Metabolism of resorcinylic compounds by bacteria. Purification and properties of acetylpyruvate hydrolase from Pseudomonas putida 01.

Acetylpyruvate hydrolase, the terminal inducible enzyme of the pathway of orcinol catabolism in Pseudomonas putida, catalyzes the quantitative conversion of acetylpyruvate into acetate and pyruvate. The enzyme has been purified approximately 40-fold from extracts of Ps. putida grown on orcinol. Disc gel electrophoresis of the preparations show one major and one minor band of protein. The molecular weight of the enzyme is approximately 38,000 by sodium dodecyl sulfate electrophoresis. Acetylpyruvate is the only known substrate for the enzyme; maleylpyruvate, fumarylpyruvate, acetoacetate, oxalacetate, and acetylacetone are not hydrolyzed by acetylpyruvate hydrolase. Several divalent cations, includ-Mg2+, Mn2+, Co2+, Ca2+, and Zn2+, enhanced hydrolytic activity, but Cu2+ was inhibitory. The enzyme shows a sharp pH optimum at 7.4. Acetylpyruvate hydrolase has an apparent K-m of 0.1 mM for acetylpyruvate with a molecular activity of 36 min minus 1 at 25 degrees. Pyruvate, oxalacetate, and oxalate are competitive inhibitors of acetylpyruvate hydrolysis by the enzyme with K-i values of 6.0, 4.5, and 0.45 mM, respectively.     

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.     

Purification and properties of a second enzyme catalyzing the splitting of carbon-mercury linkages from mercury-resistant Pseudomonas K-62.

An enzyme (splitting enzyme 2) which catalyzes the splitting of carbon-mercury linkage of arylmercury compounds was found in extracts of mercury-resistant Pseudomonas K-62. This enzyme was purified about 725-fold by treatment with streptomycin, precipitation with ammonium sulfate, and successive chromatography on Sephadex G-75 and diethylaminoethyl-cellulose. A purified preparation of the enzyme showed a single band in electrophoresis either on polyacrylamide or sodium dodecyl sulfate-containing polyacrylamide gels. The molecular weight of the enzyme was estimated to be 20,000 (determined by Sephadex G-75 gel filtration) 17,000 (determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis). The enzyme showed a Km of 180 micron and a Vmax of 3.1 mumol/min per mg for p-chloromercuribenzoic acid and a Km of 250 micron and a Vmax of 20 mumol/min per mg for phenylmercuric acetate. The optimum temperature and pH for the reaction were 40 degrees C and 5.0, respectively.     

Purification and Characterization of a Keratinase from a Feather-Degrading Bacillus licheniformis Strain

A keratinase was isolated from the culture medium of feather-degrading Bacillus licheniformis PWD-1 by use of an assay of the hydrolysis of azokeratin. Membrane ultrafiltration and carboxymethyl cellulose ion-exchange and Sephadex G-75 gel chromatographies were used to purify the enzyme. The specific activity of the purified keratinase relative to that in the original medium was approximately 70-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Sephadex G-75 chromatography indicated that the purified keratinase is monomeric and has a molecular mass of 33 kDa. The optimum pH and the pI were determined to be 7.5 and 7.25, respectively. Under standard assay conditions, the apparent temperature optimum was 50°C. The enzyme is stable when stored at −20°C. The purified keratinase hydrolyzes a broad range of substrates and displays higher proteolytic activity than most proteases. In practical applications, keratinase is a useful enzyme for promoting the hydrolysis of feather keratin and improving the digestibility of feather meal.     

Affinity purification and some molecular properties of human liver alkaline phosphatase.

Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.     

Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.     

Purification of N-hydroxy-2-acetylaminofluorene reductase from rabbit liver cytosol

N-Hydroxy-2-acetylaminofluorene reductase was purified from rabbit liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose, Sephadex G-200 and hydroxylapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 34,000 by the electrophoresis and by gel filtration on Sephadex G-200. The enzyme required cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, NADPH or NADH as an electron donor. The enzyme activity was inhibited by p-chloromercuribenzoic acid, N-ethylmaleimide, cupric sulfate or disulfiram, but little by oxygen.     

Purification and characterization of an abscisic acid-inducible anionic peroxidase associated with suberization in potato (Solanum tuberosum)

An anionic peroxidase (EC 1.11.1.7), thought to be involved in suberization, was purified 110-fold from wound-healing slices of Solanum tuberosum by a combination of ammonium sulfate fractionation, Sephadex G-100 gel filtration, isoelectric focusing, and phenyl-Sepharose CL-4B chromatography in 24% yield. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and horizontal thin-layer polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 47,000 by both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This peroxidase was found to be a glycoprotein containing about 17% carbohydrate, approximately one-quarter of which was shown to be glucosamine residues. It was found to have an isoelectric point of 3.15. An anionic peroxidase was also isolated from abscisic acid-treated callus tissue culture of S. tuberosum by the above purification procedure. The two enzymes were shown to be immunologically similar, if not identical, based on their cross-reactivity with rabbit antibody prepared against the peroxidase from wound-healing slices, whereas the major cationic peroxidase from wound-healing slices did not cross-react with this antibody. The anionic enzyme from both sources showed very similar specific activities when assayed with a range of substrates, whereas the specific activities found for the cationic isozyme isolated from wound-healing slices were quite different.     

Purification and Properties of Arginase from Soybean, Glycine max, Axes

Arginase (EC 3.5.3.1) was purified to homogeneity from cytosol of soybean, Glycine max, axes by chromatographic separations on Sephadex G-200, DEAE-sephacel, hydroxyapatite, and arginine-affinity columns. The molecular weight of the enzyme estimated by pore gradient gel electrophoresis was 240,000, while sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a single band at the molecular weight of 60,000. The optimal pH for activity was 9.5 and the K_m value was 83 millimolar. The enzyme was stimulated by polyamines such as putrescine.     

Complete purification and general characterization of FAD synthetase from rat liver

Flavin adenine dinucleotide synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2) was purified about 10,000-fold from the high-speed supernatant of rat liver by a sequence of ammonium sulfate fractionation and column chromatographies on DEAE-Sephadex (A-50), chromatofocusing, FMN-agarose affinity, and Sephadex G-200. The specific activity of the purified enzyme was 133 units (nanomoles of FAD formed per min at 37 degrees C)/mg of protein. This preparation was free from contaminating FAD pyrophosphatase. The apparent molecular weight was estimated to be 97,000 by gel filtration on Sephadex G-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 53,000. Hence, the enzyme is a dimer of approximately 100,000. The enzyme was found most active at pH 7.1, requires Mg2+, and is essentially irreversible in the direction of FAD formation. Kinetic analysis gave Km values of 9.6 microM for FMN and 53 microM for ATP.     

Purification and properties of GM1 ganglioside beta-galactosidases from bovine brain

Two GM1-beta-galactosidases, beta-galactosidases I, and II, have been highly purified from bovine brain by procedures including acetone and butanol treatments, and chromatographies on Con A-Sepharose, PATG-Sepharose, and Sephadex G-200. beta-Galactosidase I was purified 30,000-fold and beta-galactosidase II 19,000-fold. Both enzymes appeared to be homogeneous, as judged from the results of polyacrylamide disc gel electrophoresis. Enzyme I had a molecular weight of 600,000-700,000 and enzyme II one of 68,000, as determined on gel filtration. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under denaturing conditions, enzyme II gave a single band with a molecular weight of 62,000, while enzyme I gave two minor bands with molecular weights of 32,000 and 20,000 in addition to the major band at 62,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide but not from galactosylceramide. Enzyme I showed a pH optimum of 4.0 and was heat stable, while enzyme II showed a pH optimum of 5.0 and lost 50% of its activity in 15 min at 45 degrees C. Enzyme I showed a pI of 4.2 and enzyme II one of 5.9.     

Guanidoacetate methyltransferase. Purification and molecular properties.

Guanidoacetate methyltransferase has been purified about 140-fold from pig liver. Polyacrylamide gel electrophoresis of the purified enzyme showed four protein bands, each of which is associated with guanidoacetate methyltransferase activity. During gel electrophoresis at pH 3 in 8 M urea, guanidoacetate methyltransferase migrated as a single component. The molecular weight of the purified guanidoacetate methyltransferase was estimated to be 31,000 by sodium dodecyl sulfate-gel electrophoresis, which also showed only one protein component with guanidoacetate methyltransferase activity. This molecular weight is in agreement with that estimated by Sephadex G-75 chromatography. Guanidoacetate methyltransferase is inhibited by adenosylhomocysteine, 3-deazaadenosylhomocysteine, and sinefungin with Ki values of 16 microM, 39 microM, and 18 microM, respectively.     

Purification and characterization of a carboxypeptidase-transpeptidase of Bacillus megaterium acting on the tetrapeptide moiety of the peptidoglycan.

The enzyme carboxypeptidase-IIW of Bacillus megaterium incorporates free diaminopimelate into purified bacterial walls. This enzyme can be solubilized from toluene-treated cells by LiCl extraction and has now been purified 106-fold to one major band on polyacrylamide gel electrophoresis. The enzyme has an apparent molecular weight of approximately 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Sephadex G-100 gel filtration. Carboxypeptidase-IIW requires divalent cations and thiol group(s) for optimal activity. Product analysis indicates that the enzyme can hydrolyze the terminal D-alanine from the tetrapeptide of the peptidoglycan or replace it with a variety of amino acids with D-asymmetric centers for transpeptidation. Substrate specificity studies reveal that the enzymatic activity depends on the presence of N-acetyl-D-glucosamine of the GlcNAc-MurNAc-tetrapeptide. This specificity of carboxypeptidase-IIW for the N-acetyl-D-glucosamine explains in part the affinity of the enzyme for the cell wall of B. megaterium. The enzyme is compared to the carboxypeptidases-transpeptidases of other organisms with the similarities and differences discussed.     

Purification and characterization of dihydrobenzophenanthridine oxidase from elicited Sanguinaria canadensis cell cultures.

Upon treatment of Papaveracea cells with fungal elicitors, the biosynthesis of benzo[c]phenanthridine alkaloids is induced. Dihydrobenzophenanthridine oxidase, which catalyzes a later step in the biogenesis of these alkaloids, is one of the enzymes whose activity is elevated in the process. Here we report the 211-fold purification of the oxidase from elicited Sanguinaria canadensis by a combination of ammonium sulfate fractionation, DEAE-Sephadex, CM-Sephadex, Sephadex G-200, and either phenyl Superose or gel filtration chromatography. The purified enzyme utilized molecular oxygen to oxidize dihydrosanguinarine to sanguinarine with concomitant formation of hydrogen peroxide. A pH optimum of 7.0, Vmax of 27 nkat/mg protein, and apparent Km of 6.0 microM for dihydrosanguinarine were determined. Dihydrochelerythrine was also found to be a substrate for the purified enzyme, displaying an apparent Km of 10 microM. However, neither dihydronorsanguinarine nor the indole alkaloid ajmalicine was oxidized, indicating that the enzyme has some substrate specificity. Apparent molecular weight estimates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the most purified enzyme preparation obtained contained a major component of 77 kDa and two minor components between 59 and 67 kDa that can be associated with oxidase activity. Purified enzyme preparations possessed activity that was inhibited by sodium diethyldithiocarbamate, sodium azide, potassium cyanide, 1,4-DL-dithiothreitol, and mercaptoethanol.     

The isolation and characterization of sphingomyelinase from human placental tissue.

Human placental sphingomyelinase activity was eluted as a single symmetrical peak from Sephadex G-200 with a molecular weight of 290000; however, the enzyme behaved heterogeneously on ion exchange chromatography. A specific species of sphingomyelinase was purified approx. 10 000-fold to a constant specific activity of 274 000 nanomol of sphingomyelin hydrolyzed per mg protein per h. When the purified enzyme was examined on sodium dodecyl sulfate disc gel electrophoresis, two distinct protein bands in approximately equal proportions with molecular weights of 36 800 and 28 300 were found. The specificity of the enzyme is directed towards both the hydrophilic phosphocholine and the hydrophobic ceramide moieties of sphingomyelin. Possible interrelationships between the heterogenous forms of placental sphingomyelinases are discussed.     

Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: II. Characterization of the Phosphate Starvation Inducible-Excreted Acid Phosphatase

Three-day-old suspension cultured cells of Lycopersicon esculentum transferred to a Pi-depleted medium had 2.7 times the excreted acid phosphatase (Apase) activity of cells transferred to a Pi-sufficient medium. Cell growth during this time period was identical for the two treatments. Excreted Apase activity was resolved into two fractions on a Sephadex G-150 column. Most of the phosphate starvation inducible (psi) enhancement in activity was in the lower molecular weight fraction. These two fractions exhibited different substrate versus pH activity profiles. With a native polyacrylamide gel electrophoresis assay, the lower molecular weight fraction resolved into two bands of activity. Both column fractions resolved into the same single band of activity with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of this enzyme was 57 kilodalton. These data indicate that L. esculentum has at least two isozymes of the psi-excreted Apase and that these isozymes may associate to form high molecular weight aggregates. Labeling studies using [³⁵S]methionine show that the psi response in tomato cells is complex and involves changes in the steady state levels of several excreted proteins.     

Purification and characterization of saccharopine dehydrogenase from baker's yeast.

Saccharopine dehydrogenase (N6-(glutar-2-yl)-L-ly-sine:NAD oxidoreductase (L-lysine-forming)) from baker's yeast was purified to homogenicity. The overall purification was about 1,200-fold over the crude extract with a yield of about 24%. The purified enzyme had a sedimentation coefficient (S20,w) of 3.0 S. The molecular weight determinations by sedimentation equilibrium, Sephadex G-100 gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a value of about 39,000 and, therefore, saccharopine dehydrogenase is a single polypeptide chain enzyme. A Stokes radius of 27 A and a diffusion constant of 7.9 X 10(-7) cm2 s-1 were obtained from Sephadex gel filtration chromatography. The enzyme had a high isoelectric pH of 10.1. The NH2-terminal sequence was Ala-Ala----. The enzyme possessed 3 cysteine residues/molecule; no disulfide bond was present. Incubation of saccharopine dehydrogenase with p-chloromercuribenzoate or iodoacetate resulted in complete loss of enzyme activity. Whereas the coenzyme and substrates were ineffective in protecting from inactivation by p-chloromercuribenzoate, iodoacetate inhibition was protected by excess coenzyme.     

Purification and characterization of mouse kidney beta-glucuronidase.

Beta-Glucuronidase has been purified from mouse kidneys previously induced by gonadotrophin to a specific enzyme activity 15 times higher than the non-induced kidney. The purification procedure includes ultrasonication to solubilize the enzyme, acid and ammonium sulfate precipitations, gel filtration in Sephadex G-200, DEAE-ion exchange chromatography, and isoelectric focusing. The resulting product has a specific activity of 284,000 Fishman units/mg of protein, representing a 1,090-fold purification and is 17,000-fold higher than the level in the non-induced kidney. The purified beta-glucuronidase is apparently homogeneous by criteria of gel filtration, sodium dodecyl sulfate gel electrophoresis, and immunodiffusion. Characterization of the purified enzyme showed that it is identical with the lysosomal isoenzymic from electrophoretically, has subunit molecular weight of 74,000 (estimated by sodium dodecyl sulfate gel electrophoresis) and oligomer molecular weight of 300,000. The purified enzyme is stable at high temperature (up to 55 degrees) and at wide range of pH (from 4 to 11). It has a pH optimum for its activity at 4.7 and a Km of 1.18 times 10- minus 4 M. The purification and characterization of this enzyme from mouse kidney will have significance in the understanding of the molecular nature of the isoenzymes of beta-glucuronidase and will be useful in future studies on the mechanism of intracellular transport and distribution of this hydrolase.     

Partial purification and properties of acid sphingomyelinase from rat liver

Acid sphingomyelinase was purified approximately 5,200-fold from the mitochondria-lysosome-enriched particles of rat liver by sequential chromatography on DEAE-cellulose, octyl-Sepharose, Sephacryl S-300, Concanavalin A-Sepharose, and CM-cellulose. The specific activity of this highly purified enzyme was 3.2 mmol per hr per mg protein. The enzyme was active against 2-hexadecanoylamino-4-nitrophenylphosphorylcholine, but bis-4-methylumbelliferyl-phosphate and bis-p-nitrophenyl-phosphate were poor substrates. The preparation was free of Mg2+-dependent neutral sphingomyelinase and eight lysosomal enzymes except for the trace amount of acid phosphatase and beta-galactosidase. Apparent molecular weight of the enzyme was 200,000, estimated by Sephadex G-200 filtration in 0.1% Triton X-100. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed three major bands corresponding to molecular weights of 45,600, 44,500, and 40,000 with several minor bands. Characterization of the enzyme revealed almost the same properties as those of human tissues reported by other investigators, including pH optimum, requirement of Triton X-100, effects of metal divalent cations, phosphate ion, EDTA, some thiol blocking reagents, and amphophilic drugs.     

Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi.

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.