Systematic Fractionation of Swine Pancreatic Hydrolases. I. Fractionation of Enzymes Soluble in Ammonium Sulfate Solution at 0.40 Saturation<sup>*</sup>

Uriel, J; Avraméas, Stratis

doi:10.1021/bi00885a010

Cited by 19 publications

(10 citation statements)

References 12 publications

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“…The enzymatic hydrolysis of acetyl phenylalanine-@-naphthyl ester was determined using a spectrophotometric technique similar to that developed previously for the quantification of carboxypeptidase-A activity toward carbonaphthoxy-DL-phenylalanine [14]. (The use of a relatively high concentration (200/,) of solvent was necessitated by the low solubility ofused substrate in water.)…”

Section: Assays For Enzymementioning

confidence: 99%

Isolation and Some Properties of a Proteolytic Enzyme from Escherichia coli (Protease I)

Pacaud

Uriel

1971

Eur J Biochem

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By the use of electrophoretic and spectrophotometric methods two types of endopeptidases were demonstrated in crude extracts of Escherichia coli: one was active on N‐acetyl‐dl‐phenyl‐alanine‐2‐naphtyl ester and the other on N‐benzoyl‐l‐arginine‐p‐nitroanilide. The two activities were separated by gel filtration on Sephadex G‐100. After gel electrophoresis of crude extracts, three bands of activity toward acetyl‐phenylalanine‐naphthyl ester were visualized. The enzyme corresponding to the band with the strongest esterolytic activity was also responsible for casein‐hydrolytic activity in gel between pH 6 and 8; it was isolated and characterized. Starting with 450 g of wet cells, about 3 mg of a purified enzyme were obtained. This represented a recovery of 16% and almost 1000‐fold purification. The isolated enzyme, designated as protease I, was homogeneous by electrophoresis and sucrose‐gradient centrifugation. Its molecular weight was estimated by two different methods to be about 43000. The enzyme hydrolysed N‐acetyl‐dl‐phenylalanine‐2‐naphthyl ester, a chymotrypsin substrate, but was inactive upon several synthetic substrates for enzymes with carboxypeptidase‐A and trypsin‐like specificity. The esterolytic activity was inhibited by DFP, whereas it was resistant to phenyl methyl‐sulfonylfluoride even after prolonged treatment. Metal chelating agents, sulfhydryl reagents and several metal ions were without effect. The proterolytic activity of the purified enzyme was confirmed by its ability to breakdown native E. coli polynucleotide phosphorylase.

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Section: Assays For Enzymementioning

confidence: 99%

Isolation and Some Properties of a Proteolytic Enzyme from Escherichia coli (Protease I)

Pacaud

Uriel

1971

Eur J Biochem

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“…One fraction (P 0.40) contained enzymes precipitating in ammonium sulfate solution up to 0.40 saturation; the second fraction was salted out between 0.40 and 0.75 saturation. The systematic fractionation by ionexchange chromatography of the fraction S 0.40-0.75 was reported in the proceeding paper (Uriel and Avrameas, 1965). In the present work, a similar fractionation of the P 0.40 fraction has been carried out.…”

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confidence: 69%

“…The preparation of the P 0.40 fraction from "Pancreatine" and of the rabbit antiserum against pancreatic extract have been described in article I of this series (Uriel and Avrameas, 1965). Furthermore, the same ion exchangers were utilized, and the techniques for electrophoresis, immunoelectrophoresis, and gel diffusion, as well as the identification reactions on antigen-antibody precipitates, are also described in the preceding paper.…”

Section: Methodsmentioning

confidence: 99%

Systematic Fractionation of Swine Pancreatic Hydrolases. II. Fractionation of Enzymes Insoluble in Ammonium Sulfate Solution at 0.40 Saturation^*

Avraméas¹,

Uriel²

1965

Biochemistry

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t The material presented herein has been taken, in part, from a thesis submitted to the University of Paris in partial fulfillment of the requirement for the degree of Doctor of Sciences.in Ammonium properties, designated as elastases 1 and 2, have been isolated, as well as the pancreatic protease 1, an enzyme showing hydrolytic activity against (V-acetyl-L-tyrosine ethyl ester, native egg albumin, and poly-L-glutamic acid. The carboxypeptidases A and B have also been obtained in immunologically pure form. The five isolated enzymes are immunochemically distinct. alanine and hippuryl-L-arginine were purchased from Mann Research Laboratories (New York, N.Y.), and poly-L-glutamic acid from Yeda (Israel).Natural Substrates. Elastine was obtained from Sigma Chemical Co.

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“…ImmunoClectrophorBse L'immunoBlectrophorBse est effectuee dans un tampon vbronal sodique, acetate de sodium 0,05M contenant de la benzamidine-HC1 (Aldrich) 1 mM, A pH 8,6. Les gels sont prepares avec 1'Agar Noble A 0,05°/0 en poids, dans un tampon Tris-HC1 50 mM, NaC!…”

Section: Matfiriei Et Iill?thodes Prkparation Des Immunskrumsunclassified

“…Ces techniques immunochimiques ont dbja 6th utilisees avantageusement pour 1'6tude des enzymes pancreatiques obtenus par broyat tissulaire dans le cas du pore [6,7], du boeuf [8], et du rat [SJ, et un travail prdiminaire a 6th rbalisi: sur les enzymes du tissu pancrbatique humain [lo]. Cependant, aucune etude n'a 6th effectube B partir du sue pancrbatique de I'homme ou d'aucune espece animale, bien que ce materiel de depart soit plus simplc, depourvu d'enzymes non seeretoires et de proteines peu specifiques de l'organe.…”

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Composition du suc pancréatique humain

Clémente¹,

De²,

Figarella³

1972

European Journal of Biochemistry

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The enzyme composition of human pancreatic juice has been studied by immunological techniques, using non‐activated and activated juice. Antisera were prepared with activated and non‐activated pancreatic juice. Same samples of antisera were adsorbed with human serum. Immunoelectrophoresis was carried out at pH 8.6 and disc immunoelectrophoresis at pH 8.9 and 5.0. Gel‐diffusion media contained benzamidine, to prevent zymogen activation. Precipitation lines were characterized by activating the zymogens on the plates with trypsin, and identifying the enzymes produced using specific synthetic substrates. Immunoelectrophoresis showed 15–20 antigenic constituents. Although disc immunoelectrophoresis gave slightly different results, those were consistent with the results of immunoelectrophoresis. The following active enzymes were identified and localized: α‐amylase, lipase and carboxylester hydrolase, which is a lipolytic enzyme structurally and catalytically different from lipase. Several zymogens were identified: two immunologically distinct trypsinogens, one procarboxypeptidase B, two forms of procarboxypeptidase A, which are immunologically identical, and two proelastases with some common antigenic determinants. After activation, both proelastases showed a weak but different degree of chymotryptic activity. Chymotrypsinogen was localized in two major anionic lines, and one, minor, cationic line. The possibility that one anionic immune precipitate represents a polymer is discussed. No chymotryptic activity was associated with procarboxypeptidase A. Immunoelectrophoresis of activated pancreatic juice demonstrates electrophoretic and antigenic changes. Activation of one anionic trypsinogen gave rise to cationic trypsin, whereas after activation chymotrypsinogens and procarboxypeptidases A showed no important change.

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Systematic Fractionation of Swine Pancreatic Hydrolases. I. Fractionation of Enzymes Soluble in Ammonium Sulfate Solution at 0.40 Saturation^*

Abstract: t The material presented herein has been taken, in part, from a thesis submitted to the University of Paris in partial fulfillment of the requirement for the degree of Doctor of Sciences.

Cited by 19 publications

References 12 publications

Isolation and Some Properties of a Proteolytic Enzyme from Escherichia coli (Protease I)

Isolation and Some Properties of a Proteolytic Enzyme from Escherichia coli (Protease I)

Systematic Fractionation of Swine Pancreatic Hydrolases. II. Fractionation of Enzymes Insoluble in Ammonium Sulfate Solution at 0.40 Saturation^*

Composition du suc pancréatique humain

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Systematic Fractionation of Swine Pancreatic Hydrolases. I. Fractionation of Enzymes Soluble in Ammonium Sulfate Solution at 0.40 Saturation*

Abstract: t The material presented herein has been taken, in part, from a thesis submitted to the University of Paris in partial fulfillment of the requirement for the degree of Doctor of Sciences.

Cited by 19 publications

References 12 publications

Isolation and Some Properties of a Proteolytic Enzyme from Escherichia coli (Protease I)

Isolation and Some Properties of a Proteolytic Enzyme from Escherichia coli (Protease I)

Systematic Fractionation of Swine Pancreatic Hydrolases. II. Fractionation of Enzymes Insoluble in Ammonium Sulfate Solution at 0.40 Saturation*

Composition du suc pancréatique humain

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Systematic Fractionation of Swine Pancreatic Hydrolases. I. Fractionation of Enzymes Soluble in Ammonium Sulfate Solution at 0.40 Saturation^*

Systematic Fractionation of Swine Pancreatic Hydrolases. II. Fractionation of Enzymes Insoluble in Ammonium Sulfate Solution at 0.40 Saturation^*