The Purification and Properties of an Amidohydrolase from Soybean

Hoagland, Robert E.; Graf, George

doi:10.1139/o74-127

Cited by 13 publications

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“…It appears from the present study and that on the vetch enzyme that the BAPAases are specific for small peptides containing basic amino acids and cleave them on the carboxyl side of the basic residue. The reported wider specificities for other BAPAases (6,14,22,24) are probably due to the presence of other peptidases. The pea BAPAases can be extracted from seeds even after H20 imbibition in the presence of actinomycin D or cycloheximide, inhibitors of RNA and protein synthesis, respectively, suggesting that the enzymes are present, in potentially active form, in the dormant seed.…”

Section: Resultsmentioning

confidence: 93%

“…To this end it will be first necessary to characterize the proteinases and peptidases involved in this enzymic hydrolysis. Proteinases and peptidases have been partially purified from a number of plant seeds (1,3,9,14,20,22,28), but as yet no such enzymes have been purified from legume seeds to an extent sufficient for characterization with the exception of the aminopeptidases from pea (9). This paper describes the isolation from pea seeds of two peptide hydrolases active against the trypsin substrate BAPA' and their extensive purification and characterization.…”

Section: Introductionmentioning

confidence: 99%

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Partial Purification and Characterization of Two Peptide Hydrolases from Pea Seeds

Caldwell¹,

Sparrow²

1976

Plant Physiol.

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Two peptide hydrolases have been found in pea seeds (Pisum sativum var. Greenfeast) and extensively purified by ion exdchage chromatography using beazoyl-DL-argiaine-p-nitroaniide as substrate. The enzymes which both have molecular weights of 65,000 can be separated by anion exchange chromatography but are otherwise virtually identical in the properties tested. They did not hydrolyze several common protease substrates but readily hydrolyzed small peptides containing basic amino acids on the carboxyl side of these residues. They are completely inhibited by diisopropylfluorophosphate and are inhibited to varying extents by thiol reagents.The seeds of various plants are useful sources of dietary protein and those of legumes are particularly good in this respect. The bulk of the proteins of legume seeds are storage proteins, and these are mobilized by enzymic hydrolysis during germination of the seed providing a source of amino acids for the embryonic plant.An understanding of the processes involved in the mobilization of the storage proteins would be useful information for plant geneticists aiming to improve the quality of these proteins. To this end it will be first necessary to characterize the proteinases and peptidases involved in this enzymic hydrolysis. Proteinases and peptidases have been partially purified from a number of plant seeds (1,3,9,14,20,22,28), but as yet no such enzymes have been purified from legume seeds to an extent sufficient for characterization with the exception of the aminopeptidases from pea (9). This paper describes the isolation from pea seeds of two peptide hydrolases active against the trypsin substrate BAPA' and their extensive purification and characterization.MATERIALS AND METHODS Peas (Pisum sativum var. Greenfeast) were purchased from Brunnings Ltd., Melbourne, Australia. Broad beans (Vicis faba) and soybeans (Glycine max) were supplied by the CSIRO Divisions of Plant Industry and Tropical Agronomy, respectively. Malted wheat was a gift from Joe White, Maltings Ltd., Melbourne, Australia.BANA, BAPA, alanine-p-nitroanilide and glycyl-L-phenylalanine-,B-naphthylamide were obtained from Cyclo Chemical Corp.; other peptide-,3-naphthylamides and L-BAPA were from

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Section: Resultsmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Partial Purification and Characterization of Two Peptide Hydrolases from Pea Seeds

Caldwell¹,

Sparrow²

1976

Plant Physiol.

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“…Aryl acylamidase has been purified from monkey brain [17], human erythrocytes and liver [19] and rat serum [18], or to homogeneity from human serum [18] and sheep platelets [20], and their relationship to acetylcholinesterase in the corresponding tissue has been investigated. The enzyme has also been partially purified [25] [24]; P. acidovorans, 55-57 kDa [15]. Compared to them, our enzyme has much higher molecular m a s (126 kDa) and is composed of two subunits.…”

Section: Discussionmentioning

confidence: 95%

Purification and characterization of aryl acylamidase from Nocardia globerula

Yoshioka

Nagasawa

Yamada

1991

European Journal of Biochemistry

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Aryl acylamidase was purified from an extract of N-acetyl-o-toluidine-induced cells of Nocardiu globerulu I F 0 13510 in ten steps. The purified enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis. The enzyme has a molecular mass of approximately 126 kDa and consists of two subunits which are identical in molecular mass. The purified enzyme catalyzed the hydrolysis of N-acetyl-o-toluidine to o-toluidine and acetic acid at a rate of 47.7 pmol . min-' . mg-' at 35°C. It also catalyzed the hydrolysis of various anilide derivatives and esters, as well as the transfer of an acetyl group to aniline as an acetyl acceptor. The purified enzyme was sensitive to thiol reagents such as HgClz and p-chloromercuribenzoate. The amino-terminal sequence (28 amino acid residues) of the enzyme was determined. Based on the substrate specificity of this enzyme, the pathway intermediates involved in the conversion of n-acetyl-o-toluidine to 4-hydroxy-N-acetyl-o-toluidine are discussed Microbial hydroxylation of aromatic compounds catalyzes position-specific and stereospecific reactions efficiently. This reaction has been utilized in efforts to produce useful compounds in the field of medicine and for chemical industries 6wc0cH3 -t 1/2 0, -no &c0cH3 ( 1 1 Recently, we surveyed the hydroxylation of N-acetyl-otoluidine to 4'-hydroxy-N-acetyl-o-toluidine by various bacteria and actinomycetes. Nocardia globerula I F 0 13510 was selected as the best strain to accumulate 4-hydroxy-N-acetylo-toluidine [lo]. As soon as N-acetyl-o-toluidine was added to culture broth of N. globerulu, o-toluidine was formed. Subsequently, in accordance with the decrease in o-toluidine, an accumulation of 4'-hydroxy-N-acetyl-o-toluidine in the culture broth was observed. Therefore, we presumed that the first step may be the hydrolysis of N-acetyl-o-toluidine to o-toluidine by aryl acylamidase, being associated with the conversion of N-acetyl-o-toluidine into 4-hydroxy-N-acetylo-toluidine.In the present study, in order to elucidate the accumulation mechanism of 4-hydroxy-N-acetyl-o-toluidine, we attemptedCorrespondence to

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“…Synthetic protease substrates were used as L forms also. One of these substrates, benzoyl-L-arginine-p-nitroanilide (L-BAPA), was found to be hydrolyzed by trypsin (10), trypsinlike enzymes (7), various peptidases of plants (6,7,9,15,18,21,26,28), and the peptidase of Corynebacterium matruchotii (11). The D isomer, D-BAPA, is known as a competitive inhibitor for trypsin (10) and plant peptidases (5, 7).…”

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confidence: 99%

Bacteria of the genus Bacillus have a hydrolase stereospecific to the D isomer of benzoyl-arginine-p-nitroanilide

1988

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A stereospecific enzyme activity capable of cleaving the amide bond of the synthetic substrate N-benzoyl-Darginine-p-nitroanilide (D-BAPA) has been found in all aerobic and anaerobic members of the family Bacillaceae tested by us. Cells of nonsporeforming gram-positive or gram-negative bacteria contain a hydrolase activity stereospecific to N-benzoyl-L-arginine-p-nitroanilide. The D-BAPA-hydrolyzing enzymes (DBAPAases) of mid-logarithmic-phase cells of Bacillus subtilis 168 and B. cereus T were compared. These enzymes had the same molecular weight of approximately 66,000 in gel filtration and the same electrophoretic mobility after electrophoresis on polyacrylamide gels. The D-BAPAases of B. subtilis 168 and B. cereus T differed in the effect of inhibitors on enzymatic activity. While both hydrolases were inhibited by tosyl-L-lysine chloromethyl ketone and tosyl-L-arginine-methyl ester as well as leupeptin, only the D-BAPAase of B. Most proteolytic enzymes are capable of discriminating between the D and L forms of molecules. Naturally occurring amino acids found in proteins are generally L stereochemical isomers. Synthetic protease substrates were used as L forms also. One of these substrates, benzoyl-L-arginine-p-nitroanilide (L-BAPA), was found to be hydrolyzed by trypsin (10), trypsinlike enzymes (7), various peptidases of plants (6,7,9,15,18,21,26,28), and the peptidase of Corynebacterium matruchotii (11). The D isomer, D-BAPA, is known as a competitive inhibitor for trypsin (10) and plant peptidases (5, 7). It has been shown that germinated spores of Bacillus cereus T hydrolyzed the racemate DL-BAPA more effectively than the L isomer of BAPA alone (3). This result suggested hydrolysis of the D isomer of BAPA. The present investigation revealed that the D-BAPA-hydrolyzing enzyme is more active in vegetative cells than in germinating spores of bacilli. As no peptide-hydrolyzing enzyme stereospecific to the D isomer of BAPA has been described in the literature, we decided to study the prevalence and some properties of this enzyme in a variety of bacteria. In the following paper we refer to the D-BAPA-hydrolyzing enzymes as D-BAPAase. MATERIALS AND METHODSBacterial strains and culture conditions. Bacillus subtilis 168 trpC2 and B. subtilis 43.4 (spoOA43 leu-8) were used throughout this study. Additional strains tested were B. cereus T; B. thuringiensis var. israelensis; 6A10, an aerobic sporeforming marine bacterium (31) The majority of the work was carried out with B. subtilis 168 and B. cereus T. B. subtilis 168 cells were grown at 30°C in LB medium (11) and B. cereus T cells were grown at 37°C in brain heart infusion (BHI) medium (Difco Laboratories), both with shaking. Starter inocula were prepared from individual colonies on nutrient agar followed by cultivation of B. subtilis 168 in nutrient broth (Difco Laboratories) medium at 30°C and cultivation of B. cereus T in BHI medium at 37°C. The log-phase starters were used as 1% (vol/vol) of the final cultivation medium. To obtain semisynchronized cultures, cu...

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The Purification and Properties of an Amidohydrolase from Soybean

Cited by 13 publications

References 0 publications

Partial Purification and Characterization of Two Peptide Hydrolases from Pea Seeds

Partial Purification and Characterization of Two Peptide Hydrolases from Pea Seeds

Purification and characterization of aryl acylamidase from Nocardia globerula

Bacteria of the genus Bacillus have a hydrolase stereospecific to the D isomer of benzoyl-arginine-p-nitroanilide

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