bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Method for the Rapid Isolation of Nuclear Membranes from Rat Liver
Authors: Kay Robert R. , Fraser Derek , Johnston Irving R. 
Published: March 2005

A rapid, mild method for isolating nuclear membranes from isolated rat liver nuclei is described. The method employs a double digestion of nuclei with a low level of DNAase I at approximately 0.1 mM Mg2+ and slightly alkaline pH, to release the membranes.
Electron microscopy shows excellent preservation of the nuclear membrane morphology. Large membrane fragments consisting of a double membrane with ribosomes adhering to the outer layer are produced. The layers are connected at nuclear pores, which have distinct annular subunits and occasional central granules. Contamination by other subcellular structures is minimal.
The recoveries of nuclear protein, DNA, RNA and phospholipid in the membrane are approximately 8%, 1–3%, 8% and 55%, respectively. The DNA associated with the membrane does not appear to result from the reassociation of released DNA. The enzymic content of the membrane is similar to that of the microsomes. It is concluded that the nuclear membrane is the sole nuclear location of glucose‐6‐phosphatase.
About 1% of the total nuclear DNA polymerase was recovered associated with the nuclear membrane. By three criteria this enzyme was identical with pure authentic nuclear DNA polymerase. This finding is discussed against the background of the suggestions that the nuclear membrane is involved in DNA replication.
Properties of Glycogen Synthetase in Erythrocytes
Authors: Moses Shimon W. , Bashan Nava , Gutman Alisa 
Published: March 2005

Erythrocyte glycogen synthetase activity was found to be absolutely dependent on the presence of glucose‐6‐phosphate (Glc‐6‐P). The Km, for UDPG and the Ka for Glc‐6‐P were not altered by preincubation of the enzyme. Glucose‐6‐phosphate protected the enzyme against thermal in‐act:ivation. Enzyme activity was inhibited by P1, SO4=, ATP, theophylline and caffeine. Galactose‐6‐phosphate could replace Glc‐6‐P as activator.A study to the effect of various combinations of Glc‐6‐P, ATP and P1 at concentrations prevailing in the red blood cell suggested that Glc‐6‐P concentration, rather than the effect of inhibitors, was the main factor limiting the activity of erythrocyte glycogen synthetase.The low rate of glycogen synthesis in erythrocytes can be related to the lack of conversion of the enzyme to a glucose‐6‐phosphate independent form.
Structure of the Parasporal Inclusion of Bucillus thuringiensis Berliner: Characterization of a Repetitive Subunit
Authors: Glatron Marie‐Françoise , Lecadet Marguerite‐Marie , Dedonder Raymond 
Published: March 2005

The parasporal inclusion of Bacillus thuringiensis var. Berliner was solubilized by several processes. Action of denaturing and reducing agents on the crystal protein was studied and the dissolution products analyzed in three cases: (a) after treatment with mercaptoethanol and guanidine hydrochloride at pH 7,5, (b) after treatment with a reducing agent at pH 9.5 and (c) after treatment with 0.1 N and 1 N sodium hydroxide.By the first treatment, one N‐terminal and one C‐terminal amino acid were characterized whereas four N‐terminal residues appeared by the other processes performed at pH 9.5 and above pH 12. It is suggested that peptide bonds were disrupted in these latter cases. Ultracentrifugation experiments revealed the existence of only one repetitive subunit of molecular weight 80000 in the solution obtained after treatment by mercaptoethanol and guanidine hydrochloride at pH 7.5. Furthermore reassociated particles obtained from this solution were observed under phase‐contrast microscope.
Studies on the Interaction of Ligands with Phosphorylase b Using a Spin‐Label Probe
Authors: Campbell Iain D. , Dwek Raymond A. , Price Nicholas C. , Radda George K. 
Published: March 2005

Phosphorylase b can be specifically spin labelled at one SH group per subunit without affecting either the specific activity or the co‐operativity between the 5‐AMP sites.
The electron spin resonance spectrum indicates that the mobility of the spin label is greater than that of the whole enzyme. This, together with measurements of the hyperfine coupling constant suggests that the spin‐labelled SH is exposed to the solvent.
The conformational change induced by 5‐AMP is detected by the spin label. This transition occurs over the same concentration range as that for activation of the enzyme, and shows similar co‐operativity.
The previously postulated fourth conformation in the presence of 5‐AMP and glucose 1‐phosphate can be confirmed by a study of the spin‐label spectrum in the presence of both ligands.
IMP and glucose have almost no effect on the spin label on the enzyme.
Glucose 6‐phosphate causes a conformational change, and the binding of glucose 6‐phosphate to the spin‐labelled enzyme is hyperbolic.
Addition of Mn2+ to the spin‐labelled enzyme results in a diminution of the electron spin resonance signal by approximately 18%, while addition of Mg2+ causes no change in the spectrum.
The broadening of the proton resonances of AMP, glucose 1‐phosphate, glucose 6‐phosphate and adenosine, resulting from interaction with the unpaired electron spin on the labelled enzyme, has been studied.
The distance between the unpaired electron and selected protons on these ligands have been calculated.
A brief outline of the calculations is given in the Appendices.
Adaptive Changes of Phosphofructokinase and Aldolase in Adipose Tissue
Authors: Orevi Miriam , Gorin Erela , Shafrir Eleazar 
Published: March 2005

The activity of several enzymes of the glycolytic pathway was investigated in rat epididymal adipose tissue. Determination of phosphofructokinase and aldolase activity with an improved assay involving homogenization of the tissue in the presence of fructose 6‐phosphate showed that the activity of aldolase is in the same range or lower than that of hexokinase and phosphofructokinase. Both aldolase and phosphofructokinase exhibited an adaptive behavior. The activity at optimal assay conditions decreased in fasting or alloxan diabetic rats and rebounded on refeeding or upon treatment of diabetic rats with insulin. These changes are similar to those of hexobase and glucose‐6‐phosphate dehydrogenase, whereas the activity of pyruvate kinase was not influenced by these situations. Changes in the steady state concentrations of glycolytic intermediates in vivo provided further evidence that the activity of aldolase may intluence the rate of glycolysis in adipose tissue. A rise in fructose 1,6‐bisphosphate concentration relative to that of fructose 6‐phosphate was observed, especially in conditions of increased glucose flow. A rise in both fructose &phosphate and fructose 1,6‐bisphosphate was observed in diabetic rats.The pattern of activities of glycolytic enzymes and of metabolite concentrations in adipose tissue is compared with that in other tissues, and the regulatory characteristics of glycolysis peculiar to adipose tissue are discussed.
Determination of the Primary Structure of a Mouse γG2a Immunoglobulin Identification of the Disulfide Bridges
Authors: de Préval Claude , Fouegreau Michel 
Published: March 2005

All cysteine‐containing peptides of the MOPC 173 (γG2a) monoclonal immunoglobulin were isolated and sequenced, allowing characterization of the twelve intrachain disulfide bonds, and extension of previous work concerning the interchain disulfide bridges [3]. Positioning of the various bonds was deduced from homology with the known sequences of the mouse x chains [6,21], MOPC 173 VH region [2], and human γ chains [8], and allowed corroboration of the previous proposed order of the CNBr fragments isolated from the whole molecule [1].The general pattern of the γG2a mouse immunoglobulin seems in agreement with the accepted view that this molecule contains two heavy chains and two light chains linked by disulfide bonds, and having intrachain bridges disposed in a strictly linear manner [4,8].
Regulation of Inositol Biosynthesis in vivo by Pyrophosphate in Neurospora crassa
Authors: Piña Marta Z. , Brunner Aurora , Chagoya Victoria , Piña Enrique 
Published: March 2005

Pyrophosphate has previously been shown to be a competitive inhibitor of d‐glucose‐6‐phosphate cycloaldolase (NAD+‐dependent) in vitro. By growing wild‐type Neurospora crassa in a medium with 1/100 of the normal concentration of Pi a 4‐fold diminution in the PPi pool was obtained. In addition to this decrease in the PPi pool the following cellular changes were detected: a 100% increase in the water‐soluble free inositol pool; a 60% decrease in the activity in vitro of d‐glucose‐6‐phosphate cycloaldolase (NAD+‐dependent) tested under optimal conditions; no change in the utilization of inositol, measured either by its utilization as a carbon source or by its incorporation into lipids; and a 4 to 5 fold increase in the conversion of uniformly labelled [14C]‐glucose into [14C]inositol in short‐time experiments with intact N. crassa cells depleted of endogenous substrates.It is concluded that within the N. crassa cell, PPi, by inhibiting the activity of the d‐glucose‐6‐phosphate cycloaldolase participates in the regulation in vivo of inositol biosynthesis.
Cytochrome b in Neurospora crassa Mitochondria A Membrane Protein Containing Subunits of Cytoplasmic and Mitochondrial Origin
Authors: Weiss Hanns 
Published: March 2005

A membrane protein containing about 16μmol cytochrome b/g protein was prepared from Neurospora crassa mitochondria by means of chromatography on oleyl‐polymethacrylic acid resin.
At room temperature the oxidized from absorbs with maxima at 562nm, 530nm and 415nm, the reduced form with maxima at 561 nm, 530 nm and 429 nm. At low temperature these bands are shifted to 561 nm, 525 nm and 414 nm, for the oxidized form and to 558 nm, 527 nm and 427 nm for the reduced form.
The apparent molecular weight of this membrane protein is 55000 as determined by means of gel chromatography in a bile acid medium.
By means of gel electrophoresis and gel chromatography in sodium dodecylsulfate medium this protein was resolved into two smaller subunits with apparent molecular weights of 10000 and 11000 and into a larger subunit with an apparent molecular weight of 32000. The heme b group was found to be associated with the smaller subunits.
The two smaller subunits are synthesized at the cytoplasmic ribosomes, the larger subunit at the mitochondrial ribosomes, as revealed by incorporation of radioactive leucine in the presence of cycloheximide or chloramphenicol, respectively.
Pulse‐labeling experiments indicated that the subunits are derived from precursor polypeptides with at least two separate pools. The pool size of the precursor polypeptides of cytoplasmic origin is about 1% of the amount of the corresponding subunits in the membrane protein. The pool size of the precursor polypeptide of mitochondrial origin is about 5%.
Studies on the Reconstitution of Apotransketolase with Thiamine Pyrophosphate and Analogs of the Coenzyme
Authors: Heinrich Peter C. , Steffen Hans , Janser Peter , Wiss Oswald 
Published: March 2005

The binding of thiátmine pyrophosphate to apotransketolase from baker's yeast has been studied by measuring the cofactor‐dependent activity after passage through a column of Sephadex G‐25 or dialysis, respectively. At pH values >6.5, the cofactors Mg2+ and thiamine pyrophosphate were lost upon gel filtration or dialysis, indicating reversible binding. Thiamine pyrophosphate alone was able to activate and partially reconstitute transketolase activity in the absence of divalent cations. Using activity and fluorescence‐quenching measurements the reconstitution process of transketolase was studied as a function of different parameters such as pH, temperature, ionic strength, donor and acceptor substrates and various divalent cations. The rate of recombination of transketolase from its components, apotransketolase, thiamine pyrophosphate and divalent cations, increases in the order Ni < Mg < Co < Mn < Ca, whereas the catalytic activity of the enzyme does not depend on the nature of the complexing metal ion. Extensively dialyzed apotransketolase is activated by SO2−4 ions in 10—20 mM concentration. The binding constants for thiamine pyrophosphate determined by activity measurements were 4.8 μM in the absence and 1.0 μM in the presence of 4.4 mM Mg2+. A Km of 1.1 μM (5 mM Mg2+) was obtained from fluorescence quenching experiments. A Km of 5.5 μM was obtained for 2′‐ethylthiamine pyrophosphate. The V for the latter compound was 27% of that of thiamine pyrophosphate. For the following competitive inhibitors of thiamine pyrophosphate the inhibitor constants (Ki) were determined: oxythiamine pyrophosphate, 0.03 μM; tetrahydrothiamine pyrophosphate, 0.40 μM; thiochrome pyrophosphate, 6.3 μM; pyrithiamine pyrophosphate, 110 μM; inorganic pyrophosphate, 4200 μM.The relevance of the charge transfer interaction between thiamine pyrophosphate and apotransketolase is discussed.
Broad‐Specificity Protease‐Isoinhibitors for Trypsin, Chymotrypsin, Plasmin and Kallikrein from Snails (Helix pornatia)
Authors: Tschesche Harald , Dietl Thomas 
Published: March 2005

A low‐molecular‐weight fraction of 88% pure inhibitors, inhibitor preparation I, could be obtained from aqueous extracts of snails (Helix pomatia) using a series of steps involving ammonium sulfate precipitation at 63% saturation, affinity chromatography on trypsin resin and gel filtration on Sephadthx G‐50 (fine). A mixture of heat‐ and acid‐stable inhibitors of broad specificity inhibiting the proteolytic activity of bovine α‐ and β‐trypsin, bovine α‐ and β‐chymotrypsin, porcine plasmin and porcine pancreatic and serum kallikrein could thus be separated from a heat‐ and acid‐instable trypsin‐plasmin inhibitor, inhibitor preparation 11, which is of higher molecular weight and is derivcd from the albumin gland of the snail.Inhibitor preparation I is a mixture of at least 17 active components, which could be separated by equilibrium chromatography using a pH gradient from pH 4.9 to 8.0. Three of the predominant, most active components were obtained as pure homogenous proteins and identified as isoinhibitors composed of 58 amino acid residues. The three purified isoinhibitors closely resemble the basic trypsin‐kallikrein inhibitor of bovine organs with respect to amino acid composition and inhibitory specificity. They inhibit the proteolytic activity of trypsin, chymotrypsin, plasmin and organ kallikrein, but not serum kallikrein. They have been characterized according to their interaction with the enzymes, time dependence of inhibition and reactive‐site residues. Their locus of biosynthesis are the external organs, probably the subepithelial glands of the skin.
The Effect of Limited Proteolysis of Chicken Ovoinhibitor by Bovine Chymotrypsin on the Inhibitory Activities against Trypsin, Chymotrypsin and Elastase
Authors: Feinstein Gad , Gertler Arieh 
Published: March 2005

Incubation of chicken ovoinhibitor with catalytic amounts of bovine α‐chymotrypsin at low pH caused partial losses in the inhibitory activities against bovine chymotrypsin and porcine elastase, but not against bovine trypsin. Similar incubation with trypsin or elastase, even followed by incubation with carboxypeptidase A or B, had no effect on any inhibitory activity. The losses of the inhibitory activities were pH dependent and had different pH profiles for the effect on the inhibitory activities against chymotrypsin and elastase. The maximal losses were observed around pH 3, while incubation at pH 5 still resulted in 40% loss of chymotrypsin‐inhibiting activity with no effect on elastase‐inhibiting activity. The inhibitory activity against chymotrypsin was also lost faster, thus indicating that the inhibitory sites against these enzymes are distinct and differ from each other.
The N‐ and C‐terminal amino acid analyses of ovoinhibitor treated with chymotrypsin at pH 3 and 5, as compared to the native ovoinhibitor, indicate a cleavage of Phe‐Ile bond that could be related to the partial loss of chymotrypsin‐inhibiting activity. The cleavage of Tyr‐X bond was observed mainly after treatment at pH 3 and therefore was related to the loss of elastase‐inhibiting activity. The N‐terminal of the native ovoinhibitor was found to be isoleucine.
The inhibitory activities of chicken ovoinhibitor that was isolated by affinity chromatography on chymotrypsin‐Sepharose were compared to those of ovoinhibitor that was isolated by salt fractionation. The trypsin‐inhibitory activities of both preparations were equal, but the chymotrypsin and elastase‐inhibitory activities were lower than those of salt‐fractionated ovoinhibitor. These results, together with the N‐ and C‐terminal amino acid analyses, indicate that during the affinity chromatography on insoluble chymotrypsin, there occurred a limited proteolysis of the ovoinhibitor.
Polyribosome‐Bound and Free‐Cytoplasmic‐Hemoglobin‐Messenger RNA in Differentiating Avian Erythroblasts
Authors: Spohr Georges , Kayibanda Boniface , Scherrer Klaus 
Published: March 2005

Cytoplasmic messenger‐like RNA not associated with ribosomes has been investigated in immature duck erythrocytes and compared with polyribosome‐bound messenger RNA. It is shown that 9‐S RNA of the same electrophoretic mobility as the polyribosomal 9‐S globin messenger is present free in the cytoplasm. Its rate of synthesis and decay is consistent with a role as a precursor of polyribosomal 9‐S globin messenger.mRNA species different from 9‐S RNA, among them a predominant 12‐S species, are found associated with polyribosomes as well as in the free cytoplasmic pool. The free cytoplasmic mlRNA pool is more heterogeneous compared to the polyribosomal mRNAs, suggesting a translational control qualitatively and quantitatively selecting among the free cytoplasmic mlRNAs for those to be translated.
Effects of Calcium Ions on Pyruvate Kinase in Pigeon Liver
Authors: Gabrielli Franco , Baldi Silvia 
Published: March 2005

Pigeon liver pyruvate kinase shows allosteric properties similar to those of the yeast and the rat liver enzyme. These properties include cooperative interaction with phosphoenol pyruvate, allosteric inhibition by ATP and allosteric activation by fructose 1,6‐bisphosphate.The Ca2+ effect on pigeon liver pyruvate kinase has been studied on the Mg2+‐activated enzyme at different phosphoenol pyruvate concentrations and in presence of both fructose 1,6‐bisphosphate and ATP. The Ca2+ activation effect is absolutely dependent on the Mg2+ concentration: in the absence of Mg2+, pyruvate kinase has no catalytic activity and it cannot be replaced by Ca2+.Ca2+ has a double and opposite effect on the liver enzyme activity: Ca2+ is an activator at low phosphoenol pyruvate concentrations, however at increased phosphoenolpyruvate concentrations, Ca2+ becomes an inhibitor.The Ca2+ activation effect is present also in the ATP‐inhibited enzyme and at low phosphoenolpyruvate concentrations Ca2+ is able to remove completely the ATP inhibitory effect. On the enzyme activated by fructose 1,6‐bisphosphate, Ca2+ has only an inhibitory effect at all the concentrations of phosphoenolpyruvate tested.The Ca2+ inhibitory effect on the liver pyruvate kinase is observed at phosphoenolpyruvate concentrations that are above the content of phosphoenolpyruvate in the liver, indicating that in vivo Ca2+ does not play a role in the negative control of pigeon liver pyruvate kinase; the different sensitivity to calcium ions showed by hepatic and muscular enzyme suggests that Ca2+ may function differently in the control of the liver and muscle glycolytic pathways.
Effect of Progesterone on Respiration and Oxidative Phosphorylation
Authors: Aleksandrowicz Zenon , ŚWierczyński Julian , ŻElewski Leon 
Published: March 2005

Progesterone inhibited oxidation of succinate by intact rat liver mitochondria in metabolic state 3. The degree of inhibition did not depend on succinate and ADP concentration. Inhibition was less pronounced in the presence of EDTA. The respiration has been slightly inhibited by progesterone if mitochondria were damaged by sonication.
Inhibition of succinate (+ rotenone) oxidation by progesterone in intact rat liver mitochondria in metabolic state 3 was reversed by 2,4‐dinitrophenol, calcium ion, gramicidin + NH4Cl, EDTA, some anions and partially by gramicidin + K+. The degree of reversal by anions was pH‐dependent.
Progesterone inhibited ATP synthesis with succinate (+ rotenone) as substrate. This inhibition has not been observed in the presence of citrate and other anions (in contrast to inhibition caused by oligomycin).
Progesterone was without effect on ATPase activity stimulated by 2,4‐dinitrophenol. Under nonrespiring conditions progesterone did not induce ATPase activity.
The experiments described suggest that inhibition of succinate oxidation by progesterone in the intact rat liver mitochondria is exerted by interference with the uptake of protons into the mitochondrion.
The O8 Antigen of Escherichia coli
Authors: Reske Konrad , Jann Klaus 
Published: March 2005

The polysaccharide moiety of the lipopolysaccharide (polysaccharide I) from Escherichia coli F492 (O8:K27−:H−) and the polysaccharide (polysaccharide II) from the rfa mutant F612 (derived from F492) were isolated by extraction with 45% aqueous phenol at 65°C. Polysaccharide I was obtained in 1–2% yield (based on dry bacteria). It contained mannose (83.5%), glucose (5.7%), galactose (3.4%), heptose (4.6%) and 2‐keto‐3‐deoxy‐mannosulonic acid (0.85%). Polysaccharide II was obtained in 0.8—1% yield (based on dry bacteria). It contained 99.2% mannose.With the method of Yphantis it was found that the molecular weights of polysaccharides I and II were 12400 and 10400, respectively. The difference accounted for the core oligosaccharide which was present in polysaccharide I and absent in polysaccharide II. The specific optical rotation was 43°C for polysaccharide I and 42°C for polysaccharide II.Both polysaccharides were permethylated. Subsequent hydrolysis, reduction and acetylation, followed by gas‐liquid chromatography and mass spectrometry indicated that in both polysaccharides two‐thirds of the mannose units were substituted at C‐2 and one‐third at C‐3. These results were confirmed by periodate oxidation.From polysaccharide II nine oligosaccharides were obtained after partial acid hydrolysis by chromatography on Biogel P2, paper chromatography and paper electrophoresis of the borate complexes. The oligosaccharides were reduced with sodium borodeuterate (which labelled the reducing mannose units) and methylated. After hydrolysis, reduction and acetylation the products were analyzed by gas chromatography and mass spectrometry.It is concluded that the polysaccharides I and II contain about 20 repeating units of α‐mannosyl‐1,2‐α‐mannosyl‐1,2‐α‐mannose which are joined through α‐1,3 linkages.