cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue

Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80,751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. [3H]Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.     

Characterization of a prolyl endopeptidase from Flavobacterium meningosepticum. Complete sequence and localization of the active-site serine.

A prolyl endopeptidase was purified from Flavobacterium meningosepticum. It was digested with trypsin. Two oligonucleotides, based on tryptic peptide sequences and used in PCR experiments, amplified a 300-base pair (bp) fragment. A 2.4-kilobase EcoRI fragment that hybridized to the 300-bp probe was cloned in lambda ZAP and sequenced from both strands. It contains a reading frame of 2115 bp, encoding the complete protein sequence of 705 amino acids. Ion-spray mass spectrometry experiments demonstrated the presence of an NH2-terminal signal peptide: the periplasmic mature protease is 685 residues in length for a molecular mass of 76784 Da. The prolyl endopeptidase showed no general sequence homology with known protein sequences except with that of porcine brain prolyl endopeptidase. In order to identify the active-site serine, the prolyl endopeptidase was labeled with [3H]diisopropyl fluorophosphate. One labeled peptide was purified and sequenced. The active-site serine was located in position 536 within the sequence GRSNGG. This sequence is different from the active-site sequence of the trypsin (GDSGGP) and subtilisin (GTSMAS) families.     

Inhibition of Rabbit Brain Prolyl Endopeptidase by N-Benzyloxycarbonyl-Prolyl-Prolinal, a Transition State Aldehyde Inhibitor

Prolyl endopeptidase cleaves peptide bonds on the carboxyl side of proline residues within a peptide chain. The enzyme readily degrades a number of neuropeptides including substance P, neurotensin, thyrotropin-releasing hormone, and luteinizing hormone-releasing hormone. The finding that the enzyme is inhibited by benzyloxycarbonyl-prolyl-proline, with a Ki of 50 microM, prompted the synthesis of benzyloxycarbonyl-prolyl-prolinal as a potential transition state analog inhibitor. Rabbit brain prolyl endopeptidase was purified to homogeneity for these studies. The aldehyde was found to be a remarkably potent inhibitor of prolyl endopeptidase with a Ki of 14 nM. This Ki is more than 3000 times lower than that of the corresponding acid or alcohol. By analogy with other transition state inhibitors, it can be assumed that binding of the prolinal residue to the S1 subsite and the formation of a hemiacetal with the active serine of the enzyme greatly contribute to the potency of inhibition. The specificity of the inhibitor is indicated by the finding that a variety of proteases were not affected at concentrations 150 times greater than the Ki for prolyl endopeptidase. The data indicate that benzyloxycarbonyl-prolyl-prolinal is a specific and potent inhibitor of prolyl endopeptidase and that consequently it should be of value in in vivo studies on the physiological role of the enzyme.     

A human serine endopeptidase, purified with respect to activity against a peptide with phosphoserine in the P1' position, is apparently identical with prolyl endopeptidase

The present work describes the detection, purification, and characterization of a serine endopeptidase with preference for a phosphoserine in the P1' position of the substrate. During probing for the enzyme in crude extracts, as well as during its 64,000-fold purification, 32P-labeled guanidovaleryl-Arg-Ala-Ser(P)-isobutyl amide (I) was used to measure the cleavage of the Ala-Ser(P) bond. With this substrate, kcat was 1.7 s-1 and Km was 30 microM at the pH optimum, 7.5. The enzyme was classified as a serine peptidase from its reaction with a set of inhibitors, among which diisopropyl fluorophosphate was effective at low (20 microM) concentration. The endopeptidase showed an Mr of 74,000 under native as well as denaturing and reducing conditions, indicating that the native enzyme consists of only one major polypeptide chain. The molecular size and inhibition profile suggested identity of this enzyme with prolyl endopeptidase (EC 3.4.21.26). This was supported by its activity against specific substrates, such as succinyl-Gly-Pro-Leu-Pro-7-amido-4-methylcoumarin (kcat = 7.2 s-1 and Km = 290 microM), and by the inhibition of the latter activity by I. Compared with the cleavage of 100 microM I, Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu, after phosphorylation by cAMP-dependent protein kinase, was cleaved at the Ala-Ser(P) bond at a relative rate of 0.43, while cleavage of the Ala-Ser bond of the unphosphorylated undecapeptide was undetectable, i.e. less than 0.03. The pentapeptide Arg-Arg-Pro-Ser-Val was rapidly cleaved at the Pro-Ser bond (relative rate, 2.2). Still, the cleavage of the Pro-Ser(P) bond of the corresponding phosphorylated pentapeptide was even higher (relative rate, 4.0). These data suggest that phosphorylation of a serine residue in the P1' position of at least a few substrates of prolyl endopeptidase will increase the rate of their cleavage.     

Prolyl endopeptidase from Flavobacterium meningosepticum: cloning and sequencing of the enzyme gene.

The prolyl endopeptidase [EC 3.4.21.26] gene of Flavobacterium meningosepticum was cloned in Escherichia coli with the aid of an oligonucleotide probe which was prepared based on the amino acid sequence. The hybrid plasmid, pFPEP1, with a 3.5 kbp insert at the HincII site of pUC19 containing the enzyme gene, was subcloned into pUC19 to construct plasmid pFPEP3. The whole nucleotide sequence of an inserted HincII-BamHI fragment of plasmid pFPEP3 was determined by the dideoxy chain-terminating method. The purified prolyl endopeptidase was labeled with tritium DFP, and the sequence surrounding the reactive serine residue was found to be Ala (551)-Leu-Ser-Gly-Arg-*Ser-Asn(557). Ser-556 was identified as a reactive serine residue. The enzyme consists of 705 amino acid residues as deduced from the nucleotide sequence and has a molecular weight of 78,705, which coincides well with the value estimated by ultra centrifugal analysis. The amino acid sequence was 38.2% homologous to that of the porcine brain prolyl endopeptidase [Rennex et al. (1991) Biochemistry 30, 2195-2203] and 24.5% homologous to E. coli protease II, which has substrate specificity for basic amino acids [Kanatani et al. (1991) J. Biochem. 110, 315-320].     

Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme.

Protease II gene of Escherichia coli HB101 was cloned and expressed in E. coli JM83. The transformant harboring a hybrid plasmid, pPROII-12, with a 2.4 kbp fragment showed 90-fold higher enzyme activity than the host. The whole nucleotide sequence of the inserted fragment of plasmid pPROII-12 was clarified by the dideoxy chain-terminating method. The sequence that encoded the mature enzyme protein was found to start at an ATG codon, as judged by comparison with amino terminal protein sequencing. The molecular weight of the enzyme was estimated to be 81,858 from the nucleotide sequence. The reactive serine residue of protease II was identified as Ser-532 with tritium DFP. The sequence around the serine residue is coincident with the common sequence of Gly-X-Ser-X-Gly, which has been found in the active site of serine proteases. Except for this region, protease II showed no significant sequence homology with E. coli serine proteases, protease IV and protease La (lon gene), or other known families of serine proteases. However, 25.3% homology was observed between protease II and prolyl endopeptidase from porcine brain. Although the substrate specificities of these two enzymes are quite different, it seems possible to classify protease II as a member of the prolyl endopeptidase family from the structural point of view.     

Porcine muscle prolyl endopeptidase and its endogenous substrates

Prolyl endopeptidase [EC 3.4.21.26] was purified 4,675-fold with a yield of 26.3% from porcine muscle. The purified enzyme was shown to be very similar to the liver enzyme with respect to its molecular weight (72,000-74,000), antigenicity, substrate specificity, and susceptibility to protease inhibitors. Among several bioactive peptides, angiotensins I, II, and III had the lowest Km of 0.6 to 3 microM with the lowest kcat of 0.19 to 0.85 s-1, while thyrotropin-releasing hormone had the highest Km of 98 microM with the highest kcat of 14.4 s-1. Interestingly, mastoparan was hydrolyzed at alanyl bonds, but insulin was only slightly hydrolyzed and glucagon was not hydrolyzed although the latter two peptides contain prolyl and/or alanyl bonds. Muscle prolyl endopeptidase failed to hydrolyze proteins with high molecular weight such as albumin, immunoglobulin G, elastin, collagen, and muscle soluble and insoluble proteins. However, 8 of 14 peptides with molecular weights lower than 3,000, which were isolated from muscle extract, were digested by this enzyme, and they were proved to contain prolyl and/or alanyl residues in their molecules. The data suggest that they are probable endogenous substrates for prolyl endopeptidase.     

Study on a prolyl endopeptidase from the skeletal muscle of common carp (Cyprinus carpio)

A prolyl endopeptidase (PEP) was purified to homogeneity from the skeletal muscle of common carp using a procedure involving ammonium sulfate fractionation and column chromatography involving DEAE-Sephacel, Phenyl-Sepharose, DEAE-Sepharose Fast Flow, and hydroxyapatite. The molecular weight of the PEP was 82 kDa as determined by SDS-PAGE. Using Suc-Gly-Pro-MCA as a substrate, the optimal pH and temperature of the purified enzyme were pH 6.0 and 35 °C, respectively, and the K_m and k_cat were 8.33 μM and 1.71 S^?1, respectively. The activity of the PEP was inhibited by SUAM-14746, a specific inhibitor of prolyl endopeptidases, and was partially inhibited by the serine proteinase inhibitors PMSF and Pefabloc SC. According to peptide mass fingerprinting, 12 peptide fragments with a total of 134 amino acid residues were obtained, which were highly identical to prolyl endopeptidases from zebrafish (Danio rerio) and sponge (Amphimedon queenslandica), confirming the purified enzyme was a prolyl endopeptidase. Our present study for the first time reported the existence of a prolyl endopeptidase in fish muscle.     

PURIFICATION AND PROPERTIES OF A PROLYL ENDOPEPTIDASE FROM RABBIT BRAIN

Abstract— An enzyme with the specificity of a prolyl endopeptidase was purified about 880-fold from rabbit brain. The enzyme hydrolyzes peptidylprolyl-peptide and peptidylprolyl-amino acid bonds. Several biologically active peptides such as angiotensin, bradykinin, neurotensin. substance P and thyrotropin releasing hormone are degraded by hydrolysis of the bond between the carboxyl group of proline and the adjacent amino acid or ammonia respectively. The enzyme is activated by dithiothreitol and inhibited by heavy metals and thiol blocking agents. The serine protease inhibitor phenylmethanesulfonylfluoride has no effect on activity; however, inhibition was obtained with diisopropylfluorophosphate. Prolyl endopeptidase has a molecular weight of about 66,000 and a pH optimum of about 8.3. A new chromogenic substrate, N -benzyloxycarbonylglycyl-L-prolylsulfamethoxazole, was used for determination of enzyme activity. The substrate is hydrolyzed to N -benzyloxycarbonylglycyl-L-proline and free sulfamethoxazole which can be conveniently determined by a colorimetric procedure.     

A prolyl endopeptidase from murine macrophages, its assay and specific inactivation

The presence of a prolyl endopeptidase in the soluble fraction of murine peritoneal macrophages is reported. The prolyl endopeptidase is apparently highly specific for cleaving peptides after proline residues. A sensitive new fluorogenic assay substrate matching this specificity, benzyloxycarbonyl-Ala-Ala-Pro beta-methoxynaphthylamide, is described. The enzyme is rapidly inactivated by benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone, one of a class of reagents specific for cysteine proteinases, and by diisopropyl fluorophosphate, an inhibitor of serine proteinases. Culture of macrophages with the addition of low levels of benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone to the media allows the selective inhibition of the cytoplasmic enzyme as measured in lysates at the termination of culture. After exposure to inhibitor, macrophages resynthesize the enzyme over a period of days, a process which is inhibited by cycloheximide. Similar amounts of activity were found in both normal peritoneal macrophages and those elicited by prior injection of thioglycollate media. The enzyme from murine macrophages appears similar to that reported in bronchopulmonary lavage fluid and lung tissue and to those isolated from brain and pituitary tissues.     

Slow tight-binding inhibition of prolyl endopeptidase by benzyloxycarbonyl-prolyl-prolinal.

Prolyl endopeptidase is a serine proteinase that specifically cleaves peptides on the carboxy side of proline residues. Wilk & Orlowski [(1983) J. Neurochem. 41, 69-75] have shown that benzyloxycarbonyl-prolyl-prolinal (Z-prolyl-prolinal) is a potent inhibitor of prolyl endopeptidase. We show that Z-prolyl-prolinal is a slow-binding inhibitor of mouse brain prolyl endopeptidase with Ki 0.35 +/- 0.05 nM. Kinetic analysis indicates that the mechanism is a simple, but slow, reversible equilibrium between free and bound enzyme (E + I in equilibrium EI) with rate constants for association (kon) and dissociation (koff) of 1.6 X 10(5) M-1.s-1 and approx. 4 X 10(-5) s-1 respectively. Slow-binding inhibition is dependent on the presence of the aldehyde group since the alcohol (Z-prolyl-prolinol) is a rapid and 50,000-fold poorer inhibitor (Ki 19 microM). Prolyl endopeptidase from human brain is also inhibited by Z-prolyl-prolinal with kinetics similar to those of the mouse brain enzyme.     

Distribution of prolyl endopeptidase activities in rat and human brain.

Prolyl endopeptidase is a proteolytic enzyme which could have a neuropeptide catabolising role in the central nervous system. Although prolyl endopeptidase has been described as a cytosolic enzyme, it has become clear that it can also be found in particulate form. The regional and subcellular distribution of this enzyme was evaluated in rat and human brain. The activity of the enzyme was higher in the human than in the rat brain. In the human brain, the activity levels of both soluble and particulate prolyl endopeptidase were the highest in frontal, parietal and occipital cortices and the lowest in the cerebellum. In the rat brain, the regional distribution of the enzyme was more homogeneous. The activity in all the areas of the central nervous system is higher than in peripheral tissues. Subcellular distribution of the enzyme in the brain indicates that prolyl endopeptidase was higher in the cytosolic fraction than in the particulate fractions. The particulate form was enriched in the synaptosomal and the myelinic membranes. The high activity of prolyl endopeptidase in the human cortex suggests that prolyl endopeptidase could play a role in the functions of this brain area.     

The purification and characterization of a proline dipeptidase from guinea pig brain

A proline dipeptidase (EC 3.4.13.9) from guinea pig brain was purified to over 90% homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, calcium phosphate-cellulose chromatography, chromatofocusing, and gel filtration on Sephadex G-200. A purification factor of 2718-fold was obtained with a yield of 7%. The purified enzyme was found to have an apparent molecular weight of 132,000 and to consist of two dissimilar subunits of molecular weights 64,000 and 68,000. The substrate specificity of the enzyme is not that of a strict proline dipeptidase. Although it preferentially hydrolyzes proline dipeptides (Leu-Pro) it also hydrolyzes prolyl dipeptides (Pro-Leu) and dipeptides not containing proline (Leu-Leu). The purified enzyme preparation exhibited weak aminoacylproline aminopeptidase activity against Arg-Pro-Pro but it did not exhibit any post-proline dipeptidyl aminopeptidase, post-proline cleaving endopeptidase, proline iminopeptidase, prolyl carboxypeptidase or carboxypeptidase P activities when tested with a large variety of peptides and arylamides. With all of the proline and prolyl dipeptides examined the enzyme exhibited biphasic kinetics (two distinct slopes on Lineweaver-Burk plots). However, with Leu-Leu as substrate normal Michaelis-Menten kinetics were obeyed.     

Regulation of prolyl endopeptidase activity by the intracellular redox state

The activity of prolyl endopeptidase was markedly decreased during incubation of intact murine erythroleukemia cells at 45 degrees C, but not during incubation of sonicated cells or during incubation at 42 degrees C. The thermal inactivation of prolyl endopeptidase in situ required neither the synthesis of proteins and polynucleotides nor the synergistic activation of inhibitors. Moreover, inhibition of lysosomal proteinases and calpains or depletion of ATP did not affect the thermal inactivation of prolyl endopeptidase. This specific inactivation of prolyl endopeptidase was also observed following the addition to the culture medium of menadione or diamide, compounds known to increase intracellular oxidized glutathione levels. The activity of prolyl endopeptidase in the cell lysate was also dose-dependently decreased by the addition of glutathione disulfide and the decrease of the activity was prevented by coexistence of reduced glutathione. Furthermore, the level of intracellular oxidized glutathione was increased during incubation at 45 degrees C for 15 min, but not at 42 degrees C for 30 min. These results strongly suggest that the activity of prolyl endopeptidase is regulated by changes in the intracellular redox potential.     

Purification and characterization of prolyl endopeptidase from the Pacific herring, Clupea pallasi, and its role in the activation of sperm motility

Protease activities with specificity toward synthetic substrates, Suc-Gly-Pro-Leu-Gly-Pro-MCA for prolyl endopeptidase or collagenase-like peptidase, and Suc-Ala-Ala-Pro-Phe-MCA for chymotrypsin were identified in the detergent-soluble fraction of herring spermatozoa. The enzyme activities increased in the presence of herring sperm-activating protein (HSAP). Among them a prolyl endopeptidase [EC. 3. 4. 21. 26] was purified to near homogeneity from herring testis. The molecular mass of the enzyme was 79 kDa and the properties of the enzyme were quite similar to prolyl endopeptidase from other tissues or cells. Both the enzyme activation and the sperm motility activation by HSAP were inhibited by benzyloxycarbonyl-L-thioproline-thioprolinal, a specific inhibitor for prolyl endopeptidase. Furthermore, the motility activation by HSAP was inhibited by substrates of the prolyl endopeptidase. Western blotting with mouse anti-prolyl endopeptidase serum revealed the presence of 79 kDa prolyl endopeptidase in the tail fraction of herring sperm. These results suggest that prolyl endopeptidase exists on the surface of the sperm tail and interacts with the HSAP.     

Changes in Prolyl Endopeptidase During Maturation of Rat Brain and Hydrolysis of Substance P by the Purified Enzyme

We determined changes in prolyl endopeptidase activity in developing rat brain. A new and highly sensitive fluorogenic substrate, 7-(succinyl-Gly-Pro)-4-methylcoumarinamide, was used for determination of the enzyme activity. The enzyme activity per brain increased until 2 weeks of age, and then decreased during maturation. The enzyme was purified about 7800-fold from the brain of the rat at 2 or 3 weeks of age. The enzyme has a pH optimum of 5.8 to 6.5, and an approximate molecular weight of 70,000. The enzyme activity was completely inhibited by low concentrations of diisopropylfluorophosphate and partially inhibited by high concentrations of phenylmethanesulphonylfluoride, which are potent serine protease inhibitors. Moreover, thiolblocking agents and some heavy metals also have a strong effect on the activity. Bacitracin was found to be a potent inhibitor, with an IC50 value of 2.5 x 10(-6) M at 0.5 mM of the substrate. The enzyme was proved to hydrolyze the NH2-terminal tetrapeptide. Arg1-Pro2-Lys3-Pro4, from substance P to produce the heptapeptide, Gln5-Gln6-Phe7-Phe8-Gly9-Leu10-Met11-CONH2. The Km value of the hydrolysis of substance P was 1.0 mM. This enzyme may be related to the regulation of substance P in the brain, and to the development of neurones by forming the tetrapeptide because the tetrapeptide has almost the same effect as substance P on the neurite extension of neuroblastoma.     

Effects of hydrosaline treatments on prolyl endopeptidase activity in rat tissues

Enzymatic cleavage of some peptide hormones, neurotransmitters and neuromodulators could be implicated in the regulation of extra- and intracellular fluid volume and osmolality. Prolyl endopeptidase is known to hydrolyze several peptides, which act on hydromineral balance, such as angiotensins, bradykinin, vasopressin, oxytocin, thyrotropin-releasing hormone, neurotensin and opioids. In this work, we analyzed the effects of certain volume and/or osmotic changes in the activity of the soluble and membrane-bound prolyl endopeptidase in several brain areas, heart, lungs, kidney and adrenal and pituitary glands of the rat. Soluble prolyl endopeptidase activity was higher in the renal cortex of the chronic salt-loaded rats than in the control rats. In the water-deprived and polyethylene glycol-treated rats, heart particulate prolyl endopeptidase was lower than in the control rats. Particulate prolyl endopeptidase was also lower in the adrenal gland of the acute salt-loaded rats and in the brain cortex of the water-loaded rats than in the control rats. Data suggest that tissue-dependent peptide hydrolysis evoked by prolyl endopeptidase activity is involved in the water-electrolyte homeostasis.     

alpha-Chymotrypsin inhibition studies on the lignans from Vitex negundo Linn

The lignans (1-8) isolated from the roots of Vitex negundo Linn. were screened against the serine proteases alpha-chymotrypsin, thrombin and prolyl endopeptidase. Compounds 3 and 4 were found to be active only against alpha-chymotrypsin and were noncompetitive and competitive inhibitors of the enzyme, respectively. Ki values were found to be in the range 31.75-47.11 microM.     

A Kazal prolyl endopeptidase inhibitor isolated from the skin of Phyllomedusa sauvagii.

Searching for bioactive peptides, we analyzed acidic extracts of Phyllomedusa sauvagii skin and found two new proteins, PSKP-1 and PSKP-2, of 6.7 and 6.6 kDa, respectively, which, by sequence homology, belong to the Kazal family of serine protease inhibitors. PSKP-1 and PSKP-2 exhibit the unprecedented feature of having proline at P(1) and P(2) positions. A gene encoding PSKP-1 was synthesized and expressed in Escherichia coli. Recombinant PSKP-1 was purified from inclusion bodies, oxidatively refolded to the native state, and characterized by chemical, hydrodynamic and optical studies. PSKP-1 shows inhibitory activity against a serum prolyl endopeptidase, but is unable to inhibit trypsin, chymotrypsin, V8 protease, or proteinase K. In addition, PSKP-1 can be rendered active against trypsin by active-site site-specific mutagenesis, has bactericidal activity, and induces agglutination of red cells at micromolar concentrations. PSKP-1 might protect P. sauvagii teguments from microbial invasion, by acting as an inhibitor of an as-yet unidentified prolyl endopeptidase or directly as a microbicidal compound.     

Bacterial extracellular protease activities in field soils under different fertilizer managements

The major extracellular endopeptidase from Bacillus subtilis PF212 (isolated from paddy field soil) and B. subtilis CF80 (isolated from upland field soil) belongs to the group of serine proteases produced by Bacillus spp. known as subtilisins (optimum pH 7.0, optimum temperature 60 degrees C, and molecular mass 28 kDa). The NH2-terminal amino acid sequence (20 amino acids) of the endopeptidase from (i) strain CF80 was identical with that of subtilisin BPN' and (ii) strain PF212 was identical with that of subtilisin Amylosacchariticus. The properties (i.e., effect of inhibitors) of these endopeptidases were similar to those of the overall soil endopeptidase and soil endopeptidases extracted from paddy field soil. From the numbers of B. subtilis we isolated from paddy fields and found to produce a subtilisin-like serine protease, it seemed possible to consider that subtilisin was one of the soil endopeptidases in paddy field soils. The major extracellular endopeptidase from Serratia marcescens (strains 4-12-132, 4-12-131, and 4-60-110) isolated from upland field soils applied with animal slurry is a serratial metalloprotease (optimum pH 9.5, optimum temperature 40 degrees C, and molecular mass 50 kDa). The NH2-terminal amino acid sequence (20 amino acids) of the endopeptidase from strain 4-12-132 was identical with that of serratial metalloprotease, and partial DNA sequence of the endopeptidase gene of S. marcescens 4-12-132 had high homology with that of the serratial metalloprotease gene. The properties (i.e., effect of inhibitors) of this endopeptidase were similar to those of the overall soil endopeptidase in upland fields applied with animal slurry. Thus, it was possible to consider that serratial metalloprotease was one of the soil endopeptidases in upland fields applied with animal slurry.