Cascade stacking and cascade electrofocusing: their interconversion and fundamental unity.

The composition of a stack [an isotachophoresis (ITP) system] containing multiple trailing buffer constituents (“cascade stack”) was computed using a modification of the program of Routs. On electrophoresis, such a buffer mixture gave rise to multiple moving boundaries in which either buffer constituents or proteins could be stacked. Buffer zones within the stack served as “spacers.” The cascade stack exhibits a pH gradient, sharp zone boundaries, and constant zone width irrespective of the duration of electrophoresis, just as in the case of a stack comprising a single leading and trailing constituent. The pH gradient, sharp zone boundaries, and the sequential order of protein zones were maintained when the cascade stack was transposed between strongly acidic and basic electrolytes. Such a transposed isotachophoretic gel functioned as an electrofocusing system, indistinguishable from electrofocusing gels made in either buffers (buffer electrofocusing, or BEF) or Ampholine (isoelectric focusing, or IF). In the converse experiment, a cascade electrofocusing gel, formed in the same buffer mixture used to form a cascade stack, was subjected to electrofocusing until the steady-state was attained and then it was transposed between the appropriate upper and lower buffers of the corresponding cascade stacking system. Such transposition gave rise to moving zones with the typically sharp boundaries of a stack, a transient state pH gradient, and an order of protein zones within the cascade stack identical to the cascade electrofocusing system. These studies indicate the essential physical-chemical identity between these two types of electrophoretic systems and indicate the need for continued development of a unified theory for isoelectric focusing and steady-state stacking (isotachophoresis).     

The use of poly(2-acrylamido-2-methyl-1-propanesulfonic acid) polymers as spacers for isotachophoresis in sieving gel matrices

The electric field strength gradients generated in isotachophoresis (ITP) may be used for the separation of biomolecules. Poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (polyAMPS) polymers of a uniform distribution of molecular mass were synthesized and used as novel spacers in ITP. Since these polymeric spacers are strongly acidic species, their ionic charges remain constant over a wide pH range, so that their ionic mobilities are governed solely by their molecular masses and not by the pH of the milieu. A modification of ITP known as telescope electrophoresis was used to separate a number of acidic dyes of varying ionic mobility, using polyAMPS polymers as spacers. The resolution obtained was superior to that obtained by polyacrylamide gel electrophoresis (PAGE), due to the focusing effect of the electric field strength gradient. Since these novel polymeric spacers are designed to operate within sieving medium, it was decided to test their suitability for the separation of DNA molecules. DNA molecules up to 1000 bp long were successfully resolved, with a similar resolution to that obtained with conventional PAGE.     

Recovery of proteins on a milligram scale from polyacrylamide electrophoresis gels,exemplified by purification of a retinol-binding protein

An apparatus suitable for the recovery of proteins from polyacrylamide gels on a milligram scale by displacement electrophoresis (isotachophoresis) is described along with a buffer system that is suitable for this purpose with most proteins. The technique is illustrated by the recovery of a protein from a 15% polyacrylamide gel. The recovery was almost quantitative and the eluted protein showed little contamination upon quantitative amino acid analysis and automatic Edman degradation.     

Improved polyacrylamide gel electrophoresis with different amino acids as the trailing constituent

Using a polyacrylamide disc gel electrophoretic system similar to that described by J. T. Clarke (1964, Ann. N. Y. Acad. Sci.121, 428–436), we have achieved an improved separation of hemoglobins from Rana catesbeiana tadpoles by substituting one of several amino acids in the place of glycine in the electrode chamber buffer. The relative migrations (R_f) and degree of separation of these similar hemoglobins are proportional to the pK′ of the α-amino group of the amino acid used in the buffer. Specifically, for these proteins, log (R_f × 100) was found to be directly proportional to the pK′₂ of the amino acid divided by the volume conductivity (specific conductance) of the electrode chamber buffer. For example, improved separation of these hemoglobins in short electrophoretic times can be achieved, at low cost, by using dl-alanine instead of glycine in the buffer. Improved separation of other proteins which migrate at basic pH might be achieved by a similar approach.     

Nonisoelectric focusing in buffers.

Stable pH gradients were formed and focusing of proteins was carried out in polyacrylamide gels containing mixtures of simple, amphoteric buffers, replacing the Ampholine hitherto used in isoelectric focusing (IF). Stable pH gradients can also be formed between acid anolyte and basic catholyte if Ampholine is replaced by nonamphoteric buffers. The fact that focusing can be carried out with nonampholytes shows that focusing in this case is, and in all other cases may be, nonisoelectric. It is postulated that the pH gradient in IF forms by steady-state stacking (isotachophoresis) and forms within the stack. In distinction to ordinary steady-state stacking, however, the stack remains confined within the gel (or density gradient) since the strong acid and base in the electrolyte reservoirs bar by deprotonation or electrostatic repulsion migration into the electrode chambers.     

A new discontinuous buffer system for the electrophoresis of cationic proteins at near-neutral pH

A simple, discontinuous buffer system for polyacrylamide gel electrophoresis near neutral pH is described. The buffer is MOPS (3-[N-morpholine]propanesulfonic acid), the leading ion K⁺ and the trailing ion histidine. The system offers improved resolution of cationic proteins.     

A re-evaluation of the surface complexity of the intact erythrocyte

Surface proteins and glycoproteins of intact human red blood cells were labelled with ¹²⁵I by the lactoperoxidase method. The radioactive proteins were then separated in each of the Fairbanks and Laemmli one-dimensional polyacrylamide gel electrophoresis systems. The radioactive polypeptides had different mobilities in the two systems, largely due to the anomalous migration of glycoproteins in polyacrylamide gels. A two-dimensional system was therefore developed using the Fairbanks and Laemmli buffer systems to exploit these anomalies. This procedure clearly resolved radioactive glycoproteins and proteins and enabled the identification of many more surface components than had previously proved possible.     

Electrophoretic separation of histones and high-mobility-group proteins on acid-urea-Triton gels

The electrophoretic mobilities of calf thymus histones and high-mobility-group (HMG) nonhistone proteins were studied on a newly modified polyacrylamide gel containing acetic acid, urea, and the nonionic detergent Triton X-100 in combination with glycine in the electrode buffer. This gel system avoids stacking gel, photopolymerization of acrylamide, and preelectrophoresis. Under extremely low Triton concentrations some H3 variant forms (H3.1) were preferentially separated by their slower migration from bulk H3. Under increasing concentrations of Triton in the gel in the presence of 3 or 6 M urea, the mobilities of H2A.1, H3.2, H2A.2, H4, and H2B were sequentially retarded. The mobilities of H1 and HMGs remained virtually unchanged under all conditions. This gel system is able to resolve charge-modified histones.     

Gel electrophoretic isolation, in the hundred microgram range, of recombinant SDS-syntaxin from sea urchin egg cortical vesicles.

Recombinant urchin syntaxin [Xa cut], electrophoresed at pH 9.0 (25 degrees C) or 10.2 (0 degrees C) in a discontinuous Tris-chloride-glycinate buffer system in the presence of 0.03% SDS in the catholyte, exhibits a multicomponent pattern in gels of a polyacrylamide concentration of 12% and 3% crosslinking. The position in the pattern of the syntaxin band was identified by reference to electropherograms of a previous study (P. Backlund, pers. comm.). The complexity of the protein composition of the preparation was reduced by selective stacking of proteins with mobilities greater than that of syntaxin. This provides a gel pattern consisting of two bands with mobilities close to that identified as syntaxin, as well as a minor, more slowly migrating, contaminant. The two major components are designated as S1 and S2, the latter being the larger species. In the absence of SDS, the preparation exhibits two pairs of protein components. Three of the proteins are charge isomers, i.e., of equal size, differing only in net charge, assumed to be forms of S1, while the fourth component is larger and is assumed to be S2. Aliquots of the preparation, containing 150 microg of protein were loaded on a cylindrical polyacrylamide gel of 18 mm diameter, and separated S1 and S2 were excised in a position defined by their characteristic values of relative mobility (Rf). Two or three gel slices, corresponding in Rf to S1 or S2, were pooled and loaded onto a Stacking Gel (5% polyacrylamide, 20% cross-linked) of 18 mm diameter, equipped with a collection chamber of 200 microL volume. The protein was electroeluted from the gel slices and concentrated into a stack by electrophoresis. The stack, marked by bromphenolblue, was allowed to migrate into the collection chamber, was collected and analyzed by protein assay and re-electrophoresis. Re-electrophoresis of S1 shows that it consists of at least three components. Recovered S1 constitutes 47% of the preparation, based on protein assay, S2 4%. S1, isolated from SDS-PAGE, exhibits an apparent Mr of 22.7 kDa, S2 one of 34.5 kDa, similar to the value of 32.6 kDa expected from the structure of syntaxin. The absence of S2 from the electroeluate re-electrophoresed at 0 degrees C and their molecular weight relationship suggest a proteolytic transformation of S2 to S1.     

Use of benzyldimethyl-n-hexadecylammonium chloride (“16-BAC”), a cationic detergent,in an acidic polyacrylamide gel electrophoresis system to detect base labile protein methylation in intact cells

A discontinuous polyacrylamide gel system operating at pH 4.0–1.5 which resolves proteins bearing base labile groups extracted from intact cells is described. It uses potassium phosphate buffer in the running and stacking gel and glycine as the trailing ion component. Proteins are solubilized with urea and benzyldimethyl-n-hexadecylammonium chloride, a cationic detergent. The utility of the system is illustrated by fluorographs of the pattern of protein methylation in blood platelets and the HL60 promyelocyte cell line.     

Hemoglobin components and plasma proteins in Pitymys duodecimcostatus

Six different hemoglobins have been demonstrated by polyacrylamide disc gel electrophoresis in a species of vole Pitymys duodecimcostatus. Plasma proteins of the Pitymys duodecimcostatus were analyzed by polyacrylamide disc gel electrophoresis and there was no significant difference with the electrophoretic patterns of the rat (Rattus norvegicus). The hematological values, hematocrit, hemoglobin concentration and erythrocyte number have been analyzed.     

Inhibitor-“resistant” labelling of rat ventral prostate nuclear proteins : Further evidence consistent with protein synthesis associated with cell nuclei

When minced rat ventral prostate was incubated with labelled amino acids and cycloheximide or puromycin, the specific radioactivity of proteins associated with Triton X 100-washed nuclei exceeded that of the 105 000 g cytosol. The distribution of radioactive proteins from incubated mince, examined by SDS polyacrylamide gel electrophoresis was also consistent with labelling of some nuclear proteins that was resistant to inhibitors. Highly purified prostate nuclei, washed with detergent, labelled proteins of from 1–6 × 10⁴ D with radioactive amino acids. When these proteins were fractionated according to solubility, NaOH-soluble ‘acidic’ proteins, examined by SDS polyacrylamide gel electrophoresis, were highly labelled, with a distribution of radioactivity that differed from the patterns of 0.4 N H₂SO₄-soluble basic proteins (including histones), and proteins soluble in Krebs-Ringer-phosphate buffer. Although these results cannot be interpreted unambiguously, they are consistent with the synthesis of certain nuclear proteins at a site(s) sequestered from cycloheximide and puromycin. Nuclei may represent one such site.     

Molecular-sieve chromatography and electrophoresis in polyacrylamide gels

1. The absolute electrophoretic mobilities of eight proteins have been measured at pH8.76, I 0.05, in polyacrylamide gels of 20 different compositions at 10 degrees C. 2. The partition coefficients of these proteins have been determined chromatographically under the same conditions by using columns of granulated polyacrylamide gel prepared simultaneously. 3. The electrophoretic mobilities are an exponential function of the gel concentrations when the latter are corrected for water uptake. The constants of this function have been determined by curvefitting methods. They have been shown to be related to the free solution mobility and to the mean molecular radius respectively. 4. The reduced mobilities have been shown to be a linear function of the partition coefficients by statistical analyses. 5. The physical significance of the relation between electrophoretic mobility and chromatographic phase distribution in gel media is discussed in the context of these results.     

Comparison of non-histone proteins from hamster Kirkman-Robbins hepatoma and liver

Three classes of non-histone proteins were obtained from hamster Kirkman-Robbins hepatoma and liver nuclei following separation of nucleic acids with the polyethylene glycol-dextran mixture and fractionation of nuclear proteins on hydroxylapatite in a salt-glycerol-phenylmethylsulphonyl fluoride system at increasing concentrations of Na+ and K+ phosphate buffer, pH 6.8. Two-dimensional polyacrylamide gel electrophoresis of these proteins documented their high heterogeneity; many spots were common but some spots specific only for neoplastic or normal tissue were also observed.     

Fractionation of Jerusalem artichoke phenolase by immobilized copper affinity chromatography

Phenolase from tubers of Jerusalem artichoke was fractionated by metal chelate affinity chromatography using copper conjugated to iminodiacetic acid Sepharose 6B. Four fractions obtained after chromatography showed various specific activities, with an increase in activity of 160-fold for the first unbound enzymatic fraction, and 18-fold for a fraction eluted with glycine buffer. Electrophoresis of phenolase fractions in gradient polyacrylamide gel resulted in a pattern consisting of three major groups of bands differing in relative mobilities.     

Determination of the molecular weight of proteins by electrophoresis in slab gels with a transverse pore gradient of crosslinked polyacrylamide in the absence of denaturing agents

The molecular weight of proteins under nondenaturing conditions can be determined through polyacrylamide electrophoresis by comparing their relative mobilities at different gel concentrations with the relative mobilities of standard proteins under the same conditions (J. L. Hedrick and A. J. Smith (1968) Arch. Biochem. Biophys. 126, 155). This work describes a procedure that eliminates the need for several gels of different acrylamide concentrations with the use of a slab gel with a transverse pore gradient of crosslinked polyacrylamide.     

Cys2/His2 zinc-finger protein family of petunia: evolution and general mechanism of target-sequence recognition.

The EPF family is a group of Cys2/His2zinc-finger proteins in petunia. In these proteins, characteristically long spacer regions have been found to separate the zinc fingers. Our previous DNA-binding studies demonstrated that two-fingered proteins (ZPT2-1 and ZPT2-2), which have spacers of different lengths, bind to two separate AGT core motifs in a spacing specific manner. To investigate the possibility that these proteins might distinguish between the target sequences on the basis of spacing between the core motifs, we screened petunia cDNA library for other proteins belonging to this family. Initial screening by PCR and subsequent cloning of full-length cDNAs allowed us to identify the genes for 10 new proteins that had two, three or four zinc fingers. Among the two-fingered proteins the spacing between zinc fingers varied from 19 to 65 amino acids. The variation in the length of spacers was even more extensive in three- and four-fingered proteins. The presence of such proteins is consistent with our hypothesis that the spacing between the core motifs might be important for target sequence recognition. Furthermore, comparison of diverse protein structures suggests that three- and two-fingered proteins might have resulted due to successive loss of fingers from a four-fingered protein during molecular evolution. We also demonstrate that a highly conserved motif (QALGGH) among the members of EPF family and other Cys2/His2 zinc-finger proteins in plants is critical for the DNA-binding activity.     

Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1

The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a specific large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis. Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20-200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identified as a pseudouridylate synthase. Additional RNA constituents were not detected. The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed.     

PURIFICATION OF 2'',3''-CYCLIC NUCLEOTIDE 3''-PHOSPHOHYDROLASE FROM BOVINE BRAIN BY IMMUNOAFFINITY CHROMATOGRAPHY: FURTHER BIOCHEMICAL CHARACTERIZATION OF THE PROTEIN

Abstract— The purification of small amounts of 2',3'-cyclic nucleotide 3'-phosphohydrolase from bovine white matter by ion-exchange techniques (D rummond et al. , 1978) has been used to provide antigen for the production of specific rabbit antibodies to this enzyme. Specific antibody has been purified from immune serum by affinity chromatography on a column of Sepharose to which the enzyme has been attached, and the purified antibody has been coupled to cyanogen bromide-activated Sepharose. Affinity chromatography on the immunoadsorbent effectively purifies 2',3'-cyclic nucleotide 3 -phosphohydrolase in one step from an extract of an acetone powder made from bovine white matter. This modified purification procedure has reduced the time required for purification and increased the yield of the enzyme to 57%. In SDS-gel electrophoresis in phosphate buffer the enzyme migrates as an aggregate of about 98,000MW. When the buffer is Tris-glycine, the apparent MW is about 44,000 and under specific conditions two proteins of only slightly different mobilities can be discerned. Within experimental error the amino acid compositions of the proteins in the two bands are indistinguishable. Peptide patterns obtained by polyacrylamide gel electrophoresis following proteolytic digestion with Straphylococcus aureus V8 protease or papain show extensive structural homology between the two proteins, but detectable differences are apparent.     

Surface hydrophobicity of hemoglobins A, F and S determined by gel filtration column chromatography in high-phosphate buffer

We found that hemoglobins A, F and S could be separated on TSK-GEL-SW columns by differences in surface hydrophobicity when eluted with 1.8 M phosphate buffer, pH 7.4. The elution pattern of the oxy- and deoxy-forms of hemoglobins A, S and F from a TSK-GEL-SW-type gel filtration column is useful for measuring surface hydrophobicity. The elution volumes of oxyhemoglobins F, A and S on the TSK-GEL-SW column in 1.8 M potassium phosphate buffer, pH 7.4, related linearly to the log of their solubility; the higher the surface hydrophobicity, the lower the solubility. There was no linear relationship between the solubilities and the elution volumes of these hemoglobins in the deoxy-form; deoxy-Hb S was far from the lines formed by deoxy-Hb A and deoxy-Hb F. These data suggest that the solubility of oxyhemoglobins is related to simple hydrophobic interactions caused by the total surface hydrophobicity, but the extremely low solubility of deoxy-Hb S must be the result of a stereospecific strong hydrophobic interaction between amino acids at the contact regions of deoxy-Hb S molecules.