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Discrete mobility of single-stranded DNA in non-denaturing gel electrophoresis

Liu, Qiang; Scaringe, William A.; Sommer, Steve S.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em 15/02/2000 EN
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Gel electrophoresis is the standard method to separate, identify and purify nucleic acids. SSCP detects single base changes by altered mobility of single-stranded segments electrophoresed through non-denaturing polyacrylamide gels. Herein, changes in electrophoretic mobilities due to single base substitutions were measured for single-stranded segments of lengths ranging from 333 to 547 nt. A 484 nt segment in exon H of the human factor IX gene was studied most intensively. After SSCP, mobilities were determined by scanning autoradiograms at very high resolution (1200 d.p.i.), which allowed precise measurement of mobilities. When the mobilities of 46 single base substitutions were characterized, the distribution of mutant segments relative to a wild-type control was found to be discrete, i.e. the observed mobility values occurred in distinct ranges. Discrete mobility distributions were seen at different electrophoretic temperatures, buffer concentrations, segment lengths and segment sequences. In addition: (i) single base substitutions caused discontinuous distributions between highly dispersed and sharp bands; (ii) at least one single-stranded segment produced two sharp bands of similar intensity. These observations suggest that: (i) the single base changes in DNA segments in the size range 333–547 nt result in discrete conformational changes; (ii) individual DNA molecules of the same DNA segment can occasionally adopt two or more discrete conformations.

Evidence that bacteriophage T4 eph1 is a missense hoc mutation.

Childs, J D; Pilon, R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1983 EN
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An electrophoretic mutation of bacteriophage T4, eph1, appears to code for a missense hoc (highly antigenic outer capsid) protein. This is based on the observation that particles lacking hoc protein (hoc- particles), after incubation in a crude extract of Escherichia coli infected with phage carrying the eph1 mutation acquired the electrophoretic mobility of the eph1 strain (the electrophoretic mobility of the eph1 strain itself is slower than that of hoc- particles). Thus, it is likely that during infection of E. coli with the eph1 strain, a hoc protein is made that has a lower negative charge than normal hoc protein but can nevertheless bind to particles lacking hoc protein. These results confirm that eph1 is a hoc mutation.

Precursor Protein for Newcastle Disease Virus

Samson, A. C. R.; Fox, C. F.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1973 EN
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The course of viral protein synthesis during infection of chicken embryo fibroblasts with Newcastle disease virus (NDV) L. Kansas has been followed by using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Of the three major virion polypeptide molecular weight classes, I (78,400 daltons), II (53,500 daltons), and III (37,600 daltons), only II, having the same electrophoretic mobility as nucleocapsid polypeptide, appears to be the cleavage product of a precursor polypeptide PII (64,800 daltons) detected in NDV-infected cells after brief labeling with radioactive amino acids. Nucleocapsids were isolated from NDV-infected cells which had been pulse-labeled with radioactive amino acids or pulse-labeled and further incubated with unlabeled amino acids. Gel electrophoretic analysis of proteins derived from nucleocapsids showed that an increase in the period of incubation with unlabeled amino acids resulted in an increase in the amount of radioactivity in nucleocapsid protein. Polypeptide PII was not detected as a transient component of the isolated nucleocapsid fraction. These results are consistent with two interpretations. The product of PII cleavage is (i) nucleocapsid polypeptide, or (ii) a nonvirion or minor envelope polypeptide having the same electrophoretic mobility as nucleocapsid polypeptide.

Electrophoretic analysis of multiple forms of myosin in fast-twitch and slow-twitch muscles of the chick.

Hoh, J Y; McGrath, P A; White, R I
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/07/1976 EN
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283.04373%
1. A method is described for the electrophoretic analysis of intact myosin in polyacrylamide gel in a buffer system containing 0.02 M-pyrophosphate and 10% (v/v) glycerol, pH 8.8. 2. In this system chicken skeletal-muscle myosins reveal five distinct electrophoretic components, three components from the fast-twitch posterior latissimus dorsi muscle and two slower-migrating components from the slow-twitch anterior latissimus dorsi muscle. 3. The Ca2+-activated ATPase (adenosine triphosphatase) activity of myosin components was measured by densitometric scanning of the gel for the Ca3(PO4)2 precipitate formed during the ATPase reaction and subsequently for stained protein. Each component from the same muscle appears to have identical ATPase activity, but components from the fast-twitch muscle had an activity 2.2 times higher than those from the slow-twitch muscle. 4. On re-electrophoresis in the same buffer system, individual fractions of fast-twitch myosin did not reproduce the three-band pattern of the original myosin, but migrated at rates consistent with their original mobility. 5. Analysis of the mobility of the three fast-twitch myosin components in gels of different concentrations suggests that they are not stable oligomers of each other. 6. It is suggested that these components of fast-twitch myosin and slow-twitch myosin are isoenzymes of myosin.

Study of soluble lipoprotein in rat liver mitochondria

Koppikar, S. V.; Fatterpaker, P.; Sreenivasan, A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1971 EN
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1. A water-soluble lipoprotein was isolated and purified from osmotically shocked preparations of rat liver mitochondria by using a technique of Sephadex-sandwich disc electrophoresis. 2. The purified lipoprotein migrates as a distinct sharp zone in high-resolution electrophoretic systems, indicating high degree of purity. 3. The lipoprotein resembles mitochondrial membranes with respect to lipid composition and lipid/protein ratio. 4. The lipoprotein and its apoprotein fraction obtained by delipidization at −18°C to −20°C have common properties with respect to their fluorescence spectra, instability to storage and electrophoretic mobility. 5. The purified lipoprotein has an excitation maximum at 325nm and a fluorescence maximum at 418nm. 6. Storage at 4°C for 4 days or repeated freezing and thawing results in 15–30% decrease in electrophoretic mobility. 7. The patterns of incorporation in vitro of [1-14C]leucine into proteins of the soluble lipoprotein and of mitochondrial membrane of isolated rat liver mitochondria suggest a probable precursor role for the apoprotein in the formation of mitochondrial membrane protein. 8. Lipoprotein preparations isolated from mitochondrial fractions of rat kidney, brain and heart and of chicken and mouse liver resemble closely that obtained from rat liver mitochondria...

Molecular-sieve chromatography and electrophoresis in polyacrylamide gels

Morris, C. J. O. R.; Morris, Peggy
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1971 EN
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283.04373%
1. The absolute electrophoretic mobilities of eight proteins have been measured at pH8.76, I 0.05, in polyacrylamide gels of 20 different compositions at 10°C. 2. The partition coefficients of these proteins have been determined chromatographically under the same conditions by using columns of granulated polyacrylamide gel prepared simultaneously. 3. The electrophoretic mobilities are an exponential function of the gel concentrations when the latter are corrected for water uptake. The constants of this function have been determined by curvefitting methods. They have been shown to be related to the free solution mobility and to the mean molecular radius respectively. 4. The reduced mobilities have been shown to be a linear function of the partition coefficients by statistical analyses. 5. The physical significance of the relation between electrophoretic mobility and chromatographic phase distribution in gel media is discussed in the context of these results.

Ribonucleic acid synthesis in vitro in primary spermatocytes isolated from rat testis

Grootegoed, J. Anton; Grollé-Hey, Anne H.; Rommerts, Focko F. G.; Van Der Molen, Henk J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/10/1977 EN
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286.73658%
The incorporation of [3H]uridine into RNA was studied quantitatively (by incorporation of [3H]uridine into acid-precipitable material) and qualitatively (by phenol extraction and electrophoretic separation of RNA in polyacrylamide gels) in preparations enriched in primary spermatocytes, obtained from testes of rats 26 or 32 days old. The rate of incorporation of [3H]uridine into RNA of isolated spermatocytes was constant during the first 8h of incubation, after which it decreased, but the decreased rate of incorporation was not reflected in a marked change in electrophoretic profiles of labelled RNA. In isolated spermatocytes, [3H]uridine was incorporated mainly into heterogeneous RNA with a low electrophoretic mobility. Most of this RNA was labile, as shown when further RNA synthesis was inhibited with actinomycin D. Spermatocytes in vivo also synthesized heterogeneous RNA with a low electrophoretic mobility. A low rate of incorporation of [3H]uridine into rRNA of isolated spermatocytes was observed. The cleavage of 32S precursor rRNA to 28S rRNA was probably retarded in spermatocytes in vitro as well as in vivo. RNA synthesis by preparations enriched in early spermatids or Sertoli cells was qualitatatively different from RNA synthesis by the spermatocyte preparations. It is concluded that isolated primary spermatocytes maintain a specific pattern of RNA synthesis...

Hidden Electrophoretic Variation at the Xanthine Dehydrogenase Locus in a Natural Population of DROSOPHILA MELANOGASTER

Ann Buchanan, Barbara; Johnson, Diana L. E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1983 EN
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283.04373%
Sixty-two isochromosomal lines of D. melanogaster were screened for cryptic electrophoretic variation at the xanthine dehydrogenase (XDH) locus. Sequential polyacrylamide vertical slab gel electrophoresis was performed using four electrophoretic criteria. A total of 15 classes of electromorphs were revealed. D. melanogaster appears to exhibit as much polymorphism at this locus as other extensively studied Drosophila species.—No evidence for loci on the X or second chromosomes which modified XDH mobility was found. Six of the electromorphs were mapped to the Xdh ( ry) structural locus. Eight of the remaining nine classes exhibited mobility variation consistent with structural variation at the Xdh locus. The final class exhibited aberrant patterns and is under further study.

Examination of Allelic Variation at the Hexokinase Loci of DROSOPHILA PSEUDOOBSCURA and D. PERSIMILIS by Different Methods

Beckenbach, Andrew T.; Prakash, Satya
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1977 EN
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283.04373%
Recently a number of electrophoretic techniques have been applied to reveal the presence of additional genetic variation among the electrophoretic mobility classes of the highly polymorphic xanthine dehydrogenase (XDH ) and esterase-5 (est-5) loci. We examined the hexokinase loci of Drosophila pseudoobscura and D. persimilis using a variety of techniques to determine whether further allelic variation could be revealed for these much less polymorphic loci and to analyze the nature of the known variation at the hexokinase-1 (hex-1) locus. The following studies were conducted: 135 strains of the two species from six localities were examined with buffer pH ranging from 5.5 to 10.0; 40 strains of D. pseudoobscura and 9 strains of D. persimilis from Mather were studied using starch gel concentrations ranging from 8.5 to 15.5% and were examined for differences in heat stability and reactivity to the thiol reagent pCMSA; strains were also tested for susceptibility to urea denaturation and differences in relative activities. Major findings of the work are: (1) No additional allelic variation could be detected at any of the hexokinase loci by applying these techniques. The finding of abundant hidden genetic variation in XDH and est-5 does not extend to all enzyme loci. (2) Evidence from studies using pCMSA indicates that the hex-1 alleles 0.6...

The Genetics of DROSOPHILA SUBOBSCURA Populations. Xvii. Further Genic Heterogeneity within Electromorphs by Urea Denaturation and the Effect of the Increased Genic Variability on Linkage Disequilibrium Studies

Loukas, M.; Vergini, Y.; Krimbas, C. B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1981 EN
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283.04373%
Urea denaturation of allozymes was used to provide finer resolution of allelic states within classes of different electrophoretic mobility. This method gives perfectly repeatable results. About 170 isogenic strains for the O chromosome of Drosophila subobscura, derived from two natural populations, were constructed. Their gene arrangements were studied, as well as eight polymorphic genes located on the O chromosome (Est-5, Odh, Ao, ME, Xdh, Lap, Pept-1 and Acph). Crosses performed indicate that differences in urea sensitivity are genetically controlled by the same genes that control electrophoretic mobility. Twice as many alleles have been detected in comparison to the usual electrophoretic method. However, the effective number of alleles did not increase considerably.

Protein-polysaccharides of pig laryngeal cartilage

Muir, Helen; Jacobs, S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1967 EN
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285.1282%
1. Protein–polysaccharides of chondroitin 4-sulphate were extracted with neutral calcium chloride from pig laryngeal cartilage that was not completely homogenized. The protein–polysaccharides were purified by precipitation with 9-aminoacridine. On zone electrophoresis in compressed glass fibre at pH7·2 it was separated into two fractions, although two distinct zones were not obtained. These fractions, which had already been shown to differ in their antigenic determinants, also differed considerably in amino acid composition, total protein, hexose and glucosamine contents. 2. The fraction of higher mobility contained approx. 2% of protein and only traces of glucosamine. Serine and glycine accounted for over half the total amino acid residues, but aromatic, basic and sulphur-containing amino acids were not detected. The weight-average molecular weight, determined by sedimentation, was 230000. 3. Assuming that there was the same sequence of neutral sugars at the linkage points as in PP-L fraction (protein–polysaccharide light fraction), the approximate molar ratio of hexose to serine suggested that most of the serine residues were linked to chondroitin sulphate chains. Support for this was derived from the agreement between the weight-average molecular weight of the chondroitin sulphate–peptide after proteolysis...

Hemolytic anemia due to pyruvate kinase deficiency: characterization of the enzymatic activity from eight patients.

Black, J A; Rittenberg, M B; Bigley, R H; Koler, R D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1979 EN
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We have studied the red cell pyruvate kinase (PK) variants from eight patients representing five families with pyruvate kinase deficiency-associated hemolytic anemia. The kinetic properties, electrophoretic mobilities, and immunological reactivity with anti-normal red cell pyruvate kinase were determined. The patients differ in the severity of their clinical condition and in the molecular properties of their red cell pyruvate kinase variants. The most seriously affected patient (PK Beaverton) has no electrophoretically demonstrable red cell isozymes. The activity present is due to the M2 isozyme, however red cell isozyme can be detected immunologically. PK Molalla and PK Lake Oswego are thermolabile variants with normal kinetic parameters. PK Molalla, in addition, has altered electrophoretic mobility. PK Multnomah and PK Milwaukie have decreased affinity for the substrate phosphoenolpyruvate, and PK Multnomah also has altered electrophoretic mobility. PK Coos Bay shows electrophoretic variation and a slightly decreased affinity for phosphoenolpyruvate consistent with an increased modulating effect of fructose-1,6-diphosphate.

A NUCLEAR MEMBRANE CHANGE AFTER PARTIAL HEPATECTOMY

Kishimoto, Susumu; Lieberman, Irving
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/12/1964 EN
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285.1282%
Partial hepatectomy (67 per cent extirpation) of the rat leads to a change in the membrane of liver nuclei (purified with citric acid) detectable as an increase in electrophoretic mobility. No change is detectable 2 hours after the operation, but between 2 and 6 hours about a 1.4-fold increase in mobility occurs after which the mobility becomes constant at the elevated level. Removal of only 10 per cent of the liver causes no detectable change in 6 hours. Bilateral adrenalectomy immediately before partial hepatectomy does not affect the development of the nuclear change. Actinomycin D and p-fluorophenylalanine, but not noradrenalin, ionizing radiation, or EDTA, suppress the increase in electrophoretic mobility. The level of actinomycin D required to block the nuclear membrane change is 6 times greater than that necessary to prevent the rate increase in hepatic RNA metabolism that follows removal of part of the liver. This discrepancy and the difference in the response to noradrenalin indicate that, at least initially, the nuclear membrane change and the change in the rate of RNA synthesis are independent processes. The inability of EDTA to block the nuclear membrane change shows that the Zn++ requirement for DNA replication is not related to the events that lead to the alteration in the electrokinetic properties of liver nuclei.

THE SURFACE CHARGE OF RAT LIVER MITOCHONDRIA AND THEIR MEMBRANES : Clarification of Some Controversies Concerning Mitochondrial Structure

Heidrich, Hans-G.; Stahn, Roland; Hannig, Kurt
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/07/1970 EN
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283.04373%
The surface charge of intact mitochondria and submitochondrial particles was examined by the technique of preparative free flow electrophoresis. When submitochondrial preparations obtained by a swelling-contraction procedure were examined with this technique, two fractions were observed. One of these fractions exhibited the same electrophoretic properties as intact mitochondria, which indicated that it was derived from the outer limiting membrane of the mitochondrion. This fraction was found to contain the enzymes monoamine oxidase and rotenone-insensitive NADH-cytochrome c reductase which have been reported to be localized in the outer mitochondrial membrane. The other fraction exhibited an electrophoretic mobility which was different from that of intact mitochondria, and this fraction contained enzymes characteristic of the inner membrane-matrix fraction such as soluble and particulate enzymes of the Krebs cycle. Microsomes exhibited an electrophoretic mobility which was almost identical with that of the outer mitochondrial membrane. In addition to resolving the localization of enzymes in mitochondrial membranes, these data indicate that the outer limiting membrane of the mitochondrion is the sole determinant of the surface charge of mitochondria.

TRITON HYPERLIPEMIA IN DOGS : I. IN VITRO EFFECTS OF THE DETERGENT ON SERUM LIPOPROTEINS AND CHYLOMICRONS

Scanu, Angelo; Oriente, Pasquale
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 31/03/1961 EN
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Triton WR-1339, a non-ionic detergent, added to canine serum or to ultracentrifugally separated lipoproteins, induced changes in the lipoproteins which were dependent upon concentration of detergent and class of lipoproteins. D 1.063 to 1.21 lipoprotein (α-LP) was especially sensitive to the action of triton. Addition of 2 mg. of triton to 1 mg. of α-LP (based on protein content), induced only slight changes in the electrophoretic and flotation characteristics of the lipoprotein. With a tenfold increase of the detergent (triton:α-LP, 20:1), the mixture, analyzed by starch gel and paper electrophoresis, yielded a tritonlipid complex which remained close to the origin, and a nearly lipid-free protein with electrophoretic mobility higher (starch gel) or lower (paper) than native α-LP. The splitting of the lipid and protein moieties of α-LP could not be clearly shown when the same mixture was analyzed by free boundary electrophoresis. Triton alone moved only slightly in an electrical field (paper, starch gel, Tiselius); it sedimented during ultracentrifugation at D 1.006 and D 1.063 and floated at D 1.21. D 1.006 to 1.063 lipoproteins (β-LP), required larger amounts of triton to show changes. These were evident in 40 to 80:1 mixtures of triton and β-LP. In starch gel and paper electrophoresis triton retained...

STRUCTURE AND SPECIFICITY OF GUINEA PIG 7S ANTIBODIES

Edelman, G. M.; Benacerraf, B.; Ovary, Z.
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/08/1963 EN
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Additional evidence has been obtained to show that different guinea pig anti-hapten antibodies differ in the structure of their L polypeptide chains. Antibodies from animals immunized with the same hapten conjugated to different carrier proteins gave similar starch gel electrophoretic patterns after dissociation of their chains. In a study of fine differences of specificity, cross-reacting antibodies were found to have some L chains with the same electrophoretic mobility. The multiplicity of L chain bands found in the characteristic starch gel electrophoretic patterns of dissociated anti-DNP antibodies was shown to be a reflection of the heterogeneity of antibodies of slightly different specificities. Reduction and alkylation of the active fragment produced by digestion of antibodies with papain yielded starch gel electrophoretic bands corresponding in mobility to L chains. The results are consistent with the notion that L chains are involved in the acquisition of immunologic specificity.

ELECTROKINETIC PHENOMENA : X. ELECTRIC MOBILITY AND CHARGE OF PROTEINS IN ALCOHOL-WATER MIXTURES

Daniel, Janet
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 20/01/1933 EN
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285.1282%
1. The electrophoretic velocities of gelatin-, egg-albumin-, and gliadin-covered quartz particles in various alcohol-water solutions are, within the limits employed in usual experimental procedures, proportional to the field strength. 2. The electrophoretic mobilities of small, irregularly shaped quartz particles covered with an adsorbed film of protein in alcohol-water solutions are equal to the electroosmotic mobilities of the liquid past similarly coated flat surfaces. Hence the size and shape of such particles does not influence their mobilities, which depend entirely on the protein film. 3. The corrected mobility and hence presumably the charge of gelatin-covered quartz particles in solutions containing 35 per cent ethyl alcohol is proportional to the combining power of the gelatin; therefore the gelatin is adsorbed with the active groups oriented toward the liquid. The same is true in 60 per cent alcohol. 4. The charge calculated by means of the Debye-Henry approximation from the mobility of gelatin in solutions containing up to 35 per cent ethyl alcohol is, in the neighborhood of the isoelectric point, proportional to the combining power of the gelatin. Therefore the dielectric constant and the viscosity of the bulk of the medium may be used in the Debye-Henry approximation Q = 6 π η r vm (1 + κ r) to predict changes in charge from mobility. 5. In the neighborhood of the isoelectric point gelatin is probably completely ionized in buffered ethyl alcohol-water mixtures up to 60 per cent alcohol. 6. In the presence of ethyl alcohol the isoelectric point of gelatin is shifted toward smaller hydrogen ion activities. This shift...

AN ELECTROPHORETIC STUDY OF MIXTURES OF OVALBUMIN AND YEAST NUCLEIC ACID

Longsworth, Lewis G.; MacInnes, D. A.
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 20/03/1942 EN
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283.04373%
Electrophoretic patterns of mixtures of ovalbumin and yeast nucleic acid indicate that the constituents migrate independently of each other in buffer solutions of 0.1 ionic strength and at pH values somewhat higher than the isoelectric point of the protein. In the isoelectric region, however, the patterns from the two sides of the channel exhibit asymmetries that can be explained by assuming the existence in the mixture of appreciable concentrations of a reversibly dissociable complex between the components. Formation of this complex is favored by increasing concentrations of the components and decreasing ionic strength. At pH values below the isoelectric point partial precipitation of the complex occurs. The patterns obtained from each side of the channel in the electrophoresis of a mixture of two components, which form a dissociable complex, indicate only two boundaries, aside from the δ and ε effects. One of these is a normal boundary whose displacement is proportional to the mobility of a component that has separated from the mixture. In the other boundary, however, dissociation of the complex occurs and consequently the displacement of this boundary corresponds to the mobility of neither component nor to that of the complex. Moreover...

Phosphorylation of Yeast Phosphatidylserine Synthase by Protein Kinase A: IDENTIFICATION OF SER46 AND SER47 AS MAJOR SITES OF PHOSPHORYLATION*

Choi, Hyeon-Son; Han, Gil-Soo; Carman, George M.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
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The CHO1-encoded phosphatidylserine synthase from Saccharomyces cerevisiae is phosphorylated and inhibited by protein kinase A in vitro. CHO1 alleles bearing Ser46 → Ala and/or Ser47 → Ala mutations were constructed and expressed in a cho1Δ mutant lacking phosphatidylserine synthase. In vitro, the S46A/S47A mutation reduced the total amount of phosphorylation by 90% and abolished the inhibitory effect protein kinase A had on phosphatidylserine synthase activity. The enzyme phosphorylation by protein kinase A, which was time- and dose-dependent and dependent on the concentration of ATP, caused a electrophoretic mobility shift from a 27-kDa form to a 30-kDa form. The two electrophoretic forms of phosphatidylserine synthase were present in exponential phase cells, whereas only the 27-kDa form was present in stationary phase cells. In vivo labeling with 32Pi and immune complex analysis showed that the 30-kDa form was predominantly phosphorylated when compared with the 27-kDa form. However, the S46A/S47A mutations abolished the protein kinase A-mediated electrophoretic mobility shift. The S46A/S47A mutations also caused a 55% reduction in the total amount of phosphatidylserine synthase in exponential phase cells and a 66% reduction in the amount of enzyme in stationary phase cells. In phospholipid composition analysis...

A gel electrophoresis study of the competitive effects of monovalent counterion on the extent of divalent counterions binding to DNA.

Li, A Z; Huang, H; Re, X; Qi, L J; Marx, K A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1998 EN
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The behavior of alkaline earth metal cations (Mg2+ and Ca2+) and transition metal cations (Zn2+ and Cu2+) interacting with lambda-DNA-HindIII fragments ranging from 2,027 to 23,130 bp in Tris-borate-EDTA buffer solutions was investigated. The divalent counterions competed with Tris+ and Na+ for binding to polyion DNA, and the competition binding situations were investigated by measuring the reduction of the DNA mobility, by pulsed- or constant-field gel electrophoresis. The interaction of Mg2+ with DNA was intensively studied over a wide range of Mg2+ concentrations. In addition, we examined the competition binding as a function of ionic strength and DNA size. To compare valence effects, we studied Co(NH3)6(3+) interaction with DNA fragments under conditions similar to that of Mg2+. At relatively low Mg2+ concentration, the normalized titration curves of DNA mobility were well fit by Manning's two-variable counterion condensation (CC) theory. The agreement between the predicted value (total charge neutralization fraction theta) from Manning's CC theory and the data based on our measured DNA electrophoretic mobility reduction was consistent under our experimental conditions. In contrast to alkaline earth metal cations (Mg2+ and Ca2+)...