Aminoacylase from Aspergillus oryzae. Comparison with the Pig Kidney Enzyme

1980 ◽  
Vol 35 (7-8) ◽  
pp. 544-550 ◽  
Author(s):  
Ingrid Gentzen ◽  
Hans-G. Löffler ◽  
Friedh. Schneider
Keyword(s):  
Aspergillus Oryzae ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Kinetic Properties ◽  
Gel Chromatography ◽  
Ph Optimum ◽  
Pig Kidney ◽  
Hydrophobic Amino Acids ◽  
Derivatives Of

Aminoacylase (EC 3.5.1.14) from Aspergillus oryzae was purified from a commercially available crude material by heat treatment, precipitation by polyethyleneimine and ammoniumsulfate, gel chromatography and preparative disc-gel-electrophoresis. The purified product was homogenous as judged by polyacrylamide gel electrophoresis. SDS-gel electrophoresis, polyacrylamide-gel-gradient electrophoresis, gel chromatography and amino acid analysis demonstrated the enzyme to be composed of two subunits with Mr of 36 600. The kinetic properties of the enzyme were studied with chloroacetyl derivatives of alanine, phenylalanine, methionine, leucine, norleucine and tryptophan. The pH optimum of the acylase activity with chloroacetyl-alanine as substrate is at pH 8.5. Acyl derivatives of hydrophobic amino acids are preferred substrates. The enzyme has no dipeptidase activity. Aminoacylase is not inhibited by SH-blocking agents and no SH-groups could be detected with Ellman’s reagent in the native and denatured enzyme. The enzyme activity is insensitive to phenylmethylsulfonyl fluoride and N-α-p-tosylʟ-lysine chloromethyl ketone. The microbial acylase is a zinc metallo enzyme. Metal chelating agents are strong inhibitors; it is further inhibited by Cd2+, Mn2+, Ni2+, Cu2+ and activated by Co2+. The properties of pig kidney and Aspergillus acylase are compared.

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

scholarly journals Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with α2-macroglobulin

1980 ◽  
Vol 191 (3) ◽  
pp. 799-809 ◽  
Author(s):  
R G Sutcliffe ◽  
B M Kukulska-Langlands ◽  
J R Coggins ◽  
J B Hunter ◽  
C H Gore
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Plasma Protein ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Carbohydrate Content ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Tryptic Fragment

Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000–820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A.

scholarly journals Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments

1969 ◽  
Vol 113 (3) ◽  
pp. 489-499 ◽  
Author(s):  
C. R. Parish ◽  
G. L. Ada
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Cyanogen Bromide ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
Lysine Residues

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as ∈-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ‘C,D’ complex and a presumed ‘AB’ fragment. 4. The sum of the amino acid analyses of fragments A and B and the ‘C,D’ complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 ∈-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [125I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ‘C,D’ complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B–A–D–C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.

scholarly journals Purification of 2-oxoaldehyde dehydrogenase and its dependence on unusual amines

1975 ◽  
Vol 149 (3) ◽  
pp. 609-617 ◽  
Author(s):  
J Dunkerton ◽  
S P James
Keyword(s):  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
General Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Ph Values ◽  
Sheep Liver ◽  
Living Tissues

1. 2-Oxoaldehyde dehydrogenase was purified from sheep liver and gave one band on polyacrylamide-gel electrophoresis. 2. The enzyme was completely dependent for its activity on the presence of Tris or one of a number of related amines, all of general structure: (See article). When more than one R group was hydrogen no enzyme activity was observed. 3. Only one of these amines is known to exist in living tissues and large concentrations of all amines were required for maximum activity. L-2-Aminopropan-1-ol was the most effective amine on the basis of substrate Km and Vmax. values and the amine Km values. 4. The enzyme was activated by phosphate which lowered the Km values for methylglyoxal, amine and NAD+. 5. The pH optimum of the enzyme was 9.3 and there was no activity at pH values below 7.8. A search for activators that might produce activity at pH 7.4 proved unsuccessful. 6. The enzyme was inhibited by rather large concentrations of barbiturates (6-46 mM) and nitro-alcohol analogues of the activating amines (66-139 mM).

scholarly journals Purification and properties of β-mannanases I and II from the germinated seeds of Trifolium repens. Mode of galactomannan degradation in vitro

1978 ◽  
Vol 175 (3) ◽  
pp. 1079-1087 ◽  
Author(s):  
H Villarroya ◽  
J Williams ◽  
P Dey ◽  
S Villarroya ◽  
F Petek
Keyword(s):  
Trifolium Repens ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Electrophoretic Method ◽  
Germinated Seeds

Two beta-mannanases (beta-mannosidases, EC 3.2.1.25) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide-gel-electrophoresis. The final purification step completely resolved two beta-mannanases with distinct specificities, which were termed beta-mannanase I and beta-mannanase II. beta-Mannanase I was purified 1400-fold and beta-mannanase II 1000-fold. The purified enzymes showed a single protein band when examined by polyacrylamide-gel disc electrophoresis. beta-Mannanase I, apparent mol.wt. 43 000, accounted for 49% of the total activity recovered from the final step of purification. beta-Mannanase II, apparent mol.wt. 38 000, accounted for the remaining 51% of activity. Molecular-weight determinations were by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by the electrophoretic method of Hendrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. The substrate specificities of both enzymes were examined with the galactomannans of T. repens and of Medicago sativa, as well as with manno-oligosaccharides. The pH optimum was between pH 5.1 and 5.6 for both enzymes.

scholarly journals Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5′-phosphoramidate from adenosine 5′-phosphosulphate and ammonia

1981 ◽  
Vol 195 (3) ◽  
pp. 545-560 ◽  
Author(s):  
Heinz Fankhauser ◽  
Jerome A. Schiff ◽  
Leonard J. Garber
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Low Ph ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Unknown Compound ◽  
Reactive Blue 2

Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5′-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5′-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5′-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5′-phosphoramidate. The enzyme that catalyses the formation of adenosine 5′-phosphoramidate from ammonia and adenosine 5′-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH4)2SO4 precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2–agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000–65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3′-phosphate 5′-phosphosulphate will not replace adenosine 5′-phosphosulphate, and the apparent Km for the last-mentioned compound is 0.82mm. The apparent Km for ammonia (assuming NH3 to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5′-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5′-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme does not catalyse any of the known reactions of adenosine 5′-phosphosulphate, including those catalysed by ATP sulphurylase, adenosine 5′-phosphosulphate kinase, adenosine 5′-phosphosulphate sulphotransferase or ADP sulphurylase. Adenosine 5′-phosphoramidate is found in old samples of the ammonium salt of adenosine 5′-phosphosulphate and can be formed non-enzymically if adenosine 5′-phosphosulphate and ammonia are boiled. In the non-enzymic reaction both adenosine 5′-phosphoramidate and AMP are formed. Thus the enzyme forms adenosine 5′-phosphoramidate by selectively speeding up an already favoured reaction.

Immunoadsorbent isolation of pregnancy-specific β1-glycoprotein from maternal serum

1983 ◽  
Vol 61 (2-3) ◽  
pp. 130-136 ◽  
Author(s):  
Bertram W. Griffiths ◽  
André Godard
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maternal Serum ◽  
Polyacrylamide Gel ◽  
Relative Mass ◽  
Gel Chromatography ◽  
Polymeric Form ◽  
Step Procedure ◽  
New Findings

A three-step procedure for the purification of pregnancy-specific β1-glycoprotein (PSβ1G) on a milligram scale from maternal serum has been developed. The principal purification was achieved by the use of an immunoadsorbent and the remaining impurities were removed by hydroxylapatite chromatography and negative affinity chromatography. The overall procedure resulted in the purification of approximately 10 mg of PSβ1G which represented about 21% of PSβ1G in 300 mL of serum. The PSβ1G was of high purity as shown by analytical polyacrylamide gel electrophoresis, sodium dodecyl sulfate – polyacrylamide gel electrophoresis, and immunochemical tests. Experiments by immunoelectrophoresis and gel chromatography indicate that the electrophoretic mobility and relative mass of the purified PSβ1G are very similar to those of the native serum protein. Structural analysis of PSβ1G suggests that it is composed of two identical subunit chains bonded noncovalently. However, a trimeric structure for PSβ1G cannot be ruled out based on the uncertainty of relative mass estimates by gel chromatography in nondenaturing solvent. The anomalous characteristics of a previous purified polymeric form of PSβ1G (PSβ1G-I) are discussed in relation to the new findings presented here.

scholarly journals Purification and properties of a cellulase from Aspergillus niger

1977 ◽  
Vol 165 (1) ◽  
pp. 33-41 ◽  
Author(s):  
P L Hurst ◽  
J Nielsen ◽  
P A Sullivan ◽  
M G Shepherd
Keyword(s):  
Amino Acid ◽  
Aspergillus Niger ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Cellulolytic Enzyme ◽  
Protein Amino Acid ◽  
Ph Optimum

A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.

scholarly journals Purification and characterization of a tartrate-resistant acid phosphatase from human osteoclastomas

1989 ◽  
Vol 261 (2) ◽  
pp. 601-609 ◽  
Author(s):  
A R Hayman ◽  
M J Warburton ◽  
J A S Pringle ◽  
B Coles ◽  
T J Chambers
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Tartrate Resistant Acid Phosphatase ◽  
Bovine Bone ◽  
Ph Optimum ◽  
Original Tumour

Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources.

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.

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