bluuebunny/crossref_metadata_2025_split huggingface dataset:
Specific involvement of calmodulin and non‐specific effect of tropomyosin in the sensitivity to ouabain of Na+, K+‐ATPase in murine plasmocytoma cells
Authors: LELIEVRE Lionel G. , POTTER James D. , PIASCIK Michael , WALLICK Earl T. , SCHWARTZ Arnold , CHARLEMAGNE Daniele , GENY Blandine 
Published: March 2005

The Kd for ouabain for inhibition of Na+, K+‐ATPase isolated from murine plasmocytoma MOPC 173 cells is 120 μM, but when isolated in the presence of EDTA, it is 100‐fold lower (1.2 μM). Simultaneous addition of muscle tropomyosin and calcium to sensitive membranes restored the original insensitivity (tropomyosin bound to the membranes in an irreversible and saturable manner).For comparison 86Rb influx into intact cells, mediated by the Na+, K+‐pump, is half‐maximally inhibited at 50 μM ouabain.Calcium converts the enzyme to an insensitive form. This appeared to involve calmodulin because after extraction of calmodulin with EDTA and EGTA from sensitive membranes, they could not be made insensitive by the addition of tropomyosin and Ca2+. Addition of exogenous calmodulin to these calmodulin‐depleted membranes was required, in addition to tropomyosin and Ca2+, to decrease the ouabain sensitivity.The involvement of calmodulin was further assessed by measuring the range of Ca2+ concentrations required to convert to the insensitive form. At saturating concentrations of tropomyosin, increasing free [Ca2+] up to 3 μM led to an heterogenous population of Na+, K+‐ATPase forms. The calcium dependency was a saturable process. The shift to the insensitive form was half maximal at 0.65 + 0.11 μM free Ca2+ and was abolished by the addition of troponin I or trifluoroperazine (0.1 mM).These results suggest that, in murine plasmocytoma cells, the intrinsic sensitivity of Na+, K+‐ATPase to ouabain might be regulated by a calmodulin‐dependent process within a submembrane contractile‐like environment.
The widespread distribution of α‐N‐trimethylalanine as the N‐terminal amino acid of light chains from vertebrate striated muscle myosins
Authors: HENRY Gillian D. , TRAYER Ian P. , BREWER Susan , LEVINE Barry A. 
Published: March 2005

Identical tripeptides of the sequence X‐Pro‐Lys, where X is an unknown blocking group, were isolated from trypsin digests of bovine cardiac alkali light chain and the LC2 light chain of rabbit fast muscle. Chemical, electrophoretic and 1H‐NMR evidence characterized X as an unusual amino acid, α‐N‐trimethylalanine (Me3Ala), which was earlier reported as the N‐terminal amino acid of the A1 alkali light chain of rabbit fast muscle [Henry et al. (1982) FEBS Lett. 144, 11–15]. The narrow line width and chemical shift position (δ= 3.23 ppm) of the –N+ ‐(CH3) protons of Me3Ala made 1H‐NMR spectroscopy a convenient method to search for this residue in other light chains. A survey of many different light chains showed that this signal was present in all vertebrate striated muscle light chains of the A1‐type (LC1, ‘essential’ light chains) and LC2‐type (‘DTNB’‐light chains, ‘phosphorylatable’ light chains) but was absent from all invertebrate muscle and vertebrate smooth muscle light chains tested. It was also absent from the vertebrate fast‐muscle‐specific A2‐type (LC3) light chains. The spectral characteristics of these signals were consistent with their having arisen from the protons of an – N+ ‐(CH3)3 grouping. Since no ɛ‐trimethyllysine could be detected in acid hydrolysates of these proteins, it appears that Me3Ala is a general feature as the N‐terminal amino acid in these light chains. 1H‐NMR studies on bovine cardiac myosin subfragment 1 (S1) showed that the Me3Ala methyl proton signal was clearly visible and that the spectrum more closely resembled that of a rabbit S1 isoenzyme, S1(A1), than S1(A2), suggesting that the 40‐residue N‐terminal segment of the alkali light chain in cardiac S1 also possesses a high segmental mobility. Addition of actin caused the same gross changes to the cardiac S1 spectrum as noted earlier for rabbit S1(A1) [Prince et al. (1981) Eur. J. Biochem. 121, 213–219]. In particular, a marked reduction in the segmental mobility of the N‐terminal region of the alkali light chain was noted, consistent with a direct interaction of this area with actin.
The binding of vanadium (V) oligoanions to sarcoplasmic reticulum
Authors: VARGA Sandor , CSERMELY Peter , MARTONOSI Anthony 
Published: March 2005

The binding of monovanadate and decavanadate anions to sarcoplasmic reticulum vesicles was measured by equilibrium sedimentation. The affinity of vanadate binding and the molar amount of vanadium (V) bound at equilibrium is much greater with decavanadate than with monovanadate. The binding data can be rationalized in terms of one binding site per ATPase molecule for monovanadate and two sites per ATPase for decavanadate.The Ca‐ATPase crystals formed with monovanadate and with decavanadate are similar in appearance, but decavanadate is particularly effective in promoting the crystallization of Ca2+‐ATPase at low V concentration (10–100 μM) in a Ca2+‐free medium.
Control of ureogenesis
Authors: MEIJER Alfred J. , LOF Cor , RAMOS Inès C. , VERHOEVEN Arthur J. 
Published: March 2005

Control of urea synthesis was studied in rat hepatocytes incubated with physiological mixtures of amino acids in which arginine was replaced by equimolar amounts of ornithine. The following observations were made.

Intramitochondrial carbamoyl phosphate was always below 0.1 mM. Only when ornithine was absent and when, in addition, the concentration of amino acids was higher than four times their plasma concentration, intramitochondrial carbamoyl phosphate rose up to about 3 mM; under these conditions ammonia accumulated in the medium.
The relationship between ornithine‐cycle flux and the concentration of the cycle intermediates at varying amino acid concentration indicated that under near‐physiological conditions the ornithine‐cycle enzymes are far from being saturated with their substrates.
Moderate concentrations of norvaline had no effect on the rate of urea synthesis unless the cells were severely depleted of ornithine.
Activation of carbamoyl‐phosphate synthetase (ammonia) by addition of N‐carbamoylglutamate only slightly stimulated urea production at all amino acid concentrations. However, in the presence of the activator the curve relating ornithine‐cycle flux to the steady‐state ammonia concentration was shifted to lower concentrations of ammonia.
The intramitochondrial concentration of carbamoyl phosphate in rat liver in vivo was below 0.1 mM. This value is far below the concentration required for substantial inhibition of carbamoyl‐phosphate synthetase.
It is concluded that in vivo the function of activity changes in carbamoyl‐phosphate synthetase, via the well‐documented alterations in the intramitochondrial concentration of N‐acetylglutamate, is to buffer the intrahepatic ammonia concentration rather than to affect urea production per se. At constant concentration of ammonia the rate of urea production is entirely controlled by the activity of carbamoyl‐phosphate synthetase.
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.
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.
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.