Preparation and kinetic properties of gel entrapped urate oxidase.

Urate oxidase from hog liver (urate: oxygen oxidoreductase, EC 1.7.33) has been entrapped in a crosslinked 2-hydroxyethyl methacrylate gel with a 47% retention of activity. The kinetic behavior of the gel entrapped enzyme has been studied in a slurried tank reactor using uric acid as substrate. Internal diffusion effects were found to be negligible for particle sizes below 128 mum. A threefold increase in Km (app) was observed for the 128 mum particles and attributed to diffusional effects. The pH activity profile of the gel entrapped enzyme was bell-shaped at high substrate concentration and could be fitted to a titration curve of two ionizable groups, a basic group having a pK of 7.9 and an acidic group with a pK of 11.0. The gel entrapped enzyme showed excellent stability between pH 6.5 and 10.5.     

Microstructures of an amelogenin gel matrix.

The thermo-reversible transition (clear <--> opaque) of the amelogenin gel matrix, which has been known for some three decades, has now been clarified by microstructural investigations. A mixed amelogenin preparation extracted from porcine developing enamel matrix (containing "25K," 7.4%; "23K," 10.7%; "20K," 49.5%; and smaller peptides, 32.4%) was dissolved in dilute formic acid and reprecipitated by adjusting the pH to 6.8 with NaOH solution. Amelogenin gels were formed in vitro by sedimenting the precipitate in microcentrifuge tubes. The gels were fixed with Karnovsky fixative at 4 and 24 degrees C, which was found to preserve their corresponding clear (4 degrees C) and opaque (24 degrees C) states. Scanning electron microscopy, atomic force microscopy, and transmission electron microscopy were employed for the microstructural characterization of the fixed clear and opaque gels. The amelogenin gel matrix was observed to possess a hierarchical structure of quasi-spherical amelogenin nanospheres and their assemblies. The nanospheres of diameters 8-20 nm assemble to form small spherical assemblies of diameters 40-70 nm that further aggregated to form large spherical assemblies of 70-300 nm in diameter. In the clear gel, most of the large assemblies are smaller than 150 nm, and the nanospheres and assemblies are uniformly dispersed, allowing an even fluid distribution among them. In the opaque gel, however, numerous spherical fluid-filled spaces ranging from 0.3 to 7 microm in diameter were observed with the majority of the large assemblies sized 150-200 nm in diameter. These spaces presumably result from enhanced hydrophobic interactions among nanospheres and/or assemblies as the temperature increased. The high opacity of the opaque (24 degrees C) gel apparently arises from the presence of the numerous fluid-filled spaces observed compared to the low-temperature (4 degrees C) preparation. These observations suggest that the hydrophobic interactions among nanospheres and different orders of amelogenin assemblies are important in determining the structural integrity of the dental enamel matrix.     

Identification of initiation factors and ribosome-associated phosphoproteins by two-dimensional polyacrylamide gel electrophoresis.

A two-dimensional polyacrylamide gel electrophoresis procedure has been used to identify initiation factors rapidly in the high-salt-wash fraction from reticulocyte ribosomes. Initiation factors are identified by relative mobility and by co-electrophoresis with purified factors. A creatine phosphate/ATP/GTP/Pi exchange system is described which has been used to maintain [gamma-32P]ATP and [gamma-32P]GTP at constant specific activity in the cell-free protein-synthesizing system. Phosphorylated proteins associated with the protein-synthesizing complex have been identified using a combination of the two procedures. The salt-wash fraction contains eight major phosphorylated proteins and a number of minor ones. Two phosphorylated proteins are observed to comigrate with two of the three subunits of eukaryotic initiation factor 2 (eIF-2), the initiation factor involved in binding Met-tRNAf onto the 40-S subunit and promoting dissociation of 80-S ribosomes. eIF-4B, one of the proteins involved in binding mRNA to 40-S subunits is also phosphorylated. The remainder of phosphorylated proteins in the high-salt-wash fraction are not previously characterized initiation factors and have not been identified further. Two of the six phosphoproteins associated with the salt-washed ribosomes comigrate with ribosomal proteins; one is the major phosphorylated protein in 40-S ribosomal subunits, the other is an acidic protein.     

Enzymes of the human intestinal brush border membrane. Identification after gel electrophoretic separation.

The postition of a number of human intestine brush border membrane enzyme activities in polyacrylamide gels after electrophoresis has been determined. These activities are, in order from the origin, maltase/glucoamylase, lactase/phlorizin hydrolase, maltase/sucrase/isomaltase, enteropeptidase, trehalase and gamma-glutamyl-transferase. Leucylnaphthylamide hydrolyzing activity was inactivated by sodium dodecylsulfate and its position was not determined. The positions of the activities have been correlated with the positions of protein bands previously determined. One such band situated between enteropeptidase and alkaline phosphatase has not been identified.     

Two-dimensional gel analysis of soluble proteins. Charaterization of guinea pig exocrine pancreatic proteins.

A two-dimensional gel technique using slab gel isoelectric focusing in the first dimension and sodium dodecyl sulfate gradient gel electrophoresis in the second dimension has been developed for the separation of soluble proteins larger than 10,000 daltons. The technique is sensitive to 0.6 mug of protein and recovery of radiolabeled proteins averages 90%. Analysis of secretory protein from the guinea pig exocrine pancreas shows the presence of 19 distinct high molecular weight proteins. Each of these proteins has been characterized by isoelectric point, molecular weight, and proportionate mass. Thirteen of the 19 proteins have been identified by actual or potential enzymatic activity,accounting for 96% of the protein mass resolved by the two-dimensional gels.     

Two-dimensional SDS-polyacrylamide gel electrophoresis of heat-modifiable outer-membrane proteins.

An examination has been made of the effect which temperature of solubilization has upon the subsequent migration in SDS-polyacrylamide gel electrophoresis of proteins from the cell envelopes of Escherichia coli K12 and Neisseria sicca ATCC 9913. Conventional electrophoresis in tubes revealed substantial differences in the staining patterns of gels, depending upon whether the envelope samples were solubilized at 37 degrees C or 100 degrees C; in the case of N. sicca at least 6 of 13 discernible bands displayed heat-modifiable behavior. The relationship of the bands produced by each of the two temperatures was investigated by a two-dimensional electrophoresis procedure, in which a sample was solubilized at 37 degrees C and run in a usual cylindrical gel; the entire gel was then resolubilized at 100 degrees C, and laid along an acrylamide slab for electrophoresis in the second dimension. It was found that "free endotoxin" of both organisms examined contained the same major proteins as the total envelope fraction, and that these free endotoxin proteins showed the same heat-modifiable properties as when present in total envelopes.     

Plasmid migration using orthogonal-field-alternation gel electrophoresis.

The migration properties of a series of supercoiled plasmids ranging in size from 4 to 16 kilobases (kb) have been analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE). These circular DNAs enter the gel and are well resolved. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a wide range of pulse times, from 10 to 120 seconds, as well as over a broad range of total running times, from 6 to 24 hours. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of pBR322 shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.     

The differentiation of fluorescamine-modified kinins by gel electrophoresis.

A technically simple procedure has been found for the identification and differentiation of bradykinin, leukokinin-H, and lysyl-containing analogs of bradykinin. By reacting the kinins with fluorescamine and thus altering the charges, the modified kinins may be separated by polyacrylamide gel electrophoresis. At pH 4.2 fluorescamine-modified (FM) bradykinin may be separated from FM-leukokinin-H and from FM-Lys-bradykinin and FM-Met-Lys-bradykinin. Similarly at pH 9.8, FM-Lys-bradykinin and FM-Met-Lys-bradykinin can be separated from FM-bradykinin and FM-leukokinin-H. FM-Lysyl-bradykinin and FM-Met-Lys-bradykinin are not separable at either pH. This procedure may prove to be extremely useful in the identification and purification of kinin peptides.     

Monitoring intracellular protein profiles with two-dimensional gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is a method of separating complex protein mixtures, such as whole cell extracts, on the basis of protein isoelectric point and molecular weight. In bioprocess engineering, conventional 2D PAGE has tremendous potential to yield detailed information on the intracellular effect of various process conditions. It has been used in our work to examine global intracellular changes occurring in a typical cycloheximide fermentation and to look at the feedback regulatory behavior of cycloheximide biosynthesis. Application of the technique for bioprocess monitoring will require that the time necessary for preparation of a 2D electropherogram be substantially shortened. This may be accomplished by performing the separation on a miniature scale or eventually by use of capillary electrophoresis for one or more of the separations. Advantages and disadvantages of these two approaches are discussed.     

Enzymes of the human intestinal brush border membrane. Identification after gel electrophoretic separation

The position of a number of human intestine brush border membrane enzyme activities in polyacrylamide gels after electrophoresis has been determined. These activities are, in order from the origin, maltase/glucoamylase, lactase/phlorizin hydrolase, maltase/sucrase/isomaltase, enteropeptidase, trehalase and γ-glutamyltransferase. Leucylnaphthylamide hydrolyzing activity was inactivated by sodium dodecylsulfate and its position was not determined. The positions of the activities have been correlated with the positions of protein bands previously determined. One such band situated between enteropeptidase and alkaline phosphatase has not been identified.     

Protein staining and pH gradient determination on the same gel in isoelectric focusing.

The apparent isoelectric point of a component focused on polyacrylamide gels is normally estimated by extrapolating a pH gradient determined on one gel to another gel which has been stained for protein in order to locate the position of the component (1). The pH gradient is determined by slicing the gel transversely and reading the pH of the eluate after soaking the segments for 1–2 hr in a small amount of degassed water. It is assumed that the gradients in both gels are identical. Alternatively, an antimony microelectrode has been used to measure pH gradients directly in unsectioned gels (2). Similar techniques have been applied to polyacrylamide gel slabs and are reviewed by Vesterberg (3). Righetti and Drysdale (4) have recently reviewed these and other aspects of isoelectric focusing in gels.I report here a very precise method for the determination of a protein “isoelectric point” that can be accomplished with a single gel. The technique is demonstrated with yeast phosphoglycerate kinase and the very low density lipoprotein (VLDL) fraction from human plasma.     

SDS-polyacrylamide gel electrophoresis of lipopolysaccharides.

Lipopolysaccharides (LPS) prepared from four different species of Neisseria have been separated by SDS-polyacrylamide gel electrophoresis. Each LPS possessed a characteristic mobility on gels. Examination of the effect of acrylamide concentration on migration illustrated that the basis of the separation was molecular size and not intrinsic charge.     

Large-scale isolation of equine liver alcohol dehydrogenase on a blue agarose gel.

Equine liver alcohol dehydrogenase (EC 1.1.1.1) has been purified by a new scheme using a blue agarose gel (Blue Sepharose) as an affinity sorbent. Starting amounts of 0.6 to 10 kg liver have been processed to enzyme possessing 1.5 U/mg average specific activity, in about three to four days. Some parameters concernining adsorption of enzyme to the blue gel as well as recovery therefrom have been explored.     

Unhooking dynamics of U-shaped DNA molecule undergoing gel electrophoresis.

It has been found that DNA molecules are often hooked around obstacles in a U-shaped configuration in gel electrophoresis. To understand the dynamics of the unhooking of U-shaped DNA molecules undergoing gel electrophoresis, we have examined the length changes of the longer and shorter arms of the U-shape as a function of time. Two types of unhooking have been found. In one type, the length changes of both arms are expontential in time but with different time constants. In another type, the length changes of the shorter arm is exponential and that of the longer one is linear with time. The interpretation is that the extent of stretch of the spring-like DNA chain decreases as the length difference between the two arms increases during the unhooking processes, and that the frictions at the pivot point can be relatively large depending upon the local structure of the gel. The friction coefficient at the pivot point is estimated to be nu 0 = (2.98 +/- 1.42)x10(-5) g/sec.     

Estimation of ruminal bacteriophage numbers by pulsed-field gel electrophoresis and laser densitometry.

To investigate phage activity in the rumen, a method for quantifying phage has been developed. By differential centrifugation and ultrafiltration, phage particles were separated and concentrated from ruminal fluid. Linear double-stranded DNA from this fraction containing predominantly tailed phage was isolated and separated by size, using pulsed-field gel electrophoresis (PFGE). Laser densitometry of gel photographs allowed the numbers of phages with DNA in each size region to be calculated and, therefore, the total numbers per milliliter of ruminal fluid to be estimated. Phage numbers were estimated to be between 3 x 10(9) and 1.6 x 10(10) particles ml of ruminal fluid-1. The phage population, as gauged by the appearance of DNA on PFGE gels, had two major components. A broad region of DNA between 30 and 200 kb was always present on PFGE gels. It appears this region comprises DNA from a great many different phages and would include most of the temperate phages. In addition, discrete DNA bands ranging in size from 10 to 850 kb were frequently observed. DNA from one such band, of 12 kb in size, was shown to consist primarily of a single DNA type, suggesting that it originated from a specific phage. It is postulated that the discrete bands are due to epidemics or blooms of phage activity from specific, probably lytic, phages. The method that has been developed will greatly enhance future investigations into the interactions between the ruminal phage population, the ruminal bacterial population, and animal nutrition and growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cosmid mapping of the human chorionic gonadotropin beta subunit genes by field-inversion gel electrophoresis.

A cosmid clone containing the entire hCG beta gene cluster has been isolated. The restriction map of this clone has been determined by an indirect-end-label FIGE (field inversion gel electrophoresis) method. Analysis of this cosmid clone shows that there are 6 hCG beta genes in human genomic DNA. A previously uncloned portion of the hCG beta cluster, termed the "gap" region, has been shown not to contain any sequences homologous to the hCG beta cDNA. The restriction mapping method employed in this study takes advantage of the superior resolution of FIGE for high molecular weight DNA fragments in the size range 15-50 kb. This method is broadly applicable and permits rapid and accurate restriction mapping for extended regions of genomic DNA that have been cloned into cosmid or lambda vectors.     

Polyacrylamide gel electrophoresis of intact bacteriophage T4D particles.

A method for the electrophoresis of intact bacteriophage T4D particles through polyacrylamide gels has been developed. It was found that phage particles will migrate through dilute polyacrylamide gels (less than 2.1%) in the presence of a low concentration of MgCl2. As few as 5 x 10(9) phage particles can be seen directly as a light-scattering band during the course of electrophoresis. The band can also be detected by scanning gels at 260 to 265 nm or by eluting viable phage particles from gel slices. A new mutant (eph1) has been identified on the basis of its decreased electrophoretic mobility compared with that of the wild type; mutant particles migrated 14% slower than the wild type particles at pH 8.3 and 35% slower at pH 5.0. The isoelectric points of both the wild type and eph1 mutant were found to be between pH 4.0 and 5.0. Particles of T4 with different head lengths were also studied. Petite particles (heads 20% shorter than normal) migrated at the same rate as normal-size particles. Giant particles, heterogenous with respect to head length (two to nine times normal), migrated faster than normal-size particles as a diffuse band. This diffuseness was due to separation within the band of particles having mobilities ranging from 8 to 35% faster than those of normal-size particles. These observations extend the useful range of polyacrylamide gel electrophoresis to include much larger particles than have previously been studied, including most viruses.     

Simplified polyacrylamide gel electrophoresis and silver staining for analysis and preparation of influenza virus RNA segments.

The influenza virus has a genome consisting of eight RNA segments. A simplified technique to study the RNA segmental pattern by silver staining after gel electrophoresis has been developed. In addition, individual RNA segments could be isolated by a combination of polyacrylamide gel electrophoresis and isotachophoresis.     

Isoelectric focusing in polyacrylamide gel using buffer electrode solutions.

Isoelectric focusing in ampholytes of pH 6–8 range has been carried out in polyacrylamide gels using ammonia buffer at pH 10.0 and acetate buffer at pH 4.0 for the cathode and anode solutions, respectively. This system requires low voltages but compares well with isoelectric focusing using strong acid and strong base electrode solutions. The advantages of this method are the less extreme pH's in the electrode solutions and lower resistance in the neutral region of the pH gradients.     

Multiple forms of oviduct progesterone receptors analyzed by ion exchange filtration and gel electrophoresis.

Resolution of the multiple forms of steroid receptors in small samples has been improved by two new techniques: preparative ion exchange filtration and electrophoresis in highly cross-linked polyacrylamide gels of varied concentration. These techniques were used in conjunction with protamine precipitation, gel filtration, and density gradient centrifugation to separate five forms of the progesterone receptor of chick oviduct cytosol. These complexes, numbered I to V in order of elution from agarose gel columns, have been characterized with respect to apparent molecular weight, shape, and relative net charge. Form I, which is eluted in the void volume after gel filtration of cytosol in hypotonic media, is heterodisperse with respect to sedimentation coefficient and electrophoretic mobility (Rf). Form I is converted to form III by KC1. Form II has the highest axial ratio and the highest Rf at pH 10.2. This 4.2S complex can be extracted from DEAE filters, but not from protamine-precipitated cytosol, by 0.3 to 0.5 M KC1. Form III is slightly smaller (3.9S) and less asymmetric than form II. It is relased from DEAE filters and protamine-precipitated cytosol by 0.15 M KC1 and displays increased Rf upon purification. Forms II and III correspond to the B and A components described by W. T. Schrader and B. W. O'Malley ((1972), J. Biol. Chem 247, 51). Form IV may result from the proteolytic cleavage of forms II and/or III. Form V is a globular polypeptide obtained in the presence of certain divalent cations. This complex has been named the "mero-receptor" since it is the smallest part or fragment of the receptor that contains the steroid-binding site.