Nucleotide sequence and structural relationships of a chloramphenicol acetyltransferase encoded by the plasmid pSCS6 from Staphylococcus aureus.

The 4.6 kb chloramphenicol resistance (Cm) plasmid, pSCS6, isolated from a naturally occurring Staphylococcus aureus biotype C encoded an inducible chloramphenicol acetyltransferase (CAT). The respective cat gene and its regulatory region were cloned. Sequence analyses revealed two open reading frames: one for a 9-amino acid leader peptide and the other for the 215-amino acid CAT monomer. Comparisons of the predicted CAT amino acid sequences revealed a high degree of similarity between CAT from pSCS6 and the CAT variants encoded by Cm plasmids of the pC221 family. These close structural relationships suggested an intraspecific exchange of Cm-determinants between Staph. aureus of human and bovine biotype.     

Nucleotide sequence and structural relationships of a chloramphenicol acetyltransferase encoded by the plasmid pSCS6 from Staphylococcus aureus

M. CARDOSO AND S. SCHWARZ. 1992. The 4.6 kb chloramphenicol resistance (Cm^R) plasmid, pSCS6, isolated from a naturally occurring Staphylococcus aureus biotype C encoded an inducible chloramphenicol acetyltransferase (CAT). The respective cat gene and its regulatory region were cloned. Sequence analyses revealed two open reading frames: one for a 9-amino acid leader peptide and the other for the 215-amino acid CAT monomer. Comparisons of the predicted CAT amino acid sequences revealed a high degree of similarity between CAT from pSCS6 and the CAT variants encoded by Cm^R plasmids of the pC221 family. These close structural relationships suggested an intraspecific exchange of Cm^R-determinants between Staph. aureus of human and bovine biotype.     

Nucleotide sequence of the chloramphenicol resistance determinant of the streptococcal plasmid pIP501

We have sequenced the chloramphenicol resistance determinant (cat) of plasmid pIP501 from Streptococcus agalactiae to investigate its relationship with other cognate cat determinants. Sequence analysis revealed that it exhibits a high degree of similarity with the cat genes of plasmids pC221 and pUB112 from Staphylococcus aureus and pSCS1 from Staphylococcus intermedius. These genes, however, display several differences in their regulatory and coding regions. These results demonstrate that the cat determinant of plasmid pIP501 belongs to the pC221 subgroup of CAT variants.     

Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei.

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.     

Detection of 10 variants of biliverdin reductase in rat liver by two-dimensional gel electrophoresis

We have identified and characterized multiple forms of biliverdin reductase (BVR) in control rat liver cytosol. Two-dimensional electrophoresis of the purified BVR resolved a minimum of 10 discrete protein zones. All 10 proteins were BVR as judged by immunological cross-reactivity toward rabbit anti-rat BVR. Based on the isoelectric focusing pattern of separation, the BVR variants could be organized into five net-charge groups designated as BVR-IEF1 to BVR-IEF5 and three molecular mass groups designated as BVR-MW1-BVR-MW3, respectively. The pI values of the net-charge groups were: BVR-IEF1, 6.23; IEF2, 5.91; IEF3, 5.76; IEF4, 5.61; IEF5, 5.48. The Mr values of the molecular mass groups were: BVR-MW1, 30,400; MW2, 30,700; MW3, 31,400. Single dimension slab gel isoelectric focusing offered greater resolution of the net charge variants, and BVR-IEF3 was further resolved into two variants, IEF3a and IEF3b, with pIs of 5.77 and 5.75, respectively. The six net-charge variants also resolved on a preparative chromatofocusing column and were designated as BVR-CF1-BVR-CF6. The pH values of the peak fractions were: BVR-CF1, 6.91; CF2, 6.33; CF3, 6.03; CF4, 5.82; CF5, 5.45; CF6, 5.27. Correspondence between the isoelectric focusing net-charge variants and the chromatofocusing net-charge variants was established. The Mr and net-charge variants did not represent partially degraded forms of biliverdin reductase produced during purification since the pattern of resolution of variants on slab gel isoelectric focusing or two-dimensional electrophoresis did not change by purifying the proteins in the presence of protease inhibitors and 5 mM EDTA. BVR-CF2 and BVR-CF4 were purified and examined for pH-dependent cofactor requirements for activity. Both net-charge variants and two pH optima that were cofactor-dependent; maximum activity with NADPH, however, was at pH 8.5 and with NADH at pH 6.7. With both variants, however, a higher catalytic rate was observed with NADH than with NADPH at their respective pH optima. Furthermore, BVR-CF2 exhibited a higher catalytic rate than did BVR-CF4 with either cofactor throughout the pH range of 5-9.     

Purification and some properties of a chloramphenicol acetyltransferase mediated by plasmids from Vibrio anguillarum

Vibrio anguillarum strains were isolated from chloramphenicol-resistant bacteria in diseased fish. Plasmid Rms418, which confers chloramphenicol resistance, was transferred from V. anguillarum GN11379 to Escherichia coli K12 by conjugation. The Rms418-encoded chloramphenicol acetyltransferase (CAT) [EC 2.3.1.99] was isolated and purified to homogeneity using affinity chromatography on immobilized p-amino-chloramphenicol or ATP. The general CAT could be adsorbed by a matrix with a chloramphenicol base ligand (Zaidenzaig, Y. & Shaw, W.V. (1976) FEBS Lett. 62,266-271), but the Rms418-encoded CAT was not bound under these conditions. The specific activity of the enzyme, when measured by the spectrophotometric assay, was 71.4 units/mg protein at 37 degrees C. The molecular weight of the enzyme treated with SDS and 2-mercaptoethanol was shown to be approximately 22,000. The molecular weight of the native enzyme, as determined by gel filtration, was approximately 69,000, and the optimal pH was 7.8. The Km values for chloramphenicol and CoASAc were 34.5 and 150 microM, respectively. Enzyme activity was inhibited by HgCl2, p-chloromercuribenzoate (p-CMB), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and ethylendiaminotetraacetic acid (EDTA). The half life at 53 degrees C was approximately 100 min.     

菜豆幼苗EPSP合成酶的分离纯化和它的部分性质

利用硫酸铵分级沉淀,SephedexG-50凝胶柱层析,FPLCMono-Q和磷酸纤维素离子层析法从菜豆幼苗中分离提纯了EPSP合成酶。该酶被纯化2961.6倍,比活性达到6219.4nmolmg-1蛋白min-1。该酶分子量经SDS-PAGE检测为51kD,等电点为pH5.7,酶促反应最适pH7.5,最适温度45℃。6.2μmol/L的除草剂草甘膦能抑制EPSP合成酶活性的50%。     

Polyamine synthesis in mammalian tissues. Isolation and characterization of spermidine synthase from bovine brain.

Spermidine synthase (EC 2.5.1.16) was purified to apparent homogeneity (about 11 000-fold) from bovine brain by affinity chromatography, with S-adenosyl-(5')-3-thiopropylamine linked to Sepharose as the adsorbent. The enzyme preparation was free from S-adenosylmethionine decarboxylase (EC 4.1.1.50) and spermine synthase (EC 2.5.1.22) activities. The native enzyme had an apparent Mr of 70 000, was composed of two subunits of equal size, and had an isoelectric point at pH 5.22. The apparent Km values for putrescine and decarboxylated adenosylmethionine [S-adenosyl-(5')-3-methylthiopropylamine] were 40 microM and 0.3 microM respectively. Cadaverine and 1,6-diaminohexane could replace putrescine as the aminopropyl acceptor, although the reaction rates were only 6% and 1% respectively of that obtained with putrescine. Ethyl, propyl and carboxymethyl analogues of decarboxy-S-adenosylmethionine could act as propylamine donors. Both the reaction products, spermidine and 5'-methylthioadenosine, were mixed-type inhibitors of the enzyme. On the basis of initial-velocity and product-inhibition studies, a ping-pong reaction mechanism for the spermidine synthase reaction was ruled out.     

Selection of a thermostable variant of chloramphenicol acetyltransferase (Cat-86).

The moderate thermophile Bacillus stearothermophilus was used as a host in which to detect more thermostable variants of the B.pumilus chloramphenicol acetyltransferase (Cat-86) protein. Seventeen mutants were isolated and detected by their ability to grow in the presence of chloramphenicol at a previously restrictive temperature (58 degrees C). The genes encoding these proteins were sequenced; all 17 mutants carried the same C to T transition that conferred an amino acid substitution of alanine by valine at position 203 of the protein sequence. The wild-type and one mutant Cat-86 protein were purified to homogeneity using affinity chromatography, and kinetic and thermal stability studies were undertaken. Both enzymes had similar sp. act. in the region of 215 U/mg, with Km values for chloramphenicol in the range 13.8-15.4 microM and for acetyl CoA in the range 13.6-15.5 microM. The A203V mutant shows greater stability than the wild-type Cat-86 protein at temperatures above 50 degrees C and appears to pass through a transition state between 48 and 50 degrees C.     

Acidic glutathione transferase from human heart. Characterization and N-terminal sequence determination

The anionic glutathione transferase of human heart has been purified to homogeneity by using DEAE-cellulose, affinity chromatography, and FPLC. The enzyme has an isoelectric point at pH 4.75 and has an electrophoretic mobility on SDS-PAGE identical to placental transferase pi, indicating that the heart enzyme is formed by two similar subunits of 23,000 Mr. Upon isoelectric focusing on ampholine PAG plates the enzyme recovered from FPLC gave two bands of activity at pH 4.75 and 4.9 which were reduced to essentially a single band at pH 4.75 after incubation with dithiothreitol. In the immunodiffusion experiment, the heart enzyme gave a positive precipitin line with the antibodies against transferase pi but not with antibodies prepared against the "basic" transferase of human skin or against the "near-neutral" transferase of human uterus. The substrate specificities, the sensitivities to characteristic inhibitors, the amino acid composition, together with the immunological studies, strongly indicate that the anionic enzyme of human heart is closely related to the transferase pi of human placenta. The N-terminal amino acid sequence of the first 48 residues was determined and compared with the N-terminal region of other reported human glutathione transferase sequences. The heart enzyme differs from the placental enzyme in a single residue (Trp instead of Arg in the 28th position) further supporting their similarity.     

Purification of adenine phosphoribosyltransferase from Brassica juncea

Adenine phosphoribosyltransferase was purified from Brassica juncea leaves approximately 4000-fold, to homogeneity. The native enzyme is a homodimer, with a Mr of 54,000. The purification involved (NH4)2SO4 fractionation, differential ultracentrifugation, and anion-exchange, hydrophobic, dye-ligand, and affinity chromatography. The purified enzyme has a pH optimum of 9.15 and a temperature optimum of 60 degrees C. Activity of the enzyme is stimulated by Mg2+ and is inhibited by sulfhydryl reagents. At the optimum pH and 37 degrees C, the apparent Km values for adenine and 5-phosphoribosyl-1-pyrophosphate were 3.8 and 15 microM, respectively. Analysis of the purified protein by isoelectric focusing revealed the presence of two isozymes with approximate isoelectric points of 5.3 and 5.4.     

A simple method for the purification of murine corticosteroid binding globulin: characterization of a monomer

A fast and reproducible two-step method with high resolution was developed for purification of murine corticosteroid-binding globulin (CBG). The first step was liquid chromatography on a Sephacryl-S-200 column, and the CBG-containing residual was subsequently chromatographed by fast protein liquid chromatography (FPLC). This enabled us to quickly obtain a highly purified protein and the apparently isolated CBG was tested for its purity by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and sedimentation equilibrium centrifugation. The CBG concentration in pregnant mouse serum was estimated to 0.78 g/l (1.5% of the total protein). The monomeric organization of the protein was demonstrated by mercaptoethanol treatment. No NH2-terminal amino acid could be detected, probably owing to a blocked amino acid. The mol. wt (Mr) of murine CBG was determined to be 52,000 and the sedimentation constant S20 degrees, w to 3.9 S by analytical ultracentrifugation. The protein showed 5 bands when subjected to isoelectric focusing: 3 bands with apparent isoelectric points (pI) between pH 3.15-3.25 and two between pH 3.40-3.50.     

Purification and characterization of UDP-glucuronate: baicalein 7-O-glucuronosyltransferase from Scutellaria baicalensis Georgi. cell suspension cultures

UDP-glucuronate: baicalein 7-O-glucuronosyltransferase (UBGAT) catalyzes the transfer of glucuronic acid from UDP-glucuronic acid to the 7-OH of baicalein. UBGAT was purified from cultured cells of Scutellaria baicalensis Georgi (Lamiaceae). It was purified 95-fold using various chromatography and chromatofocusing procedures to apparent homogeneity. The Mr was estimated to be 110 kDa by gel filtration chromatography with a 52 kDa subunit by SDS-PAGE. The isoelectric point was pH 4.8. UBGAT was specific to UDP-glucuronic acid as a sugar donor and flavones with substitution ortho- to the 7-OH group such its baicalein (6-OH), scutellarein (6-OH) and wogonin (8-OMe).     

Purification and characterization of an inducible aromatic amino acid-sensitive form of chorismate mutase from Solanum tuberosum L. tubers

An amino acid-sensitive form of chorismate mutase (CM) has been purified over 1000-fold from disks excised from tubers of Solanum tuberosum L. cv White Rose. Purification was accomplished by chromatography on Matrix Blue A followed by affinity chromatography with tryptophan as ligand. CM assays performed in the absence of tryptophan yielded pH-dependent sigmoidal kinetics. At pH 8.0, sigmoidal kinetics were observed with a Hill coefficient of 1.66 (S0.5 = 188 microM). However, a shift from sigmoidal to hyperbolic kinetics was observed when assays were performed at pH 8.5. Addition of 9 microM tryptophan to the assay resulted in maximum activation of the enzyme with a Ka of 1.2 microM. When assayed in the presence of tryptophan, hyperbolic kinetics were observed over the pH range 6.0-8.0. Addition of tryptophan also decreased the Km for chorismate from 185 to 45 microM. Tryptophan (0.1 mM) completely protected CM from inhibition by phenylalanine (1.8 mM) and tyrosine (1.8 mM). However, in the absence of the activator, phenylalanine and tyrosine exhibited 50% inhibition at 0.80 and 0.68 mM concentrations, respectively. Both phenylalanine and tyrosine competitively inhibited CM activity with Ki values of 550 and 440 mM, respectively. Arogenate (1.0 mM) had no effect on CM activity in either the presence or absence of tryptophan. Analytical isoelectric focusing yielded an isoelectric point of 4.73.     

Purification and properties of NADP(+)-dependent isocitrate dehydrogenase from the corpus luteum

Cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) was purified 290-fold from the 15,000 x g supernatant fraction of porcine corpora lutea. The major purification step was by anion-exchange chromatography with an FPLC mono P column. Enzyme lability was overcome by including Mg2+, DL-isocitrate, dithiothreitol and glycerol in the elution buffers. The molecular weight of the denatured enzyme was found to be 48,000 by SDS-polyacrylamide gel electrophoresis. The Stokes' radius was estimated to be 3.7 nm by gel filtration and the isoelectric point was 4.8 as determined by chromatofocusing. The purified enzyme had a specific activity of 57.8 units/mg and a broad optimal pH for activity from 7.5 to 9.0. The Km for the substrates DL-isocitrate and NADP+ were 13 and 12 microM, respectively. Polyclonal antibodies were raised against the purified enzyme. Protein (Western) blotting showed an immunological similarity between the cytoplasmic enzyme of the ovary, liver, adrenal gland and heart. A difference was demonstrated between the ovarian enzyme and the heart mitochondrial enzyme. The substrate turnover number and Mr of the ovarian enzyme were similar to those found for the enzyme from the liver and adrenal gland.     

Purification and properties of a polyol dehydrogenase from the phototrophic bacterium Rhodobacter sphaeroides

A polyol dehydrogenase was detected in cell extracts of the facultative phototrophic bacterium Rhodobacter sphaeroides strain Si 4 grown on D-glucitol (sorbitol) as the sole carbon source. The enzyme was purified 150-fold to apparent homogeneity by steps involving fractionated (NH4)2SO4 precipitation, chromatography on Q-Sepharose and phenyl-Sepharose, and FPLC on Superose 12. The relative molecular mass (Mr) of the native polyol dehydrogenase was 47,200 as calculated from its Stokes' radius (rs = 2.76 nm) and sedimentation coefficient (s20, w = 4.15 S). SDS/PAGE resulted in one single band representing a polypeptide with a Mr of 52,200, indicating that the native protein is a monomer. The isoelectric point of the polyol dehydrogenase was determined to be pH 4.3. The enzyme was specific for NAD+ and oxidized both D-glucitol and D-mannitol to D-fructose, as well as D-arabinitol to D-ribulose. The pH optimum of substrate oxidation was pH 9.0 in 0.1 M Tris/HCl and that of substrate reduction was pH 6.5 in 0.1 M potassium phosphate. The reactions exhibited normal Michaelis-Menten kinetics allowing the estimation of KM values for NAD+ (0.18 mM) in the presence of D-glucitol, and for D-glucitol (31.8 mM), D-mannitol (0.29 mM) and D-arabinitol (1.8 mM), respectively. The KM value for D-fructose was 16.3 mM and that for NADH 0.02 mM. The equilibrium constants determined for the conversion of D-mannitol, D-glucitol and D-arabinitol were 4.5 nM, 0.58 nM and 80 pM, respectively. Based on the catalytic preference of the polyol dehydrogenase for D-mannitol, an enzymatic assay for D-mannitol was elaborated.     

Characterization of deglycosylated human urinary colony-stimulating factor (CSF-1)

1. Isolation of CSF-1 from human urine was performed through five purification steps. These include concentration by dialysis, silica gel absorption, hydrophobic chromatography and phenyl-Sepharose CL-6B, Fast Protein Liquid Chromatography (FPLC) and finally preparative electrophoresis on polyacrylamide gels. These methods have been reported in a previous paper (Tao et al., 1987). 2. The isolated CSF-1 which exhibits a one band pattern on SDS-PAGE under non-reducing conditions after Coomassie Blue and silver stainings. CSF-1 was purified 100,000-fold and has a specific activity of 2.16 x 10(7) units/mg protein. Its apparent Mr is 57,000 with an isoelectric point pI = 5.8-6.0 CSF-1 is a glycoprotein with 40% of carbohydrate (w/w). 3. An almost complete removal of the carbohydrate moiety from CSF-1 was obtained after treatment with trifluoromethanesulfonic (TFMS) acid followed by gel filtration on Sephadex G-25 (Fine). The deglycosylated (DG) CSF-1 possesses an apparent Mr of 38,000 and an isoelectric point, pI: 6.2 as compared to native-CSF-1 (N-CSF-1), Mr = 57,000 and pI = 5.8 respectively. 4. The TFMS treatment did not alter the activities of CSF-1 as shown by biological assay and receptor binding assay. The thermostability experiment revealed that DG-CSF-1 was less stable than N-CSF-1. The circular dichroism spectra (CD) of N-CSF-1 and DG-CSF-1 were different. 5. The features of interaction of iodinated-N-CSF-1 and iodinated-DG-CSF-1 with single cell suspensions from human peritoneal macrophage were studied. The binding activity of peritoneal macrophage was the highest among all cells examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and initial characterization of the 71-kilodalton rat heat-shock protein and its cognate as fatty acid binding proteins

The major rat heat-shock (stress) protein and its cognate were purified to electrophoretic homogeneity from livers of heat-shocked rats. Both proteins exhibited similar behavior on a variety of column chromatography matrices but were separable by preparative isoelectric focusing under nondenaturing conditions by virtue of a 0.2 pH unit difference in isoelectric point. Both purified proteins had similar physical properties, suggesting the possibility that they may have similar biological functions as well. Both proteins were homodimers under nondissociative conditions (Mr 150 000) with isoelectric points of 5.0 (cognate) and 5.2 (major stress protein). After denaturation, both proteins had an increase in isoelectric point of 0.6 pH unit, and the resulting polypeptide chains had apparent molecular weights of 73 000 (cognate) and 71 000 (major stress protein). Similarities in the electrophoretic properties of these two proteins and serum albumin, which also undergoes a large basic shift in isoelectric point due to loss of fatty acids and conformational changes accompanying denaturation, prompted us to search for lipids associated with the purified 71-kilodalton stress protein and its cognate. Thin-layer chromatography of chloroform/methanol extracts of these two proteins revealed nonesterified fatty acids bound to both proteins. Palmitic acid, stearic acid, and a small amount of myristic acid were identified by gas chromatography/mass spectroscopy. Both proteins contained approximately four molecules of fatty acid per dimer with palmitate and stearate present in a one to one molar ratio. Possible roles of the major stress protein and its cognate as fatty acid associated proteins in cellular responses to stress are discussed.     

Purification and substrate specificity of an endopeptidase from the human oral spirochete Treponema denticola ATCC 35405, active on furylacryloyl-Leu-Gly-Pro-Ala and bradykinin.

An endopeptidase was purified to homogeneity from the cell extracts of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised dialysis, anion exchange fast protein liquid chromatography (FPLC), hydroxylapatite FPLC, immobilized metal affinity FPLC, FPLC chromatofocusing, and two consecutive gel permeation FPLC steps. The enzyme is a 62-kDa protein with an isoelectric point of 6.5-7.0. Experiments with enzyme inhibitors suggest that this enzyme is a metallopeptidase and that its activity is not dependent on sulfhydryl or serine residues. The enzyme is active on furylacryloyl-Leu-Gly-Pro-Ala (FALGPA; pH optimum near 6.25), bradykinin (Bk), and several Bk-related peptides. In FALGPA, the cleavage site is the Leu-Gly bond. An imino acid is absolutely necessary in position P'2. The shortest hydrolyzed peptide was FALGPA, the hydrolysis of which is strongly and competitively inhibited by Bk (Ki = 5.0 microM). The pyrophosphate ion and phosphoramidon also inhibited the hydrolysis of FALGPA. The enzyme does not hydrolyze all typical synthetic collagenase substrates, Azocoll, Azocasein, or Type I and Type IV collagens, or any other proteins tested. In Bk-related peptides, the hydrolyzed bond was Phe5-Ser6. Since a Bk antagonist and a Bk-potentiating pentapeptide also were good substrates, it is possible that the enzyme hydrolyzes Bks and related peptides only because of the coincidental, specific amino acid sequence of those substrates. A proposal is made that since a substantial portion of the amino acid sequence of FALGPA is present in collagen (and additionally acknowledging that the furylacryloyl residue structurally resembles that of proline), the natural substrates of this enzyme may be small, soluble collagen fragments produced by other enzymes from periodontal connective tissue, and that such peptides are important for the nutrition and pathogenicity of T. denticola.     

Purification and partial characterization of DNA-dependent RNA polymerase from Rhodomicrobium vannielii

DNA-dependent RNA polymerase has been isolated from Rhodomicrobium vannielii. Like those from other eubacteria, the enzyme contained four subunits: beta and beta prime (Mr 155,000), alpha (Mr 38,000), and sigma (Mr 98,000). Analysis by isoelectric focussing showed that both alpha and sigma had several forms with different isoelectric pH values. The enzyme was sensitive to rifampicin (5 ng rifampicin ml-1 gave 50% inhibition) and capable of specific promoter selection with DNA from R. vannielii, calf thymus and phage T7D111.