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1.
A bacterial dipeptidyl carboxypeptidase inhibitor was isolated from the culture broth of a bacterium identified as Bacillus subtilis. The inhibitor was purified 33-fold from the culture supernatant of B. subtillis 3-16-20 strain by Q-, and S-Sepharose fast flow, C18 column chromatography, ethanol treatment, and ODS column chromatography. The purified inhibitor has an amino acid sequence of glycyl-prolyl-phenylalanyl-prolylisoleucine. IC50 values of the inhibitor were 177 μM (rabbit lung ACE) and 35 μM (bacterial DCP).  相似文献   

2.
Treatment of the recombinant bovine factor B with trypsin yielded a fragment (amino acid residues 62-175) devoid of coupling activity. Removal of the N-terminal Trp2-Gly3-Trp4 peptide resulted in a significant loss of coupling activity in the FBΔW2−W4 deletion mutant. Sucrose density gradient centrifugation demonstrated co-sedimentation of recombinant factor B with the ADP/ATP carrier, which is present in preparations of H+-translocating F0F1-ATPase, but not in preparations of complex V. The N-terminally truncated factor B mutant FBΔW2−W4 did not co-sediment with the ADP/ATP carrier. Recombinant factor B co-sedimented with partially purified membrane sector F0, extracted from F1-stripped bovine submitochondrial particles with n-dodecyl-β-d-maltoside. Factor B inhibited the passive proton conductance catalyzed by F0 reconstituted into asolectin liposomes. A factor B mutant, bearing a photoreactive unnatural amino acid pbenzoyl-l-phenylalanine (pBpa) substituted for Trp2, cross-linked with F0 subunits e and g as well as the ADP/ATP carrier. These results suggest that the N-terminal domain and, in particular, the proximal N-terminal amino acids are important for the coupling activity and protein-protein interactions of bovine factor B.  相似文献   

3.
L-β-Aminoisobutyrate served as an amino donor for purified β-alanine-oxo-glutarate aminotransferase from rat liver when 2-oxoglutarate was employed as an amino acceptor, but he D-isomer did not. L-β-Aminoisobutyrate acted as a competitive inhibitor with respect to β-alanine and had a Ki of approximately 2.6 mM, which is the same value as the Km of 2.7 mM. When the crude extract was applied to a DEAE-Sepharose CL-6B column, L-β-aminoisobutyrate aminotransferase and β-alanine-oxo-glutarate aminotransferase activities were found in the same fractions with a single peak. Antiserum to rat liver β-alanine-oxo-glutarate aminotransferase inhibited L-β-aminoisobutyrate aminotransferase activity in rat liver in the same way as β-alanine-oxo-glutarate aminotransferase activity.  相似文献   

4.
We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a Mr value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.  相似文献   

5.
Streptococcus milleri NMSCC 061 was screened for antimicrobial substances and shown to produce a bacteriolytic cell wall hydrolase, termed millericin B. The enzyme was purified to homogeneity by a four-step purification procedure that consisted of ammonium sulfate precipitation followed by gel filtration, ultrafiltration, and ion-exchange chromatography. The yield following ion-exchange chromatography was 6.4%, with a greater-than-2,000-fold increase in specific activity. The molecular weight of the enzyme was 28,924 as determined by electrospray mass spectrometry. The amino acid sequences of both the N terminus of the enzyme (NH2 SENDFSLAMVSN) and an internal fragment which was generated by cyanogen bromide cleavage (NH2 SIQTNAPWGL) were determined by automated Edman degradation. Millericin B displayed a broad spectrum of activity against gram-positive bacteria but was not active against Bacillus subtilis W23 or Escherichia coli ATCC 486 or against the producer strain itself. N-Dinitrophenyl derivatization and hydrazine hydrolysis of free amino and free carboxyl groups liberated from peptidoglycan digested with millericin B followed by thin-layer chromatography showed millericin B to be an endopeptidase with multiple activities. It cleaves the stem peptide at the N terminus of glutamic acid as well as the N terminus of the last residue in the interpeptide cross-link of susceptible strains.  相似文献   

6.
The positions of the inter- and intra-chain disulfide bonds of human plasma α2HS-glycoprotein were determined. α2HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)-Cys-14 (A-chain), Cys-71-Cys-82, Cys-96-Cys-114, Cys-128-Cys-131, Cys-190-Cys-201 and Cys-212-Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of α2HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the SS bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two SS bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second SS loops and the end of domains A and B, and the positions of the ordered structures.  相似文献   

7.
A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40 °C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45 °C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M− 1. Apparent Km was 1.02 μM and kcat was 148 S− 1 for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5′-RACE and 3′-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.  相似文献   

8.
N-Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group of l-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designated natA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putative N-acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamine N-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from known N-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity for l-2-phenylglycine and its chloro- and hydroxyl-derivatives. The Km and Vmax values for l-2-phenylglycine were 0.145 ± 0.026 mM and 43.6 ± 2.39 μmol · min−1 · mg protein−1, respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamine N-acetyltransferase and an arylalkylamine N-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups to l-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1).  相似文献   

9.
Three intracellular proteinases termed A, B and C were purified to homogeneity from the unicellular form of the yeast Candida albicans. Enzyme A is an aspartic proteinase that acts on a variety of proteins. Its optimal pH is around 5 and it is displaced to 6.5 by KSCN. It is not significantly inhibited by PMSF, TLCK (Tos-Lys-CHCl2) or soybean trypsin inhibitor but it is inhibited by pepstatin. Its molecular weight is 60 000. Enzyme B is a dipeptidase that acts on esters or on dipeptides without blocks in either the carboxyl or amino ends. Its pH optimum is around 7.5 and the molecular weight is 57 000. It is inhibited by PMSF, TLCK and DANME (N2Ac-Nle-OMe). Proteinase C is an aminopeptidase with an optimum pH around 8. Its molecular weight was 67 000 when determined by SDS gel electrophoresis and 243 000 when determined by gel filtration. It is active towards dipeptides in which at least one amino acid is apolar and is not active when the N-terminal amino acid is blocked. It is inhibited by EDTA or o-phenanthroline and activated by several divalent cations.  相似文献   

10.
The membrane-associated proteinase of Streptococcus lactis strain 3 hydrolyzed αs, 1-casein B into 11 peptide fragments. Eight of the 11 peptides were purified and partially characterized. Each peptide contained several, but not all six, essential amino acids required for growth. The culture was able to utilize one peptide as the sole source for the essential amino acid leucine. Leucine, serine, valine, and glycine were found to be NH2-terminal residues. Two of the peptides were phosphopeptides. The data support the functional role of the membrane-associated proteinase as being involved in the initial breakdown of proteins to peptides.  相似文献   

11.
Hack E  Kemp JD 《Plant physiology》1980,65(5):949-955
A single enzyme catalyzes the synthesis of all four N2-(1-carboxyethyl)-amino acid derivatives found in a crown gall tumor tissue induced by Agrobacterium tumefaciens (E. F. Sm. and Town.) Conn strain B6 on sunflower (Helianthus annuus L.). This enzyme, octopine synthase, has been purified by ammonium sulfate fractionation and chromatography on diethylaminoethylcellulose, blue agarose, and hydroxylapatite. The purified enzyme has all the N2-(1-carboxyethyl)-amino acid synthesizing activities found in crude preparations, and the relative activities with six amino acids remain nearly constant during purification. Although the maximum velocities (V) and Michaelis constants (Km) differ, the ratio V/Km is the same for all amino acid substrates. Thus an equimolar mixture of amino acids will give rise to an equimolar mixture of products. The kinetic properties of the enzyme are consistent with a partially ordered mechanism with arginine (NADPH, then arginine or pyruvate). Octopine synthase is a monomeric enzyme with a molecular weight of 39,000 by gel filtration and 38,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.  相似文献   

12.
Cytochrome b5 (cyt b5) is an amphipathic membrane-bound heme protein found in the endoplasmic reticulum of eukaryotes. It consists of three domains, an N-terminal cytosolic, hydrophilic domain containing the heme, a short flexible linker and an α-helical membrane-spanning domain. This study investigated whether there are specific side chain helix–helix packing interactions between the COOH-terminal membrane anchor of cyt b5 and cytochrome P450 (cyt P450) 2B4 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b5. Insertion of alanine into an α-helix causes all amino acids at its carboxyl terminus to be rotated by 100°. The ability of the alanine insertion mutants of cyt b5 to bind to cyt P450 2B4 was similar to that of the wild-type protein as was the ability of the mutant cyts b5 to stimulate the metabolism of the anesthetic, methoxyflurane. These results demonstrate that the C-terminal hydrophobic α-helix of cyt b5 does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions.  相似文献   

13.
Optimization experiments with response surface statistical analysis were performed with Schizophyllum commune to obtain high β-glucosidase yields. The factors in the optimization experiment were the concentrations of cellulose, peptone, and KH2PO4. Their optimal values were 3.2, 3.0, and 0.2 g/100 ml, respectively. Enzyme assays revealed very high β-glucosidase (22.2 U/ml) and cellobiase (68.9 U/ml) yields. The avicelase yield was low as compared with that from Trichoderma reesei. Mixtures of S. commune and T. reesei culture filtrates caused faster and more extensive saccharification of Avicel than could be achieved by either filtrate alone. A β-glucosidase was isolated and purified from the optimized culture filtrate of S. commune. The electrophoretic mobility of the purified β-glucosidase indicated a molecular weight of 97,000. The amino acid composition was similar to that of β-glucosidase from T. reesei. The acidic (aspartate and glutamate) residues or their amides or both made up approximately 20% of the protein. The NH2-terminal amino acid of the enzyme was histidine.  相似文献   

14.
Polyadenylated mRNA isolated from mineral oil-induced plasmacytoma (MOPC)-321 was injected into Xenopus laevis oocytes that were incubated in 3H-labeled amino acids. The MOPC-321 k chain was purified from an oocyte homogenate by immunoprecipitation, followed by preparative gel electrophoresis. To determine whether the precursor segment had been properly and precisely cleaved in the oocyte, the amino acid sequence of the NH2terminal end of the purified k chain was investigated. The NH2-terminal sequence obtained was identical to that of the mature, secreted form of the protein. Thus the specificity of the enzyme performing the cleavage of precursor to mature chain is similar in frog oocytes and in mammalian cells. Therefore, the enzymatic specificity has been highly conserved during evolution and evidently performs an essential role in cellular metabolism.  相似文献   

15.
《Phytochemistry》1986,25(10):2279-2282
Hevains b and l, isolated respectively from the serum and lutoids of freeze-dried latex from Hevea brasiliensis, were purified to homogeneity and compared with hevain a from commercial, ammonia-treated latex. The Mrs of hevains a and b are 69 000 and 58 000, respectively, and both exist in several charged forms. The amino acid compositions of the two enzymes differ significantly, but the reactivities to a variety of ester and protein substrates are similar, as are the pH optima. Hevain l is a distinct protease of Mr 80 000 and unique amino acid composition. It displays esterolytic activity and will digest insulin B chain, but is not proteolytic to azocollagen, azocasein, bovine serum albumen or haemoglobin. The activities of all three enzymes are dependent on the presence of serine and histidine residues.  相似文献   

16.
Genes dhaF and dhaG encoding the α and β subunits of glycerol dehydratase reactivation factor (GDHtR) were amplified from the genomic DNA of Klebsiella pneumoniae XJPD-Li. The identity of the deduced amino acid sequence of the β subunit was relatively low compared with that of K. pneumoniae (U30903), where the 96th amino acid residue was found to be the more active amino acid histidine instead of glutamine in K. pneumoniae (U30903). A specific GDHtR activity of approximately 30 U/mg was attained in Escherichia coli BL21 (pET-28a (+)-dhaFG). His6-tagged GDHtR was purified by Ni-nitrilotriacetate chromatography, and the enzyme was purified 2.6-fold in a yield of 20.7%. The study showed that both glycerol and O2-inactivated glycerol dehydratase (GDHt) could be quickly reactivated by GDHtR in the presence of ATP, Mg2+ and coenzyme B12. However, the glycerol-inactivated GDHt was more easily reactivated than O2-inactivated GDHt. In the first 10 min of the reactivation reaction, the average reactivation rate was 0.18 and 0.12 μmol/min for glycerol and O2-inactivated GDHt, respectively.  相似文献   

17.
《Phytochemistry》1987,26(10):2699-2704
Purification of cysteine synthase from seedlings of pea (Pisum sativum) reveals the presence of three forms of this enzyme, separated by chromatography on DEAE-Sephadex A-50, and also differences between the cysteine- and uracilylalanine-synthases. Isoenzymes A and B of pea cysteine synthase were purified about 1200-fold and had specific activities of 933 U/mg protein and 892 U/mg protein, respectively. Both isoenzymes were found to have the same Mr (52 000) and to dissociate into two identical subunits (Mr 26 000). The Km value of isoenzyme A is 2.1 mM for O-acetyl-l-serine (OAS) and 36 μM for sulphide, while that of isoenzyme B is 2.3 mM for OAS and 38 μM for sulphide. None of the three isoenzymes from pea seedlings catalyses the formation of the uracilylalanines l-willardiine and l-isowillardiine from OAS and uracil, although isoenzyme A catalyses the formation of β-cyano-l-alanine, and isoenzyme C catalyses the formation of l-quisqualic acid and l-mimosine. Other significant differences occur in the substrate specificity of the three isoenzymes. Several properties, including the amino acid composition of the purified cysteine synthase isoenzymes, are also described.  相似文献   

18.
Phenylalanyl-tRNA synthetase (EC 6.1.1.20) has been purified to homogeneity from a 100-fold overproducing Escherichia coli strain carrying a hybrid pBR322 plasmid containing the pheS-pheT locus. The purified enzyme is identical to the phenylalanyl-tRNA synthetase isolated from an haploid strain. The enzyme was found to dissociate in the presence of 0.5 M NaSCN and the α- and β-subunits composing the native α2β2 enzyme were separated by gel filtration. Neither isolated subunit showed significant catalytic activity. A complex indistinguishable from the native enzyme with full catalytic activity is recovered upon mixing the subunits. The N- and C-terminal sequences and the amino acid composition of each subunit were determined. They are compared to the available data concerning the primary structure of the subunits, as deduced from nucleotide sequencing of the pheS-pheT operon.  相似文献   

19.
A high-isoelectric-point (pI), alkaline endo-1,4-β-glucanase (Egl-257) of Bacillus circulans KSM-N257 was purified to homogeneity and crystallized. The purified enzyme hydrolyzed carboxymethyl cellulose (CMC) with optima of pH 8.5 and 55 °C. The molecular mass was 43 kDa, and the pI was pH 9.3. The structural gene contained a single open reading frame of 1221 bp, corresponding to 407 amino acids (aa), including a 30-aa signal peptide (377 aa and 41,680 Da for the mature enzyme). Egl-257 hydrolyzed lichenan and showed 76.3% aa identity to a lichenase from B. circulans WL-12 belonging to glycosyl hydrolase family 8 but did not hydrolyze laminarin, curdran, and xylan at all. This indicates that Egl-257 is a true endo-1,4-β-glucanase. However, this enzyme was not active on p-nitrophenyl β-d-cellotrioside and p-nitrophenyl β-d-cellotetraoside. It was crystallized by the hanging-drop vapor-diffusion method with phosphate plus CdCl2 as precipitant. Pyramid-like crystals were formed, and they diffracted X-rays beyond 2.2 Å resolution. It belongs to the space group P212121 with unit cell parameters of a=62.5 Å, b=71.7 Å, and c=88.6 Å.  相似文献   

20.
Winter HC  Dekker EE 《Plant physiology》1989,89(4):1122-1128
Aspartate aminotransferase (glutamate-oxalacetate transaminase) was partially purified from extracts of germinating seeds of peanut (Arachis hypogaea), honey locust (Gleditsia triacanthos), soybean (Glycine max), and Sophora japonica. The ability of these enzyme preparations, as well as aspartate aminotransferase purified from pig heart cytosol, to use 4-substituted glutamic acids as amino group donors and their corresponding 2-oxo acids as amino group acceptors in the aminotransferase reaction was measured. All 4-substituted glutamic acid analogs tested were poorer substrates than was glutamate or 2-oxoglutarate. 2-Oxo-4-methyleneglutarate was least effective (lowest relative Vm/Km) as a substrate for the enzyme from peanuts and honey locust, which are the two species studied that accumulate 4-methyleneglutamic acid and 4-methyleneglutamine. Of the different aminotransferases tested, the enzyme from honey locust was the least active with 2-oxo-4-hydroxy-4-methylglutarate, the corresponding amino acid of which also accumulates in that species. These results suggest that transamination of 2-oxo-4-substituted glutaric acids is not involved in the biosynthesis of the corresponding 4-substituted glutamic acids in these species. Rather, accumulation of certain 4-substituted glutamic acids in these instances may be, in part, the result of the inefficacy of their transamination by aspartate aminotransferase.  相似文献   

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