the enzyme in a half‐chair like conformation.
Studies on Polysome‐Membrane Interactions in Mouse Myeloma Cells
Authors: HARRISON Timothy M. , BROWNLEE George G. , MILSTEIN Cesar
Published: March 2005

The nature of the interaction of membrane‐bound polysomes with the endoplasmic reticulum has been studied in MOPC 21 mouse plasmocytoma tissue‐culture cells which secrete an immunoglobulin G. Treatment of microsomes at high salt concentrations (500 mM KCl) results in the detachment of 40–45% of the bound ribosomes. This released (“loose”) fraction is predominantly monosomal in contrast to the remaining “tight” polysomes. It is shown that both ribosomal fractions contain immunoglobulin light‐chain mRNA which argues against a functional distinction between them in the cell.

Polysomes have been dissociated to apparently undegraded ribosomal subunits at 0°C by incubation with puromycin in the presence of a magnesium ion buffer. Application of this procedure to membrane‐bound polysomes results in partial release of ribosomal subunits from the membrane. The ribosomal material which is not so released sediments predominantly at 60 S and this fraction can be detached by subsequent washing at 500 mM KCl. Thus, by use of the polysome‐dissociating procedure together with high salt concentrations, essentially total release of the ribosomal components can be obtained.

The results indicate that the salt‐labile interactions are mediated via the large ribosomal subunits and are the only anchoring forces to the membrane at early stages of protein synthesis. The tight membrane‐bound polysomes result from the additional anchoring effect of the polypeptide chains as they grow through the membrane.
The Reversible Reduction of Testosterone to Androst‐4‐ene‐3α, 17β‐diol by a Soluble Enzyme of Rat Liver
Authors: SANYAL Mrinal K. , ORR James C. , ENGEL Lewis L.
Published: March 2005

When testosterone was incubated with a soluble enzyme preparation of male rat liver in the presence of NADPH, androst‐4‐ene‐3α, 17β‐diol and 5β‐androstane‐3α, 17β‐diol were formed. Incubation of [3β‐2H]androst‐4‐ene‐3α, 17β‐diol with the same enzyme preparation and a large excess of NADPH resulted in the production of testosterone, and 5β‐androstane‐3α, 17β‐diol devoid of deuterium. It is concluded that testosterone is converted reversibly to the allylic alcohol and that reduction of the allylic alcohol to the saturated diol probably does not occur directly, but only through the conjugated enone. The overall reduction of [2,2,4,6,6‐2H5]testosterone to 5β‐androstane‐3α, 17β‐diol involves no loss of deuterium.
Characterisation of RNA Synthesis in vitro in Neurone‐Rich, Oligodendroglial and Liver Nuclei
Authors: MIZOBE Fumio , TASHIRO Tomoko , KUROKAWA Masanori
Published: March 2005

In neurone‐rich nuclei isolated from the adult guinea‐pig cerebral cortex, DNA‐dependent RNA polymerase B reaction is several times more active than in liver nuclei. This may be attributable to the unusual wealth of euchromatin in mature neuronal nuclei.

Nuclear RNA labelled in vitro was hydrolysed by alkali, and the number as well as the average length of nascent RNA chains were determined by measuring [3H]uridine and [3H]UMP/[3H]uridine ratio, respectively. In neurone‐rich nuclei, the number of growing RNA chains is greater, and the average chain length longer than in liver nuclie. In oligodendroglial nuclei which exhibit RNA polymerase B reaction of intermediate activity, the number of growing RNA chains is found comparable to, but the average chain length longer, than that in liver nuclei. Thus, the presence of a greater number of growing RNA chains appears to be a unique feature of neuronal nuclei.

In neuronal chromatin, mean residue ellipticity in circular dichroism spectrum in the wavelength range of 260–300 nm is significantly higher than in liver and oligodendroglial chromatins, and this is in apparent correlation with the number, rather than the average length, of growing RNA chains.

Digestion of chromatin with trypsin leads to reversion of the circular dichroism spectrum toward that of free DNA, and completely abolishes the difference in ellipticity pattern that is observed among native neurone‐rich, oligodendroglial and liver chromatins.

In liver nuclei, RNA polymerase B reaction is greatly enhanced with trypsin, which is primarily attributable to the facilitated elongation of RNA chains that is otherwise restricted. In neuronal nuclei where the steric restriction imposed on RNA synthesis is though to be intrinsically weakened, digestion with trypsin is more than necessary in disclosing the RNA polymerase B activity.

RNA synthesis in neuronal and oligodendroglial nuclei in vitro is discussed in correlation with that reported in vivo.