Purification and characterization of the nuclear cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from rat liver.

N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CAMP-NeuAc synthetase) from rat liver catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid from CTP and NeuAc. We have purified this enzyme to apparent homogeneity (241-fold) using gel filtration on Sephacryl S-200 and two types of affinity chromatographies (Reactive Brown-10 Agarose and Blue Sepharose CL-6B columns). The pure enzyme, whose amino acid composition and NH2-terminal amino acid sequence are also established, migrates as a single protein band on non-denaturing polyacrylamide gel electrophoresis. The molecular mass of the native enzyme, estimated by gel filtration, was 116 +/- 2 kDa whereas its Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 58 +/- 1 kDa. CMP-NeuAc synthetase requires Mg2+ for catalysis although this ion can be replaced by Mn2+, Ca2+, or Co2+. The optimal pH was 8.0 in the presence of 10 mM Mg2+ and 5 mM dithiothreitol. The apparent Km for CTP and NeuAc are 1.5 and 1.3 mM, respectively. The enzyme also converts N-glycolylneuraminic acid to its corresponding CMP-sialic acid (Km, 2.6 mM), whereas CMP-NeuAc, high CTP concentrations, and other nucleotides (CDP, CMP, ATP, UTP, GTP, and TTP) inhibited the enzyme to different extents.     

CMP-N-acetylneuraminic acid synthetase of Escherichia coli: high level expression, purification and use in the enzymatic synthesis of CMP-N-acetylneuraminic acid and CMP-neuraminic acid derivatives.

The gene encoding CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) in Escherichia coli serotype O7 K1 was isolated and overexpressed in E.coli W3110. Maximum expression of 8-10% of the soluble E.coli protein was achieved by placing the gene with an engineered 5'-terminus and Shine-Dalgarno sequence into a pKK223 vector derivative behind the tac promoter. The overexpressed synthetase was purified to greater than 95% homogeneity in a single step by chromatography on high titre Orange A Matrex dye resin. Enzyme purified by this method was used directly for the synthesis of CMP-NeuAc and derivatives. The enzymatic synthesis of CMP-NeuAc was carried out on a multigram scale using equimolar CTP and N-acetylneuraminic acid as substrates. The resultant CMP-NeuAc, isolated as its disodium salt by ethanol precipitation, was prepared in an overall yield of 94% and was judged to be greater than 95% pure by 1H NMR analysis. N-Carbomethoxyneuraminic acid and N-carbobenzyloxyneuraminic acid were also found to be substrates of the enzyme; 5-azidoneuraminic acid was not a substrate of the enzyme. N-Carbomethoxyneuraminic acid was coupled to CMP at a rate similar to that observed with NeuAc, whereas N-carbobenzyloxyneuraminic acid was coupled greater than 100-fold more slowly. The high level of expression achieved with the E.coli synthetase, together with the high degree of purity readily obtainable from crude cell extracts, make the recombinant bacterial enzyme the preferred catalyst for the enzymatic synthesis of CMP-N-acetylneuraminic acid.     

Sequence of the cloned Escherichia coli K1 CMP-N-acetylneuraminic acid synthetase gene

The Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase gene is located on a 3.3-kilobase (kb) HindIII fragment of the plasmid pSR23 which contains the genes for K1 capsule production (Vann, W. F., Silver, R. P., Abeijon, C., Chang, K., Aaronson, W., Sutton, A., Finn, C. W., Lindner, W., and Kotsatos, M. (1987) J. Biol. Chem. 262, 17556-17562). The CMP-NeuAc synthetase gene expression was increased 10-30-fold by cloning of a 2.7-kb EcoRI-HindIII fragment onto the vector pKK223-3 containing the tac promoter. The complete nucleotide sequence of the gene encoding CMP-NeuAc synthetase was determined from progressive deletions generated by selective digestion of M13 clones containing the 2.7-kb fragment. CMP-NeuAc synthetase is located near the EcoRI site on this fragment as indicated by the detection of an open reading frame encoding a 49,000-dalton polypeptide. The amino- and carboxyl-terminal sequences of the encoded protein were confirmed by sequencing of peptides cleaved from both ends of the purified enzyme. The nucleotide deduced amino acid sequence was confirmed by sequencing several tryptic peptides of purified enzyme. The molecular weight is consistent with that determined from sodium dodecyl sulfate-gel electrophoresis. Gel filtration and ultracentrifugation experiments under nondenaturing conditions suggest that the enzyme is active as a 49,000-dalton monomer but may form aggregates.     

Cloning and Overexpression of a Tagged CMP-N-Acetylneuraminic Acid Synthetase from E. coli Using a Lambda Phage System and Application of the Enzyme to the Synthesis of CMP-N-Acetylneuraminic Acid

The gene coding from CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase (Ec 2.7.7.43) was amplified from total DNA of E. coli strain K-235 through a primer-directed polymerase chain reaction. The gene was fused with a modified ribosome binding site of the original CMP-NeuAc synthetase gene and a decapeptide tag sequence which served as a marker for screening of expressed proteins. The gene was cloned into lambda ZAP vector at EcoRI and XbaI sites and overexpressed in E. coli Sure at a level approximately 1000 times that of the wild type. The decapeptide-containing enzyme retained almost the same specificity as indicated by the V_max and K_m values using CTP and NeuAc as substrates. A preparative synthesis of CMP-NeuAc based on the recombinant enzyme was demonstrated.     

Structural characteristics of prostaglandin synthetase from sheep vesicular gland

Prostaglandin synthetase contains both oxygenase and peroxidase activity and catalyzes the first step of prostaglandin synthesis. Aspirin (acetylsalicylic acid) inhibits oxygenase activity by acetylating a serine residue of the enzyme. In the current study, we have investigated the subunit structure of this complex enzyme and the stoichiometry of aspirin-mediated acetylation of the enzyme. The enzyme was purified to near homogeneity in both active and aspirin-acetylated forms. The purified protein was analyzed for enzymatic activity, [3H]acetate content following treatment with [acetyl-3H]aspirin, NH2-terminal sequence, and amino acid composition. The results show first, that the enzyme can be purified to near homogeneity in an active form; second, that the enzyme consists of a single polypeptide chain (molecular weight 72,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis) with a unique NH2-terminal sequence (Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-Met-Gly-); and third, that aspirin inhibits the enzyme by transfer of one acetate per enzyme monomer. Therefore, the two distinct enzymatic activities, oxygenation and peroxidation, are present in a single polypeptide chain. Experiments with a cross-linking agent indicate that in nonionic detergent the enzyme is a dimer of two identical subunits.     

Purification and properties of UDP-glucuronyltransferase from kidney microsomes of beta-naphthoflavone-treated rat

Rat kidney microsomal UDP-glucuronyltransferase activities toward phenoic xenobiotics were enhanced about 4-5-fold by treatment of the animal with beta-naphthoflavone. The transferase activity toward serotonin, an endogenous substrate, was also enhanced about 7.5-fold. A form of UDP-glucuronyltransferase was purified from kidney microsomes of beta-naphthoflavone-treated rat by solubilization with sodium cholate and two steps of column chromatography, the first with DEAE-Toyopearl (fast flow rate liquid chromatography:FFLC) and the second with UDP-hexanolamine Sepharose 4B (affinity chromatography). These procedures gave about 39-fold purification and 11.5% yield of the transferase activity toward 1-naphthol. The preparation, tentatively termed "GT-2," was highly purified as judged from the single protein band (Mr 54,000) on sodium dodecylsulfate (SDS)-polyacrylamide slab gel electrophoresis. It catalyzed the glucuronidation of not only phenolic xenobiotics such as 1-naphthol, 4-nitrophenol, and 4-methylumbelliferone but also serotonin. From the result that apparent molecular weight of GT-2 was reduced to 50,000 by endo-beta-N-acetylglucosaminidase H (Endo H)-treatment, GT-2 was found to be a 50,000 Da polypeptide carrying "high mannose" type oligosaccharide chain(s). The NH2-terminal sequence of 20 residues of GT-2 was determined to be Asp-Lys-Leu-Leu-Val-Val-Pro-Gln-Asp-Gly-Ser-His-Trp-Leu-Ser-Met-Lys-Glu- Ile-Val . It was observed that there are two amino acids substitutions in the seven NH2-terminal residues in comparison with GT-1, which was purified from liver microsomes of 3-methylcholanthrene-treated rat. The NH2-terminal sequence of GT-2 was found to be homologous with the NH2-terminal sequence from the 26th to 46th amino acid residue of various UDP-glucuronyltransferase cloned by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cathepsin D isozymes from porcine spleens. Large scale purification and polypeptide chain arrangements.

Six cathepsin D isozymes have been purified from porcine spleen using a large scale purification procedure. Five isozymes, I to V, have an identical molecular weight of 50,000 and are similar in specific activity. Isozymes I to IV contained two polypeptide chains each. The light and heavy chains have Mr = 15,000 and 35,000, respectively. Isozyme V is a single polypeptide. The molecular weight of the sixth isozyme is about 100,000 and it has only 5% of the specific activity of the other isozymes. On Ouchterlony immunodiffusion, an antiserum formed precipitin lines against the urea-denatured isozyme with Mr = 100,000. This immunoreactivity showed immunoidentity with those formed against other isozymes. The NH2-terminal sequence of light chains was identical for the isozymes. This sequence is homologous to the NH2-terminal sequence of other acid proteases, especially near the region of the active center aspartate-32. The NH2-terminal sequence of the single chain, isozyme V, Is apparently the same as the light chain sequence. The NH2-terminal sequence analysis of the heavy chain from isozyme I produced two sets of related sequences, suggesting the prescene of structural microheterogeneity. The carbohydrate analysis of the isozymes, the light chain, and the heavy chain revealed the presence of possibly four attachment sites, with one in the light chain and three in the heavy chain. Each carbohydrate unit contains 2 residues of mannose and 1 residue of glucosamine. The results suggest that the high molecular weight cathepsin D (Mr = 100,000) is the probable precursor of the single chain (Mr = 50,000), which in turn produces the two-chain isozymes. These are likely in vivo processes.     

Purification and molecular characterization of bovine pregastric lipase

A pregastric lipase was purified from calf pharyngeal tissues. The purification procedure was based on chromatographies on octyl-Sepharose and lentil-lectin-Sepharose followed by gel filtration. The final preparation, with an overall recovery of 26% of activity, gave a single protein band on dodecyl sulfate/polyacrylamide gel electrophoresis with a Mr of 55000. The Mr on gel filtration was 44-48000. The discrepancy may be due to the fact that pregastric lipase is a glycoprotein containing approximately 10% (w/w) of carbohydrate. The pI was around 7.0 and the enzyme protein is characterized by a high content of branched, aliphatic amino acid residues. The NH2-terminal amino acid sequence is: H2N-Phe-Leu/(Ile)-Gly-. Rabbit antibodies to the purified preparation detected only one component in the crude starting material in immuno-blotting experiments. Preincubation with antiserum resulted in loss of enzyme activity, showing that the antibodies were directed against the lipase.     

Purification and characterization of recombinant human leukocyte interferon (IFLrA) with monoclonal antibodies

Recombinant human leukocyte interferon produced in bacteria (IFLrA) was purified to homogeneity with the use of monoclonal antibodies against leukocyte interferon. The purified interferon exhibited a single band of Mr = approximately 19,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis and the NH2-terminal sequence were consistent with the sequence predicted from the DNA. Some of the purified product contained NH2-terminal methionine; the terminal methionine was removed from the rest of the chains.     

Sodium- and potassium-activated adenosine triphosphatase of the nasal salt gland of the duck (Anas platyrhynchos). Purification, characterization, and NH2-terminal amino acid sequence of the phosphorylating polypeptide.

Sodium- and potassium-activated adenosine triphosphatase (NaK-ATPase) was purified from nasal salt glands of the duck (Anas platyrhynchos). Enzyme of specific activity 2,000 to 2,300 mumol of Pi/mg/hour was routinely obtained by sodium dodecyl sulfate treatment of a microsomal fraction of gland homogenate in the presence of 3 mM ATP followed by pelleting of the enzyme through a sucrose density gradient. Purified NaK-ATPase was stable for over 3 months at -20 degree. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography purified NaK-ATPase was shown to contain two polypeptide chains of molecular weight 94,000 and 60,000, the smaller of which was a glycoprotein. Purified enzyme of activity 2,300 mumol of Pi/mg/hour bound 3,600 pmol of ouabain/mg of enzyme protein. Reaction with [gamma-32P]ATP in the presence of Mg2+ and Na+ gave 7,025 pmol of acyl phosphate/mg of enzyme protein. The turnover number calculated from phosphorylation data was 5,460 min-1. Amino acid analysis of the polypeptide components of duck salt gland enzyme after separation by gel filtration chromatography in sodium dodecyl sulfate demonstrated strong compositional homology with highly purified NaK-ATPase preparations from other organs and species. The NH2-terminal amino acid of the 94,000-dalton component was glycine and of the 60,000-dalton component, alanine. With a combination of manual sequencing and automated Edman degradation, the NH2-terminal amino acid sequence of the 94,00-dalton catalytic subunit was found to be Gly-Arg-Asn-Lys-Tyr-Glu-Thr-Thr-Ala-()-Ser-Glu.     

The monomeric glutamyl-tRNA synthetase from Bacillus subtilis 168 and its regulatory factor. Their purification, characterization, and the study of their interaction

The glutamyl-tRNA synthetase from Bacillus subtilis has been purified to homogeneity. It is a monomer of Mr = 65,500 whose NH2-terminal sequence is Met-Asn-Glu-Val-Arg-Val-Arg-Tyr-Ser-Pro-Ser-Pro-Thr-Gly-His-Leu. The number of tryptic peptides indicates the absence of a significant amount of sequence duplication. Under certain conditions, this monomeric enzyme is co-purified with a polypeptide beta of Mr = 46,000, which increases the affinity of the enzyme about 10-fold for glutamate and for ATP, and stabilizes it against heat inactivation. gamma-Globulins prepared against the monomeric enzyme can inhibit completely the glutamyl-tRNA synthetase activity of a B. subtilis extract and precipitate from this extract both the monomeric enzyme and the regulatory factor beta. These anti-alpha immunoglobulins do nt precipitate pure beta. These results show that the glutamyl-tRNA synthetase of B. subtilis has a structure similar to that of the Escherichia coli enzyme (Lapointe, J., and S?ll, D. (1972) J. Biol. Chem. 247, 4966-4974) and indicate that the beta factor has a function in the regulation of glutamyl-tRNA biosynthesis in vivo.     

Purification of CMP-N-acetylneuraminic acid synthetase from bovine anterior pituitary glands

CMP-beta-N-acetylneuraminic acid (CMP-neuNAc) is the substrate for the sialylation of glycoconjugates by sialyltransferases in microbes and higher eukaryotes. CMP-neuNAc synthetase catalyzes the formation of this substrate, CMP-neuNAc, from CTP and neuNAc. In this report we describe the purification of CMP-neuNAc synthetase from bovine anterior pituitary glands. The enzyme was purified by ion exchange, gel filtration, and affinity chromatography. The protein was homogeneous on SDS-PAGE with a molecular weight of 52 kDa, a subunit size similar to that of the E.coli K1 (48.6 kDa). The identity of the 52 kDa protein band was confirmed by native gel electrophoresis in that the position of the enzyme activity in gel slices coincided with the position of major bands in the stained gel. Photoaffinity labeling with 125I-ASA-CDP ethanolamine resulted in the modification of a 52 kDa polypeptide that was partially protected against modification by the substrate CTP. Enzyme activity in crude fractions could be adsorbed onto an immunoadsorbent prepared from antibody against the purified 52 kDa protein. Taken together these data suggest that the 52 kDa polypeptide purified by this procedure described in this report is indeed CMP-neuNAc synthetase. The active enzyme chromatographed on a gel filtration column at 158 kDa suggesting it exists in its native form as an oligomer.     

Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin.

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.     

Bovine angiotensin-converting enzyme: amino-terminal sequence analysis and preliminary characterization of a hybridization-selected primary translation product

Bovine lung angiotensin-converting enzyme was isolated in pure form and the sequence of the first twenty-two NH2-terminal amino acids determined. Oligonucleotides, complementary to a selected portion of the NH2-terminal amino acid sequence of the bovine glycoprotein (Mr 145,000), were synthesized and used for hybridization selection of angiotensin-converting enzyme mRNA. The hybridization-selected mRNA programmed the in vitro synthesis of a single polypeptide (Mr 130,000) that was specifically immunoadsorbed by anti-bovine enzyme antibodies. Preliminary sequence analysis of the primary translation product suggests that bovine angiotensin-converting enzyme is synthesized without a transient NH2-terminal signal sequence.     

Cloning and sequencing of the gltX gene, encoding the glutamyl-tRNA synthetase of Rhizobium meliloti A2.

The gltX gene, coding for the glutamyl-tRNA synthetase of Rhizobium meliloti A2, was cloned by using as probe a synthetic oligonucleotide corresponding to the amino acid sequence of a segment of the glutamyl-tRNA synthetase. The codons chosen for this 42-mer were those most frequently used in a set of R. meliloti genes. DNA sequence analysis revealed an open reading frame of 484 codons, encoding a polypeptide of Mr 54,166 containing the amino acid sequences of an NH2-terminal and various internal fragments of the enzyme. Compared with the amino acid sequence of the glutamyl-tRNA synthetase of Escherichia coli, the N-terminal third of the R. meliloti enzyme was strongly conserved (52% identity); the second third was moderately conserved (38% identity) and included a few highly conserved segments, whereas no significant similarity was found in the C-terminal third. These results suggest that the C-terminal part of the protein is probably not involved in the recognition of substrates, a feature shared with other aminoacyl-tRNA synthetases.     

Structural analysis of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes. Sequences of proteolytic fragments, cysteine-containing peptides, and a NADPH-protected cysteine peptide

Detergent-solubilized NADPH-cytochrome P-450 reductase was purified from porcine hepatic microsomes and compared to the rabbit enzyme isolated under identical conditions. The porcine enzyme had an equivalent specific activity toward cytochrome c compared to the rabbit enzyme. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the porcine enzyme exhibited a major band at Mr = 80,000 and two additional bands at Mr = 20,000 and 60,000. The 20-kDa fragment was shown to be the COOH-terminal portion of the protein which contains a hydrophobic sequence of 28 residues homologous to the pyrophosphate-binding portion of the FAD-binding protein p-hydroxybenzoate hydroxylase. The 60-kDa fragment corresponded to the NH2-terminal portion of the protein since this peptide and the intact protein have blocked NH2 terminal. The trypsin-solubilized porcine enzyme has an NH2-terminal sequence which is homologous to the equivalent trypsin-solubilized enzymes from rat and rabbit (80% sequence homology). Eight cysteine-containing peptides were isolated from a tryptic digest of the S-carboxymethylated pig enzyme. Significant sequence homology was not found between these peptides and other flavoproteins, except for one peptide (Glu-Val-Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg) which exhibited partial homology with the known NADPH-binding site of glutathione reductase. When the NADPH-protected enzyme was first S-alkylated with unlabeled iodoacetate, NADPH depleted, and further alkylated with 14C-labeled iodoacetate, the above radiolabeled peptide was isolated from a tryptic digest. The equivalent peptide was also isolated by a similar procedure from rabbit liver cytochrome P-450 reductase.     

Purification of high and low molecular weight plasminogen activator inhibitor 1 from fibrosarcoma cell-line HT 1080 conditioned medium

Functionally active (high-Mr) and inactive (low-Mr) plasminogen activator inhibitor 1 (PAI) have been purified from fibrosarcoma cell-line HT 1080 conditioned medium, containing 1% fetal calf serum. The two forms were first purified by affinity chromatography on heparin-Sepharose and then separated from each other by gel filtration on Sephadex G-150. The final purification was achieved by affinity chromatography on insolubilized monoclonal antibodies towards human PAI. Alternatively, the low-Mr form was purified by chromatography on carboxymethyl-cellulose. Low-Mr PAI purified in this way, could be almost fully reactivated by treatment with guanidinium chloride. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by immunoblotting revealed that the low-Mr form contained nothing but PAI at an Mr of about 50,000. In addition to PAI, the high-Mr form contained a component, which was not antigenically related to PAI. This compound had a molecular weight of about 75,000 and its NH2-terminal amino acid sequence corresponded to that of human vitronectin. We conclude that the high-Mr form of PAI constitutes a complex between 50,000 Mr PAI and vitronectin from fetal calf serum.     

Purification, characterization, and the complete amino acid sequence of porcine pancreatic deoxyribonuclease

Porcine pancreatic DNase has been purified to homogeneity. The polypeptide exhibits a single band of Mr = 34,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is a glycoprotein containing glucosamine. The results of end group analyses show leucine at the NH2 terminus and alanine at the COOH terminus. The enzymatic properties of the purified porcine DNase are very similar to those of bovine and ovine DNases. The sequence data on the tryptic and chymotryptic peptides derived from CNBr fragments of porcine DNase, along with the results of automated Edman degradation of the intact polypeptide and of the two largest CNBr fragments, indicate the complete amino acid sequence of porcine DNase to be as follows:L-R- I-A-F-N-I-R-T-F-G-E-T-K-M-S-N-A-T-S-N-Y-I-V-R-I-L-S-R-Y-D-I-A-L-I-Q- E-V-R-D-S-H-L-T-A-V-G-K-L-L-N-E-L-N-Q-D-D-P-N-N-Y-H-H-V-V-S-E-P-L-G-R- S-T-Y-K-E-R-Y-L-F-V-F-R-P-N-Q-V-S-V-L-D-S-Y-L-Y-D-D-G-C-E-P-C-G-N-D-T- F-N-R-E-P-S-V-V-K-F-S-S-P-F-T-Q-V-K-E-F-A-I-V-P-L-H-A-A-P-S-D-A-A-A-E- I-N-S-L-Y-D-V-Y-L-N-V-R-Q-K-W-D-L-Q-D-I-M-L-M-G-D-F-N-A-G-C-S-Y-V-T- T-S-H-W-S-S-I-R-L-R-E-S-P-P-F-Q-W-L-I-P-D-T-A-D-T-T-V-S-S-H-T-C-A-Y- D-R-I-V-V-A-G-P-L-L-Q-R-A-V-V-P-D-S-A-A-P-F-D-F-Q-A-A-F-G-L-S-Q-E-T- A-L-A-I-S-D-H-Y-P-V-E-V-T-L-K-R-A. The polypeptide consists of 262 amino acid residues. One of the two disulfide loops links Cys-101 and Cys-104 and the other Cys-173 and Cys-209. Two carbohydrate side chains are attached at Asn-18 and Asn-106.     

Cloning and nucleotide sequence of a heat-stable amylase gene from an anaerobic thermophile, Dictyoglomus thermophilum

A highly heat-stable amylase gene from an obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, was cloned and expressed in Escherichia coli. The nucleotide sequence of the amylase gene predicts a 686-amino-acid protein of relative molecular mass 81,200, which is consistent with that determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified enzyme. The NH2-terminal sequence determined using the enzyme purified from E. coli cells corresponds precisely to that predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein. When the amylase gene was expressed in E. coli cells, the enzyme was localized in the cytoplasmic fraction; this is probably explained by the absence of the signal sequence for secretion. By using the amylase purified from the E. coli transformant, some enzymatic properties, such as optimum pH, optimum temperature, pH-stability and heat-stability, were examined. The amylase was found to be a highly liquefying-type.