bluuebunny/crossref_metadata_2025 huggingface dataset:
The protein phosphatases involved in cellular regulation
Authors: PELECH Steven , COHEN Philip
Published: March 2005

The phosphorylase phosphatases in rat and rabbit liver cytosol that are markedly stimulated by histone H1, protamine and polylysine were identified as protein phosphatases‐2Ao, 2A1 and 2A2 by anion‐exchange chromatography, gel‐filtration and immunotitration experiments. Histone H1 and protamine also stimulated the dephosphorylation of phosphorylase kinase, glycogen synthase, fructose‐1, 6‐bisphosphatase, pyruvate kinase, acetyl‐CoA carboxylase and phenylalanine hydroxylase by phosphatases‐2A1 and 2A2, and with several of these substrates activation was even more striking (20–100‐fold) than that observed with phosphorylase (∼ 5‐fold). Activation by basic polypeptides did not involve dissociation of these phosphatases to the free catalytic subunit.

The dephosphorylation of phosphorylase by protein phosphatase‐1 was suppressed by basic polypeptides, protamine and polylysine being the most potent inhibitors. However, the dephosphorylation of glycogen synthase, pyruvate kinase and acetyl‐CoA carboxylase were markedly stimulated by histone H1 and protamine (2–13‐fold). Consequently, with the appropriate substrates, protein phosphatase‐1 can also be regarded as a basic‐polypeptide‐activated protein phosphatase.

Heparin stimulated (1.5–2‐fold) the dephosphorylation of phosphorylase by phosphatases‐2Ao and 2A1, provided that Mn2+ was present, but phosphatase‐2A2 and the free catalytic subunit of phosphatase‐2A were unaffected. Heparin, in conjunction with Mn2+, also stimulated (1.5‐fold) the dephosphorylation of glycogen synthase (labelled in sites 3abc), phosphorylase kinase and phenylalanine hydroxylase by phosphatase‐2A1, but not by phosphatase‐2A2. By contrast, the dephosphorylation of phosphorylase and phosphorylase kinase by protein phosphatase‐1 was inhibited by heparin. However, dephosphorylation of glycogen synthase and pyruvate kinase by phosphatase‐1 was stimulated by this mucopolysaccharide.

The studies demonstrate that basic proteins can be used to distinguish protein phosphatase‐1 from protein phosphatase‐2A, but only if phosphorylase is employed as substrate. Optimal differentiation of the two phosphatases is observed at 30 μ/ml protamine or at heparin concentrations < 150 μM.
The protein phosphatases involved in cellular regulation
Authors: TUNG H. Y. Lim , ALEMANY Susana , COHEN Philip
Published: March 2005

Protein phosphatases‐2Ao, 2A1and 2A2have been purified to homogeneity from rabbit skeletal muscle. Approximately 1 mg of phosphatase‐2A0and 2A1, and 0.5 mg of phosphatase‐2A2, was isolated from 4000 g muscle within 10 days. Protein phosphatases‐2Aoand 2A1each comprised three subunits, termed A, B' and C (2Ao) or A, B and C (2A1), while phosphatase‐2A2contained only two subunits, A and C. The A and C components of phosphatases‐2Ao, 2A1and 2A2had indistinguishable mobilities on sodium dodecyl sulphate/ polyacrylamide gels and identical peptide maps. By these criteria, the C component was also identical to the catalytic subunit of phosphatase‐2A purified from ethanol‐treated muscle extracts. The electrophoretic mobilities of the B and B′ subunits were slightly different, and their peptide maps were distinct.

The molecular masses of the native enzymes determined by sedimentation equilibrium centrifugation were 181 ± 6 kDa (2Ao), 202 ± 6 kDa (2A1) and 107 ± 5 kDa (2A2), while those of the subunits estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis were 60 kDa (A), 55 kDa (B), 54 kDa (B′) and 36 kDa (C). These values, in conjunction with molar ratios estimated by densitometric analyses of the gels, suggest that the subunit structures of the enzymes are AB'C2(2Ao), ABC2(2A1) and AC (2A2). Protein phosphatase‐2A2appears to be derived from 2Aoand/or 2A1during purification through degradation or dissociation of the B′ and/or B subunits.

Protein phosphatases‐2Ao, 2A1and 2A2were the only phosphorylase phosphatases in rabbit skeletal muscle that were activated by the basic proteins, protamine (A0.5= 0.25 μM), histone H 1 (A0.5= 0.3 μM) and polylysine (A0.5= 0.04 μM). Activation by protamine varied over 5–20‐fold for phosphatase‐2Aoand 5–7‐fold for phosphatases‐2A1and 2A2. The dephosphorylation of glycogen synthase was activated by basic proteins in a similar manner to the phosphorylase phosphatase activity. The isolated C subunit was also stimulated by histone H1 and protamine, but 5–10‐fold higher concentrations were required, and with phosphorylase as substrate, maximum activation was only about 2‐fold. Activation by basic proteins appears to involve their interaction with the A and/or C subunits, but not with the B or B′ subunits, or substrates phosphorylase and glycogen synthase.
Does UGA suppressor tRNATrp from Escherichia coli have a unique CCA anticodon sequence?
Authors: DELAMARCHE Christian , BUCKINGHAM Richard H.
Published: March 2005

The properties of the UGA‐suppressor tRNATrp (anticodon CCA) from Escherichia coli has greatly influenced ideas about the specificity of codon‐anticodon interactions, showing that the anticodon sequence is not the sole determinant. However, a recent hypothesis for the mechanism of suppression by this tRNA proposes that the base change in position 24, in the dihydrouridine stem, leads to a change in translational specificity of the tRNA by increasing post‐transcriptional modification of cytidine 34, in the anticodon wobble position [M. Yarus (1982) Science (Wash. DC) 218, 646–652]. The enzyme postulated to do this normally modifies C34 of a minor isoleucine isoacceptor specific for AUA codons. This modification should reduce reading of G in the third codon position: affinity chromatography on columns containing immobilised tRNAPro (anticodon VGG) has therefore been employed to isolate an enriched population of the putative suppressor species, if such a sub‐population exists. The results obtained are difficult to reconcile with the presence of a subfraction, modified post‐transcriptionally in C34, responsible for the suppression of UGA. This argues in favour of the previously advanced hypothesis for the mechanism of suppression, which depends on events outside the codon‐anticodon interaction itself.
Characterization of an enzyme which catalyzes isomerization and epimerization of d‐erythrose 4‐phosphate
Authors: TERADA Tomoyuki , MUKAE Hisayoshi , OHASHI Kazufumi , HOSOMI Saburo , MIZOGUCHI Tadashi , UEHARA Kihachiro
Published: March 2005

The enzyme which catalyzes the conversion of d‐erythrose 4‐phosphate to d‐erythrulose 4‐phosphate and d‐threose 4‐phosphate has been purified to homogeneity from a crude extract of beef liver. Analysis of the purified enzyme by Sephadex G‐100 gel filtration and sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed it to be a dimer of relative molecular mass 43000. From the gas chromatography/mass spectrometry analyses of the enzymatic reaction products, it appeared that about 90% of the total amount of tetrose 4‐phosphate was present as d‐erythrulose 4‐phosphate after equilibration. The purified enzyme, which is tentatively called ‘erythrose‐4‐phosphate isomerase’ had no significant isomerase activities on d‐glyceraldehyde 3‐phosphate, d‐ribose 5‐phosphate, d‐glucose 6‐phosphate and d‐fructose 6‐phosphate, but a strong d‐ribulose‐5‐phosphate 3‐epimerase activity was co‐purified with the erythrose‐4‐phosphate isomerase activity through every step in the isolation. Both the erythrose‐4‐phosphate isomerase and d‐ribulose‐5‐phosphate 3‐epimerase activities were inactivated at the same rate at the elevated temperature, and also inhibited to the same extent by various inhibitors. It is likey, that both activities are catalyzed by the single enzyme protein.
Immunoreactive and biologically active somatostatin in human and sheep milk
Authors: WERNER Haim , AMARANT Tanchum , MILLAR Robert P. , FRIDKIN Mati , KOCH Yitzhak
Published: March 2005

The presence of immunoreactive and biologically active somatostatin in sheep and human milk has been demonstrated. Milk somatostatin exhibits similar chromatographic behavior to that of synthetic somatostatin‐14 on both reversed‐phase C18 and cation‐exchange high‐performance liquid chromatography columns. Milk, in contrast to plasma, contains only somatostatin‐14‐like material. Milk somatostatin was capable of inhibiting the basal and the prostaglandin‐induced release of growth hormone from anterior pituitary cell cultures in a pattern similar to synthetic somatostatin‐14. The concentrations of the peptide, as determined by radioimmunoassay, were found to be 113pg/ml in human milk and 150 ± 4.8pg/ml (mean ± range) in sheep milk. These values are severalfold higher than the corresponding concentration of the peptide in the plasma of these species. These findings are analogous to our previous observations concerning two other hypothalamic hormones, luliberin and thyroliberin [Baram, T., Koch, Y., Hazum, E. and Fridkin, M. (1977) Science (Wash. DC) 198, 300–302]. The high concentration of somatostatin and other neuropeptides in milk implies either an active concentrating mechanism in the mammary gland or an additional extrahypothalamic source for the synthesis and release of these peptides.
Incorporation and metabolism of ganglioside GM2 in skin fibroblasts from normal and GM2 gangliosidosis subjects
Authors: SONDERFELD Sybille , CONZELMANN Ernst , SCHWARZMANN Günter , BURG Josef , HINRICHS Ute , SANDHOFF Konrad
Published: March 2005

Ganglioside GM2, 3H‐labeled in the sphingoid base, was added to the culture medium of normal and GM2 gangliosidosis fibroblasts. Ganglioside was found to adsorb rapidly to the cell surface, most of it could however be removed by trypsination. The trypsin‐resistant incorporation was about 10 nmol/mg cell protein, after 48 h. The rates of adsorption and incorporation depended strongly on the concentration of fetal calf serum in the medium, higher serum concentrations being inhibitory. After various incubation times, the lipids were extracted, separated by thin‐layer chromatography and visualized by fluorography. In normal cells a variety of degradation products as well as sphingomyelin was found whereas in GM2 gangliosidosis cells, only trace amounts of such products (mainly GA2) were found. In contrast, the higher gangliosides GM1 and GD1a were formed in comparable amounts (2.2–3.6% of total radioactivity after 92 h) in normal and pathologic cell lines. Supplementation of cells from GM2 gangliosidosis, variant AB, with purified GM2‐activator protein restored ganglioside GM2 degradation to almost normal rates but had no effect on its glycosylation to gangliosides GM1 and GD1a. From these results we conclude that the synthesis of higher gangliosides from incorporated GM2 can occur by direct glycosylation and not only via lysosomal degradation and resynthesis from [3H]sphinganine‐containing degradation products. Preliminary studies with subcellular fractionation after various times of [3H]ganglioside incorporation indicated biphasic kinetics for the net transport of membrane‐inserted ganglioside to lysosomes, compatible with the notion that a portion of the glycolipids can also escape from secondary lysosomes and migrate to Golgi compartment or cell surface.
Determination of the complete amino‐acid sequence of porcine miniplasminogen
Authors: MARTI Thomas , SCHALLER Johann , RICKLI Egon E.
Published: March 2005

The complete amino acid sequence of porcine miniplasminogen (Mr 37600), comprising 341 residues, was determined by automated Edman degradation in a liquid‐phase or solid‐phase sequenator. Selected fragments were produced by cleavage with 2‐(2‐nitrophenylsulfenyl)‐3‐methyl‐3′‐bromoindolenine (BNPS‐skatole), cyanogen bromide, hydroxylamine, Staphylococcus aureus protease or trypsin or with combinations thereof and by activation with urokinase. The sequence obtained was compared with the known sequences of human and bovine miniplasminogen, indicating that the porcine molecule apparently contains the same structural and functional domains as the protein of the other two species. Porcine miniplasminogen has a sequence homology of 83% with human and of 79% with bovine miniplasminogen; 74% of the amino acids are identical in all three species. The results show a higher degree of evolutionary conservatism in the structurally and/or functionally vital regions of the molecule (active site residues, kringle 5).
Purification and properties of an enterotoxin from a coatless spore mutant of Clostridium perfringens type A
Authors: LINDSAY James A. , SLEIGH Robert W. , GHITGAS Christine , DAVENPORT James B.
Published: March 2005

A method is described for isolating an enterotoxin from a coatless spore mutant (8–6) of Clostridium perfringens type A. The characteristics of this enterotoxin only slightly resembled those of previously isolated enterotoxins of C. perfringens. The type A (8–6) enterotoxin was found to be composed of two subunits of Mr 18000 with isoelectric points of 3.8 and 4.3. The LD50 for mice was 39 μg/kg with 0.10 μg corresponding to one erythemal unit. The type A (8–6) enterotoxin was inactivated by heating for 10 min at 60°C. The amino acid composition data of type A (8–6) and delta toxins was similar, but type A (8–6) and type A enterotoxins showed less similarity. This lack of similarity between type A and type A (8–6) enterotoxins was confirmed by the failure of anti‐sera to type A enterotoxin to neutralize the type A (8–6) enterotoxin, in both the mouse and erythemal tests.
Human placental neuraminidase
Authors: VERHEIJEN Frans W. , PALMERI Silvia , HOOGEVEEN André T. , GALJAARD Hans
Published: March 2005

Supernatant of homogenized human placenta hardly contains lysosomal neuraminidase activity. It is, however, possible to generate remarkably high activity by concentration of a partially purified glycoprotein fraction. This activity is labile to dilution, but can be stabilized by incubation at 37°C and acid pH.

Using β‐galactosidase specific affinity chromatography and immunotitration, we show that the activated and stabilized human lysosomal neuraminidase exists in a complex with β‐galactosidase. Sucrose density gradient centrifugation experiments demonstrate that the neuraminidase activity is exclusively present in a high density multimeric form of β‐galactosidase.

The formation of multimeric forms of β‐galactosidase is known to require a 32000‐Mr‘protective’ protein. Monospecific antibodies against this ‘protective’ protein were purified from a conventional antiserum containing a mixture of antibodies against the 64000‐Mrβ‐galactosidase protein and against the 32000‐Mr‘protective’ protein, using a nitrocellulose blot immunoaffinity purification procedure. Immunotitration experiments with these antibodies show that the 32000‐Mr‘protective’ protein is present both in association with the β‐galactosidase multimer and with the high‐density multimeric form together with neuraminidase.

Our data further suggest that association of the 32000‐Mr‘protective’ protein and another yet unidentified subunit is essential for the catalytic activity of lysosomal neuraminidase. These results explain the absence of neuraminidase activity in the autosomal recessive human lysosomal storage disorder galactosialidosis, where the 32000‐Mr‘protective’ protein is known to be absent.
Ca 125 and Ca 19–9: two cancer‐associated sialylsaccharide antigens on a mucus glycoprotein from human milk
Authors: HANISCH Franz‐G. , UHLENBRUCK Gerhard , DIENST Cornelius , STOTTROP Maria , HIPPAUF Elke
Published: March 2005

The cancer‐associated antigens Ca 125 and Ca 19–9 were demonstrated by radioimmunoassay to form structural units of a mucus glycoprotein in human milk taken from healthy women four days after parturition. The glycoprotein precipitated with the casein fraction at pH 4.6 and was completely absent in the whey as judged from Ca 19–9 assay. It could be effectively enriched by phenol‐saline extraction from soluble milk proteins and further purified by gel filtration on Sephacryl S300 and Sephacryl S400.

The active component with a bouyant density of 1.41 g/ml in isopycnic density gradient centrifugation (CsCl) shared common physico‐chemical and chemical characteristics of mucus glycoproteins. Carbohydrates representing about 68% by weight were conjugated to protein by alkali‐labile linkages, exclusively and were essentially free of d‐mannose.

Activities of Ca 125 and Ca 19–9 were both destroyed by treatment with periodate, mild alkali or neuraminidase suggesting the antigens are sialylated saccharides bound to protein by alkali‐labile linkages. The fraction of monosialylated saccharide alditols isolated after reductive β‐elimination from the mucus glycoprotein was shown to inhibit monoclonal antibodies anti‐(Ca 125) and anti‐(Ca 19–9) in radioimmunoassay.
Determination of the autocorrelation orders of proteins
Authors: MACCHIATO M. Francesca , CUOMO Vincenzo , TRAMONTANO Anna
Published: March 2005

A statistical analysis of the autocorrelation characteristics of active polypeptides has been carried out by means of the correlogram method. It is shown that the amino acid sequences of the analysed proteins, considered as a whole, are autocorrelated and that the correlograms characterize each protein reflecting its three‐dimensional structure.
Reacquisition of quaternary structure by fully reduced and denatured seminal ribonuclease
Authors: PARENTE Augusto , D'ALESSIO Giuseppe
Published: March 2005

Air‐regenerated monomers of bovine seminal ribonuclease have been found capable of reassociating into native dimers, whereas monomers refolded in the presence of a glutathione redox mixture do not reassociate into dimers [Smith, K. G., D'Alessio, G. and Schaffer, S. W. (1978) Biochemistry 17, 2633–2638]. The crucial step in the process of regeneration of dimers is an isomerization step, which the newly refolded monomers undergo in order to reassociate into dimers. The two sulfhydryls at sequence positions 31 and 32 of the seminal RNAase chain, forming in the native dimer the intersubunit disulfides, have been found to have an important role in the refolding of the monomeric intermediates, as well as in the regeneration of dimers.
Carbon‐13 nuclear magnetic resonance analysis of [1‐13C]glucose metabolism in Crithidia fasciculata
Authors: de los SANTOS Carlos , BULDAIN Graciela , FRYDMAN Benjamín , CANNATA Joaquin J. B. , CAZZULO Juan J.
Published: March 2005

The non‐invasive technique of 13C nuclear magnetic resonance was applied to study glucose metabolism in vivo in the insect parasite Crithidia fasciculata. It was found that under anaerobic conditions [1‐13C]glucose underwent a glycolytic pathway whose main metabolic products were identified as [2‐13C]ethanol, [2‐13C]succinate and [1,3‐13C2]glycerol. These metabolites were excreted by C. fasciculata into the incubation medium, while in the cells [3‐13C]phosphoenolpyruvate was also detected in addition to the aforementioned compounds. The C3 acid is apparently the acceptor of the primary CO2 fixation reaction, which leads in Trypanosomatids to the synthesis of succinate. By addition of sodium bicarbonate to the incubation mixture l‐[3‐13C]malate was detected among the excretion products, while the ethanol:succinate ratio of 2.0 in the absence of bicarbonate changed to a ratio of 0.6 in the presence of the latter. This was due to a shift of the balance between carboxylation of phosphoenolpyruvate, leading to succinate, and pyruvate decarboxylation leading to ethanol. The addition of 25%2H2O to the incubation mixture led to the formation of [2‐13C, 2‐2H]ethanol derived from the prior incorporation of 2H+ into pyruvate in the reactions mediated by either pyruvate kinase or malic enzyme. However, no 2H+ incorporation into l‐malate was detected, excluding the possibility that the latter was formed by carboxylation of pyruvate, and lending support to the idea that l‐malate results from the carboxylation of phosphoenolpyruvate to oxaloacetate by phosphoenolpyruvate carboxykinase. The formation of [2‐13C, 2‐2H]‐succinate under the same conditions reflected the uptake of 2H+ during the reduction of fumarate. When the incubations were carried out in the presence of 100%2H2O, several [1‐13C, 1‐2H]ethanol species were detected, as well as [2‐13C, 2‐2H]malate and [1,3‐13C2, 1,3‐2H2]glycerol. The former deuterated compounds reflect the existence of NAD2H species when the incubations were carried out in 100%2H2O, while the incorporation of 2H+ into [1,3‐13C2]glycerol must be attributed to the phosphoglucose‐isomerase‐mediated reaction during glycolysis.
Chick oviduct glucocorticosteroid receptor
Authors: GROYER André , LE BOUC Yves , JOAB Irène , RADANYI Christine , RENOIR Jack‐Michel , ROBEL Paul , BAULIEU Etienne‐Emile
Published: March 2005

The glucocorticosteroid receptor (GR) has been studied in oviduct cytosol prepared from estrogen‐primed, 4‐week‐withdrawn chicken. The equilibrium dissociation constant was 6 nM for dexamethasone, and 18300 receptor sites/cell were measured assuming that all cells contain identical concentrations of GR. Dexamethasone, used in most studies investigating glucocorticosteroid action, was found not to be the best GR ligand. The affinities of several natural and synthetic glucocorticosteroids for GR increased in the following order: cortisol < deoxycorticosterone < dexamethasone < corticosterone < triamcinolone acetonide. The synthetic steroid RU 486 was the most specific ligand of GR (its affinity was ∼ 10‐fold higher than that of triamcinolone acetonide), while it did not bind either to plasma transcortin (which binds dexamethasone nor, surprisingly, to progesterone receptor (PR), contrary to what occurs in mammalian species.

The molybdate‐stabilized, 8‐S form of GR was prepared from withdrawn chick oviduct, whole chick embryo or cultured chick embryo fibroblasts (which do not contain PR), and was labeled with either [3H]dexamethasone or [3H]RU 486. The sedimentation coefficient of radioactive ligand–8‐S GR complexes was shifted towards heavier forms after incubation with polyclonal (IgG‐G3) or monoclonal (BF4) antibodies generated against the molybdate‐stabilized, 8‐S form of the chick oviduct PR. Since neither IgG‐G3 nor BF4 interacted with the steroid binding 4‐S form of GR, it is suggested that these antibodies recognized a non‐steroid binding protein common to molybdate‐stabilized, 8‐S forms of GR and PR.
Thrombin‐reactive polypeptides of human blood
Authors: CHELLADURAI Mohanathasan , FOSSETT Nancy G. , GANGULY Pankaj
Published: March 2005

Two polypeptides of 74 kDa and 55 kDa have been isolated from human platelets by immunoaffinity and lectin affinity chromatography and their effects on thrombin reactivity have been examined. These proteins in combination enhanced the aggregation of platelets by thrombin while aggregation induced by trypsin, collagen and adenosine diphosphate was not significantly affected. An enhancement in the action of thrombin on fibrinogen, N‐benzoylarginine ethyl ester and H‐d‐phenylalanyl‐l‐pipecolyl‐l‐arginine‐p‐nitroanilide dihydrochloride was also observed in the presence of the platelet proteins. Under similar conditions, the proteins did not influence the esterolytic activity of trypsin or plasmin. Studies at different thrombin and protein concentrations showed maximum enhancement of enzyme reactivity when the ratio between the peptides and thrombin was optimal. In the presence of these proteins, the affinity of thrombin for N‐benzoylarginine ethyl ester was about twofold higher than in the control. Two polypeptides with properties similar to those described above have also been isolated from human plasma.

Antibodies to the above proteins isolated from either platelets or plasma were raised in rabbits. Intact platelets solubilized in Triton X‐100 or plasma showed two precipitin lines in immunoelectrophoresis against both of the above antisera and a similar pattern was observed with the isolated polyeptides. The polypeptides did not interact in immunoelectrophoresis with antisera to whole serum, antithrombin, C4 binding protein or protein S. These 74‐kDa and 55‐kDa polypeptides contained radioactivity when radioiodinated platelets were used suggesting that they are located on the cell surface. Fresh plasma was analyzed by gel electrophoresis under nondenaturing and denaturing conditions and the proteins were transferred to nitrocellulose sheets. Staining with antibody to these thrombin‐reactive proteins and 125I‐protein A showed several reactive plasma proteins under nondenaturing conditions with the major band migrating in the albumin area. In plasma treated with sodium dodecyl sulfate, the 74‐kDa and 55‐kDa components were observed. A prominent 74‐kDa band and a fainter 55‐kDa component were again observed when platelets solubilized in sodium dodecyl sulfate were analysed by the above procedure. It is proposed that human platelets and plasma contain polypeptides which may directly modulate thrombin reactivity.
Glycosyltransferase activities in liver mitochondria
Authors: ARDAIL Dominique , GATEAU Odile , MORELIS Renée , LOUISOT Pierre
Published: March 2005

The role of phospholipids in the activity of inner mitochondrial mannosyltransferase was investigated. This enzyme catalyzes the direct transfer from GDP‐mannose to lipidic acceptor.

Inner mitochondrial membranes from purified mice liver mitochondria are prepared by digitonin treatment. Swelling of mitoplasts leads to the formation of inner membrane vesicles, which are then purified on a discontinuous sucrose gradient. The validity of this fractionation procedure is controlled by measurements of specific enzymatic activities and by electron microscopy.

Measurement of mannosyltransferase activity in native inner mitochondrial membranes is unsuccessful, even in the presence of exogenous dolichyl monophosphate. Treatment of inner membranes with specific phospholipid liposomes in the presence of exogenous dolichyl monophosphate is essential in order to measure this enzymatic activity. Addition of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin in the presence of Mg2+ results in a high degree of activation of the mannosyltransferase system. Maximal enzymatic activity is obtained with an approximate 3:7 mass ratio of exogenous phospholipid to inner membrane proteins. These experiments establish that sensitivity to activation by phospholipids is an inherent property of inner membrane mannosyltransferase.

Another approach to this problem was to reconstitute an in vivo lipidic environment of the inner membrane. The results of this procedure suggest that the activity of inner mitochondrial mannosyltransferase may be subject to modulation by outer membrane lipidic extract treatment.
Cooperation among electron‐transfer complexes in ATP synthesis in chloroplasts
Authors: HANGARTER Roger , ORT Donald R.
Published: March 2005

We have investigated the extent to which redox reactions of thylakoid membranes cooperate in ATP synthesis. This was done by measuring the onset of ATP synthesis following a series of single‐turnover light‐flashes at various levels of electron transport inhibition. In the presence of antibiotics that prevent the formation of a membrane potential, the onset of ATP synthesis seems to depend entirely on the formation of an adequately large ΔpH. Under our conditions, the accumulation of about 60 mmol H+mol chlorophyll−1is then necessary to form the requisite ΔpH, which in turn requires about 15 saturating flashes in uninhibited thylakoid samples. Inhibition of some of the electron transfer centers by limiting the light intensity of the flahes, by dichlorophenyldimethylurea, by heat treatment, or by NH2OH‐treatment caused an incease in the number of flashes required for the onset of ATP synthesis. The increase in the requisite number of flashes reflected the decreased number of electrons transferred in each flash, almost exactly the same number of electrons being transferred before ATP synthesis could begin. This effect of inhibitors was true when the two photosystems were operating in unison and when either of the two photosystems was acting alone. However, when either photosystem acted alone, there was an increase in the number of flashes required for the onset of ATP synthesis, an increase which was consistent with the observed lower flash‐induced proton accumulation.

A mathematical analysis of the onset of flash‐induced ATP synthesis shows that at least several hundred proton‐translocating electron transport complexes must be cooperating to form the threshold ΔpH. In spite of this evidence for extensive cooperation among different electron transport complexes in ATP formation, the implied pooling of H+ions does not seem to involve inner vesicle regions accessible to exogenous buffers. Thus, even when the number of H+ions accumulated per flash was reduced by 70% through attenuation of the intensity of the flashes, exogenous hydrogen ion buffers present within the lumen of the thylakoid vesicle had no effect on the number of flashes required for the onset of ATP synthesis.
Protonmotive force and photophosphorylation in single swollen thylakoid vesicles
Authors: CAMPO Maria Luisa , TEDESCHI Henry
Published: March 2005

Swollen vesicles generally 40 μm in diameter were prepared from spinach chloroplasts. These vesicles appear to originate from thylakoids. The present study reports results obtained with individual vesicles using micromanipulative procedures. The electric potential across the membrane was measured with microelectrodes and the pH of the internal space was calculated from the fluorescence of the pH indicator pyranine. The individual vesicles photophosphorylate as measured with luciferin‐luciferase. Impalement with microelectrodes did not affect the ability of individual vesicles to photophosphorylate. However, there was no significant membrane potential either with continuous illumination or light flashes. In contrast, we found a ΔpH of 3.7 under photophosphorylative conditions and the incubation with the appropriate buffers blocked photophosphorylation presumably by preventing formation of a pH gradient. We propose that, in these vesicles, the membrane potential plays no role in photophosphorylation, whereas a pH gradient is obligatory.
Biochemical research on oogenesis
Authors: DENIS Herman , le MAIRE Marc
Published: March 2005

The ester bond of aminoacyl tRNA is protected against hydrolysis in the 42‐S particles (thesaurisomes) present in Xenopus laevis previtellogenic oocytes. Deacylation of tRNA is very slow in vitro, unless ATP is present. ATP causes a partial turnover of aminoacyl tRNA in vitro, with no detectable decrease in the overall aminoacylation level of tRNA, which remains close to 100%. tRNA in the particles turns over rapidly in vivo. Since the ester bond of aminoacyl tRNA is stabilized inside the 42‐S particles, this turnover cannot be a consequence of spontaneous deacylation of tRNA, followed by reacylation by the aminoacyl‐tRNA synthetases associated with the particles. We rather consider this turnover as reflecting a true metabolic activity of the particles, and a direct or indirect involvement of these particles in the oocyte's protein‐synthesizing system.
Overproduction of staphylokinase in Escherichia coli and its characterization
Authors: SAKO Tomoyuki
Published: March 2005

A recombinant plasmid which directs the overproduction in Escherichia coli of staphylokinase from Staphylo‐coccus aureus has been constructed by placing the staphylokinase gene, sak, under the control of bacteriophage λ PR promoter in the plasmid. When an E. coli strain having the plasmid was induced, the staphylokinase activity in the periplasmic fraction increased about 60‐fold and the 15.5‐kDa protein corresponding to the mature form reached about 25% of the periplasmic proteins. At the same time the 18.5‐kDa protein corresponding to the precursor form was accumulated in the membrane fraction, showing that the processing and translocation of the sak gene product were restricted during high level of its synthesis.

By using this strain, the mature staphylokinase has been easily purified to near homogeneity. The purification steps consisted of extraction of the periplasmic proteins by osmotic shock and CM‐cellulose column chromatography. Two species of staphylokinase were identified after CM‐cellulose column chromatography. Although their isoelectric points and NH2‐terminal amino acid sequences were different, their specific activities were almost equal. These results strongly suggest that the NH2‐terminal portion of staphylokinase is not important for its activity.
Incomplete polypeptides are formed in vitro by premature chain termination
Authors: CHROBOCZEK Jadwiga
Published: March 2005

The mechanism of incomplete polypeptides formation during protein synthesis was studied in the wheat germ cell‐free system programmed with brome mosaic virus RNA 4. The synthesis of coat protein, the complete product of RNA 4 translation, was accompanied by the appearance of polypeptides of lower molecular mass. It was shown that incomplete products are formed by translation of different lengths of RNA 4, always from the first 5′ AUG codon, and were due neither to proteolysis of coat protein nor to the translation of nucleolytic fragments of mRNA. The molecular masses of incomplete products were determined and the nucleotide sequence of RNA 4 was examined in the regions where wheat germ ribosomes stop translating. It was found that they contained, on average, a slightly higher guanosine content than the total coding part of RNA 4. Translation of RNA 4 in the reticulocyte lysate resulted in a marked diminution of incomplete polypeptides. Addition of high‐speed supernatant from reticulocyte lysate prevented the formation of incomplete products during translation of RNA 4 in the wheat germ system. This suggests that reticulocyte lysate contains some factor(s) which facilitate the movement of ribosomes beyond the regions where the elongation is retarded.
The complete 30‐base‐pair origin region of bacteriophage φ X174 in a plasmid is both required and sufficient for in vivo rolling‐circle DNA replication and packaging
Authors: FLUIT Adriaan C. , BAAS Pieter D. , JANSZ Hendrik S.
Published: March 2005

The origin of replication of the isometric single‐stranded DNA bacteriophages is located in a specific sequence of 30 nucleotides, the origin region, which is highly conserved in these phage genomes. Plasmids harboring this origin region are subject to rolling‐circle DNA replication and packaging of single‐stranded (ss) plasmid DNA into phage coats in φ X 174 or G4‐phage‐infected cells.

This system was used to study the nucleotide sequence requirements for rolling‐circle DNA replication and DNA packaging employing plasmids which contain the first 24, 25, 26, 27, 28 and the complete 30‐base‐pair (bp) origin region of φ X174. No difference in plasmid ss DNA packaging was observed for plasmids carrying only the 30‐bp origin region and plasmids carrying the 30‐bp origin region plus surrounding sequences (i.e. plasmids carrying the HaeIII restriction fragment Z6B of φ X 174 replicative‐form DNA). This indicates that all signals for DNA replication and phage morphogenesis are contained in the 30‐bp origin region and that no contribution is made by sequences which immediately surround the origin region in the φ X 174 genome. The efficiency of packaging of plasmid ssDNA for plasmids containing deletions in the right part of the origin region decreases drastically when compared with the plasmid containing the complete 30‐bp origin region (for a plasmid carrying the first 28 bp of the origin region to approximately 5% and 0.5% in the φ X 174 and G4 systems respectively).

Previous studies [Fluit, A. C., Baas, P. D., van Boom, J. H., Veeneman, G. H. and Jansz, H. S. (1984) Nucleic Acids Res. 12, 6443–6454] have shown that the presence of the first 27 bp of the origin region is necessary as well as sufficient for cleavage of the viral strand in the origin region by φ X 174 gene A protein. Moreover, Brown et al. [Brown, D. R., Schmidt‐Glenewinkel, T., Reinberg, D. and Hurwitz, J. (1983) J. Biol. Chem. 258, 8402–8412] have shown that omission of the last 2 bp of the origin region does not interfere with φ X 174 rolling‐circle DNA replication in vitro.

Our results therefore suggest that for optimal phage development in vivo, signals in the origin region are utilized which have not yet been noticed by the in vitro systems for φ X 174 phage DNA replication and morphogenesis.
Reconstitution and fusogenic properties of Sendai virus envelopes
Authors: HARMSEN Marco C. , WILSCHUT Jan , SCHERPHOF Gerrit , HULSTAERT Caesar , HOEKSTRA Dick
Published: March 2005

Sendai virus membranes were reconstituted by detergent dialysis, using the non‐ionic detergents Triton X‐100 and octyl glucoside. Membrane reassembly was determined by measuring the surface‐density‐dependent efficiency of resonance energy transfer between two fluorescent phospholipid analogues, which were co‐reconstituted with the viral envelopes. The functional incorporation of the viral proteins was established by monitoring the ability of the reconstitution products to fuse with erythrocyte membranes, utilizing assays based on either resonance energy transfer or on relief of fluorescence selfquenching. The persistent adherence of residual Triton X‐100 with the reconstituted membrane was revealed by an artificial detergent‐effect on the resonance energy transfer efficiency and the occurrence of hemolysis of human erythrocytes under conditions where fusion does not occur. Properly reconstituted Sendai virus envelopes were obtained with octyl glucoside. The fusion activity of the viral envelopes was dependent on the initial concentration of octyl glucoside used to disrupt the virus and the rate of detergent removal. Rapid removal of detergent by dialysis against large volumes of dialysis buffer (ratio 1:850) or by gel filtration produced reconstituted membranes capable of inducing hemagglutination but significant fusion activity was not detected. By decreasing the volume ratio of dialysate versus dialysis buffer to 1:250 or 1:25, fusogenic viral envelopes were obtained. The initial fusion kinetics of the reconstituted viral membrane and the parent virus were different in that both the onset and the initial rate of fusion of the reconstituted membranes were faster, whereas the extents to which both particles eventually fused with the target membrane were similar. The differences in the initial fusion kinetics lead us to suggest that the details of the fusion mechanism between Sendai virus and the target membrane involve factors other than the mere presence of glycoproteins F and HN in the viral bilayer.

Finally, the results also indicate that determination of the viral fusion activity in a direct manner, rather than by an indirect assay, such as hemolysis, is imperative for a proper evaluation of the functional properties retained upon viral reconstitution.
Natural‐abundance 13C nuclear magnetic resonance studies of regulation and overproduction of l‐lysine by Brevibacterium flavum
Authors: INBAR Livia , KAHANA Zvi E. , LAPIDOT Aviva
Published: March 2005

Natural‐abundance 13C NMR spectroscopy has been used to study the metabolism of the l‐lysine‐producing bacterium, Brevibacterium flavum. Relationships of biomass formation, precursor uptake, and product excretion, as a function of culture medium, oxygen supply and specific cell membrane permeability, were rapidly determined using 67.89‐MHz 13C NMR. The induction of lysine production throughout the growth cycle was studied. Intracellular and extracellular levels of free metabolites and unconsumed precursor were quantitatively measured as a function of growth culture conditions. Limited availability of oxygen resulted in accumulation and excretion of unfavorable products: lactate, succinate, alanine and valine. However, under optimal aeration conditions l‐lysine was the sole metabolite detected extracellularly. Various important long‐lived intermediates and storage compounds were detected in the intact cells (by NMR measurements). Carbon resonances of carbohydrates and amino acids were resolved and easily identified. Of particular interest are those of trehalose carbons, a storage carbohydrate.

Natural‐abundance 13C NMR spectroscopy seems most suitable for biotechnological processes where high concentrations of intermediates and end‐products can be observed. We anticipate that this approach will be employed to screen overproducing bacterial strains.
Differential effects of high‐lysine mutations on the accumulation of individual members of a group of proteins encoded by a disperse multigene family in the endosperm of barley (Hordeum vulgare L.)
Authors: LÁZARO Ana , BARBER Domingo , SALCEDO Gabriel , MENDEZ Enrique , GARCÍA‐OLMEDO Francisco
Published: March 2005

The CM proteins are a group of major salt‐soluble endosperm proteins encoded by a disperse multigene family. The effects of high‐lysine mutations on the net accumulation in barley endosperm of three members of this group (CMa, CMb, and CMe) have been investigated. Genes CMa, CMb and CMe are located in chromosomes 1, 4, and 3 respectively. Protein CMe has been found to be identical with a previously described trypsin inhibitor. The three proteins have been quantified in the different genetic stocks by HPLC. The different high‐lysine mutations have different effects on the expression patterns of the three genes: CMe is markedly decreased and CMa and CMb are increased in mutant Risø 1508, whereas all three proteins are decreased in Risø 527 and increased in Risø 7 with respect to the wild‐type Bomi; CMa and CMb are increased and CMe is unaffected in mutant Risø 56 with respect to the wild‐type Carlsberg II; and protein CMe is markedly decreased in Hiproly barley as compared with its sister line CI4362. The implications of these results in connection with the evolution of CM proteins and with the characterization of high‐lysine mutations are discussed.
Lateral proton conduction at a lipid/water interface
Authors: PRATS Michel , TOCANNE Jean‐François , TEISSIÉ Justin
Published: March 2005

Fast lateral proton conduction along the lipid/water interface has recently been experimentally demonstrated in our laboratory [Teissié, J., Prats, M., Soucaille, P. & Tocanne, J. F. (1985) Proc. Natl Acad. Sci. USA, in the press].

The present study gives a more precise description of the way various physical parameters can affect this process. The dependence of the distance covered by the proton on time is demonstrated to be quadratic. Increasing the speed of stirring in the injection compartment or the amount of injected acid or the contact between the monolayer and the acidic subphase increased the efficiency of the proton transfer. Raising the strength of the buffer in the bulk phase inhibited proton conduction.

Results from experiments where the transfer of protons from the bulk phase to the interface was modified, suggested the occurrence of an ‘energy barrier’ limiting the access of protons from the bulk phase to the lipid polar head region.
Hysteretic behaviour of citrate synthase
Authors: LÖHLEIN‐WERHAHN Gerlinde , GOEPFERT Petra , EGGERER Hermann
Published: March 2005

Citrate specimens derived from chiral acetates were converted to the CoA derivatives. These were reconverted with citrate synthase to citrates under conditions of either predominating hydrolytic burst or predominating steady‐state period. The stereochemical purity of substrates and products was determined.

Reversal of the synthase condensation step occurs under both conditions but is markedly increased during the steady‐state period. The results indicate that citryl‐CoA‐derived acetyl‐CoA and oxaloacetate create the steady‐state conditions.

The hydrolase state and the ligase/lyase state of the synthase predominate under burst and steady‐state conditions, respectively. This result indicates a conversion of the hydrolase state into the ligase/lyase state during the transition.
The pho1 mutation
Authors: SOMLO Marie , CLAVILIER Lea , DUJON Bernard , KERMORGANT Michèle
Published: March 2005

The pho1 mutation belongs to the OLI2 gene on mitochondrial DNA of Saccharomyces cerevisiae, which codes for a membrane factor subunit of the mitochondrial ATPase (apparent molecular mass 20 kDa). We analysed the ATPase complex from the pho1 mutant and from three revertants, after immunoprecipitation from mitochondrial extracts, by dodecyl sulphate/acrylamide gel electrophoresis. In two revertants the OLI2 gene product appeared as an abundant slower migrating peptide, while in the pho mutant, two bands appeared in very low amounts. For the third revertant, a strong band appeared at the normal level. Sequencing of the OLI2 gene from these strains gave the following results: the pho1 mutation is a frameshift, arising by insertion of an extra thymidine into a group of three. Two of the revertants contain the same group of four thymidines, but genetic compensation of the frameshift occurs 24 base pairs downstream by the loss of four bases, implying a deficit of one codon. The third revertant has recovered the normal three‐thymidine sequence. There is excellent correlation between the modified sequences and electrophoretic migration of the peptide product. Owing to the leakiness of the pho1 phenotype (reduced but not nil growth rate on oxidizable carbon sources, 5–10% highly oligomycinsensitive ATPase complex, low amounts of OLI2 gene product peptides), some translational correction of the frameshift is bound to occur. Based on these results, the compatibility of abnormal ATPase architecture with modified energetic efficiency is discussed.
Identification of a 60‐kDa phosphoprotein that binds stored messenger RNA of Xenopus oocytes
Authors: DEARSLY Anne L. , JOHNSON Rosalyn M. , BARRETT Perry , SOMMERVILLE John
Published: March 2005

Rapidly labelled, polyadenylated RNA is contained in three distinct fractions isolated from homogenized amphibian oocytes: (a) in ribonucleoprotein particles that are associated with a fibrillar matrix, the complexes sedimenting at > 1500S; (b) in ribonucleoprotein particles that sediment at 20–120S and have the characteristics of stored (maternal) messenger ribonucleoprotein (mRNP) and (c) in polyribosomes that sediment at 120–360S. We have compared the RNA and protein components of the first two of these RNP fractions. The polydenylated RNA extracted from the two RNP fractions differs in that the RNA from fibril‐associated RNP contains a much higher content of repeat sequences than does the RNA from mRNP. In other words, the RNA from fibril‐associated RNP is largely unprocessed and may constitute a premessenger state, which for convenience is referred to as premessenger RNP (pre‐mRNP). RNA‐binding experiments demonstrate that the polypeptide most tightly bound in pre‐mRNP is a 54‐kDa component (p54), whereas the polypeptide most tightly bound in mRNP is a 60‐kDa component (p60). Antibodies raised against p60 are used to show that this polypeptide is a common major component of pre‐mRNP and mRNP and that it is also located in oocyte nuclei. However the state of p60 is modified between the premessenger and stored message levels: the polypeptide in mRNP is heavily phosphorylated whereas the equivalent polypeptide in pre‐mRNP is completely unphosphorylated. The relative roles of the presence of repeat sequences and phosphorylation of mRNA‐associated protein in blocking translation are discussed.