Human‐Polymorphonuclear‐Leucocyte Neutral Protease and Its Inhibitor

Abstract

Human polymorphonuclear leucocytes were obtained from the synovial fluids of patients with inflamed knee joints suffering either from Reiter's syndrome or from rheumatoid arthritis. The polymorphonuclear leucocytes were collected by gentle centrifugation followed by disruption and their subcellular fractionation by centrifugation in 0.34 M sucrose to provide a granule fraction and a post-granule supernatant fraction. 0.5 M KCl extraction of the granule fraction yielded neutral protease activity, similar to trypsin, when assayed against fluorescein-labelled polymeric collagen fibrils. The post-granule supernatant fraction contained an inhibitor towards the neutral protease and trypsin. The inhibition of the neutral protease was found to be time-dependent, this inhibition being released after 1.5 -2 h. In contrast, the inhibition of trypsin was irreversible and this property was used to devise an assay procedure for the inhibitor. Kopitar and Lebez [l] demonstrated the presence of a neutral protease associated with the granule fraction obtained from pig polymorphonuclear leucocytes. They also showed that this enzyme could be inhibited by the addition of a protein fraction present in the post-granule supernatant fraction. At the same time, a similar neutral protease was obtained from extracts of polymorphonuclear leucocges present in the synovial fluid of patients with rheumatoid arthritis [2]. This neutral protease behaved similarly to trypsin when assayed with fluorescein-labelled polymeric collagen fibrils as substrate [2]. These collagen fibrils contained three fluorescein-labelled amino groups located in the telopeptide regions of each tropocollagen molecule within the polymer. Kinetic studies showed that two fluorescein-labelled groups per tropocollagen molecule were removed from the telopeptide regions by both the neutral protease and by trypsin.Since the neutral protease was assayed in the presence of the soluble fraction of the cell sonicate [2], the question arose as to how this enzyme could be active in the presence of the inhibitor described by Kopitar and Lebez [l]. This system has now been re-examined, the presence of the inhibitor has been confirmed [l] and the reason for the lack of inhibition with the polymorphonuclear leucocyte sonicates [2] has been elucidated. The present study shows this neutral protease inhibition to be time dependent and to be reversed after a period of 1.5-2.0 h at 37 "C with the subsequent total recovery of neutral protease activity towards fluorescein-labelled polymeric collagen fibrils. MATERIALS AND METHODSCrystalline trypsin (treated with L-2-tosylamido-2-phenylethyl chloromethyl ketone) was obtained from Worthington. Synovial fluid was obtained from the knee of a patient with Reiter's syndrome; 22 ml of fluid containing a total of approximately 1.4 x lo5 polymorphonuclear leucocytes was obtained.A similar number of cells were obtained from the synovial fluid removed from the knee of a rheumatoid arthritic patient. Both preparations of cells provided a neutral prote...

Evidence for the Inhibition of Trypsin by Thiols. The Mechanism of Enzyme-Inhibitor Complex Formation

“…Work with sonicates of tumor cells demonstrated the presence of latent forms of neutral protease and collagenase [3], activated by trypsin, and an inhibitor of this enzyme in the post-granule supernatant fraction [4]. This inhibitor was shown to inactivate trypsin [4] by the assay system previously described for the inhibitor of the polymorphonuclear leucocyte neutral protease [5]. We wish to present evidence for the presence of one or more significant disulphide bond(s) within both trypsin and the neutral protease required for the maintenance of the active form of these enzymes and to report the common Abbreviations.…”

Determination of Leukocyte Elastase-Inhibitor Complexes and Leukocyte Neutral Proteinase Inhibitor by Enzyme Immunoassays. Leukocyte Elastase-Inhibitor Complexes in Porcine Blood, III.

“…This is in contrast to human leukocytes, in which only small amounts of inhibitors have been described (10).…”