A B S T R A C T Extracts of human peripheral blood polymorphonuclear leukocyte granules, and two purified proteases derived from such extracts, an elastase and a chymotrypsin-like enzyme, degrade isolated bovine nasal cartilage proteoglycan at neutral pH. Viscosity studies indicate that the leukocyte granule extracts lack hyaluronidase activity and that their degradative effect on proteoglycan at physiological pH is due entirely to proteolytic action. Sepharose 4B gel chromatography and SDS-polyacrylamide gel electrophoresis of proteoglycan fractions treated with leukocyte granule enzymes at pH 7.0 indicate that they degrade one of the proteoglycan link proteins, release a fragment from the hyaluronic acid-binding portion of the proteoglycan subunit core protein, and break down the remainder of the proteoglycan subunit molecule into peptide fragments with varying numbers of chondroitin sulfate chains. Immunodiffusion studies indicate that the antigenic determinants of the proteoglycan subunit core protein and the link proteins survive treatment with granule proteases. Similar degradation of human articular cartilage proteoglycan by granule neutral proteases can be presumed to occur, in view of the similarity of structure of human articular and bovine nasal cartilage proteoglycans. The release of granule enzymes in the course of neutrophil-mediated inflammation can thus result in the Dr. Feinstein's permanent address is:

The purification of human proteolytic enzymes, i.e. two trypsins, two chymotrypsins and a t least one elastase from activated extracts of human pancreas has been accomplished by a combination of a,ffinity chromatography and conventional ion-exchange chromatography on XE-Xephadex. The enzymes thus obtained were homogeneous by several criteria : gel electrophoresis, equilibrium sedimentation and immunological assay. The molecular weight and amino-acid composition of each preparation was determined and compared with those of other mammalian enzymes. The two trypsins showed only partial immunological identity while the two chymotrypsins were identical immunologically. The data raises the question of the number of the zymogens present in the human pancreas.I n addition to the proteolytic enzymes, four protein inhibitors of proteolytic enzymes were also isolated from human pancreas. The procedure consisted of several steps that included gel filtration, affinity chromatography and isoelectric focusing. The two major components which accounted for most of the trypsin inhibitory activity were characterized and were apparently of the Kazal type. The more basic human pancreatic inhibitor had a p I of 8.7 and contained 48 amino acids with a molecular weight of 5200. The more acidic inhibitor had a PI of 6.5 and contained 39 amino acids with a molecular weight of 4300. Both inhibitors were capable of inhibiting bovine and two human trypsins. They did not inhibit two human chymotrypsins but had a weak capacity to inhibit bovine chymotrypsin.A large body of information has been gathered during the past decade concerning the enzymatic composition of pancreatic juices from various mammalian sources. However, there are only a few reports describing the composition and properties of human pancreatic proteins. This is mainly due to the scarcity of human pancreas and to the difficulties arising from the autolysis of the proteases.Buck et al. Abbreviations. EAhx-Trp-OMe, e-aminocaproyl-mtryptophan methyl ester ; Tos-Arg-OMe, N-p-tosyl-L-argininemethyl ester; Ac-Tyr-OEt, N-acetyl-L-tyrosine ethyl ester; Tos-Phe-CH,CI, ~-l-tosylamido-2-phenylethylchloromethylketone; Tos-Lys-CH,Cl, l-chloro-3-tosylamido-7-amino-2-heptanonc ; Dip-I?, di-isopropylfluorophosphate.Enzymes. Trypsin (EC 3.4.4.4) ; chymotrypsin (EC 3.4.4.5); elastase (ED 3.4.4.7).Definition. A,,, unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 a t 280 nm when measured in a 1-cm pathlength -cell.trypsin. Silberberg and Hadron [3] also reported the presence of two chymotrypsin isoenzymes, while Coan and Travis [5,6] found the presence of three forms of chymotrypsin. Differences in the isolation procedures employed, as well as the inherent autodigestabiliby of the pancreatic proteases, seem to be the main reason for these conflicting reports. The work presented here describes a method for the simultaneous isolation of human pancreatic enzymes : trypsin, chymotrypsin and elastase from frozen whole pancreas. The purification schemes were ...

A B S T R A C T The present work was undertaken to explore the effect of two purified neutral proteases derived from human peripheral blood polymorphonuclear leukocytes (PMN) on articular cartilage as a model of joint injury. Human leukocyte elastase and chymotrypsin-like enzyme, purified by affinity chromatography, released 'SO4 from labeled rabbit articular cartilage slices in vitro. Release of isotope was initially delayed, suggesting that either a lag in enzyme penetration occurs or that size of degradation fragments is a limiting factor in diffusion of label out of the tissue. The release of 'SO4 was inhibited by preincubation of elastase and chymotrypsin-like enzyme with human alpha 1-antitrypsin, or with their specific chloromethyl ketone inactivators, and the action of elastase was also inhibited by a monospecific antiserum to PMN elastase, freed of major serum proteinase inhibitors. Immunohistochemical staining procedures revealed the presence of PMN elastase inside the matrix of cartilage slices after a 20-min exposure of tissue to either the pure enzyme or crude PMN granule extract. Serum alpha 1-antitrypsin failed to penetrate into the cartilage slices under identical in vitro conditions. In association with the results reported in the accompanying paper, these findings suggest a model of cartilage matrix degradation by PMN neutral proteases in which local protease-antiprotease imbalance, coupled with different rates of penetration of protease and antiprotease into target tissue, plays a key role in accounting for matrix damage.Dr. Feinstein's permanent address is:

I. Turkey ovomucoid and chicken ovoinhibitor which have been found to be inhibitors of bovine trypsin and a-chymotrypsin can inhibit the esterolytic, proteolytic and elastolytic activities of porcine elastase. The formation of the complex between the elastase and each inhibitor could be demonstrated by means of electrophoresis a t p H 6.9. The complexes are formed in molar ratios of one to one. They are stable and no temporary inhibition could be observed.2. The complexes of porcine elastase with turkey ovomucoid and chicken ovoinhibitor inhibit bovine trypsin a t the same molar ratio as free inhibitors. The complexes of trypsin with these inhibitors inhibit elastase as do the free inhibitors. The complex of porcine elastase with turkey ovomucoid does not inhibit bovine chymotrypsin and the complex of chymotrypsin with this inhibitor does not inhibit elastase. The complex of porcine elastase with chicken ovoinhibitor inhibits bovine chymotrypsin but not the alkaline proteinase from Aspergillus sojae. The complex of this inhibitor with bovine chymotrypsin inhibits the porcine elastase while the complex iwith alkaline proteinase does not.It was concluded, therefore, that turkey ovomucoid has two independent binding sites, one for trypsin and the other for chymotrypsin or elastase and that chicken ovoinhibitor has three independent binding sites, one for trypsin, another for chymotrypsin and a third one for either elastase or alkaline proteinase.3. The complexes between porcine elastase and turkey ovomucoid or chicken ovoinhibitor are not adsorbed on elastin whereas elastase is. 4.Kunitz soybean and pancreatic trypsin inhibitors, Birk-Bowman soybean trypsin and chymotrypsin inhibitor, groundnut and h a bean trypsin and chymotrypsin inhibitors do not inhibit the esterolytic, proteolytic and elastolytic activities of porcine elastase, even in weight ratios in excess of 10 : 1.Although the tertiary structure and the catalytic site of porcine elastase are similar to those of bovine trypsin and chymotrypsin, its binding site and substrate specificity are quite different [I, 21. Elastase is not inhibited by most of the natural trypsin and chymotrypsin inhibitors [3]. Several reports described inhibition of porcine elastase by trypsin inhibitors from plants or animals 14-81 but in most cases only the non-specific proteolytic activity was inhibited while the elastolytic activity was almost unaffected An introduction of a highly specific esterase substrate for porcine elastase, N-acetyl-L-alanyl-Lalanyl-L-alanine methyl ester [I21 enhanced our studies on specific inhibition of porcine elastase by natural inhibitors.The present study describes the inhibition of porcine elastase by turkey ovomucoid and chicken ovoinhibitor and partial characterization of their complexes with this enzyme.

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