bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein A Isolated from Staphylococcus aureus after Digestion with Lysostaphin
Authors: Sjöquist John , Meloun Bedrich , Hjelm Hans
Published: March 2005

Previous methods which have been used for the preparation of protein A, such as thermal release of protein A with lysozyme from the bacteria, have given a heterogenous product in a low yield. The present publication reports the isolation procedure of protein A from Staphylococcus aureus digested by lysostaphin for 2 h at 37°C. 450—500 mg of protein A was obtained from 300 g of wet bacteria. The preparation is homogenous as seen from its disc electrophoretic pattern.

Amino acid composition was determined and based on a molecular weight of 42000. The number of amino acid residues found was 378 ± 6. Less than 0.2% hexosamines was present. The ultraviolet spectrum and the absorption coefficient, A1%275nm= 1.65 of protein A is given. Less than 0.1 mol amino terminal amino acid per mol protein was detected which indicates that the protein has a blocked N‐terminal amino acid. The protein was slowly digested with a mixture of carboxypeptidase A and B and 2 mol lysine/mol protein was released.

Precipitation of protein A with normal human γ‐globulin gave an IgG: protein A molar ratio of 2.1:1 within the equivalence zone.

The results are discussed with regard to the subunit structure of the protein and the type of attachment of protein A to the peptidoglycan part of the cell wall.
Zymogen‐Enzyme Transformations
Authors: Abita Jean‐Pierre , Lazdunski Michel , Bonsen Pieter P. M. , Pieterson Wim A. , de Haas Gerard H.
Published: March 2005

Transformation of prophospholipase A into phospholipase A is triggered by the tryptic hydrolysis of the Arg7‐Ala8 bond of the zymogen. This bond is a better substrate for trypsin than the corresponding strategic bonds of other zymogens such as trypsinogen and chymotrypsinogen A or B: Km= 2.2 mM, kcat= 7 s−1 at pH 8, 1°C. Calcium ions which have an essential role in phospholipase catalysis play no role in prophospholipase activation.

A comparative physico‐chemical analysis of the zymogen and of the enzyme shows that activation produces only very limited changes in the overall folding of prophospholipase. A structural rearrangement occurs at the N‐terminal end of the precursor. An excellent fluorescence reporter group to follow this conformational modification is the side‐chain of tryptophan‐3 (in phospholipase sequence) which passes from a polar to an apolar environment during activation.

The α‐amino group of alanine‐1 (in the phospholipase sequence) which appears on activation is essential for enzyme activity. Selective chemical modification of this function by a number of reagents abolishes phospholipase activity.

By analogy with the chymotrypsinogen‐chymotrypsin and the proelastase‐elastase transformations, it is proposed that the α‐amino group is involved in the formation of a salt‐bridge which stabilizes the adequate geometry of the active site. Evidences in favor of this hypothesis include the high pK and the low chemical reactivity of the α‐amino group, together with the resistance of native phospholipase to aminopeptidase degradation.
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.
Implication of Two Histidines in Transition‐Metal Binding in Concanavalin A
Authors: Gachelin Gabriel , Goldstein Leon , Hofnung Daniel , Kalb A. Joseph
Published: March 2005

We have performed potentiometric titrations and chemical modifications of demetallized concanavalin A and of the Ni2+ concavalin A complex. Two protons are released upon binding of one Ni2+ ion per concanavalin A subunit. The pH‐dependence of proton release indicates that two ionizable groups with pKa 6.8 are ligands for binding of the transition‐metal ion. Ethoxyformylation of histidines causes the destruction of the transition‐metal‐binding site unless it is occupied by a transition‐metal ion. For each transition‐metal site thus protected two otherwise ethoxyformylated histidines are rendered inert to the chemical modification. On photooxidation in the presence of rose bengal, protection of histidines and of the transition‐metal‐binding capacity was also observed to occur when Ni2+ ions were present. Two histidine residues in concanavalin A are, therefore, very likely metal‐ion ligands in the binding site.
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.
Studies on a Bromouracil‐Labelled DNA Fraction Induced by Antigenic Stimulation
Authors: Souleil Claude , Panijel Jacques
Published: March 2005

When primed guinea‐pig lymph‐node cells are incubated in vitro with homologous antigen and 5‐bromo‐2‐deoxy‐[3H]uridine, a peak of intermediate density appears between hybrid and normal DNA. This peak does not result from the attachment of a low density substance to DNA, since it is still present in denaturation conditions or after extensive digestion by pronase or phospholipase. By contrast, it disappears after DNA breakage by sonication, indicating that it results from end‐to‐end joining of labelled and unlabelled DNA segments.
Mathematical Treatment of Enzyme‐Catalyzed Isotope‐Exchange Reactions
Authors: Flossdorf Josef , Kula Maria‐Regina
Published: March 2005

A simple method of treating the kinetics of enzyme‐catalyzed isotope exchange reactions in systems at equilibrium is given. It depends on the analogy between the transfer of a label between species in a reacting chemical system and the flow of charge between capacitors in an electrical circuit.
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.
Complex Formation between 3‐Phospho‐d‐glyceric Acid and Divalent Metal Ions
Authors: Larson‐raźnikiewicz Märtha
Published: March 2005

Stoichiometric dissociation constants of complexes between the fully deprotonated 3‐phospho‐d‐glyceric acid and Mg2+, Mn2+, Ca2+, Co2+, Zn2+, and Ni2+, respectively, were determined in 0.25 M NaCl at 25°C. Potentiometric titrations of the ligand with these divalent metal ions were performed over a pH range close to the pK for the final deprotonation step of the ligand. The observed pH changes were utilized to estimate the dissociation constants; both algebraic and graphical techniques were employed in the estimations. Under the experimental conditions 1:1 complexes were the predominant products.
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.
Studies on the Heterogeneity of Mouse‐Globin Messenger RNA
Authors: Lanyon W. George , Paul John , Williamson Robert
Published: March 2005

Mouse reticulocyte messenger (9‐S) RNA has been prepared by treatment of polysomes with sodium dodecylsulphate. On 6% polyacrylamide gels, two sharp and one more diffuse component can be resolved in the 9‐S region. Messenger RNA prepared from 14‐S mRNA · protein from EDTA‐treated polysomes contains only the diffuse component. This component has priming activity for both α‐ and β‐globin chains, and evidence is presented that it can be partially resolved into separate messengers for each chain. The two sharp components, which migrate more slowly than the globin mRNA, are associated with the 30‐S ribosomal subunit after EDTA treatment, have no translational activity in a duck reticulocyte cell‐free protein‐synthesis system, and (unlike the diffuse component) are not destroyed by gentle sonication of polysomes to monosomes. The non‐coding components comprise approximately one‐third of the RNA in the 9‐S region of most RNA preparations from mouse reticulocytes. The diffuse (coding) 9‐S component from 14‐S mRNA · protein runs as a sharp symmetrical peak in formamide‐acrylamide gel electrophoresis, suggesting that there may be variation in secondary structure in aqueous solution rather than in molecular weight.
Reconstitution of Qβ Replicase Lacking Subunit α with Protein‐Synthesis‐Interference Factor i
Authors: Kamen Robert , Kondo Masatoshi , Römer Werner , Weissmann Charles
Published: March 2005

We have isolated a form of Qβ replicase lacking subunit α and shown that it is deficient in the utilization of Qβ‐plus strand RNA as template but competent to utilize Qβ‐minus strands, poly(C) and “6‐S” RNA. The functional identity of subunit α and protein‐synthesis‐inhibition factor i was proven by their equivalent capacity to restore full Qβ RNA‐directed activity of α‐less replicase.
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.
Interpretation of Incomplete Reactions in tRNA Aminoacylation
Authors: Bonnet Jacques , Ebel Jean‐Pierre
Published: March 2005

A study has been performed in order to try to explain some anomalies which are observed in aminoacylation reactions: the extent of aminoacylation often depends upon the experimental conditions, such as the enzyme concentration or the ionic strength.

We have demonstrated that these anomalies can be explained, in the case of yeast tRNAValII—valyl‐tRNA synthetase system, by the existence of two reactions occurring beside the aminoacylation reaction: the spontaneous deacylation of the valyl‐tRNAVal and a valyl‐tRNA synthetase‐catalyzed deacylation of the valyl‐tRNA Val. Consequently, any aminoacylation plateau merely reflects the existence of an equilibrium between the aminoacylation reaction and the deacylation reactions.

Sometimes the aminoacylation kinetics do not present a plateau but a maximum value followed by a decrease with time. This can be explained by an additional reaction: a valyl‐tRNA synthetase‐catalyzed degradation of ATP into AMP and PP1 in the presence of tRNAVal and of valine. This reaction leads to an accumulation of AMP and PP1 favouring the reverse reaction of the aminoacylation.

The kinetic constants of these reactions have been determined. Knowing these constants, it is possible to calculate theoretical aminoacylation plateau values which are in good agreement with the experimental ones. These interpretations can probably be extended to all the tRNA aminoacylation systems.
Regulation of the Catabolic Ornithine Carbamoyltransferase of Pseudomonas fluorescens
Authors: Halleux Paulette , Legrain Christiane , Stalon Victor , Piérard André , Wiame Jean‐Marie
Published: March 2005

The catabolic ornithine Carbamoyltransferase of Pseudomonas is a high molecular weight enzyme which, in vivo, catalyzes the phosphorolysis of citrulline. The enzyme exhibits pronounced allosteric properties which have been studied in great detail for the reverse direction of the reaction, the carbamoylation of ornithine. The interpretation of the kinetic data according to the “concerted transition” model of Monod, Wyman and Changeux led to the suggestion that the enzyme is an octamer built up by the aggregation of identical subunits. The present work was aimed at verifying that point and provided the following observations.

Sucrose density gradient sedimentation and polyacrylamide gel electrophoresis of the dissociated protein showed that the enzyme is made up of eight identical subunits with respect to both charge and size. The apparent molecular weight of the subunits is 39000.

The number of subunits is eight as determined by crosslinking of the enzyme in the presence of glutaraldehyde followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl‐sulfate.

The enzyme undergoes a self‐associating reaction which is under the control of its substrates, products and allosteric effectors.

The study of the effect of those ligands on the kinetic behaviour and on the distribution of the aggregation states of ornithine Carbamoyltransferase suggests that the active form of this enzyme is an octamer but that several inactive or less active octameric, tetramcric and dimeric states exist.
The Effect of Acylation on the Molecular Size of Insulin
Authors: Massey Derek E. , Smyth Derek G.
Published: March 2005

The tendency to aggregate at neutral pH exhibited by zinc insulin, insulin and some N‐acyl derivatives was studied with the aid of gel filtration. On Sephadex G‐75 and at an initial concentration of 1 mg/ml, zinc insulin appeared to possess an average molecular weight of 24000 and insulin an average molecular weight in the region of 12000. In contrast, NαA1, NαB1, NαB29‐tri‐succinylinsulin and NαA1, NαB1, NαB29,‐tricarbamylinsulin appeared predominantly monomeric with a molecular weight in the region of 6000. NαA1‐Monocarbamylinsulin resembled the tricarbamyl derivative whereas, NαA1‐monocarbainylinsulin aggregated like insulin. The results appear to reflect conformational changes undergone by the insulin molecule as a consequence of acylation at the A1 glycine.
X‐Ray Studies of Nucleoproteins Depleted of Lysine‐Rich Histone
Authors: Bradbury E. Morton , Molgaard Harald V. , Stephens Ronald M. , Bolund Lars A. , Johns Ernest W.
Published: March 2005

A method is described for the clean and quantitative removal of lysine‐rich histones from chicken‐erythrocyte and calf‐thymus nucleoproteins. X‐ray diffraction studies have been made of the whole nucleoproteins and those depleted of lysine‐rich histones. The whole nucleoproteins show the series of low‐angle rings characteristic of the native form of nucleohistone which have been attributed by Wilkins (1964) to a supercoiled structure. Removal of the lysine‐rich histones from the calf‐thymus and chicken‐erythrocyte nucleoproteins results in a sharpening of the low‐angle X‐ray rings. It appears that the lysine‐rich histones are not essential for the formation of the supercoiled structure and upon their removal the depleted nucleoprotein forms a more regular structure.
Presence of Trimers in Glucagon Solution
Authors: Gratzer Walter B. , Creeth J. Michael , Beaven Gilbert H.
Published: March 2005

Glucagon in solution at pH 10.2, at a concentration of 5 mg/ml, at which profiles of optical parameters against concentration show that it is substantially in its associated state, was covalently cross‐linked with dimethyl suberimidate. At a suberimidate concentration of 1 mg/ml the formation of high‐molecular‐weight aggregates, which is observed at higher reagent concentrations, is suppressed, and a largely homogeneous product is eluted from a gel‐filtration column in 6 M guanidine hydrochloride. This is also homogeneous as judged by polyacrylamide‐gel electrophoresis in the presence of sodium dodecylsulphate. Sedimentation equilibrium in 6 M guanidine hydrochloride led to a molecular weight of about 9600, showing that the unique cross‐linked form is the trimer; there is no evidence for dimers or hexamers, which have been inferred from sedimentation‐equilibrium distributions. It is suggested that the trimeric form of high α‐helix content may be equated with the rod‐like trimers making up the asymmetric unit in the crystal.
Effects of Alcohols on Hydrolysis Catalyzed by Beef‐Liver Esterase
Authors: Wynne David , Shalitin Yechiel
Published: March 2005

Transfer reactions catalyzed by beef‐liver esterase were studied. In the presence of alcohols as nucleophiles, transfer of the substrate acyl group to the nucleophile took place in parallel to hydrolysis. When substituted phenyl acetates were hydrolyzed in the presence of a given concentration of ethanol, the same partitioning ratio between hydrolysis and alcoholysis products was obtained irrespective of the nature of the substrate leaving group; this strongly indicates formation of a common acetyl‐enzyme intermediate during solvolysis. Kinetic analysis of the partitioning ratio as a function of the alcohol concentration indicates that there is an alcohol‐binding site on the enzyme. The acyl‐enzyme · alcohol complex undergoes hydrolysis in addition to the alcoholysis. The latter process is reduced in the presence of inert organic compounds. In the lower alcohol series, the effect of the alcohol increased with its chain length, whereas branched alcohols were less effective.
Eukaryotic Ribosomal Proteins
Authors: Delaunay Jean , Mathieu Claude , Schapira Georges
Published: March 2005

The degree of similarity of ribosomal proteins from various species or from various organs within a given species is not clearly known. Using two‐dimensional polyacrylamide‐gel electrophoresis, we have compared liver ribosomal proteins from adult chicken, adult rabbit, fetal and adult calf and ewe. In the rabbit, we also studied ribosomal proteins from kidney, caecal appendix and reticulocytes.

In all the cases which we investigated, more than 60 strongly basic proteins are visible. For one given organ, ribosomal proteins do not vary according to the stage of differentiation. Within one given species, there exists few differences from one tissue to another. From one species to another, there are also few differences but the unexpected resemblance of most of the proteins is remarkable.
The State of Aggregation of α‐Crystallin Detected after Large‐Scale Preparation by Zonal Centrifugation
Authors: Bloemendal Hans , Berns Ton , Zweers Anneke , Hoenders Herman , Benedetti Ennio L.
Published: March 2005

The protein α‐crystallin from calf eye lenses can be isolated in very high yield by zonal centrifugation. No other purification steps are required in order to obtain preparations the purity criteria of which allow sequence studies after dissociation into polypeptide chains. By this method evidence is provided that α‐crystallin is a population of aggregates built up by four different polypeptide chains, occurring in an almost constant ratio independent on the size of the aggregate.
Identification and Characterization of Pituitary Triose‐Phosphate Isomerase
Authors: Greenberg Mark M. , Yen Terence T. T. , Bobbitt Jesse L.
Published: March 2005

Several pituitary preparations from various species, including two purified follicle‐stimulating hormone preparations, inhibited the D‐glyceraldehyde‐3‐phosphate dehydrogenase reaction. The active factor in those preparations that caused the apparent inhibition has been identified as an enzyme, triose phosphate isomerase. Using D‐glyceraldehyde‐3‐phosphate as the substrate, the optimum pH for the isomerization reaction is 8.4. The partially purified enzyme has a specific activity of 34 μmol dihydroxyacetone phosphate isomerized × min−1× mg protein−1. The Km of the enzyme for D‐glyceraldehyde 3‐phosphate is 0.79 mM, and the Km for dihydroxyacetone phosphate is 1.53 mM. Half of the enzyme activity is lost in 7 min at 55°C. The molecular weight of the enzyme is estimated to be 56000. The electrophoretic mobility of the enzyme differs from that of the muscle enzyme supplied by Sigma indicating a difference in amino acid composition between the two enzymes. Electrophoresis revealed the presence of two to three isozymes of the pituitary triose phosphate isomerase.
Distribution of Proteins between Core and Split Fractions from 50‐S Ribosomal Subunits
Authors: Allet Bernard
Published: March 2005

The 50‐S ribosomal subunits were sedimented through 2 M LiCl or 3 M CsCl at low Mg2+ concentration (0.5–1 mM), and proteins in both pellet (cores) and supernatant (split proteins) were identified. No difference was detected between the two treatments: about half of the proteins were found specifically in cores or in split fraction, while the other species (common proteins) were distributed in different proportions between the two fractions. This distribution behavior was shown to be due to dynamic exchange of proteins occurring between the core particles and the surrounding medium. By the double‐labeling technique, the exchange of each protein was analyzed both in cores and in split fraction: this allowed us to estimate the partition coefficient of the common proteins. Furthermore, the analysis of the exchanges showed that the different common proteins approach at individual rates to the state of thermodynamic equilibrium in the test situation used (1 h incubation time).
Isolation of a Plasma‐Membrane Subfraction from Rat Liver Containing an Insulin‐Sensitive Cyclic‐AMP Phosphodiesterase
Authors: House Paul D. R. , Poulis Parissa , Weidemann Maurice J.
Published: March 2005

A procedure for the isolation and separation of three different subfractions of rat liver plasma membranes is described. The subfractions have different enzyme activities and sucrose buoyant densities of 1.18, 1.16 and 1.13 g/ml respectively. It was found that the low‐density subfraction had the following properties:

It appeared as smooth membrane vesicles approximately 0.1–0.2 μm in diameter when visualized under positive and negative staining with the electron microscope.

Its contamination by microsomes on a protein basis was less than 3% as measured by glucose‐6‐phosphatase activity, and no succinic dehydrogenase activity or DNA were detected.

The low‐density subfraction contained high relative specific activities of 5′‐nucleotidase, (Na+, K+)‐Mg2+‐stimulated ATPase, phosphodiesterase, and a glucagon and adrenalin‐sensitive adenylate cyclase. This fraction yielded over 30% of the total 5′‐nucleotidase, adenylate cyclase and phosphodiesterase activities of the homogenate.

A specific 3′: 5′‐AMP phosphodiesterase activity of this subfraction was stimulated by insulin. The half‐maximal concentration required to produce this stimulation was 2.3 nM. The stimulation was already observed at the shortest time interval tested (30 sec).

The localization in situ of the low‐density membrane subfraction on the surface of the hepatic cell is discussed, and it is suggested that it is derived mainly from the microvillar surface of hepatic parenchymal cells facing the perisinusoidal space.
Fate of Protein‐Containing Liposomes Injected into Rats
Authors: Gregoriadis Gregory , Ryman Brenda E.
Published: March 2005

[3H]Amyloglucosidase and 131I‐labelled albumin were entrapped into liposomes (lipid spherules) composed of phosphatidyl choline, cholesterol and dicetyl phosphate (7:2:1 molar ratio). 131I‐labelled albumin was also entrapped in [3H]cholesterol‐liposomes.

Investigations on the fate in vivo following intravenous injection of such protein‐containing liposomes into rats, showed that the bulk of liposomal radioactivity is removed from the blood within minutes. There is no measurable leakage of entrapped 131I‐labelled albumin while liposomes are in the circulation.

Most of the removed liposomal radioactivity was recovered in the liver. Autoradiographic examination of the liver 3 min after injection of albumin‐containing [3H]cholesterol‐liposomes suggests that hepatocytes and probably Kupffer cells are involved in the uptake of liposomes. It appears that liposomes enter this tissue intact but subsequently liposomal membranes and entrapped protein are catabolized.

These catabolic processes are probably occurring in the liver lysosomes, as most of the liposomal radioactivity in this tissue was recovered in the mitochondrial‐lysosomal fraction. Liposomes appear to be promising as a vehicle for the administration of enzymes and possibly also drugs to patients with storage diseases.
The Membrane Structure Studied with Cationic Dyes
Authors: Dell'antone Paolo , Colonna Raffaele , Azzone Giovanni Felice
Published: March 2005

Cationic dyes interact weakly with de‐energized submitochondrial particles incubated in a KCl medium. Energization of the particles results in a marked increase of binding. The Scatchard plots reveal, in the case of neutral red, and at pH 7.0, binding sites of 150–200 μmol per g protein with a dissociation constant of about 2.5 μM. In the case of acridine orange at the same pH, the value for binding sites is about 50 μmol per g protein with a dissociation constant of about 2 μM.

The binding of the cationic dyes to the energized particles is accompanied by a large decrease of absorbance which is reversed on addition of uncouplers. The change of absorbance is stoichiometrically dependent on the utilization of ATP, but independent of whether energy is supplied from respiration or from ATP. Within a certain range of protein/dye concentration ratio the reaction kinetics appears of pseudo first‐order character.

The change of absorbance is proportional to the amount of particles until a saturation is obtained. This permits titration the binding sites of the membrane for the dye, using the “end‐point” of the absorbance change or using the method of continuous variations. The number of binding sites found through these procedures is in agreement with those obtained from the binding experiments.

The pH of the medium has a marked effect on the dye‐membrane interaction. Increasing the pH from 6 to 8 results in an increase of the rate of binding, in a decrease of the dissociation constant and in a decrease of the number of binding sites.

The lipophilic cation, nupercaine, exerts a marked inhibition on the binding of the cationic dye and the concomitant decrease of absorbancy. The inhibition is of competitive type with a Ki of 20 μM.

The presence of electrolytes in the medium is required for the high affinity binding and the change of absorbance. Replacement of KCl with sucrose results in abolition of both phenomena.

The results are discussed in relation to the problem of the proposed inverted polarity of the ion translocation mechanism in the submitochondrial particles. The suggestion is made that sonication of mitochondria results in a “loss of polarity” rather than in “inversion of polarity”. The kinetics of the interaction indicates a limiting step which is probably that required for the interaction of the dye with the energized site.