The metabolism of neuropeptides. Hydrolysis of peptides by the phosphoramidon-insensitive rat kidney enzyme ''endopeptidase-2'' and by rat microvillar membranes.

Endopeptidase-2, the second endopeptidase in rat kidney brush border [Kenny & Ingram (1987) Biochem. J. 245, 515-524] has been further characterized in regard to its specificity and its contribution to the hydrolysis of peptides by microvillar membrane preparations. The peptide products were identified, after incubating luliberin, substance P, bradykinin and angiotensins I, II and III with the purified enzyme. The bonds hydrolysed were those involving a hydrophobic amino acid residue, but this residue could be located at either the P1 or P1' site. Luliberin was hydrolysed faster than other peptides tested, followed by substance P and bradykinin. Human alpha-atrial natriuretic peptide and the angiotensins were only slowly attacked. Oxytocin and [Arg8]vasopressin were not hydrolysed. No peptide fragments were detected on prolonged incubation with insulin, cytochrome c, ovalbumin and serum albumin. In comparison with pig endopeptidase-24.11 the rates for the susceptible peptides were, with the exception of luliberin, much lower for endopeptidase-2. Indeed, for bradykinin and substance P the ratio kcat./Km was two orders of magnitude lower. Since both endopeptidases are present in rat kidney microvilli, an assessment was made of the relative contributions to the hydrolysis of luliberin, bradykinin and substance P. Only for the first named was endopeptidase-2 the dominant enzyme; for bradykinin it made an equal, and for substance P a minor, contribution.     

The hydrolysis of alpha-human atrial natriuretic peptide by pig kidney microvillar membranes is initiated by endopeptidase-24.11.

alpha-Human atrial natriuretic peptide, a 28-amino-acid-residue peptide, was rapidly hydrolysed by pig kidney microvillar membranes in vitro, with a t1/2 of 8 min, comparable with the rate observed with angiotensins II and III. The products of hydrolysis were analysed by h.p.l.c., the pattern obtained with membranes being similar to that with purified endopeptidase-24.11 (EC 3.4.24.11). No hydrolysis by peptidyl dipeptidase A (angiotensin I converting enzyme, EC 3.4.15.1) was observed. The contribution of the various microvillar membrane peptidases was assessed by including specific inhibitors. Phosphoramidon, an inhibitor of endopeptidase-24.11, caused 80-100% suppression of the products. Captopril and amastatin (inhibitors of peptidyl dipeptidase A and aminopeptidases respectively) had no significant effect. Hydrolysis at an undefined site within the disulphide-linked ring occurred rapidly, followed by hydrolysis at other sites, including the Ser25--Phe26 bond.     

The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase A (angiotensin-converting enzyme).

Both endopeptidase-24.11 and peptidyl dipeptidase A have previously been shown to hydrolyse the neuropeptide substance P. The structurally related peptide neurokinin A is also shown to be hydrolysed by pig kidney endopeptidase-24.11. The identified products indicated hydrolysis at two sites, Ser5-Phe6 and Gly8-Leu9, consistent with the known specificity of the enzyme. The pattern of hydrolysis of neurokinin A by synaptic membranes prepared from pig striatum was similar to that observed with purified endopeptidase-24.11, and hydrolysis was substantially abolished by the selective inhibitor phosphoramidon. Peptidyl dipeptidase A purified from pig kidney was shown to hydrolyse substance P but not neurokinin A. It is concluded that endopeptidase-24.11 has the general capacity to hydrolyse and inactivate the family of tachykinin peptides, including substance P and neurokinin A.     

Synaptosomal Membrane-Bound Form of Endopeptidase-24.15 Generates Leu-Enkephalin from Dynorphin 1-8, α- and β-Neoendorphin, and Met-Enkephalin from Met-Enkephalin-Arg6-Gly7-Leu

Brain contains a membrane-bound form of endopeptidase-24.15, a metalloendopeptidase predominantly associated with the soluble protein fraction of brain homogenates. Subcellular fractionation of the enzyme in rat brain showed that 20-25% of the total activity is associated with membrane fractions including synaptosomes. Solubilization of the enzyme from synaptosomal membranes required the use of detergents or treatment with trypsin. The specific activity of the enzyme in synaptosomal membranes measured with tertiary-butoxycarbonyl-Phe-Ala-Ala-Phe-p-aminobenzoate as substrate was higher than that of endopeptidase-24.11 ("enkephalinase"), a membrane-bound zinc-metalloendopeptidase believed to function in brain neuropeptide metabolism. Purified synaptosomal membranes converted efficiently dynorphin1-8, alpha- and beta-neoendorphin into leucine enkephalin and methionine-enkephalin-Arg6-Gly7-Leu8 into methionine enkephalin in the presence of captopril, bestatin, and N-[1-(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate, inhibitors of angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase (EC 3.4.11.2), and membrane-bound metalloendopeptidase (EC 3.4.24.11), respectively. The conversion of enkephalin-containing peptides into enkephalins was virtually completely inhibited by N-[1-(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, a specific active-site-directed inhibitor of endopeptidase-24.15, indicating that this enzyme was responsible for the observed interconversions. The data indicate that synaptosomal membranes contain enzymes that can potentially generate and degrade both leucine- and methionine-enkephalin.     

The kinetics of hydrolysis of some synthetic substrates containing neutral hydrophilic groups by pig pepsin and chicken liver cathepsin D.

1. Several peptides containing either of the sequences -Phe(NO2)-Trp- and -Phe(NO2)-Phe- and an uncharged hydrophilic group were synthesized, and the steady-state kinetics of their hydrolysis by pig pepsin (EC 3.4.23.1) and chicken liver cathepsin D (EC 3.4.23.5) were determined. Despite the presence of a hydrophilic group to increase substrate solubility, it was not possible to achieve the condition [S]0 much greater than Km, and, in some cases, only values of kcat./Km could be determined by measuring the first-order rate constant when [S]0 much less than Km. 2. Occupancy of the P2 and P3 sites considerably enhanced the specificity constant, and alanine was more effective than glycine at site P2. 3. The specificity constants for the hydrolysis by pepsin of those substrates in the present series that contain an amino acid residue at site P3 are considerably lower than for comparable substrates containing a cationic group. This difference does not apply to cathepsin D. 4. Hydrolyses with cathepsin D commonly exhibited a lag phase, and a possible explanation for this is given.     

New substrates for enteropeptidase. I. Biologically active hepta-nonapeptides

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue provided that less than four negatively charged amino acid residues are in positions P2-P5 of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR decreases VYIHPF) and the Hb(2-8) (LTAEEK decreases A) and Hb(1-9) (MLTAEEK decreases AA) peptides of the cattle hemoglobin beta-chain. The Km values for all the substrates (approximately 10(-3) M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the -DDDDK- full-size linker (Km approximately 10(-4) M). The kcat values for AT and Hb(2-8) were also close and low (approximately 30 min-1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2-8) peptide by one amino acid residue from its N- or C-terminus results in a dramatic increase in the catalytic efficiency of the hydrolysis: the kcat value for Hb(1-9) is 1510 min-1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.     

The metabolism of neuropeptides. Endopeptidase-24.11 in human synaptic membrane preparations hydrolyses substance P.

Synaptic membrane preparations from human striatum and human diencephalon were shown to contain a phosphoramidon-sensitive metalloendopeptidase that appeared identical with endopeptidase-24.11. The activity of endopeptidase-24.11 was determined with an enzymic assay employing [D-Ala2,Leu5]enkephalin as substrate, and its distribution in human brain was similar to that in pig brain, with the striatum containing the highest levels. The choroid plexus and pons also contained substantial activity. A good correlation (r = 0.97) was obtained for the distribution of the endopeptidase in pig brain and pituitary by the enzymic assay and by an immunoradiometric assay specific for pig endopeptidase-24.11. Synaptic membrane preparations from human striatum and diencephalon hydrolysed substance P at the same sites as did preparations of pig striatal synaptic membranes, and hydrolysis was substantially abolished by phosphoramidon. These results suggest that endopeptidase-24.11 is the principal enzyme hydrolysing substance P in human synaptic membrane preparations.     

Subsite mapping of an acidic amino acid-specific endopeptidase from Streptomyces griseus, GluSGP, and protease V8.

The substrate specificities of an acidic amino acid-specific endopeptidase of Streptomyces griseus, GluSGP, and protease V8 [EC 3.4.21.19] were investigated with peptide p-nitroanilide substrates which have a Glu residue at the P1 position. GluSGP and protease V8 favored Pro and Leu residues at S2, respectively, while the S3 subsite of GluSGP preferred Phe over either Ala or Leu. The S3 subsite of protease V8 preferred Leu over either Ala or Phe. The best substrates for GluSGP and for protease V8 were Boc-Ala-Phe-Pro-Glu-pNA with a Km value of 0.41 mM (0.1 M Tris-HCl, pH 8.8) and Boc-Ala-Leu-Leu-Glu-pNA with a Km value of 0.25 mM (0.1 M phosphate, pH 7.8), respectively. The kcat/Km values for these substrates obtained with GluSGP were about one hundred to twenty thousand times larger than those obtained with protease V8. Protease V8 exhibited a single optimal pH of around 8 for the hydrolysis of Boc-Ala-Ala-Leu-Glu-pNA and Boc-Ala-Leu-Leu-Asp-pNA.     

Enzyme-substrate interactions in the hydrolysis of peptide substrates by thermitase, subtilisin BPN', and proteinase K

Peptide substrates of the general structure acetyl-Alan (n = 2-5), acetyl-Pro-Ala-Pro-Phe-Alan-NH2 (n = 0-3), and acetyl-Pro-Ala-Pro-Phe-AA-NH2 (AA = various amino acids) were synthesized and used to investigate the enzyme-substrate interactions of the microbial serine proteases thermitase, subtilisin BPN', and proteinase K on the C-terminal side of the scissile bond. The elongation of the substrate peptide chain up to the second amino acid on the C-terminal side (P'2) enhances the hydrolysis rate of thermitase and subtilisin BPN', whereas for proteinase K an additional interaction with the third amino acid (P'3) is possible. The enzyme subsite S'1 specificity of the proteases investigated is very similar. With respect to kcat/Km values small amino acid residues such as Ala and Gly are favored in this position. Bulky residues such as Phe and Leu were hydrolyzed to a lower extent. Proline in P'1 abolishes the hydrolysis of the substrates. Enzyme-substrate interactions on the C-terminal side of the scissile bond appear to affect kcat more than Km for all three enzymes.     

Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin

The inhibitory constants of a series of synthetic N-carboxymethyl peptide inhibitors and the kinetic parameters (Km, kcat, and kcat/Km) of a series of model synthetic substrates were determined for the membrane-bound kidney metalloendopeptidase isolated from rabbit kidney and compared with those of bacterial thermolysin. The two enzymes show striking similarities with respect to structural requirements for substrate binding to the hydrophobic pocket at the S1' subsite of the active site. Both enzymes showed the highest reaction rates with substrates having leucine residues in this position while phenylalanine residues gave the lowest Km. The two enzymes were also inhibited by the same N-carboxymethyl peptide inhibitors. Although the mammalian enzyme was more susceptible to inhibition than its bacterial counterpart, structural variations in the inhibitor molecules affected the inhibitory constants for both enzymes in a similar manner. The two enzymes differed significantly, however, with respect to the effect of structural changes in the P1 and P2' positions of the substrate on the kinetic parameters of the reaction. The mammalian enzyme showed the highest reaction rates and specificity constants with substrates having the sequence -Phe-Gly-Phe- or -Phe-Ala-Phe- in positions P2, P1, and P1', respectively, while the sequence -Ala-Phe-Phe- was the most favored by the bacterial enzyme. The sequence -Gly-Gly-Phe- as found in enkephalins was not favored by either of the enzymes. Of the substrates having an aminobenzoate group in the P2' position, the mammalian enzyme favored those with the carboxyl group in the meta position while the bacterial enzyme favored those with the carboxyl group in the para position.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endopeptidase-24.11 Is the Integral Membrane Peptidase Initiating Degradation of Somatostatin in the Hippocampus

Abstract: The membrane metalloenzyme endopeptidase-24.11 has been localized by immunocytochemistry in the porcine hippocampus in the stratum oriens and stratum radiatum. Endopeptidase-24.11 was found to be ∼10-fold more abundant in a striatal than a hippocampal membrane preparation. Both somatostatin-28 and somatostatin-14 were metabolized by endopeptidase-24.11, but the kinetics of hydrolysis markedly favoured the smaller form of the neuropeptide. After phase separation with Triton X-114 of striatal and hippocampal membrane preparations, and by using selective inhibitors, the major (>80%) somatostatin-metabolizing activity was found to partition into the detergent-rich phase and was attributable predominantly to endopeptidase-24.11. The residual activity observed in the presence of the selective endopeptidase-24.11 inhibitor phosphoramidon was blocked by Pro-Ile or N -[1-( RS )-carboxy-3-phenylpropyl]-Ala-Ala-Phe- p -aminobenzoate, inhibitors of endopeptidase-24.16 and endopeptidase-24.15, respectively. However, Pro-Ile, at comparable concentrations, was shown to inhibit endopeptidase-24.11, challenging the validity of its use as a selective inhibitor of endopeptidase-24.16. The immunocytochemical and Triton X-114 phase-separation data implicate endopeptidase-24.11, rather than endopeptidase-24.16 or endopeptidase-24.15, as the major physiological somatostatin-degrading neuropeptidase in the striatum and hippocampus.     

Substrate specificity of human cathepsin D using internally quenched fluorescent peptides derived from reactive site loop of kallistatin

Kallistatin, a serpin that specifically inhibits human tissue kallikrein, was demonstrated to be cleaved at the Phe-Phe bond in its reactive site loop (RSL) by cathepsin D. Internally quenched fluorescent peptides containing the amino acid sequence of kallistatin RSL were highly susceptible to hydrolysis by cathepsin D. Surprisingly, these peptides were efficiently hydrolyzed at Phe-Phe bond, despite having Lys and Ser at P2 and P2' positions, respectively, which was reported to be very unfavorable for substrates for cathepsin D. Due to the importance of cathepsin D in several physiological and pathological processes, we took the peptide containing kallistatin RSL sequence, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, as a reference substrate for a systematic specificity study of S3 to S3' protease subsites (EDDnp=N-[2,4-dinitrophenyl]-ethylenediamine and Abz=ortho-amino benzoic acid). We present in this paper some internally quenched fluorescent peptides that were efficient substrates for cathepsin D. They essentially differ from other previously described substrates by their higher kcat/Km values due, mainly, to low Km values, such as the substrate Abz-Ala-Ile-Ala-Phe-Phe-Ser-Arg-Gln-EDDnp (Km=0.27 microM, kcat=16.25 s(-1), kcat/Km=60185 microM(-1) x s(-1)).     

Purification of endopeptidase-24.11 (''enkephalinase'') from pig brain by immunoadsorbent chromatography.

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.     

Neurokinin B is hydrolysed by synaptic membranes and by endopeptidase-24.11 (enkephalinase) but not by angiotensin converting enzyme

The major site of hydrolysis was the Gly8-Leu9 bond. Angiotensin converting enzyme (peptidyl dipeptidase A, EC 3.4.15.1) from pig kidney hydrolysed substance P releasing the C-terminal tripeptide Gly-Leu-MetNH2 but failed to hydrolyse neurokinin B. Pig brain striatal synaptic membranes hydrolysed neurokinin B producing a similar pattern of products as did endopeptidase-24.11. Substantial inhibition of this activity was achieved with the selective inhibitor phosphoramidon. A combination of phosphoramidon and bestatin abolished the hydrolysis of neurokinin B by synaptic membranes. Thus, a bestatin-sensitive aminopeptidase may play a role in the synaptic metabolism of neurokinin B in addition to endopeptidase-24.11. This aminopeptidase appears to be distinct from aminopeptidase N (EC 3.4.11.2).     

The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.     

Metabolism of neuropeptides. Hydrolysis of the angiotensins, bradykinin, substance P and oxytocin by pig kidney microvillar membranes.

Microvillar membranes derived from the brush border of the renal proximal tubule are very rich in peptidases. Pig kidney microvilli contain endopeptidase-24.11 associated with a battery of exopeptidases. The manner by which some neuropeptides are degraded by the combined attack of the peptidases of this membrane has been investigated. The contribution of individual peptidases was assessed by including inhibitors (phosphoramidon, captopril, amastatin and di-isopropyl fluorophosphate) with the membrane fraction when incubated with the peptides. Substance P, bradykinin and angiotensins I, II and III and insulin B-chain were rapidly hydrolysed by kidney microvilli. Oxytocin was hydrolysed much more slowly, but no products were detected from [Arg8]vasopressin or insulin under the conditions used for other peptides. The peptide bonds hydrolysed were identified and the contributions of the different peptidases were quantified. For each of the susceptible peptides, the main contribution came from endopeptidase-24.11 (inhibited by phosphoramidon). Peptidyl dipeptidase A (angiotensin-I-converting enzyme) was of less importance, even in respect of angiotensin I and bradykinin. When [2,3-Pro3,4-3H]bradykinin was also investigated at a lower concentration (20 nM), the conclusions in regard to the contributions of the two peptidases were unchanged. The possibility that endopeptidase-24.11 might attack within the six-residue disulphide-bridged rings of oxytocin and vasopressin was examined by dansyl(5-dimethylaminonaphthalene-1-sulphonyl)ation and by reduction and carboxymethylation of the products after incubation. Additional peptides were only observed after prolonged incubation, consistent with hydrolysis at the Tyr2-Ile3 and Tyr2-Phe3 bonds respectively. These results show that a range of neuropeptides are efficiently degraded by microvillar membranes and that endopeptidase-24.11 plays a key role in this process.     

The hydrolysis of brain and atrial natriuretic peptides by porcine choroid plexus is attributable to endopeptidase-24.11.

The hydrolysis of the porcine 26-residue brain natriuretic peptide (BNP-26) and its counterpart human 28-residue atrial natriuretic peptide (alpha-hANP) by pig membrane preparations and purified membrane peptidases was studied. When the two peptides were incubated with choroid plexus membranes, the products being analysed by h.p.l.c., alpha-hANP was degraded twice as fast as BNP. The h.p.l.c. profiles of alpha-hANP hydrolysis, in short incubations with choroid plexus membranes, yielded alpha hANP' as the main product, this having been previously shown to be the result of hydrolysis at the Cys7-Phe8 bond. In short incubations this cleavage was inhibited 84% by 1 microM-phosphoramidon, a specific inhibitor of endopeptidase-24.11. BNP-26 was hydrolysed by choroid plexus membranes, kidney microvillar membranes and purified endopeptidase-24.11 in a manner that yielded identical h.p.l.c. profiles. In the presence of phosphoramidon, hydrolysis by the choroid plexus membranes was 94% inhibited. Captopril had no effect and, indeed, no hydrolysis of BNP-26 by peptidyl dipeptidase A (angiotensin-converting enzyme) was observed even after prolonged incubation with the purified enzyme. The stepwise hydrolysis of BNP-26 by endopeptidase-24.11 was investigated by sequencing the peptides produced during incubation. The initial product resulted from hydrolysis at Ser14-Leu15, thereby opening the ring. This product (BNP') was short-lived; further degradation involved hydrolysis at Ile12-Gly13, Arg8-Leu9, Gly17-Leu18, Val22-Leu23, Arg11-Ile12 and Cys4-Phe5. Thus endopeptidase-24.11 is the principal enzyme in renal microvillar and choroid plexus membranes hydrolysing BNP-26 and alpha-hANP.     

Substrate specificity of human pancreatic elastase 2

The substrate specificity of human pancreatic elastase 2 was investigated by using a series of peptide p-nitroanilides. The kinetic constants, kcat and Km, for the hydrolysis of these peptides revealed that this serine protease preferentially hydrolyzes peptides containing P1 amino acids which have medium to large hydrophobic side chains, except for those which are disubstituted on the first carbon of the side chain. Thus, human pancreatic elastase 2 appears to be similar in peptide bond specificity to the recently described porcine pancreatic elastase 2 [Gertler, A., Weiss, Y., & Burstein, Y. (1977) Biochemistry 16, 2709] but differs significantly in specificity from porcine elastase 1. The best substrates for human pancreatic elastase 2 were glutaryl-Ala-Ala-Pro-Leu-p nitroanilide and succinyl-Ala-Ala-Pro-Met-p-nitroanilide. However, there was little difference among substrates with leucine, methionine, phenylalanine, tyrosine, norvaline, or norleucine in the P1 position. Changes in the hydrolysis rate of peptides with differing P5 residues indicate that this enzyme has an extended binding site which interacts with at least five residues of peptide substrates. The overall catalytic efficiency of human pancreatic elastase 2 is significantly lower than that of porcine elastase 1 or bovine chymotrypsin with the compounds studied.     

Extended investigation of the substrate specificity of dipeptidyl peptidase IV from pig kidney.

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney was investigated, using a series of substrates, in which the amino-acid residue in position P1, a structural derivative of proline, was altered with respect to ring size and substituents. It was demonstrated that dipeptidyl peptidase IV hydrolyses substrates of the type Ala-X-pNA, where X is proline (Pro), (R)-thiazolidine-4-carboxylic acid (Thz), (S)-pipecolic acid (Pip), (S)-oxazolidine-4-carboxylic acid (Oxa), or (S)-azetidine-2-carboxylic acid (Aze). The ring size and ring structure of the residue in the P1 position influence the rate of enzyme-catalysed hydrolysis of the substrate. The highest kcat value (814 s-1) was found for Ala-Aze-pNA. In contrast, the kcat value for Ala-Pro-pNA is nearly 55 s-1. With all substrates of this series, the rate-limiting step of the hydrolysis by dipeptidyl peptidase IV is the deacylation reaction. Compounds of substrate-like structure, in which the P2 residue has an R-configuration, are not hydrolysed by dipeptidyl peptidase IV.     

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

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.