Sodium Dodecyl Sulfate-disc Gel Electrophoresis Research Articles

Purification and properties of an endo-α-mannan hydrolase from the mushroom Volvariella volvacea

The enzyme, endo-α-mannanase, from culture filtrate of a mushroom Volvariella volvacea has been purified 73-fold by acetone precipitation, ion-exchange chromatography (DEAE-Sephadex), and gel-permeation chromatographies on Bio-Gel P-300 and on Sephacryl S-200 columns. The enzyme preparation gave a single protein band on sodium dodecyl sulfate-disc gel electrophoresis at pH 6.8 and has a molecular weight of approx. 56,000. It has no α- or β-mannosidase activity and does not act on β-gluco-or galactomannan. The enzyme shows maximum activity on baker's yeast α-mannan at pH 5.0 and at 55 °C, and is fairly stable between pH 3 and 6 and temperatures up to 50 °C. The K m is 32.25 mg mannan/ml. Enzyme activity is inhibited by Hg 2+, sodium azide, iodoacetic acid, EDTA, and Ag +, in decreasing order.

Caractérisation biochimique et biophysique du collagène de l'insecte Tenebrio molitor L. (coleoptera, tenebrionidae)

The collagen from the mesenteric sheath of the tenebrionid insect Tenebrio molitor was extracted by limited pepsin digestion and purified. This collagen was characterized using CM-cellulose chromatography, sodium-dodecylsulfate disc-gel electrophoresis and aminoacid analysis. This molecule was found to be assembled from three identical alpha chains and could be represented by the formula (alpha) 3. The amino acid composition is characteristic of collagen (one-third glycine, high iminoacid content), with high content of hydroxylysine and low content of alanine. Cyanogen bromide digests of these chains indicated that they are not related to any of the known invertebrate or vertebrate chains of interstitial collagens. The molecular weight (M = 280000D) and length (290 nm) were typical, and the banding patterns of the segment-long-spacing crystallites (SLS) and of the reconstitued fibrils were very similar to type I collagen. The denaturation temperature (Td) was 30.7 degrees C and correlated with the total pyrrolidine content as observed in other collagens (von Hippel & Wong's relation). It was concluded that the collagen from this insect showed the classical biochemical and biophysical features of other invertebrate interstitial "primitive" collagens.

Immunochemical localization of phenylalanine ammonia-lyase and chalcone synthase in anthers

Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) from anthers of the garden tulip "Apeldoorn" have been purified to apparent homogeneity as revealed by sodium dodecyl sulfate disc-gel electrophoresis. Phenylalanine ammonia-lyase was either purified by successive chromatography on Sephacryl S 300 Superfine, HA Ultrogel and on diethylaminoethyl Sephacel or by immunoaffinity chromatography in a single step. Purification of CHS was achieved by chromatography on Sephadex G 200 and on HA Ultrogel followed by chromatofocusing. The purified enzymes were used for the immunization of rabbits. The specificity of the antisera against both PAL and CHS was tested by diverse methods. Antisera against PAL and CHS were employed to detect the localization of the enzymes in cross sections of tulip anthers using an indirect immunofluorometric method. The results show that PAL and CHS are located predominantly in the tapetum cells. These observations strengthen the view that the tapetum plays an important role in the regulation of phenylpropanoid metabolism within the loculus of anthers.

The folate-binding protein of rat kidney. Purification, properties, and cellular distribution.

Folate-binding protein (FBP) from rat kidney was isolated, and its properties and location in the kidney were determined. The particulate fraction of rat kidney homogenate was freed of its bound folate, solubilized with Triton X-100, and the FBP was purified using a combination of DEAE-cellulose and affinity chromatography. The purified protein migrated as a single band on sodium dodecyl sulfate-disc gel electrophoresis, has an isoelectric point of 5.7, contains 21.7% carbohydrate, and has an Mr of 28,500-30,000. The purified protein retained its affinities for different folate derivatives and its sensitivity to inorganic anions. Inorganic anions enhanced the binding of 5-methyltetrahydrofolate; chloride ion was the most effective, followed by Br- greater than I- greater than SO2-4. Chloride ion was also found to lower the dissociation constant of the folic acid-FBP complex at 50 degrees C by about 10-fold. This effect is thought to derive from the formation of a ternary FBP-folic acid-Cl- complex which is more stable than the binary FBP-folic acid complex. An antiserum raised against the purified protein in rabbits was used to determine the location of FBP in the kidney by immunofluorescence. Intense fluorescence staining for FBP was localized at the apices (brush border) of proximal tubules. The choroid plexus, an organ previously shown to contain FBP, also exhibited intense fluorescent staining.

6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase from rat liver.

Rat liver 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was phosphorylated by [gamma-32P]ATP plus fructose-6-P or [2-32P]fructose-2,6-P2. The radioactivity co-migrated with homogeneous 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase during sodium dodecyl sulfate-disc gel electrophoresis and the phosphoenzyme was acid-labile and base-stable. Hydrolysis of the phosphate group from phosphoenzyme prepared with either donor and the hydrolysis of their tryptic phosphopeptides depended on pH similarly. The pH dependence suggested that phosphate was linked to the N3 of a histidine residue. Co-electrophoresis and co-chromatography of alkaline hydrolysates of the labeled phosphoenzyme prepared from either substrate allowed the definitive identification of 3-phosphohistidine in the degradation products. Modification of the enzyme with diethylpyrocarbonate inactivated both the phosphotransferase and phosphohydrolase activities and suppressed the phosphorylation of the enzyme by ATP and fructose-6-P or by fructose-2,6-P2. Trypsin digestion of the phosphoenzyme formed upon incubation with ATP or fructose-2,6-P2 yielded an identical phosphopeptide after high pressure liquid chromatography. All the above data are consistent with this enzyme catalyzing both phosphohydrolase and phosphotransferase reactions with the mediation of phosphohistidine in the reaction scheme(s).

Rabbit skeletal muscle calcium-dependent protease requiring millimolar CA2+. Purification, subunit structure, and Ca2+-dependent autoproteolysis.

Rabbit skeletal muscle calcium-dependent protease which requires millimolar Ca2+ concentration for activity is a dimer composed of Mr = 73,000 and 30,000 subunits. The subunit structure has been confirmed by co-elution of the two polypeptide bands on Sephadex G-200 chromatography, and by cross-linking the enzyme with dimethyl suberimidate followed by sodium dodecyl sulfate-disc gel electrophoresis. Preincubation of the enzyme with p-chloromercuribenzoate prior to nondenaturing electrophoresis resulted in dissociation of the subunits. The rabbit muscle calcium-dependent protease was rapidly inactivated when incubated in the presence of 6 mM Ca2+. The half-life of protease activity at 30 degrees C was independent of protease concentrations over the range of 0.005 to 0.272 mg/ml. The rate of inactivation was not affected by a 270-fold molar excess of a substrate protein succinylated lysozyme. Protease activity also rapidly decreased (t1/2 = 8.4 min) during the assay at 37 degrees C as determined by a decrease in linearity of the time course when substrate was limiting. The rate of protease inactivation during the assay was essentially the same as that observed when the protease was incubated at 37 degrees C in the absence of substrate (t1/2 = 7.2 min). The addition of either leupeptin or the calcium-dependent protease inhibitor protein from dog heart prevented protease inactivation. The protease displayed an increase in activity during the time course of autoproteolysis at 30 degrees C when activity was measured in the presence of 0.2 mM Ca2+ instead of 5 mM Ca2+.

Bakers' yeast protease a purification and enzymatic and molecular properties

It has been previously demonstrated in our laboratory that uridine nucleosidase (EC 3.2.2.3) is inactivated by yeast protease A (EC 3.4.23.8). A complete purification procedure for protease A from bakers' yeast, which lacks the acidic activation step used by other workers, and the major properties of the enzyme are shown. The enzyme is homogeneous as judged by disc gel electrophoresis. Its molecular weight, calculated from both sodium dodecyl sulfate-disc gel electrophoresis and gel filtration experiments, is around 45,000. The protein does not possess quaternary structure. The isoelectric point is 4.1. Carbohydrate content is around 8%. Amino acids analysis and sulfur analysis reveal the presence of 1-SH group and two disulfide bridges. The free-SH group does not seem to be involved in catalysis. Amino terminal analysis shows that isoleucine is at the amino terminal position. The pH optima are 2.4 for the hydrolysis of azocasein and casein, and 3.3 for the hydrolysis of hemoglobin. The K m value for hemoglobin is 1.7 × 10 −5 m. The inhibition exerted by pepstatin on the proteolytic activity of protease A is pH dependent. Among various yest enzyme substrates only uridine nucleosidase is inactivated by protease A.

Protease digestion of colonic mucin. Evidence for the existence of two immunochemically distinct mucins.

Colonic mucin was prepared by phenol-water partition extraction of th colonic mucosa, followed by ethanol precipitation of the water-soluble material, molecular sieve chromatography, and reductive sodium dodecyl sulfate-disc gel electrophoresis in an agarose-polyacrylamide gradient gel. Although this material as observed to be relatively homogeneous by physical criteria, it was shown to contain at least two components by immunodiffusion. Molecular sieve, ion exchange, adsorption, lectin affinity, and electrophorectic techniques failed to separate the two components. However, by protease digestion employing either 1% w/w pronase or papain, followed by molecular sieve chromatography, we were able to separate at least the portions of the molecules containing the immunodeterminants. Physicochemical and immunologic properties of both components were characteristic of mucins. Mucin A (pronase digest fraction 1 from molecular sieve chromatography) was enriched in threonine, proline, and sialic acid, while mucin B (pronase digest fraction 3 from molecular sieve chromatography) was enriched in serine, alanine, and fucose. Mucin A and mucin b were shown to be immunologically distinct. The evidence suggests that the protease-digested mucin fractions originate from two different mucins, one a sialomucin, the other a fucomucin; however, the alternative explanation, that these two mucin fragments may originate from a single mucin molecule, cannot be ruled out at present.

31] Preparation of human leukocyte interferon in the presence of tunicamycin and its characterization

This chapter presents methodology to show that the molecular size heterogeneity of human leukocyte interferon (HLIF) can be eliminated when interferon is produced in the presence of tunicamycin, a specific glycosylation inhibitor. The evidence suggests that the molecular basis of this heterogeneity may be the relative extent of glycosylation of its molecular components. Tunicamycin is used as a specific inhibitor, to probe the glycosylation status of HLIF. Tunicamycin is shown to alter the properties of HLIF molecules that are released into the production media. The use of tunicamycin as a probe, is particularly revealing, because its inhibitory action suggests the N-glycosidic nature of linkage between the polypeptide and its saccharide moiety. HLIF is known to contain two distinct molecular weight components when subjected to sodium dodecyl sulfate-disc gel electrophoresis: 21,000 and 16,000 dalton components in the approximate ratio of 3:7. If HLIF contains sialic acid, then impairment of its glycosylation by tunicamycin should alter its behavior on isoelectric focusing.

Purification and properties of sarcosine oxidase from Cylindrocarpon didymum M-1.

Sarcosine oxidase was purified to homogeneity from the cell extract of Cylindrocarpon didymum M–1, aerobically grown in medium containing choline as the carbon source. The molecular weight of the enzyme was estimated to be 45,000 by gel filtration method and 48,000 by the sodium dodecylsulfate disc gel electrophoresis method. The enzyme exhibited an absorption spectrum with maxima at 277 and 450 run and shoulders at 370 and 470 nm. The anaerobic addition of sarcosine to the enzyme resulted in the disappearance of the peak at 450 nm. The enzyme contained one mol of covalently bound FAD per mol of enzyme. Enzyme activity was inhibited by Ag+, Cu2+, Hg2+, p-chloromercuribenzoate and iodoacetate. The enzyme oxidized sarcosine but was inert toward choline, betaine, dimethylglycine and N-methyl amino acids. Km and Vmax values for sarcosine were 1.8 ihm and 26.2 μmol/min/mg, respectively. The enzyme catalyzed the following reaction: Sarcosine+O2+H2O→glycine +formaldehyde+H2O2.

Boar malpha-acrosin. Purification and characterization of the inital active enzyme resulting from the conversion of boar proacrosin to acrosin.

The preparation of highly purified malpha-acrosin is described. Purification was achieved by controlled activation of partially purified proacrosin, followed by gel chromatography over Sephadex G-100 at pH 3.0. The final malpha-acrosin preparation resulted in a single protein band with a molecular weight of 49,000 as determined by sodium dodecyl sulfate-disc gel electrophoresis. Disc arginine naphthylamide hydrolyzing band with a relative migration of 0.39 malpha-acrosin catalyzed the hydrolysis of synthetic substrates containing arginine and lysine, but not phenylalanine. Although calcium ions were not required for enzymatic activity, the addition of calcium chloride stimulated the activity through an increased substrate affinity and an increased maximal velocity. Polyamines stimulated the maximal velocity of the reaction, but were without effect on the substrate affinity. malpha-Acrosin was inhibited by lima bean, ovo-mucoid, and seminal plasma proteinase inhibitors. Diisopropyl fluorophosphate and 1-chloro-3-tosylamide-7-amino-L-2-heptanone treatment resulted in an irreversible inhibition, while L-arginine, benzamidine, and p-aminobenzamidine were competitive inhibitors with respect to substrate. These properties of malpha-acrosin are very similar to those previously reported for mbeta-acrosin and suggest that the portion of the molecule lost during the conversion of malpha-acrosin to mbeta-acrosin contributes little to the topography of either the active site or regulatory sites of the enzyme.

Purification and some properties of an extracellular alpha-amylase from Bacteroides amylophilus.

A medium was developed to obtain maximum yields of extracellular amylase from Bacteroides amylophilus 70. Crude enzyme preparation, obtained by ammonium sulfate precipitation of cell-free broth, contained six amylolytic isoenzymes that were detected by isoelectric focusing and polyacrylamide gel electrophoresis. One of these amylases was purified by diethylaminoethyl-Sephadex A-50 ion-exchange chromatography and Sephadex G-200 gel filtration techniques. Some properties of the purified extracellular alpha-amylase were: optimum pH, 6.3; optimum temperature, 43 degrees C: PH stability range, 5.8 to 7.5; isoelectric point, pH 4.6; molecular weight, 92,000 (by sodium dodecyl sulfatedisc gel electrophoresis); and sugars causing inhibition, cyclomaltoheptaose, cyclomaltohexaose, and alpha-d-phenylglucoside. In addition, Ca2+ and Co2+ were strong activators,and Hg2+ was a strong inhibitior; all other cations were slightly stimulatory. Dialysis against 0.01 M ethylenediaminetetraacetic acid caused a 58% loss of activity that was restored to 92% of the original by the addition of 0.04 M Ca2+. The enzyme affected a blue-value-reducing-value curve characteristic of alpha-type amylases. The relative rates of hydrolysis of amylose, soluble starch, amylopectin, and dextrin were 100, 97, 92, and 60%, respectively; Michaelis constants for these substrates were 18.2, 18.7, 18.2, and 16.7 mumol of d-glucosidic bond/liter, respectively. The enzyme degraded maize (corn) starch granules to some extent and had relatively little activity on potato starch granules.

Sodium dodecyl sulfate-disc gel electrophoresis patterns of bovine lung surfactant

Bovine lung surfactant material, isolated by three steps of centrifugation from lung washings, had a phospholipid: protein ratio of 4.98 ± S.D. 0.62 μmol/mg. Sucrose continuous density gradient centrifugation showed only one visible band at a density of 1.081 g/ml. Sodium dodecyl sulfate-disc gel electrophoresis patterns of the apoprotein extracted from this material revealed that the main component (70–80%) of the protein subunits of bovine lung surfactant material has a molecular weight of 36 000. It was suggested that the apoprotein of bovine lung surfactant material exists as a combination of a few protein subunits, most of which have a molecular weight of 36 000.

Structure of plant cell walls. Purification and characterization of a beta-1,4-galactanase which degrades a structural component of the primary cell walls of dicots.

Wild type Bacillus subtilis, when grown on a soybean arabinan-galactan, secretes a beta-1,4-galactanase which has been purified more than 200-fold from the culture fluid. Affinity chromatography was the most effective step in a purification procedure which resulted in a preparation that contained only a single 40,000 molecular weight protein band upon sodium dodecyl sulfate-disc gel electrophoresis. The purified galactanase digests a beta-1,4-galactan purified from citrus pectin and digests partially the isolated cell walls of suspension-cultured sycamore cells. The predominant product of the enzymic degradation of the substrates tested is a 4-linked tetragalactose. Evidence is presented to support the hypothesis that the galactanase attacks its substrates in both an exo- and endo-manner. The products obtained upon galactanase digestion of the soybean arabianin-galactan demonstrate that the earlier proposal concerning the structure of this polysaccharide must be incorrect.

Soluble adenylate cyclase from the culture medium of Bordetella pertussis: purification and characterization

Culture medium of exponentially growing Bordetella pertussis (strain 114) contains significant quantities of soluble (100,000 X g for 1 h) adenylate cyclase. The enzyme was purified by chromatography on diethylaminoethyl-cellulose and Sephadex G-200. The purest material yielded a single band on sodium dodecyl sulfate-disc gel electrophoresis. It is heat labile, has a temperature optimum of 30 degrees C, a pH optimum of pH 7 to 8, and a Km for adenosine 5'-triphosphate of 0.4 mM, and requires Mg2+ for maximum activity. The molecular weight, by sodium dodecyl sulfate-disc gel electrophoresis and sucrose density gradient, is approximately 70,000. The enzyme is markedly inhibited by fluoride and weakly inhibited by monovalent salts, but its activity is not altered by alpha-keto acids of nonsubstrate nucleoside triphosphates. Thus, but its presence in the culture supernatant, its smaller molecular weight, and its insensitivity to alpha-keto acids and nucleotides, this enzyme differs from the bacterial adenylate cyclases previously described.

Measurement of apolipoprotein A-I in rat high density lipoprotein and in rat plasma by radioimmunoassay

A double antibody radioimmunoassay (RIA) for rat apolipoprotein A-I is reported. The ApoA-I isolated from delipidated HDL by gel filtration yielded a single band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS), and its amino acid composition resembled that reported by others. ApoA-I was iodinated by lactoperoxidase and the resulting 125I-apoA-I was purified by gel filtration. Up to 93% of 125I-apoA-I was precipitable by antibody and greater than 99% of bound 125I-apoA-I was displaced by "cold" apoA-I. Other rat lopoproteins and apolipoproteins did not react in this system. Human plasma were also not reactive, nor were dog, goat, and sheep plasmas.

Light-induced changes of enzyme activities in parsley cell suspension cultures: Increased rate of synthesis of phenylalanine ammonia-lyase

When dark-grown cell suspension cultures of parsley ( Petroselinum hortense) were illuminated for increasing periods of time, increasing amounts of phenylalanine ammonialyase activity were obtained 5 hr after the onset of light. Pulses of [ 35S]methionine of varying duration from 1 to 150 min were given to cell cultures in the dark period subsequent to a light period of 2.5 hr. The cells were harvested 5 hr after the onset of light. Analysis of the soluble proteins by polyacrylamide gel electrophoresis revealed a distinct peak of radioactivity coinciding with the activity of phenylalanine ammonia-lyase. The results of experiments in which radioactive methionine was administered for 10 min to dark-grown or light-induced cells at different times after the light period were compared. An efficient incorporation of radioactivity into the fractions possessing the enzyme activity was observed 5 hr after induction, while no significant labeling was detected either after 1.5 or 25 hr, or in extracts from nonilluminated cells. The radioactive fractions containing the enzyme activity were further analyzed by sodium dodecyl sulfate-disc gel electrophoresis. Significant amounts of radioactivity at the molecular weight of the subunits of phenylalanine ammonia-lyase (84,000) were found only in the extracts from cells which had been labeled 5 hr after induction. These results suggest that the light-induced increase in phenylalanine ammonia-lyase activity is due to de novo synthesis, but not to an activation of preformed, inactive enzyme.