The acute-phase response in (NZB X NZW)F1 and MRL/l MICE.

The acute-phase plasma protein response to disease activity in murine models of autoimmune lupus-like disease was investigated by measurement of the concentration of serum amyloid P component (SAP) in NZB X W and MRL/l mice. The levels of SAP, which is a major acute-phase protein in mice, did not rise at all in response to progression of disease in NZB X W mice between the ages of 1 and 9 mo. This resembles the behavior of acute-phase proteins such as C-reactive protein and serum amyloid A protein in human systemic lupus erythematosus, and just as in human lupus, where the occurrence of intercurrent microbial infection can stimulate an acute-phase response, so injection of bacterial lipopolysaccharide or casein into the NZB X W mice stimulated "normal" acute-phase SAP production. In marked contrast, MRL/l mice developed greatly increased levels of SAP, which correlated closely with progression of their pathology as they aged. The disease profile of the MRL/l strain includes rheumatoid factors and spontaneous polyarthritis and their SAP response resembles the behavior of acute phase proteins in human rheumatoid arthritis. Different patterns of acute-phase response in different autoimmune disorders may thus be a reflection of the genetic predisposition to particular diseases and/or contribute to their pathogenesis. The existence of animal counterparts for the various clinical patterns of human acute-phase protein production will assist in experimental investigation of the underlying mechanisms and of the biological role of the acute-phase response.

Demonstration of ricin-binding sites on the outer face of azurophil and specific granules of rabbit polymorphonuclear leukocytes.

The presence of carbohydrate residues on the outer surface of PMN granules has been demonstrated by the use of ricin-conjugated ferritin. The binding of the lectin was inhibited by alpha-lactose. No difference in the binding densities of azurophil or specific granules was observed.

Anti-neutrophil elastase defense of the normal human respiratory epithelial surface provided by the secretory leukoprotease inhibitor.

Secretory leukoprotease inhibitor (SLPI), a 12-kD nonglycosylated serine antiprotease with a high capacity for inhibiting neutrophil elastase (NE), is produced by cells of mucosal surfaces including the human lung. The molar concentrations of SLPI in total respiratory tract epithelial lining fluid (ELF) were 56 +/- 10% that of alpha 1-antitrypsin, suggesting SLPI may be more important for the anti-NE protection of the pulmonary epithelial surface than previously thought. However, evaluation demonstrated that SLPI in respiratory ELF was only one-third functional. Studies aerosolizing recombinant SLPI (rSLPI) to sheep demonstrated that in the short term, neither aerosolization and alveolar deposition nor the lavage procedure inactivated the SLPI molecule. In vitro studies with rSLPI demonstrated that exposure to oxidants did not modify the form of the molecule, while exposure to oxidants and NE caused the molecule to be cleaved from 12 to 8 kD. Consistent with this, evaluation of SLPI in lavage fluid of individuals with cystic fibrosis (a condition with oxidants and NE on the respiratory epithelium) showed that the SLPI was degraded. However, evaluation of SLPI in normal ELF by molecular sieve analysis and Western analysis demonstrated an intact 12-kD molecule, suggesting that the partial inactivation of SLPI in normals in vivo is not because it is complexed to NE or exposed to oxidants + NE. Together, these observations demonstrate that SLPI is present in large amounts in respiratory ELF, but since the majority of the SLPI is inactive, it likely does not play a significant role in protecting the normal respiratory epithelium, except perhaps in the upper airways where the levels of SLPI are the highest.

Control of exogenous proteinases and their inhibitors at the macrophage cell surface.

The actions and availability of human neutrophil elastase and its protein inhibitor, Eglin, when co-incubated with macrophages were investigated. Eglin did not induce radical production by mouse peritoneal macrophages; nor were specific binding sites for Eglin detected on these cells. Mouse peritoneal macrophages could inactivate both elastase and Eglin extensively, when these targets were used at concentrations appropriate to the extravascular fluids. Two methods were used for assessing such inactivation: one, as in previous literature, only took account of molecules remaining in the supernatant after interaction with the cells; the other (lacking from most previous studies) took into account all target molecules, including those associated with the cells. From an analysis of both types of experiment, it was shown that the cell-derived inactivators were stable products, whose quantity was not significantly influenced by the induction of a macrophage oxidative burst and its associated free radicals. They were probably mainly proteinases and proteinase inhibitors. Thus, mouse peritoneal macrophages restrict the activity of proteinases and inhibitors by means of stable molecules, such as proteins. Other mononuclear phagocytes may use free radicals and oxidants more extensively in this respect.

Isolation and characterization of an enkephalin-degrading aminopeptidase from rat brain.

An enkephalin-degrading aminopeptidase from rat brain extracts has been purified to apparent homogeneity. This enzyme cleaves the N-terminal tyrosine from Leu-enkephalin and hydrolyzes some beta-naphthylamides and p-nitro-anilides of neutral, basic and aromatic, but not acidic, amino acids. The enzyme requires a free amino group on the substrate and has a neutral pH optimum. After dialysis against EDTA, the enzyme requires a divalent cation (Zn2+, Co2+ greater than Mn2% greater than Mg2+) for activity. The enzyme is inhibited by puromycin, o-phenanthroline, p-chloromercuribenzoate and EDTA, but not by puromycin, methylsulfonyl fluoride or a specific peptide inhibitor of leucine amino-peptidase. The aminopeptidase consists of two subunits and has a molecular weight of about 100 000.

Selective oxidation of Met-192 in bovine alpha-chymotrypsin. Effect on catalytic and inhibitor binding properties.

Catalytic and inhibitor binding properties of bovine alpha-chymotrypsin, in which the Met-192 residue has been converted by treatment with chloramine T to the sulfoxide derivative (Met(O)192 alpha-chymotrypsin), have been examined relative to the native enzyme (alpha-chymotrypsin), between pH 4.5 and 8.0 (mu = 0.1), and/or 5.0 degrees C and 40.0 degrees C. Values of kcat, k+2 and/or k+3 for the hydrolysis of all the substrates examined (i.e., tMetAcONp, ZAlaONp, ZLeuONp, ZLysONp and ZTyrONp) catalyzed by native and Met(O)192 alpha-chymotrypsin are similar, as well as values of Km for the hydrolysis of ZLeuONp, ZLysONp and ZTyrONp. On the other hand, Ks and Km values for the hydrolysis of ZAlaONp and tMetAcONp are decreased by about 5-fold. Met-192 oxidation does not affect the kinetic and thermodynamic parameters for the (de)stabilization of the complex formed between the proteinase and the bovine basic pancreatic trypsin inhibitor. On the other hand, the recognition process between between alpha-chymotrypsin and the recombinant proteinase inhibitor eglin c from the leech Hirudo medicinalis is influenced by the oxidation event. Considering known molecular models, the observed catalytic and inhibitor binding properties of native and Met(O)192 alpha-chymotrypsin were related to the inferred stereochemistry of the proteinase-substrate and proteinase-inhibitor contact region(s).

Preliminary crystallographic data on the complex of bovine alpha-chymotrypsin with the recombinant proteinase inhibitor eglin c from Hirudo medicinalis.

The molecular complex built by bovine alpha-chymotrypsin and the recombinant proteinase inhibitor eglin c from Hirudo medicinalis has been crystallized from polyethylene glycol solutions, using a twofold molar excess of the inhibitor with respect to the serine proteinase. The optimum pH for crystal growth is 6.5. The crystals belong to the monoclinic space group P2(1), with unit cell constant: a = 55.3 A, b = 59.4 A, c = 42.5 A, beta = 99.0 degrees; one complex moiety is present per asymmetric unit. The crystals diffract to 2.0 A resolution and are suitable for detailed X-ray crystallographic investigations.

Crystal and molecular structure of the bovine alpha-chymotrypsin-eglin c complex at 2.0 A resolution.

The crystal structure of the complex between bovine alpha-chymotrypsin and the leech (Hirudo medicinalis) protein proteinase inhibitor eglin c has been refined at 2.0 A resolution to a crystallographic R-factor of 0.167. The structure of the complex includes 2290 protein and 143 solvent atoms. Eglin c is bound to the cognate enzyme through interactions involving 11 residues of the inhibitor (sites P5-P4' in the reactive site loop, P10' and P23') and 17 residues from chymotrypsin. Binding of eglin c to the enzyme causes a contained hinge-bending movement around residues P4 and P4' of the inhibitor. The tertiary structure of chymotrypsin is little affected, with the exception of the 10-13 region, where an ordered structure for the polypeptide chain is observed. The overall binding mode is consistent with those found in other serine proteinase-protein-inhibitor complexes, including those from different inhibition families. Contained, but significant differences are observed in the establishment of intramolecular hydrogen bonds and polar interactions stabilizing the structure of the intact inhibitor, if the structure of eglin c in its complex with chymotrypsin is compared with that of other eglin c-serine proteinase complexes.

Crystal and molecular structure of the inhibitor eglin from leeches in complex with subtilisin Carlsberg.

The crystal structure of the molecular complex of eglin, a serine proteinase inhibitor from leeches, with subtilisin Carlsberg has been determined at 2.0 A resolution by the molecular replacement method. The complex has been refined by restrained-parameter least-squares. The present crystallographic R factor (Formula: see text) is 0.183. Eglin is a member of the potato inhibitor 1 family, a group of serine proteinase inhibitors lacking disulfide bonds. Eglin shows strong structural homology to CI-2, a related inhibitor from barley seeds. The structure of subtilisin Carlsberg in this complex is very similar to the known structure from barley seeds. The structure of subtilisin Carlsberg in this complex is very similar to the known structure of subtilisin novo, despite changes of 84 out of 274 amino acids.

Structure of the proteinase inhibitor eglin c with hydrolysed reactive centre at 2.0 A resolution.

The inhibition of serine proteinases by both synthetic and natural inhibitors has been widely studied. Eglin c is a small thermostable protein isolated from the leech, Hirudo medicinalis. Eglin c is a potent serine proteinase inhibitor. The three-dimensional structure of native eglin and of its complexes with a number of proteinases are known. We here describe the crystal structure of hydrolysed eglin not bound to a proteinase. The body of the eglin has a conformation remarkably similar to that in the known complexes with proteinases. However, the peptide chain has been cut at the 'scissile' bond between residues 45 and 46, presumed to result from the presence of subtilisin DY in the crystallisation sample. The residues usually making up the inhibiting loop of eglin take up a quite different conformation in the nicked inhibitor leading to stabilising contacts between neighbouring molecules in the crystal. The structure was solved by molecular replacement techniques and refined to a final R-factor of 14.5%.

Aerosolization of recombinant SLPI to augment antineutrophil elastase protection of pulmonary epithelium.

In a variety of lung diseases the respiratory epithelial surface must contend with an increased burden of neutrophil elastase (NE). One candidate for augmenting epithelial anti-NE protection is the secretory leukoprotease inhibitor (SLPI). In vitro evaluation demonstrated that 96 +/- 1% of the recombinant SLPI (rSLPI) molecules were capable of inhibiting NE, with an association rate constant of 7.1 +/- 0.1 X 10(6) M-1.s-1. Evaluation of rSLPI after in vitro and in vivo aerosolization showed that aerosolization did not alter rSLPI. Aerosolization of a single dose of 50 mg rSLPI to sheep resulted in a fourfold increase of the anti-NE capacity in epithelial lining fluid (ELF) at 3 h, with a half-life in ELF of 12 h. After aerosolization some rSLPI appeared in lung lymph. Simultaneous aerosolization of rSLPI and recombinant alpha 1-antitrypsin (rAAT) demonstrated a molar ratio of the concentration in lymph to the concentration in ELF 3 h after the aerosol eightfold higher for rAAT than for rSLPI. Overall, these observations demonstrate that it is feasible to use aerosolized rSLPI to directly augment the anti-NE capacity of the lung, particularly on the pulmonary epithelial surface.

Comparison of specificity of human and horse leucocyte proteinases with synthetic peptide substrates.

Highly purified horse leucocyte proteinases 1, 2A and 2B hydrolyze synthetic substrates which are decomposed also by human leucocyte elastase but they are unable to hydrolyze typical substrates of cathepsin G. Thus in distinction to other mammalian species horse leucocytes are devoid of cathepsin G and contain only elastases.

Adjuvant-induced inflammatory disease in the rat: plasma levels of peptide hydrolases and protease inhibitors reflect disease activity.

Both during the primary (localized) inflammation and the development of the secondary (generalized) inflammation in adjuvant-treated rats, the plasma level of functional alpha-macroglobulins increases while proteases (measured as peptide hydrolases) sharply decrease. The decreased peptide hydrolase levels during episodes when protease 'spillage' into the bloodstream is elevated, suggests a more rapid clearance of alpha-macroglobulin-protease complexes associated with inflammation.