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The isolation of viable hepatocytes from rat liver was found to depend on the source of collagenase (EC 3.4.24.3) more than any other single factor examined. Collagenase purified from Achromobacter iophagus/Bacillus polymyxa (collagenase/dispase) gave reproducibly high viability without the use of complex perfusion protocols.
We have developed a closed-flow system that continuously sweeps away gases evolved in enzyme assay mixtures into a commercially available oxides of nitrogen (NOx) analyzer for the quantitation of any nitric oxide present in these gases. The system enabled us to study both the stoichiometry and the kinetics of NO production by a copper-containing nitrite reductase (EC 1.7.99.3) purified from Achromobacter cycloclastes IAM 1013. In addition to its much greater sensitivity in comparison with standard gas chromatographic (GC) techniques, the method offers the advantage that NO, a very reactive free radical, is immediately swept away and quantitated, obviating the necessity for periodic manipulations and disturbances of the reaction mixture characteristic of other GC quantitations. The characteristics of the system are discussed and its utility in studies of the kinetics and stoichiometry of NO production from nitrite is confirmed by comparison with results obtained using manometric and GC techniques.
A spectrophotometric assay for dissimilatory nitrite reductases has been developed utilizing mammalian cytochrome c (equine heart) as reductant and spectrophotometric agent. The copper-containing nitrite reductase from Achromobacter cycloclastes has been shown to have apparent Km's for reduced cytochrome c and nitrite of 86 +/- 5 and 5.63 +/- 0.03 microM, respectively. The heme cd-containing enzyme from Pseudomonas stutzeri was shown to have apparent Km's for reduced cytochrome c and nitrite of 260 +/- 60 and 1.8 +/- 0.8 microM, respectively. This assay represents a simple, general method for consistently evaluating the activity of the copper- and heme cd-containing nitrite reductases that are capable of utilizing readily available mammalian cytochrome c as electron donor and should be useful for mechanistic studies of these enzymes.
A methodological study of practical importance to protein sequencing has been carried out. Peptide mapping and sequence analysis of the cleavage products of reduced and carboxymethylated ribonuclease have been applied to the study of the activity and specificity of trypsin, chymotrypsin, elastase, lysyl endopeptidase (Achromobacter protease I), endoproteinase Arg-C (from mouse submaxillary gland), Staphylococcus aureus V8 protease, pepsin, and thermolysin in the presence of 20% methanol, ethanol, 2-propanol, and acetonitrile at 22 and 37 degrees C. The peptide bond specificities were retained, and the activities were generally unaffected or moderately reduced at 22 degrees C and pH 8. At 37 degrees C the activity of chymotrypsin, endoproteinase Arg-C, V8 protease at pH 4, and pepsin was substantially reduced and decreased in the order methanol, ethanol, 2-propanol, and acetonitrile. The activity of thermolysin at 55 degrees C was reduced very little in the presence of 20% organic solvent and 50 mM Ca2+. In low calcium and 20% 2-propanol at 22 degrees C the activity of thermolysin was restricted to the complete and specific cleavage of peptide bonds N-terminally of Phe, Ile, and Leu. The experiments suggest that secondary proteolytic digestions can be carried out directly in reversed-phase-HPLC fractions, and that organic cosolvents can be applied to control the degree of proteolysis. Moreover, the denaturing potential of these solvents might be useful in the degradation of proteins resistant to proteolysis, for example, in studies aimed at identification of disulfide bridges.
Homoserine dehydrogenase of Saccharomyces cerevisiae has been rapidly purified to homogeneity by heat and acid treatments, ammonium sulfate fractionation, and chromatography on Matrex Gel Red A and Q-Sepharose columns. The final preparation migrated as a single entity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr of 40,000. The Mr of the native enzyme was 81,000 as determined by gel filtration, suggesting that the enzyme is composed of two identical subunits. This feature was also confirmed by cross-linking analysis using the bifunctional reagent dimethyl suberimidate. Feedback inhibition by L-methionine and L-threonine was observed using the purified enzyme. The enzyme was markedly stabilized against heat treatment at high salt concentrations. Additions of feedback inhibitors or high concentrations of salts failed to cause any dissociation or aggregation of the enzyme subunits unlike enzymes from other sources such as Rhodospirillum rubrum. The enzyme denatured in 3 M guanidine-HCl was refolded by simple dilution with a concomitant restoration of the activity. Cross-linking analysis of the renaturation process suggested that the formation of the dimer is required for activity expression. Amino acid sequence analysis of peptides obtained by digestion of the enzyme protein with Achromobacter lyticus protease I revealed that several amino acid residues are strictly conserved among homoserine dehydrogenases from S. cerevisiae, Escherichia coli, and Bacillus subtilis.
Modification of 12 arginine residues per molecule of formate dehydrogenase (formate : NAD+ oxidoreductase, EC 1.2.1.2.) from the methylotrophic bacterium, Achromobacter parvulus I, by 2,3-butanedione results in complete inactivation of the enzyme. Inactivation of the enzyme is reversible and proceeds in two steps via formation of the intermediate enzyme-butanedione complex. Coenzymes but not formate effectively protect formate dehydrogenase from inactivation. Complete maintenance of enzyme activity and specific protection of one arginine residue per enzyme subunit are achieved on formation of the binary complex, enzyme-NAD, or the ternary complex, enzyme-NAD-azide. One arginine residue is supposed to be located at the NAD-binding site of the formate dehydrogenase active centre.
The reactions of nitrogen monoxide (NO) with the blue copper-containing nitrite reductases from Alcaligenes sp. NCIB 11015 and Achromobacter cycloclastes IAM 1013 were investigated spectroscopically. The electron paramagnetic resonance (EPR) signals of the blue coppers vanished in the presence of NO at 77 K, being fully restored by the removal of NO. The additions of NO to the enzyme solutions resulted in the substantial bleaching of the visible absorption bands at room temperature. The reactions were also completely reversible. These results suggest the formation of a cuprous nitrosyl complex (Cu+-NO+), which is likely the intermediate in the enzymatic nitrite reduction.
Two denitrifying bacteria, Pseudomonas stutzeri and Achromobacter cycloclastes, were incubated with Na15NO2 and NaN3 under conditions that allowed catalysis of nitrosyl transfer from nitrite to azide. This transfer, which is presumed to be mediated by the heme- and copper-containing nitrite reductase of P. stutzeri and A. cycloclastes, respectively, leads to formation of isotopically mixed 14,15N2O, whereas denitrification leads to 15N2O. The conditions that emphasized nitrosyl transfer also partially inhibited the nitric oxide reductase system and led to accumulation of 15NO. Absorption of NO from the gas phase by acidic CrSO4 in a sidewell largely abolished nitrosyl transfer to azide. With these two organisms, which are thought to be representative of denitrifiers generally, catalysis of nitrosyl transfer seemed to depend on NO.
Catechol 2,3-dioxygenases were cloned from Alcaligenes sp. KF711, Pseudomonas putida KF715, and Achromobacter xylosoxidans KF701 which are biphenyl/polychlorinated biphenyls-degrading bacteria. All of the cloned enzymes were purified by preparative polyacrylamide gel electrophoresis (PAGE). The purified catechol 2,3-dioxygenases were significantly different from one another in ring-fission activities to catechol and its derivatives. The catechol 2,3-dioxygenase from Alcaligenes sp. KF711 exhibited higher ring-fission activity to 4-chlorocatechol than those from P. putida KF715 and A. xylosoxidans KF701. In electrophoretic mobilities, the three enzymes were different from one another on nondenaturing PAGE but the same on SDS-PAGE.
Three catechol 2,3-dioxygenases for biphenyl, naphthalene/salicylate, and toluene/xylene oxidation were cloned from Achromobacter xylosoxidans KF701, Pseudomonas putida (NAH7), and Pseudomonas sp. (pWWO). The cloned catechol 2,3-dioxygenases were identified by enzymatic activity assay in addition to yellow bands on polyacrylamide gel after electrophoresis and activity staining. All of the cloned catechol 2,3-dioxygenases exhibited their highest activities on catechol as a substrate compared with catechol derivatives including 4-chlorocatechol, 3-methylcatechol, and 4-methylcatechol. The cloned catechol 2,3-dioxygenases are not fused proteins but were significantly different from one another in their electrophoretic mobilities on nondenaturing 7.5%-polyacrylamide gel.
The complete amino acid sequence of a blue copper protein from Alcaligenes faecalis S-6 has been determined. This protein is clearly homologous to pseudoazurins in Achromobacter cycloclastes and Pseudomonas AM1, more distantly related to plant plastocyanins, and markedly different from the azurin of Pseudomonas aeruginosa. Yet all of these proteins bind copper, and analogous ligands appear to be involved.
The three-dimensional structure of pseudoazurin, a single copper-containing protein from Alcaligenes faecalis strain S-6, has been determined at 2.9 A resolution by X-ray crystallography. The sequences of two other pseudoazurins from Pseudomonas AM1 and Achromobacter cycloclastes may also be accommodated in this structure. The structure, an eight-stranded beta-barrel, resembles closely those of plastocyanin and azurin. It possesses two extra alpha-helices at the C-terminus, whereas azurins have an alpha-helical flap in the middle of their sequences.
The complete amino acid sequence of prostaglandin endoperoxide synthase from sheep vesicular gland has been deduced by cloning and sequence analysis of DNA complementary to its messenger RNA. The results were confirmed by digestion of the enzyme with carboxypeptidase Y and by automated Edman degradation of the intact enzyme polypeptide and peptide fragments obtained by limited proteolysis of the enzyme with Achromobacter proteinase I. Mature sheep prostaglandin endoperoxide synthase is shown to be composed of 576 amino acids with an Mr of 66,175. The precursor peptide is predicted to contain a 24-residue signal peptide. The serine residue susceptible to acetylation by aspirin is found to be located near the C-terminus of the enzyme polypeptide.
A basic protein (pI 10.2), named basic protein I, was purified to homogeneity from the venom of Trimeresurus flavoviridis (Habu snake) after four chromatographic steps. The amino acid sequence of this protein was determined by sequencing the S-pyridylethylated derivative of the protein and its peptides produced by chemical (cyanogen bromide and formic acid) and enzymatic (chymotrypsin, Achromobacter protease I, and Staphylococcus aureus V8 protease) cleavages. The protein consisted of 122 amino acid residues and was similar in sequence to phospholipases A2 from the venoms of crotalid and viperid snakes. A most striking feature of this protein is that aspartic acid at the 49th position common in phospholipases A2 is replaced by lysine. When the protein acted on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine, oleic acid was preferentially released, indicating that the protein has phospholipase A2 activity. Its molar activity toward 1,2-dilauroyl-sn-glycero-3-phosphorylcholine, however, was 1.5% that of T. flavoviridis phospholipase A2 isolated previously. The fact that both affinity to Ca2+ and reactivity to p-bromophenacyl bromide of basic protein I are approximately one order of magnitude lower than those of T. flavoviridis phospholipase A2 might explain the low activity of basic protein I.
Two phospholipases A2, named phospholipases A2-I and A2-II, were purified to homogeneity from the venom of Trimeresurus gramineus (green habu snake). The complete amino acid sequence of phospholipase A2-I was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I, clostripain, and chymotrypsin) and chemical (hydroxylamine) cleavages of the S-pyridylethylated derivative of the protein. The protein consisted of 122 amino acid residues and was similar in sequence to phospholipases A2 from the venoms of crotalid snakes which belong to the category of Group II. A most striking feature of this protein is that tyrosine at the 28th position which is common in phospholipases A2 and is assumed to be a part of the Ca2(+)-binding loop is replaced by phenylalanine. Such replacement is the first finding in Group II phospholipases A2. Secondary structure compositions of phospholipase A2-I are similar to those of Crotalus atrox phospholipase A2. No appreciable Ca2(+)-induced difference spectrum was observed, due probably to the absence of the effective chromophoric groups in the neighborhood of the Ca2+ binding site although Ca2+ is bound with affinity similar to that for T. flavoviridis phospholipase A2.
In addition to phospholipase A2-I (PLA2-I) reported previously (ODA et al., 1991, Toxicon 29, 157), a new PLA2 named PLA2-II was isolated from Trimeresurus gramineus (green habu snake) venom, and its amino acid sequence was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I and clostripain) cleavages of the carboxamidomethylated derivative of the protein. The protein consisted of 122 amino acid residues and His-47, Asp-48, and Asp-98 which have been assumed to be essential for PLA2 activity were conserved. Its sequence similarity to PLA2-I was 79%, with 26 residual differences. In contrast to the unique presence of Phe-28 in PLA2-I, PLA2-II contains Tyr-28 as seen in most of other PLA2s. There was no significant difference between the dissociation constants of PLA2-I and PLA2-II for Ca2+. Secondary structure compositions of PLA2-II were similar to those of PLA2-I and Crotalus atrox PLA2. A striking difference was found between these isozymes in contractile activity of isolated smooth muscle preparation of guinea-pig ileum. PLA2-II was over ten times more potent than PLA2-I, although its lipolytic activity toward egg-yolk was even slightly weaker (73%) than that of PLA2-I. The difference in contractile activities of PLA2-I and PLA2-II could be assumed to be due to discriminative lipid recognition brought about by different amino acid residues at the 58th position (Asp for PLA2-I and Asn for PLA2-II).
Two phospholipases A2 named PLA2-III and IV were newly isolated from Trimeresurus gramineus (green habu snake) venom in addition to PLA2-I and II reported previously [ODA et al. (1991) Toxicon 29, 157; Fukagawa et al. (1992) Toxicon 30, 133]. Their isoelectric points were determined to be about 4.5. PLA2-III and IV exhibited almost unchanged lipolytic activity toward egg-yolk when compared with PLA2-I. The amino acid sequences were determined by sequencing the native proteins and the peptides produced by enzymatic (Achromobacter protease I and clostripain) and chemical (hydroxylamine) cleavages of the S-carboxamidomethylated derivative of the proteins. Both proteins consisted of 122 amino acid residues. When compared with PLA2-I, PLA2-III showed only a single amino acid substitution at the N-terminal position; namely from His to Asn. PLA2-IV also showed a single substitution from Ala to Asp at position 72. It was inferred that these amino acid substitutions between PLA2-I and PLA2-III or IV are due to the single base substitution at the corresponding codons of genes, which might be preserved independently. The unique presence of Phe at position 28, where Tyr is commonly located and assumed to be a part of the Ca(2+)-binding loop, was conserved in both PLA2-III and IV as in PLA2-I. There was no significant difference in the dissociation constants (4.3-5.2 x 10(-4) M) for Ca2+ between these PLA2S and Tyr-28-containing PLA2S. These results suggested that the p-hydroxy group of Try-28 does not play a crucial role in binding of PLA2S to Ca2+.
Four acidic phospholipase A2 (PLA2) isozymes named PLA2-I, II, III and IV have previously been isolated from Trimeresurus gramineus (green habu snake) venom and sequenced [Oda et al. (1991) Toxicon 29, 157; Fukagawa et al. (1992) Toxicon 30, 1131; Fukagawa et al. (1993) Toxicon 31, 957]. They contain aspartate-49 which is known to bind Ca2+, essential for catalysis. In the present study, a basic PLA2 named PLA2-V containing lysine-49 was newly isolated from the same snake venom. Its isoelectric point was 9.4 and considerably higher than those (c. 4.5) of PLA2-I-IV. PLA2-V was 1.1% as active as PLA2-I toward egg-yolk emulsion but exhibited strong myotoxicity. The amino acid sequence of PLA2-V was determined by sequencing the S-carboxamidomethylated derivative and its peptide fragments produced by enzymatic (clostripain, chymotrypsin, Achromobacter protease I and Staphylococcus aureus V8 protease) cleavages. PLA2-V consists of 122 amino acid residues and is highly homologous (72-78%) to Lys-49 PLA2s so far isolated from Viperidae snake venoms but less homologous (52%) to PLA2-I. The presence of Asn-28, which is characteristic of Lys-49 PLA2s, was confirmed.