Polygalacturonase from Rhizopus stolonifer, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings

Apparently homogeneous polygalacturonase-elicitor purified from the filtrates of Rhizopus stolonifer cultures stimulates germinating castor bean seedlings to produce greatly increased levels of casbene synthetase activity. The purification procedure involved gel-filtration chromatography on Sephadex G-25 and G-75 columns followed by cation-exchange chromatography on a Sephadex CM C-50 column. Homogeneity of the purified preparation was indicated by the results of cationic polyacrylamide disc gel electrophoresis and isoelectric focusing (pI = 8.0). The identity of the casbene elicitor activity and polygalacturonase were indicated by the coincidence of the two activities at all stages of purification, the coincidence of both activities with the single protein-staining band detected on a cationic polyacrylamide disc gel and an isoelectric focusing gel, and the identical behavior of both activities on an agarose gel affinity column. The purified polygalacturonase-elicitor is a glycoprotein with approximately 20% carbohydrate content and an estimated molecular weight of 32,000 by polyacrylamide disc gel electrophoresis.     

Preparative Electrophoresis of Isotopically Labeled L-Cell Interferons

The preparative method of polyacrylamide gel electrophoresis was adapted for purification and characterization of isotopically labeled L-cell interferons. Re-covery of interferon activity was quantitative, and purification and resolution were comparable to those obtained by analytical polyacrylamide gel electrophoresis. Ultimate specific activities attainable ranged from 2 x 10(6) to 3 x 10(6) international units per mg of protein.     

Purification and characterization of extracellular proteinases excreted by a strain of Bacillus subtilis BS2, isolated from fermented African locust bean 'iru'

Proteinases were excreted by strains of Bacillus subtilis during fermentation of African locust bean cotyledons. Those excreted by one strain were purified and characterized by ammonium sulphate precipitation, ion-exchange chromatography (IEC), gel filtration, inhibition tests and polyacrylamide gel electrophoresis (PAGE). Three proteinases and an esterase without proteolytic activity were identified. A serine proteinase which showed a high degree of hydrophobicity and a neutral proteinase were present. The third proteinase showed both proteolytic and esterolytic activities, and had multiple electrophoretic mobilities on polyacrylamide gel.     

Detection of DNA and RNA polymerase activities in situ following electrophoresis in polyacrylamide gels.

A procedure is described for the detection of DNA dependent DNA and RNA polymerase activities in intact polyacrylamide gels that contain DNA. After electrophoresis under non-denaturing conditions, the intact gels are incubated with DNA or RNA polymerase reaction mixture in which one of the four deoxyribonucleoside or ribonucleoside triphosphates is radioactively labeled. The acid insoluble radioactivity associated with the intact gel is then analyzed by autoradiography of the intact gel or by liquid scintillation spectrometry of the sliced gel. Inhibition of the enzymatic activities by low molecular weight compounds such as N-ethylmaleimide or rifampin can be demonstrated by this procedure.     

Soluble proteins in alfalfa roots as related to cold hardiness

Soluble proteins extracted from alfalfa roots of hardy and nonhardy varieties were studied in relation to cold hardiness with polyacrylamide gel electrophoresis and quantitative enzyme analysis. Soluble protein content of alfalfa roots increased during hardening in all varieties. Two new isoenzymes with peroxidase activities were found in the fully hardened samples but no large shifts in the electrophoretic pattern were detected with polyacrylamide gel electrophoresis. Peroxidase and catalase activities increased during hardening in all varieties, but only small differences among hardy and nonhardy varieties were detectable. The studies indicated that protein metabolism was altered during the hardening process.     

Purification and characterization of alpha-toxin of Clostridium oedematiens type A

The alpha-toxin of Clostridium oedematiens type A was purified from culture filtrate by two steps of column chromatography and repeated gel filtration. The purified alpha-toxin proved homogeneous in polyacrylamide gel electrophoresis and agar gel double diffusion. The molecular weight of the alpha-toxin was estimated at 280,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and at 260,000 by gel filtration on a Sephadex G-200 column. The isoelectric point determined by isoelectric focusing polyacrylamide gel electrophoresis was 6.1. No dissociation of the purified alpha-toxin into subunits was demonstrated in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 50% lethal and edematizing doses per mg protein of the purified alpha-toxin were 5.9 X 10(4) and 5.9 X 10(5), respectively. The L +/50 doses per mg protein of the toxin was 4.6 X 10(3). The purified alpha-toxin, when injected intradermally into the rabbit skin, induced increased vascular permeability. The toxin contained little or no hemolytic or lecithinase activity. These results attest that the lethal, edematizing and vascular permeability-enhancing activities elicited by C. oedematiens type A culture reside on the same protein molecule.     

Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.     

Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: chromatographic characterization of the biologically active proteins.

Biochemical characterization of hemagglutinin and sialidase activities from Clostridium perfringens strain CN3870 revealed that this strain produced three sialidase enzymes that were separable to gel filtration, ion exchange chromatography, and polyacrylamide gel electrophoresis. The molecular weights of sialidase I, II, and III activities were 310,000 +/- 10,000, 105,000 +/- 4,000 and 64,000 +/- 2,000, respectively, the first figure being an approximate value only.     

Proteins in the luminal fluid from the bovine oviduct.

Oviducal fluid was collected by cannulation from four cows and by irrigation from fifteen slaughtered cows.The proteins in the fluid were examined by polyacrylamide gel electrophoresis at pH 4-5 and pH 8-9, isoelectric focusing on polyacrylamide, immunodiffusion, immunoelectrophoresis, affinity chromatography and gel filtration. The macromolecular components found were mainly serum proteins but small amounts of other proteins were detected in oestrous and dioestrous samples by electrophoresis at pH 8-9 following fractionation of the fluid by gel filtration or affinity chromatography. Small amounts of cathodically migrating proteins were detected directly by electrophoresis at pH 4-5 in dioestrous samples but not in oestrous samples. Determination of glycosidase activities revealed that the levels at oestrus were similar to the levels detected in serum. At dioestrus, the activities of B-N-acetylgalactosaminidase and beta-N-acetylglucosaminidase were elevated.     

Identity of acyl-CoA oxidase with glutaryl-CoA oxidase

Rats fed on clofibrate- and DEHP-containing diets showed virtually proportional increases in hepatic acyl-CoA oxidase and glutaryl-CoA oxidase activities. The solubilization profiles of the two activities from the light mitochondrial fraction of the liver homogenate of DEHP-treated rats were the same, and the glutaryl-CoA oxidase/acyl-CoA oxidase activity ratio remained constant through the purification. The final preparation obtained was a single protein based on the result of polyacrylamide gel electrophoresis. The evidence indicates that the two activities are associated with the same protein.     

Intracellular distribution of ribonuclease activity during erythroid cell development.

Five ribonuclease activities, separable by polyacrylamide gel electrophoresis, have been detected in erythroid bone marrow cells from anaemic rabbits. Their intracellular distribution has been investigated and compared with that of the ribonucleases in reticulocytes. Both the acid and alkaline ribonuclease activities of reticulocytes are much lower (30--50 fold) than those of bone marrow erythroid cells. The most marked decrease in enzyme activity occurs in the fractions containing ribosomes and mitochondria plus lysosomes. In these subcellular organelles there was also a qualitative change in the ribonuclease electrophoretic pattern, whereas the cytosol enzymes of marrow erythroid cells and reticulocytes remained largely unchanged. Several ribonucleases released from reticulocyte membranes with urea were similar to those present in the lysosomal plus mitochondrial fraction, as shown by detection of enzyme activity after polyacrylamide gel electrophoresis. The decline in ribonuclease activity was found to begin in the orthochromatic cells, which have a highly condensed nucleus and are no longer active in DNA and RNA synthesis, and to coincide with a decrease in acid phosphatase activity and loss of lysosomes.     

黑线姬鼠与大林姬鼠组织中超氧化物酶和过氧化物酶的分布与活性比较

以黑线姬鼠(Apodemus agrarius)和大林姬鼠(A. peninsulae)为研究对象,采用聚丙烯酰胺凝胶电泳(PAGE)不连续体系的方法,比较分析了心、肝、肾、肌肉、脑、肺6种器官和组织中超氧化物酶(SOD)和过氧化物酶(POD)活性,并建立了2种酶的电泳图谱。结果显示,上述2种酶在黑线姬鼠和大林姬鼠的6种器官和组织中均有表达并表现出明显的特异性,其中,2种鼠中超氧化物酶共分离出迁移率由0.15～0.66的9条电泳谱带,过氧化物酶共分离出迁移率由0.09～0.83的20条电泳谱带。在肝和肺中酶的活性最强,黑线姬鼠6种器官和组织中超氧化物酶活性均强于大林姬鼠,2种鼠组织中过氧化物酶的活性和分布相似,但在同一物种不同器官和组织间过氧化物酶的活性及分布存在明显差异。     

Purification and partial characterization of arylsulphatase C from human placental microsomes

Arylsulphatase C (EC 3.1.6.1) has been purified 300-fold from human placental microsomes using a four step procedure involving solubilization with Triton X-100, chromatography on hydroxyapatite, column chromatofocussing and ion-exchange chromatography on DEAE-Sepharose. The purified enzyme is electrophoretically homogeneous and has a molecular weight of 440 000 as determined by polyacrylamide gradient gel electrophoresis. On analysis of the preparation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate a polypeptide of molecular weight 74 000 was observed, suggesting that the enzyme as purified may be a hexamer. The behaviour of the enzyme during chromatofocussing indicates the enzyme has a pI of 6.56. Steroid sulphatase, as measured by activity towards dehydroepiandrosterone sulphate, co-purifies with arylsulphatase C suggesting that both activities are due to a single enzyme.     

Scanning of polyacrylamide gel electrophoresis columns for detection of unstained protein zones and for their localization relative to enzyme activities.

Background disturbances which often confuse 280-nm scans of polyacrylamide gels, can be distinguished from true protein peaks by scanning also at a wavelength where the proteins do not absorb (for instance 310 nm).A scanning technique has been used also for precise localization of spectrophotometrically detectable enzyme activities relative to protein zones. After electrophoresis the gel is transferred to a specially designed quartz cuvette and scanned at 280 nm for protein detection. The substrate is then allowed to diffuse into the gel and the activity is located by scanning at a wavelength absorbed by the product.Scanning of polyacrylamide gel electrophoresis columns can be used for the study of solute-solute interactions, as illustrated by a simple model experiment on the binding of bilirubin to albumin.     

The purification and characterization of a phospholipase A in hamster heart cytosol for the hydrolysis of phosphatidylcholine

Phospholipases A1 and A2 catalyze the hydrolysis of acyl groups of phospholipids at C-1 and C-2, respectively. These phospholipases are important in phospholipid catabolism and the remodeling of the acyl groups of phospholipids. Phospholipase A from hamster heart cytosol was purified by a combination of ion-exchange and gel filtration chromatography. The purity of the enzyme was assessed by nondenaturing polyacrylamide gel electrophoresis, two-dimension polyacrylamide gel electrophoresis, and immunological studies. The purified enzyme exhibited both phospholipase A1 and A2 activities toward phosphatidylcholine and had the ability to hydrolyze the acyl groups of phosphatidylethanolamine. However, the enzyme was not active toward lysophosphatidylcholine, diacylglycerol, or triacylglycerol. By Sepharose 6B chromatography, the molecular weight of the purified enzyme was estimated to be 140,000. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was composed of identical Mr 14,000 subunits. At least six subunits in the native enzyme could be cross-linked by dimethyl suberimidate. Both phospholipase A1 and A2 activities showed similar pH profiles, exhibited no absolute requirements for divalent metallic cations, but displayed a high degree of specificity for the acyl groups of phosphatidylcholine at both C-1 and C-2. The Km of phospholipases A1 and A2 for 1-palmitoyl-2-arachidon-ylglycerophosphocholine was found to be identical (0.5 mM).     

Organization of a multifunctional protein in pyrimidine biosynthesis. Analyses of active, tryptic fragments

The multifunctional protein which catalyzes the first three steps of pyrimidine biosynthesis in hamster cells can be cleaved by trypsin into enzymatically active fragments. When the fragments are separated by nondenaturing polyacrylamide gel electrophoresis, three major polypeptide bands appear. Carbamyl phosphate synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3) activities are associated with 129,000-, 660,000-, and 94,000-dalton bands, respectively. Further analysis of these fragments by denaturing polyacrylamide gel electrophoresis has shown that the aspartate transcarbamylase band seen on the nondenaturing gel is actually a large aggregate of 39,000-dalton fragments and the dihydroorotase band is a dimer of 44,000-dalton fragments. The data reported here indicate that (i) this multifunctional protein is composed of three enzymatically independent domains, (ii) the sum of the molecular weights of these three domains (129,000 + 39,000 + 44,000 = 212,000) is similar to that of the undigested monomer (220,000 daltons), and (iii) a site important to the formation of the native multimeric protein is probably near the aspartate transcarbamylase domain.     

Purification and specificity of carboxypeptidase from Streptomyces griseus K-1.

A carboxypeptidase of St. griseus K-1 (CPase S) was found to possess the specificities of both mammalian pancreatic CPase A and B. Three adsorbents for affinity chromatography were prepared by coupling l-Leu, d-Leu, and d-Arg with CH-Sepharose 4B. d-Arg-CH-Sepharose and l-Leu-CH-Sepharose retained the purified CPase S but d-Leu-CH-Sepharose did not. The activities of CPase S toward CGL and BGA were eluted in the same position. CPase S migrated as a single band on polyacrylamide gel electrophoresis and the two activities were both extracted from this band on the gel.     

Studies on the Molecular Weights of Esterase and Peroxidase Isozymes of Maize

The molecular weights of esterase and peroxidase isozymes of maize seedlings were directly determined by improved polyacrylamide gradient gel electrophoresis. The different isozyme bands developed in polyacrylamide slab gel electrophoresis (uniform gel) were identified in polyacrylamide gradient gel electrophoresis by means of isozyme variants. The molecular weights of esterase isozymes E1, E2, E3F, E3S, a, b, c, named according to isozyme patterns in uniform gel, are <20000, 35200, 33000, 38500, 29900, 28500, 34000 doltons respectively. The molecular weights of peroxidase isozymes PX4F and PX4S are 131000 and 149000 doltons respectively. According to the band location in uniform gel and in gradient gel, some biochemical properties of the isozyme bands and relationships between the isozyme bands were analyzed. The possible errors in the determination of smaller molecular weight isozymes are discussed.     

The localization of galactosyltransferases in polyacrylamide gels by a coupled enzyme assay

A coupled enzyme assay for GlcNAc¹: UDP-galactose galactosyltransferase has been developed that allows this enzyme to be assayed spectrophotometrically and in nondenaturing polyacrylamide gels. Utilizing three, intermediate enzymes, galactosyltransferase activity has been coupled to the production of NADH with a stoichiometry of 2 mol of NADH produced for each mol of galactose transferred to GlcNAc. The enzyme reactions coupled to the production of UDP by galactosyltransferase can be summarized as follows:

The activities of partly purified bovine milk galactosyltransferase and galactosyltransferase in dialyzed fetal calf serum have been determined spectrophotometrically by measuring NADH production at 340 nm. The reaction is dependent on N-acetylglucosamine, UDP-galactose, and Mn²⁺. For both enzyme sources, activities calculated from NADH production are similar to those determined from assays that use radioactive sugar nucleotide substrates. Both galactosyltransferase activities have been localized on 7.5% nondenaturing polyacrylamide gels after electrophoresis by incubating the gel with an agarose indicator gel containing the coupled enzyme system. Enzyme activity is marked by NADH fluorescence, which is dependent on the presence of N-acetylglucosamine in the indicator gel. The intensity of fluorescence increases with increasing galactosyltransferase activity applied to the gel.     

C-terminal truncation of bovine protein disulfide isomerase increases its activity.

Protein disulfide isomerase as usually purified by the method of Lambert and Freedman (Biochem. J., 1983, 213, 225-234) although appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis can be separated into two major components on a size-exclusion high performance liquid chromatography column or by polyacrylamide gel electrophoresis. These two components have the same N-terminal sequences but the C-terminal sequences are different, suggesting that one is the C-terminal slightly truncated protein. The shortened protein is more active in both the isomerase and the thiol-protein oxidoreductase activities.