Purification of protein A, an outer membrane component missing in Escherichia coli K-12 ompA mutants.

Outer membrane materials prepared from an Escherichia coli ompA (tolG) strain do not contain one of the major outer membrane proteins found in ompA+ strains. This protein has been purified in high yield from detergent-solubilized cell envelope material prepared from an ompA+ strain by preparative electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The purified protein is homogeneous in three electrophoretic systems, contains 2 mol of reducing sugar/mol of peptide and has alanine as the N-terminal amino acid. The amino acid composition is nearly identical to outer membrane protein II or B purified by others from incompletely solubilized cell envelope material. Thus, the fraction of outer membrane protein II or B that is difficult to solubilize is identical with the more readily solubilized fraction.     

Caproyl esterase from rat testis: Purification and action on cumulus cells

Rat caproyl esterase (E.C.3.1.1.1), extracted from testis with Tween 80, was purified by cation exchange and lectin affinity chromatography. The 104-fold purified enzyme had an activity of 840 μmol/hr per mg protein. The purified esterase did not contain any hyaluronidase or N-acetyl-glucosaminidase activity. Electrophoresis on sodium dodecyl sulfate polyacrylamide gels revealed a single band of approximately 60,000 molecular weight. The esterase had an isoelectric point of 5.1. Inhibition experiments showed high sensitivity of the enzyme to sulfhydryl agents and complete inactivation by sodium aurothiomalate. The purified caproyl esterase was shown to digest the cumulus matrix from mouse ova.     

Subunit structure of anthranilate synthetase from Neurospora crassa.

Freshly purified preparations of anthranilate synthetase complex from Neurospora crassa appeared to be homogeneous on polyacrylamide disc gels and were composed of two distinct subunits, 94,000 and 70,000 daltons, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Carboxymethylation of the complex or treatment with guanidine hydrochloride and urea before sodium dodecyl sulfate treatment did not alter the subunit pattern. When the purified complex was iodinated with 125I- or methylated with [14C]dimethylsulfate, no labeled components other than the two subunits stained with Coomassie blue were detected after electrophoresis in the presence of sodium dodecyl sulfate. Although some purified preparations were stable, most were unstable upon storage. Analysis of the unstable preparations on nondenaturing and sodium dodecyl sulfate polyacrylamide disc gels revealed that the complex in these preparations was progressively fragmented to smaller components and subunits upon repeated freeze-thaw treatment or prolonged incubation at or above 4 degrees. Distinct fragments were generated ranging in size down to 25,000 daltons, and some fragments retained some of the activities associated with the anthranilate synthetase complex. On the basis of these and earlier studies, we conclude that anthranilate synthetase from Neurospora crassa is composed of two distinct subunits in an alpha2beta2 structure; one subunit is a trifunctional peptide which contains the catalytic sites for the phosphoribosylanthranilate isomerase and indoleglycerol phosphate synthetase reactions, and associates with the second subunit to form glutamine-dependent anthranilate synthetase. The smaller subunits and components previously reported for this complex are apparently due to protease activity present in purified preparations.     

Detection of chitinase activity after polyacrylamide gel electrophoresis

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.     

Subunit composition of human liver alpha-L-fucosidase.

The subunit composition of human liver alpha-L-fucosidase was studied by using three different procedures for treating and electrophoresing the purified enzyme. Although the presence on only one subunit was found by conventional procedures using sodium dodecyl sulphate and urea, two non-identical subunits with mol. wts. of 59 000 and 54 000 were found when the alpha-L-fucosidase was reduced and S-carboxymethylated with iodoacetate followed by electrophoresis on polyacrylamide gels containing 0.1% sodium dodecyl sulphate and 8.0 M-urea.     

Two-dimensional peptide mapping by polyacrylamide-gel electrophoresis with limited proteolysis in SDS

The peptide mapping method described by Cleveland, et al. (1) was improved to a two-dimensional analysis applicable to minute amounts (less than 0.5 microgram) of proteins. Radioiodinated proteins for analysis were purified by electrophoretic elution of the proteins from polyacrylamide gels into buffer containing 0.1% sodium dodecyl sulfate. The proteins were digested enzymatically in the presence of 0.1% sodium dodecyl sulfate and an excess of nonlabeled bovine serum albumin (0.2 mg/ml) relative to labeled substrate in order to attain reproducibility by maintaining a consistent substrate concentration among different samples. The peptides of these limited proteolytic products were resolved by two-dimensional polyacrylamide gel electrophoresis (isoelectric focusing followed by SDS-gels). The resulting 2D-peptide maps of murine and bovine albumin and a murine lymphocyte membrane protein, Tp100, showed excellent resolution and reproducibility.     

The identification of myosin in rabbit hepatocytes.

A myosin-like protein was identified in isolated rabbit liver cells. It was extracted with high-ionic-strength buffer containing ATP, and purified by gel filtration in the presence of iodide. The myosin polypeptide was indistinguishable in size from the heavy chain of muscle myosin as determined by electrophoresis on polyacrylamide gels and gel filtration in the presence of sodium dodecyl sulfate. The hepatic myosin had an amino acid composition similar to that of muscle myosin, but lacked 3-methylhistidine. The Mg2+ -ATPase of the myosin was not activated by muscle actin. At low ionic strength, in the presence of Mg2+, the protein aggregated to form bipolar filaments 0.3 mum in length. A protein which resembled muscle actin in size and amino acid composition was extracted along with the myosin. Based on scans of stained sodium dodecyl sulfate polyacrylamide gels, the myosin content was estimated as 0.3% to 0.4% of the cell protein. The actin-like component was present in approximately ten-fold excess by weight. This ratio suggests that the organization and function of myosin in the hepatocyte is very different from that in the muscle cell.     

Purification and characterization of bovine tissue factor

Tissue factor (tissue thromboplastin, factor III), an initiator of coagulation, has been purified 142,000-fold to homogeneity from bovine brain. The protein is an integral membrane glycoprotein with an apparent molecular weight of 43,000 as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The apoprotein was first purified by extraction with Triton X-100 and repeated preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Antiserum was produced against a few micrograms of purified apoprotein and was used to construct an immunoadsorbent column. The column was then used for affinity purification of the apoprotein directly from the Triton X-100 extract, thereby significantly increasing the amount of purified protein produced. The purification scheme may be generally useful for the rapid and large scale purification of membrane proteins. Tryptic digestion of the apoprotein in Triton X-100 cleaved a peptide of approximately 3000 daltons without affecting the activity. The activity was recovered directly from stained SDS polyacrylamide gels, and the profile of recovered activity corresponded directly with the stained bands. The activity shifted along with the protein band following tryptic digestion, thus demonstrating that the protein observed on the gels is tissue factor. The coagulant activity of the purified apoprotein was reconstituted by the addition of phospholipid. Optimal activity was observed at phospholipid to protein ratios (w/w) greater than 450:1.     

Ribosomal proteins from rabbit reticulocytes: number and molecular weights of proteins from ribosomal subunits.

The ribosomal proteins from 40 S and 60 S subunits of rabbit reticulocytes were separated by two-dimensional polyacrylamide gel electrophoresis. The protein spots stained with Coomassie brilliant blue were cut out and the proteins were extracted. The material extracted from each spot was mixed with proteins of known molecular weight and then analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Both the total number and the molecular weights of each of the proteins were determined by these procedures. Thirty-two proteins were identified in the 40 S subunits; their molecular weights ranged from 8000 to 39,000 (average mol. wt = 25,000). Thirty-nine proteins were identified in the 60 S subunit; their molecular weights ranged from 9000 to 58,000 (average mol. wt = 31,000). The sum of the molecular weights of the individual proteins from each subunit is in agreement with previous estimations, derived from physico-chemical measurements of the total protein in mammalian ribosomal subunits. The molecular weight distribution obtained for the isolated proteins was nearly identical to that derived from spectrophotometric analysis of polyacrylamide-sodium dodecyl sulfate gels of the total protein mixtures from each subunit stained with Coomassie brilliant blue. The results are consistent with the hypothesis that reticulocyte ribosomes contain one copy of most of their protein constituents.     

Tricarboxylate carrier of bovine liver mitochondria. Purification and reconstitution

The tricarboxylate carrier of bovine liver mitochondria has been solubilized by Triton X-114 and purified by chromatography on hydroxylapatite and Silica Gel 60. The purified carrier could be visualized as a single band in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with Mr 37,000-38,000. The carrier, after reconstitution in phospholipid vesicles, catalyzed the exchange of [14C]citrate against citrate, malate, and threo-D8-isocitrate and was inhibited by the specific tricarboxylate carrier inhibitor 1,2,3-benzenetricarboxylic acid.     

Partial purification and characterization of the multiple molecular forms of staphylococcal clotting activity (coagulase).

The clotting activity of Staphylococcus aureus strain 104 was purified 46,000-fold, but absolute purity was not achieved. Carbohydrate content of the purified material was not more than 5%. Elution of clotting activity from denaturing and nondenaturing polyacrylamide gels revealed the presence of four distinct molecular forms. Molecular weights of the forms were approximately 31,500, 34,800, 44,800, and 56,800 as determined by gel filtration in 8 M urea, by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis, and by calculation with determined values for the Stokes radius and sedimentation coefficient. Molecular weights determined on sodium dodecyl sulfate-urea gels were found to decrease as the gel concentration increased, suggesting that the amount of sodium dodecyl sulfate bound was less than normal. Estimated frictional ratios for the forms showed that they differ in shape from one another and that they are all highly asymmetrical. Each of the forms had an isoelectric point between pH 5.44 and 5.47 when focused in 6% polyacrylamide gels for 9 h; however, prolonged focusing altered the isoelectric point of the forms to within the range of pH 4.35 to 4.65. The multiple clotting forms were not artifacts of the purification procedure and did not appear to be products of the proteolytic degradation of a larger protein.     

Rat liver cytosolic azoreductase. Purification and characterization.

An azoreductase has been purified to apparent homogeneity from the hepatic 105,000 x g supernatant fraction of 3-methylcholanthrene-treated rats. In the presence of sodium dodecyl sulfate, the purified enzyme preparation electrophoreses on polyacrylamide gels as a single protein band with a molecular weight of 30,000. In the absence of detergent, chromatography of the azoreductase on Sephadex G-100 gives a molecular weight of about 52,000 suggesting that the native enzyme may exist as a dimer. The purified azoreductase has a typical flavoprotein absorption spectrum and contains 2 mol of FAD/mol of enzyme. The enzyme catalyzes the reductive fission of methyl red (2'-carboxy-4-N,N-dimethylaminoazobenzene) and a structure-activity study indicates that the 2'-carboxyl group of methyl red is essential for catalysis since other structurally related analogs are totally inactive.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens.

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

Histone gene expression in early development of Xenopus laevis

Abstract. This study comprises the hybridization analysis of electrophoretically separated histone mRNAs from oocytes and embryos of Xenopus laevis , and analysis of in vitro translation products of these mRNAs on polyacrylamide gels containing sodium dodecyl sulfate (SDS) or Triton X-100. In oocytes and embryos up to the tailbud stage, four types of mRNAs complementary to histone H2B DNA and two complementary to histone H4 DNA can be discriminated by their different electrophoretic mobilities on polyacrylamide gels. Electrophoretic heterogeneity was not detected for messengers for histones H2A and H3.
Histone mRNA, purified by hybridization under stringent conditions with a cloned histone gene cluster, was used to direct histone protein synthesis in a wheat-germ cell free system. The proteins synthesized comigrate with purified marker histones when electrophoresed on SDS-gels or acid-urea gels containing Triton X-100. When hybrid-selected histone mRNAs from oocytes and embryos in different developmental stages are translated, the proteins made by the mRNA from one stage can not be discriminated from those made by the mRNA from another stage after electrophoresis on SDS-gels or acid urea Triton X-100 gels.     

Histone H5 in nucleated erythrocytes of fish as determined by radioimmunoassay

Tissue-specific histone H5 in the nucleated erythrocytes of dogfish, scup, skate, tautog, sea robin and toad fish was studied. The presence of this histone was inferred by its electrophoretic mocility on polyacrylamide gels containing either acid-urea or sodium dodecyl sulfate. By radioimmunoprecipitation assays, cross reaction was observed between fish histones and an anti-H5chicken antibody. The antibody was specific to chicken histone H5; purified chicken histone H1 and calf thymus total histones did not cross react. It is concluded that fish histone H5 shares common antigenic determinants with the chicken H5 histone.     

Polypeptide structure of DNA polymerase I from Saccharomyces cerevisiae

DNA polymerase I of the yeast Saccharomyces cerevisiae has been purified to near homogeneity. The enzyme sediments under high salt conditions as a band at 7.4 S and two polypeptides of Mr = 140,000 and 110,000 are resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both polypeptides react with rabbit anti-yeast DNA polymerase I serum and can be shown to be enzymatically active by renaturation in situ after electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This high molecular weight form of yeast DNA polymerase I is very sensitive to inhibition by aphidicolin. The biochemical properties of the enzyme and inhibitors that may aid in distinguishing yeast DNA polymerases I and II are also described.     

Analysis of rat serum apolipoproteins by isoelectric focusing. I. Studies on the middle molecular weight subunits.

Analytical isoelectric focusing (IEF) has been applied to the study of the apolipoprotein components of rat serum high density and very low density lipoproteins. The apolipoproteins were separated on 7.5% polyacrylamide gels containing 6.8% urea, with a pH gradient of 4-6. The middle molecular weight range apolipoproteins were identified on IEF gels by the use of apolipoproteins purified by electrophoresis on gels containing sodium dodecyl sulfate (SDS). The A-1 protein focused as 4 to 5 bands from pH 5.46 to 5.82; the A-IV protein and the arginine-rich protein each focused as 4 to 6 bands from pH 5.31 to 5.46. The low molecular weight proteins focused from pH. 4.43 to 4.83 and are the subject of a separate communication. Comparisons of the IEF method with SDS gel electrophoresis, polyacrylamide gel electrophoresis in urea, and Sephadex chromatography are also reported. Additional studies were also carried out that tend to rule out carbamylation or incomplete unfolding of the proteins in the presence of urea as the causes of the observed heterogeneity.     

Presence of two subunit types in ribulose 1,5-bisphosphate carboxylase from Thiobacillus intermedius.

Ribulose bisphosphate carboxylase (EC 4.1.1.39) has been purified to homogeneity from glutamate-CO2-thiosulfate-grown Thiobacillus intermedius by pelleting the protein from the 93,000 X g supernatant fluid followed by ammonium sulfate fractionation and sedimentation into a discontinuous sucrose density gradient. The molecular weight of the native protein approximated that of the higher plant enzyme (550,000) based on its relative electrophoretic mobility in polyacrylamide disc gels compared with that of standards of known molecular weight, including crystalline tobacco ribulose bisphosphate carboxylase. Sodium dodecyl sulfate electrophoresis in 12% polyacrylamide disc gels and Sephadex G-100 chromatography in the presence of sodium dodecyl sulfate indicated that the purified Thiobacillus protein, like the tobacco enzyme, consisted of two types of nonidentical subunits. The molecular weights of the large and small subunits were estimated to be about 55,000 and 13,000, respectively, by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase activity of the protein purified from spinach leaves and T. intermedius responded similarly to the effectors reduced nicotinamide adenine dinucleotide phosphate and 6-phosphogluconate. Contrary to a previous report (K. Purohit, B. A. McFadden, and A. L. Cohen, J. Bacteriol. 127:505-515, 1976), these results indicate that ribulose bisphosphate carboxylase purified from Thiobacillus intermedius closely resembles the higher plant enzyme with respect to quaternary structure, molecular weight, and regulatory properties.     

The purification of nuclease-free T4-RNA ligase.

RNA ligase has been highly purified in good yields from bacteriophage T4-infected Escherichia coli by a rapid and reproducible procedure. The enzyme is free of phosphomonoesterase and ribonuclease activities and is therefore suitable for the synthesis of oligoribonucleotides and for the labeling of the 3'-terminus of RNA. Greater than 90% of the protein in the enzyme preparation migrates as a single band on gradient polyacrylamide gels containing sodium dodecyl sulfate during electrophoresis. For use as a DNA synthesis reagent the enzyme may be reliably freed of deoxyribonuclease activity by an additional chromatographic procedure using a commercially avialable resin.     

DNA-dependent RNA polymerases from Artemia salina. Subunit structure of polymerase II.

RNA polymerase II from larvae of the brine shrimp, Artemia salina, was highly purified by two cycles of DEAE-cellulose chromatography followed by centrifugation through discontinuous sucrose gradients. Gradient fractions were subjected to elctrophoresis is polyacrylamide gels containing sodium dodecyl sulfate. The subunit structure of RNA polymerase II was determined by quantitative comparison of the polypeptides and enzyme activity present in each gradient fraction. The enzyme contains one copy of each of four subunits with estimated molecular weights of 170,000, 130,000, 36,000 and 24,000. The total molecular weight agrees well with the molecular weight estimated for the native enzyme by density gradient centrifugation.