Method of detection of protein kinase activity in polyacrylamide gel using two-dimensional protein separating system

Method for detection of protein kinase activity in polyacrylamide gel have been developed. After separation of proteins by isoelectric focusing in non-denaturing condition, gel was incubated in a reaction buffer containing [gamma-32P]ATP. 32P-labeled proteins were separated by subsequent SDS/PAGE electrophoresis in second dimension. The proposed method was used for detection of protein kinase activity in human blood serum and triton X-100 soluble proteins of heads of Drosophila melanogaster.     

Two-dimensional electrophoresis of plasma proteins without denaturing agents.

A technique of two-dimensional polyacrylamide gel electrophoresis for the separation of plasma proteins is described. Human plasma proteins were separated by isoelectric focusing followed by electrophoresis in a 4 to 21% linear gradient gel slab. No denaturing agent was used throughout the procedure, so that the analysis of native proteins is possible. Two-dimensional patterns obtained from normal human plasma samples were recorded as "staining density maps," which are similar to contour line maps, and more than 230 protein spots were counted reproducibly on each "staining density map." This technique permits the simultaneous estimation of pI's and approximate molecular weights of native proteins on the slab gel. Applications of this technique to an IgA myeloma plasma sample and a porcine serum sample are described.     

Microheterogeneity in purified broad bean polyphenol oxidase

Polyphenoloxidase was purified from chloroplasts of broad bean leaves (Vicia faba L.) to apparent homogeneity. The enzyme was composed of two proteins with an apparent mass of 65 and 68 kilodaltons after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme contained covalently attached carbohydrates and bound concanavalin A, Phaseolus vulgaris erythroagglutinin, and Ricinus communis agglutinin lectins. Under native isoelectric focusing, several charged isoforms were present in the pH range of 4 to 6. Many, if not all, of the isoforms separated by isoelectric focusing were glycosylated and bound concanavalin A. All these isoforms shared a 65 kilodalton protein in common, and some of the isoforms were associated with both a 65 and 68 kilodalton protein. Isoforms separated by isoelectric focusing in the presence of 9 molar urea followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a similar pattern of proteins within a slightly higher pH range from 5 to 6.5.     

Partial purification and molecular characterization of a lymphokine (costimulator) required for the mitogenic response of mouse thymocytes in vitro.

We have partially purified a lymphokine, costimulator, which is necessary to induce mitogenesis in mouse thymocytes in vitro. Costimulator is released from mouse leukocytes exposed to Con A for 12 to 18 hr. It has been purified more than 100 X by gel exclusion chromatography and isoelectric focusing. Thymocytes from CBA/J mice respond to the mitogenic lectin Con A only if the costimulator concentration is above a certain level. Culturing such cells with Con A at a density below 1 X 10(6) cells/ml produces costimulator concentrations too low for mitogenesis. This system has been developed into a quantitative assay for costimulator, to monitor purification, recovery, and biologic activity in various methods of molecular characterization. The activity is trypsin sensitive, and has a buoyant density characteristic of protein or glycoprotein. However, for a protein, it is relatively heat stable. Its m.w., established by carrying out sedimentation, gel filtration, and buoyant density measurements, is 30,500, and its frictional coefficient is 1.45. Costimulator purified by isoelectric focusing is active at 10(-10) M or lower in tissue culture.     

De novo synthesis of peroxidase in cotton ovule culture medium

The biosynthesis of cotton ( Gossypium hirsutum L. 'Stoneville 208') peroxidase (EC 1.11.1.7) has been investigated in an organ culture system, since this enzyme may play a role in cell wall biogenesis or host defense mechanisms. Electrophoretic analysis of proteins from cotton ovule cultures indicated relatively few proteins being released into the surrounding medium. De novo synthesis of released peroxidase and other medium proteins was determined by in vivo labeling of ovule cultures with [³⁵S]-methionine. Analysis of labeled culture medium by denatured gel electrophoresis followed by fluorography showed incorporation of isotope into 2 major proteins with molecular weights of 30 kD and 56 kD, as well as a limited number of minor proteins. Similar analysis of native isoelectric focusing gels coupled with autoradiography demonstrated [³⁵S]-methionine incorporation into 2 major proteins with pI values of 4.3 and 5.0. The pI 5.0 protein was shown to have a molecular weight of 30 kD. The pI 4.3 protein had a molecular weight of 56 kD and was shown to be peroxidase by activity staining. Minor radiolabeled proteins were observed in the cationic region of the isoelectric focusing gels.     

Expression of human insulin-like growth factor I in bacteria: use of optimized gene fusion vectors to facilitate protein purification

Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone. After IgG affinity purification of the fusion proteins from the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized expression system based on a modified synthetic IgG-binding domain (z), resistant to hydroxylamine, gave the highest yield of fusion protein. After cleavage, the hormone could be separated from the IgG-binding moiety and from noncleaved fusion protein by a second passage through the IgG affinity column. The biological activity and the purity of the IGF-I obtained were confirmed by a radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography.     

Purification and partial characterization of a mitogenic protein released from preadipocytes of massively obese subjects

A protein released into the culture medium by omental preadipocytes of massively obese persons, which stimulates the replication of rat perirenal preadipocytes, has been purified to a high degree. By gel filtration chromatography, the molecular mass of the mitogenic protein was approximately 66,000 daltons (Da), while on sodium dodecyl sulfate - polyacrylamide gel electrophoresis, two subunits were obtained, relative masses (Mr) of approximately 31,000 and approximately 35,000. The isoelectric point of the approximately 66,000 Da entity was 5.6 +/- 0.2. By specific radioreceptor assay, the purified protein was related to epidermal growth factor and transforming growth factor alpha. It was not related to insulin-like growth factors I and II by radioimmunoassay and radioreceptor assay. We propose that the approximately 66,000 Mr protein, and other mitogenic proteins released by preadipocytes from massively obese persons, act through paracrine-autocrine mechanisms and may play a role in the development of the hyperplasia of enlarged fat cells characteristic of massive corpulence.     

Isoelectric focusing of high-molecular-weight protein complex under native conditions using agarose gel

The isolation and characterization of protein complexes are essential steps toward understanding cellular functions. A method for separating and characterizing high-molecular-weight protein complexes using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with native agarose gel isoelectric focusing (IEF) is described. Using this method, fractions containing high-molecular-weight protein complexes were analyzed. The advantages of using native agarose gel IEF include the ability to concentrate the protein complexes and the ease of handling when performing 2D separations. Although limited with respect to the size of molecules and particles that may be separated, this method is useful for the isolation and characterization of high-molecular-weight protein complexes.     

Cyclic nucleotide phosphodiesterase in rat neostriatum: multiple isoelectric forms with similar kinetic properties

Separation of multiple forms of cyclic nucleotide phosphodiesterase from the soluble supernatant fraction of rat neostriatum by isoelectric focusing yielded five separate peaks of cyclic nucleotide hydrolysing activity. Each separated enzyme form displayed a complex kinetic pattern for the hydrolysis of both cyclic AMP and cyclic GMP, and there were two apparent Km's for each nucleotide. At 1 microM substrate concentration, four enzyme forms exhibited higher activity with cyclic AMP than with cyclic GMP, while one form yielded higher activity with cyclic GMP than with cyclic AMP. Cyclic AMP and cyclic GMP were both capable of almost complete inhibition of the hydrolysis of the other nucleotide in all the peaks separated by isoelectric focusing; the IC50's for this interaction correlated well with the relative rates of hydrolysis of each nucleotide in each peak. The ratio of activity at 1 microM substrate concentration for the five enzyme forms separated by isoelectric focusing was 10:10:5:15:1 for cyclic AMP hydrolysis; and 6:6:4:8:2 for cyclic GMP hydrolysis; and the isoelectric points of the five peaks were 4.3, 4.45, 4.7, 4.85, and 5.5, respectively. Known phosphodiesterase inhibitors did not preferentially inhibit any of the separated forms of activity for either cyclic AMP or cyclic GMP hydrolysis, at either high (100 microM) or low (1 microM) substrate concentrations. Preliminary examination of the subcellular distribution of the different forms of enzyme activity indicated a different degree of attachment of the various forms to particulate tissue components. Isoelectric focusing of the soluble supernatant of rat cerebellum gave rise to a slightly different pattern of isoelectric forms from the neostriatum, indicating a different cellular distribution of the isoelectric forms of PDE in rat brain. Polyacrylamide disc gel electrophoresis of the soluble supernatant of rat neostriatum also generated a characteristic pattern of five separate peaks of cyclic nucleotide phosphodiesterase activity, each of which hydrolysed both cyclic AMP and cyclic GMP. Polyacrylamide gel electrophoresis of single enzyme forms previously separated by isoelectric focusing gave single peaks, with a marked correspondence between the enzyme forms produced by isoelectric focusing and those produced by gel electrophoresis, suggesting that both protein separation procedures were isolating the same enzyme forms. The results indicate the existence of multiple isoelectric forms of cyclic nucleotide phosphodiesterase in the soluble supernatant fraction of rat neostriatum, all of which exhibit similar properties. In this tissue a single kinetic form of this enzyme appears to exist displaying complex kinetic behaviour indicative of negative cooperativity and hydrolysing both cyclic AMP and cyclic GMP, with varying affinities.     

Purification and Properties of Dextransucrase from Streptococcus mutans

The dextransucrase (EC 2.4.1.5) activity from cell-free culture supernatants of Streptococcus mutans strain 6715 has been purified approximately 1,500-fold by ammonium sulfate precipitation, hydroxylapatite chromatography, and isoelectric focusing. The enzyme was eluted as a single peak of activity from hydroxylapatite, and isoelectric focusing of the resulting preparation gave a single band of dextransucrase activity which focused at a pH of 4.0. The final enzyme preparation contained two distinct, enzymatically active proteins as judged by assay in situ after polyacrylamide gel electrophoresis. One of the proteins represented 90% of the total dextransucrase activity and 53% of the total protein. The molecular weight of the enzyme was estimated by gel filtration to be 94,000. The temperature optimum of the enzyme was broad (34 to 42 C) and its pH range was rather narrow, with optimal activity at pH 5.5. The K(m) for sucrose was 3 mM, and fructose competitively inhibited the enzyme reaction with a K(i) of 27 mM.     

Factors in the rat submaxillary gland that stimulate growth of cultured glioma cells: identification and partial characterization

The effect of rat submaxillary extract on the growth of rat C6 glioma cells in serum-free culture has been examined. Extracts (10-15 microgram/ml) of submaxillary glands from both male and female rats markedly enhanced the growth of serum-deprived C6 cells and, in combination with insulin, transferrin, and NIH-LH (a source of fibroblast growth factor), were able to stimulate C6 cell growth to an extent comparable to that achieved with an optimal amount of fetal calf serum. The mitogenic activity of rat submaxillary extracts was found to be heat-labile, acid-stable, and partially inactivated by protease and 2-mercaptoethanol. Under our assay conditions, biologically active preparations of purified mouse submaxillary gland epidermal growth factor (EGF) or nerve growth factor (NGF) were not mitogenic for C6 cells, nor was the mitogenic activity of rat submaxillary extracts inhibited by antiserum to these mouse submaxillary gland growth factors. These results suggest that the active component(s) of rat submaxillary extracts is unrelated to either EGF or NGF. The growth-enhancing effect also appears unrelated to esteropeptidase activity present in these extracts since the mitogenic activity was unaffected by several protease inhibitors. Moreover, two purified mouse submaxillary gland arginylesteropeptidases, EGF-binding protein and gamma-subunit of 7 S NGF, were unable to elicit a comparable growth response even when added to cell culture medium at unreasonably high concentrations. The C6 cell mitogenic activity of crude submaxillary extracts could be separated into two biologically similar components by either gel filtration on Sephadex G-100, preparative isoelectric focusing in a pH gradient of 3-10, or adsorption to DEAE-cellulose followed by elution with a sodium chloride gradient. One of the active components was acidic in nature and had an apparent molecular weight of 40,000, while the other was near neutral in charge and possessed a molecular weight of approximately 20,000. The relationship between these two C6 cell mitogenic components and the rat submaxillary gland component responsible for stimulating Balb/c-3T3 cell growth in serum-free, factor supplemented medium (McClure et al., 1979, J. Cell Biol. 83:96a) is also discussed.     

Purification and physical characterization of nucleic acid helix-unwinding proteins from calf thymus.

We have devised a general protein fractionation procedure which selects for eukaryotic DNA-binding proteins, some of which resemble DNA-unwinding proteins from prokaryotes. Proteins were selected which (a) pass through a native DNA-cellulose column, (b) bind to a denatured DNA-cellulose column, and (c) remain bound to the latter column during a rinse with a dilute solution of the sodium salt of the polyanion dextran sulfate. When this fractionation was applied to the soluble proteins fo calf thymus, three major protein species were recovered. The predominant one has an apparent molecular weight of about 24,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is isoelectric near neutrality, and elutes as a monomer from denatured DNA-cellulose at moderate NaCl concentrations. This protein, designated calf-unwinding protein 1 (UP1), has been purified to homogeneity. However, isoelectric focusing reveals four or five subspecies (apparently separated by single-charge differences) which differ appreciably in their affinities for DNA. Two other major proteins are obtained which have apparent molecular weights in sodium dodecyl sulfate of 33,000: the first, which elutes with low salt from DNA-cellulose as a homogeneous preparation, appears to be a basic protein (although it is clearly not a histone); the other, which elutes from DNA-cellulose as the major component of a "high salt eluting fraction," is an acidic protein which co-purifies with less prominent species of higher molecular weights. Proteins similar to each of these three major calf thymus proteins have been observed by us and others in tissue culture cells of mouse, hamster, monkey, and humans, suggesting their wide occurrence among eukaryotes.     

Purification and Some Properties of Tetanolysin

Tetanolysin was purified from the culture fluid of a strain of Clostridium tetani by ammonium sulfate fractionation, acetone precipitation and repeated gel filtration. Two hemolysins with different molecular weights were separated by gel filtration, and the smaller one, tetanolysin, was further purified. The purification raised the specific activity of tetanolysin 1,050-fold to 500 HU/μg of protein. The purified preparation gave a single, relatively broad band on polyacrylamide gel electrophoresis, in which the activity was roughly parallel with the protein concentration. However, on sodium dodecylsulfate-gel electrophoresis it gave two bands with nearly equal amounts of proteins, showing molecular weights of 53,000 and 48,000±3,000. Furthermore, isoelectric focusing revealed four peaks of the activity whose isoelectric pHs were 6.1, 5.6, 5.3, and 6.6 in decreasing order of the activity. These findings suggest that the preparation contains four hemolysins with different pis, which are classifiable into two groups by molecular size. The preparation was completely free of tetanus neurotoxin and proteases. Tetanolysin was more strongly inhibited by cholesterol and more rapidly adsorbed onto erythrocytes than θ-toxin of Cl. perfringens.     

Oxidation of fatty alcohol in the cotyledons of jojoba seedlings.

An externally accessible polypeptide has been purified from hepatoma tissue culture cells. The purification involves four steps: deoxycholate extraction of whole cells, isoelectric focusing of deoxycholate-insoluble material in the presence of 8 m urea and Triton X-100, hydroxylapatite chromatography in the presence of sodium dodecyl sulfate, and preparative acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The final preparation is homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by isoelectric focusing in polyacrylamide. The polypeptide has an apparent molecular weight of 55,000 and is labeled following in situ lactoperoxidase-catalyzed iodination of the hepatoma tissue culture cells. The polypeptide can also be labeled by growing cells in the presence of labeled amino acids, but is not labeled by growth in labeled sugars. The purified protein does not react with the periodate-Schiff reagent. Hence, it does not appear to be a glycoprotein that contains mannose, fucose, glucosamine, or sialic acids.     

Carbohydrate composition of rat hepatocyte nuclear membrane as compared to normal, Morris hepatoma 7800, and phenobarbital-induced microsomal membranes

The composition of the nonhistone nuclear proteins of rat liver, brain, thymus, and kidney has been analyzed by isoelectric focusing in polyacrylamide gel. Approximately 20–30 components were separated with a wide range of isoelectric points (pl) in the 3- to 10-pH region.Different extraction procedures applied to liver nuclei removed protein mixtures with similar components present in varying amounts. 8 m Urea 50 mm phosphate, pH 7.6, was the most successful and removed most of the nonhistone protein.The thiol groups of proteins extracted from the nuclei of liver, brain, thymus, and kidney with 8 M urea, 50 mM phosphate were labeled with [¹⁴C]N-ethylmaleimide. Although there was a slight variation in the overall thiol content of these tissue proteins, separation of the mixture by isoelectric focusing and SDS polyacrylamide gel electrophoresis showed complex patterns indicating greater heterogeneity than was apparent from the Coomassie blue dye binding.     

Two-dimensional separation of chloroplast membrane proteins by isoelectri focusing and electrophoresis in sodium dodecyl sulphate

Proteins of chloroplast subfragments enriched in Photosystem I and Photosystem II electron flow activity have been analyzed by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, polyacrylamide gel isoelectric focusing (pH 5–7) was used in the presence of Triton X-100, followed at right angle by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Characteristic fingerprints were obtained for the Photosystem I and II fractions and a correlation between the major proteins separated by isoelectric focusing and the major polypeptides separated by undimensional SDS electrophoresis was established. Two dominant spots of 68 000 and 60 000 daltons appeared in the two-dimensional patterns of Photosystem I fractions $\text{p}\text{I}$ values about 5.6; two spots with molecular weights of 33 000 and 23 000 were characteristics for Photosystem II fractions $\text{p}\text{I}$ values about 5.3 and 6.3). Photosystem I fractions were furthermore characteristics by a series of spots in the 44 000–33 000 range $\text{p}\text{I}$ values from about 5.9 to 6.8). The two-dimensional system revealed that (a) several SDS-polypeptides have multiple forms differing in charge only, (b) some proteins separated by isoelectric focusing are resolved in the second dimensional into polypeptides of different size. The two-dimensional method combining Triton X-100 isoelectric focusing' and SDS electrophoresis provides a higher degree of resolution than either of the unidimensional methods thus allowing a detailed analysis of chloroplast membrane proteins.     

Purification of the Atlantic salmon hepatic 21 kDa protein and classification as a high mobility group chromatin protein.

The 21 kDa protein of liver from Atlantic salmon (Salmo salar) has been purified. Hepatic nuclei were extracted with 0.75 M HClO4. The extracted proteins were fractionated using reversed phase high performance liquid chromatography. The purity of the protein was analysed by isoelectric focusing in the first, and SDS-polyacrylamide gel electrophoresis in the 2nd dimension. Isoelectric focusing separated the protein into 5 spots. In gel trypsin digestion after isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis resulted in identical migration of the tryptic peptides. The amino acid composition of the 21 kDa protein was similar to that of high mobility group (HMG) proteins C and D from rainbow trout (Oncorhynchus mykiss). The N-terminal sequence of the amino acids 1-19 revealed a conserved region characteristic for HMG 14/17 proteins of mammals and avians, and their equivalents in rainbow trout. Considering the electrophoretic mobility, amino acid composition and N-terminal amino acid sequence it is concluded that the 21 kDa protein of Atlantic salmon is a member of the HMG protein family resembling the HMG D protein of rainbow trout.     

Investigations on the outer membrane proteins of Salmonella typhimurium.

The number, nature and organization of the outer membrane proteins of Salmonella typhimurium have not yet been resolved. Therefore these proteins were isolated using a concentrated solution of guanidine hydrochloride and studied using different analytical techniques. Upon chromatography on Sephadex G-200 four fractions were obtained. Only the fraction containing a protein of molecular weight 13,000 produced immunoprecipitation reactions with the antisera raised against the whole bacteria. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, 7 major proteins were found, with molecular weights between 13,000 and 43,000. Isoelectric focusing on 4.6% polyacrylamide gels resolved the outer membrane proteins into 10 bands with apparent isoelectric points between 5.0 and 8.4. Finally these proteins could be further resolved into as many as 50 spots where a two-dimensional electrophoresis was carried out with isoelectric focusing in the first dimension, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate in the second dimension. These results demonstrated that the outer membrane proteins of S. typhimurium are extremely heterogeneous. To investigate the mode of organization of lipopolysaccharides in the outer membrane, the membrane proteins were separated by the liquid isoelectric focusing technique. Lipopolysaccharides were primarily found to be associated with a protein of isoelectric point 7.8.     

Purification and characterization of neuraminidase from Clostridium perfringens.

Clostridium perfringens cells were cultivated on a large scale using an automatic system. Neuraminidase secreted by the cells into the culture medium was purified 380 000-fold by: precipitation with ammonium sulfate between 50 and 85% saturation, filtration on Sephadex G-75, electrophoresis on polyacrylamide gel, and by isoelectric focusing. Three enzyme fractions with different migration rates were obtained by preparative disc electrophoresis in polyacrylamide gel, and five fractions with isoelectric points between pH 4.7 and 5.4 were observed after isoelectric focusing. This microheterogeneity disappeared after denaturation of the enzyme in 0.1% sodium dodecylsulfate or 8M urea. The isoelectric point of the denatured enzyme corresponded to pH 4.3. All enzyme fractions were identical with regard to their immunological and kinetic properties; they had the same molecular weights. The origin of the different "conformers" of neuraminidase is discussed. The existence of genuine isoenzymes could largely be excluded. The yield of neuraminidase was 65%, which corresponded to about 10 mg of pure enzyme from 100 l of culture medium. The enzyme was free of protease and various other glycosidase activities. The neuraminidase preparation appeared not to be contaminated by other proteins as judged by electrophoretic analysis using either the native enzyme or the enzyme denatured by sodium dodecylsulfate or urea; ultracentrifugation; chromatography on Sephadex G-200; and immunological methods. The molecular weights of the native or denatured enzyme were found to be in the range between 60 000 and 69 000 (on an average 63 750) using four independent methods. The existence of subunits of neuraminidase was excluded. The neuraminidase exhibited a spec. act. of 580 or 615 U/mg protein with glycopeptides from edible birds' nests or sialyllactose, respectively, as substrates. Additional kinetic properties and the UV-absorption spectrum of the enzyme are described.