bluuebunny/crossref_metadata_2025_split huggingface dataset:
Substrate Specificity Studies on a Calf Thymus Nuclear Phosphoprotein Kinase
Authors: SCULLEY Thomas B. , MACKINLAY Antony G. 
Published: July 2010

the protein kinase activity associated with the phosphoprotein fraction of calf thymus nuclei has been examined for its ability to phosphorylate exogenous phosphoprotein substrates. β‐Casein and phosvitin are both efficient phosphate acceptors. Phosphorylation of αs1 and β‐caseins was shown, indirectly, to occur at threonyl‐49 and threonyl‐41 respectively. Both threonyl residues occur within the sequence Thr‐Glu‐Asp.
αs1‐Casein becomes a much better substrate for the kinase when partially dephosphorylated. A similar response is shown by a phosphopeptide containing the αs1‐casein phosphate cluster and is due to a new phosphorylation site becoming available. Efficient phosphorylation of β‐casein requires that the phosphate cluster (residues 15 ‐ 19) be intact and results are presented which are consistent with there being a similar requirement for phosphorylation of the site created in αs1‐casein by partial dephosphorylation.
Comparison of genetic variants of β‐casein as phosphate acceptors for the kinase shows that the presence of lysyl residues close to the phosphorylation site markedly depresses the rate of phosphorylation. Maleylation of β‐ casein increases the rate of phosphorylation for all of the variants tested, although to varying extents. Treatment of maleylated β‐casein with trypsin to remove the N‐terminal phosphopeptide inhibits phosphotylation by the kinase.
The structural determinants of β‐casein allowing it to be efficiently phosphorylated by the kinase are concluded to be the presence of a sequence surrounding the phosphorylation site, which is rich in acidic amino acid residues and from which basic residues are absent. The acidic phosphate cluster of β‐casein also promotes phosphorylation either by interacting directly with the enzyme or through its influence on the conformation of β‐casein.
Relation of Oxidation of Long‐Chain Fatty Acids to Gluconeogenesis in the Perfused Liver of the Guinea Pig:
Authors: TUTWILER Gene F. , BRENTZEL H. James 
Published: July 2010

The potent, specific inhibitor of long‐chain fatty acid oxidation, 2‐tetradecylglycitiic acid (McN‐3802), at 10μM totally abolished ketogenesis from endogenous substrates in the isolated perfused guinea pig liver. This effect was accompanied by a marked inhibition of gluconeogenesis from lactate plus pyruvate and by a shift toward a more oxidized state of the mitochondrial (3‐hydroxybutyrate/acetoacetate ratio) and cytoplasmic (lactate/pyruvate ratio) compartments.
The addition of octanoate (88–500 μM) almost completely reversed the inhibitor! etyect of 2‐tetradecyl‐ glycidic acid on gluconeogenesis. Octanoate oxidation, measured by the rate of ketogenesis, was not inhibited. This protective effect of octanoate against inhibition of gluconeogenesis by 2‐tetradecylglycidic acid was seen even though in some experiments the mitochondrial redox state remained two to three trines the magnitude observed prior to octanoate addition.
Gluconeogenesis from 4 mM glycerol was not inhibited and gluconeogenesis from 3 inM propionate was only slightly inhibited by 2‐tetradecylglycidic acid.
Thus, it would appear that fatty acid oxidation in guinea pig liver is essential for maintaining maximal rates of gluconeogenesis, especially from substrates dependent on pyruvate carboxylation for conversion to glucose.
A single dose of 2‐tetradecylglycidic acid (orally, 10–25 mg/kg) given to guinea pigs previously fasted 72 h produced a highly significant decrease of total ketones, of the 3‐hydroxybutyrate/acetoacetate ratio and of plasma glucose. A smaller hypoglycemic effect was seen when the drug was administered to animals fasted for only 24 h or 48 h.
It appears from evidence in vivo and in vitro that the guinea pig and rat respond similarly to inhibition of fatty acid oxidation. This may be important since it has been suggested that the role of fatty acid oxidation in glucose synthesis is markedly different in these two species.
Circular‐Dichroism Study of the Interaction of Aspartate‐Aminotransferase Isoenzymes with a Coenzyme Analog
Authors: CAROTTI Daniela , RIVA Francesca , SANTUCCI Roberto , ASCOLI Franca , FASELLA Paolo 
Published: July 2010

The interaction between a coenzyme derivative, 4′‐N‐(2,4‐dinitro‐5‐fluorophenyl)‐pyridoxamine 5′‐phosphate, and the apoenzyme of cytoplasmic and mitochondrial aspartate aminotransferase, was studied by circular dichroism.The specific complexes initially formed were characterized by their circular dichroic spectra. The spectra indicate that the complex is very probably the same for the two isoenzymes. In contrast the spectra recorded during further reaction, in agreement with previous results, monitor different reaction paths and characterize the irreversible labeling at the active site of the cytoplasmic enzyme and regeneration of pyridoxal 5′‐phosphate in the mitochondrial enzyme.By following circular dichroic changes in the mitochondrial enzyme, initial kinetic characterization of the cleavage of 4′‐N‐(2,4‐dinitro‐5‐fluorophenyl)‐pyridoxamine 5′‐phosphate to form pyridoxal 5′‐phosphate at the active site, is provided.
The Role of Lactoperoxidase‐H2O2 Compounds in the Catalysis of Thyroglobulin Iodination and Thyroid Hormone Synthesis
Authors: COURTIN Françoise , DEME Danielle , VIRION Alain , MICHOT Jean Luc , POMMIER Jacques , NUNEZ Jacques 
Published: July 2010

Lactoperoxidase catalyzes successively, as thyroid peroxidase, the iodination of several tyrosyl residues of thyroglobulin and the coupling of some of them to thyroid hormones. We show the following results in this paper.

The iodination reaction is catalyzed by lactoperoxidase compound I whereas lactoperoxidase ‘compound II’ catalyzes the coupling reaction. Lactoperoxidase compound III catalyzes neither reaction.
Titration of lactoperoxidase ‘compound II’ by ferrocyanide showed that this enzyme‐H2O2 species is two oxidizing equivalents above the native enzyme, and therefore constitutes a second form of lactoperoxidase ‘compound I’. These two forms of lactoperoxidase ‘compound I’ differ probably from one another in the localization of one of the two oxidizing equivalents either on porphyrin ring: π‐compound I (compound I), or on the apoprotein: R°‐compound 1 (‘compound II’). The difference in specificity between π‐compound I and R°‐compound I in catalyzing the iodination and the coupling reaction respectively, might therefore depend differences both in the distribution of the two oxidizing equivalents and in the structure of the substrates.
In the presence of free diiodotyrosine, a halophenol which stimulates at very low concentrations the coupling reaction but has no effect on the iodination reaction, the transfer of electrons between the substrate and lactoperoxidase R°‐compound I is nearly stoichiometric: 1 mole of hormone is produced for each mole of lacto‐peroxidase R°‐compound I. Thus free diiodotyrosine seems to play the role of a very specific co‐factor of the coupling reaction.
Iodide and SCN− also stimulate the coupling reaction when the H2O2/enzyme ratio used to prepare lactoperoxidase R°‐compound I is higher than one. They do so by preventing the accumulation of the inactive derivative of lactoperoxidase, compound III, which is formed in the presence of excess H2O2. Iodide prevents the formation of compound III whereas SCN− very rapidly decomposes compound III back to the native enzyme.
Molecular Properties of Lipoprotein Lipase
Authors: OLIVECRONA Thomas , BENGTSSON Gunilla , OSBORNE James C. 
Published: July 2010

The monomer molecular size of bovine lipoprotein lipase was evaluated by sedimentation equilibrium measurements and by gel permeation chromatography in 6 M guanidinium chloride. To establish molecular weight unequivocally we determined the partial specific volume (v̄) experimentally. This was done by analyzing equilibrium concentration profiles from analytical ultracentrifugation in 6 M guanidinium chloride using buffers made up in H2O and 2H2O. The combined results gave a v̄ of 0.71 ± 0.007 ml/g and a molecular weight of 41700 ± 1000 for monomeric bovine lipoprotein lipase. This value did not change upon mild tryptic digestion; the elution volume upon gel permeation chromatography in 6 M guanidinium chloride was also unaffected by treatment with trypsin.Sedimentation equilibrium measurements of the trypsin‐treated material in the presence of reducing agents gave limiting molecular weights of 19000 and 23000, demonstrating that mild trypsin digestion cleaved lipoprotein lipase into two polypeptide chains of similar size held together by disulfide bonds. Mild trypsin digestion also resulted in a loss of secondary structure as determined by circular dichroic measurements. Discussion centers around the correlation between these effects of trypsin on the molecular properties of lipoprotein lipase and the previously reported effects on the kinetic properties of the enzyme.
Purification and Partial Characterization of Two Acidic Proteases from the White‐Rot Fungus Sporotrichum pulverulentum
Authors: ERIKSSON Karl‐Erik , PETTERSSON Bert 
Published: July 2010

Two acidic proteases from the white‐rot fungus Sporotrichum pulverulentum have been purified and partially characterized. The enzymes were purified in four steps, protease I 152‐fold and protease II 127‐fold. The purity of the enzymes was investigated by analytical isoelectric focusing and by dodecylsulfate/polyacrylamide gel electrophoresis.The isoelectric points of protease I and protease II were at pH 4.7 and 4.2 respectively. The molecular weights were 28000 and 26000 and the pH optima 5.0 and 5.2. Both enzymes were inhibited by heavy metal ions such as Ag+, Hg2+ and to some extent also by Cu2+. Partial inhibition was also observed with EDTA and αα′‐ dipyridyl.The specificities of protease I and II were investigated using human fibrinopeptide A as substrate. Protease splits off the arginine in the C‐terminal position, while protease II splits at the carboxyl side of both Phe‐8 and Leu‐9 in fibrinopeptide A.A physiological effect of the two proteases has also been demonstrated. Thus, if a culture solution from S. pulverulentum grown on cellulose was treated with the individual proteases or a mixture of the enzymes a considerable increase in endo‐l,4‐β‐glucanase activity was obtained. It may be that the endo‐glucanases are produced in a zymogenic form and activated by the proteases. However, other explanations for the phenomenon are also possible.
Purification and Characterization of β‐Galactoside (α2→6)sialyltransferase from Rat Liver and Hepatomas
Authors: MIYAGI Taeko , TSUIKI Shigeru 
Published: July 2010

Asialofetuin sialyltransferase from Triton X‐100 extracts of rat liver was resolved by phosphocellulose chromatography into two fractions, designated I and II in order of elution. When previously treated with Arthrobacter ureafaciens neuraminidase, fraction I eluted at about the same position as II while no alteration occurred in II. Primary rat hepatomas contained only a single asialofetuin sialyltransferase, identical to fraction I in chromatographic behavior.Transferases I and II were purified to near homogeneity. Transferase II, as well as neuraminidase‐treated I, could be sialylated auto‐catalytically, indicating that the lack of sialic acid in II is not due to the lack of a sialic‐acid‐accepting site. Both enzymes formed an (α2→6)sialylgalactoside linkage with asialo‐glycoproteins of the glycosylamine‐type and with lactose, and were indistinguishable immunologically. Nevertheless, the transferases exhibited different molecular weights of 37000 (I) and 43000 (II). When heated at 50°C, transferase I lost half its original activity within 20 min while II was scarcely inactivated. Kinetically, transferase I showed three‐times higher affinity than II for CMP‐N‐acetylneuraminic acid and for desialylated plasma membrane.Asialofetuin sialyltransferase was also purified from primary rat hepatoma. The purified enzyme was identical to transferase I in every respect examined. We conclude that hepatomas contain transferase I but lack transferase II.
The ppp(A2′p)nA and Protein Kinase Systems in Wild‐Type and Interferon‐Resistant Daudi Cells
Authors: SILVERMAN Robert H. , WATLING Diane , BALKWILL Frances R. , TROWSDALE John , KERR Ian M. 
Published: July 2010

Daudi cells, a human lymphoblastoid line, are exceptionally sensitive to the growth inhibitory effects of interferon, 1 unit/ml being sufficient to inhibit cell growth. In addition, interferon treatment of these cells severely inhibits the incorporation of exogenous thymidine into DNA and causes cells to accumulate in the G1(G0) at the expense of the S phase of the cell cycle. The possible involvement of ppp(A2′p)nA (n= 2 to 4) in these effects has been investigated. No (< 1 nM) ppp(A2′p)nA or (A2′p)nA or alternative products of the ppp(A2′p)nA synthetase [e.g. NAD (2′pA)2] were detected in interferon‐treated cells. In addition no evidence was obtained for the occurrence of ppp(A2′p)nA‐mediated ribosomal RNA cleavage in these cells even after several days of treatment with relatively high doses of interferon.A line of Daudi cells which is resistant to all three of the above effects of interferon was selected. The wild type and resistant lines were compared with respect to the ppp(A2′p)nA and interferon and double‐stranded RNA (dsRNA)‐mediated protein kinase systems. The resistant line was not receptor‐negative as it responded to interferon by the production of elevated levels of the ppp(A2′p)nA synthetase similar to those observed in extracts from wild‐type cells. There was no detectable difference between the lines in the levels of the (2′‐5′)phosphodiesterase responsible for the degradation of ppp(A2′p)nA. There was, however, about a twofold increase in the ppp(A2′p)nA‐dependent endoribonuclease activity in response to interferon with extracts from the wild‐type but not the resistant cells. In addition, although the dsRNA‐dependent protein kinase activity increased in both types of cell there was a striking reduction in the level of protein phosphorylation in general in response to interferon with material from the wild‐type but not the resistant cells.
Non‐equilibrium Thermodynamic Assessment of Redox‐Driven H+ Pumps in Mitochondria
Authors: PIETROBON Daniela , ZORATTI Mario , AZZONE Giovanni F. , STUCKI Jörg W. , WALZ Dieter 
Published: July 2010

Isolated mitochondria suspended in an aerobic medium with 3‐hydroxybutyrate or succinate serving as electron donor attain a stationary state with vanishing net flow of H+ ions (state 4). Adding valinomycin to such a suspension in the presence of various concentrations of K+ ions and a weak acid system such as acetate or phosphate creates new stationary states for the mitochondria which are characterized by a constant influx of K+ ions, while the net flow of H+ ions again vanishes due to the recycling of these ions by the weak acid system.Sufficiently low concentrations of K+ ions (less than 4 mM) cause these stationary states to last long enough for a separation of the mitochondria by centrifugation. The difference in electrochemical potential for H+ ions can then be determined by means of the partitioning of radioactively labelled markers. Suitable procedures to correct for binding of the markers are described.It is found that, for a constant affinity of the electron in the suspending medium, electron flow and the flow of K+ ions, which indicates the flow of pumped H+ ions, are linearly dependent on the electrochemical potential difference of H+ ions. The phenomenological coefficients obtained from these correlations are discussed with respect to the contributions of additive constants in the linear relations. It is found that, under the present experimental condition, such constants most likely vanish thus yielding symmetric flow‐force relations. It is concluded that the redox‐driven H+ pumps are not tightly coupled due to molecular slipping in the pumps and that the molecular stoichiometry is 2 H+ ions/electron for coupling site I and 4 H+ ions/electron for coupling sites II and III together.
Disulfide Bridges in Tropomyosin
Authors: WILLIAMS David L. , SWENSON Charles A. 
Published: July 2010

The effect of rabbit skeletal tropomyosin and tropomyosin‐troponin on the ATPase activity of actomyosin was compared for tropomyosins with the Cys‐190 disulfide bridge intact (crosslinked tropomyosin) or broken and blocked with N‐ethylmaleimide (MalNEt‐tropomyosin). Experiments were carried out at 25°C under the following solution conditions: 30 mM KCl, 5 mM MgCl2, 1 mM EGTA or 0.1 mM Ca2+, 2 mM ATP, 0.02% NaN3, 10 mM imidazole at pH 7.0 (or 10 mM Tris at pH 8.0).At pH 7.0 both MalNEt‐tropomyosin and crosslinked tropomyosin had an inhibitory effect on the ATPase activity of actomyosin subfragment‐1, with MalNEt‐tropomyosin inhibiting more strongly than crosslinked tropomyosin.Under the same conditions the inhibitory effect of both tropomyosins on the ATPase activity of acto‐(heavy meromyosin) was less pronounced but the inhibitory effect of MalEt‐tropomyosin was still approximately twice that of crosslinked tropomyosin at the stoichiometric ratio of tropomyosin to actin. At pH 8.0 MalNEt‐tropomyosin had an inhibitory effect on the ATPase of acto(heavy meromyosin) while crosslinked tropomyosin had a potentiating effect, demonstrating the dual effect of tropomyosin at one set of solution conditions and protein ratios.In the presence of excess troponin and 1 mM EGTA at pH 7.0 MalNEt‐tropomyosin and crosslinked tropomyosin were indistinguishable in their ability to inhibit the ATPase of acto‐(heavy meromyosin). In the presence of excess troponin and 0.1 mM Ca2+, crosslinked tropomyosin enhanced the ATPase of acto‐(heavy meromyosin) to a slightly greater degree than MalNEt‐tropomyosin. Our results suggest that potentiation and inhibition are occurring simultaneously. We have previously shown that the presence or absence of a disulfide bridge has a profound effect on the stability of the –COOH terminal half of the molecule [Williams and Swenson (1981) Biochemistry, 20, 3856]. The results presented here indicate that the presence of the disulfide bridge in tropomyosin also affects the ATPase activity of actomyosin and suggest that MalNEt‐tropomyosin and crosslinked tropomyosin may be useful tools in studying the kinetic mechanism of skeletal muscle control.
Polymères hydrosolubles de NAD+ et d'ADP
Authors: SICSIC Sames , MAVAMBU Yakini , BRÉMONT Béatrice , LE GOFFIC François 
Published: July 2010

Hydrosoluble Polymers of NAD+ and ADP. Coenzymatic Properties as a Function of their Size Hydrosoluble polymers of NAD+ and ADP were synthesized according to a described method with some modifications. The cofactor was bound to the matrix by a spacer group of five atoms at the exocyclic adenine C‐6 amino group. Cofactor incorporations were very high. The loading of NAD+ polymers were: 420 μmol NAD+/g polymer and 330 μmol ADP ribose/g polymer arising from degradation of NAD+; the loading of ADP polymers were: 1.40 and 1.43 mmol ADP/g polymer. Each polymer obtained in a same polymerization step was fractionated in two parts by gel filtration in such a manner that these two parts had the same framework but different molecular weights. The biological properties of the fractionated polymers were compared. The small sized polymers of NAD+, assayed with four dehydrogenases, were more reduced enzymatically than the larger ones and had the higher reduction rates relative to free NAD+. In a coupled system (l‐lactate dehydrogenase, formate dehydrogenase) with coenzyme regeneration, the rate of production of l‐lactate at the steady state of the system using NAD+ polymers of small size was 80% that of the system using free NAD+. The small‐sized polymers of ADP were more phosphorylated by pyruvate kinase and creatine kinase than the larger ones. But the relative rates of transformation by pyruvate kinase of large sized polymers were higher than those of smaller ones.
A Genetic Approach to Reveal the Action of the Opiate Receptor in Selected Neuroblastoma‐Glioma Cells
Authors: SIMANTOV Rabi , NADLER Hana , LEVY Rivka 
Published: July 2010

Three clones of neuroblastoma‐glioma cells that contain low amounts of calmodulin were selected from the NG108‐15 cells after several treatments with high concentrations of chlorpromazine. Purified membranes of the three clones had decreased numbers of both α‐adrenergic and opiate receptors, monitored with [3H]yohimbine and [3H,d‐Ala2]methionine encephalinamide, respectively. No changes were observed in the affinity of these radioactive ligands to the receptors of the selected cells as compared to the parent cells. Addition of bovine brain calmodulin did not affect the binding of [3H,d‐Ala2]methionine encephalinamide to the membranes of the selected cells and they had the same number of acetylcholine receptors, determined with 1‐quinuclidinyl‐[phenyl‐4‐3H]‐benzilate, as the parent NG108‐15 cells.The basal ATPase activity in the membranes of the selected cells was 35–50% of the parent cells, with a decreasedVvalue and no significant change in the affinity constantKato ATP. Addition of Ca2+to the purified membranes increased theVof the ATPase in the selected as well as the parent cells but theVof the selected cells remained lower than that of the parent cells. Ca2+had no effect on theKato ATP in either cell type. The Ca2+‐dependent ATPase activity of both the parent and the selected cells was also calmodulin‐dependent since it was blockedin vitroby chlorpromazine. The co‐regulation of opiate and adrenergic receptors and their interaction with calmodulin and Ca2+‐ATPase is discussed in view of recent observations indicating biochemical and physiological association between opiates, Ca2+and adrenergic compounds.
Wild‐Type and Mutant Forms of Homoisocitric Dehydrogenase in the Yeast Saccharomycopsis lipolytica
Authors: GAILLARDIN Claude M. , RIBET Anne‐Marie , HESLOT Henri 
Published: July 2010

Homoisocitric dehydrogenase (EC 1.1.1.155) has been purified 525‐fold from the yeast Saccharomycopsis lipolytica with a yield of 25%. The preparation was judged to be homogeneous by electrophoresis under denaturing and non‐denaturing conditions and by isoelectric focusing; it consisted of a single protein with molecular weight of 48000. In the presence of homoisocitric acid, a higher molecular weight was observed, suggesting a dimeric structure for the native enzyme.Complementing mutants devoid of homoisocitric dehydrogenase activity mapped at two closely linked loci (lys9 and lys10). Lvs10 mutants displayed NAD‐reducing activity, whereas lys9 mutants retained some carboxylating activity.Our results are best explained by the assumption that the active enzyme is a dimer of identical subunits involved in successive dehydrogenation and decarboxylation steps.
Protein Synthesis by Isolated Acetabularia Chloroplasts
Authors: GREEN Beverley R. 
Published: July 2010

Isolated chloroplasts of Acetabularia incorporate radioactive amino acids into more than 30 polypeptides in the light, including the apoprotein of the P700‐chlorophyll a protein complex, the reaction centre core of photo‐system I [Biochim. Biophys. Acta, 609, 107–120 (1980)]. In this paper it is shown that the apoproteins of the two minor chlorophyll a complexes, thought to be part of photosystem II reaction centre core, are also synthesized by isolated chloroplasts. Furthermore, they are integrated correctly into the thylakoid membrane in the absence of any cytoplasmic contribution, such that they can be isolated as chlorophyll‐protein complexes indistinguishable from those already in the membrane.In contrast, the minor chlorophyll a + b complex ‘CP 29’ [Camm, E. L. and Green, B. R. (1980) Plant Physiol. 66, 428–432] and its dimers are not synthesized by isolated chloroplasts. In this they resemble the other chlorophyll a + b complex, the light‐harvesting complex (LHC). It may be significant that the LHC, which is not essential for photosynthetic activity, is under nuclear control, while the reaction centre polypeptides, cytochrome b559, and cytochrome f, are synthesized on chloroplast ribosomes.