Synthesis and processing of mammalian protamines and transition proteins

Mouse and rat seminiferous tubule fragment cultures were used to examine synthesis and processing of mammalian protamines and transition proteins. The tubule fragments were incubated with [³H]-arginine, [³H]-histidine, [³⁵S]-cysteine, or [³²P]-PO₄, and radiolabeled proteins were analyzed by acid/urea polyacrylamide gel electrophoresis and fluorography or autoradiography. Newly synthesized protamines were recovered from sonication-resistant nuclei (SRN) and could not be detected in cytoplasmic fractions, indicating that protamines are deposited into nuclei immediately after synthesis. Newly synthesized mouse protamine 1 (mP1) and the precursor to mouse protamine 2 (pre-mP2) migrated more slowly during electrophoresis than their predominant testicular forms, identified by staining with Coomassie blue R-250. Within 1 hour of synthesis, the electrophoretic mobilities of mP1 and pre-mP2 increased to match those of their predominant forms. These changes are consistent with initial charge-neutralizing modifications of the newly synthesized protamines, followed by removal of at least some of the modifying ligands, to unmask protamine basicity. Steady-state phosphorylation rates were high for rat protamine 1 (rP1) and were independent of phosphate content; both rP1 molecules of low and high phosphate content were rapidly phosphorylated. Pre-mP2-3, a major processing intermediate derived by proteolysis of pre-mP2, was also rapidly phosphorylated. Like the protamines, transition protein 2 (TP2) was rapidly phosphorylated and increased in electrophoretic mobility soon after synthesis. In contrast, transition protein 1 (TP1) was not phosphorylated and did not exhibit multiple electrophoretic forms. © 1994 Wiley-Liss, Inc.     

A new continuous, monodimensional electrophoretic system for the separation and quantitation of individual glycosaminoglycans

A rapid and sensitive method for agarose gel electrophoresis is described. By simply miniaturizing a conventional gel electrophoresis apparatus, we have decreased the time necessary for the separation of nucleic acid molecules by a factor of 10. The ability to detect DNA molecules by ethidium bromide fluorescence has simultaneously been increased fivefold. Transfer of DNA from these “minigels” onto nitrocellulose filters followed by hybridization using the procedure of C. M. Southern (1975, J. Mol. Biol.98, 503–517) was found to be efficient and rapid. This technique is sufficiently sensitive to detect radioactive quantities of [³²P]phosphate-labeled DNA or RNA microinjected into 500 chick embryo fibroblasts.     

Characteristics of Membrane Protein Phosphorylation in Plasmodium berghei-Infected Mouse Erythrocytes1

ABSTRACT. Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (³²P)orthophosphate and incubating isolated membrane with (γ-³²P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with M, 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb¹. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits². During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel''s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight³. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along⁴. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation⁵; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments       

Preparative two-dimensional polyacrylamide gel electrophoresis of 32 P-labeled RNA

Complex mixtures of RNA molecules may be separated by two-dimensional electrophoresis on polyacrylamide gel slabs. The first dimension of the separation is carried out on acid gels in the presence of a high urea concentration, the second on more concentrated gels buffered at pH 8. The method has been applied to the complete separation of RNA fractions obtained after a preliminary gel electrophoresis of partial enzymic digests of ³²P-labeled bacteriophage RNA. Another application is the fractionation of partial digests as obtained in sequence determination of RNA molecules. Spots are detected by autoradiography and extracted by a simple micro procedure which yields the material in a concentrated form suitable for sequence analysis by fingerprinting.     

Direct microinjection of rabbit globin mRNA into mouse 3T3 cells. Analysis of the polypeptides synthesized in vivo

3T3 cells grown attached to 9 mm² coverslips have been microinjected in the cytoplasm with total rabbit globin mRNA and the polypeptides synthesized after injection have been labelled with [³⁵S]-methionine under conditions in which the product of as few as 100 cells could be analysed by high resolution two-dimensional gel electrophoresis followed by 10 days' fluorography. Microinjection of rabbit globin mRNA results in the synthesis of a basic polypeptide of mol. wt 15 K that is not present in control cells, and that co-migrates with purified [³H]leucine-labelled globin as determined by high resolution two-dimensional gel electrophoresis (NEPHGE). Visual inspection of the fluorograms revealed that the injection of globin mRNA (up to 14000 molecules/cell) does not alter significantly the relative intensity of the major acidic (IEF) and basic (NEPHGE) polypeptides synthesized by the cells.     

Direct determination of the specific activity of RNA uniformly labeled with 32P

A simple method for the direct determination of the specific activity of RNA uniformly labeled with ³²P is described. The procedure is based on the premise that upon disintegration of ³²P to ³²S, the phosphodiester bond is broken. Analysis of the rate of decay of the full-length molecule by gel electrophoresis and autoradiography can accurately determine the “intramolecular specific activity” of the RNA. An equation that predicts the relative intensity of the intact RNA molecules remaining as a function of time is presented. These predictions are confirmed using in vitro-synthesized RNA labeled at a known specific activity. This procedure has been used to determine the intramolecular specific activity of RNA labeled in vivo in yeast. It can also be employed to choose the best conditions for experiments utilizing uniformly labeled RNA or single-stranded DNA and requiring the detection of intact molecules.     

Processing of RNA in Escherichia coli is limited in the absence of ribonuclease III,ribonuclease E and ribonuclease P

A strain of Escherichia coli lacking RNAase III and containing thermolabile RNAase E and RNAase P was labeled with ³²P_i at a non-permissive temperature. RNA molecules were separated by two-dimensional polyacrylamide gel electrophoresis. Most of the small RNA species were isolated and analyzed for the presence of 5′ nucleoside triphosphates. In 16 of the 22 species analyzed a significant number of the individual molecules contained 5′ di or triphosphates. We conclude, therefore, that very little endonucleolytic RNA processing occurs in the absence of the three RNA processing enzymes RNAase III, RNAase E and RNAase P.     

Surface glycoprotein of human natural killer cells recognized by wheat germ agglutinin

We analyzed surface glycoproteins of human natural killer (NK) cells by utilizing lectins. Among the lectins tested, wheat germ agglutinin (WGA) was found to bind preferentially to CD16(Leu11)-positive lymphocytes as determined by two-colour flow cytometry. Analysis of glycoproteins in the lysate prepared from NK cells with sodium dodecyl sulfate (SDS) gel electrophoresis followed by Western blotting and¹²⁵I labeled WGA staining revealed that a glycoprotein with anM _r of 65 kDa was strongly bound to the lectin, but no corresponding glycoprotein was detected in the lysate of T lymphocytes. This glycoprotein (GP65) gave several spots in the pI range 4.1–4.6 on 2-dimensional gel electrophoresis. Sialidase treatment of GP65 resulted in a single spot on the 2-dimensional gel, suggesting that GP65 is heterogeneous in the degree of sialylation. GP65 was shown to be exposed on the cell surface, since it was radiolabeled with¹²⁵I by the lactoperoxidase-catalyzed method. We next isolated GP65 from human peripheral blood lymphocytes by a combination of chromatography on a cation-exchange column and a WGA-agarose column and preparative SDS gel electrophoresis. It is suggested that GP65 is a novel surface glycoprotein on human NK cells.     

Protein production and release by dispersed rat submandibular gland cells in vitro after adrenergic stimulation

Enzymatically dispersed cell aggregates were prepared from rat submandibular glands. Cells were responsive to α- and β-adrenergic agonists, as measured by net K⁺ release and radiolabeled protein secretion, respectively. Protein production by submandibular gland cells was constant during the 90 min experimental period. Specific agonist and antagonist experiments demonstrated that both α- and β-adrenergic receptor stimulation were required for maximum secretion of newly synthesized protein. Proteins were radiolabeled with [³⁵S] methionine and both soluble cell and secreted proteins examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography. A broad size range of newly synthesized proteins was detected (Mr～10⁴?5 × 10⁵). Adrenergic stimulation (1-epinephrine) specifically increased the secretion of certain radiolabeled proteins and, in addition, resulted in both cellular and secreted proteins with electrophoretic characteristics distinct from that of control preparations.     

Resolving Power: A Quantitative Measure of Electrophoretic Resolution

Resolving power is a quantitative measure of the ability of an electrophoretic system to separate DNA (and other) molecules of similar size. It is a dimensionless quantity, and hence facilitates comparison of the performance of electrophoretic systems that operate very differently. Resolving power can be determined as a function of molecular length from experimental data consisting of a series of completely resolved bands on a gel or blot; closely spaced bands are not required. We discuss factors such as the mass of DNA in a particular band and the spatial resolution of the system used to image the distribution of DNA on a gel or blot that, while not an intrinsic part of the electrophoretic system, may influence the observed resolving power. We derive an empirical global dispersion function that applies both to images of gels obtained after a fixed time of electrophoresis of all the samples and to images obtained as each species reaches a detector located at a fixed distance from the starting well. We use this dispersion function to show that the improvement in resolving power produced by extending the time or distance of electrophoresis in a static, uniform electric field asymptotically approaches a limiting value that is a function of the length of the DNA. When plotted as a function of molecular length, this limiting value defines an envelope that characterizes the intrinsic limits of performance of a particular electrophoretic system (e.g., electric field strength, gel type and concentration, buffer, temperature). Comparing the resolving power of static field agarose gel electrophoresis as routinely practiced for separating DNA molecules from 10³ to 10⁵ bp long with other electrophoretic schemes suggests that significant improvements should be achievable.     

Technetium-99m-labeled antibodies

It is essential in any method for radiolabeling antibody with^99mTc that the labeling procedure is rapid and reliable, producing a highly stable^99mTc-antibody complex with minimal effect on the immunoreactivity of the antibody. In the present study, analysis of the stability and homogeneity of radiolabeled (^99mTc and¹²⁵I) antibodies (HMFG1 and PR1A3) was carried out by fast protein liquid chromatography (FPLC) using superose-6 and S-200 columns, and by polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. Superose 6 and S-200 gel filtration analysis showed the radiolabel (^99mTc or¹²⁵I) eluting with a retention time identical to that of native antibody. No peaks of relative molecular size (Mr) corresponding to possible antibody fragments were seen in either the UV or the radioactive FPLC elution profiles. PAGE analysis of^99mTc labeled antibody, however, revealed the presence of a number of radiolabeled antibody fragments (Mr<IgG) that were not detected by the same analysis of¹²⁵I labeled antibody. The stability of the radiolabeled antibodies in serum in vitro was also studied. FPLC (superose-6) analysis after 45 h incubation in normal serum in vitro revealed 3.3% (HMFG1), and 20% (PR1A3) of the^99mTc on a molecule or aggregate with a Mr greater than that of IgG. There is also the appearance of small amounts of^99mTc-labeled material with a Mr<IgG in the later fractions (2.2% for HMFG1 and 4.9% for PR1A3). Similar results were obtained using radioiodinated antibody, although the small amount of low molecular size material detected as a single peak with a longer retention time than the^99mTc equivalent corresponds to free iodide.     

Chemical Characterization and Pronase Susceptibility of the Na:K Pump-Associated Phosphoprotein of Human Red Blood Cells

The phosphoproteins formed by incubation of red cell ghosts with [γ-³²P]ATP in the presence of Mg and Na + Mg have been characterized by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The ³²P-labeled phosphoprotein was seen as a single peak confined to the region of the diffuse 90,000 dalton polypeptide band; labeling with Na + Mg considerably increased the quantity of ³²P-phosphoprotein contained in this band relative to labeling with Mg alone. Treatment of intact cells with Pronase known to partially hydrolyze the glycoproteins and the 90,000 daltons polypeptide did not change either the amount or the position of the ³²P-phosphoprotein present in the gels. The molecular weight of the ³²P-phosphoprotein is estimated to be 103,000. Pronase treatment of intact cells also did not significantly alter any of the transport parameters of the membrane such as the K pump flux, ouabain binding, or Na,K-ATPase. In contrast, treatment of ghosts with Pronase not only resulted in drastic alteration of the transport parameters but also inhibited the formation of the phosphoprotein under all conditions. Thus, while the Na:K pump is not intrinsically resistant to Pronase, those elements of the pump which are susceptible are not accessible from the outside of the cell. Further, SDS-polyacrylamide gel electrophoresis after Pronase treatment of intact cells results in a substantial increase in the purification of the phosphoprotein relative to that which was previously possible in ghosts.     

Schwann Cell Surface Proteins and Glycoproteins

Abstract: To identify surface sialoglycoproteins of rat Schwann cells and to compare molecular weights of these sialoglycoproteins with those present in rat peripheral nervous system myelin, we prepared Schwann cells from sciatic nerves of 1–3-day-old rats and cultured them in monolayer. Surface sialoglycoproteins of the cultured cells were tritium-labeled by the periodateborohydride procedure and compared with sialoglycoproteins of adult rat peripheral nervous system myelin by fluorography following polyacrylamide slab gel electrophoresis in sodium dodecyl sulfate. Three radioactive bands with apparent molecular weights of 114,000–132,000, 105,000–115,000, and 44,000–56,000 were observed in both the Schwann cell and myelin preparations. Bands of similar apparent molecular weights were noted in Schwann cells metabolically radiolabeled with d -[1,6-³H]glucosamine. A band co-migrating with myelin P₀ glycoprotein was the most intensely radiolabeled of all peptides in periodate-B³H₄^?treated myelin, but was present in only trace amounts in periodate-B³H₄^? or d -[1,6-³H]glucosamine radiolabeled Schwann cells. Many presumably non-myelin glycoproteins were identified in the cultured Schwann cells by the periodate-borohydride procedure and by incubation of the cells with d -[1,6-³H]glucosamine. An immunoprecipitation technique was used to detect radiolabeled peptides in a nonionic detergent extract of freshly prepared, surface-radioiodinated Schwann cells that were bound by a rabbit anti-Schwann cell serum preabsorbed with rat fibroblasts. Many radioactive peptides were detected in the immunoprecipitate, but the two most intensely radiolabeled had apparent molecular weights of 105,000–115,000 and 95,000–106,000. This study has identified a number of glycoproteins synthesized by cultured rat Schwann cells which resemble in apparent molecular weight the glycoproteins expressed in rat peripheral nervous system myelin and has defined Schwann cell surface proteins recognized by a specific anti-rat Schwann cell antiserum.     

The Biochemical characterization of syrian hamster cell-surface alloantigen

The first alloantiserum to be described in Syrian hamsters has been characterized for its ability to react with externally and internally radiolabeled antigens derived from normal hamster lymphoid cells. Utilizing conventional biochemical techniques, radioiodinated and³H-leucine labeled cellular extracts have been prepared, partially purified by lentil lectin affinity chromatography, and indirectly immunoprecipitated with experimental alloantisera. Analysis of the precipitated radiolabeled antigens by polyacrylamide gel electrophoresis in SDS has identified two prominent cell-surface proteins at 39,000 (p39) and 29,000 daltons (p29) on 2-mercaptoethanol reduced gels. Further analysis of the radiolabeled extract has demonstrated the existence of hamster cell-surface proteins at 43,000 and 12,000 daltons which are immunoprecipitated by a xenoantiserum directed against human ₂ microglobulin. Coelectrophoretic studies indicate the independent identity of these four species of hamster cellsurface proteins. These results suggest that between two hamster lines, derived from animals caught 40 years apart from different geographic locations in Syria, polymorphism of cell-surface antigens is restricted to p39 and p29 molecular species.     

Studies on the bioactivity of radiolabeled,highly-purified bovine thyrotropin

Bovine thyrotropin radiolabeled stoichiometrically with chloramine T was subjected to high pressure liquid chromatography on a Waters' Protein I-125 column. More than 80% of the radioactivity eluted after the Na¹²⁵I (“salt”) peak. In contrast, thyrotropin bioactivity eluted before the salt peak. Radiolabeled thyrotropin affinity-purified with thyroid plasma membranes eluted after the salt peak. Discordance between the thyrotropin bioactivity and radioactivity elution profiles was confirmed by labeling thyrotropin with ¹²⁵I by lactoperoxidase and then measuring both bioactivity and radioactivity in each chromatographic fraction. These data suggest that the bioactivity in radiolabeled thyrotropin may not be inherent in the radiolabeled molecules.     

A method for measuring anion transfer across red cell membranes by continuous monitoring of fluorescence

A method was developed for the identification of Ca²⁺-binding proteins after electrophoresis on polyacrylamide gels. The method involves equilibration of the gel with ⁴⁵Ca either during or after electrophoresis, followed by visualization of the ⁴⁵Ca-binding proteins by autoradiography.     

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method¹. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 °C during the gel run allows for the separation of unstructured DNA or RNA molecules.In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol ^1,2.