Wilson B. Knight

Comparison of the Proteoglycanolytic Activities of Human Leukocyte Elastase and Human Cathepsin G in Vitro and in Vivo

In this study, we evaluated the in vitro and in vivo potency of human leukocyte elastase (HLE) and human cathepsin G (HCG) as proteoglycanases. In vitro evaluation was done using bovine nasal septum aggrecan and aggrecan/hyaluronan aggregate as substrates. Enzyme activity was assessed by the ability of the proteinases to abrogate the ability of aggrecan to aggregate with hyaluronan. In vivo activity of the proteinases was tested by injecting purified HLE and HCG intra-articularly into rabbit stifle joints and quantifying the levels of proteoglycan released into synovial fluids. On a molar basis, HCG was at least tenfold more potent than HLE as a proteoglycanase in vitro. Moreover, HCG was twofold more potent as a proteoglycanase in vivo. In contrast, HLE hydrolyzed elastin approximately 22-fold faster than HCG, but was only slightly more rapid than HCG when [3H]-transferrin was used as substrate. These data indicate that HCG is more potent than HLE as a proteoglycanase both in vitro and in vivo. Thus, HCG could be more important in the pathogenesis of rheumatoid arthritis than previously suspected.

PMN elastases: A comparison of the specificity of human isozymes and the enzyme from other species toward substrates and inhibitors

The human elastases isolated from polymorphonuclear neutrophils (PMN) and purulent sputum displayed identical kinetic constants toward substrates and inhibitors. The elastases from the two sources yield identical N-terminal sequences and were recognized by antiserum prepared against human sputum elastase (HSE) isozyme-4 (I-4). The data support the proposal put forth by Twumasi and Liener (1977, J. Biol. Chem. 252, 1917-1926) that the human elastase from sputum is of PMN origin. PMN elastases from other species displayed kinetic constants toward both substrates and inhibitors significantly different from the human enzyme. Therefore, extrapolation of inhibitor profiles from these elastases to the human source should be avoided. Four groups of isozymes were resolved from HSE by FPLC. Only the most basic isozyme (I-4) was obtained as a single species. The isozymes displayed identical macroscopic kinetic constants toward several substrates and two classes of inhibitors. The similar partition ratios observed with a cephalosporin-derived inhibitor suggest that the microscopic rate constants are also identical. The data support the proposal suggested by Baugh and Travis (1976, Biochemistry 15, 836-841) that HLE isozymes differ only in carbohydrate content. Whatever the source of human PMN elastase heterogeneity, it does not result in heterogeneous catalytic properties. In addition, a new protein was identified in elastase preparations derived from human sputum. This protein displayed homology to serine proteases and properties suggesting that it is identical to azurocidin.

Inhibition of human leukocyte elastase by C-2 substituted cephalosporin sulfones

Abstract Cephalosporin sulfones with a number of substituents at the C-2 position were prepared and tested as inhibitors of human leukocyte elastase (HLE), an enzyme implicated in the tissue destruction associated with pulmonary emphysema. Nearly all substituents gave a substantial increase in activity against the enzyme over the unsubstituted parent. The enzyme can accommodate a number of functional groups at this position, but is not very discriminating. Both α- and β-methyl compounds have comparable activity, as do α-phenylthiomethyl and α-methoxy. Substitution at this position has led to the preparation of several compounds with exceptional potency against HLE.

SYNTHESIS OF NEW THIAZINOINDOLE DERIVATIVES AND THEIR EVALUATION AS INHIBITORS OF HUMAN LEUKOCYTE ELASTASE AND OTHER RELATED SERINE PROTEASES

A novel thiazinoindole tricyclic ring system was designed as potential inhibitors of serine proteases. The compounds were synthesized by ring closure at 80–90°C in poliphosphoric acid of the appropriate N′-alkyl or aryl substituted indolylthiourea derivatives. Members of this class of compounds inhibited human leukocyte elastase (Ki=30–40 μM) and α-chymotrypsin.

Phosphorous acid analogs of L-680,833, a potent monocyclic β-lactam inhibitor of human leukocyte elastase

Abstract Analogs of the monocyclic β-lactam human leukocyte elastase (HLE) inhibitor L-680,833 in which the carboxyl group is replaced by phosphorous acid moieties were synthesized and found to be potent inhibitors of the enzyme ( k inact / K i in the range 217,000–1,326,000 M −1 s −1 ). Cellular activity was demonstrated by inhibition of the generation of the N-terminal cleavage product Aα-(1–21) from the Aα chain of fibrinogen.

PROTEOGLYCAN-DEGRADING ACTIVITY OF HUMAN STROMELYSIN-1 AND LEUKOCYTE ELASTASE IN RABBIT JOINTS. QUANTITATION OF PROTEOGLYCAN AND A STROMELYSIN-INDUCED HABR FRAGMENT OF AGGRECAN IN SYNOVIAL FLUID AND CARTILAGE

The objective of this study was to compare the specificity and potency of recombinant human SLN-1 (rhSLN) and human leukocyte elastase (HLE) as proteoglycan (PG)-degrading enzymes after intraarticular injection into rabbits. Another objective was to evaluate the elicitation of a rhSLN-induced hyaluronan-binding region (HABR) fragment from rabbit aggrecan in joints using a polyclonal antiserum (anti-FVDIPEN) against the synthetic peptide, Phe-Val-Asp-Ile-Pro-Glu-Asn (FVDIPEN). The intraarticular injection of either activated rhSLN or HLE resulted in enzyme-specific quantitative release of PG fragments into synovial fluid. Based on the criteria used herein, HLE appears to be a more potent PG-degrading enzyme than SLN. Intraarticular injection of rhSLN also resulted in time- and dose-dependent release of a new HABR fragment of aggrecan (HABR-FMDIPEN) into both articular cartilage and synovial fluid. HABR-FVDIPEN is likely to be a good marker of matrix metalloproteinase (MMP)-induced degradation of aggrecan.