scholarly journals Purification of endopeptidase-24.11 (‘enkephalinase’) from pig brain by immunoadsorbent chromatography

1983 ◽  
Vol 215 (3) ◽  
pp. 519-523 ◽  
Author(s):  
J M Relton ◽  
N S Gee ◽  
R Matsas ◽  
A J Turner ◽  
A J Kenny
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Deae Cellulose ◽  
Single Step ◽  
Endopeptidase 24.11 ◽  
Pig Brain ◽  
Endopeptidase Activity ◽  
Hydrolysis Of ◽  
The Brain ◽  
Subunit Size

Membrane preparations from striatum of pig brain contain endopeptidase activity towards iodoinsulin B-chain. Only 50% of the hydrolysis of insulin B-chain is inhibitable by phosphoramidon, and DEAE-cellulose chromatography can resolve the phosphoramidon-sensitive and -insensitive activities. The former activity (now designated ‘endopeptidase-24.11’) is responsible for hydrolysis of [D-Ala2,Leu5]enkephalin and is identical with an enzyme in brain that has previously been referred to as ‘enkephalinase’. Pig striatal endopeptidase-24.11 has now been purified to homogeneity in a single step by immunoadsorbent chromatography using a monoclonal antibody. The overall purification was 23 000-fold, with a yield of 30%. The brain enzyme appears to be identical with kidney endopeptidase-24.11 in amino acid composition as well as by immunological and kinetic criteria. However, it differs slightly in apparent subunit size (Mr = 87 000), attributable to differences in glycosylation.

scholarly journals A monoclonal antibody to kidney endopeptidase-24.11. Its application in immunoadsorbent purification of the enzyme and immunofluorescent microscopy of kidney and intestine

1983 ◽  
Vol 214 (2) ◽  
pp. 377-386 ◽  
Author(s):  
N S Gee ◽  
R Matsas ◽  
A J Kenny
Keyword(s):  
Monoclonal Antibody ◽  
Fluorescence Microscopy ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Specific Activity ◽  
Cell Types ◽  
Kidney Tubules ◽  
Pig Kidney ◽  
Endopeptidase 24.11 ◽  
Endopeptidase Activity

Hybridoma methodology has been used to produce a monoclonal antibody, GK 7C2, that binds specifically to microvillar endopeptidase-24.11 (EC 3.4.24.11). The antibody (an immunoglobulin G) was generated by fusion of mouse plasmacytoma cells with splenocytes from a Balb/c mouse immunized with pig kidney microvillar membranes. The identity of the antigen recognized by GK 7C2 was established by immuno-precipitation from detergent-solubilized pig kidney microvilli. The protein had an apparent Mr of 90 000 and contained endopeptidase activity sensitive to phosphoramidon. The identity was confirmed by immunoadsorbent purification of endopeptidase-24.11 by a column to which GK 7C2 had been attached. The endopeptidase, purified in a yield of 40%, was electrophoretically homogeneous and of specific activity comparable with that purified by other means. Fluorescence microscopy established that GK 7C2 bound specifically to the luminal membranes of kidney tubules and the intestinal mucosa. Thus endopeptidase-24.11 is located in the brush-border membranes of both cell types.

8 Functional Role of Histidine in Diets of Young Pigs

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 13-13
Author(s):  
Jaap van Milgen ◽  
Nathalie Le Floc’h
Keyword(s):  
Amino Acid ◽  
Dose Response ◽  
The Body ◽  
Whole Body ◽  
Body Protein ◽  
Constituent Amino Acid ◽  
Young Pigs ◽  
Hydrolysis Of ◽  
The Brain

Abstract Histidine is a constituent amino acid of body proteins and, once incorporated in protein, histidine can be methylated post-translationally to methyl-histidine. Histidine is also a precursor of histamine, a neurotransmitter and involved in the immune response. Histidine and histamine are constituents of a number of dipeptides, which act as pH buffers, metal chelating agents, and anti-oxidants, especially in skeletal muscles and in the brain. A considerable fraction of whole-body histidine is present as carnosine, the dipeptide of histidine and β-alanine. In the longissimus muscle, about 40% of the total histidine content is present as carnosine. The histidine in carnosine can be methylated to anserine or balenine, and the pig is among the few species that synthesize both forms. Hydrolysis of body protein and of histidine-containing dipeptides results in the release of the constituent amino acids. However, only the histidine of protein and carnosine can be reused for protein synthesis. Methyl-histidine is either excreted in the urine or remains bound in the dipeptides and accumulates in the body. Because carnosine represents such a large histidine reservoir, a dietary histidine deficiency may not directly lead to a reduction in growth, especially if growth is given a higher priority for histidine utilization than maintaining or depleting the histidine-containing dipeptide reserves. Few histidine dose-response studies have been done in piglets and differences in the estimated requirements may be due to differences in diluting or depleting the dipeptide reserves. However, at low histidine intakes, both feed intake and growth are reduced and a reduction of the histidine-to-lysine supply by 1 percentage point results in a growth reduction of 4%. Histidine dose-response studies need to consider the role of histidine as a constituent amino acid of body protein as well as its role in dipeptides.

scholarly journals Structure of the β-Galactosidase Gene fromThermus sp. Strain T2: Expression in Escherichia coli and Purification in a Single Step of an Active Fusion Protein

1998 ◽  
Vol 64 (6) ◽  
pp. 2187-2191 ◽  
Author(s):  
Alejandro Vian ◽  
Alfonso V. Carrascosa ◽  
José L. García ◽  
Estrella Cortés
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Bacillus Stearothermophilus ◽  
Specific Activity ◽  
Active Form ◽  
Deae Cellulose ◽  
Single Step ◽  
Amino Acid Sequences ◽  
Glycosyl Hydrolases ◽  
Flanking Regions

ABSTRACT The nucleotide sequence of both the bgaA gene, coding for a thermostable β-galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (M r, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bgaAgene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the β-galactosidase ofBacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric β-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70°C and aKm value of 1.6 mM witho-nitrophenyl-β-d-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70°C.

scholarly journals THE ENZYMATIC HYDROLYSIS OF AMINO ACID β-NAPHTHYLAMIDES II. PARTIAL PURIFICATION AND PROPERTIES OF A PARTICLE-BOUND COBALT-ACTIVATED RAT KIDNEY AMINOPEPTIDASE

1966 ◽  
Vol 14 (5) ◽  
pp. 401-413 ◽  
Author(s):  
K. FELGENHAUER ◽  
G. G. GLENNER
Keyword(s):  
Amino Acid ◽  
Rat Kidney ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Disc Electrophoresis ◽  
Partial Purification ◽  
Soluble Enzyme ◽  
Solvent Fractionation ◽  
Biochemical Systems ◽  
Hydrolysis Of

In order to determine the relationship and characteristics of the soluble and tissue-bound (so-called "lyo" and "desmo") components of enzymes in histochemical and biochemical systems, a biochemical investigation of the hydrolysis of l-leucyl β-naphthylamide in rat kidney was undertaken. By ultracentrifugation, autolysis, salt and solvent fractionation procedures and DEAE-cellulose column chromatography primary soluble and tissuue-bound enzymes were separated and partially purified. The soluble enzyme was found to be inhibited by p-chloromercuribenzoate and reactivated by cysteine. The tissue-bound enzyme was found to be inhibited by o-phenanthroline and reactivated by Co++, and hydrolyzed peptides and amino acid β-naphthylamides at rates differing markedly from those of the soluble enzyme and commercial leucine aminopeptidase. Disc electrophoresis of the tissue-bound enzyme preparation demonstrated two protein bands, both revealing enzymic activity with almost identical hydrolytic characteristics, i.e., isozymes. This evidence established the identity of the tissue-bound Co++-activated enzyme with that found histochemically in the brush border of the proximal tubuli, and indicated its functional capacity as that of an aminopolypeptidase. It was also concluded that in the system investigated the "lyo" and "desmo" components represented, in reality, distinct enzymes with unique characteristics.

scholarly journals Hydrolysis of human and pig brain natriuretic peptides, urodilatin, C-type natriuretic peptide and some C-receptor ligands by endopeptidase-24.11

1993 ◽  
Vol 291 (1) ◽  
pp. 83-88 ◽  
Author(s):  
A J Kenny ◽  
A Bourne ◽  
J Ingram
Keyword(s):  
Atrial Natriuretic Peptide ◽  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Natriuretic Peptides ◽  
Receptor Ligands ◽  
Prolonged Incubation ◽  
Endopeptidase 24.11 ◽  
Pig Brain ◽  
Hydrolysis Of ◽  
Atrial Natriuretic

Endopeptidase-24.11 (E-24.11, EC 3.4.24.11) is widely believed to play a physiological role in metabolizing atrial natriuretic peptide (ANP). Since the discovery of ANP, new natriuretic peptides have been isolated and other peptides synthesized as receptor ligands. The hydrolysis in vitro of six related peptides by the endopeptidase has been studied, mainly by h.p.l.c. The initial attack on the 32-residue form of pig brain natriuretic peptide (pBNP-32) was shown to be at the Ser20-Leu21 bond, as had been previously shown for the 26-residue form. In contrast, human brain natriuretic peptide-32 (hBNP-32), which differs in ten residues from pBNP-32, was attacked first at the Met4-Val5 bond, releasing the N-terminal tetrapeptide, and only later at bonds within the ring: at Arg17-Ile18 and subsequently at four other sites. Urodilatin, which has a four-residue extension at the N-terminus compared with alpha-human atrial natriuretic peptide-28 (alpha-hANP), was degraded at about half the rate of the latter, though the C-terminal Phe-Arg-Tyr was released at the same rate. The 22-residue C-type natriuretic peptide was hydrolysed more rapidly than alpha-hANP, as were two C-receptor ligands (peptides with deletions within the ring): C-ANP4-23 (rANP4-23 des-Gln18,Ser19,Gly20,Leu21,Gly22) and SC 46542 (hANP5-28 des-Phe8,Gly9,Ala17,Gln18). Angiotensin-converting enzyme failed to hydrolyse pBNP-32, hBNP-32 or 125I-rat (r) ANP, even after prolonged incubation. Km and kcat values were determined for the hydrolysis of alpha-hANP, porcine BNP-26, porcine BNP-32 and 125I-rANP by E-24.11. Ki values were determined for six peptides, alpha-hANP, urodilatin, hBNP-32, C-type natriuretic peptide (CNP), SC 46542 and C-type natriuretic peptide (C-ANP4-23), in radiometric assays of E-24.11 with either [125I] insulin B chain or [125I] rANP as substrate. The Ki values (2.5-13 microM) for CNP were the lowest of any of the group, whereas those for hBNP-32 (151-172 microM) were the highest. The physiological significance of these results is discussed, especially in regard to the relative resistance of hBNP-32 to attack and the ability of the C-receptor ligands to compete with natriuretic peptides for hydrolysis by E-24.11.

scholarly journals A monoclonal antibody to bovine brain inositol monophosphatase. Immunoaffinity purification of the brain and kidney enzymes and evidence for their structural identity

1989 ◽  
Vol 264 (3) ◽  
pp. 793-798 ◽  
Author(s):  
N S Gee ◽  
S Howell ◽  
G Ryan ◽  
C I Ragan
Keyword(s):  
Monoclonal Antibody ◽  
Specific Activity ◽  
Binding Capacity ◽  
Bovine Brain ◽  
High Yield ◽  
Brain Extract ◽  
Kidney Cortex ◽  
Inositol Monophosphatase ◽  
Hydrolysis Of ◽  
The Brain

A monoclonal IgG2b(K) antibody, G-2A4, has been generated against bovine brain myo-inositol monophosphatase (EC 3.1.3.25). The identity of the antigen recognized by the antibody was established by using e.l.i.s.a. and Western blotting procedures, and by immunoprecipitation of enzyme activity from crude brain supernatant. In addition, the hydrolysis of Ins1P by crude brain extract was inhibited by up to 83% by the pure antibody. Under identical conditions, the hydrolysis of Ins(1,4)P2 was unaffected. An immunoadsorbent column containing monoclonal antibody G-2A4 covalently attached to CNBr-activated Sepharose 4B has been used for rapid purification of the brain enzyme. Elution conditions have been optimized to allow isolation of the enzyme in high yield (54%) with full retention of column-binding capacity. The enzyme was electrophoretically homogeneous, Mr 30,000 and of higher specific activity than that purified conventionally. Chromatography of the pure enzyme on high resolution ion-exchange columns revealed some charge heterogeneity, possibly indicative of some type of post-translational modification. The immunoadsorbent column has also been used to purify the bovine kidney cortex enzyme to homogeneity. Partial proteolytic fragmentation patterns of the brain and kidney enzymes using endoprotease glu-C were identical, suggesting that they are almost certainly products of the same gene.

Effects of D-amino acid substituents on degradation of LHRH analogues by proximal tubule

1987 ◽  
Vol 252 (3) ◽  
pp. E320-E326 ◽  
Author(s):  
G. Flouret ◽  
T. Majewski ◽  
D. R. Peterson ◽  
A. J. Kenny ◽  
F. A. Carone
Keyword(s):  
Amino Acid ◽  
Specific Inhibitor ◽  
Multiple Bonds ◽  
Lhrh Analogues ◽  
Angiotensin I Converting Enzyme ◽  
Endopeptidase 24.11 ◽  
Renal Brush Border Membranes ◽  
Hydrolysis Of

Less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, the luteinizing hormone-releasing hormone, LHRH, is degraded in renal proximal tubules (PT) in vivo (rat) and in vitro (rabbit) to less than Glu-His (2), less than Glu-His-Trp (3), and less than Glu-His-Trp-Ser (4). LHRH may be cleaved by endopeptidases simultaneously at multiple bonds, or initially at Ser4-Tyr5 followed by carboxypeptidase hydrolysis of 4 to 3 and then 2. To distinguish between these mechanisms, [3H]LHRH analogues were incubated with rabbit renal brush-border membranes (BBM), microinfused into PT in vivo or in vitro, and products were analyzed by HPLC. [D-Ser4]LHRH was not cleaved at D Ser4-Tyr5 but yielded less than Glu-His-Trp-D-Ser-Tyr-Gly as the major metabolite plus 2 and 3. [D-Trp6]LHRH was cleaved by BBM and PT to 2 and 3, but not to 4. [D-Ser4, D-Trp6]LHRH was not cleaved by BBM, but was degraded to 2 by PT in vivo. Thus, D-amino acid substituents altered the expected cleavage pattern of these analogues. [3H]LHRH was cleaved by BBM or by endopeptidase-24.11 from porcine PT to metabolites 2, 4, small amounts of 3, and less than Glu-His-Trp-Ser-Tyr-Gly, but cleavage was strongly inhibited by the specific inhibitor phosphoramidon. Thus, normally LHRH may be cleaved in PT by endopeptidase-24.11 to 2 and 4, and by angiotensin I-converting enzyme to 3, its known cleavage site.

scholarly journals Kidney neutral endopeptidase and the hydrolysis of enkephalin by synaptic membranes show similar sensitivity to inhibitors

1982 ◽  
Vol 203 (2) ◽  
pp. 519-522 ◽  
Author(s):  
I S Fulcher ◽  
R Matsas ◽  
A J Turner ◽  
A J Kenny
Keyword(s):  
Neutral Endopeptidase ◽  
Synaptic Membranes ◽  
General Role ◽  
Leucine Enkephalin ◽  
Pig Kidney ◽  
Pig Brain ◽  
Endopeptidase Activity ◽  
Hydrolysis Of

Neutral endopeptidase (EC 3.4.24.11) from pig kidney hydrolyses [125I]iodo-insulin B-chain and leucine-enkephalin. Both activities were equally sensitive to inhibition by phosphoramidon [N-(alpha-L-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptophan] and thiorphan [N-(DL-2-benzyl-3-mercaptopropionyl)glycine]. Thermolysin hydrolysis of insulin B-chain was also sensitive to both inhibitors. The hydrolysis of the Gly3-Phe4 bond of Leu-enkephalin by synaptic membranes prepared from pig brain was partially inhibited by phosphoramidon and thiorphan. Synaptic membranes appear to contain another endopeptidase activity that is insensitive to these reagents. These observations suggest that enzymes similar to the kidney endopeptidase may play a general role in neuropeptide metabolism.

scholarly journals The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11

1984 ◽  
Vol 223 (2) ◽  
pp. 433-440 ◽  
Author(s):  
R Matsas ◽  
A J Kenny ◽  
A J Turner
Keyword(s):  
Amino Acid ◽  
Amino Acid Residues ◽  
Peptide Substrate ◽  
Peptide Substrates ◽  
Pig Kidney ◽  
Endopeptidase 24.11 ◽  
C Terminus ◽  
Wide Range ◽  
Amidated Peptide ◽  
Hydrolysis Of

Endopeptidase-24.11 (EC 3.4.24.11), purified to homogeneity from pig kidney, was shown to hydrolyse a wide range of neuropeptides, including enkephalins, tachykinins, bradykinin, neurotensin, luliberin and cholecystokinin. The sites of hydrolysis of peptides were identified, indicating that the primary specificity is consistent with hydrolysis occurring at bonds involving the amino group of hydrophobic amino acid residues. Of the substrates tested, the amidated peptide substance P is hydrolysed the most efficiently (Km = 31.9 microM; kcat. = 5062 min-1). A free alpha-carboxy group at the C-terminus of a peptide substrate is therefore not essential for efficient hydrolysis by the endopeptidase. A large variation in kcat./Km values was observed among the peptide substrates studied, a finding that reflects a significant influence of amino acid residues, remote from the scissile bond, on the efficiency of hydrolysis. These subsite interactions between peptide substrate and enzyme thus confer some degree of functional specificity on the endopeptidase. The inhibition of endopeptidase-24.11 by several compounds was compared with that of pig kidney peptidyldipeptidase A (EC 3.4.15.1). Of the inhibitors examined, only N-[1(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate inhibited endopeptidase-24.11 but not peptidyldipeptidase. Captopril (D-3-mercapto-2-methylpropanoyl-L-proline), Teprotide (pGlu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) and MK422 [N-[(S)-1-carboxy-3-phenylpropyl]-L-Ala-L-Pro] were highly selective as inhibitors of peptidyldipeptidase. Although not wholly specific, phosphoramidon was a more potent inhibitor of endopeptidase-24.11 than were any of the synthetic compounds tested.

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