In vitro polymerization of microtubules from HeLa cells

Although the purification of microtubules from brain by alternate cycles of polymerization and depolymerization in vitro has become routine, the application of this method to non-neural cultured cells has been less successful. Previous investigations have suggested that it was necessary to use substrate-grown cells and 4 M glycerol to obtain microtubules from cultured cells. We have developed a method for preparing microtubules from HeLa cells in spinner cultures without the use of glycerol. Microtubules can be readily carried through two complete cycles of polymerization at 37 degrees C and depolymerization at 4 degrees C in vitro. The microtubules obtained are morphologically similar to brain microtubules in electron micrographs, and the tubulin subunits have mobilities similar to those of brain tubulins on polyacrylamide gels. Typical yields in the second polymerization pellet are about 1 mg protein/ml of packed cells or 2.5-3.0% of the total protein in the soluble cell extract. The major nontubulin protein present after two cycles of polymerization and depolymerization has an apparent mol wt of 68,000 daltons. If glycerol is used during polymerization, this band is virtually absent.     

Affinity electrophoresis: New simple and general methods of preparation of affinity gels

Two simple and generally applicable methods of preparation of affinity gels for affinity electrophoresis in agarose and polyacrylamide gels are described. In the first method, amino ligands are coupled to periodate-oxidized agarose gel beads (Sepharose 4B), and homogeneous affinity gels are obtained after mixing the melted substituted beads with either melted agarose solution or with the polymerization mixture used for the preparation of polyacrylamide gels. This type of affinity gel was used for affinity electrophoresis of lectins (immobilized p-aminophenyl glycosides), ribonuclease (immobilized uridine 3′,5′-diphosphate 5′-p-aminophenyl ester), trypsin (immobilized p-aminobenzamidine), and double-stranded phage DNA fragments (immobilized acriflavine). Alternatively, heterogeneous affinity gels are prepared from the suspension of ligand-substituted agarose, dextran, or polyacrylamide gel beads in the polymerization solution normally used for preparation of polyacrylamide electrophoretic gels. This technique was used for affinity electrophoresis of lectins, ribonuclease, and trypsin on affinity gels containing appropriate ligands coupled to the gel beads “activated” by various methods. Applicability of affinity gels prepared by the two methods described above for affinity isoelectric focusing is demonstrated.     

An improved method for arylsulfatase A detection on polyacrylamide slab gels.

An improved method for the detection of arylsulfatase A on polyacrylamide slab gels has been developed using a four-step reaction sequence which leads to the formation of a stable, localized, visible product. The reaction sequence involves the formation of nitrocatechol which, in turn, reduces cupric ferricyanide to form Hatchett's brown, an insoluble brown compound. The next steps involve polymerization of 3,3′-diaminobenzidine and osmication of the polymer. Since the initial reaction product, nitrocatechol, reacts quickly with the cupric ferricyanide to form the Hatchett's brown precipitate, little diffusion occurs and the observed bands of Hatchett's brown are highly localized. The formation of the Hatchett's brown can be observed as the arylsulfatase A reaction proceeds. The final osmium-stained product is very stable and has been stored for weeks without appreciable loss of light absorption or increase in band width. Analysis of arylsulfatase A activity in human leukocyte lysates after discontinuous electrophoresis in polyacrylamide slab gels demonstrated the presence of four bands of activity.     

Poly-N-acryloyl-Tris gels as anticonvection media for electrophoresis and isoelectric focusing

We describe in detail the synthesis of an acrylic monomer, N-acryloyl-tris(hydroxy-methyl)aminomethane (NAT), which was successfully used for the preparation of gels for electrophoresis and isoelectric focusing. The polymerization kinetics and transparency of the poly(NAT) gels crosslinked by N,N'-methylenebisacrylamide (Bis) are also shown. Poly(NAT)-Bis gradient (4-24%) gel resolves proteins according to their size. The exclusion limit of this gel is slightly over 3 X 10(6), which is more than threefold higher than the exclusion limit of the polyacrylamide gradient gel of the same concentration. The gel made of 6% NAT and 3% Bis represents a suitable matrix for isoelectric focusing. These results demonstrate that poly(NAT)-Bis gels could be advantageously used in those applications where the extensive sieving by the polyacrylamide matrix is not desir desirable.     

An apparatus for the simultaneous casting of large numbers of uniform cylindrical polyacrylamide gels

An apparatus for the simultaneous casting of a large number of cylindrical polyacrylamide gels is described. Gels can be cast that are uniform with respect to length, loading-surface flatness, and internal polymerization properties. The basis of the method is casting the gels as an inverted single block which totally excludes oxygen from gel-loading surfaces during polymerization.     

A method for the quantitative determination of protein incorporated in solubilized polyacrylamide gels

Dense and light polyacrylamide gels containing N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) or N,N′-diallyltartardiamide (DATD) as the crosslinker have been tested for their solubilization properties. Both types of gel can be dissolved in 10 mm periodic acid. The time and temperature required for complete dissolution of slabs of DHEBA crosslinked gels (12 h at 50°C), however, greatly exceed those required for dissolving slabs of DATD-polyacrylamide gel (0.5 h at 22°C). Bovine serum albumin kept under the respective dissolving conditions gave a lower response in the Lowry protein assay in instances where hot periodic acid had been used. Nearly independent of the type and concentration of their constituents, the different dissolved polyacrylamide gels interfere slightly with the Lowry assay by causing some “aspecific” color development. A method is outlined enabling a reliable quantitative determination of protein incorporated in DHEBA or DATD crosslinked polyacrylamide gels.     

A method for the quantitative determination of protein incorporated in solubilizable polyacrylamide gels

Dense and light polyacrylamide gels containing N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) or N,N′-diallyltartardiamide (DATD) as the crosslinker have been tested for their solubilization properties. Both types of gel can be dissolved in 10 m periodic acid. The time and temperature required for complete dissolution of slabs of DHEBA crosslinked gels (12 h at 50°C), however, greatly exceed those required for dissolving slabs of DATD-polyacrylamide gel (0.5 h at 22°C). Bovine serum albumin kept under the respective dissolving conditions gave a lower response in the Lowry protein assay in instances where hot periodic acid had been used. Nearly independent of the type and concentration of their constituents, the different dissolved polyacrylamide gels interfere slightly with the Lowry assay by causing some “aspecific” color development. A method is outlined enabling a reliable quantitative determination of protein incorporated in DHEBA or DATD crosslinked polyacrylamide gels.     

Formation of the formate-nitrate electron transport pathway from inactive components in Escherichia coli.

When Escherichia coli was grown on medium containing 10 mM tungstate the formation of active formate dehydrogenase, nitrate reductase, and the complete formate-nitrate electron transport pathway was inhibited. Incubation of the tungstate-grown cells with 1 mM molybdate in the presence of chloramphenicol led to the rapid activation of both formate dehydrogenase and nitrate reductase, and, after a considerable lag, the complete electron transport pathway. Protein bands which corresponded to formate dehydrogenase and nitrate reductase were identified on polyacrylamide gels containing Triton X-100 after the activities were released from the membrane fraction and partially purified Cytochrome b1 was associated with the protein band corresponding to formate dehydrogenase but was not found elsewhere on the gels. When a similar fraction was prepared from cells grown on 10 mM tungstate, an inactive band corresponding to formate dehydrogenase was not observed on polyacrylamide gels; rather, a new faster migrating band was present. Cytochrome b1 was not associated with this band nor was it found anywhere else on the gels. This new band disappeared when the tungstate-grown cells were incubated with molybdate in the presence of chloramphenicol. The formate dehydrogenase activity which was formed, as well as a corresponding protein band, appeared at the original position on the gels. Cytochrome b1 was again associated with this band. The protein band which corresponded to nitrate reductase also was severely depressed in the tungstate-grown cells and a new faster migrating band appeared on the polyacrylamide gels. Upon activation of the nitrate reductase by incubation of the cells with molybdate, the new band diminished and protein reappeared at the original position. Most of the nitrate reductase activity which was formed appeared at the original position of nitrate reductase on gels although some was present at the position of the inactive band formed by tungstate-grown cells. Apparently, inactive forms of both formate dehydrogenase and nitrate reductase accumulate during growth on tungstate which are electrophoretically distinct from the active enzymes. Activation by molybdate results in molecular changes which include the reassociation of cytochrome b1 with formate dehydrogenase and restoration of both enzymes to their original electrophoretic mobilities.     

Satellite DNAs contain sequences that induced curvature

The repeating units of mouse, rat, and alpha-monkey satellites have been cloned. All three show properties that are characteristic of curved DNA: (i) their migration in polyacrylamide gels is slower than predicted from their sequences, and (ii) they appear as curved molecules when visualized by electron microscopy. All three satellite repeats contain runs of d(A.T)n greater than or equal to 3 residues that are likely to be responsible for their curvature. From analysis of 20 different satellite DNA sequences, we conclude that, in satellite DNA, adenine residues show a high tendency to cluster in groups of three or more.     

Mapping and length measurements of restriction enzyme fragments by electron microscopy.

1. We have mapped by electron microscopy the DNA-fragments formed by the action of the restriction endonuclease from Arthrobacter luteus of phi X 174 replicative form DNA. These fragments were separated by polyacrylamide gel electrophoresis and hybridized to phiX 174 single stranded DNA. The partial duplex molecules were inspected in the electron microscope. In this way the relative order of eleven fragments ranging in size from approximately 100 to 1000 nucleotide pairs has been established and compared with that deduced from reciprocal digestion studies. 2. The measured lengths of the fragments agreed well with the lengths found by gel electrophoresis. 3. The purity of the isolated fragments was checked. Most of the contaminating fragments derive from nearest neighbours in the preparative polyacrylamide gels.     

Electrophoretic separation and immunological identification of type 2X myosin heavy chain in rat skeletal muscle

One slow and three fast myosin heavy chains have been described in typical skeletal muscles of the adult rat using immunocytochemical analysis. Electrophoretic isolation and immunochemical identification of these four isoforms has not been achieved. An electrophoretic procedure is described which, by altering the cross-linkage and polymerization kinetics of 5% polyacrylamide gels, allows resolution of these four distinct myosin heavy chains. Using specific monoclonal antibodies and double immunoblotting analysis, the identity and electrophoretic migration order of the myosin heavy chains was established to be: 2A less than 2X less than 2B less than beta/slow.     

Identification of the major enzymic activities of the mitochondrial inner membrane in terms of their migration in sodium dodecyl sulfate polyacrylamide gel electrophoresis

An attempt has been made to identify the large number of components separated in sodium dodecyl sulfate polyacrylamide gels of a mitochondrial inner membrane preparation with the enzymic activities they represent. Individual enzymes and enzyme complexes (electron transfer and the ATP synthesizing and hydrolyzing complexes) were purified and coelectrophoresed with the inner membrane preparation on 10% polyacrylamide gels. Different bands in the densitometric profile of the inner membrane preparation were thus identified with one or more components of one or more of the electron transfer or ATP-synthesizing and -hydrolyzing complexes. Only one major component (band 14), of molecular weight 29,000, was not represented in any of these complexes. It is a hydrophobic protein of as yet unknown function.     

Characterization of a proteolytic-enzyme inhibitor with allergenic activity. Multiple functions of a parasite-derived protein.

1. A trypsin inhibitor from the tick Boophilus microplus was purified by ion-exchange chromatography and gel filtration. 2. It is pure by the criteria of constant specific activity on gel filtration and by electrophoresis on polyacrylamide gels containing sodium dodecyl sulphate. 3. The protein undergoes reversible polymerization, dissociating at low pH. 4. The apparent molecular weight measured by electrophoresis on polyacrylamide gels containing sodium dodecyl sulphate is 18,500. 5. Inhibition of trypsin occurs by formation of a 1 :1 molar complex. 6. Chymotrypsin is also inhibited, though the dissociation constant of the complex formed is larger than with trypsin. The protein possesses independent sites for the inhibition of chymotrypsin and trypsin. 7. The inhibitor preparation gives an immediate hypersensitivity reaction on intradermal injection into cattle that have been exposed to the tick. The allergenic activity is due to the inhibitor protein itself and not to contaminating material, since the two activities were not separated during purification or in two subsequent affinity-chromatography procedures. 8. The hypersensitivity reaction is a true immunological response, since it is found in almost all cattle that have been exposed to the tick, but not in unexposed animals. In addition, passive cutaneous anaphylaxis can be demonstrated with serum from exposed, but not from unexposed, animals.     

The prevention of distortion in ultrathin-layer polyacrylamide gel isoelectric focusing

Although isoelectric focusing patterns in ultrathin layers of polyacrylamide gel are distorted by the presence of salts, such as ammonium persulfate, this reagent is commonly used to promote polymerization of the gel. The amount of ammonium persulfate can be reduced but this causes the formation of sloppy gels or incomplete polymerization. A method is described here in which an ultrathin polyacrylamide gel is formed on a polyester sheet using ammonium persulfate in the absence of ampholyte. After complete polymerization, the ammonium persulfate is washed out and ampholyte is allowed to diffuse into the gel. Subsequent isoelectric focusing is then free from distortion caused by the presence of ammonium persulfate.     

Genetic variants detected among 106 lymphocyte polypeptides observed in two-dimensional gels.

Peripheral blood lymphocytes from 40 children have been examined for genetic variation in their protein constituents by two-dimensional polyacrylamide gel electrophoresis. One hundred six polypeptides chosen without respect to genetic variability were scored in gels from the 40 children. For each child, gels from both parents were also examined to substantiate the genetic basis of variants observed. Of the total of 4,240 polypeptides, 23 could not be scored unambiguously. Fourteen of the polypeptides showed genetic variants in one or more of the children. One hundred twenty-nine of 4,217 polypeptides scored exhibited the combination of a normal and a variant polypeptide. All variants were present in at least one of the parents of the subject. The index of heterozygosity observed (3.05% +/- .23%) indicates substantial genetic variation in cellular protein constituents.     

Use of immobilized cells of Rhizopus nigricans for the 11α-hydroxylation of progesterone

The filamentous fungus, Rhizopus nigricans, was immobilized in polyacrylamide, alginate, and agar gels and its ability to 11α-hydroxylate progesterone was examined. No activity was detected using polyacrylamide gel but both agar and alginate gels have proved capable of hydroxylation. Agar gels displayed faster rates and higher yields. It was possible to induce hydroxylase synthesis within agar and alginate gels, and microscopical examination provided evidence for hyphal growth within these gels. The concept of increased biomass was used to explain the observed increase in the rates of hydroxylase activity of the immobilized cells. Conversely, hyphal overcrowding was postulated for the rapid inactivation observed under some operating conditions.     

A sensitive polyacrylamide disc gel method for detection of proteinases

To enable direct detection of proteinase activities subsequent to electrophoresis, a technique utilizing the incorporation or diffusion of protein substrates into polyacrylamide disc gels was developed. Denatured insoluble substrates, casein or hemoglobin, were added to acrylamide solutions prior to polymerization of the gel mixture. Alternatively, soluble protein substrates were diffused into gels after electrophoresis. In either case, an incubation period ensued at the pH optimum of the proteinases to allow for their detection. Classification of resolved proteinases was accomplished subsequent to electrophoresis by incubation of gels in media containing either synthetic substrates, as the naphthylamide derivatives, or specific inhibitors of the enzymes. Separation of purified trypsin from chymotrypsin, and proteinases in preparations of seminal plasma and mouse blastocysts homogenates demonstrated the efficacy of the method at the submicrogram enzyme level.     

On the Identification of α- and β-Tubulin Subunits

Abstract: Confusion appears to have arisen in the literature regarding the designation of α-and β-tubulin in polyacrylamide gels. The presence or absence of 8 M-urea in sodium dodecyl sulfate (SDS) polyacrylamide gels leads to different patterns for unalkylated tubulin subunits (and other proteins), making difficult the designation of the α and β subunits by original definition using electrophoretic mobility in the molecular weight dimension. The specific biochemical property of posttranslational tyrosylation of the α subunit has been used to identify further this subunit. Under all conditions tested, the β subunit has been found to be more acidic than the α subunit, with isoelectric point differences that agree with theoretical and published values. If the tubulin subunits are reduced and alkylated, the β subunit migrates more rapidly in SDS polyacrylamide gels, with or without urea present. However, unalkylated tubulin subunits can comigrate or even reverse their relative mobility if 8 M-urea-SDS polyacrylamide gels are used for subunit separation. The results also confirm the earlier reports that the post-translational tyrosylation of protein appears exclusively restricted to α-tubulin and can be demonstrated in an in vivo situation. In addition, the results suggest that only the α₂ subunit of tubulin is tyrosylated.     

Improving immobilized biocatalysts by gel phase polymerization

A new method is presented for the treatment of gel-type supports, used for immobilizing microbial cells and enzymes, to obtain high mechanical strength. It is particularly useful for ethanol fermentation over gel beads containing immobilized viable cells, where the beads can be ruptured by gas production and the growth of cells within the gels. This method consists of treating agar or carrageenan gel with polyacrylamide to form a rigid support which retains the high catalytic activity characteristic of the untreated biocatalysts. The size and shape of the biocatalyst is unaffected by this treatment. The method involves the diffusion of acrylamide, N,N'-methylenebisacrylamide and beta-dimethylaminopropionitrile (or N,N,N',N'-tetramethyl-ethylenediamine) into the performed biocatalyst beads followed by the addition of an initiator to cause polymerization within the beads. Treated gels have been used for the continuous fermentation of glucose to ethanol in a packed column for over two months. During this operation, the gel beads maintained their rigidity, and the maximum productivity was as high as 50 g h(-1) L(-1) gel. There was no appreciable decay of cell activity.     

Photopolymerization with EDTA and Riboflavin for Proteins Analysis in Polyacrylamide Gel Electrophoresis

The Protein Journal - A new method for photosensitized polymerization of polyacrylamide gels was proposed. Photopolymerization of acrylamide/N,N′-methylenebisacrylamide (AM/Bis) was assisted...