Sodium Dodecyl Sulfate-acrylamide Gel Research Articles

Purification and characterization of high-molecular-weight forms of adrenocorticotropic hormone of ovine pituitary glands

A highly purified preparation of high-molecular-weight adrenocorticotropic hormone (ACTH) was prepared from ovine pituitary glands by dilute acetic acid extraction, oxycellulose fractionation. Sephadex gel filtration, and affinity chromatography on immobilized alphap(1-39)ACTH antibodies. Two ACTH peptides of molecular weights of 24 000 and 34 000 were detected by sodium dodecyl sulfate-acrylamide gel electrophoresis in this preparation. It appeared that the immobilized antibodies adsorbed two forms equally well and could not distinguish between them under the conditions used. These two ACTH peptides were found to be present in crude extracts of ovine pituitary glands, indicating that they were not artifacts produced by the purification procedure. The high-molecular-weight forms of ACTH were found to be susceptible to degradation by tissue enzymes. They could be easily destroyed during the extraction, if precautions were not taken. Moreover, they were poorly adsorbed by oxycellulose which had been used for the adsorption of ACTH activity from crude preparations by most investigators. These properties probably accounted for the fact that high-molecular-weight forms of ACTH remained undetected until very recently.

Nuclear matrix. Isolation and characterization of a framework structure from rat liver nuclei.

A nuclear framework structure termed the nuclear matrix has been isolated and characterized. This matrix forms the major residual structure of isolated nuclei and consists largely of protein with smaller amounts of RNA, DNA, carbohydrate, and phospholipid. The nuclear matrix can be further resolved by combined treatment with DNase and RNase. The remaining nuclear protein structure, after extraction of 90 percent of the nuclear protein, 99.9 percent of the DNA, and 98 percent of the RNA and phospholipid, is termed the nuclear protein matrix. Electron microscopy of this final nuclear protein matrix reveals an interior framework structure composed of residual nucleolar structures associated with a granular and fibrous internal matrix structure. The internal matrix framework is derived from the interchromatinic structures of the nucleus, and is connected to a surrounding residual nuclear envelope layer containing residual nuclear pore complex structures. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the nuclear matrix proteins demonstrates three major polypeptide fractions, P-1, P-2, and P-3, with average molecular weights of approximately 69,000, 66,000 and 62,000, as well as several minor polypeptides which migrate at approximately 50,000 and at higher molecular weights (>100,000). Polypeptides with molecular weights identical to those of P-1, P-2 and P-3 are also components of isolated nuclear envelopes and nucleoli, whereas isolated chromatin contains no detectable matrix polypeptides. This suggests that the major matrix polypeptides are localized in specific structural regions of the nucleus, i.e., nuclear envelope, nucleoli, and interchromatinic structures. The presence of cytochrome oxidase activity in the isolated nuclear matrix indicates that at least some integral proteins of the nuclear membrane are associated with the matrix.

Characterization of human β2-microglobulin by isoelectric focusing

β 2-Microglobulin (B 2M) isolated from the urine of normal subjects and patients with cadaveric renal transplantation, showed 2 homologues by isoelectric focusing, one with a p I 5.3, the other with a p I 5.7. These proteins show identical molecular weights by dextran gel filtration and sodium dodecyl sulfate acrylamide gel electrophoresis. The immunogenic reactivity demonstrates partial identity using antiserum to human B 2M from 2 different sources.

Precursors of the T4 internal peptides.

The precursors of the two T4 internal peptides have been identified by in vitro cleavage of individual phage proteins eluted from sodium dodecyl sulfate-acrylamide gels. The precursor of internal peptide VII is p22, the product of T4 gene 22 and an essential component of the morphogenic core. The precursor of peptide II is a protein with a molecular weight of approximately 13,000, whose gene has yet to be defined by mutation. A newly detected protein of approximately 15,000 molecular weight is found to be cleaved and is, therefore, likely to be a component of precursor head structures.

Steric orientation of hapten groups and the effect on antibody reactivity

Antibodies to the II isomeric form of the fluorescyl hapten group were elicited in rabbits. IgG antibodes specific for the Fl(II) group were purified by immunoadsorption and their ligand-binding properties measured by fluorescence quenching and equilibrium dialysis. Anti-Fl(II) IgG antibodies were compared to IgG antibodies to the I isomeric form at the protein and active site levels. Comparative H- and L-chain patterns in electrophoresis, using sodium dodecyl sulfate acrylamide gels, indicated small but significant differences. Fluorescence quenching studies with isomeric ligands, in which the side group was small [e.g. FlNH 2(I)], suggested that the active sites of the two anti-isomeric populations were quite similar. However, when the fluorescyl group was attached to a large antibodies to discriminate the isomeric forms was affinity dependent. Minor but significant differences in the active sites of the two antibody populations were noted in quantum yield, spectral shift and circular dichroism studies monitoring bound ligand. The studies suggest that steric isomers of large hapten groups may stimulate common anti-fluorescyl clones, and the differences noted in the antibody populations may reflect a differential quantitative enrichment of specific clones.

Hormonal regulation of ornithine aminotransferase biosynthesis in rat liver and kidney

The relative rates of ornithine aminotransferase (OAT) synthesis in vivo were studied by pulse-labeling rats with [4,5- 3H]leucine, isolating the mitochondrial enzyme protein by immunoprecipitation with a monospecific antibody, dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels, and determining the radioactivity in OAT. After 4 days of treatment with triiodothyronine (T 3), both the enzyme activity level and the relative synthetic rate of OAT in rat kidney were elevated over twofold. The level of hepatic OAT activity was unaffected by this treatment. Thyroidectomy caused a 50% drop in the basal level of OAT activity and synthesis in kidney but not in liver. Although the basal levels of activity and synthesis of both renal and hepatic OAT were unaffected by adrenalectomy, the glucagon induction of the enzyme in liver was enhanced by about one-third and the T 3 induction in kidney was suppressed 50% by this operation. After 4 days of treatment with estrogen, both the enzyme activity level and the relative synthetic rate of OAT in male rat kidney were elevated nearly 10-fold. Hepatic OAT activity and synthesis were unaffected by this regimen. Thyroidectomy almost completely abolished the estrogen induction of OAT in kidney. OAT induction by estrogen could be restored by treating thyroidectomized rats with T 3. Simultaneous administration of T 3 plus estrogen to intact rats produced a multiple effect, resulting in a striking 20-fold induction of renal OAT. Although administration of either T 3 or estrogen causes an increase in the synthesis of immunoprecipitable OAT protein in rat kidney, each of these hormones may induce OAT by a different mechanism.

Human erythrocyte pyruvate kinase Total purification and evidence for its antigenic identity with L-type enzyme

Erythrocyte pyruvate kinase (ATP:pyruvate 2-0-phosphotransferase, EC 2.7.1.40) has been purified 40 000 times from human erythrocytes, according to an original method. The whole purification procedure included toluene extraction, ammonium sulphate fractionation, DEAE-Sephadex batchwise chromatography and affinity chromatography on a Dextran Blue-Sepharose column with specific elution by fructose 1,6-diphosphate. The final preparation had specific activity of 290 I.U./mg of proteins and the overall yield was about 30%. Pyruvate kinase showed only one protein band as judged by sodium dodecyl sulphate acrylamide gel electrophoresis. Pure enzyme was injected into rabbits and monospecific antiserum was obtained able to neutralize, per ml, 150 I.U. of erythocyte-type pyruvate kinase as well as of l-type enzyme. l-type and erythrocyte-type pyruvate kinases showed reactions of complete identity when tested in immunodiffusion against anti-erythrocyte type pyruvate kinase sera; in all cases a single precipitation line could be detected. l-type pyruvate kinase when mixed with anti-erythocyte pyruvate kinase serum suppressed all ability of that antiserum to react immunological with erythocyte enzyme. Finally the microcomplement fixation curves using anti-erythrocyte pyrurate kinase serum were identical for erythrocyte and l-type enzymes. From these results it appeared that no antigenic difference between L-type and erythocyte enzyme could be detected. Consequently the most likely hypothesis is that both these enzymes are coded by the same single gene, the slight electrophoretic differences between them being due to post-synthetic tissuespecific changes.

Chromatin-like structures obtained after alkaline disruption of bovine and human papillomaviruses

Four low-molecular-weight polypeptides migrating like H2a, H2b, H3, and H4 calf liver histones were detected by sodium dodecyl sulfate-acrylamide gel electrophoresis of highly purified preparations of bovine papillomavirus (BPV) and human papillomavirus (HPV). Complexes of these polypeptides and viral DNA were isolated by agarose-gel filtration of the alkaline disruption products of both viruses. When observed under the electron microscope, these complexes appeared as circular structures composed of nucleosomes with a diameter of about 8.0 nm interconnected by a naked DNA filament. The maximal frequency of nucleosomes per molecule was 30 for both viruses, corresponding to a condensation ratio of the viral DNA of 2.5.

Protein kinase and phosphoproteins of avian myeloblastosis virus.

A protein kinase associated with purified virions of avian myeloblastosis virus, BAI strain A, was highly purified by ion-exchange chromatography and gel filtration. On the basis of molecular sieving on Sephadex G-200, the enzyme protein appeared to have a molecular weight of about 50,000 to 60,000; disc gel electrophoresis in sodium dodecyl sulfate-acrylamide gels revealed the presence of at least two polypeptide chains; and isoelectric focusing on acrylamide gels revealed two protein bands with activity. Of the nonviral proteins used as phosphate acceptors, the greatest rate of phosphorylation was obtained with alpha-casein. Potential physiological substrates for this activity included specific virion polypeptide of avian myeloblastosis virus. One of the virion polypeptides found in association with reverse transcriptase activity from avian myeloblastosis virus accepted more phosphate than any of nonviral or viral polypeptides examined on the basis of nanomoles of 32P incorporated per milligram of protein.

The solubilization of bone and dentin collagens by pepsin Effect of cross-linkages and non-collagen components

Bone and dentin collagen are less susceptible to solubilization by pepsin digestion than is skin collagen. Digestion at 4°C for 72 h solubilized only 35.3% of bovine cortical bone and 5.6% of bovine dentin compared with nearly 100% dissolution of bovine skin. Sodium dodecyl sulfate-acrylamide gel electrophoresis and molecular sieve chromatography showed that, for bone and dentin, intact α chains and cross-linked aggregates of β, γ and higher weight remained intact after pepsin solubilization but lower molecular weight fragments also were prevalent indicating chain scission in helical regions. Electron microscopic examination of segment long spacing precipitates of the soluble collagens confirmed the presence of solubilized polymerized collagen. The principal reducible cross-link in both bone and dentin was the precursor of dihydroxylysinonorleucine and this cross-link was also present in the solubilized collagens. Small amounts of non-collagenous proteins and glycosaminoglycans of different compositions in dentin and bone resisted extraction before pepsin digestion. However, the differences in solubilization of the collagens have been related to differences in cross-linkage placement.

Purification, molecular weight, amino acid, and subunit composition of arylsulfatase A from human liver

Arylsulfatase A (EC 3.1.6.1) from human liver self-associates at pH 5.0. This behavior was used to purify the enzyme to a specific activity of 2872 μmol nitrocatechol sulfate hydrolyzed per hour per milligram of protein, a 7000-fold increase in specific activity over the crude homogenate. No evidence of heterogeneity can be detected by acrylamide gel electrophoresis at pH 8.9 and 6.8 or by sedimentation equilibrium centrifugation at pH 8.1 and 5.0. The purified enzyme also exhibits one precipitin line on immunodiffusion and immunoelectrophoresis against antibody prepared to an impure enzyme sample. The amino acid analysis of the enzyme reveals a high content of proline and the presence of carbohydrate. The molecular weight, measured by sedimentation equilibrium centrifugation, is 104,500 at pH 8.1 and 413,000 at pH 5.0. Centrifugation in 6 m guanidine hydrochloride indicated the presence of two components of molecular weights 53,000 and 66,000. The presence of two components was verified by acrylamide gel electrophoresis of denatured enzyme in 6 m urea and in sodium dodecyl sulfate. The molecular weights of the components, determined from protein standards, by sodium dodecyl sulfate acrylamide gel electrophoresis are 49,000 and 59,000.

Synthesis of type II collagen in vitro by embryonic chick neural retina tissue.

Collagen produced by chick neural retina tissue in vitro has the properties of type II collagen. Isolated neural retina tissue from stage 29-32 chick embryos incorporated [14C]glycine and [3H]proline into collagen consisting of alpha1 chains and a higher molecular weight species. The peptides produced by cyanogen bromide digestion of the collagen are identical to those from authentic type II collagen from cartilage in charge properties, as determined by carboxymethyl-cellulose chromatography, and size distribution, as determined by sodium dodecyl sulfate-acrylamide gel electrophoresis.

Purification and characterization of squid brain myosin.

Myosin was extracted from frozen squid brain and purified by a modification of the procedure of Pollard et al. (Pollard, T.D., Thomas, S.M., and Niederman, R. (1974) Anal. Biochem. 60, 258-266). Myosin was eluted from Bio-Gel A-15m column as a single peak of (K+-EDTA)-activated ATPase ((K+-EDTA)-ATPase) activity with an average partition coefficient (Kav) of 0.22. In sodium dodecyl sulfate-acrylamide gel electrophoresis, the purified myosin showed a predominant band with similar electrophoretic mobility as the heavy chain of rabbit skeletal muscle myosin, and two less intense bands near the bottom of the gel. No actin band was seen. The properties of the (K+-EDTA)-ATPase activity were: (a) the time course of the reaction was biphasic at 25 degrees but linear at 32 degrees; (b) the optimum rate of reaction was obtained between 0.3 and 0.8 M KCl; (c) the pH optimum was between 8.0 and 9.0; (d) the reaction was specific for ATP with an apparent Km of 0.19 mM. ATPase activity in 0.06 M KCl and 5 mM MgCl2 was increased about 1.5 times by a 10-fold excess of rabbit skeletal muscle F-actin and about 5 times by a 40-fold excess. The actin activation was inhibited slightly by the addition of 0.2 mM CaCl2 and completely by the addition of 10 mM CaCl2. Myosin formed arrowhead patterns with rabbit skeletal muscle F-actin as observed by electron microscopy of negatively stained samples. It also aggregated in bipolar filaments which attached to decorated actin filaments at different angles, as well as formed cross-connections and ladder-like patterns between actin filaments. These two forms of interactions between myosin and actin were abolished by treatment with MgATP.

Protein Bodies from the Endosperm of Castor Bean

Protein bodies from the storage endosperm of dry castor bean (Ricinus communis L.) were isolated by successive nonaqueous linear density gradient centrifugation. The isolated protein bodies were lysed by the addition of water, and the various structural components of the organelles were separated by sucrose gradient centrifugation. The matrix protein remained at the top of the gradient while the membrane, the crystalloids, and the globoids migrated to densities 1.15 g/cm(3), 1.30 g/cm(3), and > 1.46 g/cm(3), respectively. The protein of the protein bodies was distributed evenly between the crystalloids and the matrix, and little protein was present in the globoids or the membrane.The proteins of the protein bodies were resolved into protein components of diverse molecular weights in sodium dodecyl sulfate-acrylamide gel electrophoresis. The protein components of the organelle matrix were distinct from those of the crystalloids. Whereas the matrix proteins had very diverse molecular weights, the crystalloid proteins were mainly composed of several proteins with molecular weights between 50,000 and 60,000 daltons. Also, the matrix proteins were soluble in water while the crystalloid proteins were insoluble in water but soluble in salt solution, thus representing albumins and globulins, respectively. Two of the matrix proteins with molecular weights approximately 120,000 and 65,000 daltons were identified as the phytohemagglutimin and the toxic protein ricin, respectively.During germination, the crystalloid proteins served as the storage protein and went through rapid degradation with smaller polypeptides formed as intermediates. In contrast, the proteins of the matrix under-went a much slower degradation during the same period and did not appear to be storage protein.

Bacteriophage T4 prehead proteinase: II. Its cleavage from the product of gene 21 and regulation in phage-infected cells

Using antiserum prepared against phage T4 prehead proteinase with a new technique for detecting antigens separated on sodium dodecyl sulphate-acrylamide gels, we demonstrate that the enzyme is a fragment of the protein coded for by phage gene 21 . T4PPase is derived from P21 by proteolytic cleavage. P21, T4PPase and several intermediate cleavage products accumulate in lysates of head gene mutants, but are present in very low amounts in a wild-type infection. The conversion of P21 to T4PPase is autocatalytic in vitro , since purified T4PPase converts P21 to itself. We show that P21, active enzyme, and several intermediate cleavage products are associated with the aberrant preheads made by the temperature-sensitive mutant in gene 23 , tsA78. A few molecules of inactive T4PPase are also found per purified phage particle after capsid maturation. We propose a model for the regulation of protein cleavage during T4 development in which about 100 molecules of inactive P21 zymogen are incorporated into the prehead. The zymogen is autocatalytically activated to T4PPase, cleaves the capsid precursor proteins, and finally self-inactivates by autodigestion.

Collagen cross-linking. Purification and substrate specificity of lysyl oxidase.

Lysyl oxidase is a specific amine oxidase that catalyzes the formation of aldehyde cross-link intermediates in collagen and elastin. In this study, lysyl oxidase from embryonic chick cartilage was purified to constant specific activity and a single protein band on sodium dodecyl sulfate acrylamide gel electrophoresis. This band had an apparent molecular weight of 62,000. The eluted protein cross-reacted with inhibiting antisera developed against highly purified lysyl oxidase. The highly purified enzyme was active with both insoluble elastin and embryonic chick skin or bone collagen precipitated as reconstituted, native fibrils. There was low activity with nonhydroxylated collagen, collagen monomers, or native fibrils isolated from lathyritic calvaria. The maximum number of aldehyde intermediates formed per molecule of collagen that became insoluble was two. These results indicate that lysyl oxidase has maximum activity on ordered aggregates of collagen molecules that may be overlapping associations of only a few collagen molecules across. Formation of aldehyde intermediates and cross-links during fibril formation may facilitate the biosynthesis of stable collagen fibrils and contribute to increased fibril tensile strength in vivo.

Regulation of protein synthesis in reticulocyte lysates: phosphorylation of methionyl-tRNAf binding factor by protein kinase activity of translational inhibitor isolated from hemedeficient lysates.

A previous study demonstrated that the translational inhibitor from lysates of heme-deficient rabbit reticulocytes is associated with a protein kinase activity. Chromatography of this inhibitor preparation on phosphocellulose yields two distinct protein kinase activities, PC1 and PC2. PC1, which consitutes about 90% of the activity in the unresolved preparation, does not inhibit protein synthesis in lysates, but actively phosporylates calf thymus histone II in a 3':5'-cyclic AMP-denpendent reaction. PC2 contains the translational inhibitor, phosphorylates histone poorly, and is not cyclic AMP-dependent. While [gamma-32P]ATP as the phosphate donor, the two kinase fractions were analyzed with the putative substrates, salt-washed 40S ribosomal subunits, and the initiation factor that mediates the binding of Met-tRNAf to the 40S subunit. PC1 is inactive with the initiation factor, but phosphorylates 40S subunits at a single major site that migrates as a 31,000-dalton band in sodium dodecyl sulfate-acrylamide gels; phosphorylation requires cyclic AMP. Similar phosphorylation of the reticulocyte 40S site (31,000 daltons) can be demonstrated with other cyclic AMP-dependent kinases from reticulocytes, rat liver, and bovine heart muscle. PC2 phosphorylates the small subunit (38,000 daltons) but not the large subunit(s) of the initiation factor; the reaction does not require cyclic AMP. PC2 does not phosphorylate 40S subunits. In the presence of 40S subunits, the initiation factor appears to be rapidly bound in a manner that effectively blocks phosphorylation of the initiation factor by PC2; under the same conditions phosphorylation of the 40S subunit by PC1 is not affected. The initiation factor has been shown to reverse the inhibitions of protein chain initiation induced in lysates by heme deficiency, double-stranded RNA, oxidized glutathione, or the purified translational inhibitor. The observation that the Met-tRNAf binding factor is phosphorylated by PC2 supports the hypothesis that this initiation factor is a target for the action of the translational inhibitor activated in heme deficiency.

Isolation and initial characterization of glial fibrillary acidic protein from chicken, turtle, frog and fish central nervous systems

The purification procedure for mammalian glial fibrillary acidic protein allowed the isolation of related proteins from the brain and spinal cord of the chicken, turtle, frog and fish. With the exception of the turtle, the proteins so isolated were homogeneous and migrated as a single band on sodium dodecyl sulfate-acrylamide gel electrophoresis, displaying the same mobility as bovine glial fibrillary acidic protein, 54 000 mol. wt. In the turtle an additional slower migrating band was constantly present, together with the main species. Mammalian and submammalian proteins were similar in amino acid composition and appeared to be susceptible to the same type of in situ proteolysis, with degradation of the major species into multiple polypeptides ranging down to 40 500 mol. wt. Unless degraded, the proteins isolated from submammalian vertebrates were excluded from sodium dodecyl sulfate-acrylamide gels if a reducing agent was not added to the electrophoretic sample, thus suggesting the existance of disulfide bridges between polypeptide chains, as demonstrated for the mammalian protein. The purified submammalian antigens cross-reacted with antisera to human glial fibrillary acidic protein with formation of spurs not only at the junction between mammalian and submammalian precipitation lines, but also between submammalian lines. The antisera produced against chicken antigen did not react with the human antigen and the antichicken sera could not be absorbed with human antigen. An immunologically active cyanogen bromide peptide in the myoglobin range (17 200 mol. wt.) characteristic of the mammalian protein, degraded and nondegraded, was not present in the digest of the submammalian proteins.

An electrophoretic comparison of the histones of various strains of Tetrahymena pyriformis

Histones were extracted from isolated macronuclei of several strains of the ciliated protozoan Tetrahymena pyriformis and compared by electrophoresis on both urea-acrylamide and sodium dodecyl sulfate-acrylamide gels. High resolution urea-acrylamide-gel electrophoresis resolves Tetrahymena histones into five main classes. The lysine-rich histone H1 exhibits microheterogeneity within each strain, mostly due to phosphorylation, and varies extensively in electrophoretic mobility and apparent molecular weight among the strains. Both H3 and H4 are constant among Tetrahymena strains and consist of several secondarily modified subspecies. However, while the electrophoretic constancy of H4, observed in higher organisms, extends to this lower eukaryote, H3 of each Tetrahymena strain migrates faster than calf thymus H3 in both gel systems. This suggests that H3 is not as rigidly conserved as H4. Fraction HX has no electrophoretic counterpart in calf thymus histone. It consists of five subfractions, each of which displays a remarkably constant electrophoretic mobility among the various strains. H2B is electrophoretically variable among Tetrahymena strains. The intersyngen and interphenoset diversity of Tetrahymena histone is shown to be comparable to that found among vertebrate classes.

L-type pyruvate kinase from human liver: Purification by double affinity elution, electrofocusing and immunological studies

L-type pyruvate kinase (ATP:pyruvate 2-O- phosphotransferase , EC 2.7.1.40) was highly purified from adult human liver. This purification included ammonium sulphate fractionation, DEAE-Sephadex batchwise absorption and two CM-Sephadex chromatographies with selective elution by ligands; in the former chromatography pyruvate kinase was eluted by ATP, in the latter one by phospho enolpyruvate and fructose 1,6-diphosphate. The last step of the purification procedure involved a hydroxyapatite column chromatography. This purification procedure allowed us to obtain 3.6 mg of protein with a specific activity 190 I.U./mg, i.e. a 1200-fold purification with an overall yield of about 8%. This preparation was homogeneous as judged by immunodiffusion, acrylamide and sodium dodecyl sulphate acrylamide gel electrophoresis. Anti L-type pyruvate kinase antibodies were obtained from rabbits and the antigenic properties of L-type pyruvate kinase were studied. The enzyme appeared to be a tetramer (molecular weight 220 000—240 000) with subunits of similar molecular weight about 60 000). Two interconvertible major forms were found by isoelectrofocusing in a sucrose gradient and in an acrylamide slab gel: one had an isoelectric point of 5.85 ± 0.09 and was the major enzymatic form after incubation with fructose 1,6-diphosphate or high concentrations or SH reagents. The other form (isoelectric point 6.28 ± 0.03) was the major form of L-type pyruvate kinase in liver crude extract, and after incubation of purified enzyme with a proteic fraction isolated from liver extract by ammonium sulphate precipitation.