Sodium Dodecyl Sulfate-acrylamide Gel Electrophoresis Research Articles

THE COMPONENT FRAGMENTS OBTAINED BY ACID DISSOCIATION OF PAPAIN‐SOLUBILIZED H‐2 MOLECULES*

H-2Kk and H-2Dd molecules were specificially purified from a radioiodinated H-2a preparation obtained by papain digestion of spleen cell membranes of A/J strain mice. The molecules were isolated by binding to H-2 alloantisera of the corresponding private specificity followed by precipitation with rabbit anti-mouse IgG antiserum. The specifically precipitated radioiodinated H-2Kk and H-2Dd molecules were dissociated by acid treatment into large and small components of about 37,000 and 11,000 respectively. These were separated by gel filtration at acid pH or by gel isoelectric focusing in the presence of 6 M urea. Each component separated by gel filtration of the acid-dissociated H-2 molecules showed a high degree of size homogeneity as determined by sodium dodecyl sulphate-acrylamide gel electrophoresis. Upon gel isoelectric focusing, however, the small components showed two peaks of radioactivity closely located together at pH 7-8, both of which had a restricted pH range, while the large components gave one peak of a relatively wide pH range of pH 5-6. The H-2Kk and H-2Dd molecules gave essentially the same pattern in terms of the numbers and the positions of the radioactivity bands. Under the iodination conditions used the large components of H-2Kk molecules contained more radioactivity than the small components, while the reverse was true in case of H-2Dd molecules. Such a difference was also found with H-2Kk and H-2Dd molecules isolated by use of alloantisera of the respective public specificity. The assay of binding of the isolated components with H-2 alloantisera of defined specificity revealed that the large components retain most of the allospecificities of the parental H-2 molecules. No H-2 allospecificities were found on the small components. The small components showed extensive binding with rabbit antiserum against mouse beta2-microglobulin. The same antiserum did not show any binding with the large components. On the other hand, both of the components did bind with rabbit antiserum against papain-solubilized H-2 molecules.

Pre-proparathyroid hormone: fidelity of the translation of parathyroid messenger RNA by extracts of wheat germ.

Pre-proparathyroid hormone is the major protein synthesized in wheat-germ extracts in response to addition of an 8-15S fraction of parathyroid RNA. The accuracy of the translation of the mRNA from parathyroid tissue was examined by analysis of the carboxyl-terminal tryptic peptide and the amino-terminal amino acid of the protein, by analysis of the size distribution of the mRNA, and by translation of the mRNA in a second cell-free extract. When 8-15S RNA was fractionated on a sucrose gradient containing formamide, RNA that supported the synthesis of pre-proparathyroid hormone was present in a single symmetrical peak, suggesting that it was homogeneous. Analyses by paper chromatography and electrophoresis of the proline-containing tryptic peptides of pre-proparathyroid hormone indicate that they are identical with the corresponding proline-containing peptides of parathyroid hormone. Because the COOH-terminal tryptic peptide of parathyroid hormone contains proline, the data indicate that the COOH termini of pre-proparathyroid hormone and parathyroid hormone are identical. Methionine from initiator [35S]Met-tRNAfMet was rapidly incorporated into pre-proparathyroid hormone by the wheat-germ extract, and a single-step Edman degradation selectively removed almost all of the initiator [35S]methionine present in pre-proparathyroid hormone. Translation of the 8-15S RNA in a cell-free extract from Krebs-II ascites cells resulted in a protein that comigrated with pre-proparathyroid hormone on sodium dodecyl sulfate-acrylamide gel electrophoresis. These data support the conclusion that the wheat-germ system accurately translates the mRNA for parathyroid hormone, and they strengthen the contention that pre-proparathyroid hormone is the initial biosynthetic product.

Purification and properties of a β- N-acetylaminoglucohydrolase from malted barley

β- N-Acetylaminoglucohydrolase (β-2-acetylamino-2-deoxy- D-glucoside acetylaminodeoxyglucohydrolase, EC 3.2.1.30) was extracted from malted barley and purified. The partially purified preparation was free from α-and β-glucosidase, α- and β-galactosidase, α-mannosidase and β-mannosidase. This preparation was free from α-mannosidase only after affinity chromatography with p-amino- N-acetyl-β- D-glucosaminidine coupled to Sepharose. The enzyme was active between pH 3 and 6.5 and had a pH optimum at pH 5. A MW of 92000 was obtained by sodium dodecyl sulfate-acrylamide gel electrophoresis and a sedimentation coefficient of 4.65 was obtained from sedimentation velocity experiments. β- N-Acetylaminoglucohydrolase had a K m of 2.5 × 10 −4 M using the p-nitrophenyl N-acetyl β- D-glucosaminidine as the substrate.

Pregrowth hormone: product of the translation in vitro of messenger RNA coding for growth hormone.

Membrane fraction RNA isolated from rat pituitary tumor (GC) cells has been translated in a wheat germ extract. A product was synthesized which was immunologically related to growth hormone, but which migrated more slowly than growth hormone upon sodium dodecyl sulfate-acrylamide gel electrophoresis. The mobility of the cell-free product on gels of this type was unchanged by treatment with either KOH or RNase. The mobilities during paper electrophoresis of the methionine-containing tryptic peptides obtained from the cell-free product were identical to those obtained from growth hormone synthesized and secreted by the GC cells. Molecular weights for growth hormone and the cell-free product of 19,500 and 24,000, respectively, were determined by gel electrophoresis of these proteins together with marker proteins of known molecular weights. No protein with the properties of the cell-free product was detected after a 2 min incubation of the GC cells with [35S]methionine. However, treatment of the GC cells, with a protease inhibitor, L-1-tosylamide-2-phenyl-ethylchloromethyl ketone (TPCK), led to the appearance of a new polypeptide, immunologically related to growth hormone, and with a mobility on gels identical to that of the cell-free product. These results strongly imply that the cell-free product represents a growth hormone precursor (pregrowth hormone) which is rapidly converted to growth hormone in pituitary cells.

Zur näheren Charakterisierung von Trypsininhibitoren bei dem Polychaeten Sabellastarte indica (Savigny). Zweite Mitteilung

In this paper we discribe a method for the isolation of trypsin inhibitors from the tentacles of the annelid Sabellastarte indica Savigny. These inhibitors - now homogeneous in their molecular weight - can be characterised by sodium dodecylsulfate-acrylamide gel-electrophoresis, in their inhibitory activity towards trypsin, plasmin and chymotrypsin. The inhibitors from Sabellastarte indica possess a stoichiometric binding relation of 2:1 for trypsin, lysine being the amino-acid in the reactive centre of the inhibitor responsible for interaction with trypsin. The reactive centre for trypsin is not identical with the reactive centre which binds chymotrypsin and does not influence the binding of chymotrypsin. These newly described inhibitors are therefore multiheaded, a type not previously described for invertebrates.

Serine transhydroxymethylase isoenzymes from a facultative methylotroph

Two serine transhydroxymethylase activities have been purified from a facultative methylotrophic bacterium. One enzyme predominates when the organism is grown on methane or methanol as the sole carbon and energy source, whereas the second enzyme is the major isoenzyme found when succinate is used as the sole carbon and energy source. The enzyme from methanol-grown cells is activated by glyoxylate, is not stimulated by Mg2+, Mn2+, or Zn2+, and has four subunits of 50,000 molecular weight each. The enzyme from succinate-grown cells is not activated by glyoxylate and is stimulated by Mg2+, Mn2+, and Zn2+, and sodium dodecyl sulfate-acrylamide gel electrophoresis indicates that this enzyme has subunit molecular weight of 100,000, the same as the molecular weight obtained for the active enzyme. Cells grown in the presence of both methanol and succinate incorporate less methanol carbon per unit time than cells grown on methanol and have a lower specific activity of the glyoxylate-activated enzyme than methanol-grown cells. Adenine, glyoxylate, or trimethoprim in the growth medium causes an increased level of serine transhydroxymethylase in both methanol- and succinate-grown cells by stimulating the synthesis of the glyoxylate-activated enzyme.

Relaxation complexes of plasmid DNA and protein. II. Characterization of the proteins associated with the unrelaxed and relaxed complexes of plasmid ColE1.

The proteins of the DNA-protein relaxation complex of plasmid ColE1 were labeled with [3H]leucine by growth of ColE1 containing Escherichia coli cells in the presence of this radioactive labeled amino acid. Three major [3H]leucine-labeled proteins are found associated with the supercoiled DNA in the ColE1 relaxation complex. The molecular weights of these proteins, determined by sodium dodecyl sulfate-acrylamide gel electrophoresis, are 60,000, 16,000, and 11,000, respectively. Induction of relaxation of the supercoiled DNA by treatment of the complex with sodium dodecyl sulfate results in a dissociation of the two smaller proteins from the DNA. The 60,000 protein, however, remains associated specifically with the nicked strand of the open circular DNA. The strand-specific association of this protein with the relaxed DNA resists heat denaturation of the DNA, sedimentation through an alkaline (pH 12.5) sucrose gradient, and centrifugation to equilibrium in an alkaline (pH 12.5) CsCl gradient.

Deoxyribonucleic acid-envelope complexes from Escherichia coli. A complex-specific protein and its possible function for the stability of the complex.

The different Escherichia coli envelope fractions (cell wall, cytoplasmic membrane, and DNA-envelope complex fragments) were isolated by free-flow electrophoresis and analyzed by sodium dodecylsulfate-acrylamide gel electrophoresis. The DNA-envelope complex fragments possess a specific protein (mol wt 80,000-90,000). Upon treatment with trypsin, this protein disappears and the complex breaks down, thus releasing DNA, cell wall, and cytoplasmic membrane. Disaggregation of the complex can also be achieved by high salt concentrations. Lysozyme treatment dissolves the murein layer within the complex but does not disaggregate the complex. From these and other results on the stability of the DNA-envelope complex, conclusions can be drawn about the possible linkage within the described envelope particles.

Studies on structural proteins of the rat liver ribosomes I. Molecular weights of the proteins of large and small subunits

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this ‘three-dimensional’ polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.

Purification and preliminary characterization of human leukocyte elastase

Affinity chromatography permits the purification of 1–3 mg of human leukocyte elastase from the leukocytes contained in 500 ml of whole blood. Lysosomal granule proteins are extracted from polymorphonuclear leukocytes and subjected to chromatography on a column of elastin-Sepharose. Contaminating proteins are eluted with buffer containing 1 m NaCl and then elastase activity is eluted with buffer containing 8 m urea. The enzyme retains all of its esterase activity against N-t-BOC- l-alanine p-nitrophenyl ester after exposure to 8 m urea and retains 22% of its activity in the presence of 1% sodium dodecyl sulfate. In sodium dodecyl sulfate and 2-mercaptoethanol leukocyte elastase undergoes autolysis giving rise to several low molecular weight fragments. The molecular weight of the native enzyme is found to be 22.000 by both gel filtration and sodium dodecyl sulfate—acrylamide gel electrophoresis. A characteristic set of four isozymes is seen after acrylamide disc gel electrophoresis at pH 4.5. All bands are active against elastin and also contain carbohydrate by the periodic acid-Schiff stain. On the basis of stain intensity, the slower moving isozymes appear to be richest in carbohydrate. Active leukocyte elastase forms a complex with α 1-antitrypsin in a 1:1 molar ratio. The elastase must be enzymatically active for complex formation to occur.

Isolation and characterization of two hydroxyproline-containing glycoproteins from normal animal lung lavage and lamellar bodies.

Two glycopeptides, present in particulate material obtained by pulmonary lavage from normal rabbits, were isolated and characterized. The same two glycopeptides were present in preparations of lamellar bodies from rabbit lung. The estimated molecular weights of the two glycopeptides by sodium dodecyl sulfate-acrylamide gel electrophoresis were found to be 62,000 and 36,000 and both were found to contain hydroxyproline and relatively high amount of glycine (11 and 15 per cent, respectively). Carbohydrate analysis of the two glycopeptides demonstrated the presence of glucosamine, sialic acid, mannose, Fucose, and galactose. Similar glycopeptides of the same molecular weights, and amino acid and carbohydrate compositions have been found in layage mateial isolated from lungs of patients with alveolar proteinosis. The data indicate that thses two collagen-like glycopeptides are major intra-alveolar proteins in many mammals, including humans.

Fractionation and Characterization of Surface Antigens from Group a Neisseria Meningitidis

Group A meningococcal surface components were first subjected to fractionation with a mixture of chloroform-methanol. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the aqueous phase containing 30 to 40% of the original material revealed only two polypeptide components and a slowly migrating carbohydrate component. The soluble fraction of the interphase was found to contain most of the bacterial surface proteins and the chloroform-methanol phase essentially all of the lipid components. The components of the aqueous phase were further fractionated by use of the hydrophobic affinity column, 4-phenylbutylamino-Sepharose and gradient elution with NaCl to yield fractions I and II. Fraction II was further separated into a minor and a major component (IIb) with Sepharose G-200. Fraction I contained the group A polysaccharide in ionic linkages with a minor polypeptide component (6%). It elicited bactericidal antibodies in rabbits and protected mice against homologous challenge, whereas the polysaccharide alone was non-immunogenic in these animals. Fraction IIb was a polysaccharide-polypeptide complex with unknown linkages; it induced a low concentration of rabbit antibodies that were bactericidal to group A and C meningococci. Mice vaccinated with fraction IIb were most resistant to homologous challenge and the resistance was also extended to challenges with group B and C cells. Fractions I and IIb appeared to be useful alternatives to the currently employed group-specific polysaccharide vaccines for the protection against drug-resistant meningococci. A simplified procedure for the preparation of group-specific polysaccharide was presented.

Purification of cardiac myosin: Application to hypertrophied myocardium

A method is described for the preparation of high purity myosin from small amounts of cardiac muscle. The method employs homogenization and prolonged extraction of the cardiac tissue. Purification is achieved through three successive precipitation-dissolution cycles and without the use of column chromatographic techniques. Purity of the myosin preparation is assessed at various stages of the purification procedure by sodium dodecylsulfate-acrylamide gel electrophoresis and by measurement of RNA and nucleoprotein content. With 1.5–2.0 g of rabbit right ventricle as the starting tissue, this method yields 4–6 mg myosin per g wet tissue. The method is also shown to give similar results with rabbit right ventricles hypertrophied by pulmonary stenosis.

Isolation and characterization of type III collagen from chick stain.

A collagen species was isolated from the skin of 8 week-old chicks by pepsin treatment at 4 degrees C and was identified as type III collagen by analogy to human and calf type III collagen. Like human type III collagen, the type III collagen molecule from chick skin is composed of three alpha1 (III) chains which are linked together by disulfide bridges. The amino acid composition of [alpha1 (III)]3 purified by CM-cellulose chromatography and agarose-gel chromatography includes two cysteine residues per chain, and the hydroxyproline/proline ratio is greater than 1.0. The cyanogen bromide peptide pattern of [alpha1 (III)]3 on sodium dodecylsulfate acrylamide gel electrophoresis differed from that of alpha1 (I), alpha1 (II) and alpha2 chains. Segment-long-spacing crystallites from chick skin type III collagen showed the same cross striation pattern as segment-long-spacing crystallites from calf skin type III collagen. Unlike human type III collagen, type III collagen from chick skin was highly soluble in 1.0M NaCl, 0.05M Tris/HCl buffer, pH 7.5, and could not be separated from type I collagen by fractional salt precipitation. On CM-cellulose chromatography, [alpha1 (III)]3 molecules from chick skin eluted in the same position as alpha2 chains, in contrast to human [alpha1 (III)]3, which elutes in the position of beta12.

Stimulation of Collagen Synthesis in a Cell-Free System by mRNA from Chick embryos

Embryonic chick RNA was translated in a cell-free system derived from wheat germ. One of the products synthesized in vitro under the direction of this RNA could be identified as collagen on the basis of collagenase digestion experiments and sodium dodecylsulfate-acrylamide gel electrophoresis. By submitting the RNA to chromatography on oligo(dT)-cellulose, a 26-30-fold enrichment of the mRNA coding for collagen was achieved.

The molecular properties of the copper enzyme galactose oxidase

Galactose oxidase (EC 1.1.3.9) has been purified 140-fold by DEAE- and CM-cellulose chromatography from cultures of Polyporus circinatus. The enzyme has a molecular weight of 68,000 ± 3,000 as determined by sedimentation equilibrium, sodium dodecyl sulfate-acrylamide gel electrophoresis, Sephadex G-150 chromatography, and osmometry. Galactose oxidase is a single-chain protein which does not self-associate. Charge isozymes of the enzyme are detected by ion-exchange chromatography and gel electrophoresis. The amino acid composition determined herein is significantly different from that previously reported (Kelly-Falcoz, F., Greenberg, H., And Horecker, B. L. (1965) J. Biol. Chem. 240, 2966–2970). The enzyme contains 1% by weight of neutral carbohydrate. Galactose oxidase contains 1 g-atom of copper per 70,000 g of protein. The metal does not contribute to the electrophoretic or isozymic properties of the protein. However, the sedimentation coefficients of the holo- and apoenzymes, 4.76S and 4.83S, respectively, do suggest that small differences in protein conformation accompany the removal of the copper from the holoenzyme. Attempted sulfhydryl group titration of galactose oxidase shows that the holoenzyme is resistant to denaturation. However, in β-mercaptoethanol-guanidine HCl 5 half-cystine residues are titrated in the apoenzyme. On a dry-weight basis, the E 1cm 1% value for galactose oxidase at 280 nm is 15.4. Galactose oxidase has an isoelectric point above pH 10 which is a probable source of some of its anomalous behavior in physical measurements and enzyme-activity determinations.

Role of uridine diphosphate glucose in the biosynthesis of starch. Mechanism of formation and enlargement of a glucoproteic acceptor.

A potato particulate preparation, which is constituted mainly by proplastids, was capable of glucose transfer from micromolar concentrations of uridine diphosphate [14C]glucose into an endogenous protein molecule to form a 14C‐labelled glucoprotein. This glucoprotein had short radioactive glucan chains and could act as acceptor for the synthesis of larger radioactive glucan chains from labelled uridine diphosphate glucose, either at millimolar concentrations or in the presence of glucose 1‐phosphate or adenosine diphosphate glucose at micromolar concentrations. This last step required Mn2+ or Mg2+ ions.The enzyme responsible for the formation of the glucoproteic acceptor was named uridine diphosphate glucose‐protein transglucosylase I and its properties were studied.The uridine diphosphate glucose‐protein transglucosylase activity, that was detected at micromolar uridine diphosphate glucose concentrations in the presence of glucose 1‐phosphate, could be distinguished from uridine diphosphate glucose–protein transglucosylase I by the following criteria: The glucose 1‐phosphate dependent activity required Mn2+ or Mg2+ ions, was sensitive to temperature and formed radioactive glucan chains totally hydrolyzed by β‐amylase. Conversely, uridine diphosphate glucose–protein transglucosylase I was active in the absence of Mn2+ or Mg2+ ions, was resistent to temperature and formed radioactive glucan chains scarcely attacked by β‐amylase.Sequential analysis of the glucose incorporation from uridine diphosphate [3H]glucose and uridine diphosphate [14C]glucose into the glucoprotein and sodium dodecylsulphate–acrylamide gel electrophoresis of the labelled glucoproteins formed in the presence and in the absence of glucose 1‐phosphate were carried out.On the basis of the data obtained a simple scheme of reactions is proposed to explain the involvement of the uridine diphosphate glucose–protein transglucosylase I and glucose 1‐phosphate dependent activities in the synthesis of α‐1,4 glucan chains of the starch type.

Bovine Erythrocyte Superoxide Dismutase: SUBUNIT STRUCTURE AND SEQUENCE LOCATION OF THE INTRASUBUNIT DISULFIDE BOND

The subunit assembly of bovine erythrocyte superoxide dismutase has been studied, through analysis of the effects of heat, removal of metal ion cofactors, and disulfide bond reduction, upon the dissociation of the subunits. Investigations utilizing sodium dodecyl sulfate-acrylamide gel electrophoresis and sedimentation equilibrium analysis demonstrate that the 2 polypeptide subunits of the protein are not covalently joined, but are associated through unusually strong noncovalent interactions. This absence of intersubunit disulfide bonds, in conjunction with the titration of a single free sulfhydryl group per subunit polypeptide chain, necessitates that the 3 half-cystine residues per subunit exist as 1 residue of cystine, forming an intrasubunit disulfide bond, and 1 residue of cysteine. The specific half-cystine residues participating in the disulfide linkage and contributing the free thiol function were identified. Analysis of the products of cyanogen bromide cleavage, with acrylamide gel electrophoresis, demonstrates that the intrasubunit disulfide links 1 half-cystine residue in the sequence NH2-terminal to Met-115, to another half-cystine residue in the sequence COOH-terminal to Met-115. Through conventional procedures of enzymatic digestion, peptide isolation, and peptide characterization, Cys-55 and Cys-144 are shown to participate in the intrasubunit disulfide linkage, and Cys-6 to exist as free cysteine.

Purification on hydroxyapatite of liver ribosomes and polysomes from unfasted mice

Batchwise purification of liver ribosomal and polysomal preparations on hydroxyapatite removes glycogen, bound hemoglobin, ribonuclease and other proteins contaminating ribosomal preparations. This procedure eliminates mandatory fasting and thus provides pure ribosomes and polysomes from animals maintained under normal physiological conditions. Ribosomes and polysomes purified on hydroxyapaptite and eluted at low salt concentrations (0.4 M KH 2 PO 4) can be dissociated into subunits in the presence of puromycin and 0.3–0.5 M KCl. These subunits are active in poly(U)-directed phenylalanine synthesis. Sodium dodecylsulfate—acrylamide gel electrophoresis detected no degradation of the ribosomal RNA. The ability of polysomes to direct amino acid incorporation into hot trichloroacetic acid precipitate is not lost during purification on hydroxyapatite. In addition, the resolution of polysome profiles was greatly improved following hydroxyapatite purification. Incubation of purified murine liver polysomes in the absence of ribonuclease inhibitor resulted in no polysome breakdown. Identical treatment of unpurified polysomes resulted in a partial conversion to 80-S monomers, and suggested that ribonuclease was washed from the polysome preparations during purification. Ribosomes and polysomes from both mouse liver and rat liver can be purified batchwise on hydroxyapatite. In addition, this procedure can be used to purify ribosomes from both fed animals, with large amounts of cellular glycogen, and fasted animals with significantly less glycogen contamination.

Analysis of Proteinuria in Health and Disease Using Sodium Dodecyl Sulphate—Acrylamide Gel Electrophoresis

Abstract. Sodium dodecyl sulphate—acrylamide gel electrophoresis (SDS‐AGE) separates proteins according to their molecular weight (MW) without being influenced by their electrostatic charge. It was carried out on the urine of 17 healthy control subjects and 92 ambulatory patients with suspected or known renal impairment. The SDS‐AGE patterns were classified as physiological, low MW predominance, middle MW predominance, high MW predominance, or mixed low and high MW. Patients were separately classified as having either normal kidneys or glomerular, tubular or mixed renal lesions according to the results of clinical investigation. Comparison of both classifications revealed that SDS‐AGE allowed a correct diagnosis of the site of renal involvement in 83 per cent of cases. Predominantly low MW protein excretion correlated with tubular damage. Middle and high MW patterns correlated with glomerular disease, and a relationship between nephrotic syndrome with minimal glomerular changes and middle MW pattern was found in two cases, suggesting that SDS‐AGE could be used to evaluate the selectivity of proteinuria. However, the estimation of selectivity by SDS‐AGE should be studied in a sufficient number of cases and compared with measurements of selectivity by the protein clearance method.