Interleukin 1 production by human polymorphonuclear neutrophils

The purpose of this study was to determine whether human polymorphonuclear neutrophils (PMN), which share a common cell lineage with macrophages, could produce factors such as IL 1. Other properties which these two cell types share are their phagocytic nature and the common receptor and antigens on their cell surfaces. IL 1, in many of its physical, biochemical, and functional characteristics, is found to resemble endogenous pyrogen (EP). PMN have been cited as a possible cell source of EP, but there have also been reports in which the capacity of PMN to produce EP has been questioned. This study shows that normal human PMN can be stimulated by particulate agents such as zymosan and soluble agents such as phorbol myristic acetate to produce a factor(s) which induces proliferation of mouse thymocytes, i.e., PMN IL 1. This PMN IL 1 was released from PMN in a dose- and time-dependent fashion. PMN IL 1 was nondialyzable, was heat-labile, and was inactivated at pH below 5 and above 8. PMN IL 1 stimulated the proliferation of normal human synovial fibroblasts and caused release of a neutral protease (plasminogen activator) from synovial cells. The synovial and thymocyte-proliferating capacity of PMN IL 1 was not affected by the protease inhibitor aprotinin or by soybean trypsin inhibitor. Gel filtration studies estimate the m.w. of PMN IL 1 to be approximately 13,000 to 17,000.     

Detection and preliminary characterization of Taenia solium metacestode proteases.

The metacestode of Taenia solium persists for years in the human central nervous system. As proteolytic enzymes play an important role in the survival of tissues helminths, we examined extracts of T. solium metacestodes for proteolytic activity using 9 synthetic peptide substrates and 3 proteins (hemoglobin, albumin, and immunoglobulin G). The proteolytic enzymes were classified based on their inhibitor profiles. At neutral pH, aminopeptidase(arginine-7-amino-4-trifluoromethylcoumarin) and endopeptidase(benzyloxy-carbonyl-glycine-glycine-arginine-7-amino-4- trifluoromethylcoumarin) substrates were cleaved. Hydrolysis of both substrates was inhibited by chelating agents, which inhibit metalloproteases. Peak activity with both substrates eluted in gel filtration fractions corresponding to a molecular weight of about 104 kDa. Cysteine protease activity was identified, which cleaved benzyloxy-carbonyl-phenylalanine-arginine-7-amino- 4-trifluoromethylcoumarin (Z-Phe-Arg-AFC) and hemoglobin. Cleavage of Z-Phe-Arg-AFC was maximal at acid pH, was stimulated by thiols, and was inhibited by leupeptin and Ep459. Peak cysteine protease activity eluted in gel filtration fractions corresponding to a molecular weight of 32 kDa. Aspartic protease activity was identified by specific inhibition with pepstatin of acid digestion of hemoglobin and immunoglobulin G. Immunoglobulin digestion occurred at acid pH, with preferential degradation of the heavy chain. Upon gel filtration chromatography, the aspartic protease activity eluted as a broad peak with maximal activity at about 90 kDa. No serine protease activity was detected. None of the parasite enzymes digested albumin. Proteolytic enzymes of T. solium may be important for parasite survival in the intermediate host, by providing nutrients and digesting host immune molecules.     

Normal human neutrophils are a source of a specific interleukin 1 inhibitor

In the course of our study on neutrophil production of an interleukin 1 (IL-1)-like factor, we found that the addition of polymorphonuclear neutrophils (PMN) to monocytes cultured in the presence of zymosan resulted in decreased IL 1 activity of the resultant supernatant, suggesting that PMN may contain an inhibitor of IL 1. The objective of this investigation was to study this IL 1 inhibitor which normal human PMN contain. The inhibitor is constitutively present in the PMN because 0 hr PMN lysates and unstimulated PMN supernatants also show inhibitory activity. The PMN inhibitor inhibits IL 1 (crude and partially purified) in a dose-response manner and does not affect basal [3H]thymidine incorporation in the presence or absence of PHA-P. The PMN inhibitor does not have any effect on interleukin 2 (IL 2)-induced proliferation of the IL 2-dependent CTLL cells. The inhibitor can be generated in the absence of serum and is not produced as a result of proteolytic activity from PMN enzymes. The inhibitor is heat-labile and is most stable at neutral pH. Gel filtration studies on Sephadex G-200 indicate that the inhibitor is heterogeneous in size. Two inhibitory peaks, at 45,000 to 70,000 m.w. and at greater than 160,000 m.w., were observed. When zymosan-stimulated PMN supernatant was chromatographed, there was separation of inhibitory factor from a 17,000 m.w. proliferating factor. Presence of this PMN inhibitor may be important in negative regulation of IL 1.     

Priming activity for chemiluminescence reaction of PMN in the culture supernatant of streptococcal preparation (OK-432)-stimulated spleen cells

We investigated the effect of supernatant from human spleen cell culture stimulated with a streptococcal preparation, OK-432 (OK sup), on the luminol-dependent chemiluminescence (CL) of human polymorphonuclear leukocytes (PMN). The fMLP-stimulated CL of PMN was markedly enhanced by the pretreatment with OK sup. This result indicates that OK sup contained the factor(s) that primes fMLP-stimulated CL of PMN. The priming factor(s) in OK sup was partially inactivated by the treatment at 56 C for 30 min and pH 2 or pH 10 treatments. Since the enhancing effect of OK sup on the CL was inhibited by the treatment of sodium azide and the addition of catalase or taurine, it was assumed that OK sup augments the activity of MPO-H2O2-HOCl system of fMLP-stimulated PMN.     

Acid-activated insulin-like growth factor binding protein protease activity of Cathepsin D in normal and malignant prostatic epithelial cells and seminal plasma

In this study, we demonstrate insulin-like growth factor binding protein (IGFBP) acid proteolysis in conditioned media (CM) from normal and malignant primary cultures of prostatic epithelial cells, prostatic cell lines, and in seminal plasma. We further demonstrate the absence of such activity in CM from prostatic stromal cells. Radio-labeled IGFBPs (1–6) were incubated with various acidified CM and seminal plasma. None of these media showed IGFBP proteolytic activity at neutral pH, but all CM from prostatic epithelial cells (PC-E) demonstrated strong IGFBP proteolysis at acidic pH. No acid-activated proteolysis was observed in the CM from stromal cell cultures. In order to ascertain the role of cathepsin D, anti-cathepsin antibodies were used to immunodeplete the media of the selected enzymes prior to incubation with IGFBPs. Depletion of cathepsin D greatly reduced the proteolytic activity of the PC-E CM. Additionally, purified cathepsin D yielded a digestion pattern identical to that produced by prostatic cell CM and seminal plasma, following acidic incubation with IGFBP-3. Remarkably, the proteolytic pattern generated by seminal plasma, when incubated with IGFBP-3 at neutral pH, corresponded to that produced by prostate-specific antigen (PSA), demonstrating the interpolation of both neutral and acid proteases from prostate cells into seminal plasma. In conclusion, prostatic epithelial cells secrete acid-specific IGFBP protease(s) related to cathepsin D. Although no significant statistical difference was observed in the degree of acid-specific proteolysis in the media from normal versus malignant primary epithelial cell cultures, physiologicalcharacteristics of the malignant state might facilitate increased cathepsin D activity. We suspect this proteolysis may play a role in prostatic cell proliferationand invasive tumor growth. J. Cell. Physiol. 171:196–204, 1997. © 1997 Wiley-Liss, Inc.     

A stopped-flow calorimeter for biochemical applications.

In human skin fibroblast cultures a fraction of the procollagen that was processed to collagen and remained in the cell layer was further proteolytically modified by removal of both N- and C-terminal telopeptides. The proteolytic activity was associated with the cell layer, since secreted collagens were found always to contain intact telopeptides. The inclusion of neutral polymers, which caused the accumulation of the collagen in the cell layer [Bateman, Cole, Pillow & Ramshaw (1986) J. Biol. Chem. 261, 4198-4203], made the telopeptide cleavage more apparent in those cells which expressed the proteolytic activity. The extent of this cleavage was variable from cell culture to cell culture and between experiments with the same fibroblast line. The proteolytic activity was pH-dependent; cleavage was greatest at a culture-medium pH of 7.5 and 8.0 and was completely inhibited at a culture-medium pH of 7.0 and 6.5. The activity was significantly inhibited by soybean trypsin inhibitor, an elastase-specific inhibitor (N-acetylalanylalanylprolylvalylchloromethane) and the thrombin inhibitor hirudin. This cell-associated proteolytic activity may play a role in collagen degradation by removing the telopeptides, which are the primary sites of collagen cross-linking, thus destabilizing the collagen matrix sufficiently to render it susceptible to further proteolytic breakdown.     

Cloning of the nprA gene for neutral protease A of Bacillus thuringiensis and effect of in vivo deletion of nprA on insecticidal crystal protein.

The nprA gene, encoding Bacillus thuringiensis neutral protease A, was cloned by the use of gene-specific oligonucleotides. The size of neutral protease A deduced from the nprA sequence was 566 amino acids (60,982 Da). The cloned nprA gene was partially deleted in vitro, and the deleted allele, designated nprA3, was used to construct an nprA3 strain (neutral protease A-deficient strain) of B. thuringiensis. Growth and sporulation of the nprA3 strain were similar to those of an isogenic nprA+ strain, although the extracellular proteolytic activity of the nprA3 strain was significantly less than that of the nprA+ strain. The nprA3 strain produced insecticidal crystal proteins that were more stable than those of the isogenic nprA+ strain after solubilization in vitro, and sporulated cultures of the nprA3 strain contained higher concentrations of full-length insecticidal crystal proteins than did those of its isogenic counterpart. The absence of neutral protease A did not affect the insecticidal activity of a lepidopteran-specific crystal protein of B. thuringiensis. These results indicate that crystal protein stability and yield may be improved by deletion of specific proteases from B. thuringiensis.     

Purification of several proteolytic enzymes by tosyl- and carbobenzoxy-triethylene-tetramine-sepharoses.

Tosyl-triethylenetetramine-Sepharose (Tos-T-Sepharose) and carbenzoxytriethylenetetramine-Sepharose (Z-T-Sepharose) were found to be adsorbents utilizable in the purification of several microbial and animal proteases. The former Sepharose derivative adsorbed alpha-chymotrypsin, trypsin, subtilisin, thermolysin and neutral subtilopeptidase at neutral pH range, and acid proteases such as pepsin and Rhizopus niveus protease at pH 3.5-6.5. alpha-Chymotrypsin and trypsin were eluted with 0.1 N acetic acid and Rhizopus protease with 0.5 N acetic acid, thermolysin with 1 M guanidine-HCl or 33% ethyleneglycol, whilst pepsin was recovered by elution with 2 M guanidine-HCl at pH 3.5. The binding of neutral subtilopeptidase and subtilisin to this adsorbent was comparatively weak and both the enzymes were recovered by elution with 0.5 M NaCl at neutral pH. On the other hand, Z-T-Sepharose was found to bind tightly to these proteolytic enzymes except neutral subtilopeptidase. Trypsin and alpha-chymotrypsin were released from the adsorbent column with 1 M p-toluenesulfonate, and subtilisin with 1 M guanidine-HCl or 33% ethyleneglycol at neutral pH region. By these chromatographic procedures, the specific activities of these proteolytic enzymes increased effectively. Comparison of the binding abilities of acetyl-, benzoyl-, tosyl- and carbobenzoxy-T-Sepharoses to these enzymes suggests that hydrophobicity of tosyl and carbobenzoxy groups plays an important role in the enzyme-adsorbent interaction.     

The B12 anti-tryptase monoclonal antibody disrupts the tetrameric structure of heparin-stabilized beta-tryptase to form monomers that are inactive at neutral pH and active at acidic pH

The novel tetrameric structure of human beta-tryptase faces each active site into the central pore, thereby restricting access of most biologic protease inhibitors. The mechanism by which the anti-tryptase mAb B12 inhibits human beta-tryptase peptidase and proteolytic activities at neutral pH, but augments proteolytic activity at acidic pH, was examined. At neutral pH, B12-beta-tryptase complexes are inactive. At acidic pH, B12 (intact and Fab) minimally affects peptidase activity when added to beta-tryptase tetramers, but does induce susceptibility to inhibition by soybean trypsin inhibitor and antithrombin III. Surprisingly, B12 Fab-beta-tryptase complexes formed at both neutral and acidic pH exhibit the apparent molecular mass of a complex with 1 beta-tryptase monomer and 1 Fab by gel filtration. B12 does not compete with heparin for binding to tryptase at either neutral or acidic pH. Thus, B12 directly disrupts beta-tryptase tetramers to monomers that are inactive at neutral pH, whereas at acidic pH, are active and more accessible to protein inhibitors and substrates.     

Enzyme inhibitors from plants. Isolation and characterization of a protease inhibitor from arrow root (Maranta arundinaceae) tuber

A protease inhibitor from arrow root (Maranta arundinaceae) tuber has been isolated in a homogeneous form. The inhibitor has a Mr of 11,000-12,000; it inhibited bovine trypsin, bovine enterokinase, bovine α-chymotrypsin and the proteolytic activity of human and bovine pancreatic preparations. The inhibitor is resistant to pepsin, and elastase. It could withstand heat treatment at 100°C for 60 min and exposure to a wide range of pH (1.0–12.5) for 72 h at 4°C without loss of activity. Arginyl groups are essential for the action of the inhibitor. Preincubation of the inhibitor at pH 3.7 with trypsin or chymotrypsin caused nearly a two-fold increase in inhibitor potency     

Recombinant cathepsin E has no proteolytic activity at neutral pH

Cathepsin E (CatE) is a major intracellular aspartic protease reported to be involved in cellular protein degradation and several pathological processes. Distinct cleavage specificities of CatE at neutral and acidic pH have been reported previously in studies using CatE purified from human gastric mucosa. Here, in contrast, we have analyzed the proteolytic activity of recombinant CatE at acidic and neutral pH using two separate approaches, RP-HPLC and FRET-based proteinase assays. Our data clearly indicate that recombinant CatE does not possess any proteolytic activity at all at neutral pH and was unable to cleave the peptides glucagon, neurotensin, and dynorphin A that were previously reported to be cleaved by CatE at neutral pH. Even in the presence of ATP, which is known to stabilize CatE, no proteolytic activity was observed. These discrepant results might be due to some contaminating factor present in the enzyme preparations used in previous studies or may reflect differences between recombinant CatE and the native enzyme.     

An alkaline protease from Bacillus circulans BM15, newly isolated from a mangrove station: characterization and application in laundry detergent formulations

An investigation on the properties of an alkaline protease secreted by Bacillus circulans BM15 strain isolated from a mangrove sediment sample was carried out in order to characterize the enzyme and to test its potency as a detergent additive. The protease was purified to apparent homogeneity by ammonium sulphate precipitation and was a 30-kDa protease as shown by SDS-PAGE and its proteolytic activity was detected by casein zymography. It had optimum activity at pH 7, was stable at alkaline pH range (7 to 11), had optimum temperature of activity 40°C and was stable up to a temperature of 55°C after incubation for one hour. Hg²⁺, Zn²⁺, Co²⁺, and Cu²⁺completely inhibited the enzyme activity, while Ca²⁺, Mg²⁺, K⁺ and Fe³⁺ were enhancing the same. The serine protease inhibitor PMSF and metal chelator EDTA inhibited the activity of this protease while the classic metalloprotease inhibitor 1, 10 phenanthroline did not show inhibition. The enzyme was stable in SDS, Triton-X-100 and H₂ O₂ as well as in various commercial detergents after incubation for one hour. The extracellular production of the enzyme, the pH and temperature stability and stability in presence of oxidants, surfactants and commercial detergents suggest its possible use as a detergent additive.     

Two-step prorenin-renin conversion. Isolation of an intermediary form of activated prorenin

Prorenin is an inactive form of the aspartic protease renin. Like pepsinogen, it is activated at low pH. The kinetics of acid activation of prorenin were studied in human amniotic fluid and plasma and in preparations of purified prorenin isolated from amniotic fluid and plasma. Conversion of prorenin (pR) into active renin (R) appeared to be a two-step process involving the generation of an intermediary form of activated prorenin (pRa). The pR----pRa step is an acid-induced reversible change in the conformation of the molecule, and the pRa----R step is proteolytic. pRa----R conversion occurred in amniotic fluid at low pH by the action of an endogenous aspartic protease. In plasma pRa----R conversion occurs after restoration of pH to neutral and is caused by the serine protease plasma kallikrein. pRa----R conversion did not occur in purified preparations of prorenin. Thus, in contrast to pepsinogen, the acid-induced reversible conformational change is not followed by autocatalysis. pRa of amniotic fluid and plasma could be separated from R by affinity chromatography on Cibacron blue F3GA-agarose, and R but not pRa was detected by an immunoassay using monoclonal antibodies reacting with R and not with pR. The first-order rate constant for pR----pRa conversion depends on the protonation of a polar group (or groups) with pK approximately 3.4, the rate constant being proportional to the fraction of pR molecules that have this group protonated. This is analogous to the reversible acid-induced conformational change of pepsinogen that occurs before its proteolytic conversion into pepsin. kcat/Km for pRa----R conversion by plasmin and plasma kallikrein at pH 7.4 and 37 degrees C was 7.8 X 10(6) and 5.2 X 10(6) M-1 min-1, respectively, which was about 50-70 times greater than for pR----R conversion. The susceptibility of pRa to proteolytic attack is high enough for the intrinsic factor XII-kallikrein pathway to cause rapid pRa----R conversion at 37 degrees C even in whole blood with its abundance of serine protease inhibitors. Formation of pRa may occur in vivo in an acidic cellular compartment, such as exo- or endocytotic vesicles.     

Cytoplasmic origin of the so-called nuclear neutral histone protease.

1. We have investigated the origin of proteolytic activity which causes degradation of histones in chromatin isolated from Xenopus liver and the rat liver at neutral pH. Polyacrylamide disc gel electrophoresis was used for detection of proteolytic products of histones. 2. No proteolytic degradation of histones occurs in chromatin isolated from Xenopus erythrocytes and rat liver according to our procedure even after prolonged incubation at pH 8.0 and pH 5.0. However with chromatin isolated from Xenopus liver a high level of histone degradation is observed under similar conditions. 3. Mixing isolated nuclei from Xenopus erythrocytes with a crude cytoplasmic fraction from Xenopus liver causes histone proteolysis in isolated chromatin at pH 8.0. In similar experiments with corresponding fractions from rat liver histone proteolysis can be introduced only after repeated freezing and thawing of the cytoplasmic fraction. 4. A purified lysosomal preparation from rat liver causes a similar type of histone degradation upon incubation with chromatin from Xenopus erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal fraction from rat liver is inhibited by sodium bisulphite. 6. We conclude that the neutral proteolytic activity which causes degradation of histones in isolated chromatin is due to a contamination with neutral protease(s) originating from cytoplasmic organelles.     

Serum calcium-decreasing factor (caldecrin) from porcine pancreas has proteolytic activity which has no clear connection with the calcium decrease.

We purified a serum calcium-decreasing factor, which showed chymotrypsin-like protease activity, from porcine pancreas to homogeneity. The factor administered to mice intravenously at a dose of 20 micrograms/kg b.w. decreased serum calcium by 15%. Treatment of the factor with the serine protease inhibitor, PMSF, caused a leftward shift in the dose-response curve, showing strengthened activity. It also caused a decrease in serum calcium and hydroxyproline levels in rats. At a dose of 10 ng/ml, the factor inhibited 45Ca release from cultured fetal long bone stimulated by parathyroid hormone (PTH) and PTH-related protein, but not by interleukin-1 alpha, prostaglandin E1 and 1,25-dihydroxy vitamin D3. No other well-known pancreatic proteases had these effects. In view of the results of experiments using protease inhibitor and pancreatic proteases, and in view of the specificity of this factor in vitro, we propose that the factor exerts its serum calcium-decreasing activity most probably not through proteolytic degradation of PTH, but through an inhibition of PTH action on bones by a yet undefined mechanism.     

The enzyme that cleaves apolipoprotein A-II upon in vitro incubation of human plasma high-density lipoprotein-3 with blood polymorphonuclear cells is an elastase

The proteolytic activity directed against apolipoprotein A-II (apo-A-II) which is released from human blood polymorphonuclear cells (PMN) when they are incubated with human plasma high-density lipoprotein-3 (HDL3) was studied to assess the properties and site specificity of the enzyme. When 125I-apo-A-II-labeled HDL3 was incubated with the PMN protease at 37 degrees C, a complete cleavage of apo-A-II was observed which paralleled the formation of bands of approximately 11,000 and 7,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 7,000-dalton component had the following N-terminal sequence: NH2-Thr-Asp-Tyr-Gly-Lys-Asp-Leu-Met-Glu-Lys. This corresponds to residues 19 through 28 of the intact apo-A-II monomer. Methoxysuccinyl (MeO-Suc)-Ala-Ala-Pro-Val-chloromethylketone-(CH2Cl) caused a 90% inhibition of apo-A-II hydrolysis at the highest concentration tested (6 X 10(-4)M). Besides apo-A-II, the PMN enzyme also hydrolyzed a synthetic substrate, MeO-Suc-Ala-Ala-Pro-Val-4-nitroanilide and its 4-methylcoumaryl-7-amide analogue. The protease appeared to have a mass of 28,000 daltons as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]diisopropylfluorophosphate-labeled PMN enzyme. That the PMN enzyme which cleaves apo-A-II is an elastase was derived from the following criteria: 1) cleavage at the Val-X bond in apo-A-II and in the two synthetic substrates studied; 2) prevention of the cleavage by MeO-Suc-Ala-Ala-Pro-Val-CH2Cl, a known specific elastase inhibitor; and 3) a mass comparable to that reported for a pure PMN elastase. These studies establish that apolipoproteins can be suitable substrates for enzymes of the elastase family.     

Fibronectin-degrading proteases from the membranes of transformed cells

The local degradation of fibronectin substrata by Rous sarcoma virus-transformed chick embryonic fibroblasts requires cell-contact-related metalloendoprotease and serine-protease activities. Using fibronectin-containing SDS gels, two large proteases with apparent molecular weights of 120K and 150K were found only in the membrane fraction of transformed cells and were absent in normal cells. Both 120K and 150K proteases were active at neutral pH, but showed preferential inhibitor sensitivities of serine and metal proteases, respectively. The 150K protease appeared to account for most of the proteolytic activity since metalloendoprotease inhibitors completely blocked proteolytic activity of the 150K in fibronectin gels, more than 80% of the fibronectin-degrading activity of solubilized membranes, and largely suppressed the appearance of fibronectin degradation spots in cultures of transformed cells.     

Protease production by the ontjom fungus,Neurospora sitophila

Summary The production of protease and mycelium byNeurospora sitophila cultured on solid and liquid potato dextrose media was studied. Maximal activity of protease extracted from 4-day-old cultures occurred at pH 6.5 when an unfractionated peanut (groundnut) protein substrate was used. The greatest protease activity and mycelium production occurred during the first day of the 4-day test period. Potato dextrose media containing more than 0.2 M NaCl resulted in decreased protease and mycelium production, while tapioca starch was without affect at concentrations up to 1.4%. Addition of up to 0.3 M sucrose to growth media greatly stimulated protease production and mycelial growth. Maximal proteolytic activity was observed in extracts from mycelium cultured in potato dextrose media adjusted from pH 6.0 to 7.5. Activity was greater when soluble peanut protein was used as a substrate, compared to unfractionated or globulin protein substrates.     

Neutral metal chelator-sensitive protease in insect moulting fluid

Proteolytic activity in moulting fluid from the sphingid Manduca sexta has at least two pH optima; these occur at pH 7 and at pH 7·7. The latter activity is shown to be trypsin-like in that it is susceptible to inhibition by diisopropylfluorophosphate. By contrast, the peak at neutral pH consists of proteolytic activity not hitherto described in invertebrates. This activity shows little or no inhibition with diisopropylfluorophosphate or p-hydroxymercuribenzoate but is strongly inhibited by chelators such as 1,10-phenanthroline, 8-hydroxyquinoline, and EDTA. The neutral metal chelator-sensitive activity requires calcium but the inhibitor data permit the conclusion that the metal ion inhibited by the chelators belongs to the first transition series and thus cannot be calcium. The neutral protease appears to be similar to proteases previously characterized from bacteria and snake venom. In moulting fluid from Manduca, proteolytic activity in vitro is very low in the presence of 1,10-phenanthroline at every pH studied except pH 7·7; in vivo, ecdysis is inhibited in Manduca larvae fed on diet containing a sufficient level (0·02 per cent or higher) of 1,10-phenanthroline. The metal chelator-sensitive proteolytic activity appears to be an essential moulting protease in Manduca.     

Neutral proteinase inhibitors in PMN leukocytes. II. Isolation and characterization of a neutral proteinase inhibitor from porcine PMN leukocytes

An inhibitor of neutral proteinases was purified from porcine PMN leukocytes by gel filtration on Sephadex G-75 superfine and ion-exchange chromatography on Mono S. Thus an inhibitor preparation with a specific inhibitory activity against chymotrypsin of 10 IU/mg was obtained. In dodecyl sulfate gel electrophoresis a single protein band with an apparent molecular mass of 40 kDa was found under reducing conditions. Under non-reducing conditions the inhibitor forms higher molecular mass aggregates. On isoelectric focusing several protein bands with isoelectric points between pH 7.0 and 7.5 could be separated. The amino-acid composition of the inhibitory protein was determined. The inhibition mechanism was studied and association rate constants (kon) were measured and calculated for the reaction with chymotrypsin as well as leukocyte and pancreatic elastase. In Western blot analysis and in enzyme immunoassay studies crossreactivity between antibodies directed against porcine leukocyte neutral proteinase inhibitor and the corresponding inhibitor of bovine PMN leukocytes could be demonstrated.