A purified nucleoprotein fragment of the 30 S ribosomal subunit of Escherichia coli.

A '13 S' nucleoprotein fragment was isolated from a nuclease digest of Escherichia coli 30-S ribosomal subunits and purified to gel electrophoretic homogeneity. It contained two polynucleotides, of about 1.1 . 10(5) and 2.5 . 10(4) daltons, which separated when the fragment was deproteinized. The major protein components were S4, S7 and S9/11, with S15, S16, S18, S19 and S20 present in reduced amount.     

Purification of rabbit and human serum paraoxonase.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.     

Purification of membrane proteins from Acholeplasma laidlawii by agarose suspension electrophoresis in Tween 20 and polyacrylamide and dextran gel electrophoresis in detergent-free media.

Four of the membrane proteins from Acholeplasma laidlawii that are soluble in the nonionic detergent Tween 20 have been purified by preparative electrophoretic techniques utilizing different supporting media. The last purification step for two of the major proteins was a preparative polyacrylamide gel electrophoresis performed in the absence of any detergent. The proteins were recovered by continuous elution. The purity of the fractions was examined by analytical polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. Two of the minor proteins were purified by dextran gel electrophoresis as the final step, which was also performed in a detergent-free buffer. The separation was followed by scanning the dextran gel in ultraviolet light. The proteins were recovered by slicing the gel and degrading the gel slices with dextranase. The homogeneity of the fractions was checked by electroimmunoassay.     

S1 nuclease as a probe of yeast ribosomal 5 S RNA conformation.

5 S RNA was isolated from Saccharomyces cerevisiae grown in the presence of 32P-phosphate and digested with nuclease S1, a single-strand specific nuclease. Two different procedures were employed to determine the sites of attack on the RNA. First, 5 S RNA was isolated from nuclease S1 digests, digested to completion with ribonuclease T1, and then 'fingerprinted' by two-dimensional electrophoresis. Quantitation of each of the characteristic RNAase T1-derived oligonucleotides was employed to determine the relative susceptibility of various regions of the molecule to nuclease S1. A second procedure to define nuclease S1-susceptible sites in the molecule employed polyacrylamide gel electrophoretic fractionation of nuclease S1 digests followed by identification of the nucleotide sequences of the released RNA fragments. Both procedures showed that the region of the molecule between residues 9 and 60 was most susceptible to nuclease S1, with preferential cleavage occurring between residues 12-25 and 50-60. These results are discussed in relation to a proposed model for the secondary structure of yeast 5 S RNA.     

Isolation, characterization, and activation of the magnesium dependent endodeoxyribonuclease from Bacillus subtilis.

A major endodeoxyribonulcease was isolated from a mutant of the transformable Bacillus subtilis 168. The magnesium-dependent endonuclease was purified approximately 750-fold to electrophoretic homogeneity. The enzyme had a molecular weight of about 31 000, as determined by gel filtration and polyacrylamide gel electrophoresis. The protein appears to be composed of two subunits. The nuclease was dependent on magnesium or maganese ions for hydrolytic activity. The purified nuclease degraded DNA from several species of Bacillus, as well as Escherichia coli DNA, alkylated, depurinated, and thymine-dimer containing B. subtilis DNA, and hydroxymethyluracil-containing phage DNA. The enzyme also hydrolyzed single-stranded DNA, although native DNA was the preferred substrate. However, the nuclease was unable to degrade ribosomal RNA. The cleavage products of the DNA hydrolysis have 5'-phosphate and 3'-hydroxyl ends. The enzyme could be activated in crude extracts by heat treatment or treatment with guanidine hydrochloride. The nuclease activity was inhibited by phosphate and by high concentrations of NaCl. A possible function for this endonuclease in bacterial transformation is discussed.     

Isolation of Two Acetyl Esterases from Extracts of Bacillus subtilis

Acrylamide gel electrophoresis of crude cellular extracts of Bacillus subtilis revealed the presence of two acetyl esterases. Esterase A, the slower migrating enzyme, was found to be present in both vegetative and sporulating cells, whereas esterase B activity was more abundant after exponential growth ceased. Both esterases were present in the supernatant fraction of lysed spheroplasts and in a disrupted spore preparation. Of four pleiotropic asporogenous mutants tested, three exhibited decreased esterase B activity. Esterases A and B were partially purified by differential precipitation and co-chromatographed on diethylaminoethyl (DEAE)-cellulose (pH 7.5) and DEAE-Sephadex (pH 8.5). By employing gel filtration chromatography, the two esterases were separated, and molecular weights of 160,000 and 51,000 were estimated for esterases A and B, respectively. Esterase A was further purified to electrophoretic homogeneity by differential heating and preparative starch block electrophoresis. Sodium dodecyl sulfate-acrylamide gel electrophoresis of purified esterase A yielded a single protein band with a molecular weight of 31,000. The pI values of esterases A and B were determined to be 6.4 and 5.4, respectively.     

Similarity in structure and function of the 3′-terminal region of the four brome mosaic viral RNAs

A 3′-terminal fragment, about 160 nucleotides long, was cleaved by limited nuclease digestion from each of the four RNA components of brome mosaic virus, and purified by two cycles of gel electrophoresis. These fragments accepted tyrosine in reactions catalyzed by wheat germ aminoacyl-tRNA synthetase. Analyses of nuclease digests suggested that the sequences of the fragments from brome mosaic virus RNA 3 and 4 were identical and that the fragments from RNA 1 and 2 differed from that of RNA 4 only in the positions of two and one nucleotides, respectively. A fragment isolated in a similar way from cowpea chlorotic mottle virus was similar in size to the brome mosaic virus RNA fragments, accepted tyrosine in the presence of wheat germ aminoacyl-tRNA synthetase, but had a substantially different nucleotide sequence.     

Purification of larvicidal protein from Bacillus sphaericus 1593

Coat proteins from the spores of Bacillus sphaericus 1593 were separated by preparative polyacrylamide gel electrophoresis. Neutralising antibodies were raised against a single protein band exhibiting toxicity to mosquito larvae. IgG was purified and coupled to CNBr-activated Sepharose 4B to be used as an immunoaffinity matrix. The larvicidal protein was purified to electrophoretic homogeneity using this immunoaffinity column. The single protein species resolved into four peptides of molecular weights 42.6, 44.1, 50.7 and 51.3 KDa on polyacrylamide gel electrophoresis under denaturing conditions. This protein contained 12% carbohydrates. The purified protein exhibited an LC50 value of 8.3 +/- 1.6 ng/ml when tested against early third instar larvae of the mosquito Culex pipiens var quinquefasciatus.     

Immunochemical reactivity of simian sarcoma virus polypeptides isolated by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis

The polypeptides associated with a zonal centrifugation purified simian sarcoma virus propagated in lymphoblastoid NC-37 cells were isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) using a procedure designed to minimize the loss of immunochemical reactivity. The proteins p10, p15, p28, p36, p44, p75, and p86 were obtained in large yield and high degree of homogeneity. The electrophoretically purified p28 was analyzed by competition radioimmunoassay using antiserum to a pore exclusion and ion exchange purified simian sarcoma virus p28. Complete competition was observed with extracts of simian sarcoma virus infected cells. No competition was observed with uninfected or unrelated, infected cell extracts. The antigen-antibody affinity as measured by the slope of the competition curve using antiserum to p28 and 125I-labeled and electrophoretically purified p28 was the same as that for the p28 released from sonication-disrupted simian sarcoma virus. The data indicates that preparative purifications by polyacrylamide gel electrophoresis in the presence of SDS may be generally applicable for the isolation of proteins with essentially the same immunospecificities and affinity for a specific antiserum as proteins isolated by procedures that avoid the use of SDS and electrophoresis.     

Solubilization, isolation, and immunochemical characterization of the major outer membrane protein from Rhodopseudomonas sphaeroides

Solubilization of the major outer membrane protein of Rhodopseudomonas sphaeroides, and subsequent isolation, has been achieved by both non-detergent- and detergent-based methods. The protein was differentially solubilized from other outer membrane proteins in 5 M guanidine thiocyanate which was exchanged by dialysis for 7 M urea. The urea-soluble protein was purified to homogeneity by a combination of DEAE-Sephadex chromatography and preparative electrophoretic techniques. Similar to the peptidoglycan-associated proteins of other Gram-negative bacteria, the protein was also purified by differential temperature extraction of the outer membrane in the presence of sodium dodecyl sulfate (SDS) followed by preparative SDS-polyacrylamide gel electrophoresis. Immunochemical analysis of the proteins isolated by the two techniques established the immunochemical identity and homogeneity of each preparation. Immunoblots of SDS-polyacrylamide gels revealed that antibody directed against the major outer membrane protein reacted with the three high molecular weight aggregates present in the outer membrane which we have previously shown to be composed of the major outer membrane protein and three nonidentical small molecular weight proteins.     

Isolation and purification of a neurodepressing hormone from the eyestalk of Procambarus bouvieri (Ortmann).

1. A neurodepressing hormone has been isolated and purified to homogeneity from aqueous extracts of 2000 eyestalks of the Mexican crayfish Procambarus bouvieri (Ortmann). 2. Purification was achieved by gel filtration on Sephadex G-25 and G-15, and preparative paper electrophoresis at four pH valueimately 1200 molecular weight and composed of neutral amino acids. 5. No N-terminal group could be found. From its electrophoretic behavior it is concluded that the C-terminal group is also blocked.     

Purification and Characterization of Clathrin from Bovine Brain

Abstract: Clathrin has been purified to electrophoretic homogeneity by initial extraction of clathrin from purified coated vesicle fraction, followed by column chromatographies with gel filtration. DEAE-cellulose, and hydroxylapatite and finally by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Antibody specific to clathrin has also been obtained. Two forms of native clathrin, fast and slow components, have been prepared to about 95% purity by hydroxylapatite column chromatography. Both fast and slow components are believed to represent two different aggregates of clathrin subunit because they comigrate in agarose electrophoresis. pH 7.4, and also migrate as clathrin subunit on SDS-PAGE with a molecular weight of 175,000. Furthermore, both components cross-react with antibody against purified clathrin and compete for antibody binding site with labeled fast component. The fast component can also be converted to the slow component. In addition to clathrin, two proteins of about 38,000 and 35,000 M.W. that consistently co-purified with native clathrin are probably also intrinsic to coated vesicle.     

Sequence of a specifically encapsidated RNA fragment originating from the tobacco-mosaic-virus coat-protein cistron.

When 25-S tobacco mosaic virus (TMV) protein aggregate and TMV RNA, which has been partially digested by T1 RNase, are mixed under conditions suitable for reconstitution, only a few RNA fragments are encapsidated. These fragments were isolated and purified by polyacrylamide gel electrophoresis. The sequence of the three main fragments, the longest of which (fragment 1) was estimated to contain 103 nucleotides, has been determined. The two smaller fragments are portions of the longer chain produced by an additional specific scission. Because of the great affinity of 25-S TMV protein for this nucleotide sequence, it will be referred to as the "specifically encapsidated RNA fragment". The occurrence of a "hidden break" in the sequence has been demonstrated: fragment 1, purified by electrophoresis on a polyacrylamide gel without 8 M urea, gives rise upon further electroporesis in the presence of urea to two new bands corresponding to the two halves of the molecule. A stable hair-pin secondary structure has been derived from the base sequence which can account for the specificity of action of the enzyme. Because of its properties, we have suggested elsewhere that the sequence of fragment 1 might correspond to the disk recognition site for reconstitution, which is known to be located at the 5' end of the intact RNA. But experiments with TMV RNA whose 5'-OH end has been radioactively phosphorylated with polynucleotide kinase show that this is not the case. Analysis of the amino acid coding capacity of the fragment has instead revealed that fragment 1 is a portion of the TMV coat protein cistron.     

Methods for the Rapid Purification of Trehalase from Dictyostelium Discoideum

A rapid and reliable method for the preparation of homogeneous trehalase from the cellular slime mold, Dictyostelium discoideum for usage in enzyme characterization studies and trehalose assays was developed. This procedure takes advantage of the fact that trehalase activity is secreted by Dictyostelium during the course of development, the major fraction being released late in fruiting body formation. Purification of trehalase to electrophoretic homogeneity was accomplished utilizing the techniques of ultrafiltration, streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephacel chromatography and preparative disc gel electrophoresis. Analysis of the purified enzyme by analytical polyacrylamide disc gel electrophoresis demonstrated the presence of a single protein band which was stainable with Coomassie blue. Assay of trehalase activity in eluates from segments of a companion gel indicated that all of the recovered trehalase activity was associated with this band of protein. Examination of the substrate specificity of the purified enzyme indicated absolute specificity for trehalose.     

Purification and characterization of B-protein from human serum

B-Protein, present in the serum of individuals with cancer, has been purified to electrophoretic homogeneity. The purification procedure consisted of chromatography on Sephacryl S-200, Affi-Gel Blue, Con A--Sepharose 4B, wheat germ lectin--Sepharose and preparative polyacrylamide gel electrophoresis. The molecular weight of B-Protein is estimated to be 100 000 to 120 000. It is a glycoprotein which appears to be composed of two subunits, each with a molecular weight of approximately 52 000. Analytical polyacrylamide gel electrophoresis and analytical ultracentrifugation data indicate that purified B-Protein is homogeneous. Isoelectric focusing studies also show the purified B-Protein to be homogeneous in composition consisting of a single band of pI = 4.8. Amino acid analysis is consistent with this acidic isoelectric point. Other analyses indicate that B-Protein contains 7% carbohydrate and 7% lipid in the form of triglycerides.     

Isolation of human platelet and red blood cell plasma membrane proteins by preparative detergent electrophoresis

High resolution polyacrylamide gel electrophoretic techniques have been applied to the preparative isolation and analysis of plasma membrane proteins and glycoproteins from human platelets and red blood cells. The techniques presented allow relatively simple, direct, rapid and quantitative purification of a broad molecular weight range of membrane proteins, by means of continuous elution preparative gel electrophoresis of proteins solubilized with sodium dodecyl sulfate. Spectrophotometric and fluorophotometric (fluorescamine) profiling, and high resolution gel electrophoretic analysis (SDS-acrylamide gradient slab gels, and gel electrofocusing) of eluted protein species indicate that purified membrane proteins of a broad molecular weight range may be obtained in a one step procedure, and in quantities and concentrations sufficient for further analytical or experimental procedures.     

Isolation of human platelet and red blood cell plasma membrane proteins by preparative detergent electrophoresis.

High resolution polyacrylamide gel electrophoretic techniques have been applied to the preparative isolation and analysis of plasma membrane proteins and glycoproteins from human platelets and red blood cells. The techniques presented allow relatively simple, direct, rapid and quantitative purification of a broad molecular weight range of membrane proteins, by means of continuous elution preparative gel electrophoresis of protein solubilized with sodium dodecyl sulfate. Spectrophotometric and fluorophotometric (fluorescamine) profiling, and high resolution gel electrophoretic analysis (SDS-acrylamide gradient slab gels, and gel electrofocusing) of eluted protein species indicate that purified membrane proteins of a broad molecular weight range may be obtained in a one step procedure, and in quantities and concentrations sufficient for further analytical or experimental procedures.     

Analysis of polyoma virus DNA replicative intermediates by agarose gel electrophoresis.

Agarose gel electrophoresis has been used to fractionate polyoma virus DNA replicative intermediates (RI) according to maturity. Approximate electrophoretic mobility versus maturity relationships were obtained for both intact (supercoiled) and nicked (relaxed) RI. There was considerable overlap between the supercoiled and relaxed RI populations after electrophoretic fractionation. Intact RI could be recovered from preparative agarose gels for further analysis by centrifugation, electron microscopy, re-electrophoresis, or nuclease digestion.     

Purification from Cephalosporium acremonium of the initial enzyme unique to the biosynthesis of penicillins and cephalosporins

The stability of the unstable enzyme, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase from Cephalosporium acremonium C-10, was increased 10-fold which facilitated its purification. The active enzyme was purified over 100 fold to electrophoretic homogeneity by protamine sulfate treatment, ammonium sulfate fractionation, gel filtration and hydrophobic interaction chromatography. It appears to have a minimal size of 360 kDa based on SDS-polyacrylamide gel electrophoresis.     

Isolation and characterization of highly purified alpha-1-antitrypsin.

Highly purified human alpha-1-antitrypsin (phenotype MM) was obtained by an original method of preparative electrophoresis. The criteria of homogeneity were assured by one arc in crossed immunoelectrophoresis and one band on polyacrylamide gel. A unique N-terminal amino acid (pyroglutamic acid) and a unique C-terminal residue (lysine) were identified. Determined by gel electrophoresis, its molecular weight was 47,000 daltons.