Purification and characterization of Pz-peptidase B, a neutral metalloendopeptidase from bovine spermatozoa

We have demonstrated previously that the Pz-peptide synthetic substrate is cleaved by two distinct spermatozoal peptidases, Pz-peptidases A and B. To facilitate further investigations, Pz-peptidase B was purified from bovine spermatozoa. The soluble extract from 81 grams of washed epididymal spermatozoa was fractionated by a five-step procedure consisting of anion-exchange, lectin affinity, hydrophobic interaction, chromatofocusing, and gel filtration chromatography. This method yielded 1 mg of 170-fold purified Pz-peptidase B with a 26% recovery. The purified Pz-peptidase B was electrophoretically homogeneous and possessed a monomeric molecular weight of 90,700. Isoelectric focusing revealed microheterogeneity with pIs ranging from 5.02 to 5.09. Pz-peptidase B was irreversibly inactivated at pH 3.5 or below, and activity was reduced at moderate ionic strengths. Hydrolysis of the Pz-peptide was maximal at pH 7.5. Pz-peptidase B was strongly inhibited by a metal chelator and phosphoramidon. Reactivation of metal-depleted enzyme by various metal ions suggested that Pz-peptidase B was a zinc metallopeptidase. Pz-peptidase B hydrolyzed a wide variety of synthetic substrates and physiologically activity peptides at the amino side of hydrophobic amino acids. These results established that Pz-peptidase B should be classified as a neutral metalloendopeptidase. Overall, the properties of Pz-peptidase B were very similar to those of previously described neutral metalloendopeptidases implicated in degradation of regulatory peptides.     

Identification and preliminary characterization of two distinct bovine seminal Pz-peptidases

Two peptidases hydrolyzing the Pz-peptide substrate were identified in bovine semen. Each Pz-peptidase was strongly inhibited by chelating agents, suggesting both were metallopeptidases. However, these peptidases could be distinguished by other properties and were designated Pz-peptidases A and B. Pz-peptidase A hydrolyzed the Pz-peptide at the Leu-Gly bond, was inhibited by tosylphenylethylchloromethylketone (TPCK) but not by phosphoramidon and had a pH optimum near 6, whereas Pz-peptidase B cleaved the Pro-Leu bond, was inhibited by phosphoramidon but not by TPCK and had a pH optimum near 7. Seminal plasma, light particulates and cytoplasmic droplets contained almost exclusively Pz-peptidase A, and Pz-peptidase A predominated in sperm extracts. Pz-peptidase B was found primarily in sperm extracts, but Pz-peptidase B activity was also present in ultralight particulates. Pz-peptidase A of spermatozoa required Triton X-100 for complete extraction, but Pz-peptidase B was solubilized from spermatozoa by nitrogen decompression without detergents. Pz-peptidase B was inhibited by several detergents. In particular, addition of 0.1% Hyamine 2389 to sperm extracts inhibited 99% of the Pz-peptidase B activity. Thus, Pz-peptidase B may have been overlooked in previous studies employing extraction of spermatozoa with Hyamine 2389. The properties of both seminal PZ-peptidases were different from those of purified bovine testicular PZ-peptidase, suggesting that PZ-peptidases from these sources were not identical.     

Distribution of Pz-peptidase in bovine epididymal and ejaculated semen

Bovine epididymal or ejaculated semen was fractionated by density gradient centrifugation in Percoll, and seminal components recovered from the gradients were subjected to additional separation and washing steps. This procedure resulted in isolation of four major seminal constituents: particle-free extracellular fluid, washed light particulates, washed cytoplasmic droplets, and washed spermatozoa. When assayed using the Pz-peptide substrate, all the isolated seminal fractions contained substantial Pz-peptidase activity. The extracellular fluid Pz-peptidase was present in soluble form, but Triton X-100 was required for complete extraction of the Pz-peptidase activity from the spermatozoa, cytoplasmic droplets, and light particulates. The greatest Pz-peptidase activities were observed in the cytoplasmic droplet and epididymal sperm extracts, whereas the activities in extracellular fluid, extracts of light particulates, and extracts of ejaculated spermatozoa were relatively low. Most of the Pz-peptidase activity in extracts of epididymal spermatozoa was attributable to cytoplasmic droplets. The specific Pz-peptidase activities found by regression analysis were 6.1 mU/billion attached cytoplasmic droplets and 1.1 mU/billion spermatozoa. These results established that in the bovine, cytoplasmic droplets were the major source of Pz-peptidase activity in semen and that Pz-peptidase was not primarily a spermatozoal enzyme.     

Isolation of the major glycoprotein from fat cell plasma membranes

The major glycoprotein of rabbit fat cell plasma membranes has been solubilized by Brij 99 extraction and purified to homogeneity by preparative polyacrylamide gel electrophoresis and concanavalin A-Sepharose affinity chromatography. The isolation procedure yielded a glycoprotein with an apparent molecular weight of 79,000 which appeared as a single component by Coomassie blue and periodic acid-Schiff staining as well as by distribution of radioactivity after ¹²⁵I labeling. The lectin chromatography was effective in removing polypeptides and Schiff-nonreactive glycoproteins which migrated in close proximity to the major glycoprotein during electrophoresis but were not retained on the concanavalin A column. Determination of the amino acid and sugar composition of the purified glycoprotein indicated that it contained 18% carbohydrate by weight which occurred in the form of 30 mannose, 14 galactose, 23 glucosamine, 3 galactosamine, 6 N-acetylneuraminic acid, and 1 fucose residues per molecule. Approximately one-fifth of the total protein-bound saccharide of the adipocyte plasma membrane was accounted for by this glycoprotein and its composition suggested that it was the source of some of the previously identified (Y. Kawai, and R. G. Spiro, 1977, J. Biol. Chem.252, 6236–6244) asparagine- and serine (threonine)-linked carbohydrate units of the fat cell surface.     

Purification and properties of bovine myocardial phosphorylase phosphatase (protein phosphatase C).

Phosphorylase phosphatase was purified to homogeneity from bovine myocardium by the procedure of Brandt et al. (Brandt, H., Capulong, Z. L., and Lee, E. Y. C., (1975) J. Biol. Chem.250, 8038–8044). The purified enzyme consists of a single polypeptide chain of M_r, 34,800 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The K_m for phosphorylase a was 2.9 μm and at V_max, the enzyme had a turnover number of 11.7 mol phosphorylase a (dimer) converted/mol phosphatase/s. Phosphorylated histone and protamine were also substrates for this phosphatase. The K_m for histone was 46 μm (based on incorporated ³²P_i and at V_max a turnover number of 3.3 mol phosphate released/mol phosphatase/s was found. In general, the properties of the bovine phosphorylase phosphatase closely resemble those found for the rabbit liver enzyme.     

Purification and characterization of Pz-peptidase from rabbit muscle

Pz-peptidase was purified from rabbit muscle by acid precipitation of tissue homogenate followed by cation- and anion-exchange chromatography, gel chromatography, and immunoadsorption. In analytical gel chromatography, one single peak of protein with corresponding Pz-peptidase activity was obtained. The enzyme had an apparent Mr of 74,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was eluted at pH 4.8 in chromatofocusing. No metals were detectable in the protein by neutron activation analysis. Purified Pz-peptidase hydrolyzed Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys (Km 7.2 microM) most effectively in the presence of 5 mM 2-mercaptoethanol and 10 mM CaCl2. No inhibition was observed with inhibitors of serine proteinases, aspartic proteinases, or metalloproteinases, apart from some nonspecific reversible inhibition by 1,10-phenanthroline. The activation by Ca2+ was reversed by EDTA. The enzyme was not inhibited by E-64, cystatin, or leupeptin, but was irreversibly inactivated by iodoacetate, iodoacetamide, and N-ethylmaleimide. It was therefore concluded that rabbit muscle Pz-peptidase is not a typical member of any of the four recognized catalytic classes of proteinases, but may be an atypical cysteine endopeptidase. The peptidase was not bound by alpha 2-macroglobulin. No hydrolysis of gelatin or fibronectin by the enzyme was detected, nor was there any activation of latent collagenase.     

Further purification of epidermal growth factor by high-performance liquid chromatography

Epidermal growth factor (EGF) purified by the method of Savage and Cohen (J. Biol. Chem.247, 7601–7611 (1972) using DEAE-cellulose chromatography as the final purification step was further resolved into two major uv-absorbing components by reverse-phase high-performance liquid chromatography (HPLC). Both components, referred to as α-EGF and β-EGF, competed with ¹²⁵I-labeled EGF for the EGF receptor, induced premature eye opening in neonatal mice, and had an amino acid composition similar to that published by Savage et al. (J. Biol. Chem.247, 7612–7621 (1972). β-EGF migrated slightly faster than α-EGF during sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. α-EGF was fourfold more potent than β-EGF and was 10-fold more potent than DEAE-purified EGF in stimulating DNA synthesis in quiescent Rat-1 cells. HPLC purification of EGF can replace the DEAE-cellulose chromatography step currently used and produces a more potent and less heterogeneous EGF species.     

The Exquisite Structure and Reaction Mechanism of Bacterial Pz-peptidase A toward Collagenous Peptides: X-RAY CRYSTALLOGRAPHIC STRUCTURE ANALYSIS OF PZ-PEPTIDASE A REVEALS DIFFERENCES FROM MAMMALIAN THIMET OLIGOPEPTIDASE*

Pz-peptidase A, from the thermophilic bacterium Geobacillus collagenovorans MO-1, hydrolyzes a synthetic peptide substrate, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg (Pz-PLGPR), which contains a collagen-specific tripeptide sequence, -Gly-Pro-X-, but does not act on collagen proteins themselves. The mammalian enzyme, thimet oligopeptidase (TOP), which has comparable functions with bacterial Pz-peptidases but limited identity at the primary sequence level, has recently been subjected to x-ray crystallographic analysis; however, no crystal structure has yet been reported for complexes of TOP with substrate analogues. Here, we report crystallization of recombinant Pz-peptidase A in complex with two phosphinic peptide inhibitors (PPIs) that also function as inhibitors of TOP and determination of the crystal structure of these complexes at 1.80–2.00 Å resolution. The most striking difference between Pz-peptidase A and TOP is that there is no channel running the length of bacterial protein. Whereas the structure of TOP resembles an open bivalve, that of Pz-peptidase A is closed and globular. This suggests that collagenous peptide substrates enter the tunnel at the top gateway of the closed Pz-peptidase A molecule, and reactant peptides are released from the bottom gateway after cleavage at the active site located in the center of the tunnel. One of the two PPIs, PPI-2, which contains the collagen-specific sequence, helped to clarify the exquisite structure and reaction mechanism of Pz-peptidase A toward collagenous peptides. This study describes the mode of substrate binding and its implication for the mammalian enzymes.     

Purification,partial structural characterization and health benefits of exopolysaccharides from potential probiotic Pediococcus acidilactici NCDC 252

Probioceuticals are probiotic-derived biologically active compounds that positively influence human health. Exopolysaccharides (EPS) are long chain polymers of sugars and their derivatives with diverse biological functions. The objective of this work was to study EPS from Pediococcus acidilactici (PA) NCDC 252, a potential probiotic. In silico analysis of PA NCDC 252 genome revealed an EPS gene cluster (10 genes) and genes were further analysed for their functional domains and products. EPS from PA NCDC 252 were purified by ethanol precipitation and gel filtration chromatography on Sephadex G-100. Molecular mass of purified EPS was 89.1 KDa as determined by gel filtration chromatography. Structural and component analysis by FTIR, NMR and UPLC revealed purified EPS to be linear homopolysaccharides (α-glucans) with few α-(1→3) branches. in vitro studies showed anti-oxidant, reduction potential and anti-cancer activity in dose dependent manner. Total antioxidant potential of EPS was 11.9 %. In-vitro cell viability assay revealed anti-proliferative effect of EPS on human colon cancer cell line (HCT116). At 10 and 100 μg/mL, EPS inhibited HCT116 cells up to 67.1 % and 87.3 % respectively. These results suggest that PA EPS can be therapeutically used as anti-oxidant and anticancer agent after in vivo studies.     

Studies on a peptidase acting on a synthetic collagenase substrate Developmental pattern in rat granuloma tissue and distribution of enzyme in the tissues of various animals

The developmental pattern in experimental rat granuloma tissue and the distribution in the tissues of a few animals (monkey, rabbit, guinea pig anrat) of a peptidase acting on a synthetic collagenase substrate, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (Pz-peptide) has been studied. Maximum enzyme activity was found in 4-month-old rats and on the fourth day of implantation of the cotton wick. Pz-peptidase appears to have a ubiquitous distribution in animal tissues; the highest enzyme activity was generally found in liver, intestine and kidney of the animals. The total activity in other organs (spleen, heart, lungs and brain) was much less compared to that of liver, intestine or kidney.     

Further characterization and thiophosphorylation of smooth muscle myosin

(i) Myosin from chicken gizzards was purified by a modification of an earlier procedure (M. N. Malik, 1978,Biochemistry17, 27–32). When this myosin, as well as that prepared by the method of A. Sobieszek and R. D. Bremel (1975,Eur. J. Biochem.55, 49–60), was analyzed by gradient slab gel using the discontinuous buffer system of Neville (1971,J. Biol. Chem.246, 6328–6334), a closely spaced doublet in the heavy chain and four light chains were observed as opposed to one heavy chain and two light chains with the method of Weber and Osborn (1969, J. Biol. Chem.244, 4406–4412). These findings raise the possibility of the existence of myosin isoenzymes in smooth muscle. (ii) The purified gizzard myosin was found to be free of kinase and phosphatase. Phosphorylation or thiophosphorylation of myosin was observed only by exogenously adding kinase. A maximum of 1.2 mol of ³²P/mol of myosin and 2.3 mol of ³⁵S/mol of myosin were obtained. The actin-activated ATPase activity depended upon the extent of thiophosphorylation of myosin; a four- to fivefold increase in the activity was observed when myosin was fully thiophosphorylated. Thiophosphorylated myosin was found to be more stable than phosphorylated myosin.     

Botulinum Neurotoxin Type A: Limited Proteolysis by Endoproteinase Glu-C and α-Chymotrypsin Enhanced Following Reduction; Identification of the Cleaved Sites and Fragments

Botulinum neurotoxin (NT) serotype A is a ～150-kDa dichain protein. Posttranslational nicking of the single-chain NT (residues Pro 1–Leu 1295) by the protease(s) endogenous to Clostridium botulinum excises 10 residues, leaving Pro 1–Lys 437 and Ala 448–Leu 1295 in the ～50-kDa light (L) and ～100-kDa heavy (H) chains, respectively, connected by a Cys 429–Cys 453 disulfide and noncovalent bonds [Krieglstein et al. (1994), J. Protein Chem. 13, 49–57]. The L chain is a metalloprotease, while the amino- and carboxy-terminal halves of the H chain have channel-forming and receptor-binding activities, respectively [Montecucco and Schiavo (1995), Q. Rev. Biophys. 28, 423–472]. Endoproteinase Glu-C and α-chymotrypsin were used for controlled digestion at pH 7.4 of the ～150-kDa dichain NT and the isolated ～100-kDa H chain (i.e., freed from the L chain) in order to map the cleavage sites and isolate the proteolytic fragments. The dichain NT appeared more resistant to cleavage by endoproteinase Glu-C than the isolated H chain. In contrast, the NT with its disulfide(s) reduced showed rapid digestion of both chains, including a cleavage between Glu 251 and Met 252 (resulting in ～30- and ～20-kDa fragments of the L chain) which was not noted unless the NT was reduced. Interestingly, an adjacent bond, Tyr 249–Tyr 250, was noted earlier [DasGupta and Foley (1989), Biochimie 71, 1193–1200] to undergo “self-cleavage” following reductive separation of the L chain from the H chain. The site Tyr–Tyr–Glu–Met (residues 249–252) appears to become exposed following reduction of Cys 429–Cys 453 disulfide. Identification of Glu 669–Ile 670 and Tyr 683–Ile 684 as protease-susceptible sites demonstrated for the first time that at least two peptide bonds in the segment of the H chain (residues 659–684), part of which (residues 659–681) is thought to interact with the endosomal membranes and forms channels [Oblatt-Montal et al., (1995), Protein Sci. 4, 1490–1497], are exposed on the surface of the NT. Two of the fragments of the H chain we generated and purified by chromatography are suitable for structure–function studies; the ～85- and ～45-kDa fragments beginning at residue Leu 544 and Ser 884, respectively (both extend presumably to Leu 1295) contain the channel-forming segment and receptor-binding segments, respectively. In determining partial amino acid sequences of 10 fragments, a total of 149 amino acids in the 1275-residue NT were chemically identified.     

The activities of ''Pz-peptidase'' and ''endopeptidase 24.15'' are due to a single enzyme.

It was found that Pz-peptidase (assayed with 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys) and endopeptidase 24.15 (assayed with benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate) were co-purified from rat skeletal muscle, were co-eluted in high-resolution gel chromatography and co-existed in a homogeneous preparation of rat testis endopeptidase 24.15. The action of partially purified Pz-peptidase from rat testis on 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg was blocked by an inhibitor of endopeptidase 24.15, and also by a substrate of this enzyme. The partially purified enzyme hydrolysed two substrates of endopeptidase 24.15 with Km values similar to those published previously, and its action on 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys was inhibited by compounds that are considered specific for endopeptidase 24.15. We conclude that the activities previously attributed to two distinct enzymes are due to only one, and that the merging of the two literatures may lead to new lines of research.     

Analysis of cytogenetic stability in natural populations of terrestrial mollusks (based on DNA comet assay)

Alkaline gel electrophoresis of isolated cells (comet assay) was used to assess degree of nuclear DNA damage in populations of terrestrial mollusks Bradybaena fruticum Müll., Chondrula tridens Müll., Cepaea vindobonensis Fer., and Stenomphalia ravergieri Fer. living in the forest-steppe landscape of the southern part of the Mid-Russian Upland. Evidence of differences in the parameters studied was found. The age dynamics of the degree of damage of the genetic apparatus was observed. Possible causes of the identified differences are discussed.     

Characterization of a Novel Plasma Membrane Protein, Expressed in the Midgut Epithelia of Bombyx mori, That Binds to Cry1A Toxins

We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of Bombyx mori. P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and K_d constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc. Binding assays of P252 with various lectins indicated the presence of three antennal N-linked high-mannose-type as well as O-linked mucin-type sugar side chains. While the function of P252 is not yet clear, we propose that it may function with Cry1A toxins during the insecticidal response and/or Cry toxin resistance mechanism.     

Large-scale purification and characterization of calmodulin from ram testis its metal-ion-dependent conformers

Calmodulin was isolated in large quantities from ram testis by a simple procedure involving sequentially ammonium sulfate fractionation, heat treatment, anion exchange chromatography on DEAE-cellulose and gel filtration on Sephacryl S-200. Divalent cations (Mg²⁺ and/or Ca²⁺) were present throughout the purification which was entirely performed in the absence of chelators. The final yield was approx. 90 mg per kg testis. Ram testis calmodulin appears to be essentially identical to the brain homologous protein by the following criteria: ultraviolet absorption spectrum, amino acid composition showing a single residue of ?-N-trimethyl lysine, and tryptic peptide maps obtained by high performance liquid chromatography. Turkey gizzard myosin light-chain kinase, the activation of which is extremely specific for calmodulin (Walsh, M.P., Vallet, B., Cavadore, J.C. and Demaille, J.G. (1980) J. Biol. Chem. 255, 335–337), was indeed activated by ram testis calmodulin in the presence of calcium. The isolated protein migrated at different rates upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, depending on the absence or presence of divalent metals which probably induce different conformations. The relative migration rates were Ca²⁺ > Mn²⁺ > Mg²⁺ > EDTA. In the presenceof divalent metals, the observed doublet may be ascribed to the equilibrium between ion-free and ion-saturated forms, which exhibited different Stokes radii, as already suggested (Grab, D.J., Berzins, K., Cohen, R.S. and Siekevitz, P. (1979) J. Biol. Chem. 254, 8690–8696).     

The molecular properties of the copper enzyme galactose oxidase

Galactose oxidase (EC 1.1.3.9) has been purified 140-fold by DEAE- and CM-cellulose chromatography from cultures of Polyporus circinatus. The enzyme has a molecular weight of 68,000 ± 3,000 as determined by sedimentation equilibrium, sodium dodecyl sulfate-acrylamide gel electrophoresis, Sephadex G-150 chromatography, and osmometry. Galactose oxidase is a single-chain protein which does not self-associate. Charge isozymes of the enzyme are detected by ion-exchange chromatography and gel electrophoresis. The amino acid composition determined herein is significantly different from that previously reported (Kelly-Falcoz, F., Greenberg, H., And Horecker, B. L. (1965) J. Biol. Chem.240, 2966–2970). The enzyme contains 1% by weight of neutral carbohydrate.Galactose oxidase contains 1 g-atom of copper per 70,000 g of protein. The metal does not contribute to the electrophoretic or isozymic properties of the protein. However, the sedimentation coefficients of the holo- and apoenzymes, 4.76S and 4.83S, respectively, do suggest that small differences in protein conformation accompany the removal of the copper from the holoenzyme.Attempted sulfhydryl group titration of galactose oxidase shows that the holoenzyme is resistant to denaturation. However, in β-mercaptoethanol-guanidine HCl 5 half-cystine residues are titrated in the apoenzyme. On a dry-weight basis, the E_1cm^1% value for galactose oxidase at 280 nm is 15.4. Galactose oxidase has an isoelectric point above pH 10 which is a probable source of some of its anomalous behavior in physical measurements and enzyme-activity determinations.     

Isolation and complete amino acid sequences of eclosion hormones of the silkworm,Bombyx mori

Four closely related eclosion hormones (EHs), EH-I, -II, -III and -IV, were purified from 770,000 pharate adult heads of the silkworm, Bombyx mori. The complete amino acid sequence was determined by Edman degradation of peptide fragments generated by enzymatic digestion. Preliminary results have been reported (Kono et al., Agric. biol. Chem.51, 2307–2308, 1987). Accumulated data suggested that EH-IV has the longest sequence consisting of 62 residues. EH-I and -III have N-termini truncated by two residues. EH-I and -II terminate at position 61. The removal of the C-terminal leucine in EH-I and -II might have occurred artificially during extraction.     

An alternative quenched fluorescence substrate for Pz-peptidase

7-Methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-Lys(2,4-dinitr oph enyl) is introduced as a new quenched fluorescence substrate for assaying Pz-peptidase (also known as soluble metallo-endopeptidase and endo-oligopeptidase). The value of Km for partially purified Pz-peptidase from rat muscle was 8.6 microM. High protein concentrations did not interfere with the assay, so that for the first time continuous assays of Pz-peptidase in crude tissue extracts became possible.