scholarly journals Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus

1989 ◽  
Vol 263 (3) ◽  
pp. 913-919 ◽  
Author(s):  
R M Chalmers ◽  
C A Fewson
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Maximum Velocity ◽  
Acinetobacter Calcoaceticus ◽  
Polyacrylamide Gel ◽  
Prosthetic Group ◽  
Gel Filtration Chromatography ◽  
Ph Optimum ◽  
Benzaldehyde Dehydrogenase

Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.

The reaction between hemoglobin α-subunit and haptoglobin

1976 ◽  
Vol 54 (11) ◽  
pp. 1011-1015 ◽  
Author(s):  
Frank A. Terpstra ◽  
David B. Smith
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Human Hemoglobin ◽  
Gel Filtration Chromatography ◽  
Separation Techniques ◽  
Α Subunit ◽  
Α Subunits

The interaction between human hemoglobin α-subunit and porcine haptoglobin was investigated by polyacrylamide gel electrophoresis, gel filtration chromatography, sedimentation through excess α-subunit and gel filtration in an α-subunit-containing medium. No interaction was detected by the first two methods indicating dissociation of the complex during the application of these separation techniques. The latter two methods, in which the complex is studied in a medium of excess subunits, showed that haptoglobin became saturated with the binding of two α-subunits.

scholarly journals The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing

1987 ◽  
Vol 245 (2) ◽  
pp. 575-582 ◽  
Author(s):  
H W Davidson ◽  
J C Hutton
Keyword(s):  
Secretory Granule ◽  
Gel Electrophoresis ◽  
Secretory Granules ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Carboxypeptidase B ◽  
Carboxypeptidase H ◽  
Insulin Secretory Granule

A carboxypeptidase B-like enzyme was detected in the soluble fraction of purified insulin secretory granules, and implicated in insulin biosynthesis. To investigate the role of this activity further, we purified the enzyme from rat insulinoma tissue by gel-filtration chromatography and affinity elution from p-aminobenzoyl-arginine. A yield of 42%, with a purification factor of 674 over the homogenate, was achieved. Analysis of the purified carboxypeptidase by SDS/polyacrylamide-gel electrophoresis under either reducing or non-reducing conditions showed it to be a monomeric protein of apparent Mr 55,000. The preparation was also homogeneous by high-performance gel-filtration chromatography. The enzyme bound to concanavalin A, showing it to be a glycoprotein. Amino acid analysis or chemical deglycosylation and SDS/polyacrylamide-gel electrophoresis indicated a protein Mr of 50,000, suggesting a carbohydrate content of approx. 9% by weight. The purified enzyme was able to remove basic amino acids from the C-terminus of proinsulin tryptic peptides to generate insulin, but did not further degrade the mature hormone. It was inhibited by EDTA, 1,10-phenanthroline and guanidinoethylmercaptosuccinic acid, and stimulated 5-fold by CoCl2. The pH optimum of the conversion of diarginyl-insulin into insulin was in the range 5-6, with little activity above pH 6.5. Activity was also expressed towards a dansylated tripeptide substrate (dansyl-phenylalanyl-leucyl-arginine; Km = 17.5 microM), and had a pH optimum of 5.5. These properties are indistinguishable from those of the activity located in secretory granules, and are compatible with the intragranular environment. The insulin-secretory-granule carboxypeptidase shared several properties of carboxypeptidase H from bovine adrenal medulla and pituitary. We propose that the carboxypeptidase that we purified is the pancreatic isoenzyme of carboxypeptidase H (crino carboxypeptidase B; EC 3.4.17.10), and is involved in the biosynthesis of insulin in the pancreatic beta-cell.

scholarly journals Isolation of a human hepatic 60 kDa aspartylglucosaminidase consisting of three non-identical polypeptides

1989 ◽  
Vol 262 (1) ◽  
pp. 189-194 ◽  
Author(s):  
M Baumann ◽  
L Peltonen ◽  
P Aula ◽  
N Kalkkinen
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Inherited Disease ◽  
Ph Optimum ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Molecular Masses ◽  
Polypeptide Chains

We have characterized the properties of human aspartylglucosaminidase (EC 3.5.1.26), the lysosomal enzyme which is deficient in the human inherited disease aspartylglucosaminuria. The purification procedure from human liver included affinity chromatography, gel filtration, strong-anion- and strong-cation-exchange h.p.l.c., chromatofocusing and reverse-phase h.p.l.c. In a denaturing SDS/polyacrylamide-gel electrophoresis, the 6600-fold purified enzyme was shown to be composed of three non-identical inactive polypeptide chains of molecular masses 24, 18 and 17 kDa. In a native polyacrylamide-gel electrophoresis, these polypeptide chains ran as one active enzyme complex. As judged from the elution position of the native enzyme in a Biogel P-100 gel filtration, the approximate molecular mass of this complex was 60 kDa. The enzyme had a pI of 5.7, a pH optimum at 6, of 0.48 mM and a specific activity of 200,000 nkat for the substrate 2-acetamido-1-beta-(L-aspartamido)-1,2-dideoxy-D-glucose. The enzyme showed a 57% loss of activity at 60 degrees C after 45 h but was practically inactive after incubation at 72 degrees C for a few minutes. The molecular structure, Km and specific activity as well as the thermostability of the enzyme described here are different from those reported previously for human aspartylglucosaminidase.

scholarly journals Purification and properties of S-adenosylmethionine: aldoxime O-methyltransferase from Pseudomonas sp. N.C.I.B. 11652

1985 ◽  
Vol 226 (1) ◽  
pp. 147-153 ◽  
Author(s):  
D B Harper ◽  
J T Kennedy
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Pseudomonas Sp ◽  
Ph Optimum

An enzyme catalysing the O-methylation of isobutyraldoxime by S-adenosyl-L-methionine was isolated from Pseudomonas sp. N.C.I.B. 11652. The enzyme was purified 220-fold by DEAE-cellulose chromatography, (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and chromatography on calcium phosphate gel. Homogeneity of the enzyme preparation was confirmed by isoelectric focusing on polyacrylamide gel and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme showed a narrow pH optimum at 10.25, required thiol-protecting agents for activity and was rapidly denatured at temperatures above 35 degrees C. The Km values for isobutyraldoxime and S-adenosyl-L-methionine were respectively 0.24 mM and 0.15 mM. Studies on substrate specificity indicated that attack was mainly restricted to oximes of C4-C6 aldehydes, with preference being shown for those with branching in the 2- or 3-position. Ketoximes were not substrates for the enzyme. Gel filtration on Sephadex G-100 gave an Mr of 84 000 for the intact enzyme, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated an Mr of 37 500, suggesting the presence of two subunits in the intact enzyme. S-Adenosylhomocysteine was a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 0.027 mM. The enzyme was also susceptible to inhibition by thiol-blocking reagents and heavy-metal ions. Mg2+ was not required for maximum activity.

scholarly journals cis, cis-Muconate cyclase from Trichosporon cutaneum

1980 ◽  
Vol 191 (1) ◽  
pp. 37-43 ◽  
Author(s):  
A Gaal ◽  
H Y Neujahr
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Trichosporon Cutaneum ◽  
Thiol Groups ◽  
Divalent Metal Ions ◽  
Ph Optimum

The inducible enzyme catalysing the conversion of cis, cis-muconate to (+)-muconolactone was purified 300-fold from the yeast Trichosporon cutaneum, grown on phenol. The enzyme has a sharp pH optimum at pH 6.6. It reacts also with several monohalogen derivatives and with one monomethyl derivative of cis, cis-muconate, but not with cis, trans- or trans, trans-muconate or 3-carboxy-cis, cis-muconate. In contrast with the corresponding enzymes in bacteria, the yeast enzyme does not require added divalent metal ions for activity and is not inhibited by EDTA. The purified enzyme can be resolved into two peaks by isoelectric focusing. The two forms have pI 4.58 (cis, cis-muconate cyclase I) and pI 4.74 (cis, cis-muconate cyclase II), respectively. Each of these is homogenous on polyacrylamide-gel electrophoresis in the absence or presence of sodium dodecyl sulphate. The two enzyme forms have the same molecular weight (50000) as determined by gel filtration and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. They have the same Km value (25 microM) for cis, cis-muconate. They differ with respect to their content of free thiol groups. cis, cis-Muconate cyclase I contains one thiol group, essential for activity, but relatively stable upon storage. cis, cis-Muconate cyclase II contains two thiol groups that are readily oxidized during storage with concomitant loss of activity.

scholarly journals Fungal degradation of aromatic nitriles. Enzymology of C-N cleavage by Fusarium solani

1977 ◽  
Vol 167 (3) ◽  
pp. 685-692 ◽  
Author(s):  
David B. Harper
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Fusarium Solani ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Sole Source ◽  
Polyacrylamide Gel ◽  
Ph Optimum

1. A strain of the fungus Fusarium solani able to use benzonitrile as sole source of carbon and nitrogen was isolated by elective culture. 2. Respiration studies indicate that the nitrile, after degradation to benzoate, is catabolized via catechol or alternatively via p-hydroxybenzoate and 3,4-dihydroxybenzoate. 3. Cell-free extracts of benzonitrile-grown cells contain an enzyme mediating the conversion of benzonitrile into benzoate and ammonia. 4. The nitrilase enzyme was purified by DEAE-cellulose chromatography, (NH4)2SO4 precipitation and gel filtration on Sephadex G-200. The homogeneity of the purified enzyme preparation was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing on polyacrylamide gel. 5. The enzyme showed a broad pH optimum between pH7.8 and 9.1 and a Km with benzonitrile as substrate of 0.039mm. The activation energy of the reaction deduced from an Arrhenius plot was 48.4kJ/mol. 6. The enzyme was susceptible to inhibition by thiol-specific reagents and certain heavy metal ions. 7. Gel filtration gave a value of 620000 for the molecular weight of the intact enzyme. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that the enzyme was composed of eight subunits of mol.wt. 76000. 8. Rates of enzymic attack on various substrates indicated that the nitrilase has a fairly broad specificity and that the fungus probably plays an important role in the biodegradation of certain nitrilic herbicides in the environment.

scholarly journals Synthesis of procollagen by matrix-free cells from embryonic-chick arteries

1977 ◽  
Vol 166 (3) ◽  
pp. 437-446 ◽  
Author(s):  
J. David Schofield ◽  
Richard Harwood ◽  
David S. Jackson
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Helical Conformation ◽  
Type I ◽  
Embryonic Chick ◽  
Gel Filtration Chromatography ◽  
Free Cells ◽  
Matrix Free

Cells were isolated from the major arteries of 17-day chick embryos by digestion of the tissue with collagenase and trypsin. The cells, when examined immediately after isolation, exhibited a high degree of viability and they were shown to synthesize and secrete procollagen at a high and constant rate for several hours when incubated in suspension in modified Krebs medium. Continuous labelling of the cells with [14C]proline demonstrated a lag of about 30min between the time at which the synthesis of non-diffusible peptide-bound hydroxy[14C]proline became linear and the time at which its secretion into the medium became linear. This lag time compares with that of 18min observed for freshly isolated matrix-free cells from embryonic-chick tendon, which synthesize and secrete the same type of collagen. Gel-filtration chromatography and polyacrylamide-gel electrophoresis indicated that the collagenous polypeptides secreted into the medium were in the precursor form, known as procollagen, and that the constituent pro-α-chains were linked by interchain disulphide bonds and were also in a triple-helical conformation. Characterization of the secreted procollagen by gel-filtration chromatography, polyacrylamide-gel electrophoresis, DEAE-agarose chromatography, and polyacrylamide-gel electrophoresis of peptides obtained by CNBr cleavage, indicated that the predominant form was type-I procollagen. This work extends the range of freshly isolated matrix-free cell systems, which have been characterized for use in studies on the biosynthesis and secretion of procollagen, and it indicates differences in the rates of secretion of procollagen in different cell types secreting the same type of procollagen.

Purification and characterization of a chymosinlike protease from the gastric mucosa of harp seal (Pagophilus groenlandicus)

1984 ◽  
Vol 62 (8) ◽  
pp. 699-708 ◽  
Author(s):  
K. Shamsuzzaman ◽  
N. F. Haard
Keyword(s):  
Gastric Mucosa ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Porcine Pepsin ◽  
Milk Clotting

Four zymogens of acidic proteases A, B, C, and D were isolated from the gastric mucosa of harp seals by ion-exchange chromatography on a diethylaminoethyl-Sephadex A-50 column. The major zymogens were A and C, and the ratio of zymogen A to zymogen C was greater in extracts from 1-week-old animals than in extracts from adult animals. Zymogens A and C were further purified by affinity chromatography using carbobenzoxy-D-phenylalaninetriethylene tetramine Sepharose and gel filtration on a Sephadex G-100 column. Certain physical and catalytic properties of proteases A and C were compared with those of calf chymosin (EC 3.4.23.4) and porcine pepsin (EC 3.4.23.1). Zymogen C and the corresponding enzyme were homogeneous on analytical polyacrylamide gel electrophoresis. Zymogen A was homogeneous as judged by sodium dodecyl sulphate (SDS) – polyacrylamide gel electrophoresis and high performance liquid chromatography, but was heterogenous by polyacrylamide gel electrophoresis at pH 8.3. Zymogens A and C had molecular weights of 33 800 and 44 000, respectively, as estimated by SDS–polyacrylamide gel electrophoresis. Protease A had an isoelectric point of 4.90. Protease A was similar to calf chymosin with respect to several criteria. It had a higher ratio of milk-clotting to proteolytic activity than those of seal protease C and porcine pepsin and had a pH optimum of 2.2–3.5 for hemoglobin hydrolysis. It did not inactivate ribonuclease, had very low activity on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine and lost activity in 6 M urea. These results indicate protease A is chymosinlike.

scholarly journals Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Purification and preliminary characterization

1988 ◽  
Vol 250 (3) ◽  
pp. 743-751 ◽  
Author(s):  
R W MacKintosh ◽  
C A Fewson
Keyword(s):  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Reduction Reaction ◽  
Oxidation Reactions ◽  
Benzaldehyde Dehydrogenase ◽  
Blue Sepharose

A quick, reliable, purification procedure was developed for purifying both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from a single batch of Acinetobacter calcoaceticus N.C.I.B. 8250. The procedure involved disruption of the bacteria in the French pressure cell and preparation of a high-speed supernatant, followed by chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose CL-6B and Matrex Gel Red A, and finally gel filtration through a Superose 12 fast-protein-liquid-chromatography column. The enzymes co-purified as far as the Blue Sepharose CL-6B step were separated on the Matrex Gel Red A column. The final preparations of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II gave single bands on electrophoresis under non-denaturing conditions or on SDS/polyacrylamide-gel electrophoresis. The enzymes are tetramers, as judged by comparison of their subunit (benzyl alcohol dehydrogenase, 39,700; benzaldehyde dehydrogenase II, 55,000) and native (benzyl alcohol dehydrogenase, 155,000; benzaldehyde dehydrogenase II, 222,500) Mr values, estimated by SDS/polyacrylamide-gel electrophoresis and gel filtration respectively. The optimum pH values for the oxidation reactions were 9.2 for benzyl alcohol dehydrogenase and 9.5 for benzaldehyde dehydrogenase II. The pH optimum for the reduction reaction for benzyl alcohol dehydrogenase was 8.9. The equilibrium constant for oxidation of benzyl alcohol to benzaldehyde by benzyl alcohol dehydrogenase was determined to be 3.08 x 10(-11) M; the ready reversibility of the reaction catalysed by benzyl alcohol dehydrogenase necessitated the development of an assay procedure in which hydrazine was used to trap the benzaldehyde formed by the NAD+-dependent oxidation of benzyl alcohol. The oxidation reaction catalysed by benzaldehyde dehydrogenase II was essentially irreversible. The maximum velocities for the oxidation reactions catalysed by benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were 231 and 76 mumol/min per mg of protein respectively; the maximum velocity of the reduction reaction of benzyl alcohol dehydrogenase was 366 mumol/min per mg of protein. The pI values were 5.0 for benzyl alcohol dehydrogenase and 4.6 for benzaldehyde dehydrogenase II. Neither enzyme activity was affected when assayed in the presence of a range of salts. Absorption spectra of the two enzymes showed no evidence that they contain any cofactors such as cytochrome, flavin, or pyrroloquinoline quinone. The kinetic coefficients of the purified enzymes with benzyl alcohol, benzaldehyde, NAD+ and NADH are also presented.

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