Generation of methionine and leucine-enkephalin from precursor molecules by cation-sensitive neutral endopeptidase of bovine pituitary

Highly purified preparations of cation-sensitive neutral endopeptidase, from bovine pituitary, and also rabbit brain, generate methionine-enkephalin, from α-endorphin, a peptide containing the amino acid sequence 61–76 of β-lipotropin (β-LPH), The enzyme also catalyzes the hydrolysis of the Leu-Thr bond in the synthetic peptide Tyr-Gly-Gly-Phe-Leu-Thr-2-naphthylamide with the release of leucine-enkephalin and Thr-2-naphthylamide. Neither Met- nor Leu-enkephalin are degraded. The data indicate that the presence of a free N-terminal group of tyrosine inhibits the further degradation of Leu- and Met-enkephalin by the endopeptidase. It is suggested that cation-sensitive neutral endopeptidase is one of the enzymes capable of generating Met- and Leu-enkephalin $\text{in}\text{,}\text{vivo}$.     

The purification and specificity of a neutral endopeptidase from rabbit kidney brush border

1. A neutral peptidase, previously shown to be located in the brush border of the proximal tubule, and assayed by its ability to hydrolyse [(125)I]iodoinsulin B chain was purified from rabbit kidney. 2. The starting material for the purification was a microsomal pellet prepared from a homogenate of cortical tissue. The membrane-bound enzymes were solubilized by treatment with toluene and trypsin. About half the neutral peptidase activity was released by this treatment in a form that no longer sedimented with the microsomal pellet and which penetrated polyacrylamide gels when subjected to disc electrophoresis. Other treatments with detergents or proteolytic enzymes either inactivated the peptidase or failed to convert it into a genuinely soluble form. 3. Chromatography with successive columns of Sephadex G-200, DEAE-cellulose and hydroxyl-apatite yielded an enzyme that was free of other brush-border peptidase activities and which was homogeneous on disc electrophoresis and ultracentrifugation. 4. The purified enzyme attacked [(125)I]iodoglucagon at a rate comparable with that for [(125)I]iodoinsulin B chain. It did not appear to attack proteins (insulin, albumin and casein) that had been similarly iodinated. 5. Unlabelled insulin B chain and unlabelled glucagon were substantially hydrolysed by the endopeptidase, whereas insulin and albumin released only trivial amounts of ninhydrin-reacting material. The resistance of insulin to attack by endopeptidase, even after prolonged incubation, was confirmed by biological and immunoassay. 6. The specificity of the peptidase was determined by analysis of the products after incubating unlabelled insulin B chain, and some oligopeptide substrates, including pentagastrin, with the enzyme. All of the bonds readily cleaved were those involving the alpha-amino group of hydrophobic residues, i.e. x-Leu-, x-Val-, x-Tyr-, x-Phe- and x-Met-, provided that the residues were not C-terminal. 7. The enzyme showed only endopeptidase activity. Substrates suitable for aminopeptidases, carboxypeptidases or esterases were not attacked.     

Lens neutral endopeptidase occurs in other bovine and human tissues.

Lens neutral endopeptidase (EC 3.4.24.5) was previously thought to be unique to the eye lens. We report here the finding of a neutral endopeptidase, in a variety of bovine and human tissues, which is very similar both biochemically and immunologically to the lens endopeptidase. SDS/polyacrylamide-gel electrophoresis of partially purified enzyme fractions from various bovine tissues shows the characteristic pattern of at least eight bands with Mr values ranging from 24,000 to 32,000 which was described for the bovine-lens neutral endopeptidase. The relative activity of the enzyme varies from tissue to tissue with lung having the highest activity. Partially purified enzyme fractions from these tissues cross-react with antiserum raised in rabbit against bovine lens endopeptidase showing apparent identity when examined side by side in Ouchterlony double-diffusion tests. The human enzyme also cross-reacts with the antiserum but when tested by double-diffusion against the bovine enzyme the precipitin lines show spurring at the joining edges indicating a structural difference between the human and the bovine enzymes. It was also found by Western blot experiments, after denaturing polyacrylamide-gel electrophoresis of the enzyme, that the polypeptide components of the human and bovine enzymes show somewhat different banding patterns.     

Adenosine deaminase from bovine brain: purification and partial characterization.

Bovine brain adenosine deaminase cytoplasmatic form was purified about 450 fold by salt fractionation, column chromatography on DEAE-cellulose, octyl-sepharose 4B and affinity chromatography on CH-sepharose 4B 9-(p-aminobenzyl)adenine. The purified enzyme was homogeneous on disc gel electrophoresis; the enzyme had a molecular mass of about 65 kDa with an isoelectric point at pH 4.87. The Km values for adenosine and 2'-deoxyadenosine were 4 x 10(-5) and 5.2 x 10(-5) M, respectively. The enzyme showed a great stability to temperature with a half life of 15 hours at 53 degrees C significantly different compared to that known for other mammalian forms of this enzyme. Aza and deaza analogs of adenosine and erythro-9-(2-hydroxy-3-nonyl) adenine were good inhibitors of the bovine brain enzyme with little difference with respect to those reported for the adenosine deaminases purified from other sources. Kinetic constants for the association and dissociation of coformycin and 2'-deoxycoformycin with the bovine brain adenosine deaminase are reported.     

Identification and partial purification of a dipeptidyl aminopeptidase from Streptococcus faecalis

A dipeptidyl aminopeptidase was identified in Streptococcus faecalis JH2SS and was partially purified (approximately 245-fold) by HPLC. Gel filtration chromatography indicated an Mr of 140 000. The partially purified enzyme exhibited a requirement for Co2+. The pH optimum for the hydrolysis of L-Val-L-Ala-p-nitroanilide was approximately 9.5. The apparent Km for this substrate was 0.22 mM. The enzyme preferentially hydrolysed X-Ala-Y substrates, but also utilized X-Pro-Y substrates, and therefore is most closely related to the mammalian dipeptidyl aminopeptidase II (EC 3.4.14.-). The enzyme was inhibited by p-chloromercuribenzoate, but not by iodoacetate, N-ethylmaleimide or the serine protease inhibitor phenylmethylsulphonyl fluoride.     

A neutral endopeptidase in the microvillar membrane of pig intestine. Partial purification and properties.

An enzyme hydrolysing [125I]iodo-insulin B chain was enriched in preparations of intestinal microvilli. The activity could be solubilized by Triton X-100 and was partially (76-fold) purified. It was very sensitive to inhibition by phosphoramidon and was also inhibited by chelating agents. In its enzymic, molecular and immunological properties the intestinal enzyme closely resembled kidney microvillar neutral endopeptidase (kidney-brush-border neutral proteinase, EC 3.4.24.11).     

Properties and partial purification of sialate-O-acetyltransferase from bovine submandibular glands

The O-acetylation of sialic acids in various positions is a frequent modification of these residues in glycoproteins and glycolipids of higher animals and some bacteria. Sialic acid O-acetylation is involved in the regulation of many cell biological and pathophysiological events. Since the properties and the structural and molecular genetic aspects of the eukaryotic sialate O-acetyltransferases are not yet known, we attempted to isolate the enzyme from bovine submandibular glands. O-Acetyltransferase was solubilised from its microsomal location with a zwitterionic detergent and enriched by approximately 50-fold in three steps, including affinity chromatography on coenzyme A. It exhibits a molecular mass of 150-160 kDa. Evidence was obtained for the putative existence of a low-molecular-mass, dialysable enzyme activator. The enzyme showed best activity with CMP-N-acetylneuraminic acid (CMP-Neu5Ac), followed by N-acetylneuraminic acid (Neu5Ac). These compounds, as well as AcCoA, have high affinity for both the microsome-bound and the partially purified O-acetyltransferase. CoA is a strong inhibitor. N-Acetyl-9-O-acetylneuraminic acid was found to be the main reaction product. No evidence was obtained for the involvement of an isomerase that might be responsible for the migration of O-acetyl groups within the sialic acid side chain.     

Solubilization and partial purification of GABAB receptor from bovine brain

gamma-Aminobutyric acid (GABA)B receptor has been solubilized and partially purified by an affinity column chromatography. GABAB receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of asolectin. The solubilized GABAB receptor was adsorbed on baclofen-coupled epoxy-activated Sepharose 6B. The affinity matrix adsorbed 80% of the solubilized [3H]GABA binding activity to GABAB receptor, and approximately 75% of the adsorbed activity could be eluted with 1 M KC1. GABAB receptor binding in the fraction eluted from affinity column was displaced by GABA, baclofen and 2-hydroxy saclofen in a dose-dependent manner. Furthermore, the purified GABAB receptor showed approximately 2800-fold purification as compared with the original solubilized fraction and possessed the specific binding activity of 17.68 p mol/mg of protein. This binding consisted of a single binding site with a dissociation constant of 64.4 nM. The present results indicate that affinity column chromatographic procedures using baclofen-coupled epoxy-activated Sepharose 6B are suitable for the partial purification of GABAB receptor from cerebral tissues.     

Subcellular fractionation, partial purification and characterization of neutral triacylglycerol lipase from pig liver.

The subcellular distributions of acidic (pH 4.5) and neutral (pH 7.5) longchain triacylglycerol lipases (glycerol ester hydrolase, EC 3.1.1.3) of pig liver have been determined. The distribution of the acidic lipase closely paralleled that of the lysosomal marker enzyme, cathepsin D. Approx. 60% of the neutral lipolytic activity resided in the soluble fraction;the distribution of this activity failed to parallel that of marker enzymes for mitochondria, lysosomes, microsomes, or plasma membranes. A method has been developed for purification of the neutral lipase from the soluble fraction by ultracentrifugation. An approximate 90-fold purification was achieved, with recovery of 16% of the initial activity. The partially purified neutral lipase exhibited a pH optimum between 7.25 and 7.5. It required 30 mM emulsified triolein for optimal activity and ceased to liberate fatty acids after 30 min of incubation. The enzymatic activity was destroyed by heating at 60 degrees C. Neutral lipase was inhibited by sodium deoxycholate, Triton X-100 and iodoacetamide. The activity was not inhibited by sodium taurocholate, EDTA, heparin and diethyl-p-nitrophenyl phosphate. Neutral lipase failed to exhibit activity in assay systems specific for lipoprotein lipase, monoolein hydrolase, tributyrinase, and methyl butyrate esterase and showed little or no capacity to hydrolyze chyle chylomicrons or plasma very low density lipoproteins. It is suggested that the function of neutral lipase may be to supply the liver with fatty acids liberated from endogenously synthesized or stored triacylglycerols.     

Identification and partial purification of a thiol endothelin-converting enzyme from porcine aortic endothelial cells.

Endothelin is a potent peptide vasoconstrictor. The final step in the processing of endothelin has been postulated to be the cleavage of the Trp21-Val22 peptide bond in proendothelin by a putative endothelin-converting enzyme. A soluble extract of primary porcine aortic endothelial cells was found to contain an enzyme activity that converted proendothelin-1 (proET-1) to an endothelin-1 (ET-1)-like peptide as determined by the rabbit aortic ring contraction assay. This enzyme was partially purified by DE52 ion-exchange chromatography. Incubation of proET-1 with the partially purified enzyme generated a product which had a retention time on HPLC identical to that of authentic ET-1. Further analysis of the product showed that it caused contraction of rabbit aortic rings, had a molecular weight identical to ET-1 as measured by fast atom bombardment mass spectrometry, and competed for [125I]ET-1 binding in an RIA using specific antibodies which recognize the carboxy terminal tryptophan of ET-1. The enzyme activity could be inhibited by thiol protease inhibitors such as Z-phe-pheCHN2 and p-hydroxymercuribenzoate, but not by serine- or metalloprotease inhibitors. The optimal pH for the enzymatic activity was between 7.0 and 7.5, and no activity was detected at pH 4.0. These results demonstrate that this thiol protease is a potential endothelin-converting enzyme.     

Cation-sensitive neutral endopeptidase: isolation and specificity of the bovine pituitary enzyme

A highly purified preparation of a cation-sensitive neutral endopeptidase was obtained from bovine pituitaries. The enzyme constitutes almost 0.1% of the protein in bovine pituitary homogenates. Polyacrylamide gel electrophoresis of the enzyme showed a single protein band, and in gel filtration experiments on calibrated Sepharose 6B columns the enzyme eluted slightly ahead of thyroglobulin, suggesting an apparent molecular weight of about 700,000. Polyacrylamide gel electrophoresis in SDS-containing buffers indicated the presence of three major components with molecular weights ranging from about 24,000 to 28,000. The enzyme hydrolyzes bonds between hydrophobic and small neutral amino acids in both model synthetic substrates and biologically active peptides such as substance P, LH-RH, and bradykinin. Peptide bonds in which the carbonyl group is contributed by a glutamyl or arginyl residue are also hydrolyzed, especially if they are preceded in the sequence by hydrophobic amino acids. Leupeptin exclusively inhibited enzymatic activity toward the arginine-containing substrates. This observation, together with the high molecular weight and broad specificity of the enzyme, raised the possibility that the isolated enzyme represents a proteolytic complex composed of units with distinctly different activities. Preliminary attempts to dissociate the enzyme into catalytic units of lower molecular weight were not successful and led to loss of activity.     

Identification and purification of a functional amine transporter from bovine chromaffin granules

The amine transporter from bovine chromaffin granules has been purified in a functional state. Two isoforms with different pI values have been separated and shown to be active. One with an unusually acidic pI (approximately 3.5) has been shown to be a glycoprotein with an apparent Mr of 80,000. The purified polypeptide catalyzes transport of serotonin upon reconstitution with an apparent Km of 2 microM and a Vmax of 140 nmol/mg/min, 150-200-fold higher than the one determined in the native system. Transport is inhibited by reserpine and tetrabenazine, ligands which bind to two distinct sites on the transporter. These findings suggest that the binding sites for both drugs reside on a single polypeptide. The reconstituted purified transporter binds [3H]reserpine with a biphasic kinetic behavior, KD values of 0.3 and 30 nM and Bmax of 310 and 4200 pmol/mg protein, respectively. In addition, binding of [3H]reserpine is accelerated upon imposition of a pH gradient across the proteoliposome. From these findings it is evident that a single polypeptide catalyzes the various functions of the transporter.     

Identification and purification of a cytosolic phosphotyrosyl protein phosphatase from bovine spleen

A protein tyrosine kinase with an apparent Mr of 60,000 was highly purified from bovine spleen and used to phosphorylate poly(Glu, Tyr) (4:1) on tyrosine residues for the study of phosphotyrosyl protein phosphatases from this tissue. About 70% of the phosphotyrosyl protein phosphatase activity in extracts of bovine spleen was adsorbed on DEAE-Sepharose. Chromatography of the eluted phosphotyrosyl protein phosphatases on phosphocellulose indicated the presence of at least two species, one that did not bind to the phosphocellulose and a second species that did bind and was eluted at about 0.5 M NaCl. The phosphatase that did not bind to phosphocellulose was further purified by successive chromatography on poly(L-lysine)-Sepharose, L-tyrosine-agarose, poly(Glu,Tyr)-Sepharose, and Sephacryl S-200. The enzyme had an apparent Mr of 50,000 as estimated by gel filtration and 52,000 as estimated by NaDodSO4- polyacrylamide gel electrophoresis. The phosphatase exhibited a pH optimum of 6.5-7.0, was inhibited by Zn2+ and vanadate ions, and was stimulated by EDTA. Sodium fluoride and sodium pyrophosphate, inhibitors of phosphoseryl protein phosphatases, had no effect on the enzyme. Protein inhibitors of type 1 phosphoseryl/threonyl phosphatase were also ineffective.     

Partial purification and characterization of a rat kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide

The activity for the hydrolysis of succinyl trialanine-4-nitroanilide was higher in kidney homogenates of female rats and mice than in those of male rats and mice. An enzyme hydrolyzing the above substrate was extracted from female rat kidney homogenate and partially purified by means of gel filtration on Sepharose 4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme cleaved the bond between succinyl dialanine and alanine-4-nitroanilide of the substrate and showed a Km value of 3.3 mM at the optimal pH of 7.5. The activity was increased by Ca2+ and Mg2+, but inhibited by EDTA. With oxidized insulin B chain as a substrate, the enzyme cleaved the carbonyl bonds of Ala-14, Tyr-16 and Gly-23 efficiently, and those of His-5 and His-10 less efficiently.     

Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go.

A guanine-nucleotide-binding protein (G-protein) was purified from cholate extracts of bovine brain membranes by sequential DEAE-Sephacel, Ultrogel AcA-34, heptylamine-Sepharose and Sephadex G-150 chromatography. Guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S])-binding activity copurified with a 25,000 Da peptide and a 35,000-36,000 Da protein doublet. Neither pertussis toxin nor cholera toxin catalysed the ADP-ribosylation of a protein associated with the GTP[35S]-binding activity. Photoaffinity labelling of the purified protein with 8-azido[gamma-32P]GTP indicated that the GTP-binding site resides on the 25,000 Da protein. The 35,000-36,000 Da protein doublet was electrophoretically indistinguishable from the beta-subunits of other GTP-binding proteins, and the 36,000 Da protein was recognized by antiserum to oligomeric Gt. The purified protein specifically bound 17.2 nmol of GTP[35S]/mg of protein. The Kd of the binding site for radioligand was approx. 15 nM. The brain GTP-binding protein co-migrated during SDS/polyacrylamide-gel electrophoresis with a GTP-binding protein, named Gp, purified from human placenta [Evans, Brown, Fraser & Northup (1986) J. Biol. Chem. 261, 7052-7059], and cross-reacted with antiserum raised against the placental protein, but not with antiserum raised to brain Go. SDS/polyacrylamide-gel electrophoresis of the brain and placental GTP-binding proteins in the presence of Staphylococcus aureus V8 protease yielded identical peptide maps.     

Identification and partial purification of a novel milk clotting enzyme from Onopordum turcicum

Summary Seeds, flowers and leaves of Onopordum turcicum were found to contain proteolytic enzymes able to coagulate milk. Extraction, concentration and identification of the operational parameters affecting the activity of the enzyme complex were followed by partial purification steps involving gel-filtration and ion-exchange chromatography. Milk clotting activity of the enzyme complex was tested in several steps of its purification and an increase of almost 200 fold was obtained. Molecular weight of the proteolytic enzyme fraction having the maximum activity was determined to be about 19000–24000. Isoelectric point (pI) of the enzyme complex with maximum activity was estimated to be in the range 3.3–3.7.