Purification and properties of the extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1299.

Dextransucrase [EC 2.4.1.5] activity from cell-free culture supernatant of Leuconostoc mesenteroides NRRL B-1299 was purified by (NH4)2SO4 fractionation, adsorption on hydroxyapatite, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The extracellular enzyme was separated into two principal forms, enzymes I and N, and the latter was shown to be an aggregated form of the protomer, enzyme I. Enzymes I and N were both electrophoretically homogeneous and their relative activities reached 820 and 647 times that of the culture supernatant, respectively. On sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, enzyme N dissociated into the protomer enzyme I, with a molecular weight of 48,000. Enzyme I was gradually converted into enzyme N upon aging, and this conversion was stimulated in the presence of NaCl. The optimum pH and temperature of enzyme I activity were pH 6.0 and 40 degrees, respectively, while those of enzyme N were pH 5.5 and 35 degrees. The Km values of enzymes I and N were 13.9 and 13.1 mM, respectively. Ca2+, Mg2+, Fe2+, and Co2+ stimulated the activity of enzyme N, and EDTA showed a potent inhibitory effect on this enzyme. Moreover, the activity of enzyme N was more effectively stimulated by exogenous dextrans as compared with enzyme I.     

Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ

A rapid and simple, large-scale method for the purification of DNA-dependent RNA polymerase III (EC 2.7.7.6) from wheat germ is presented. The method involves enzyme extraction at low ionic strength, polyethyleneimine fractionation, (NH₄)₂SO₄ precipitation, and chromatography on DEAE-Sepharose CL-6B, DEAE-cellulose, and heparin agarose. Milligram quantities of highly purified enzyme can be obtained from kilogram quantities of starting material in 2 to 3 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that RNA polymerase III contains 14 subunits with molecular weights of: 150,000; 130,000; 94,000; 55,000; 38,000; 30,000; 28,000; 25,000; 24,500; 20,500; 20,000; 19,500; 17,800; and 17,000. Subunit structure comparison of wheat germ RNA polymerases I, II, and III indicates that all three enzymes may contain common subunits with molecular weights 20,000, 17,800, and 17,000. In addition, RNA polymerases II and III may contain a common subunit with a molecular weight of 25,000, and RNA polymerases I and III may contain a common subunit with a molecular weight of 38,000.     

Properties and subunit composition of RNA polymerase II from plant cell cultures.

The purification of DNA-dependent RNA polymerase II (EC 2.7.7.6) from plant cell cultures of Petroselinum (parsley) is described. The procedure during which enzyme I is eliminated includes initial precipitation with (NH4)2SO4, an ultracentrifugation step, gel filtration on Sepharose 4B, chromatography on DEAE-cellulose, DNA-agarose and DEAE-Sephadex. The enzyme purified almost to homogeneity exhibits maximal activity with denatured DNA, and is activated preferentially by Mn2+; alpha-amanitin acts as a strong inhibitor. Electrophoresis of the enzyme in the presence of dodecylsulphate indicates that it is composed of seven subunits with mol. wts of 200 000, 180 000, 140 000, 43 000, 26 000, 25 000 and 16 000. The results of molecular weight and molar ratio determinations suggest that Petroselinum RNA polymerase II may exist in two active forms differing only in the composition of their high molecular weight subunits.     

Purification and characterization of glutamate synthase from Azospirillum brasilense.

Growth conditions for Azospirillum brasilense Sp6 were devised for maximal expression of glutamate synthase. The enzyme levels were largely affected by the type and concentration of the nitrogen source. A 10-fold increase in the synthesis of the enzyme was observed at a limiting concentration of ammonia. The enzyme was purified to homogeneity by a procedure which was fairly rapid and allowed a good recovery of enzyme (30%). Azospirillum glutamate synthase is a complex iron-sulfur flavoprotein with a stoichiometry of 1 flavin adenine dinucleotide:1 flavin mononucleotide:8 Fe:8 S per protomer with a molecular weight of 185,000. The protomer is composed of two dissimilar subunits with molecular weights of 135,000 and 50,000. Kinetic parameters were determined. Km values for NADPH, 2-oxoglutarate, and L-glutamine were 6.25, 29, and 450 microM, respectively. The optimum pH was about 7.5. Complete reduction of the enzyme under anaerobic conditions was obtained either by NADPH (in the presence of a regenerating system) or dithionite or by photochemical reduction (in the presence of EDTA and 5-deazariboflavin). No stable long-wavelength intermediates were observed.     

Subunit topography of RNA polymerase from Escherichia coli. A cross-linking study with bifunctional reagents.

The quaternary structures of Escherichia coli DNA-dependent RNA polymerase holenzyme (alpha 2 beta beta' sigma) and core enzyme (alpha 2 beta beta') have been investigated by chemical cross-linking with a cleavable bifunctional reagent, methyl 4-mercaptobutyrimidate, and noncleavable reagents, dimethyl suberimidate and N,N'-(1,4-phenylene)bismaleimide. A model of the subunit organization deduced from cross-linked subunit neighbors identified by dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the large beta and beta' subunits constitute the backbone of both core and holoenzyme, while sigma and two alpha subunits interact with this structure along the contact domain of beta and beta' subunits. In holoenzyme, sigma subunit is in the vicinity of at least one alpha subunit. The two alpha subunits are close to each other in holoenzyme, core enzyme, and the isolated alpha 2 beta complex. Cross-linking of the "premature" core and holoenzyme intermediates in the in vitro reconstitution of active enzyme from isolated subunits suggests that these species are composed of subunit complexes of molecular weight lower than that of native core and holoenzyme, respectively. The structural information obtained for RNA polymerase and its subcomplexes has important implications for the enzyme-promoter recognition as well as the mechanism of subunit assembly of the enzyme.     

Subunit structure and activity of glyceraldehyde-3-phosphate dehydrogenase from spinach chloroplasts.

Glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NADP+ oxidoreductase (phosphorylating), EC 1.2.1.13) from spinach chloroplasts is a polymeric protein of approx. 600,000 daltons and sodium dodecyl sulphate gel electrophoresis shows that it consists of two subunits of molecular weight 43,000 and 37,000. Comparison of amino acid analyses and tryptic peptide maps indicates that the two subunits have a different primary structure. The native enzyme contains 0.5 mol of NADP+ and 0.5 mol of NAD+ per protomer of 80,000 daltons, no reduced pyridine nucleotides have been detected. Almost complete inactivation is obtained by reaction of two cysteinyl residues per 80,000 daltons with tetrathionate or iodo[14C2]acetic acid; since the same amount of radioactivity is incorporated in the two subunits it is likely that they are both essential for the catalytic activity. Charcoal stripping of native glyceraldehyde-phosphate dehydrogenase produces an apoprotein which still retains most of the enzymatic activity but, unlike the holoenzyme, is gradually inactivated by storage at 4 degrees C and does not react with iodoacetate under the same conditions in which the holoenzyme is completely inactivated.     

The C-S Lyases of Higher Plants : Direct Comparison of the Physical Properties of Homogeneous Alliin Lyase of Garlic (Allium sativum) and Onion (Allium cepa)

Garlic and onion alliin lyases, although from closely related species, have many differences. The two enzymes differ in their K_m values, pH optima, and isoelectric points. There is a major difference in their molecular weight and subunit structure. The garlic holoenzyme has a molecular weight of 85,000 and consists of two subunits of molecular weight 42,000. The onion enzyme has a holoenzyme molecular weight of 200,000 composed of four subunits of molecular weight 50,000. The onion enzyme is much more difficult to dissociate into its subunits which suggests differences in subunit interaction between the two enzymes. The dimeric stucture of the garlic and the tetrameric structure of the onion enzyme is consistent with a coenzyme content (pyridoxal-5′-phosphate) equivalent to one mole per subunit. The two enzymes vary vastly in their spectra, the onion enzyme having a lower pyridoxal-5′-phosphate absorbance at 430 nanomoles and an inability to react with l-cysteine. Both enzymes are glycoproteins and bind to concanavalin A-Sepharose columns. The onion alliin lyase binds more tightly than the garlic enzyme. The amino acid content of both enzymes is similar as is the carbohydrate content. However, upon hydrolysis the onion lyase does yield more mannose units than the garlic enzyme which is consistent with the former's stronger affinity for concanavalin A.     

Studies on the functions of the RNA polymerase components by means of mutations

Summary It had been shown earlier, that RNA polymerase 13 S particles contain the large components with a molecular weight of about 3–10⁵ and small subunits with a molecular weight of 4·10⁴-1·10⁵. These polymerase components easily dissociate and reassociate with restoration of the enzyme activity.Both temperature-sensitive (tsX) and rifamycin-resistant (rif-r-I) mutations proved to affect the large polymerase component without changing the small subunits. These mutations were mapped at different, though closely linked, loci of metB-thi region of E. coli K12 chromosome. These results as well as certain literature data allow to conclude that the large RNA polymerase component consists of at least two polypeptides, one being altered by ts mutation, and the other—by rif-r mutation.The large polymerase component when separated from the small subunits retain the ability to bind to T2 phage DNA while the separate small subunits lack this property. Rifamycin does not affect RNA polymerase-T2 DNA binding while ts mutation leads to inability of the enzyme to form stable complexes with DNA. Therefore, it is likely that the polypeptide affected by ts mutation is responsible for the attachment of RNA polymerase to specific sites of DNA template. On the other hand, the small subunits as well as polypeptide of the large component, which determines RNA polymerase sensitivity to rifamycin, seem not to participate in the enzyme binding to DNA template. It is suggested, that the catalytic site of RNA polymerase is located in the large component and formed by rifamycin-binding polypeptide. The small subunits are supposed to have regulatory function and activate the large components.     

Subunit size and paracrystal structure of avian liver acetyl-CoA carboxylase.

The subunit molecular weight of chicken liver acetyl-CoA carboxylase has been redetermined by immunoprecipitation and sodium dodecyl sulfate gel electrophoresis. In the presence of parotid trypsin inhibitor, the immunoprecipitate gave a single band corresponding to a molecular weight of 230,000, which was also found to contain bound biotin. From the biotin content of the protomer (1.0 prosthetic group per 480,000 daltons) it appears that it consists of two non identical subunits, both with molecular weights of approximately 230,000.Electron microscopy has been carried out on the active filamentous form of the enzyme and on paracrystals formed under high-salt conditions. These indicate that the filaments are readily distortable helical ribbons, with an approximate axial repeat of 1100 Å, containing eight protomers. The paracrystals are made up of a staggered lateral packing of filaments.     

Characterization of an endoribonuclease, RNase N, from Escherichia coli.

The properties of the enzyme ribonuclease N were investigated. By comparing the distribution in the cell of RNase N with the bonafide intracellular beta-galactosidase, and the periplasmic alkaline phosphatase enzymes, we showed that RNase N is an intracellular enzyme. Since previous studies suggested that it is an endoribonuclease, it was compared to RNase III, the only other known intracellular endoribonuclease in Escherichia coli. Using homopolymers and co-polymers we found that, while RNase III could digest double-stranded RNA only, RNase N digested single-stranded and double-stranded RNA with similar efficiency. Furthermore, all RNAs used, natural as well as synthetic, were substrates for the enzyme. Using 5 S rRNA as a substrate it was confirmed that the enzyme is an endonuclease. The final products of the reaction of this enzyme are 5'-mononucleotides. The molecular weight of the enzyme is about 120,000 and it seems to contain two subunits which are similar in size. These properties thus differentiate this enzyme from all other known ribonucleases in E. coli.     

2-Hydroxyisonicotinate dehydrogenase isolated from Mycobacterium sp. INA1

2-Hydroxyisonicotinate dehydrogenase from Mycobacterium sp. INA1 was purified 26-fold to apparent homogeneity. The enzyme is involved in isonicotinate degradation by Mycobacterium sp. INA1 and catalyzes the conversion of 2-hydroxyisonicotinate to 2,6-dihydroxypyridine-4-carboxylate. The purified protein exhibited a native molecular mass of 300 kDa and subunits of 97, 31 and 17 kDa, respectively, indicating an α₂β₂γ₂ structure. The absorption spectrum of the homogeneous enzyme was characteristic for an iron/sulfur flavoprotein. 3.8 mol of iron, 3.7 mol of acid labile sulfur, 0.94 mol of FAD and 0.75 mol of molybdenum were determined per mol of protomer. The molybdenum cofactor was identified as molybdopterin cytosine dinucleotide. 2-Hydroxyisonicotinate dehydrogenase was inactivated in the presence of cyanide. According to these basic properties the protein seems to belong to the class of molybdo-iron/sulfur flavoproteins of the xanthine oxidase family.     

The A protomer of islet-activating protein,pertussis toxin,as an active peptide catalyzing ADP-ribosylation of a membrane protein

Islet-activating protein (IAP), pertussis toxin, is an oligomeric protein composed of an A protomer and a B oligomer. IAP and its A protomer were equipotent, on a molar basis, in enhancing GTP-dependent adenylate cyclase activity and in causing ADP-ribosylation of the 41,000 M_r protein when directly added to the cell-free membrane preparation from rat C6 glioma cells. Similar actions of IAP observed upon its addition to intact C6 cells were not mimicked by its A protomer, indicating that the A protomer had to be associated with the B oligomer to become accessible to its site of action on the inner surface of the membrane of intact cells. The A protomer, but not IAP, exhibited NAD-glycohydrolase activity in the reaction mixture lacking cellular components but containing dithiothreitol. Their actions on membranes were not accelerated by dithiothreitol, but markedly suppressed by oxidized glutathione. Thus, C6 cell membranes may possess certain “processing” enzyme(s) responsible for releasing the A protomer from the IAP molecule and for reductive cleavage of an intrachain disulfide bond in the released protomer, thereby producing an active peptide which functions to cause ADP-ribosylation of one of the subunits of guanine nucleotide regulatory protein in the receptor-adenylate cyclase system.     

Nonidentical subunits of pyrocatechase from Pseudomonas arvilla C-1.

Pyrocatechase [catechol:oxygen, 1,2-oxidoreductase (decyclizing), EC 1.13.11.1] from Pseudomonas arvilla C-1 has been reported to contain 2 g atoms of iron/mol of enzyme, based on a molecular weight of 90,000, determined by sedimentation and diffusion constants (Y. Kojima, H. Fujisawa, A. Nakazawa, T. Nakazawa, F. Kanetsuna, H. Taniuchi, M. Nozaki, and O. Hayaishi, 1967, J. Biol. Chem., 242, 3270–3278). The molecular weight was estimated again by sedimentation equilibrium and Sephadex G-200 gel filtration and found to be 63,000 and 60,000, respectively. The enzyme was also found to contain 1 g atom of iron/mol of enzyme, based on a molecular weight of 63,000. The enzyme was dissociated into two bands on polyarcylamide gel electrophoresis in the presence of either sodium dodecyl sulfate or 8 m urea, and was separated into two subunits, α and β, by CM-cellulose chromatography using a buffer solution containing 8 m urea. The molecular weights of the α and β subunits were determined to be 30,000 and 32,000, respectively, by sodium dodecyl sulfate-gel electrophoresis. The NH₂-terminal sequences of these subunits determined by Edman degradation were as follows: α subunit, Thr-Val-Asn-Ile-Ser-His-Thr-Ala-Gln-Ile-Gln-Gln-Phe-Phe-Gln-Gln-(X)-(X)-Gly -Phe-Gly; β subunit, Thr-Val-Lys-Ile-Ser-His-Thr-Ala-Asp-Ile-Gln-Ala-Phe-Phe-Asn-Gln-Val-(X)-Gly-Leu-Asx. The COOH-terminal amino acid residues were determined to be alanine for the α subunit and glycine for the β subunit by three different methods: carboxypeptidase digestion, tritium labeling, and hydrazinolysis. These results indicate that the enzyme consists of two nonidentical subunits, α and β.     

Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase II from the mouse plasmacytoma, MOPC 315.

DNA-dependent RNA polymerase II was purified from the mouse plasmacytoma, MOPC 315. Soluble enzyme was obtained from a nucleoplasmic fraction and subjected to chromatography on phosphocellulose, DEAE-cellulose, and DEAE-Sephadex ion exchange resins and was subjected to sedimentation in sucrose density gradients. A chromatographically homogeneous enzyme was obtained which was purified about 25,000-fold relative to whole cell extracts and which had a specific activity (on native DNA) similar to those reported for other purified eukaryotic class II RNA polymerase preparations. Analysis of purified RNA polymerase II by polyacrylamide gel electrophoresis under nondenaturing conditions revealed three protein bands, designated II-O, II-A, and II-B in order of electrophoretic mobility. The subunit compositions of these nondenatured bands were subsequently analyzed by electrophoresis under denaturing conditions. Each enzyme II form contained subunits with molecular weights of 140,000 (II-c), 41,000 (II-d), 30,000 (II-e), 25,000 (II-f), 22,000 (II-g), 20,000 (II-h), and 16,000 (II-i). Molar ratios were unity for all subunits except subunit II-h which had a molar ratio of 2. Each enzyme form was distinguished by its highest molecular weight subunit. II-O contained subunit II-o (molecular weight 240,000), II-A contained subunit II-a (molecular weight 205,000), and II-B contained subunit II-b (molecular weight 170,000). Total molecular weights for II-O, II-A, and II-B were calculated as 554,000, 519,000, and 484,000, respectively. In addition, the number of RNA polymerase II molecules per MOPC 315 tumor cell was calculated to be about 5 times 10-4.     

Subunits of cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis.

Cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis have been purified to a homogeneous state as judged from their electrophoretic behavior and their subunit structures studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The T. novellus enzyme is composed of two kinds of subunits of 32,000 and 23,000 daltons and its minimum molecular weight is 55,000 on the basis of heme content and amino acid composition. The N. agilis enzyme also has two kinds of subunits of 40,000 and 27,000 daltons and its minimum molecular weight is 66,000 on the basis of heme content and amino acid composition. Therefore, the molecule of each enzyme is composed of two kinds of subunits which resemble the subunits of the eukaryotic cytochrome oxidase biosynthesized in the mitochondrion at least with respect to molecular weight.     

Bovine liver monoamine oxidase: A modified purification procedure and preliminary evidence for two subunits and one FAD

Bovine liver mitochondrial monoamine oxidase was isolated in a more active state and in higher yields by an improved purification method which utilized β-mercaptoethanol and which contained several other important modifications. The subunit structure of the purified enzyme components was investigated by chemical and enzymatic methods. The subunit molecular weight of the three enzyme components isolated was estimated to be 52,000 by sodium dodecyl sulfate disc electrophoresis and by exclusion-diffusion chromatography on Biogel A-5m with 6 m guanidine HCl as the solvent. The number of peptides observed in the peptide map of the tryptic digest of the S-β-carboxymethylcysteine derivative of the enzyme also showed that the subunit molecular weight was about 52,000. Since it was previously reported that the monomer molecular weight of the enzyme was about 110,000, the active enzyme is made up of two subunits. The NH₂-terminus of the enzyme of both subunits is blocked since Edman degradation and aminopeptidase failed to release an NH₂-terminal amino acid. The COOH-terminal amino acid of both subunits was shown to be leucine by carboxypeptidase digestion of the enzyme since it was liberated quantitatively. From the FAD content of the enzyme and the subunit data, it is proposed that the enzyme probably consists of two subunits which differ possibly in that only one subunit contains 8-α-cysteinyl FAD.     

Molecular properties of cis,cis-muconate cycloisomerase from Pseudomonas putida

cis,cis-Muconate cycloisomerase (cis,cis-muconate lactonizing enzyme, EC 5.5.1.1.) was purified in crystalline form from Pseudomonas putida. Ultracentrifugation studies, as well as gel filtration chromatography and electrophoresis, indicate that the enzyme is an oligomeric protein of molecular weight 252,000 (s_20,w 12.20 × 10^?13 s), which is built of six homologous protomers of molecular weight 42,000. Studies of enzyme crystals and enzyme molecules in the electron microscope suggest that the cis,cis-muconate cycloisomerase is a hexamer in which the six protomers are arranged in a dihedral point-group symmetry 32 (D₃). Each protomer has a diameter of 42.5Åand six protomers are associated in a structure with a trigonal antiprismatic geometry (a hexamer D₃ octahedron). This model could account for the dimensions most frequently observed by negative staining of the enzyme in solution. A model for the three-dimensional structure of enzyme crystals in which each hexameric enzyme molecule is surrounded by eight neighbouring enzyme molecules, is described.