Rapid degradation of D- and L-succinimide-containing peptides by a post-proline endopeptidase from human erythrocytes

We have been interested in the metabolic fate of proteins containing aspartyl succinimide (Asu) residues. These residues can be derived from the spontaneous rearrangement of Asp and Asn residues and from the spontaneous demethylation of enzymatically methylated L-isoAsp and D-Asp residues. Incubation of the synthetic hexapeptide N-Ac-Val-Tyr-Pro-Asu-Gly-Ala with the cytosolic fraction of human erythrocytes resulted in rapid cleavage of the prolyl-aspartyl succinimide bond producing the tripeptide N-Ac-Val-Tyr-Pro. The rate of this reaction is equal for both L- and D-Asu-containing peptides and is 10-fold greater than the rate of cleavage of a corresponding peptide containing a normal Pro-Asp linkage. When the aspartyl succinimide ring was replaced with an isoaspartyl residue, the cleavage rate was about 5 times that of the normal Pro-Asp peptide. The tripeptide-producing activity copurified on DEAE-cellulose chromatography with an activity that cleaves N-carbobenzoxy-Gly-Pro-4-methylcoumarin-7-amide, a post-proline endopeptidase substrate. These two activities were both inhibited by an antiserum to rat brain post-proline endopeptidase, and it appears that they are catalyzed by the same enzyme. This enzyme has a molecular weight of approximately 80,000 and is covalently labeled and inhibited by [3H]diisopropyl fluorophosphate. The facile cleavage of the succinimide- and isoaspartyl-containing peptides by this post-proline endopeptidase suggests that it may play a role in the metabolism of peptides containing altered aspartyl residues.     

Human lung mast cells: purification and characterization

Detailed studies of the biochemistry and pharmacology of mast cell-mediated inflammatory disorders have been hampered by the inability to purify human mast cells. We now report techniques to purify human lung mast cells to apparent homogeneity. The major purification steps are: 1) dispersion of lung fragments into a single-cell suspension with enzyme combinations (pronase-chymopapain, collagenase-elastase); 2) partial purification by countercurrent centrifugation elutriation (CCE); and 3) affinity column chromatography. Enzymatic dispersion yielded suspensions with congruent to 10(6) mast cells per gram of lung parenchyma in purities of 1.2 to 9.7%. Dispersed mast cells responded comparably to those in parent lung fragments to challenge with anti-human IgG and pharmacologic agonists. Elutriation of lung cell suspensions yielded mast cell-enriched fractions with purities up to 70%. High purity mast cell fractions were combined, passively sensitized with purified human penicillin (BPO)-specific IgE, and purified by a BPO-affinity column chromatography procedure. Post elutriation mast cell purities of 29 +/- 3.5% were increased to 84 +/- 3% (range 65 to 98%) by the affinity column. Short-term (24 hr) culture of column-purified mast cells allowed adherence of non-mast cell contaminants to tissue culture plates, further increasing purity (up to 100%). Purified mast cells were intact and functional as assessed by dye exclusion, survival in short-term culture, IgE-mediated histamine release, and modulation of release by the pharmacologic agonists adenosine, IBMX, prostaglandin E2, and fenoterol.     

Receptor binding of guinea pig and pig vasoactive intestinal peptides by rat lung.

Guinea pig vasoactive intestinal peptide (gpVIP) differs from other mammalian VIPs in four of its 28 amino acid residues. In the present study, the gpVIP displaced 125I-labelled pig VIP (pVIP) binding by rat lung membranes with 7.7-fold lower potency than pVIP. Degradation of gpVIP by rat lung membranes, assessed by radioimmunoassay and h.p.l.c., was 1.9-fold greater than that of pVIP. This difference in degradation of the two peptides was not large enough to account for the lower receptor-binding potency of gpVIP. The amino acid residues that distinguish pVIP from gpVIP are likely to contribute to the interaction of VIP with receptors and peptide hydrolases in lung membranes.     

Studies on prolyl endopeptidase from shakashimeji (Lyophyllum cinerascens): purification and enzymatic properties

High prolyl endopeptidase (post-proline cleaving enzyme) [EC 3.4.21.26] activity was detected in fruit bodies of shakashimeji (Lyophyllum cinerascens), tsukuritake (mushroom: Agaricus bisporus), hirohachichitake (Lactarius hygrophoroides), and yaburebenitake (Russula lepida) which belong to the genus Basidiomycetes. Cell-free extract of shakashimeji showed high activities of proline iminopeptidase and arylamidase as well as prolyl endopeptidase. The prolyl endopeptidase was purified from the extract of shakashimeji by sequential chromatographies on DEAE-Toyopearl, DEAE-Sephadex and hydroxyapatite, and high-performance liquid chromatography with a DEAE-5PW column. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 6.8 as checked with Z-Gly-Pro-beta-naphthylamide as a substrate and was stable in the range of pH 5.8-7.4. The isoelectric point of the enzyme was 5.2 and the molecular weight was estimated to be 76,000 by gel filtration on Sephadex G-150 and by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme was a monomer. The enzyme was completely inhibited by diisopropyl fluorophosphate (DFP), Z-Gly-Pro-CH2Cl, and Z-Pro-prolinal, while it was not inhibited by p-chloromercuribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), or metal chelators. It was estimated that at least five subsites were concerned with the enzyme-substrate binding. Among them, the S1, S2, and S1' sites showed high stereospecificity, as in mammalian, microbial, and plant enzymes. The enzyme hydrolyzed TRH at the carboxyl side of the proline residue. The mushroom enzyme, that was sensitive to DFP, Z-Pro-prolinal, and Z-Gly-Pro-CH2Cl, but not to PCMB, were quite similar in characteristics to the Flavobacterium enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human skin tryptase: purification, partial characterization and comparison with human lung tryptase

Human skin tryptase was isolated using stepwise low- and high-salt extraction and further purified 448-fold with 33% yield using octyl-Sepharose CL-4B hydrophobic affinity chromatography, Sephacryl S-200 gel filtration and finally octyl-Sepharose CL-4B or cellulose phosphate ion exchange chromatography. The skin tryptase, which has an apparent Mr of 120,000 by gel filtration in high-salt buffer, consisted of polypeptide chains of Mr 34,000 and 38,000 when resolved on SDS gels. Both polypeptide chains, labelled with [3H]diisopropyl fluorophosphate, indicated that they were representative of subunits and that the native proteinase was an aggregate of subunits. However, in some preparations only one band with Mr 34,000 was seen. In low-salt buffer the enzyme was labile and at least 1.4 M KCl was needed to keep the enzyme stabile when incubated at 37 degrees C for 30 min. Heparin glycosaminoglycan partially stabilized the tryptase but addition of protein (e.g. albumin, 80 micrograms/ml) to the tryptase-heparin mixture was needed to keep the enzyme stabile. Tryptases purified by exactly the same method from human lung tissue and from human skin had identical molecular size in gel filtration and in SDS-polyacrylamide gel electrophoresis. They also revealed identical enzyme kinetic parameters with several synthetic peptide substrates. The inhibition profile was identical for both enzymes, and they also crossreacted completely in immunodiffusion plates. These studies strongly indicate that mast cells found in skin as well as lung contain closely related, possible identical trypsin-like proteinases.     

Porcine liver succinyltrialanine p-nitroanilide hydrolytic enzyme. Its purification and characterization as a post-proline cleaving enzyme

Succinyltrialanine p-nitroanilide(STANA)-hydrolytic enzyme was purified 5,200-fold from porcine liver soluble fraction with a yield of 75% by ammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Sephadex G-150, and hydroxylapatite columns. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate (SDS). The pI of the enzyme was 4.9 by dis gel electrofocusing and the molecular weight was calculated to be 72,000 by gel filtration on a Sephadex G-150 column and 74,000 by SDS-polyacrylamide gel electrophoresis. Acidic amino acids amounted to 17.2% of the total amino acid residues, and the basic ones, 12.9%. No hexosamine was detected. The STANA-hydrolytic enzyme showed maximal activity at pH 7.4 against succinyltrialanine p-nitroanilide and at pH 6.5 against succinyl-Gly-Pro-4-methylcoumaryl 7-amide (MCA), and was stable between pH 6 and 7 in the presence of dithiothreitol. This enzyme hydrolyzed succinyl-Gly-Pro-Leu-Gly-Pro-MCA, succinyl-Gly-Pro-MCA, succinyl-Ala-Pro-Ala-MCA, and several proline-containing natural peptides in addition to succinyltrialanine p-nitroanilide, but was unable to hydrolyze the substrates of aminopeptidases, dipeptidylaminopeptidase IV, trypsin, and chymotrypsin. Elastatinal and chymostatin were effective inhibitors and their IC50 values were 8.7 micrograms/ml and 18.2 micrograms/ml, respectively. The enzyme was completely inhibited by 10(-7) M p-chloromercuribenzoic acid (pCMB), 10(-7) M p-chloromercuriphenylsulfonic acid (pCMPS), and 10(-4) M diisopropyl phosphofluoridate (DFP), but not by 1 mM E-64, which is known as an inhibitor specific to thiol proteinase. The enzyme was easily inactivated by agitation in a Vortex mixer, and its activity was recovered by the addition of thiol compounds such as dithiothreitol, 2-mercaptoethanol and cysteine. The effects of inhibitors and thiol compounds were substantially identical when the enzyme activity was measured with either succinyltrialanine p-nitroanilide or succinyl-Gly-Pro-MCA as a substrate. These results indicate that the STANA-hydrolytic enzyme in the liver soluble fraction is a post-proline cleaving enzyme [EC 3.4.21.26].     

Synthesis and purification of amyloidogenic peptides

The polypeptide hormone amylin forms amyloid deposits in patients with type 2 diabetes mellitus. Amyloid-forming peptides are often very difficult to synthesize and purify. Amylin and fragments of amylin are no exception. In this paper we describe the efficient synthesis and purification of two amyloidogenic fragments of human amylin. One fragment corresponds to residues 17 to 37 of the full-length hormone and the other corresponds to residues 24 to 37. These fragments have previously been identified in vivo and have been shown to form amyloid in vitro. The strategy used to elucidate appropriate conditions for the synthesis and purification of these peptides is generally applicable to other peptides that are difficult to synthesize. These peptides were prepared using solid-phase peptide synthesis with Fmoc alpha-amino protection. The effects of varying the solvent, side-chain-protecting group and choice of cleavage conditions were examined. The use of NMP as the main solvent and cleavage with trifluoroacetic acid, phenol, ethanedithiol, thioanisole, and water proved to be optimal. 1,1,1,3, 3,3-Hexafluoro-2-propanol (HFIP) was found to be the best solvent for solubilizing the crude peptides. A wide range of HPLC conditions for the purification of the peptides were examined and an acetonitrile-based solvent system with HCl as the ion pairing agent provided efficient purification.     

Human interleukin 1: purification and properties

Human interleukin 1 (IL 1) has been purified to homogeneity by a procedure of molecular weight fractionation, isoelectric focusing, and preparative polyacrylamide gel electrophoresis. The homogeneity of the purified material has been demonstrated by silver staining of analytical polyacrylamide gels. The homogeneous IL 1 retains only a trace of its original biological activity because of the denturing effects of the sodium dodecyl sulfate used in the final step of purification. Very highly purified IL 1, retaining strong biological activity, has been eluted from nondenaturing polyacrylamide gels. This IL 1 has been demonstrated to stimulate human and mouse T and B lymphocytes, fibroblasts, and synovial cells. In addition, in vivo treatment of animals with IL 1 resulted in the immunologically relevant symptoms of fever, increased plasma levels of acute phase proteins, and increased numbers of circulating neutrophils.     

Human antimicrobial peptides: analysis and application

Antimicrobial peptides are innate host defense molecules that have a direct effect on bacteria, fungi and enveloped viruses. They are found in evolutionarily diverse species ranging from prokaryotes and plants to invertebrate and vertebrate animals. Humans express several families of antimicrobial peptides in myeloid cells and on various epithelial surfaces where they are poised to defend against pathogens. Recently, antimicrobial peptides from animals and plants have served as templates for the design of new therapeutic antibiotics. This review provides an introduction to the biology of human antimicrobial peptides, followed by a more detailed discussion of their isolation from tissues and biological fluids, their purification by gel electrophoresis and chromatography and assays of their antimicrobial activities.     

Human eosinophil peroxidase: purification and characterization

Human eosinophil peroxidase (EPO) was isolated from granules from granulocytes of a patient with hypereosinophilia. The granules were extracted by means of 0.2 M NaAc, pH 4.0. The purification steps included gel filtration chromatography on Sephadex G-75 superfine and ion-exchange chromatography on CM-Sephadex G-50. The purified protein showed one band on agarose-electrophoresis, a high peroxidase activity, and a 415-nm/280 nm ratio of 1.15. After reduction, EPO showed two bands on SDS-PAGE of m.w. 52,000 and 15,000, respectively. On gel filtration, the unreduced protein had a m.w. of approximately 77,000. Amino acid analyses showed a high content of arginine and aspartic acid. Monospecific antibodies to EPO were prepared in rabbits, and a specific radioimmunoassay was developed. There was an almost linear correlation between the content of EPO measured by the radioimmunoassay and the number of eosinophils in a mixed cell extract from reference material, indicating the eosinophil origin of EPO. The content of EPO was estimated to be 15.0 micrograms/10(6) eosinophils.     

Human erythrocyte myosin: identification and purification

Human erythrocytes contain an Mr 200,000 polypeptide that cross-reacts specifically with affinity-purified antibodies to the Mr 200,000 heavy chain of human platelet myosin. Immunofluorescence staining of formaldehyde-fixed erythrocytes demonstrated that the immunoreactive myosin polypeptide is present in all cells and is localized in a punctate pattern throughout the cell. Between 20-40% of the immunoreactive myosin polypeptide remained associated with the membranes after hemolysis and preparation of ghosts, suggesting that it may be bound to the membrane cytoskeleton as well as being present in the cytosol. The immunoreactive myosin polypeptide was purified from the hemolysate to approximately 85% purity by DEAE-cellulose chromatography followed by gel filtration on Sephacryl S-400. The purified protein is an authentic vertebrate myosin with two globular heads at the end of a rod-like tail approximately 150-nm long, as visualized by rotary shadowing of individual molecules, and with two light chains (Mr 25,000 and 19,500) in association with the Mr 200,000 heavy chain. Peptide maps of the Mr 200,000 heavy chains of erythrocyte and platelet myosin were seen to be nearly identical, but the proteins are distinct since the platelet myosin light chains migrate differently on SDS gels (Mr 20,000 and 17,000). The erythrocyte myosin formed bipolar filaments 0.3-0.4-micron long at physiological salt concentrations and exhibited a characteristic pattern of myosin ATPase activities with EDTA, Ca++, and Mg++-ATPase activities in 0.5 M KCl of 0.38, 0.48, and less than 0.01 mumol/min per mg. The Mg++-ATPase activity of erythrocyte myosin in 0.06 M KCl (less than 0.01 mumol/min per mg) was not stimulated by the addition of rabbit muscle F-actin. The erythrocyte myosin was present in about 6,000 copies per cell, in a ratio of 80 actin monomers for every myosin molecule, which is an amount comparable to actin/myosin ratios in other nonmuscle cells. The erythrocyte myosin could function together with tropomyosin on the erythrocyte membrane (Fowler, V.M., and V. Bennett, 1984, J. Biol. Chem., 259:5978-5989) in an actomyosin contractile apparatus responsible for ATP-dependent changes in erythrocyte shape.     

Partial purification and characterization of post-proline cleaving enzyme: enzymatic inactivation of neurohypophyseal hormones by kidney preparations of various species.

The inactivation of the neurohypophyseal hormones arginine vasopressin and oxytocin, both 14C-labelled in the C-terminal glycine residue, by enzymes present in kidney homogenates of various species has been investigated, and some of the enzymes responsible have been partially purified and characterized. The Leu-Gly peptide bond of oxytocin is generally most effectively cleaved by kidney homogenates, although with certain species enzymic activity hydrolyzing the Pro-Leu bond is significant. Degradation of arginine vasopressin is slower than oxytocin in all species studied, and appears to occur by a different overall mechanism since cleavage of the Pro-Arg bond is more significant than hydrolysis of the Arg-Gly bond. The enzyme releasing glycinamide from oxytocin and the "Post-Proline Cleaving Enzyme", which releases C-terminal dipeptide from oxytocin and arginine vasopressin, were partially purified from lamb kidney by ammonium sulfate fractionation and column chromatography. The two enzymes are shown to be separate entities with different pH profiles. The prolyl peptidase activity released the C-terminal dipeptides from oxytocin and arginine vasopressin at similar rates and was inhibited by p-chloromercuriphenylsulfonic acid, 1,10-phenanthroline, L-1-tosylamido-2-phenylethylchloromethyl ketone, Co2+, Ca2+, and Zn2+, but significantly enhanced by dithiothreitol. The prolyl peptidase preparation cleaves proline-containing peptide substrates at the Pro-X bond. The rate of cleavage is dependent on the nature of residue X and with the conditions used there is no cleavage when X equals Pro; however, cleavage occurs when X is a D isomer: [Mpr1, D-Arg8] vasopressin is inactivated at a rate similar to [Mpr1, Arg8]- and [Mpr1, Lys8] vasopressin, suggesting that the known prolonged biological action of [Mpr1, D-Arg8] vasopressin is not due to resistance to the prolyl peptidase. In all characteristics tested the lamb kidney prolyl peptidase was identical to the post-proline cleaving enzyme isolated earlier from human uterus. In vivo experiments in the cat suggested that both the glycinamide-releasing enzyme and post-proline cleaving enzyme are present and effective in inactivating neurohypophyseal hormones in the intact animal.     

Human beta-adrenergic receptors. Simultaneous purification of beta 1- and beta 2-adrenergic-receptor peptides.

Monoclonal antibodies to insulin secretory granule membranes were obtained following immunization of mice with granule membranes purified from a rat transplantable insulinoma. The specificities of the antibodies were investigated by using binding assays with different insulinoma subcellular fractions, by indirect immunofluorescence studies with intact and permeabilized cells, and by immunoblotting of granule membrane proteins fractionated by SDS/polyacrylamide-gel electrophoresis. Fifty-six antibodies were characterized initially, and 21 representative cell lines were cloned. The antibodies fell into four categories: (1) binding preferentially to secretory granules, and reacting with a component of approx. 80,000 Da on immunoblots (antigen designated SGM 80); (2) binding preferentially to secretory granules, and reacting with components of approx. 110,000 and 50,000 Da on immunoblots (antigen designated SGM 110); (3) binding preferentially to secretory granules but unreactive on immunoblots; (4) binding to membrane antigen(s) with a widespread intracellular distribution which included granules and plasma membranes. The antigens SGM 80 and SGM 110 were studied in more detail and both were shown to be integral membrane glycoproteins with antigenic determinants located on the internal face of the secretory granule membrane. These antigens were also present in normal rat islets of Langerhans and similar components were detected by immunoblotting in secretory granules from anterior pituitary and adrenal medulla. Proteins which were immunologically related to SGM 80 and SGM 110, but distinct in molecular size, were also identified in liver. It is concluded that secretory granules contain specific components which are restricted in subcellular location but widespread in tissue distribution. The antibodies obtained will be valuable reagents in the further investigation of the biogenesis and turnover of insulin secretory granules.     

Effect of vasoactive peptides on prostacyclin formation in cerebromicrovascular cellular elements and glia: a comparative study

The aim of the present study was to determine basal and stimulated release of prostacyclin from the separately cultured endothelial and smooth muscle cells derived from rat brain microvessels and from glial cells.The basal release of PGI₂ (measured as a 6-keto-PGF_1α formation by radioimmunoassay method) was significantly greater in cultured endothelial cells than in cultured smooth muscle or glial cells (254 ± 32, 140.7 ± 17 and 76.8 ± 5.8 pg/mg protein, respectively). Prostacyclin formation stimulated by angiotensin I, angiotensin II and bradykinin was significantly increased in the smooth muscle cells. A significant enhancement of PGI₂ formation was also observed in the glial cells exposed to angiotensin II or bradykinin. Vasoactive peptides did not affect prostacyclin production in the endothelial cells.Presented results indicate that the smooth muscle cells represent the most sensitive site of prostacyclinpeptide interaction. These data also suggest that the endothelial and the glial cells may protect the cerebromicrovascular smooth muscle by inactivating vasoactive peptides derived from either the blood or the brain.     

Human placental steroid sulfatase: purification and properties

Steroid sulfatase is recovered quantitatively from the 105,000 g h supernatant of human placental microsomes extracted with Triton X-100. The solubilized enzyme has been purified using conventional techniques. Throughout the purification procedure, steroid sulfatase appears to be heterogeneous as evidenced by certain, but not all, criteria. Following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the final preparation exhibits a major component and varying amounts of two minor ones. Antibodies raised in rabbits with the heterogeneous immunogen give rise to a single precipitation line when the native enzyme is analyzed by double immunodiffusion or by immunoelectrophoresis. In addition, using aged preparations of microsomes and immunoaffinity techniques, steroid sulfatase activity was found to be associated with the fastest migrating minor component. This finding would suggest that the apparent heterogeneity of purified steroid sulfatase is linked to degradation processes occurring within the microsomal preparations. Steroid sulfatase has a Stokes radius of 56 A, a sedimentation coefficient of 4.85 +/- 0.15S (in Triton-containing buffers) and binds 1.3 g of Triton X-100-per g of protein. The molecular weight of the Triton-protein complex was calculated to be 166,000 in which the glycoprotein portion contribution is about 43% (72,000). In contrast, the apparent molecular weight of the major polypeptide determined on calibrated SDS-gels is 62,000. The purified enzyme exhibits two pH optima with cholesterol sulfate as substrate, an acidic one at pH 5.0 and a second one at pH 7.5. The Km values for cholesterol sulfate, dehydroandrosterone sulfate and p-nitrophenylsulfate were 5.26, 14 and 1,320 microM, respectively.