Enzymes In Polyacrylamide Gels Research Articles

The Multiple Forms of α-Amylase Enzyme of the Araucaria Species of South America: A. araucana (Mol.) Koch and A. angustifolia (Bert.) O. Kutz

alpha-Amylase is one of the major enzymes present in the seeds of both Araucaria species of South America and it initiates starch hydrolysis during germination and early seedling growth. The pattern of the multiple forms of alpha-amylase of the two Araucaria species was investigated by electrophoresis and isoelectrofocusing of the native enzyme in polyacrylamide gels. The enzyme forms were compared in the embryo and megagametophyte of quiescent seeds and of seeds imbibed for 18, 48, and 90 hours. Specific alpha-amylase enzyme forms appear and disappear during these imbibition periods showing both similarities and differences between tissues and species. Before imbibition, there are five alpha-amylase forms identical in both tissues, but different between species. After 18 hours of imbibition, there are two enzyme forms in both tissues of Araucaria araucana seeds, only one form in the embryo of Araucaria angustifolia but two forms in the megagametophyte of this specie. After 48 hours of seed imbibition, most of the enzyme forms present in quiescent seeds reappear. At 90 hours of imbibition different enzyme forms are detected in the embryo with respect to the gametophyte. The changes in form patterns of alpha-amylase are discussed according to a possible regulation of gene expression by endogenous gibberellins.

Molecular properties of phosphoenolpyruvate carboxylase from C3, C3?C4 intermediate, and C4 Flaveria species

Phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) from Flaveria trinervia Mohr (C4), F. floridana Johnston (C3-C4), and F. cronquistii Powell (C3) leaves were compared by electrotransfer blotting/enzyme-linked immunoassay (Western-blot analysis), mobility of the native enzyme in polyacrylamide gels and in isoelectric focusing (IEF) gels, peptide mapping, and in-vitro translation of RNA isolated from each plant. The PEPCases from the C3 and C3-C4 plants were very similar to each other in terms of electrophoretic mobilities on gels and isoenzyme patterns on IEF gels, and identical in peptide mapping. Quantitative differences were noted, however, in that the C3-C4 intermediate plant contained more PEPCase overall and that the relative activity of individual isoenzymes shifted between the C3 and C3-C4 intermediate PEPCases. The PEPCase from the C4 plant had a different isoenzyme pattern, a different peptide map, and was far more abundant than the other two enzymes. Western blot analysis demonstrated the cross-reactivity of PEPCases from all three Flaveria species with antibody raised against maize PEPCase. The results provide evidence, at the molecular level, that supports the view of C3-C4 intermediate species as C3-like plants with some C4-like photosynthetic characteristics, but there are differences from the C3 plant in the quantity and properties of the PEPCase from the C3-C4 intermediate plant.

Electrophoretic Transfer as a Technique for the Detection and Identification of Plant Amylolytic Enzymes in Polyacrylamide Gels

An electrophoretic transfer technique was developed for the specific identification of isozymes of starch debranching enzyme, alpha-amylase, and beta-amylase. Amylolytic enzymes are separated by native polyacrylamide slab gel electrophoresis and proteins in gels are electrophoretically transferred through starch-containing polyacrylamide gels. Each amylolytic enzyme degrades starch in the transfer gel to its characteristic limit dextrin as it moves through the gel. Various limit dextrins in the starch gel are identified by their characteristic color development in KI/I solution. Isozymes of starch debranching enzyme, alpha-amylase, and beta-amylase can be easily identified in the same gel.

Purification and characterization of glucosidase II, an endoplasmic reticulum hydrolase involved in glycoprotein biosynthesis.

Rat liver glucosidase II, an endoplasmic reticulum hydrolase involved in the biosynthesis of the N-linked class of glycoproteins, has been purified in good yield to a state approaching homogeneity. The purified enzyme hydrolyzes p-nitrophenyl-alpha-D-glucopyranoside, 4-methylumbelliferyl-alpha-D-glucopyranoside, maltose, and the precursor oligosaccharides glucose1-2mannose9N-acetylglucosamine, but it does not act on glucose3mannose9N-acetylglucosamine or p-nitrophenyl-beta-D-glucopyranoside. The ratio of the rate at which glucose is released from p-nitrophenyl-alpha-D-glucopyranoside to that from glucose2mannose9N-acetylglucosamine or glucose1mannose9N-acetylglucosamine remains constant throughout the 8-step purification procedure; thus it appears that a single enzyme is responsible for the activities toward both the artificial and oligosaccharide substrates. The fact that the enzyme cleaves both of the inner 1,3-linked glucosyl residues from the precursor oligosaccharides supports the view that they are linked in the alpha-configuration. The pH dependence of enzymatic activity is quite similar for different substrates, showing a broad optimum between pH 6 and 7.5. Activity toward p-nitrophenyl-alpha-D-glucopyranoside is enhanced by 12 mM 2-deoxy-D-glucose (260-300% activation) and 25 mM mannose (150% activation), but these two compounds inhibit the action of the enzyme toward the precursor oligosaccharides. By isoelectrofocusing the purified enzyme exhibits one form, which has a pI of 3.5-3.8. Reductive polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicates that glucosidase II has a subunit molecular weight of 65,000. Ferguson plot analysis of the behavior of native enzyme in polyacrylamide gels indicates that it is a 262,000-dalton tetramer. Gel filtration gives a molecular weight of 288,000. Several lines of evidence indicate that the enzyme is a glycoprotein.

Renaturation and localization of enzymes in polyacrylamide gels: studies with UDPglucose pyrophosphorylase of Dictyostelium.

We have developed procedures for renaturing, in polyacrylamide isoelectric focusing gels, homomeric enzymes (i.e., enzymes with identical subunits) that have been denatured with sodium dodecyl sulfate or urea or both. The renatured enzymes can then be localized as discrete species by conventional histochemical staining. One of these enzymes, uridine diphosphoglucose pyrophosphorylase (UTP:alpha-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.9) of Dictyostelium discoideum, was studied in detail. Conditions have been established for renaturing and localizing this enzyme and for quantitating the amount of activity recovered. Up to 40% of the activity can be recovered after renaturation. This procedure is widely applicable because several enzymes, including alcohol dehydrogenase (EC 1.1.1.1) and lactate dehydrogenase (EC 1.1.1.27), can be localized. It is sensitive enough to resolve isozymes and enzyme variants that differ by a single charged amino acid. It can be used to localize enzymes in crude cell extracts that have been resolved in two-dimensional slab gels by sodium dodecyl sulfate electrophoresis and isoelectric focusing. These methods should allow detailed analysis of genes and their enzyme proteins that, though present in small amounts in eukaryotic cells, perform important metabolic or developmental functions.

Genetic variability in deep-sea echinoderms

The electrophoretic mobility of enzymes in polyacrylamide gels was used a measure of genetic variability in deep-sea echinoderms. Both variable and monomorphic species were found. The levels of polymorphism and heterozygosity were similar to those of species living in shallow-water and terrestrial environments, evidence that these are unrelated to climatic stability. We propose that polymorphism is more probably related to the intensity of selection pressures and a reflection of biological fluctuations. A tendency toward a deficiency of heterozygotes from the number expected in Hardy-Weinberg equilibrium suggests that populations of the ophiuroid Ophiomusium lymani may be genetically isolated.

Nucleotide pyrophosphatase of rat liver. A comparative study on the enzymes solubilized and purified from plasma membrane and endoplasmic reticulum.

Studies on the subcellular distribution of rat liver nucleotide pyrophosphatase activity revealed its presence in the plasma membrane and the endoplasmic reticulum only. The enzymes from either source were solubilized specifically with trypsin without an apparent change of their catalytic properties. A 200-fold and 1600-fold purification, respectively, was achieved by a procedure including DEAE-cellulose and affinity-chromatography with AMP as ligand, gel filtration on Sephadex G-200 and gel electrophoresis. Both nucleotide pyrophosphatases were isolated as electrophoretically homogeneous soluble proteins. They were shown to contain carbohydrate moieties. The electrophoretic mobility of both enzymes in polyacrylamide gels was identical at three pH values. Dodecylsulfate gel electrophoresis indicated a molecular weight of 137 000 for both glycoproteins. The enzymes hydrolyze a variety of purine and pyrimidine nucleotides yielding a 5'-nucleoside monophosphate. Adenosine 3':5'-monophosphate, nucleic acids and phosphate monoesters are not cleaved, but p-nitrophenyl-thymidine5'-monophosphate is readily hydrolyzed. In view of their substrate and inhibitor specificities the enzymes are considered nucleotide pyrophosphatases rather than phosphodiesterases.

The detection of hexokinase, glucosephosphate isomerase and phosphoglucomutase activities in polyacrylamide gels after electrophoresis: A novel method using immobilized glucose 6-phosphate dehydrogenase

A new method has been developed for the histochemical detection of enzymes in polyacrylamide gels, in which the auxiliary enzyme is polymerized into the gel prior to electrophoresis. After electrophoresis of the sample, the immobilized auxiliary enzyme remains present and active throughout the length of the gel to enable zones of sample activity to be detected with high sensitivity and resolution. The particular auxiliary enzyme used was glucose 6-phosphate dehydrogenase, which allowed detection of hexokinase, glucosephosphate isomerase, and phosphoglucomutase; however, it seems likely that the immobilization method is applicable to a large number of proteins. To retain a maximum of activity, the enzyme must be in contact with acrylamide monomer for as short a time as possible before polymerization occurs. After polymerization, the enzyme is completely immobilized, and bound covalently to the gel matrix via a free sulfhydryl or amino group.

Methods for Visualization of Enzymes in Polyacrylamide Gels

White bands resulting from precipitation of dodecan-1-ol liberated by hydrolysis of sodium dodecyl sulfate and decan-5-ol released by hydrolysis of decan-5-yl sulfate produced zymograms of the primary and secondary alkylsulfatases from Pseudomonas C(12)B. Gas-liquid chromatographic analyses of ether extracts of the precipitate-containing segments of the zymograms confirmed the identity of the alcohols which were not discerned in extracts of segments of the gels other than those containing precipitates. beta-Galactosidase from Escherichia coli was marked on zymograms by the liberation of o-nitrophenol from o-nitrophenyl-beta-D-galactoside, and arylsulfatase from Pseudomonas C(12)B was marked in gels by liberation of p-nitrophenol from p-nitrophenyl sulfate. Membrane-associated dissimilatory nitrate reductases from a nitrate respirer (Enterobacter aerogenes) and a denitrifier (Pseudomonas perfectomarinus) did not penetrate either 6.8 or 3% polyacrylamide gel but were demonstrable at the top of the gels. In the membrane-bound state, formate served as electron donor for nitrate reductase from E. aerogenes, and reduced nicotinamide adenine dinucleotide (NADH) served as donor for nitrate reductase from P. perfectomarinus. Both enzymes reduced nitrate at the expense of reduced benzyl viologen as well. Assimilatory nitrate reductase from E. aerogenes moved easily into the 6.8% gels (R(f) = 0.43 under the conditions of these experiments). The reduced dye served as electron donor for the assimilatory reductase, but formate and NADH did not. Incubation of the membrane-associated nitrate reductases with 2% Triton X-100 solubilized the enzymes and removed the capacity of formate and NADH to serve as electron donors. Both retained the ability to reduce nitrate at the expense of reduced benzyl viologen. The solubilized dissimilatory reductase from E. aerogenes moved further in the gels (R(f) = 0.49) than the soluble assimilatory reductase; the solubilized dissimilatory reductase from the denitrifier, P. perfectomarinus, moved further in the gels (R(f) = 0.64) than either of the enzymes from E. aerogenes.

Methods for Visualization of Enzymes in Polyacrylamide Gels

White bands resulting from precipitation of dodecan-1-ol liberated by hydrolysis of sodium dodecyl sulfate and decan-5-ol released by hydrolysis of decan-5-yl sulfate produced zymograms of the primary and secondary alkylsulfatases from Pseudomonas C 12 B. Gas-liquid chromatographic analyses of ether extracts of the precipitate-containing segments of the zymograms confirmed the identity of the alcohols which were not discerned in extracts of segments of the gels other than those containing precipitates. β-Galactosidase from Escherichia coli was marked on zymograms by the liberation of o -nitrophenol from o -nitrophenyl-β-D-galactoside, and arylsulfatase from Pseudomonas C 12 B was marked in gels by liberation of p -nitrophenol from p -nitrophenyl sulfate. Membrane-associated dissimilatory nitrate reductases from a nitrate respirer ( Enterobacter aerogenes ) and a denitrifier ( Pseudomonas perfectomarinus ) did not penetrate either 6.8 or 3% polyacrylamide gel but were demonstrable at the top of the gels. In the membrane-bound state, formate served as electron donor for nitrate reductase from E. aerogenes , and reduced nicotinamide adenine dinucleotide (NADH) served as donor for nitrate reductase from P. perfectomarinus . Both enzymes reduced nitrate at the expense of reduced benzyl viologen as well. Assimilatory nitrate reductase from E. aerogenes moved easily into the 6.8% gels ( R f = 0.43 under the conditions of these experiments). The reduced dye served as electron donor for the assimilatory reductase, but formate and NADH did not. Incubation of the membrane-associated nitrate reductases with 2% Triton X-100 solubilized the enzymes and removed the capacity of formate and NADH to serve as electron donors. Both retained the ability to reduce nitrate at the expense of reduced benzyl viologen. The solubilized dissimilatory reductase from E. aerogenes moved further in the gels ( R f = 0.49) than the soluble assimilatory reductase; the solubilized dissimilatory reductase from the denitrifier, P. perfectomarinus , moved further in the gels ( R f = 0.64) than either of the enzymes from E. aerogenes .

Escherichia coli Phosphoenolpyruvate Carboxylase: PHYSICAL AND CHEMICAL PROPERTIES

Abstract Phosphoenolpyruvate carboxylase of Escherichia coli was purified to homogeneity. The molecular weight of the native protein, as determined by sedimentation equilibrium analyses, is 402,000. Sedimentation equilibrium analyses on the enzyme denatured in 3.5 m, 5 m, and 7 m guanidine hydrochloride gave a single species with a molecular weight of 99,590. Electrophoretic analyses of the enzyme in polyacrylamide gels in sodium dodecyl sulfate also gave a single species with a molecular weight of 107,000. These data indicate that the carboxylase is composed of four identical subunits, each having a molecular weight of approximately 100,000. Amino acid analyses indicate the presence of 36 cysteine residues per mole, all of which appear to exist as free sulfhydryl groups. The enzyme does not appear to be a metalloprotein. The partial specific volume is 0.737, the sedimentation coefficient is 12.3, and the specific reaction rate is 3.77 x 104 moles of substrate carboxylated per min per mole of enzyme. The diffusion coefficient and the frictional coefficient (f/f0) calculated from the molecular weight, V, and S gave values of 2.84 x 10-7 cm2 sec-1 and 1.53, respectively.

Phosphatases: Applications of new methods for their separation

1. 1. Phosphomono- and phosphodiesterases from several microorganisms and two photosynthetic tissues have been examined by gel electrophoresis. Each species has a unique pattern of these two enzymes. 2. 2. A nondestructive method for locating these enzymes in polyacrylamide gels is described that enables one to differentiate between phosphomono- and phosphodiesterases and that provides a rapid method for identifying cell extracts from the patterns of phosphatases present. 3. 3. Substrate affinity chromatography on cellulose phosphate columns has been successfully used to separate phosphomonoesterases from crude cell extracts. Also columns of glyceryl cellulose phosphate retard the elution of phosphodiesterases but not other proteins. 4. 4. This affinity chromatography can be used to remove phosphatases from crude extracts or to purify phosphatases, either phosphomono- or diesterases.

Preparatin, proteolysis and reversible oxidationof highly purified Azotobacter vinelandii polynucleotide phosphorylase.

1. A new method has been developed for the preparation in good yield of highly purified Azotobacter vinelandii polynucleotide phosphorylase in its reduced form. 2. Aging or digestion with trypsin causes the enzyme to develop a primer requirement that is not eliminated by beta-mercaptoethanol. 3. The development of a primer requirement is accompanied by marked changes of the electrophoretic mobility of the enzyme in polyacrylamide gels. 4. The enzyme is inactivated by aerial oxidation or thiol-specific reagents. The lost activity is restored by beta-mercaptoethanol, but not by oligonucleotide primers.

Diphosphopyridine Nucleotide-linked Isocitrate Dehydrogenase from Bovine Heart: POLYMERIC FORMS AND SUBUNITS

Abstract The molecular weight of a DPN-linked isocitrate dehydrogenase preparation, exhibiting a single major band on polyacrylamide disc gel electrophoresis, has been examined by molecular sieve chromatography. Comparison of the elution volume (Ve) of the enzyme with reference proteins of known molecular weight on columns of Sephadex G-200 and Sepharose 6B permitted calculation of a molecular weight in the range 290,000 to 365,000. Inclusion of the positive modifier ADP (0.035 mm to 1.0 mm) in the eluting buffer (0.15 m potassium phosphate-0.1 mm dithiothreitol at pH 7.2) of columns of Sepharose 6B revealed an aggregate of the enzyme of molecular weight 670,000 ± 20,000; a species of apparent molecular weight 1,100,000 was observed with 0.8 m ammonium sulfate-5 mm potassium phosphate-0.1 mm dithiothreitol buffer (pH 7.1) containing 0.05 mm to 1.0 mm ADP. Aggregate formation is dependent on protein concentration. Thus, a monomer-dimer equilibrium could be shown when varying amounts of the enzyme were chromatographed on Sepharose 6B in 0.1 mm ADP-0.15 m potassium phosphate-0.1 mm dithiothreitol at pH 7.2. The limiting values of molecular weight were 330,000 and 680,000 at low and high protein concentrations, respectively. The dissociation constant of the monomer-dimer equilibrium was found to be K = 0.25 ± 0.12 mg per ml. The electrophoretic mobility of the enzyme in polyacrylamide gels containing sodium dodecyl sulfate at pH 7.1 or 8 m urea at pH 2.9 indicated polypeptide chains of average molecular weight 40,800 ± 500; a value of 40,900 ± 700 was obtained by chromatography on Bio-Gel A-5 M in 6 m guanidine hydrochloride. This suggests that the enzymically active monomer of molecular weight 330,000 is composed of eight subunits.