Presence Of Dodecyl Sulfate Research Articles

Isolation of a five-polypeptide cytochrome b-f complex from spinach chloroplasts.

A simple rapid purification of a cytochrome b-f complex from spinach chloroplasts is described. Novel features of the method include: 1) EDTA treatment of thylakoids prior to detergent extraction; 2) affinity chromatography over equine cytochrome c linked to Sepharose 4B; and 3) inclusion of the protease inhibitor phenylmethylsulfonyl fluoride. Cytochrome b-f complex is obtained in good yield, free of exogenous lipid, and with high plastoquinol:plastocyanin oxidoreductase activity. The complex contains 2 eq of cytochrome b-563 per eq of cytochrome f and a Rieske iron-sulfur center. Polyacrylamide gel electrophoresis in the presence of dodecyl sulfate indicates that the complex is composed of five distinct polypeptides of Mr = 37,000, 33,500, 22,000, 19,000, and 16,500. Only the Mr = 33,500 and 22,000 polypeptides stain for heme. The Mr = 37,000 component in this preparation is absent from cytochrome b-f complex isolated by another procedure (Hurt, E., and Hauska, G. (1981) Eur. J. Biochem. 117, 591-599). The complex described here also differs in its spectrum at 77 K, its stability, and its buoyant density.

Nucleotide sequence of the Escherichia coli pyrE gene and of the DNA in front of the protein-coding region.

Orotate phosphoribosyltransferase (EC 2.4.2.10) was purified to electrophoretic homogeneity from a strain of Escherichia coli containing the pyrE gene cloned on a multicopy plasmid. The relative molecular masses (Mr) of the native enzyme and its subunit were estimated by means of gel filtration and electrophoresis in the presence of dodecyl sulfate. The amino acid sequences at the N and C termini, as well as the amino acid composition, were determined. The nucleotide sequence of the structural pyrE gene, including 394 nucleotide residues preceding the beginning of the coding frame, was also established. From the results the following conclusions may be drawn. Orotate phosphoribosyltransferase is a dimeric protein with subunits of Mr 23 326 consisting of 211 amino acid residues. The pyrE gene is transcribed in a counter-clock wise direction from the E. coli chromosome as an mRNA with a considerable leader segment in front of the protein-coding region. This leader contains a structure with features characteristic for a (translated?) rho-independent transcriptional terminator, which is preceded by a cluster of uridylate residues. This indicates that the frequency of pyrE transcription is regulated by an RNA polymerase (UTP) modulated attenuation.

Improved purification and further characterization of acetyl-CoA carboxylase from cultured cells of parsley (Petroselinum hortense).

Acetyl-CoA carboxylase from irradiated cell-suspension cultures of parsley (Petroselinum hortense) has been purified to apparent homogeneity. The procedure included affinity chromatography of the enzyme on avidinmonomer--Sepharose 4B. Molecular weights of about 420000 for the native enzyme and about 220000 for the enzyme subunit were determined respectively by gel filtration or sucrose-density-gradient sedimentation and by electrophoresis in the presence of dodecyl sulfate. The purified enzyme showed an isoelectric point of 5. The enzyme carboxylated the straight-chain acyl-CoA esters of acetate, propionate, and butyrate at decreasing rates in this order. The catalytic efficiency of the carboxylase was highest when ATP existed largely as MgATP2- complex. At the optimum pH of 8 the apparent Km values for the substrates were: acetyl-CoA, 0.15 mmol/1; bicarbonate, 1 mmol/1; MgATP2-, 0.07 mmol/1. The carboxylase was inhibited by greater than 50 mmol/l NaCl, KCl, or Tris/HCl buffer. The putative allosteric activator, citrate, stimulated the enzyme only slightly at concentrations below 2 mmol/l, but strongly inhibited the carboxylase at higher concentrations. The results of these studies demonstrate that several properties of the light-inducible acetyl-CoA carboxylase of parsley cells, an enzyme of the flavonoid pathway, are remarkably similar to those of acetyl-CoA carboxylases from a variety of other organisms.

The purification and partial characterization of human salivary kallikrein

The major arginine esterase activity in human saliva has been purified. This enzyme lowers the blood pressure of a rabbit and produces kinins in acid treated dog plasma. It is therefore a kallikrein. The kallikrein has an unusual amino acid composition: aspartic acid and glutamic acid comprise 40% of the residues; the total number of basic residues is less than 5%; glycine and proline together make up more than 40% of the residues. The enzyme has a p I of 4.0 and an M r of 27000 as determined by dodecyl sulfate gel electrophoresis. On the other hand, sedimentation equilibrium data and the amino acid composition give an M r value of only 9600. The enzyme could be a rather asymmetric molecule. The circular dichroism spectrum shows a minimum at 200 nm with [ θ] = −28000 deg · cm 2 · dmol −1. The spectrum suggests that the enzyme structure contains polyproline form II helix together with β-turns. This structure is stable in the presence of dodecyl sulfate.

Polynucleotide Phosphorylase from a Cyanobacterium (Synechococcus sp.): Subunit Composition and Properties

Abstract Polynucleotide phosphorylase from cells of the cyanobacterium Synechococcus sp. has been purified 1400-fold by an improved procedure. The enzyme purified to homogeneity and lacking nuclease, phosphatase and protease contaminations reveals a single band of ADP polymerizing activity upon polyacrylamide gel electrophoresis under nondenaturing conditions which cor­responds to a molecular mass of about 275 000. The enzyme migrates as a single polypeptide of Mr ≈ 70 000 when subjected to gel electrophoresis in the presence of dodecyl sulfate indicating a composition of α4 for the native enzyme molecule. The isoelectric point of the purified enzyme as determined by isoelectric focusing was found to be at 4.2 ± 0.1. Polynucleotide phosphorylase of Synechococcus is preferentially activated by Mg2+; KCl has a significant stimulatory effect.

Solid-phase sequence analysis of polypeptides eluted from polyacrylamide gels. An aid to interpretation of DNA sequences exemplified by the Escherichia coli unc operon and bacteriophage lambda.

An approach to sequencing proteins by the solid-phase method combined with isolation of proteins and polypeptides by gel electrophoresis is described. Mixtures of proteins or polypeptides resulting from digests are fractionated in the presence of dodecylsulphate in polyacrylamide gels. They are detected with Coomassie blue, eluted, selectively reacted with porous glass derivatives and sequenced in their amino-terminal regions with the aid of a new microsequencer. Alternatively they can be analysed or digested with enzymes and fingerprinted. It is a relatively rapid method of purifying proteins for sequence analysis which we have used to provide partial protein sequence data to complement DNA sequences. Nine genes, four from the unc operon of Escherichia coli encoding the alpha, beta, gamma and epsilon subunits of ATP synthase and five for capsid proteins of bacteriophage lambda, have been identified by this method.

Autolysis studies of cathepsin D.

Incubation of the single polypeptide chain cathepsin D from bovine spleen at pH 3.5, resulted in the fragmentation of the molecule. This was followed by gel electrophoresis in the presence of dodecyl sulphate, gel filtration, circular dichroism and enzyme activity measurements. Main bands of Mr 30,000 and 15,000 appeared first, followed by bands corresponding to smaller fragments. Conformational changes of the cathepsin D molecule were observed in the near ultraviolet circular dichroism spectrum during the autolysis. Measurements of the initial inactivation rate showed apparent first order kinetics which was biphasic at lower concentrations of the enzyme. The inactivation rate of cathepsin D increases with decreasing enzyme concentration. The presented results are interpreted in terms of autolytic degradation of cathepsin D.

Cytochrome P-450 monooxygenase activities in human and rat liver microsomes.

Microsomes were prepared from human livers obtained from renal donors of various ages and both sexes. Their drug-metabolizing capacity was measured and compared to that of rat liver microsomes. The following parameters were investigated: cytochrome P-450, cytochrome B5, NADPH-cytochrome c reductase, epoxide hydrolase, aryl hydrocarbon hydroxylase, benzphetamine N-demethylase, p-nitroanisole-O-demethylase, ethoxycoumarin-O-deethylase, steroid-16 alpha-hydroxylase. In addition, the metabolism of benzo(a)pyrene, progesterone, pregnenolone, testosterone, dehydroepiandrosterone and estradiol was studied in detail in vitro. The inhibitory effect of metyrapone and alpha-naphthoflavone on 7-ethoxycoumarin-O-deethylase was measured. The microsomal proteins of both species were separated by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The following conclusions were drawn from the results obtained. Human liver microsomes can be stored under optimal conditions for the measurement of a large variety of enzymic activities. Human liver microsomes are able to metabolize the various xenobiotics used as substrates with a rate similar to that of female rat liver microsomes. No sex-linked difference in enzymic activity was observed in human microsomes. Significant differences in benzo(a)pyrene and steroid metabolism were registered when human and rat liver microsomes were compared. The monooxygenase activities, the sensitivity to in vitro alpha-naphthoflavone and metyrapone, the results of steroid metabolism, and slab gel electrophoresis are strong indications for multiplicity of human liver cytochrome P-450.

Reaction of mersalyl with mitochondrial proteins under native and denaturing conditions as exemplified by the identification and isolation of the phosphate carrier.

Pig heart mitochondria were incubated with [203Hg]mersalyl and the radioactive pattern was analyzed by fluorography after dodecyl sulfate gel electrophoresis. No differences in the radioactivity distribution were found after labeling with various mersalyl concentrations, at different pH and after labeling in the native or dodecyl sulfate-dissociated state of mitochondria. A redistribution of [203Hg]mersalyl between various proteins in the presence of dodecyl sulfate could directly be demonstrated by mixing labeled membranes with unlabeled matrix proteins, as well as by comparison of the radioactivity patterns of whole mitochondria labeled with irreversibly reacting N-([2-3H]ethyl)maleimide and reversibly binding [203Hg]mersalyl. From these data it is concluded that under native conditions mersalyl is principally bound to the phosphate carrier protein, whereas during dissociation in dodecyl sulfate the organomercurial is redistributed and mainly attached to the ADP/ATP-carrier protein.

The effect of delta-aminolevulinate on catalase T-messenger RNA levels in delta-aminolevulinate synthase-defective mutants of Saccharomyces cerevisiae.

Total RNA was isolated from mutants of Saccharomyces cerevisiae that lack active delta-aminolevulinate synthase and are therefore defective in heme biosynthesis. The RNAs were translated in the cell-free protein synthesis system from wheat germ, and the catalase T synthesized was isolated by immunoadsorption and polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. Little or no catalase T product was detected with RNA from mutant cells grown in the absence of a heme precursor. As judged from in vitro translational capacity, RNA fractions from mutant cells grown in the presence of delta-aminolevulinate contained at least 10 times more catalase T mRNA than RNA from unsupplemented cells.

Multiple structural genes for mouse amylase.

Salivary and pancreatic amylases from the mouse show both structural and quantitative genetic variation encoded within a gene complex on chromosome 3. Two fundamental questions prompted by this variation are whether salivary and pancreatic amylases are derived from different structural genes and whether multiple structural genes are causing the quantitative variation observed in each of the two amylases. These questions were approached by comparing the amylase protein from 12 congenic lines carrying amylase gene complexes derived from different origins. The amylases were purified by affinity chromatography employing the inhibitor cyclohepta-amylose and characterized in terms of amino acid composition, specific activity, molecular weight, and heat stability. They were analyzed by native electrophoresis in polyacrylamide gels and by peptide mapping employing both cyanogen bromide cleavage and restricted proteolysis in the presence of dodecylsulfate. By these techniques, many differences in the structure of pancreatic amylase that were not reflected in the salivary amylase were found among mouse strains. Likewise, a distinct salivary amylase variant was found. These results suggest that independent structural genes exist for the two amylases. Furthermore, by all criteria used, pancreatic amylase from single strains exhibits molecular heterogeneity, whereas heterogeneity was never found for salivary amylase. We conclude that at least four structural genes code for pancreatic amylase while only a single gene, different from any of the pancreatic genes, codes for salivary amylase.

Properties of the general acyl-CoA dehydrogenase from pig liver.

The properties of the general acyl coenzyme A dehydrogenase from pig liver and the stable reduced dehydrogenase . product and oxidized dehydrogenase . acetoacetyl-CoA complexes of the dehydrogenase were investigated. The enzyme has a molecular weight of 178,000 to 183,000 determined by gel filtration chromatography and by gel electrophoresis at different acrylamide concentrations. The subunit molecular weight is 45,000 based on acrylamide gel electrophoresis in the presence of dodecyl sulfate which agrees with the minimum molecular weight calculated from the flavin:protein ratio and the amino acid analysis. The subunits are identical, or very similar, as judged by quantitative NH2-terminal analysis and mapping of tryptic peptides. Immunochemical analyses by the complement fixation technique show that the structure of the oxidized enzyme is different from the structure of enzyme . acyl-CoA complexes whether the flavin in these complexes is in the oxidized or reduced state. The amino acid analysis, isoelectric point, and a procedure for crystallizing the dehydrogenase are also reported.

Molecular architecture of annelid erythrocruorins. Extracellular hemoglobin of Arenicola marina (Polychaeta).

The respiratory protein, erythrocruorin, of the annelid Arenicola marina was investigated. Spectral properties show many analogies with those of vertebrate hemoglobine. 144 heme groups (ferroprotoporphyrin) were found in the whole molecule, which has a relative molecular mass of 3.56 X 10(6), as determined by sedimentation equilibrium, and an isoelectric point of 4.69. Protein dissociation patterns were analysed by electrophoresis after denaturation in the presence of dodecylsulfate, with and without 2-mercaptoethanol. A tentative model associating molecular mass of the native molecule, heme content, molecular mass of the polypeptide chains and functional properties is proposed. A twelfth subunit of A. marina erythrocruorin would contain twelve heme groups arranged in three functional units made up of four protomers, half of these being covalently bound to non-heme chains; two structural chains would be spatially arranged as bonds between the subunits.

Release of a two-chain form of antithrombin from the antithrombin-thrombin complex.

The stable, inactive complex between antithrombin and thrombin was prepared and subsequently purified free of reactants by a procedure which minimized secondary thrombin proteolysis. Dissociation of the complex was effected by hydroxylamine or ammonia in the presence or absence of sodium dodecyl sulphate, or by prolonged treatment at 100°C in the presence of dodecyl sulphate. All these procedures released from the complex a proteolytically modified, two‐chain form of antithrombin and not the intact protein. Dissociation in the absence of denaturant also released active thrombin. Control experiments indicated that the inhibitor was indeed released from the complex as the modified form of antithrombin rather than as intact antithrombin which was subsequently modified by the dissociation agents or by thrombin. The modified form of antithrombin dissociated from the complex could not be differentiated electrophoretically from a proteolytically modified, inactive form of antithrombin which has been shown previously to be produced free in solution during the reaction between antithrombin and thrombin. These observations suggest that limited proteolysis of antithrombin by thrombin may be involved in the formation of the inactive complex. The inhibitor may thus exist in the complex as a proteolytically modified, two‐chain species. However, a state in which the scissilc peptide bond of the inhibitor only forms a tetrahedral adduct with the active site serine of the enzyme, as shown by X‐ray crystallography in other protease‐protease inhibitor complexes, cannot be excluded.

Covalent cross-linking of porcine small-intestine microvillar aminopeptidase. Subunit structure of the membrane-bound and the solubilized enzyme

The porcine intestinal microvillar aminopeptidase (EC 3. 4. 11. 2) consists of three types of subunits, α, β and γ, of molecular weights determined to 168.000, 118,000 and 54.000, respectively. The isolated detergent-form of the enzyme was cross-linked in dilute solution in the presence of Triton X-100 by diimidates varying in chain length from five to twelve carbon atoms. The tendency of the subunits to participate in inter-protomer cross-linking depended on the chain length of the reagent as shown by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. Diimidates of chain lengths from seven to twelve carbon atoms caused formation of products of molecular weights up to about 660.000, whereas the six carbon atom diimidate generated complexes lower in molecular weight and the five carbon atom diimidate introduced very few inter-protomer cross-linkages. Cross-linking of brush border membrane vesicles, with dimethylsuberimidate, followed by Triton X-100 solubilization and isolation of the microvillar aminopeptidase by immunoadsorbent chromatography on antiaminopeptidase M-Sepharose, indicated a maximum molecular weight of the membrane-bound enzyme of 330.000. The β- and γ-polypeptide chains were previously demonstrated to be formed by limited proteolysis of the α-polypeptide chain. Peptide mapping excluded the possibility of the γ-chain being generated from the β-chain. The subunit structures of the membrane-bound enzyme were suggested as α2, αβγ, and β2γ2. Small amounts of a larger polypeptide chain of molecular weight 330.000 are present in preparations of microvillar aminopeptidase. Peptide mapping indicated structural homology between this component, termed 2α, and α,β and γ. It is suggested that the large polypeptide is a precursor of the intestinal aminopeptidase.

Flavanone synthase from Petroselinum hortense. Molecular weight, subunit composition, size of messenger RNA, and absence of pantetheinyl residue.

Flavanone synthase from irradiated cell suspension cultures of parsley was purified to apparent homogeneity. Molecular weights of about 77 000 for the enzyme and about 42 000 for the subunits were determined respectively by sedimentation-equilibrium measurements and disc-gel electrophoresis in the presence of dodecyl sulfate. A specific antiserum was prepared for the enzyme and was used in an assay for flavanone synthase mRNA activity in partially purified RNA preparations. The apparent molecular size of flavanone synthase mRNA was estimated by sucrose gradient centrifugation and gel electrophoresis under partially denaturing conditions. Values of about 17 S and Mr = 0.62 X 10(6) were obtained. The fractionation patterns suggested that flavanone synthase mRNA was homogeneous in size. All together, the results support the idea that the enzyme is composed of two subunits which are probably identical. Amino acid analysis and a microbial assay were carried out to test the possible occurrence of cysteamine, beta-alanine, and pantothenate in the enzyme. The results were negative, indicating the absence of pantetheine or a similar residue. The possible similarity in mechanism between flavanone synthase and 3-oxoacyl-(acyl carrier protein) synthase is discussed.

The liver microsomal FAD-containing monooxygenase. Spectral characterization and kinetic studies.

A FAD-containing monooxygenase isolated from pig liver microsomes migrates as a single band upon electrophoresis in polyacrylamide gels in the presence of dodecyl sulfate. The minimum molecular weight based on mass of amino acids per mole of flavin is 64,000. However, the catalytically active enzyme exists as aggregating units of the monomer. Neither oxygen nor organic substrates perturbed the spectrum of the oxidized flavoprotein and their binding to this form of the enzyme could not be detected. Anaerobically NADPH bleaches the flavoprotein, and in the presence of both NADPH and oxygen a remarkably stable intermediate form of the enzyme, with an absorption band at 375 nm, is observed. The spectrum of the intermediate resembles that of a peroxyflavin. The monooxygenase catalyzes NADPH- and oxygen-dependent oxygenations of nucleophilic nitrogen- or sulfur-containing compounds. Kinetic studies carried out with a model organic nitrogen substrate (trimethylamine) and a sulfur substrate (methimazole) gave similar patterns. The kinetic data are consistent with an ordered Ter-Bi mechanism with an irreversible step between the second and third substrate where NADPH is added first, followed by oxygen, and the oxidizable organic substrate is added last. If NADPH is the first substrate added, then NADP+ must be the last product released since NADP+ is competitive with NADPH.

Effect of trypsin on the cell surface proteins of hepatoma tissue culture cells. Characterization of a carbohydrate-rich glycopeptide released from a calcium binding membrane glycoprotein.

Concentrations of trypsin that bring about aggregation of hepatoma tissue culture (HTC) cells also release from the cell surface an Mr = 55,000 glycopeptide fragment. This glycopeptide fragment also accumulates in the medium, including serum-free medium, as a normal consequence of membrane protein turnover. The trypsin-released glycopeptide is labeled when cells are grown in the presence of fucose or leucine before treatment of the cells with the protease. Similarly, the glycopeptide fragment can be labeled by reacting cells in situ by lactoperoxidase-catalyzed radioiodination or by tritiated borohydride reduction of cells treated first with neuraminidase and galactose oxidase. The tryptic glycopeptide fragment was purified by concanavalin A-Sepharose chromatography, and hydroxyapatite chromatography in the presence of dodecyl sulfate. The amino acid and carbohydrate composition was determined, as was the sensitivity of the purified glycopeptide to a variety of endo- and exoglycosidases. The purified glycopeptide contains an average of 17 sialic acid residues and hence, shows charge heterogeneity after electrophoresis in isoelectric focusing gels. The charge heterogeneity can be eliminated completely by treatment with neuraminidase. The glycopeptide after this treatment is homogeneous. The trypsin-sensitive membrane glycoprotein which is the source of the Mr = 55,000 glycopeptide was identified by two-dimensional gel electrophoretic analysis of labeled cells, treated or not treated with trypsin. This glycoprotein, which has an apparent molecular weight of 85,000 and forms a homodimer in the presence of calcium ions, was purified and its identity as the parent of the Mr = 55,000 glycopeptide was confirmed by showing that the same Mr = 55,000 fragment was released by trypsin from the purified glycoprotein as was released from the intact cells.

Characterization of Porins from the Outer Membrane of Salmonella typhimurium 2. Physical Properties of the Functional Oligomeric Aggregates

We have purified to homogeneity, from mutant strains of Salmonella typhimurium, the small oligomers of porin that confer permeability channels to artificial vesicle membranes reconstituted from phospholipids and lipopolysaccharide. The molecular weights of the porin oligomers from the strains SH5551 and SH6017 appeared to be 130000 and 125000, respectively, and those of the monomers were 41000 and 37500, respectively, when determined by sedimentation equilibrium in the presence of dodecylsulfate. It was thus concluded that the functional porin oligomers consisted of three identical subunits. The Stokes' radius of the trimer . dodecylsulfate complex was around 5 nm. The trimer bound less dodecylsulfate than the monomer. The trimer . dodecylsulfate complex retained at room temperature the native conformation of porin, which is rich in beta-structure. When the trimers were dissociated further by various treatments, only the porin monomers were recovered in significant amounts, and the permeability-conferring activity was lost simultaneously. We propose, therefore, that the trimer is the minimal functional unit of porin that is capable of forming permeability channels in the outer membrane of Salmonella typhimurium.

Canine cardiac myosin with special referrence to pressure overload cardiac hypertrophy. I. Subunit composition.

In studies of myosin from left and right ventricles of normal hearts and hypertrophic hearts at 5 weeks and 13 weeks after aortic banding, polyacrylamide gel electrophoresis shows intermediate molecular weight components which derive from heavy chains fragmented in the presence of dodecyl sulfate. The proportion of degraded heavy chains is greater in myosin from hypertrophic hearts than normal hearts, with comparable degradation in left and right ventricle myosin. The observed fragmentation of myosin results from proteolysis due to contaminant proteases or a thermally activated, heat-stable nonenzymatic process, or both. The susceptibility of heavy chains to crude myofibrillar proteases differs in normal and hypertrophic cardiac myosin; however, the kinetics of tryptic digestion are identical for both myosins. With precautions to minimize proteolytic artifacts on dodecyl sulfate-polyacrylamide gel electrophoresis, preparations of myosin from left and right ventricles of normal and hypertrophic hearts exhibit comparable subunit composition, with approximately molar ratios of heavy chains, light chain L1, and light chain L2. Comparable stoichiometry for the light chain fraction is determined by high speed sedimentation equilibrium at pH 11 and direct fractionation of the different cardiac myosins. We do not confirm reports (e.g. Wikman-Coffelt, J., Fenner, C., Smith, A., and Mason, D. T. (1975) J. Biol. Chem. 250, 1257-1262) of different proportions of light chains in left and right ventricle myosin of normal and hypertrophic canine hearts. The light chains display microheterogeneity, with L1 generating two isoelectric variants and L2 generating two major and two minor variants, but identical mobilities and isoelectric values are obtained in the different myosin preparations.