Purification and properties of chicken egg-white cobalamin-binding protein

A cobalamin-binding protein has been purified from chicken egg-white by using a combination of conventional and high performance ion-exchange chromatography. Following initial purification by DEAE-cellulose, ammonium sulphate precipitation, Sephacryl S-200 CM-cellulose and affinity chromatography, appropriate fractions were further purified using the Pharmacia fast protein liquid chromatography (FPLC) system. Using this method of purification, egg-white CBP has been purified more rapidly and with greater recovery than with conventional column chromatography. The homogeneity of this protein was verified by SDS-PAGE. The Mr was 37,000 by SDS-PAGE and 39,000 by gel filtration, which indicated that it was a glycoprotein. The stokes radius was 4.1 nm and pI was 4.3. The protein bound 57COB12 with a molar ratio of 1/1 and kd of 0.40 microM. The egg-white CBP was composed of 294 amino acid residues. Thiol groups and metal ions were not connected with the Cbl-binding activities.     

Purification and properties of aminopeptidase H from chicken skeletal muscle.

Aminopeptidase H was purified from fresh chicken breast muscle by ammonium sulfate fractionation and successive chromatographies on DEAE-cellulose, Ultrogel AcA 34, activated thiol-Sepharose 4B, phenyl-Sepharose CL-4B and DEAE-cellulose again. The purified enzyme migrated as a single band on SDS/PAGE. Aminopeptidase H exhibits activity against both L-leucine beta-naphthylamide and alpha-N-benzoyl-DL-arginine beta-naphthylamide. The molecular mass of this enzyme was found to be 52 kDa on SDS/PAGE and 400 kDa on Sepharose 6B column chromatography. The optimum pH for the hydrolysis of both substrates was 8.0 and this activity was remarkably enhanced by reducing agents. The enzyme was strongly inhibited by monoiodoacetate and leupeptin, but not affected by EDTA, phenylmethylsulfonyl fluoride, pepstatin, bestatin or puromycin. Aminopeptidase H has been shown to hydrolyze di-, tri- and tetrapeptides in the manner of an aminopeptidase, as well as the beta-naphthylamide derivatives of amino acids. However, the enzyme has not been shown to hydrolyze proteins such as hemoglobin, bovine serum albumin, myofibrillar proteins or sarcoplasmic proteins.     

Purification and properties of an aminopeptidase from rat-liver cytosol.

An aminopeptidase was purified from the rat-liver cytosolic fraction to apparent electrophoretic homogeneity. The enzyme is a monomeric protein of 95 kDa, having an isoelectric point of 4.9. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cysteine. The enzyme hydrolyzed a broad spectrum of amino acid beta-naphthylamides at a neutral pH. The enzyme also hydrolyzed di-, tri-, and oligopeptides, including physiologically active peptides such as enkephalins and Met-Lys-bradykinin. The enzyme was inhibited by metal-chelating agents, sulfhydryl-reactive reagents, N-P-tosyl-L-phenylalaninechloromethyl ketone, N-P-tosyl-L-lysinechloromethyl ketone, and puromycin but not by protease inhibitors of microbial origin. The enzyme was activated by the addition of Co2+ and sulfhydryl compounds. The aminopeptidase enhanced proteolysis when the enzyme was added to the protease assay system with purified rat-liver cytosolic neutral protease, suggesting the cooperative action of aminopeptidase in the overall process of protein degradation.     

Purification and characterization of aminopeptidase N from chicken intestine with potential application in debittering

An aminopeptidase with broad substrate specificity was purified to homogeneity (123.7-fold) with a yield of 3.43% from chicken (Gallus gallus) intestine using a combination of chromatographic separation strategies. The enzyme was identified as alanyl aminopeptidase or aminopeptidase N (APN) by Peptide Mass Fingerprinting. The molecular weight of the enzyme was estimated to be ∼180 kDa by SDS-PAGE and gel filtration chromatography. The enzyme was found to be a glycoprotein, having 40% sugar residue and a molecular mass of 108 kDa after deglycosylation. The enzymatic activity was optimal at 60 °C and pH 6.0. The enzyme preferentially hydrolyzed Leu-β-NA (K_m = 0.1 mM) followed by Ala, Phe, Tyr and Gly at N-terminal. The enzyme activity was completely inhibited by 1,10 phenanthroline (1 mM) and bestatin (1 mM) confirming it as a metalloprotease. Potential of this enzyme in combination with other endoproteases for the production of debittered protein hydrolysates has been discussed.     

Purification and properties of aminopeptidase C from porcine skeletal muscle.

1. Aminopeptidase C was purified from porcine skeletal muscle. 2. The mol. wt of the enzyme was found to be 103,000 on both Sephadex G-200 column chromatography and SDS-PAGE. 3. The optimum pH for the hydrolysis of L-leucine p-nitroanilide was around 7.0. 4. The activity of this enzyme was strongly inhibited by EDTA, bestatin and puromycin. 5. The enzyme acted on the beta-naphthylamide derivatives of amino acids and oligopeptides.     

Purification and properties of arylsulphatase A from chicken brain

1. Chicken brain arylsulphatase A was purified 2000-fold, with overall recovery 14%, by using ammonium sulphate fractionation, ethanol precipitation, Sephadex G-200 gel filtration and DEAE-Sephadex column chromatography. 2. The purified preparation was free from beta-glucuronidase, beta-galactosidase, acid phosphatase, inorganic pyrophosphatase and adenosine 3'-phosphate 5'-sulphatophosphate sulphohydrolase activities. 3. Polyacrylamide-gel electrophoresis indicated that the purified preparation was not homogeneous. 4. Chicken brain arylsulphatase was markedly inhibited by carbonyl reagents in the presence of traces of Cu(2+) in the system. Other metal ions such as Fe(2+) and Zn(2+), were inactive. 5. Ascorbic acid alone had no effect on enzyme activity but enhances the inhibition by Cu(2+). 6. Chicken brain arylsulphatase A resembled arylsulphatase A of other animal species in its kinetic properties such as K(m) value, anomalous time-activity relationship and the inhibitory effect of phosphate, sulphite and sulphate ions. However, its electrophoretic mobility, behaviour under zinc acetate fractionation and stimulation by Ag(+) were similar to arylsulphatase B of other animal species. Thus, this enzyme did not correspond to either arylsulphatase A or arylsulphatase B but properties of both. 7. The purified enzyme preparation can degrade cerebroside 3-sulphate.     

Purification and properties of beta-galactosidase from chicken seminal plasma

1. beta-Galactosidase (EC 3.2.1.23) from chicken seminal plasma was purified approx. 111-fold to homogeneity. 2. pH optimum of the enzyme ranged from 3.6 to 4.0 and its Km was 0.65 mM with p-nitrophenyl-beta-D-galactoside as substrate. 3. The enzyme was unstable at its optimal activity pH and was activated by Cl- ions. 4. The enzyme had pI value of 4.0. 5. The active enzyme had Mr approx. 100,000 by Sephacryl S-300 chromatography. SDS electrophoresis in the presence of beta-mercaptoethanol showed four bands corresponding to Mr of approx. 90,000, 75,000, 65,000 and 13,000.     

Purification and properties of human intestine alanine aminopeptidase

Human intestinal alanine aminopeptidase has been purified to greater than 90% homogeneity. The enzyme was released from mucosal cell membranes by Triton X-100 treatment. The native enzyme had a molecular weight of 206,000 in dilute buffer and 108,000 in the presence of sodium dodecyl sulfate. The enzyme was inhibited by chelators suggesting the presence of a metal ion in the enzyme. The most potent chelator inhibitor tested, o-phenanthroline, gave mixed kinetics (Ki = 67 micro M). Activity was restored by removal of the chelator. The enzyme was inhibited competitively by amino acids having hydrophobic side chains such as L-phenylalanine (Ki = 0.67 mM). Puromycin and methicillin also inhibited the enzyme in the competitive (Ki = 12.5 micro M) and noncompetitive (Ki = 4.6 mM) manner, respectively. Kinetic analysis of several amino acid beta-naphthylamides as substrates demonstrated the preference for substrates having hydrophobic or basic amino terminal residues with no beta-branching. L-Methionyl-beta-naphthylamide was the most tightly bound with L-alanyl-beta-naphthylamide was the most rapidly hydrolyzed.     

Purification and properties of rat brain dipeptidyl aminopeptidase

Dipeptidyl aminopeptidase, which hydrolyzes the 7-(Gly-Pro)-4-methylcoumarinamide, has been purified from the brains of 3 week-old rats. It was purified about 2,600-fold by column chromatography on CM-cellulose, hydroxyapatite and Gly-Pro AH-Sepharose. This enzyme hydrolyzed Lys-Ala-beta-naphthylamide well with an optimum pH of 5.5. It was inhibited by diisopropyl fluorophosphate, phenyl-methanesulfonyl fluoride, some cations, and puromycin, but was not inhibited by p-chloromercuribenzoate, N-ethylmaleimide, dithiothreitol, EDTA, iodoacetic acid, and bacitracin, indicating that rat brain dipeptidyl aminopeptidase is a serine protease. This enzyme showed a molecular weight of 220,000 by gel filtration and of 51,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The properties of purified rat brain dipeptidyl aminopeptidase were similar to those of bovine pituitary dipeptidyl peptidase II, but the molecular weight and substrate specificity of these enzymes were different.     

Purification and properties of chicken prothrombin

Prothrombin was isolated from citrated chicken plasma. The isolation depends upon the elimination of an interfering substance closely adherent to chicken prothrombin by treatment with SrCO₃. Subsequent to this, the classical adsorption to barium citrate, chromatography on DEAE-cellulose, and gel filtration on Sephadex G-200 was carried out. Prothrombin purified by this method was found to have a specific activity of 1050 Iowa units (850 N.I.H. thrombin units) per mg. Recovery from plasma averaged 40%. Molecular weight by Sephadex G-200 chromatography was 73,000 ± 5,000 and by dodecyl sulfate sodium salt acrylamide gel electrophoresis 70,000 ± 5,000. A stable dimer of M_r 138,000 was observed in some preparations. The isoelectric pH in both acetate and phosphate buffers (μ = 0.1) was 3.95. Rabbit antibody to chicken prothrombin evidenced a single line by immunoelectrophoresis against purified antigen and chicken plasma.     

Purification and some properties of an aminopeptidase from the seeds of Cannabis sativa

An aminopeptidase (HSA) with a molecular mass of 78 kDa was purified from hemp (Cannabis sativa) seeds. The activity was inhibited by monoiodeacetic acid, p-chloromercuri-phenylsulfonic acid, and Zn2+ ion. The specificity of HSA was similar to that of a leucyl aminopeptidase [EC 3.4.11.1] from mammalian cytosol. However, other enzyme properties were different from these of leucyl aminopeptidase.     

Purification and properties of human placental aminopeptidase B.

Aminopeptidase B (EC 3.4.11.6; L-arginyl-beta-naphthylamidase) was purified 1,800-fold from human placental cytoplasm and characterized. The enzyme was subjected to ammonium sulfate fractionation and a series of chromatographies on DE-52, hydroxylapatite, Bio-gel A 0.5 m and L-arginine-Sepharose. The native molecular mass of the enzyme was estimated to be 220,000 by gel filtration. The molecular mass was estimated to be about 83,000 by SDS/PAGE in the absence of 2-mercaptoethanol, suggesting that the enzyme exists in a polymeric form. The isoelectric point of the enzyme was 5.4. The purified enzyme was most active at pH 7.2 with L-arginyl-beta-naphthylamide as substrate and the Km value for this enzyme was 0.3 mmol/l. Human placental aminopeptidase B was markedly activity by Cl-. Bestatin and arphamenin, low molecular weight peptides, showed appreciable inhibition of this enzyme. However, amastatin and puromycin did not inhibit the enzyme. Bacitracin markedly activated this enzyme.     

Purification and properties of a new aminopeptidase from Escherichia COLI K12.

An aminopeptidase (EC 3.4.11.-) capmable of hydrolyzing L-alanyl-beta-naphthyl-amide and certain other aminoacyl beta-naphthylamides was purified to homogeneity from extracts of Exherichia coli K-12. The enzyme, designated aminopeptidase II, is a monomeric protein of mol. wt. 100 000. It exhibits a broad pH optimum in the range pH 7.0--9.0. Although Zn2+, Fe3+ and Cr3+ are strong inhibitors of enzyme activity, a metal requirement for catalysis could not be firmly established. Neither sulfhydryl reagents nor serine protease inhibitors affected enzyme activity.     

Rabbit intestinal aminopeptidase N. Purification and molecular properties

The detergent and protease forms of rabbit intestinal aminopeptidase N were purfied for chemical investigations and future specific immunological labeling of the enzyme in situ. The purification of the detergent form required a special technique called 'reverse immunoabsorbant chromatography'. The specific activity of the detergent form finally obtained was identical to that of the protease form. A significant charge micro heterogeneity persisted in the most purified preparations, due probably to a certain level of variability in the sugar moiety. The major proteolytic cleavage which occurred at the hydrophilic-hydrophobic junction of the detergent form during its conversion into the protease form was well defined. But additional splittings probably in C-terminal region of the molecules led to several protease forms differing by their size. The molecular weight assigned to the peptide liberated during the above conversion was overestimated due to preferential detergent binding to hydrophobic structures. The correct value, estimated by a new isotopic dilution method, was 3800 (36-38 residues) for the peptide originating from the rabbit enzyme. The real anchor plunging into the membrane core is possibly still shorter. Comparative N-terminal residue determinations in the detergent form, the protease form and the peptide difinitely confirmed that the enzyme is anchored to the bursh border membrane by its N-terminal region.     

Purification and properties of an aminopeptidase from Alaska pollack, Theragra chalcogramma, roe

An aminopeptidase was isolated from a soluble fraction of Alaska pollack roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-200. The molecular weight of the enzyme was estimated to be 125,000 and 105,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The pH optimum and temperature optimum were 7.2 and 35 degrees C, respectively. The purified enzyme hydrolyzed various alpha-aminoacyl beta-naphthylamides and cleaved L-Ala-beta-naphthylamide most rapidly. Both a sulfhydryl group and a divalent metal ion are essential for activity; however, the enzyme was inhibited when incubated with divalent metal ions. Puromycin, chelating agents, and thiol reagents were effective inhibitors. The enzyme was also inhibited by L-amino acids, in particular glutamic acid. Thus, the Alaska pollack roe aminopeptidase resembles soluble alanyl aminopeptidase [EC 3.4.11.14].