Polynucleotide kinase from rat-liver nuclei. Purification and properties.

A polynucleotide kinase, which catalyzes the phosphorylation of 5'-hydroxyl ends of deoxyribonucleic acid in the presence of adenosine triphosphate, has been purified 260-fold with a yield of 14% from 0.15 M NaCl extracts of rat liver nuclei. The purified enzyme has a pH optimum of 5.5. The enzyme is reversible inhibited by p-chloromercuribenzoate. The S0.5 value (ligand concentration required for a half-maximal activity) for ATP is 2.5 muM. A bivalent cation is essential for the reaction and S0.5 values for Mg2+, Ca2+ and Mn2+ are 3.3 mM, 4 mM and 0.05 mM respectively. Pyrophosphate remarkable inhibits the activity with I0.5 value (ligand concentration required for a half-maximal inhibition) of 0.2 mM, and sulfate, with I0.5 of 0.5 mM, whereas phosphate weakly inhibits the activity with I0.5 of about 20 mM. An apparent molecular weight of the purified enzyme is estimated to be 8 X 10(4) by gel filtration on a column of Sephadex G-150, and the Stokes radius of the enzyme molecule is shown to be about 0.36 nm. Sucrose density gradient centrifugation reveals that the enzyme has a sedimentation coefficient of about 4.4 S.     

Purification and properties of an endonuclease endogenous to rat-liver nuclei

An endonuclease endogenous to rat-liver nuclei has been purified by a series of chromatographic procedures and finally by isoelectric focusing (IEF) electrophoresis. The nuclease fraction prepared by the IEF electrophoresis (IEF fraction) showed a pI value of 5.7 and migrated as a single band to a molecular weight position of 46,000 on an SDS-polyacrylamide gel. The activity for single-stranded DNA was enhanced by 10 mM MgCl2 and/or by 5-15 mM MgCl2 in the presence of 2 mM CaCl2 (an optimum pH, 7.0), but was lowered by CaCl2 alone and inhibited strongly by ZnCl2 or MnCl2. The activity for duplex DNA was rather low, although an optimum condition was 10 mM MgCl2. In fact, even under this condition, the activity was about 40% lower than that for single-stranded DNA. Moreover, the IEF fraction formed single-strand nicks much more rapidly than double-strand cuts in pBR322 DNA, and preferentially produced deoxyadenosine 5'-monophosphate termini in the DNA. In addition, RNAase activity was also detected in this fraction.     

Purification and characterization of an aminopeptidase from Mycoplasma salivarium.

The aminopeptidase which had been shown to be present in Mycoplasma salivarium was found to be associated with the cell membranes of the organism. The enzyme was solubilized in water by papain digestion of the membranes pretreated with Triton X-100 and purified approximately 130-fold by ion-exchange chromatography on DEAE-Sephadex A-50, affinity chromatography on L-leucylglycine-AH-Sepharose 4B, and gel filtration on Sepharose CL-6B. The purified enzyme had a molecular mass of 397 kilodaltons, estimated by gel filtration through Sepharose CL-6B, and gave two bands of activity in analytical disc polyacrylamide gel electrophoresis: a dense, diffuse band and a less dense, narrow one, accounting for 90 and 5% of stained proteins in the gel, respectively. The purified protein revealed two bands with molecular masses of 50 and 46 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed selectively the cleavage of the N-terminal arginine and leucine residues of peptides; had a pH optimum at 8.5; and was inhibited remarkably by bestatin, o-phenanthroline, EDTA, and L-cysteine, but was activated nine- and twofold by MnCl2 and MgCl2, respectively. The enzyme pretreated with MnCl2 had much higher maximum velocity (Vmax) for L-leucine-p-nitroanilide than the one not treated. That is, the Michaelis constant (Km) and Vmax values of the pretreated enzyme were 10.5 mM and 12.1 microM/min, respectively, whereas those of the untreated enzyme were 5.8 mM and 1.6 microM/min, respectively.     

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 extracellular leucine aminopeptidase from the Bacillus sp. N2

A halophilic bacterium was isolated from fermented anchovy sauce and identified as Bacillus species. An extracellular leucine aminopeptidase from Bacillus sp. N2 was purified to homogeneity using four successive purification steps. The enzyme has a native molecular mass of about 57 000 Da using FPLC gel filtration analysis and a molecular mass of 58 000 Da using SDS-polyacrylamide gel electrophoresis. This monomeric leucine aminopeptidase showed maximum enzyme activity at pH 9·5. The optimum temperature was 50 °C when L -Leu- p -nitroanilide was the substrate. The leucine aminopeptidase was inactivated by 1,10-phenanthroline, dithiothreitol and sodium dodecyl sulphate. Enzyme activity was increased by addition of Co²⁺. It is likely that Co²⁺ plays an important role in the catalysis or stability of the Bacillus sp. N2 leucine aminopeptidase. Sodium chloride (0–4·5 mol l⁻¹) increased the hydrolytic activity towards L -Leu- p -nitroanilide. The N-terminal amino acid sequence was Glu-Arg-Glu-Leu-Pro-Phe-Lys-Ala-Lys-His-Ala-Tyr-Ser-Thr-Ile. The purified enzyme had a high specificity for L -Leu- p -nitroanilide.     

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 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 glutamyl aminopeptidase from chicken egg-white

Hydrolytic activities characteristic for different aminopeptidases were detected in the egg-white of unfertilized chicken eggs, and one aminopeptidase was isolated in an electrophoretically homogeneous form. The isolated aminopeptidase preferentially hydrolyzed bonds of alpha-glutamyl residue at the NH(2)-end of synthetic substrates and peptides. The enzyme is a dimer with an M(r) of 320,000 and pI of 4.2. Its optimal pH and temperature are 7.6 and 60 degrees C, respectively. EDTA, amastatin, and N-bromosuccinimide are inhibitors, while Ca2++ and Mn2+ are activators of the enzyme Ca2+ also stabilizes the enzyme. According to the observed properties, the isolated chicken egg-white aminopeptidase belongs to the glutamyl aminopeptidases.     

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].     

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 characterization of an aminopeptidase from bovine leukocytes

An aminopeptidase was purified about 4,000-fold from the clarified homogenate of bovine leukocytes by a series of column chromatographies on DEAE-cellulose, hydroxyapatite, Sephadex G-150, and DEAE-Toyopearl. The purified enzyme had a specific activity of 3.8 mumol X min-1 X mg-1 with arginine beta-naphthylamide (Arg-2-NNap) as substrate, and a minute amount of contaminating protein was found to be present by gel electrophoresis. The molecular weight of the enzyme was estimated to be 94,000 by gel filtration on Sephadex G-150. The enzyme had a broad substrate specificity and a pH optimum between 6.5 and 7.0 for the hydrolysis of alpha-aminoacyl beta-naphthylamides. It hydrolyzed beta-naphthylamides of basic, aliphatic, and aromatic amino acids, and also catalyzed the liberation of amino-terminal phenylalanine from phenylalanyl peptides. The enzyme was inhibited by bestatin, puromycin, 1,10-phenanthroline, sulfhydryl reagents, and a variety of heavy metal ions. Only the cobaltous ion stimulated the enzyme and the values of both Km and Vmax for Arg-2-NNap increased. In gross properties the present enzyme resembles porcine liver aminopeptidase reported previously (Kawata, S., et al. (1982) J. Biochem. 92, 1093-1101) very closely.     

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 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.     

Purification and properties of an alkaline ribonuclease from the hepatic cytosol fraction of bullfrog, Rana catesbeiana.

In the hepatic cytosol fraction of bullfrog, Rana catesbeiana, an alkaline RNase [EC 3.1.4.22] exists in two forms. One is the free form of RNase, which elutes from a carboxymethyl-cellulose column at a concentration of 0.2 M NaC1. The other is a masked or latent form (RNase-RNase inhibitor complex) which is not adsorbed on the carboxymethyl-cellulose column and which can be converted to the free form of RNase by the addition of p-chloromercuribenzoate. Electrophoretically pure RNase was obtained by the following procedure. The unadsorbed fraction of hepatic cytosol on a column of carboxymethyl-cellulose was treated with p-chloromercuribenzoate and then applied to a second carboxymethyl-cellulose column. The molar weight of RNase was determined to be approximately 12,000 by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. From the results of gel filtration, the molecular weight of the RNase-RNase inhibitor complex was 130,000. The RNase hydrolyzed poly C, poly U, and poly I, but not poly A or poly G. When poly C was used as a substrate, 2',3'-cyclic CMP as an intermediate and 3'-CMP as a final product were identified. The results of amino acid analysis indicated the presence of an unusual component. The general properties of the RNase and the RNase-RNase inhibitor complex are also reported.